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VIRUS
Reviews and Research
Journal of the Brazilian Society for Virology
Volume 15, October 2010, Supplement 1
Editors:
Hermann G. Schatzmayr, Editor
Ortrud Monika Barth, Co-Editor
Editorial Board
Alice K. I. Nagata
Ana Cláudia Franco
André N. Dusi
Bergmann M. Ribeiro
Célia R M Barardi
Clarissa Damaso
Cláudia L. Vitral
Cláudia M O. Simões
Cláudio A. Bonjardim
Davis F. Ferreira
Edson E. da Silva
Eurico A. Neto
Fernando R. Spilki
Francisco M. Zerbini Jr.
Iara M. Trevisol
Janice R. C. Zanella
Luciana J. da Costa
Luiz Caron
Luiz Tadeu M. Figueiredo
Paulo E. Brandão
Paulo M. Roehe
Regina M. B. Vitral
Rejane Schaefer
Viviane F. Botosso
Adress
Pavilhão Hélio e Peggy Pereira - 1º Andar - Sala B103 - Instituo Oswaldo Cruz - Fiocruz
Avenida Brasil, 4365, CEP 21045-900 - Rio de Janeiro, BRASIL
Phone 55 21 2562 1707 - Fax 55 21 2260 4866
e-mail: hermann@ioc.fiocruz.br
barth@ioc.fiocruz.br
denilde@ioc.fiocruz.br
sbv@ioc.fiocruz.br
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BRAZILIAN SOCIETY FOR VIROLOGY DIRECTIVE BOARD (2009-2010)
Officers
President
Vice-president
First Secretary
Second Secretary
First Treasurer
Second Treasurer
Paulo M. Roehe, RS
Eduardo F Flores, RS
Célia R M Barardi, SC
Cláudia M Oliveira Simões, SC
Fernando R. Spilki, RS
Rejane Schaefer, SC
Councillors
Luiz Tadeu M Figueiredo (2009-2010)
Eurico Arruda Neto (2009-2010)
Iara Trevisol (2009-2010)
Area Representatives
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Animal Virology
Janice R. C. Zanella, SC
Paulo Eduardo Brandão, SP
Basic Virology
Davis F. Ferreira, RJ
Clarissa R. A. Damaso, RJ
Human and Public Health
Regina M. B. Martins, SP
Edson E. da Silva, RJ
Plant and Invertebrates
Alice K. Inoue, RJ
Bergmann M. Ribeiro, MG
Immunobiologicals
Claudia L. Vitral, RJ
Viviane F. Botosso, SP
Board of Examiners – Hélio Gelli Pereira
Bergmann Morais Ribeiro
Célia R. M. Barardi
Davis F. Ferreira
Eurico de Arruda Neto
Regina Maria Bringel Martins
Invited Speakers
Ana Cláudia Franco, UFRGS
Andréa Faria de Almeida, UFRJ
Angélica Cristine de Almeida Campos, USP
Célia Regina Monte Barardi, UFSC
Ciro de Quadros, Sabin Vacine Institute
Clarissa R A Damaso, UFRJ
Claudia M O Simões, UFSC
Claudio A Bonjardim, UFMG
Cláudio César Cirne dos Santos, Fiocruz
Davis Fernandes Ferreira, UFRJ
Dennis T Brown, North Carolina State University
Dwight Lynn, INSell Consulting
Eckard Wimmer, Stony Brook University
Eduardo Nascimento, UFPE
Eliane Veiga da Costa, Fiocruz
Elizabeth Pacheco Batista Fontes, UFV
Elliot W Kitajima, ESALQ/USP
Erna G. Kroon, UFMG
Eurico de A. Neto, USP
Fernando Osorio, University of Nebraska
Fernando R. Spilki, FEEVALE
Flávio G. da Fonseca, UFMG
Fumio H Ito, USP
Giovanni Coelho, Ministério de Saúde
Graciela Andrei, Rega Institute For Medical
Research
Gustavo Delhon, University of Illinois at Urbana
Helio Montassier, UNESP
Jane Cook, University of Liverpool
Janice R. C. Zanella, EMBRAPA
João Galizzi Filho, UFMG
João Renato R Pinho, USP
João Trindade Marques, UFMG
Jorge A M Rezende, ESALQ/USP
Jorge Caetano Junior,
José Pascoal Simonetti, Fiovruz
Juan Cristina, Universidad de La Republica
Juliana V. C. Oliveira, USP
Kelly Lager, National Animal Disease Center
Laura H V G Gil, CPqAM - Fiocruz
Ledy do Horto dos Santos, UFF
Luciana Barros de Arruda, UFRJ
Luciana J Costa, UFRJ
Maria Inês Zanolli Sato, CETESB
Maria Paula Del Medico Zajac, INTA
Mario C. S. Brum, UNIPAMPA
Marta Rojas,
Mikael Berg, Swedish Universtity of
Agriculutural
Murilo Zerbini, UFV
Paulo J Zimmermann, UFCSPA
Poliane Alfenas, UFV
Raquel Hernandez, North Carolina State
Univesity
Renata Dezengrini, UFMT
Renato de O Resende, UNB/Brasília
Ricardo Diaz, Unifesp
Rosane Silva, UFRJ
Ruben Donis, Center for Disease Control
Sabrina E de Matos Almeida, FEPPS
Samuel Cibulski, UFRGS
Sandra Regina R Simonetti, Fiocruz
Selma Gomes, Fiocruz
Sylvie German-Retana, INRA - Bordeaux
Tereza Cristina Cardoso, UNESP
Vagner Lunge, ULBRA
Verónica Rajal, Universidade de Salta
Viviane Botosso, USP
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Financial Support
CNPQ – Conselho Nacional de Desenvolvimento Cientifico e Tecnológico
FAPERGS – Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul
FEPAGRO – Fundação Estadual de Pesquisa Agropecuária
Exhibitors
BIOSAFE – BIOSSEGURANÇA DO BRASIL LTDA
ALKA – TECNOLOGIA EM DIAGNÓSTIOS
SINAPSE BIOTECNOLOGIA
BIOSYSTEMS COM IMP EQUI LAB LTDA
FAIRPORT LTDA
DAFRATEC
UNISCIENCE
SIGMA-ALDRICH
LOBOV CIENTIFICA
BIOEASY
LOCCUS DO BRASIL LTDA
NOVA ANALITICA
BSTEC SOLUÇÕES EM BIOSSEGURANÇA
Organizers
Tribeca Eventos
Tribeca Turismo
General Information
Secretary Schedule
October, 17th – 02:0 pm to 08:00 pm
October, 18th, 19th and 20th - 07:0am to 07:00pm
Identification Card
The identification card Will be required for attending all activities in the meeting.
Identification Card – Escorts
Participantes should show the identification card for drivers/escorts in order to have access to
transport to and from accomodation as well as for accessing social activities.
Media Desk (for lecturers only)
The media desk will be open as scheduled for the secretary of the meeting.
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Data - files with presentations - must be delivered at the media desk at least 2 hours before the
scheduled time for the presentation. Please note that personal computers will not be allowed in
lectures. Presentations will be copied and made available to members of SBV after the meeting at the
institutional homepage unless unauthorised by the speakers.
VIP Room
A VPI room will be available for lecturers, invited persons and SBV staff.
Certificates
The certificates of presentation/participation will be available at the secretary of the event on the last day
of the meeting. Identification cards will be required.
Travel Agency
Tribeca Tourism is the Official Travel Agency of the meeting. For hotel information, reservations,
transportation and flights, please contact Tribeca’s office at the meeting. Information on sightseeing
tours, visits to the Vale dos Vinhedos and many other pleasant touristic activities will also be available
at Tribeca’s office, upon request.
Transfer in Gramado
A transfer service will be available from the official hotels to the Meeting Center of FAURGS, home to the
XXI ENV/V ENMercosur. Please ask for the transfers’ schedule at the reception desk of your hotel.
Transfer - Porto Alegre
All the participants that required transfer to Porto Alegre must confirm return schedules at the Tribeca’s
Office. Please bear in mind that you must leave Gramado at least 4 hours before you scheduled flight
departure.
Board of Examiners – Hélio Gelly Pereira Award
Bergmann Morais Ribeiro
Célia R. M. Barardi
Davis F. Ferreira
Eurico de Arruda Neto
Regina Maria Bringel Martins
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Telephone Numbers
Banco Bradesco – 55 (54) 3286.1955
Banco do Brasil – 55 (54) 3286.2724
Banco HSBC – 55 (54) 3286.2039
Banco Itaú – 55 (54) 3286.2464
Banco Santander – 55 (54) 3286.1308
Banco Sicredi – 55 (54) 3295.1100
Banrisul – 55 (54) 3286.8303
Brigada Militar – 190 ou 55 (54) 3286.1261
Cine Embaixador (Palácio dos Festivais) – 55 (54)
3286.1058
Corpo de Bombeiros – 193 ou 55 (54) 3286.1549
Delegacia de Polícia – 55 (54) 3286.2300
FAURGS – 55 (54) 3286.3075
Hospital Arcanjo São Miguel – 55 (54) 3286.4594
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Centro de Cultura – 55 (54) 3286.4323
Igreja do Relógio – 55 (54) 3286.3676
Igreja São Pedro – 55 (54) 3286.1187
Informações Turísticas – 55 (54) 3286.1475
Polícia Rodoviária Federal – 55 (54) 3266.2222
Posto de Saúde Central – 55 (54) 3286.1236
Prefeitura Municipal – 55 (54) 3286.0200
Rodoviária – 55 (54) 3286.1302
Secretaria de Turismo – 55 (54) 3286.0220
Sierra Park – 55 (54) 3286.9900
Tele Táxi – 55 (54) 3286.1229 e 55 (54) 3286.1230
Bombeiros: 193 ou 55 (54) 3286.1549
Posto de Saúde: 55 (54) 3286.1236
SCIENTIFIC PROGRAM
XXI National Meeting of Virology
October, 17-20, 2010
Gramado, RS, Brasil
October 17th
Sunday
October 18th
Monday
07:00pm to
09:00pm
Openning
Ceremony
Conferece #1
Theme: Influenza vírus
Speaker: Dr. Ruben O. Donis,
Chief of the molecular virology and vaccines branch at the U.S.
Centers for Disease Control and Prevention.
09:00pm to
00:00am
Welcome Cocktail
07:30am to
08:20am
Courses
Courses:
1) Novas estratégias de sequenciamento com enfoque em
genomas virais.
Speaker: Dra. Rosane Silva, UFRJ
2) Introdução sobre os mecanismos de replicação dos genomas
virais
Speaker: Dra. Luciana J. Costa, UFRJ
Dr. Davis F. Ferreira, UFRJ
3) Biossegurança e suas aplicações em virologia
Class #1: Níveis de Biossegurança. Avaliação de Risco. Agentes
Virais e Critérios de Biossegurança. Biossegurança no Trabalho em
Campo.
Speaker: Dr Edison Luiz Durigon, USP
Dra Viviane Botosso, USP e Instituto Butantan.
Class #2: Aspectos gerais sobre comissões de Biossegurança e
serviços de Inspeções em laboratórios.
Speaker: Dra Sandra Regina Rodrigues Simonetti, IOC/Fiocruz/RJ
Class #3: Gestão em Biossegurança. Equipamentos de Proteção
Individual (EPI) para os diferentes Níveis de Biossegurança-NB2 e
NB3. Aula Interativa: demonstração prática sobre o uso de diferentes
EPI.
Speaker: Dr. José Pascoal Simonetti, IOC/Fiocruz/RJ
4) Curso de extensão para professores
Dengue/Febre amarela.
Speaker: Dra Ledy do Horto dos Santos Oliveira, UFF
Viroses sexualmente transmissíveis.
Speaker: Dra Ledy do Horto dos Santos Oliveira, UFF
08:30am to
10:30am
Round Tables
Round Tables #1
Antiviral
Theme: “Topics on Antiviral Research in Brazil”
Speaker: Dr Cláudio César Cirne dos Santos, FIOCRUZ e UFF
Speaker: Dra Cláudia M. O. Simões, UFSC – “Antiherpes activity of
cardenolide derivatives.”
Speaker: Dra Clarissa Damaso, UFRJ – “Cidofovir and ST-246:
antiviral drugs against Cantagalo virus.”
Round Table #2
Human Virology
01: Vírus Influenza A – Situação no RS.
Speaker: Dr. Paulo J. Zimmermann Teixeira, UFCSPA
02: Caracterização de HIV local – importância do subtipo C no RS.
Speaker: Dra. Sabrina E. de Matos Almeida, FEPPS
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SCIENTIFIC PROGRAM
03: “Diagnóstico e Epidemiologia Molecular das Hepatites Virais B e
C no Rio Grande do Sul”
Speaker: Dr. Vagner Lunge da ULBRA-CANOAS
Round Table # 3
Invertebrates
Theme: Cultura de célula de insetos.
Speaker: Dr. Dwight E. Lynn, INSell Consulting: “Development of
insect cell lines: Methodology and some success stories”.
Speaker: Dra. Juliana Velasco de Castro Oliveira, USP: “Estudo da
rede gênica do nucleopoliedrovírus Anticarsia gemmatalis (AgMNPV2D) em linhagens de células de inseto distintos, e sua influência na
expressão dos genes do hospedeiro durante a infecção”.
Speaker:Dra. Andréa Farias de Almeida, UFRGN: “Estratégias de
Produção in vitro de baculovírus”.
Round Table #4
Animal Virology
Theme: Vírus da bronquite infecciosa aviária
Speaker: Dr Helio Montassier, UNESP Jaboticabal “Diversidade de
vírus da bronquite infecciosa no Brasil”.
Speaker:Dra Jane Cook, “Protectotipos em IBV” Universidade de
Liverpool, Reino Unido.
Speaker: Dra. Tereza Cristina Cardoso, Profa. Adjunta de Virologia da
UNESP Araçatuba, “Coronavírus de perus: diversidade molecular e
patologia”
Round Table #4.1
Animal Virology
Theme: “Chicken anemia virus-like”.
Speaker: Dra Ana Cláudia Franco, UFRGS
Round Table #5
Plant Virology - Avanços na interação-planta-patógeno
Speaker: Dra. Elizabeth Pacheco Batista Fontes, Dep. de Bioquimica
e Biologia Molecular, UFV.
Speaker: Dra. Poliane Alfenas, Dep. de Microbiologia, UFV.
Speaker: Dr. Thierry Candresse, INRA Bordeaux, França “Translational research against plant viruses: unravelling plant-virus
interactions and using the information to develop Plum pox virusresistant Prunus crops”
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10:30am to
11:00am
Coffee-break
11:00am 12:30pm
Conference
Conference #2:
Antiviral
Speaker: Dra Graciela Andrei (khatolieke Universiteit Leuven, Bélgica)
“Monitoring Drug Resistance for Herpesviruses”
12:30pm to
02:00pm
Lunch
SCIENTIFIC PROGRAM
October 19th
Tuesday
02:00pm 03:10pm
Conference
Conference #3:
Basic Virology
Speaker: Dr. Eckard Wimmer, University of Goettingen, “Molecular
biology of poliovirus replication and basis of picornaviral
pathogenesis”.
03:15pm to
04:45pm
Oral Presentation
1. Basic Virology
2. Invertebrate and Plant Virology
3. Immunobiologicals and Antiviral
4. Environmental Virology
5. Animal Virology
03:15pm to
04:45pm
Oral Presentation I - Hélio Gelli Pereira Award
04:45pm to
05:15pm
Coffee-break
05:15pm to
06:25pm
Conference
Conference #4
Animal Virology Theme: Poxvirus, viral pathogenesis - molecular
mechanisms of viral virulence and host range.Speaker: Dr. Gustavo
Delhon, University of Nebraska – Lincoln Center for Virology
06:30pm to
07:30pm
Sessão de painéis 1
07:30pm to
09:30pm
Break
09:30pm to
00:00am
Cultural Programm
07:30am to
08:20am
Courses
Courses:
1) Novas estratégias de sequenciamento com enfoque em
genomas virais.
Speaker: Dra. Rosane Silva, UFRJ
2) Introdução sobre os mecanismos de replicação dos genomas
virais
Speaker: Dra. Luciana J. Costa, UFRJ
Dr. Davis F. Ferreira, UFRJ
3) Biossegurança e suas aplicações em virologia
Class #1: Níveis de Biossegurança. Avaliação de Risco. Agentes
Virais e Critérios de Biossegurança. Biossegurança no Trabalho em
Campo.
Speaker: Dr Edison Luiz Durigon, USP
Dra Viviane Botosso, USP e Instituto Butantan.
Class #2: Aspectos gerais sobre comissões de Biossegurança e
serviços de Inspeções em laboratórios.
Lecturer: Dra Sandra Regina Rodrigues Simonetti, IOC/Fiocruz/RJ
Class #3: Gestão em Biossegurança. Equipamentos de Proteção
Individual (EPI) para os diferentes Níveis de Biossegurança-NB2 e
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SCIENTIFIC PROGRAM
NB3. Aula Interativa: demonstração prática sobre o uso de diferentes
EPI.
Speaker: Dr. José Pascoal Simonetti, IOC/Fiocruz/RJ
4) Curso de extensão para professores
Dengue/Febre amarela.
Speaker: Dra Ledy do Horto dos Santos Oliveira, UFF
Viroses sexualmente transmissíveis.
Speaker: Dra Ledy do Horto dos Santos Oliveira, UFF
Viroses de veiculação hídrica.
Speaker: Dr Fernando Spilki, Feevale
08:30am to
10:30am
Round Table
Round Table #6
Environmental Virology
Speaker: Dr. Maria Inês Zanolli Sato, CETESB “Brazilian Legislation
for Drinking Water: monitoring of enteric vírus”
Speaker: Dr. Eliane Veiga da Costa, Laboratório de Enterovírus da
Fiocruz “Environmental monitoring in the global polio eradication”
Speaker: Dr. Veronica Rajal, Universidad de Salta, Argentina
“Water and Human Health in Latin America”
Speaker: Dr.Célia Regina Monte Barardi, UFSC
“Deteccion and viability assays for fecal-oral transmitted viruses in
water and mollusks”
Round Table #7
Plant Virology
Theme: “Passado, presente e futuro da fitovirologia no Brasil”.
Speaker: Dr Elliot W. Kitajima – ESALQ/USP, Piracicaba: “Histórico
da fitovirologia brasileira”
Speaker: Dr Jorge A. M. Rezende, ESALQ/USP, Piracicaba:
“Contribuição das técnicas biológicas para estudos com vírus de
plantas”
Speaker: Dr Renato de O. Resende, UNB, Brasília: “Contribuição das
técnicas moleculares para estudos com vírus de plantas”
Round Table #8
Human Virology – Variabilidade e evolução genômica
Speaker: Dra. Selma Gomes, FIOCRUZ/RJ – “Variabilidade do HBV”
Speaker: Dr Juan Cristina, Universidad de la Republica - “Genetic
variability of Hepatitis A virus”
Speaker: Dr Ricardo Diaz, Unifesp – HIV
Speaker: Dra Rosane Silva, UFRJ - “Variabilidade genética do vírus
da Hepatite C (HCV) obtidos de pacientes virgens de tratamento e em
resposta à terapia com Interferon e Ribavirina”.
Round Table #9
Human Virology
Dengue e outras flaviviroses brasileiras
Speaker: Dra Raquel Hernandez, “Host Range Mutations to obtain
Dengue Virus Vaccine”
Speaker: Dr Giovanni Coelho, “O Plano Nacional de Controle de
Dengue”
Speaker: Dr Luiz Tadeu Moraes Figueiredo, FMRP-USP: “Outros
flavivirus causadores de doença humana no Brasil”
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SCIENTIFIC PROGRAM
Round Table #10
Animal Virology
Theme: Viroses emergentes e re-emergentes
Speaker: Porcine Reproductive and Respiratory Syndrome
Virus (PRRSV)
Speaker: Dr. Fernando Osório, University of Nebraska
“Pathogenesis of PRV isolates from wild pigs and the pathogenesis
studies of the pandemic influenza virus studies in swine”
Speaker: Dr. Kelly M. Lager, National Animal Disease Center, USDAARS
Palestra 3: West Nile Virus (WNV)
Speaker: Dra. Laura H. V. G. Gil, CPqAM – Fiocruz
10:30am to
11:00am
Coffee-break
11:00am to
12:30pm
Conference
Conference #5
Animal VirologyTheme: “Immune modulation of negative strand
RNA viruses, with special emphasis on influenza and Newcastle
disease virus”
Speaker: Dr. Hans Mikael Berg, Swedish University of Agricultural
Sciences, de Uppsala, Suécia.
12:30am to
02:00pm
Lunch
02:00pm to
03:10pm
Conference
Conference #6:Basic Virology
Theme: “Mechanism of Cell Penetration by Alpha Viruses”Speaker: Dr.
Dennis T. Brown, North Carolina State University
03:15pm to
04:45pm
Oral Presentation
1. Basic Virology
2. Invertebrate and Plant Virology
3. Immunobiologicals and Antiviral
4. Environmental Virology
5. Animal Virology
03:15pm to
04:45pm
Oral Presentation II - Hélio Gelli Pereira Award
04:45pm to
05:15pm
Coffee-break
05:15pm to
06:25pm
Conference
Conference #7
Human Virology
Theme: Comemorativa aos 30 anos de erradicação da varíola
Palestrante: Dr. Ciro de Quadros, Sabin Vaccine Institute
06:30pm to
07:30pm
Sessão de painéis 2
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SCIENTIFIC PROGRAM
October, 20th
Wednesday
07:30pm to
09:30
Break
09:30pm to
00:00am
Cultural Programm
7:30 - 8:20
Courses
Courses:
1) Novas estratégias de sequenciamento com enfoque em
genomas virais.
Speaker: Dra. Rosane Silva, UFRJ
2) Introdução sobre os mecanismos de replicação dos genomas
virais
Speaker: Dra. Luciana J. Costa, UFRJ
Dr. Davis F. Ferreira, UFRJ
3) Biossegurança e suas aplicações em virologia
Class #1: Níveis de Biossegurança. Avaliação de Risco. Agentes
Virais e Critérios de Biossegurança. Biossegurança no Trabalho
em Campo.
Speaker: Dr Edison Luiz Durigon, USP
Dra Viviane Botosso, USP e Instituto Butantan.
Class #2: Aspectos gerais sobre comissões de Biossegurança e
serviços de Inspeções em laboratórios.
Lecturer: Dra Sandra Regina Rodrigues Simonetti, IOC/Fiocruz/RJ
Class #3: Gestão em Biossegurança. Equipamentos de Proteção
Individual (EPI) para os diferentes Níveis de Biossegurança-NB2
e NB3. Aula Interativa: demonstração prática sobre o uso de
diferentes EPI.
Speaker: Dr. José Pascoal Simonetti, IOC/Fiocruz/RJ
4) Curso de extensão para professores
Dengue/Febre amarela.
Speaker: Dra Ledy do Horto dos Santos Oliveira, UFF
Viroses sexualmente transmissíveis.
Speaker: Dra Ledy do Horto dos Santos Oliveira, UFF
Viroses de veiculação hídrica.
Speaker: Dr Fernando Spilki, Feevale
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08:30am to
09:45am
Conference
Conference #8:
Plant and Invertebrates
Theme: “Functional analysis of plant-potyvirus interactions”
Speakers: Dra. Sylvie German-Retana, INRA Bordeaux-Aquitaine,
França
09:45am to
10:15am
Coffee-break
10:15am to
12:00pm
General Meeting of SBV. Helio Gelli Pereira Awards
12:00am to
13:30pm
Lunch
SCIENTIFIC PROGRAM
01:30pm to 03:30pm
Round Table
Round Table #11
Animal Virology
Theme: BoHV-1/BoHV-5
“Update about BoHV-1/5 research”.
Speaker: Dra. Maria Paula Del Medico Zajac, INTA
“Produção e caracterização de cepas recombinantes do herpesvírus
bovino tipo 5”.
Speaker: Dr. Mário Celso Sperotto Brum, UNIPAMPA
“Bovine herpesvirus 5 induces an overproduction of nitric oxide in the
brain of rabbits”.
Speaker: Dra. Renata Dezengrini, UFMT
““Detecção molecular de infecções por herpesvírus bovino no
Brasil”.
Speaker: Dra. Ana Cláudia Franco, UFRGS
Round Table #12
Basic Virology
Theme: “Interferons - da bancada a clínica”
Speaker: Dr João Renato R. Pinho, Departamento de
Gastroenterologia, Faculdade de Medicina da USP, ”Advances in the
treatment of hepatitis C virus with interferon alfa and antivirals”.
Speaker: Dr João Trindade Marques - Department Of Bichemistry &
Molecular Biology - Northwestern University - Evanston, IL, USA,
“Role played by Interferon-regulatory factor 3 (IRF-3) in the antiviral
response”.
Speaker: Dr Cláudio A. Bonjardim - Grupo de Transdução de Sinal Laboratório de Vírus- ICB – UFMG, “An update in the role played by
Interferons in the control of innate and adaptive immunity”.
Speaker: Dr João Galizzi Filho, Faculdade de Medicina – UFMG - “O
interferon alfa no tratamento das hepatites virais crônicas B e C”.
Round Table #13
Basic Virology – Imunologia das infecções virais
Speaker: Dr Flavio G. da Fonseca, UFMG “Imuno-modulação
negativa em infecções agudas, zoonóticas, cusadas pelo vaccinia
virus”
Speaker: Dr Eduardo J. M. Nascimento, University of Pittsburgh,
“Involvement of Host Innate Immune Responses on Dengue
Vasculopathy”.
Speaker: Dra Luciana Barros de Arruda, UFRJ. “Modulação do
tráfego intracelular de antígenos e utilização de células dendríticas
autólogas como potenciais estratégias de vacinação anti-HIV”
Round Table #14
Basic Virology – Patogênese das infecções virais
Componentes:
Speaker: Dra Erna Kroon, UFMG – “Patogênse de dengue-3”
Speaker: Dr Murilo Zerbini, UFV – “Patogênese em begomovírus”
Speaker: Dr Eurico de Arruda Neto, USP - “Patogênese de Infecção
por vírus Oropouche”.
Round Table #15
Animal Virology – Raiva & Aftosa
Componentes:
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SCIENTIFIC PROGRAM
Speaker: Dr Fumio H. Ito, FMVZ-USP - “Patogenia do vírus da
raiva”
Speaker: Dr Samuel Cibulski, UFRGS
Speaker: Dr. Victor Emmanoel Vieira Saraiva,
PANAFTOSA-OPS/OMS - “Perspectiva de erradicação da Febre
Aftosa no Brasil e na América Latina”
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01:30pm to
06:30pm
Workshop
Workshop
Theme: Porcine Reproductive and Respiratory Syndrome Virus
(PRRSV)
Speaker: Dr. Fernando Osorio, University of Nebraska-Lincoln, EUA.
Título: “Current Frontiers in PRRSV Research”
Speaker: Dra. Marta Rojas, Servicio Agricola y Ganadero, Chile.
Título: “Programa oficial de erradicação do PRRSV no Chile e
Diagnóstico inicial da infecção pelo PRRSV no Chile”
Speaker: Dra. Janice Zanella, EMBRAPA CNPSA
Título: “Estudos de diagnóstico da infecção pelo PRRSV no Brasil”
Speaker: Dr. Jorge Caetano Junior – MAPA
Título: “Medidas para redução do risco de ingresso da PRRS no
Brasil”.
06:30pm
Encerramento
SCIENTIFIC PROGRAM
ORAL AND POSTERS PRESENTATION
Day and Schedule of poster evaluation:
The poster must be fixed at 08:00am to 09:00am on the day of exhibition according with the
area of virology.
October 18th (Monday) – Session 01:
Animal Virology
Basic Virology
Plant Virology
Immunobiologicals
October 19th (Tuesday) – Session 02:
Environmental Virology
Human and Healty Virology
The posters will be evaluate on October 18th and 19th, at 06:30pm to 07:30pm
ORAL PRESENTATION – Approved Papers
Workshop 1: Environmental Virology
October 18th (Monday)
03:15pm to 04:45pm
OP-1
Corrêa, A.A.; Souza, D.S.M.;
Moresco, V.; Kleemann, C.R.;
Ramos, A.P.D.; Garcia, L.A.T.;
Barardi, C.R.M.
STABILITY OF HUMAN ADENOVIRUS, MURINE
NOROVIRUS, AND HEPATITIS A VIRUS IN SEAWATER WITH
AND WITHOUT U.V. IRRADIATION
OP-2
Comerlato, J.; Kluge, M.; Luz,
R.B.; Rodrigues, M.T.; Silva,
J.V.S.; Fabre, R.B.; Oliveira, L.K.;
Fontana, T.; Spilki, F.R.
COMPARISON OF VIRAL AND BACTERIAL CONTAMINATION
IN SURFACE AND GROUNDWATER SAMPLES FROM DAIRY
FARMS AT THE PARANHANA WATERSHED, SOUTHERN
BRAZIL.
OP-3
Barbosa, J.E.F.; Pereira, P.S.;
Neponuceno, A.M.; Crapez, M.;
Giongo, V.; Paixão, I.C.P.
EVALUATION OF THE INFLUENCE OF ENVIRONMENTAL
PARAMETERS IN A MARINE VIROSPHERE- EUTROPHIC
ESTUARY OF GUANABARA BAY-RJ
17
SCIENTIFIC PROGRAM
Workshop 2: Animal Virology
October 18th (Monday)
03:15pm to 04:45pm
OP-1
Simão, G.M.R.S.; Júnior, A.S.; Silva, F.;
Ferreira, H.C.C.; Lobato, Z.I.P.; Fietto,
J.L.R.; Almeida, M.R.; Fausto, M.C.
OP-2
Freitas, T.R.P.; Lyra, T.M.P.
OP-3
Felippe, P.A.N.; Silva, L.H.A.; Sakata, S.T.;
Martini, M.C.; Santos, M.M.A.B.; Arns,
C.W.
CORONAVIRUSES FROM WILD AVIAN SPECIES IN
BRAZIL BETWEEN 2005 AND 2006: DETECTION AND
CHARACTERIZATION.
OP-4
Gardinali, N.R.; Barry, A.F.; Otonel, R.A.A.;
Moraes, D.A.; Molinari, B.L.D.; Bodnar, L.;
Ribeiro, J.; Leme, R.A.; Alfieri, A.F.; Alfieri,
A.A.
DETECTION OF HEPATITIS E VIRUS IN A
SLAUGHTERHOUSE IN BRAZIL
OP-5
Campos, A.C.A.; Araújo, D.B.; Rodrigues,
C.S.; Romano, C.M.; Cunha, E.M.S.;
Sacramento, D.R.V.; Zanotto, P.M.Z.
Durigon, E.L.; Lazarini, S.R.F.
PHYLOGENETIC ANALYSIS OF RABIES VIRUS
PHOSPHOPROTEIN AND MATRIX PROTEIN FROM
BRAZILIAN
VARIANT
MAINTAINED
BY
HAEMATOPHAGOUS BATS Desmodus rotundus.
SUBCLINICAL DISEASE ASSOCIATED WITH
INFECTION BY Porcine circovirus 2 (PCV-2) ON SWINE
HERD
AFRICAN SWINE FEVER VIRUS IN BRAZIL: HISTORIC
AND ACHIEVEMENTS OVER LABORATORIAL VIRUS
AND ANTIBODY SCREENING IN THE EMERGENCE
PHASE
Workshop 3: Basic Virology
October 18th (Monday)
03:15pm to 04:45pm OP-1
Gavioli, A.F.; Vidotto, A.; Morais, A.T.S.;
Nogueira, M.L.
CHARACTERIZATION OF THE INTERACTION
BETWEEN YELLOW FEVER VIRUS NS5 WITH A
HUMAN GIPC1-PDZ PROTEIN.
OP-2
Costa, S.; Aguiar, R.; Silva, E.; Costa, L.
HUMAN IMMUNODEFICIENCY VIRUS TYPE 1 (HIV-1)
TRANSLATION IS REGULATED BY POLIOVIRUS
PROTEASE 2A
OP-3
Afonso, P.P.; Mermelstein, C.S.; Cunha e
Silva, N.L.; Damaso, C.
ANALYSIS OF VIRUS-HOST CELL INTERACTIONS
DURING COTIA VIRUS INFECTION
OP-4
Carvalho, C.A.M.; Sousa Júnior, I.P. Silva,
J.L.; Gomes, A.O.
SHEDDING LIGHT ON THE ENTRY OF A NEW WORLD
ALPHAVIRUS INTO HOST CELLS
OP-5
Figueira, M.; Mendonça, M.; Volotão, E.;
Leite, J.
MOLECULAR ANALYSIS OF VP1, VP2, VP3, VP4, AND
VP7 GENES OF GROUP A ROTAVIRUS STRAINS
GENOTYPE G5 CIRCULATING IN BRAZIL FROM 1986
TO 2005
18
SCIENTIFIC PROGRAM
Workshop 4: Human and Heath Virology
October 18th (Monday)
03:15pm to 04:45pm
OP-1
Albuquerque, A.C.C.; Pereira, A.R.S.;
Almeida, T.A.N.
VACCINATION AGAINST HEPATITIS B AMONG
DENTIST奪S SURGEON AND STUDENTS OF THE
ODONTOLOGY奪S COURSE OF ASSOCIATION
CARUARUENSE OF HIGHER EDUCATION (ASCES).
OP-2
Chagas, B.S.; Batista, M.V.A.; Crovella, S.;
Freitas, A.C.
NEW VARIANTS OF E6 AND E7 ONCOGENES OF
HUMAN PAPILLOMAVIRUS TYPE 31 IDENTIFIED IN
NORTHEASTERN BRAZIL AND THEIR RELATIONSHIP
WITH PREDICTED T-CELL EPITOPES
OP-3
Lemos, M.L.; Santos, E.O.; Pedrosa,
C.M.S.; Silva, J.L.A.; Souza, V.S.B.; Cahú,
G.G.O.M.; Coêlho, M.R.C.D.
DETECTION OF ANTIBODIES AGAINST rK39
ANTIGEN OF LEISHMANIA CHAGASI IN HIV
PATIENTS
OP-4
Arruda, L.M.F.; Silva, D.F.L.; Moraes, M.M.;
Sagica, F.E.S.; Jesus I.M.
INCIDENCE AND PREVALENCE OF CYTOMEGALOVIROSIS IN INDIVIDUALS WITH THE HIV DEMAND
INSTITUTE OF SPONTANEOUS EVANDRO CHAGASIEC/SVS/MS.
OP-5
Prazeres, A.S.C.
EVIDENCE OF IN THE AMAZON REGION (2008-2009)
BY THE DETECTION OF HI AND IGM ANTIBODIES
Workshop 5: Plant Virology
October 18th (Monday)
03:15pm to 04:45pm
OP-1
Oliveira, A.S.; Lima, R.N.; Aliaga, R.C.T.;
Melgarejo, T.; Resende, R.O.
GEOGRAPHIC DISTRIBUTION AND MOLECULAR
PHYLOGENY OF IRIS YELLOW SPOT VIRUS
(TOSPOVIRUS) ISOLATES FROM ONIONPRODUCING PROVINCES IN PERU
OP-2
Fayad-André, M.S.; Resende, AR.O.; Dusi,
A.N.
SPREAD OF VIRUSES INFECTING GARLIC CROPS
CULTIVATED UNDER DIFFERENT PRODUCTION
SYSTEMS IN BRAZIL
OP-3
Bruckner, F.P.; Cascardo, R.S.; Zerbini Jr,
F.M.; Alfenas-Zerbini, P.
VIRUS-INDUCED GENE SILENCING OF THE GENES
TCTP, SNF1 AND SUBÁ ANALYSIS BY QUANTITATIVE
RT-PCR AND EFFECT ON INFECTION OF N.
BENTHAMIANA BY PEPYMV.
OP-4
Boari, A.J.; Oliveira, A.C.S.; Sousa, C.M.;
Pantoja, K.F.C.; Souza, C.A.; Bonfim, K.
EVALUATION OF BLACK PEPPER MOTHER PLANTS
AS FOR VIRUSES IN THE STATE OF PARÁ
19
SCIENTIFIC PROGRAM
Workshop 7: Animal Virology
October 18th (Monday)
03:15pm to 04:45pm
OP-6
Jaime, J.; Vargas, D.S.; Vera, V.J.
A FIRST GENERATION RECOMBINANT ADENOVIRUS
EXPRESSING PROTEIN E2 OF THE BOVINE VIRAL
DIARRHEA VIRUS TO BE EMPLOYED AS VACCINE
OP-7
Cruz, T.F.; Kanashiro, T.M.; Araújo Junior,
J.P.
INDIRECT TRAPPING ELISA (ENZYME-LINKED
IMMUNOSORBENT ASSAY) FOR PORCINE
CIRCOVIRUS TYPE 2 ANTIBODY DETECTION
OP-8
Batista, H.B.C.R.; Petzhold, S.A.; Lima,
F.E.S.; Santos, T.B.; Gigoletti, R.; Roehe,
L.R.; Kunert Filho, H.; Pacheco, S.M.;
Franco, A.C.; Roehe, P.M.
A SANDWICH ENZYME LINKED IMMUNOSORBENT
ASSAY FOR RABIES ANTIBODY DETECTION IN
SERA OF DIFFERENT SPECIES
OP-9
Santos, S.S.; Brandão, P.E.; Barros, I.N.;
Prado,C.O.; Razera, G.A.; Alejo, C.T.; Silva,
S.O.S.; Buitrago, L.Y.V.; Richtzenhain, L.J.,
IBV VARIANTS DETECTED IN BRAZILIAN POULTRY
FLOCKS
OP-10
Lorenzetti, E.; Medeiros, T.N.S.; Bodnar, L.;
Moraes, D.A.; Molinari, B.L.D.; Ribeiro, J.;
Leme, R.A.; Alfieri, A.F.; Alfieri, A.A.
G4P[6] GENOTYPE DIVERSITY IN PORCINE
ROTAVIRUS STRAINS ISOLATED OF VACCINATED
PIG HERD
OP-11
Baldin, C.M.; Favero, C.M.; Castro,
A.M.M.G.; Taniwaki, S.A.; Castro, F.G.;
Brandão, P.E.; Richtzenhain, L.J.
MOLECULAR CHARACTERIZATION OF SWINE TTV1
AND TTV2 FROM A HERD LOCATED IN SÃO PAULO
STATE.
Workshop 8: Basic Virology
October 19th (Tuesday)
03:15pm to 04:45pm
OP-6
Hernandez, R.; Piper, A.; Ribeiro, M.;
Kononchik, J.; Vancini, R.; Hunt, S.; Brown, D.
HOST RANGE MUTATIONS OF SINDBIS VIRUS
DEFINE ALPHAVIRUS HOST ADAPTIVE FUNCTIONS.
OP-7
Valadão, A.L.; Aguiar, R.S.; Tanuri, A.;
Peterlin, B.M.
EMETINE IMPACTS ON HIV REPLICATION BY RNA
PROCESSING BODIES DISRUPTION
OP-8
Oliveira, L.C.; Alcântara, T.C.; Brasil,
B.S.A.F.; Kroon, E.G.; Ferreira, P.C.P.;
Bonjardim, C.A.
ROLE PLAYED BY THE CELLULAR GENE c-fos
DURING THE REPLICATION CYCLE OF THE
ORTHOPOXVIRUS VACCINIA VIRUS
OP-9
Pena, N.M.; Azevedo, R.G.; Castro, D.F.;
Diaz, R.S.; Komninakis, S.
HIGH LEVELS OF POLYMORPHISMS RELATED TO
RALTEGRAVIR RESISTENCE AMONG RALTEGRAVIR
NAIVE INDIVIDUALS IN BRAZIL
OP-10
Batista, M.V.A.; Ferreira, T.A.E.; Freitas,
A.C.; Balbino, V.Q.
THE USE OF ENTROPY FOR SELECTING
PHYLOGENETIC INFORMATIVE GENOMIC REGIONS
AND THE EVOLUTION OF PAPILLOMAVIRUS
20
SCIENTIFIC PROGRAM
Workshop 9: Human and Health Virology
October 19th (Tuesday)
03:15pm to 04:45pm
OP-6
Silva Júnior, J.V.J.; Bertani, G.R.; Gil,
L.H.V.G.; Almeida, S.
A RNA TRANSFECTION SYSTEM FOR GENERATION
OF VIRUS BY IN OVO ELECTROPORATION
OP-7
Vancini, R.; Paredes, A.; Ribeiro, M.;
Ferreira, D.; Hernandez, R.; Brown, D.
NEW STRUCTURE OF DENGUE-2 VIRUS REVEALED
BY CRYO-ELECTRON MICROSCOPY AND CHEMICAL
CROSS-LINKING
OP-8
Jardim, A.C.G.; Mankouri, J.; Verow, M.;
Rahal, P.; Harris, M.
EFFECTS OF BOTH NATURAL AND RATIONALLY
DESIGNED COMPOUNDS ON AMP PROTEIN KINASE
ACTIVATION AND HCV REPLICATION
OP-9
Carvalho, L.G.; Santos,D.R.L.; Marinho,
A.M.; Ramos, S.; Pinto, M.A.; Resende,
F.C.; Cajaraville, A.C.R.A.
HEPATITIS E VIRUS (HEV) EXPERIMENTAL
INFECTION STUDY IN CYNOMOLGUS MACAQUE
(MACACA FASCICULARIS)
OP-10
Mota, M.T.O.; Babeto, E.; Cândido, N.M.;
Calmon, M.F.; Bonilha, J.; Rahal, P.; Vassallo,
J.; Cunha, I.W.; Soares, F.A.; Peitl, P.
HPV AND ALTERATIONS IN GENE EXPRESSION IN
PENILE CARCINOMA
OP-11
Diaz, R.S.; Vilhena, C.; Côrtes, R.M.; Reis,
A.D.; Santos, C.; Abrão, P.
GENETIC VARIATION IN IL28B AND SUSTAINED
VIROLOGIC RESPONSE OF HEPATITIS C VIRUS
Workshop 10: Immunobiologicals
October 19th (Tuesday)
03:15pm to 04:45pm
OP-1
Silva, A.N.M.R.; Santos, J.J.S.; Almeida,
S.R.; Marques Junior, E.T.A.; Gil, L.H.V.G.
CONSTRUCTION AND CHARACTERIZATION OF
SUBGENOMIC YELLOW FEVER REPLICONS
EXPRESSING THE DOMAIN III OF DENGUE VIRUS
ENVELOPE PROTEIN
OP-2
Cruz, H.M.; Villela-Nogueira, C.A.;
Rodrigues do Ó, K.M.; Lewis-Ximenez,
L.L.; Lampe, E.; Villar, L.M.
EVALUATION OF COMMERCIAL ENZYME
IMMUNOASSAY FOR ANTIBODIES AGAINST
HEPATITIS C VIRUS (ANTI-HCV) DETECTION USING
SALIVA SAMPLES.
OP-3
Silva Junior, H.C.; Mouta Junior,S.S.; Leite,
J.P.G.; Moraes e Souza, M.T.B.
EXPRESSION OF ROTAVIRUS VP6 PROTEIN IN
MAMMALIAN HEK293-T CELLS
OP-4
Pereira, S.S.; Oliveira, G.S.; Hassen, S.R.;
Fernandes, C.F.C.; Silva, L.H.P.; Stabeli,
R.G.
GENERATION OF IMMUNE PHAGE DISPLAY LIBRARY
TO SELECT SPECIFIC RABIES ANTIBODIES
Obs.: the paper that are not listed above will be presented as poster ( except papers that were declined by
the scientific committee)
21
SCIENTIFIC PROGRAM
8 - Prêmio Hélio Gelli Pereira
A avaliação dos trabalhos selecionados ao Prêmio “Hélio Gelli Pereira” ocorrerá durante o Workshop 6 e o
Workshop 11 nos dias 18 e 19 de outubro, respectivamente, das 15:15 às 16:45 horas para todas as categorias (IC,
Mestrado e Doutorado).
Os apresentadores terão 10 minutos para apresentação oral do trabalho, sendo que ao final da apresentação, serão
reservados 5 minutos a arguição dos avaliadores.
Workshop 6: Prêmio “Hélio Gelli Pereira”, categoria Iniciação Científica
18/10/2010 (segunda-feira)
15:15-16:45
OP-1
Andrade, V.M.M.; Mesquita, M.M.A.;
Siqueira, M.M.; Souza, T.M.L.
THE NATURAL ENDOGENOUS RNA POLYMERASE
(NERP) FROM RESPIRATORY SYNCYTIAL VÍRUS.
OP-2
Rodrigues, C.S.; Araujo, D.B.; Campos,
A.C.A.; Sanfilippo, L.F.; Medina, A.O.;
Yoshihara, C.K.; Martorelli, L.F.A.; Kataoka,
A.P.; Durigon, E.L.; Favoretto, S. R.
DETECÇÃO DE ANTICORPOS NEUTRALIZANTES
PARA O VÍRUS DA RAIVA DE MAMÍFEROS
SILVESTRES PROVENIENTES DA ÁREA DE
SOLTURA NO LITORAL NORTE DO ESTADO DE S.P.
OP-3
Cruz, H.M.; Silva, E.F.; Villela-Nogueira,
C.A.; Nabuco, L.C.; Rodrigues do Ó, K.M.;
Lewis-Ximeneza, L.L.; Fumiko, C.
EVALUATION OF SALIVA SPECIMENS AS
ALTERNATIVE SAMPLE TO DETECT HEPATITIS B
SURFACE ANTIGEN.
OP-4
Macedo, P.V.
IMPACTO DA PANDEMIA DE INFLUENZA A /H1N1 EM
CRIANÇAS NA CIDADE DE S.P. NOS ANOS 2009 E 2010.
OP-5
Comparini, R.G.; Zetehaku, A.C.; Filho,
A.F.P.L.; Nahon, N.C.; Santos, N.T.;
Gagliani, L.H.; Caseiro, M.M.; Filho, D.J.A.
ANALYSIS OF CCR5 DELTA 32 HETEROZYGOSIS IN
PATIENTS WITH LONG TERM HIV-1 SUPRESSION
UNDERGOING ANTIRETROVIRAL THERAPY
OP-6
Alves, C.M.; Andrade, V.M.; Accioly, M.;
Abrantes, J.L; Ferreira, V.F.; Siqueira, M.M.;
Souza, T.M.L.
EFFECTS OF TRIAZOLIC COMPOUNDS ON
INFLUENZA VÍRUS REPLICATION.
Workshop 11: Prêmio “Hélio Gelli Pereira”, categoria mestrado e doutorado
19/10/2010 (terça-feira)
15:15-16:45
OP-7
Mendonça, L.M.; Sampaio, T.L.; Costa, L.J.
LOWER LEVELS OF INTEGRASE FOUND IN HIV-1
PARTICLES IN THE ABSENCE OF VIRAL
ACCESSORY PROTEIN NEF: EFFECT OF NEF ON
VIRAL PROCESSING.
OP-8
Ganime, A.C.; Carvalho-Costa, F.A.;
Mendonça, M.C.L.; Vieira, C.B.; Santos,
M.S.; Filho, R.C.; Miagostovich, M.P.; Leite,
J.P.G.
ENVIRONMENTAL MONITRING OF ROTAVIRUS A IN
A HOSPITAL INTENSIVE CARE UNIT IN RIO DE
JANEIRO CITY, BRAZIL.
22
SCIENTIFIC PROGRAM
OP-9
Sampaio, T. L.; Cunha, M.S.; Mendonça,
L.; Tanuri, A. Costa, L.
INVESTIGAÇÃO DA ATIVIDADE DA PROTEÍNA
ACESSÓRIA NEF DURANTE O CICLO REPLICATIVO
DO SIVCPZ ISOLADO GAB2
OP-10
Mondini, A.; Bronzoni, R.V.M.; Nunes,
S.H.P.; Chiaravalloti Neto, F.; Massad, E.;
Alonso, W.J.; Lázzaro, E.S.M.; Ferraz, A.A.;
Zanotto, P.M.A.; Nogueira, M.L.
SPATIO-TEMPORAL
TRACKING
AND
PHYLODYNAMICS OF NA URBAN DENGUE 3
OUTBREAK IN SÃO PAULO.
OP-11
Castro, T.X.; Labar the, N.V.; Garcia,
R.C.N.C.
CARACTERIZAÇÃO MOLECULAR DOS PARVOVÍRUS
ASSOCIADOS AOS CASOS DE GASTROENTERITE
EM FILHOTES DE CÃES E GATOS NO ESTADO DO
RIO DE JANEIRO.
OP-12
Véras, N.M.C.; Gray, R.R.; Brígido, L.F.M.;
Rodrigues, R.; Salemi, M.
HIGH RESOLUTION PHYLOGENETICS AND
PHYLOGEOGRAPHY OF HIV-1 SUBTYPE C
EPIDEMIC IN SOUTH AMERICA.
23
RESUMOS
Estratégias de produção in vitro de baculovírus
Andréa Farias de Almeida
Universidade Federal do Rio Grande do Norte, Programa
de Pós-Graduação em Engenharia Química, e-mail:
andreafalm@eq.ufrn.br
Os baculovírus são vírus que infectam artrópodes,
principalmente insetos da ordem Lepidoptera,
caracterizam-se por possuir corpo de oclusão, e uma
matriz proteica (poliedrina ou granulina) que envolvem
os virions protegendo-os no ambiente. Estes vírus são
considerados excelentes agentes de biocontrole devido
à especificidade oferecida aos seus hospedeiros e a
sua forma de propagação. A crescente demanda de
bioinseticidas virais (baculovírus) no mercado nacional
é incentivadora para busca de alternativas que
promovam o aumento na produção. As diferentes
estratégias de operação utilizadas normalmente nos
cultivos com células microbianas (bactérias, fungos e
leveduras) são aplicáveis também aos cultivos com
células animais (mamíferos e insetos), tais como: cultivo
descontínuo (batelada ou batch), cultivo descontínuo
alimentado (batelada-alimentada ou fed-batch), cultivo
contínuo e cultivo contínuo com retenção de células
(perfusão). Estas estratégias de produção tornam-se
ferramentas indispensáveis para os cultivos in vitro de
baculovírus seja para produção de bioinseticidas ou para
proteínas recombinantes. Existe um forte interesse no
desenvolvimento de processos de ampliação de escala
para produção de bioinseticidas baseado no cultivo de
células de inseto e subsequente infecção com
baculovírus. Portanto, a escolha do processo de
produção, bem como, o modo de operação dos sistemas
de produção é de muita importância para obtenção de
altos rendimentos com custo efetivo e visando uma
possível ampliação de escala. Assim, o modo de
operação em batelada alimentada normalmente é
utilizado como alternativa para o aumento de produção
quando se utiliza o sistema em batelada. Diante disto, a
estratégia para melhorar a produção in vitro de
baculovírus utilizando processo em batelada-alimentada
associado com baixa multiplicidade de infecção (Low
MOI) foi desenvolvida com a finalidade de aumentar a
sua produção.
Palavras-chaves: estratégias de produção, baculovírus,
processo in vitro.
Smallpox, its Eradication and the last
reservoirs, Ethiopia and Somalia
Ciro A. de Quadros, Md. MPH.
Executive Vice-President
Albert B. Sabin Vaccine Institue
Washington, DC
USA
Smallpox was highly endemic in Ethiopia in the early
seventies.
The Program in Ethiopia was a major challenge as it
was understaffed for the first 4 years, and the
communications and transportation was precarious, with
less than 5,000 kilometers of all-weather road in a country
that had a population of over 25 million people and had
1.2 million square kilometers.
Ethiopia was the first country in the world in which the
eradication program started from day one utilizing only
the surveillance and containment strategy and in the first
year of the program, with a staff of less than 50
individuals and 7 vehicles, the program investigated over
25,000 cases.
Besides that, there was civil unrest in several areas of
the country, variolation and resistance to vaccination was
widespread in several provinces and by the third year of
the program there was a violent change in Government.
Despite these difficulties, after a concerted effort that
lasted about six years, the last case of the disease was
detected in August, 1976 in the Ogaden Desert, bordering
Somalia.
Subsequently, in 1977, the last case of smallpox was
detected in Somalia.
CIDOFOVIR AND ST-246: ANTIVIRAL DRUGS
AGAINST CANTAGALO VIRUS
Damaso, C.; Jesus, D. M.; Fernandes, E.S.
Laboratório de Biologia Molecular de Vírus, Instituto de
Biofísica Carlos Chagas Filho, Universidade Federal do
Rio de Janeiro, Rio de Janeiro. E-mail:
damasoc@biof.ufrj.br
Cantagalo virus (CTGV) is a strain of vaccinia virus
(VACV; Poxviridae) isolated in 1999 from an outbreak of
pustular disease in dairy cattle and milkers in Rio de
Janeiro state. Since then, several outbreaks of CTGVlike have been frequently reported in distinct states of
Brazil. There is no antiviral therapy against poxvirus
infections. Therefore, in addition to the prevalence of
cowpox infections in Europe and the occurrence of
complications from smallpox vaccination, the spread of
these VACV in the wild imposes the need for novel,
effective anti-poxvirus drugs. Cidofovir (CDV) is an
acyclic phosphonate nucleoside analog that has proved
its efficacy against a variety of DNA virus, including
poxvirus. Our group reported the antiviral action of CDV
on the replication of Cantagalo virus (CTGV). Our data
indicate that CDV inhibited genome encapsidation and
this effect would probably lead to the severe inhibition of
virus yield. Analysis of virus DNA isolated form CDV25
treated cells by AFM demonstrated the presence of DNA
aggregates not observed in control virus DNA. These
aggregates seem to be excluded during the process of
genome encapsidation. Nevertheless, this effect was not
a result of a direct effect of CDV on virus morphogenesis.
We have also studied the effect of ST-246 on Cantagalo
virus replication. ST-246 is newly-discovered antiOrthopoxvirus drug that targets virus egress from cells.
Our data indicate that CTGV is the most susceptible
Orthopoxvirus studied so far. We observed the reduction
of plaque formation, intracellular and extracellular yields,
inhibition of B-galactosidase expression by recombinant
viruses and the reduction of comet tail formation. We
have also studied the effect of ST-246 in mice after
infection with CTGV by tail scarification. ST-246 was able
to prevent the formation of primary lesion after infection
with CTGV but not with VACV-WR. In sum, both CDV
and ST-246 are promising drugs to treat the infection by
CTGV.
Support: CNPq and Faperj
“MICROBICIDE AND ANTI-HIV-1 ACTIVITY OF
DITERPENES ISOLATED FROM THE
BRAZILIAN BROWN ALGAE”
Claudio Cesar Cirne
O constante avanço da infecção pelo Vírus da
Imunodeficiência Humana (HIV), com a modificação de
seu perfil como a interiorização, pauperização, maior
incidência em mulheres e faixas etárias mais altas, além
do aumento da resistência aos medicamentos em uso,
exige medidas profiláticas mais efetivas, além de drogas
terapêuticas com menor custo. Diversos estudos
mostram que as vacinas ainda não são realidade e
podem estar muito longe de chegar ao mercado. A
principal causa da feminilização da doença, em países
em desenvolvimento, é a contaminação de esposas por
seus maridos e que não usam ou permitem qualquer
medida preventiva na relação sexual com as mesmas.
O papel potencial dos microbicidas em impedir a
transmissão mucosa de HIV-1 tem sido claramente
demonstrado. É fato que, refere-se a um novo tipo de
produto a ser desenvolvido, para que as pessoas
possam usar via vaginal ou retal para se proteger da
infecção pelo HIV e possivelmente outras doenças
sexualmente transmissíveis. Um grande problema é que
as novas tecnologias de saúde, raramente se tornam
amplamente disponíveis nos países em
desenvolvimento, fato que pode levar até mais de uma
década após a sua aprovação nos os EUA e a Europa,
o que demonstra ser um atraso inaceitável para esta
tecnologia para salvar vidas, principalmente com
recursos públicos. Desta forma estudos com
26
substâncias, como os diterpenos, derivadas de produtos
naturais, como as algas marinhas tem demonstrado
importantes efeitos no que se refere a atividade inibitória
e mecanismo de ação para esta infecção, o que abriu a
possibilidade para pesquisas de substâncias nacionais,
que podem gerar produtos que sejam importantes como
mecanismo de proteção contra a grave infecção pelo
HIV.
AN UPDATE IN THE ROLE PLAYED BY
INTERFERONS IN THE CONTROL OF INNATE
AND ADAPTIVE IMMUNITY
CLÁUDIO A. BONJARDIM
Grupo de Transdução de Sinal - Laboratório de Vírus ICB – UFMG – 31270-901 – claubonj@icb.ufmg.br
Interferons (IFNs) were discovered as antiviral agents
50 years ago, and enormous progress has been made
since then. Nowadays, IFNs (specifically type I IFNs),
have been ascribed as the cytokines that bridge the
innate and adaptive immunity soon after the recognition
of pathogen-associated molecular patterns (PAMPs) by
the infected host. Notably, a unifying mechanism for type
I IFN production has been established upon innate
immune detection. Thus, TLR 3, 4, 7 and 9 associate
endosomal recognition of PAMPs to type I IFN responses,
a mechanism that has been shown in plasmacytoid
dendritic cells to be dependent on the PI3K/mTOR/S6K
pathway. It is worth noting that pathogen recognition
triggers a fine-tuned controlled program that not only
includes the production of antiviral (IFN) and proinflammatory cytokines to initiate the antiviral response
but also signals the cessation of the response through
the induction of suppressors of cytokine signaling (SOCS).
SOCS in turn is under tight regulation of the TAM
receptors (protein tyrosine kinase receptors TYRO3, AXL
and MER), and activation of which thereby protects the
host from the threats of autoimmune diseases.
ANTIHERPES ACTIVITY OF CARDENOLIDE
DERIVATIVES
Simões, C.M.O.1a; Bertol, J.W.2a; Pádua, R.M.3b; Kreis,
W.4; Braga, F.C.3b; aLaboratório de Virologia Aplicada,
1
Depar tamento de Ciências Farmacêuticas,
2
Departamento de Microbiologia, Parasitologia e
Imunologia, Universidade Federal de Santa Catarina,
Florianópolis, SC, Brazil; bLaboratório de Fitoquímica,
3
Faculdade de Farmácia, Universidade Federal de Minas
Gerais, Belo Horizonte, MG, Brazil; 4Friedrich-Alexander
Universität, Erlangen-Nürnberg, Germany. E-mail:
claudias@reitoria.ufsc.br
The treatment of herpes infections with conventional
nucleoside analogues (ex. acyclovir-ACV) is effective in
most cases, but drug-resistance may arise due to the
prolonged treatment. This fact supports the researches
for searching new drugs to treat these infections. Cardiac
glycosides bind and inhibit Na+/K+-ATPase (NKA) activity,
and the cardenolide drugs such as digoxin and digitoxin
are clinically used for treating heart failure and atrial
arrhythmia. However, very recently the antiherpes activity
(anti-HSV types 1 and 2) of some cardenolides has been
published (Hartley et al. Arch.Virol. 151, p.2495, 2006;
Dodson et al. Virology 366, p.340, 2007; Su et al. Antiviral
Res. 79, p.62, 2008). These results encouraged us to
perform an antiherpes screening of 68 natural and
synthetic cardenolide derivatives. Some of these
compounds showed higher activity, when compared to
that of ACV, including against one HSV-1 strain (29R)
that is resistant to this drug. We will describe here the
antiviral effects of two compounds, which have been
chosen due to their high detected anti-HSV activity.
Cytotoxicity (CC50) was evaluated on Vero or GMK-AH1
cells by using MTT assay, and antiviral activity (IC50)
was tested against HSV-1 (KOS and 29R strains) and
HSV-2 (333 strain) by viral plaque number reduction
assay. Their mechanisms of action were evaluated
through a sequence of assays to identify in which steps
of the viral replication process these compounds act.
Compound 4 showed selectivity indices (SI=CC50/IC50)
of 2,107 (HSV-1/KOS); 4,566 (HSV-1/29R); and 6,944
(HSV-2/333). Compound 12 showed selectivity indices
of 645 (HSV-1/KOS); 792 (HSV-1/29R); and 1,238 (HSV2/333). The obtained results showed that both compounds
did not display virucidal effects neither inhibited viral
attachment and entry into the cells (dextran sulphate
was used as positive control). However, these compounds
maintained the anti-HSV-1 activity even at the higher
tested MOI (0.4), showing virus yield reductions of 5.3
Log10 (compound 4) and 4.0 Log10 (compound 12), when
compared to untreated controls (8.8 Log10). It is important
to note that this MOI is a thousand times higher than the
MOI (0.0004) used for screening. They also inhibited the
viral cell-to-cell spread and interfered on viral release
inhibiting 100% (compound 4) and 64% (compound 12)
of this step, at concentrations equivalent to their IC50
values (0.13μM and 0.27μM, respectively). The Western
blotting analysis of viral protein expression showed that
both compounds inhibited á (ICP27) gene and,
consequently, â (UL42) and ã (gB and gD) genes
expression. The anti-ATPase activity was also evaluated,
and compounds 4 and 12 presented 50% NKA inhibition
at 0.92μM and 4.0μM, respectively, indicating that an
alteration of cell electrochemical gradient may be involved
in the mechanism of herpes inhibition.
Financial support: CNPq / CAPES.
DETECCION AND VIABILITY ASSAYS FOR
FECAL-ORAL TRANSMITTED VIRUSES IN
WATER AND MOLLUSKS
Célia Regina Monte Barardi*
Lab Environmental Team: PhD students: Adriana de Abreu
Corrêa, Aline Viancelli and Doris Sobral Marques Souza;
MSc student: Vanessa Moresco; Under graduate
students: Lucas Ariel Totaro Garcia, Mariana Rangel
Piloto, Mariana Nascimento; DTI/CNPq: Ana Paula Dores
Ramos (PhD) and Adriano Luiz Teixeira: Technical support:
Nicésio Delfino.
*
Laboratório de Virologia Aplicada, Departamento de
Microbiologia, Imunologia e Parasitologia, Centro de
Ciências Biológicas, Universidade Federal de Santa
Catarina, Florianópolis, Santa Catarina, Brasil, Caixa
Postal 5101 - 88040-970 cbarardi@ccb.ufsc.br
While PCR-based techniques provide rapid, sensitive,
and specific detection of environmental viruses they do
not allow us to differentiate infective from inactivated
viruses. This is mainly due to the persistence of genomes
of inactivated viruses that remain either partially or fully
intact in the environment. Therefore, some efforts have
been made to design methods for only infectious viruses
detection. Enzymatic treatments may differentiate
infective from inactivated viruses based on differences
in the ability of their proteic capsids to protect the
genomes from proteases and nucleases. With the capsid
degraded, the naked DNA/RNA is more susceptible to
nucleases degradation than capsid-enclosed DNA/RNA.
Another way to check viral viability is based on the ability
of the environmental sample to infect an in vitro cell
culture, using permissive cells before PCR assay (ICC/
PCR). Although this former method is sensitive to
evaluate the viral viability, there are suspicions if all DNA
detected in the assay comes from the cell culture
replication. One way to circumvent this problem is to
use the mRNA from infected cells to check viability of
DNA genome viruses (ICC/RT/PCR). Based on these
facts, the Laboratório de Virologia Aplicada/UFSC is
making efforts to develop and apply techniques to check
viral viability on environmental samples. Some of these
results will be presented in the present work. One matrix
studied was seawater under U.V. light and chlorine for
Human Adenovirus (HadV2), Murine Norovirus (MNV-1)
and Hepatitis A Virus (HAV) inactivation. 300L of natural
seawater were artificially seeded with HAV (108 FFU/
mL), MNV-1 (108 PFU/mL) and HAdV2 (109 FFU/mL) and
treated by 36W UV lamp, into a mollusk depuration tank,
with recirculation, for up to 120h, in three independents
assays. One liter of viral seeded seawater was harvested
every 24h and viral particles were concentrated by
flocculation method using skimmed milk. The kinetics
of viral decay was evaluated by quantification of genomic
27
copies (q(RT)PCR for the three viruses) and infectious
viral particles (ICC-RT-PCR for HAdV2). Based on qPCR
results, the kinetics observed were different for each
virus, reaching, at 120h of contact time, ~5log10 and
~3log10 reduction for HAdV2 and HAV, respectively; for
MNV-1, was observed a ~4,5log10 reduction at 72h under
UV treatment. Despite the genome detection, HAdV2
was able to remain infectious only up to 72h, according
ICC-RT-PCR results. Concerning chlorine ability to
disinfect seawater, the test seawaters were seeded with
106 PFU/mL of MNV and 1.5x 106 FFU/mL of HAdV2
and treated by adding initial free chlorine concentrations
of 2.5mg/L for up to 60min. The kinetics of disinfection
were evaluated by quantification of genomic copies
(q(RT)PCR) and infectious viral particles (Plaque Assay)
in two independents assays. Moreover, two enzymatic
treatments (DNAse for HAdV2 and RNAse for MNV-1)
of samples have been applied to inactivate free genomes
and assess viral infectivity when using q(RT)PCR. After
30 min of treatment of natural seawater (0.5mg/L of free
chlorine were present at this moment) a ~2log10 and
~3log10 reduction where observed for MNV-1 and HAdV2,
respectively, based on q(RT)PCR results. When viral
infectivity was analyzed, a reduction of more than 4log10
was observed for infectious MNV-1, while HAdV2
presented a ~2.3log10 reduction, remaining infective
viruses present after 60 min. Another matrix studied was
water samples collected from affluent, middle and effluent
from two swine manure treatment system (ETDS and
UD) from EMBRAPA - Swine and Poultry, Concordia,
Santa Catarina, to quantify porcine adenovirus (PAdV).
Samples positive on qPCR were treated with DNAse.
The combined results of qPCR from treated and no treated
samples revealed that on affluent samples from ETDS
system were found an average of 8.69x104 genome
copies (gc)/mL, from that an average of 1.31x102 gc/mL
were from viable virus. On samples collected in the middle
point were detected 5.13x104 gc/mL, from that an
average of 5.25x102 gc/mL of viable virus. On the last
sample point, the effluent from the system presented an
average of 3.92 x103 total gc and 9.74x101 gc/mL from
viable virus. On the other hand, from UD system the
average on affluent was 6.92x104 gc/mL with 2.78x102
gc/mL viable. On samples collected in the middle point
were detected 1.22x104 gc/mL from that 1.25x102 gc/
mL were viable. On the last sample point, the effluent
from the system presented an average of 3.61x102 gc/
mL where 1.93x101 gc/mL were viable. The results clearly
show the presence of viable virus particles on all samples
points from both treatment systems. Finally, a shellfish
depuration system was analysed to check its efficiency
to eliminate HAdV5 and HAV from oysters, after seawater
treatment with ultraviolet (UV). The conventional PCR
detection for both viruses, in digestive tissue, showed
that, during the depuration process, HAdV5 genome was
28
detected during all sampling periods (0h, 48h, 72h and
96h), and the presence of the HAV genome was detected
until 72h. In order to evaluate viral viability, oyster
samples were inoculated on cell monolayers at noncytotoxic dilutions and the cell supernatants were
subsequently subjected to nucleic acid extraction in order
to detect infectious viral particles. ICC-PCR assay
demonstrated a viral inactivation after 96h of seawater
recirculation submitted to UV radiation. For the HAV, the
viral inactivation was reached at 48h of depuration.
However, for the total inactivation of HAdV5, more than
96h of water recirculation under UV radiation was required.
To confirm the ICC-PCR results for HAdV5 infectivity,
all samples were tested by IFA, using A549 cells. Viral
viability by IFA positive was shown until 72h of oyster
depuration, confirming that 96h depuration were
necessary to guarantee virus-safe oysters.
Financial support: MAPA/CNPq/ 578200/2008-2 and
EMBRAPA - Macro programa 2.
INFECTION OF CELLS BY ALPHAVIRUSES
Dennis T. Brown and Raquel Hernandez
Department of Molecular and Structural Biochemistry
North Carolina State University
Sindbis Virus (SV), the prototype alphavirus in the family
togaviridae, infects both mammalian and insect cells in
its sylvatic cycle. The ability of SV to infect cells
possessing significantly different biochemical
environments suggests there may be a common receptor
and a common mode of entry into each cell type. Two
models for alpha virus entry have been proposed. The
first proposes that virus binds to receptors, is internalized
in endosomes and the creation of an acid environment
within this compartment drives the fusion of the virus
membrane with the endosome membrane. A second
model proposes that interaction of the virion with the
receptor induces the formation of a pore in the cell
membrane through which the virus RNA passes and that
membrane fusion is not necessary. The former model is
supported by evidence that agents which block the
acidification of endosomes can prevent virus RNA or
protein synthesis and the demonstration that virus can
fuse with artificial membranes on exposure to acid pH.
The second model is supported by direct observation in
the electron microscope and the observation that
alphavirus infection make the plasma membrane
permiable. We have shown that exposure to acid pH
can produce dramatic conformational changes in the virus
surface. We will describe experiments in which we have
tried to resolve the differences in the two models in an
attempt to explain all observations in a common model.
“DEVELOPMENT OF INSECT CELL LINES:
METHODOLOGY AND SOME SUCCESS
STORIES.”
Dwight E. Lynn, INSell Consulting, USA
The first continuous insect cell lines were developed 50
years ago by researchers in China (Gao) and Australia
(Grace). Since those early successes, approximately
600 cell lines have been reported from more than 125
insect species. Approximately 80% of these are from
two orders - dipteran insects from which cells are used
for arbovirus research (mosquitoes) or molecular biology
and genetics (Drosophila spp.) and lepidopterans used
primarily in baculovirus research. Cell lines have been
developed from a variety of tissues with embryonic tissue
being a favored source for many researchers. While the
basic technique has hardly changed over the 50 years,
the improvements and wide availability of media and an
improved understanding of the conditions that lead to
successful cell growth have made development of new
cell lines almost routine. Cell lines from Spodoptera
frugiperda (IPLB-Sf21 and Sf-9) and Anticarsia
gemmatalis (UFLAG-286) will be described as particular
success stories.
XXI MEETING ON VIROLOGY & V MERCOSUL
MEETING ON VIROLOGY
FAURGS Convention Center. Gramado, Rio Grande de
Sul, Brazil
October 17 to 20, 2010
Eckard Wimmer
Stony Brook University
Stony Brook, NY
sequence of the viral proteins. Such large-scale changes
yielded surprising phenotypes, particularly those related
to the specific infectivity of virus variants and to the
attenuation of virus pathogenicity in CD155 tg mice. The
strategy has been tested with influenza virus yielding
highly attenuated influenza virus strains.
Of interest is also a poliovirus variant in which sequences
of the polyprotein were “scrambled” by maximizing the
number of nucleotide changes while preserving both
codon bias and amino acid sequences. The scrambled
sequences were used to replace the domains P1
(structural proteins), P2, or P3 (non-structural proteins)
of the P1-P2-P3 polyprotein. Surprisingly, the scrambled
sequence of P1 (934 changes out of 2,643 P1
nucleotides) in a P1scrambled-P2-P3 virus did not alter the
virus’ growth properties. However, P1 scrambled-P2-P3
poliovirus loses out in competition experiments with wild
type virus. Notably, this is not the case with P1CP-Max-P2P3 poliovirus (566 synonymous mutations in the capsid
region).
We have used scrambled sequences in the non-structural
region of the poliovirus polyprotein (P2+P3) to search
for higher order structures essential for vial replication
or for the search of encapsidation signals in
morphogenesis. Moreover, poliovirus with a P1-P2scrambledP3 genome fails to recombine with a C-cluster coxsackie
virus (C-CAV20) in vivo, which is interesting because
neurovirulent PV/CAV recombinants evolve frequently
in different parts of the world from oral poliovirus
vaccines, causing small epidemics of poliomyelitis. This
risk factor of OPV could be avoided if Sabin vaccine
strains with a scrambled P2 region were used in
vaccination campaigns.
Environmental monitoring in the global polio
eradication
“Recoding viral RNA genomes through chemical
synthesis: Novel genetics and practical applications”
Costa, EV
Chemical synthesis of viral genomes is independent of
a natural template and, thus, it allows modifying the
structure and function of a virus’ genetic information to
an extent not possible before. Following our de novo
synthesis of poliovirus in 2002, the first of an organism,
we have used this new strategy to fur ther our
understanding of an organism’s properties, particularly
its pathogenic armory if it causes disease in humans,
and to make use of this new information to protect from
or treat human viral disease. Specifically, we have
recoded the genome of poliovirus, altering the capsidcoding region by introducing 600 to 1,000 nucleotide
changes. Specifically, we altered favored to unfavored
codon pairs, thereby changing the codon pair bias within
the polyprotein without changing codon bias or the
Poliomyelitis is a very ancient disease, and the global
effort to eradicate it is the largest public health initiative
in history. In 1988, polio existed in over 125 countries on
five continents, and more than 350,000 children were
paralyzed because of it. In that same year, the 41st World
Health Assembly committed the Member States of the
World Health Organization (WHO) to the global
eradication of poliomyelitis by the year 2000. Although
two excellent vaccines had been licensed by 1961, the
Global Polio Eradication Initiative (GPEI) followed
Panamerican Health Organization in choosing the oral
poliovirus vaccine (OPV) with attenuated virus over the
inactivated poliovirus vaccine (IPV), for both routine and
supplementary immunization activities. Through effective
use of OPV, the WHO-GPEI has nearly achieved its goal
29
of eradicating wild polioviruses. During the last two
decades of the 20th century, global eradication of
poliovirus appeared to be a realistic goal because polio
transmission has been interrupted in the Americas,
Europe and the Western Pacific, and type 2 wild
poliovirus was globally eradicated. Though only 4
countries (Afghanistan, Pakistan, India and Nigeria) have
remained endemic since 2005, polio has re-emerged in
countries previously declared polio-free, like Tajikistan.
This country which is part of WHO’s European Region
(certified as polio-free in 2002) had its last indigenous
case of polio in 1997. Thus, it is urgent not only to
reevaluate the tactics of stopping wild-poliovirus
circulation, but also to define post-eradication policies.
The original eradication strategy, based on OPV
immunization, was supported by the conception that,
although revertant polioviruses have increased
neurovirulence, their transmissibility remains low and they
soon disappear from populations. However, this
hypothesis was disproved in 2000, when it was shown
that numerous cases of poliomyelitis in Hispaniola Island
had been caused by circulating vaccine-derived poliovirus
(cVDPV) type 1. Also, several VDPV outbreaks were
seen in 14 countries between 2000 and 2009. These
findings caused WHO to reassess both current and future
immunization strategies. In order for the eradication
initiative to be effective, it is essential to achieve close
integration between surveillance and laboratory activities.
That guarantees the data generated from epidemiology
and virology are available as the basis for action by
immunization programme managers and others
responsible for implementing eradication strategies. In
this context, environmental surveillance has been used
successfully in monitoring Enterovirus circulation and
assessing the extent or duration of epidemic poliovirus
circulation in specific populations. In some countries,
wild polioviruses and VDPV have been detected in the
environment, despite the absence of reported Acute
Flaccid Paralysis cases. Environmental surveillance is
also a potential tool for monitoring indirectly the extent
of population immunity.
GEMINIVIRUS-HOST INTERACTIONS: NEW
INSIGHTS INTO THE NIK-MEDIATED
DEFENSE SIGNALING SUPPRESSED BY THE
GEMINIVÍRUS NUCLEAR SHUTTLE PROTEIN
Anesia A Santos, Otavio Brustoline, Fahyme CS
Almeida, Elizabeth PB Fontes
Departamento de Bioquimica e Biologia MolecularBIOAGRO- Universidade Federal de Vicosa- 36570.000,
Vicosa, MG, Brazil
The NSP-interacting kinase (NIK) receptor-mediated
30
antiviral signaling has been identified as a virulence
target of the geminivirus nuclear shuttle protein (NSP)
(1, 2). Recent progress towards elucidating the NIKmediated antiviral signaling includes the identification of
the ribosomal protein L10 (rpL10) as a specific partner
and substrate of NIK that functions as the immediate
downstream effector of NIK-mediated signaling (3).
Phosphorylation of rpL10 by NIK promotes translocation
of the ribosomal protein to the nucleus where it may
function to mount a defense response that negatively
impacts virus infection. More recently, we found that NIK1
undergoes a stepwise pattern of phosphorylation within
its activation-loop domain (A-loop) with distinct roles for
different threonine residues. The conserved Thr-474 and
Thr-469 were found to be phosphorylated in vitro and
mutations at Thr-474 impaired autophosphorylation and
were defective for kinase activation in vitro and in vivo.
In contrast, a mutation at Thr-469 did not impact
autophosphorylation and increased substrate
phosphorylation, suggesting an inhibitory role for Thr469 in kinase function. Our results establish that NIK1
functions as an authentic defense receptor as it requires
activation to elicit a defense response. Our data also
suggest a model whereby phosphorylation-dependent
activation of a plant receptor-like kinase enables the Aloop to control differentially auto- and substrate
phosphorylation. We also provide evidence that
geminivirus infection directly interferes with NIK-mediated
nuclear relocalization of rpL10 as a counterdefensive
measure. Overexpression of a constitutively activated
NIK (mutant T474D) in tomato plants, which is not
inhibited by the geminivirus NSP, conferred tolerance to
geminivirus infection. We performed global expression
profiling on geminivirus-infected leaves and on T474Doverexpressing tobacco leaves. The present geminivirusand T474D-induced transcriptional studies demonstrate
a clear predominance of shared responses over stimulusspecific positive changes on soybean leaves. These
results clearly indicate that viral infection may constitute
the predominant stimulus that triggers NIK activation.
MESA REDONDA: PASSADO, PRESENTE,
FUTURO DA FITOVIROLOGIA NO BRASIL
HISTORY OF THE PLANT VIROLOGY IN
BRAZIL
Kitajima, E.W.
Departamento de Fitopatologia e Nematologia, Escola
Superior de Agricultura Luiz de Queiroz, USP, Piracicaba,
SP, Brazil. Email: ewkitaji@esalq.usp.br
Though literature on plant virology in Brasil is present
since the beginning of the 20th century, formal
researches on plant viruses started in the 1930’s at the
Instituto Biológico de São Paulo (IB) by A.A. Bitancourt
and K.M. Silberschmidt and at the Instituto Agronomico
de Campinas (IAC), by A.S. Costa. The main viral
diseases studies were on cotton, tobacco, vegetable
crops and particular focus on citrus tristeza. Costa and
Silberschmidt started the organization of a research group
on plant viruses since 1950, which consolidated in the
1970’s. Costa’s group soon became involved at the
beginning of the graduate program at the ESALQ/USP
in 1970. At the same time some activities on plant
virology started in several parts of Brazil (UFRGS, UFPr,
UFV, UFRPe, UFCe). During the 1970’s several members
of the Costa’s group at IAC migrated to other institutions
as ESALQ, Unicamp, UNESP, IAA, UnB). In the UnB,
ex-members of the IAC plant virus group participated in
the consolidation of a graduate course on plant pathology
in 1976. Various graduate programs on plant pathology
(UFV, UFCe, UFLa, UFRPe, UFRGS) besides ESALQ
and UnB start producing new generation of plant
virologists which nucleated new research centers on plant
viruses in several centers of Embrapa (Vegetable Crops,
Biotechnology, Cerrado, Wheat, Corn and Sorghum,
Soybean, Rice and bean, Cassava and Tropical
Fruticulture, Grapevine and vine, Temperate climate
Fruticulture, Oriental Amazon) and universities (UEM,
UEL, UPF, UFAl, UFRRJ). Presently an estimate of 100
professionals and posdocs are engaged in researches
on diseases caused by plant viruses and related
pathogens, besides graduate and undergraduate
students. Many of these professionals graduated or have
postdoctoral experience in foreign centers in the US
(California, Wisconsin, Illinois, Georgia, Oklahoma,
Kentucky, Arizona, Ithaca, Florida, Louisiana, and centers
of the USDA, etc.), Japan (Tokyo, Hokkaido, Tsukuba,
etc.), Australia (Queensland, Adelaide), Germany
(Braunschweig), France (INRA, Paris, etc.), Spain
(Valencia, Malaga, etc.), Italia (Torino, Bari, etc.), Portugal
(EAN), the Netherlands (Wageningen), etc. We should
also stress the many cooperative researches involving
invited foreign scientists as R. Best (Australia), T.J.
Grant, C.W. Bennett, H.S. Fawcett M.R. Nelson (US), C.
Wetter (Germany), J.M. Bové, O. Le Gall (France), R.
Flores, M. Cambra, P. Moreno (Spain), S. Yamashita
(Japan), D. Peters (the Netherlands), O. Lovisolo (Italia),
M. Bar Joseph (Israel), V.F. Eastop (UK). Viral diseases
are quite common in Brazil, due to the large territorial
extension and the tropical climate which favor the
proliferation of the vectors and assure the constant
present of the host plants. A large number of viruses
affecting most of the cultivated and wild plants has been
studied in Brazil, belonging to practically all known genera.
Some of them were new in the literature, but most of
them were already described abroad and probably were
introduced. Many of these viral diseases caused and
still cause significant socio-economical problems such
as citrus tristeza and leprosis, “vira cabeça” caused by
Tospoviruses in vegetable and ornamental plants, bean
golden mosaic, and several begomovirus-induced
diseases in tomato and pepper, papaya ringspot, leaf
roll and sevre isolates of PVY in potato, CMV affecting
banana, pineapple wilt, soybean stem necrosis, winter
cereal yellows, etc. Other plant diseases caused by
pathogens as viroids, fastidious prokaryonts and
protozoans have been investigated by virologists in Brazil.
Viroids were found in citrus, grapevine, stone fruits and
ornamentals. Xylem invading bacteria (Xylella) were
found causing diseases in plum, citrus and coffee. A
large number of Candidatus phytoplasma and
Spiroplasma are being detected associated to witches’
broom type diseases of many cultivated and wild plants,
some of them important in the culture of passion flower,
corn, etc. The most recent menace is HLB in citrus,
caused by Candidatus Liberibacter, of which two species
were found in Brazil. Trypanosomatid flagellates
(Phytomonas) were detected in cassava causing poor
development of the roots and associated to spots in
tomato fruits and legume seeds. There are many success
stories of plant virology in Brazil as the expansion of the
strawberry culture after the discovery of that a cocktail
of viruses caused early degeneration of the plants and
their elimination, certification and mass multiplication of
virus-free plants; control of citrus tristeza by replacement
of the rootstock and premunization; control of the papaya
ringspot by an aggressive and systematic rouguing
program; continuous generation of virus resistant varieties
of several vegetable crops; control of bean golden mosaic
by zoning; consolidation of a seed potato production
program, etc. Electron microscopy and immunological
techniques for plant virus detection and identification was
started in the 1960’s, complemented by the introduction
of molecular methods in the 1980’s, and virus
identification by genomics is a routine procedure today.
Publications reporting the results of these researches
are growing at steady rate with an average of 150 new
abstracts and articles yearly. At the early years these
publications were mostly made in journals as Bragantia,
Arquivos do Inst.Biológico, O Biológico, Revista da
Agricultura, etc., but after 1970, with the advent of
specialized journals as Fitopatologia Brasileira (now
Tropical Plant Pathology) and Summa Phytopathologica,
they became the main sources for information on
Brazilian plant virology. However, many papers have also
been published in foreign specialized journals as
Phytopathology, Plant Disease, Plant Pathology, Journal
of Virology, Virology, Journal of General Virology,
Archives of Virology, Journal of Phytopathology, etc.
Brazilian researches on plant virology is becoming more
efficiente thanks to the cooperation among universities
and insititutions as well as through intense international
cooperation with centers of the Netherlands, Germany,
31
France, Spain, Japan, Australia, US and several Latin
American countries (Argentina, Paraguai, Colombia,
Costa Rica, Mexico).
DENGUE VIRUS 3 PATHOGENESIS IN MICE
MODEL
Erna Geessien Kroon
Laboratório de Vírus, Instituto de Ciências Biológicas,
Universidade Federal de Minas Gerais (UFMG), Av.
Antõnio Carlos, 6627 Belo Horizonte, Minas Gerais CEP
31-270901, Brazil
Dengue virus (DENV) may cause symptomatic infection
with mild, undifferentiated febrile illness called classical
dengue fever (DF) or a more severe disease, potentially
fatal, known as dengue hemorrhagic fever (DHF) or
dengue shock syndrome. The pathogenesis of DHF is
based on the virulence of the infecting DENV and
depends on the infecting serotypes and genotypes; it is
also based on the immunopathogenesis that is mediated
by host immune responses, including dengue viruscross-reactive antibodies that augment the severity of
infections. Involvement of central nervous system (CNS)
is extensively described. The present study describes
the virulence of DENV-3 isolates in a mouse model by
intracranial (i.c.) inoculation with genotypes I and III. Our
data suggest that, in this experimental model, DENV-3
genotype I may have the propensity to cause neurological
disease in mice, whereas the genotype III is associated
with asymptomatic infection in mice. Additionally, the
symptomatic mice show a decrease of white blood cell
count, infectious DENV in the brains and alterations in
levels of IFN-gamma, IL-6 and MCP-1. The results
confirm the mouse model as a way to study the biology
of DENV-3 isolates and to improve the knowledge about
the neurovirulence of the different genotypes of DENV.
Financial support: CNPq, FAPEMIG, INCT Dengue e
Pronex Dengue
WORKSHOP PRRSV
Fernando Osorio
Undoubtedly porcine reproductive and respiratory
syndrome (PRRS) is considered to be the most globally
significant infectious disease of swine. The awareness
on the economic significance on PRRS virus infections
for the swine industry, plus the sudden emergence of
new PRRS virus strains of unprecedented virulence in
China and S.E Asia, and the attempts at controlling the
disease at local or regional levels (in spite of the lack of
optimal, cost efficient technological tools for such
endeavor), all this emphasizes the importance that this
32
disease has for international animal agriculture. This
review will summarize the current situation regarding
PRRSV geographical circulation, the effect on the
international trade between countries that have or not
the disease, and different initiatives for eradication in
the US and elsewhere. Some of these are success stories
(Chile, Sweden), others are ongoing with encouraging
results (Stevens County, Minnesota, US) while others
are just starting (Sonora, Mx). We will also discuss
technological tools put in practice or still under
development in different locations to fight this disease
and the challenges that are yet to be overcome.
WATERBORNE VIRUS DISEASES
Fernando Rosado Spilki1
1 - Laboratório de Microbiologia Molecular, Universidade
Feevale, Novo Hamburgo, RS, Brazil
Many organisms may be excreted in feces and
transmitted by water, including bacteria protozoa and
viruses. In places with poor sanitation, low rates of sewage
treatment, as in the case of Latin America, such agents
have their way into water bodies facilitated. A wide variety
of viral agents can be transmitted by water, including
rotaviruses, noroviruses, enteroviruses, enteric
adenovirus, hepatitis viruses A and E. These agents,
the so-called enteric viruses, are very resistant in the
environment and can cause from subclinical infections
until diseases such as gastroenteritis and hepatitis,
among others. The prevention of viral diseases
transmitted by water goes through various strategies,
including vaccination for some agents, but mainly by
the use of sanitation and the spread of such knowledge
to the population in a direct and clear way. In this sense,
a short course was prepared to presented during this
meeting, aimed at teachers of public schools, with general
information about water-borne viruses. We hope this
information should be useful for preparation of lectures
and informational materials to the population.
RESUMO DE ATIVIDADE
FUMIO HONMA ITO
Prof. Titular
Departamento de Medicina Veterinária Preventiva e
Saúde Animal
Faculdade de Medicina Veterinária e Zootecnia da
Universidade de São Paulo
Desde 1976 está lotado no Departamento de Medicina
Veterinária Preventiva e Saúde Animal, da FMVZ-USP,
em regime de dedicação exclusiva com ministração de
disciplinas de Epidemiologia veterinária e de Medicina
veterinária preventiva, notadamente relacionadas aos
agentes zoonóticos. No início, as pesquisas estavam
voltadas mais para a febre aftosa, no entanto, nos anos
80, o foco de atenção foi direcionado para a raiva e
doenças zoonóticas como leptospirose e brucelose.
Desde a década de 60, o Departamento vem oferecendo
o serviço de diagnóstico da raiva como parte de
prestação de serviço à comunidade, sem ônus para os
usuários do serviço. Com a criação do curso de Pósgraduação em nível de Mestrado e Doutorado, no
programa de “Epidemiologia Experimental e Aplicadas
às Zoonoses”, as pesquisas e orientação de teses foram
mais direcionadas para a raiva, envolvendo os aspectos
de etiologia, patogenia e patologia, diagnóstico, profilaxia
e controle. A partir de 2.000, com o desenvolvimento de
técnicas moleculares, o grupo de pesquisa, em parceria
com os pesquisadores japoneses, vem realizando
estudos de caracterização genética e epidemiologia
molecular dos isolados do vírus da raiva do Brasil. É
membro da Coordenação Estadual de Controle da Raiva
do Estado de São Paulo, da Secretaria da Saúde do
Estado de São Paulo e integra o Comitê Científico
Consultivo sobre Raiva (CCR) do MAPA.
DENGUE IN BRAZIL: TRENDS, SURVEILLANCE AND THE EPIDEMICS OF 2008-2010
Joao Bosco Siqueira Jr 1,2 , Gisele Folador da
Fonseca2, Giovanini Evelim Coelho1, Ana Cristina da
Rocha Simplício1, Lívia Carla Vinhal1
1. National Dengue Control Program/Health Surveillance
Secretariat/Ministry of Health Brazil
2. Institute of Tropical Medicine and Public Health/Federal
University of Goias/Brazil
Objective: To characterize the current trends and
epidemiology of dengue in Brazil, with emphasis on the
2008 and 2010 epidemics and to evaluate the timeliness
of the dengue surveillance system
Methods: We reviewed available data from the
surveillance system (Information System for Reportable
Diseases - SINAN) and from the Hospital Information
System of the Unified Health System (SIH/SUS), from
2000 to 2010. A descriptive analysis of reported and
hospitalized cases according to sex, age, city of
residence, case classification, confirmation criteria, onset
of symptoms and date of data entry were performed
stratified by population size of the municipalities of the
country.
Results: Over 4.8 million dengue cases were reported
from 2000 to June 2010. The largest epidemics recorded
until the moment occurred in 2002, 2008 and 2010
reflecting cycles of different serotypes circulating in
Brazil. An increase in the number of reported cases, in
disease severity and hospitalizations was observed
since 2000 with a shift toward children mainly in 2008/
2009 with the predominance of serotype 2. The outbreaks
of 2008 highlighted this shift with at least 25% of reported
and hospitalized cases occurring in children under 15
years of age. Nine Brazilian municipalities with a population
larger than 1 million inhabitants accounted for around
20% of the cases during the study period. The analysis
of the timeliness of the surveillance system showed that
cases are prompt reported but the median for data entry
of the surveillance forms is ~20 days after the onset of
symptoms.
Conclusions: The epidemics of 2008 represented the
worst scenario of dengue in Brazil, regarding disease
severity and hospitalizations. These epidemics were
characterized by a shift in disease severity and
admissions towards children. This rapid change in the
epidemiology of dengue occurred similarly within the
country, despite of the population size of the
municipalities. Dengue remains one of the major public
health challenges in Brazil, and a continuous evaluation
of the disease epidemiology, surveillance and prevention
activities is mandatory to reduce the burden of the
disease in the country.
GENETIC DIVERSITY OF INFECTIOUS
BRONCKITIS VIRUSES ISOLATED IN BRAZIL
MONTASSIER, H.J. 1 1
Laboratório de Virologia e Imunologia – IMUNOVIR –
Depto.de Patologia Veterinária – FCAV – UNESP
Jaboticabal - SP;
* e-mail: heliojm@fcav.unesp.br
Infectious bronchitis virus (IBV) appeared in Latin
America by the 1950s and the first reported isolate from
that continent was of the Mass serotype in Brazil,
although isolation of the first variant was not reported in
that county until some 10 years later. In a study carried
out in the mid 1990s, IB isolates of at least 5 different
antigenic types were found in commercial chickens of
all types throughout Brazil, but mainly in the major poultry
producing area of the South. A significant number of IBV
field isolates, differing in phylogenetic analysis from the
genetic patterns of S1, N or non-structural protein genes
of American, European, Australian or even Asiatic
strains, have been detected in the Brazilian commercial
poultries, since the beginning of 2000. Additionally, these
variant isolates were grouped with earlier Brazilian variant
viruses isolated in 1980’s as well as with IBV isolates
recovered from commercial poultry flocks of Argentina,
between 2001 to 2008. Argentinean and Brazilian IBV
isolates shared 93.4% of similarity in their aminoacid
33
sequences, leading to the hypothesis that these variant
viruses seem to be indigenous to Brazil and Argentine,
and might derive from a common ancestor. These viruses
are circulating and evolving in the field for a long time
ago by infecting chickens from commercial poultries in
Brazil and might break the immunity conferred by Mass
serotype anti-IBV vaccines and cause the frequent
outbreaks of infectious bronchitis in this country. Thus,
it is also essential to further determine the immunotypes
(serotypes and protectotypes) and the pathotypes (tissue
tropism and virulence properties) of the Brazilian IBV
variants in order to define a candidate vaccine strain to
be used in IB immunoprophylaxis in Brazil.
Financial support: FAPESP e CNPq
PROTECTOTYPES OF IBV
Jane K A Cook
Consultant Microbiologist
Huntingdon, Cambridge, UK
The coronavirus, infectious bronchitis virus (IBV) causes
one of the most highly infectious diseases of poultry
worldwide. The initial site of infection is the upper
respiratory tract, but the virus can spread systemically
and cause disease in both the kidneys and the female
reproductive tract. Particularly in young birds, secondary
infection with bacteria, such as E coli, can worsen the
clinical infection, resulting in coli septicaemia with high
morbidity and variable mortality. High quality live
attenuated and inactivated vaccines are available to
control of IB infections. Only the live attenuated ones
are used in broilers, whilst both must be used to provide
life long protection of layers and breeders since
inactivated IB vaccines are only effective following live
priming.
Despite the availability of these vaccines, however, IB
cannot be considered to be under control. Apart from
the variety of diseases this virus can cause, the main
problem we face in controlling IB infections is that, being
an RNA virus, IBV undergoes both spontaneous mutation
and recombination, leading to continual alterations in the
amino acid composition of the viral genome. If the
changes that occur take place in the spike (S)
glycoprotein they may lead to the emergence of what is
defined by laboratory assays as a new IB serotype or
variant. Traditionally, novel IB variants were identified by
serum neutralisation tests (serotyping). However,
increasingly molecular methods, based on genetic
characterisation of part (usually just that which encodes
the S1 subunit of the surface glycoprotein) of the genome
are used to identify them (genotyping). Methods used
include sequencing, detection of genotype-specific parts
of the genome by RT-PCR, or determination of the
34
position of enzyme cleavage sites. These methods give
results much more quickly than serotyping and provide
useful information for epidemiological studies, but in the
case of IBV, results obtained by serotyping or genotyping
do not provide identical groupings of variants.
However, from a practical viewpoint this is not as
significant as might at first appear because we now know
that the protection provided by IB vaccines may be much
broader than the results of laboratory typing methods
might imply. This is because the IB variants are identified,
by either serotyping or genotyping, on the basis of a
very few changes in the amino acid composition of the
virus genome. Even when the changes occur in the S1
subunit of the spike glycoprotein, which is the major
induce of protective immunity and carries most of the
virus neutralizing epitopes, because so few changes
occur, most of the genome is unchanged.
This talk will discuss new IB variants of major importance
and describe ways in which protection against them may
be improved using currently available vaccines, without
the need to develop a new vaccine for each new,
significant IB variant that emerges.
DSRNA-INDUCED APOPTOSIS IS TRIGGERED BY DIRECT ACTIVATION OF BAX BY
INTERFERON REGULATORY FACTOR 3
Joao T. Marques, Saurabh Chattopadhyay, Kristi L.
Peters, Bryan R.G. Williams, Ganes C. Sen
Double stranded RNA (dsRNA) is a byproduct of viral
replication and a potent trigger for apoptosis, an
important element of innate antiviral defenses. Although
many dsRNA-activated pathways have been implicated
in apoptosis, the primary mechanism remains unclear.
Here, we characterized the mechanism of dsRNAinduced apoptosis, which requires Interferon (IFN)
Regulatory Factor (IRF) 3 but is distinct from previously
known pathways. Despite the well-characterized role of
IRF3 as a transcription factor, dsRNA-induced apoptosis
is independent of de novo gene expression and can be
triggered by forms of IRF3 that no longer translocate to
the nucleus, bind DNA or induce transcription. Instead,
dsRNA induces direct interaction between IRF3 and Bax
in the cytoplasm, which leads to the activation of the
latter. IRF3-activated Bax then induces apoptosis through
the intrinsic pathway. Our results reveal a new pathway
that directs apoptosis in response to dsRNA and relies
a transcription-independent function of IRF3.
CONTRIBUTIONS OF BIOLOGICAL APPROACHES ON THE STUDIES OF PLANT
VIRUSES.
Rezende, J.A.M.
Departamento de Fitopatologia e Nematologia, ESALQ/
USP, Piracicaba, SP. E-mail: jamrezen@esalq.usp.br
The first major contribution of biological methods on the
studies of plant viruses is associated with the beginning
of plant virology at early 1880’s. It was the demonstration
that leaf sap from Tobacco mosaic virus (TMV) infected
plants was contagious when injected with capillary glass
needles into healthy plants. At the same occasion the
causal agent of leaf variegation of Abutilon was shown
to be propagated vegetatively and graft-transmissible.
The identification of virus vectors as well as the
establishment of virus vector-relationship, whose studies
started with the classical works with Rice dwarf virus in
Japan, at the beginning of the 20th century, was also
accomplished by biological assays. The discovery of local
lesion hosts at late 1920 established a biological
quantitative assay for virus infectivity and paved the way
for the isolation and purification of plant viruses. Soon it
became clear that vector and graft transmissions were
also important tools do demonstrate the viral nature of
some virus diseases, whose agents were not transmitted
by mechanical inoculation. These biological tools were
and still are widely used to fulfill Koch’s Postulates for
plant virus diseases. This knowledge is fundamental for
the development of strategies for virus disease
management. For several species of plants, especially
fruit trees including citrus, grapevines, and apple, the
identification and maintenance of virus free matrixes rely
mainly on the use of indicator host for virus indexing
due to its high sensitivity. Any strategy of breeding for
virus resistance requires a good knowledge of the virus
and its different strains, which is achieved by biological
and molecular assays. Screening germoplasm for virus
resistance is carried out under natural infection or by
mechanical vector, and graft inoculations. The
establishment of preimmunization for virus disease
control, whose principle is the interference between
strains of the same virus, described in the 1930’s,
requires the selection of mild strains, which can be
achieved by searching outstanding plants under naturally
infected fields, or through some biological assays that
will promote virus segregation. Biological assays will
continuously contribute for further development of plant
virology.
Área: Virologia Vegetal
Tema: Mesa-redonda 7
Arquivo: RezendeJAM.doc
Apresentador: Jorge Alberto Marques Rezende
AN UPDATE ON HEPATITIS A VIRUS
BIOLOGY.
Juan Cristina
Laboratorio de Virología Molecular. Centro de
Investigaciones Nucleares. Facultad de Ciencias,
Montevideo, Uruguay.
Hepatitis A virus (HAV) is the causative agent of type A
viral hepatitis. HAV is endemic in the South American
region. Recent findings have shown that HAV possess
several characteristics that make it unique among the
family Picornaviridae. Important differences in the
mechanisms of translation initiation by comparison with
other members of the family, and a particular strategy of
codon usage in the context of virus/cell competition make
also HAV a particularly interesting virus. Interaction of
HAV with host cell innate immune system permits HAV
to interfere with interferon regulatory factors signaling
the antiviral response. HAV circulates in vivo as
distributions of closely genetically related variants referred
to as quasispecies. HAV exploits all known mechanisms
of genetic variation to ensure its survival, including
mutation and recombination. Only one serotype and six
different genetic groups (three humans and three simian)
have been described. HAV mutation rate is significantly
lower as compared to other members of the family
Picornaviridae. All these appear to contribute, at least in
part, to the presence of only one known serotype.
PATHOGENESIS STUDIES OF THE 2009
PANDEMIC INFLUENZA VIRUS AND
PSEUDORABIES VIRUS FROM WILD PIGS IN
SWINE
Lager, KM,1 Vincent,AL,1 Ciacci-Zanella JR,2 Zanella,
EL,3 Miller, LC,1 Kehrli, ME.1
1
Virus and Prion Diseases of Livestock Research Unit,
National Animal Disease Center, USDA, ARS, Ames,
IA, USA. 2Labex-USA, EMBRAPA – Brazilian Agriculture
Research Corporation, Brasilia, DF, Brazil. 3Universidade
de Passo Fundo, Campus Universitário do Bairro São
José, Caixa, Brazil.
Over the last ten years in the United States the
epidemiology and ecology of swine flu and pseudorabies
has been dynamic. Swine flu is caused by influenza A
virus and the disease was first recognized in pigs
concurrent with the 1918 Spanish flu pandemic in
humans. Pigs displayed clinical signs similar to people
affected by the Spanish flu. Reverse genetics has been
used to demonstrate that during the 1918 Spanish flu
pandemic, pigs and people were infected with a similar
virus, an H1N1 influenza virus. In contrast to the severity
of disease reported in people, experimental infection of
swine with the 1918 Spanish flu virus produced minimal
respiratory disease while it induced fatal infections in
35
mice and ferrets. Although the 2009 pandemic H1N1 virus
was not as pathogenic in people as the historical reports
from 1918, the 2009 virus induced fatal disease in mice
and ferrets and induced moderate disease in pigs.
Understanding how influenza viruses cause disease and
why there are species differences is the subject of intense
research by many around the world. Pseudorabies virus
(PRV) infection can induce respiratory disease,
reproductive failure, and affect the central nervous
system. PRV vaccines have been used to eradicate the
virus from swine herds and even from entire countries.
One weakness in PRV control and eradication programs
is the indigenous PRV infection of feral swine. Since the
eradication of PRV from the U.S. commercial swine herd,
there have been rare case reports of commercial swine
becoming infected with feral swine PRV isolates. The
difficulty in controlling the expansion of the feral swine
population in North America presents a chronic threat to
the PRV-free status of the US commercial swine herd. A
better understanding of this source of infection may lead
to improved PRV control programs. This report
summarizes studies investigating the pathogenicity and
transmissibility of influenza viruses and feral swine PRV
isolates from the U.S. in swine.
WEST NILE VIRUS: ARE WE PREPARED?
Laura Gil
West Nile virus (WNV) is a small, enveloped, arbovirus
(arthropod-borne viruses), positive RNA virus of the
Flaviviridae family. This virus is closely related to other
arthropod-borne and medically relevant viruses, such as
Dengue (DEN), Yellow fever (YF), and Japanese
encephalitis (JE) viruses. The WNV is the most
widespread arbovirus in the world, and since its
introduction into North America in 1999, the virus has
spread rapidly across the continent. In recent years, the
virus spread southward into the Central and South
America as well, and the public health impact of the
virus in those areas remains poorly understood. The virus
cycles in nature between mosquitoes and birds, but also
infects human, horses, and other vertebrates. In humans,
WNV disseminates to the central nervous system and
causes severe disease primarily in the immunosuppressed and elderly persons. Although WNV vaccines
were already approved for animal use, there is no vaccine
available for humans. Several experimental vaccines are
in development, including attenuated, chimeric
flaviviruses replaced with the WNV prM/E gene region,
plasmid DNA encoding the prM/E gene, and inactivated
whole virions. This presentation will provide an overview
and update on WNV, and will highlight how chimeric
viruses can be used as a valuable diagnostic tool and
36
for vaccine development against WNV.
CURSO DE EXTENSÃO PARA PROFESSORES - EXTENSION COURSE FOR
TEACHERS
Ledy H. S. Oliveira
Day 1 – 18th October 2010 7.30 – 8.20 am
Dengue/Yellow Fever
1. Vector born diseases approach
2. Dengue prevention as a school-connection problem
3. Health education, social responsibility, behavioral
changes, attitudes
4. Yellow fever and non-human primates
Sexually transmitted viruses
Day 2 – 19th October 2010 7.30-8.20 am
The course addresses:
1. STI prevention involves sexual education
2. STI related risk behaviors
3. Concerns about herpes, genital warts, hepatitis,
molluscum contagiosum, AIDS
RESUMO DA APRESENTAÇÃOOUTROS
FLAVIVIRUS CAUSADORES DE DOENÇA
HUMANA NO BRASIL, NA MESA-REDONDA,
DENGUE E OUTRAS FLAVIVIROSES
BRASILEIRAS.
Luiz Tadeu Moraes Figueiredo
Nesta apresentação, pretende-se mostrar evidências de
que estão ocorrendo várias outras flaviviroses no Brasil
além do dengue e da febre amarela e ressaltar a
importância das mesmas em termos de saúde pública.
Desta forma, chamaremos atenção para flavivirus
zoonóticos de aves, transmitidos por mosquitos Culex
e potencialmente causadores de infecções humanas do
sistema nervoso central. Tratam-se dos: vírus Rocio, que
infecta cavalos em diferentes regiões do Brasil e foi
detectado em 2 pacientes de Manaus com
meningoencefaliteviral; vírus da encefalite de SaintLouis
que foi detectado infectando cavalos e também,
produzindo pequenos surtos de meningoencefalite e
doença febril aguda, além de casos esporádicos, no
estado de São Paulo; do vírus Cacipacoré que é causador
de doença febril aguda em diferentes partes do Brasil e
foi encontrado infectando distintas espécies de
mosquitos e carrapato; do vírus WestNile que já foi
introduzido no país e vem infectando cavalos de
diferentes regiões, porém, sem impacto aparente em
saúde pública humana.
“IMMUNE MODULATION OF NEGATIVE
STRAND RNA VIRUSES, WITH SPECIAL
EMPHASIS ON INFLUENZA AND NEWCASTLE DISEASE VIRUS”
Mikael Berg, PhD, professor
Department of Biomedical Sciences and Veterinary Public
Health, Section of Virology, Swedish University of
Agricultural Sciences, Uppsala, Sweden
Background
During viral infection the virus immediately faces the
innate immunity in the form of type I interferon (IFN).
Viral components like the genomic RNA are recognized
by specialized receptors like Toll-like receptors (TLRs)
and helicases like RIG-I and via intracellular signaling
IFN is expressed. The IFNs are secreted and bound to
its receptor and another signaling event leads to
expression of a large number of antiviral proteins, so
called interferon stimulated genes (ISGs). As a response
to this challenge many viruses have evolved a number
of evasion strategies for the IFN mediated defense
mechanisms. This includes inhibition of the expression
of interferon itself, the signaling leading to expression of
ISGs or by direct inhibition of function the ISG proteins.
The negative strand RNA viruses form an extremely
important group of viruses and are no exception from
this rule of IFN inhibition. It appears that special genes
have evolved to handle this task. The zoonotic potential
may lie in this ability.
Materials and methods
Different isolates of influenza and NDV was studied for
their variability in the NS and P genes respectively. The
NS1 genes were cloned and tested in vitro for their ability
to inhibit an IFN reporter system and the expression of
IFN mRNA.
Results
Different NS1 have variable abilities to shut down the
IFN gene. Especially notable is that NS1s from the socalled allele B are poor in this, while most from allele A
are much better. The variability between the genes is
considerable. Furthermore, the viral genome of NDV
appears to have different ability to induce IFN.
Discussion
The ability to shut down the innate immunity of the host
is vital for a virus to be able to replicate. We have shown
that different NS1s from influenza virus have a strong
capability to do this, but it differs between different
isolates. The difference in ability to evade the IFN
response may explain the differences in crossing species
barriers between viruses. As comes for NDV different
isolates have variable ability to shut-down the IFN
response.
Conclusion
To understand the ability of viruses to evade the host
immunity is important to understand pathogenicity and
zoonotic potential of different viruses.
BEGOMOVIRUS PATHOGENESIS: UNRAVELING A COMPLEX, MULTILAYERED NETWORK OF HOST-PATHOGEN INTERACTIONS
Zerbini, F.M. 1, Alves-Junior, M. 1, Andrade, E.C. 1,
Antunes, T.F.S.1, Silva, F.N.1, Alfenas-Zerbini, P.2, Fontes,
E.P.B.3
1
Dep. de Fitopatologia/BIOAGRO, 2Dep. de Microbiologia/
BIOAGRO, 3Dep. de Bioquímica e Biologia Molecular/
BIOAGRO, Universidade Federal de Viçosa, Viçosa, MG.
*E-mail: zerbini@ufv.br
Begomoviruses (family Geminiviridae) are plant-infecting
viruses with a circular, single-stranded DNA genome
encapsidated in twinned icosahedra and transmitted by
whiteflies in a persistent, non-propagative manner.
Begomoviruses infect fully differentiated plant cells, and
replicate and accumulate in the nuclei. Begomovirus
genomes are among the smallest of all viruses (2.6 kb
for monopartite species, 5.2 kb for bipartite species)
and code for 6 to 8 proteins. Two proteins, Rep and Ren,
are directly involved in viral DNA replication, although
neither is a DNA polymerase. One protein, CP, forms
the viral capsid and is involved in virus-vector interactions.
One (AV2) or two proteins (MP and NSP), are directly
involved in viral cell-to-cell movement for mono- and
bipartite species, respectively. Two additional proteins,
Trap and AC4, plus NSP, are responsible for downregulating or suppressing host defense responses and
up-regulating host metabolic pathways which are
beneficial to the virus. In order to initiate infection,
begomoviruses must overcome defense responses from
the host, including PAMP-triggered immunity (PTI) and
effector-triggered immunity (ETI). The most common PTIbased response against viruses is RNA silencing, in
which dsRNA formed during viral genome replication is
cleaved into siRNAs which are used as a guide to detect
and degrade viral RNA. In spite of having DNA genomes
replicated without generating a dsRNA intermediate,
begomoviruses are inducers and targets of RNA silencing.
Depending on the virus, Trap or AC4 suppress RNA
silencing. Interestingly, a strong synergism occurs in a
mixed infection with one virus that uses Trap and another
that uses AC4, demonstrating the fundamental role of
RNA silencing suppression in the establishment of a
successful viral infection. Begomoviruses use several
components the host DNA replication machinery in order
to replicate their genomes. However, they infect
differentiated plant cells in which these components are
present at very low concentrations, or not present at all.
37
Therefore, begomoviruses must reprogram the cell cycle
in order to induce the expression of DNA replication
factors. This is accomplished by the interaction between
Rep and pRb, the plant homologue of the retinoblastoma
protein. Interestingly, this reprogramming of the cell cycle
does not activate cell division. Instead, cells enter the
endoreduplication cycle. Once viral infection is initiated,
its maintenance requires an intricate set of interactions
between viral and host factors. These include interactions
between Trap and the plant homologue of SNF1, a protein
involved in the regulation of plant responses to a number
of stresses, and between NSP and NIK, a member of
the LRR-containing receptor-like serine/threonine kinase
family that includes members involved in plant
development and defense. Interactions involving the MP
protein have only recently been reported and are less
understood. Probably as a consequence of the different
degrees in which these interactions occur, some
begomoviruses are phloem-restricted in their hosts, while
others are capable of invading the mesophyll. In our
laboratory, we are studying begomovirus-host interactions
using Tomato rugose mosaic virus (ToRMV), which is
phloem-restricted in tomato (Solanum lycopersicum) and
in the laboratory host Nicotiana benthamiana, and
Tomato yellow spot virus (ToYSV), which is also phloemrestricted in tomato, but invades the mesophyll in N.
benthamiana. ToYSV also infects Arabidopsis thaliana,
a model plant for which a large collection of mutants and
other genetic and molecular tools are available. We have
analyzed several aspects of viral infection in tomato
and N. benthamiana plants with single or mixed infection
by ToRMV and ToYSV. We are also constructing
recombinants between ToRMV and ToYSV to identify viral
pathogenicity determinants, and searching for plant
proteins which interact with the MP protein. Results of
these experiments will be presented and discussed in
the context of begomovirus pathogenesis.
Financial support: Research in our laboratory is
supported by the National Research Institute for PlantPest Interactions, and by grants from CNPq and
Fapemig.
POTYVIRUS-HOST INTERACTIONS DURING
INITIAL STEPS OF INFECTION
Alfenas-Zerbini, P. 1*, Bruckner, F.P. 1, Coco D. 1,
Cascardo, R.S.2, Carvalho, M.3, Zerbini, F.M.2, Maia, I.G.3
1
Dep. de Microbiologia/BIOAGRO, 2Dep. de Fitopatologia/
BIOAGRO, Universidade Federal de Viçosa, Viçosa, MG,
3
Dep. de Genética, Universidade Estadual Paulista,
Botucatu, SP. *E-mail: palfenas@ufv.br
The genomes of most plant viruses code for only 4-10
38
proteins which are required to complete the infection
cycle, including viral genome replication, cell-to-cell
movement and systemic spread. For a successful
infection, these viral proteins must interact with host
factors, modulating metabolic pathways and coordinating
a complex network of biochemical interactions in the
pathogen’s favor. A subtractive library constructed from
susceptible tomato plants infected by the plant potyvirus
Pepper yellow mosaic virus (PepYMV) identified several
genes which are putatively involved in the initial steps
of the viral infection process, including those that code
for the Translationally Controlled Tumor Protein (TCTP)
and the tomato homologue of the Saccharomyces
cerevisae SNF1 kinase (Sl-SNF1). TCTP is a highly
conserved protein in eukaryotes, and is involved in
several fundamental cellular processes such as cell
growth, cell cycle progression, programmed cell death
and protection against different types of stresses. SNF1
kinase plays an important role in transcriptional activation
and expression of genes involved in the cellular response
to different stresses, such as nutrient limitation, high
salinity, heat shock and virus infection. The objectives
of this work are to study the roles of TCTP and Sl-SNF1
during PepYMV infection in susceptible hosts. The
subcellular localization of TCTP in healthy and PepYMVinfected plants was analyzed by confocal microscopy
using a TCTP-YFP fusion. In healthy plants the
subcellular localization of TCTP is cytoplasmatic, as
described for other organisms. Forty-eight hours after
PepYMV infection, TCTP is relocated to the nucleus. In
plants in which PepYMV infection was already
established (7 days after inoculation), TCTP relocalization
to the nucleus was less efficient, with the protein
accumulating predominantly in the cytoplasm. Transgenic
tobacco plants overexpressing TCTP showed a higher
accumulation of PepYMV at 14 and 21 days after
infection compared to non-transformed controls. Together,
these results suggest that TCTP has an important role
in the infection by PepYMV. The functional role of the
TCTP during PepYMV infection is also being investigated
using virus-induced gene silencing (VIGS) and TCTPsilenced transgenic plants. In order to identify and
characterize additional viral and host factors involved in
the TCTP-mediated network of plant-potyvirus
interactions, transgenic tomato plants expressing the TAP
(tandem affinity purification) tag were produced, protein
complexes were purified using TAP, and its constituents
are being analyzed by mass spectrometry. To assess
the role of Sl-SNF1 in the PepYMV infection process,
transgenic tomatoes (cv. ‘Moneymaker’) silenced for this
gene were generated. Silenced transgenic plants were
mechanically inoculated with PepYMV and ELISA was
performed to verify the phenotype resulting from Sl-SNF1
silencing. The results showed that four out of five nontransformed plants were infected, while all ten transformed
plants remained symptomless and were ELISA negative.
Assessment of viral accumulation by quantitative RTPCR is being performed to confirm the absence of viral
replication. Silencing of Sl-SNF1 by VIGS was very
effective, and these plants also showed a reduction in
viral load at 72 hours after inoculation. These results
suggest that both TCTP and Sl-SNF1 have critical roles
in the tomato-PepYMV interaction, being necessary for
the establishment of a systemic infection. Further studies
should be conducted to confirm this hypothesis and to
improve our understanding of the nature and mechanisms
of the interactions between TCTP, Sl-SNF1 and PepYMV.
BOVINE HERPESVIRUS 5 INDUCES NA
OVERPRODUTION OF NITIC OXIDE IN THE
BRAIN OF RABBITS
O BOHV-5 INDUZ A SUPERPRODUÇÃO DE
ÓXIDO NÍTRICO NO ENCÉFALO DE
COELHOS
Renata Dezengrini1
Professora Adjunta, Laboratório de Microbiologia,
Faculdade de Ciências Médicas, Universidade Federal
de Mato Grosso - Campus Cuiabá. Av. Fernando Correa
da Costa, n. 2367, Bairro Boa Esperança, CEP 78060900.
E-mail
para
correspondência:
renatadezengrini@yahoo.com.br/
Os herpesvírus bovino tipos 1 (BoHV-1), 2 (BoHV-2) e 5
(BoHV-5) são classificados na Família Herpesviridae,
subfamília Alphaherpesvirinae e produzem perdas
econômicas em rebanhos atingidos por diminuir os
índices produtivos e reprodutivos, causando a
mortalidade de animais (ROIZMAN et al., 1992;
FRANCO; ROEHE, 2007). Essas infecções ocorrem
com frequência em rebanhos brasileiros (WEIBLEN et
al., 1989; SALVADOR et al., 1998; COLODEL et al., 2002;
RISSI et al., 2006; SILVA et al., 2007). O BoHV-1 é
associado a várias manifestações clínicas, incluindo
aborto, doença respiratória, genital e eventualmente
neurológica (SILVA et al., 2007). O BoHV-2 é responsável
pela mamilite herpética, enquanto que o BoHV-5 é
associado à enfermidade neurológica de curso
geralmente fatal em bovinos jovens (VOGEL et al., 2003;
FRANCO; ROEHE, 2007). Esses vírus têm sido alvo de
estudos de patogenia, pela importância sanitária e
econômica das infecções para rebanhos bovinos. Nesses
estudos, coelhos têm sido utilizados como modelo
experimental, pois quando inoculados pela via intranasal
com o BoHV-5 apresentam doença neurológica, e pela
via conjuntival com o BoHV-1 desenvolvem sinais
oculares semelhantes aos observados em bovinos
(ROCK; REED, 1982; SILVA et al., 1999; CARON et al.,
2002).
A doença neurológica produzida pelo BoHV-5 em coelhos
e em bovinos nem sempre é acompanhada de detecção
abundante de antígenos no encéfalo e lesões
histopatológicas, que justificassem a severidade das
manifestações clínicas (RISSI et al., 2006). Por isso,
acredita-se que outros mecanismos possam estar
envolvidos na produção de sinais neurológicos durante
a infecção por esse vírus (FLORES et al., 2009).
O óxido nítrico (NO) é sintetizado por três isoformas da
enzima óxido nítrico sintase (NOS), e regula funções
fisiológicas, como a condução de estímulos nervosos e
vasodilatação, além da imunidade inata contra patógenos
(PACHER et al., 2007). Embora a atividade
antimicrobiana do NO contra fungos e bactérias seja
bem documentada e a atividade antiviral dessa molécula
tenha sido comprovada para o herpes simplex humano
tipo 1 (HSV-1) em células de cultura (AKAIKE; MAEDA,
2000), a ocorrência de toxicidade durante a infecção por
vírus neurotrópicos in vivo, como o HSV-1, tem sido
relatada (KOPROWSKI et al., 1993; ZHENG et al., 1993;
FUJII et al., 1999; AKAIKE; MAEDA, 2000; UBOL et al.,
2001; MEYDING-LAMADÉ et al., 2002).
Durante a infecção pelo HSV-1, a resposta inflamatória
estimulada é intensa, não sendo suficiente para limitar
a progressão da encefalite. Não obstante, essa resposta
exacerbada é implicada no desenvolvimento da
encefalite fatal em camundongos, pois observa-se
estresse oxidativo relacionado à expressão de iNOS no
encéfalo desses animais infectados (LUNDBERG et al.,
2008; MARQUES et al., 2006; 2008). A toxicidade e
participação do NO na patogenia de encefalites virais é
atribuída à diminuição da síntese de ATP e interferência
com a síntese de DNA nas células, S-nitrosilação de
proteínas, além da modulação da resposta imune celular
(AKAIKE; MAEDA, 2000). Portanto, acredita-se que o
NO, produzido após a estimulação da imunidade inata,
possa apresentar participação na neuropatogenia do
BoHV-5. Nesse sentido, o tratamento com inibidores da
NOS, aliado à terapia antiviral, poderia elucidar a
participação do NO na patogenia dessa infecção.
Em um primeiro momento, aspectos da patogenia da
infecção neurológica pelo herpesvírus bovino 5 (BoHV5) foram estudados em coelhos inoculados. Investigouse a função do óxido nítrico (NO) na doença neurológica
produzida pelo BoHV-5 em coelhos. A espectrofotometria
para os produtos de degradação do NO (NO2 e NO3)
revelou um aumento significativo nos seus níveis em
várias regiões do encéfalo de coelhos infectados (F(4,
40)=3.33; P<.02). A quantificação do NO no encéfalo
nos dias seguintes à inoculação viral (2 a 6 dias pósinfecção; d.p.i.) revelou um aumento gradativo,
correlacionado temporal e espacialmente com a invasão
e disseminação viral, e precedendo o desenvolvimento
de sinais neurológicos. Por imunoistoquímica, revelouse grupos de neurônios e astrócitos expressando a
39
enzima óxido nítrico sintase (NOS) em regiões do
encéfalo próximas a neurônios positivos para antígenos
do BoHV-5. Esses resultados demonstram que a
replicação do BoHV-5 no encéfalo de coelhos induz uma
a síntese exacerbada de NO. O aumento nos níveis de
NO no início da infecção se correlaciona especial e
temporalmente com a disseminação viral no encéfalo e
precede o desenvolvimento de sinais neurológicos.
Sugere-se assim que a produção aumentada de NO em
resposta à infecção possa participar da patogenia dessa
doença neurológica.
Posteriormente, pela necessidade de um antiviral para
associação nos estudos com inibidores da NOS,
investigou-se a atividade de três fármacos antivirais frente
ao BoHV-1, BoHV-2 e BoHV-5 in vitro pelo teste de
redução no número de placas virais. Dentre os fármacos
que possuem ação inibitória sobre a replicação dos
herpesvírus, destacam-se os análogos de nucleosídeos,
como o Aciclovir (ACV) e Ganciclovir (GCV). Esses
fármacos são ativados por fosfoproteínas virais - a
timidina kinase (TK) ou a fosfotransferase (UL97) - e
inibem a replicação da cadeia nascente de DNA por
impedir a adição de outros deoxinucleotídeos trifosfatos
(REID et al., 1988; WHITLEY; ROIZMAN, 2001; COEN;
RICHMAN, 2007; HUSSEIN et al., 2008). O Foscarnet
(PFA) é um análogo sintético do pirofosfato, e impede a
conversão de nucleosídeos à nucleotídeos, impedindo
que a DNA polimerase complete o seu ciclo catalítico
(ERIKSSON et al., 1982; DE CLERCQ, 2001). Testes in
vitro demonstraram resultados contraditórios sobre a
atividade do ACV frente ao BoHV-1 (THIRY et al., 1983;
GLOTOV et al., 2004). O PFA inibiu a replicação do BoHV1 em células primárias (SCHWERS et al., 1980) e em
células do cumulus co-cultivadas com embriões bovinos
(MARLEY et al., 2006). Estudos com o BoHV-2 e BoHV5 são escassos na literatura. Por ensaio de placa,
verificou-se que o ACV foi pouco ativo frente aos três
vírus; o Ganciclovir apresentou atividade moderada frente
ao BoHV-2 e, em menor grau, contra o BoHV-5, sendo
ineficaz frente ao BoHV-1. O Foscarnet (PFA) apresentou
a atividade antiviral mais pronunciada, sendo o único
fármaco que, na concentração de 100 μg/mL, inibiu
completamente a produção de placas pelos três
herpesvírus bovinos testados.
Portanto, o PFA foi selecionado para testes de terapia
experimental de infecções pelos herpesvírus bovinos in
vivo. Para isso, coelhos inoculados pelas vias intranasal
(BoHV-5) ou no saco conjuntival (BoHV-1 e BoHV-5)
foram submetidos a protocolos de terapia com 3
aplicações intraperitoneais de PFA 100 mg/kg/dia ou
água ultrapura (controles), iniciados 8-24 horas p.i.
Coelhos inoculados com o BoHV-5 e tratados com o
PFA apresentaram índices de mortalidade (11/22; 50%)
estatísticamente inferiores aos controles não-tratados
(21/22; 93,7%). Uma redução significativa no título médio
40
de vírus foi observada no pico da excreção viral. Em
coelhos inoculados no saco conjuntival com o BoHV-1
e tratados com o PFA, foram observadas reduções na
excreção viral, na frequência, severidade e duração dos
sinais oculares, comparando-se com o grupo controle.
Por fim, com os resultados dos experimentos anteriores,
investigou-se o efeito da inibição da isoforma induzível
da enzima óxido nítrico sintase (iNOS), associada ou
não ao tratamento com o PFA, na infecção neurológica
pelo BoHV-5 em coelhos. Grupos de coelhos inoculados
com o BoHV-5 foram tratados com o inibidor da iNOS
aminoguanidina (AG); com PFA; com ambos os
fármacos; ou não receberam tratamento. Animais de
todos os grupos excretaram o vírus nas secreções nasais
entre os dias 2 e 10 pi e desenvolveram doença
neurológica, porém com diferentes freqüências e
períodos de incubação. Os índices de morbidade e
mortalidade foram de 100% (6/6) nos grupos AG e
controle; 66,7% (4/6) no grupo PFA e 83,3% (5/6) no
grupo AG+PFA. O período de incubação foi
significativamente menor e os sinais neurológicos foram
mais precoces e severos nos animais do grupo AG.
Portanto, o tratamento com PFA reduziu a morbidade e
mortalidade associadas com a infecção pelo BoHV-5; e
o tratamento com AG resultou no agravamento e na
antecipação do quadro neurológico.
Os resultados obtidos por meio dos experimentos
relatados contribuem para o conhecimento da patogenia
da doença neurológica pelo BoHV-5, demonstrando a
participação do NO na patogenia da infecção neurológica
aguda produzida por esse vírus, bem como
demonstrando a eficácia do PFA frente ao BoHV-1 e
BoHV-5. Não obstante, abrem perspectivas para estudos
adicionais de patogenia e terapêutica anti-herpesvírus.
THE 2009 H1N1 INFLUENZA PANDEMIC:
LESSONS LEARNED
Ruben Donis, Ph.D., Influenza Division, CDC, DHHS
A novel influenza subtype H1N1 discovered in April 2009
among patients with influenza-like illness in the United
States was traced to earlier outbreaks of unknown
etiology in Mexico. Despite early detection and rapid
deployment of interventions, the virus spread globally in
the human population causing a pandemic that was
officially declared by the World Health Organization on
June 11, 2009.
Genetic analyses performed within days of virus isolation
revealed that all the genes were of swine influenza origin.
Evolutionary analysis indicated that the novel 2009/
H1N1 virus contained genes from North American and
Eurasian swine influenza viruses. Such genome
composition had not been detected previously among
swine viruses anywhere, and human-to-swine
transmission may enhance the circulation of this virus
in swine. Interestingly, the most recent ancestors of
the 2009/H1N1 viruses were pig viruses that circulated
in the late 1990’s, underscoring gaps in global virologic
surveillance in this species.
Swine are highly permissive for generation of reassortant
viruses with novel properties. Half a century of sporadic
human infections with classical H1N1 viruses from swine
in the US pointed towards the pandemic potential of these
viruses should they acquire person-to-person
transmissibility. Reassortment of classical North
American H1N1 with Eurasian swine influenza viruses
resulted in the incorporation two new genes, suggesting
a linkage between these events and its transmissibility
Pandemic H1N1 vaccination in the US started in early
October 2010, just 4 months after pandemic declaration
and only 6 months after virus discovery. Despite these
laudable efforts, the second wave of pandemic H1N1
swept through the US and peaked in October/November,
reducing the public health impact of the vaccine.
Interestingly, the 2009/H1N1 virus infected primarily
infants, children and young adult age groups. Persons
older than 65 years were postulated to have protective
immunologic memory elicited by prior infection with
seasonal H1N1 influenza viruses circulating in the early
1950s. The host protection mechanisms have been
poorly defined, but cross-reactive antibodies were most
frequently detected among individuals 60 years of age
or older. Interestingly, even 2009/H1N1- susceptible
young adults achieved protective antibody titers with a
single dose of vaccine, indicating that prior seasonal
influenza infections were sufficient for immunologic
priming.
2009/H1N1 circulation peaked during the winter months
of the Southern Hemisphere (June-August 2009), and
the fall-winter (October-December) in the Northern
Hemisphere. H1N1 circulation during these peaks
reached very high levels, overshadowing the seasonal
influenza virus subtypes. Although the future evolution
of the 2009 H1N1 virus in the human population cannot
be predicted, continued circulation, particularly among
younger age groups, seems likely.
The emergence of a novel influenza virus in the human
population is a rare host switch event – as such, these
events are highly unpredictable. The 2009 influenza
pandemic taught us very important lessons from which
we must learn to better coordinate future animal and
human health preparedness to face the next pandemic.
VARIABILITY OF HEPATITIS B VIRUS:
RELATIONSHIPS BETWEEN AFRICAN AND
BRAZILIAN ISOLATES
Selma de Andrade Gomes
Laboratório de Virologia Molecular, Instituto Oswaldo
Cruz, FIOCRUZ, Rio de Janeiro, RJ, Brazil
Hepatitis B virus (HBV) is the prototype member of the
Hepadnaviridae family, and is prone to mutations due to
the replication of its genome (double-stranded DNA) via
reverse transcription of an RNA intermediate. On the
other hand, the compactness of its genome prevents a
large degree of genetic variability. Due to these
antagonistic characteristics, HBV possesses an
evolutionary rate intermediate between DNA and RNA
viruses.
The genetic variability of HBV is reflected by at least
eight genotypes (A to H, not including two new proposed
genotypes) that are related to their geographical origins.
Genotypes are based on the greater than 8% sequence
divergence for their entire genome and most of them are
divided into subgenotypes with distinct virological and
epidemiological properties.
In Brazil, we have demonstrated that genotypes A, D
and F are predominant and their distributions across the
country follow a gradient from northern to southern
regions. North, Northeast and Southeast regions showed
a higher prevalence of genotype A, the most common
genotype in Brazil. The high rate of genotype D isolates
in the South region could be explained by influx of
immigrants from Central Europe at the beginning of the
20th century. The balanced distribution of genotypes A
and D in the Central-West region reflects the delayed
occupation of that area by migration flows from South,
Southeast and Northeast regions. Interestingly, in
contrast to other Latin American countries, HBV genotype
F (native from aboriginal population) represents a low (<
15%) proportion of the Brazilian isolates, even in the
North region where descendents of the native aboriginal
population are more numerous.
HBV genotype A had been initially divided into two genetic
subgroups, A1 (from Africa) and A2 (from Europe). In
previous studies conducted by us in the general Brazilianor in the Afro-Brazilian population, HBV/A/A1 appeared
to be the most frequent subgenotype, indicating that HBV
main influx came from Africa. Genotypes G and C, rarely
detected in South America, were recently identified in
Brazil. Specific transmission route (probably linked to
homosexual behavior) of genotype G isolates and
pathogenic properties of genotype C (from Asia and more
aggressive than others) might explain their presence in
HBV infected Brazilian patients submitted to drug
treatment.
HBV genotypes A, D and E circulate in Africa. Classified
now in several subgenotypes, genotype A isolates are
more diverse in Africa than in the rest of the world,
suggesting an African origin and a long history on this
continent. Genotype E, thought to be the most recent
41
genotype originating in Africa, is extensively spread
throughout the West-Africa. Despite the African historic
slave trade, genotype E has only sporadically been found
outside Africa, indicating that this genotype was
introduced during the past 200 years. We have recently
shown that HBV genotype E was the most prevalent
genotype in Angola, followed by genotype A. We supposed
that HBV/A/A1 has been introduced into Brazil during
the slave trade. Angola was one of the main suppliers of
the slave trade, and it is reasonable to think that HBV/
A/A1 was introduced into Brazil via Angola. Yet, only
about 10% of the samples characterized in Angola
belonged to genotype A. Further studies with HBV/A/A1
from Brazil and Africa should be conducted to elucidate
the origin of subgenotype A1, which is frequently detected
in Brazilian individuals and rarely found in Luanda, the
main port of the slave trade to Brazil.
FUNCTIONAL ANALYSIS
POTYVIRUS INTERACTIONS
OF
PLANT-
Sylvie German-Retana
Equipe de Virologie, UMR GDPP 1090, INRA Université
de Bordeaux 2, BP 81, F-33883 Villenave d’Ornon,
France.
german@bordeaux.inra.fr
Phytoviruses are major plant pathogens of agriculture
worldwide and cause considerable economic damage in
various crops including vegetables, grains, and
ornamentals. In the absence of curative control methods
against plant viruses, the most cost-effective, reliable
and environmentally-friendly strategy for controlling viral
diseases, remains the breeding of genetic resistance
into crop cultivars. Though some durable resistances
exist, virus variants already present in, or arising from
the virus population, often break the resistance down.
Therefore, it is crucial to understand basic biological plantvirus interactions in order to identify tangible targets for
new resistance strategies (involving identification of host
factors required for viral infection).
The Potyvirus genus is the largest genus of plant viruses
infecting a broad range of dicot and monocot crops.
Recent discoveries begin to reveal the basis of the
molecular mechanisms associated with durable
resistances against potyviruses. These resistances are
linked to mutations of plant factors interacting with viral
proteins during the infectious cycle. Such mutations,
impairing the interaction, confer to the plant a recessive
resistance against the virus.
In recent years, components of the eukaryotic translation
initiation complex were identified as essential
determinants in the outcome of RNA virus infections,
including potyviruses. In particular, we showed that
42
recessive allelic genes mo1 1 and mo1² in lettuce,
currently used to protect lettuce crops against Lettuce
mosaic virus (LMV), correspond to mutant alleles of the
gene encoding eukaryotic translation initiation 4E (eIF4E)
(4, 6). Recently, we showed that viral resistance-breaking
determinants map not only to the Viral protein genomelinked (VPg) encoding region (identified so far as the
single potyvirus virulence determinant) but also to the
C-terminus of the CI (Cytoplasmic Inclusion helicase),
providing the first example of a potyvirus CI gene acting
as a determinant for eIF4E-mediated recessive
resistance breaking (1). By performing in vitro and in
vivo interaction assays, we revealed a complex interplay
between CI and other factors, VPg and eIF4E.
Furthermore, through a global survey of the biological
and molecular diversity of LMV isolates, we showed that
propagation of many non-lettuce isolates in mo11 plants
is accompanied by gain in pathogenicity. In particular,
we identified CI mutations systematically associated with
evolution toward resistance-breaking, which could
represent a threat for mo1 resistance durability.
Given the importance of eIF4E for potyviruses, we also
evaluated the possibility that eIF4G, a key cellular partner
of eIF4E, might also be recruited. Analysis of Arabidopsis
eIF4G KO mutants showed for the first time that eIF4G
factors are indispensable for potyvirus infection and,
more remarkably, that specific isoforms are recruited by
different potyviruses to contribute to a very early step in
viral infection (3, 5).
In parallel, we aimed to clarify the biological role of the
RNase activity associated with the 20S proteasome in
plant-potyvirus interactions. Our recent efforts have led
to the demonstration that the RNase activity is harboured
by the proteasome alpha-5 subunit and that this subunit
physically interacts in planta with the LMV Helper
Component protein (HcPro). Susceptibility analyses of
Arabidopsis mutants knocked-out for each At-PAE genes
encoding alpha-5, showed that one (KO-pae1) of the two
mutants exhibit a significantly increased susceptibility
to LMV infection. Taken together, these results extend
to A. thaliana alpha-5 the range of HcPro-interacting
proteasomal subunits, and suggest that HcPro may
modulate its associated RNase activity that may
contribute to an antiviral response (2).
1.Abdul-Razzak, A., T. Guiraud, M. Peypelut, J.
Walter, M. C. Houvenaghel, T. Candresse, O. Le Gall,
and S. German-Retana. 2009. Involvement of the
cylindrical inclusion (CI) protein in the overcoming of an
eIF4E-mediated resistance against Lettuce mosaic
potyvirus. Mol Plant Pathol 10:109-13.
2. Dielen, A. S., F. T. Sassaki, J. Walter, T. Michon,
G. Ménard, G. Pagny, R. Krause-Sakate, I. Maia, S.
Badaoui, O. Le Gall, T. Candresse, and S. GermanRetana. 2010. The 20S proteasome alpha-5 subunit of
Arabidopsis thaliana carries an RNase activity and
interacts in planta with the Lettuce mosaic potyvirus
HcPro protein. Molecular Plant Pathology in press.
3. Gallois, J. L., C. Charron, F. Sanchez, G. Pagny,
M. C. Houvenaghel, A. Moretti, F. Ponz, F. Revers,
C. Caranta, and S. German-Retana. 2010. Single amino
acid changes in the turnip mosaic virus viral genomelinked protein (VPg) confer virulence towards Arabidopsis
thaliana mutants knocked out for eukaryotic initiation
factors eIF(iso)4E and eIF(iso)4G. J Gen Virol 91:28893.
4. German-Retana, S., J. Walter, B. Doublet, G.
Roudet-Tavert, V. Nicaise, C. Lecampion, M. C.
Houvenaghel, C. Robaglia, T. Michon, and O. Le Gall.
2008. Mutational analysis of plant cap-binding protein
eIF4E reveals key amino acids involved in biochemical
functions and potyvirus infection. J Virol 82:7601-12.
5. Nicaise, V., J. L. Gallois, F. Chafiai, L. M. Allen, V.
Schurdi-Levraud, K. S. Browning, T. Candresse, C.
Caranta, O. Le Gall, and S. German-Retana. 2007.
Coordinated and selective recruitment of eIF4E and
eIF4G factors for potyvirus infection in Arabidopsis
thaliana. FEBS Lett 581:1041-1046.
6. Nicaise, V., S. German-Retana, R. Sanjuan, M. P.
Dubrana, M. Mazier, B. Maisonneuve, T. Candresse,
C. Caranta, and O. LeGall. 2003. The eukaryotic
translation initiation factor 4E controls lettuce
susceptibility to the Potyvirus Lettuce mosaic virus. Plant
Physiol 132:1272-1282.
Tereza Cristina
Poult enteritis complex (PEC) has been incriminated as
a major cause of loss amongst turkey poults in other
countries. We have observed this in Brazil, associated
with diarrhoea, loss of weight gain and, commonly, high
mortality. In this study, we have used reverse transcriptase
polymerase chain reaction (RT-PCR) to detect turkey
coronavirus (TCoV) in 30-120 days of age sick poults
from a particular producer region in Brazil. The RT-PCR
was applied to extracts of intestine tissue suspensions
(IS), and the respective intestinal contents (IC), bursa
of Fabrícius (BF), faecal droppings (FD) and cloacal
swabs (CS). After the first description TCoV infections
in our country, twenty 1-day-old specific pathogen free
chicks and 20 1-day-old commercially derived turkey
poults were inoculated with a Brazilian strain of turkey
coronavirus (TCoV) to study the pathogenicity and virus
distribution up to 14 days post-inoculation (dpi) by
histopathology, immunohistochemistry (IHC), reverse
transcriptase polymerase chain reaction (RT-PCR) and
sequencing. At 2 – 14 dpi, TCoV-antigens were detected
in the paranasal sinus and lachrymal accessory gland
(Harderian gland) of infected chicks and in the ileum,
ileocaecal junction and caecum of infected poults.
Lymphocytic inflammation was present in these tissues.
TCoV was re-isolated from pooled tissue suspensions
of nasal concha, Harderian gland and paranasal sinus
from chicks, as well as from the ileum, ileocaecal junction
and caecum of poults, after three consecutive passages
in 28-day-old embryonated turkey eggs. Viral RNA
corresponding to the spike gene region (1178 to 2073
genome position) was amplified from the upper respiratory
tract of chickens and from the intestinal tract of poults
and phylogenetic analysis confirmed the identity as TCoV.
This is the first description of TCoV antigens and mRNA
in upper respiratory tissues in experimentally infected
chickens. In order to verify the genetic variantion, The
degree of genetic and pathologic variation exhibited by
a turkey coronavirus (TCoV) strain was investigated after
nine serial passages in 25-day-old turkey embryos
obtained from wild broad-breasted bronze breeders. In
spite of spleen, liver, kidneys, bursa of Fabrícius and
thymus have been collected and analysed, the main
histolopathological changes were only documented in
the intestine sections. Microscopic lesions were
characterized as mild enteritis, low degree of enterocyte
vacuolization and detachment of the intestinal villous
after five consecutive passages and were considered
absent in the last passages. Genealogic analysis based
on partial S2 DNA sequences suggested that Brazilian
isolate might be considered as originated from TCoV
strains circulating in the United States, as its harbours
100% identity with TCoV-Gl strain. Although S2
sequences from each passage revealed no significant
point mutations, and no correlation could be speculate
between S2 nucleotide changes and pathologic features
in infected embryos. This is the first demonstration of
wild turkey embryos as a model for TCoV isolation and
propagation. Finally, these achieves are the unique
description of TCoV in Brazil, and also in South of
America.
TRANSLATIONAL RESEARCH AGAINST
PLANT VIRUSES: UNRAVELLING PLANTVIRUS INTERACTIONS AND USING THE
INFORMATION TO DEVELOP PLUM POX
VIRUS-RESISTANT PRUNUS CROPS
V. Decroocq1, T. Candresse1, D. Tricon1, G. Pagny1,
P. Paulstephenraj 1, S. Poque 1, P. Cosson 1, M.
Dalmais2, A. Bendahmane2 & H. Prieto3
1
UMR GDPP INRA-Université Bordeaux 2, 71 Avenue
Edouard Bourlaux, 33140 Villenave d’Ornon (France)
2
URGV, INRA, 2, Rue Gaston Crémieux CP 5708, 91057
EVRY Cedex (France)3 INIA, La Platina Station, Santa
Rosa 11610, La Pintana, Santiago de Chile (Chile)
43
A variety of genetical and biochemical approaches are
currently available that allow us to gradually unravel the
complex molecular interplay linking phytopathogenic
viruses and their host plants. This has lead to the
identification of plant genes that either restrict or impair
plant invasion by viruses (resistance determinants) or,
on the contrary, contribute to the invasion process
(susceptibility determinants). Remarkably, results of the
past few years have demonstrated that in some cases
allelic forms of susceptibility determinants can behave
as resistance factors. This situation is particularly well
illustrated in the case of Potyvirus-plant interactions by
the translation initiation factors eIF4E and eIF4G.
Isoforms of these proteins have been shown, in a number
of systems, to be absolutely required for successful virus
infection but, at the same time, other allelic forms
correspond to widely used recessive resistance genes.
In the case of Plum pox virus, the causal agent of the
devastating Sharka disease of stone fruit trees, we have
shown that eiF(iso)4E and eiF(iso)4G are absolutely
required for successful infection of the model plant
Arabidopsis thaliana by PPV. Resistance mapping efforts
in the natural Prunus hosts of PPV indicate that these
or other isoforms may also contribute to natural polygenic
resistance against this virus
The various strategies used in a large scale translational
research effort to transform this key information into PPVresistant Prunus crops will be illustrated, together with
current efforts to identify other plant resistance/
susceptibility factors to Potyviruses.
DIAGNÓSTICO E EPIDEMIOLOGIA MOLECULAR DAS HEPATITES VIRAIS B E C NO RIO
GRANDE DO SUL
Prof. Dr. Vagner Ricardo Lunge
As hepatites B e C são importantes problemas de saúde
pública no Rio Grande do Sul, devido à alta prevalência
dos agentes etiológicos virais, à possibilidade de
cronificação e à gravidade das doenças relacionadas. A
maioria dos casos são clinicamente assintomáticos
durante o período após a infecção, dificultando o
diagnóstico e o início dos procedimentos terapêuticos.
A taxa de cronificação varia conforme a forma de
infecção (vertical/horizontal) e o tipo de hepatite (10%
para hepatite B em adultos e 70-80% para hepatite C).
As hepatites B e C crônicas evoluem para cirrose e
carcinoma hepatocelular em parcela significativa dos
pacientes.
O vírus da hepatite B (HBV) pertence à família
Hepadnaviridae e apresenta envelope externo,
nucleocapsídeo, enzima DNA polimerase e genoma de
DNA contendo em torno de 3.200 nucleotídeos
44
parcialmente duplicados. Devido às variações genéticas,
o HBV é filogeneticamente classificado em 8 genótipos
(A, B, C, D, E, F, G e H) com diversificada distribuição
geográfica. Especificamente no nosso estado, o genótipo
D é mais freqüente, seguido dos genótipos A e F.
O diagnóstico da hepatite B envolve a análise laboratorial
de marcadores sorológicos (HBsAg, anti-HBc, etc.) e
moleculares (HBV-DNA). A detecção do HBV é realizada
com marcadores sorológicos, mas a análise do HBVDNA pode ser útil na confirmação da infecção aguda
(DNA é detectado antes do HBsAg) ou crônica (casos
de infecção oculta). A quantificação (carga viral) no
plasma é fundamental no monitoramento dos pacientes
infectados (com ou sem tratamento), pois pacientes com
níveis elevados de HBV-DNA possuem riscos
significativamente maiores de progressão para cirrose
e carcinoma hepatocelular. Técnicas laboratoriais de
quantificação do HBV (como PCR quantitativo, bDNA e
principalmente PCR em tempo real) são essenciais na
rotina clínica, incluindo testes comerciais e in house.
A análise do genótipo tem importância epidemiológica,
mas a relevância clínica ainda não é reconhecida. Já a
análise da seqüência de aminoácidos da DNA polimerase
viral é essencial na avaliação da resistência do HBV
aos análogos de nucleos(t)ídios (lamivudina, adefovir,
entecavir, telbivudina, tenofovir, etc.) utilizados no
tratamento. Estudos demonstram o rápido
desenvolvimento de resistência com o uso destes
antivirais, principalmente para lamivudina (medicamento
utilizado no tratamento do próprio HBV e na terapia
altamente ativa contra o HIV-1). Técnicas laboratoriais
de hibridização e sequenciamento do gene P do HBV
identificam as mutações responsáveis pela resistência,
permitindo a definição de medicamentos para a terapia
de cada paciente.
O vírus da hepatite C (HCV) pertence à família
Flaviviridae. O material genético é um RNA fita simples
com 9.500 nucleotídeos, protegido por um capsídeo
icosaédrico e envelope lipídico com duas glicoproteínas.
O HCV apresenta variabilidade genética, sendo
classificado em genótipos que diferem em torno de 35%
no genoma. A variação é maior nas regiões que codificam
as proteínas do envelope e menor nas extremidades
não codificantes (5´ e 3´UTR). São 6 genótipos principais
(1, 2, 3, 4, 5 e 6) e múltiplos subtipos (a, b, c, etc) com
variada distribuição geográfica no mundo. Os mais
freqüentes no Rio Grande do Sul são 1, 3 e 2,
respectivamente, mas ocorrem variações importantes
nas diferentes regiões do estado.
O diagnóstico laboratorial do HCV inclui a detecção de
anticorpos anti-HCV (ensaios de ELISA e RIBA) e
análises moleculares quali/quantitativas e de
genotipagem. A detecção do RNA viral é a principal
alternativa na identificação do HCV, seja na confirmação
da infecção ou na avaliação do sucesso do tratamento
(ribavirina mais interferon - convencional ou peguilado).
A carga viral é realizada previamente ao início e durante
o tratamento para acompanhar a eficiência do
procedimento terapêutico. A genotipagem é importante
para fins epidemiológicos e também clínicos, pois a
duração e o tipo de tratamento variam conforme o
genótipo viral. Técnicas laboratoriais de análise molecular
do HCV (RT-PCR, convencional ou em tempo real,
hibridização, RFLP e bDNA) são utilizadas na rotina
clínica.
Pesquisas recentes têm focado nos aspectos genéticos
do hospedeiro humano que estão associados às
hepatites virais B e C. Estudos de associação em escala
genômica demonstraram polimorfismos no genoma
humano que estão fortemente associados com a taxa
de eliminação espontânea viral e com o sucesso do
tratamento antiviral. O principal exemplo é a descoberta
de alguns SNPs (polimorfismos de nucleotídeo único)
próximos ao gene da interleucina 28 B (IL28B) no
cromossomo 19 humano que foram associados à
evolução e ao tratamento da hepatite C. Estudo realizado
recentemente demonstrou que um destes polimorfismos
está associado com a resolução espontânea em
indivíduos co-infectados pelo HIV-1 no nosso estado.
WATER AND HUMAN HEALTH IN LATIN
AMERICA
The World Health Organization (2006) estimated that
about 1.5 million deaths per year from diarrheal diseases,
mainly in children, are attributable to environmental
factors such as contaminated drinking water, poor
sanitation and poor hygiene. In the Americas, for the
period of 2000-2005 the children mortality due to acute
diarrheas was 3.7 % and the Andean region was the
most affected with 7.8 %.
A large portion of the total burden of diarrheal disease is
caused by fecal-oral pathogens from both human and
animal sources that are discharged into the aquatic
environments.
A great variety of pathogens, including bacteria
(Escherichia coli, Shigella, Salmonella, Campylobacter,
Aeromonas, Clostridium dificile), mycobacteria
(Mycobacterium tuberculosis and Mycobacterium aviumintracellulare), viruses (rotavirus, adenovirus, enterovirus
serotypes 40 and 41, norovirus, astroviruses), fungi (the
role is not clear but Candida is frequently associated
with persistent diarrhoea), and parasites (Giardia lamblia,
Entamoeba histolytica, Microsporidia, Strongyloides
stercoralis), can contribute to persistent, as well as to
acute, diarrheal illnesses. The pathogenic parasites and
bacteria on this non-extensive list have been well studied;
numerous drug treatments have been developed that are
accessible and affordable for most of the population.
Conversely, viruses are difficult to detect and because
of the lack of effective treatments the main strategy to
decrease viral impact must be monitoring and prevention.
Hugo Ramiro Poma 1, Mercedes Cecilia Cruz 1,
Verónica Beatriz Rajal1,2,*
1
Facultad de Ingeniería, INIQUI-CONICET, Universidad
Nacional de Salta, Av. Bolivia 5150, 4400 Salta, Argentina
2
Fogarty International Center, University of California in
Davis, USA
*
E-mail: vbrajal@gmail.com
In Latin America, economic globalization has meant the
increased movement of people and goods and changes
in environmental and occupational health hazards, often
occurring in the context of political and economic
instability. Of the estimated 183 million people living in
poverty in Latin America, more than half are children
and teenagers, 72 % of them live in urban areas, and
mortality and morbidity impact especially the infant
population. Acute respiratory infections (mainly
pneumonia) and diarrhea, followed by measles, malaria
(and frequently a combination of them) are the most
common causes of morbidity and mortality among
children under the age of five in the developing world.
Malnutrition, socioeconomic status, disruption of
traditional lifestyles, accessibility to clean water and
sanitation facilities, age and their breast-feeding status
are the main factors that influence the incidence of
diarrhea.
45
TRABALHOS CONCORRENTES AO PRÊMIO
SPATIO-TEMPORAL
TRACKING
AND
PHYLODYNAMICS OF AN Urban Dengue 3
Outbreak in São Paulo, Brazil
Adriano Mondini 1 , Roberta Vieira de Moraes
Bronzoni1, Silvia Helena Pereira Nunes1, Francisco
Chiaravalloti Neto1,2, Eduardo Massad3, Wladimir J.
Alonso4, Eduardo S. M. Lázzaro5, Amena Alcântara
Ferraz 5 , Paolo Marinho de Andrade Zanotto 6 ,
Maurício Lacerda Nogueira1*
Faculdade de Medicina de São José do Rio Preto, São
José do Rio Preto, Brazil, Superintendência de Controle
de Endemias, São José do Rio Preto, Brazil, LIM 01HCFMUSP, Faculdade de Medicina da Universidade de
São Paulo, São Paulo, Brazil, Forgarty International
Center, National Institutes of Health, Bethesda, Maryland,
United States of America, Secretaria Municipal de Saúde
e Higiene de São José´ do Rio Preto, São José do Rio
Preto, Brazil, Laboratório Evolução Molecular e
Bioinformática (LEMB), Departamento de Microbiologia,
Instituto de Ciências Biome´dicas. Universidade de São
Paulo, São Paulo, Brazil
Abstract
The dengue virus has a single-stranded positive-sense
RNA genome of ,10.700 nucleotides with a single open
reading frame that encodes three structural (C, prM, and
E) and seven nonstructural (NS1, NS2A, NS2B, NS3,
NS4A, NS4B, and NS5) proteins. It possesses four
antigenically distinct serotypes (DENV 1–4). Many
phylogenetic studies address particularities of the
different serotypes using convenience samples that are
not conducive to a spatio-temporal analysis in a single
urban setting. We describe the pattern of spread of
distinct lineages of DENV-3 circulating in SaÜo Jose´
do Rio Preto, Brazil, during 2006. Blood samples from
patients presenting dengue-like symptoms were collected
for DENV testing. We performed M-NPCR using primers
based on NS5 for virus detection and identification. The
fragments were purified from PCR mixtures and
sequenced. The positive dengue cases were geo-coded.
To type the sequenced samples, 52 reference sequences
were aligned. The dataset generated was used for iterative
phylogenetic reconstruction with the maximum likelihood
criterion.
The best demographic model, the rate of growth, rate of
evolutionary change, and Time to Most Recent Common
Ancestor (TMRCA) were estimated. The basic
reproductive rate during the epidemics was estimated.
We obtained sequences from 82 patients among 174
blood samples. We were able to geo-code 46 sequences.
The alignment generated a 399-nucleotidelong dataset
with 134 taxa. The phylogenetic analysis indicated that
all samples were of DENV-3 and related to strains
circulating on the isle of Martinique in 2000–2001. Sixty
DENV-3 from São José do Rio Preto formed a
monophyletic group (lineage 1), closely related to the
remaining 22 isolates (lineage 2). We assumed that these
lineages appeared before 2006 in different occasions.
By transforming the inferred exponential growth rates
into the basic reproductive rate, we obtained values for
lineage 1 of R0 = 1.53 and values for lineage 2 of R0 =
1.13. Under the exponential model, TMRCA of lineage 1
dated 1 year and lineage 2 dated 3.4 years before the
last sampling. The possibility of inferring the spatiotemporal dynamics from genetic data has been generally
little explored, and it may shed light on DENV circulation.
The use of both geographic and temporally structured
phylogenetic data provided a detailed view on the spread
of at least two dengue viral strains in a populated urban
area.
ENVIRONMENTAL MONITORING OF ROTAVIRUS A IN A HOSPITAL INTENSIVE CARE
UNIT, IN RIO DE JANEIRO CITY, BRAZIL.
Ana C Ganime1; Felipe A Carvalho-Costa2; Marcos
CL Mendonça1; Carmen B Vieira1; Marisa S Santos3;
Rubens Costa Filho4; Marize P Miagostovich1;José
P G Leite1*
1 Laboratório de Virologia Comparada e Ambiental,
Instituto Oswaldo Cruz/FIOCRUZ, Rio de Janeiro, Brazil.
2 Instituto Oswaldo Cruz/FIOCRUZ, Rio de Janeiro,
Brazil.
3Instituto Nacional de Cardiologia Laranjeiras, CCIH, Rio
de Janeiro, Brazil
4Unidade de terapia intensiva – Hospital Pró-Cardíaco,
Rio de Janeiro, Brazil
*Corresponding Author: José Paulo Gagliardi Leite,
Laboratório de Virologia Comparada e Ambiental, Pav.
Hélio e Peggy Pereira, Instituto Oswaldo Cruz, FIOCRUZ,
AV. Brasil 4365, Manguinhos, 21040-360, Rio de Janeiro,
RJ, Brasil. Phone: +55 21 2562 1817 – FAX +55 21
25621851. E-mail: jpgleite@ioc.fiocruz.br.
KEY WORDS: rotavirus-A, fomites, hospital infection,
VP6 gene 2
Hospital infection control is still based on a few groups
of bacteria resistant to antibiotics. The aim of this study
was to investigate, over a 6-month period, the
environmental presence of human rotavirus-A (RV-A) in
an intensive care unit (ICU) of a private hospital in Rio
de Janeiro, Brazil. Surfaces swabbing of the ICU were
undertaken in 12 sites: keyboard infusion pump (KP),
inside door handle of the room (ID), telephone (TE), coffee
table (CT), outside door handle of the bathroom (OD),
alcohol gel support (SA), chlorhexidine support (SC),
common waste cover trash (CW), TV remote control (RC),
bed control (CB) button flushing (BF), companion chair
47
(CC). By using nucleic acid amplification methodology
for VP6 gene, Reverse Transcriptase Polymerase Chain
Reaction (RT-PCR) and nested-PCR, RV-A were detected
in 73 (14,5%) out of 504 samples collected. Positive
samples were quantified by Real time PCR for NSP3
gene and inoculated in cell culture. The results showed
that all fomites at some point were positive for RV-A, the
SA had the greatest number of positive samples, 11
(15,1%); followed by BF with 9 (12,3%). CC, SC and
CW, with 8 (11,0%) positive samples for each one, ID
with 6 (8,2%) positive samples, CB and the OD 5 (6,8%);
KP 4 (5,5%) and RC, CT and TE with 3 (4,1%).
Environmental transmission of the virus was thought to
have occurred by hands contaminated with RV-A. RV-A
detection from fomites may have a role in controlling
and monitoring cleaning and hand washing, preventing
RV-A transmission.
RABIES VIRUS NEUTRALIZING ANTIBODIES
DETECTION IN WILD MAMMALS FROM A
RELEASE AREA, NORTH COAST OF SÃO
PAULO STATE, BRAZIL.
C. S. Rodrigues a, D. B. Araujoa, A. C. A. Campos a, L.
F. Sanfilippob, A. O. Medinab, C.K. Yoshiharab, L. F. A.
Mar torellic, A. P. Kataokac, E. L. Durigone S. R.
Favorettoa,d.
a – Núcleo de Pesquisas em Raiva do Laboratório de
Virologia Clínica e Molecular da Universidade de São
Paulo, São Paulo, SP, Brasil.; b – Gaia Consultoria
Ambiental –São Paulo, SP, Brasil; c – Centro de Controle
de Zoonoses,, São Paulo, SP, Brasil. ; d – Instituto
Pasteur de São Paulo, São Paulo, SP, Brasil; e Laboratório de Virologia Clínica e Molecular da
Universidade de São Paulo, São Paulo, SP, Brasil.
E-mail: camila.seabrar@hotmail.com
Rabies is an infectious disease with nervous
sintomatology that causes thousands of humans and
animals deaths worldwide annually. The vaccination
campaigns for dogs and cats allowed the control of the
urban form of the disease; however, the cycle maintained
by wild animals, mainly by the haemathofagous bat
Desmodus rotundus, as by others species of wild
mammals presents an emergent relevance in the disease
epidemiology. The use of sera samples from wildlife
animals from a release area named Acarau Farm, in
Bertioga City, north coast of São Paulo State, Brazil,
provides an excellent opportunity for the study of rabies
epidemiology in those animal species. The aim of this
study was the detection of rabies virus neutralizing
antibodies through the “Simplified fluorescent inhibition
microtest for the titration of rabies neutralizing antibodies”
– SFIMT - technique in sera of animals from the release
area. The animal species studied were: Dipelphis aurita
48
(opossum), Dasypus novemcinctus (armadillo),
Tamanduá tetradactyla (tamanduá), Nasua nasua (quati),
Oligoryzomys nigripes (wild rat), Arvicola sapidus (water
rat), Cebus apella (capuchin monkey) and Leopardus
pardalis (leopard cat). Out of 49 blood samples analyzed,
10 (20,41%) presented positive titers (considering as
positive titers e” 0,5UI – protective titers); those results
were detected in opossum, wild rat, water rat, capuchin
monkey and coati and indicates rabies virus circulation
in the area. Considering the potential risk of rabies
transmission from wild to domestic animals and even
humans and the lack of epidemiologic studies in wildlife,
the results of this study can provide a valuable information
about the rabies virus circulation in those animals as for
the establishment of better strategies of control and
prevention of the disease.
Keywords: rabies, wild mammals, epidemiology,
antibodies.
EFFECTS OF TRIAZOLIC COMPOUNDS ON
INFLUENZAVIRUS REPLICATION.
Alves, C.M.¹, Andrade, V.M.¹, Accioly, M.¹, Abrantes,
J.L.1,², Ferreira, V. F.3, Siqueira, M.M.1, Souza, T.M.L¹
¹Instituto Oswaldo Cruz/FIOCRUZ; ²Instituto de
Bioquímica Médica/UFRJ; 3Instituto
de Química/UFF, RJ, Brazil.
E-mail: crismaia@ioc.fiocruz.br / crisfarmufrj@gmail.com
The Influenza virus is the most prevalent infectious agent
that affects respiratory tract resulting in high morbidity,
mortality and major spending on public health system.
Currently, two antiviral drugs, oseltamivir and zanamivir,
are available for treatment.
However, some drug-resistant strains are emerging so it
is crucial to search for new alternative drugs for the
treatment of influenza. We studied the effects of 91
triazolic derivatives on the replication of influenza H3N1
strain and found that the compound 4 inhibited
hemagglutination and neuraminidase activity with values
of EC50 equal to 459 nM and 10 nM, respectively, and a
complete inhibition hemagglutination with 25 uM of drug.
Cytotoxicity was low, with CC50 value equal to 1811
uM. The compound 4 showed a competitive inhibition
mechanism on the viral neuraminidase. Other
experiments, such as generation of resistant strains in
presence of our compound are being developed by our
group, as well as, studies involving other strains of
influenza in 2009 (H1N1). Therefore, our findings suggest
that structure 4 has a promising antiviral activity, thus
encouraging, further studies about this compound.
EVALUATION OF SALIVA SPECIMENS AS
ALTERNATIVE SAMPLE TO DETECT
HEPATITIS B SURFACE ANTIGEN.
Helena Medina Cruz a/*, Elisangela Ferreira da Silvaa,
Cristiane A.Villela-Nogueirab, Letícia C Nabucoa, Kycia
Maria Rodrigues do Óc, Lia Laura Lewis-Ximeneza, Clara
Fumiko Tachibana Yoshidaa, Elisabeth Lampea , Lívia
Melo Villara
Laboratory of Viral Hepatitis, Oswaldo Cruz Institute,
FIOCRUZ, RJ, Brazil, bHepatology Division, Clementino
Fraga Filho University Hospital, Federal University of
Rio de Janeiro, Rio de Janeiro - Brazil.
a
*Corresponding author: Helena Medina Cruz, Laboratório
de Hepatites Virais Pavilhão Helio e Peggy Pereira - Térreo
- sala B09, Instituto Oswaldo Cruz – FIOCRUZ. Av. Brasil,
4365 Manguinhos-RJ. Cep:21045900. Rio de Janeiro, RJ,
Brazil. Fax (+ 55 21) 2270 6397. Tel: (+ 55 21) 21 2562
1918; e-mail address: lvillar@ioc.fiocruz.br
Neste estudo um ensaio imunoenzimático comercial foi
adaptado para detectar o HBsAg em amostras de saliva
coletadas por duas formas distintas. Amostras foram
coletadas de 115 indivíduos de duas formas distintas:
Salivette (Sarsted) e saliva total não estimulada.
As amostras de saliva foram testadas seguindo ELISA
modificado e os resultados comparados com aqueles
obtidos em amostras de soro correspondente que foram
testadas de acordo com as informações do fabricante.
Para otimizar o ensaio foram avaliados os seguintes
pontos: tampão de eluição das amostras de fluido oral,
volume de amostra, período de incubação amostraconjugado e determinação do ponto de corte do teste.
32 Maiores valores de sensibilidade e especificidade
foram obtidos quando a incubação amostra-conjugado
foi aumentada para 16 horas e utilizada a curva ROC
para determinar o ponto de corte do teste. Das 47
amostras reagentes para HBsAg no soro, 40 amostras
também foram reagentes para HBsAg em fluido oral e
44 em saliva total. Já das 68 amostras não reagentes
para HBsAg no soro, 64 amostras de fluido oral e 63
amostras de saliva total também não apresentaram
reatividade para HBsAg. Os resultados de concordância
entre as amostras de soro e saliva foram excelentes
(valor de kappa (k): 0,80 para fluido oral e 0,87 para
saliva total) e também apresentou ótima reprodutibilidade
do teste. Esses dados demonstram que o uso da saliva
total e fluido oral em conjunto com o ensaio
imunoenzimático comercial modificado podem ser
promissores para a vigilância da infecção pelo vírus da
Hepatite B.
Palavras-chave: Hepatite B, saliva total, fluido oral,
ensaio imunoenzimático.
LOWER LEVELS OF INTEGRASE FOUND IN
HIV-1 PARTICLES IN THE ABSENCE OF VIRAL
ACCESSORY PROTEIN NEF: EFFECT OF
NEF ON VIRAL PROCESSING.
Luiza M. Mendonça1, Thatiane L. Sampaio1, Luciana
J. Costa1
1
Departamento de Virologia, Instituto de Microbiologia
Professor Paulo de Góes, Universidade Federal do Rio
de Janeiro, Rio de Janeiro. 21.941 590.
Corresponding Author: Luciana Jesus da Costa
CCS – Bloco I, subsolo, sala I-048, Departamento de
Virologia, IMPPG, Universidade Federal do Rio de
Janeiro. Ilha do Fundão. Cidade Universitária. Rio de
Janeiro, RJ.
Zip Code: 21951-590. Phone: (21) 2560 8344, ext. 117.
ljcosta@micro.ufrj.br
Keywords: Nef, Protease, Integrase, Assembly,
Maturation, Lentivirus.
Nef, an accessory protein expressed early during the
replication cycle of the primate lentiviruses (HIV and
SIV), plays an important role in viral infectivity and
disease progression by a still not completely understood
mechanism. Although many studies have described the
role of Nef on the early stages of HIV-1 replication cycle,
its contribution on the late stages is less explored. Our
group has previously demonstrated that Nef interacts
with GagPol by its p6*-PR region. Since both p6* and
PR are involved with protein processing, we are now
exploring Nef’s role on maturation and viral protease
activity. To assess the level of protease activity in the
presence or absence of Nef, Hek293T cells were
transfected with HIV-1 NL4-3 WT (WT) or the Nef-deleted
counterpart (HIV-1Nef) in the presence of Lopinavir (LPV),
a HIV-1 protease inhibitor, at increasing concentrations
or at a fixed concentration at different time points. Then,
we observed the level of Gag processing in cell lysates
and supernatants. We also assessed virus infectivity by
the TZM-bl indicator cell line assay. In our experiments,
HIV-1Nef showed a IC50 of 15nM to LPV, in contrast to
a IC50 of 9nM of the WT, besides showing an abnormal
kinetics of Gag and GagPol processing. This data
demonstrate that viruses produced in the absence of
Nef are more resistant to protease inhibitors. Analyses
of released virions also showed that HIV-1Nef has less
unprocessed Gag. Our results indicate that in the
absence of Nef the protease activity is increased,
however, this event is deleterious to the virus, since HIV1Nef is about 5 times less infectious than WT. Therefore,
we suggest that premature activation of protease can
lead to abnormalities in virion assembly, for instance in
the ratio of protein incorporation in the budding virus. To
confirm this hypothesis, we investigated the levels of
Pol subunities incorporation into virions. We found that
49
the HIV-1Nef virus incorporate two-times less Integrase
compared to the WT virus, and also an abnormal ratio of
p66 to p51 Reverse Transcriptase subunities was found
in the HIV-1Nef. These data could explain the difference
in infectivity of the HIV-1Nef to the WT virus.
HIGH-RESOLUTION PHYLOGENETICS AND
PHYLOGEOGRAPHY OF HIV-1 SUBTYPE C
EPIDEMIC IN SOUTH AMERICA.
Nazle M.C. Véras1,2, Rebecca R. Gray2, Luis Fernando
M. Brígido3, Rosângela Rodrigues3, Marco Salemi2,
4
*
1
Pós-Graduação em Biologia Molecular, Instituto de
Biologia, Universidade de Brasília, DF, 70919-900, Brazil
2
Department of Pathology, Immunology, and Laboratory
Medicine, University of Florida College of Medicine,
Gainesville, FL 32610, USA
3
Retrovirus Laboratory, Virology Service, Adolfo Lutz
Institute, Ave. Dr Arnaldo 355, São Paulo, SP, 01246902, Brazil
4
Emerging Pathogens Institute, University of Florida,
Gainesville, FL 32610, USA
HIV-1 subtype C represents 30% to 65% of HIV infections
in Southern Brazil and, more recently, isolated cases of
HIV-1 subtype C infection were also reported in Argentina,
Uruguay, Paraguay and Venezuela. Phylogenetic studies
suggested that the Brazilian subtype C epidemic was
initiated by an introduction of closely related strains.
Nevertheless, because of sampling limitations, the point
of entry and the timing of subtype C introduction in Brazil,
as well as the origin of the founder lineage are still
controversial. The present study investigated in depth
the origin, spread and phylogeography of HIV-1C in South
America, by analyzing carefully assembled datasets
covering four South American countries, six Brazilian
states and three genomic regions (p24, reverse
transcriptase and gp41). High-resolution phylogenetic
analysis of the three different viral genes consistently
showed a well supported monophyletic clade including
all available strains from Brazil, Uruguay and Argentina.
Molecular clock and likelihood mapping analysis showed
that HIV-1C introduction in Brazil dated back to 19601970s and was followed by a nearly simultaneous starlike outburst of viral lineages indicating a subsequent
rapid spread. Phylogeographic patterns suggested Paraná
as the entrance point of subtype C and an asymmetrical
gene-flow from Paraná to Santa Catarina and Rio Grande
do Sul fostered by the strong inter-connectivity between
population centers in southern Brazil. The study illustrates
how coupling phylogeography inference with geographic
information system data is critical to understand the origin
and dissemination of HIV-1 subtype C in South America
and potentially predict its future spread.
50
PANDEMIC INFLUENZA A/H1N1 IMPACT IN
CHILDREN ON SAO PAULO CITY, BRAZIL,
DURING THE YEARS OF 2009 AND 2010
PRISCILA VASCON MACEDO
Co-autores: Macedo, P.V., Oliveira, D.B.L., Thomazelli,
L.M., Durigon, G.S., Caldeira, R.N., Stornii J.G.,
Gutierrez, V.C.R., Pirez, C.M.N., Martinez, M.B., Berezin,
E.N., Durigon,E.L.
Financial support: Fundação de Amparo a Pesquisa
do Estado de São Paulo (FAPESP) numero:
Email: priscilav.macedo@hotmail.com
H1N1 virus of swine origin had a big and unexpected
incidence in 2009 when started to infect the human being.
It arose from rearrangements suffered since 1930, and
found viral segments characteristic of human, bird and
two types of pigs. At this time there are two main strains
of H1N1 circulating in humans, a pandemic and a
seasonal. The first reported case of pandemic strain was
presented in March in Mexico, spreading since then,
causing a high morbidity and mortality worldwide. World
Health Organization (WHO) officially declared the
pandemic in June 2009, noting that most infected people,
including children, developed a disease slightly selflimiting. However, children 2-5 years and adults with
respiratory and cardiac diseases, and healthy young
people were most affected. In our study, we analyzed
655 samples of nasopharyngeal aspirates, by the
technique of Real-Time PCR, in children less than 18
years old with symptoms of respiratory tract infection
attended in Hospital Universitário da Universidade de
São Paulo and children less than 2 years old admitted at
Santa Casa de Misericórdia de São Paulo, with the same
symptoms, between January 2009 and August 2010. The
results showed that 6.41% are positive for Pandemic
Influenza A/H1N1 and 4.27% for Seasonal Influenza A.
In pandemic Influenza A/H1N1 positive samples, there
were 9.52% of co-infection with another respiratory virus
studied as (HMPV,HRSV, HPIV, HBOC,HADV and FluB).
However, with Seasonal Influenza A there was 39.28%
of co-infection. The age of more infection with Pandemic
Influenza A/H1N1 and Seasonal Influenza A were 1-6
months, but coming from ER at the first case, and from
Pediatrics at the second one. So, it’s necessary to use
better-standardization techniques to help the disease
diagnostics, specifically in cases with pandemic virus,
as Influenza A/H1N1 of 2009.
ANALYSIS OF CCR5 DELTA 32 HETEROZYGOSIS IN PATIENTS WITH LONG TERM
HIV- 1 SUPPRESSION UNDERGOING ANTIRETROVIRAL THERAPY
Rafael G. Comparini ª *, Ana C. Zetehakuª, Antônio
F. P. L. Filhoª, Natasha C. 6 Nahonª, Nivia T. dos
Santosª, Luiz H. Gaglianiª, Marcos M. Caseiroª, Dercy
J. de SA-Filhoª.
ª Centro Universitário Lusíada (UNILUS) – Núcleo
Acadêmico de Estudos e 10 Pesquisas em Virologia/
Laboratório de Biologia Molecular.
Keywords: HIV-1, CCR5, antiretroviral therapy.
Previous researches have shown that being heterozygous
for a 32 bp deletion 3 in the CCR5 alele is associated
with the duration of time to development of AIDS by
turning the HIV-1 entry slower. We examined whether
CCR5 delta 32 heterozygosis was associated with
antiretroviral therapy long-term HIV-1 suppression in
Santos/Brazil. In addition we described clinical course
of HIV-1 infection among those individuals. Patients under
antiretroviral therapy presenting 3 years of viral load less
than 50 HIV RNA copies per milliliter were defined as
presenting long-term HIV- 1 suppression. We selected
randomly 83 patients for CCR5 analysis. This analysis
was based on polymerase chain reaction (PCR), using
primers spanning the CCR5 32 bp deletion. We found
that 7.2% of study subjects were heterozygous CCR5/
CCR5 delta 32. The ethnic group comprised Caucasians
and African descendents. Pneumonia and Candidiasis
were the most frequent opportunistic infection. The
average CD4+T cells counts in CCR5 heterozygous
group was 601, 609, 719 cells per microliter in 2005,
2006 and 2007, respectively, and CD8+T average was
1461, 1353, 1515 cells per microliter. Our data
demonstrated that the frequency of CCR5 heterozygosis
in the group of patients with long-term HIV-1 suppression
is similar to those found in general Brazilian population
(6.4% to 14.2%), neither associated to a better CD4+ T
cell profile. Therefore, this suggests that HIV-1
suppression is not associated with CCR5 heterozygous.
CARACTERIZAÇÃO MOLECULAR DOS
PARVOVÍRUS ASSOCIADOS AOS CASOS
DE GASTRENTERITE EM FILHOTES DE CÃES
E GATOS NO ESTADO DO RIO DE JANEIRO”
Tatiana X. Castro 1; Norma V. Labarthe 2 ; Rita de
Cássia N.C.Garcia1
1
Departamento de Microbiologia e Parasitologia, Instituto
Biomédico, Universidade Federal Fluminense, Rua Prof.
Ernani Pires de Melo, 101- 24210-130 – Niterói, Rio de
Janeiro. Tel 55 21 2629-2432;
2
Programa de Biodiversidade e Saúde, Fundação
Oswaldo Cruz, Av. Brasil 4036 – Prédio da Expansão,
sala 214, CEP 21040-361, Rio de Janeiro.
The aim of this study was to perform the molecular
characterization of parvovirus detected in fecal samples
of dogs and cats up to one year of age with enteritis in
the State of Rio de Janeiro.
After laboratory confirmation of canine parvovirus (CPV)
infection by hemagglutination/ hemagglutination inhibition
(HA/HI) and polymerase chain reaction (PCR), 69 fecal
samples from vaccinated (37) and unvaccinate puppies
(32) were submitted to partial sequencing of the gene
coding for VP2 capsid protein. The analysis of amino
acids (AA) 297, 300, 305, 426 e 555 allowed the
characterization of CPV-2a (41 samples), CPV-2b (27)
and CPV-2c (1).
Synonymous mutations in AA 440 (12) and 574 (28) were
detected. Despite this, the mutations detected in CPV
strains detected in 37 samples from vaccinated puppies
apparently did not affect the AA related to antigenicity
of CPV, and did not compromise the overall ability of
current vaccines in protect puppies against the circulating
types. Six fecal samples from domestic cats collected
in 2004 were also subjected to sequencing of the same
region for characterization of parvovirus infection
associated with this species and all of them were
characterized as feline panleukopenia virus (FPLV). The
detection of non-synonymous mutations in AA that affect
parvovirus host range in two feline and one canine strains
indicate that CPV and FPLV are subject to important
evolutionary events and the continuous epidemiological
surveillance of this agent distribution is essential to
understanding the mechanisms that drive its evolution
in Brazil.
Key-words: Canine Parvovirus (CPV); Feline
Panleukopenia Virus (FPLV); Enteritis; Molecular
Characterization.
INVESTIGAÇÃO DA ATIVIDADE DA
PROTEÍNA ACESSÓRIA NEF DURANTE O
CICLO REPLICATIVO DO SIVCPZ ISOLADO
GAB2
Sampaio, T. L. *1; Cunha, M.S.1; Mendonça, L.1; Tanuri,
A.2, Costa, L.1
1
Departamento de Virologia, Instituto de Microbiologia e
Imunologia, UFRJ; 2Departamento de Genética, Instituto
de Biologia, UFRJ. E-mail: thati_sampaio@
yahoo.com.br
The absence of Nef protein in Simian and Human
Immunodeficiency Viruses (SIV and HIV) reduces viral
infectivity about 10 fold. Little is known about the
mechanisms underlying the gain of infectivity conferred
by Nef.
We analyzed the effect of the complete and a partial
deletion of the nef gene on the replicative cycle of
SIVcpzGAB2. Mutagenesis of the start codon of nef
resulted in the absence Nef expression (SIVcpz”Nef), or
51
insertion of two nucleotides at position 218 resulted in
the expression of a truncated N-terminus of Nef (SIVcpz
t-Nef). 293T cells were used for transfections and viral
protein expression was evaluated by Western blotting.
CA production was quantified by ELISA p24 and viral
infectivity was assayed in TZM-bl indicator cells. In cell
lysates and supernatants of SIVcpz t-Nef transfected
cells we observed a great accumulation of the Gag and
GagPol precursors and the par tially processed
intermediates, this accumulation coinciding with a
significant reduction in CA levels and absence of the
RT. Pattern of distribution of CA through a sucrose gradient
(30-70%) was different from that of SIVcpz and
SIVcpz”Nef remaining at the denser fractions, indicating
that the majority of viral particles of SIVcpz t-Nef were
immature. Infectivity of the SIVcpz t-Nef was abrogated
in TZM-bl cells and was not complemented by providing
Nef in trans. Expression of this truncated peptide from
an expression vector together with the SIVcpz”Nef clone
recapitulates the results described above. We also
determined whether this truncated peptide would act as
a dominant negative both for SIVcpz and HIV-1. t-Nef
peptide was able to reduce HIV-1 and SIVcpz infectivity
in 95 and 50%, respectively, and to lead to Gag
accumulation. These results identify the dominantnegative effect of N-terminus of Nef from SIVcpz as a
viral gene product that interferes with Gag processing
by viral protease and inhibited viral infectivity.
Key words: HIV, SIV, Nef, Gag, GagPol
THE NATURAL ENDOGENOUS RNA POLYMERASE (NERP) FROM RESPIRATORY
SYNCYTIAL VIRUS (RSV)
Andrade, VMM.1; Mesquita, MMA.1; Siqueira, MM.1;
Souza, TML.1
1
Instituto Oswaldo Cruz/Fiocruz; 2Instituto de Bioquímica
Médica/UFRJ, RJ, Brasil
RSV is a paramyxovirius and the main ethological agent
of acute lower respiratory tract infection, affecting mainly
children under two years of age and elderly population.
In the present study, we investigated the presence of an
RNA polymerase activity within RSV virions (NERP) and
the virus inhibition by triazolic compounds targeting this
enzyme. The supernatant of RSV-infected HeLa cells
was harvested and submitted to logarithmic dilutions,
up to undetectable level. These dilutions were further
incubated in a buffer containing MgCl2 with different
concentrations of ribonucleotides. The reaction was
stopped, viral RNA was extracted and submitted to qRTPCR. In antiviral assays, we also added the drugs within
the reaction of RT-PCR. We observed a 2-log
enhancement of viral RNA content, due to the above
52
mentioned incubation. To confirm that enrichment was
due to RSV specific sequences, the above mentioned
RNA was genotyped. Indeed, we observed RSV specific
sequences that clustered together in phylogenetic trees.
This technique allowed us to genotype RSV in clinical
samples that were otherwise unsequenciable. The
proposed approach did not affected RSV infectivity in
HeLa cell line. Moreover, when comparing the NERP
activity from different RSV-positive clinical samples, we
noticed different polymerase fitness, with implication for
Km and Vmax. The method was also able to detect the
polymerase inhibition with the drugs. Together, our findings
indicate that this assay might provide an useful functional
model for measuring RSV RNA polymerase activity.
Key words: Respiratory Syncytial Virus (RSV), Natural
Endogenous RNA Polymerase (NERP) and qRT-PCR.
Financial Support: FAPERJ, CNPq, POM-FIOCRUZ.
"In memory of Dr. Herrmann Gonçalves Schatzmayr, a
landmark in the development of virology in Brazil".
Brazilian Society for Virology
ABSTRACTS
SEROLOGIC EVIDENCE OF ROCIO VIRUS
INFECTION IN RURAL POPULATION IN SANTA
CATARINA STATE.
ID: 00003-00001
Área: 05 - Virologia Humana e Saúde Pública
Borges, A.A.1; Chávez, J.H.2; Reis, V.P.3; Pereira, G.W.4;
Teixeira, A.M.5; Figueiredo, L.T.M.6.
1
UFAL, Universidade Federal de Alagoas, Instituto
Ciências Biológicas e da Saúde, Praça Afrânio Jorge s/
n, Maceió-AL; 2FMRP-USP, Faculdade de Medicina de
Ribeirão Preto, Universidade de São Paulo, Centro de
Pesquisa em Virologia, Av. Bandeirantes 3900, Ribeirão
Preto; 3UNISUL, Universidade do Sul de Santa Catarina,
Av José Acácio Moreira 787, Tubarão-SC.
Rocio Virus (ROCV) was responsible for a remarkable
outbreak of epidemics encephalitis during the 1970s, in
the Ribeira Valley, in the southern coast of São Paulo
State, Brazil. This virus was first isolated in 1975 from
the cerebellum of a fatal human case of encephalitis.
ROCV belongs to the Flaviviridae family and Flavivirus
genus that comprises more than 70 members, 40 of which
of medical importance, such as Dengue, West Nile,
Yellow Fever and Saint Louis Encephalitis Virus. Virus
isolation and serological data suggest that ROCV is
maintained in a cycle in which wild birds, including some
migratory species, are the reservoirs and Aedes and
Psorophora mosquitoes are the vectors. It is intriguing
how this flavivirus appeared in 1970s and disappeared
seven years later after the encephalitis outbreak from
the Ribeira Valley. However, the presence of neutralizing
antibodies for ROCV has been detected in people living
in rural areas of southeastern and northeastern Brazil. It
is possible that this virus circulates in distinct regions of
Brazil and the reemergence of ROCV with ensuing severe
encephalitis outbreaks represents a permanent threat.
For this reason, the aim of this study was to perform a
serological survey of ROCV antibodies in Turvo county,
located southern in the state of Santa Catarina, Brazil.
Serological survey was performed using a recombinant
ELISA which detects IgG antibodies against ROCV DIII.
Anti-DIII antibodies were detected in 2 (1,0%) of 200
samples. This result suggests that ROCV may circulate
in different rural areas far from where the outbreak was
first reported in the 1970s. This finding reinforces
serological survey studies in order predict viral circulation
as well as the risk of reemergence of ROCV. Financial
support: FAPESP; CNPq
Palavras-chaves: Rocio, Serologic survey, Flavivirus
USE OF RECOMBINANT DOMAIN III PEPTIDES OF
FLAVIVIRUS E PROTEIN AS ANTIGEN FOR IGGELISA
ID: 00004-00001
Área: 01 - Imunobiológicos
Chávez, J.H.1; Figueiredo, L.T.M.2
1. USP-FMRP, Universidade de São Paulo, Avenida
Bandeirantes, 3900
The Flavivirus genus of Flaviviridae includes viruses of
great impact in public health. In Brazil, 11 flavivirus are
known and two of them are related to infections of CNS:
Saint Louis Encephalitis Virus (SLEV) and Rocio Virus
(ROCV). Recently, SLEV has been diagnosed in acute
febrile illness cases. ROCV was responsible for an
encephalitis outbreak in the Valley of Ribeira, SP, during
the 1970s and disappeared seven years later. However,
serologic evidences of ROCV antibodies suggest the
circulation of this virus in Brazil. Additionally, another
important virus is West Nile Virus (WNV), introduced in
the US 10 years ago,fast spreading in all the Americas.
WNV was isolated in Argentina in 2006 and serologic
evidences of this virus were also found in Colombia. The
possible introduction of WNV in Brazil, the emergence
of SLEV and the reemergence of ROCV reinforce the
development of diagnostic tools to identify cases to
prevent future outbreaks. Serological diagnosis of
flavivirus infections is commonly difficult due to the
extensive antigenic cross-reactivity among these
viruses, especially where two or more of these viruses
are endemic. In order to overcome this problem, it has
been proposed that recombinant DIII peptides containing
virus-specific epitopes could be used for specific
serological diagnosis of flavivirus infections. In this work,
we describe the standardization of a recombinant IgG
ELISA using the DIII from SLEV, ROCV and WNV as
antigens. Plates were coated with 4ìl/mL of purified DIII
antigen for 48h. MIAF and mice samples previously
immunized with recombinant DIII were used for
standardization. Human samples (n=96) were also tested
and a high prevalence of IgG antibodies against SLEV
(29%) and ROCV (27%) was observed. The recombinant
DIII ELISA exhibited some cross-reactivity when human
samples were tested, but not in MIAF samples. These
results support the use of DIII as tool in diagnostic
assays for flavivirus surveillance. Financial Support:
FAPESP
Palavras-chaves: Domain III antigen, ELISA, Rocio
Virus, Saint Louis Encephalitis Virus, West Nile Virus
DENTIFICATION OF MURINE NOROVIRUSES IN
DISTRICT FEDERAL, BRAZIL
ID: 00007-00001
Área: 05 - Virologia Humana e Saúde Pública
Lorga, R.N.1; Silva, R.L.V.2; Lima, L.M.P.3; Nagata, T4.;
Silva, P.A. 5
55
ABSTRACTS
2. UCB, Universidade Católica de Brasília, SGAN 916
Norte AV W5 CEP:70790-1603. LACEN-DF, Laboratório
Central do Distrito Federal, SGAN, Quadra 601, Lotes O
e P. Brasília-DF CEP: 70830-0104. UnB, Universidade
de Brasília, Laboratório de Microscpia Eletrônica,
Departamento de Biologia Celular, IB4, CEP
Murine Norovirus (MNV) is a mice virus that was first
identified in 2003. It is a non-enveloped virus that belongs
to the Caliciviridae family, has an icosahedral morphology
and measures approximately 28 to 35 nm in diameter
with a single stranded RNA genome. MNV is the only
member from the norovirus genera that could be
replicated in cell culture system what makes possible to
study the basic mechanisms of norovirus replication in
cell culture as well as its pathogenesis in a natural host.
Due to these factors, MNV is used as an analogical model
to study human noroviruses. The objective of this study
is to identify the MNV in stool samples from university
laboratory mice. We also intend to make progress on the
research by the achievement of the genomic sequencing
and replicate them in culture cell for access of bioassays.
To the extraction of the virus, we used 31 mice’s stools
samples collected from two animal facilities located in
different establishments from Brasília, DF. After the
extraction by the Trizol method, the viral genome was
submitted to the RT-PCR with the use of specific primers..
From the 31 analyzed samples, six were positive,
corresponding a total of 19,35%. By this study, we found
a significant proportion of mice infected with MNV.
Palavras-chaves: murine norovirus, calicivirus, Distrito
Federal, Brazil
DEVELOPMENT OF A LOW-COST METHOD OF
TITRATION OF COMBINED LIVE VACCINE AGAINST
NEWCASTLE DISEASE VIRUS AND INFECTIOUS
BRONCHITIS VIRUS
ID: 00008-00002
Área: 01 - Imunobiológicos
ORSI, M.A. 1 ; CAMILLO, S.C.A. 2 ; RIBEIRO, S.A.M. 3;
RAMAZZOTI, A.4; REISCHAK, D.5; Mendonça, A.O6.
2. Lanagro-SP, National Agricultural Laboratoryo, Rua Raul
Ferrari s/n- J.Sta Marcelina-Campinas-São Paulo-Brasil
Brazil has become the biggest broiler chicken exporter
and the third producer in the world. The prophylaxis of
the ND and IB are based on active immunization through
the employment of live vaccines. Veterinary vaccines
should be controlled by manufactures and, in several
countries also by independent analysts, before they are
approved for the commercial use. The success of the
vaccination procedure are linked to the amount of virus
used per dose. The conventional methodology of titration
of live combined vaccine employs neutralization of one
56
virus fraction and titration of the other. With this purpose,
a pure antibody are used in the all tubes or at least in the
last fourth dilution tubes that having spent 4-33 mL of
antibodies. It means $ 120-990 per/vaccine. The objective
of this study is developing an alternative and low-cost
methodology using only 10% of antibodies diluted in PBS
and added to the vaccine dilution (105.7,106.4,
107.1,107.8)volume/volume. After that. It was inoculated
0.2ml/egg in 9-11-day old specific pathogen-free
embryonated eggs, considering the titres presented in on
all vaccines, after we checked if the vaccine was
neutralized by the presence or absence of characteristic
lesions of the virus in the vaccinal fraction and the
presence of characteristic lesions or hemaglutination in
the vaccinal fraction that wasn’t neutralized. Over time
and with testing conducted, it was verified that
neutralization with this amount of antibodies did not meet
a good part of the fractions used in combined vaccines.
The amount was increased to 20% of antibodies diluted
in PBS, it showed good results for the vast majority of the
vaccines tested. After using this method in at least 200
batches of vaccines. The results showed be safely and
mean the use only 0,8 mL of antibodies with one cost of
$24, five time more cheaper than the conventional method.
In development countries that depends on the importation
of immunoreagents this discovery is very significant.
Palavras-chaves: VACCINE, COMBINED LIVE
VACCINE, DEVELOPMENT OF METHOD, METHOD OF
TITRATION, VACCINE AGAINST NDV AND IBV
COMPARISON OF A LOW-COST AND CONVENTIONAL METHODS FOR TITRATION OF COMBINED
LIVE VACCINES AGAINST NEWCASTLE DISEASE
VIRUS AND INFECTIOUS BRONCHITIS VIRUS.
ID: 00008-00003
Área: 01 - Imunobiológicos
ORSI, M.A.1; ZARONI, M.M.H.2; SPILKY, F.R.3; ARNS,
C.W. 4
1. Lanagro-SP/MAPA, Nat. Agric. Lab./Ministr y of
Agriculture, Livestock and Food Supply, Rua Raul Ferrari
s/n, Jdim. Sta. Marcelina, Campinas, São Paulo,SP 2.
Feevale University, Feevale University, Novo HamburgoRS 3. LVA-UNICAMP, Animal Virology LaboratoryUnicamp, Campinas, SP, Campinas, SP
The prophylaxis of Newcastle disease virus(ND) and
infectious bronchitis virus(IB) are based on the active
immunization, mainly through the employment of live
vaccines. The success of vaccination procedures are
linked to the amount of virus used per dose, among other
several factors. The objective of this study is to compare
the conventional titration, using pure antibodies with a
low-cost methodology using only 20% of antibodies
diluted in PBS and added to log10 vaccine diluents (5.7,
ABSTRACTS
6.4, 7.1, 7.8)volume/volume. After that, it was inoculated
in 9-11 day old specific pathogen-free embryonated eggs
(0.2mL/egg). Five vaccine batches of live combined
vaccine against ND and IB were selected. This sample
size is according to a test of 80% of power and 5% of
significant probability. From each batch were used 12
bottles of vaccines, being three for each titration. Taking
the batch as a unity, it was divided in two half parts. The
two titrations methods were randomly sorted to each
one. The titration was made in two repetitions. Fixing up
D the difference between the conventional method’s and
the low-cost method’s titration for the five pairs of half
batches. The effects between the two methodologies were
compared by testing the null hypothesis that the two
titrations (convectional and low-cost) have no significant
differences, i.e.Ho:D=0vs Ha:D‘“0. The test’s criterion
decision is to reject Ho when toe”t and accept Ho when
to<t, where t has a Student’s distribution with n-1 degrees
of freedom. We compute t=m/sm, where m is the average
of the n differences D, sm=s/”n, is the estimative of
standard error of the mean m and s the standard deviation
of D. The test assured no evidence that the two methods
produced different titres. The results showed that both
titration methods were similar. In this sense we
recommend choosing the low-cost methodology and have
advantage to use it in practice. Financial support:
Ministério da Agricultura, Pecuária e Abastecimento.
Palavras-chaves: combined live vaccines, comparision
of methods, vaccine
ISOLATION OF THE CAPRINE ARTHRITIS-ENCEPHALITIS VIRUS (CAEV) FROM CHOROID PLEXUS
AND SYNOVIAL MEMBRANE OF CAEV BRAZILIAN
NATURALLY INFECT GOATS.
ID: 00010-00001
Área: 03 - Virologia Animal
oligodendrocytes as well as pneumocytes, fibroblasts
and endothelial and epithelial cells. In vitro, CAEV can
replicate in primary fibroblasts derived from choroid
plexus or synovial membrane cultures. Our goal was to
isolate CAEV Brazilian strains from choroid plexus (CP)
and goat synovial membrane (GSM) from CAEV naturally
infected goats. Explants of GSM and CP were obtained
aseptically from twelve CAEV seropositive females goats
(eight Saanen, one Alpine, and two Toggenburg) of dairy
goat herds from Pernambuco, Rio Grande do Norte, and
Minas Gerais States. Small tissue fragments of CP and
GSM were cultured at 37 æ%C at 5% CO2 in a humidified
atmosphere. The double nested-PCR for gag partial
sequence of the CAEV genome was used to amplify a
fragment of 185bp. Primers specific to the fourth exon
of the human-âactin gene were used as housekeeping
gene. Specific amplification bands were detected in eight
(66.7%) of GSM and five (41.7%) of choroid plexus
cultures, and four (33.3%) in both. The â-actin gene used
as control was amplified in all cultures analyzed. Gag
DNA-proviral was detected after 15 to 60 days post
implantation of cells in culture (average 33 days), most
of them detected after 21days. All cell monolayer cultures
developed cytopathic effects, mainly in the GSM that
showed multinucleated giant cell formation, cell lyses
and cytoplasmatic vacuolization. The Culture of GSM
and CP were a good source to obtain CAEV Brazilian
strains and this methodology proved to be an alternative
method for identification of CAEV post-mortem. Financial
support: FAPEMIG & CNPq
Palavras-chaves: Caprine arthritis-encephalitis virus,
isolation, choroid plexus, goat synovial membrane.
CAPRINE ARTHRITIS-ENCEPHALITIS (CAE)
OCURRENCE IN MINAS GERAIS STATE, BRAZIL.
00010-00002
Área: 03 - Virologia Animal
Braz, G.F.1; Reis, J.K.P.2; Castro, R.S.3; Oliveira, C.H.
S.5; Rajão, D.S.6; Gouveia, A.M.G.7; Alves, F.7; Oliveira,
F.G.8; Leite, R.C.9
1. UFMG, Escola de Veterinária - DMVP/ Laboratório de
Retroviroses - Universidade Federal de Minas Gerais,
Av. Antonio Carlos, 6627 - Belo Horizonte – MG – Brasil
2. UFRPE, Lab de Virologia - DMVP/Universidade Federal
Rural de Permambuco, Av. Dom Manuel Medeiros s/n,
Recife-PE
Braz G. F.1; Reis, J.K.P.2; Nascimento, P.M.P.3; Gouveia,
A.M.G.4; Oliveira, A.P.5; Rajão, D.S.6; Alves,F.7; Oliveira,
F.G.8; Leite, R.C.9
1. UFMG, Universidade Federal de Minas Gerais, Av.
Antonio Carlos, 6627 - Belo Horizonte – MG – Brasil2.
EPAMIG, Empresa de Pesquisa Agropecuária de Minas
Gerais, Av. Cândido da Silveira, 1647 - Bairro Cidade
Nova. Belo Horizonte - MG
The caprine arthritis-encephalitis virus (CAEV) was first
isolated in 1980 from synovial membranes of adult goats
with ar thritis and from brains of kids with
encephalitis.CAEV is a goat Lentivirus that causes
persistent infection and chronic degenerative disease.
The virus infects cells of the monocyte/macrophage lines,
and as all known lentiviruses, may infect other cell lines
besides macrophages, including microglia, astrocytes,
Caprine arthritis-encephalitis (CAE) is an important viral
infectious disease worldwide. The etiologic agent CAEV,
a lentivírus of the family Retroviridae, causes
leucoencephalomyelitis and progressive polyarthritis in
young, and pneumonia and mastitis in adult goats. The
disease occurs in goat herds in several parts of Brazil
with considerable economic losses due to a progressive
and often debilitating arthritis, and decreasing in milk
57
ABSTRACTS
production associated with mastitis. Minas Gerais State
(MG) contributes with one of the biggest contingent of
goats and milk production of the southeast of Brazil.
The objective of this study was to evaluate the occurrence
of the Caprine arthritis-encephalitis in Minas Gerais,
Brazil. Seven hundred and twenty-one goats with ages
from two months to eight years Saanen, Alpine,
Toggenburg and mixed-breed from five dairy goat herds
distributed in four microregion of MG (Central, Sul de
Minas, Alto Paranaíba and Zona da Mata) were analyzed.
They were tested for detection of antibodies anti-p28 of
the caprine arthritis-encephalitis virus by agar gel immunodifusion (AGID) assay using a commercially available
test. Among the results, 389 (53.95%) animals were
positive and 332 (46.05%) were negative. High
occurrence of CAEV-seropositive animals was observed
in all tested microregions. Additionally many cases of
animals with CAE clinical signs were also found, mainly
arthritis, lameness and mastitis. CAE occurrence in
Minas Gerais State is outstanding and, considering that
the goat industry in this region has become an important
economical activity more studies are necessary for the
control and prevention of CAE in this region. Financial
support: FAPEMIG & CNPq.
Palavras-chaves: Caprine arthritis-encephalitis,
occurrence, AGID, Minas Gerais
THROMBOPOIETIN AND PLATELET RESPONSES
IN PATIENTS UNDERGOING ANTIVIRAL THERAPY
WITH PEGYLATED INTERFERON ALFA-2b PLUS
RIBAVIRIN FOR CHRONIC HEPATITIS C VIRUS
INFECTION
ID: 00015-00001
Área: 05 - Virologia Humana e Saúde Pública
DE ALMEIDA, A.J.1; MOTTA, D.J.G.2; ABREU, S.N.3;
CAMPOS-DE-MAGALHÃES, M. 4; BRANDÃO-MELLO,
C.E.5; YOSHIDA, C.F.T.6; LAMPE, E.7
1. FIOCRUZ, FUNDAÇÃO OSWALDO CRUZ, RUA
LEOPOLDO BULHÕES, 1480, MANGUINHOS, RIO DE
JANEIRO, RJ2. UNIRIO, UNIVERSIDADE FEDERAL DO
ESTADO DO RIO DE JANEIRO, RUA MARIZ E
BARROS, 775, TIJUCA, RIO DE JANEIRO, RJ
Serum levels of thrombopoietin (TPO), a major regulator
factor of platelet production, were found to increase in
patients with sustained virological response (SVR) to
interferon (IFN) therapy. However, TPO levels during
therapy with pegylated interferon (PEG IFN) with ribavirin
(RBV) are unknown. The aim of this study was to
determine TPO levels at different time periods of therapy
with PEG IFN alfa-2b plus RBV in patients chronically
infected with HCV - genotype 1 in relation to platelet
count status. Between August 2004 and August 2009 a
total of 53 patients, 21 (39.6%) males and 32 (60.4%)
58
females with a mean age of 51.6 ± 8.8 years were studied.
The infecting genotype was analyzed by restriction
fragment length polymorphism (RFLP) and TPO levels
were measured in serum samples by using a commercial
quantitative sandwich enzyme immunoassay. Patients
were treated with PEG IFN-alfa 2b plus RBV during 48
weeks. Median platelet counts at pretreatment and 24
weeks after completion of treatment were 136.000/mm3
(62.000–410.000) and 146.000/mm3 (51.000–316.000),
respectively (p=0.9709). Median TPO levels at
pretreatment and 24 weeks after completion of treatment
were 114.9 pg/ml (22.5–303.9) and 183.5 pg/ml (55.2–
339.9), respectively (p=0.0002). When compared on the
basis of platelet count, thrombocytopenic patients
(platelet count < 150.000/mm3; n=32) presented median
TPO levels of 179.1 pg/ml at 24 weeks after completion
of treatment, which was greater than median baseline
values (118.4 pg/ml) (p=0.0077). Regarding
nonthrombocytopenic patients (n=21), an increase was
noted (199.4 pg/ml) from a median baseline TPO levels
of 102.3 pg/ml (p=0.0055). Our data demonstrated a
significant increase of serum TPO levels after treatment
with PEG IFN alfa-2b plus RBV in patients infected with
HCV genotype 1, being greater in the subgroup of
nonthrombocytopenic patients. Platelet response appears
not to be related solely to increase of TPO levels in these
patients.
Palavras-chaves: THROMBOPOIETIN, PLATELETS, HEPATITIS C, INTERFERON, RIBAVIRIN
A COMPARATIVE STUDY OF TWO HEPATITIS C
VIRUS ANTIBODY/ANTIGEN ENZYME-LINKED
IMMUNOSORBENT ASSAYS.
ID: 00017-00001
Área: 01 - Imunobiológicos
BRANDÃO, C.P.U.1; MARQUES, B.L.C.2; LAMPE, E.3;
VILLELA-NOGUEIRA, C.A.4; PAULA, M.T.M.5; VILLAR,
L.M. 6
1. IOC, Instituto Oswaldo Cruz, Av. Brasil, 4365
Manguinhos - RJ2. HSE, Hospital Federal dos Servidores
do Estado do Rio de Janeiro, Rua Sacadura Cabral, 178
Rio de Janeiro - RJ3. Fiocruz, Fundação Oswaldo Cruz,
Av. Brasil, 4365 Manguinhos - RJ4. HUCFF/UFRJ,
Hospital Clementino Fraga Filho- UFRJ, Avenida
Brigadeiro Trompowski - Ramos, Rio de Janeiro - RJ,
21044-020
Hepatitis C virus (HCV) infection is currently under
diagnosed, leaving many individuals unaware of their
infection status. Routine diagnosis of HCV infection is
based on antibodies against HCV (anti-HCV) detection
using highly sensitive second- or third-generation enzyme
immunoassay (EIA). But, accurate HCV diagnosis also
requires HCV RNA detection. Therefore molecular tests
ABSTRACTS
implementation is difficult in resource-limited regions with
technical and economical deficiencies. Alternatives like
the ultrasensitive HCV antibody/antigen (HCV Ab/Ac)
EIAs have therefore been proposed. The objective of
this study was to evaluate the concordance among two
new EIAs used to detect HCV antibody/antigen using a
panel of sera samples. A group of 371 individuals were
recruited in Blood Bank and Public Health Laboratories
from Rio de Janeiro during 2009 to 2010 to provide blood
samples. Sera were tested for HCV Ab/Ac detection using
two different assays (Biorad, USA and Abbott, USA)
according manufacturer’s instructions. Initially reactive
samples were re-assayed in duplicate by the same
procedure, and were considered as positive only when
they demonstrated reactivity in two out of the three
analyses. Samples that present discordant EIA results
were submitted to Cobas Taqman HCV to detect HCV
RNA. Forty-five (11.8%) of the serum samples were
positive by both HCV Ab/Ac EIAs, while 317 (85.4%)
were negative by both tests, with a resultant concordance
of 89.5%. Three out of nine discordant HCV Ab/Ac EIA
results presented HCV RNA. Using PCR as gold
standard, sensitivities of Biorad and Abbott assays were
95.8% and 97.9%, respectively; while specificities of
Biorad and Abbott tests were 99.6% and 98.4%,
respectively. These results show that HCV Ab/Ac EIAs
present good correlation to PCR assay. These assays
are both a cost-effective and a less labor-intensive
alternative to PCR, enhancing its clinical utility. Financial
support: FAPERJ, FIOCRUZ, ABBOTT, BIORAD.
Palavras-chaves: hepatitis C virus, ELISA, ANTIBODY/
ANTIGEN
(BVDV), Porcine Parvovirus (PPV) and Torque Teno virus
1 and 2 (TTV1 and 2). The objective was to study these
contaminants in order to select cell lines with better
quality. Sixty one cell lines from a plenty of species were
evaluated to the five viruses and to Mycoplasma, using
PCR, except to PPV, in which was used the Real Time
PCR with non-specific fluorescent dyes. All of the PCRs
were previously standardized to identify the sensitivity
and analytical specificity. Thirteen samples were positive
to Mycoplasma spp, 12 to BVDV, 24 to PCV1, none to
TTV1 and 2 and 48 to PPV. This last result was confirmed
with agarose gel eletrophoresis and through others
oligonucleotide primers described in the literature. These
results show the importance of the research with cells
contaminant as the utilization of contaminated cell lines
can make the diagnostic doubtful. The high number of
positive samples to the PPV suggests the study not
only of the cells, but also of the inputs used like trypsin,
media and serum. There is no definitive parameter to
which contaminants cells cultures should be tested. The
tests proposed in this work have great importance for
animal diagnosis, because the microorganisms in
question can be harmful to the cell development and
also produce questionable results. We intend to expand
the test to detect contamination between cell lines as it
can make the diagnostic doubtful as the viruses need
specific cells to replicate.
Palavras-chaves: Cell lines, PCR, Mycoplasma ssp,
BVDV, PCV1.
DETECTION OF VIRAL STRAINS AND MYCOPLASMA
SPP BY PCR IN CELL LINES
ID: 00018-00002
Área: 03 - Virologia Animal
ID: 00018-00001
Área: 04 - Virologia Básica
Fonseca Jr., A.A. 1 , Costa, E.A. 2 , DIAS, N.L. 3 ,
Magalhães, C.G.4, GUEDES, E.O.5, Gasparini, M.R.6,
Sales, E.B.7, SALES. M.L.8, OLIVEIRA, T.F.P.9
1. LANAGRO-MG, Laboratório Nacional Agropecuário de
Minas Gerais, Rua Rômulo Joviano, s/n, Centro, Pedro
Leopoldo, Minas Gerais CEP: 336000002. UFMGVeterinária, Escola de Veterinária da UFMG, Av. Antônio
Carlos, 6627. Belo Horizonte, Minas Gerais
OLIVEIRA, T.F.P. 1 , CAMARGOS, M.F. 2 , OLIVEIRA,
A.M.3, DIAS, N.L.4, COTTORELLO, A.C.5, Heinemann,
M.B.6, Fonseca Jr, A.A.7
1. LANAGRO/MG, Laboratório Nacional Agropecuário de
Minas Gerais, Rua Rômulo Joviano, s/n, Centro, Pedro
Leopoldo, Minas Gerais CEP: 336000002. Retrolab,
Laboratório de Retroviroses;Escola de Veterinária da
UFMG, Av. Antônio Carlos, 6627, Pampulha, Belo
Horizonte, Minas Gerais CEP: 31270901
Cell cultures are very used in virology laboratories to
diagnose viral diseases. They must be handle carefully
because they are susceptible to many contaminants
such as Mycoplasma spp, transmitted in the
manipulation. Recently, other cell culture contaminats
have been reported such as expanded to the study
Porcine Circovirus 1 (PCV1), Bovine Viral Diarrhea Virus
A REAL-TIME PCR FOR DIAGNOSIS OF OVINE
HERPESVIRUS 2
Ovine herpesvirus 2 (OvHV-2) is the causative agent of
the sheep-associated malignant catarrhal fever (SA-MCF),
a frequently fatal disease primarily of certain ruminants.
The diagnose of SA-MCF disease is important as a
differential test for foot and mouth disease as well as
rabies. The aim of this work is to develop a real time PCR
for detection of OvHV-2. . The first stage of work was the
design of Plexor primers for the glycoprotein B gene of
OvHV-2. The reaction was done with kit Plexor for realtime PCR in Rotorgene 3000 Cortbett. A initial cycle of
denaturation for 5 min at 95° C was followed by 40 cycles
59
ABSTRACTS
of 15 s at 95° C and 35 s at 62° C followed by a curve with
denaturing temperature of 84° C for 1 min followed by an
increase of 1° C/4 s. The clinical tests were carried out
using 40 brain samples of cattle with clinical diagnose of
encephalitis and 10 semen samples of sheep with clinical
diagnose for OvHV-2. The denaturation peak occurred at
87,8° C. Five cattle samples and seven ovine samples
were positive. Nonspecific amplification due to primer
dimers in the negative samples were differentiated in the
denaturation curve. The real-time PCR offers several
advantages in relation to the conventional by being more
sensitive, rapid and by reducing the contamination
probability. The PCR developed in this work was capable
to detect small amounts of the. Works with the validation
of the technique are already being made.
Palavras-chaves: Ovine Herpesvirus 2, Real-Time PCR,
Diagnosis
CONTROLLING DENGUE INFECTION: RNAI TOOL
INHIBITS VIRUS REPLICATION IN HUMAN
MONOCYTIC CELLS.
ID: 00021-00001
Área: 04 - Virologia Básica
Reis, V.P.1, Gomez-Ruiz, A.C.2, Silva, B.M.3, Bonjardim,
C.A.4, Fonseca, B.A.L.5
1. USP, Universidade de São Paulo, Av. Bandeirantes
39002. UFMG, Universidade Federal de Minas Gerais,
Av. Antônio Carlos 6627
Dengue viruses (DENV) are the most important
arboviruses of public importance, with several outbreaks
every year. These viruses usually cause dengue fever,
but some patients may experienced dengue hemorrhagic
fever or dengue shock syndrome with a considerable
rate of mortality. Recently RNA interference (RNAi) has
been suggested as a potential tool against human
viruses.The RNAi is a newly described phenomena that
occurs in all eukariotic cells.The small interfering RNA
are generated by Dicer cleavage and binds to RISC
complex(RNA Induced Silence Complex) degradating a
target mRNA and inducing gene silencing at posttranscriptional level. The objective of this study was to
evaluate the efficiency of short hairpin RNAs(shRNAs)
in monocytic cells(U-937), challenged with dengue virus
type 4, and demonstrate the potential of RNAi
phenomena to inhibit dengue replication.The shRNA
fragments were designed against specific sequences of
dengue virus genome. The target sequences chosen to
be silenced were: 5´UTR, 3´UTR and prM, and all the
sequences were homologous to all four serotypes of
dengue viruses.Dengue virus type 4 strain H-241 was
previously titrated and the cells were challenged with a
MOI 0,1. The supernatants were collected at 24, 48 and
72 hours post-infection. After this time, viral RNA was
60
extracted from supernatants and analyzed by Real Time
PCR.Our results show that at 48 hours post-infection
we observed an inhibition of 15%, 54% and 77,5% to
5´UTR, 3´UTR and prM respectively.After 72 hours p.i.,
the inhibition was 57%, 70% and 53% to 5´UTR, 3´UTR
and prM respectively. Based on these results and
previously studies with same techniques, RNAi
phenomena is able to inhibit dengue virus replication in
human cells.There are no other studies using RNAi
against dengue virus type 4. This RNAi-based tool, in
near future could bring a new hope to control dengue
virus infection. Financial Support: FAPESP and CNPq.
Palavras-chaves: Dengue virus, RNAi, Flavivirus, Real
Time PCR.
EVIDENCES OF ARBOVIRUSES INFECTION IN
RIBEIRÃO PRETO POPULATION DURING DENGUE
OUTBREAK.
ID: 00021-00002
Área: 05 - Virologia Humana e Saúde Pública
Reis, V.P.1, Chavez, J.H.2, Badra, S.J.3, Figueiredo, L.T.M.4
1. USP, Universidade de São Paulo - Faculdade de
Medicina de Ribeirão Preto - Centro de Pesquisa Virologia,
Avenida dos Bandeirantes 3900.
Arthropode-born viruses are widely spread in Americas
and are responsible for large outbreaks. Mostly of them,
belongs to Flaviviridae family, genus Flavivirus. In Brazil
eleven flavivirus are described, and Dengue virus (DENV)
causes the most important disease. Therefore, during
the 70’s, Rocio virus (ROCV) caused a large outbreak in
the Ribeira Valley-SP with severe cases of neurological
manifestation. Saint Louis Encephalitis virus (SLEV) is
also responsible for disease with neurological
manifestation in Brazil and North America. In 2006, during
a Dengue outbreak, SLEV was detected causing acute
febrile disease, with positive viral isolation, but no
neurological manifestation. Although West Nile Virus
(WNV) has not been detected yet in Brazil, serologic
evidences in horses have shown its emergence in our
country. These arboviruses are responsible for
neurological manifestations or may cause acute febrile
disease. Currently, no epidemiological study shows the
prevalence of neutralizing antibodies to these viruses in
population. For this reason the aim of this study was to
perform a sero-survey to ROCV, SLEV e WNV in Ribeirão
Preto- São Paulo state, Brazil. Sero-survey was
performed using indirect ELISA to detect IgG against
DIII from ROCV, SLEV e WNV. 100 samples collected
during dengue outbreak in 2006 were tested. Our results
show a prevalence of 3% and 2% of antibodies against
DIII from ROCV e SLEV, respectively. No WNV antibodies
were detected. These findings reinforce the circulation
ABSTRACTS
of ROCV and SLEV and may suggest misdiagnosis of
these infections and the possible threat of severe
encephalitis outbreaks. Financial support: FAPESP.
Palavras-chaves: Arboviruses, Rocio virus, Saint Louis
virus, West Nile Virus, Sero-survey.
SEROPREVALENCE OF HANTAVIRUS IN HUMANS
ON THE BORDER OF BRAZIL AND ARGENTINA
ID: 00022-00001
Área: 05 - Virologia Humana e Saúde Pública
SOUZA, W.M. 1 , MACHADO, A.M. 2 , FIGUEIREDO,
L.T.M.3, BOFF, E.4
1. UNOESC, Universidade do Oeste de Santa Catariana,
Rua Oiapoc, 211, Agostini, São Miguel do Oeste, SC2.
CPV - FMRP/USP, Centro de Pesquisa em Virologia,
Faculdade de Medicina de Ribeirão Preto, Av.
Bandeirantes, 3900 - Monte Alegre - CEP 14049-900 Ribeirão Preto/SP
In the region of the western tip of Santa Catarina state,
according to reports from the Ministry of Health, no
reports of hantavirus pulmonary syndrome, zoonotic
disease transmitted by feces of infected rodents. With
aim to know and show about the presence or not of
hantavírus infections, a seroepidemiological study of
individuals, residents of this region was performed. So
were studied a total of 340 volunteers of both genders,
from towns of Belmonte and Paraíso. The serum of these
patients was collected and used for detection of IgG
antibodies against recombinant N protein of Araraquara
hantavirus, by ELISA assay, according to the protocol
developed at the Center for Research on Virology. The
positive samples were then titrated and confirmed by
immunofluorescence assay. This study demonstrated a
presence of IgG antibodies to hantavirus N protein in
3.53% of the population. The occupation of farmer was
the most frequent, and 81% had direct and indirect
contact with rodents, 91.7% of positive cases were
farmers, of the probable cause of infection was through
cleaning barns These antibodies are noteworthy, given
that the levels of antibodies are found in individuals whose
contact with hantavirus may have occurred many years.
This study shows the circulation of hantavirus in the
region, a fact that until then had not reported all the serum
reagents had contact with the pathogen, but did not
develop pulmonary and cardiovascular syndrome, but
one must be alert because it is a serious and emerging
disease with great importance.
Palavras-chaves: Hantavirus, Morbidity, Seroprevalence,
Zoonotic.
A SEROLOGICAL SURVEY OF MORBIDITY AND
ARENAVIRUSES IN THE BORDER REGION
BETWEEN BRAZIL AND ARGENTINA
ID: 00022-00002
Área: 05 - Virologia Humana e Saúde Pública
SOUZA, W.M. 1 , MACHADO, A.M. 2 , FIGUEIREDO,
L.T.M.3, BOFF, E.4
1. UNOESC, Universidade do Oeste de Santa Catariana,
Rua Oiapoc, 211, Agostini, CEP: 89900-000, São Miguel
do Oeste, SC2. CPV - FMRP/USP, Centro de Pesquisa
em Virologia da Faculdade de Medicina de Ribeirão Preto,
Av. Bandeirantes, 3900 - Monte Alegre - CEP 14049-900
- Ribeirão Preto/SP.
In Brazil, until 2010 only five cases of arenavirus
haemorrhagic fever were reported, two fatalities and three
non-fatal hemorrhagic fever in the region west of the state
of Santa Catarina, according to reports from the Ministry
of Health, no reports of arenavirus haemorrhagic fever,
a zoonotic disease transmitted by contact and biting
infected rodent excreta. With aim to know and show about
the presence or not of arenavirus infections, a
seroepidemiological study of individuals, residents of this
region was performed. So were studied a total of 340
volunteers of both genders, from towns of Belmonte and
Paraíso. The serum of these patients was collected and
used for the detection of IgG antibodies against
recombinant N protein of arenaviruses Junin, by ELISA,
according to the protocol developed at the Center for
Research on Virology. The positive samples were
confirmed by western blot. This study demonstrated the
presence of IgG antibodies against the protein in
arenaviruses initially 0.58% of the population. Since when
subjected to a confirmatory test by western blot, the
samples were proven to be negative, these individuals
would probably have an infection at the time of collection
with high antibody levels which probably gave crossreaction. Regarding the occupation of farmer was the
most frequent, and 81% had direct and indirect contact
with rodents. This study did not show movement of
arenaviruses in the region, and despite there being
reported, the characteristics of morbidity and bordering
country arenaviroses edema, suggested that there could
be movement in the region, but further studies should
be conducted at both regional and national level to better
understand the epidemiology of this serious disease
issue, emerging and very important.
Palavras-chaves: Arenavirus, Fever hemorrhagic,
Seroprevalence.
DETERMINATION OF VIRAL LOAD IN DENGUE
VIRUS 3- INFECTED PATIENTS WITH DIFFERENT
CLINICAL FORM OF DENGUE
ID: 00024-00001
Área: 05 - Virologia Humana e Saúde Pública
61
ABSTRACTS
SILVA, A.M. 1 , GOMES, A.L.V. 2 , CORDEIRO, M.T. 3,
GUIMARÃES, G.F.4, GIL, L.H.V.G.5, MARQUES, E.T.A.6
1. LaViTE-IAM, Laboratório de Virologia e Terapia
Experimental - Instituto Aggeu Magalhães, Av. Prof
Moraes Rego sn, Cidade Universitária -Recife/PE, CEP:
50.670-4202. UFPE, Universidade Federal de
Per nambuco, Av. Prof Moraes Rego sn, Cidade
Universitária -Recife/PE3. UPitt, Department of Infectious
Diseases and Microbiology, University of Pittsburgh.,
Pittsburgh, Pennsylva
Studies correlating viral load, the host’s immune status
and clinical forms of dengue are important for the
understanding of the pathogenesis, diagnosis and
prognosis of dengue. The quantification of magnitude in
viral replication is fundamental in the follow-up of disease
progression into more severe forms. In this work we
studied the viremia kinetics of dengue virus serotype 3
(DENV-3) in different infection types and clinical forms
of dengue. A total of 314 sequential samples from 85
patients with DENV-3 infections were analyzed for viremia
kinetics, in addition to another 209 unique samples
collected in febrile period. Associations of viremia levels
(determined by real-time PCR), platelet levels and
antibody responses with disease severity were evaluated.
When testing sequential samples, the levels of viremia
were similar in both primary and secondary infections,
as well as in the different clinical forms of dengue. The
median time of viremia duration was ten days. The
maximum viral load was observed between the first and
third day of onset. In the first samples of each patient
collected in the febrile period, the viremia levels were
higher in more severe forms than in dengue fever
(pFinancial support: Fiocruz, CNPq and NIH.
Palavras-chaves: viral load, dengue, flavivirus.
COMPARATION OF DIFFERENT ROUTES OF
ADMINISTRATION FOR A DENGUE VIRUS
SEROTYPE 3 VACCINE CANDIDATE
ID: 00024-00002
Área: 01 - Imunobiológicos
GUIMARÃES, G.F.1, Da SILVA, A.N.M.R.2, ALMEIDA,
S.R.3, MARQUES, E.T.A.4, GIL, L.H.V.G.5
1. UFPE, Universidade Federal de Pernambuco, Av. Prof
Moraes Rego sn, Cidade Universitária -Recife/PE2.
LaViTE, Depar tamento de Virologia e Terapia
Experimental - Centro de Pesquisas Aggeu Magalhães,
Av. Prof Moraes Rego sn, Cidade Universitária - Recife/
PE3. UPitt, Center for Vaccine Research-University of
Pittsburgh-Depar t of Infectious Diseases and
Microbiology, 9022 Biomedical Science Tower 3,
Pittsburgh, PA 15261
The increased transmission and geographic spread of
62
dengue fever make it one of the most important
mosquito-borne viral diseases of humans. Since there
is no specific treatment for dengue disease, a licensed
vaccine has become a global health priority. Thus, the
development of alternative vaccination strategies such
as DNA vaccines encoding specific dengue sequences
is considered to be a powerful innovative strategy. Into
this approach, the use of codon optimized sequences
co-expressed with Lysossome-associated membrane
protein (LAMP) molecules, which is able to drive the
new translated chimera antigen into the MHC-II, were
found to elicit enhanced immune responses when
compared to DNA vaccines encoding unmodified native
antigens. We previously described the construction of a
DNA vaccine encoding the optimized sequences for Cterminal of capsid (C), pre-membrane (prM) and envelope
(E) proteins of DENV-3, fused with LAMP-1. The aim of
this study was to compare antibody response after
subcutaneous (sc.) versus intramuscular (im.)
administration of two plasmids DNA-vaccines, p43DENV3-prME-opt and p43-DENV3-prME-opt-LAMP. Fiveweek BALB/c mice were inoculated by the s.c or i.m.
route with each construct in a total volume of 100 ul(1ug/
ul) using five doses. We observe that mice immunized
with either construct by sc. route had the highest
neutralizing antibody titers against dengue, around 1:2560
for p43-DENV3-prME-opt and 1:5120 for p43-DENV3prME-opt-LAMP. In contrast, the group of mice inoculated
by im. route gained low title, around of 1:160 for both
constructs. In summary, the results of this study
demonstrated that the immunogenicity of ours dengue
DNA vaccine was much stronger when administered
subcutaneously, and this response was improved with
the presence of LAMP. The promising results of this study
encourage the application of this strategy to the
development of an effective dengue tetravalent DNA
vaccine.
Financial support:PDTIS/FIOCRUZ,NIH-USA and
CAPES.
Palavras-chaves: dengue, codon usage, LAMP, route
of administration, DNA vaccine.
CELLULAR TOXICITY EVALUATION OF BOVINE
HERPESVIRUS 1 AND 5 MULTIEPITOPE PROTEIN
IN BOVINE PBMC
ID: 00027-00001
Área: 01 - Imunobiológicos
Ferrari, A.B. 1, Laguardia-Nascimento, M. 2, Barbosa,
A.A.S.3, Moreira, G.D.4, Fonseca, F.G.5, Barbosatancioli, E.F.6
1. UFMG, Departamento de Microbiologia - ICB, Av.
Antonio Carlos 6627. Campus Pampulha. Belo Horizonte,
MG.
ABSTRACTS
The Bovine herpesvirus 1 and 5 (BoHV-1 and BoHV-5)
has been largely isolated from dairy and beef cattle. The
bovine herpesvirus represents a major epidemiological
and economical problem for the cattle industry worldwide,
especially in Brazil that holds the largest commercial
herd in the world. The technique for production of
heterologous proteins has been developed in the 70s
and since then has become an essential tool for the
study of the structure and function of proteins, besides
the production of molecules of interest for prevention
and treatment of several diseases. This study aimed to
evaluate the cellular toxicity of recombinant proteins of
BoHV-1 and 5 (each of them containing three antigenic
epitopes [gD, gB and VP8]fused to a histidine tail) for
bovine peripheral blood mononuclear cells (PBMC). These
proteins are candidate molecules for the production of
vaccine to be used in control programs of BoHV-1 and 5
in cattle. Bovine PBMC (from 2 animals) were treated
with proteins BoHV-1, BoHV-5 and BoHV-1 + BoHV-5 in
different times at concentration 100 Micrograms by
150.000 cells. After 24, 48 and 72 hours of incubation,
cell viability was measured by MTT. The statistical
analysis obtained by ANOVA with Tukey pos-test
demonstrated that cell viability of treated PBMC in any
concentration of BoHV-5 and BoHV-1 recombinant
proteins showed no statistical difference when compared
to their respective control cells. However, the treatment
of PBMC using both recombinant proteins simultaneously
(BoHV-1 + BoHV-5) showed statistical difference.
Palavras-chaves: BoHV-5, BoHV-1, Recombinant
protein, PBMC.
EVALUATION OF CHEMOKINES AND CYTOKINES
RECEPTORS IN HTLV-1 INFECTED INDIVIDUALS.
ID: 00027-00002
Área: 05 - Virologia Humana e Saúde Pública
Souza, J.G.1, Fonseca, F.G.2, Teixeira, A.L.3, Martins,
M.L. 4 , Mar tins, C.P.S. 5 , Carvalho, L.D. 6 , BarbosaStancioli, E.F.7
1. UFMG, Departamento de Microbiologia - ICB, Avenida
Antonio Carlos, 6627. Campus Pampulha. Belo Horizonte,
MG.2. HEMOMINAS, Fundação HEMOMINAS, Alameda
Ezequiel Dias, Belo Horizonte, M.G.3. UFMG, Departamento
de Clínica Médica, Faculdade de Medicina da UFMG., Av.
Prof. Alfredo Balena, 190 Belo Horizonte - MG
The Human T-Lymphotropic virus 1 (HTLV-1) is the
etiologic agent of chronic inflammatory syndromes as
HAM/TSP, ATL and rheumatologic diseases. Many
factors contribute for the development of these
syndromes, especially a vigorous immunologic response,
although remains unclear the difference of the
pathogenesis installation among infected individuals, and
the fact of many infected people remains lifelong
asymptomatic. The objective of the present study was
to assess chemokines (chemokines CXCL9/MIG,
CXCL10/IP-10, CCL5/RANTES) and soluble TNF
receptors (sTNFR1 and sTNFR2) levels in HTLV-1
infected asymptomatic carriers (AC), oligosymptomatic
(OL), individuals with rheumatologic diseases (RD) and
with HAM/TSP (HT), comparing to noninfected individuals
(NI). Chemokines and cytokines receptors levels were
evaluated by ELISA using a commercial R&D system,
according to manufactures. The results showed that the
chemokines CXCL9/MIG, CXCL10/IP-10, CCL5/
RANTES were increased in HT group.
Palavras-chaves: HTLV-1, HAM/TSP, chemokines,
cytokines receptors.
TUMOR NECROSIS FACTOR ALPHA (TNF -308 G/A)
POLYMORPHISM IN BRAZILIAN HUMAN
IMMUNODEFICIENCY VIRUS 1 INFECTED PATIENTS
ID: 00030-00001
Área: 05 - Virologia Humana e Saúde Pública
FREITAS. F.B. 1 , ISHAK, R. 2 , ISHAK, M.O.G. 3 ,
VALLINOTO, A.C.R.4
1. UFPA, Universidade Federal do Pará, Trav. Augusto
Corrêa, n° 01
TNF-á is a pleiotropic cytokine that acts as an
inflammatory mediator. Investigations suggest that TNFá is involved in the pathogenesis of HIV-1 infection since
it is overproduced by infected individuals. The production
of TNF-á is, at least partially, genetically determined.
High production of TNF-á occurs in the presence of 308A allele while -308G allele is associated with low
concentrations. The present study investigated whether
the TNF-á gene -308 polymorphism (G/A) could be a
factor in the susceptibility to HIV-1 infection. We
investigated the frequency of this mutation in a sample
of 194 HIV-1 asymptomatic infected patients and 57
healthy control seronegative individuals. All patients in
HIV infected group were receiving antiretroviral therapy
(ARVT). The alleles identification was performed by RFLP
analysis of 107pb product using Nco I restriction enzyme.
The RFLP products followed by a 3% agarose gel
electrophoresis. The analysis of allele frequencies
revealed a higher prevalence of the mutant A allele in
the HIV-1 infected group (11,50%) when compared to
the control group (5,26%) and the G allele was most
prevalent in the healthy group (94,73%) than in the
infected group (88,40%), however the difference was not
statistically significance (÷2=3,211; p=0,0731). Genotype
frequency analysis showed that the G/G homozygous
was the largest genotype in control (89,47%) and infected
(78,86%) groups, furthermore, the presence of -308A
63
ABSTRACTS
allele (A/A or A/G) was most frequent in the seropositive
patients (21,14%) compared with control group (10,53%),
but this differences were not significant (÷2=2,528;
p=0,1070). Thus, the present study did not observe
significant associations between genetic variants and
susceptibility to HIV infection, but the previously findings
remain very discrepant and further studies are necessary
to better define the relationship between HIV-1 infection
and the TNF -308 G/A polymorphism. Support: PN-DST
& AIDS/MS, UFPA, CAPES, CNPq.
Palavras-chaves: Human immunodeficiency virus 1,
Polymorphism, Tumor Necrosis factor alpha.
MORPHOLOGICAL ANALYSIS OF A Lactococcus
Lactis BACTERIOPHAGE BY TRANSMISSION
ELECTRON MICROSCOPY
ID: 00031-00001
Área: 04 - Virologia Básica
ELLER, M.R.1, ASSIS, L.M.2, DIAS, R.S.3, PEREIRA,
A.L.4, OLIVEIRA, L.L.5, DE PAULA, S.O.6
1. UFV, Universidade Federal de Viçosa, Av. PH Rolfs,
Viçosa, MG
Infections of lactic acid bacteria (LAB) by bacteriophages
are recognized as the main cause of failure or slow
fermentation in the modern dairy industry. Therefore, the
knowledge of phage morphology and metabolism is an
important tool in the dairy factories for the study,
development and implementation of specific technologies
to improve their systems of prevention and infection
control. In this work, we analyzed morphologically
bacteriophages isolated from failure fermentation in a
dairy industry in Minas Gerais, Brazil. A solution
containing isolated phages was treated with
polyethyleneglycol (PEG 8000) 30% and centrifugated
to precipitate viral particles. Supernatant was discarded
and the pellet resuspended in a specific buffer. PEG was
extracted by using an equal volume of chloroform.
Dilutions of the sample were observed by Transmission
Electron Microscopy at the Center for Microscopy and
Microanalysis (CMM) of Federal University of Viçosa.
Phages showed an average size of 230 nm with a tail
length of 180 nm and isometric heads with a diameter of
50 nm. No basal plate was observed. These
characteristics are consistent with those of 936-type
phages, a group of Siphoviridae family, which contains
isometric heads with 45-65 nm in diameter, collar, a long
noncontract tail 100-200 nm in length, and generally no
basal plate. Variations in the morphological dimensions
are common between members of a group and are not
decisive for the classification of these viruses. However,
these data are consistent with the characteristics of the
main lactococci phages isolated. Financial support:
64
CNPq/FAPEMIG/FUNARBIC.
Palavras-chaves: Bacteriophages, Lactococcus lactis
phage, Morphological Analysis.
MOLECULAR CHARACTERIZATION OF A
LACTOCOCCAL BACTERIOPHAGE ISOLATED FROM
A FAILURE FERMENTATION IN A DAIRY FACTORY
ID: 00031-00002
Área: 04 - Virologia Básica
ELLER, M.R., 2DIAS, R.S., 3ASSIS, L.M., 4PEREIRA,
A.L., 5OLIVEIRA, L.L., 6DE PAULA, S.O.
1. UFV, Universidade Federal de Viçosa, Av. PH Rolfs,
Centro, Viçosa, MG
1
Lactic acid bacteria (LAB) can be infected and lysed by
an extensive range of bacteriophages, which is
recognized as the main cause of failure or slow
fermentation in the modern dairy industry. Contaminate
processes prejudice the quality of product or cause even
its complete loss. Although technological advances in
fermentation have reduced the incidence of
bacteriophage infections, they certainly do not eliminate
it. The objective of this work was the isolation and
molecular characterization of a Lactococcus lactis lytic
bacteriophage from failure fermentation in a Brazilian
factory. For this, one liter of serum was collected of a
Cottage cheese failure fermentation e treated with
polyethyleneglycol 6000 10%. The isolate was propagated
and was molecularly characterized using multiplex
Polymerase Chain Reaction, pulsed field electrophoresis,
restriction enzyme profile and protein profile. Phage was
isolated from a L. lactis subsp. cremoris strain and
presented a 48 kbp DNA with patterns of 4, 7, 1 and 1
fragments when cleaved by EcoR I, Hae III, Hind III and
BamH I, respectively. Phage showed a protein profile
with a major protein of 18 kDa and was classify, by
multiplex PCR, in the group of 936-type phages,
Siphoviridae family, the most abundant in dairy products
in the world. This work is the first report of a member of
this group with a genome greater than 40 kbp. Financial
support: CNPq/FAPEMIG/FUNARBIC.
Palavras-chaves: Bacteriophages, Lactococcus lactis,
Molecular Characterization.
CONSTRUCTION OF A NEW BACULOVIRUS
TRANSPOSITION VECTOR FOR THE PRODUCTION
OF POLYHEDRA CONTAINING RECOMBINANT
VIRUSES
00039-00001
Área: 03 - Virologia Animal
Ardisson-Araújo, D. M. P., 2Morgado, F. S., 3Ribeiro,
B. M.
1
ABSTRACTS
1. IB/CEL/UnB, Laboratório de Microscopia Eletrônica,
Insituto de Ciências Biológicas, Campus Universitário
Darcy Ribeiro, Brasília-DF
The commercially available Bac-to-Bac system
(Invitrogen) for the construction of recombinant
Autographa californica multiple nucleopolyhedrovirus
(AcMNPV) does not generate occlusion bodies
(phenotype occ-) during virus insect cell infection. This
is so because the polyhedrin gene (AcPH) was replaced
by the transposition target cassette in the bacmid
genome present in a plasmid form inside an E. coli strain
(DH10-Bac, Invitrogen). Thus, insect larvae infection with
this recombinant virus occurs only through hemocoel
injection. In order to construct occluded recombinant
baculovirus (occ+) we carried out modifications in the
pFastBac1® (pFB1, Invitrogen) transposition vector. First
of all, the commercial vector was digested with AccI
enzyme, blunted and religated to produce pFB1/AccI
without the AcPH promoter. An 1.100 bp region from
pSynXIV VI+ vector, called PSX (AcPH gene with its
own promoter and two viral promoters in tandem) was
amplified by PCR and cloned into the pGem®-T easy
(Promega) cloning vector to generate pGem-PSX. The
fragment PSX was removed from pGem-PSX by NotI/
SpeI digestion and ligated into the pFB1/AccI digested
with the same enzymes, generating pFB1/AccI-PSX. The
modified vector was used to generate recombinant
baculovirus according to the Bac-to-Bac manufacturer’s
instructions, called vAcwtocc+. We analyzed the
polyhedrin production by light microscopy and SDSPAGE. Polyhedra were produced when vAcwtocc+ was
used to infect insect cells from 48 h post-infection (p.i.)
until cell death. Extract cell analysis showed the 30 kDa
polyhedrin band, identical to AcMNPV band. We are
currently cloning reporter genes in order to analyze the
activity of the promoters in tandem. Before this work,
the construction of occ+ virus was only possible by the
inefficient homologue recombination method using
pSynXIV VI + vector. Therefore, the recombinant virus
generated in this work may be used for the per os
infectivity assays of recombinant viruses.
Palavras-chaves: AcMNPV, Baculovirus, Polyhedra
production, Transposition vector.
THE ENHANCER EFFECT OF THE BACULOVIRUS
AgMNPV HOMOLOGOUS REGIONS 1 AND 3 OVER
THE IE1 AND GP64 VIRAL PROMOTERS
ID: 00039-00002
Área: 03 - Virologia Animal
Morgado, F. S., 2Ardisson-Araújo, D. M. P, 3Ribeiro, B.
M.
1. IB/CEL/UnB, e Microscopia Eletrônica, Insituto de
1
Ciências Biológicas, Campus Universitário Darcy Ribeiro,
Brasília-DF
The baculovirus Anticarsia gemmatalis multiple
nuclepolyhedrovirus (AgMNPV) infects larvae of the
Anticarsia gemmatalis moth. This virus has a circular
genome of 132239 base pairs, composed of 165 open
reading frames and non-coding structures such as the 9
homologous regions (hr) dispersed in the genome. These
are redundant direct repeat sequences containing
imperfect palindromes at the core of the repeat
sequences, and they are involved in viral replication and
expression. In this study, we constructed reporter plasmid
vectors containing the hr 1 and 3 of AgMNPV, upstream
to viral promoters of the immediate early 1 (IE1) and the
budded virus envelope (GP64) proteins. These promoters
are regulating the expression of the chemiluminescent
reporter protein Firefly Luciferase (FLUC). This allows
us to quantify the enhancer effect of the hr sequences
over the isolated viral promoters during infection. The
constructs hr-promoter-FLUC plasmids were individually
transfected into UFL-Ag-286 insect cells and then the
cells were infected with AgMNPV-2D (wild-type) virus.
At different hours post infection, the transfected/infected
cells were lysed and the cells extracts were analyzed
for the presence of the FLUC protein chemilluminescence
activity, that was quantified by a luminometer. Here, we
confirmed that the homologous regions isolated presents
a marked enhancer effect on the production of Luciferase.
In the case of the GP64 promoter, hrs showed 10 times
more Luciferase expression, compared to the expression
controlled only by the GP64 promoter. The hr plus IE1
promoter showed up to 5 times more Luciferase than
the IE1 promoter alone. This effectively proves that the
AgMNPV hr1 and hr3 sequences function as enhancers
in cis over viral promoters during the course of infection,
elucidating one of the mechanisms of viral expression
in infected cells and will allows us to design new ways
to overexpress desirable proteins with the baculovirus
expression vector systems.
Palavras-chaves: Baculovirus, enhancer, homologous
region, luciferase, viral expression.
MORPHOLOGIC ANALYSIS OF BACTERIOPHAGES
OF BOVINE MASTITIS-CAUSING Staphylococcus
aureus
ID: 00040-00001
Área: 04 - Virologia Básica
Dias, R. S., 2Silva, L. C. F., 3Fonseca, L. A. B. V, 4Eller,
M. R., 5Silva, V. D., 6Silva, L. S., 7Souza, F. O., 8Oliveira,
L. L., 9Paula, S. O.
1. UFV, Universidade Federal de Viçosa, Avenida PH
Rolfs s/n Campus Universitário
1
65
ABSTRACTS
Bovine mastitis is identified as the main disease affecting
dairy herds in the world. This infectious disease causes
loss of milk quality and serious economic losses to both
manufacturer and dairy industry. This disease can be
caused by more than 150 different agents, and
Staphylococcus aureus is described as one of the most
important of them. Although antibiotic therapy is the main
procedure used to combat mastitis, it faces problems
such high costs, eradication of commensal microbiota,
presence of residues in milk and generation of resistant
strains. Thus, the request for alternatives to antibiotics
becomes essential, and phagoterapy has been showed
as a promising tool in controlling these pathogens, due
to its specificity and quickness when compared to the
time for development of new antibiotics. Thus, the
isolation and characterization of new phages is the great
importance to researchers and the population. To perform
the morphological analysis by transmission electron
microscopy were dripped 10 μL of viral suspension,
previously purified by ultracentrifugation on sucrose
cushion, in a 200 mesh grid previously covered with
FormVar. Samples were contrasted with uranyl acetate
2% for 20 seconds, and excess removed with filter paper.
Images were obtained in transmission microscope Zeiss
EM 109, operating at 80 kV. Phages isolated in this work
can be classified taxonomically as belonging to the order
Caudovirales, Myoviridae family.
Financial Support: FAPEMIG
Palavras-chaves: Staphylococcus aureus, Bacteriophages, Bovine Mastitis.
pathogenesis studies, and vaccine research. Currently,
the antigens for serological kit have been produced by
viral inoculation in mouse brain and the whole virus
particle is used. This study aimed the construction of
two recombinant multiepitope genes covering all four
serotypes of DENV and expression in E.coli for use in a
serological diagnostic kit. The multiepitope genes were
built according to the antigenic regions of the E protein
described in the literature, covering the four serotypes
of which are merged DENV in tandem by glycine linkers
in order of serotype of 4, 3, 2 and 1. The proteins were
expressed in E. coli BL21: DE3 and purified by affinity
chromatography using a Ni+-NTA resin, and the presence
of peptides were confirmed by western blot with antihistag. In order to confirm the antigenicity of recombinant
protein, DOT ELISA and Western blot analysis were
performed with sera of patients positive for DENV-1. The
positive reactions were observed when the membranes
were treated with DENV-1 positive sera. At moment, the
antigens are being tested individually by antigen-coating
ELISA with positive sera for DENV 1, 2 and 3 serotypes
more prevalent in Brazil. Financial support: FAP-DF and
CNPq.
Palavras-chaves: Dengue, diagnostic kit, recombinant protein.
EXPRESSION
OF
TWO
RECOMBINANT
MULTIEPITOPE DENGUE VIRUS ENVELOPE
PROTEINS IN E.COLI FOR SEROLOGICAL
DIAGNOSTIC KIT
ID: 00043-00001
Área: 06 - Virologia Vegetal
ID: 00041-00001
Área: 05 - Virologia Humana e Saúde Pública
MALDANER, F.R., 2DOS SANTOS, F.B., 3ARAGÃO,
F.J.L., 4 FRANCO, O.L., 5 SCHATZMAYR, H. G.,
6
RESENDE, R. O, 7NAGATA, T.
1. UnB, Universidade de Brasília, Brasília-DF2. UCB,
Universidade Católica de Brasília, Brasília-DF3. IOCFIOCRUZ, Instituto Oswaldo Cruz, Rio de janeiro4.
EMBRAPA-Cenargen, Empresa Brasileira de Pesquisa
Agropecuária, Asa Norte-Brasília
GEOGRAPHIC DISTRIBUTION AND MOLECULAR
PHYLOGENY OF IRIS YELLOW SPOT VIRUS
(TOSPOVIRUS) ISOLATES FROM ONIONRODUCING
PROVINCES IN PERU
Oliveira, A.S., 2Lima, R.N., 3Torres, R.C.A., 4Melgarejo,
T.A., 5Resende, R.O.
1. UnB, Universidade de Brasília, Intituto de Ciências
Biológicas (IB-04) 2. UNALM, Universidad Nacional
Agraria La Molina, Departamento de Fitopatología
1
1
Dengue is the most common mosquito-borne viral disease
of humans and in recent years, has become a major
international public health concern. The virus group
consists of 4 serotypes that manifest similar symptoms,
ranging from a mild febrile illness to a dengue
hemorrhagic fever. The accurate and efficient diagnosis
of dengue is important for clinical care, surveillance,
66
Iris Yellow spot virus (IYSV) is reported worldwide,
mainly infecting onion plants. It is responsible for
significant yield reductions of this vegetable. In South
America, IYSV was already reported in Brazil, Chile and
Peru. In the latter, the export market of fresh and dries
onions was increased over the past years. In this work,
onion plants with typical symptoms of IYSV infection
were collected in three onion-producing provinces in Peru.
Several samples were collected and the viruses were
transferred onto Nicotiana benthamiana by mechanical
inoculation. Total RNA from each sample was extracted
and used as template for amplification of the nucleocapsid
gene by RT-PCR with the primers J13 and IYSV-NBH-R.
The expected amplified fragments of approximately 0.9
Kb were cloned into pGEM-Teasy and sequenced.
ABSTRACTS
Sequences of four Peruvian-IYSV isolates were
compared with those available in the GeneBank and
multiple alignments of nucleoprotein amino acid
sequences were used as input for phylogenetic analysis.
These chosen isolates originated from the more
important onion-producing areas in the country as Lima
(Barranca),Ica (San Jacinto) and Arequipa (Congata and
Alata). The results showed that IYSV is widely spread in
all sampled regions in Peru. The amino acid sequence
identity when compared among the four isolates ranged
from 96% to 99%. The phylogenetic tree based on the
amino acid sequences showed that, although variability
can be seen in the N-amino acid sequences, the Peruvian
isolates were placed in the same cluster. The four new
isolates sequenced in this work also clustered with USA
isolates and other Peruvian isolates previously
characterized. However, they differ from other isolates
characterized in South America (eg. Brazil, Chile). The
genetic similarity between American and Peruvian
isolates is probably explained by the onion trade route
between these countries and the similar growing
conditions.
Financial support: UnB,CNPq,FAP-DF
Palavras-chaves: Cebola, Filogenia, Iris Yellow spot
virus, Peru, Tospovirus.
THE VP1 GENE OF A PORCINE GROUP A
ROTAVIRUS IN PIGLETS IS HOMOLOGOUS TO
NCDV STRAIN VP1 GENE
ID: 00045-00002
Área: 04 - Virologia Básica
BARROS, I.N., 2BALDIN, C.M., 3CASTRO, A.M.M.G.,
ASANO, K.M., 5OLIVEIRA, C.P., 6RODRIGUEZ, D.,
7
PARRENO, V., 8 AYRES, G.R., 9 SILVA, S.O.S.,
10
RICHTZENHAIN, L.J., 11BRANDÃO, P.E.
1. VPS - FMVZ / USP, Depar tment of Preventive
Veterinary Medicine and Animal Health, School of
Veterinary Medicine, USP, Av Prof Dr Orlando Marques
de Paiva, 87 CEP 05508 270 -Cidade Universitária SP2.
INTA, National Institute of Agricultural Technology,
Argentina., Instituto de Virología, CICV y AINTA,CC25(1712)Castelar, B. Aires, Argentina
1
4
Outbreaks of rotavirus in porcine are sporadic and it is
believed that porcine rotavirus diseases may be
associated with host immune and environmental.
However, porcine group A rotavirus (PGAR) is one of
the main causative agents of acute diarrhea in neonatal
and preweaning piglets. Therefore, this study aimed to
detect PGAR in piglet feces from São Paulo State by
ELISA assay and perform a molecular characterization
by an heminested RT-PCR and DNA sequencing.
Seventy-seven piglet feces samples from different herds
in São Paulo State with and without diarrhea were tested
by a polyclonal antibody ELISA specific for group A
rotavirus detection. Positive samples were next
submitted to an heminested RT-PCR targeted to the VP1
protein gene (228-bp), a conserved region of rotavirus
genome and the amplicons were submitted to DNA
sequencing and aligned with sequences retrieved from
the GenBank for the construction of a Neighbor-Joining
trees for nucleotides (MCL model). As results, 2.59%
(2/77) samples were positive by ELISA assay and the
partial VP1 gene of one strain of PGAR was successfully
sequenced. The result obtained by BLAST/nt showed
100% identity with a bovine Rotavirus A strain NCDV,
adapted to cell culture. In the phylogenetic tree, distance
genealogy showed the porcine strain obtained herein is
included in the same cluster of group A rotavirus of
different species and the strain diverged from bovine
vaccine strains cluster and other two group A rotavirus
clusters, one with monkey strains and other with human
strains. As a conclusion, this study showed that the
PGAR found herein and NCDV rotavirus strains are
homologous in relation to the VP1 gene, a data not yet
reported for PGAR studies, suggesting a possible
interspecies transmission. Therefore, these results
contribute to the molecular characterization of group A
rotavirus, and for the understanding of the rotavirus
molecular epidemiology.
Financial support: FAPESP nº 2008/56620-9 Palavraschaves: Characterization, pigletes, rotavirus, VP1 protein.
CHARACTERIZATION OF THE ARG1 STRAIN OF
EQUINE HERPESVIRUS 1 IN ADULT MICE USING A
NESTED-PCR TARGETING ORF64-REGION
ID: 00045-00001
Área: 03 - Virologia Animal
OLIVEIRA, C.P., 2CASTRO, A. M. M. G., 3MORI, E.,
GALOSI, C., 5Ayres, G.R., 6Santos, S.S., 7Barros, I.N.,
8
TORRES, C.A., 9SILVA, S. O. S., 10BRANDÃO, P.E.,
11
RICHTZENHAIN, L.J.
1. VPS - FMVZ / USP, Faculdade de Medicina Veterinaria
e Zootecnia / Universidade de Sao Paulo, Av. Prof. Dr.
Orlando Marques de Paiva, 87 CEP 05508 270 - Cidade
Universitária2. FCV/UNLP, Universidad Nacional de La
Plata, Facultad de Ciencias Veterinarias, Calle 60 y 118 S/
N (1900) La Plata-Casilla de Correo 296-Buenos AiresArgentina
1
4
Equine herpesvirus-1 (EHV-1) causes respiratory,
neurological and reproductive problems, leading to
economic losses to horse breeders. Arg1 is an Argentinean
strain of EHV-1 isolated from an outbreak of neurologic
and reproductive disorders in mares. Previous studies
demonstrated that Arg1 shows the ORF30 single point
mutation (neuropathogenic marker) for horses. The mouse
model has been used to reproduce the EHV-1 patogenicity
67
ABSTRACTS
and shows similar cell tropism, comparing to natural host.
The present study was performed in order to evaluate the
neuropathogenicity of the Arg1 strain of EHV-1 in the adult
mouse model, using a nested-PCR (nPCR) with primers
targeting ORF 64 region of viral genome, which is involved
in virus latency. Six C3H/HePAS mice (6 weeks old) were
used: five of them were infected with EHV-1 strain Arg1
(103 TCID50/25 μL, via intra nasal) and one was inoculated
with culture medium by the same route (negative control).
Mice were observed over four days, and then were
necropsied. Spleen, lung, thymus, liver and brain were
collected and tested to detect EHV-1 by nested-PCR
targeting ORF64 region (PCR F: 5‘ACGCCCCCTTCG
TTCCTC3‘, PCR R: 5‘ CGCTCCACCTCGGTCCTG3‘.
Nested F: 5‘ GGCGGAGTCTGCGTTGTG 3‘ Nested R:
5‘CCGGAGCCCGACGACGAG3‘). No clinical signs and
macroscopic lesions were observed in all inoculated mice.
However, virus DNA was detected by PCR in lung and
brain of all five inoculated mice, and in the thymus of two
mice. No viral DNA was detected in spleen and liver of all
inoculated mice. These results show that Arg1 strain of
EHV-1 replicates in the lung, thymus and brain tissues of
adult mice without apparent pathogenicity. Additional
histopathological studies must be conducted to evaluate
tissue damage. The ORF 64 nPCR is an efficient tool for
viral detection in experimentally infected adult mice,
suggesting its feasibility as a screening test in clinical
samples. Finalcial support: CAPES.
Palavras-chaves: Adult mice, equine herpesvirus, nestedPCR, ORF 64.
DETECTION AND MOLECULAR CHARACTERIZATION
OF NOROVIRUS ASSOCIATED TO ACUTE
GASTROENTERITIS
ID: 00046-00001
Área: 05 - Virologia Humana e Saúde Pública
BARLETTA, V.H, 2CARVALHO, I.P, 3BARLETTA, R.H,
TIBIRIÇÁ, S.H.C, 5FERREIRA , M.S.R, 6Xavier, M.P.T.P, 7
MIAGOSTOVICH, M.P, 8LEITE, J.P.G, 9ROSA E SILVA, M.L
1. UFJF, UNIVERSIDADE FEDERAL DE JUIZ DE FORA,
Rua José Lourenço Kelmer, s/n - Campus Universitário,São
Pedro.JUIZ DE FORA,MG2. FIOCRUZ-RJ, FUNDAÇÃO
OSWALDO CRUZ-RJ, Av. Brasil, 4365 - Manguinhos, Rio
de Janeiro, RJ, Brasil.3. UFRJ, UNIVERSIDADE
FEDERAL DO RIO DE JANEIRO, Centro de Ciências da
Saúde- Bloco I, Cidade Universitária -Ilha do Fundão, RJ
was to analyze the infections rate and distribution of
genogroups and genotypes of NoVs strains detected.
From January 2008 to December 2009, 224 diarrheic
stool samples were obtained from children up to 5 years,
attended with gastroenteritis in health services in Juiz
de Fora city, Minas Gerais state, Southeastern Brazil.
All stool samples were previously screened by a latex
agglutination test for detection of group A rotavirus. To
investigate NoV infection viral RNA was extracted using
the guanidine isothiocya¬nate-silica method and the
complementary DNA (cDNA) was obtained using a
random primer (pd(N)6™). CDNA was submitted to PCR
using degenerate primers for region B (MON
431,432,433,434) that detect GI and GII NoVs strains.
Genogroups GI and GII and genotypes were determined
using PCR based on region D (primers Cap A, B1 e B2,
Cap C, D1, D3) following sequencing and phylogenetic
analysis, respectively. NoVs were detected in 9,8% (22/
224) of the fecal samples. From those 4 (18,2%) NoV
strains were classified as GI and 18 (81,8%) as GII. The
genogroups of 12 of these GII NoVs strains were carried
out with primers of region D. Six positive NoVs strains
only could be classified as GII using primers of region B
(MON 431/433). Data from sequencing and phylogenetic
analysis characterized 12 NoV strains in GII/4 (58,3%)
and GII/6 (41,7%). Thus, these results confirms previous
data that have been demonstrated that GII as the most
prevalent genotypes associated to GE worldwide.
Financial Support: FAPEMIG, PROPESQ/UFJF.
Palavras-chaves: Epidemiology, Gastroenteritis,
Genotyping, Noroviruses, RT-PCR.
HLA ALLELES ASSOCIATIONS WITH AMINO ACIDS
PEPTIDES PRESENT IN THE ENVELOPE PROTEIN
OF THE DENGUE 3 VIRUS
ID: 00048-00001
Área: 05 - Virologia Humana e Saúde Pública
1
4
Gastroenteritis is one of commonest childhood disease,
constituting an important public health problem in
developing countries. Among viruses,Noroviruses(NoVs)
are recognized as the cause of outbreak and sporadic
cases of acute gastroenteritis (GE) in children and adults.
NoVs are classified in five different genogroups (G) which
GI, GII and GIV infect humans. The aim of this study
68
ALENCAR, L.X.E., 2NASCIMENTO, E.J.M, 3CORDEIRO, M.T., 4 Gil, L.H.V.G, 5 Braga-Neto, U.,
6
MONTENEGRO, S.M.L, 7MARQUES JÚNIOR, E.T.A.
1. CPqAM/FIOCRUZ, Departamento de Virologia e Terapia
Experimental, Centro de Pesquisas Aggeu Magalhães,
Av. Professor Moraes Rego, s/n Cidade UniversitáriaRecife-PE cep50670-4202. Texas A&M University,
Depar tment of Electrical Engineering, Texas A&M
University College Station, 407 College Main, College
Station, TX 77840-1274, United States3. University of Pitt,
Department of Infectious Diseases and Microbiology,
University of Pittsburgh, 3550 Terrace Street Pittsburgh,
PA 15213-2500, United States4. CPqAM/FIOCRUZ,
Departamento de Imunologia do Centro de Pesquisas
Aggeu Magalhães, Av. Professor Moraes Rego, s/n Cidade
Universitária-Recife-PE cep50670-420
1
ABSTRACTS
Dengue virus infection has emerged as one of the most
important arthropod-borne diseases. In some dengueinfected individual, the disease progresses to its severe,
life-threatening form, dengue hemorrhagic fever (DHF).
Host genetic factors may be relevant and predispose
some individuals to the severe dengue disease. Samples
collected from 61 individuals that developed dengue fever
(DF), dengue fever complicated (DFC) and DHF were
mapped by ELISPOT assay using peptides synthesized
based on the deducted sequence of the genome of a
Dengue 3 virus (DENV-3) isolated from Brazil. The
relationship between the peptides identified with those
predicted by bioinformatics associate the responses with
different HLA supertype of the patients. HLA was
genotyped using polymerase chain reaction–sequencespecific primers. We assessed statistical significance
by means of a one-sided Fisher’s test for an odds ratio
larger than 1. Currently, we have assayed 18 HLA-typed
patients and 10 peptides were considered reactive.
Among these amino acids peptides present in the
envelope protein of the dengue 3 virus, the majority were
able to bind to the HLA class I and only one for HLA
class II. The results indicate the presence of 8 promising
peptide-allele associations, with the class I allele HLAA34, -B8, -B15, -B39, -B45, -C5, -C8, -C16 and the class
II HLA-DR12. These findings allowed the characterization
of HLA class I and class II epitopes in the envelope
protein of the DENV-3 in human infection.
Financial Support: CPqAM/CNPq/NIH/CAPES
Palavras-chaves: Dengue virus, Elispot, Epitope, HLA
class I, HLA class II.
DEVELOPMENT AND VALIDATION OF HIGH
THROUGHPUT SCREENING ASSAY FOR
INHIBITORS OF YELLOW FEVER VIRUS
REPLICATION
ID: 00049-00001
Área: 01 - Imunobiológicos
difficult to use in high-throughput assays. The aim of
this work was to evaluate the antiviral properties of
several natural substrates from a Fiocruz-MG library
using a stable cell line expressing a bicistronic replicon
of yellow fever virus. The replicon named rYFVLucNeoIres, was previously constructed in our laboratory,
which express the firefly luciferase reporter and neomycin
phosphotransferase genes in a replication-dependent
manner. The replication inhibition of the replicon leads to
a decrease of Luciferase expression level. In this study
we analyzed about 1000 extracts from a large library
comprising 4000 different Brazilian natural substrates.
The high throughput assay was developed on 96-well
plate and the cell line BHK-21-rYFV-LucNeoIres were
seeded at densities at 2x104 cells per well. The antiviral
activity of the substrates was identified by the evaluation
of luciferase expression, after 48 hours of treatment with
20 μg/mL of each substrate. The possible cytotoxicity of
each substrate was also evaluated during the experiment.
The Luciferase values obtained in raw lights units for
each treatment were analyzed and compared with the
negative control. Thirty-two extracts showed an inhibition
of 50% or more of luciferase activity, demonstrating that
the method developed is sensitive, fast and very efficient
for antiviral drugs screening. None of 32 extracts proved
to be cytotoxic. In summary, the innovative technique
described in this work will greatly facilitate the screening
of antivirals drugs against flaviviruses.
Financial support: FACEPE, CNPq, Fiocruz. Palavraschaves: Antiviral, Cell Line, Replicon, Yellow Fever Virus.
ASSOCIATION OF KIR GENE CLUSTER POLYMORPHISMS IN PATIENTS WITH CHRONIC
HEPATITIS C VIRUS INFECTION
ID: 00052-00002
Área: 05 - Virologia Humana e Saúde Pública
Vasconcelos, JM, 2Tamegão-Lopes, BP, 3Móia, LJMP,
Amaral, ISA, 5Takeshita, LYC, 6Medeiros, ZL, 7Bandeira,
CL, 8Soares,MCP, 9Sena, L, 10Demachki, S, 11Santos,
EJM
1. ICB-LGHM/UFPA, Instituto de Ciências BiológicasLaboratório de Genética Humana e Médica, Cidade
Universitária Prof. José da Silveira Netto. Av. Augusto
Correa, 01. Guamá2. ICS-UFPA, Instituto de Ciências
da Saúde, Travessa Generalissimo Deodoro, s/n
Umarizal3. IEC-PA, Instituto Evandro ChagasHepatologia, Avenida Almirante Barroso, 492. Marco.
Belém-PA4. CCBS-UEPA, Centro de Ciências Biológicas
e da Saúde, Rua Perebebuí, 2623. Marco. Belém-PA
1
4
OLIVEIRA, A. G., 2ALMEIDA, S. R., 3DA SILVA, A. N.
M. R., 4BERTANI, G. R., 5GIL, L. H. V. G.
1. IAM, Instituto Aggeu Magalhães, Av. Prof. Moraes
Rego, s/n Cd. Universitária, Recife - PE - Brazil Cx Post
74722. UFPE, Universidade Federal de Pernambuco, Av.
Prof. Moraes Rego, 1235 Cd Universitária, Recife - PE,
Brazil. CEP 50670-901
1
The re-emergence of flaviviruses and its significance as
a public health problem has generated the interest for
the development of new vaccines approaches and
antiviral agents. Antiviral drugs are not available to the
population and about 200.000 cases of yellow fever are
estimated per year in the world. Traditional methods for
antiviral screening are laborious, time-consuming and
Hepatitis C virus (HCV) is a major health problem
worldwide, infecting an estimated 170 million people. The
current therapy uses pegylated interferon (PEG-IFN) plus
69
ABSTRACTS
Ribavirin (RBV), but just half of the treated patients can
eradicate the virus (sustained virological response, SVR).
The presence of Killer Immunoglobulin-like Receptor
(KIR) KIR2DL3 has been associated with both
spontaneous resolution of viremia following HCV infection
and satisfactory response to the treatment. In our study
we investigated 60 chronic hepatitis C patients, infected
with HCV genotypes 1 and 3, and compared to 345
healthy controls in order to investigate putative
association of HCV infection with KIR polymorphism.
All patients were positive for anti-HCV antibodies and
HCV RNA in serum. Individuals were genotyped for 14
KIR genes using PCR-SSP and acrylamide gel
electrophoresis. The frequency of KIR2DL2 gene was
higher in chronic infected patients than in controls (OR=
6.4; p=0.0009), while the frequency of the KIRD2DL3/
KIRD2DL3 genotype was higher in controls (OR: 0.17;
p=0.0023) than in patients. This result has been
previously reported as strongly related with spontaneous
viral clearance and with SVR. In fact, KIR2DL3 is a
weaker inhibitor than KIR2DL2, which could result in more
activating NK cells during the antivirus response, possibly
allowing viral resolution and a better prognostic to
treatment. In conclusion, presence of KIR2DL2 gene
predisposes to HCV chronic infection, while homozygous
KIR2DL3 have a better prognostic for spontaneous
healing or SVR.
Palavras-chaves: KIR, HCV, KIR2DL2, KIR2DL3,
HEPATITIS C VIRUS.
in Natural Killer cells as part of the innate immune
response, and the first line defense against viruses. In
the present study, KIR polymorphisms were investigated
for association with HTLV-1 infection in asymptomatic
carriers. Controls (345 individuals) and HTLV-1 carriers
(53 individuals) were genotyped for 14 KIR genes using
PCR-SSP and acrylamide gel electrophoresis. The
frequencies of KIR2DS1 (p=0.043), KIR2DS3 (p=0.026),
KIR2DS4 (p=0.0117) genes were statistically higher in
HTLV-1 infected patients than in controls. The profile
constituted by KIR2DS1+/KIR2DS3+ predispose strongly
to HTLV-1 infection (p=0.0048). The HLA-C molecule is
the ligand of both KIR2DS1 and KIR2DS4; however this
interaction is weak, suggesting that cells bearing these
receptors are hiporesponsive, explaining our results. To
the present date, there are no studies on association
between KIR genes and HTLV-1 infection.
Palavras-chaves: HTLV-1, KIR2DS1, KIR2DS3,
KIR2DS4.
GENETIC CHARACTERIZATION AND PHYLOGENETIC ANALYSIS OF PORCINE CIRCOVIRUS
TYPE 2 (PCV2) DETECTED IN SWINE DIARRHEAL
AND NON-DIARRHEIC FECES
ID: 00053-00001
Área: 03 - Virologia Animal
Oliveira, T.H.N., 2CASTRO, A.M.M.G., 3BALDIN, C.M.,
FAVERO, C.M., 5BRANDÃO, P.E., 6RICHTZENHAIN,
L.J.
1. FMVZ-USP, Faculdade de Medicina Veterinária e
Zootecnia da Universidade de São Paulo, Av. Prof. Dr.
Orlando Marques de Paiva, 87 CEP 05508 270 - Cidade
Universitária
1
4
ASSOCIATION OF KIR GENE CLUSTER POLYMORPHISMS WITH HTLV-1 INFECTION
ID: 00052-00001
Área: 05 - Virologia Humana e Saúde Pública
Tamegão-Lopes, BP, 2 Vasconcelos, JM, 3Gomes,
STM, 4Mendes, LAM, 5Ewerton, PD, 6Sena, L, 7Santos,
EJM, 8Lemos, JAR
1. UFPA, Instituto de Ciências Biológicas - Laboratório
de Genética Humana e Médica, Rua Augusto Corrêa,
01, Guamá. Belém, Pará, Brasil.2. Fundação HEMOPA,
Centro de Hemoterapia e Hematologia do Estado do Pará,
Travessa Padre Eutíquio, 2109, Batista Campos. Belém,
Pará, Brasil.
1
HTLV-1 is a retrovirus that can infect T-lymphocytes and
be transmitted by breastfeeding, sexual contact, blood
transfusion, syringes and needles sharing. This retrovirus
is distributed worldwide, exhibiting high geographic
heterogeneity, and is directly associated to diseases in
1-5% of the carriers, such as HTLV-1 Tropical Spastic
Paraparesis, Adult T-cell Leukemia/Lymphoma, uveitis,
dermatitis, strongiloidosis, and arthritis. The Killer
Immunoglobulin-like Receptor (KIR) genes are expressed
70
Porcine circovirus type 2 (PCV2) belong to the genus
Circovirus and is divided into subtypes PCV2-1 (1A-1C)
and PCV2-2 (2A-2E). This small non-enveloped virus is
responsible for the PCV2 associated diseases. The
occurrence of granulomatous enteritis, one of the PCV2
associated disease, is increasing and is characterized
by diarrhea and growth retardation that has a great impact
on swine industry. PCV2 single-stranded circular DNA
presents at least three functional open reading frames
(ORFs) encoding proteins for viral replication (ORF1 Rep gene), nucleocapsid (ORF2 - Cap gene) and
apoptosis and pathogenesis in vivo (ORF3). The aim of
this study was evaluate the molecular diversity of PCV2
detected in diarrheal and non-diarrheic feces. Seven
samples (6 diarrheal and 1 non-diarrheic feces) were
sequenced with four pairs of primers (P1/P2, P3/P4, P5/
P6 and P7/P8) by the dideoxynucleotide chain termination
method. The sequences obtained from the 7 PCV2
positive samples had 1767 nt in length (6 diarrheal and 1
ABSTRACTS
non-diarrheic feces) and were closely related to each
other showing 98.1-99.6 % and 95.4-99.6 % nucleotide
and amino acid homology, respectively; the lowest values
were showed between diarrheal and non-diarrheic
samples. ORF1 and ORF2 nucleotide identities of the 7
sequences ranged 97.7-100 % and 96.5-100 %,
respectively. As expected, ORF2 has more mutations
points. The 7 sequences alignment with 45 available in
GenBank showed nucleotide identity ranged 93.7-99.6
%. Phylogenetic analysis classified the six diarrhea
samples as PCV2-1B and the non-diarrheic sample as
PCV2-1A. Although only one non-diarrheic sample was
sequenced, it was classified in another subgroup,
suggesting that the PCV2 detected in diarrheal and nondiarrheic cases, although very similar, showed significant
nucleotide differences that permits classify them in
different subgroups.
Palavras-chaves: FECES, GENETIC CHARACTERIZATION, PCV2, PHYLOGENETIC ANALYSIS,
SWINE.
DETECTION OF TTV1 AND TTV2 IN PIGLETS WITH
AND WITHOUT DIARRHEA FROM PIG HERDS
LOCATED IN SÃO PAULO STATE.
primers used to 305 pb TTV1, 252 pb TTV2 fragment
amplification: TTV1F (5´CGG GTT CAG GAG GCT CAA
T 3´), TTV1R (5´GCC ATT CGG AAC TGC ACT TAC T
3´), TTV2F (5´TCA TGA CAG GGT TCA CCG GA 3´)
and TTV2R (5´CGT CTG CGC ACTT ACT TAT ATA CTC
TA 3´). The rate of TTV1 elimination was higher, 42%
(47/111) than TTV2, 35% (39/111) and co-infection rate
was 19.8 % (22/111). Nursery piglets eliminated more
frequently TTV2 while growing piglets eliminated more
TTV1. Diarrheic animals were more often infected by
both TTV species alone or co-infected. In conclusion
TTV1 and TTV2 are widespread in different pig herds
from São Paulo state and feces proved an important
route of viral shedding. TTV alone causing diarrhea in
piglets are not describe but it is possible that diarrheic
animals are more susceptible to TTV infection than non
diarrheic.
Palavras-chaves: Torque teno virus, piglets, viral
shedding, diarrhea
HUMAN CORONAVIRUS IN HOSPITALIZED PATIENT
SUSPECTED AS 2009 H1N1 INFLUENZA A
INFECTION.
ID: 00054-00001
Área: 05 - Virologia Humana e Saúde Pública
ID: 00053-00002
Área: 03 - Virologia Animal
CABEÇA, T.K, 2 CARRARO,E., 3 CAMARGO, C.N,
PUERARI,D, 5 GUATURA, S.B, 6 SOUZA, J.M.S,
7
GRANATO, C.F.H, 8BELLEI, N.C.J
1. UNIFESP, Universidade Federal de São Paulo, Rua
Pedro de Toledo, 781 15ºandar
1
Favero, C.M., Castro, A.M.M.G., Baldin, C.M.,
Taniwaki, S.A., 5 Miyashiro, S., 6 Brandão, P.E.,
7
Richtzenhain, L.J.
1. FMVZ-USP, Faculdade de Medicina Veterinária e
Zootecnia da Universidade de São Paulo, Av. Prof. Dr.
Orlando Marques de Paiva, 87 CEP 05508 270 - Cidade
Universitária2. IB-UNESP, Instituto de Biociências de
Botucatu, Distrito de Rubião Jr., s/nº 18618-970 Botucatu
- SP3. IB, Instituto Biológico, São Paulo, Avenida
Conselheiro Rodrigues Alves, 1.252 - CEP 04014-002.
1
2
3
4
Torque teno virus is a small non enveloped virus with
circular single strand DNA genome, classified within the
Anelloviridae family. So far two species of TTV were
described infecting pig herds, TTV1 and TTV2. Swine
TTV has been detected in serum, plasma, nasal swab,
rectal swab and feces, indicating fecal-oral transmission
as the most significant way of spreading. Although the
pathogenesis of TTV remains unclear a higher frequency
of TTV2 infection was associated with pigs showing
signs of enteric disease associated with PCV2. The aim
of this work was verify the rate of TTV1, TTV2 elimination
in feces samples from nursery and growing piglets with
and without diarrhea. One hundred and eleven feces
samples were collected from pig herds, from São Paulo
state, Brazil. DNA extraction was carried out by
phenol:chloroform:proteinase K protocol and the following
4
Human Coronavirus (HCoV) cause upper respiratory
illness and occasionally lower tract disease in
susceptible populations. Four HCoVs are currently known
to infect the respiratory tract: human coronavirus OC43
(HCoV-OC43) and 229E (HCoV-229E), SARS associated
coronavirus (SARS-CoV) and the recently identified
human coronavirus NL63 (HCoV-NL63) and HKU-1
(HCoV-HKU1). Since HCoV and other respiratory viral
pathogens cause very similar clinical respiratory
symptoms, differential diagnosis of pathogens is required
for adequate case management. Indeed, many different
viral infections could occur simultaneously. In this study,
we investigated HCoVs infection among 118 hospitalized
patients suspected as 2009 H1N1 Influenza A infection,
but not confirmed on follow-up, during August to October,
2009 at Hospital Sao Paulo. Tests were conducted using
a Pancoronavirus RT-PCR screening assay to detection
of all coronaviruses in clinical samples (nasal swab) and
a second step specific duplex RT-PCR assay to diagnosis
HCoV-OC43 and HCoV-229E. Pancoronavirus assay
detected 1/118 (0.8%) of HCoVs, diagnosed as HCoVOC43 by duplex RT-PCR on September. This patient was
71
ABSTRACTS
female, 2 years old, hospitalized for intestinal surgery
intervention and after some days a suspect viral infection
acquired inside the hospital ward occurred: the child had
fever (>38°C), cough and others symptoms of pulmonary
disease. The child was admitted to an intensive care
unit (UTI) and left the hospital in better situation after 1
month. No coinfection with Influenza B, Rinovirus,
Metapneumovirus and Adenovirus was detected with this
case. We conclude that HCoV could be a relevant
pathogen causing lower respiratory infection, can cause
severe disease among children and even during a
pandemic wave of Influenza virus H1N1 hospital
infections by other viruses can occur. Pancoronavirus
RT-PCR showed to be valuable tool in the investigation
for the presence of all coronaviruses.
Palavras-chaves: Human Coronavirus, Lower
Respiratory Infection, Nosocomial Infection,
Pancoronavirus
CHARACTERIZATION OF THE INTERACTION
BETWEEN YELLOW FEVER VIRUS NS5 WITH A
HUMAN GIPC1-PDZ PROTEIN.
are related to the RNA metabolism that interacts with
NS5. The purpose of this study is to characterize the
interaction between GIPC1 and NS5. Initially we
confirmed by plasmid linkage using two-hybrid assays
that GIPC1 interacts with NS5. Various NS5 deletion
mutants were constructed and co-transformed with gipc1
in yeast to determine the minimal domain of NS5 required
for this interaction. We will further characterize this
interaction using GST-pull down, CO-IP and by analyzing
viral replication in absence of GIPC1-PDZ using iRNA.
Financial Support: FAPESP/CNPq.
Palavras-chaves: YELLOW FEVER VIRUS, GIPC1PDZ, PROTEIN INTERACTION
GENETIC ANALYSIS OF Pepper yellow mosaic virus
AND Potato virus Y ISOLATES COLLECTED FROM
PEPPER.
ID: 00058-00001
Área: 06 - Virologia Vegetal
MOURA, M.F., 2MITUTI, T., 3MARUBAYASHI, J.M.,
GIORIA, R., 5kOBORI, R., 6PAVAN, M.A., 7KRAUSESAKATE, R.
1. UNESP/FCA - Botucatu, Universidade Estadual
Paulista Julio de Mesquita Filho - Faculdade de Ciências
Agronômicas, Rua José Barbosa de Barros, 17802.
Sakata LTDA., Sakata Seed Sudamerica, Rua Plínio
Salgado, 4320 - Bragança Paulista
1
4
ID: 00057-00001
Área: 04 - Virologia Básica
GAVIOLI, A.F., 2 VODOTTO, A., 3 MORAIS, A. T.
SILVEIRA DE, 4NOGUEIRA, M. L.
1. FAMERP, FACULDADE DE MEDICINA DE SÃO
JOSÉ DO RIO PRETO, AV. BRIGADEIRO FARIA LIMA,
5416, VILA SÃO PEDRO, 15090-000
1
Yellow Fever (YF) is a mosquito-borne hemorrhagic fever
caused by Yellow Fever Virus (YFV), prototype of the
genus Flavivirus. YF is characterized by severe hepatitis,
renal failure, hemorrhage, and rapid terminal events that
lead to shock and death. The mechanism of Flaviviruses
replication is not completely known but it includes
interactions of viral RNA with cellular and viral proteins.
The nonstructural protein 5 (NS5) is the largest and the
most conserved Flavivirus protein and comprises the
methyltransferase and the RNA-dependent RNA
polymerase (RNApol) domains. NS5 is critical for many
functions, including replication, capping of RNA and hostcell gene regulation. The protein human GPIPC1/synectin,
a single PDZ domain protein, was originally recognized
as ~90 aminoacid–long repeated sequences in the
synaptic protein, acts as a scaffolding protein to function
in multiple biological processes such as protein
trafficking, endocytosis, and receptor clustering.
Accumulating evidence further indicates that GIPC1
plays a role in regulating cell polarity and motility. We
previously report a Yeast 2-hybrid screening using YFV
NS5 as bait and a human cDNA library as a prey. In this
previous study we identified several human proteins that
72
Pepper yellow mosaic virus and Potato virus Y are
potyviruses found infecting pepper in Brazil. A primer
pair named PepNib (5’ GWTSGYYGMMTTGGATGATG
3’) and PepUTR (5’ AGTAGTACAGGAAAAGCC 3’) was
constructed to amplify the complete coat protein gene
from both virus and was used in this work. Nineteen
isolates of potyvirus collected from pepper in São Paulo
State during 2007-2008 and four provided by Sakata Seed
Sudamérica were selected for sequencing the coat protein
gene. Through the 23 potyvirus, 20 belong to PepYMV
and 3 to PVY, indicating that PepYMV was the prevalent
species of potyvirus found infecting sweet pepper. The
PVY was only found at the Lins region. The amino acid
identity between the PepYMV isolates was 93% to 100%
while for the PVY isolates 94% to 98% for the capsid
region. The CP amino acid identity between PepYMV
and PVY isolates was 73% to 79%. The DAG motif
implicated in the aphid transmission is replaced by DAA
on all the sequences of PepYMV analyzed. At least one
isolate of PepYMV is aphid transmitted, indicating that
this motif is not essential for this properties. The motif
VW/TMMDGD is present on all PepYMV sequences,
while for PVY, the amino acid tyrosine is replaced by
valine. These differences help to identify the best regions
to design specific primers for each virus and to better
ABSTRACTS
characterize the recent specie PepYMV. Financial
support: FAPESP/CAPES.
Palavras-chaves: Potyvirus, RT-PCR, Motifs.
PROPOSED CLASSIFICATION OF CapsicumISOLATES OF Pepper yellow mosaic virus IN
PATHOTYPES
ID: 00058-00002
Área: 06 - Virologia Vegetal
MOURA, M.F., 2MARUBAYASHI, J.M., 3MITUTI, T.,
GIORIA, R., 5KOBORI, R., 6PAVAN, M.A., 7KRAUSESAKATE, R.
1. UNESP/FCA - Botucatu, Universidade Estadual
Paulista Julio de Mesquita Filho - Faculdade de Ciências
Agronômicas, Rua José Barbosa de Barros, 17802.
Sakata LTDA., Sakata Seed Sudamerica LTDA., Rua
Plínio Salgado, 4320 - Bragança Paulista (SP)
1
4
Pepper yellow mosaic virus and Potato virus Y are
potyvirus that infect Capsicum spp. in Brazil. The main
method for controlling these pathogens is the genetic
resistance conferred by alleles located in the locus pvr2
and Pvr4. PVY isolates can be classified in pathotypes
based on the differential resistance response in a
differential series of Capsicum composed by Yolo Wonder
(pvr2), Yolo Y (pvr21), Florida VR2 (pvr22), Puerto Rico
Wonder (pvr23), Serrano Vera Cruz (pvr24) and Serrano
Criollo de Morelos 334 (Pvr4). Twenty-five PepYMV
isolates were inoculated in this series and the
symptomatology observed during 60 days. Viral infection
was confirmed by indirect ELISA with anti-potyvirus
antiserum. A wide range of reactions was observed,
showing that the classification proposed to PVY not
covered the biological diversity of the PepYMV isolates.
PepYMV isolates could be classified into pathotypes P
(1.2), P (1.2.3) and P (1.3) also observed for PVY isolates.
But nine isolates of PepYMV could not be classified in
the different pathotypes proposed for PVY. Here we
propose the pathotypes P (2), P (1.3.P4), P (1.2.3.P4),
P (2.3) and P (3) for the additional response for the
PepYMV isolates. Financial support: FAPESP/CAPES.
Palavras-chaves: Potyvirus, Indirect E.L.I.S.A.,
Resistance
1. CPqAM-FIOCRUZ, Centro de Pesquisas Aggeu
Magalhães, Av. Prof. Moraes Rego, s/n, CDU, RecifePE, Brasil. Caixa Postal: 74722. UFPE/Dept. Genética,
Universidade Federal de Pernambuco, Av. Prof. Moraes
Rego, 1235, CDU, Recife-PE, Brasil. CEP:50670-9013.
UFPE/LIKA, Universidade Federal de Pernambuco, Av.
Prof. Moraes Rego, 1235, CDU, Recife-PE, Brasil.
CEP:50670-901
The interferon system is the first line of defense against
viral infection in mammals, and the innate cellular antiviral
mechanisms mediated by type I interferon (IFN-I; IFN-á/
â) are potentially the most important pathways of the
host defense limiting viral replication. The aim of this
work was to establish a sensitive nonviral bioassay in
order to detect and quantify human IFN-I, based in a cell
line that carries a luciferase gene that is controlled by
the IFN-stimulated response element (ISRE) promoter.
Baby hamster kidney cells (BHK-21) were transfected
with pISRE-Luc-Hygro plasmid and at 48 hours posttransfection the cells were selected by hygromicin drug
(300μg/ml) for 10 days. After selection several individual
clones were isolated by limiting dilution and amplified in
the presence of hygromycin. Cloned cells were then
tested by the induction of ISRE promoter with human
IFN-á and the luciferase activity was measured.
Luciferase activity was strongly stimulated when the
ISRE-Luc reporter cell line was incubated with human
IFN-á, thus demonstrating that the reporter cell line was
responsive to human IFN-á. The clone with lowest
background and highest sensitivity to IFN-I were amplified
and stored for further use. In the future, this stable
reporter cell line will be used for IFN-I quantification,
and as a tool for elucidating the mechanisms of IFN-I
induction and signaling in response to flavivirus infection
and other viruses. Financial support: CNPq, FIOCRUZ.
Palavras-chaves: Dengue, Imunidade inata, Linhagem
celular
HUMAN PLATELET POLYMORPHISM (HPA) IN
PATIENTS COINFECTED HIV/HCV
ID: 00061-00001
Área: 05 - Virologia Humana e Saúde Pública
PICELLI, N., 2SOUZA, L.R., 3SILVA, G.F., 4SILVEIRA,
L.V.A, 5PARDINI, M.I.M.C., 6GROTTO, R.M.T.
1. FMB- Unesp, Faculdade de Medicina de Botucatu,
Universidade Estadual de São Paulo - Botucatu, Distrito
de Rubião Junior s/n - Botucatu2. UNIP - BAURU,
Universidade Paulista - UNIP - Bauru, Rua Luiz Levorato,
2-140 - Chácaras Bauruenses5. IB, Departamento de
Bioestatística -Instituto de Biociências - Unesp, Distrito
de Rubião Junior s/n - Botucatu
1
ESTABLISHMENT OF AN INTERFERON SPECIFIC
REPORTER CELL LINE TO INVESTIGATE VIRUSINDUCED INTERFERON SIGNALING IN VITRO.
ID: 00059-00001
Área: 01 - Imunobiológicos
Silva, M.M.C., 2 Oliveira, A.G., 3Bertani, G. R., 4Gil,
L.H.V.G.
1
73
ABSTRACTS
The coinfection HIV/HCV is frequent and, 40% of the
HIV infected patients are VHC co-infected. The viral
genomes variability leads several VHC genotypes and
HIV subtypes existence. Although, the HCV target cells
are hepatocytes, this virus was already associated with
platelets. However, the HCV entry receptor is the CD81,
which it isn´t express in platelets, suggesting the
existence of the other protein associated with the virus
in platelets. Platelets express polymorphic antigenic
determinants on their surface, named HPA. Some HPA
resides in transmembrane glycoprotein of integrin family,
which are adhesion proteins are already associated with
other virus entry in their target cells. Preview study
demonstrated an association between HPA -5b and HCV
infection in monoinfected. There aren´t evidence if in the
HIV/VHC coinfection this association is preserved. Then,
this study performed the HPA genotyping, HCV
genotyping and HIV subtyping to evaluate if there is
association among HPA profile and the HCV presence
in coinfected patients and, if this association are affected
by HCV genotype and HIV subtype. The HPA -1a/1b
genotype was more frequent.
Palavras-chaves: HPA, HCV, HIV
ASSOCIATION OF THE HEPATITIS C VIRUS (HCV)
VIRAL LOAD IN PLASMA AND PLATELETS OF THE
INFECTED PATIENTS.
ID: 00062-00001
Área: 05 - Virologia Humana e Saúde Pública
BARRETO, S.F.D, 2ARIEDE, J.R., 3GROTTO, R.M.T.,
4
SILVA, G.F., 5PARDINI, M.I.M.C.
1. FMB - UNESP, FACULDADE DE MEDICINA DE
BOTUCATU - UNESP / LABORATÓRIO DE BIOLOGIA
MOLECULAR - HEMOCENTRO, Distrito de Rubião
Junior s/n Cep: 18603-9702. FMB - UNESP, FACULDADE
DE MEDICINA DE BOTUCATU - UNESP /
DEPARTAMENTO DE CLÍNICA MÉDICA, Distrito de
Rubião Junior s/n Cep: 18603-970
1
The Hepatitis C Virus (HCV) viral load is a useful marker
in the monitoring of infection. The viral RNA quantification
in serum or plasma has been used to evaluate the therapy
response and to define rapid and slow responders. The
undetectable viral load in four weeks after the introduction
of antiviral therapy can be predictive of the sustained
virologic response (SVR). Spite of this, some patients
can be back to present detectable viral load in serum
characterizing therapeutic failure. Although the HCV
presents hepatic tropism, the platelets are carriers of
virus in infected patients but it is unknown if the quantity
of virus associated with platelets have any relation with
the plasma viral load. Then, the goal of this study was
evaluated the HCV viral load in plasma and platelets in
50 infected patients. The HCV viral load was performed
74
using qRT-PCR using in house methodology described
by Dexter et al. (2009). The results showed that when
the plasma viral load was undetectable the HCV RNA
quantification in platelets was undetectable too,
suggesting that the antiviral therapy suppress the HCV
in plasma and platelets in the same way. However, when
the plasma viral load was lower 10,000UI/mL the HCV
RNA was undetectable in platelets. The plasma viral load
was higher than in platelets, suggesting that the
therapeutic response can be compartmentalized.
Palavras-chaves: Hepatitis C Virus (VHC), Platelets,
qRT-PCR
EXPOSITION IN VITRO OF THE PLATELETS FROM
PERSON NON-INFECTED TO HEPATITIS C VIRUS
(HCV): EVALUATION OF THE HPA -1, -3 AND -5
POLYMORPHISMS.
ID: 00062-00002
Área: 05 - Virologia Humana e Saúde Pública
Padovani, J.L., 2BARRETO, S.F.D, 3CORVINO, S.M.,
SILVA, G.F., 5PARDINI, M.I.M.C, 6GROTTO, R.M.T.
1. FMB - UNESP, Faculdade de Medicina de Botucatu UNESP / Laboratório de Biologia Molecular - Hemocentro,
Distrito de Rubião Junior s/n Cep:2. FMB - UNESP,
Faculdade de Medicina de Botucatu - UNESP /
Departamento de Clínica Médica, Distrito de Rubião
Junior s/n Cep:
1
4
Hepatitis C is a chronic disease caused by a virus, the
HCV, which usually infects hepatic cells by interaction
with a cell surface receptor, a tetraspanin (CD81).
However, adhesion proteins already were associated with
the virus entry in hepatocytes and have been related
with the entry of the others virus in their host cell, too.
The HCV RNA already found in platelet but it doesn’t
express CD81, although this protein is present in
megakaryocytes. Platelets express polymorphic
antigenic determinants on their surface, named HPA.
Some HPA resides in transmembrane glycoprotein of
integrin family. Preview study demonstrated an
association between HPA -5b and HCV infection. The
goal of this study was to determine the presence of HCV
RNA in platelets collected from peripheral blood from 50
blood donors with RT-PCR negative for the virus. These
platelets were infected in vitro with HCV and incubated
at 37oC for 8-48 hours. RNA was then extracted and
used for RT-PCR and qRT-PCR to detect the presence
of the virus and evaluate quantitative differences in viral
load according with the HPA profile. All platelets were
positive for HCV RNA after incubation with the virus,
demonstrating that the virus interacts with the platelets
in vitro. There was deviation from Hardy–Weinberg
equilibrium in the HPA-1 system. The allele frequency of
ABSTRACTS
HPA-1b was found to be significantly lower.
Palavras-chaves: Hepatitis C Virus (HCV), Platelets,
HPA
A FULL-LENGTH cDNA CLONE OF A BRAZILIAN
ISOLATE OF BOVINE VIRAL DIARRHEA VIRUS
(BVDV)
ID: 00063-00001
Área: 03 - Virologia Animal
Arenhart, S., 2Gil, L.H.V.G., 3Flores, E.F, 4Weiblen, R.
1. UFSM, Universidade Federal de Santa Maria, Santa
Maria-RS2. CPqAM-FIOCRUZ, Centro de Pesquisas
Aggeu Magalhães, Recife-PE
1
Bovine viral diarrhea virus (BVDV) is the causative agent
of an economically important disease affecting cattle
worldwide. This virus presents wide antigenic variation
among isolates around the world. Immediate implications
of these variations are observed in the failure of vaccine
protection and diagnosis. For these reasons, the aim of
this study was to construct an infectious clone of the
BVDV Brazilian pair isolate - IBSP-4. A full-length cDNA
clones of the BVDV Brazilian isolate IBSP-4 cytophatic
and non-cytophatic was assembled in three fragments
into a low-copy plasmid vector pBSCHDR using the yeast
homologous recombination system. To rescue infectious
virus RNA was transcribed in vitro from three cytophatics
and three non-cytophatics clones and transfected in
MDBK cells. The supernatant of monolayers were serially
passaged in MDBK cells. Virus recovered from the
infectious clones was characterized biologically by plaque
assay, replication kinetics and immunofluorescence
assay. The clones exhibited similar characteristics to
those of the parental IBSP-4, including replication
kinetics, plate morphology and reactivity to monoclonal
antibodies against BVDV. These preliminary results
showed that these infectious clones can be used for
further applications as a powerful tool to study BVDV.
Likewise, infectious clone technology provides an
opportunity to study host-virus interactions and
development of safe and effective live vaccines
possessing defined attenuation mutations to control the
virus infection.
Financial support: CNPq/UFSM
Palavras-chaves: Bovine Viral Diarrhea virus, Infectious
clone, Molecular Virology
Fernandes, M.J.B., 2Simoni, I.C., 3Harakava, R., 4Rivas,
E. B., 5Arns, C.W.
1. IB, Instituto Biológico, Av. Conselheiro Rodrigues Alves
1252 Caixa Postal 128982. UNICAMP, Instituto de
Biologia, Caixa Postal 6109
1
Infectious bursal disease virus (IBDV) causes a disease
among young chickens of great economic importance
to the poultry industry worldwide both by the mortality
as by immunosuppression. IBDV is classified according
to the antigenicity and/or pathogenicity into classical
virulent (cv), very virulent (vv), and antigenic variant
strains. This classification is based mainly on mutations
in the VP2 gene hypervariable region of the genomic
segment A. However, studies with VP1 gene (RNAdependent RNA-polymerase) in the genomic segment B
have demonstrated the role of this gene in the
pathogenicity too. Its phylogeny suggests a possible
reassortment event originating the vvVP1. The aim of
this study was to compare the nucleotide sequences
and to perform phylogenetic analysis of VP1 gene from
Brazilian IBDV samples. VP1 gene from seventeen
samples was amplified by RT-PCR resulting in 594-bp
fragments. Previously, three of these samples were
characterized as cv strains and the others as vv strains
through VP2 gene sequence and phylogenetic analysis.
Comparisons among our samples and published strains
in relation to deduced amino acid (aa) from VP1 showed
that three cvVP2 samples were also classified as cvVP1
strains and thirteen vvVP2 samples presented all four
common aa substitutions of vvVP1 strains. In the
phylogenetic tree, all these sixteen samples clustered
together with the respective cv or vvIBDV strains. Only
one sample classified as vvVP2 presented the VP1
sequence and phylogeny most closely related to the cv
strains. This sample carrying vvVP2 but cvVP1 could
be a descendant of IBDV ancestors before the
reassortment of genome B or simply is the result of a
genetic exchange between segments of the different
strains or same with a live attenuated vaccine. Anyway,
this is a first report of natural genetic reassortment of
IBDV in Brazil.
Palavras-chaves: gene VP1, genetic reassortment,
infectious bursal disease virus, phylogeny, sequencing
IN VITRO ANTIVIRAL EVALUATION OF BRAZILIAN
PLANTS EXTRACTS AGAINST AVIAN METAPNEUMOVIRUS
NATURAL BRAZILIAN REASSORTANT OF INFECTIOUS BURSAL DISEASE VIRUS
ID: 00065-00001
Área: 04 - Virologia Básica
ID: 00064-00001
Área: 03 - Virologia Animal
1
Barros, A.V., 2Padilla, M.A., 3Conceição, A.O., 4Simoni,
I.C., 5Fernandes, M.J.B., 6Arns, C.W.
1. IB, Instituto Biológico de São Paulo, Av. Conselheiro
75
ABSTRACTS
Rodrigues Alves 12522. UNICAMP - IB, Universidade
Estadual de Campinas - Instituto de Biologia, Rua
Monteiro Lobato, 2553. FCM, Faculdade de Ciências
Médicas, Programa Pós Graduação, UNICAMP,
Campinas/SP, Rua: Tessália Vieira de Camargo, 1264.
UESC, Universidade Estadual de Santa Cruz, Ilhéus/
Ba, Rod. Ilhéus-Itabuna, Km 16
Avian pneumovirus (aMPV), also called turkey
rhinotracheitis virus (TRTV), is a RNA virus that causes
an upper respiratory tract infection in turkeys and
chickens of all ages and is present in a wide range of
countries. The search for antiviral drugs more effective
and affordable is one of strategies to control the viral
infections. Plants and their derivatives have potential
use in treatment against viruses. The objective of the
present study was to evaluate the citotoxicity and antiviral
activity in vitro of leaves aqueous extracts of
Banisteriopsis variabilis B. Gates, Bumelia sartorum var.
latifolia Miq., Byrsonima intermedia A. Juss., Campomanesia xanthocarpa O. Berg., Coffea arabica L.,
Gochnatia polymorpha (Less.) Cabrera, Guettarda
angelica Mart., Lithrea molleiodes (Vell.) Engl., Xylopia
aromatica (Lam.) Mart. and Uncaria tomentosa var. dioica
Bremek. against aMPV. The citotoxicity evaluation of
each extract was determined by cellular morphology
alterations in Vero cells determining the maximum noncitotoxic concentration (MNCC). These concentrations
were used in antiviral assay. The antiviral activity was
determined by a titration technique that depends on the
ability of plant extract (MNCC) to inhibit the produced
cytopathic effect 5 days after infection. The titles were
calculated by Reed & Muench method. The antiviral
activity was expressed as viral inhibition index (VII) and
considered positive when the VII was e” 1.5. The results
showed that the MNCC of extracts ranged between 500
to 125 &mumg/mL. The B. variabilis and B. sartorum
presented antiviral activity against aMPV with VII of 2.87
and 2.63, respectively. Byrsonima intermedia, L.
molleoides and G. polymorpha also obtained activity with
VII of 1.56. Previous antiviral studies with these extracts
showed similar results against animal herpesviruses
indicating that they can be promising as antiviral
composts source by presenting antiviral activity against
RNA and DNA viruses.FAPESP.
Palavras-chaves: antiviral, aMPV, plants
VIRUS-INDUCED GENE SILENCING OF THE GENES
TCTP, SNF1 AND SUBÁ ANALYSIS BY
QUANTITATIVE RT-PCR AND EFFECT ON INFECTION
OF N. BENTHAMIANA BY PEPYMV.
1
4
1. UFV, Universidade Federal de Viçosa, Av P. H. Rolfs,
s/n. Viçosa, MG 36570-000
The genomes of most plant viruses code for 4-10 proteins
which are required to complete the infection cycle,
including viral genome replication, cell-to-cell movement
and systemic spread. For a successful infection, these
viral proteins must interact with host factors, modulating
metabolic pathways and coordinating a complex network
of biochemical interactions in the pathogen¡¯s favor. A
subtractive library constructed from susceptible tomato
plants infected by the plant potyvirus Pepper yellow
mosaic virus (PepYMV) identified several genes which
are putatively involved in the viral infection process.
Among these genes are those that code for the
translationally controlled tumor protein (TCTP), sucrose
non-fermenting-1 (SNF1) and subunit alpha of the 26S
proteasome (Subá). However, the exact role of these
genes in the context of viral infection remains
undetermined. To assess whether these genes are directly
related to the viral infection process, functional analysis
by virus-induced gene silencing (VIGS) using a Tobacco
rattle virus (TRV)-based vector was carried out. The
phenotype of healthy (not virus infected) plants which
were silenced for each of the three genes consisted of a
slight reduction in size. Differences in symptom severity
were observed in silenced plants inoculated and non
inoculated with PepYMV. Confirmation of the silenced
state was performed by qRT-PCR. For this, five candidate
genes were tested as normalizers, and the gene encoding
Gyrase A was chosen. Silencing in plants infiltrated with
TRV-SNF1 was very effective and these plants showed
a reduction in viral load 72 hours after inoculation. These
results suggest that the SNF1 gene plays a direct role in
the viral infection, favoring its establishment. Further
studies should be conducted to confirm this hypothesis
and improve our understanding of the nature and
mechanisms of interaction between SNF1 and PepYMV.
Silencing and viral load analyses are ongoing for the
genes TCTP and Subá. Financial support: FAPEMIG,
CNPq Palavras-chaves: PepYMV, Potyvirus, SNF1,
Subalpha, TCTP
MONITORING THE DENGUE VIRUS CIRCULATION IN
SETE LAGOAS CITY, MINAS GERAIS, BRAZIL
ID: 00069-00001
Área: 05 - Virologia Humana e Saúde Pública
1
ID: 00066-00001
Área: 06 - Virologia Vegetal
76
Bruckner, F. P., 2 Cascardo, R. S., 3Zerbini, F. M.,
Alfenas-Zerbini, P.
4
Franco, S.Q, 2 Andrade, R.P., 3 Pessanha, J.E.M.,
Caldas, S., 5Cecílio, A.B.
1. FUNED, Fundação Ezequiel Dias, rua Conde Pereira
Carneiro, 80, Gameleira, Belo Horizonte2. PUC Minas,
ABSTRACTS
Pontifícia Universidade Católica de Minas Gerais, Av.
Dom José Gaspar, 500, Coração Eucarístico, Belo
Horizonte3. UFMG, Universidade Federal de Minas
Gerais, Av. Prof. Alfredo Balena, 190, Santa Efigênia,
Belo Horizonte
Dengue virus (DENV) is a widely prevalent arbovirus in
tropical and subtropical regions. The World Health
Organization estimates that over 2.5 billion people are
currently at risk from Dengue viruses infection globally.
The disease is transmitted to humans commonly by
Aedes aegypti mosquitoes. The DENV genome is a single
stranded RNA molecule of positive polarity and about
11 kb in length that encode a single open reading frame.
There are four distinct serotypes, DENV-1, DENV-2,
DENV-3, and DENV-4, and among them specific
genotypes. The objective of this study was monitoring
the circulation of DENV in the population using larvae
collected in nature to confirm the serotypes circulating
in the area of study. The serum sample from patients
with dengue fever or dengue shock syndrome living in
Sete Lagoas city, Minas Gerais State, Brazil and also
larvae collected in the same region were inoculated in
C6/36 cells for viral isolation. The supernatants of
infected cells showing typical cytopathic effect were used
for viral RNA extraction, and the product was used as
template in a reverse transcription – PCR reaction. The
detection and serotyping of the virus were made by Real
Time PCR using Power SYBR® Green PCR Master Mix.
The results showed the circulation of sorotypes DENV-2
and DENV-3 among humans in the area of study which
were confirmed identifying the same serotypes circulating
em larvae. Material from larvae is easier to obtain and
maintain being a good source of DENV serotypes
circulating in a region of study. The next step will be the
determination of nucleotide sequences in the CprM region
and construction of phylogenetic trees to study the
genotypes of isolated viruses.
Financial support: FIP/PUC Minas, FAPEMIG,
FUNED,INCT em Dengue
Palavras-chaves: Dengue virus, larvas, sorotipo
EVALUATION OF THE INHIBITORY ACTIVITY OF
CRUDE AQUEOUS EXTRACT OF Byrsonima
intermedia AGAINST SUID AND EQUID
HERPESVIRUS
Instituto Biológico, Av. Cons. Rodrigues Alves, 1252, São
Paulo
The alphaherpesviruses are able to establish neuronal
latency with reactivation remains. The emerging
resistance to the available drugs against herpesviruses
led the need for new therapeutic agents against these
diseases. Brazil represents one of the world’s wealthiest
sources of plant material with pharmacological activity
including the Cerrado region that is the second greater
biome of Brazil. Byrsonima intermedia A. Juss, is very
common in Cerrado and the leaves already showed
several properties against some diseases. This study
aimed to evaluate the inhibition of suid (SuHV-1) and
equid (EHV-1) herpesvirus in cell lines (MDBK and VERO,
respectively) with different concentrations of crude
aqueous extract of Byrsonima intermedia. The
cytotoxicity of the extract was evaluated through cellular
morphology alterations by microscopic examination
determining the maximum non-toxic concentrations
(MNTC). For viral inhibition assay the extract in different
concentrations and the viral logarithmic dilutions were
inoculated at the same time in monolayers, after being
together for 1 hour in CO2 incubator at 37ºC. Some
qualitative tests standardized with different solvents were
done to identify the presence of some chemical
compounds. Inhibitory activities were calculated as the
difference of virus titre between treated and untreated
control cultures. The concentration of the extract was
considered inhibitor when the viral inhibition index (IIV)
was e” 1.5. The qualitative tests revealed the presence
of phenolic compounds, steroids and saponins. The
cytotoxicity assay showed the same MNTC of 250μg/
ml in both cell lines. In the viral inhibition, the extract
tested proved to be inhibitor until the concentration 7,8μg/
ml against EHV-1 with IIV= 6,12 in MNTC, and up to
concentration 3,9μg/ml against SuHV-1 with IIV= 6,15
in MNTC. These results showed the importance of further
studies to determine the actives compounds responsible
for the activity.
Palavras-chaves: Antiviral, Byrsonima intermedia,
Herpesvirus equino, Herpesvírus suíno
DIVERSITY OF ENDOGENOUS RETROVIRUS IN
NEOTROPICAL FELIDS
ID: 00071-00001
Área: 03 - Virologia Animal
ID: 00070-00002
Área: 04 - Virologia Básica
MATA, H., 2GONGORA, J., 3EIZIRIK, E., 4OLIVEIRA,
C.S., 5RAVAZZOLO, A. P.
1. UFRGS, UNIVERSIDADE FEDERAL DO RIO
GRANDE DO SUL, AV. Bento Gonçalves, 90902. USYD,
THE UNIVERSITY OF SYDNEY, Rm 546, RMC Gunn
Building (B19). NSW, 2006, Sydney, Australia3. PUCRS,
PONTIFÍCIA UNIVERSIDADE CATÓLICA DO RIO
1
PADILLA, M. A., HOE, V. M. H., ROSSI, M. H.,
BARROS, A. V., 5SIMONI, I. C., 6FERNANDES, M. J.
B., 7ARNS, C. W.
1. UNICAMP, Universidade Estadual de Campinas, Rua
Monteiro Lobato, 225, Campinas2. Instituto Biológico,
1
4
2
3
77
ABSTRACTS
GRANDE DO SUL, Av. Ipiranga, 6681, prédio 12
Endogenous retroviruses (ERVs) are inherited copies or
remnants of exogenous retroviruses derived from past
infections of germ cells and subsequent integration into
the host genome and passed on to subsequent
generations. ERVs are thought to be ubiquitous in the
genomes of most, if not all ver tebrate species.
Knowledge of endogenous retroviruses in felids is
practically restricted to the domestic cat, and their
distribution among wild species is unclear. Here we
analyse the distribution, potential to function and
phylogenetic relationships of ERVs in four wild felid
species (Leopardus tigrinus, L. pardalis, Puma
yagouarondi and Panthera onca) from Brazil. The ERV
reverse transcriptase (pro-pol) gene fragment (0.7-1 kb)
was amplified using degenerate primers, cloned and
sequenced. Preliminary analyses of 100 clone inserts
identified 17 ERV pro-pol DNA sequences which were
translated into putative amino acid sequences showing
that feline ERVs possess stop codons and deleterious
mutations. Thus, these feline ERVs are generally, if not
universally, defective as has been observed in many
other hosts. Phylogenetic analyses of these novel
sequences along with representative ERV sequences
from seven retroviruses show that the former clustered
into three minor clades within Gammaretrovirus. Further
analyses of the evolutionary relationships and evidence
for ERV functionality within wild felines are underway.
Financial support: CNPq
Palavras-chaves: BRAZIL, ERV, FELIDS, NEOTROPICAL, RETROVIRUSES
member of the family Flaviviridae and is classified in 04
serotypes (DENV-1-4). There is no vaccine available
against Dengue virus and vector control strategies that
seek to minimize human-mosquito contact, has not being
effective. In many cases of infection, the differential
diagnosis is important for the therapy of the patient. This
study aimed to develop the real-time PCR to increase
sensitivity of detection and typing of Dengue virus.
Detection and typing of Dengue virus by real-time PCR,
“Sybr Green” system. Samples of reference of the DENV1 to 4 were multiplied in C6/36 cells. To optimized the
real-time PCR technique, we used primers specific to
the region NS5 already tested in conventional PCR. The
assay were optimized testing different concentrations
of primers, cDNA and varying annealing temperature.
The melting temperature and the amplification curve
obtained for each serotype were efficient for the detection
and typing of the Dengue virus. Considering the time of
viral RNA extraction and cDNA preparation, the result of
real-time PCR can be obtained in a single day (about 6
hours). The system used in this study was rapid and
effective for the detection and typing of reference
samples, and the technique should be tested with clinical
samples.
Financial support: FAPEMIG,FUNED,INCT em
DENGUE.
Palavras-chaves: Aedes mosquitoes, Dengue virus,
Sybr Green, Real-time PCR
EVALUATION OF CRUDE AND FRACTIONATED
EXTRACTS OF MEDICINAL PLANT AS ANTIVIRAL
AGAINST ROTAVIRUS
DETECTION AND TYPING OF DENGUE VIRUS USING
“SYBR GREEN” SYSTEM
ID: 00073-00001
Área: 05 - Virologia Humana e Saúde Pública
ID: 00072-00001
Área: 05 - Virologia Humana e Saúde Pública
ATAIDE, A.C.Z, 2OLIVEIRA, P.C, 3CALDAS,S, 4SILVA,
F.O, 5Cecílio, A.B.
1. FUNED, Fundação Ezequiel Dias, Rua Conde Pereira
Carneiro n.80 - Gameleira- Belo Horizonte- Minas Gerais2.
PUC Minas, Pontifícia Universidade Católica de Minas
Gerais, Av Dom José Gaspar n.500 -Coração
Eucarístico- Belo Horizonte- Minas Gerais
Reis, R.R, 2Caldas, S, 3Cecílio, A.B
1. PUC Minas, Pontíficia Universidade Católica de Minas
Gerais, Dom José Gaspar, 500 Coração Eucaristíco Belo Horizonte - MG2. FUNED, Fundação Ezequiel Dias,
Conde Pereira Carneiro, 80 - Gameleira Belo Horizonte MG
1
Each year, 100 million people are infected with Dengue
virus. Dengue is an alarming public health problem in
tropical and subtropical regions of the world. The disease
has a spectrum ranging from asymptomatic infection to
hemorrhagic fever and shock, and in some cases could
progress to death. Dengue is an arbovirus transmitted
by the bite of the Aedes mosquitoes. In Brazil, dengue
epidemics are related to increased number of vectors,
which occurs during the rainy season. The virus is a
78
1
Rotaviruses belong to the family Reoviridae and are
identified in 7 groups (A-G). The group A predominates
in nature and causes diarrhea in humans. Rotavirus is a
major causative agent of diarrhea and mortality in children
under 5 years with an annual incidence of 100 million
cases. Transmission occurs through fecal-oral, human
to human or by ingestion of food/water contaminated.
The virus infects the enterocytes of the small intestine
and spreads to the entire gastrointestinal tract. The
conventional treatment of fluid replacement is palliative.
However many herbal medicines may enhance
ABSTRACTS
processes of phagocytosis and microbicidal activity. A
search for antiviral drugs effective and affordable is one
of the strategies to control this type of diarrhea, and
plants and natural compounds have potential use in the
therapy against rotavirus. Study of activity of crude and
fractionated extracts of aroeira “in vitro” as antiviral. The
reference sample SA-11 were multiplied in MA-104 cells.
For the antiviral assay, virus was used in serial dilutions
and were pre-incubated with crude and fractionated
extracts, followed by inoculation in a monolayer of cells
with 80% of confluence. For assessing the percentage
of inhibition (PI), the inoculated material was collected
and titrated for the detection of virus titers after the
antiviral assay. The crude extract showed antiviral activity
of a 100% against rotavirus. The crude extract were
fractionated in 05 fractions, and four of them showed
partial inhibition of rotavirus. The PI ranged from 21% to
75% and one fraction had an opposite effect of increasing
the virus titer. Among the crude extract and fractions
tested, the crude extract has higher potential antiviral
action against rotavirus as it showed a 100% of PI, and
the antiviral action decreased as the crude extract is
fractionated.
Financial Support: FAPEMIG, FUNED.
Palavras-chaves: Rotaviruses, Antiviral, Medicinal plant
vectors. Here we report the construction of subgenomic
replicons, derived from a full-length cDNA of the 17D
strain of yellow fever virus, encoding the domain III of
dengue virus envelope protein. For construction of a
replicon, an in frame deletion of nearly the entire structural
prM and envelope protein was made, which retained the
first six residues of the prM protein for correct clivage of
the capside protein. The deleted structural protein coding
sequence region was used for cloning the domain III of
DENV envelope protein, using two different approaches:
first, for tetravalent construct, the DIII of all four DENV
serotypes were connected sequentially to construct the
tandem domain. Second, for monovalent constructs, the
domain III of each dengue virus was cloned individually,
being developed four constructs (DENV 1-4). All of them
were generated by standard yeast recombinant
techniques using appropriately designed sets of primers
with overlapping sequences. Autonomous replication of
the replicons were confirmed by indirect immunofluorescence assay of BHK-21 transfected cells. The
replicons described in this work can be used to be transcomplemented into virus like particles and should have
broad applications for vaccine development.
Financial support: FIOCRUZ-PDTIS,CNPq.
Palavras-chaves: dengue, domínio III, envelope,
replicon, vacina.
CONSTRUCTION AND CHARACTERIZATION OF
SUBGENOMIC YELLOW FEVER REPLICONS
EXPRESSING THE DOMAIN III OF DENGUE VIRUS
ENVELOPE PROTEIN
CHARACTERIZATION OF FIBROPAPILLOMAASSOCIATED TURTLE HERPESVIRUS IN GREEN
TURTLES FROM BRAZIL
ID: 00074-00001
Área: 01 - Imunobiológicos
ID: 00075-00001
Área: 03 - Virologia Animal
da Silva, A.N.M.R, 2Santos, J.J.S., 3Almeida, S.R.,
Marques Junior, E.T.A., 5Gil, L.H.V.G.
1. CPqAM-FIOCRUZ, Centro de Pesquisas Aggeu
Magalhães-FIOCRUZ, Campus da UFPE Av. Moraes
Rego s/n, CEP:50670-4202. CVR, University of
Pittsburgh, Center for Vaccine Research, Pennsylvania,
United States of America
Rodenbusch, C.R., 2 Baptistotte, C., 3Melo, M.T.D.,
Pires, T.T., 5Werneck, M.R., 6Torezani, E., 7Canal, C.W.
1. UFRGS, Universidade Federal do Rio Grande do Sul,
Av. Bento Gonçalves, 9090 Porto Alegre - RS2. TAMAR
- ICMBio, Centro TAMAR - ICMBio, Av. Paulino Muller
1111, Vitória - ES3. TAMAR, Fundação Pró-Tamar, Rua
Antônio Athanásio, 273 Ubatuba - SP
The Flavivirus genus of enveloped positive-sense RNA
viruses constitutes over 70 members and almost 40 are
capable of causing human disease. Dengue virus (DENV)
is an important member of the genus that has a
significant impact on public health throughout the tropics.
Flavivirus structural proteins are dispensable for RNA
genome replication. These proteins can be completely
deleted and such RNAs, termed replicons, posses the
complement of genetic elements necessary for
autonomous amplification of their genomes in susceptible
cells. The amplification of replicon RNA in the cytoplasm
of cells makes them excellent vectors for expression of
heterologous genes, being the basis for effective vaccine
The fibropapillomatosis is an emerging disease with high
prevalence in turtles and characterized by multiple
papillomas, fibromas and fibropapillomas in the skin or
viscera. This disease is called “green turtle fibropapillomatosis” (GTFP) because it was first recorded in
green turtles. In the Brazil, the first record of GTFP was
in 1986 in the state of Espírito Santo (ES), and during
the period of 2000-2004, 14.96% of the 4471 green turtles
examined had tumors. The etiologic agent of GTFP is
still uncertain, but the patterns of disease spread during
outbreaks among captive green turtles have shown a
pattern of infectious etiology. A herpesvirus has been
isolated from fibropapillomas and is present in 95% of
1
4
1
4
79
ABSTRACTS
natural infections and in 100% of experimentally induced
tumors. The aim of this work was to detect the
fibropapilloma-associated turtle herpesvirus in marine
turtles from Brazil by PCR, to characterize the virus by
DNA sequencing and characterize tumors according to
their location, appearance and pigmentation. A total of
122 tumors from green turtles from Bahia (BA), Ceará
(CE) and ES states were analyzed; the green turtle was
the only species of sea turtle captured; all turtles were
juveniles and sexually immature. On the basis of the
number and size of tumors, each animal was assigned
a tumors severity score: mild (36.9%); moderate (31.2%)
and heavy (32%). As for location 82% were in the anterior
part of the animal, 14.8% on the back and 3.2% in the
carapace or plastron. The tumors were collected from
healthy (70.5%), 9.8% from weakened and 19.7% from
dead turtles. As for the appearance of the tumor, 73.8%
were papillary and 40.1% were pigmented. When analyzed
by PCR, 69.7% (85/122) were positive, with 21/38, 44/
60 and 20/24, respectively, for BA, CE and ES. Three
samples of ES were sequenced and aligned with the
five virus variants described in the literature. The three
sequences are distinct from each other and more closely
related to the variant HA.
Palavras-chaves: herpesvirus, green turtle, fibropapillomatosis.
CORRELATION ANALYSIS OF SEASONALITY OF
ADENOVIRUS GENE DETECTION AND WATER
QUALITY PARAMETERS BASED ON A YEARLY
MONITORING
ID: 00078-00002
Área: 02 - Virologia Ambiental
SILVA, H.D., 2 SANTOS, S.F.O., 3 GARCÍA-ZAPATA,
M.T.A., 4ANUNCIAÇÃO, C.E.
1. UFG, Universidade Federal de Goiás, Universidade
Federal de Goiás Campus, Samambaia, Caixa Postal
131, Goiânia, GO.
1
Enteric viruses are generally disseminated in the
environment via faecal-oral transmission. Among them,
adenoviruses (AdVs) have been attracting great interest
because of their high prevalence in water environments
and high stability in the environment. Adenoviral infections
are among the leading causes of a variety of human
diseases, especially respiratory illnesses, acute
conjunctivitis, and gastroenteritis. In Brazil, there are few
studies on environmental virology, and in the state of
Goiás in the midwest region of Brazil, they are
inexpressive. The goal of this study was to detect and
monitor the presence of adenovirus in rivers and lakes
used for recreation and as a source for the public water
supply in Goiânia-city, Brazil, and to carry out a correlation
80
analysis of the seasonality of adenovirus gene detection
and water quality parameters based on yearly monitoring.
We collected 54 water samples from two lakes and two
rivers. The samples were concentrated using a positivelycharged membrane and DNA was extracted by a phenolchloroform-isoamyl alcohol method, followed by PCR and
sequencing of the positive amplicons. Adenovirus DNA
was detected in 44.4% (24 of 54) of samples collected.
Physicochemical and bacteriological tests were carried
out according to water quality monitoring standard
procedures. Significant differences over the study period
were observed for the presence of AdVs in water and
the values for nitrites, phosphates and fixed and total
residues. Significant differences were not observed in
the bacteriological tests. The occurrence of adenovirus
in the state of Goiás showed a seasonal trend. This is
the first study that to detect and monitor adenovirus in
water sources in Goiânia-city. The present results may
be useful to generate an eco-epidemiological profile of
adenoviruses or even identify the transmission routes
of some neglected diseases, highlighting the need to
define a virus marker.
Palavras-chaves: adenovirus, bacteriological test, PCR,
physical-chemical tests, water.
STUDIES ON THE BEHAVIOR OF THE CONTAGIOUS
ECTHYMA VIRUS IN CAPRINE CORNEAL CELLULAR
CULTIVATION
ID: 00079-00001
Área: 03 - Virologia Animal
SANTANA, R.L., 2 SILVA Jr, L.C., 3 KASSAR, T.C.,
GOMES, A.L.V., 5CAMPINHO, D.S.P., 6NASCIMENTO,
M.C.O., 7NASCIMENTO, S.A., 8GIL, L.H.G., 9 MAIA,
R.C.C., 10CASTRO, R.S.
1. UFRPE, Universidade Federal Rural de Pernambuco,
Rua Dom Manoel de Medeiros s/n - Dois Irmãos - Recife
- PE2. CPqAM - Fiocruz, Centro de Pesquisas Aggeu
Magalhães - Fiocruz, Rua Professor Moraes Rêgo, s/n Cidade Universitária - Recife - PE
1
4
Contagious Ecthyma (EC) is described as an acute and
proliferative disease that affects sheeps and goats,
caused by the Contagious Ecthyma virus (ECV) of the
Parapoxvirus genera. It is widely distributed around the
world, including in Brazil, with a especial relevance to
the Northeast region. The disease control in endemic
regions is performed through vaccination, although the
limitations on the production of vaccines, such as the
replication of the virus in cellular cultures, has caused
the commercialization of a vaccine in Brazil obtained
from crusts of the lesions of infected animals, which
favors the dissemination of many other diseases,
specially viral, since the vaccine is not inactivated. In
ABSTRACTS
the state of Pernambuco, the disease is endemic and
can be mistaken by other vesicular diseases such as
Foot and Mouth disease, being necessary to differentiate
them both to facilitate de National Foot and Mouth
Eradication Program (PNEFA). The present study was
conducted mainly to evaluate the behavior of ECV in
primary caprine fetal corneal (CFC) cellular culture never
before tested for replicating ECV. Crust samples from
nine sheeps and two goats showing classic EC
symptoms, originated from different states of the
Northeast region of Brazil, were inoculated in the CFC
monolayers for seven consecutive weekly passages.
Cytopathic effect was observed in all passages initiated
at 24 hours post-infection, characterized by rounding of
the cells, fusion with syncytia formation, vacuolization
and cytoplasmic corpuscle inclusion with 25-100%
monolayer detachment, varying with the sample. This
results show that primary caprine fetal corneal cells
showed to be extremely permissive to ECV and that
ECV field samples isolates adapted easily to the culture,
with little variation among samples. In conclusion, primary
CFC can be utilized to isolate and evaluate ECV
cytopathic effects, being shown useful to scale
production of this virus.
Financial Support: CNPq.
Palavras-chaves: Contagious Ecthyma Virus, Caprine
Corneal Cell, Cytopathic Effect.
Polymerase Chain Reaction (PCR) technique was used,
having as the positive control the vaccine strain virus
ATCC VR-Lederle 128TM, and primers targeting the virus
nucleoprotein. Serum samples were submitted to RNA
extraction, RT-PCR and cDNA quantification prior to PCR,
and the sensitivity of the test was determined by serial
dilution of the cDNA of the positive control, along with
different negative controls. Briefly, it was performed the
1:10 dilution of the samples to establish the primer
detection limit, which was still observed at 0.4 ng / μL of
cDNA. Later in the project samples collected from animals
with clinical signs, including systemic and neurological
symptoms, suggestive of Canine Distemper were tested.
The samples that tested negative to the virus coincidently
were originated from animals with an advanced state of
neurological signs, which have been presented as a
difficult phase for virus detection in the blood. Most
samples were tested positive for the virus, indicating
the endemic profile of the disease. The results obtained
in this study have been shown very promising as an
attempt to establish a more comprehensive and fast
assessment of the canine distemper cases attended in
the Veterinary Hospital of the Federal Rural University of
Pernambuco. Financial support: UFRPE
Palavras-chaves: Distemper virus, Molecular diagnosis,
Infectious disease.
OPTIMIZATION OF THE MOLECULAR DIAGNOSIS
FOR THE DETECTION OF DISTEMPER VIRUS AT
THE VETERINARY HOSPITAL - UFRPE
PHYLOGENETIC ANALYSIS OF COMPLETE
GENOME SEQUENCES OF HEPATITIS B VIRUS
GENOTYPE F OF CHILEAN PATIENTS WITH
CHRONIC INFECTION
ID: 00079-00002
Área: 03 - Virologia Animal
ID: 00083-00001
Área: 05 - Virologia Humana e Saúde Pública
NASCIMENTO, M.C.O., 2SILVA Jr, L.C., 3KASSAR, T.C.,
GOMES, A.L.V., 5CAMPINHO, D.S.P., 6SANTANA, R.L.,
7
NASCIMENTO, S.A., 8 GATI, M.H., 9FRANCO, L.O.,
10
ANDRADE, L.S.S., 11MAIA, R.C.C.
1. UFRPE, Universidade Federal Rural de Pernambuco,
Rua Dom Manoel de Medeiros s/n - Dois Irmãos - Recife
- PE2. CPqAM - Fiocruz, Centro de Pesquisas Aggeu
Magalhães - Fiocruz, Rua Professor Moraes Rêgo, s/n Cidade Universitária - Recife - PE
VENEGAS, M., 2ALVARADO-MORA, M.V., 3VILLANUEVA, R., 4PINHO, J.R.R., 5CARRILHO, F.J., 6BRAHM,
J.
1. Universidad de Chile, Sección de Gastroenterología,
Departamento de Medicina, Hospital Clínico Universidad
de Chile, Santos Dumont 999 - Sección de
Gastroenterología 3er piso Sector D2. FMUSP,
Laboratory of Gastroenterology and Hepatology, School
of Medicine, University of São Paulo, Av.Dr. Eneas de
Carvalho Aguiar, 500 segundo andar3. Universidad de
Chile, Instituto de Ciencias Biomédicas, Facultad de
Medina, Universidad de Chile, Santos Dumont 999 Sección de Gastroenterología 3er piso Sector D
1
4
The Canine Distemper is a disease caused by a virus of
the Morbillivirus genus and Paramyxoviridae family. The
Morbillivirus are enveloped viruses with negative nonsegmented single stranded RNA genome, covered by a
nucleocapsid of helical symmetry. It causes a disease
that affects dogs of all ages, but with a higher incidence
in young dogs. Considering that it is a widespread disease
in Recife, this work aimed to optimize the molecular
diagnosis of Canine Distemper in order to obtain a better
detection of the virus in animals and establish this type
of diagnosis at the Veterinary Hospital of UFRPE. The
1
Hepatitis B virus (HBV) is estimated to cause chronic
infection in more than 350 millions people worldwide and
1 million deaths per year. Sequence analysis of HBV is
a useful tool for the management of HBV infected
patients, and there are only very few data available about
genotypes and drug resistance mutations in the viruses
81
ABSTRACTS
circulating in Chile. Since the HBV genotype F (HBV/F)
is the most prevalent in the country, the goal of this
study was to obtain HBV full genome sequences from
chronic HBV/F infected patients, and to determine their
subgenotypes in an effort to correlate these results with
the presence of resistance mutations, and clinical data.
Twenty-one serum samples of chronic infected patients
were subjected to full-length PCR amplification, and both
strands of the whole genomes were completely
sequenced. Phylogenetic analyses were performed along
with reference sequences available from GenBank
(n=290). Sequences were aligned using Clustal X
software, and edited in the SE-AL software. Bayesian
phylogenetic analyses were conducted using Markov
Chain Monte Carlo (MCMC) approach for 10 millions
generations for obtaining the substitution tree using
BEAST v.1.5.3. Value of posterior probability was obtained
by Tree Annotator v.1.5.3. All the analyzed Chilean
chronic patients were HBV subgenotype F1b carriers and
the viral sequences clustered into four different groups,
suggesting that different viral strains are circulating within
the population. Additionally, we identified primary drug
resistance mutations using CodonCode Aligner Software.
Only one naïve patient presented primary drug resistance
mutation to Lamivudine, Emtricitabine and Clevudine due
to the occurrence of V173L, L180M and M204V
mutations. In conclusion, this study is the first analysis
of HBV complete genome sequences circulating in Chile,
and its phylogenetic analysis suggests that HBV/F1b is
the most frequently found among people with HBV chronic
infection. Furthermore, we determined that several
different viral entries into the country are present in the
Chilean population. Finally, we have found one patient
with drug resistance mutations that did not referred
previous treatment. Financial support: Proyecto OAIC
362/09 Hospital Clínico, Universidad de Chile, and
FAPESP 07/53457-7 - 08/50461-6.
Palavras-chaves: Chile, Complete Genome, Drug
resistance mutation, Genotype F, Hepatitis B virus.
MOLECULAR CHARACTERIZATION OF THE
HEPATITIS B VIRUS GENOTYPES CIRCULATING IN
COLOMBIA: AN INFERENCE OF THE GENOTYPE F
GEOGRAPHICAL DISTRIBUTION
ID: 00083-00002
Área: 05 - Virologia Humana e Saúde Pública
ALVARADO-MORA, M.V., 2ROMANO, C.M., 3GOMESGOUVEA, M.S., 4GUTIERREZ, M.F., 5BOTELHO, L,
6
CARRILHO, F.J., 7PINHO, J.R.R.
1. FMUSP, Laboratory of Tropical Gastroenterology and
Hepatology, School of Medicine, University of São Paulo,
Av.Dr. Eneas de Carvalho Aguiar, 500 segundo andar2.
FMUSP, Laboratory of Virology, School of Medicine,
University of São Paulo, Av.Dr. Eneas de Carvalho Aguiar,
1
82
5003. PUJ, Depar tament of Microbiology, Pontificia
Javeriana University, Cr 7 No 45-05
Hepatitis B is a worldwide health problem, infecting about
2 billion people, with more than 350 million chronic
carriers. The aim of this study was to characterize the
viral sequences in 143 HBsAg positive volunteer blood
donors from Colombia. A fragment of 1306bp partially
comprising HBsAg and polymerase coding regions (S/
POL) was amplified and sequenced. Viral sequences were
genotyped by phylogenetic analysis using reference
sequences from each genotype obtained from GenBank.
Sequences were aligned using Clustal X software and
edited in the SE-AL software. Bayesian phylogenetic
analyses were conducted using Markov chain Monte Carlo
(MCMC) approach to obtain the MCC tree using BEAST
v.1.5.3. Value of posterior probability was obtained by
Tree Annotator v.1.5.3. Of all samples, 78 were positive
by PCR and 52 were successfully sequencing. Genotype
F3 was the most prevalent in this population (75%).
Genotypes G (7.7%), A2 (15.3%) and F1b (2%) were
found for the first time in this population. Since the
genotype F (HBV/F) was the most prevalent, we
estimated the time of the most recent common ancestor
(TMRCA) for each sub-genotype (F1, F2, F3 and F4)
and also for Colombian F3 sequences using two different
datasets: (i) 77 sequences comprising 1300bp of S/POL
region and (ii) 283 sequences comprising 681bp of S/
POL region. Since there is no consensus on the HBV
substitution rate, we estimated TMRCA using two other
previously estimated evolutionary rates: (i) 2.60x10-4 s/
s/y and 1.5x10-5 s/s/y. Bayesian Skyline plot (BSL) was
applied under strict and relaxed uncorrelated lognormal
molecular clock. TMRCA in years was estimated using
MCMC implemented in BEAST v.1.5.3. As already
expected, a huge variation of TMRCAs was found for
HBV/F by using the two different substitution rates in
the two dataset. TMRCA of genotype F were around 151
years under the higher rate and 2418 years under the
slow rate. The analysis of each F subgenotype suggests
that the subgenotype F3 is the older between the four F
subgenotypes and the subgenotype F4 is the most
recent. In conclusion, we reported for the first time the
characterization of the HBV genotypes circulating in
Colombia and the TMRCA for the four different
subgenotypes of genotype F based on different
substitution rates.
Financial support: FAPESP 07/53457-7 - 08/50461-6.
Palavras-chaves: Bayesian analysis, Colombia,
Genotyping, Geographical Distribution, Hepatitis B virus.
ASSESSMENT OF METHODS FOR CONCENTRATION
AND MOLECULAR DETECTION OF ADENOVIRUS IN
UNTREATED WATER: A META-ANALYSIS
ABSTRACTS
ID: 00085-00001
Área: 02 - Virologia Ambiental
SANTOS, S.F.O., 2SILVA, H.D., 3ANUNCIAÇÃO, C.E.,
QUEIROZ, C.C.B.D., 5GARCÍA-ZAPATA, M.T.A.
1. UFG, Universidade Federal de Goiás, Universidade
Federal de Goiás, Campus Samambaia, Caixa Postal
131, Goiânia-Go.
1
4
Molecular methodologies based on PCR have been used
for the detection of adenovirus (AdVs) in environmental
samples. It is a consensus among researchers that these
methodologies offer some advantages compared with
traditional methods for the isolation of virus by cell
culture, since they are more sensitive and specific and
also require less processing time. However, the method
to be used for virus concentration in environmental
samples is still controversial. Consequently, we carried
out a meta-analysis, aiming at responding the following
question: “How effective is the use of methods of
concentration with microfiltration (MF), ultracentrifugation
(UC), ultrafiltration (UF), and associations of MF+UC and
MF+UF coupled to molecular detection of AdVs in
samples of untreated water?”. We selected articles based
on a systematic review of articles available at
specialized databases, using key words previously
specified by the authors. Initially, 293 articles were
selected and sorted out after reading the abstracts
(Relevance Test I). Posteriorly, the selected articles were
submitted to a Relevance Test II, when they were fully
read, the results were collected and digitalized in a
database worksheet. All the phases of analysis and
allocation of the articles were performed using double
entrance by applying specific criteria of exclusion and
inclusion. As a final result, we collected data from 33
studies and concluded that: a) PCR should not be the
method of choice for the detection of AdVs in
environmental samples; instead, the use of qPCR or
Nested-PCR should be prioritized; b) for the detection of
AdVs in water samples collected in rivers or lakes, the
method of choice should be an association of
ultracentrifugation and Nested-PCR; c) it is advisable to
use an association of microfiltration membrane,
ultrafiltration, and qPCR for the detection of AdVs in
treated and untreated sewage samples.
Palavras-chaves: adenovirus, untreated water,
concentration, meta-analysis, PCR
PREVALENCE OF HEPATITIS C INFECTION IN
SICKLE CELL ANEMIA PATIENTS IN SALVADOR,
BAHIA.
ID: 00088-00001
Área: 05 - Virologia Humana e Saúde Pública
PACHECO, S.R, 2 REIS, A.F, 3 SANTANA, N.P,
ZANETTE, A.M.D, 5ZARIFE, M.A.S, 5 GONÇALVES,
M.S, 7REIS, M.G, 8SILVA, L.K
1. CPqGM-FIOCRUZ-BA, CENTRO DE PESQUISA
GONÇALO MONIZ-FUNDAÇÃO OSWALDO CRUZBAHIA, RUA WALDEMAR FALCÃO,121-CANDEALSALVADOR-BAHIA2. HEMOBA, FUNDAÇÃO DE
HEMOTERAPIA E HEMATOLOGIA DA BAHIA, AV.
VASCO DA GAMA, SN-RIO VERMELHO-SALVADORBAHIA3. LACEN, LABORATÓRIO CENTRAL DE
SAÚDE PÚBLICA PROFESSOR GONÇALO MONIZ,
RUA WALDEMAR FALCÃO,123-CANDEAL-SALVADORBAHIA
1
4
Hepatitis C virus (HCV) is the most common bloodborne
infection in sickle cell anemia patients. This study aims
to determine the prevalence of HCV infection among
sickle cell anemia patients before and after the
introduction of blood screening test and to assess the
contribution of other risk factors besides blood
transfusion, especially among young patients. A total of
553 sickle cell anemia patients who attended the
HEMOBA center starting in 2008 up to the present time
were included and an informed consent agreement was
mandatory for participants. Data from serological and
molecular tests was obtained using a fourth generation
ELISA test and Amplicor (Roche) at LACEN. The overall
seroprevalence of anti-HCV was 6.5% (36/553). HCV
seropositivity was associated with residence in Greater
Salvador, age older than 16 years old, a history of ten or
more transfusions, infection in other family members and
use of non-disposable syringes or needles. The
prevalence of infection confirmed by detection of HCV
RNA was 3.5% (19/545). Genotype 1 was predominant
in 86.7%. Among patients younger than 16 years
prevalence of HCV infection was 0.8% (2/265). According
to these patients the only risk factor they were exposed
to was blood transfusion in other centers. In conclusion,
the risk of acquiring HCV has been reduced by the
implementation of the anti-HCV serological screening
test but residual risk remains. The use of more sensitive
tests and quality control in other centers may be required
to reduce transmission during transfusion in the
immunological window.
Palavras-chaves: HEPATITIS C, SICKLE CELL
ANEMIA, PREVALENCE, VHC-RNA.
TLRS EXPRESSION IN THE TRIGEMINAL GANGLIA
AND BRAIN OF WILD TYPE AND KNOCKOUT MICE
INFECTED WITH HSV-1
ID: 00094-00001
Área: 05 - Virologia Humana e Saúde Pública
1
ZOLINI, G.P.P., 2 LIMA, G.K., 3DIAS, M.F., 4 SILVA,
83
ABSTRACTS
M.G.A., 5Oliveira, G. P;, 6GAZZINELLI, R.T., 7KROON,
E.G., 8CAMPOS, M.A.
1. CPqRR/ FIOCRUZ, Centro de Pesquisas René Rachou,
Avenida Augusto de Lima, 1715 Barro Preto - Belo
Horizonte - MG 30190-0022. UFMG, Universidade
Federal de Minas Gerais, Depar tamento de
Microbiologia, Dep. de Microbiologia, UFMG cidade
universitária, Belo Horizonte - MG 31270-901
After infection, Herpes simplex virus type 1 (HSV-1)
usually become latent and can be reactivated, causing
manifestations like cold sores, or more serious injuries
such as encephalitis. Once infected, the host uses the
cells of innate immune system, that through “Toll Like
Receptors” (TLRs) recognize the “pathogen associated
molecular patterns” (PAMPs) to mount the immune
response. When activated, TLRs transduce a signal,
triggering a phosphorylation cascade that culminates in
the activation of genes related to the innate immune
defense (NO, cytokines and chemokines). TLR2KO,
TLR9KO, TLR2/9KO and C57BL/6 WT mice were
intranasally infected with 106 p.f.u. of HSV-1, and their
respective controls received PBS. Mice euthanasia was
at the 5th day post infection, previously showed to be
the viral multiplication peak, and then trigeminal ganglia
(TG) and brain were collected. Total RNA from TG and
brain were extracted and then reverse transcription was
done to obtain cDNA and subsequently perform Real
Time PCR, to verify the expression of TLRs 1, 2, 3, 6, 7
and 9. WT infected mice showed significant difference
of the expression of these TLRs compared to respective
controls in TG, but not in brain, what did not occurred
with KOs mice. TLR9KO infected mice had a decreased
expression of the TLRs 1 and 6 in TG and increased
expression of TLRs 1, 2, 6 and 7 in brain, when compared
to WT infected mice. TLR2KO infected mice had a
decreased expression of the TLRs 1, 7 and 9 in TG and
increased expression of TLRs 1, 6, 7 and 9. TLR2/9KO
infected mice had a decreased expression of the TLRs 1
and 6 in TG and increased expression of TLRs 3 and 7.
Palavras-chaves: Brain, Encephalitis, Herpes Simplex
Virus 1, Trigeminal Ganglia, Toll Like Receptors.
GENOTYPING OF HEPATITIS C VIRUS IN THE STATE
OF CEARA
ID: 00096-00001
Área: 05 - Virologia Humana e Saúde Pública
LIMA, F.E.S, 2 SÁ, R.C.A, 3 PERDIGÃO, A.C.B.,
KIMURA, R.Y., 5KIMURA, R.Y, 6ARAÚJO, F.,M.C.
1. LACEN-CE, Laboratório Central de Saúde Pública do
Ceará, Av. Barão de Studart, 2405
1
4
The estimated prevalence of hepatitis C virus (HVC)
84
infection in the world is 2,2% (approximately 130 million).
It is one of the most important causes of liver cirrhosis
and cancer, with 80% of infected people being chronic,
leading 20% to live cirrhosis. The HCV, as a RNA virus
presents high genomic variability which contains six
genotypes and more than 70 subtypes. These genotypes
identification allow to define the treatment strategy for
the disease. That’s why is important to know the HCV
genotypes of each region. The aim to study the
prevalence of HCV genotypes in the state of Ceara,
between the years 2005 and 2009. A retrospective study
of 439 patients was done. All of them were anti-HCV
positives, confirmed by PCR qualitative and/or
quantitative and had the genotypes determined at the
Central Laboratory of Public Health of Ceara. Out of 439
patients, 284 (64,7%) were female whose ages ranged
from 22 to 77 years and 155 (35,3%) were male with
ages ranged from 24 to 75 years old. The genotype 1
was found in 303 cases (69%), the genotype 2 in 12
(2,8%) cases and the genotype 3 in 124 (28,2%0 of the
total number of patients studied. The genotype 1 had the
bigger prevalence, followed by genotypes 3 and 2. The
genotypes 4, 5 and 6 were not found. The results showed
that the molecular epidemiology of HCV in Ceara is similar
to what is known to be present in the rest of the Brazil.
Financial Support: LACEN/Ministério da Saúde
Palavras-chaves: Ceara, Genotyping, HCV
CIRCULATION OF RESPIRATORY VIRUSES IN THE
STATE OF CEARA
ID: 00096-00002
Área: 05 - Virologia Humana e Saúde Pública
PERDIGÃO, A.C.B., 2ARAÚJO, F.M.C., 3MELO, M.E.L.,
RORIZ, M.L.F.S., 5MIRALLES, I.S., 6ARAÚJO, R.M.C,
7
SOUSA, A.S.S., 8SILVA, L.A.B., 9SANTOS, M. C., 10SÁ,
R.C.A., 11MELLO, W. A.
1. LACEN-CE, Laboratório Central de Saúde Pública,
For taleza2. UFC, Universidade Federal do Ceará,
Faculdade de Medicina-Sobral3. IEC, Instituto Evandro
Chagas, Belém
1
4
Acute respiratory infections (ARI) are the main reason
for visits to doctor’s offices and account for high index
of morbidity worldwide, especially in children up to five
years old. In order to obtain information about the
circulation of respiratory viruses in For taleza,
nasopharyngeal specimens were collected from patients
with ARI, treated at two sentinel units and from patients
with severe suspected ARI, influenza A/H1N1 pandemic,
mainly at São José Hospital and others hospitals of the
State from January to July 2010. The specimens that
came from the sentinel units were analyzed by indirect
immunofluorescence assay (IFA) in the virology section
ABSTRACTS
of the Central Laboratory of Public Health of Ceara using
a commercial kit (Biotrin) containing monoclonal
antibodies against respiratory syncytial virus (RSV),
influenza A and B, parainfluenza 1, 2 and 3 and
adenovirus. The specimens collected at the hospitals
were sent to Evandro Chagas Institute for real time RTPCR processing. A total of 272 samples were collected,
66 (24,3%) positive for any of the studied respiratory
viruses. RSV was responsible for 23 (8,5%) of the
investigated cases, 11 (4%) adenovirus, 9 (3,3%)
influenza B, 7 (2,6%) influenza A, 7 (2,6%) parainfluenza
3, 6 (2,2%) parainfluenza 2 and 5 (1,8%) parainfluenza
1. A total of 191 samples were collected from hospitals,
30 (15,7%) had positive results for influenza A/H1N1
pandemic and one (0,5%) for seasonal influenza.
Negative results by IFA accounted for 69,2% (188/272)
and negative results by real time RT-PCR were 83,8%
(160/191), which suggests possible circulation of other
respiratory viruses such as rhinoviruses and
coronaviruses. The results showed that all respiratory
viruses circulating surveyed during the first half of 2010
in Ceará, with the predominance of RSV over the others
seasonal respiratory viruses and predominance of
influenza A/ H1N1 pandemic over the influenza A
seasonal.
Financial Support: LACEN/Ministério da Saúde.
Palavras-chaves: Respiratory viruses, Circulation,
Ceara.
MOLECULAR CHARACTERIZATION OF N, HE AND S
PARTIAL GENES OF BRAZILIAN NEONATAL BOVINE
CORONAVIRUS STRAINS
ID: 00097-00002
Área: 03 - Virologia Animal
BARROS, I.N., 2 AYRES, G.R., 3 SILVA, S.O.S.,
SANTOS, S., 5 TORRES, C.A., 6 ASANO, K.M.,
7
SOUZA, S.P., 8OLIVEIRA, C.P., 9RICHTZENHAIN, L.J.,
10
BRANDÃO, P.E.
1. FMVZ / USP, Department of Preventive Veterinary
Medicine and Animal Health, School of Veterinary
Medicine, USP, Av Prof Dr Orlando Marques de Paiva,
87 CEP 05508 270 -Cidade Universitária SP2. CRG,
Coronavirus Research Group, Av Prof Dr Orlando
Marques de Paiva, 87 CEP 05508 270 -Cidade
Universitária SP
1
4
Different bovine coronavirus (BCoV) strains show many
degrees of deletions or substitutions, resulting in altered
antigenicity and pathogenicity of the virus. In previous
study, it was demonstrated a deletion within the
hypervariable region of the S1 subunit of the spike (S)
glycoprotein in Brazilian strains of bovine coronavirus
from Minas Gerais and São Paulo, also found in human
coronavirus OC43. Therefore, this study aimed to analyze
these fifteen samples named USP01 (with deletion in
S1) to USP14 and LYVB (no deletion in S1), based on
the hemagglutinin esterase (HE) protein and the
nucleocapsid (N) protein genes and compare the
sequences obtained herein with others known BCoV and
one OC43 strain (GenBank) based on the HE, N and S
partial genes. To this end, these strains were submitted
to RT-PCRs targeted to the N gene (306-bp fragment)
and HE gene (441-bp) and then submitted to DNA
sequencing. For each gene, the studied strains
sequences were aligned with sequences retrieved from
the GenBank for the construction of Neighbor-Joining
trees for nucleotides (MCL model). The USP01, USP03
and USP05 were successfully sequenced for the HE
gene, and USP01 and USP05 for the N gene. Distance
genealogy for each gene showed that all strains obtained
herein were included in the same cluster and were closer
to São Paulo BCoV strains and sharply distinct from the
BR-UEL strains, suggesting association with the
geographical origin of the samples and the absence of
recombinations in these strains, as the tree had no
significant change regardless the genes studied. This
study contributes to greater understanding of the
molecular characterization and epidemiology of BCoV
and to the definition of genetic markers useful on
diagnostics and prevention measures future as effective
immunogens.
Financial support: FAPESP nº 2008/56620-9 Palavraschaves: bovine, characterization, coronavirus, molecular,
neonatal.
CORONAVIRUS FOUND IN BRAZILIAN EQUINES IS
INDISTINGUISHABLE FROM BOVINE CORONAVIRUS (BCoV)
ID: 00097-00001
Área: 03 - Virologia Animal
BARROS, I.N., 2 NOGUEIRA NETO, F.S>, 3AYRES,
G.R., 4TORRES, C.A., 5SILVA, S.O.S., 6 Santos, S.,
7
ASANO, K.M., 8SOUZA, S.P., 9RICHTZENHAIN, L.J.,
10
BRANDÃO, P.E.
1. FMVZ / USP, Department of Preventive Veterinary
Medicine and Animal Health, School of Veterinary
Medicine, USP, Av Prof Dr Orlando Marques de Paiva,
87 CEP 05508 270 -Cidade Universitária SP2. VET,
Veterinarian of São Paulo, Av Prof Dr Orlando Marques
de Paiva, 87 CEP 05508 270 -Cidade Universitária SP3.
CRG, Coronavirus Research Group, Av Prof Dr Orlando
Marques de Paiva, 87 CEP 05508 270 -Cidade
Universitária SP
1
Equine coronavirus (ECoV) and Bovine coronavirus
(BCoV) are members of the Betacoronavirus genus
(Nidovirales: Coronaviridae). Coronaviruses (CoVs) are
85
ABSTRACTS
especially associated with enteric and respiratory
disease. In Cattle, coronavirus infection causes severe
diarrhea and respiratory diseases in neonatal animals
and contributes to the winter dysentery in adults. In
horses, coronavirus causes mainly neonatal enterocolitis
that is an economically significant disease for horse
breeders, whereas chronically infected adult animals are
often a source of the coronavirus. In this study, two
equine feces samples (E19 and E17) previously
diagnosed as coronavirus positive by a nested RT-PCR
targeted to the pol gene were analyzed by RT-PCR
targeted to the nucleocapsid (N) protein gene (306-bp
fragment), the hypervariable region of the spike (S)
glycoprotein gene (488-bp) and the hemagglutinin
esterase (HE) gene (441-bp) and then submitted to DNA
sequencing. For each gene, the studied strains
sequences were aligned with sequences retrieved from
the GenBank for the construction of Neighbor-Joining
trees for nucleotides (Maximum Composite Likelihood
model). Distance genealogy for each gene showed that
both equine strains are included in the same cluster of
BCoV strains and in a divergent cluster from ECoV
strains. As a conclusion, these results show that the
coronavirus found in these two equines field strains is
indistinguishable from BCoV strains and distinct from
the GenBank ECoV strains, suggesting interspecies
transmission. Besides, these results not only contribute
to the molecular characterization of CoVs, but also for
the understanding of the coronavirus molecular
epidemiology.
Financial support: FAPESP nº 2008/56620-9
Palavras-chaves: bovine, characterization, coronavirus,
equine, molecular.
CANTAGALO VIRUS ENTRY IS INHIBITED BY
SULFATED GALACTAN ISOLATED FROM THE RED
ALGA BOTRYOCLADIA OCCIDENTALIS
ID: 00098-00001
Área: 04 - Virologia Básica
MEDAGLIA, MLG, 2 Oliveira, SN, 3 MOURÃO, PA,
DAMASO, C
1. IBCCF, Instituto de Biofísica Carlos Chagas Filho UFRJ, av. Carlos Chagas, Ilha do Fundão - Rio de Janeiro
- RJ2. IBqM, Instituto de Bioquímica Médica - UFRJ, av.
Carlos Chagas, Ilha do Fundão - Rio de Janeiro - RJ
1
4
Cantagalo virus (CTGV) is a strain of vaccinia virus
(VACV; Poxviridae), originally isolated from lesions on
cows in 1999 in Brazil. Despite the frequent occurrence
of outbreaks, there are no antiviral therapies available.
Therefore, the search for new anti-poxvirus compounds
is of extreme relevance. Sulfated galactan (SG) is a
highly anionic polysaccharide extracted from the red
algae Botryocladia occidentalis. Its linear backbone is
composed of repeated galactose disaccharide units,
similar to animal glycosaminoglycans (GAGs). It has
been previously reported that VACV entry is partially
mediated by the interaction of viral envelope proteins
with GAGs. Therefore, here we evaluated the effect of
SG on the entry of CTGV into host cells. Non-cytotoxic
concentrations of SG were incubated with CTGV and
BSC-40 cells during the adsorption period (2 hours). Then,
after 48 hours in the absence of SG, we observed that
virus plaque formation was inhibited in nearly 80% at
2.5 ug/ml, with an IC50 of aproximately 0,03 ug/ml. The
use of an SG isolated from Gelidium crinale inhibited
virus replication similarly. Pre-treatment of cells with
20ug/ml SG showed no expressive inhibition in virus
replication. The analysis of infectious progeny production
after 24 hours indicated that 5ug/ml SG led to nearly
80% inhibition in BSC-40, C6 and BHK-21 cells, but no
effect in PK-15 and RK-13 cells was observed. We used
recombinant CTGV expressing â-galactosidase under
control of a viral early/late promoter. The addition of 15
ug/ml SG during adsorption led to an inhibiton of 80% in
â-galactosidase activity after 6 hours of infection. The
addition of GAGs during virus adsorption showed that
heparin inhibited virus plaque formation in 80%, but at
20 ug/ml. Addition of SG together with 20ug/ml heparin
showed no significant synergism. Fragmentation of SG
slightly reduced its inhibitory effect. Experiments are in
progress to evaluate the effect of desulfating SG and
analyze virus entry by immunofluorescence assays.
Palavras-chaves: Antiviral, Cantagalo Virus, Sulfated
galactan, Virus entry
TLR2 AND TLR9 EXPRESSED IN TRIGEMINAL
GANGLIA ARE CRITICAL TO VIRAL CONTROL
DURING HERPES SIMPLEX VIRUS 1 INFECTION
ID: 00101-00001
Área: 05 - Virologia Humana e Saúde Pública
Lima, G.K., 2Zolini, G.P.P., 3Mansur, D.S., 4Lima, B.H.,
Wischhoff, U., 6Astigarraga, R.G., 7Dias, M.F., 8Silva,
M.G.A., 9Kroon, E.G., 10Campos, M.A.
1. UFMG, Universidade Federal de Minas Gerais, Av.
Antônio Carlos, 6627 - Pampulha - Belo Horizonte - MG2.
CPqRR, Centro de Pesquisas René Rachou, Fiocruz,
Belo Horizonte, MG3. UFSC, Universidade Federal de
Santa Catarina-SC, Santa Catarina
1
5
Herpes simplex virus 1 (HSV-1) triggers toll-like receptors,
86
ABSTRACTS
which elicit cytokine production. Kinetics of viral
multiplication and cytokine expression in C57BL/6 wild
type (WT) mice infected intranasally with 106 PFU of
HSV-1 was evaluated. Virus was found in the trigeminal
ganglia (TG), but not in the brains of the animals without
encephalitis signs, between the 2nd and 6th days postinfection (d.p.i.). Cytokine expression in TG peaked on
the 5th d.p.i. TLR9-/- and TLR2/9-/- mice were more
susceptible to the virus, with 60% and 100% mortality,
respectively, as opposed to 10% in the WT and TLR2-/mice. Increased levels of CXCL10/IP-10 and CCL2/MCP1 and reduced levels of IFN-gamma and IL1-beta
transcripts, measured in TG and brains on the 5th d.p.i.,
in addition to the virus presence in the brain, were
correlated with total mortality in TLR2/9-/-. Cytokine
alterations in TLR2/9-/- mice coincided with
histopathological changes in their brains, which did not
occur in WT and TLR2-/- and occurred only slightly in
TLR9-/- mouse brain. We hypothesize that control of HSV1 infection depends on immune responses in the TG
and brains and an uncontrolled and inefficient immune
response is induced after deregulation of the cytokine
profile due to TLR deficiency and virus invasion of the
brain. Financial Support: CNPq/Brazil, Fapemig/Brazil,
INCTV/Brazil, PAPES IV/V/Fiocruz.
Palavras-chaves: HSV-1, Immunity, TLR
The Natural Endogenous RNA Polymerase (NERP)
from Respiratory Syncytial Virus (RSV)
ID: 00102-00001
Área: 04 - Virologia Básica
Andrade, V.M.M., 2Mesquita, M.M.A., 3Siqueira, M.M.,
Souza, T.M.L.
1. FIOCRUZ/IOC, Fundação Oswaldo Cruz/Instituto
Oswaldo Cruz, Rua Leopoldo Bulhões, 1480, 1º andar,
sala B105.
RNA was genotyped. Indeed, we observed RSV specific
sequences that clustered together in phylogenetic trees.
This technique allowed us to genotype RSV in clinical
samples that were otherwise unsequenciable. The
proposed approach did not affected RSV infectivity in
HeLa cell line. Moreover, when comparing the NERP
activity from different RSV-positive clinical samples, we
noticed different polymerase fitness, with implication for
Km and Vmax. The method was also able to detect the
polymerase inhibition with the drugs. Together, our
findings indicate that this assay might provide an useful
functional model for measuring RSV RNA polymerase
activity.
Financial Support: FAPERJ, CNPq, POM-FIOCRUZ.
Palavras-chaves: Respiratory Syncytial Virus, NERP,
qRT-PCR.
DETECTION OF HEPATITIS B VIRUS GENOTYPE A1
IN A QUILOMBO ISOLATED COMMUNITY FROM
MARANHÃO, BRAZIL
ID: 00103-00001
Área: 05 - Virologia Humana e Saúde Pública
Alvarado-Mora, M.V., 2Botelho, L, 3De Souza, V.A.U.F.,
Pannuti, C.S., 5Carrilho, F.J., 6Pinho, J.R.R.
1. FMUSP, Laboratory of Gastroenterology and
Hepatology, School of Medicine, University of São Paulo,
Av. Dr. Enéas Carvalho de Aguiar, 500 CEP 05403-000 São Paulo - SP - Brazil2. FMUSP, Laboratory of Virology,
School of Medicine, University of São Paulo, Av. Dr.
Enéas Carvalho de Aguiar, 500 CEP 05403-000 - São
Paulo - SP - Brazil
1
4
1
4
RSV is a paramyxovirius and the main ethological agent
of acute lower respiratory tract infection, affecting mainly
children under two years of age and elderly population.
In the present study, we investigated the presence of an
RNA polymerase activity within RSV virions (NERP) and
the virus inhibition by triazolic compounds targeting this
enzyme. The supernatant of RSV-infected HeLa cells
was harvested and submitted to logarithmic dilutions,
up to undetectable level. These dilutions were further
incubated in a buffer containing MgCl2 with different
concentrations of ribonucleotides. The reaction was
stopped, viral RNA was extracted and submitted to qRTPCR. In antiviral assays, we also added the drugs within
the reaction of RT-PCR. We observed a 2-log
enhancement of viral RNA content, due to the above
mentioned incubation. To confirm that enrichment was
due to RSV specific sequences, the above mentioned
The Brazilian population is mainly descendent from
European colonizers, Africans and Amerindians. Some
Afro-descendants lived in small isolated communities
since the slavery period. These runaway-slaves
descendents stayed in culturally isolated communities,
called Quilombos, without significant additional admixture
since their establishment. The prevalence and genotypes
of hepatitis B virus (HBV) have distinct geographical
distribution, especially in Brazil, where wide variation of
HBV infection was found. The epidemiological status of
HBV infection in Quilombos communities in Maranhão
state remains unknows. The aim of this study was
characterized the HBV genotypes circulating inside a
Quilombo isolated community from Maranhão, Brazil.
Seventy-two samples from Frechal, Maranhão, were
collected between 2000 to 2001. All serum samples were
screened by enzyme-linked immunosorbent assays (EIA)
for the presence of hepatitis B surface antigen (HBsAg).
HBsAg positive samples were submitted to DNA
extraction. A fragment of 1306bp partially comprising
HBsAg and polymerase coding regions (S/POL) was
87
ABSTRACTS
amplified by nested PCR and sequenced. Viral sequences
were genotyped by phylogenetic analysis using reference
sequences from each genotype obtained from GenBank
(n=363). Sequences were aligned using Clustal X
software and edited in the SE-AL software. Bayesian
phylogenetic analyses were conducted using Markov
chain Monte Carlo (MCMC) approach to obtain the MCC
tree using BEAST v.1.5.3. Value of posterior probability
was obtained by Tree Annotator v.1.5.3. Of the 72
individuals, 9 (12.5%) were HBsAg-positive and 4 of
them were successfully sequenced for the 1306bp
fragment. These samples were genotype A1 and grouped
a single cluster. The present study represents the first
report on the HBV genotype characterization of this
community in the Maranhão state in Brazil. A high HBsAg
prevalence was found. The present findings emphasize
the urgent need for HBV vaccination in quilombo remnant
communities. Additionally, genotype A1 has been shown
to be common in African populations and in the AfroBrazilian population, suggesting a common introduction
of HBV during the slave trade from Africa to Brazil.
Financial support: FAPESP 07/53457-7 - 08/50461-6.
Palavras-chaves: Afro-Brazilian population, Bayesian
Analisys, Genotype A1, Hepatitis B virus, Quilombo
isolated community.
samples were tested using third-generation ELISA tests
for HBV markers (HBsAg, anti-HBc and anti-HBs), antiHCV and anti-HDV. The results showed the prevalence
of Anti-HBc varied from 3.95% to 54.55% (p 51 years.
HBsAg varied from 1.97% to 8.39% in the four regions
(p = 0.033). Anti-HBs prevalence ranges from 17.48% to
62.5% among the four regions (p< 0.001). In summary,
although the process of implementing a vaccine against
HBV has been successful, new strategies are necessary
to increase immunization in the rural population since
we found a high prevalence of HBV. Also, the high
prevalence of HCV probably is not known in the country
and suggests that there is a lot infected people untreated
for HCV.
Palavras-chaves: HBV, HCV, HDV, Colombia,
Epidemiology
STUDY OF THE COINFECTION OF HEPATITIS B
VIRUS WITH HUMAN IMMUDEFICIENCY VIRUS,
HEPATITIS A AND C VIRUS
ID: 00105-00001
Área: 05 - Virologia Humana e Saúde Pública
De-Farias, R.P., 2Figueiredo, D.D.L.G., 3Bastos, A.G.S.,
Amaral, F.N., 5 Sobral-Filho, R.G., 6 Silva, J.R.C.,
7
Cavalcanti, B.B., 8 Oliveira, V.F., 9 Alves, C.R.M.S.,
10
Nascimento, A.C.H.D., 11Melo, M.M.M.
1. FIP, Faculdades Integradas de Patos, Patos, Paraíba
2. LACEN, Laboratório Central de saúde Pública, Recife,
Pernambuco
3. FMN, Faculdade Maurício de Nassau, Recife,
Pernambuco
1
4
EPIDEMIOLOGICAL STUDY OF HEPATITIS B (HBV),
HEPATITIS C (HCV) AND HEPATITIS DELTA (HDV)
VIRUSES IN THE COLOMBIAN POPULATION
ID: 00103-00002
Área: 05 - Virologia Humana e Saúde Pública
ALVARADO-MORA, M.V., 2GOMES-GOUVÊA, M.S.,
GUTIERREZ, M.F., 4DE AZEVEDO, R.S., 5CARRILHO,
F.J., 6PINHO, J.R.R.
1. FMUSP, Gastroenterology, School of Medicine,
University of São Paulo, Av Dr Eneas de Carvalho Aguiar,
500 2. andar2. PUJ, Depar tment of Microbiology,
Pontificia Javeriana University, Cr 7# 45-023. USP,
Pathology, School of Medicine, University of São Paulo,
Av Dr Eneas de Carvalho Aguiar, 500 2. andar
1
3
There are more than 500 million people are chronically
infected with one of the human hepatitis viruses
worldwide. In Colombia, there are some areas where more
than 70% of the population has been infected with HBV
and there are not studies of prevalence in the general
population about HCV and HDV. The aim of this study
was determine the prevalence of HBV, HCV and HDV in
populations from four different Colombian places:
Amazonian Region, Quibdó, Santa Marta and San
Andres Island (n= 618 samples), with ages varying from
12 to 72 years old. The samples were collected from
people of different risk groups for hepatitis infection. All
88
Hepatitis B is an infectious disease caused by Hepatitis
B virus (HBV), a hepadnavirus with circular DNA-based
genome featuring double helical structure, with a strong
preference for liver cells. The coinfection of HBV with
other viruses is of great clinical importance, leading to
an alarming prognosis, as well as interfering with the
results of therapy. This study selected 70 (100%) sera
of patients referred to the Department of Molecular
Biology of the Central Public Health Laboratory of
Pernambuco (LACEN-PE) to detect the rate levels of
HBV-DNA. We used the COBAS Amplicor kit (Roche)
for detection of HBV DNA in sera of patients. Serologic
detection kits (HIV, HCV, HAV) were tested using the
methodology of the supplier (DiaSorin) ELISA. All
samples analized had a positivity of 100% for the test of
HBV-DNA, showing values between 38 000 IU / ml. Nine
(12.8%) patients, with ages between 6 and 66 years,
presented positive results for coinfection with HIV virus,
eight (88.8%) of them were male and one (11.1%) was
female. Three (33.3%) sera from patients aged 28 to 68
years were positive for coinfection with hepatitis C virus
ABSTRACTS
(HCV), also with male predominance on that, and 2
(22.2%) sera from patients with 25 and 34 years old
showed to be positive for coinfection witn hepatitis A
virus (HAV ), in this case, 1 (50%) male and 1 (50%)
female. Therefore, co-infection HBV / HIV occurred in
considerable numbers in relation to others, which may
be explained by the fact that these two viruses have
common routes of transmission, mainly sexual,
parenteral and vertical. Our results support existing
research literature, making it clear the necessity of
obtaining data relating to any infection that exists parallel
to the patient, enabling thus to determine an
individualized, fast and effective treatment against these
co-infections.
Financial support: LACEN-PE
Palavras-chaves: Hepatitis, coinfection, HIV, ELISA,
PCR.
IDENTIFICATION OF DENGUE VIRUS SEROTYPES
THROUGH THE TEST OF MOLECULAR BIOLOGY IN
CASES OF DEATH, THE PERIOD JANUARY 2008 JULY 2010 IN PERNAMBUCO STATE
ID: 00105-00002
Área: 05 - Virologia Humana e Saúde Pública
De-Farias, R.P., 2Figueiredo, D.D.L.G., 3Melo, M.M.M.,
Santiago R.G., 5 Sobral-Filho, R.G, 6 Silva, J.R.C.,
7
Cavalcanti, B.B., 8Oliveira, V.F.
1. FIP, Faculdades Integradas de Patos, Patos, Paraíba2.
LACEN, Laboratório Central de Saúde Pública, Recife,
Per nambuco3. UFPE, Universidade Federal de
Pernambuco, Recife, Pernambuco
1
4
Dengue is an acute febrile infectious disease caused by
a family of viruses named Flaviridae, transmitted through
the mosquito Aedes aegypti, also infected by the virus.
Currently, dengue is considered a major public health
problem in the country. The virus has four serotypes:
DENV-1, DENV-2, DENV-3 and DENV-4. According to
Guzman et al. evidence suggests that the occurrence of
sequential infection increases the risk of dengue
hemorrhagic fever. According to the Ministry of Health,
Brazil, the four serotypes are circulating already, and
the type 4 was recently confirmed in the city of Boa
Vista-RR. There are different methods that can be
adopted to confirm the diagnosis of the disease, however,
the technique of molecular biology is the most specific
and rapid confirmation of the serotype in samples of
serum, blood, or viscera. Our study analyzed viscus
fragments of 90 suspected cases of death caused by
dengue, received at the Central Laboratory of Public
Health LACEN-PE from January 2008 to July 2010. The
virus RNA was extracted from fragments of viscera by
the technique of magnetic silica extraction kit BioMerieux,
later, viral serotypes were detected by RT-PCR using
the technique of Lanciotti. 68 (75.5%) of the processed
samples were negative for the disease and 22 (24.4%)
positive, 4 (18.1%) serotype DENV-1, 10 (45.4%) DENV2 and 8 (36.3%) DENV-3, in relation to gender 14 (63.6%)
were male and 8 (36.3%) female. It was verified the
presence of three serotypes DENV-1, DENV-2 and
DENV-3 circulating in the state of Pernambuco, which
agrees with data reported for the period. The PCR proved
to be very effective in disease diagnosis and serotypes
identification.
Palavras-chaves: Serotypes, Virus, Dengue, Test of
molecular.
EVALUATION OF ENTEROVIRUS 71 IMMUNE
STATUS IN SÃO PAULO STATE, BRAZIL
ID: 00108-00001
Área: 05 - Virologia Humana e Saúde Pública
Luchs, A, 2Cilli, A, 3Russo, D.H., 4Costa, F.F., 5Carmona,
R.C.C, 6Timenetsky, M.C.S.T.
1. IAL, Instituto Adolfo Lutz, Avenida Dr. Arnaldo, 355
1
Enterovirus 71 (EV71) infections are the most important
type, except poliovirus, because they are frequently
complicated by the neurologic diseases, in fact EV71
has emerged as a significant pathogen with potencial to
cause large outbreaks, and the last 8-10 years have seen
shift frequency of repor ted EV71 associated to
encephalitis fatalities. For the reason that limited
information is available about the level of immunity
against EV71 in São Paulo State, Southeastern region,
especially after the Asian outbreaks, serological data
from 1999 – 2005 are presented in this study. The level
of immunity against EV71 was investigated in total of
442 sera samples oppor tunistically selected, and
representing the general population. The individuals were
divided in the following age data: 0 – 5 years (preschool
and kindergarden children), 6 – 15 years (schooling
children), and e” 16 years (adult individuals). The titer of
neutralizing antibodies against EV71 was determined by
microneutralization assay, and titer of e” 1:8 was defined
as indicative of protected immunity. Overall, 12.4% of
tested samples had neutralizing antibodies to EV71, and
87.6% were negative. No correlation between age groups
and seropositive were observed. There was no significant
difference in EV71 immunity in preschool children aged
0 – 5 years (11.0%), schooling children aged 6 – 15
years (14.6%) or adults e” 16 years (12.5%). The
serological data obtained showed a low rate of protective
EV71 antibodies, suggesting that EV71 infections is
uncommon in this region, and relatively high susceptibility
of the inhabitants to EV71 related diseases.
Palavras-chaves:
Enterovirus
71,
soroneu89
ABSTRACTS
tralization, Brasil.
VACCINATION AGAINST HEPATITIS B AMONG
DENTISTâ•™S SURGEON AND STUDENTS OF THE
ODONTOLOGYâ•™S COURSE OF ASSOCIATION
CARUARUENSE OF HIGHER EDUCATION (ASCES).
ID: 00110-00001
Área: 05 - Virologia Humana e Saúde Pública
Albuquerque, A.C.C, 2Pereira, A.R.S, 3Almeida. T.A.N
1. ASCES, Faculdade Associação Caruaruense do
nsino Superior, Av. Europa, N. 584 Santa Maria Gorete
Caruaru-PE
1
The vaccine against hepatitis B virus (HBV) in dentists
is of utmost necessity, since they have a high risk of
infection by viruses. The aim of this study was to
evaluate the vaccination status of dental teachers and
students of the ASCES faculty. The study was
accomplished in the clinical practice laboratories of the
ASCES in the October to November 2009 period. The
sample size was 48 participants, 35 of these were
students and 13 teachers. Was applied to each participant
a questionnaire to collect data as knowledge on HBV,
HBV vaccine, care taken with the biosecurity in the
workplace and other aspects. Data were stored and
analyzed by Excel 2007. In relation to the vaccine for
hepatitis B, 95.8% (46/48) had at least one dose and
45.7% of these (21/46) took the 03 doses recommended
by the Ministry of Health.Health professionals are
exposed to HBV infection, suggesting that contact with
patients and handling of body fluids are risk factors of
occupational transmission of this virus,recommending
complete vaccination of these professionals against
hepatitis B.
Financial support: PIBIC/ASCES.
Palavras-chaves: Biossegurança, Dentistas,
Estudantes, Hepatite B, Vacina.
QUANTIFICATION OF PORCINE ADENOVIRUS BY
QPCR IN RESIDUAL WATER FROM TWO MANURE
TREATMENT SYSTEM
alternative has to be carefully evaluated concerning
microbiological parameters, avoiding environment
contamination and transfer of pathogens to animals or
to humans. The presence of enteric viruses can be used
as parameter to assess the performance of treatment
technologies in sewage and water plants. Among the
viral pathogens that can be chosen as a biological marker
for water treatment, are porcine adenoviruses (PAdV),
an abundant and prevalent virus in swine feces. The
present study aimed to quantify PAdV in samples of
residual water from two swine manure treatment system
(ETDS and UD) from EMBRAPA - Swine and Poultry,
Concordia, SC, during different steps of water treatment
through qPCR. Twelve sampling events were performed
from March 2009 to May 2010, with a sum of 31 water
samples analyzed. Samples were concentrated by
adsorption-elution-precipitation method and polyethylene
glycol precipitation. Nucleic acid extraction was
performed using a QIAmp MinElute Virus Spin (Qiagen)
and quantified by qPCR using specific primers and
Taqman® probe. A PAdV sequence fragment was cloned
into TOPO TA cloning plasmid and used as standard.
The results showed that the average number of genome
copies (gc)/mL in the water collected from the affluent,
meddle and effluent from ETDS were: 8.69E+04,
5.13E+04 and 3.92E+03, respectively. On the other hand,
from UD, the average on affluent, meddle and effluent
was 6.92E+04, 1.22E+04 and 3.61E+02 gc/mL,
respectively. Although, further studies are necessary to
attest viral viability, since the results presented here only
quantified virus genome copies, according the results
pointed here was possible to infer that these swine
manure treatment systems need additional steps for virus
removal.
Financial support:CNPq/EMBRAPA.
Palavras-chaves: adenovírus porcino, água de reuso,
suinocultura.
DENGUE NS1 ANTIGEN DETECTION IN CEREBRAL
SPINAL FLUID SAMPLES BY TWO COMMERCIALLY
AVAILABLE ENZYME-LINKED IMMUNOSORBENT
ASSAYS
ID: 00112-00001
Área: 05 - Virologia Humana e Saúde Pública
ID: 00111-00001
Área: 02 - Virologia Ambiental
ARAÚJO, F.M.C, 2ARAÚJO, R. M. C., 3RAMALHO, I. L.
C., 4RORIZ, M. L. F. S., 5MELO, M. E. L., 6MIRALLES,
I.S., 7ARAÚJO, L. C., 8DANTAS, C. A., 9CAVALCANTI,
L. P. G. C., 10SIDRIM, J. J. C.
1. LACEN-Ce, Laboratório Central de Saúde Pública, Rua
Barão de Studart, 24052. PPGCM, Programa de Pós
Graduação em Ciências Médicas, Faculdade de
Medicina3. UFC, Universidade Federal do Ceará,
Faculdade de Medicina4. NUVEP, Núcleo de Vigilância
1
Viancelli, A., Garcia, L.A.T, Steinmetz, R., Kunz, A.,
Esteves, P.A., 6Barardi, C.R.M.
1. UFSC, Universidade Federal de Santa Catarina,
Florianopolis2. EMBRAPA - CNPSA, EMBRAPA - Suínos
e Aves, Concórdia - SC
1
2
3
4
5
Swine production in Santa Catarina plays an important
role on society, environment and economy. Considering
this, it has been proposed the reuse of this water on
swine production process or on agriculture. However, this
90
ABSTRACTS
Epidemiológica, Rua Almirante Barosso, 6005. UFC,
Universidade Federal do Ceará, Campus do Pici
Dengue fever is a mosquito-borne disease caused by
one of four dengue viruses –DENV-1, DENV-2, DENV-3
and DENV-4 – which belong to the genus Flavivirus,
family Flaviviridae. Dengue’s symptoms can vary from
mild fever, the most common, to potentially fatal forms,
such as dengue hemorrhagic fever and dengue shock
syndrome. Unusual manifestations such as
myocardiopathy, hepatic insufficiency, hepatitis,
encephalopathy and encephalitis are also becoming
increasingly common. The involvement of the central
nervous system in patients with dengue has been
reported lately in countries where the disease is endemic.
There is no antiviral therapy or vaccine approved for use
against dengue, so patient management requires good
laboratory and clinical support. Specific and rapid
diagnosis can help in directing the proper treatment to
patients. The purpose of this study was to verify
sensitivity and specificity of two ELISA kits for detection
of the dengue NS1 antigen in cerebral spinal fluid samples
from patients with fatal outcomes We used 74 spinal
fluid samples from patients who had died of dengue,
obtained during autopsies performed between 2005 and
2008. There was an outbreak of dengue in the state of
Ceará in this period, with a peak in 2008. The Pan-E
Dengue Early ELISA kit was able to detect the NS1 Ag
in the CSF with a sensitivity of 45.8% (CI: 34.5-57.2%)
and specificity of 94% (CI: 88.6-99.4%). The results for
the Platelia Dengue NS1 Ag kit were sensitivity of 16.7%
(CI: 8.2-25.2% and specificity of 100% (CI: 100-100%).
Both kits were able to detect the NS1 Ag in the cerebral
spinal fluid of individuals who had died of dengue.
However, the Pan-E Dengue Early ELISA kit was more
sensitive, and can be recommended for use with spinal
fluid samples. This study facilitates research into the
involvement of the central nervous system by dengue,
enabling speedier intervention in patients suffering from
non-classical dengue, thus reducing mortality.
Financial support: LACEN/ MS
Palavras-chaves: Cerebral spinal fluid, Dengue, NS1
antigen.
NEUROLOGICAL MANIFESTATIONS OF DENGUE IN
PATIENTS WITH FATAL OUTCOMES IN CEARA,
BRAZIL
ID: 00112-00002
Área: 05 - Virologia Humana e Saúde Pública
ARAÚJO, F. M. C., 2ARAÚJO, R. M. C., 3PERDIGÃO,
A. C. B., 4RORIZ, M. L. F. S., 5MELO, M. E. L., 6MIRALLE,
I. S., 7 VILAR, D. C. L. F., 8 HOLANDA, S. G. S.,
9
OLIVEIRA, D. N., 10SÁ, R. C. A., 11SIDRIM, J. J. C.
1
1. LACEN-CE, Laboratório Central de Saúde Pública,
Rua Barão de Studart, 24052. PPGCM, Programa de
Pós Graduação em Ciências Médicas, Faculdade de
Medicina3. UFC, Universidadde Federal do Ceará,
Faculdade de Medicina4. SVO-CE, Sistema de
Verificação de Óbitos, Rua Madre Ana Couto, 6725.
NUVEP, Núcleo de Vigilância Epidemiológica, Rua
Almirante Barosso, 600
Dengue infection presents classically is dengue fever or
dengue hemorrhagic fever. In recent years unusual
manifestations are becoming increasingly common. The
involvement of the central nervous system (CNS) in the
patients with dengue has been reported in countries where
the disease is endemic. The use of cerebral spinal fluid
(CSF) as biological sample helps to clarity if there is a
direct infection of the CNS or if the neurological
manifestations are due to other organs injury. The aim of
this study was to determine the frequency of the CNS
infection by dengue virus in individuals with fatal
outcomes, during a dengue fever epidemic period from
2005 to 2008 in the State of Ceara, Brazil. The sampling
was made up of: CSF, blood and tissue specimens from
163 corps where the had been autopsied the coroner’s
office. The 163 CSF samples were processed by viral
isolation, RT-PCR, AgNS1, AcIgM and IgG for dengue
virus. The ether biological samples had already been
processed and the results were used to classify the
cases. Out of 163 individuals, 90 were infected with
dengue viruses. From those, 57,8% were male and 42,2%
female. The mean age was 33 years varying from 2
months to 86 years old. Mean days between the onset
of symptoms and the death was 5 days varying from
one to 14 days. The diagnostic results, in CSF, yielded
by the 7 distinct approaches were: 7 RT-PCR positives,
4 for DENV-3 and 3 DENV-2; 1 isolation of DENV-3; 26
AgNS1 positive; 27 AcIgM positive and 38 AcIgG positive
for dengue. Out 90 dengue positive individuals, 46 had
evidence of CSF infection by dengue viruses. The
following neurological diagnosis were mode: 11
encephalitis; 14 menigoencephalitis, 9 bacterial
meningitis, 4 viral meningitis, 8 encephalopaties. Thus,
37 individual with neurological features were due to
dengue virus infection, given a positive rate of 41% of
the 90 dengue positive cases and 22,7% of the fatal
outcomes studied.
Financial Support: LACEN/Ministério da Saúde
Palavras-chaves: CSF, Dengue, Fatal outcomes,
Neurological manifestations.
THE MUTATIONS PROFILE IN INTEGRASE GENE IN
INTEGRASE INHIBITORS-NAÏVE HIV-1 INFECTED
PATIENTS.
ID: 00117-00001
91
ABSTRACTS
Área: 05 - Virologia Humana e Saúde Pública
TANIKAWA, A.A., 2ALHO, M.J.O., 3FIRMIANO, R.C.,
SOUZA, L.R., 5GROTTO, R.M.T., 6PARDINI, M.I.M.C.
1. FMB-UNESP, Faculdade de Medicina de Botucatu UNESP / Laboratório de Biologia Molecular - Hemocentro,
Distrito de Rubião Júnior, s/nº, cep 18603-9702. FMBUNESP, Faculdade de Medicina de Botucatu - UNESP /
Departamento de Doenças Tropicais, Distrito de Rubião
Júnior, s/nº, cep 18603-9703. FMB-UNESP, Faculdade
de Medicina de Botucatu - UNESP / Departamento de
Clínica Médica, Distrito de Rubião Júnior, s/nº, cep
18603-970
1
4
The antiretroviral resistance remains one of the most
obstacles to sustained suppression of HIV during highly
active antiretroviral therapy (HAART). Several studies
have evaluated the impact of these mutations in protease
and reverse transcriptase genes, major targets of the
antiretroviral therapy. The high prevalence of these HIV
resistant variants showed the necessity of introduction
of new drugs as alternative to antiretroviral therapy, as
integrase (IN) inhibitors, drugs low available in Brazil yet.
The integrase inhibitors (INIs) have demonstrated
impressive potency in both treatment-naïve and
treatment experienced patients. In this study, we
evaluated the presence of polymorphisms and resistance
mutations associated with IN inhibitors in 18 IN inhibitorsnaïve HIV-1 infected patients from Botucatu region.
Plasma viral RNA was used as source to amplification
and sequencing of the IN gene. Preliminary results show
prevalence of subtype B based on analysis of pol IN
sequences, the presence of the IN minor resistance
mutations associated with Raltegravir and Elvitegravir:
V151I (27,8%), Q95E/S (11,1%), I203M (5,6%), G163K
(5,6%), E138D (5,6%), M154L (5,6%) and other mutations
as V201I, T124N/A, I72V, S17N, T206S. These
preliminary results indicate that although mutations in
IN gene are presented in naïve patients, these mutations
aren’t associated with loss of susceptibility to IN
inhibitors, suggesting that this drugs can be improve the
antiretroviral therapy in the region studied. More samples
are being analyzed to evaluate the impact of these
findings.
Palavras-chaves: HIV-1, Integrase Inhibitor, Resistance.
TWO INTERFERON GAMMA ALLELIC VARIANTS
(A256G and A325G) AND HAPLOTYPES PROTECT
AGAINST SEVERE DENGUE CASES
ID: 00118-00001
Área: 05 - Virologia Humana e Saúde Pública
Pastor, A.F., 2Acioli-Santos, B., 3Oliveira, T.W.F., 4Gil,
L.H.V.G., 5Marques Jr, E.T.A
1
92
1. LaViTE/IAM/Fiocruz, Virology and Experimental
Therapy Laboratory (IAM/FIOCRUZ), Av. Prof. Moraes
Rego, s/n Cidade Universitária, Recife, BR. CEP:50.670420.2. UFPE, Department of Genetics, Federal University
of Pernambuco, Recife, Brazil., Av. Moraes Rego, 1235,
Cidade Universitária 50670-901 Recife PE3. UP,
Department of Infectious Diseases and Microbiology,
University of Pittsburgh, Pittsburgh, PA 15260, United
States of America
Dengue is a vector-borne disease that endangers an
estimated 2.5 billion people in the world. While the majority
of infected people develop dengue fever (DF), a small
group develops severe cases including dengue fever with
complications (DFC) and dengue hemorrhagic fever
(DHF). Altogether, dengue account for approximately
24,000 deaths per year. Despite dengue pathogenesis
being only partially understood, it is known that host
genetic factors must play a role in susceptibility/
resistance to DFC/DHF. Interferon gamma (IFNã) is a
cytokine secreted by human immune cells that are
involved in the antiviral response. Some SNPs (Single
Nucleotide Polymorphisms) in the IFNã gene have been
associated with gene expression level modification and
viral disease development, such as Hepatitis B virus
(HBV) infection. The aim of this work was to study the
biological consequences of A256G (intron) and A325G
(3´ UTR) polymorphisms on dengue clinical outcome in
a Brazilian cohort established in Recife. The Illumina
Genotyping System was used to genotype 30 DF and
74 DFC/DHF patients as well as 98 control patients. The
allele distribution of control and dengue-infected patients
was in Hardy-Weinberg equilibrium for both SNPs. A256G
genotype frequencies were 0.894 AA and 0.106 AG (GG
genotype was not found), with G allele being correlated
with DFC/DHF protection (OR=3.32; p= 0.013). In the
A325G locus the frequencies were 0.375 AA, 0.481 AG
and 0.144 GG. A significant correlation was found
between the 325G allele and protection to DHF/DFC
(OR=8.65; p = 3x10-9). Three haplotypes (AA, AG and
GG) and five genotypes (haplotypes pairs) were found.
The heterozygous group AA/AG + AA/GG was
associated with severe dengue protection (OR=9.72;
p=3x10-9). Here, for the first time, a significant protective
effect against severe dengue is presented among 256G
and 325G IFNã variant alleles as well as the respective
AG and GG heterozygous haplotypes in the IFNã gene.
Palavras-chaves: Severe dengue, Interferon gamma,
Polymorphism, Haplotype, Protection.
TWO GENOTYPES CLONING OF HUMAN C-257T CFH
PROMOTER POLYMORPHISM: A TOOL TO
STUDYING THE DENGUE INFECTION RESPONSE
THROUGH THE LUCIFERASE EXPRESSION LEVELS
ABSTRACTS
ID: 00118-00002
Área: 05 - Virologia Humana e Saúde Pública
Área: 05 - Virologia Humana e Saúde Pública
ZEMINIAN, L.B., 2PADOVANI, J.L., 3CORVINO, S.M.,
FERNANDES, R.E., 5FIRMIANO, R.C., 6SILVA, G.F.,
7
PARDINI, M.I.M.C., 8GROTTO, R.M.T.
1. FMB/UNESP, Faculdade de Medicina de BotucatuUNESP/ Laboratório de Biologia Molecular do
Hemocentro, Distrito de Rubião Junior s/n2. FMB/UNESP,
Faculdade de Medicina de Botucatu - UNESP/
Departamento de Clínica Médica, Distrito de Rubião
Junior s/n
1
Pastor, A.F., Acioli-Santos, B., Guimarães, G.F.,
Marques Jr, E.T.A., 5Gil, L.H.V.G.
1. LaViTE/IAM/FIOCRUZ, Virology and Experimental
Therapy Laboratory, Aggeu Magalhães Institute- IAM/
FIOCRUZ, Av. Prof. Moraes Rego, s/n Cidade
Universitária - Recife- BR CEP: 50.670-4202. UFPE,
Depar tment of Genetics, Federal University of
Pernambuco, Recife, Brazil., Av. Moraes Rego, 1235,
Cidade Universitária 50670-901 Recife PE3. UP,
Department of Infectious Diseases and Microbiology,
University of Pittsburgh, Pittsburgh, PA 15260, United
States of America
1
2
3
4
Dengue virus infection is a global public health concern
that endangers an estimated 2.5 billion people in the
world, with an estimated incidence of 50–100 million
cases resulting in 24,000 deaths per year. The Human
Complement Factor H, codified by CFH gene, is a
negative regulator of the alternative pathway that appears
to play a role in determining dengue severity. The SNP
C-257T, located in the CFH gene promoter, has been
associated with several diseases and recent studies on
dengue have shown that high CFH expression levels is
associated with protection against dengue hemorrhagic
fever development. The aim of this work was to clone C257T CFH promoter genotype in a plasmid that contain
the Luciferase reporter gene in order to confirm the T
allele higher expression on dengue infected cells. A 1.245kb DNA fragment encompassing the CFH gene promoter
was amplified by PCR and cloned into the pDRIVE cloning
vector (Quiagen). Afterwards, the 1.24-kb SacII/VspI
fragment was ligated to the SacII/NdeI of reporter vector
(pNFKB_Luc, Clontech) to allow transcription of firefly
Luciferase gene under the control of CFH promoter. Two
plasmids were constructed: one with the C allele
(pCFHC_Luc) and another one with the T allele
(pCFHT_Luc) in a -257 position of the CFH promoter.
These plasmids will be valuable research tools for
studying the true role of the SNP C-257T on gene
expression level and the effects of this expression during
dengue infection. To conclude this work, the group intends
to evaluate the activity of each promoter reporter plasmid
constructed by comparing the Luciferase expression
levels in transfected and dengue virus-infected cells.
Palavras-chaves: Dengue response, CFH promoter,
Cloning, Luciferase, Expression.
EVALUATION OF PROTEASE INHIBITORS RESISTANCE IN UNTREATED PATIENTS HCV INFECTED
LIVING IN BOTUCATU REGION, SP, BRAZIL.
4
The hepatitis C virus (HCV) is an important pathogen
associated with chronic hepatic disease and some
infected patients can develop liver cirrhosis in 20-25 years
after infection, which may progress to hepatocellular
carcinoma. The treatment of chronic Hepatitis C is aimed
at sustained virological response (SVR), defined as
having undetectable HCV RNA at the end of therapy for
at least 6 months. Currently, the gold standard therapy
is a combination of pegylated interferon-á and the ribavirin
but this treatment present efficacy in 50% of patients
infected with genotypes 1, the most prevalent in Brazil.
Then, new drugs more effective and less toxic have been
developed to improve the attendance of the HCV infected
patients as protease inhibitors. The third nonstructural
region of the HCV genome encode the protease, enzyme
essential to HCV replication and because main target of
new antiviral therapies. However, the emergence of drug
resistant variants has been the major obstacle to
therapeutic successful. Resistant variants have already
been isolated in patients treated with protease inhibitors
and, these resistant variants are associated with non
response to treatment. But the impact of the resistant
variants in naïve protease inhibitors patients is unclear
yet and, this information can evaluate the impact of
protease inhibitors in antiviral therapeutic. The goal of
this study was evaluate the primary resistance in 50
patients untreated with protease inhibitors. Serum RNA
viral was used as source to amplification and sequencing
to HCV NS3 genomic region and, evaluates the presence
of resistance mutations. Preliminary results showed that
substitution A156V was present in 85% of the virus
circulating in protease inhibitors naive patients. This
mutation is associated with less susceptibility to SCH6,
VX-950 and telaprevir. More samples are being analyzed
to evaluate the impact of these findings.
Palavras-chaves: HCV, Protease Inhibitors, Resistance.
EXPERIMENTAL INFECTION OF RODENTS WITH
FAECES FROM EXPERIMENTALLY Vaccinia virus
INFECTED COWS: IMPLICATIONS IN THE
MAINTENANCE CYCLE DURING OUTBREAKS OF
VACCINIA BOVINE
ID: 00119-00002
93
ABSTRACTS
ID: 00120-00001
Área: 03 - Virologia Animal
Área: 04 - Virologia Básica
MORAIS, A.T.S, 2BRONZONI, R.V.M, 3NOGUEIRA,
M.L
1. FAMERP, Faculdade de Medicina de Sao Jose do Rio
Preto, Av.Brigadeiro Faria Lima, 5416; 15090-0002. Unesp
Ibilce, Universidade Estadual Paulista Julio de Mesquita
Filho, Rua Cristovao Colombo,2265; 15054-000
1
D’Anunciação, L., ABRAHÃO, J.S., BONJARDIM,
C.A., 4REHFELD, I.S, 5FERREIRA, P.C.P;, 6TRINDADE,
G.S.,, 7LOBATO, Z.I.P., 8GUEDES, M.I.M., 9Kroon, E.G.
1. UFMG, Universidade Federal de Minas Gerais, Av.
Antônio Carlos, 6627 - Pampulha, Belo Horizonte - Minas
Gerais
1
2
3
The Vaccinia virus (VACV), used in the vaccine to
eradicate smallpox, is the prototype virus of the genus
Orthopoxvirus and has been implicated in outbreaks of
bovine vaccinia in several properties in Brazil over the
past 11 years. The transmission of VACV between cattle
occurs mainly through the hands of the milkers or milking
equipment that have contact with sick animals. The
contact of the milker with teats and udder can cause
injuries in people hands and forearms. Viral particles can
be detected in faeces of infected animals for several
weeks after infection. This could be a possible route of
spread of the virus in the environment, like on a farm,
for example. The concern study of this pathway of viral
shed increases, as cattle faeces are widely used as
organic fertilizer in small plantations. This study
evaluates whether BALB/c mice exposed to bovine
faeces from cows infected with VACV could be infected
with this virus. For this, a group of 5 mice were exposed
for 20 days to shavings added with 2.3g of bovine faeces,
obtained from cows infected with VACV, sample Guarani
P2 (GP2V) and who were known to shedding the virus in
faeces. Samples of faeces and blood of mice were
collected periodically and submitted to PCR for vgf and
ha genes. Another group was similarly exposed to wood
shavings containing faeces from bovine not infected with
VACV and then were subjected to the tests listed. After
20 days, the animals were sacrified to collect serum for
neutralization assays. Viral DNA was detected in faeces
and blood samples of mice exposed to bovine faeces
contaminated with VACV during the whole period from
1st to 17th days post-infection, in contrast to the control
group. Moreover, antibodies anti-VACV were detected in
all animals exposed to bovine faeces with VACV at a
mean titer of 1/160, at day 20 post-infection. This study
shows that rodents can become infected with VACV
present in feaces of experimentally infected cattle, and
corroborate the theory that these animals may actively
participate in the chain of bovine vaccinia in rural Brazil.
Palavras-chaves: Route of spread of Vaccinia virus,
Vaccinia virus, viral maintenance cycle.
KNOCKING-DOWN THE EXPRESSION OF EIF3L
DECREASE YELLOW FEVER VIRUS REPLICATION
Yellow fever virus (YFV) is a mosquito-borne member of
the Flavivirus genus and causes an important disease.
NS5 is a nonstructural viral dual protein that presents
the methyltransferase and RNA-dependent RNA
polymerase (RNApol) domains, which are essential during
viral replication. Despite the existence and efficient
vaccine, there is no antiviral drug for the treatment of
yellow fever or other flaviviruses. Interactions among NS5
and cellular proteins have been studied for the
understanding of viral replication. Recently, we
demonstrated that NS5 physically interacts with
eukaryotic protein EIF3L using a two-hybrid system, in
vitro binding assay and an in vivo co-immunoprecipitation
method. The aim of this study was evaluate the role of
eIF3L in yellow fever infected cells on virus multiplication
using RNA interference mechanism. For this, the
expression of eIF3L was down regulated by transfecting
LLC-MK2 cells prior to infection with specific shRNA
duplex target to eIF3L. Knocking-down expression of
eIF3L significantly decreased the production of infectious
YFV. These results indicate that the interaction of eIF3L
with YF NS5 plays an important role in viral replication
and is discussed. The knowledge of such interactions
might provide a better understanding of viral replication
and might be useful in the rational development of drugs.
Financial support: Fapesp
Palavras-chaves: interaction protein-protein, yellow fever
virus, eIF3L protein.
ANTIVIRAL ACTIVITY OF THE Lentinula edodes ON
BOVINE HERPESVIRUS AND POLIOVIRUS.
ID: 00124-00001
Área: 04 - Virologia Básica
Rincão, V.P., 2 Yamamoto, K.A., 3 Galhardi, L.C.F.,
Bernardi, A.L.S., 5Soares, S.A., 6 Meirelles, L.D.P.,
7
Nozawa, C., 8Linhares, R.E.C.
1. UEL, Universidade Estadual de Londrina, Rod. Celso
Garcia Cid, Pr 445 Km 380, CEP 86051-980, Londrina PR2. UFC, Universidade Federal do Ceará, Av. da
Universidade, 2853 - Benfica - Fortaleza - CE, CEP
60020-181.
1
4
Fungi comprise a vast source of new pharmaceutical
ID: 00122-00001
94
ABSTRACTS
products. Among them, the basidiomycete Lentinula
edodes have distinguished itself by presenting
substances with antimicrobial, antitumor and antiviral
activity. The polysaccharides, derived from mushrooms,
have among others, proven antiviral activity. Therefore,
the objective of this study was to evaluate the antiviral
activity of aqueous (AqE) and ethanol (EtOHE) extracts
as well as a polysaccharide (LeP) isolated from L.
edodes on the replication of poliovirus-1 (PV-1) and
bovine herpesvirus-1 (BoHV-1). Cytotoxicity was
evaluated by MTT test. The virucidal activity, inhibition
of adsorption and antiviral activity at different times of
addition of extracts and polysaccharide (-2h,-1h, 0h, +1
h and +2 h), were monitored by plaque assay, at
concentrations of the 3.1 - 25 mg/ml (AqE), 0.375 - 3
mg/ml (EtOHE) and 0.025 - 0.2 mg/ml (LeP). The
evaluation of cytotoxicity of AqE, EtOHE and LeP
resulted in values of CC50 of 74.0 mg/ml, 29.9 mg/ml
and >4.0 mg/ml, respectively. The best results for the
antiviral activity of AqE and LeP were obtained for the
time of 0h with inhibition of, 82.5 and 63.8% for PV-1,
and 89.9 and 74.2% for BoHV-1, respectively. EtOHE
showed greater inhibition at the time of +1h with 94.6%
for PV-1 and 90.6% for BoHV-1. The substances tested
showed no significant virucidal activity and inhibition of
viral adsorption. This data were confirmed by indirect
immunofluorescence. The results suggest that extracts
and LeP has activity in the early stages of replication,
after adsorption step, for both viruses. However, new
experiments should be undertaken in order to find the
exact stage of viral replication where these substances
were acting.
Financial support: CNPq, Capes, Fundação Araucária,
Proppg/UEL.
Palavras-chaves: Antiviral activity, Lentinula edodes,
Bovine herpesvirus, Poliovirus.
estimates, 80% of the world’’s population use
phytomedicinal sources to supplement their health care.
In Brazil, due to high cost of industrialized drugs,
medicinal plants are still widely used as a treatment option.
Guazuma ulmifolia Lam., is popularly used in the tretment
of diarrhea, hemorrhages, fever, bronchitis, asthma, and
hypertension. Presently, despite vaccination campaigns,
poliovirus is still endemic in some countries in Africa
and Asia. It therefore represents a worldwide threat to
the reintroduction of the disease in countries where it is
under control. In this study, we investigated the in vitro
antiviral effect of a fraction (F14) from G. ulmifolia against
poliovirus type 1. The cytotoxity assay was determined
using de colorimetric dimethylthiazolyl diphenyl
tetrazolium bromide method (MTT), and the antiviral
activity by plaque assay. Different concentrations of the
substance were added before (-1h and -2h), during (0h)
and after (+1h and +2h) the infection. Twenty micrograms
per ml to 2.5ìgD ml of the test substance were added in
different stages of the viral infection. The 50% cytotoxic
concentration (CC50) was 40ìgD ml. The high percentage
of viral inhibition (%VI) occurred when the substance
was added at the moment of infection (time 0h), with
100% inhibition, at the concentrations of 20, 10 and 5ìgD
ml. The substance added 1 hour after the infection
showed an inhibition of 64.7% with a selectivity index
(SI) of 4.57 for the highest concentration. At the times 2h, -1h and +2h there was no inhibitory effect. The
virucidal test presented an inhibition of 78.8% and SI of
4.73 at the concentration of 20ìgD ml. We suggested
that the extract of G. ulmifolia acts in the initial stage of
viral replication and also presented direct effect on the
viral particle.
Financial support: CNPq, Capes, ProppgD UEL.
Palavras-chaves: Antiviral activity, Guazuma ulmifolia,
Poliovirus.
ANTIVIRAL ACTIVITY OF AN EXTRACT FROM
Guazuma ulmifolia AGAINST POLIOVIRUS TYPE 1
THE COMPLETE GENOME SEQUENCE OF A
SAPOVIRUS ISOLATED IN BRASILIA, BRAZIL
ID: 00124-00002
Área: 04 - Virologia Básica
ID: 00125-00001
Área: 05 - Virologia Humana e Saúde Pública
Czer nisz, E.S., 2 Albuquerque, D., 3 Rincão, V.P.,
Galhardi, L.C.F., 5 Mello, J.C.P., 6Linhares, R.E.C.,
7
Nozawa, C.
1. UEL, Universidade Estadual de Londrina, Rod. Celso
Garcia Cid, Pr 445 Km 380, Londrina - PR, CEP 860519802. UEM, Universidade Estadual de Maringá, Av.
Colombo, 5.790, Jd. Universitário, Maringá - PR - Brasil,
CEP 87020-900
1
1
4
According to the World Health Organization (WHO)
Anjos K, 2Nagata T, 3Lima L M P, 4Silva P A
1. UCB, Universidade Católica de Brasília, QS 07 lote 1
EPCT2. UnB, Universidade de Brasília, Asa Norte
Sapporo virus is a member of the genus Sapovirus within
Caliciviridae family, and a causative agent of acute
gastroenteritis. The Sapporo virus genome is linear,
positive-sense, single-stranded RNA, of approximately
7,5 kb that is polyadenylated at the 3’ terminus. At
moment, Sapporo virus is divided into five genogroups,
95
ABSTRACTS
whereas GIII is a porcine infecting one. In Brazil it is
known that the most causative gastroenteritis viruses
are rotavirus and norovirus, whereas the occurrence of
sapovirus seems sporadic. In the Federal District (DF),
a sapovirus was identified recently by RT-PCR targeting
partial capsid protein gene from stool sample. To
determine the sequence of complete genome, cDNA
fragments of genome were recovered by RT-PCR dividing
into two genomic regions, cloned and sequenced. After
sequencing using primer walking the results were
compared with all human Sapporo virus complete genome
sequences deposited in EMBL/DDBJ/GenBank
database, and the best matched partner was one of
Japanese (HM002617) and one of German (AY694184)
isolates with 72% identities. Both isolates were of
Genogroup I, genotype 1 (GI-1). As determined
previously, the DF sapovirus isolate was identified as
genotype 2 of Genogroup I (GI-2). In our knowledge this
is the first report of complete genome sequence of this
genotype.
Palavras-chaves: calicivirus, molecular epidemiology, sapovirus.
expressed by recombinant CTGV and VACV-WR in the
presence of ST-246 confirmed these results. Comet tail
formation in CTGV-infected cells was drastically inhibited
by 0.01 uM ST-246, whereas comet tails were still
observed for VACV-WR at 0.05 uM. CTGV isolates
collected in distinct outbreaks over the last decade
presented similar response pattern and susceptibility. In
vivo assays were performed by infecting mice with CTGV
and VACV-WR by tail scarification. Treatment of infected
mice with ST-246 by oral gavage confirmed the increased
susceptibility of CTGV to ST-246, which was able to
prevent lesion formation. These results strengthen the
promising potential of ST-246 to treat CTGV infection.
CTGV presented lower levels of F13 accumulation during
infection, as well as lower levels of F13 inserted into
extracellular particles when compared with VACV-WR.
The relationship of these data with the increased
susceptibility of CTGV to ST-246 effect is currently under
investigation.
Support: CNPq, Faperj, Capes.
Palavras-chaves: antiviral, poxvirus, vírus cantagalo,
vírus vaccinia.
THE ANTI-ORTHOPOXVIRUS DRUG ST-246 INHIBITS
CANTAGALO VIRUS REPLICATION WITH
INCREASED EFFICACY
ANTIVIRAL EFFECT OF POLYSACCHARIDES
ISOLATED FROM MARINE ALGAE ON CANTAGALO
VIRUS REPLICATION
ID: 00130-00001
Área: 04 - Virologia Básica
ID: 00130-00002
Área: 04 - Virologia Básica
1
Santos-Fernandes, E., 2Beltrame, C.O., 3Hruby, D.E.,
Jordan, R., 5Damaso, C.
1. UFRJ, Instituto de Biofísica Carlos Chagas FilhoUniversidade Federal do Rio de Janeiro, CCS, bloco C,
sala 28, Rio de janeiro, RJ 21941-5902. SIGA, SIGA
technologies, Corvallis, OR, USA
1
4
4
Cantagalo virus (CTGV) is a strain of vaccinia virus
(VACV; Poxviridae) that was isolated from pustular lesions
on cows and dairy workers in Rio de Janeiro State in
1999. CTGV-like outbreaks have been reported frequently
over the last decade in several Brazilian states. No
antiviral therapy is currently available, which makes the
need for an effective anti-CTGV treatment extremely
relevant. ST-246 is a potent inhibitor of orthopoxvirus
egress from cells and has proved its efficacy in cell
culture and in animal models. It targets VACV F13 protein,
leading to a block in the production of extracellular virus.
In this work, we evaluated the effect of ST-246 on CTGV
replication. Virus plaque formation assays in BSC-40
cells revealed that CTGV is 6 to 38 times more
susceptible to the drug than VACV strain WR and cowpox
virus, respectively. Titration of cell-associated viruses
indicated IC50 values of 0.00146 uM for CTGV and
0.0108 uM for VACV-WR. The analysis of â-gal activity
96
Barbosa, A.V.C., 2 Duar te, M.E., 3 Noseda, M.D.,
Gonçalves, A.G., 5Ducatti, D.R.B., 6Damaso, C.
1. UFRJ, Instituto de Biofísica Carlos Chagas Filho Universidade Federal do Rio de Janeiro, CCS - bloco C
sala C1-028 Cidade Universitária, Rio de Janeiro, RJ2.
UFPR, Depar tamento de Bioquímica e Biologia
Molecular- Universidade Federal do Paraná, Setor de
Ciências Biológicas, Centro Politécnico, Jardim da
Américas, Curitiba
Cantagalo virus (CTGV) was characterized as a vaccinia
virus strain (VACV; Poxviridae) in 1999. It is the etiologic
agent of a pustular disease in dairy cattle and milkers,
and frequent outbreaks have been reported in distinct
states of Brazil lately. No antiviral therapy is commercially
available for poxvirus infections, which accounts for the
increasing efforts to study novel anti-poxvirus drugs.
VACV entry is partially mediated by the interaction of
virus envelope proteins with the negative charges of
sulfates on glycosaminoglycans. In this study we have
analyzed the antiviral effect of four polysaccharides
isolated from marine algae on the replication of Cantagalo
virus: Iota-Gy, Lambda-2T, ECW and UV2S. We evaluated
their effects on virus plaque formation after the addition
of distinct concentrations of the polysaccharides during
the adsorption stage. After 48 hours of infection (hpi),
ABSTRACTS
the cells were stained with 0.1% crystal violet to visualize
viral plaques. All four substances inhibited the formation
of virus plaques, but Iota-Gy, Lambda-2T and ECW were
more effective, leading to nearly 80%, 88% and 77%
inhibition at 10ug/ml, respectively, whereas UV2S
reached 78% inhibition at 80 ug/ml. The elevation of the
MOI did not alter its effect when we investigated the
production of infectious particles by plaque assay on
BSC-40 cells. Virus yield after 24 hpi was inhibited by
approximately 90% when 10 ug/ml Lambda-2T was
added during adsorption. On the other hand, the addition
of Lambda-2T after virus internalization at 3 hpi did not
affect virus production and 100% of virus yield was
recovered after 24 hours. A preliminary assay indicate
that the addition of Lambda-2T to the cells at increasing
times prior to virus infection led to similar levels of
inhibition of virus yield. These results indicate that
Lambda-2T could represent a promising antiviral
substance of natural origin to treat Cantagalo virus
infection. Support: CNPq, Faperj, PIBIC, PRONEXCarboidratos.
Palavras-chaves: agentes antivirais, poxvírus, virus
cantagalo.
PERFORMACE ASSESSMENT OF IMMUNOLOGICAL TESTS USED FOR DETECTION
SURFACE ANTIGEN OF HEPATITIS B (HBsAg)
ID: 00131-00001
Área: 05 - Virologia Humana e Saúde Pública
Silva, J.R.C., 2 De-Farias, R.P., 3 Cavalcanti, B.B.,
Sobral-Filho, R.G ., 5Alves, C.R.M.S., 6Oliveira, V.F.,
7
Nascimento, A.C.H.D., 8Melo, M.M.M.
1. LACEN, Laboratório Central de Saúde Pública de
Pernambuco, Recife, Pernambuco2. FIP, Faculdades
Integradas de Patos, Patos, Paraíba3. UFPE,
Universidade Ferderal de Per nambuco, Recife,
Pernambuco
1
4
One of the most important markers for detection of
infection by hepatitis B is the surface antigen (HBsAg).
The aim of this study was to evaluate the performance
of four methods of immunoassay for the detection of
HBsAg. We studied 250 serum samples obtained from
the serum bank of the Central Laboratory of Pernambuco
(LACEN) in the period from January to June 2009, with
125 positive and 125 negative, each sample was
analyzed in four methodology (electrochemical/
ELECSYS, chemiluminescence/LIASON, by
microparticle enzyme immuno assay-MEIA/AxSYM,
enzyme immuno assay-ELISA/ETI-MAK-4) . In the
second phase of our study five positive serum samples
with different viral loads were diluted, in which nonreactive
plasma, were tested in 11 replicates per sample with
different titles. The study revealed 100% of sensitivity in
the methodologies used in the ETI-MAK-4 and AxSYM,
whereas the LIAISON and ELECSYS obtained a
sensitivity of 99.2% and 95.4% respectively. The
specificity of the four assay was similar, with few
discrepancies between the obtained results. With respect
the detections limit, the ELECSYS obtained a
performance equivalent to the AxSYM. The kit ETI-MAK4 reached a good detection limit and LIAISON was linear
remained even with the change in viral load in different
amount.
Palavras-chaves: Hepatitis, HBsAg, methods of
immunoassay.
IDENTIFICATION OF OCCULT HEPATITIS B IN
PATIENTS ATTENDING IN THE PUBLIC NETWORK
OF THE STATES OF PERNAMBUCO IN THE PERIOD
OF JANUARY TO JUNE 2010
ID: 00131-00002
Área: 05 - Virologia Humana e Saúde Pública
Cavalcanti, B. B., 2De-Farias, 3Silva, J. R. C, 4SobralFilho, R.G., 5Alves, C.R.M.S., 6Nascimento, A.C.H.D.,
7
Bastos, A.G.S., 8Melo, M.M.M.
1. FIP, Faculdades Integradas de Patos, Patos, Paraíba3.
UFPE, Universidade Federal de Pernambuco, Av. Prof
Moraes Rêgo, 1235, Cidade Universitária, Recife - PE4.
LACEN, Laboratório de Saúde pública do Estado de
Pernambuco, Rua João Fernandes Vieira, S/N - Bairro
Boa Vista, Recife - PE
1
The occult Hepatitis B, according to some studies, is
defined by the presence of HBV-DNA, but without the
detection of HBsAg in serum of patients infected with
viral load usually around 100-1000 IU / mL. In this work,
our objective was to identify occult hepatitis B in patients
treated in public health in Pernambuco during the period
January to June 2010. We used two groups of patients:
the first was of samples with positive serology for antiHBc and the second was isolated from samples with
HBV-DNA positive new treatment. Of 100 patients with
these characteristics were observed in only four results
compatible with the definition of occult hepatitis B. In
patients 1, 2 and 3 might notice a low viral load and
seroconversion of HBeAg to anti-HBe. Probably the virus
remains inside the cell leading to a state of chronic
infection. The fourth patient is with low viral load, but
with anti-HIV positive. It is a possible cause of occult
hepatitis B because the co-infection between HIV and
the hepatotropic virus suppresses the appearance of
surface antigen. It is very important the diagnosis occult
hepatitis B, because the maintenance of viral replication,
even at low levels, results in the development of
hepatocellular carcinoma in the patient, besides the
potential risk of disease transmission. More research is
97
ABSTRACTS
needed to answer open questions about the standard
isolated anti-HBc and occult HBV.
Palavras-chaves: Hepatitis B occult, ELISA, HBV DNA.
EARLY DIAGNOSIS DENGUE VIRUS INFECTION:
COMPARISON BETWEEN A REAL TIME RT-PCR AND
THE COMMERCIAL PLATELIA DENGUE NS1
ID: 00133-00001
Área: 05 - Virologia Humana e Saúde Pública
Dornas, F. P., 2Poloni, T. R. R. S., 3Alfonso, H. L.,
4
Amarilla, A. A., 5 Müller, V.D.M., 6 Oliveira, A. S.,
7
Figueiredo, L.T.M., 8Aquino, V.H.
1. FCFRP - USP, Departamento de Análises Clínicas,
Toxicológicas e Bromatológicas da Faculdade de Ciências
Farmacêu, Av. do Café, s/nº. - Campus Universitário da
USP - Ribeirão Preto - SP - 14040-92. CPV - FMRP,
Centro de Pesquisa em Virologia da Faculdade de
Medicina de Ribeirão Preto, Av. Bandeirantes, 3900 Monte Alegre - CEP 14049-900 - Ribeirão Preto/SP
1
Dengue, the most common human arboviruses, is
nowadays an important public health problem worldwide
with 2,5 billion people living in more than 100 endemic
countries. Dengue virus (DENV), belongs Flavivirus
genus, Flaviviridae family, includes four antigenically
distinct viruses (DENV 1-4) and it is transmitted by the
biting of mosquitoes of the Aedes genus. Infection with
any of the four serotypes can be asymptomatic or causes
illness ranging from mild viral syndrome, classic dengue
fever (DF) to hemorrhagic disease (DHF). The clinical
diagnosis is difficult in the acute phase of the disease
when the symptoms are very similar to other febrile
illness, corresponding to the laboratory the definitive
diagnosis. Recently, a new ELISA that detects NS1
protein in serum has been developed for early diagnosis.
In this study, we have compared the sensitivity of an in
house real time RT-PCR with a commercial ELISA NS1
for diagnosis of dengue virus infection. We have tested
86 serum samples from patients with dengue virus
infection and 6 samples from patients without infection
with an in house real time RT-PCR and the PLATELIA
DENGUE NS1 AG (BIO-RAD, France). The NS1 was
positive in 60/86 (70%), negative in 11/86 (12.7%) and
equivocal in 15/86 (17.4%), while the real time RT-PCR
was positive in 77/86 (89.5%) and negative in 9/86
(10.5%). NS1 and real time RT-PCR were negative in the
6 samples from patients without dengue infection. Thus,
the sensitivity for diagnosis of dengue infection in the
acute phase was 70% for NS1 and 89.5% for real time
RT-PCR, difference was statistically significant (p =
0.0013). All negative or equivocal samples (n = 26) in
the NS1 were those who had a viral load d”60 PFU/ml,
except one of them that had a viral load of 639 PFU/ml.
98
The real time RT-PCR showed to be more sensitive than
the NS1, principally, among those samples with low viral
load. Thus, we suggest the use of the real time RT-PCR
for early diagnosis in the dengue suspected cases.
Financial support: FAPESP Área: Virologia Humana e
Saúde Pública Tema: Virologia Molecular Apresentador:
Fábio Pio Dornas.
Palavras-chaves: Real Time RT-PCR, Dengue virus,
diagnosis.
LACK OF VIRULENCE DETERMINANT ASSOCIATED
WITH
DENGUE
FEVER
AND
DENGUE
HEMORRHAGIC FEVER IN THE E GLYCOPROTEIN
SEQUENCE OF DENGUE VIRUSES SEROTYPES 1,
2 AND 3
ID: 00135-00001
Área: 04 - Virologia Básica
Moreira, E. A., 2Paula, S. O. de, 3Oliveira, L. L. de,
Yotoko, K. S. C.
1. USP, Centro de Pesquisa em Virologia. Faculdade de
Medicina de Ribeirão Preto. Universidade de São Paulo.,
Av. Bandeirantes, 3.900. Monte Alegre - Ribeirão Preto SP. CEP: 14040-9002. UFV, Departamento de Biologia
Geral. Universidade Federal de Viçosa., Avenida Peter
Henry Rolfs, s/n . Campus Universitário. 36570-000
VIÇOSA - MG
1
4
Dengue is considered the most important arboviral
disease of humans, and four serotypes (DENV-1 to 4)
are known. Dengue fever has emerged as a serious
international public health threat with almost half of the
world’s population at risk of infection. The disease can
be classified as dengue fever (DF) or dengue hemorrhagic
fever (DHF). The reason some infected persons develop
DHF while others do not remains unknown. We performed
phylogenetic analyses of dengue virus (DENV) E
glycoprotein nucleotide sequences from patients with DF
or DHF to analyze the existence of a viral virulence
determinant correlated with the disease manifestation.
In this study, we used 140 sequences of DF including
virus from serotypes DENV-1, 2, 3 and 4, and 62
sequences of DHF from DENV-1, 2 and 3, available at
GenBank/NCBI. An E glycoprotein nucleotide sequence
from yellow fever was used as outgroup. The topology of
the consensus tree shows that haplotypes represent DF
and DHF are disperse in the phylogeny and appears
independent of the virus sequences. Nevertheless, some
ancestral states could be identified with significance as
causing DHF or DF. Our results show that with
phylogenetic analyses of E glycoprotein nucleotide
sequences we can not identify a marker correlated with
the clinical manifestations of dengue.
Palavras-chaves: Bayesian Inference, Phylogenetic
ABSTRACTS
Analysis, Dengue Hemorrhagic Fever, BayesTraits,
Multistate comparative method.
DETECTION OF DENGUE VIRUS IN FIELD-CAUGHT
AEDES AEGYPTI MOSQUITOES IN CAMPUS
PAMPULHA, UFMG, BRAZIL.
ID: 00136-00001
Área: 05 - Virologia Humana e Saúde Pública
VILELA, A.P.P., 2 SANTOS, J.R., 3 EIRAS, A.E.,
BONJARDIM, C.A., 5FERREIRA, P.C.P., 6KROON, E.G.
1. UFMG - LabVirus, Universidade Federal de Minas
Gerais - Laboratório de Vírus, Av. Antônio Carlos, 6627 Pampulha - Belo Horizonte - MG2. UFMG - LABEQ,
Universidade Federal de Minas Gerais - Laboratório de
Ecologia Química, Av. Antônio Carlos, 6627 - Pampulha
- Belo Horizonte - MG
1
4
Dengue virus (DENV) is the causative agent of dengue,
the most prevalent arbovirus in the world, and responsible
for approximately 50 million cases per year. Aedes aegypti
is the principal vector mosquito in DENV cycle.
Identification and DNA sequencing of DENV from fieldcaught mosquitoes is therefore important for
epidemiological investigations. The current dengue
epidemiologic situation in Minas Gerais State, Brazil, is
characterized by co-circulation of DENV-1, DENV-2 and
DENV-3 serotypes. Mosquitoes collected at campus
Pampulha, UFMG, were processed and analyzed for the
presence of viral genome. The collection period was from
April 2008 to February 2009 using the MosquiTRAP(R)
for catching mosquitoes. A total of 230 traps were
installed in 37 units of the campus, resulting in 7269
mosquitoes catched throughout the period. A total of 576
pools were formed containing from one to 20 mosquitoes
each, and the pools were separated by month and
geographical location. Viral genome was detected in
8,33% of pools of Ae. Aegypti pools by RT-PCR. Of the
48 positive pools, 34 pools were serotyped. Fifteen pools
were positive for DENV-2 and 19 for DENV-3. This work
presents an analysis of C-prM gene sequences from
mosquitoes naturally infected with DENV-2 and DENV-3
in campus Pampulha, UFMG, Brazil.
Palavras-chaves: Dengue virus, A. aegypti, detection.
EVOLUTION OF A 24 NUCLEOTIDES INSERTION AT
THE 69 REVERSE TRANSCRIPTASE (RT) CODON OF
HIV-1 PATIENTS ON HAART, SAO PAULO -BRAZIL.
ID: 00138-00001
Área: 05 - Virologia Humana e Saúde Pública
1
4
Souza, L.O., 2 Lopes, G.I.S.L, 3 Batista, J.P.G,
Cavalcanti, J.S., 5Souza, O.N., 6Brigido, L.F.M.
1. IAL, Instituto Adolfo Lutz - São Paulo, Av. Dr. Arnaldo
,355 Prédio da Virologia Cep. 01246-902 São Paulo/SPBrasil2. PUCRS, Pontificia Universidade Católica do Rio
Grande do Sul, Av. Ipiranga 6681 - Partenon- Porto Alegre
Cep: 90619-900 RS - Brasil
The 69 codon is one of the main sites for mutations in
the RT gene. Insertions occur in about 1-2% of patients
on failure HAART. Two or more amino-acids (aa)
insertions have a lower frequency. The simultaneous
occurrence with TAMs (41/210/215) is associated with
resistance to all NRTIs. The aim of this study is to
evaluate the effect of a 24 nucleotides insertion at codon
69RT in one patient, which the insert has an aa change
inside of it after two years. Two sequences of plasma
samples (March/07 and July/09) from a patient, attended
at a public health service, were submitted to Stanford
algorithm to resistance test and locate the insertion. The
alignment was carried out using the BioEdit software
with the HXB2 reference strain and subtype B consensus
sequences. Both samples present an insertion of eight
aa at 69RT. The first sample presents the insertion
STGKKDST (CD4 381cell/mm3 and Viral Load-VL
5,64log) and the second with an aa change (E) at the
first T (CD4 893cell/mm3 and VL 4,32log) this fragment
has similarity of human genome by Blast. Inserts larger
than two aa at 69RT are rarely described. We found no
reports in the literature of insertions longer than seven
aa. In this case we note that it is not a duplication of
bases which is more commonly. To the best of our
knowledge, this is the first 69RT insertion of part of human
genome in HIV genome. The distance between the
sequences was 2% in two years. Despite the insertion,
patient VL decreased over a log and CD4 increased
slightly, albeit the presence of other mutations that
produce impact on resistance profile. Insertion of multiple
aa at 69RT, and the change of one aa to other with distinct
physical-chemical characteristics demonstrate great
flexibility of this region. Further studies on the presence
of insertions at 69RT must be performed to identify the
actual impact of these changes in the mechanisms of
drug resistance.
Palavras-chaves: HIV-1, Antiretroviral therapy, Insertion,
Reverse transcriptase.
MONITORING OF BVDV STATUS LEVEL HERDS
ANALYZED THROUGH BULK MILK ELISA
ID: 00139-00001
Área: 03 - Virologia Animal
Almeida, L.L., 2Hein, H.E., 3Marks, F.S., 4Rodenbusch,
C.R., 5Costa, E.F., 6Corbellini, L.G., 6Canal, C.W.
4. Lab.Virologia, FAVET, Laboratorio de Virologia,
Faculdade de Veterinaria, Av. Bento Goncalves, 9090.
1
99
ABSTRACTS
Porto Alegre, RS.5. UFRGS, Universidade Federal do
Rio Grande do Sul, Av. Bento Goncalves, 9090. Porto
Alegre, RS.6. EPILAB/UFRGS, Laboratorio de
Epidemiologia, Faculdade de Veterinaria, Av. Bento
Goncalves, 9090. Porto Alegre, RS.
Analysis of bulk tank milk for antibodies against bovine
viral diarrhea virus (BVDV) is a practical approach to
monitor infection in dairy herds. The knowledge of the
infection status is impor tant to prevent viral
dissemination to non-infected herds. The objective of the
present study was to monitor BVDV infection in herds
assessing their antibody levels in bulk tank milk. Samples
were collected twice with a one-year interval, from 90
dairy herds non-vaccinated against BVDV. Additionally,
location data of the herds was obtained to elaborate maps
illustrating the herd infection-status using the geographic
information system (GIS) software ArcMap (ESRI). Bulk
milk samples were tested for antibodies against BVDV
using a commercial indirect ELISA kit. Samples were
considered positive or negative according to the level of
antibodies detected using a cut off previously
established. Milk samples collected in 2009 presented
69 negative and 21 positive results. In 2010, 76 samples
were negative and 14 positive. It was possible to visualize
the spatial distribution of the herd infection-status;
apparently there was no cluster of infection. Monitoring
with two consecutive tests showed that 68 herds
remained negative, suggesting that they are free from
BVDV. On the other hand, 13 herds remained positive
and are suspicious of harboring active infection, needing
complementary testing. Seroconversion was found in
only one herd during the period, evidencing recent
infection and an annual incidence of BVDV of 1.4% (1/
69). Other 8 herds showed an accentuated decline of
antibody levels, and this finding suggests viral clearance.
The present study estimated a low prevalence and low
incidence in a population of dairy herds not using specific
BVDV control measures and semi-intensively reared.
Herd monitoring for BVDV infection can be performed
through bulk milk ELISA and complemented by
geographic information system.
Financial support: CNPq, CAPES, FAPERGS.
Palavras-chaves: antibody, bovine, bvdv, epidemiology,
milk.
ENTEROVIRUS IDENTIFICATION AFTER INCREASE
IN VIRAL MENINGITIS REPORTS DUE TO
INTENSIFICATION OF EPIDEMIOLOGICAL
SURVEILLANCE ACTIONS IN SÃO JOSÉ DO RIO
PRETO, SÃO PAULO, 2009
ID: 00145-00001
Área: 05 - Virologia Humana e Saúde Pública
B. C. Machado, 2Calux, S.J., 3Russo, D.H., 4 Reis,
A.F.N., 5Antônio, L.E.A., 6Reina, M.C.F.P., 7Carvalhanas,
T.R.M.P., 8Carmona, R.C.C., 9Timenetsky
1. NDE-IAL, Núcleo de Doenças Entéricas - Centro de
Virologia - Instituto Adolfo Lutz, Av. Dr. Arnaldo, 355 Cerqueira César São Paulo SP2. SMS-SJRP, Departamento
de Vigilância Epidemiológica - Secretaria Municipal da
Saúde de São José do Rio Preto, Av. Alberto Andaló, 30303.
GVE-SJRP, Grupo de Vigilância Epidemiológica XXIX São
José do Rio Preto, Rua das Palmeiras, 1404. CVE-SP,
Centro de Vigilância Epidemiológica - Secretaria de Estado
da Saúde de São Paulo, Av. Dr. Arnaldo, 351 - Cerqueira
César - São Paulo SP
1
The Human Enterovirus (HEV) genus of the
Picornaviridae family, are the most commonly agents
associated with viral meningitis in Brazil and all over the
world. In 2009, 267 meningitis cases were notified in
São José do Rio Preto (SJRP), an increase of 42%
compared with 2008, with 188 cases. In the same period
an increase in the number of viral meningitis cases was
detected corresponding to 60%, 114 in 2008 and 182 in
2009. The objective of the study was to describe the
laboratory aspects and epidemiological profile of viral
meningitis cases diagnosed in SJRP in the period
between March and October 2009 after intensified
surveillance. Clinical samples of 20 cases were sended
to the Enteric Diseases Branch of Adolfo Lutz Institute
in São Paulo (NDE-IAL). Viral isolation in RD, Hep-2 and
VERO cell cultures was made. Immunofluorescence Assay (IFA), Polymerase Chain Reaction Reverse Transcription (RT-PCR) and Paired Sera Titration
by Microneutralization Technique were employed for
identification. In total, 55% (11/20) of the cases presented
positive results. Predominant serotype was echovirus 6
(7/11; 63.6%) (one case concluded by seraconversion
observation) followed by echovirus 4 (3/11; 27.3%). One
case was positive for HEV genus(1/11; 9.1%). Results
reveal a co-circulation of two distinct serotypes of HEV
in SJRP in 2009. This epidemiological pattern despite
the fact that it is not so uncommom among the HEV is a
prerequisite to lead to recombination between co-infection
serotypes. It is important to highlight that the increase
in viral meningitis cases in 2009 is direct related to
implantation of intensified surveillance that brought better
quality in diagnosis mainly with improving capability of
public health officials. The integrated work between the
three surveillance levels (city, region and state) and the
NDE-IAL resulted with viral meningitis cases in SJRP
elucidated and collaborates with data for further studies
of HEV circulation in São Paulo State.
Palavras-chaves: Enterovirus, Viral Meningitis,
Epidemiological Surveillance.
EXPERIMENTAL INFECTION OF RABBITS WITH
100
ABSTRACTS
VACCINIA VIRUSES ISOLATED FROM HORSES IN
SOUTHERN BRAZIL
ID: 00146-00001
Área: 03 - Virologia Animal
Cargnelutti, J.F., 2 Masuda, E.K., 3 Schmidt, C.,
Nogueira, P.R.K., 5Weiblen, R., 6Flores, E.F.
1. UFSM, Universidade Federal de Santa Maria, Av.
Roraima, 1000 CEP97105-900 Santa Maria - RS
1
4
Two genotypically and phenotypically distinct isolates
of vaccinia virus (VACV) were recovered from an
outbreak of severe cutaneous disease in horses in Rio
Grande do Sul State in 2008 (Pelotas 1 and 2). Both
viruses were inoculated individually in 35 white New
Zealand rabbits, divided in 7 groups, to evaluate the
susceptibility of rabbits to VACV. The animals were
inoculated in the paranasal sinuses with different doses:
Pelotas 1 groups: 1)102.5TCID50, 2)104.5TCID50,
3)106.5TCID50; Pelotas 2 groups: 4)102.5TCID50, 5)
104.5TCID50, 6)106.5TCID50; and Group 7) 1mL of
culture medium. The animals were evaluated for 21 days
for clinical signs. Nasal swabs for virus isolation and
blood samples for virus detection by PCR and serology
were collected. Necropsy and histopathology was
performed after death or euthanasia. All animals from
groups 2, 3, 4, 5 and 6 showed serous to hemorrhagic
nasal discharge, eye serous discharge, severe
respiratory distress, diarrhea and 100% lethality. Only
two animals from group 1 remained healthy. Two animals
from groups 1 and 4 showed secondary lesions on the
ears and eyelids. Viral shedding was observed from day
1pi to death in groups 2, 3, 5 and 6, and was intermittent
in groups 1 and 4. Viremia was detected in alternate days
in all Pelotas 1 inoculated animals, and at days 4-9pi in
Pelotas 2 groups. Neutralizing antibodies were detected
in all VACV inoculated animals that survived more than
9 days. At necropsy, severe interstitial pneumonia and
liquid intestinal content were observed. Histological
examination of lungs revealed severe diffuse interstitial
fibrosing pneumonia with necrossupurative bronchopneumonia. The results showed that, regardless of viral
dose inoculation, rabbits develop systemic severe
disease after inoculation of both VACV isolates.
Palavras-chaves: Equine, Pathogenesis, Poxvirus,
VACV, variola.
BIANCHI, E., 2MARTINS, M., 3 NOGUEIRA, P.R.K.,
WEIBLEN, R., 5FLORES, E.F.
1. UFSM, Universidade Federal de Santa Maria, Av.
Roraima prédio 20 sala 4200 bairro Camobi CEP
97105900 Santa Maria
1
4
Field isolates of bovine viral diarrhea virus (BVDV) display
a high genetic and antigenic diversity, that can difficult
diagnosis and vaccine formulation. The present study
characterized genetically and antigenically 20 isolates of
BVDV from Rio Grande do Sul (2000 - 2010). The isolates
were associated with a variety of clinical conditions that
included respiratory or gastroenteric disease, skin lesions,
abortions, animals with retarded growth, and persistently
infected animals. Most isolates (19 or 95%) belong to the
non-cytopathic biotype (NCP), one isolate (5%) had a
mixture of viruses NCP and cytopathic (CP). The
sequencing based on the region of 260 - 360 nucleotides
of the untranslated 5’UTR of the viral genome followed by
phylogenetic analysis identified 13 isolates of BVDV-2
(65%) and seven BVDV-1 (35%). It was not possible to
associate genotypes or subgenotypes with clinical
conditions: both BVDV-1 and BVDV-2 were involved in
different clinico-pathological syndromes. Analysis of
reactivity with a panel of 20 monoclonal antibodies (MAbs)
revealed a marked variability in the major envelope
glycoprotein (E2) among viruses of the same genotype,
but especially between different virus genotypes. Virus
neutralization assays (VN) with anti-sera of BVDV-1 and
BVDV-2 reference strains against the isolates revealed
variable levels of cross-reactivity between viruses of the
same genotype, and lack or very low reactivity between
viruses of different genotypes. These results suggest a
higher prevalence of genotype 2 viruses in the cattle
population of Rio Grande do Sul, confirms the remarkable
antigenic diversity and reinforce the need to include viruses
of genotypes BVDV-1 and BVDV-2 in vaccines.
Palavras-chaves: BVDV, antigenic difference,
phylogenetic analysis, serum neutralization.
HEPATITIS B VIRUS INFECTION IN RURAL
COMMUNITIES OF THE PANTANAL WETLANDS,
MATO GROSSO DO SUL STATE, CENTRAL BRAZIL
ID: 00149-00001
Área: 05 - Virologia Humana e Saúde Pública
BIGATON, G., 2TELES, S.A.
1. UFMS, UNIVERSIDADE FEDERAL DE MATO
GROSSO DO SUL, FILINTO MULLER S/N2. UEMS,
UNIVERSIDADE ESTADUAL DE MATO GROSSO DO
SUL, AV. PRESIDENTE VARGAS S/N3. IPTSP, Instituto
de Patologia Tropical e Saúde Pública, AV. AFONSO
PENNA S/N
1
GENOTYPIC AND ANTIGENIC PROFILE OF BOVINE
VIRAL DIARRHEA VIRUS ISOLATES FROM RIO
GRANDE DO SUL (2000-2010)
ID: 00148-00001
Área: 03 - Virologia Animal
SOROEPIDEMIOLOGIA DA INFECÇÃO PELO VÍRUS
101
ABSTRACTS
DA HEPATITE B EM POPULAÇÃO PANTANEIRA DE
MATO GROSSO DO SUL Bigaton, G.1; Teles, S.A.3;
Matos, M. F. C.1; Mousquer, G. M.1; Murat, P. G.1;
Nascimento D.1; Froes, I. B.1; Pires, F. R.1; Martins, R.
M. B.3; Oliveira, R. D.2; Dorval, M. E. C.1; Motta-Castro,
A. R. C.1 1Universidade Federal de Mato Grosso do Sul,
Campo Grande, MS; 2Universidade Estadual de Mato
Grosso do Sul, MS; 3Instituto de Patologia Tropical e
Saúde Pública, Goiânia, GO, Brazil .E-mail:
gaubigaton@hotmail.com The hepatitis B virus infection
(HBV) is spread worldwide and its prevalence varies
largely in several geographical regions in the world. In
Brazil, varied prevalence rates have been found in
different areas and population groups. The objective of
this paper is to investigate the prevalence of the
serological markers for hepatitis B in the riverside
population living in Pantanal sul-mato-grossense, a region
considered to be socioeconomically poor. The study
involved 321 individuals living in four remote communities
in the Alto Paraguai Basin: Serra do Amolar/São
Lourenço(n=74), Paraguai Mirim (n=100), Porto da Manga
(n=74) and Passo do Lontra ( n=73). The samples were
tested for detection of HBsAg, total anti-HBc and antiHBs markers using immune enzymatic essay. The
positive HBsAg samples were analyzed for HBeAg and
anti-HBe. PCR was used to detect the HBV DNA of the
HBsAg reagent samples. Out of 321 riverside dwellers
with ages ranging from 1 to 89 years (mean 26.7 and
standard deviation ± 19.3), 52% (167/321) were males,
54.2% were Caucasians and 43.3% reported to have a
steady relationship. Most individuals (93.4%) presented
low socioeconomic and educational levels, poor hygiene
and housing conditions and their houses did not did not
have proper sewage system. Fishing was the main
subsistence activity. The global prevalence of HBV
infection was 36.5%, ranging from 15.1% (Passo do
Lontra community) to 61% (Paraguai Mirim community).
Positivity for total anti-HBc associated with HBsAg was
1.6% and the association of anti-HBc with anti-HBs,
indicating former infection and immune response, was
found in 32.1% (103/321). Isolated anti-HBc was seen in
2.8% (9/321). The presence of anti-HBs as an isolated
marker, indicating antecedent of vaccine response was
detected in 32.4% (104/321) of the individuals. From the
two HBsAg positive samples, four were anti-HBe reagent.
HBV DNA was detected in 40% (02/05) of the HBsAg
reagent samples and in 80% (04/05) of the positive antiHBe. The HBV isolates were identified as genotypes D
and F. The findings reinforce the need for additional
programs of health education and alternative schemes
against hepatitis B with a view to increasing the vaccine
coverage in isolated communities of Central Brazil.
Financial support:FUNDECT/MS
Área: Virologia Humana
Tema: HBV
102
Arquivo: HBV populações rurais MS
Apresentador: Gláucia Bigaton
Palavras-chaves: HEPATITIS B, PANTANAL,
SOROEPIDEMIOLOGY.
SEROPREVALENCE OF HEPATITIS B VIRUS
INFECTION AND ASSOCIATED RISK FACTORS
AMONG PRISON INMATES IN MATO GROSSO DO
SUL, BRAZIL
ID: 00149-00002
Área: 05 - Virologia Humana e Saúde Pública
Stief, A.C.F., 2 Mar tins, R.M.B., 3 Andrade, S.M.O.,
Castro-Souza L, 5Murat, P.G., 6Mousquer, G.J., 7Teles,
S.A., 8Bigaton, G., 9Francisco, R.B.L., 10Pompilio, M.A.,
11
Motta-Castro, A.R.C.
1. UFMS, Universidade Federal de Mato Grosso do Sul,
Campo Grande, MS, Brasil, AV. Filinto Muller s/nº2.
IPTESP, Instituto de Patologia Tropical e Saúde Pública,
Universidade Federal de Goiás, Goiânia, GO, B, Campus
Universidade3. UFG, Faculdade de Enfermagem,
Goiânia, GO, Brasi, Goiânia4. LACEM, Laboratório
Central de Saúde Pública, Campo Grande - MS
1
4
Inmates are at high risk for infection with hepatitis B
virus (HBV) because of the risk behavior related to
confinement conditions. The aim of this study was to
estimate the prevalence of HBV infection and associated
factors among prison inmates in Campo Grande, MS,
Brazil. A total of 408 individuals were interviewed on
socio-demographic characteristics, associated factors
and HBV vaccination by means of a standardized
questionnaire. Blood samples were collected from all
participants and serological markers for HBV were
detected by enzyme-linked immunosorbent assay.
Hepatitis B surface antigen (HBsAg) and/or antibodies
against hepatitis B core antigen (anti-HBc) positive
samples were tested for HBV-DNA by polymerase chain
reaction. The overall prevalence of HBV infection was
17.9% (95% CI: 14.4-22.0). The HBsAg carrier rate was
0.5%. Anti-HBc and anti-HBs were detected in 56
individuals (13.7%), indicating that they had been infected
and developed natural immunity, and 15 (3.7%) were
positive for anti-HBc only. Ninety eight prisoners (24%)
had only anti-HBs, suggesting that this population had
low vaccine coverage. An occult HBV infection rate of
0% was found among anti-HBc-positive individuals.
Multivariate analysis of associated factors showed that
age > 35 years, low schooling level, and illicit drug use
are significantly associated with HBV infection. Our data
show that HBV infection prevalence is similar or slightly
lower than those reported in other prison inmates in Brazil.
Independent predictors of HBV infection in this population
include older age, low schooling level and illicit drug use.
ABSTRACTS
Financial support: FUNDECT/MS Área: Virologia Humana
Tema: Hepatitis B Arquivo: HBV-prison MS Apresentador:
Gláucia Bigaton Palavras-chaves: associated factors,
Hepatitis B, prevalence, prisoners.
ANTIHERPES ACTIVITY AND MODE OF ACTION OF
A CARDENOLIDE DERIVATIVE
ID: 00150-00001
Área: 04 - Virologia Básica
Bertol, J.W., 2Pádua, R.M., 3Rigotto, L.F., 4Kreis, W.,
Braga, F.C., 6Barardi, C.R.M., 7Simões, C.M.O.
1. UFSC, Universidade Federal de Santa Catarina,
Campus Reitor João David Ferreira Lima - Trindade 88040-970 - Florianópolis2. UFMG, Universidade Federal
de Minas Gerais, Av. Antônio Carlos, 6627 - Pampulha Belo Horizonte - MG CEP 31270-9013. FAU, FriedrichAlexander Universität, Schlossplatz 4 - 91054 Erlangen,
Germany
analysis of viral protein expression showed that
compound 12 inhibited á (ICP27) gene and, consequently,
â (UL42) and ã (gB and gD) genes expression. The antiATPase activity evaluation presented NKA activity
inhibition of 50% at 4ìM, suggesting that the alteration
of cell electrochemical gradient may be involved in the
mechanism of viral inhibition. Additional experiments are
now under development in our laboratory to better
understand its complete mode of action. Financial
support: CNPq/CAPES Palavras-chaves: HSV, Cardiac
Glycoside, Antiviral.
1
5
Cardiac glycosides belong to a naturally derived
compounds group that bind and inhibit Na+K+-ATPase
(NKA). Its members have been used for treatment of
heart failure and atrial arrhythmia, such as digoxin and
digitoxin. The antiherpes activity (anti-HSV types 1 and
2) of some cardenolides was recently published. The HSV
are responsible for oral, ocular and genital regions
infection and may also establish it in nervous system.
The treatment of herpes infections with conventional
nucleoside analogues (acyclovir) is effective in most
cases, but drug-resistance may arise due to prolonged
treatment. Thus, based on the results of an antiherpes
screening of 68 cardenolide derivatives realized in our
laboratory, one compound (number 12) has been chosen
due to its high anti-HSV activity [selectivity indices
(SI=CI50/IC50) of 645 (HSV-1 KOS); 792 (HSV-1 29-R);
and 1.238 (HSV-2 333)]. Cytotoxicity (CC50) was evaluated
on Vero cells by using MTT assay, and antiviral activity
(IC50) was tested against HSV-1 (KOS strain) by plaque
reduction assay. The mechanism of action was evaluated
through a sequence of assays to identify in which steps
of the viral replication process this compound acts. The
results showed that compound 12 did not display virucidal
effects neither inhibition on viral attachment and entry
into the cells (dextran sulphate used as positive control).
However, this compound maintained its anti-HSV-1
activity even at the higher tested MOI (0.4) showing a
virus yield reduction of 4 Log10 in comparison to untreated
controls. This MOI is thousand times higher than MOI
(0.0004) used for the screening. This compound also
inhibited the viral cell-to-cell spread and interfered in viral
release inhibiting 64% of this step, at the concentration
equal to its IC50 value (0.27ìM), and showing a dependent
concentration-response inhibition. The western blotting
ANTIHERPES SCREENING OF CARDENOLIDE
DERIVATIVES
ID: 00150-00002
Área: 04 - Virologia Básica
Bertol, J.W., 2Carvalho,A., 3Pádua, R.M., 4Kreis, W.,
Braga, F.C., 6Barardi, C.R.M., 7Simões, C.M.O.
1. UFSC, Universidade Federal de Santa Catarina,
Campus Reitor João David Ferreira Lima - Trindade 88040-970 - Florianópolis2. UFMG, Universidade Federal
de Minas Gerais, Av. Antônio Carlos, 6627 - Pampulha Belo Horizonte - MG CEP 31270-9013. FAU, FriedrichAlexander Universität, Schlossplatz 4 - 91054 Erlangen,
Germany
1
5
Cardiac glycosides are natural compounds found in
specific plant species. Some of them, like digoxin, are
clinically employed for the treatment of congestive heart
failure due to their positive inotropic effect, which occurs
by the inhibition of the Na+/K+- ATPase. Meanwhile,
recent papers reported novel therapeutic possibilities for
cardiac glycosides including antiherpes activity. Human
infections associated to HSV are spread around the globe
being responsible for significant morbidity and mortality.
HSV are able to cause litic, latent and transformed
infections. Although effective, the conventional treatment
of HSV infections with nucleoside analogues may display
drug resistance, if prolonged. Likewise, there is a resistant
viral strain dissemination problem, especially among
immunocompromised individuals. Hence, the present
study intended to evaluate the antiherpes activity of 68
cardenolides, including natural compounds and synthetic
derivatives. Cytotoxicity was evaluated on Vero cells by
using MTT assay, and antiviral activity was tested against
HSV-1 (KOS strain) by viral plaque number reduction
assay. Results were expressed as 50% cytotoxic
concentrations (CC50) and 50% viral replication inhibitory
concentrations (IC50), respectively, in order to calculate
the selectivity index (SI=CC50/IC50) of each tested sample.
From this screening, it was found that three compounds
(numbers 66, 67 and 68) showed the highest antiherpes
activity when compared to the tested reference drug
103
ABSTRACTS
acyclovir (ACV). The SI values of these compounds were
10.095, 3.289 and 1.786, respectively,whereas ACV
presented SI of 580. These results will be further explored
in our laboratory to elucidate the mechanism of
antiherpes activity.
Financial support: CNPq/CAPES.
Palavras-chaves: HSV, Cardiac Glycoside, Antiviral.
PRELIMINARY EVALUATION OF ANTIHERPES
ACTIVITY OF PREPARATIONS FROM Echinodorus
grandiflorus LEAVES
ID: 00151-00002
Área: 04 - Virologia Básica
SINCERO, T.C.M., 2SILVA, I.T., 3GARCIA, E.F., 4DE
OLIVEIRA, M.A., 5 Braga, F.C., 6 Barardi, C.R.M.,
7
SIMÕES, C.M.O.
1. UFSC, Universidade Federal de Santa Catarina,
Depar tamento de Ciências Farmacêuticas, UFSC,
Florianópolis, SC, Brazil2. UFMG, Universidade Federal
de Minas Gerais, Faculdade de Farmácia, UFMG, Belo
Horizonte, MG, Brazil
activity with SI values of 3630, 6591 and 1571,
respectively, pointing out diterpenes as antiherpes
constituents of E. grandiflorus. Further studies are
required to identify the bioactive compounds, as well as
their mechanism of action, which are in course in our
laboratory. Financial support: CNPq, CAPES/PNPD.
Palavras-chaves: Herpes Simplex Viruses, Echinodorus
grandiflorus, antiviral, diterpens.
PRELIMINARY ANTIHERPES AND ANTIGENOTOXIC
ACTIVITY OF Uncaria tomentosa AND Uncaria
guianensis
ID: 00152-00001
Área: 03 - Virologia Animal
1
Herpes Simplex Viruses (HSV) are known to be
responsible for many human diseases, and efforts have
been made to find new drugs for their treatment. Natural
products are an inexhaustible source of bioactive
molecules with promising pharmacological activities,
including antiviral activity, which justifies the research
in this area. Echinodorus grandiflorus (Alismataceae) is
a medicinal plant popularly known as “chapéu-de-couro”,
whose leaves are traditionally used as tonic, diuretic,
and anti-inflammatory. The chemistry of the species
comprises diterpenes, flavon-C-glycosides and
hydroxycinnamoyltartaric acids (HCTA), classes of
compounds which present inhibitory effects of viral
replication. In this study, we evaluated the antiherpes
activity of six preparations from leaves of E.grandiflorus
[EG1 (30% EtOH extract ), EG2 (CH2Cl2 extract), EG3
(EtOAc diterpene-rich fraction), EG4 (CH2Cl2 diterpenerich fraction), EG5 (HCTA-rich fraction) and EG6
(flavonoid-rich fraction)]. Cytotoxicity was evaluated on
Vero cells by using MTT assay, and antiviral activity
was tested against HSV-1 (KOS strain) by viral plaque
number reduction assay. Results were expressed as 50%
cytotoxic concentrations (CC50) and 50% viral replication
inhibitory concentrations (IC50), respectively, in order to
calculate the selectivity index (SI=CC50/IC50) of each
tested sample. The addition of these samples to HSVinfected cells had no effect on virus replication. However,
all of them exhibited significant antiherpes activity when
cells were pretreated with these samples prior to virus
infection showing high SI values. Among the tested
samples, EG2, EG3 and EG4 showed more promising
104
SINCERO, T.C.M., 2SCHNEIDER, N.F.Z., 3CAON, T.,
BORRÉ, G.L., 5PAVEI, C., 6KAISER, S., 7MORAES,
R.C., 8ORTEGA, G., 9BARARDI, C.R.M., 10SIMÕES,
C.M.O.
1. UFSC, Universidade Federal de Santa Catarina,
Florianópolis, SC.2. UFRGS, Universidade Federal do
Rio Grande do Sul, Porto Alegre, RS.
1
4
Herpes Simplex Viruses (HSV) are responsible for many
human infectious diseases persisting latent during the
lifetime of the host. Prolonged therapy with the available
antiherpes drugs had induced drug-resistance, hence,
the development of new antiherpes agents are needed.
Uncaria tomentosa and U.guianensis (cat’s claw) have
shown anti-inûammatory, antioxidant and antitumoral
effects. Alkaloids, triterpenes and tannins have been
isolated from both species, and their biological activities
could be correlated to these compounds. This study
investigated the cytotoxicity, antiherpes and
antigenotoxic effects of one crude ethanolic extract (UT1)
and four fractions purified from U. tomentosa (UT2 to
UT5), and two samples prepared with U. guianensis +
U. tomentosa (UT6 and UT7). The enriched fractions of
triterpenes are UT2; UT4; UT5 and alkaloids UT3. The
cytotoxicity was evaluated on Vero cells by using MTT
assay and antiherpes activity was tested against HSV
type 1 (KOS strain) by viral plaque reduction assay.
Results were expressed as 50% cytotoxic
concentrations (CC50), 50% viral replication inhibitory
concentrations (IC50) and selectivity index (SI=CC50/IC50).
The antigenotoxic effect was evaluated by Comet assay
using pre-, simultaneous and post-treatment, after
irradiation with ultraviolet (UV) light to induce DNA
damage. The extracts UT1, UT6 and UT7 showed the
most promising antiviral activity in the pre-treated assay,
showing SI values of 1.779, 1.890 and 3.841,
respectively. The antigenotoxic effects were
concentration-dependent and were higher during the
simultaneous treatment, and a reduction on DNA repair
ABSTRACTS
capacity was observed for UT6 and UT7. UT1 showed
the highest DNA repair effect. Once UV-induced damage
may accentuate herpetic symptoms, the protective effect
of Uncaria products is desirable for an antiherpes
formulation. Further in vitro and in vivo studies are
necessary in order to confirm these results.
Financial support: CNPq,CAPES.
Palavras-chaves: Herpes Simplex Viruses, genotoxicity,
cytotoxicity, Uncaria guianensis, Uncaria tomentosa.
IN VITRO ANTIVIRAL EFFECTS OF DITERPENE
ISOLATED FROM THE BRAZILIAN BROWN ALGA
Canistrocarpus cervicornis (DICTYOTACEAE)
ID: 00153-00001
Área: 05 - Virologia Humana e Saúde Pública
BARBOSA, J.E.F., 2V, M.A., 3 PEREIRA, P.S., 4
CAVALCANTI, D.N., 5DE PAULA, J.C., 6TEIXEIRA, V.L.,
7
GIONGO, V., 8PAIXAO, I.C.P.
1. UFF, DEPARTAMENTO DE BIOLOGIA CELULAR E
MOLECULAR, UNIVERSIDADE FEDERAL FLUMINENSE, RUA OUTEIRO JOÃO BATISTA, CENTRO NITERÓIRJ3. UFF, DEPARTAMENTO DE BIOLOGIA MARINHA,
UNIVERSIDADE FEDERAL FLUMINENSE, RUA
OUTEIRO JOÃO BATISTA, CENTRO-NITERÓI, RJ4.
UNIRIO, Instituto de Biociências, UNIVERSIDADE
FEDERAL DO ESTADO DO RIO DE JANEIRO, AVENIDA
PASTEUR, 458, URCA, RJ
obtained with HSV-1 control according to TCID50 method.
Although both compounds have shown viral inhibition
rates above 90% we observed specifically for diterpene
2 the same inhibitory rate of acyclovir. The virucidal
activity of the diterpenes 2 from C. cervicornis, showed
only after 4 hours of incubation a virucidal activity at the
dilution of 106 (78%) and for this reason we cannot say
that these compounds may be presented as possible
virucidal biomolecules. Therefore, we considered these
substances promising as anti-HSV-1 and concluded that
it was possible to advance to the in vivo antiviral studies.
Palavras-chaves: ANTIVIRAL AGENTS, Canistrocarpus
cervicornis, Cytotoxicity, Dolastane diterpene.
HEPATITIS “A” OPTIMIZED DNA-BASED VACCINES
EVALUATION: ANTIGEN EXPRESSION, PROCESSING AND SECRETION ANALYSIS
1
Canistrocarpus cervicornis is an important and abundant
alga from the Brazilian coast. Although many diterpenes
have been isolated from Brazilian C. cervicornis, the
antiviral activity of this type of compound is unknown.
Human simplex virus type-1 (HSV-1) are etiologic agents
of diseases involving infection that varies in severity
from sub-clinical infections to fatal ones. Drug-resistant
HSV strains frequently emerge during a long ACV-based
treatment due to the reduced expression or the non
functioning of timidine kinase. Therefore, the search for
new antiviral agents, especially with different
mechanisms of actions, is a crucial goal. The present
study evaluated Dolastane diterpenes (1 and 2) isolated
from the Brazilian brown alga Canistrocarpus cervicornis,
collected in Praia do Forno, Armação de Búzios, Rio de
Janeiro State, Brazil and this study aims to evaluate its
antiviral activity against HSV-1. A MTT-based assay on
cytotoxicity was used to evaluate the diterpenes 1 and
2 and it was observed that both compounds were not
cytotoxic for VERO cell lineage presenting cytotoxicity
values of 1423,3μM and 706 μM for diterpene 1 and 2
respectively, besides we observed that the CC50 of the
diterpene 1 was 48% higher than acyclovir’s and
presented in vitro a reduction of 3 logs of infectivity for
diterpene 2 of C. cervicornis when compared to the values
ID: 00154-00001
Área: 01 - Imunobiológicos
Sousa, R.C.V., 2Viana, I.F.T., 3Cruz, F.S.P., 4Melo, A.B.,
Marques Jr., E.T.A., 6Dhalia, R.
1. IAM - FIOCRUZ, Fundação Oswaldo Cruz, Instituto
Aggeu Magalhães, Laboratório de Virologia e Terapia
Experimental, Av. Prof. Moraes Rego. Cidade
Universitária, Recife - PE, Brasil. CEP:50670420
1
5
Hepatitis A virus (HAV) infection affects million of people
worldwide causing a disease that ranges from a lack of
symptoms to severe cases. Although commercial viral
inactivated vaccines are available, delivery of these
formulations for developing countries massive
vaccination campaigns is still impracticable, as
consequence of the highly expensive manufacturing cost.
One strategy to overcome this limitation consists on
development of DNA-based vaccines that take potential
advantages as more stability, safety and lower
manufacturing costs. HAV genome could be divided into
three parts: 1-5‘ Untranslated region (UTR); 2-a single
ORF that encodes 4 structural proteins (VP4, VP2, VP3
and VP1) and 7 non-structural proteins (2A, 2B, 2C, 3A,
3B, 3C and 3D); 3-3’ polyadenilated UTR. The polyprotein
is processed by the viral 3C protease generating the
viral capsid particle. From capsid proteins, VP1 and VP3
are target for diagnostic kits and vaccines, considering
their highly immunogenicity. Here we described an
optimized DNA-based HAV truncated polyprotein (truncHAV) vaccine construct, that we developed to induce
non-infective virus like particles production. We also fused
trunc-HAV to the N-terminal type 1 human Lysosome
Associated Membrane Protein – hLAMP1, generating
LAMP-trunc-HAV construct, aiming to facilitate antigens
secretion. Firstly we expressed VP1, VP3 and 3C
individual proteins in bacteria in order to raise specific
105
ABSTRACTS
polyclonal antibodies in rabbit. All purified antibodies were
able to recognize, specifically, their respective antigens.
Currently we are transfecting eukaryotic cells with both
DNA-based vaccines to check antigens expression,
processing and secretion marking antigens with the
polyclonal antibodies obtained. According our results we
are intending to immunize mice with both vaccines, in
order to evaluate their capacity to induce satisfactory Tcell and B-cell responses, before advance to non-human
primate models.
Financial support: IAM/FIOCRUZ.
Palavras-chaves: Hepatitis A, DNA-based vaccine,
Expression analysis.
MODELING DENGUE CLINICALS FORM CLASSIFICATOR APPLYING ARTIFICIAL NEURAL
NETWORK USING GENOMICS DATA
ID: 00154-00002
Área: 05 - Virologia Humana e Saúde Pública
Oliveira, T.W.F., 2 Sousa, R.C.V., 3 Acioli-Santos, B.,
Buarque de Lima-Neto, F., 5Marques, E.T.A.
1. IAM/FIOCRUZ, Departamento de Virologia e Terapia
Experimental, Centro de Pesquisas Aggeu Magalhães,
FIOCRUZ, Av. Prof. Moraes Rego. Cidade Universitária.
Recife-PE, Brasil.2. POLI-UPE, Depar tamento de
Sistemas e Computação, Escola Politécnica de
Pernambuco, Universidade de Pernambuco,, Rua
Benfica, 455 - Madalena - Recife/PE CEP: 50.750-4703.
UP, Department of Infectious Diseases and Microbiology,
University of Pittsburg, University of Pittsburg, Pittsburg,
PA 15260. USA
1
4
The time between initial symptoms and reaching a
diagnosis of dengue hemorrhagic fever can be the cause
of death for some patients. According to recent
recommendations by the World Health Organization
(WHO) the dengue infection can be classified as dengue
and severe dengue. The differentiation between those
clinical forms is based on clinical aspects and laboratorial
exams, but these are not always available and
sometimes might be inaccurate. Because that, this
research try to minimize this problem, developing a tool
that uses artificial neural networks (ANN) to better
interpret the risk of a subject to develop the severe form
of dengue using genomic data (genetic polymorphism).
The polymorphism data was obtained from application
of mass genotyping technique from Illumina. The
univariable approach showed the participation of 6 loci
(IFNg - A/G, MBL2 – A/G, OASL – C/T, STAT1 C/T,
FAAD – C/T, INDO – A/G) related to the innate immunity
as important on the phenotype severe dengue. The ANN
was trained using genetic data from 105 patients, 26
DHF, 49 CDF and 30 DF. Based on the ANN model of the
106
genotypic data, the ANN model could predict the clinical
outcome correctly in 85% the dengue cases. These
results are encouraging, and if validated in a larger
independent cohort this method can be used to identify
individuals with high risk to develop severe dengue and
could facilitate the implementation of public programs
for Prevention, Control and Treatment of Dengue.
Financial support: CNPq/NIH
Palavras-chaves: bioinformatics, classificator,
genomics.
RECOMBINATION AND PSEUDORECOMBINATION
DRIVING THE EVOLUTION OF TWO BEGOMOVIRUS
SPECIES IN BRAZIL
ID: 00155-00001
Área: 06 - Virologia Vegetal
LIMA, ATM, 2 SILVA, FN, 3 ROCHA, CS, 4 ALVESJÚNIOR, M, 5ZERBINI, FM
1. UFV, Universidade Federal de Viçosa, Avenida Peter
Henry Rolfs, Campus Universitário, 36570-000 VIÇOSA
- MG
1
The genus Begomovirus (family Geminiviridae) includes
dicot-infecting, whitefly-transmitted viruses with the
genome comprised of one or two molecules of circular,
single-stranded DNA. In Brazil, tomato-infecting
begomoviruses have emerged as serious pathogens due
to the introduction of a new biotype of the insect vector.
Tomato rugose mosaic virus (ToRMV) and Tomato severe
rugose virus (ToSRV) are often found in tomato fields.
The complete sequence of the DNA-Bs of ToSRV
(NC_009612) and ToRMV (NC_002556) show an identity
of 98%. Additionally, the high nucleotide identity (97%)
between their common regions suggests the possibility
that the two viruses share the same DNA-B. The objective
of this work was to study how recombination and
pseudorecombination modulated the evolution of these
two begomoviruses. Recombination analysis were
performed using the RDP3 package. Infectious clones
corresponding to the DNA-A and DNA-B of the ToSRV
and ToRMV were used for biolistic inoculation in all
possible combination between the DNA-A and DNA-B
components. The formation of viable pseudorecombinants
was verified by rolling circle amplification following restrict
enzyme analyses for the DNA-A and DNA-B of the two
viruses at 28 days after inoculation. Recombination
analysis showed that the common region and part of the
gene encoding the replication-associated protein were
transferred from ToSRV to ToRMV. The formation of viable
pseudorecombinants supports the hypothesis that the
DNA-A of recombinant ToRMV captured the DNA-B of
its parental virus (ToSRV). Symptom severity was
equivalent in single or mixed infections, indicating that
ABSTRACTS
synergism does not occur between these two viruses.
However, ToRMV negatively affect the accumulation of
ToSRV when the two viruses were inoculated together,
indicating that the recombinant is more adapted than
the parental virus. The results presented here demonstrate
the importance of these mechanisms for the evolution
and adaptation of begomoviruses. Financial support:
FAPEMIG, INCT em Interação Planta-Praga.
Palavras-chaves: Begomovirus, Geminiviridae,
Pseudorecombination, Recombination.
it in relation to others. The presence of two point
mutations in a small conserved sequence is the possible
explanation for the relative distance from this phage
compared to the others 936-type phages.
Financial support: CNPq, FUNARBIC, FAPEMIG Área:
Virologia Básica Tema: Lactococci Bacteriophages
Apresentador: Lucas Mayrink Assis Palavras-chaves:
BACTERIOPHAGE, BRAZILIAN DAIRY FACTORY,
LACTOCOCCAL, LACTOCOCCAL BACTERIOPHAGE,
PHYLOGENETIC ANALYSIS.
PHYLOGENETIC ANALYSIS OF A LACTOCOCCAL
BACTERIOPHAGE ISOLATED FROM A BRAZILIAN
DAIRY FACTORY
PRELIMINARY EVALUATION OF ANTIHERPES
ACTIVITY OF SULFATED POLYSACCHARIDES
FROM Lithothamnion calcareum (Hapaladiceae)
ID: 00161-00001
Área: 04 - Virologia Básica
ID: 00162-00001
Área: 04 - Virologia Básica
Assis, L.M, 2 Eller, M.R, 3 Dias, R.S, 4 Pereira, A.L,
Oliveira, L.L, 6Paula, S.O
1. UFV, UNIVERSIDADE FEDERAL DE VIÇOSA,
Avenida Peter Henry Rolfs, s/n Campus Universitário
36570-000 VIÇOSA - MG
Cardozo, F.T.G.S, 2 Malagoli, B. G., 3 Braga, F. C.,
Barardi, C.R.M, 5Simões, C.M.O.
1. UFSC, Universidade Federal de Santa Catarina,
Campus Universitário. CEP: 88040-900. Trindade,
Florianópolis-SC. Brazil.2. UFMG, Universidade Federal
de Minas Gerais, Av. Antônio Carlos, 6627. Pampulha,
Belo Horizonte-MG. Brazil.
1
5
PHYLOGENETIC ANALYSIS OF A LACTOCOCCAL
BACTERIOPHAGE ISOLATED FROM A BRAZILIAN
DAIRY FACTORY Assis, L.M.; Eller, M.R.; Dias, R.S.;
Pereira, A.L.; Oliveira, L.L.; Paula, S.O. Laboratório de
Imunovirologia Molecular e Glicoimunologia,
Departamento de Biologia Geral, Universidade Federal
de Viçosa, Viçosa, MG, Brazil. E-mail:
lucas.assis@ufv.br Bacteriophages that infect Lactic
Acid Bacteria (LAB) are the main problem in the industrial
processing of dairy products in the world. The infection
of starter cultures by these agents results in significant
economic losses. Few information is known about the
endemic species on Brazilian factories due to lack of
research in the area. In this work, we perform the
sequencing and analysis of a conserved sequence of
the genome of a Lactococcus lactis bacteriophage
isolated from failure fermentation in a Brazilian factory.
Total DNA was extracted and a Polymerase Chain
Reaction was conducted to amplify a 318 bp fragment,
which was cloning in DH5á Escherichia coli in a pGEM®
T Easy Vector System. The plasmid was purified in
accordance to manufactures recommendations to
posterior sequencing. Phage sequence was compared
to sixteen related sequences retrieved from GeneBank.
Alignment was performed with multiple alignment program
CLUSTALW and an unrooted tree was constructed using
the distance-based neighbor-joining method. Phage
sequence was clustered with others of the 936-type
phages, a group of Siphoviridae family and showed high
identity (96 %) to CB19, a 936-type phage isolated from
Canada in 2009, which shares a common mutation with
1
4
The exploration of marine environment represents a
promising strategy for searching new active antiviral
compounds. Marine algae are a rich source of naturally
occurring sulfated polysaccharides, for which many
biological activities have been described, including
antiviral action. Herein, we report the in vitro antiherpes
activity of Lithothamnion calcareum (Hapaladiceae)
seaweed. This species is found in deep waters and
commercialized in Brazil as a mineral supplement. The
samples were acquired from a commercial source
(Phoster Algamar). Fractions enriched in polysaccharides
were obtained by extraction with 1% and 2% w/v Na2CO3
solution (fractions B1 and B2, respectively), at 60 °C,
followed by precipitation with ethanol and dialysation (cut
off 10,000 Da). Fractions B1 and B2 were characterized
as sulfated polysaccharides by spectroscopic data and
chemical reactions. The cytotoxicity of these samples
on Vero and GMK-AH1 cells was assessed by MTT
assay, and the potential antiherpes activity was evaluated
by viral plaque assay. The tested viruses were Herpes
Simplex Virus types 1 (HSV-1, KOS strain) and 2 (HSV2, strain 333), and an acyclovir resistant strain (HSV-1
29R). Two treatment strategies were used: (1)
simultaneous treatment, when the samples were added
simultaneously with viruses; and (2) post-infection
treatment, when samples were added after virus infection.
Results were expressed as selectivity index (SI=50%
cytotoxic concentration/50% viral replication inhibitory
concentration). The tested samples did not show
107
ABSTRACTS
considerable activity by treatment 2, but induced an
efficient inhibition of viral replication in treatment 1. The
obtained SI values varied from 20 to 42 for HSV-1 strains,
while HSV-2 was more sensitive to the samples, with SI
values of 993 and 990 for B1 and B2, respectively. These
findings suggest that B1 and B2 fractions may act
inhibiting the initial steps of HSV replication, or by a
virucidal mechanism.
Palavras-chaves: antiviral activity, Lithothamnion
calcareum, HSV, sulfated polysaccharides.
ANTIHERPES ACTIVITY OF TWO SULFATED
POLYSACCHARIDES FROM Agaricus blazei WITH
DIFFERENT SULFATION DEGREES
ID: 00162-00002
Área: 04 - Virologia Básica
respectively. The obtained SI values for S-P1 were 78
(HSV-1 KOS) and 64 (HSV-1 29-R), whereas S-P2 was
significantly (test t) more active with SI values of 393
(HSV-1 KOS) and 242 (HSV-1 29-R). These findings
corroborate the fact that the antiviral activity of sulfated
polysaccharides can be related to their sulfate content.
Palavras-chaves: Agaricus blazei, antiviral activity,
HSV-1, sulfated polysaccharides.
STABILITY OF HUMAN ADENOVIRUS, MURINE
NOROVIRUS, AND HEPATITIS A VIRUS IN
SEAWATER WITH AND WITHOUT U.V. IRRADIATION
ID: 00163-00001
Área: 02 - Virologia Ambiental
Corrêa, A. A., 2 Souza, D. S. M., 3 Moresco, V.,
Kleemann, C.R., 5Ramos, A. P. D., 6Garcia, L. A. T.,
7
Barardi, C. R. M.
1. ufsc, universidade federal de santa catarina, ufsc,
ccb, mip, lab. virologia aplicada, campus trindade.
88040970
1
4
Cardozo, F.T.G.S., 2Camelini, C.M., 2Mascarello, A.,
Rossi, M.J., 4Nunes, R.J., 6Barardi, C.R.M., 7Simões,
C.M.O.
1. UFSC, Universidade Federal de Santa Catarina,
Campus Universitário. CEP: 88040-900. Trindade,
Florianópolis-SC. Brazil.
1
4
Agaricus blazei Murill (syn A. subrufescens, A.
brasiliensis) is a Basidiomycete fungus native to the
Atlantic forest of Brazil. This mushroom is a potential
source of glucans with special conformational features
and high molecular weight, such as glucans with (1’!6)beta- and (1’!3)-beta- linkages. Additionally, it is well
known that sulfated polysaccharides present antiviral
action. In this sense, this work describes the chemical
sulfation of A. blazei fruiting bodies polysaccharides by
the chlorosulfonic acid-pyridine method, and the
evaluation of their potential antiherpes activity. In order
to analyze the influence of sulfation degree on antiviral
activity, we performed this reaction in 30 and 90 minutes
to obtain two sulfated derivatives: S-P1 and S-P2,
respectively. S-P1 and S-P2 were characterized by
infrared spectroscopy (IR) and elemental analysis (CHN).
The cytotoxicity of these samples on Vero cells was
assessed by MTT assay, and the potential antiherpes
activity was scrutinized by viral plaque number reduction
assay. The tested viruses were Herpes Simplex Virus
types 1 (HSV-1, KOS strain) and an acyclovir resistant
strain (HSV-1 29R). Results were expressed as 50%
cytotoxic concentrations (CC50) and 50% viral replication
inhibitory concentrations (IC50), in order to calculate the
selectivity index (SI=CC50/IC50) of each tested sample.
Their IR spectra presented two new absorption bands at
1.253 cm-1 and 810 cm-1. These bands are related to
sulfate groups, confirming that sulfation had actually
occurred. Elemental analysis revealed the presence of
11% and 14% sulfur content for S-P1 and S-P2,
108
Viruses have been linked to episodes of illnesses related
to consumption of contaminated shellfish. The risk may
be reduced by depuration, which is a method that reduces
the levels of microorganisms present in mollusk meat,
decreasing the potential for associated infections. The
aim of this study was to investigate the stability of Human
Adenovirus (HadV2), Murine Norovirus (MNV-1) and
Hepatitis A Virus (HAV), in natural seawater with and
without UV irradiation. 300L of natural seawater were
artificially seeded with HAV (108 FFU/ml), MNV-1 (108
PFU/ml) and HAdV2 (109 FFU/ml) and treated by 36W
UV lamp, into the depuration tank, with recirculation, for
up to 120h, in three independents assays. One liter of
viral seeded seawater was harvested every 24h and viral
particles were concentrated by flocculation method using
skimmed milk. The kinetics of viral decay was evaluated
by quantification of genomic copies (q(RT)PCR for the
three viruses) and infectious viral particles (ICC-RT-PCR
for HAdV2). Based on qPCR results, the kinetics
observed were different for each virus, reaching, at 120h
of contact time, 5log10 and 3log10 reduction for HAdV2
and HAV, respectively; for MNV-1, was observed a
4,5log10 reduction at 72h under UV treatment. Despite
the genome detection, HAdV2 was able to remain
infectious only up to 72h, according ICC-RT-PCR results.
Assays involving viral stability without UV irradiation,
demonstrated a progressive reduction of viral load along
the 120h of seawater recirculation for three viruses (2log10
for HAdV2; 2,5log10 for HAV and 3log10 for MNV. In
conclusion, the natural decreasing of viral load can be
due to seawater composition, viral aggregation and the
existence of environmental factors, such as ionic strength
ABSTRACTS
and compounds naturally found in seawater. This can
also interfere with the disinfection process by UV
treatment. These data will be subsequently useful to plan
water disinfection in shellfish depuration tanks.
Financial support: MAPA/CNPq
Palavras-chaves: viral stability, desinfection, enteric
viruses.
EVALUATION OF TROPICAL SOURCE WATERS AND
MOLLUSKS IN SOUTH BRAZIL USING MICROBIOLOGICAL AND CHEMICAL PARAMETERS
ID: 00163-00002
Área: 02 - Virologia Ambiental
Ramos, A. P. D., 2Souza, D. S. M., 3Moresco, V., 4Pilotto,
M. R., 5Delfino, N., 6Leal, D. A. G., 7Franco, R.M.B.,
8
Taniguchi, S., 9Bícego, M. C., 10Montone, R. C., 11Barardi,
C. R. M.
1. UFSC, Universidade Federal de Santa Catarina, CCB,
MIP, Lab. de Virologia Aplicada2. UNICAMP,
Universidade Estadual de Campinas, Inst. de Biologia,
Depto. de Biologia Animal, Lab. de Protozoologia3. USP,
Universidade de São Paulo, Depto. de Oceanografia
Física, Química e Geológica.
1
When seawater is contaminated with human feces and
chemical products, the mollusks growing in this water
are able to concentrate contaminants as a consequence
of their filter-feeding process. The aim of the present
study was to detect Human Adenovirus (HAdV),
Salmonella, Cryptosporidium sp oocysts, Giardia sp
cysts and chemical contaminants in oysters allocated
in different sites in Florianopolis. Cultivated Pacific
oysters (Crassostrea gigas) were distributed in four
places. Animals were analyzed at time zero (before
distribution) and after 14 days post-allocation (in this
case, oysters and 10 liters of water samples). For
virological analyses in oysters, viral particles were eluted
by organic method previously described. For water
samples, the flocculation method using acidified
skimmed milk was used for viral concentration.
Cryptosporidium sp oocysts and Giardia sp cysts were
analyzed by direct immunofluorescence in the oysters
and water samples. Chemical contamination by total
aliphatic hydrocarbons, polycyclic aromatic hydrocarbons
and linear alkylbenzene were also measured in the
animals. At time zero no genome copies/gram (gc/g) of
HAdV were detected in the oysters from the farm. After
14 days, the results obtained for all the parameters
analyzed were: site 1: 5.0 x 104 gc/g (HAdV) and 250
cfu/g (total coliforms); site 2: 43 gc/g and 610 cfu/g; site
3: 7.0 x 104 gc/g, 350 cfu/g and 8.3 Cryptosporidium
oocysts/animal and site 4: 3.65 x 105 gc/g and 4.1 x
103 cfu/g. For water samples; site 1: no HAdV detection;
site 2: 1.9 x 105 gc/L; site 3: 3.0 x 108 gc/L and site 4:
1.7 x 107 gc/L and 25 Giardia cysts/L. According to
chemical analyses, the site 4 was the most contaminated
site of this study. These results had confirmed the human
contamination impact on various ocean water sites as a
consequence of the sewage discharge that contributes
for the oyster’s contamination and reinforces the need
for oyster’s depuration.CNPq 578200/2008-2.
Palavras-chaves: Human adenovirus, microbiological contamination, mollusks.
DNA REPLICATION OF CANTAGALO VIRUS IS THE
MAJOR TARGET FOR THE ANTIVIRAL ACTION OF
BREQUINAR
ID: 00164-00001
Área: 04 - Virologia Básica
SCHNELLRATH, L.C., 2DAMASO, C.
1. IBCCF, Instituto de Biofísica Carlos Chagas Filho, Av.
Carlos Chagas - Ilha do Fundão
1
Cantagalo virus (CTGV; Poxviridae) was isolated in 1999
from pustular lesions in cows and milkers in the State of
Rio de Janeiro. Since then many similar outbreaks in
several states of Brazil have been reported. Therefore
the study of new compounds with anti-poxvirus activity
is extremely important, because there are no antiviral
therapies available today. The immunosupressor agent
Brequinar (BQR) blocks de novo pyrimidine biosynthetic
pathway through the inhibition of the fourth enzyme,
dihydroorotate dehydrogenase (DHO-DHase), preventing
the formation of pyrimidine nucleotides and,
consequently, the synthesis of RNA and DNA.
Leflunomide, another DHO-DHase inhibitor, has been
reported to inhibit the replication of several human viruses.
Therefore, our goal in this work is to evaluate the antiviral
activity of BQR on CTGV replication, analyzing the steps
of the virus cycle. BQR at 1uM already led to a complete
inhibition in virus plaque formation in BSC-40 cells 48
hours post-infection. Cell viability was inhibited only 20%
at 75 μM BQR. The IC50 was 0,014 μM for CTGV progeny
production, and concentrations higher than 1 μM were
able to inhibit more than 90% of viral titers. Other
Orthopoxvirus were sensitive to BQR. When we used 30
μM BQR, virus titers, viral protein and DNA accumulation
revealed a strong inhibition over 99% when compared to
control cells with virtually no detection of virus
macromolecules and an inhibition of virus progeny
production exceeding 2-log. The severe effect of BQR
on the late phase of the viral cycle was confirmed after
observing the inhibition of â-galactosidase expression
under control of a virus initial-late promoter. Immunofluorescence assays confirmed that BQR was able to
severely inhibit CTGV DNA replication. Addition of 100
109
ABSTRACTS
uM uridine was able to reverse the inhibition of virus
DNA accumulation, as well as to recover progeny
production and the accumulation of viral proteins to
control levels, indicating that this drug is probably
targeting the stage of DNA synthesis.
Palavras-chaves: Cantagalo virus, Brequinar, de novo
pyrimidine biosynthetic pathway.
ANALYSIS OF VIRUS-HOST CELL INTERACTIONS
DURING COTIA VIRUS INFECTION
ID: 00165-00001
Área: 04 - Virologia Básica
AFONSO, P.P., 2MERMELSTEIN, C.S., 3CUNHA-ESILVA, N.L., 4DAMASO, C.
1. UFRJ, Universidade Federal do Rio de Janeiro, Av.
Carlos Chagas Filho, 373 - Ilha do Fundão - Rio de Janeiro
1
Cotia virus SPAn 232 (COTV) was isolated in 1961 from
sentinel mice in Brazil. Our group has recently
characterized COTV and suggested its classification as
a new genus of the Poxviridae. We are currently interested
in studying novel aspects of virus-host cell interactions.
BSC-40 (monkey) and C6 (rat) cells infected with COTV
generate similar kinetics of intra- and extracellular progeny
production and accumulate viral proteins and DNA at
similar levels. Nevertheless, virus-induced CPE is not
observed in BSC-40 cells up to 96 hpi, whereas cell
rounding is noticed in C6 cells at 24 hpi, although less
intense than VACV-induced CPE. In addition, COTV
produces tiny plaques in BSC-40 monolayers only after
8 days of infection. Wound-healing assays in BSC-40
monolayers revealed no migration of COTV-infected cells
up to 40 hpi. Disorganization of microtubule network is
observed at 24 hpi in BSC-40 cells with no alterations in
cell morphology. In some cells, microtubule
rearrangement colocalizes with virosomes and anti-COTV
staining. At 16 hpi in BSC-40 cells, we observe partial
preservation of actin stress fibers and the presence of
small virus-tipped actin tails. The number of actin tails
per cell is nearly 58% and 85% of those produced in
VACV-infected BSC-40 cells and in COTV-infected C6
cells, respectively. Nevertheless, actin tails are mostly
circular in COTV-infected BSC-40 cells and nearly 50%
shorter than those observed for VACV. In addition, tails
projecting from cell borders are rarely detected, as
opposed to what is observed in COTV-infected C6 cells
and VACV-infected BSC-40 cells. Support: CNPq, Faperj.
Palavras-chaves: Citoesqueleto, Poxvírus, Vírus Cotia.
THE EFFECT OF THE TIME-OF-ADDITION OF THE
FRACTIONS P1 E P1S FROM Azadirachta indica IN
THE REPLICATION OF HERPES SIMPLEX TYPE 1
ID: 00166-00001
Área: 04 - Virologia Básica
Faccin, L.C., 2Yamamoto, K.A., 3Hattori, L., 4Rincao,
V., 5Ray, B., 6Linhares, R.E.C., 7Nozawa, C.
1. UEL, Universidade Estadual de Londrina, Rodovia
Celso Garcia Cid, Pr445, Km380, Campus Universitário,
Londrina - PR2. UB, University of Burdwan, Bardhaman,
West Bengal, India
1
Azadirachta indica, commonly known as neem, is used
in India for gastrointestinal disorder such as diarrhea.
Anti-inflammator y, anti-pyretic, hypoglycemic,
antimicrobial and anti-cancerous activities were also
reported. The antiviral property was also described for
measles, bovine herpes and human immunodeficiency
agent. Herpes simplex-1 (HSV-1) is the cause of orofacial
lesions, keratoconjunctivitis and encephalitis, and
characterized by the establishment of latency. Currently,
several compounds have been studied to deal with HSVresistant strains. In this work we investigated the effect
of A. indica at different stages of HSV-1 replication. P1
is the native polysaccharide from A. indica leaves and
P1S its sulfated derivative. The MTT assay determined
the toxicity of the test substances and showed that 50%
cytotoxic concentration to P1 was 1440 μg/ml and to
P1S was higher than 1600 μg/ml. The antiviral activity
was evaluated by plaque assay by the addition of various
concentrations (25-200ìg/ml) of the fractions, before (-2
and -1 hour), during (0 hour) and after viral infection (1
and 2 hour). The time-of-addition assay showed the
greatest percentage of viral inhibition (%VI) when the
substances were added at time 0 hour, 63.5% for P1
and 91.2% for P1S at 200 ìg/ml. The 50% inhibitory
concentration (IC50) for P1 was 177 ìg/ml and the
selectivity index (SI) 8.13. For P1S the IC50 was < 25
ìg/ml and the SI > 64. When the substances were added
before or after the infection a decrease in the %VI was
observed. We demonstrated that P1S is the more
effective than P1. We conclude that the sulfate group
acts an important hallmark in anti-viral activity. Therefore,
we suggest that the main mode of action of the sulfated
derivative is the interference with envelope structures
by masking structures necessary for virus attachment
or entry into host cells.
Suport: CNPq, CAPES, Fundação Araucária and
Proppg/UEL.
Palavras-chaves: Herpes simplex virus, Azadirachta
indica, antiviral.
VARIATION AND DIVERSIFICATION OF HIV-1
SUBTYPES B AND C PROTEASE: AN EVOLUTIONARY ANALYSIS
ID: 00167-00001
110
ABSTRACTS
Área: 05 - Virologia Humana e Saúde Pública
Área: 03 - Virologia Animal
Maletich, DJ, 2Araújo, LAL, 3Medeiros, RM, 4Matte,
MCC, 5Almeida, SEM, 5Almeida, SEM
1. PPGBM-UFRGS, Programa de Pós-Graduação em
Genética e Biologia Molecular UFRGS, Av. Bento
Çonçalves, 9500 Prédio 43323 Porto Alegre, RS , Brasil
Cx Postal 150532. CDCT-FEPPS, Centro de
Desenvolvimento Científico e Tecnológico FEPPS, Av.
Ipiranga, 5400 - Porto Alegre, RS, Brasil
1
1
The protease activity of human immunodeficiency virus
type 1 (HIV-1) is required for the maturation of virions
into infectious particles, making this protein an important
target for antiretroviral drugs. Indiscriminate use of
antiviral therapy and the rapid evolution of viral proteins
is an important issue in the emergence of resistance
mutations. As a genetic barrier, HIV-1 may not exceed a
number of amino acid variations, then the spectrum of
possible viral variants appears to be limited by patterns
of nucleotide substitution. We analyzed patterns of
nucleotide and amino acid variation in 537 protease
sequences from HIV-1 subtypes B and C circulating in
Brazil. Maximum likelihood and Bayesian approaches
were used to explore the phylogenetic relationships and
selecting the sequences that harbor a high number of
nucleotide variations within their subtype. Positiveselected sites were inferred from viral phylogenies by
multiple evolutive likelihood models using PAML software
and as a tool for recognizing and comparing the
evolutionary rates; it was also implemented to assess
the rates of mutation between the subtypes. Evolutionary
and phylogenetic analyses show that although both
subtypes are spreading exponentially in Brazil, the HIV1 subtype C has a higher growth rate than that of subtype
B. Furthermore, analysis in progress show that there is
a difference between the rates of mutation, comparing
subtype B and C. These preliminary data show a slower
mutation rate for subtype C, but in contrast, a high level
of genetic variability in amino acids (76,5% variable sites
for subtype C and 63,5% variable sites for subtype B).
Subtypes B and C co-circulate in Brazil and are the
targets of antiretroviral drugs distributed by the National
Health System (SUS). Thus, understanding the variability
and the spectrum of these variation may have important
consequences for the effective treatment of patients
infected by HIV-1.
Palavras-chaves: Brazil, Evolução, HIV, Protease,
Subtipos.
MOLECULAR ANALYSIS OF BOVINE CORONAVIRUS
(BCoV) STRAINS FROM A WINTER DYSENTERY
OUTBREAK IN DAIRY COWS BASED ON THE
PARTIAL N, HE AND S GENES
BARROS, I.N., 2 TORRES, C.A., 3 SANTOS, S.,
AYRES, G.R., 5 SILVA, S.O.S., 6 ASANO, K.M.,
7
OLIVEIRA, C.P., 8SOUZA, S.P., 9RICHTZENHAIN, L.J.,
10
BRANDÃO, P.E.
1. FMVZ / USP, Department of Preventive Veterinary
Medicine and Animal Health, School of Veterinary
Medicine, USP, Av Prof Dr Orlando Marques de Paiva,
87 CEP 05508 270 -Cidade Universitária SP2. CRG,
Coronavirus Research Group, Av Prof Dr Orlando
Marques de Paiva, 87 CEP 05508 270 -Cidade
Universitária SP
4
Bovine coronavirus (BCoV) belongs to Betacoronavirus
genus and causes severe diarrhea in new-born calves,
winter dysentery (WD) in adult cattle and is associated
with respiratory tract infections. In addition, the main
economic losses caused by BCoV infections are the
decrease in milk production and the body condition score.
The virion contains five major structural proteins, the
spike (S) protein, the hemagglutinin esterase (HE) protein,
the transmembrane (M) protein, the small membrane (E)
protein and the nucleocapsid (N) protein. Molecular
analysis of BCoV genes has been conducted and
compared in different countries, and demonstrated
several degrees of substitutions or deletions especially
in the S protein, being in greater potential for future
mutations. Therefore, this present study aimed to analyze
BCoV strains from a WD outbreak in a farm of dairy
cows based on the partial genes N, HE and S. The eleven
feces samples of dairy cows studied herein were
previously diagnosed as rotavirus negative by RT-PCR
for the VP1 protein gene and coronavirus positive by a
nested RT-PCR for RdPd gene and then submitted to
RT-PCRs targeted to the N protein gene (306-bp
fragment), the hypervariable region of S glycoprotein gene
(488-bp) and HE protein gene (441-bp). The amplicons
were submitted to DNA sequencing and the sequences
obtained were aligned with sequences retrieved from the
GenBank for the construction of Neighbor-Joining trees
for nucleotides (MCL model). In the HE and S genes
analysis, the studied strains shared high nucleotide
identity among them and segregated in an exclusive
cluster. Other Brazilian strains included in the analysis
segregated in another cluster. However, they were
homologous to other different strains from the GenBank
in the N gene analysis and were significantly distinct
from Mebus and Kakegawa strains. These results
contribute to the molecular characterization and
epidemiology of BCoV.
Financial support: FAPESP nº 2008/56620-9.
Palavras-chaves: bovine, characterization, coronavirus,
outbreak, dysentery.
ID: 00169-00002
111
ABSTRACTS
CORONAVIRUS IN QUAILS: THE ROLE OF QUAILS
AS RESERVOIRS FOR CHICKENS
ID: 00169-00001
Área: 03 - Virologia Animal
TORRES, C.A., 2VILLARREAL, L.Y, 3 AYRES, G.R.,
BARROS, I.N, 5SANTOS, S, 6OLIVEIRA, C.P., 7SILVA,
S.O.S, 8RICHTZENHAIN, L.J., 9BRANDÃO, P.E.
1. FMVZ / USP, Department of Preventive Veterinary
Medicine and Animal Health, School of Veterinary
Medicine, USP, Av. Prof. Dr. Orlando Marques de Paiva,
87 CEP 05508 270 - Cidade Universitária2. Intervet,
Intervet Schering- Plough Animal Health, Av. Sir Henry
Wellcome, 335 CEP 06714-050 Moinho Velho – Cotia/
SP3. CRG, Coronavirus Research Group, Av. Prof. Dr.
Orlando Marques de Paiva, 87 CEP 05508 270 - Cidade
Universitária
1
4
Quail breeding is currently highly important in the
Brazilian agricultural market, but little is known on
infectious diseases in these animals. In relation to the
coronaviruses that might affect quails, there is still a
lack of information about the molecular diversity in this
host worldwide and on the role of quails as reservoirs for
infectious bronchitis of chickens. Therefore, the purpose
of this study was to investigate the occurrence of
coronavirus in quails kept close to laying hens. For this,
samples (pools) of trachea, lung, reproductive tract,
kidney and enteric content of 3 flocks of quails
(immunized with attenuated vaccine of the
Massachusetts serotype) and 3 of chickens were
collected in a same farm in 2010 and tested with a nestedRT-PCR, targeted to the 3’UTR of the Gammacoronavirus
genus. The positive samples were submitted to the
sequencing reaction for phylogenetic analysis. In the
samples of quails, viral RNA was detected in enteric
contents, kidney, trachea and lungs of one of the pools
and in the reproductive tract of another. In samples from
chickens, viral RNA was detected in kidney and trachea
in the samples studied. The result obtained by BLAST/n
showed 97% identity with the Gammacoronavirus genus,
but were divergent from a putative quail coronavirus
previously reported. Based on these results, one can
infer that quails are highly important in the epidemiology
of avian coronaviroses and might share a same virus
lineage when in close contact as in the present study.
Further studies are under way in order to assess the
genetic identities of the viruses detected herein for the
generation of preventive measures and for the further
clarification of the genetic diversity of coronaviruses in
quails.
Financial support: CNPQ
Palavras-chaves: Chicken, Coronavirus, Quail,
Reservoirs.
112
HIGH PREVALENCE OF NOROVIRUS AND
ROTAVIRUS AS THE CAUSES OF ACUTE
GASTROENTERITIS IN HOSPITALIZED CHILDREN IN
SÃO PAULO STATE
ID: 00170-00001
Área: 05 - Virologia Humana e Saúde Pública
Ribeiro, C D, 2Morillo, S G, 3Luchs, A, 4Vilanova, B C,
Eid, V R T, 6Eduardo, M B P, 7Suzuki, E, 8Barcelos, M
D, 9Solera, A, 10Timenetsky, M C S T, 11Carmona, R C C
1. IAL, Instituto Adolfo Lutz, Av Dr Arnaldo 3552. CVE,
Centro de Vigilância Epidemiológica do Estado de São
Paulo, Av Dr Arnaldo 3513. CVE Rio Preto, Vigilância
Epidemiológica da Secretaria Municipal da Saúde de São
José do Rio Preto, São José do Rio Preto4. GVE 1,
Grupo de Vigilância Epidemiológica I - Capital, São Paulo
- SP5. GVE 29 S.J.R.P, Grupo de Vigilância
Epidemiológica XXIX - São José do Rio Preto, São José
do Rio Preto
1
5
Rotavirus (RV) and more recently noroviruses (NoV) are
recognized as the main cause of moderate to severe
acute diarrhea in children. In 2006, RV vaccination was
introduced into the Brazilian National Immunization
Program for all infants, and since then a reduction in RV
detection in children with gastroenteritis has been
observed. The aim of this study was to determine the
prevalence of RV and NoV and to assess the severity of
illness associated with these viral agents of
gastroenteritis in hospitalized children. A total of 286 stool
specimens were collected from children under 5 years
of age, hospitalized with acute diarrhea, at three pediatric
hospitals in São Paulo State, from September 2009 to
August 2010. Commercial enzyme immunoassay kits
were performed routinely to detect rotavirus
(RIDASCREEN® Rotavirus, R-Biopharm AG, Darmstadt,
Germany) and NoV (RIDASCREEN® Norovirus 3rd
Generation EIA, R-Biopharm AG, Darmstadt, Germany)
among RV- negative samples. Of the 286 documented
hospitalized cases with acute gastroenteritis, 131 (45.8%)
were identified as viral infections. RV was detected in
26.2% (75"286), and NoV detected in 26.5% (56/211) of
the samples analyzed. Rotavirus infections were the
leading cause of children’s hospitalization in autumn and
winter months whereas Norovirus infections, during spring
and summer time. These findings highlight the need to
implement Norovirus ELISA detection assays in
association with rapid EIA RV detection assays for the
clinical diagnosis and prevention of gastroenteritis viral
infections and monitoring the changing etiology of acute
infections in the population. An effective Norovirus
vaccine would be of significant additional benefit to the
current RV vaccine so as to decrease the disease burden.
ABSTRACTS
Palavras-chaves: CHILDREN,
TERITIS, ROTAVIRUS, NOROVIRUS.
GASTROEN-
PERFORMANCE OF SEROLOGICAL TESTS FOR
IDENTIFICATION OF DAIRY HERDS WITH BOVINE
VIRAL DIARRHOEA VIRUS INFECTION
ID: 00171-00001
Área: 03 - Virologia Animal
STURZA, D.F., 2 FLORES, E., 3 WEIBLEN, R.,
WENDLANT, A., 5PERIM, F.
1. UFSM, Universidade Federal de Santa Maria, Avenida
Roraima, 1000, bairro Camobi, Santa Maria - RS, 97105900
Pereira-Oliveira, G., 2Silva-Fernandes, A.T., 3Ferreira,
V.M., 4Borges, I.A., 5Abrahão, J.S., 6Bonjardim, C.A.,
7
Ferreira, P.C.P., 8Travassos, C.E.P.F., 9Trindade, G.S.,
10
Kroon, E.G.
1. UFMG, Universidade Federal de Minas Gerais, Av.
Antônio Carlos, 6627, CEP: 31270-901. Belo Horizonte,
Minas Gerais, Brasil2. UENF, Universidade Estadual do
Nor te Fluminense Darcy Ribeiro, Av. Alber to
Lamego,2000,CEP: 28015-620. Campos, Rio de Janeiro,
Brasil
1
1
4
The success of control/eradication programs of bovine
viral diarrhea virus (BVDV) infection necessarily includes
the identification and elimination of persistently infected
(PI) animals. Since these animals continuously shed virus
in secretions and excretions, the prevalence of antibodies
in herds with PI animals is often high, and with high
titers. Because of these characteristics, bulk milk
samples were subjected to two serological techniques
in order to establish the most appropriate in conducting
screening of herds. For this, 767 bulk milk samples were
analyzed by a commercial ELISA kit (reference test)
and by an adapted virus neutralization (VNT) assay
(proposed test). The toxic effects of milk on cell culture
were reduced by increasing the final volume. One hundred
seventy seven and 139 samples were positive in ELISA
and VNT, respectively. Thus, the adapted VNT had a
sensitivity of 98% and a specificity of 93%. The Kappa
index (k) was 0.82, demonstrating an excellent agreement
between the two techniques. The analysis of the
coefficient of correlation between the absorbance values
(OD) and VNT titers demonstrated a moderate positivity
(r = 0.61). However, a significant part of samples with
VNT titers e” 80 did not show high OD values. On the
other hand, some samples with low VNT titers presented
high ODs. VNT titers e” 80 are suggestive of the presence
of PI animals in the herd. Therefore we conclude that the
adapted VNT is more appropriate for herd screening when
searching for herds with high antibody titers. Financial
support: CNPq
Palavras-chaves: animals persistently infected, BVDV,
bulk milk, ELISA, virus neutralization.
CANTAGALO VIRUS - A NEW ISOLATION IN RIO DE
JANEIRO STATE: BIOLOGIC AND MOLECULAR CHARACTERIZATION
ID: 00172-00001
Área: 03 - Virologia Animal
Bovine Vaccinia (BV) is a zoonosis caused by Vaccinia
virus (VACV), the prototype member of the family
Poxviridae. In 1999, a new sample of VACV, named
Cantagalo virus (CTGV) was isolated from bovine dairy
cattle during an outbreak in the Cantagalo County, Rio
de Janeiro State. Molecular analysis suggested that
CTGV has probably originated from the vaccine strain
VACV-IOC used in the Brazilian smallpox eradication
campaign. The clinical symptoms of Cantagalo infection
are often indistinguishable from those associated with
other Orthopoxvirus (OPV), especially Cowpox virus
(CPXV), Buffalopox virus (BPXV) and other Brazilian
VACV isolates. Vesicular and pustular lesions are the
major signals of the disease. In October 2001, during a
new outbreak of BV in the same city, a second sample
of CTGV was isolated and sent to the Laboratory of Virus
/UFMG and named CTGV-II. In order to better
characterize this sample, biologic study and molecular
analysis was performed. The sample was isolated in
choriallantoic membranes (CAMs) and in Vero cells. DNA
samples from the CTGV-II were extracted and used as
template for the amplification of ha gene. When exposed
to the CTGV-II we observed that CAMs presented pocks
formation and Vero cell monolayers showed evident
cytopathic, typical to that classically observed for OPV.
Subsequently, to better identify this etiologic agent, ha
gene was amplified by PCR and sequenced. CTGV-II ha
gene sequence was compared to VACV-WR, VACV-IOC,
VACV-LST, CPXV-BR, BPXV, CTGV and other Brazilian
VACV strains. Phylogenetic analysis showed 100%
similarity between CTGV-II and CTGV. This study
confirms the re-isolation of CTGV in Rio de Janeiro State
after two years of the first isolation.
Financial support: CNPq, CAPES, FAPEMIG, MAPA.
Palavras-chaves: Bovine Vaccinia, Cantagalo virus,
Zoonotic disease.
CLONING AND EXPRESSION OF A FRAGMENT OF
BOVINE HERPESVIRUS TYPE 1 GLYCOPROTEIN E
ID: 00176-00001
Área: 03 - Virologia Animal
1
Oliveira, S.A.M, 2 Dellagostin, O.A, 3 Flores, E.F,
113
ABSTRACTS
4
Weiblen, R
1. UFPel, Universidade Federal de Pelotas, Bairro Capão
do Leão, Pelotas, RS2. UFSM, Universidade Federal de
Santa Maria, Av. Roraima, bairro Camobi CEP: 97105900, Santa Maria, RS
XXIX-SJRP, Grupo de Vigilância Epidemiológica XXIX
– São José do Rio Preto, São José Rio Preto-SP6.
COVISA/SMS, Coordenadoria de Vigilância em Saúde,
Secretaria Municipal da Saúde de São Paulo, São PauloSP
Bovine herpesvirus type 1 (BoHV-1) is a member of the
family Herpesviridae, subfamily Alphaherpesvirinae. BoHV-1 infection is associated with
rhinotracheitis (IBR), genital disease (IPB/IPV) and
abortions. The BoHV-1 genome encodes several
glycoproteins that are expressed in the viral envelope.
Among them, glycoprotein E (gE) has been targeted for
deletion for the development of BoHV-1 and BoHV-5
differential vaccines. Our group recently developed a
marker vaccine using a BoHV-5 strain with deletion on
the gE gene. The objective of this work was to clone and
express a fragment of BoHV-1 gE to be used in conjunct
with a BoHV-5 gE-deleted vaccine to differentiate
vaccinated from naturally infected animals. The gene was
amplified by polymerase chain reaction (PCR) and
cleaved with enzymes BamHI and NdeI to be ligated to
the vector. The vector used was the pET 16b that contains
an histidine tail and express the product as a fusion
protein. Cloning and expression were performed in
Escherichia coli, using BL21 (DE3) codon plus. Protein
purification was achieved by applying cell extracts to
nickel columns. Preliminary results showed the success
of cloning and expression. We conducted a Western blot
using an anti-histidine antibody that demonstrates the
presence of the recombinant protein. Along with this, an
SDS-PAGE showed the expression of a protein of
approximately 25 kDa, correspondent to the cloned
fragment. Additional tests are underway to further
characterize the protein and to use it as antigen in an
ELISA test.
Financial support: CAPES.
Palavras-chaves: cloning, differential vaccines,
expression, glycoprotein E, herpesvirus.
Rotavirus (RV) infections are recognized as a major cause
of severe gastroenteritis in infants and young children
worldwide. Approximately 90% of all rotavirus diseases
have been shown to be caused by rotaviruses with
P[8]G1, P[4]G2, P[8]G3, P[8]G4, and P[8]G9
specificities. In March 2006, Brazil introduced a
monovalent P[8]G1 human RV vaccine (Rotarix®
GlaxoSmithKline Biologicals) into its national Expanded
Program for Immunization. The aim of this study was to
assess the impact of immunization on the incidence of
severe rotavirus acute gastroenteritis. Surveillance of
rotavirus diarrhea was conducted involving 263 children
under 5 years of age who were admitted for treatment of
diarrhea at 3 sentinel hospitals in the cities of São Paulo
and São José Rio Preto, from September 2009 to July
2010. RV was detected in 65 (25%) of the fecal
specimens by using enzyme-linked immunosorbent
assay. Of all episodes of rotavirus diarrhea, 79% occurred
during the first 2 years of life, peaking at 7-12 months of
age. Rotavirus isolates were characterized by reversetranscriptase polymerase chain reaction to determine P
and G genotypes. G2 (65.5%) was the most prevalent
serotype followed by G1 (17.2%) and G3 (1.7%). P[4]
was the most common genotype of rotavirus. The most
common P-G association identified in this study was
P[4]G2 (65.0%). Mixed infections with common
genotypes P-G were also identified. Preliminary data
obtained from hospitals in São Paulo State and
information concerning the program of monitoring acute
diarrheal diseases (MADD) show that hospitalizations
for diarrhea in children under 5 years had a significant
reduction after the introduction of rotavirus vaccine.
Sentinel hospital-based surveillance is essential to
monitor changes in the epidemiology of rotavirus disease
and the impact of vaccination after introduction,
considering change in frequency, severity of disease,
and circulating rotavirus types.
Palavras-chaves: Rotavirus, Genotypes, Sentinel
Hospital, Gastroenteritis.
SENTINEL HOSPITAL SURVEILLANCE FOR
ROTAVIRUS IN SÃO PAULO STATE
ID: 00178-00001
Área: 05 - Virologia Humana e Saúde Pública
Carmona, RCC, 2Luchs, A, 3Morillo, SG, 4Ribeiro, CB,
Eduardo, MBP, 7Susuki, E, 8Barcelos , MD, 9Solera, A,
10
Eid, VRT, 11Madalosso, G., 11Timenetsky, MCST
1. IAL, INSTITUTO ADOLFO LUTZ, Ave. Dr. Arnaldo,
3552. CVE - SP, CENTRO DE VIGILANCIA
EPIDEMIOLÓGICA DO ESTADO DE SÃO PAULO, Av.
Dr. Arnaldo, 3513. CVE-SJRP, CENTRO VIGILÂNCIA
EPIDEMIOLÓGICA DE SÃO JOSÉ DO RIO PRETO,
São José Rio Preto - SP4. GVE 1-SP, Grupo de Vigilância
Epidemiológica I – São Paulo, São Paulo, SP5. GVE
1
6
114
EXTRACTS OF THE TRICHILIA CATIGUA (CATUABA/
CATIGUA) INHIBIT BOVINE HESPESVIRUS-1
REPLICATION IN CELL CULTURE.
ID: 00181-00001
Área: 04 - Virologia Básica
BERNARDI, A.L.S., 2Faccin-Galhardi, L.C, 3Rincão, V.P.,
MELLO, J.C.P., 5Linhares, R.E.C., 6NOZAWA, C.
1
4
ABSTRACTS
1. UEL, Universidade Estadual de Londrina, Rod. Celso
Garcia Cid. Pr 455 - Km 380 Londrina-PR2. UEM,
Universidade Estadual de Maringá, Av. Colombo 5790
Jd. Universitário, Maringá-PR
Some economically relevant diseases of the cattle are
caused by bovine hespesvirus 1 (BoHV-1), such as,
rhinotracheitis, pustular vulvovaginitis, balanoposthitis
and abortion. As a member of the Herpesviridae family
BoHV-1 shares the capability to establish latency in the
nervous ganglia. This is one of the main causes that
hinder the eradication of the diseases worldwide. The
antimicrobial effects of Trichilia catigua (T. catigua) have
been already demonstrated. The effects of crude extract
(CE), acetate (AcF) and aqueous fractions (AqF) from
bark of T. catigua were evaluated in the replication of
BoHV-1. Plaque assay was used to evaluate their
antiviral activity. The citotoxicity was performed by MTT
assay in HEp-2 cells. The following protocols were used:
a) the substances were added before (-1 and -2 h), during
(0 h) and after (1 and 2 h) viral infection; b) the virucidal
activity (virus plus concentrations of the substances)
and c) viral adsorption inhibition assay (virus plus
substances incubated at 4° C for 1 hour). The MTT test
resulted in a CC50 > 400μg/mL for all the substrates.
The assays were done in concentrations of 100μg/mL,
50 μg/mL, 25 μg/mL and 12,5 μg/mL. At the time 0h and
in the virucidal test of the CE the inhibition was 100%
for all the concentrations. The virus inhibition was total
at the 100μg/mL and 50 μg/mL of AcF and AqF at the
time 0h. The AcF inhibited 100% the replication for the
virucidal and the inhibition of adsorption tests at all the
concentrations. For the virucidal treatment, AqF at the
concentration of 100μg/mL to 25μg/mL showed similarly
100% inhibition, however, the inhibition of adsorption
presented an IC50 of 57 μg/mL and the SI >7. The results
suggested that the compounds of T. catigua act on the
early stages of the BoHV-1 replication and also directly
on the viral particle. Financial Support: CNPq; Fundação
Araucária, PROPPG/UEL. Área: Virologia Básica.
Palavras-chaves: Antiviral, Bovine herpesvirus-1, Trichilia
catigua.
Research, Rua Prof. Antônio Prudente, 109 4º andar
01509-010 São Paulo, SP Brasil3. Famerp, Faculty of
Medicine of Sao Jose do Rio Preto, Av. Brigadeiro Faria
Lima, 5416 - Vila São Pedro - 15090-000 São José do
Rio Pre4. USP, Faculty of Medicine of Ribeirao Preto,
Avenida Bandeirantes, 3900 - Monte Alegre – CEP:
14049-900 - Ribeirão Preto - SP
Recurrent respiratory papillomatosis (RRP) is a disease
characterized by the formation of benign papillomas in
the epithelium of the upper respiratory tract, including
the larynx. These pathological changes of the mucosa
have high recurrence rate so thus at its therapy often
requires multiple procedures. This disease is more
common in children, but can also occur in adults. Infection
with Human Papillomavirus (HPV) has been considered
the main cause of laryngeal papillomas. HPV-6 and HPV11 are the most frequently viral types detected in
respiratory papillomatosis. This study aims to expand
the knowledge concerning genetic variability of HPV
types commonly found in laryngeal papillomas, besides
to identify the impact of nucleotide variations in the LCR
and E6 regions upon the HPV transcriptional activity.
HPV detection was conducted by PCR using the
PGMY09/11 primer system followed by RFLP
genotyping. The presence of HPV was analyzed in 31
biopsy specimens of laryngeal papillomas. HPV-6 was
found in 19 (61%) samples and HPV-11 in 12 (39%)
samples. Our results confirm that laryngeal papillomas
are caused mostly by HPV- 6 and HPV-11. Thus, it is
evident that HPV is an important risk factor for
papillomatosis and characterization of viral genotypes
associated with RRP is important to establish the most
appropriate therapies to infections and lesions caused
by this virus. In the next stage of the study we will amplify
the LCR and E6 regions of the virus in order to analyze
the genetic variability of HPV detected.
Financial support: Fapesp.
Palavras-chaves: Genetic variability, HPV-6 e HPV-11,
Human Papillomavirus, Laryngeal papillomas, Recurrent
respiratory papillomatosis.
ANALYSIS OF HPV GENETIC VARIABILITY IN
LARYNGEAL PAPILLOMATOSIS
NS3 PROTEASE EXPRESSION FROM VIROLOGICAL
SUSTAINED RESPONDER AND NON-RESPONDER
PATIENTS.
ID: 00182-00001
Área: 05 - Virologia Humana e Saúde Pública
ID: 00183-00001
Área: 05 - Virologia Humana e Saúde Pública
BONFIM, C.M, 2 NOGUEIRA, R.L., 3 KUPPER,D.S.,
VALERA, F.C.P., 5 NOGUEIRA, M.L., 6 VILLA, L.L.,
7
SICHERO, L., 8Rahal, P.
1. Unesp, São Paulo State University, R. Cristóvão
Colombo, 2265 Bairro: Jd. Nazareth CEP:15054-000 S.
J. do Rio Preto2. Ludwig, Ludwig Institute for Cancer
1
1
4
Provazzi, P. J. S., 2Cabral, H., 3Canduri, F., 4Nogueira,
M., 5Rahal, P.
1. UNESP-IBILCE, Instituto de Biociências, Letras e
Ciências Exatas, Depto de Biologia, Rua Cristóvão
Colombo, 2265. São José do Rio Preto/SP. CEP:150540002. USP - Ribeirão Preto, Universidade de São Paulo,
115
ABSTRACTS
The Hepatitis C virus (HCV) infects 130 million people
worldwide and is responsible for acute and chronic hepatitis,
cirrhosis and hepatocelular carcinoma. Nevertheless most
effective treatment options are not yet available. The HCV
Genotype 3 has a high frequency in Brazil and in clinical
evaluations it is associated with a mild illness manifestation
and a better response to the antiviral therapy. Since the
processing of the viral polyprotein is essential for HCV
replication, the NS3 protease has been considered to be a
primary target for the development of anti-HCV drugs. In
previous work we identified amino acids substitutions on
active site and zinc ion binding site of NS3 protease
genotype 3 of virological sustained responder patient and
non-responder patients. With that the objective of this work
is to evaluate the enzymatic activity of NS3 Protease
genotype 3 variants sequences from virological sustained
responder and non-responder patients. The Protease NS3
variants sequences was cloned into expression vector and
expressed in E. coli cells to assess their level of expression,
which are in the process of purification. We believe that
evaluation of the NS3 Protease’s enzymatic activity from
sensible and resistant to treatment patients will provide
key information about the NS3 and consequently the
replication viral and the Hepatitis C establishment.
Additional studies will provide more conclusive results.
Financial support: FAPESP, CAPES and CNPq.
Palavras-chaves: Expressão de proteínas, HCV genótipo
3, Hepatite C.
dissemination of pathogens in waters and shellfish
growing areas. Bacterial indicators such as faecal
coliforms have been used as indicators of water quality.
However the correlation between bacterial and viral
indicators is not always established. The aim of this study
was to evaluate human adenoviruses (HAdV), hepatitis
A virus (HAV), polyomaviruses (JCPyV) noroviruses (NV)
contamination in seawater samples (10 liters), collected
during one year (August 2009 to July 2010), in eleven
beaches of Florianópolis city, classified in two zones
according to the faecal coliforms (FC) presence:
acceptable (A) and non-acceptable (B) levels. The
samples were concentrated by flocculation methodology,
based on the direct binding of the viruses to acidified
skimmed milk. Nucleic acids extraction was performed
using a QIAmp MinElute Virus Spin Kit (Qiagen). The
quantification of the viral genomes was performed by
qPCR and RT-qPCR, using a TaqMan assay, for the
viruses HAdV, JCPyV and HAV and qualitative RT-PCR
for NV, followed by a semi-nested reaction, for serotypes
GI and GII determination. 10-fold dilutions of the nucleic
acids were used in all samples to prevent possible
inhibition. For samples evaluated by real time PCR, 37%
and 45% were positive for HAdV genome, with an average
of 1.86 x 106 and 8.17 x 105 GC/L for zones A and B
respectively. For HAV genome, 59% and 56% were
positive, with a mean value of the 3.44 x 102 and 6.45 x
101 GC/L for zones A and B respectively. For JCPyV,
no sample for zone A, and 4% for zone B were positive,
with a mean value of the 3.20 x 104. For NV, 7% of the
samples were positive for serotype GI (33% in zone A
and 67% in zone B) and 8% for serotype GII (20% in
zone A and 80% in zone B). In conclusion, we could
observe the massive presence of viral contamination on
Florianópolis beaches without a clear relationship
between FC and viruses.
Palavras-chaves: viruses, seawater, bacterial,
environmental contamination.
ANALYSIS OF THE SANITARY CONDITIONS OF THE
SEAWATER IN FLORIANÓPOLIS BEACHES BY
VIRAL AND BACTERIAL PARAMETERS
OCCURRENCE OF BEGOMOVIRUS INFECTING
PEPPER CROPS (Capsicum annum) IN MINAS
GERAIS STATE.
ID: 00184-00001
Área: 02 - Virologia Ambiental
ID: 00187-00001
Área: 06 - Virologia Vegetal
MORESCO, V., 2 NASCIMENTO, M.A., 3 GARCIA,
L.A.T., 4KLEEMANN, C.R., 5RAMOS, A.P D., 6SOUZA,
D.M.S., 7SIMÕES, C.M.O., 8BARARDI, C.R.M.
1. UFSC, Universidade Federal de Santa Catarina, Depto.
MIP, Campus Trindade, CEP 88040900, Florianópolis,
SC, Brasil
1
Faculdade de Ciências Farmacêuticas de Ribeirão Preto.,
Av. do Café s/nº, Campus Universitário da USP 14040903 - Ribeirao Preto/SP3. USP - São Carlos, Universidade
de São Paulo. Depto de Química e Física Molecular,
Avenida do Trabalhador Sãocarlense, 400 13560-970 Sao Carlos/SP4. FAMERP, Faculdade de Medicina de
São José do Rio Preto. Depto Doenças Infecciosas e
Parasitárias, Av Brigadeiro Faria Lima, 5416 15090-000
- Sao Jose do Rio Preto/SP
1
Coastal waters are continually saturated by
contamination of faecal origin, contributing for the
116
Nozaki, D.N., 2Rocha, K.C.G., 3Gioria, R., 4Kobori, R.F.,
Krause-Sakate, R., 6Pavan, M.A.
1. UNESP/FCA, Universidade Estadual Paulista “JÚLIO
DE MESQUITA FILHO” Campus Botucatu, Depto Prod.
Vegetal- Defesa Fitossanitaria. R José Barbosa de Barros,
nº 17802. SAKATA, Sakata Seed Sudamerica Ltda, Av.
Plínio Salgado nº4320, , C.P. 427, 12906-840, Bragança
Paulista -SP
5
ABSTRACTS
Viruses belonging to the genus Begomovirus family
Geminiviridae, are transmitted by the whitefly Bemisia
tabaci Gennadius and causes important losses in
different crops. In Brazil, the infection of begomovirus in
Capsicum sp. was only reported in Pernambuco, Bahia
and São Paulo States. The symptoms described involves:
mosaic, leaf distortion and yellowing. In the first semester
of 2010 leaf samples of two different fields of sweet
pepper were received for pathogen diagnosis. The areas
were located in Minas Gerais State: regions of Carmópolis
de Minas and Araguari. Sweet pepper leaves presented
symptoms similar to those described for begomoviruses.
PCR analysis using universal oligonucleotides
(PAL1v1978, PAR1c496), amplified a fragment that was
sequenced. The nucleotide sequence revealed high
identity with Tomato severe rugose virus (ToSRV EU600238.1). This is the first report of natural infection
of ToSRV in sweet peppers in Minas Gerais State.
Palavras-chaves: Begomovirus, Sweet pepper, Minas
Gerais State, Tomato severe rugose virus, Ocurrence.
of samples were analyzed using analysis of variance
(ANOVA) test (Prism 4 software) and comparisons of
means were conducted using Tukey test with significance
indicated by a probability of P < 0.05. It was not detected
a significant correlation between viral load and number
of copies of bacterial DNA for the different categories of
M. hyopneumoniae related microscopic lesions. Also,
was not detected a significant correlation between the
number of copies of bacterial DNA and the different types
of lesions found in samples of lungs (P>0.05).
Financial support: FAPEMIG and CNPq.
Palavras-chaves: MYCOPLASMA HYOPNEUMONIAE, PORCINE CIRCOVIRUS-2, REAL TIME PCR.
INTERACTION OF PORCINE CIRCOVIRUS-2 (PCV-2)
AND MYCOPLASMA HYOPNEUMONIAE IN LUNGS
OF PIGS EVALUATED BY REAL TIME PCR.
Calux, SJ, 2Machado, BC, 3Russo, DH, 4Sousa, CA,
Misaki, IH, 6Timenetsky, MCST, 7Carmona, RCC
1. IAL, Instituto Adolfo Lutz - São Paulo, Av. Dr. Arnaldo,
355 - São Paulo, SP. CEP: 01246-0002. VE-Guarulhos,
Vigilância Epidemiológica de Guarulhos, Rua Íris, 300 Guarulhos, SP. CEP: 07051-080
ID: 00188-00001
Área: 03 - Virologia Animal
SILVA, F.M.F., 2 LIMA, S., 3 BEZERRIL, J.E.,
MYRRHA, L.W., 5 VIDIGAL, P.M.P., 6 PETERNELLI,
E.F.O., 7 VARGAS, M.I., 8 FIETTO, J.L.R., 9 SILVA
JÚNIOR, A., 10ALMEIDA, M.R.
1. UFV, Universidade Federal de Viçosa, Av. PH Rolfs,
s/n Campus Universitário Cep:36570000 Viçosa-MG
1
4
Porcine circovirus 2 (PCV2) has been linked with Porcine
circovirus associated diseases (PCVAD) during the last
decade. However, it is known that not all pigs infected
with PCV2 will develop PMWS and that the incidence of
PMWS is higher when co-infecting viral and bacterial
pathogens are present. The aim of this study was to
evaluate the effects of co-infection of Mycoplasma
hyopneumoniae and PCV2 from the evaluation of viral
and bacterial DNA by real time PCR. A TaqMan-based
real time for quantitation of PCV2 and a SYBR Green
real time for quantitation of M. hyopneumoniae were used
in this study. DNA extracted by Wizard SV Genomic
Purification System (Promega) from 108 samples of lung
pigs. The tissue samples were fixed in 10% neutral
buffered formalin, embedded in paraffin wax, and
processed routinely for histopathological investigation.
A plasmid containing the viral and bacterial genome was
quantified by spectrophotometry and was used to create
a standard curve for PCV2 and M. hyopneumoniae
quantification, respectively. Viral load and bacterial DNA
ECHOVIRUS 30 ASSOCIATED TO INTRAFAMILIAL
MENINGITIS OUTBREAK IN GUARULHOS, SÃO
PAULO, BRAZIL - 2010
ID: 00191-00001
Área: 05 - Virologia Humana e Saúde Pública
1
5
Outbreaks of meningitis caused by Human Enterovirus
(HEV) frequently occur in the summer and the autumn.
In Brazil, echovirus serotype 30 (E-30) is the most
common virus related with these outbreaks. In the period
from January to May 2010, there were 76 cases
diagnosed as viral meningitis by cerebrospinal fluid (CSF)
cytochemical examination in the municipality of
Guarulhos. In March, the Epidemiological Surveillance
recorded the occurrence of three cases of probable viral
meningitis in children from the same family, with two
brothers and a cousin infected. According to
epidemiological data, the younger brother had the first
symptoms (fever and vomiting), followed by his older
brother and later his cousin, featuring an intrafamilial
transmission. CSF samples from the two brothers were
sent to the Enteric Diseases Branch of Adolfo Lutz
Institute. The diagnosis of viral meningitis for the cousin
was completed only by cytochemical analysis. The viral
isolation in RD (human rhabdomyosarcoma) cell culture
was positive from both CSF samples and the isolated
virus was identified as E-30 by Indirect
Immunofluorescence Assay and Reverse Transciption –
Polymerase Chain Reaction. The main transmission path
of EVH is fecal-oral and when associated with
inappropriate sanitary conditions, favors the spread of
these viruses, allowing the intrafamilial transmission. The
source of contamination may be associated with the fact
117
ABSTRACTS
that children often played near a stream with a history of
recent flooding. The aim of this study is to report the
circulation of E-30 in cases of meningitis in an intrafamilial
outbreak, as well as to emphasize the importance of
preserving hygienic and sanitary conditions and having
greater commitment to implement educational and
preventive actions together with the population.
Palavras-chaves: Viral Meningitis, Human Enterovirus,
Echovirus.
ANALYSIS IN VIVO OF THE ANTIVIRAL ACTIVITY
OF THE PHARMACOLOGICAL INHIBITOR SP600125
ID: 00192-00001
Área: 04 - Virologia Básica
Cruz, A. F. P., 2Leite, G. G. F., 3Ferreira, P. P. C., 4Kroon,
G. E., 5Bonjardim, A. C.
1. GTS, Grupo de Transdução de Sinal, Avenida Antonio
Carlos, Pampulha, Belo Horizonte, MG2. LABVIRUS,
Laboratório de Vírus, Departamento de Microbiologia,
Instituto de Ciências Biológicas - UFMG, Avenida
Antonio Carlos, Pampulha, Belo Horizonte, MG3. UFMG,
Universidade Federal de Minas Gerais, Avenida Antonio
Carlos, Pampulha, Belo Horizonte, MG
1
The Orthopoxvirus genus encompasses eight members
of the Poxvirus family and they have the ability to infect
vertebrate and invertebrate hosts. Vaccinia virus (VACV)
is the prototype of this genus, and Brazilian isolates of
VACV have been associated with outbreaks of bovine
vaccinia (VB) in many rural areas in Brazil, causing
ulcerative lesions in cows and in humans, leading to
high economic and social impact. Our studies verified a
significant reduction in viral yields when analyzing
various cell lines pretreated with the pharmacological
inhibitor of JNK pathway, SP600125 (SP), and infected
with VAVC or with Cowpox virus (CPXV). Our studies
were extended to analyze the antiviral activity of the SP
inhibitor in an in vivo model. For this end, 4-week-old
BALB/c mice were separate into 5 groups. The 1st and
2nd group were inoculated with VACV, the 3rd and 4th
were inoculated with CPXV and 5th group was inoculated
with PBS. All groups were inoculated intranasally with a
dose of 5x105 pfu/mL of the respective virus and the
control group was inoculated with 1.0 mL of PBS. Mice
of the 2nd and 4th groups were treated with SP at the
concentration of 30mg/kg/day via intraperitoneal route.
The animals were followed for 7 days and observed for
weight loss, ruffling fur, arching back, balanopostitis,
facial edema, plantar edema, difficulty breathing and
periocular alopecy. To evaluate the progression of the
disease we used a method adapted from Pulford et al.
2004. We observed a delay in disease progression in the
animals treated with SP. On day 7th, the mice that were
118
still alive were euthanized and some organs were
collected to analyze viral replication. For this end, the
organs were macerated in MEM. Supernatant fluids from
the macerate were collected and virus titer (PFU/g) was
determined by plaque forming assay in Vero cells. A
significant reduction in viral yields was verified only in
the animals treated with SP. These data point out
SP600125 as a potential antipoxviral.
Palavras-chaves: antipoxviral, pharmacological inhibitor
sp600125, Vaccinia virus.
THE HOST FACTOR C-JUN PLAYS A ROLE IN THE
RELEASE OF ENVELOPED FORMS OF Vaccinia
virus
ID: 00192-00002
Área: 04 - Virologia Básica
Torres, A. A., 2Leite, G. G. F., 3Cruz, A. F. P., 4Pereira,
A. C. T. C., 5Ferreira, P. P. C., 6Kroon, G. E., 7Bonjardim,
A. C.
1. GTS, Grupo de Transdução de Sinal, Avenida Antonio
Carlos, Pampulha, Belo Horizonte, MG2. LABVIRUS,
Laboratório de Vírus, Departamento de Microbiologia,
Instituto de Ciências Biológicas - UFMG, Avenida
Antonio Carlos, Pampulha, Belo Horizonte, MG3. UFMG,
Universidade Federal de Minas Gerais, Avenida Antonio
Carlos, Pampulha, Belo Horizonte, MG4. UFPI,
Universidadade Federal do Piauí, Bairro Ininga - Teresina
- PI
1
Poxvirus encompasses the larger and more complex
family of DNA viruses. The Orthopoxvirus Vaccinia
(VACV) is a large double-stranded DNA virus (H”200 Kpb)
that replicates exclusively in the cytoplasm of infected
host cells. As virus-host cell interaction plays a decisive
role in viral biology, our group has been studying the
activation of protein kinases in response to VACV
infection. Previously, we demonstrated that VACV induces
the activation of the transcription factor c-Jun, from early
until late times, and both MEK/ERK and JNK pathways
are involved. Using cell lines stably-expressing c-Jundominant-negative mutation (DN), we observed that the
expression of viral proteins was not affected in these
cells, but a significant reduction in virus yields and plaque
size were verified, also through fluorescence microscopy.
However, through transmission electron microscopy, we
observed no defects in VACV morphogenesis that could
explain their diminished proliferation in DN cells. As the
size of the plaque is determined by the release of viral
enveloped forms (EV), we decided to investigate whether
c-Jun was involved in viral spread. By fluorescence
microscopy, we did not observe a decreased formation
of actin tails in DN cells. However, through plaque assay
of EVs, we found a smaller amount of virus in the
ABSTRACTS
supernatant of DN cells. Together, these data suggest
that the absence of c-Jun in A31 cells, somehow affect
the release of extracellular enveloped particles, without
affecting actin tails formation.
Palavras-chaves: FACTOR C-JUN, ENVELOPED
FORMS, Vaccinia virus.
RETROSPECTIVE CLINICAL CASES OF CANINE
DISTEMPER IN THE VETERINARY HOSPITAL OF
UNIVERSIDADE FEDERAL DE VIÇOSA-MG
ID: 00193-00001
Área: 03 - Virologia Animal
Botelho, C.V.
1. UFV, UNIVERSIDADE FEDERAL DE VICOSA,
AVENIDA PH ROLFS S/N
prevalence in other categories was 30,77%; 11,88%;
4,88%; 1,05% and 0,006% respectively. The age factor
was inversely proportional to the occurrence of the
disease. These results suggest that canine distemper is
a disease with significant prevalence, one of the major
infectious diseases of dogs. Young animals fall into the
category most susceptible to infection by the virus and
there is no distinction between the sexes.
Financial support: FAPEMIG, FUNARBE Área: Virologia
Animal Tema: Cinomose canina Arquivo: CINOMOSE
CANINA-Botelho.doc Apresentador: Clarisse Vieira
Botelho.
Palavras-chaves: CINOMOSE, RETROSPECTIVO,
UFV, DIAGNOSTICO, FAIXA ETARIA.
1
RETROSPECTIVE CLINICAL CASES OF CANINE
DISTEMPER IN THE VETERINARY HOSPITAL OF
UNIVERSIDADE FEDERAL DE VIÇOSA-MG Botelho,
C.V.¹; Moretti, D.T.¹; Santos, C.V.¹; Carvalho, F.G.¹;
Campos, E.C.; Costa, P.R. S.¹; Almeida, M.R.2; Peternelli,
E.F.O 2; Júnior, A.S.¹ . ¹Departamento de Veterinária,
Laboratório de Virologia, UFV, Viçosa, MG;
²Departamento de Bioquímica e Biologia Molecular,
Laboratório de Infectologia Molecular Animal – LIMA.
BIOAGRO - UFV, Viçosa, MG - Brazil. E-mail:
clarisse.botelh@ufv.br The canine distemper is a highly
contagious disease caused by a virus of family
Paramyxoviridae which affects preferentially young dogs
under the age of one year. This virus is endemic in the
microregion of Viçosa and is manifested by signs of
gastrointestinal, respiratory, skin and neurological
problems. The prevalence of the disease and
epidemiological variables are still unclear. This study
aimed to evaluate the prevalence of distemper in dogs
Veterinary Hospital/DVT/UFV at the months between
January 2000 and December 2009. In this study were
evaluated 16.636 clinical cases of animals attended with
various pathologies. It was adopted as a criterion for
identification of disease, the presumptive diagnosis
(animals treated with classic signs of the disease) and
definitive diagnosis (classic signs of illness associated
with the presence of corpuscle Lentzs in leukocytes).
The animals were categorized into age groups (d” 1 year>
1st d” 3 years,> 3 to d” 6,> 6 to d” 9,> 9 to d” 12,> 12
years) and sex. Of these cases, 737 (4,43%)
corresponded to cases of distemper in accordance with
the criteria for presumptive diagnosis and 99 (0,59%)
according to the criteria of definitive diagnosis. Males
and females showed the prevalence of 55.02% and
44.98% respectively, both adopted of the diagnostic
criteria. The age of animals younger than one year (d” 1
year) matched the highest prevalence (50,72%). The
ASSESSMENT OF “IN VITRO” CILIARY MOVEMENT
OF BOVINE OVIDUCT EPITHELIAL CELLS
EXPERIMENTALLY EXPOSED TO BOVINE
HERPESVIRUS TYPE 1 (BoHV-1).
ID: 00194-00001
Área: 03 - Virologia Animal
Batista, M.L, 2 Alves, M.F, 3Pavão, D.L, 4Souza, F,
Queiroz, R.K.R, 6D’Angelo, M
1. IB, Instituto Biológico, Av. Conselheiro Rodrigues
Alves, 1252
1
5
Studies on the interaction of bovine oviduct epithelial
cells)#BOEC*# with pathogens, has led us to clarify
possible changes of bovine herpesvirus type 1 )#BoHV1*# in the beats of ciliated epithelium cells. The process
of capture and transport of oocytes through the oviduct
depends on the movement of cilia, which is responsible
for targeting them. The aim of this study was to evaluate
in an experimental model of in vitro oviduct, the BoHV-1
virus action on the ciliary movement of BOEC. The
oviducts were collected from slaughtered cows,
dissected, washed, sliced lengthwise and then fixed with
pins in sterile petri dishes containing a 1 cm layer of
sterile paraffin, immersed in 50mL of 199 medium and
divided into control group )#n = 15*# and exposed to the
pathogen )#n = 15, 0.5 mL of virus at 10v ,w TCID50D
mL*#. The plates were incubated in 5% CO2, 92%
humidity and 38.5 °C for 5 hours. After the incubation
period, black latex microspheres were placed on the
luminal surface of the oviduct and, for 1 minute, the ciliary
movement was observed in a stereomicroscope )#20X*#.
The control oviducts showed directions of movement
consistent with the in vivo patterns, with no movement
of the ball in the middle region of the oviduct, movement
directed toward the uterus in the region of the infundibulum
and drive directed to the ovary in the region of the isthmus.
Already the oviducts exposed, although they haven´t
shown any change in the direction of motion, there was
119
ABSTRACTS
a drive slower balls in the control group. These preliminary
results allow us to point out that possibly there is a
change in ciliary cells according to the adsorption and
viral penetration and consequently the use of systems
for biosynthesis of the cell by the virus. However, the
continuity of these tests will have a more precise method
of measuring the displacement of the microspheres for
a better representation of the results.
Financial support: CNPq
Palavras-chaves: BoHV-1, bovine oviduct epithelial cells,
ciliary movement.
(VACV Copenhagen B13R, E3L, F1L and N1L
homologues) presented no polymorphism in VBH genome
when compared to VACV-WR. Probably, their gene
products are fully functional. Taken together, these data
point out for the profound biological divergences of VBH
and VACV-WR, albeit the genetic similarity between them.
These intriguing differences will certainly reflect in the
pathogenesis of these VACV strains in vivo.
Palavras-chaves: Vaccinia virus, Brazilian Vaccinia virus,
Vaccinia virus Belo Horizonte, Virus-host interaction,
Signaling pathways.
DIFFERENTIAL ROLES PLAYED BY HOST
SIGNALING PATHWAYS DURING INFECTION WITH
Vaccinia virus STRAINS WESTERN RESERVE AND
BELO HORIZONTE
ADVERSE EVENTS POST SMALLPOXACCINATION:
INSIGHTS FROM TAIL SCARIFICATION INFECTION
IN MICE WITH Vaccinia virus (VACV)
ID: 00196-00001
Área: 03 - Virologia Animal
ID: 00195-00001
Área: 04 - Virologia Básica
Mota, BEF, 2Gallardo-Romero, N., 3de Souza Trindade,
G., 4Shannon-Klecker, M., 7Campos, MA, 8Vieira, LQ,
9
da Fonseca, FG, 10Damon, IK, 10Ferreira, PCP, 11Kroon,
EG, 11Bonjardim, CA
1. UFMG, Universidade Federal de Minas Gerais, Av
Antônio Carlos, 6627, Belo Horizonte, MG. CEP 312709012. CDC, Centers for Disease Control and Prevention,
1600 Clifton Road NE, Atlanta, GA, EUA. 303333.
CPqRR/FIOCRUZ, Centro de Pesquisas René Rachou,
Av Augusto de Lima 1715, Belo Horizonte, MG. CEP
30190-002
1
Albarnaz, J.D., Augusto, P.C.F., Ferreira, P.C.P.,
Kroon, E.G., 5Bonjardim, C.A.
1. UFMG, Universidade Federal de Minas Gerais, Av.
Antônio Carlos, 6627, ICB bl. F4 sl. 258, CEP 31270901, Belo Horizonte, MG
1
2
3
4
During infection, viruses hijack cell biosynthetic
machinery and evade from host antiviral response aiming
to proper progeny formation and dissemination. Although
Vaccinia virus (VACV) encodes several proteins related
to immune evasion, it also manipulates the activity of
cellular signaling pathways to create an intracellular milieu
more favorable to its replication. In Brazil, infections with
VACV are characterized as emerging zoonosis, affecting
rodents, cattle and humans. Several VACV strains have
been isolated from these natural infections and constitute
a useful tool to investigate VACV-host interaction. This
work evaluated activation and functional relevance of
cellular signaling pathways during infection with VACV
Belo Horizonte (VBH), isolated during a mousepox
outbreak in UFMG’s animal facility, when compared to
reference strain, VACV Western Reserve (VACV-WR).
Activation of MEK/ERK, JNK, p38 MAPK and PI3K/Akt
was temporally regulated along infection by both viruses.
However, contrary to the observed with VACV-WR,
pharmacological inhibition of MEK (UO126) and PI3K
(LY294002) did not affect significantly VBH replication
(inhibition d” 50%). On the contrary, inhibition of p38
MAPK (SB202190) reduced VBH yield in 90%, but did
not VACV-WR yield. In presence of LY294002, VBH protein
synthesis and progeny formation were delayed but not
inhibited and VBH morphogenesis proceeded normally.
Regardless prosurvival signals transmitted via PI3K/Akt
and MEK/ERK pathways, apoptosis was triggered during
VBH infection. Four known anti-apoptotic VACV genes
120
Adverse events upon smallpox vaccination with live
strains of Vaccinia virus (VACV) comprise an array of
clinical manifestations that occur primarily in
immunocompromised patients leading to significant host
morbidity and mortality. The increasing numbers of an
immune-suppressed population and the possible release
of Variola virus (VARV) as a bioterrorist act have given
rise to concerns over vaccination complications should
vaccination be reinitiated. Our goal was to evaluate the
components of the host immune system that are
sufficient to prevent morbidity/mortality in a murine model
of tail scarification, which mimics immunological and
clinical features of smallpox vaccination in humans.
Infection of C57BL/6 mice led to a strictly localized
infection, with complete viral clearance by day 28 p.i.
On the other hand, infection of T and B-cells deficient
mice (Rag1-/-) produced a severe disease, with
uncontrolled viral replication in the inoculation site and
dissemination to internal organs. Surprisingly, infection
of B-cell deficient animals (μMT) produced no mortality.
However, viral clearance in μMT animals was delayed
compared to WT animals, with detectable viral titers in
organs late in infection. Treatment of Rag1-/- with antivaccinia rabbit serum had little effect on the morbidity/
ABSTRACTS
mortality of this strain, but it was effective in reduce
viral titers in ovaries. Finally, NUDE athymic mice showed
a similar outcome of infection as Rag1-/-, and passive
transfer of WT T cells to Rag1-/- animals proved fully
effective in preventing disease and mortality. These
results strongly suggest that both T and B cells are
important in the immune response to primary VACV
infection in mice, and that T-cells are required to control
the infection at the inoculation site and providing help
for B-cells to produce antibodies, which help to prevent
viral dissemination. These insights might prove helpful
to design new, safer vaccines, and to develop a
treatment for the post-smallpox vaccination.
Palavras-chaves: Smallpox vaccination, Adverse
events, Vaccinia virus, Immune response.
CALICIVIRUS DETECTION IN CHILDREN FROM
GOIÂNIA, GOIÁS WITH OR WITHOUT DIARRHEA
SYMPTOMS
ID: 00197-00001
Área: 05 - Virologia Humana e Saúde Pública
BORGES, A.M.T., 2FIACCADORI, F.S., 3MENDANHA,
D.M., 4CARDOSO, D.D.P., 5SOUZA, M.D.
1. UFG, Universidade Federal de Goiás, Rua 235 esq.
1ª Av., Setor Universitário, Goiânia, Goiás
1
The human calicivirus have high prevalence in both
developed and developing countries, including Brazil.
These viruses are responsible for high hospitalization
rates and cause great burden to the public and private
health systems. In order to evaluate the occurrence of
these viruses among day-care children, we have collected
237 fecal samples from children under five years of age,
with or without diarrhea symptoms. These children
attended eight different day-care centers (Centros
Municipais de Educação Infantil-CMEIs) in Goiânia,
Goiás, and all the samples were collected from February2008 to January-2009. The viral detection was performed
by RT-PCR using the primer pairs: P289/290 and Mon381/
383. The overall positivity rate for calicivirus was 24.5%
(58/237). Positive samples were detected in children from
all CMEIs, and in every month, during the period of the
study, with a higher positivity rate being detected during
February and March. The caliciviruses were detected
among children with or without diarrhea symptoms,
however, the highest positivity rates (26.7%) were
detected from samples collected from children without
diarrhea, showing that calicivirus infection also circulate
asymptomatic children. Future studies will be conducted
to further characterize these positive calicivirus samples,
and also to evaluate the role of these asymptomatic
children in the epidemiology of the human caliciviruses
in our region.
Palavras-chaves: calicivirus, day-care centers,
developing countries.
QUASISPECIES DIVERSITY OF NONSTRUCTURAL 5A GENOMIC REGION OF HEPATITIS C VIRUS
GENOTYPE 1
ID: 00198-00002
Área: 05 - Virologia Humana e Saúde Pública
Jardim, A.C.G., 2Bittar, C., 3Matos, R.P.A., 4Yamasaki,
L.H.T., 5de Carvalho-Mello, I.M.V.G., 6Rahal, P.
1. UNESP-IBILCE, UNESP - IBILCE - Campus São José
do Rio Preto - Departamento de Biologia, Rua Cristóvão
Colombo, 2265,CEP 15054-010, São José do Rio Preto
-SP2. USP, USP - Universidade de São Paulo, Rua do
Matão, trav 14, n° 321, Cidade Universitária, CEP 05508900, São Paulo-SP3. USP, Universidade de São Paulo,
Instituto de Medicina Tropical, Av Dr Arnaldo, 455,
Cerqueira César, CEP: 012469034. HIAE, Hospital
Israelita Albert Einstein, Av Albert Einstein, 627/701,
CEP 05652-000 São Paulo-SP5. Butantan, Instituto
Butantan - Laboratorial de Imunologia, Av Vital Brasil n°
1500, CEP 05503-900, Butantã, São Paulo, SP
1
The NS5A protein has been implicated in the resistance
of HCV to interferon therapy. In this study we analyzed
the genetic variability of quasispecies on the coding region
of HCV NS5A in patients who developed infection with
HCV genotype 1:a non-responder patient (EAO) and two
end-of-treatment responders (CSM and PCZ). The
collection points for EAO patient were pre-treatment, 14
weeks of treatment, 14 days, 2 and 6 months after end
of treatment. For end-of-tretament responders samples
were collected at pre-treatment, the rebound point and
monthly until 6 months after end of treatment. Samples
were submitted to RT-PCR, cloned and sequenced. Ten
sequences were used in the analysis of NS5A for each
collection point. The analysis revealed that the genetic
distance decreased gradually in after treatment samples,
showing the lower value on the 6 months sample for
patient PCZ. Though the genetic distance of patient CSM
decreased gradually until the 3 months collection and
increase afterwards on 4th, 5th and 6th months gradually.
EAO patient showed decreased values in samples
collected at 12 weeks of treatment, a subsequent gradual
increase up to 2 months and a further decrease in 6
month. Regarding the NS5A quasispecies diversity, a
strain started to be predominant on 4 months sample for
patient PCZ and 2 months sample for patient CSM, in
both cases the predominant quasispecies were detected
until the last point. On patient CSM, the predominant
quasispecie emerged at the rebound point. Alternatively,
in patient PCZ the predominant quasispecie was detected
in the 2nd month. For the patient EAO, a predominant
121
ABSTRACTS
variant could be identified on 12 weeks, this variant
remained predominant in the next point and in 6th month
another variant was identified as predominant. These
results suggest that the dynamic of quasispecies might
be particular for each patient. This can be due to
differential selective pressure of the treatment and
individual immune system pressure.
Financial support: FAPESP
Palavras-chaves: Genotype 1, HCV, Hepatitis C, NS5A,
Quasispecies.
QUASISPECIES COMPOSITION OF NS5A PROTEIN
FROM HEPATITIS C VIRUS GENOTYPE 3A
ID: 00198-00001
Área: 05 - Virologia Humana e Saúde Pública
Bittar C, 2Jardim, ACG, 3Yamasaki, LHT, 4Queiróz, ATL,
Carareto, CMA, 6Pinho, JRR, 7de Carvalho-Mello, IMVG,
8
Rahal, P
1. UNESP-IBILCE, UNESP - IBILCE - Campus São José
do Rio Preto - Departamento de Biologia, Rua Cristóvão
Colombo, 2265,CEP 15054-010, São José do Rio Preto
-SP2. USP, USP - Universidade de São Paulo, Rua do
Matão, trav 14, n° 321, Cidade Universitária, CEP 05508900, São Paulo-SP3. USP, Universidade de São Paulo,
Instituto de Medicina Tropical, Av Dr Arnaldo, 455,
Cerqueira César, CEP: 012469034. HIAE, Hospital
Israelita Albert Einstein, Av Albert Einstein, 627/701,
CEP 05652-000 São Paulo-SP5. Butantan, Instituto
Butantan - Laboratorial de Imunologia, Av Vital Brasil n°
1500, CEP 05503-900, Butantã, São Paulo, SP
1
5
Hepatitis C is a global health issue, with a prevalence of
130 million seropositive persons worldwide. Because of
the high mutation rate of the RNA polymerase, this virus
presents a vast genetic variability that is presented in a
variety of levels, the genotypes, subtypes and the
quasispecies. The variability of one of the viral proteins,
NS5A, is believed to be related to the response to the
IFN therapy, the adopted treatment for the infection. This
study aimed to analyze the quasispecies composition
of the NS5A from HCV in patients infected with the
genotype 3a during and after treatment in patients Nonresponder (NR) and End-of-treatment responder (ETR).
Samples from 8 patients (4NR and 4ETR) were collected.
Viral RNA was extracted, cDNA synthesized, the NS5A
region was amplified and cloned. Until now 151
sequences from 6 patients were generated. The analysis
of the nucleotide sequences revealed that the same
quasispecies is found in two consecutive collections (4m
e 5m) from patient RF119 (ETR). The frequency of this
quasispecies is higher at 4 months (14%) than at 5
months (7%). When analyzing the amino acid sequences
of another ETR patient RF109, one quasispecies
122
emerges on the 2nd month after treatment (10%
frequency), it is found with a 8,33% frequency at 3
months, at 4 months with 13,33% and in the 5th month
the frequency raises to 60%. The analysis of the amino
acid sequences of patient RF119 revealed a
quasispecies that emerges at 3 months after treatment
at a 6,67% frequency, is also found at 4 months with
37,71% frequency and at 5 months with 28,57%
frequency. One amino acid sequence of non-responder
patient RF60 is found at 2 months (10% frequency) and
is also found at 5 months after treatment with an 8%
frequency. The phylogenetic tree shows the grouping of
all sequences of each patient in a monophyletic branch.
Further data can reveal if the predominant quasispecies
appears in other collection points and if other strains
becomes predominant during infection. Financial
Support: FAPESP
Palavras-chaves: Genotype 3, HCV, Hepatitis C, NS5A,
Quasispecies.
HPV AND ALTERATIONS IN GENE EXPRESSION IN
PENILE CARCINOMA
ID: 00199-00001
Área: 05 - Virologia Humana e Saúde Pública
Mota, M.T.O., 2Babeto, E., 3Cândido, N.M., 4Peitl, P.,
Calmon, M., 6Bonilha, J., 7Soares, F.A., 8Cunha, I.W.,
9
Vassallo, J., 10Rahal, P.
1. IBILCE/UNESP, Instituto de Biociências, Letras e
Ciências Exatas - UNESP, R. Cristóvão Colombo, 2265.
São José do Rio Preto, SP2. FAMERP, Faculdade de
Medicina de São José do Rio Preto, Av. Brigadeiro Faria
Lima, 5416. São José do Rio Preto, SP3. HCANC,
Hospital A. C. Camargo, R. Professor Antônio Prudente,
211. São Paulo, SP
1
5
Penile squamous cell carcinoma (PSCC) is an invasive
epithelial tumor with high morbidity. It’s rare in developed
countries, but in Brazil has incidences ranging from 2.9
to 6.8 per 100,000 inhabitants. Early diagnosis, treatment
and follow-up are impaired by cultural and socio-economic
status of the patients. Without treatment patients die in
two years after the diagnosis of the first lesions, however,
the cure rate is high when diagnosis is done earlier. The
etiology and molecular factors that affect the
development of this tumor still remains not fully
understood. In the last years several studies has
implicated human papillomavirus (HPV) in
carcinogenesis. The goals of this work are to study the
possible role of different HPV genotypes in PSCC
development and to identify gene expression alterations
in tissue samples. Penile tissues from patients harboring
PSCC were assessed by PCR for HPV prevalence and
by RaSH (Rapid Subtraction Hybridization) for gene
ABSTRACTS
expression. It was found a high prevalence of HPV
(73.8%) in tumoral sample tissues. The most prevalent
genotype found are HPV16 (92%) followed by HPV11
(4%) and HPV35 (4%). RaSH revealed 57 genes
differentially expressed in normal and tumoral tissues:
30 in tumoral tissues and 27 in normal tissues. Most
relevant genes in tumoral tissues are PBEF1 (pre-B-cell
colony enhancing factor 1) and ANXA1 (annexin A1),
both related to cellular proliferation and anti-apoptotic
effects; RPL6 (ribosomal protein L6) linked to transcription
and translation regulation and WASH7 (WASH complex
subunit 7), that plays a role in endosome sorting. In
normal tissues p16, a known tumor suppressor gene is
the most relevant gene. Gene alterations found could
improve our knowledge of molecular mechanisms of
penile carcinoma and to help the diagnosis, treatment
and prognostic. The high prevalence of HPV suggests
an important role of HPV in the development of PSCC.
Financial support: FAPESP
Palavras-chaves: Alterações genéticas, Câncer de
pênis, Papilomavírus, RaSH.
FUNCTIONAL ANALYSIS OF THE GENE THAT
ENCODES THE TOMATO HOMOLOGUE OF SNF1,
DIFFERENTIALLY EXPRESSED DURING THE
TOMATO-PepYMV INTERACTION
which transformation was confirmed and mechanically
inoculated these plants with PepYMV. ELISA was
performed to verify the phenotype resulting from SlSNF1
silencing (increased level of susceptibility or resistance
to PepYMV). The results showed that four out of five
non-transformed plants were infected, while all ten
transformed plants remained symptomless. Assessment
of viral accumulation by quantitative RT-PCR is being
done to confirm the absence of viral replication. These
results suggest that the tomato homologue of SNF1 has
a role in supporting the establishment of viral infection.
Further studies should be conducted to confirm this
hypothesis and improve our understanding of the nature
and mechanisms of the interaction between SlSNF1 and
PepYMV.
Financial support: FAPEMIG, CNPq.
Palavras-chaves: Functional analysis, PepYMV,
Potyvirus, SNF1, Tomato.
PARTIAL GENOME SEQUENCING OF Papaya lethal
yellowing virus (PLYV) ISOLATES FROM RIO
GRANDE DO NORTE STATE, BRAZIL.
ID: 00200-00002
Área: 06 - Virologia Vegetal
Pereira, A.J., 2 Cascardo, R.S., 3 DALTRO, C.B.,
ANDRADE, E.C., 5Zerbini, F.M.
1. UFV, Universidade Federal de Viçosa, Av. P.H. Rolfs,
s/n. Viçosa, MG 36570-0002. Embrapa, Embrapa
Mandioca e Fruticultura Tropical, R. Embrapa, s/n. Cruz
das Almas, BA 44380-000
1
ID: 00200-00001
Área: 06 - Virologia Vegetal
CASCARDO, R.S., 2BRUCKNER, F.P., 3ZERBINI, F.M.,
ALFENAS-ZERBINI, P.
1. UFV, Universidade Federal de Viçosa, Av. P.H. Rolfs,
s/n
1
4
Viruses that infect plants have different mechanisms to
infect the host. The interaction between host defense
responses and viral infection leads to changes in the
transcriptional pattern of the plant. Knowledge of the
functional significance of these changes may help us to
understand the mechanisms of viral infection and plant
defense, contributing to the development of effective
strategies to control plant viruses. Previous work
demonstrated the induction of the tomato homologue of
the Saccharomyces cerevisae SNF1 kinase during the
early stages of tomato infection by the potyvirus Pepper
yellow mosaic virus (PepYMV). However the exact role
of this protein within the context of viral infection remains
undetermined. We generated transgenic tomatoes (cv.
‘Moneymaker’) silenced for the gene that encodes the
SNF1 homologue (SlSNF1). Transformations were
performed via Agrobacterium tumefaciens using the
Gateway vector pK7GWIWG2, which allows the inclusion
of inver ted repeats that generate dsRNA when
transcribed. We vegetatively propagated the plants on
4
The papaya (Carica papaya) is a fruit crop of great
economical importance throughout the Brazilian
Northeast, which is responsible for 60% of the national
output. Papayas in the states of Ceará and Rio Grande
do Norte are affected by lethal yellowing disease, caused
by Papaya lethal yellowing virus (PLYV). Previous work
indicated that PLYV is a putative sobemovirus. However,
the complete viral genome has not yet been sequenced,
and very few studies have been carried out on the genetic
diversity of PLYV isolates. The objectives of this work
were to obtain a full-length sequence of the viral genome
and to assess the genetic diversity of the virus. Foliar
samples were collected during January of 2009 in fields
from Rio Grande do Norte state, and total RNA was
extracted. A pair of degenerate primers based on the
sequences of known sobemoviruses was used for the
RT-PCR-based amplification of an approximately 900 bp
fragment corresponding to the central region of the viral
genome. Fragments corresponding to a total of 16 viral
isolates were cloned and sequenced. Sequence analyses
indicated >97% identity among the isolates, 94-100%
identity with a previously sequenced PLYV isolate from
123
ABSTRACTS
Ceará state, and a lower but significant identity with the
sobemoviruses Rice yellow mottle virus (RYMV),
Sesbania mosaic virus (SeMV) and Southern bean
mosaic virus. These results suggest a low degree of
genetic diversity among PLYV isolates, and are in
agreement with the provisional placement of PLYV in
the genus Sobemovirus. Definitive taxonomic
conclusions, however, require the full-length genomic
sequence. With that purpose, specific primers were
designed based on the sequences of the 5’ and 3’ ends
of the 900 bp fragment, and additional degenerate primers
were designed based on the regions of the 5’ and 3’ ends
of sobemoviruses. These primers were used to amplify
the remaining portions of the genome. Cloning and
sequencing of these fragments is under way.
Financial support: CNPq, FAPEMIG
Palavras-chaves: Genome Sequencing, PLYV,
Sobemovirus.
EVALUATION OF THE ROTARIX VACCINATION
IMPACT ON ENTERIC VIRUS PREVALENCE AMONG
CHILDREN IN GOIÂNIA, GOIÁS
ID: 00201-00001
Área: 05 - Virologia Humana e Saúde Pública
MORAES, T.C., 2BORGES, A.M.T., 3ALMEIDA, T.N.V.,
SOUZA, M.D., 5JUNQUEIRA, I.C., 6CARDOSO, D.D.P.,
7
FIACCADORI, F.S.
1. UFG, Universidade Federal de Goiás, Rua 235 s/nº,
esq. 1ª Av, Setor Universitário, Goiânia
1
4
Acute gastroenteritis constitutes an important cause of
morbidity and mortality among children less than five
years of age. Among children, the Rotavírus A (RVA)
are the main etiological agents of the viral gastroenteritis,
however; other enteric viruses, such as caliciviruses,
astroviruses and enteric adenoviruses, are frequently
associated to this syndrome. The worldwide impact of
RVA infections had led to the development of many
vaccination strategies aiming at the reduction of the
morbid-mortality rates associated to this agent. In Brazil,
the RotarixÒ was included, in March 2006, in the National
Immunization Program. After the introduction of the
vaccine, as an alternative for the control of rotavirus
infections, it became of great importance monitoring of
the viral gastroenteritis cases in order to evaluate the
vaccine efficacy, and its effect on the circulation of other
viral agents among children in the country. In this context,
our study aimed at the evaluation of the RotarixÒ impact
on the viral gastroenteritis cases in Goiânia, Goiás. For
this, 65 fecal samples were collected from children less
than five years of age, suffering from acute diarrhea that
sought care in two hospitals located in Goiânia. All
samples were analyzed by EIA for the detection of
124
adenoviruses and by RT-PCR form the detection of
caliciviruses and astroviruses. From the total samples
collected, 24.6%; 6.1% and 3.1% were positive for
calicivirus, enteric adenoviruses and astroviruses,
respectively. When we consider the recorded symptoms
we observed that calicivirus positivity rates were
significantly higher among children without fever. Our
results also demonstrated an increase in the prevalence
rates of calivirus and adenoviruses infections after the
introduction of the RotarixÒ in Goiânia, whereas the
astrovirus positivity rates remained the same as
observed in studies previously conducted in the region.
Palavras-chaves: prevalence, enteric virus, children.
ROTAVIRUS A INFECTION IN GOIÂNIA, GOIÁS,
POST-VACCINATION
ID: 00201-00002
Área: 05 - Virologia Humana e Saúde Pública
ALMEIDA, T.N.V., 2BORGES, A.M.T., 3SOUZA, M.D.,
MORAES, T.C., 5 JUNQUEIRA, I.C., 6 CARDOSO,
D.D.P., 7FIACCADORI, F.S.
1. UFG, Universidade Federal de Goiás, Rua 235, esq.1ª
Av., Setor Universitário, Goiânia, Goiás
1
4
The Rotavírus A (RVA) are ubiquitous gastroenteric
viruses that infect human and animals. These viral agents
are recognized around the world as important etiological
agents of acute gastroenteritis among children less than
five years of age. Because of the impact of the rotavirus
infections in children morbi-mortality, various preventive
measures, including vaccination strategies, have been
developed and implemented in various parts of the world.
In Brazil, the viral oral human attenuated vaccine
RotarixÒ was included in the National Immunization
Program in March 2006. Due to use of the vaccine as a
control and prevention measure against RVA, a careful
monitoring of the effects of the vaccine on the prevalence
and circulation of these agents, among the children
population of different parts of Brazil, became necessary.
Therefore, this study aimed at the evaluation of the
effects of the RotarixÒ on the circulation and prevalence
of RVA in Goiânia. For this, 65 fecal samples were
collected from children less than five years of age, with
diarrhea symptoms, that had been previously vaccinated
or not, that sought medical care in two hospitals in
Goiânia. All fecal samples were screened for rotavirus
by an enzyme immune assay (EIA) and by
Polyacrilamide Gel Electrophoresis (PAGE), with a total
positivity rate for rotavirus of 16.9%. Our results show
that a higher prevalence was observed among nonvaccinated children (23.1%). When we consider the symptomatology, 90.9% of the positive samples were collected
from children presenting diarrhea associated with fever
ABSTRACTS
and vomit. The 11 positive samples were genotyped by
Multiplex RT-PCR, and nine could be genotyped for VP7
gene (four were typed as G2, one as G8 and four as G2/
G8), and five for the VP4 gene (four were P[4] and one
was P[9]/P[11]). The results show a decrease in the RVA
prevalence after the introduction of the vaccine in this
population, as well as a shift in the circulating genotypes.
Palavras-chaves: Rotavirus, Infection, Children.
PHYLOGENETIC ANALYSIS OF THE GENE SEGMENTS OF HEMAGGLUTININ AND NEURAMINIDASE
OF INFLUENZA B VIRUSES CIRCULATING IN
BRAZIL FROM 2004 TO 2008.
ID: 00202-00001
Área: 05 - Virologia Humana e Saúde Pública
influenza B virus, and are of great relevance for the
continuing suitability and implementation of policies and
strategies aimed at controlling and preventing influenza
infections in our population.
Palavras-chaves: Influenza B, Reassor tment,
Hemagglutinin, Neuraminadase, antigenic drift.
MENSSUREMENT OF SEASONAL INFLUENZA
VIRUS SUSCEPTIBILITY TO NEURAMINIDASE
INHIBITORS, N1 AND N2 SUBTYPES, CIRCULATING
IN BRAZIL.
ID: 00202-00002
Área: 05 - Virologia Humana e Saúde Pública
Lima, C.H.A, 2Souza TML, 3Pinhão AT, 4Machado DBB,
Resende PC, 6Motta FC, 7Baccala GP, 8Siqueira MM
1. IOC/FIOCRUZ, Instituto Oswaldo Cruz/FIOCRUZ, Av.
Brasil, 4365 - Manguinhos - Rio de Janeiro - RJ - Brasil
- HPP - sala B1052. BMX, Fundação BioMerrieux, França
1
5
Resende PC, 2Born PS, 3Motta FC, 4Siqueira MM
1. IOC/FIOCRUZ, Instituto Oswaldo Cruz/FIOCRUZ, Av.
Brasil, 4365 - Manguinhos - Rio de Janeiro - RJ - Brasil
- HPP - sala B105
1
Infections caused by influenza viruses are a major
challenge for public health worldwide. Influenza B viruses
belong to the Orthomyxoviridae family, their segmented
genome is composed of a single strand RNA and negative
polarity. The evolutionary mechanisms of reassortment
and antigenic drift favor the continuous emergence of
new variants, which require annual reformulation of the
vaccine. Since the early 80s, two lineages antigen and
phylogenetically distinct circulating: B/Victoria/2/87-like
(Vic87) and B/Yamagata/16/88-like (Yam88). Since the
last decade these co-circulating in different countries,
along with a viral variant which has rearranged the gene
segments hemagglutinin (HA) of Vic87 and
neuraminidase (NA) of Yam88. Not enough is known about
the circulation of these viruses in Brazil. So the purpose
of this study was to identify and molecularly characterize
the gene segments of HA and NA of influenza B
circulating in different regions of Brazil during epidemics
from 2004 to 2008. The methodology utilized was Sanger
sequencing for determination of viral variants. We
detected different substitutions in HA and NA genes in
relation to vaccine strains, and demonstrated the cocirculation of both lineages from 2005. However, we did
not observe the occurrence of reassortment nor the
emergence of strains with markers of resistance to
neuraminidase inhibitors, based on the genes
investigated. Despite the co-circulation, in the period
2006-2008, we observed agreement between the
circulating lineages and vaccine strains recommended
for use in the Southern Hemisphere, however, the same
was not true for the period 2004-2005. This set of
information contribute to a better understanding of the
mechanisms involved in the molecular evolution of
Clinical use of the NAI oseltamivir has been associated
with the emergence of viral resistance resulting from
subtype specific neuraminidase mutations. Due to this
fact the LVRS/IOC/FIOCRUZ started a surveillance study
of seasonal influenza virus susceptibility to
neuraminidase inhibitors (NAI) in order to evaluate the
impact of the frequent oseltamivir-resistant NA mutations.
These are the preliminary results of this study. It has
been conducted using a NA inhibition assay (NA-Star®
Influenza Neuraminidase Inhibitor Resistance Detection
Kit, Applied Biosystems, Foster City, CA) on samples
isolated from different States in Brazil. The virus was
isolated and propagated in Madin-Darby canine kidney
(MDCK) cells from clinical specimens. The 50% inhibitory
concentration values (IC50) were determined based on
mean IC50. To identify potentially resistant viruses
(outliers), a threshold of the IC50 value was defined for
subtype by the upper bound of the 95% confidence. The
viruses were divided in two groups, sensitive and
resistant. The Influenza A H3N2 viral sensitive (0,25 and
012 nM) and resistant (158,59 and 158,59 nM) (0.44 nM)
had mean and median lower to oseltamivir than Influenza
A(H1N1) viruses sensitive (0,36 and 0,35 nM) and
resistant (146,16 and 145,37 nM). Palavras-chaves:
Neuraminidase, Influenza, Susceptibility.
PROPERTIES OF A Turnip ringspot virus ISOLATE
FROM PARANÁ STATE
ID: 00203-00001
Área: 06 - Virologia Vegetal
PICOLI, M.H.S., 2SILVA, J.M., 3CARNELOSSI, P.R.,
PELISSON, N., 5BIJORA, T., 6FACCO, C.U., 7ALMEIDA,
1
4
125
ABSTRACTS
A.M.R., 8SOUTO, E.R.
1. UEM, Universidade Estadual de Maringá, Av. Colombo,
5,790, Jardim Universitário, Maringá, Paraná. CEP 870209002. EMBRAPA, Empresa Brasileira de Pesquisa
Agropecuária, Rod. Carlos João Strass. Caixa Postal 231.
CEP 86001-970, Londrina, PR.
Comoviruses have positive-sense, single-stranded,
bipartite RNA genomes designated RNA1 e RNA2. This
genus infects predominantly Leguminosae. Turnip ringspot
virus (TuRSV) is a Comovirus that infects Brassicaceae
plants, causing a variety of symptoms ranging from
chlorotic spots and rings on leaves to complete chlorosis
and death. Two fragments of RNA1 and RNA2 of TuRSV
isolated from Eruca sativa, in Paraná state, were amplified
by RT-PCR using random primers for reverse transcription.
The amplified product of 369 bp from RNA1 and the 168
bp product from RNA2 were cloned and sequenced. After
comparisons with other sequences from the GenBank,
the RNA1 sequence showed 83% identity with RNA1 of a
TuRSV isolate from Toledo-USA, and the RNA2 showed
80% identity with the RNA2 of the same virus. Another
set of primers was designed with PrimerDesign®, to
amplify the 32 -terminal region of TuRSV-Paraná RNA1
genome. The 559 bp RT-PCR product was sequenced,
showing 82% homology with the isolates TuRSV-Toledo
and TuRSV-M12 from Russia. The isolate of TuRSV from
Paraná has a narrow host range, infecting Eruca sativa,
Brassica rapa, Nicotiana benthamiana and Chenopodium
amaranticolor. The thermal inactivation point (TIP) of the
virus was determined as 65ºC and the dilution end point
(DEP) as 10-3. The virus could infect radish plants after
being for 25 days in crude sap of E. sativa at room
temperature. Polyacrylamide gel electrophoresis of the
purified virus revealed several protein bands, including
those of 27 and 41 kDa, tipical of TuRSV. Transmission
electron microscopy revealed isometric particles ranging
from 25-30 nm in diameter.
Palavras-chaves: Comovirus, RT-PCR, Rúcula,
Sequenciamento parcial.
Group A rotavirus is the major cause of acute infectious
diarrhea in children and a serious public health problems
in the world. It is an important cause of mortality in
developing countries and morbidity in developed
countries. The virus genome is composed by 11
segments of double-strand RNA. Viral particle is formed
by a triple capsid; the outer layer is composed of VP7
and VP4 proteins, which define the G and P types,
respectively. Rotavirus presents a great antigenic
variability, and different combinations of G and P
genotypes may arise on a given region over the years.
In Brazil, a monovalent vaccine against rotavirus
(G1P[8]) was introduced into the childhood immunization
program in 2006. A survey on the frequency and
distribution of rotavirus genotypes causing gastroenteritis
in infants and children in the Triângulo Mineiro region of
Minas Gerais has been underway since September 2005;
so, the objective of this study was detect and
characterize rotaviruses on stools of children with
gastroenteritis at Uberaba and Uberlândia, MG, in 2009
and 2010 years. The virus detection was made by latex
agglutination and visualization of rotavirus genome on
poliacrilamide gel electrophoresis after extraction from
stools by silica method; the positive samples were
genotyped by RT-PCR. From 315 samples analysed, 14
(4,4%) specimens were positive. The age group most
affected by diarrhea was children between 7 and 12
months. The results showed the same prevalence of the
rotavirus in outpatients and inpatients. We also observed
a marked increase in rotavirus prevalence from 2009
(1,0%) to 2010 (10,3%). The two samples found in 2009
showed long electropherotypes and genotypes
G5G10Pnon-typed and G3Pnon-typed. All the specimens
detected in 2010 had short electropherotypes. Continuous
monitoring rotavirus genotypes circulating in the region
is crucial to better understand the dynamics of rotavirus
strains especially after the vaccine implementation.
Financial support: CNPQ;FAPEMIG;CAPES;UFTM
Palavras-chaves: Human Rotavirus, Genotyping,
Epidemiology.
ROTAVIRUS-ASSOCIATED DIARRHEA AT
TRIÂNGULO MINEIRO-MG, BRAZIL, IN 2009/2010
PERIOD
SHEDDING LIGHT ON THE ENTRY OF A NEW
WORLD ALPHAVIRUS INTO HOST CELLS
ID: 00205-00001
Área: 05 - Virologia Humana e Saúde Pública
ID: 00208-00001
Área: 04 - Virologia Básica
Dulgheroff, A.C.B., 2FIGUEIREDO, E.F., 3MOURA,
L.M.S., 4MOREIRA, K.C., 5GOUVEA, V., 6DOMINGUES,
A.L.S.
1. UFTM, Universidade Federal do Triângulo Mineiro,
Av.Frei Paulino, 30 - Bairro Abadia - Uberaba-MG2. UFRJ,
Universidade Federal Rio de Janeiro, Av. Brigadeiro
Trompowsky, SN – C.C.S/IMPPG, Ilha do Fundão, RJ,
RJ
CARVALHO, C.A.M., 2SOUSA JR., I.P., 3SILVA, J.L.,
4
GOMES, A.M.O.
1. IBqM/UFRJ, Instituto de Bioquímica Médica/
Universidade Federal do Rio de Janeiro, Av. Carlos
Chagas Filho, 373 - Cid. Universitária, Rio de Janeiro,
RJ, 21941-902
1
126
Mayaro virus (MAYV) is a New World alphavirus
perpetuated in nature by alternating replication in a
ABSTRACTS
mosquito and a mammal. Virus entry into target cells
occurs by receptor-mediated endocytosis followed by
fusion between the viral envelope and the endosomal
membrane. The aim of this work was to analyze the
dynamics of the endocytic route taken by MAYV particles
during their entry into mammalian and mosquito cells. To
achieve that, the virus was fluorescently labeled on its
envelope proteins without impairment to viral infectivity
and the fluorescent signal was tracked in the host cells
by laser-scanning confocal fluorescence microscopy and
spatiotemporal image correlation spectroscopy. Our
results show that, after a brief period of similarity, the
distribution of the fluorescent signals became different
between the distinct host cells lines, which correlated to
differences in the dynamics of the intracellular movement
of these signals. We suggest that MAYV recruits different
endocytic routes for entry into mammalian and mosquito
cells.
Financial support: CAPES, CNPq, FAPERJ, FINEP,
INBEB and PRONEX.
Palavras-chaves: Alphavirus, Entry, Mayaro, Tracking.
INVESTIGATION OF THE ANTIVIRAL ACTIVITY OF
LACTOFERRIN
ID: 00208-00002
Área: 04 - Virologia Básica
CARVALHO, C.A.M., 2GONÇALVES, R.B., 3 SILVA,
J.L., 4GOMES, A.M.O.
1. IBqM/UFRJ, Instituto de Bioquímica Médica/
Universidade Federal do Rio de Janeiro, Av. Carlos
Chagas Filho, 373 - Cid. Universitária, Rio de Janeiro,
RJ, 21941-9022. DBQ/UNIRIO, Depar tamento de
Bioquímica/Universidade Federal do Estado do Rio de
Janeiro, R. Frei Caneca, 94 - Centro, Rio de Janeiro, RJ,
20211-040
1
Lactoferrin (Lf) is a multifunctional iron-binding
glycoprotein which is known to exert a broad-spectrum
primary defense activity against bacteria, fungi, protozoa
and viruses. In order to address the way by which Lf
exerts its antiviral activity, we evaluated the effects of
Lf treatment in the infection process of Mayaro virus
(MAYV). MAYV is an alphavirus endemically spread in
South America, where it is responsible for sporadic
outbreaks of human infections. Our results show that Lf
was able to promote a large inhibition of MAYV infection
in Vero cells without lead to cytotoxic effects. Tracking
the early steps of infection of fluorescently labeled MAYV
by laser-scanning confocal fluorescence microscopy, we
could observe that virus entry into host cells was strongly
inhibited by the presence of Lf. Our findings suggest
that Lf inhibits virus entry into cells rather than later
phases of viral replication and point out the potential of
the protein against alphavirus infections. Financial
support: CAPES, CNPq, FAPERJ, FINEP, INBEB and
PRONEX.
Palavras-chaves: Alphavirus, Antiviral, Lactoferrin,
Mayaro.
PURIFICATION AND A POLICLONAL ANTISERUM
PRODUCTION FOR CASSAVA COMMON MOSAIC
VIRUS.
ID: 00210-00001
Área: 06 - Virologia Vegetal
CARNELOSSI, P.R., 2 BIJORA, T., 3 SILVA, J.M.,
PICOLI, M.H.S., 5 PELISSON, N., 6 FACCO, C.U.,
7
ALMEIDA, A.M.R., 8SOUTO, E.R.
1. UEM, Universidade Estadual de Maringá, Avenida
Colombo, 5790, Jardim Universitário, CEP: 87020900,
Maringá-PR Brasil2. Embrapa-Soja, Empresa Brasileira
de Pesquisa Agropecuária - Embrapa Soja, Rod. Carlos
João Strass - Distrito de War ta; CEP 86001-970;
Londrina-PR Brasil
1
4
A great diversity of viruses can be detected in cassava
culture, however, in Brazil, Cassava common mosaic
virus (CsCMV), Potexvirus, is the most frequent and
important in the south and southeast cassava producing
regions. Considering this virus is harmful, and infected
plants usually do not show symptoms, the application
of serological diagnostic tests allows indexing plant
material free of viruses. These methods are useful for
plant virus detection for being sensitive, efficient and
fast. Nevertheless, the most significant limitation of
serology is the availability of a virus specific antiserum.
A CsCMV isolate from Paranavaí-PR was efficiently
purified from Nicotiana benthamiana plants and a
polyclonal antiserum was produced. The yield of virus
purification was 3,84 and 3,15 mg of virus/ml for each
purification conducted. Through indirect-ELISA tests the
antiserum titre was determined as 1/10.000, not reacting
with healthy plants. The immunoglobulins G were also
purified resulting in a titre of 1/1.000 providing greater
sensitivity than the raw antiserum in indirect-ELISA tests.
Palavras-chaves: CsCMV, Cassava, Potexvirus, indirectELISA.
EVALUATION OF CASSAVA COMMON MOSAIC
VIRUS ELIMINATION FROM MICROPROPAGATED
PLANTS WITH ANTIVIRAL.
ID: 00210-00002
Área: 06 - Virologia Vegetal
1
4
BIJORA, T., 2 CARNELOSSI, P.R., 3 SILVA, J.M.,
FACCO, C.U, 5 PICOLI, M.H.S., 6 SANTANA, P.P.,
127
ABSTRACTS
SOUTO, E.R.
1. UEM, Universidade Estadual de Maringá, Av. Colombo,
5.790; Jd. Universitário; Maringá-PR Brasil; CEP 87020900
7
In order to obtain virus free cassava plants, meristem
tip culture associated with thermotherapy is used.
However, in many cases, this method alone is not
sufficient for virus elimination, and it would justify the
complementary use of chemotherapy. In this study, to
evaluate the effect of different concentrations of
Ribavirine®, this antiviral was added to MS culture
medium, associated with a previous thermotherapy to
obtain cassava plants, free from Cassava Common
Mosaic Virus (CsCMV). Cassava stems were segmented
in cuttings of 20 cm in length and, after being sealed
with paraffin, were cultivated for 30 days in a growth
chamber, under 16h of light at 38ºC and 8h of dark at
26ºC. Shoots from cuttings were disinfected, and the
apical meristems measuring 0,5 to 0,7 mm were removed
from the shoots, and cultivated in a MS medium with
Ribavirine® at the concentrations of 0,5, 10, 15, 20, 30
and 40 mg/L, using 20 plants per treatment. The
meristems were maintained in the growth chamber, under
temperature of 24±2°C in a photoperiod of 16 hours.
Concentrations of 15 to 40 mg/L induced fitotoxic effects,
and death of plantlets, after 10 days of culture. Some
cassava plantlets could develop, and leaves were
removed from these plants for CsCMV indexing through
indirect-ELISA. Three out of 20 plants regenerated in the
5 mg/L Ribavirine® medium, and 4 out of 20 regenerated
in the medium concentration of 10 mg/L. The absence of
virus was confirmed in 3 out of the 7 indexed plants.
Palavras-chaves: Apical meristems, CsCMV,
Ribavirine®, Thermotherapy.
in their hosts. In this study, we investigated the
reactivation of latent infection and the distribution of
BoHV-5 DNA in the brain of latently, experimentally
infected sheep. Fifteen lambs were inoculated intranasally
with BoHV-5 SV-507/99 strain, with a titer of 106.8
TCID50/ml and nasal swabs for viral isolation were
collected on a daily basis up to day 15 post-inoculation
(dpi). Thirty days pi, five of these animals were euthanized
for collection of the brain to investigated the presence of
viral DNA by using PCR. During acute infection all animals
shed virus in nasal secretions up to day 11 pi. The peak
of virus excretion occurred between days 2 and 3 pi. On
day 85 pi, dexamethasone (Dx) was administered to
animals, resulting in viral reactivation in 62.5% of animals
(5/8), with virus shedding starting at the third day after
Dx administration. Viral shedding lasted 1-5 days, with
titers lower than those observed during acute infection.
At 30 dpi, latent viral DNA was detected by PCR in the
trigeminal ganglia (5/5) and olfactory bulb (5/5); in lower
frequency in the pons (2/5), cerebellum (2/5), anterior/
ventro-lateral/posterior cortex (1/5) and olfactory cortex
(1/5). Therefore, we conclude that sheep are susceptible
to BoHV-5, and that latent infection is established in
trigeminal ganglia and also in other parts of the brain.
Thus, lambs may be used as model to study latent BoHV5 infection.
Palavras-chaves: BoHV-5, latent infection, PCR, sheep.
A RNA TRANSFECTION SYSTEM FOR GENERATION OF VIRUS BY IN OVO ELECTROPORATION
ID: 00213-00001
Área: 05 - Virologia Humana e Saúde Pública
Silva Júnior, J. V. J., 2Almeida, S. R., 3Bertani, G.R.,
Gil, L. H. V. G.
1. CPqAM/Fiocruz, Centro de Pesquisas Aggeu
Magalhães/Fundação Oswaldo Cruz, Av. Professor
Moraes Rego s/n, Campus da UFPE. Cidade
Universitária,50670-4202. LIKA/ UFPE, Laboratório de
Imunopatologia Keizo Asami/ Universidade Federal de
Per nambuco, Av. Moraes Rego S/N, Campus da
UFPE.Cidade Universitária,50670-420
1
REACTIVATION AND DISTRIBUTION OF BOVINE
HERPESVIRUS TYPE 5 DNA IN THE CENTRAL
NERVOUS SYSTEM OF LATENTLY INFECTED
LAMBS
ID: 00212-00001
Área: 03 - Virologia Animal
CADORE, G.C., 2 ANZILIERO, D., 3HERMES, B.N.,
WEIBLEN, R., 5FLORES, E.F.
1. UFSM, Universidade Federal de Santa Maria, Santa
Maria 97105-900, RS, Brasil
4
1
4
Bovine herpesvirus type 5 (BoHV-5) is an important
pathogen of cattle with ability to establish latent infections
128
Reverse genetics or de novo synthesis, in molecular
virology, comprises the generation of viruses from cloned
cDNA by plasmid-encoded or in vitro synthesized RNA
transfection. Today, this system is important tool to study
the virus life cicle, to understand the roles of viral proteins
in virus-host interplay and pathogenicity, besides allowing
the generation of recombinant viruses to live vaccines.
However, these viruses were only recovered (rescue) in
a limited number of permissive cells. Moreover, the DNA
transfer into chick embryos by in ovo electroporation
ABSTRACTS
has been applied since 1996 and today is used in
embryology study, mainly to neural tissue. Thus, to
recognize the dependence/limit of the reverse genetics
to permissive cells and the successful electroporation
of chick tissue, we create the first system for virus rescue
in ovo through of the RNA virus [recombinant Yellow Fever
virus expressing the Yellow Fluorescent Protein (YFP),
YFV-YFP] transfection into chicken embryos. The YFVYFP used was generated by homologous recombination
in yeast and its full-length genome was in vitro
transcribed. The RNA obtained was transfected into chick
embryos by in Specific Pathogen Free ovo
electroporation. Three days post-electroporation, the
evaluation of the viral rescue was performed by YFP
reporter gene expression in fluorescence microscope.
After the fluorescence microscope, the embryo was
crushed and the product was inoculated in Baby Hamster
Kidney 21 cell (BHK-21). Three days post-inoculated,
RT-PCR from supernatant RNA and direct
imunofluorescence (DIF) were performed. Although the
RT-PCR and the DIF are still in process, the positive
result obtained of the YFP expression indicates the
successful of the system created to recovery virus.
Financial support: Fiocruz/ CNPq.
Palavras-chaves: Chick, in Ovo, Reverse genetic, RNA
transfection, Yellow Fluorescent Protein.
BOHV-1 AND BOHV-5 DETECTION IN DIFFERENT
CLINCAL SAMPLES OBTAINED FROM CATTLE IN
GOIÁS, BRAZIL BY MULTIPLEX PCR.
ID: 00214-00002
Área: 03 - Virologia Animal
Silva, D.A., 2Silva, A.M., 3Souza, K.M., 4Soares, P.,
Castro, M.C., 6Moraes, P.C.G., 7Roehe, P.M., 8Brito,
W.M.E.D.
1. UFG, Universidade Federal de Goiás, Campus
Samambaia. Caixa postal 131 - CEP: 74001-970 - Goiânia
- GO2. UPIS, União Pioneira de Integração Social, SEP
Sul EQ 712/912 Conjunto A - Asa Sul - 70390-125 Brasília - DF3. IPVDF - FEPAGRo, Instituto de Pesquisas
Veterinárias Desidério Finamor, Estrada do Conde,
n.°6000 – Sans Souci. 92990-000 Eldorado do Sul – RS
vaginal secretion and 23 samples of respiratory
discharge were collected. All samples were submitted
to standard phenol-chloroform protocol for DNA
extraction. BoHV 1 and 5 were detected by a multiplex
PCR for glycoprotein C gene detection. The expected
fragment amplified was 354 bp for BoHV-1 e 159 bp for
BoHV-5. Out of brain samples, 45 (50.0%) were positive
for at least one of the two viruses: BoHV-1 was identified
in 12 samples (26.7%); BoHV-5 were identified in 21
samples (46.7%) and both viruses occurred in 12 (26.7%)
samples. Twenty two semen samples (36.1%) showed
positive results. BoHV-1 was detected in seven (31.8%)
samples; BoHV-5 were detected in eight (36.4%) samples
and both viruses were detected in seven (31.8%)
samples. The virus was detected in 34 (39-1%) vaginal
samples and only BoHV-5 were detected. Two samples
(8.7%) of respiratory discharge showed positivity, one
for BoHV-1 and one for BoHV-5. The results here
presented demonstrate a high frequency of BoHV-5 not
only in CNS samples but also in the other clinical samples
analyzed. Despite the small sample size, a change in
the epidemiological pattern of distribution of these viruses
occurs in Goiás. The high rates of concomitant detection
of BoHV-1 and -5 may demonstrate that the infection by
one virus does not exclude infection by the other.
Palavras-chaves: Bovinos, herpesvirus bovinos,
multiplex PCR, Centro oeste.
TITRATION OF RABIES ANTIBODIES IN CANINES
LIVING IN URBAN AREAS OF CUIABÁ, STATE OF
MATO GROSSO, BRAZIL
ID: 00214-00001
Área: 03 - Virologia Animal
1
5
Bovine herpesviruses type 1 (BoHV-1) and type 5 (BoHV5) are two important closely related pathogens of cattle.
BoHV-1 has been historically associated with upper
respiratory and genital tract infections while BoHV-5 leads
to severe encephalitis in calves. The present study
reports the occurrence of BoHV-1 and -5, in different
clinical specimens of bovines from Goiás, Brazil. Between
August/2008 and July/2009, 90 frozen brain samples of
bovine that died with compromised neurological functions,
61 semen samples from healthy bulls, 87 samples of
Saadi, T.M., 2 Guimarães, F.R., 3 Martorelli, L.F.A.,
Kataoka, A.P., 5Souto, F.J.D, 6Brito, W.M.E.D., 7Nociti,
D.L.P.
1. UFMT, Universidade Federal do Mato Grosso, Av.
Fernando Correa da Costa, 2367 - Bairro Boa Esperança.
Cuiabá-MT - 78060-9002. CCZ-SP, Centro de Controle
de Zoonoses - SP, Rua Santa Izabel, 181 - Vila Buarque.
São Paulo, SP - 01221-0103. UFG, Universidade Federal
de Goiás, Rua 235 s/n Setor Leste Universitário, Goiânia,
GO - 74605-050
1
4
Rabies is one of the most important zoonosis because
their fatal evolution and wide geographical distribution.
The widespread dog rabies vaccination campaign had
the aim to reduce the incidence of rabies in urban areas
of the country. Vaccination coverage, recommended by
the World Health Organisation (WHO), is at least 80% of
canine population. Although there is no report of the
occurrence of rabies in humans and in pets in the State
of Mato Grosso in recent years, it is important to check
129
ABSTRACTS
the protection rates of animals. In order to evaluate the
immune response against the rabies virus in dogs living
in urban areas of Cuiabá, Mato Grosso, Brazil, we have
analyzed serum from 450 dogs collected in four different
vaccination locations during the National Rabies
Vaccination Campaign for Animals, carried out by the
Municipal Center for the Control of Zoonosis. The
samples were submitted to a Rapid Fluorescent Focus
Inhibition Test, and it was verified that 54.9% of the
animals in the city had protection titers. When the regions
were analyzed separately, we were able to observe that
the seroprotection reached 33.6% of the animals in the
Southern region, while in the Northern, Eastern, and
Western regions of the city the animals showed indices
varying from 64.4% to 64.6%. Our data revealed an
heterogeneous coverage of the vaccination protection
in different regions of the city as well as an overall low
vaccination coverage. Efforts must be made to enhance
vaccination coverage in future campaigns.
Palavras-chaves: Zoonose, Raiva, Cão, RFFI, Centro
Oeste.
PCR DETECTION AND NUCLEOTIDE SEQUENCE
ANALYSIS FROM gag GENE FROM CAPRINE
ARTHRITS ENCEPHALITIS VIRUS OBTAINED FROM
MILK OF NATURALLY INFECTED GOATS
ID: 00215-00001
Área: 03 - Virologia Animal
Caldart, E.T., 2 Chiappetta, C.M., 3 Lopes, E.F.,
Ravazzolo, A.P.
1. UFRGS, Universidade Federal do Rio Grande do Sul,
Av. Bento Gonçalves, 9090 Porto Alegre/RS CEP:91540000
1
4
The caprine arthritis encephalitis virus (CAEV) is a
member of the Retrovirus family pertained to the genus
Lentivirus. It is responsible for a disease mainly
characterized by arthritis and, more rarely, encephalitis
in young animals. Economic losses have been described
due to lower milk quality and yield. Diagnosis has been
a challenge for years and could be limited due to genomic
Lentivirus variability. Therefore, characterize local viral
variants is required to improve programs for prevention
and control of small ruminant lentivirus (SRLVs)
infections. In this study, we detected and analyzed
sequences of circulating variants of CAEV from a herd
in Southern Brazil. Genomic DNA was extracted from
milk (n=10) cells by DNAzol® and was used in a seminested PCR to detect gag gene. The amplified DNA (n=3)
was purified, pooled and subsequently cloned with the
TOPO TA Cloning® kit. Four clones were chosen, plasmid
DNA extracted using Wizard Plus minipreps Kit and
submitted to sequencing in the ACTGene Laboratory
130
(Centro de Biotecnologia, UFRGS). An alignment of the
sequences (430 nucleotides) obtained in this work and
sequences from Genbank were generated with
CLUSTALW1.6 in order to verify the phylogenetic
relationships among SRLVs. Pairwise genetic distances
were calculated using MEGA4.1 with the Tamura-Nei
substitution model. Phylogeny construction was carried
out using the Neighbour-joining (NJ) method (bootstraps
replicates = 1000). Synonymous and non-synonymous
substitution rates were calculated with SNAP program.
Sequence comparison using the prototypic strains of
SRLVs revealed that the Brazilian caprine isolates
showed less divergence from CAEV-CO sequence (0.078
- 0.089 % nucleotide pairwise distance) than from the
MVV (0.173 – 0.306 %) prototypic strains, which was
confirmed by the phylogeny.
Financial support: PIBIC-CNPq.
Palavras-chaves: CAEV, diagnose, gag, molecular
variability, SRLV.
STUDIES ON THE MECHANISM OF ACTION OF
CIDOFOVIR ON THE REPLICATIVE CYCLE OF
VACCINIA VIRUS
ID: 00216-00001
Área: 04 - Virologia Básica
JESUS, D. M., 2 COSTA, L.T., 3GONÇALVES, D. L.,
ACHETE, C. A., 5 ATTIAS, M., 6 Moussatché, N.,
7
DAMASO, C.
1. UFRJ, Universidade Federal do Rio de Janeiro, Av.
Brigadeiro Trompowsky, s/n. Cidade Universitária.2.
Inmetro, Instituto Nacional de Metrologia, Xerém, RJ3.
UF, University of Florida, Gainesville, FL, USA
1
4
Vaccinia virus (VACV) is is a member of the Poxviridae.
For the last decade, several outbreaks of vaccinia-virus
related disease have been reported in dairy cattle in
Brazil. Cantagalo virus is a VACV strain isolated in the
first reported outbreak in 1999. Cidofovir (CDV) is a
phosphonate nucleoside analog that has proved its
efficacy against a variety of DNA virus, including poxvirus.
Presently, there is no treatment for poxvirus infections.
The goal of this work is to study the mechanism of action
of CDV on the replicative cycle of VACV. After an analysis
of CDV effect on virus yield in a one-step cycle, the
concentration of 20 ug/mL of CDV was chosen for its
inhibition of about 90% of virus titers. At those conditions,
DNA replication was inhibited in 30% and late proteins
accumulation was inhibited by 25%. Analysis of genome
resolution revealed that telomeres were resolved
successfully. Proteolysis of core proteins was severely
inhibited and, as expected, maturation of virus particles
was also repressed. Nevertheless, shift experiments
using rifampicin showed that CDV did not affect
ABSTRACTS
morphogenesis directly. Analysis of purified viral particles
formed in presence of CDV indicated the presence of
the complete set of proteins. However, the DNA level
inside those particles was 64% lower than in the control.
AFM analyses showed that DNA molecules bearing CDV
had an altered structure and were not encapisaded into
the particles. That leads to the hypothesis that CDV could
impair DNA encapsidation due to changes in DNA
conformation. We are currently working on the hypothesis
that DNA bearing CDV may not bind efficiently to proteins
important for DNA encapsidation. To test this, we are
perfoming DNA binding assays using total cytoplasmic
extract of infected cells or purified viral telomere-binding
protein I6. I6 is being expressed in prokaryote system
and purified by affinity column. We will also evaluate I6DNA binding using the AFM. Support: CNPq and Faperj.
Palavras-chaves: Vaccinia virus, Poxvirus, Cidofovir.
from 2 to 16 at day 30 pv. A boost vaccination performed
at day 240 pv resulted in a rapid and strong anamnestic
antibody response (titers from 16 to 256). Selected serum
samples from vaccinated animals showed a broad VN
activity against nine BoHV-5 and eight BoHV-1 field
isolates. These results show that the recombinant virus
is attenuated and immunogenic for calves, and induces
an antibody response differentiable from that induced
by natural infection. Thus, the recombinant BoHV-5gE/
TK” is an adequate candidate strain for a modified live
vaccine.
Palavras-chaves: Herpesvírus bovino tipo 5, vacina
diferencial, recombinante, atenuação, imunogenicidade.
A RECOMBINANT BOVINE HERPESVIRUS 5
DEFECTIVE IN THYMIDINE KINASE AND
GLYCOPROTEIN E IS ATTENUATED AND
IMMUNOGENIC FOR CALVES
ID: 00221-00001
Área: 04 - Virologia Básica
ANTIVIRAL ACTIVITY OF Agaricus brasiliensis
POLYSACCHARIDE IN REPLICATION OF HUMAN
AND BOVINE HERPESVIRUS
YAMAMOTO, K.A., 2RINCÃO, V.P., 3SOARES, S.A.,
LINHARES, R.E.C., 5NOZAWA, C.
1. UEL, Universidade Estadual de Londrina, Rod. Celso
Garcia Cid, PR 445, Km 380, Londrina-PR, Cx. Postal
60012. UFC, Universidade Federal do Ceará, Av. da
Universidade, 2853 - Benfica, Fortaleza - CE
1
4
ID: 00218-00001
Área: 03 - Virologia Animal
ANZILIERO, D., 2SANTOS, C.M.B., 3BAUERMANN,
F.V., 4 CAROZO, L., 5BERNARDES, L.M., 6STURZA,
D.A.F., 7BRUM, M.C.S., 8WEIBLEN, R., 9FLORRES, E.F.
1. UFSM, UNIVERSIDADE FEDERAL DE SANTA
MARIA, Avenida Roraima, nº 1000 Cidade Universitária
Bairro
Camobi
Santa
Maria2. UNIPAMPA,
UNIVERSIDADE FEDERAL DO PAMPA - CAMPUS
URUGUAIANA, BR 472, km 592, Caixa Postal 118. Curso
de Medicina Veterinária, Hospital Veteri
1
We herein report an investigation on the attenuation and
immunogenicity of a recombinant bovine herpesvirus 5
(BoHV-5), defective in glycoprotein E (gE) and thymidine
kinase (tk) genes (BoHV-5gE/TK”) constructed out of a
Brazilian strain. First, 80 to 90-days-old seronegative
calves (n=6) inoculated intranasally with the recombinant
(107.5TCID50) shed virus in low titers for up to 6 days,
and did not develop clinical signs. At day 30 post-infection
(pi) all calves had virus-neutralizing (VN) antibodies in
titers of 4 to 8 and were negative for anti-gE antibodies.
Administration of dexamethasone (0.1mg/kg/day during
5 days) at day 42 pi did not result in virus shedding or
increase in VN titers. Secondly, a group of 8 months-old
calves (n=9) vaccinated intramuscularly (IM) with the
recombinant virus (107.5TCID50) did not shed virus in
nasal secretions, remained healthy and developed VN
titers from 2 to 8 post-vaccination (pv). Lastly, 21 calves
maintained under field conditions vaccinated IM with the
recombinant virus (107.3TCID50) developed VN titers
Agaricus brasiliensis is a basideomycete traditionally
used as health food source in Brazil for the prevention
of
cancer,
hyperlipidaemia,
ar teriosclerosis and chronic hepatitis. Moreover, its
polysaccharide has been described as antimutagenic, antidiabetic and antiviral. In this study, we
evaluated the cytotoxicity of a polysaccharide (PLS) and
its sulfated (SPLS) and carboxymethylated (CPLS)
derivatives of Agaricus brasiliensis in HEp-2 cell cultures,
as well as, their antiviral activity against human (HSV-1)
and bovine (BoHV-1) herpesvirus. The 50% cytotoxic
concentrations of PLS, SPLS and CPLS were above
2.0, 2.5 and 4.0 mg/ml, respectively, by the use of
dimethylthiazolyl diphenyl tetrazolium bromide method
(MTT). Antiviral activity was monitored by plaque assay
and the compounds were added at different
concentrations, before (-2 and -1h), during (0h) and after
(+1 and +2h) viral infection. The results showed that when
the PLS and SPLS were added to cells at 0h and +1 and
+2h there was a reduction in the number of plaques,
dependent on the concentration, up to 69% for BoHV-1.
For HSV-1 the highest inhibition of 82.7% was detected
only at 0h. These compounds were ineffective when
added at -2 and -1h before infection and did not inhibit
adsorption either suggesting an action at the initial stage
of HSV-1 and BoHV-1 replication. For the CPLS, the
major inhibition was 37.5% for HSV-1 and 49.1% for
131
ABSTRACTS
BoHV-1, at 0h, even at high concentrations. This showed
that carboxymethylation reduced the antiviral activity of
the polysaccharide. Further analyses are under
development in order to elucidate the steps affected by
these substances.
Palavras-chaves: human herpesvirus, bovine
herpesvirus, Agaricus brasiliensis polysaccharide,
antiviral activity.
PHYLOGENETICAL CLUSTERING OF INFECTIOUS BURSAL DISEASE VIRUS (IBDV) STRAINS
Financial support: FAPEMIG and CNPq.
Palavras-chaves: IBDV, phylogenetic, virus.
DEVELOPMENT AND VALIDATION OF AN ENZYME
LINKED IMMUNOSORBENT ASSAY (ELISA) FOR
DETECTION OF PCV-2 ANTIBODIES IN SERUM
SAMPLES FROM SWINE.
ID: 00223-00001
Área: 03 - Virologia Animal
FAUSTO, M.C., 2 SALGADO, R.L., 3CRISPIM, J.S.,
MYRRHA, L.W., 5 Silva, F.M.F, 6 Peternelli, E.F.O.,
7
Lobato, Z. I. P., 8Fietto, J.L.R, 9Silva Júnior, A, 10Almeida,
M. R.
1. UFV, Universidade Federal de Viçosa, Avenida Peter
Henry Rolfs, s/n Campus Universitário 36570-000
VIÇOSA - MG2. UFMG, Universidade Federal de Minas
Gerais, Av. Antônio Carlos, 6627 - Pampulha - Belo
Horizonte - MG CEP 31270-901
1
ID: 00222-00001
Área: 03 - Virologia Animal
POLÊTO, M.D., 2PETERNELLI, E.F.O., 3 VIDIGALl,
P.M.P, 4SILVA, F.M.F, 5 MYRRHA, L.W, 6FÉLIX, F.S,
7
BRESSAN, G.C., 8FIETTO, J.L.R, 9SILVA JÚNIOR, A,
10
ALMEIDA, M.R
1. UFV, Universidade Federal de Viçosa, Avenida Peter
Henry Rolfs, s/n Campus Universitário 36570-000
VIÇOSA - MG
1
Infectious bursal disease virus (IBDV) is considered one
of the most important pathogens in the poultry industry.
The IBDV causes a highly contagious immunosuppressive disease in chickens called Infectious bursal
disease (IBD). In IBDV genome, the coding region of the
external capsid protein (VP2) is the phylogenetic signal
most used to assess the genetic diversity of viral strains.
In this work, a phylogenetic clustering for IBDV strains
was proposed by analyses of VP2 sequences. A search
for VP2 sequences was performed into GenBank and
537 sequences were selected. From these, the complete
sequences (96) were selected for define the clusters in
phylogenetic analyses. The phylogenetic hypotheses
were inferred by Bayesian Inference (BI) on the MrBayes
3.1 program with 10,000,000 of generations. In order to
speed up the construction of the trees, a basic model of
nucleotide substitution was inferred with the aid of the
MrModeltest program. Another phylogenetic hypothesis
was obtained from the hypervariable region of the 96
selected sequences for test the reability of phylogenetic
groupings. Five major groups were defined by topological
analyses of the phylogenetic trees. These groups
separated strains of IBDV with well defined
characteristics of pathogenicity. From these results, this
group was tested in all the 537 sequences obtained from
GenBank, selecting the hypervariable region of VP2. All
five phylogenetic clusters were reproduced in this set of
data and these groupings represent a more efficient
classification of IBDV strains. Furthermore, this
determination can enable the development of more
effective diagnostic methods and vaccination programs
more efficient.
132
4
The Porcine circovirus 2 (PCV2) has recently emerged
as a major cause of production losses in the swine
industry worldwide. The term PCVAD (Porcine circovirus
2 associated diseases) was introduced to summarize
the various clinical manifestations related to the virus in
swine, as Postweaning multisystemic wasting syndrome
(PMWS), Porcine dermatits nephropathy syndrome
(PDNS), pneumonia, enteritis, reproductive failure and
nervous manifestations. The capsid protein (Cap) is the
most immunogenic portion of the PCV2, consisting of
an interesting antigen for serological studies and vaccine
development. Since serologic studies are primordial for
monitoring viral, an immunoenzymatic assay (ELISA)
was developed in this study for the detection of antibodies
to PCV2 in serum samples of swine. The recombinant
capsid protein of PCV2 (rCAP PCV2) was expressed in
a heterologous expression system using Escherichia coli,
purified, confirmed as its antigenicity by Western blotting
and used to standardize the technique. To determine the
cutt off were used 29 negative serum samples for antibodies
to PCV2 by ELISA and for validation of the test, 155 serum
samples were evaluated by ELISA and by Immunoperoxidase Monolayer Assay (IPMA). Of the 125 samples
positive by IPMA, 124 were positive and one negative by
ELISA, while of the 30 samples negative by IPMA, 29
were negative and 1 positive by ELISA, showing a
concordance of 98.70% between the two tests. The
sensitivity and specificity of the IPMA, the positive and
negative predictive values of ELISA were determined as
99.20%, 96.66%, 99.20% and 96.66%, respectively.
Therefore, the ELISA that was developed and validated in
this study proved to be an alternative test to the IPMA and
it can be considered a simple, fast and secure for the
detection of antibodies to PCV2 in serum samples of swine.
ABSTRACTS
Financial support: FAPEMIG/CNPq.
Palavras-chaves: DETECTION, ELISA, PCV-2.
PROPERTIES OF Cucumber mosaic virus ISOLATES
FROM DIFFERENT HOSTS IN PARANÁ STATE.
ID: 00224-00001
Área: 06 - Virologia Vegetal
Pelisson, 2Silva, 3Picoli, 4Carnelossi, 5Souto
1. UEM, Universidade Estadual de Maringá, Av. Colombo,
5790, 87020-900, Maringá-PR
1
Cucumber mosaic virus (CMV) is the type member of
the Cucumovirus genus. It is distributed worldwide and
has the largest host range of all plant viruses, causing
diseases in vegetable, fruit and ornamental crops, with
severe economic losses. Numerous isolates differing in
host range and pathogenicity have been reported. They
fit into two major serotypes, subgroups I and II. Five
isolates of CMV from Commelina sp., periwinkle, melon,
and cucumber, collected in the northwest region of
Paraná, were evaluated for their biological and molecular
properties. All isolates were maintained in Nicotiana
benthamiana and were inoculated in different hosts. The
CMV isolate from periwinkle induced severe symptoms
in most of the plants, while the isolate from Commelina
induced only mild symptoms in Nicotiana tabacum,
tomato, and Petunia hybrid, and the isolate from melon
did not induced symptoms on these same hosts. All
isolates reacted with an antiserum for CMV in indirectELISA tests. The reverse transcriptase reaction and PCR
amplifications using CMV primer 1 (downstream) and
CMV primer 2 (upstream) for the 3´ region of the viral
coat protein gene, amplified DNA fragments ranging from
486 to 488 bp for all isolates. The sequencing of the RTPCR products of isolates Commelina and periwinkle
indicated they were 95 to 98% identical to those of
subgroup I CMV isolates.
Palavras-chaves: ELISA, RT-PCR, CUCUMOVIRUS.
THE SUCCESS OF TWO VIDEOS ABOUT AEDES
MOSQUITOES AS ALLIES IN THE FIGHT AGAINST
DENGUE FEVER
ID: 00225-00002
Área: 05 - Virologia Humana e Saúde Pública
Dengue fever (DF) is an acute illness of sudden onset
that usually follows a benign course. Frequently, these
infections occur in urban areas because people have
been creating mosquitoes in their houses. In Brasil, Public
Health Authorities and other Organizations have
promoted campaigns aiming to inform the population
about the risks of this disease and how to avoid it.
Unfortunately, dengue fever is maintained as an endemic
disease in the country. This situation suggests that we
might consider that infections as behaviour diseases. In
order to increase among the citizens the knowledge about
this educational issue, create favourable attitudes and
change overt behaviour, the Laboratory for production
and treatment of images-IOC, Fiocruz, with collaboration
of others institutions, have produced two educational
scientific videos about the life cycle of the mosquitoes
that are involved in the DF transmission. The first one
was “The macro and micro world of Aedes aegypti – For
to fight it is needful to know it” and the second was “Aedes
aegypti and Aedes albopictus - the threat in the tropics”.
Both have been presented in Portuguese, English and
Spanish. Their successful is evidenced by the awards
from several festivals around the world. (Festival de Cine
Y Vídeo Cientifico MIF-SCIENCES 2006 – Habana;
VIDEOMED 2006– Badajoz; CINECIEN 2006, Mercosul;
Mif-Sciences.net Special Techfilm 2007, Prague; 2nd
International Science Film Festival 2007, Athens; Vedere
la Scienza Festival 2008 – Milano; Internacional Health
film festival 2008, Liège; Toronto Portuguese Film Festival
2009; Videomed Santiago 2010; IX International
Videomed Tucumán 2010). As a result of their success,
in Brazil and in many others countries these videos are
been used as educational tools to promote citizenship
attitudes in students of different school levels. The
message of the videos is: to combat urban DF occurrence
is necessary to know well the biologic characteristics of
the arthropod vectors.
Palavras-chaves: Aedes aegypti, Aedes albopictus,
Arthropod Borne viruses, Educational approach.
AN IMMUNOCAPTURE RT-PCR PROTOCOL FOR
Cassava common mosaic virus DETECTION FROM
MICROPROPAGATED PLANT MATERIAL AND IN
THE FIELD
ID: 00227-00001
Área: 06 - Virologia Vegetal
SILVA, J.M., 2PICOLI, M.H.S., 3CARNELOSSI, P.R.,
PELISSON, N., 5ALMEIDA, A.M.R., 6SOUTO, E.R.
1. UEM, Universidade Estadual de Maringá, Av. Colombo,
5.790, Jd. Universitário, Maringá, Paraná, Brasil, CEP
870209002. Embrapa Soja, Empresa Brasileira de
Pesquisa Agropecuária, Rod. Carlos João Strass,
Londrina, Paraná, Brasil, CEP 8600-970
1
VIEIRA, G.J., PERIM, L., CABRAL, M.C., LIBERTO,
M.I.M.
1. FIOCRUZ, Instituto Oswaldo Cruz, Laboratório de
Produção e tratamento de imagens- FIOCRUZ. Av. Brasil
RJ2. IMPPG/UFRJ, Inst. de Microbiologia/Universidade
Federal do Rio de Janeiro, Laboratório de estruturas de
superfície de Vírus. IMPPG, CCS.
1
2
3
4
4
133
ABSTRACTS
Several viruses infect cassava, where the most frequent
is Cassava common mosaic virus (CsCMV), Potexvirus,
occurring in warm temperatures in the South and
Southeast regions of Brazil. Even CsCMV infected plants
sometimes do not show any kind of symptoms, being a
drawn back for plant virus indexing. The objective of this
work was to optimize an immunocapture RT-PCR (ICRT-PCR) for CsCMV detection from micropropagated
plants and from plants collected in the field. Samples of
61 plants in the field were previously tested by indirectELISA, indicating that 54 were CsCMV infected. From
the 15 plantlets from meristem tissue cultures, 8 were
ELISA positive and 7 negative. After, the material of 7
plants from the field, and the 7 plantlets that were ELISA
negative, they were IC-RT-PCR tested. Microcentrifuge
tubes of 0,2 ìl were coated with CsCMV virus-specific
purified IgG (2ìg/ml) and after incubation they were
covered with plant crude sap diluted at 1/10, followed by
a RT-PCR reaction with a set of primers that amplify
part of the polymerase gene of CsCMV. The amplified
RT-PCR products were cloned, sequenced and compared
with other sequences in the GeneBank, showing 86%
homology with the sequence of a Brazilian isolate of
Cassava common mosaic virus (U23414.1), confirming
that all samples tested negative through indirect-ELISA
where infected with CsCMV. These results indicate that
ELISA tests alone are not sufficient to guarantee that a
plant material is free from CsCMV.
Palavras-chaves: CsCMV, indirect-ELISA, Potexvirus,
RT-PCR.
ELISA DETECTION OF CASSAVA COMMON MOSAIC
VIRUS IN CASSAVA PRODUCING AREAS FROM THE
NORTHWEST REGION OF PARANÁ STATE
ID: 00227-00002
Área: 06 - Virologia Vegetal
CARNELOSSI, P.R., 2 SILVA, J.M., 3 BIJORA, T.,
PICOLI, M.H.S., 5 PELISSON, N., 6 FACCO, C.U.,
7
SOUTO, E.R.
1. UEM, Universidade Estadual da Maringá, Av. Colombo,
5.790, Jd. Universitário, Maringá, Paraná, Brasil, CEP
87020-900
1
4
Cassava is vegetatively propagated and is subjected to
the dissemination of systemic diseases through
successive generations such as those caused by
viruses. For a Cassava common mosaic virus (CsCMV)
survey conducted in the northwest region of Paraná State,
samples of varieties Olho Junto, Fécula Branca, IAC
90, IAC 12, IAC 13, IAC 14, and Baianinha were collected
for indirect-ELISA tests. Leaf crude saps were diluted
from 1/20 to 1/2500 and were tested against a specific
polyclonal antiserum for CsCMV. ELISA results revealed
134
that all field cassava collected samples were infected.
Samples of ‘Baianinha´ gave the highest values of
absorbance readings, indicating in that variety the virus
had its highest concentration. However, the absorbance
readings for ´Baianinha´, ‘IAC 90´, ‘Fécula Branca´ and
‘IAC 13´ did not differ statistically in relation to the viral
concentration at the 5% level of significance, while the
varieties IAC 14 and Olho Junto presented significant
differences (probably in these varieties the virus was
less concentrated). As a result, indirect-Elisa was
efficient for CsCMV detection in cassava propagative
material.
Palavras-chaves: CsCMV, indirect-ELISA, polyclonal
antiserum, propagative material.
EVALUATION OF IMMUNE STATE AGAINST
ORTHOPOXVIRUS IN RURAL POPULATIONS FROM
AMAZONIA AND SOUTHEAST , BRAZIL
ID: 00228-00001
Área: 05 - Virologia Humana e Saúde Pública
Figueiredo,P.O, 2Silva-Fernandes, A.T, 3Mota, B.E.F,
E.M, Braga, 5Bonjardim, C.A, 6Ferreira, P.C.P, 7Kroon,
E.G, 8Trindade, G.S.
1. UFMG, Universidade Federal de Minas Gerais, Av.
Antônio Carlos, 6627, Campus Pampulha, CEP: 31270901. Belo Horizonte, MG
1
4
Vaccinia virus (VACV), historically important because
of its use in the global campaign to eradicate smallpox,
circulates in the country since the 60s. Infection by this
virus affect both cattle and humans, characterizing them
as zoonoses of importance to public and animal health.
The virus is transmitted to humans through contact with
lesions of cattle and cause localized infections with the
development of typical vesicular-pustular lesions. With
the aim to screen the spread of Orthopoxvirus and
evaluate the immune status of rural populations in the
Amazon and southeastern Brazil against VACV, we
analyzed samples from Mantena and Terra Nova do Norte,
owned by the states of Minas Gerais and Mato Grosso,
respectively. In none of the locations analyzed bovine
VACV outbreaks had been reported until the time of sera
collection (1995 and 1996, respectively). These samples
were tested by ELISA and serum neutralization (PRNT)
for anti-orthopoxvirus detection. Of the 70 samples tested
belonging to the Amazon 27% were positive by ELISA.
27% of positive samples are from people who were not
vaccinated against smallpox, or were born after 1977,
closing date of vaccination in Brazil. A collection of 62
samples from Mantena were analyzed and seropositivity
was 11% and 29% of those who are unvaccinated
persons. Of the 26 ELISA positive sera from both
populations, six were also positive by PRNT thus
ABSTRACTS
indicating the effectiveness of the immunity given by
vaccination as these samples are from patients
vaccinated against smallpox. The detection of
orthopoxvirus antibodies in unvaccinated people indicates
a possible circulation of VACV in human populations in
the absence of bovine vaccinia outbreaks. Our results
support the need for more research on the subject,
especially as regards the identification of potential animal
reservoirs and novel ways of disease transmission.
Financial support: CNPq, PRPq/UFMG.
Palavras-chaves: Vaccinia vírus, Orthopoxvirus,
seropositivity.
that 5% milk blocking produces less unspecific reactions
than BSA and that no statistical differences are seen
among 1:50 and 1:100 equine sera dilution. A few more
dilutions will be tested for the conjugate, but good results
were obtained with 1:25,000 dilution. Once final settings
are done, over 100 equine sera will be simultaneously
tested in ELISA and PRNT. Data will be crossed and
the sensitivity and specificity of the standardized test
will be calculated. Support: CNPq, Fapemig, CAPES,
UFMG.
Palavras-chaves: Bovine Vaccinia, epidemiology,
EQUINE.
STANDARDIZATION OF AN ELISA-IgG TO DETECT
ANTI-ORTHOPOXVIRUS ANTIBODIES IN EQUINE
SERA.
THE PROTEIN KINASE PAK1 IS ASSOCIATED WITH
THE DISSEMINATION OF BOTH Vaccinia AND
Cowpox virus
ID: 00228-00002
Área: 03 - Virologia Animal
ID: 00229-00001
Área: 04 - Virologia Básica
Borges, I.A, 2Figueiredo, P.O, 3Martins, A.P.S, 4SilvaFernandes, A.T, 5Moreira, A.P, 6Crepalde,K.R, 7Bonjardim,
C.A, 8 Reis, J.K.P, 9 Ferreira, P.C.P., 10 Kroon, E.G,
11
Trindade, G.S
1. UFMG, Universidade Federal de Minas Gerais, Av.
Antônio Carlos, 6627, Campus Pampulha, CEP: 31270901. Belo Horizonte, MG
Mügge, F.L.B., 1Andrade, L.G., 2Ferreira, P.C.P, 3Kroon,
E.G., 4Bonjardim, C.A.
1. GTS, Grupo de Transdução de Sinal, Av Antonio Carlos
6627, CEP 31270-901 Belo Horizonte2. Lab Vírus,
Laboratório de Vírus, Av Antonio Carlos 6627, CEP
31270-901 Belo Horizonte3. ICB, Intituto de Ciências
Biológicas, Av Antonio Carlos 6627, CEP 31270-901 Belo
Horizonte4. UFMG, Universidade Federal de Minas
Gerais, Av Antonio Carlos 6627, CEP 31270-901 Belo
Horizonte
1
The circulation of Vaccinia virus (VACV) in Brazil is
usually associated to the Bovine Vaccinia (BV), a
zoonosis that affects bovine herds and dairy workers
generating serious economical and social impacts. In
the past eleven years many efforts have been made to
better comprehend BV epidemiology, but the range and
role of hosts involved are some of the gaps that remain.
Recent data concerning a VACV outbreak in Crioulo
horses from a breeding center in Rio Grande do Sul (RS)
State highlighted the lack of information about the
importance of these animals in BV epidemiology. The
regular use of equine transportation in dairy production
reinforces the need to study their role in VACV
transmission cycle. Therefore, an ELISA is being
standardized to detect anti-OPV IgG in equine sera. A
collection of equine sera was double screened in BSC40 and Vero cells Plaque Reduction Neutralizing Test
(PRNT) to identify anti-OPV positive and negative sera.
ELISA was then performed with a 100ng of purified and
UV-inactivated VACV-WR per well, 1% bovine serum
albumin (BSA) and 5% milk blocking and equine sera
dilutions of 1:50 and 1:100. Different dilutions of antiequine IgG conjugated to a horseradish peroxidase were
also tested. TMB was used as a substrate and the plate
was read at the absorbance of 450nm. Positive control/
negative control ratios (P/N) were used to compare
different conditions tested. Partial results demonstrate
1
Interfering with cellular signal transduction pathways is
a common strategy employed by viruses to create a
propitious intracellular environment that fulfills the
requirements for a whole and efficient virus multiplication
cycle. The interactions between host cells and the
members of the Poxviridae family have been studied by
our group. Previous studies showed that cellular infection
with the Orthopoxvirus Vaccinia (VACV) or Cowpox
(CPXV) viruses promotes the activation of MAPKs
ERK1/2 and JNK1/2 signaling pathways, which play
important and differential roles in both Orthopoxvirus
biology. With the aim to characterize another important
cellular constituent during VACV and CPXV cycle, we
investigated the possible involvement of PAK1 on the
infection by these viruses. Thus, mouse embryo
fibroblasts (MEFs) derived from wild-type (PAK1+/+) and
PAK1-null (PAK1-/-) mice were infected with VACV or
CPXV at different times. We showed, through Western
blot assay, that PAK1 is not critically involved in the
early steps of the cycle of both viruses, such as virus
entry. On the other hand, we found that the absence of
PAK1 led to a severe reduction in plaque phenotype and
infection of MEFs PAK1-/- carried out at low-multiplicity
was followed by a greatly reduced production of both
135
ABSTRACTS
intracellular mature virus (IMV) and extracellular
enveloped virus (EEV), suggesting that virus spread was
drastically impaired. Immunofluorescent staining of actin
cytoskeleton showed a reduction in the amount of VACV
or CPXV-induced actin tails in MEFs PAK1-/- .
Furthermore, confocal microscopy of MEFs PAK1+/+
infected with VACV or CPXV revealed a colocalization
of p-PAK1 with actin tails tips as well as CEVs (B5R)
particles. Indeed, these results show that PAK1 plays a
role in the viral dissemination of both VACV and CPXV,
being an important cellular constituent to assure the
efficiency of this process.
Palavras-chaves: Poxvirus, poxvirus-host cell
interactions, Vaccinia virus, Cowpox virus, PAK1.
intradermal, and 12.5% by intranasal route. In mouse
model 70.0% of animals died after intramuscular and
intradermal inoculation routes and 10.0% by intranasal
challenge. Paralytic manifestation was found with this
strain in all animals in both animal models. By sequencing
of glycoprotein gene, several substitutions were detected
at antigenic domains such as: AI (position 231), AII (34
– 42 and 198- 200), domain of fusion dependent on low
pH (102 – 179), transmembrane domain (440 – 461) and
residue 242. These viruses showed contrasting biological
behaviors that can be linked to those substitutions at
antigenic domains previously described. Agencia: CNPq.
Palavras-chaves: bat, glycoprotein, pathogenicity,
phylogeny, rabies.
PATHOGENIC PROFILE OF A RABIES VIRUS ISOLATE
FROM INSECTIVOROUS BAT LASIURUS EGA IN
HAMSTER AND MOUSE MODEL AND DETECTION OF
MULTIPLE AMINOACIDS SUBSTITUTIONS ON
BIOLOGICALLY ACTIVE DOMAINS OF VIRAL
GLYCOPROTEIN
Prevalência de vírus respiratórios em pacientes
acompanhados pelo Hospital de Clínicas de Porto
Alegre
ID: 00230-00001
Área: 03 - Virologia Animal
1
ESTÉVEZ GARCIA, A. I, 2 BRANDÃO, P. E,
MOCHIZUKI, N, 4ALBAS, A, 5ITO, F.H
1. FMVZ/USP, Faculdade de Veterinária e Zootecnia da
Universidade de Sao Paulo, Av. Prof. Orlando Marques
de Paiva, 87 Cidade Universitária SP, cep 05508-2702.
NU, Nihon University, Veterinary Research Center.,
Fujisawa, Kanagawa, Japan3. APTA, Pólo Regional de
Desenvolvimento Tecnológico dos Agronegócios da Alta
Sorocabana, Presidente Prudente, São Paulo
1
3
Pathogenic profile of a rabies virus isolated from an
insectivorous bat Lasiurus ega was compared with a
rabies fixed virus strain (Challenge Virus Standard
CVS&frasl32) in hamster and mouse model in terms of
length of incubation and clinical periods, clinical
manifestation and death rates. Animals were challenged
using 104.0 LD50&frasl0.05mL by three different
inoculation routes: intramuscular, intradermal, and
intranasal. Viral titer estimation was obtained by Reed
and Müench method in mice. Presence of viral antigen
in brains of animals manifesting signals compatible with
rabies was confirmed by the Direct Immunofluorescence
Test. Rabies virus isolate from L. ega bat was able to
cause disease in hamster only by intramuscular (2.1%
of mortality) and intranasal (8.3%) challenge. In mice,
50.0% and 30.0% of mortality were observed after
intramuscular and intranasal inoculation, respectively. The
clinical signs observed were predominantly furious. In
hamster model CVS&frasl32 strain induced 62.5% of
mortality by intramuscular challenge, 78.1% by
136
ID: 00234-00001
Área: 05 - Virologia Humana e Saúde Pública
de-Paris, F., 2Beck, C., 3Pinheiro Machado, A. B. M.,
Menezes, D. S., 5Paiva, R. M., 6Barth, A. L.
1. HCPA, Hospital de Clínicas de Porto Alegre, Ramiro
Barcelos, 2350 Porto Alegre2. UFRGS, Universidade
Federal do Rio Grande do Sul, Ramiro Barcelos, 2400
Porto Alegre
4
Introdução: Infecções do trato respiratório são causa
significativa de morbidade e mortalidade no RS. Os vírus
são a causa mais comum de infecção aguda do trato
respiratório e os mais frequentemente relatados têm sido
o Vírus Respiratório Sincicial (VRS), Parainfluenza tipo
1, 2 e 3 (PIV-1, PIV-2 e PIV-3), Adenovírus (AdV),
Influenza A e B. (FluA e FluB). O Influenza A, por
exemplo, esteve recentemente em evidência durante a
pandemia de (H1N1) suíno. Objetivo: Avaliar a prevalência
de vírus respiratórios de pacientes internados e em
emergência no Hospital de Clínicas de Porto Alegre neste
período pós-pandemia de Influenza A (H1N1) suíno, e
campanha de vacinação para este agente. Metodologia:
Foram incluídas amostras de secreção de nasofaringe
encaminhadas à Unidade de Microbiologia no Serviço
de Patologia Clínica em um período de 30 dias e
analisadas por Imunofluorescência indireta para a
presença de vírus respiratórios (pesquisa para VRS, PIV1, PIV-2, PIV-3, AdV e FluA, FluB). Resultados: De 250
amostras avaliadas, 140 (56%) foram positivas para os
vírus pesquisados, sendo que destas 134 (95,71%) eram
positivas para o VRS. As faixas etárias mais afetadas
foram crianças menores de um ano (81,4%) e de 1 a 2
anos (10,7%). Conclusão: Na população analisada pelo
estudo o vírus mais frequentemente isolado foi o VRS
(+95%), afetando mais significativamente crianças de
ABSTRACTS
até 12 meses (+81%). Esses dados estão de acordo
com o esperado para a sazonalidade do VRS, nos meses
que antecedem o início do inverno, e reforça a
necessidade de estratégias de prevenção e manejo de
infecções respiratórias agudas, inclusive por Vírus
Respiratório Sincicial, neste hospital além de alertar para
a necessidade de estratégias de prevenção de epidemias
sazonais por outros agentes virais.
Palavras-chaves: vírus respiratórios, vírus sincicial
respiratório, vírus influenza.
DETECTION OF HUMAN BOCAVIRUS IN FECAL
SAMPLES FROM DISTRITO FEDERAL, BRAZIL
is no identification program and surveillance of bocavirus,
and to our knowledge this is the first report of bocavirus
occurrence in this region.
Palavras-chaves: Human bocavirus, fecal samples,
Distrito Federal, Brazil.
IDENTIFICATION OF AICHIVIRUS IN FECAL
SAMPLES FROM INDIVIDUALS OF THE FEDERAL
DISTRICT, BRAZIL.
ID: 00235-00002
Área: 05 - Virologia Humana e Saúde Pública
Silva RLV, 2Lorga RN, 3Lima LMP, 4Nagata T, 5Silva PA
1. UCB, Universidade Católica de Brasília, SGAN 916
Norte AV W5 CEP:70790-1602. UnB, Universidade de
Brasília, Laboratório de Microscpia Eletrônica,
Departamento de Biologia Celular, IB4, CEP3. LACENDF, Laboratório Central do Distrito Federal, SGAN, Quadra
601, Lotes O e P. Brasília-DF CEP: 70830-010
1
ID: 00235-00001
Área: 05 - Virologia Humana e Saúde Pública
Silva RLV, 2Dias TRS, 3Lima LMP, 4Nagata T, 5Silva PA
1. UCB, Universidade Católica de Brasília, SGAN 916
Norte AV W5 CEP:70790-1602. UnB, Universidade de
Brasília, Laboratório de Microscpia Eletrônica,
Departamento de Biologia Celular, IB4, CEP3. LACENDF, Laboratório Central do Distrito Federal, SGAN, Quadra
601, Lotes O e P. Brasília-DF CEP: 70830-010
1
In 60s, the animal bocavirus was described in bovine
and canine samples. Recently, bocavirus was detected
in human samples. Firstly, it was identified in samples
from respiratory tract and then from fecal samples from
individual with acute gastroenteritis. The human bocavirus
(HBoV) was provisionally classified in the genus
Bocavirus, family Parvoviridae. This project aims to
identify and characterize circulating bocavirus in the
Distrito Federal, Brazil. We analyzed randomically 50
samples of diarrhea stool suspensions stored at the
Virology Department of LACEN-DF, for the years 2006
to 2010. The viral DNA was extracted by PureLink ™
viral RNA/DNA Purification Kit and then was subjected
to the method of PCR amplification for detection of a
bocavirus genotype 1 and 3, and a nested PCR for
genotype 2. We identified 11 positive samples of
Bocavirus, in which 10 were HBoV-1 and 01 HBoV-3.
This result strengthens the importance of bocavirus as
an agent of viral gastroenteritis, since it was detected in
patients with diarrhea symptoms that were negative for
rotavirus. It was observed that most positive samples
(08 of 11) were collected during July to October,
corresponding to the period of winter and drought in the
Distrito Federal, but to confirm this seasonality it is
necessary additional studies. Some positive samples
were diagnosed as bocavirus in outbreak of food-borne
illness caused by Salmonella group D. In this case, the
causative agent of gastroenteritis was considered
bacterial, but the possibility of co-infection aggravating
the case can not be discarded. In Distrito Federal, there
Aichivirus, a member of the family Picornavidae, is one
of the responsible of gastroenteritis. The virus particle
has icosahedral morphology without envelope. The
genome of this virus consists of a single strand RNA of
approximately 8300 nucleotides with a positive polarity.
The importance of aichivirus in acute gastroenteritis has
been described mainly by studies in Asian countries,
being previously associated to oyster‘s consumption.
There were also reports in other countries out of the
Asian continent, like Germany and France. In Brazil this
virus was first described, in 2005, in five stool samples
of children from Goiânia-Goiás. It has currently been
described three genotypes: A and B are associated with
human gastroenteritis and C is associated with infection
in porcine. The objective of this study is to identify the
circulation of aichivirus in the Federal District (DF). We
analyzed 50 stool samples that were storaged in the
Virology Department of LACEN-DF between the years
2006 and 2010. By using the PureLink viral RNA/DNA
Purification Kit (Invitrogen), total DNA containing genetic
material of virus was extracted and submitted to the
method RT/PCR with specifics primers. The aichivirus
was detected in two samples, corresponding 4 % of the
analyzed samples. They were collected in the years of
2007 and 2010. The samples were from female patients,
with diarrhea, in which one of them was positive for
Salmonella group D. In this case the etiologic agent may
be from bacterial origin, however the presence of
aichivirus could have aggravated the infection. Because
of the lack of information about its genotype, we intend
to do further studies about it. Palavras-chaves:
Aichivirus, gastroenteritis, Distrito Federal, Brazil.
GENETIC VARIABILITY OF Tomato severe rugose
137
ABSTRACTS
virus AND THE VECTOR Bemisia tabaci IN PEPPER
AND TOMATO PLANTS IN SÃO PAULO STATE,
BRAZIL
ID: 00236-00001
Área: 06 - Virologia Vegetal
ROCHA, K.C.G., 2 MARUBAYASHI, J.M., 2 NAVASCASTILLO, J., 2KRAUSE-SAKATE, R., 3PAVAN, M.A.,
4
YUKI, V.A.
1. FCA/UNESP, Faculdade de Ciências Agronômicas/
UNESP, Rua José Barbosa de Barros, 1780, CP 237 CEP 18610-307, Botucatu, SP, Brazil2. CSIC, Estación
Experimental “La Mayora”, CSCI, 29760 - AlgarroboCosta, Málaga - Spain3. IAC, Instituto Agronômico de
Campinas, 13012-970 - Campinas, SP - Brasil
1
The incidence of begomoviruses has sharply increased
in Brazil following the introduction of the B biotype of the
whitefly vector in the early 1990s. A survey was carried
out to evaluate the genetic variability of begomoviruses
and B. tabaci populations collected in pepper and tomato
crops from São Paulo State, Brazil. Total DNA was
extracted from 710 pepper and 103 tomato samples, and
the presence of begomoviruses was tested by
Polymerase chain reaction (PCR) and Rolling Circle
Amplification (RCA) followed by PCR. Positive samples
were selected and analyzed by RCA-RFLP and cleaved
by the restriction enzyme HpaII. By PCR, 99 (13.94%)
samples collected from pepper and 39 (37.86%) from
tomato were positives for the presence of begomovirus,
while by RCA-PCR 333 (46.90%) and 82 (79.61%) from
pepper and tomato, respectively, indicating higher
sensitivity of this technique. The 5’ region of the coat
protein (CP) gene and a segment of the intergenic region
was analyzed indicating the presence of Tomato severe
rugose virus (ToSRV) in pepper and tomato plants.
However, the partial sequencing of clones from RCA
products from a tomato sample indicated mixed infection
of ToSRV with Tomato yellow vein streak virus (ToYVSV).
Different restriction profiles for isolates of ToSRV were
obtained by RCA-RFLP. Higher genetic variability was
observed for begomovirus found in tomato compared to
that from pepper plants. The variability of B. tabaci
populations was performed by sequencing of DNA
fragments corresponding to mitochondrial cytochrome
oxidize I (mtCOI) gene and by restriction pattern by PCRRFLP with TaqI enzyme. Based on the comparison with
mtCOI reference sequences, all specimens were
classified in the Middle East-Asia Minor genetic group
that comprises the B biotype. The specimens could be
divided in four haplotypes. By PCR-RFLP with TaqI, a
typical biotype B profile was obtained for all samples.
Financial support: CAPES/CNPq.
Palavras-chaves: Geminiviridae, Vegetables, Whiteflies,
138
Cytochrome oxidase I gene, RCA-RFLP.
MOLECULAR AND BIOLOGICAL CHARACTERIZATION OF TWO ISOLATES OF BIDENS MOSAIC
VIRUS (BiMV).
ID: 00237-00001
Área: 06 - Virologia Vegetal
Spadotti D.M.A., 2 De Marchi B.R., 3Sanches M.M.,
Pavan M.A., 5Krause-Sakate, R.
1. FCA, UNESP-Faculdade de Ciências Agronômicas,
Rua José Barbosa de Barros, 1780 Botucatu
1
4
The BiMV is a tentative species of the genus Potyvirus.
By the analysis of the coat protein sequence, BiMV was
recently regarded as a strain of Potato virus Y (PVY). It
is commonly found infecting pea, sunflower and Bidens
pilosa, but in São Paulo State, BiMV can also naturally
infect lettuce. Two isolates, one from lettuce and other
collected from Bidens pilosa called BiMV-13 and BiMVSP, respectively, were studied. The virus was sap
transmitted to different plant species using Potassium
Phosphate buffer 0.01 M pH 7.0. The BiMV-13 isolate
showed a very restricted host range, involving the species
of Chenopodium quinoa, C. amaranthicolor, pea and
lettuce, while the BiMV-SP isolate could infect the same
plants as BiMV-13 but also G. globosa (assymptomatic),
Nicotiana benthamiana, N. occidentalis, N. clevelandii,
N. edwardsonii, N. tabacum ‘Xanthi’, N. rustica, N.
glutinosa and Zinnia elegans. Curiously BiMV-13 could
not infect Bidens pilosa. The presence of the virus was
confirmed by the extraction of total RNA and RT-PCR
using specifics primers for BiMV. Comparison between
the coat protein sequence of the BiMV-13 and BiMV-SP
revealed identities of 97% at the aminoacid level and
98% between BiMV-SP and BiMV from pea (accession
number AY960150). The coat protein aminoacid identity
between BiMV and Sunflower chlorotic mottle virus
(SCMoV) is 83% to 84%, compared to 80 to 83% with
PVY. The genome of SCMoV was recently complete
sequenced indicating that SCMoV should be considered
as a new species of potyvirus. The complete genome
sequence of BiMV will clarify their taxonomic position.
Palavras-chaves: Potyvirus, BiMV, Alface.
ANALYSIS OF THE OCCURRENCE OF Lettuce
mosaic virus, Lettuce mottle virus and Bidens mosaic
virus IN LETTUCE PRODUCTION AREAS FROM SÃO
PAULO STATE.
ID: 00240-00001
Área: 06 - Virologia Vegetal
1
DE MARCHI, B.R., 2SPADOTTI, D.M.D.A, 3KRAUSE-
ABSTRACTS
SAKATE, R., 4SANCHES, M.M, 5PAVAN, M.A.
1. UNESP/FCA, UNIVERSIDADE ESTADUAL
PAULISTA, Fazenda Experimental Lageado CEP 18603970 - Botucatu - SP. - Caixa Postal 237
Three viruses cause mosaic symptoms visually
indistinguishable on lettuce: Lettuce mosaic virus (LMV,
potyvirus), considered the most important virus in this
culture, Lettuce mottle virus (LeMoV, sequivirus) and
Bidens mosaic virus (BiMV, potyvirus). In order to verify
the importance of each virus in this culture, samples of
lettuce and weeds, found nearly the cultivated areas,
were collected from the regions of Bauru, Campinas,
Mogi das Cruzes between the years 2008 and 2010. Total
RNA was extracted and the presence of the virus verified
by RT-PCR using specific primers for each virus. A total
of 268 lettuce and 145 weed plants were analyzed.
Curiously, LeMoV was the virus found more frequently
on lettuce, followed by LMV. The occurrence of BiMV in
lettuce was extremely low and restricted to the regions
of Campinas and Bauru, where it also was found in weeds
such as Bidens pilosa and Galinsoga parviflora. The three
viruses were found infecting G. parviflora, indicating that
this plant is an important reservoir for the viruses in
nature.
Palavras-chaves: Alface, LeMoV, LMV, BiMV, sequivirus.
MOLECULAR ANALYSIS OF VP1, VP2, VP3, VP4,
AND VP7 GENES OF GROUP A ROTAVIRUS
STRAINS GENOTYPE G5 CIRCULATING IN BRAZIL
FROM 1986 TO 2005
ID: 00241-00001
Área: 04 - Virologia Básica
Silva, MFM, 2Mendonça, MCL, 3Volotão, EM, 4Leite,
JPG
1. Fiocruz, Fundação Oswaldo Cruz, Av. Brasil 4365,
Manguinhos, 21040-360, Rio de Janeiro, RJ, Brasil
1
The group A rotavirus (RV-A) genotype G5, a common
pathogen in pigs, but also able to infect horses and cattle,
was detected in a high frequency in stool samples
collected in the 1980s and the early 1990s in Brazil. After
1996, the G5 has disappeared as an endemic strain,
becoming only sporadically detected. On the other hand,
the RV-A G9 has showed a broad geographic distribution
in Brazil at this period. Recently, the G5 was detected in
children with severe diarrhea in Argentina, Brazil,
Cameroon, Paraguay, People’s Republic of China, and
Vietnam. It suggests that the G5, although uncommon
overall in humans, is present worldwide. In this work we
analyzed and determined the genetic variability of VP1,
VP2, VP3, VP4 and VP7 genes of twenty-eight G5P[8]
human RV-A strains isolated from a 19-year long sample
collection (from 1986 to 2005), representing four different
Brazilian States. Based on the sequence and
phylogenetic analysis of the VP7 gene all strains were
clustered with other Brazilian G5 strains. The VP4 genes
analyzes demonstrated that three P[8] genetic lineages:
(P[8]-1, P[8]-2 and P[8]-3), circulated in Brazil between
1986 and 2005, in association with genotype G5. Partial
sequence analysis of VP1, VP2 and VP3 revealed a high
degree of identity with Human RV-A strains, suggesting
that the Brazilian strains, probably, might have been
originated from a human RV-A strain. Our results
demonstrate that the inner RV-A genotype G5 proteins
have been adapted in humans for at least 20 years, and
emphasize the importance of the continuous virological
surveillance of circulating RV-A to detect new variants
and possible antigenic changes with potential effect on
vaccine effectiveness. In addition this work contributes
to a better understanding of the dynamics and pattern of
RV-A G5 evolution. Palavras-chaves: Gastroenteritis,
Rotavirus A, Genotype G5, Sequencing, Viral evolution.
OUTBREAK OF PANDEMIC INFLUENZA A H1N1 2009
IN A DAY CARE CENTER OF SAO PAULO-BRAZIL IN
2010.
ID: 00243-00001
Área: 05 - Virologia Humana e Saúde Pública
Guatura, S. B., 2 Camargo,C.N., 3 Parmezan, S. N.,
Cabeça,T.K., 5 Watanabe, A.S.A., 6 Granato, C.F.H,
7
Bellei, N.C.J.
1. UNIFESP, Federal University of Sao Paulo, SP, Brazil.,
Rua Pedro de Toleto, 781- 15º andar
1
4
Pandemic H1N1 2009 influenza was of great concern for
vaccine program in 2010 due to great impact on morbidity
among Brazilian people. National Immunization program
contemplated several risk groups including children up
to 5 years old.The purpose of the study was influenza
infection surveillance among symptomatic children aged
up to 12 years attended in a day care center of Sao
Paulo hospital. Virological tests were performed during
the 6 months of the study period, March to August 2010.
Nasal swabs were collected and tested by in house RTPCR protocol for amplification of a segment M and NS
of influenza A and B gene, respectively. Samples positive
for influenza A were diagnosed as H1N12009 or seasonal
influenza according to Real Time RT-PCR CDC protocol
instructions and then samples were sequenced with
commercial Kit. Eighty-two samples were performed.
Children median age was three years and 42.7% (35/82)
were vaccinated for influenza A H1N1 2009. Influenza
H1N1 2009 were detected in 17.1% (14 /82) of samples,
7.3% (6/82) were positive for influenza B and 62/82
(75.6%) were negative. The median age of positive
139
ABSTRACTS
influenza A/B cases was 6 (range 1-11years). An
outbreak of H1N1 2009 occurred from April to May and a
30% (11/37) of rate attack was observed. The first five
children, median age 6 (6-7years), were from the same
classroom and presented illness on the first week.
Influenza B (3 cases) were also detected during this time
among other school children (range 6-8 years). Three
sequenced positive samples showed 100% of similarity
with Influenza A virus (A/Mississippi/AF2471/2010/
H1N1). This data and the school outbreak confirmed
circulation of influenza among older children than those
included on National Immunization Program
Recommendation. Surveillance is especially important
in the post-pandemic period, when the behavior of the
H1N1 virus (2009) as a seasonal virus can not be
predicted and others interventions may be required.
Palavras-chaves: Influenza A H1N1, Real Time RT-PCR,
Day care center, outbreak.
MOLECULAR EPIDEMIOLOGY OF NOROVIRUS AND
ASTROVIRUS IN STOOL SAMPLES ISOLATED
FROM CHILDREN WITH ACUTE DIARRHEA IN
NITEROI-RJ.
ID: 00245-00001
Área: 05 - Virologia Humana e Saúde Pública
Xavier, MPTP, 2Ferreira, MSR, 3Ribeiro, RLA, 4Oliveira,
S.A, 5Miagostovich, M. P., 6Cubel Garcia R.C.N, 7Leite,
J.P.G
1. FIOCRUZ-IOC-LVCA, Laboratório de Virologia
Comparada, Depar tamento de Virologia, Instituto
Oswaldo Cruz, FIOCRUZ, AV. Brasil 43652. MIP-UFF,
Departamento de Microbiologia e Parasitologia, Instituto
Biomedico, Universidade Federal Fluminense, Rua Prof.
Hernani Melo 101, CEP 24210-130, Niteroi, RJ3. DIPUFF, Depar tamento de Doenças Infecciosas e
Parasitarias, Universidade Federal Fluminense, Rua
Marquês do Paraná 303 - 2º andar - HUAP, Centro Niterói
(RJ) - CEP 24033-9
collected from inpatients and outpatients attending a
public pediatric hospital. A total of 87 fecal samples were
collected from April to September 2003. All samples were
previously tested negative for rotavirus group A. NoV
and HAstV were detected by reverse transcriptionpolymerase chain reaction (RT-PCR) using primers MON
431432/433/434 (region B) and MON 269/270
respectively. NoV was observed in 29/87 (25.2%) and
HAstV in 8/58 (13.8%). The genotype characterization
of NoV strains was carried out by nucleotide sequencing
using a single PCR amplification with a set of primers
that encodes the capsid region (region D). Circulation of
GII.4 and GII.6 genotypes was observed. HAstV
genotyping was performed by sequence using the same
primers for detection and only HAstV-1 genotype was
observed. Our results demonstrate NoV and HAstV
infections causing acute gastroenteritis in children
attending a public pediatric hospital in the city of Niterói,
RJ and it will be helpful for designing prevention and
transmission strategies of these important etiologic
agents.
Palavras-chaves: Norovirus, Astrovirus, Diarrhea,
Viruses.
RISK ASSESSMENT OF NOROVIRUS INFECTION IN
RECREATIONAL WATER (RODRIGO DE FREITAS
LAGOON, RIO DE JANEIRO).
1
Viral gastroenteritis is one of the most common illnesses
in humans and it has a great impact mainly in infants
and young children. Norovirus (NoV), previously called
Norwalk-like virus, and human astrovirus (HAstV)
constitute important groups of human pathogens
associated with cases of gastroenteritis in people
worldwide. Epidemiological investigations have shown
that the most important forms of transmission are personto-person contact and contaminated food. Adults and
children of semi-closed and closed institutions are
affected, as well as hospitals and groups of families. In
order to study molecular epidemiology of NoV and HAstV
among children under two years old with acute diarrhea
in the municipality of Niteroi, RJ, stool specimens were
140
ID: 00246-00001
Área: 02 - Virologia Ambiental
Vieira, C.B., 2 Mendes, A.C.O., 3 Guimarães, F.R.,
Fumian, T.M., 5Gaspar A.MC., 6Miagostovich, M.P.
1. IOC-FIOCRUZ, Instituto Oswaldo Cruz - Fundação
Oswaldo Cruz, Av. Brasil 4365, Manguinhos - Rio de
Janeiro/RJ; Pav Helio e Peggy Pereira-B203
1
4
Microbial contamination of aquatic environments poses
a potential public health risk when improperly managed.
Recreational activities in water have contributed to the
transmission of waterborne diseases. Although evidence
from epidemiological studies and outbreak reports have
shown a relationship between adverse health effects and
immersion in water recreation of poor quality, the
difficulties associated with assigning an infection by
contact with water are numerous. Norovirus (NoV) are
responsible for 60-80% of outbreaks of gastroenteritis
around the world and have been detected in different
types of recreational waters. However, it is difficult to
estimate the waterborne risk of NoV infection by
epidemiologic studies because sporadic cases are
seldom reported. The aim of this study was to evaluate
the risk of infection by waterborne NoV through ingestion
of water in recreational activities in Rodrigo de Freitas
Lagoon. From August 2007 to July 2008, 2L of surface
ABSTRACTS
water were monthly collected in 10 points of the Lagoon,
totalizing 120 samples. The samples were concentrated
by an adsorption-elution method using a negatively
charged
membrane
and
reconcentrated to a final volume of 2mL in Centriprep®YM-50.
NoV was detected by conventional and quantitative
polymerase chain reaction preceded by reverse
transcription. NoV was detected in 17.5% (21/120) of
the studied samples, ranging from 15.7 to 265 cg/L. The
estimated risk of infection based on the probability of
infection after primary contact recreation was 1.9 x 103. Considering each site of collection, the risk of infection
ranged from 1.8 x 10-3 to 2.8 x 10-3 and just at one site
the risk presented lower than the acceptable risk proposed
by the U.S. EPA (10-3 [infection/bathers.day]). Data
obtained in this study demonstrated the need for
measures to prevent human contamination of people
whose have direct contact with waters from Rodrigo de
Freitas Lagoon. Financial support: IOC-FIOCRUZ,
PGBCM, CNPq.
Palavras-chaves: Norovirus, Recreational water, Risk
Assessment, Rodrigo de Freitas Lagoon.
RISK ASSESSMENT ANALYSIS OF ADENOVIRUS
INFECTION IN ENGENHO NOVO STREAM, ATLANTIC
FOREST FIOCRUZ CAMPUS, RIO DE JANEIRO.
ID: 00247-00001
Área: 02 - Virologia Ambiental
HAdV were detected in 6.5% (7/108) of the studied
samples, with 1.7x103 cg/L of a mean of concentration.
Quantitative concentration values were determined for
the water samples, assuming a random distribution of
the viruses within and between samples, which is
described by a Poisson distribution. The exponential
model was used to assess the daily risk of HAdV
infection associated with the ingestion of water. The
estimated risk based on the probability of infection ranged
from 1.1 x 10-2 to 5.4 x 10-3 and the risk presented
higher than the acceptable risk proposed by the USEPA
(10-3[infection/bathers.day]). The results revealed that
the most contaminated sites were those corresponding
to higher human occupancy. It demonstrates the need
for measures to prevent contamination and remediation
of the water body assessed, particularly in sites which
represent the area of highest occupancy human.
Financial support: PROBIO II -FIOCRUZ
Palavras-chaves: Adenovirus, Atlantic Forest,
Environmental Virology, Quantitative Risk Assessment.
SURVEILLANCE OF AMANTADINE AND
OSELTAMIVIR RESISTANCE AMONG VARIANTS
INFLUENZA VIRUSES A OF SÃO PAULO CITY
ID: 00248-00001
Área: 05 - Virologia Humana e Saúde Pública
Vedovello, 2Comone, P, 3Sacramento, PR, 4Durigan, MS,
Ana Pricila Perine, 6Vieira, SE, 7Leal, DB, 8Durigon, EL,
9
Botosso VF
1. IB, Instituto Butantan, Av. Vital Brasil, 1500. Butantan,
São Paulo - SP, 05503-0002. ICB III, Instituto de Ciências
Biomédicas, Avenida Prof. Lineu Prestes -Cidade
Universitária, SP - SP - CEP 05508-900.3. HU, Hospital
Universitário - USP, Av. Professor Lineu Prestes, 2565 Cidade Universitária, SP - SP - 05508-000
1
5
PORTO, N., 2GUIMARÃES, F., 3Fumian, T.M., 4Victoria,
M., 5LEITE, J.P.G, 6MIAGOSTOVICH, M.P., 7 VIEIRA,
C.B
1. IOC-Fiocruz, Instituto Oswaldo Cruz - Fundação
Oswaldo Cruz, Av. Brasil, 4365 - cep: 21040-360,
Manguinhos - Rio de Janeiro, RJ
1
Human Adenovirus (HAdV) is of major public health
impor tance and can cause a variety of clinical
manifestations associated with the gastrointestinal,
respiratory and urinary tracts, as well as the eyes. The
aim of this study was to evaluate the risk of HAdV
infection through ingestion of water in recreational
activities in Engenho Novo Stream, located at Atlantic
Forest Fiocruz Campus, Rio de Janeiro, and correlate
the risk with human occupation. From June 2008 to May
2009 2L of water samples were monthly collected in nine
GPS established sites. Water samples were concentrated
by an adsorption-elution method using a negatively
charged membrane and reconcentrated by Centriprep®
YM-50. All water samples enrolled in this study were
tested for the presence of HAdV by quantitative real time
PCR (qPCR) that target the genome region encoded for
hexon protein. Previously, it was demonstrated that there
were no correlation to HAdV detection and ,E. Coli level.
Influenza viruses are still associated with significant rates
of morbidity and mortality. Adamantane derivatives,
Amantadine and Rimantadina, innibitors of M2 ion
channel have been used as first choice antiviral drugs
against community outbreaks of influenza A viruses. But
the emergence of transmissible drug-resistant strains
have been increasing globally and their clinical use are
limited. A single specific point of mutation in M2
sequence coding for the amio acid position 26,27,30,31
or 34 confers resistance to these drugs. An alternative
for the treatment and prevention of influenza were the
neuraminidase inhibtors, Oseltamivir and Zanamivir, that
are effective for both IA and IB, and low frequency of
drug-resistence are reported among post treatment
isolates. To investigate the frequency of drug-resistant
Influenza A viruses circulating in São Paulo city we
sequenced both genes, Matrix and Neuraminidase that
141
ABSTRACTS
are targets of adamantane derivatives and neuraminidase
inibitors, respectively. We collected 526 nasopharyngeal
aspirates from children younger than 5 years old
hospitalized at University Hospital of University of São
Paulo with respiratory symptoms, during 2006. Twentyfive samples were positive for Influenza A virus: 17
H3N2, 4 H1N1 and 4 were not typed. The M2 gene of 14
Influenza A viruses (13 H3N2 and 1 H1N1) was
sequenced and all of them contained a changed at amino
acid 31 (serine to asparagine [S31N]) known to be
correlated with adamantane resisistance. The NA gene
of 16 samples (15 H3N2 and 1 H1N1) was sequenced
and no mutations were found related to the emergence
of antiviral resistance to neuraminidase inhibitors.
Virological surveillance for patterns of drug resistance
is essential for determination of antiviral treatment
strategies and for composition of pandemic preparedness
stockpiles.
Palavras-chaves: Influenza, Neuraminidase protein, Drug
resistance, Matriz Protein.
GENETIC DIVERSITY OF INFLUENZA A VIRUS
(H3N2) IN SÃO PAULO, CITY, BRAZIL, 1999 TO 2006
analysis was carried out for 24 HA genes (HA1).
Characterization of the hemagglutinin gene revealed
conserved sequences at the receptor-binding site as well
as variations due to amino acid substitutions at the
antigenic sites. The changes were limited to some codons
at, or near the antigenic sites B and D on the HA1
molecules that were recognized as antibody binding sites,
which are critical for antigenic drift. During the 8 studied
flu seasons, mutations of HA1 genes occurred
progressively and were responsible for about 3 times of
antigenic drift of influenza A H3N2 viruses in the city. It
was also found a good similarity between the circulating
strains and vaccine strains recommended for the
corresponding period. Palavras-chaves: Influenza,
Hemagglutinin, Genome, Genetic diversity, Respiratory
Viruses.
One hour and 24 hours neutralization assays for
BOVINE VIRAL DIARRHEA VIRUS (BVDV) antibodies.
ID: 00251-00001
Área: 03 - Virologia Animal
KUNERT FILHO, H.C., 2 LIMA, F.E.S., 3 BATISTA,
H.B.C.R., 4CENCI, A., 5FRANCO, A.C., 6ROEHE, P.M.
1. UFRGS, UNIVERSIDADE FEDERAL DO RIO
GRANDE DO SUL, Av. Sarmento Leite, nº 500. Porto
Alegre, RS. CEP 90050-1702. FEPAGRO - IPVDF,
Equipe de Virologia - FEPAGRO Saúde Animal. Instituto
de Pesquisas Veterinárias Desidério Finamor., Estrada
do Conde nº 6000. Eldorado do Sul, RS. CEP 92990000.
1
ID: 00248-00002
Área: 05 - Virologia Humana e Saúde Pública
Comone, P, 2Vedovello, D, 3Sacramento, PR, 4Vieira,
SE, 5Tenório, ECN, 6Stewien, KE, 7Durigon, EL, 8Botosso,
VF
1. IB, Instituto Butantan, Avenida Vital Brasil, 1500 São
Paulo - SP, 05503-0002. ICB III, Instituto de Ciências
Biomédicas, Avenida Prof. Lineu Prestes, 2415 - São
Paulo - SP - CEP 05508-9003. HU, Hospital Universitário
- USP, Av. Prof. Lineu Prestes, 2565 - São Paulo, SP,
05508-000
1
Influenza A viruses can be classified into subtypes based
on the antigenic properties of the surface glycoproteins,
hemagglutinin (HA) and neuraminidase (NA). All influenza
A virus infect avian species, but only a few subtypes
have been found in humans. An extensive genetic and
antigenic variability, especially in their surface proteins,
NA and HA, and its segmented genome are striking
features of these viruses. The high variability of the virus
can generate new strains able to escape from the
previous population immunity, which are related to annual
epidemics or pandemics. On present study we performed
molecular analysis of influenza A virus isolated from
infants and children with respiratory illness attended at
the University Hospital from São Paulo University, from
1999 to 2005. Among 1944 samples analyzed by duplex
RT-PCR, 3.8% (n=74) were influenza A viruses, 86% of
them (n = 61) were subtyped as H3, 4.1% (n = 3) as H1
and 13,5 (n = 10) were not subtyped. Phylogenetic
142
This study was carried out to determine whether the
sensitivity of serum neutralization (SN) tests would be
affected by the use of distinct periods of incubation of
the serum/virus mixture. Tests were performed in
microtiter plates; sera were diluted twofold (1:5 and 1:10).
One hundred TCID50 of the cytopathic NADL strain of
BVDV-1 were mixed with serum and incubated for either
1h (1hSN) or 24h (24hSN) at 37° C before the addition of
MDBK cells. The test performed with 24hSN revealed a
slightly higher sensitivity, detecting 1,18% (8/674) more
antibody positive sera than the 1hSN. However, the
difference between the tests was not significant. The
difference in detection of positive sera after a 24hSN
suggests that the 24h incubation did not increase
sensitivity in the test significantly.
Palavras-chaves: Bovine Viral Diarrhea Virus, Serum
neutralization, 1hSN, 24hSN.
ISOLATION AND PCR DETECTION OF BOVINE
HERPESVIRUSES IN BRAIN TISSUES OF CATTLE
SUBMITTED TO RABIES DIAGNOSIS.
ABSTRACTS
ID: 00251-00002
Área: 03 - Virologia Animal
KUNERT FILHO, H.C., 2LIMA, F.E.S., 3CAMPOS, F.S.,
DEZEN, D., 5 ROSA, J.C.A., 6 BRITO, W.M.E.D.,
7
FRANCO, A.C., 8ROEHE, P.M.
1. UFRGS, UNIVERSIDADE FEDERAL DO RIO
GRANDE DO SUL, Av. Sarmento Leite nº 500. Porto
Alegre, RS, Brasil. CEP 900150-170.2. FEPAGRO IPVDF, FEPAGRO Saúde Animal - Instituto de Pesquisas
Veterinárias Desidério Finamor., Estrada Municipal do
Conde nº 6000. Eldorado do Sul, RS, Brasil; CEP 92990000.3. UFG, UNIVERSIDADE FEDERAL DO GOIÁS, Rua
235, s/n. Goiânia, GO, Brasil. CEP 74605-050.
1
4
Bovine herpesviruses 1 and 5 (BoHV-1; BoHV-5) are
associated to a variety of clinical manifestations in cattle,
including meningoencephalitis. However, rabies virus is
the most prevalent agent of viral encephalitis in cattle in
Brazil, though in many cases the diagnosis is not
confirmed by laboratory investigation. The aim of the
present study was to search for infectious BoHV-1 and
BoHV-5 in cattle brain specimens submitted to rabies
diagnosis in the state of Rio Grande do Sul in the period
2009-2010. Virus isolation attempts were carried out in
97 specimens (36 rabies-infected and 61 rabiesuninfected specimens). After three passages in MDBK
cells, infectious herpesviruses were not detected in any
of the specimens. An additional search was conducted
for the presence of viral DNA in some brain tissue
samples by PCR. This detected the presence of viral
DNA in 10/41 samples. Subsequently, a nested PCR
will be performed to differentiate BoHV-1 from BoHV-5
DNA in such tissues. These findings provide evidence
that although animals may be latently infected with BoHV,
the encephalitis which led animals to death could not be
associated to these viruses. Therefore, although these
viruses are widely disseminated among Brazilian cattle,
these could not be associated to neurological disease in
the population examined.
Palavras-chaves: Isolation, Bovine herpesvirus 1 and
5, Rabies, PCR, Nested PCR.
IMMUNOGENICITY OF PLASMIDS ENCODING THE
NUCLEOPROTEIN AND GLYCOPROTEIN GENES OF
RABIES VIRUS
ID: 00253-00001
Área: 03 - Virologia Animal
Lima, F.E.S, 2Batista, H.B.C.R, 3Roehe, L.R, 4Schaefer,
R., 5Rijsewijk, F.A.M., 6Franco, A.C., 7Roehe, P.M.
1. UFRGS, Universidade Federal do Rio Grande do Sul,
Rua Sarmento Leite, 5002. IPVDF, Instituto de Pesquisas
Veterinárias Desidério Finamor, Estrada do Conde, 60003.
Embrapa, Embrapa Suínos e Aves, Concórdia, SC
1
The nucleoprotein (N) and glycoprotein (G) are the most
immunogenic proteins of rabies virus (RABV). In order
to compare the immunogenicity of N and G genes,
plasmids containing these genes were constructed under
the control of CMV promoter (pREC- G and pVR1012-N)
and used in a vaccination challenge experiment in mice.
Twenty days-old mice received three doses of vaccination
with the plasmids, by the intramuscular route with a 21
days interval. Twenty-two mice were divided into four
groups: group 1 (n=6) received 100ìg of pREC- G, group
2 (n=6) received 100ìg of pVR1012-N, group 3 (n=6)
received an association of pREC- G (50ìg) and pVR1012N (50ìg) and group 4 (n=4), the control group, received
buffer solution. Fourteen days later the mice were
challenged by intramuscular route, with 100 fifty per cent
lethal doses (LD50) of the RABV CVS-11 strain. In groups
2 and 3 the survival rate was 33.3%, in group 1 the
survival rate was 16.6%. All inoculated mice in control
group (group 4) died with clinical signs of rabies. The
presence of RABV in the brains of mice was confirmed
by direct imunofluorescence test (DFAT) in animals that
died after the challenge. These results show that the
plasmids codifying the N and G of RABV were able to
induce a partial degree of protection in inoculated mice.
The insertion of these genes into viral vectors as well
as the use of adjuvants or the use of intranasal route for
vaccination could be tested to improve the protein
expression, the immunogenicity of plasmids and survival
rates of vaccinated mice.
Palavras-chaves: Rabies virus, nucleoprotein,
glycoprotein, plasmids, immunogenicity test.
FIRST DESCRIPTION OF ENTEROVIRUS GENOMES
IN WATER SAMPLES COLLECTED FROM THE BROA
RESERVOIR, SP, BRAZIL.
ID: 00254-00001
Área: 02 - Virologia Ambiental
KLUGE, M., 2 COMERLATO, J., 3 FABRES, R. B.,
FONTANA, T., 5 LUZ, R. B., 6 SILVA, J. V. S.,
7
STAGGEMEIER, R., 8RODRIGUES, M. T., 9TUNDISI,
J. G., 10TUNDISI, T. M., 11SPILKI, F. R.
1. Feevale, Universidade Feevale, RS-239, 2755, Novo
Hamburgo, RS.2. IIE, Instituto Internacional de Ecologia,
Rua Bento Carlos, 750. São Carlos, SP.
1
4
Broa reservoir, located in São Carlos, state of São Paulo,
Brazil, is an artificial lake built in 1936 by a hydroelectric
company, and it is used nowadays for recreation and
research purposes only. Several studies have been made
regarding the physical and chemical aspects of this
manmade lake, the topography and hydrology of the
region and also the biological analyses of numerous live
organisms present in this environment. However, there
143
ABSTRACTS
are no virological studies in these waters that evaluate
the occurrence of enteric viruses; the presence of such
microorganisms may pose a risk for human health, since
the lake is often used for recreation by the local
population. In this sense, the aim of this study was to
investigate, in water collected in a single event in the
Broa reservoir, the presence of enterovirus genomes.
Enteroviruses are components of an enteric virus genus
within the Picornaviridae family, which are associated
with distinct clinical manifestations in human beings and
other animals. Four samples of 500 mL each were
collected from the same point of Broa reservoir, which
passed through a concentration process using
electronegative filters. The viral RNA extraction was
performed, followed by a reverse transcription for cDNA
synthesis. PCR reactions were carried out using primers
that were originally designed with potential alignment in
highly conserved regions of enteroviruses genome in the
genomic fragment of 5’-nontranslated region (5'-NTR).
The PCR products were analyzed by electrophoresis on
a 2% agarose gel in a TBE buffer, stained and visualized
under UV light. Two of the four samples resulted positive
for enterovirus genome. To our knowledge, this is the
first description of enteroviral genomes on samples
collected on this well-studied water body. Further
research aiming longitudinal monitoring for both
enteroviruses, as well as for other enteric viruses should
be conducted on the near future on this same area.
Financial support: CNPq/Fapergs/Feevale.
Palavras-chaves: enteric viruses, water environment, enterovirus.
ENTEROVIRUS OCCURRENCE IN SURFACE
WATERS LOCATED IN OSÓRIO, RS, BRAZIL.
RNA genome. The aim of this study was to evaluate the
presence of enterovirus in Maquiné river and ponds
Pinguela and Peixoto, a complex watershed of
interconnected freshwater ponds, located in Osório, RS,
Brazil. Those water bodies are surrounded by both urban
and rural areas, being affected by different levels of
effluent disposal charges. The samples were collected
in 500 ml sterile flasks and were screened for the
presence of enterovirus genomes, using polymerase
chain reaction (PCR) assay. First, the samples were
subjected to a concentration process, in which they were
passed through an electronegative membrane. Later, the
extraction of viral RNA was performed, followed by a
reverse transcription for cDNA synthesis. PCR reactions
were carried out using primers that were originally
designed with potential alignment in highly conserved
regions of enteroviruses genome in the genomic fragment
of 5’-nontranslated region (5'-NTR). The reaction products
were submitted to electrophoresis on 2% agarose gel in
a TBE buffer, stained with BlueGreen Loading Dye (LGC)
and visualized by UV light. Two samples were collected
from Maquiné river and Peixoto pond, which showed
negative for enterovirus. On the other hand, the sample
collected from Pinguela pond resulted positive for
enterovirus. The Pinguela pond has a higher impact due
the discharge of raw sewage from local population of
surrounding areas.
Financial support: CNPq, Fapergs, Feevale.
Palavras-chaves: enteric viruses, surface waters,
enterovirus.
EFFECTS OF TRIAZOLIC COMPOUNDS ON
INFLUENZA VIRUS REPLICATION.
ID: 00255-00001
Área: 04 - Virologia Básica
ID: 00254-00002
Área: 02 - Virologia Ambiental
Alves,C.M., 2Andrade, V.M., 3Accioly, M., 4Abrantes,
J.L., 5Ferreira, V. F., 6Siqueira, M.M., 7Souza, T.M.L
1. IOC, Instituto Oswaldo Cruz, Rua Leopoldo Bulhões
1480, primeiro andar, sala B1052. UFRJ, Universidade
Federal do Rio de Janeiro, Dpto. de Bioquímica Médica3.
UFF, Universidade Federal Fluminense, Niterói
1
KLUGE, M., COMERLATO, J., ARANTES, T. S., LUZ,
R. B., 5FABRES, R. B., 6FONTANA, T., 7SILVA, J. V. S.,
8
OLIVEIRA, L. K., 9STAGGEMEIER, R., 10RODRIGUES,
M. T., 11SPILKI, F. R.
1. Feevale, Universidade Feevale, RS-239, 2755, Novo
Hamburgo, RS.
1
2
3
4
Enteric viruses are frequently found in water
environments associated with sewage discharge. They
are transmitted via the fecal-oral route and show great
resistance to adverse environmental conditions. Among
these viral pathogens, enteroviruses have been pointed
as a potential indicator of fecal contamination, and are
associated with distinct clinical manifestations such as
mild febrile illness, meningitis and myocarditis. They
belong to the Picornaviridae family, which are small and
nonenveloped viruses with a single-stranded positive
144
The Influenza virus is the most prevalent infectious agent
that affects respiratory tract resulting in high morbidity,
mortality and major spending on public health system.
Currently, two antiviral drugs, oseltamivir and zanamivir,
are available for treatment. However, some drug-resistant
strains are emerging so it is crucial to search for new
alternative drugs for the treatment of influenza. We
studied the effects of 91 triazolic derivatives on the
replication of influenza H3N1 strain and found that the
compound 4 inhibited hemagglutination and
neuraminidase activity with values of EC50 equal to 459
ABSTRACTS
nM and 10 nM, respectively, and a complete inhibition
hemagglutination with 25 uM of drug. Cytotoxicity was
low, with CC50 value equal to 1811 uM. The compound 4
showed a competitive inhibition mechanism on the viral
neuraminidase. Other experiments, such as generation
of resistant strains in presence of our compound are
being developed by our group, as well as, studies
involving other strains of influenza in 2009 (H1N1).
Therefore, our findings suggest that structure 4 has a
promising antiviral activity, thus encouraging, further
studies about this compound.
Palavras-chaves: Influenza, Triazolic Compounds,
Neuraminidase.
order to detect and genotype GI and GII genogroups.
The results obtained in this study revealed that twenty
samples (14.1%) were positive for NoV, being one GI
(0.7%) and the others GII (13.4%). Samples were
characterized as GII-3 (n=3), GII-4 (n=5), GII-6 (n=1)
and GII-12 (n=2). Nine samples were not characterized
yet. This study demonstrated the circulation of NoV since
1994 in Rio de Janeiro as well as the circulation of
different genotypes during the study period.
Financial Support: CGLab - Ministry of Health, Brazil
and CNPq.
Palavras-chaves: Acute gastroenteritis, Daycare center,
Norovirus, Rio de Janeiro.
NOROVIRUS AS THE ETIOLOGIC AGENT OF CASES
OF ACUTE GASTROENTERITIS IN A DAYCARE
CENTER IN THE CITY OF RIO DE JANEIRO
ECLOSION AND IDENTIFICATION OF AEDES
(DIPTERA,: CULICIDAE) EGGS COLLECTED IN BELO
HORIZONTE, BRAZIL.
ID: 00256-00001
Área: 05 - Virologia Humana e Saúde Pública
ID: 00257-00001
Área: 05 - Virologia Humana e Saúde Pública
TINGA, A.C.C., 2 FERREIRA, M.S.R., 3 XAVIER,
M.P.T.P., 4MOTTA, S.L., 5RIBEIRO, A.M., 6LEITE, J.P.G.,
7
MIAGOSTOVICH, M.P.
1. LVCA - IOC, Lab. of Comparative and Environmental
Virology - Oswaldo Cruz Institute, Av. Brasil, 4365 CEP:21040-360, Manguinhos - Rio de Janeiro - Brasil2.
Daycare - Fiocruz, Bertha Lutz Daycare Center - Oswaldo
Cruz Foundation, Av. Brasil, 4365 - CEP:21040-360,
Manguinhos - Rio de Janeiro - Brasil
1
1
Noroviruses (NoV) are considered the first agents
involved in acute gastroenteritis outbreaks of viral etiology
and the second in sporadic cases. They belong to the
family Caliciviridae, genus Norovirus. NoV are divided
into five genogroups (GI - GV) and 29 genotypes, being
the GII-4 genotype the most prevalent worldwide. NoV
are transmitted via fecal-oral route: direct contact,
vomiting aerosol, contaminated surfaces or ingestion of
contaminated food or water. Outbreaks commonly occur
in closed environments such as daycare centers, cruise
ships, nursing homes, schools and military camps and
affect individuals of all ages and both genders. This study
was performed with 142 fecal samples obtained between
1994 and 2008 from children under five years old with
acute gastroenteritis that attended a daycare center in
Rio de Janeiro city. At that period the Comparative and
Environmental Virology Laboratory just performed
diagnosis for rotavirus A (RV-A), being these samples
negative. The aim of the current study was to determine
NoV´s presence in the samples. Genomic sRNA was
extracted from the fecal suspension followed by reverse
transcription (RT). A multiplex quantitative PCR (qPCR)
and a conventional PCR targeting the ORF-1 “ ORF-2
junction and region D, respectively, were performed in
ZEYMER, B.R., 2CURSINO, A.E., 3FIGUEIREDO, L.B.,
VILELA, A.P.P., 5NASCIMENTO, J.C., 6SONODA, I.V.,
7
SILVA, A.C., 8 BRANDÃO, S.T., 9 PESSANHA, J.E.,
10
KROON, E.G.
1. UFMG, Universidade Federal de Minas Gerais Instituto de Ciências Biológicas - Laboratório de Vírus,
Av. Antônio Carlos, 6627 - Pampulha - Belo Horizonte MG2. SMSBH-LZ, Secretaria Municipal de Saúde de Belo
Horizonte - Laboratório de Zoonoses, R. Rio Grande do
Norte, 1179 - Funcionários - Belo Horizonte - MG3.
SMSBH-GCZ, Secretaria Municipal de Saúde de Belo
Horizonte - Gerência de Controle de Zoonoses, Av Afonso
Pena, 2236 4º andar - Centro - Belo Horizonte - MG
4
Dengue is caused by infection with one of the four closely
related virus serotypes DENV-1, DENV-2, DENV-3 and
DENV-4. They are transmitted to humans by mosquitoes
of the Aedes (Diptera: Culicidae) genus. In Brazil A.
aegypti is the main vector of dengue altough A.
albopictus larvae have been found infected with DENV3. The vertical viral transmission in mosquitoes occurs
when the infected female lays eggs infected with DENV.
This study aimed to assess the eclosion rate of Aedes
eggs under controled conditions and to identify larvae.
Ovitraps containing pallets were installed by the Health
Department of Belo Horizonte (HD) in nine regional of
the city. Pallets of the Barreiro (Bar.), East (E), Northwest
(NW) and Pampulha (Pam.) regional corresponding to
15th epidemiological week (April 11 to 17, 2010), were
kindly provided by the Laboratory of Entomology. The
pallets were submersed in water in plastic glasses and
covered with tulle. On the fifth day of larvae eclosion,
stages L3 and L4 larvae were identified as A. aegypti or
A. albopictus and stored at -70°C for later detection and
145
ABSTRACTS
isolation of DENV. The HD has collected 831 pallets, but
only the samples containing eggs (457), were sent to
our laboratory, from which 96, 86, 134 and 141 were from
Bar., E, NW and Pam. respectively. A total of 25,120
eggs were analyzed, among them, 11,643 (46.3%)
ecloded and were identified. The Bar., E, NW and Pam.
areas showed 56.1%, 46.0%, 49.7% and 37.3% eclosion
rates, respectively. The larvae identification resulted in
4,218 (36.2%) A. aegypti, 280 (2.4%) A. albopictus and
7,113 (61.1%) Aedes sp. The higher number of received
samples from NW and Pam. regional are closely related
to previously notified cases of dengue in Belo Horizonte,
where dengue outbreaks have been reported, indicating
that this work can be a useful tool to assess the
epidemiological condition of the disease and viral ecology.
Financial support: CAPES-MEC/MS/SCTIE/Decit/
FAPEMIG/CNPq/Pronex Dengue/INCT-Dengue.
Palavras-chaves: Dengue virus, A. aegypti, A.
albopictus, eclosion.
DETECTION OF DENGUE VIRUS FROM LARVAES
OF AEDES AEGYPTI AND AEDES ALBOPICTUS IN
BELO HORIZONTE, BRAZIL.
ID: 00257-00002
Área: 05 - Virologia Humana e Saúde Pública
CURSINO, A.E., 2ZEYMER, B.R., 3FIGUEIREDO, L.B.,
SANTOS, J.R., 5SONODA, I.V., 6NASCIMENTO, J.C.,
7
SILVA, A.C., 8 BRANDÃO, S.T., 9 PESSANHA, J.E.,
10
KROON, E.G.
1. UFMG, Universidade Federal de Minas Gerais Instituto de Ciências Biológicas - Laboratório de Vírus,
Av. Antônio Carlos, 6627 - Pampulha - Belo Horizonte MG2. SMSBH-LZ, Secretaria Municipal de Saúde de Belo
Horizonte - Laboratório de Zoonoses, R. Rio Grande do
Norte, 1179 - Funcionários - Belo Horizonte - MG3.
SMSBH-GCZ, Secretaria Municipal de Saúde de Belo
Horizonte - Gerência de Controle de Zoonoses, Av Afonso
Pena, 2236 4º andar - Funcionários - Belo Horizonte MG
and centrifugated. RNA from each pool was extracted
using RNA isolation the Qiamp Viral RNA Kit (QIAGEN,
USA) and used as template in reverse transcription
polymerase chain reaction (RT-PCR) as described by
Lanciotti et al. (1992). A total of 4220 Aedes larvae were
processed and separated in 135 pools. One hundred
nineteen (88%) of these pools were A.aegypti and sixteen
(12%) were A.albopictus. DENV-1, DENV-2 and DENV-3
was detected in 23 pools (17%). We also detected the
presence of DENV-1 and DENV-3 in 4 pools from
A.albopictus. These data suggests that A.albopictus
could have participated as vector in the dengue outbreaks
occurred in Belo Horizonte. The infection of A.aegypti
and A.albopictus larvae is an evidence of transovarial
transmission of DENV in nature that confirms that DENV
can be vertically transmitted. Moreover, DENV vector
surveillance is an important system for the early detection
of epidemics, introduction of serotypes and genotypes.
The vector surveillance could be used as instrument of
vector control intensification measures.
Financial support: CAPES-MEC/MS/SCTIE/Decit/
FAPEMIG/CNPq/PRONEX Dengue/INCT-Dengue.
Palavras-chaves: Dengue virus, Aedes, Detection.
ON THE EVOLUTION OF A BRAZILIAN GENOTYPE
STRAIN OF AVIAN INFECTIOUS BRONCHITIS VIRUS
IN VERO CELLS
1
4
Dengue virus (DENV) is the most important arboviruses
disease in humans in tropical areas of the world and is
the causative agent of dengue fever and dengue
hemorrhagic fever. DENV belongs to the genus Flavivirus
of the family Flaviviridae. It consists of four serotypes,
DENV-1, DENV-2, DENV- 3 and DENV-4. It is horizontally
transmitted to humans by infected Aedes mosquitoes,
but mosquitoes and larvae may be infected by vertical
transmission. In this study, we aim to present the data
of DENV surveillance in A.aegypti and A.albopictus
larvaes collected during the months April, May and June
of 2010 from nine districts in the Belo Horizonte city.
Pools of mosquitoes formed with up to 50 mosquitoes
were triturated, macerated in 300 μl Leibowitz L15 medium
146
ID: 00259-00001
Área: 03 - Virologia Animal
Brandão, P. E., 2Torres, C. A., 3Oliveira, C. P., 4Barros,
I. N., 5Ayres, G. A., 6Santos, S., 7Estevez, A., 8Souza,
S. P., 9Richtzenhain, L.J., 10Sandri, T. L., 11Villarreal,
L.Y.B.
1. VPS FMVZ USP, Fac. Medicina Veterinária e Zootecnia
USP, Av. Prof. Dr. Orlando M. Paiva 87 CEP 055082702.
ISPAH, Intervet Schering Plough, Av. Sir Henry Wellcome
3353. CRG, Coronavirus Research Group, Av. Prof. Dr.
Orlando M. Paiva 87 CEP 05508270
1
Avian infectious bronchitis virus (IBV) (Nidovirales:Coronaviridae) is a chicken Gammacoronavirus with the highest evolution rates in the genus and
intra and inter-host diversity is a well documented
phenomenon. This study aimed to assess the genetic
variation of serial passages of a Brazilian genotype IBV
strain in vitro. To this end, strain CRG33, propagated in
chicken embryos, was inoculated in VERO cells
monolayers up to the 4th passage and each passage
was monitored with a-) an RT-PCR targeted to the S1
gene (nt 705 to 1094) and b-) an RT-PCR to the protein
5a mRNA (170bp). The S1 amplicons were next submitted
to DNA sequencing. All passages were positive to both
RT-PCRs and S1 sequences and showed no
ABSTRACTS
polymorphism. The finding of IBV mRNA in the cell
cultures demonstrates that the CRG33 IBV strain is
replicating in the VERO cells and that the RNA detected
is not due to the original inoculum. As for the S1 sequence
analysis, the lack of nucleotide mutations shows that,
regarding the genome area under study, CRG33 has
reached a fixed status. The resultant quasi-species after
passages in different biologic systems, be it in a bird-tobird-system or from embryos to cells, for instance, is
dependent on the initial quasi-species constitution of the
original IBV population and the mode it interacts with
the new cellular environment and is modulated by both
membrane elements such as viral proteins-receptor
affinities and intracellular components such as
transcription and translation of viral components and the
interaction between viral and cellular proteins. This
difference could be speculated as the basis for the earlier
fixation of CRG33 in comparison to previous reported,
as this strain belongs to the Brazilian genotype of the
virus, which is genetically divergent from all the known
genotypes. As a conclusion, different genotypes of IBV
present different evolutionary patterns not only in vivo
as previously known, but also in vitro, as described
herein.
Palavras-chaves: coronavirus, evolution, cells.
TYPE 2 CORONAVIRUS DETECTION IN CASES OF
DIARRHEA AND ACUTE PNEUMONIA IN YOUNG
PERUVIAN ALPACAS (Vicugna pacos)
ID: 00259-00002
Área: 03 - Virologia Animal
Luna, L. R., 2 Brandão, P.E., 3 Richtzenhain, L. J.,
Maturrano, L., 5Rosadio, R.
1. VPS FMVZ USP, Fac. Medicina Veterinária e Zootecnia
USP, Av. Prof. Dr. Orlando M. PAiva 87 CEP 055082702.
CRG, Coronavirus Research Group, Av. Prof. Dr. Orlando
M. PAiva, 87 CEP 055082703. UNMSM, Facultad de
Medicina Veterinaria, Universidad Nacional Mayor de San
Marcos, Lima, Peru
1
detection of viral nucleic acids in fecal and lung tissue
samples with a Nested PCR to the RdRp gene. Thirty
fecal samples (15 fecal samples from animals suffering
diarrhea and 15 fecal samples from apparently healthy
animals) and 16 lung tissue samples from animals with
fatal acute pneumonia were included in the survey. Type
2 Coronavirus was detected in 10 diarrheic fecal samples
bur not detected in samples from apparently healthy
animals, and 7 of 16 lung tissues samples. These findings
may confirm the pathogenic role and the susceptibility
of the alpaca regarding coronaviruses. This study
represents the first report of type 2 Coronavirus
associated with cases of diarrhea and fatal acute
pneumonia in Peruvian young alpacas. Area: Virologia
Animal Tema: Coronavirus Apresentador: Paulo Eduardo
Brandão.
Palavras-chaves: coronavirus, alpaca, Peru, enteritis,
pneumonia.
SEROPREVALENCE OF ANTIBODIES AGAINST
HEPATITIS D VIRUS (HDV) IN SAMPLES FROM AN
INDIGENOUS POPULATION OF THE BRAZILIAN
WESTERN AMAZON.
ID: 00260-00001
Área: 05 - Virologia Humana e Saúde Pública
Miguel JC, 2 Villar, LM, 3 Silva, EF, 4 Sousa, PSF,
Fernandes, CAS, 6Ginuino CF, 7Lampe, E, 8LewisXimenez, LL
1. FIOCRUZ, Fundação Oswaldo Cruz, Avenida Leopoldo
Bulhões, 1480 - Pavilhão Hélio e Peggy Pereira - Térreo2.
Noel Nutels, Laboratório Central Noel Nutels, Rua do
Resende, 118 - Centro - Rio de Janeiro
1
5
4
The alpaca (Vicugna pacos) is the most important
domestic South American camelid in Peru, are major
fiber and meat producers in the Andes. New born alpacas
are highly susceptible to infectious disease, and major
mortality from enteric and acute pneumonia occurs within
the first month of life. Relatively little is known about
virus infections in acute pneumonia in alpaca except
that they are susceptible to Parainfluenza type 3 (PI-3),
Adenovirus and Respiratory sincitial (RSV) virus infection.
In viral enteritis, rotavirus, in Peruvian young alpacas,
and Coronavirus, related to fatal cases in camelids of
other countries, have been reports. This study aimed to
investigate Type 2 Coronavirus infection in young alpacas
between 3 – 5 weeks of age from southern Peru by the
Hepatitis D virus (HDV) is a defective hepatotropic virus
that relies on hepatitis B virus (HBV) for viral assembly
and secretion and is always associated with HBV
infection. In Brazil, high endemicity of HBV infection is
found in the Amazon Region, principally among
indigenous populations due to household transmission
and occasionally by sexual contact or religious practices
that involve percutaneous injuries. In the present study,
the presence of antibodies against hepatitis D virus (antiHD) was evaluated among 433 serum samples from
indigenous populations located in the Brazilian Western
Amazon to better understand the distribution of HDV in
this population. Antibody against hepatitis B core (antiHBc total) had been identified in 405 serum samples
and an additional 28 were hepatitis B surface antigen
(HBsAg) positive. As for demographic information, 190
were females and 215 were males, with a mean age of
41 years. The presence of HDV marker (anti-HDV) was
detected using the commercial test ETI-AB-DELTAK-2
(DiaSorin, Italy), according to the manufacturer’s
147
ABSTRACTS
instructions. Reactive or indeterminant results were
retested in duplicate to confirm the results. Anti-HDV
was detected in ten serum samples, six being reactive
and four indeterminant. Among the 28 serum samples
HBsAg positive, two (7%) were reactive to anti-HDV. Of
the 405 serum samples anti-HBc positive/HBsAg
negative eight (1.9%) were reactive to anti-HDV. The ten
anti-HDV reactive samples were predominantly from male
native indians (7/10) with a mean age of 48 years (range
from 7 to 76). In conclusion, these findings demonstrate
that HDV prevalence is high in this population and still a
serious problem in the Brazilian Western Amazon. Further
studies will be done to give a better panel of HDV infection
in this population.
Palavras-chaves: Hepatite Delta, População indígena,
anti-HDV, Soroprevalência.
repeatedly indeterminate in the RIBA assay. We observed
that gender was relevant in the positivity to anti-HCV
among the 20 positive blood donors, as 18 (90%) were
male. The prevalence of anti-HCV in blood donors in the
Province of Bié was higher than that observed previously
in Angola which was 1%. Risk factors should be assessed
for this group and molecular tests for hepatitis C are yet
to be carried for a better understanding of the
epidemiology in this population.
Palavras-chaves: anti-HCV, Angola, Prevalência.
Prevalence of antibodies against hepatitis C virus
(anti-HCV) in the province of Bié, Angola
ID: 00261-00001
Área: 05 - Virologia Humana e Saúde Pública
ID: 00260-00002
Área: 05 - Virologia Humana e Saúde Pública
1
Peliganga, L.B., 2Miguel, J.C., 3Silva, E.F., 4Sousa,
P.S.F., 5Melgaço, J.G., 6 Morgado, L.N., 7Lampe, E.,
8
Lewis-Ximenez,L.L.
1. FIOCRUZ, Fundação Oswaldo Cruz, Av. Leopoldo
Bulhões, 1480 - Manguinhos - Rio de Janeiro - RJ
1
Infection with hepatitis C virus (HCV) is considered an
important public health problem as most cases become
chronic which may lead to the development of liver
cirrhosis and hepatocellular carcinoma. Africa is
considered of varied endemicity for hepatitis C with
highest prevalence observed in Egypt. In this study, we
evaluated the prevalence of anti-HCV among 1512 serum
samples (500 blood donors and 1012 pregnant women)
collected between February 2007 and August 2007 in
the province of Bié, Angola. These samples were stored
at -20C until they were shipped in dry ice to the Viral
Hepatitis National Reference Laboratory in Brazil.
Samples were initially tested using the commercial
enzyme immunoassay HCV Ultra (BioMerieux, USA) with
results reactive or indeterminate being retested in
duplicate. However, the samples that remained
indeterminate were additionally tested with CHIRON RIBA
HCV 3.0 SIA (Ortho, USA), which is an immunoblot test
that uses HCV recombinant antigens (c33c and NS5)
and synthetic peptides (c100p, c22p and 5 - 1-1p). Among
the 500 blood donors, 412 were males and 88 were
females. The average age among the blood donors and
pregnant women was 29 and 24 years, respectively.
Positivity was identified in 20 (4%) blood donors and 6
pregnant women (0.6%). Indeterminate results were
observed in 4 blood donors samples of which only 1 was
148
EVALUATION OF THREE HEPATITIS C VIRUS
ANTIBODY (ANTI-HCV)TESTS BASED ON ENZYMELINKED IMMUNOSORBENT ASSAY METHODS USED
IN THE DIAGNOSIS OF HEPATITIS C INFECTION IN
BRAZIL.
Silva,E.F., 2Miguel,J.C., 3Oliveira,J.C., 4Sant’Ana,I.C.,
Martins,P.P., 6Lampe,E., 7Villar,L.M.
1. FIOCRUZ, Fundação Oswaldo Cruz, Avenida Leopoldo
Bulhões número 1480 manguinhos Cep:21041-210
5
The routine diagnosis of hepatitis C virus (HCV) infection
is based on anti-HCV antibodies detection by two main
methods (enzyme immunoassay [EIA] and
chemiluminescence immunoassay [CIA]) but falsepositives are a problem. The aim of this study was to
evaluate the performance of three commercial anti-HCV
EIAs. Two other anti-HCV tests were also performed as
supplementary and confirmatory tests, respectively: a
recombinant strip immunoblot assay (CHIRON RIBA
HCV 3.0 SIA) and a quantitative HCV-RNA detection
(COBAS TAQMAN). In this study, sera samples were
collected from 191 healthy individuals and suspected
cases of hepatitis C who were referred to the National
Reference of Laboratory for Viral Hepatitis (Rio de Janeiro,
Brazil) during 2008 to 2010. Sera were tested by fourth
generation EIAs from three different manufacturers: (A1)
HCV Ultra (BioMerieux, USA), (A2) ETI-AB-HCVK-4
(DiaSorin, Italy) and (A3) - HCV Ab (Radim, Italy).
Discordant results among EIAs were tested by
supplementary and confirmatory tests. Total anti-HCV
seropositivity rates according to the three different
screening tests were: 45% (n=85) with A1 test, 42%
(n=79) with A2 and 46% (n=86) with A3. Moreover, 77
samples were reactive and 99 were not reactive in all
EIAs. Eleven samples presented discrepant results
among EIAs. Of the 11 RIBA HCV tests that were
performed, six were indeterminate and only one of that
was negative by HCV RNA. HCV RNA was detected in
nine samples among 11 discordant results and mean
level of HCV RNA was 177.72 UI/mL (range 25 – 747 UI/
ABSTRACTS
mL). These results showed that A3 assay presented more
concordant results according to HCV RNA since all
reactive samples by EIA were also HCV RNA reactive
and two samples that presented negative results by this
EIA were also negative by COBAS TaqMan. In
conclusion, to eliminate doubts related to false positive
findings in the initial HCV screening tests, additional
confirmatory HCV RNA assay should be performed.
Palavras-chaves:
HEPATITEC,
ESTUDO
COMPARATIVO, KITS DIAGNÓSTICO, ANTI-HCV, HCVRNA.
Rapid tests for HBsAg and anti-HCV antibody as initial
screening for hepatitis B and C in a hepatitis clinic.
not been observed with the anti-HCV rapid tests. Further
large scale testing must be carried out to validate rapid
tests especially for anti-HCV.
Palavras-chaves: Hepatite C, Hepatite B, Teste Rápido,
teste imunocromatografico, ELISA.
ROTAVIRUS A GENOTYPE P[4]G2: GENETIC
DIVERSITY AND REASSORTMENT EVENTS AMONG
STRAINS CIRCULATING IN BRAZIL BETWEEN 2005
AND 2009.
ID: 00266-00001
Área: 05 - Virologia Humana e Saúde Pública
GOMEZ, M. M., 2 MENDONCA, M.C.L., 3VOLOTAO,
E.M., 4 TORT, L.F.L., 5 SILVA, M. F. M., 6 SA, A.C.C.,
7
CRISTINA, J., 8LEITE, J. P. G.
1. FIOCRUZ, Fundação Oswaldo Cruz, Av. Brasil 4365,
Manguinhos, 21040-360, Rio de Janeiro, RJ2. UdelaR,
Laboratory of Molecular Virology, Nuclear Research
Center, School of Sciences, Iguá. 4225, 11400,
Montevideo, Uruguay
1
ID: 00262-00001
Área: 05 - Virologia Humana e Saúde Pública
Sant’Ana,I.C., 2 Almeida,A.J., 3Peres,L.R., 4Hasselmann, B., 5 Miguel,J.C., 6 Silva,E.F., 7 Ginuino,C.F.,
8
Lampe,E., 9Ximenez,L.L.
1. Fiocruz, Fundação Oswaldo Cruz, Av. Leopoldo
Bulhoes, 1480, manguinhos, Rio de janeiro, Rj Cep:
21041-210
1
Hepatitis B and C virus infections have been recognized
as major public health problems worldwide. Diagnostic
tests using immunochromatographic methods have been
developed to devise rapid tests that do not require
sophisticated laboratory settings and yield quick results.
The Viral Hepatitis Clinic has recently included rapid
testing for HBsAg and anti-HCV as initial screening
among patients referred for serological or clinical
investigation. However, the applicability in different clinical
and serological settings must be assessed. To evaluate
the utility of rapid tests for hepatitis B and C an initial
evaluation was carried out for HBsAg using the
commercial Vikia HBsAg (BioMerieux, France) assay and
for hepatitis C two anti-HCV rapid test assays were used:
1) Rapid HCV Test Bioeasy (Bioeasy Diagnostica, Brazil)
and 2) Rapid Immuno-HCV (Wama Diagnostica, Brazil).
Results obtained were compared with those obtained
from the automated commercial microparticle enzyme
immunoassays AxSym HBsAg and anti-HCV tests
(Abbott Laboratories, USA). Thirty nine patients were
submitted to rapid tests for HBsAg and 52 were submitted
to rapid tests for anti-HCV. The results for the Vikia HBsAg
rapid test were 100% concordant with the AxSym HBsAg
Abbott tests, being 5 positive and 34 negative. As for
anti-HCV rapid testing, 37 were tested by Bioeasy antiHCV and 17 by Rapid Immuno-HCV. The sensitivity and
specificity for the Bioeasy anti-HCV were 63% and 90%,
respectively, and for the Rapid Immuno-HCV 60% and
75%, respectively. For initial screening rapid tests by
Vikia HBsAg seemed to yield reliable results which have
Group A rotaviruses (RV-A) are the leading cause of
severe gastroenteritis in infants and young children
worldwide. Due to the epidemiologic complexity of RVA, especially in developing countries, it is important to
determine which genotypes are circulating, principally
after the introduction of the monovalent (P[8]G1) vaccine
Rotarix™ in Brazil by the National Immunization Program.
In Phase III trials with Rotarix™, the prevalence of
genotype P[4]G2 was extremely low, and therefore,
evaluation of heterotypic immunization against this
genotype was performed by meta-analysis statistics
tests. Different studies have shown the re-emergence of
genotype P[4]G2 in Brazil, since 2005, as well as in
other countries, suggesting that it could be a continental
phenomenon related to the temporal variability in the
genotypes naturally occurring distribution. It is important
to note that genotype P[4]G2 does not share VP4 or
VP7 antigens with the vaccine strain. Therefore, we
performed a phylogenetic analysis based on VP4 (VP8*),
VP7, VP6 and NSP4 genes of RV-A genotype P[4]G2
samples isolated from the five region of Brazil between
2005 and 2009. This study revealed that different genetic
variants of RV-A genotype P[4]G2 circulated in Brazil
between 2005 and 2009, and that this variability is highly
influenced by: a) occurrence of point mutations; b)
reassortment events; and c) widespread global gene flow.
The results obtained in this study are fundamental to
our understanding of the epidemiology and evolution of
RV-A genotype P[4]G2 and demonstrate the importance
of continuous monitoring and molecular characterization
of RV-A strains circulating in human and animal
populations.
149
ABSTRACTS
Financial support: CNPq, FAPERJ, CGLAB/MS.
Palavras-chaves: genetic variability, P[4]G2 genotype,
phylogenetic analysis, reassortment, rotavirus A.
ID: 00267-00002
Área: 03 - Virologia Animal
CORBELLINI, A.O., 2WEBER, M.N., 3Souza, C.K.,
PINTO, L.D., 5Budaszewski, R. F, 6BOABAIA, F., 7SILVA,
S.C., 8DRIEMEIER, D., 9CANAL, C. W.
1. UFRGS, Universidade Federal do Rio Grande do Sul,
Bento Gonçalves, 9090
1
POLYMERASE CHAIN REACTION FOR DETECTION
OF BOVINE HERPESVIRUS TYPE 1 IN BLOOD
SERUM AND SWAB SAMPLES
ID: 00267-00001
Área: 03 - Virologia Animal
WEBER, M.N, 2 CORBELLINI, A.O., 3SOUZA, C.K.,
PINTO, L.D., 5RODENBUSCH, C.R, 6CANAL, C.W.
1. UFRGS, Universidade Federal do Rio Grande do Sul,
Bento Gonçalves, 9090
1
4
Bovine herpesvirus type 1 (BoHV-1) is a member of the
subfamily Alphaherpesvirinae, which causes clinical
syndromes such as rhinotracheitis, pustular
vulvovaginitis or balanoposthitis, abortion, infertility,
conjunctivitis and encephalitis in bovine species. BoHV1 establishes a latent infection that can be reactivated
by stressful situations, keeping the virus circulating in
the herd. The polymerase chain reaction (PCR) is an
effective alternative for the detection of this agent that
can be used to analyze samples from different sources.
The present study aimed to stablish a PCR protocol for
BoHV-1 in blood serum and swab samples from clinical
samples submitted to our laboratory. Swab samples were
stored in phosphate-buffered saline (PBS) pH 7.2 and
subjected to centrifugation to obtain the supernatant. To
determine the protocol’s sensitivity in blood serum and
swab, negative samples were artificially contaminated
with BoHV-1 LA strain in ten-fold serial dilutions. DNA
extraction was performed using one silica-based protocol
and the PCR primers amplified a 265 bp fragment from
the glycoprotein E gene. Blood serum (69 samples), nasal
swabs (16) and genital swabs (4) of eigthy-one animals
were analysed. Blood serum and swabs had a detection
limit of 101.2 and 102.2 TCID50/mL, respectively. Nine
animals were found positive for BoHV-1, accounting for
11.11% of the cattle tested. Different sensitivities among
the samples were probably due to the presence of PCR
inhibitors in the diverse samples. The present results
provide a better understanding of the different
sensitivities obtained when diverse samples are tested
by PCR for BoHV-1, assisting in a better diagnosis of
the disorders caused by BoHV-1.
Palavras-chaves: Bovine herpesvirus type 1 (BoHV-1),
polymerase chain reaction (PCR), blood serum, SWAB
SAMPLES.
IDENTIFICATION OF BVDV IN PERSISTENTLY
INFECTED ANIMALS IN RIO GRANDE DO SUL
150
4
Improved productivity and sanitary control are necessary
to increase national competitiveness in the global meat
market. Bovine viral diarrhea virus (BVDV) belongs to
the Flaviviridae family of viruses from the genus
Pestivirus, and are an important cause of diseases
responsible for reducing reproduction rates in cattle. The
infection of pregnant cattle 40 to 120 days into gestation
can result in the birth of persistently infected (PI) cattle,
which are the main source of infectious viral particles
within a herd. Therefore, the identification and removal
of infected animals is crucial in controlling outbreaks of
BVD disease. This study evaluates two different tests,
reverse transcription PCR and the ELISA immunoassay,
that have been used to identify PI animals in herds
suspected of carrying a BVD viral infection. Blood serum
samples were collected from eight cattle herds, totalling
55 animals, suspected of harboring PI individuals. We
used RT-PCR to test for the presence of the BVD virus
via the amplification of a 157 bp fragment in the 5’UTR
region of the viral genome, after extraction of the total
RNA using the TRIzol® LS protocol. Serum from animals
that tested positive for the BVD virus were then subjected
to ELISA (Herdcheck BVDV Ag/Serum Plus-IDEXX)
tests to evaluate its diagnostic performance relative to
RT-PCR. Both tests identified 9 individual cattle (16.36%)
positive for BVD virus, suggesting no diagnostic
differences between RT-PCR and ELISA under the testing
conditions. Given that ELISA testing kits, such as the
one used in this study, are faster, cheaper and much
easier to use than RT-PCR, our study suggests a broader
adoption of these tests to ensure agricultural
competitiveness in the international market.
Palavras-chaves: Bovine viral diarrhea virus (BVDV),
reverse transcription PCR, ELISA immunoassay.
PERSISTENT PARVOVIRUS B19 INFECTION IN
MULTIPLE-TRANSFUSED
PATIENT
WITH
HEMOGLOBIN H DISEASE (Hb H) CONNECTED WITH
MULTIPLE GENOME ALTERATIONS AND
SUBCLINICAL COURSE (A CASE REPORT)
ID: 00270-00001
Área: 05 - Virologia Humana e Saúde Pública
Slavov SN, 2Kashima S, 3Wagatsuma VDM, 4Silva Pinto
AC, 5Rodrigues ES, 6Covas DT
1
ABSTRACTS
1. HCFMRP-USP, Fundação Hemocentro Ribeirão Preto,
FMRP, USP, Rua Tenente Catão Roxo 2051
Parvovirus B19 (B19V) is a significant human pathogen
causing wide spectrum of clinical conditions ranging from
mild to life-threatening. One of the most important
aspects of B19V infection is the risk of its transfusion
transmission, especially among patients suffering from
various hemoglobinopathies. Three consecutive plasma
samples from a patient with Hemoglobin H Disease (Hb
H) disease, previously defined by PCR as B19V positive
were collected at regular time intervals. B19V was
detected using primers for the NS1 region, generating
an amplicon of 442 bp, which was subjected to
sequencing. All samples were positive for B19V and with
low viral load. The sequencing and phylogenetic analysis
demonstrated that our sequences cluster with highest
similarity (99% BPs) into sub-genotype 1A group as a
distant branch. They had multiple mutations: substitutions
(22.3%) and deletions (5%). The microcytic/hypocromic
anemia remained stable (mean Hb 8.2 gm/dL; MCV=66%;
MHC=21%) as well as the leucocytes (4.400) and
platelets (479.103/ìl). Patients with Hb H disease can
suffer from increased rates of B19V infections/
reinfections, mainly due to the constant need of blood
transfusions and the absence of routine B19V screening
of the collected blood. The clinical picture in such patients
may vary depending on the type of hemoglobinopathy.
Our results, showed persistent viral replication not
connected with B19V symptomatology probably due to
the compensation of constant blood transfusions.
Nevertheless, the prolonged viral persistence raises
important questions: why B19V is not cleared despite it
is not a latent virus and can mutations modulate the
immune response and development of virus-host
“tolerance”? These results demonstrate also the need of
quantitative PCR assays for routine B19V screening of
blood donations. This will not only improve NAT but also
will prevent from unwanted spread of B19V among highrisk patient groups.
Palavras-chaves: Parvovirus B19, Hemoglobinopathies, Phylogenesis.
DETECTION AND GENOTYING OF CANINE
PARVOVIRUS IN SOUTHERN BRAZIL
ID: 00271-00001
Área: 03 - Virologia Animal
Pinto, L.D., 2Gonçalves, K.R., 3Streck, A.F., 4Souza,
C.K., 5Corbellini, A. O., 6Weber, M.N., 7Gava, D., 8Júnior,
N.B., 9Canal, C.W.
1. UFRGS, Universidade Federal do Rio Grande do Sul,
Av. Bento Gonçalves 9090 Porto Alegre RS Brasil
1
Canine parvovirus (CPV) was described in the late 1970s
and is currently one of the major causes of diarrhea and
death among puppies. In the 1990s, CPV-2a and CPV2b antigenic variants completely replaced original type
2 and rapidly spread to the canine population worldwide.
The CPV-2c strain was described in Italy in 2001, with
one amino acid change (Asp-426 to Glu-426) on a major
antigenic site. This mutation was also detected in Vietnam
(2004), Spain (2006), the United States (2007) and
Uruguay (2007). In 2009, our research group detected
type 2c in fecal samples of dogs in the metropolitan
region of Porto Alegre, in southern Brazil. The aims of
the present study were to detect CPV-2 in different
Brazilian regions and to determine the predominant
antigenic types. Fecal samples or rectal swabs obtained
from male and female dogs of different breeds aged 1
month to 1 year, from different towns in the state of Rio
Grande do Sul and from different Brazilian states, were
analyzed. The total DNA of the samples was extracted
using a commercially available silica-based kit, with PCR
amplification of a fragment of 583 VP2 gene base pairs.
The amplification products were purified, sequenced,
aligned by the Clustal method by the Bioedit 7.0.0
software and submitted to GenBank. The analysis
revealed that 40 (28.6%) out of 140 samples were positive
for CPV-2. A total of 40 CPV-2 strains from the states of
Rio Grande do Sul, Paraná and Santa Catarina were
sequenced, with detection of 77.5% (31/40) of type 2c,
17.5% (7/40) of type 2b and 2.5% (1/40) of type 2a. Our
results indicate that the CPV-2c strain is widely
disseminated in southern Brazil.
Palavras-chaves: canine parvovirus, characterization, detection, diagnosis, genotyping.
GENETIC DIVERSITY OF NOROVIRUSES IN BRAZIL
ID: 00272-00001
Área: 05 - Virologia Humana e Saúde Pública
FIORETTI, J.M., 2FERREIRA, M.S.R, 3VICTORIA, M.,
LEITE, J.P.G., 5MIAGOSTOVICH, M.P.
1. Fiocruz, Fundação Oswaldo Cruz, Av. Brasil, 4365 Manguinhos, Rio de Janeiro - RJ, Brasil.
1
4
Acute gastroenteritis is one of major public health
problems in developing and developed countries.
Norovirus (NoV), a member of the family Caliciviridae,
is recognized as important etiological agent of diarrhea
outbreaks affecting children, adults, and seniors. NoV is
classified into five genogroups (G), which GI, GII, and
GIV were described in infected humans. This study aimed
to assess the diversity of NoV circulating in different
States of Brazil including Acre, Alagoas, Bahia, Ceará,
Maranhão, Minas Gerais, Pernambuco, Rio de Janeiro,
Rio Grande do Norte, Rio Grande do Sul, Sergipe and
Distrito Federal. All strains evaluated in this study were
obtained from cases of acute gastroenteritis screened
by polymerase chain reaction preceded by reverse
151
ABSTRACTS
transcription (RT-PCR) using a set of degenerate primers
target to amplified a region of genome that encodes a
RNA polymerase RNA dependent (region B), that is able
to detect NoV GI and GII, simultaneously. For NoV
genotyping, another PCR using specific primers for GI
and II which amplify part of the coding region for viral
capsid protein (region D) was performed. A total of 63
nucleotide (nt) sequences were obtained by sequencing
the products obtained by amplification of the region D.
All strains analyzed were grouped in GII and the
genotypes found were: GII.4 (50), GII.6 (5), GII.7 (1),
GII.12 (4), GII.16 (1) and GII.17 (2). The nucleotide identity
for GII.4 ranged from 89.7 to 100%. Two groups of strains
sharing 100% of nt identity included samples from an
outbreak occurred in Bahia in 2009. Another group
included strains circulating in 2006 to 2008 in the States
of Bahia and Rio Grande do Sul. Although 79% of NoV
belonged to genotype GII.4, corroborating with other
studies that show this as the most prevalent genotype
in the world, the results demonstrated the circulating and
dissemination of different genotypes revealing the
importance of performing molecular diagnosis NoV in
the country.
Palavras-chaves: Norovirus, PCR, GII.4, Brazil,
Genotyping.
HCV GENOTYPE PREVALENCE IN DIFFERENT RISK
GROUPS
distribution of HCV genotype in the 5648 patients
analyzed, was follow: 82% for genotype 1 (4618/5648),
1.8% for genotype 2 (106/5648), 16% for genotype 3
(899/5648), 0.1% for genotype 4 (1/5648), 0.01% for
genotype 5 (1/5648); 19 samples had indeterminate
results. Within the groups considered at risk for HCV
infection, in HD patients (n=113), 63%, 27% and 8.8%
presents genotype 1, 2 and 3 respectively; in DU patients
(n=30), 66.6%, 6.6% and 26.6% % were infected with
genotype 1, 2 and 3 respectively; in HCV/HIV (n=286)
genotype 1, 3 and 4 was found in 78%, 21%, 0.6%
respectively, and in biological accidents the prevalence
of genotype 1, 2, 3 and 4 was 83%, 1.4%, 15% and
0.4% respectively. The high frequency of genotype 2 in
the HD group was due an outbreak in a HD unit.
Conclusion: Genotype 1 was more prevalent in our
region with near 80% the decades from 2000 to 2010.
Genotype 3 was more frequent in the group of drug users
and co-infected with HIV (21-26%). Financial
support:FIOCRUZ,SUS,FAPERJ.
Palavras-chaves: HCV, Genótipo, Prevalência.
EVALUATION OF PERFORMANCE TESTS FOR
COMMERCIAL GENOTYPING OF HEPATITIS C
ID: 00274-00001
Área: 05 - Virologia Humana e Saúde Pública
Amaral, F.N, 2 De-Farias, R.P., 3 Figueiredo, F.M.,
Bastos, A.G.S., 5Silva-Neto, A.M., 6Sobral-Filho, R.G.,
7
Oliveira, V.F., 8Nascimento, A.C.H.D., 9Silva, J.R.C.,
10
Cavalcanti, B.B., 11Melo, M.M.M.
1. FIP, Faculdades Integradas de Patos, Patos, Paraíba2.
LACEN, Laboratório Central de Saúde Pública, Recife,
Pernambuco3. FMN, Faculdade Maurício de Nassau,
Recife, Pernambuco4. UFPE, Universidade Federal de
Pernambuco, Recife, Pernambuco
1
ID: 00273-00002
Área: 05 - Virologia Humana e Saúde Pública
Muniz, C.C.L, 2Marques, V.A, 3Silva, L.D.A, 4Barros,
J.J.F, 5Lampe, E.
1. IOC/Fiocruz, Fundação Oswaldo Cruz, Rua Leopoldo
Bulhões, 140. Manguinhos- RJ
1
The HCV genotype has been shown to have an important
role in clinical and histological feature and on the
response to antiviral treatment. Patients infected with
HCV genotype 2 and 3 isolates have higher rates of
response to therapy than type 1 isolates. In Brazil, the
type of interferon distributed to hepatitis C depends on
infecting genotype. Aim: To determine the prevalence of
HCV genotypes among different risk groups such as
chronic hemodialysis patients (HD), co-infected with HIV
(HCV/HIV), drug users (DU) and biological accidents (AB)
attending at the Viral Hepatitis Laboratory/FIOCRUZ, Rio
de Janeiro, Brazil, in the period from 2000 to 2010.
Material and Methods: Between 2000 and 2010 a total
of 5648 HCV RNA positive sera samples of patients
attending at the Viral Hepatitis Laboratory/FIOCRUZ were
genotyped by restriction fragment length polymorphism
(RFLP) of PCR products or by Versant-Lipa HCVII (Bayer)
or by LINEAR ARRAY HCV (ROCHE).Results: The
152
4
Infection with hepatitis C virus (HCV) is considered
responsible for most cases of liver diseases in the world.
While there are many data on the prevalence of infection,
it is estimated that about one million of new cases appear
every year worldwide and that approximately 1-2% of
the population is infected with the prevalent genotypes
1, 2 and 3. The identification of these genotypes is an
important step in the infection treatment. Thereby, it is
important to consider the efficiency of the chosen
methodology and the differences between commercial
kits available to define the best option. Considering the
lack of comparative study between the performance of
commercial kits, we propose to compare genotyping kits
Line Probe Assay (INNO-LiPA ®) (Siemens Medical
Solutions Diagnostics) and LINEAR ARRAY ® HCV
Genotyping Test, v2.0 (Roche), and evaluate the
correlation between the tests and the characteristics of
ABSTRACTS
the kits. In this study, 47 sera samples obtained from
the serum bank of the Central Laboratory of Public Health
- LACEN / PE were evaluated. The samples were
amplified through the system COBAS Amplicor ®. From
the 47 samples analyzed, 15 (31.25%) were negative
controls, 27 (56.25%) genotype 1, 1 (2.08%) genotype 2
and 4 (8.3%) genotype 3, showing agreement in both
tests, except for two samples that were not detected by
the kit INNO-LiPA ®. Somethig like that can occur when
viral load is low. Regarding the visibility of the outcome
and handling of the tapes, LINEAR ARRAY ® performed
best. Concerning amount of sample required to perform
the tests, the INNO-LiPA ® uses a smaller amount,
allowing a greater number of repetitions. The INNO-LiPA
® also has the advantage of establishing the viral
subtypes, although with no clinical significance. The
precautions in handling, time and number of steps is
similar in both tests. Both tests performed well, and can
be utilized without jeopardizing the results of diagnosis.
This study was conducted without conflict of interests.
Palavras-chaves: Hepatitis C, Tests, Genotyping.
PREVALENCE OF ADENOVIRUS IN SAMPLES
RECEIVED IN THE PUBLIC HEALTH NETWORK OF
PERNAMBUCO IN THE PERIOD FROM JANUARY TO
JULY OF 2010
ID: 00274-00002
Área: 05 - Virologia Humana e Saúde Pública
Bastos, A.G.S., 2Amaral, F.N., 3Silva-Neto, A.M., 4DeFarias, R.P., 5 Figueiredo, F.M., 6Sobral-Filho, R.G.,
7
Ferreira, M.V., 8Alves, C.R.M.S., 9Oliveira, V.F., 10Melo,
M.M.M.
1. FIP, Faculdades Integradas de Patos, Patos, Paraíba2.
LACEN, Laboratório Central de Saúde Pública, Recife,
Pernambuco3. FMN, Faculdade Maurício de Nassau,
Recife, Pernambuco4. UFPE, Universidade Federal de
Pernambuco, Recife, Pernambuco
1
In 2006, the Brazilian Ministry of Health created a
program of epidemiological surveillance for rotavirus. Due
to difficulty in clinical differentiation between diarrhea
caused by both rotavirus and adenovirus, the
epidemiological surveillance of Pernambuco includes the
detection of adenovirus on all suspect samples of
rotavirus diarrhea. Due to the epidemiological importance
of these virological diseases, the prevalence of
adenovirus on these samples was evaluated. Sample
faeces of children and adult, attended in the public
network health from the state of Pernambuco, in the
period from January to July of 2010, were used in this
study. The detection of the virus was carried through by
using the commercial ELISA (RIDASCREEN-Adenovirus
R-biopharm- ALKA) kit. The results were analyzed with
the software “Epi info”, version 6.04, adopting a
significance level of 0,05 (p 4 days, 1 (25%) the duration
of vomiting was ignored. Concerning to the episodes of
diarrhea, 2 (50%) presented four episodes in 24 hours, 1
(25%) presented three episodes in 24 hours and 1 (25%)
presented six episodes in 24 hours, 2 (50%) diarrhea
whit duration of 3 days, 2 (50%) > 7 days. 3 (75%) did
not present fever, 1 (25%) experience fever greater than
38 degrees Celsius. 3 (75%) of the positive patients,
took rotavirus vaccine and 1 (25%) the vaccine was
ignored. None of the adult samples studied was positive
in tests. The data obtained in this study reinforce the
importance of both differential diagnosis and to supervise
viral gastroenteritis in the child population.
Palavras-chaves: Adenovirus, diarrhea and virus,
ELISA.
MOLECULAR CHARACTERIZATION OF NOROVIRUS STRAINS DETECTED IN CHILDREN FROM
ASUNCION, PARAGUAY, DURING 2004-2005.
ID: 00276-00001
Área: 05 - Virologia Humana e Saúde Pública
GALEANO, M.E., 2AMARILLA, A.A., 3MARTINEZ, I.M.,
CARVALHO-COSTA, F.A., 5 RUSSOMANDO, G.,
6
PARRA, G.I., 7MIAGOSTOVICH, M., 8LEITE, J.P.
1. LVCA. IOC-FIOCRUZ, Laboratory of Comparative and
Environmental Virology, Oswaldo Cruz Institute, Oswaldo
Cruz Foundatio, Av. Brasil, 4365. Manguinhos - Rio de
Janeiro - RJ - Brasil CEP: 21040-3602. IICS-UNA,
Molecular Biology and Genetics Department, Health
Sciences Research Isntitute., Cnte. Gamarra y
Lagerenza. Sajonia. Asuncion, Paraguay.
1
4
Acute gastroenteritis(AGE) is an important cause of
morbidity and mortality of children.
Palavras-chaves: Norovirus, Gastroenteritis,
Epidemiology.
DETECTION OF CAPRINE HERPESVIRUS (CpHV-1)
IN SMALL RUMINANTS IN THE SOROCABA REGION,
SÃO PAULO, BRAZIL
ID: 00277-00001
Área: 03 - Virologia Animal
Nogueira, A.H.C., 2Okuda, L.H., 3 De Stefano, E.,
Chiebao, D.P, 5Ribeiro, C.P., 6Lara, M.C.H., 7Villalobos,
E.M.C., 8Cardoso, M.V., 9Pituco E.M., 9Pituco E.M.
1. IB, Instituto Biológico, Av. Conselheiro Rodrigues
Alves, 1252,CEP 04014-002, São Paulo/SP, Brasil2.
UPDSorocaba-APTA, 2Unidade de Pesquisa e
Desenvolvimento de Sorocaba, APTA, Rua Antonio
1
4
153
ABSTRACTS
Gomes Morgado, 340 CEP: 18013-440 - Sorocaba/SP
Considering that there are low studies about caprine
herpesvirus 1 (CpHV-1) in small ruminants in Brazil, the
objective was to detect antibodies against CpHV-1 in
blood serum of 100 sheep and 51 goats in the Sorocaba
region, São Paulo, Brazil. Virus neutralization (VN) was
used as described for BoHV-1 in the Manual of Diagnostic
Tests and Vaccines for Terrestrial Animals (OIE, 2009),
with some changes. Initially, samples were tested in
quadruplicate in a 1:4 dilution. Positive samples were
analyzed by titration in 96-well plates. Antibody
quantification was carried out using serial two-fold
dilutions, from 1:4 to 1:512. After that, CpHV-1 strain VR
262, supplied by the American Type Cell Collection, was
added (200 TCID50/50mL) to the microplates and
incubated at 37oC with 5% CO2 for 2 hours. Later on,
3x105 MDBK cells/mL were added to the culture and
microplates were incubated again for 96 hours. Reading
was carried out in an inverted microscope, considering
as positive those samples that showed titers equal or
greater than 4. Validate the test by checking the back
titration of virus (which should give a value of 100 TCID50
with a permissible range of 30–300 TCID50), the control
sera and the cell control wells. Seventeen per cent (25/
151) of the samples analyzed were positive: 17% (17/
100) from sheep and 16% (8/51) from goats. Due to the
genetic and antigenic similarity of CpHV-1 and other
alphaherpesviruses from ruminants, such as BoHV-1, 2
and 5, the presence of antibodies against these other
viruses was also assessed by VN in animals positive
for CpHV-1. No neutralizing antibody against these
viruses was found. Based on the results conclude, these
animals were infected with CpHV-1. This is the first
description of natural infection by caprine herpesvirus in
small ruminants in the Sorocaba region. More studies
should be carried out in order to assess the distribution
and negative impact of caprine herpesvirus infection in
small ruminants.
Palavras-chaves: ANTICORPOS, SOROLOGIA,
HERPESVIRUS, CAPRINOS, OVINOS.
SYNTHETIC
AMINOMETHYLNAPHTHOQUINONES INHIBIT EARLY AND LATE STAGES OF IN
VITRO REPLICATION OF BOVINE HERPESVIRUS -5
ID: 00278-00002
Área: 03 - Virologia Animal
Pinto,A.M.V., 2Leite, J. P.G., 3Neves, A.P., 4Vargas, M.
D, 5Cirne-Santos, 6Paixão I. C. N P P
1. UFF, Departamento de Microbiologia e Parasitologia,
Instituto Biomédico Universidade federal Fluminense,
Rua Hernani de Mello n° 101 Niterói-RJ2. FIOCRUZ,
Laboratório de Virologia Comparada/ Instituto Oswaldo
Cruz, Avenida Brasil n° 4365-Rio de Janeiro-RJ3. UFF,
1
154
Departamento de Biologia Celular e Molecular, Instituto
de Biologia Universidade Federal Fluminense, Outeiro
de São João Batista s/n Valonguinho Niterói-RJ4. UFF,
Instituto de Química da Universidade Federal
Fluminense, Outeiro de São João Batista s/n Valonguinho
Niterói-RJ
Bovine herpesvirus 5 (BHV-5) is DNA virus belonging to
the Family Herpesviridae, They have been associated
with different clinical manifestations in cattle, including,
conjunctivitis, genital disease, abortions, respiratory and
neurological diseases. The objectives of our study were
to evaluate the in vitro antiviral activity and mechanism
of action of three synthetic naphthoquinones,
2((R1amino)(R2) methyl-3-hydroxi-1,4-naphthoquinones
R423 (R1=2,4-C6H3Cl2, R2 =n-Bu), R424 (R1=2,4C6H3Cl2, R2=Bn) and R425((R1=4-C6H3NO2, R2 = Bn)
in BHV-5 replication. Cytotoxic effect (CE) were measured
in MDBK cells treated with different drug concentrations
(25, 50, 250, 500 and 1000μM) by MTT and 50% cytotoxic
concentration were, respectively, 989 ± 2, 31±2,5,
36±3,5, 1654± 4,3 for ACV, R423, R424, R425. Antiviral
analyses of compounds against BHV-5 virus were
measured by inhibition of CE on infected cells by MTT
and plaque assay (PA). The IC50 values were determined
for ACV (MTT 125±1.8; PA 166 ± 2), R423 (MTT 2.7±1.8;
PA3.2±3.2) R424 (MTT1.9± 3; PA 3.2±5.0) and R425
(MTT 2.3 ± 0.9; PA 3.8±5.0). The low virucidal ability of
all compounds including ACV was evaluated and the
residual infectivity determined by (PA). For the attachment
and penetration assays, plates were pre-chilled at 4°C
for 1h and then some plates were infected with 200 PFU
BHV-5 in the presence and absence of different virus
concentrations. Another set of plates were infected with
200 PFU BHV-5 and, after incubation at 4°C, different
concentrations of compounds (0- 25 and 50μM) were
added, followed by incubation for 1 h at 37°C. For the
time of addition studies monolayers were infected with
1x104 PFU BHV-5 and various concentrations of
compounds added at time 0h or at intervals of 1, 2, 3, 4,
5, and 6 h post-infection. Ours results showed that all
compounds slightly affected virus attachment and
penetration, but time of addition studies revealed that all
compounds assayed suppressed BHV-5 replication in
MDBK cells in all steps of the virus replication cycle.
Hence, the range of antiviral effect of these naphthoquinones is considerable and theirs targets are most likely
some viral enzymes. Further investigations of the
mechanism of the antiviral activity of these
naphthoquinones are underway. Financial support: UFF,
CNPq, FAPERJ, IOC/Fiocruz Área:Virologia Anima.
Palavras-chaves: AMINOMETHYLNAPHTHOQUINONES, HERPESVIRUS, INHIBIT.
DETECTION
OF
ANTIBODIES
AGAINST
ABSTRACTS
PSEUDORABIES VIRUS, PORCINE CIRCOVIRUS
TYPE 2 AND INFLUENZA VIRUSES IN FERAL PIGS
FROM PANTANAL, MATOGROSSO DO SUL, BRAZIL
ID: 00283-00001
Área: 03 - Virologia Animal
SCHAEFER, R., 2SILVEIRA, S., 3SCHIOCHET, M.F.,
SOZO, J.S., 5 SIMON, N.L., 6 RITTERBUSCH, G.,
7
PIOVEZAN, U., 8JULIANO, R.S., 9PELLEGRIN, A.O.,
10
ZANELLA, J.R.C.
1. CNPSA, Embrapa Suínos e Aves, BR153, Km 110
Vila Tamanduá, CEP: 89700-000 Concórdia, SC2. UnC,
Universidade do Contestado, Rua Victor Sopelsa, 3000.
CEP: 89700-000 Concórdia - SC3. CPAP, Embrapa
Pantanal, Rua 21 de Setembro, 1880. CEP: 79320-900,
Corumbá, MS4. Bolsista DTI2- CNPq, CNPq, BR153,
Km110 Vila Tamanduá CEP: 89700-000 Concórdia, SC5.
Bolsista PIBIC-CNPq, CNPq, BR153, Km110, Vila
Tamanduá CEP: 89700-000 Concórdia, SC
1
4
Populations of feral pigs are present in eleven Brazilian
states where they are considered invasive species and
causing serious damage to the ecosystem. In the
Pantanal wetland, feral pigs are considered an established
animal population where is estimated the existence of
about 9.800 groups of animals. Monitoring and
surveillance of wild or domestic pig’s populations are
important for disease control, especially for diseases
controlled by eradication programs, as Pseudorabies virus
(PRV), avoiding the reintroduction of PRV in areas
considered free of disease. Aiming to detect PRV in wild
pigs, this study analyzed swabs samples (nasal, vaginal
and / or preputial) and serum collected in 2009 from 42
feral pigs that inhabit the Pantanal region. The samples
were inoculated in SK6 cells for virus isolation attempts,
submitted to viral DNA extraction and amplification of a
fragment of PRV gD gene (glycoprotein D) by PCR, and
the sera was analyzed by the neutralization test. In order
to investigate other viral agents that could be present in
these populations, we also analyzed sera by
immunocytochemistry test for detection of antibodies to
porcine circovirus type 2 (PCV2) and by
haemagglutination inhibition test for detection of
antibodies against swine influenza (SIV) viruses. Eleven
samples were positive by PCR to PRV. However, all
samples resulted negative by tissue culture viral isolation,
probably due to transportation logistics and loss of
infectious viral particles. We detected antibodies in sera
to PRV from 31/42 (73.8%) samples, to PCV2 from 39/
39 (100%) samples, to SIV, subtype H1N1 from 12/39
(30.76%) and to subtype H3N2 from 36/39 (92.3%)
samples. These results confirm the presence of PRV,
PCV2 and SIV in feral pigs in the Pantanal region,
showing the importance of these animal populations as
reservoirs of such pathogens. Studies are underway to
determine molecular variability of different PRV isolates
from Pantanal wild pigs. Financial support: CNPq (578102/
2008-0).
Palavras-chaves: Feral pigs, pseudorabies, porcine
circovirus type 2, swine influenza, serology.
SEARCHING FOR NEW DRUGS TO INHIBIT THE
NEURAMINIDASE ACTIVITY OF INFLUENZA
VIRUSES
ID: 00284-00001
Área: 05 - Virologia Humana e Saúde Pública
Tavares, M.I.A., 2Santos, M.G.M., 3Ferreira, A.M.A.,
Lima, L.M., 5Couceiro, J.N.S.S.
1. UFRJ, Universidade Federal do Rio de Janeiro, Av.
Carlos Chagas Filho, 373, Lab.I0-63, Dept. Virologia,
IMPPG, CCS, I. Fundão2. UFRJ, Universidade Federal
do Rio de Janeiro, Av. Carlos Chagas Filho, 373, LASSBio,
CCS, I. Fundão
1
4
Since ancient times, the influenza A and B viruses has
been responsible for respiratory syndromes with 250,000
to 500,000 deaths per year and, severe worldwide
pandemics. The elderly and young adults are the most
important groups of risk for these infections, which has
revealed the subtypes H1N1 and H3N2 of influenza A
and influenza B viruses as causal agents. The current
pharmacology therapy of the infections caused by
influenza viruses is primarily based on drugs that inhibit
the sialidase activity of the neuraminidase (NA). Two
neuraminidase-inhibitors drugs were licensed: the
zanamivir (Relenza®, Laboratories GSK) and oseltamivir
(pro-drug Tamiflu, Roche), this last one setting up as the
unique available pharmacological option for treatment of
A flu. However, the prevalence of the influenza A
infections and the indiscriminate use of the oseltamivir
(TamifluÒ) are responsible by an increasing number of
cases of drug resistance, holding in check the treatment
of the patients that present flu. Thereafter, exhibiting the
discover of new neuraminidase inhibitors as target, this
Project was developed by studies of 28 non analog-tosialic acid molecules, using fluorescence microtechnique
methodologies in which 4-methyl umbelliferyl as revealer,
and analyses of their inhibition potential on the virus
infectivity in MDCK cell cultures (canine kidney cells).
In this way, it was possible the initial identification of a
new compound (LASSBIO-1183), that was able to inhibit
the subtype H3N2 of influenza A viruses with IC50 of
135 micromolar. With these results in hands we decide
to study the inhibitory activity of 10 new LASSBio-1183
analogues, with identification of three molecules able to
present antiviral activity at IC50 = 100, 90 and 80
micromolar. These results are observed as clearly
155
ABSTRACTS
promising for definition of a new prototype drug for
treatment of the influenza virus infections, attending to
the interests of the institutions responsible for the public
healthy.
Financial Support: INCT-INOFAR - CNPq / FAPERJ.
Palavras-chaves: influenza viruses, drugs, inhibition,
neuraminidase activity.
HIGH SUSCEPTIBILITY OF CHILDREN TO
HEPATITIS A VIRUS IN A PREVIOUSLY HEPATITIS
A ENDEMIC AREA IN THE NORTH REGION OF RIO
DE JANEIRO CITY, BRAZIL
ID: 00285-00001
Área: 05 - Virologia Humana e Saúde Pública
NAVARRO, F.L.O., 2ARTIMOS, S.O., 3MELGAÇO, J.G.,
ROCHA, A.M., 5CRUZ, O.G., 6GASPAR, L.P., 7LIMA,
S.M.B., 8 FREIRE, M., 9GASPAR, A.M.C., 10VITRAL,
C.L.
1. Fiocruz, Fundação Oswaldo Cruz, Av Brasil, 4365,
Manguinhos, RJ.2. UFF, Universidade Federal
Fluminense, R. Ernani de Melo, 101, Niterói, RJ.
1
4
Results of seroprevalence studies conducted in several
Brazilian regions have shown an increase in the number
of susceptible individuals to hepatitis A virus (HAV)
infection, increasing the chance for outbreaks of the
disease. Herein, the prevalence of HAV infection was
evaluated among 686 children and adolescents (aged 1
to 18 years old) living in the community of Manguinhos
and assisted at the Health Unit Sinval Germano Faria,
ENSP, Fiocruz in 2008. After formal consent, capillary
blood was drawn through a middle finger puncture and
blood samples were stored in filter paper for subsequent
anti-HAV testing by a commercial EIA (Bioelisa HAV,
Biokit). Each participant or legal tutor was also submitted
to an interview using a standardized questionnaire.
Statistical analysis was carried out to investigate
possible associations between anti-HAV seropositivity
and risk factors. The overall prevalence of anti-HAV was
40,4%, being 90% of children under the age of five
susceptible to HAV infection. Risk factors associated
with seropositivity were: age (>5 years old).
Palavras-chaves: epidemiology, hepatitis A, anti-HAV,
seroprevalence, vaccine.
DETECTION OF A NEW CIRCOVIRUS IN POULTRY
ICBS/UFRGS sala 208, Por to Alegre/RS2. IPVDF,
Instituto de Pesquisas Veterinárias Desidério Finamor,
Estrada min. do conde 6000, Eldorado do Sul/RS
In search for the etiological agent of a clinical condition
in chickens described by apathy and lack of appetite,
Phi 29 polymerase amplification of DNA from sera of
number of diseased chickens was applied. This resulted
in the amplification of DNA with a unit length of 2.4 kb.
Sequence analysis of the amplified product showed about
40% nucleotide identity with the genome of chicken
anemia virus (CAV), which causes anemia and
immunosuppresion in newly hatched chickens. The newly
discovered genome was considered to belong to a yet
non-described virus species, which has tentatively been
named Chicken gyrovirus 2 (CGV2). Based on sequencing
results of the 2.4 kb amplification product a
5´untranscribed region (5´UTR) and the coding regions
for three viral proteins, VP1, VP2 and VP3, were identified
in CGV2. The amino acid identities between the predicted
viral proteins VP1, VP2 and VP3 of CGV2 and those of
CAV are 38.8%, 40.3%, and 32.2%, respectively. It was
concluded that the newly discovered 2.4 kb Phi 29
product represents the genome of a new member of the
Circoviridae. Because its homology with CAV, which
belongs to the genus Gyrovirus, it has been allocated to
this genus as well. The discovery of this new Gyrovirus
species in chickens may have important implications
for the poultry industry and for our basic understanding
of the biology of gyroviruses. Future studies are needed
to understand the dissemination of this virus and its role
in diseases of chickens.
Financial support: CNPq/UFRGS Área: Virologia Animal
Tema: Circovirus Arquivo: CGV2_HFSantos.
rtf Apresentador: Helton F. dos Santos.
Palavras-chaves: Circovirus, chicken, gyruviruses,
poultry.
USING THE SEROLOGICAL TESTING ALGORITHM FOR HIV SEROCONVERTION (STARHS) FOR
ESTIMATING HIV-1 INCIDENCE IN A VOLUNTARY
AND COUNSELING TESTING CENTERS (VCT) IN THE
CITY OF PAULISTA, PERNAMBUCO - BRAZIL.
ID: 00288-00001
Área: 05 - Virologia Humana e Saúde Pública
LIMA, K.O., 2SALUSTIANO, D.M., 3CORDEIRO, G.B.,
FERREIRA, M.E.R, 5 COÊLHO, M.R.C.D., 6 MELO,
H.R.L
2. CTA / COAS/PE, Centro de Testagem e
Aconselhamento, R. Inanimada s/n. - Arthur Lundreg I
Paulista-PE CEP:53417-5803. DFF/CCB/UFPE, DEPTO.
DE FISIOLOGIA E FARMACOLOGIA/UFPE, Av. Prof.
Moraes Rego, 1235 - Cidade Universitária, Recife - PE -
1
4
ID: 00287-00001
Área: 03 - Virologia Animal
Santos, H.F., 2Rijsewijk, F.A.M., 3Teixeira, T.F., 4Dezen,
D., 5Franco, A.C., 6Roehe, P.M.
1. UFRGS, Universidade Federal do Rio Grande do Sul,
1
156
ABSTRACTS
CEP: 50670-9014. DMC/CCS/UFPE, DEPTO DE
MEDICINA CLÍNICA /UFPE, Av. Prof. Moraes Rego, 1235
- Cidade Universitária, Recife - PE - CEP: 50670-9015.
PPGMEDTROP/UFPE, PROGRAMA DE PÓSGRADUAÇÃO EM MEDICINA TROPICAL, Av. Prof.
Moraes Rego, S/N, Cidade Universitária, Recife-PE
CEP:50.670-420
The voluntary and counseling testing centers (VCT)
provides an important epidemiological information of
prevalence and incidence of HIV infection among the
population served, revealing the impact of the dynamics
of the epidemic locally.This study aimed to estimate the
annual incidence of HIV-1 infection by the serological
testing algorithm (STARHS). It was used the enzyme
immunoassay BED -CEIA (Calypte Biomedical
Corporation - USA), according to the manufacturer’s
protocol. This work was enrolled individuals of the
voluntary and counseling testing center (VCT) at city of
Paulista, Pernambuco – Brazil. It was analyzed from 2008
to 2009, 81 seropositive HIV individuals and estimated
incidence and prevalence of HIV-1 infection.The results
demonstrated that the global incidence was 0.47% per
year (95% CI: 0.26-0.69) and the prevalence of 1.1%
(95% CI: 0.87-1.33). The incidence was significantly
higher among men (1.16% per year, 95% CI: 0.54-1.79)
than into the women group (0.15%, 95% CI: 0:02 to 0:27).
Palavras-chaves: BED-CEIA, CTA, HIV, RECENT
INFECTION.
continue replicating the virus and transmit infection, and
that donors HBsAg/anti-HBc in chronic phase may
contain amounts of HBV-DNA low and not be detected
by molecular tests. There is a risk of transmission that
occurs if the donor is in the early phase of infection,
before detection of HBsAg and that can be avoided by
searching the HBV-DNA. Tests for detecting nucleic acid
(NAT) for HIV, HCV and HBV in donors already occurs in
some countries and is being implemented for HIV and
HCV in Brazil. The objectives of this study were: to
perform nested-PCR for HBV-DNA in the anti-HBc
positive donors with and without anti-HBs, determine the
viral load in donors HBsAg/anti-HBc positive by the
AMPLICOR test and estimate the frequency HBV-DNA
in these serological profiles. Three hundred and twenty
donors anti-HBc/anti-HBs positive, 39 HBsAg/anti-HBc
positive and 41 anti-HBc positive alone, from Foundation
HEMOPE, participated in the study. The HBV-DNA was
not detected in anti-HBc positive donors with and without
anti-HBs and were quantified in 64% of HBsAg/anti-HBc.
These results suggest that using a more sensitive
technique would increase the chance of detecting HBVDNA in a larger number of donors. Consider replacing
the test by HBsAg tests for detection of HBV-DNA,
requires caution and further studies, however, it is useful
to detect infection during the window period. Palavraschaves: HEPATITIS B, HBV-DNA, BLOOD DONORS,
FREQUENCY.
FREQUENCY OF HBV-DNA IN BLOOD DONORS ANTIHBc POSITIVE
SEROPREVALENCE OF HEPATITIS B AND C VIRUS
IN PATIENTS WITH SYSTEMIC LUPUS
ERYTHEMATOSUS
ID: 00288-00002
Área: 05 - Virologia Humana e Saúde Pública
ID: 00289-00001
Área: 05 - Virologia Humana e Saúde Pública
BARROS, E.A., 2 LEMOS, M.F, 3 VALENÇA, M.I.B.,
MELO, J.H., 5MOREIRA, R.C., 6COÊLHO, M..R.C.D
1. PPGMEDTROP/CCS/UFPE, PROGRAMA DE PÓSGRADUAÇÃO EM MEDICINA TROPICAL/CCS/UFPE,
Av. Prof. Moraes Rego - S/N. 50.670-901 - Cidade
Universitária, Recife-PE2. IAL/SP, INSTITUTO ADOLFO
LUTZ DE SÃO PAULO, Av. Dr. Arnaldo, 355- SÃO PAULO
01246-9023. HEMOPE, FUNDAÇÃO DE HEMATOLOGIA
E HEMOTERAPIA DE PERNAMBUCO, Rua Joaquim
Nabuco, 171 - Derby, Recife - 52011-0404. DFF/CCB/
UFPE, DEPARTAMENTO DE FISIOLOGIA E
FARMACOLOGIA DA UFPE, Av. Prof. Moraes Rego,
1235 - Cidade Universitária, Recife - PE - CEP: 50670901
MELO, J.H.L., 2 SOUZA,V.S.B., 3 CAHÚ, G.G.O.M.,
SILVA,J.L.A., 5ALBUQUERQUE, A.C.C., 6KOSMINSKY, S., 7COÊLHO, M.R.C.D.
1. LIKA, Setor de Virologia, Laboratório de Imunopatologia
Keizo Asami, Universidade Federal de Pernambuco, Av.
Prof. Moraes Rego, S/N, Cidade Universitária, RecifePE CEP: 50670-9012. PPGMEDTROP, Programa de
Pós-Graduação em Medicina Tropical, Centro de Ciências
da Saúde, UFPE, Av. Prof. Moraes Rego, S/N., Cidade
Universitária, Recife-PE, CEP: 50.670-4203. ASCES,
Associação Caruaruense de Ensino Superior, Avenida
Portugal, 584, Bairro Universitário, Caruaru-PE CEP:
55016-9014. DMC/CCS, Departamento de Medicina
Clínica, Centro de Ciências da Saúde, UFPE, Av. Prof.
Moraes Rego, S/N, Cidade Universitária, Recife-PE CEP:
50670-9015. DFF/CCB, Departamento de Fisiologia e
Farmacologia, Centro de Ciências Biológicas, UFPE, Av.
Prof. Moraes Rego, S/N, Cidade Universitária, RecifePE CEP: 50670-901
1
4
The major cause of rejection in blood centers is the
detection of anti-HBc, which causes lack of blood donors
and dispose of bags. However, research shows that
donors anti-HBc positive with or without anti-HBs, may
1
4
157
ABSTRACTS
Systemic Lupus Erythematosus (SLE) is an autoimmune
disease of unknown etiology. Genetic predisposition,
hormonal and environmental factors are associated with
its pathogenesis. Infections disease causing frequent
problems in lupus patients, especially those hospitalized
with complications of the disease, being responsible for
30 to 50% of deaths and considered the second cause
of death. However, information on the seroprevalence of
HBV and HCV infection in SLE patients is very scarce.
In this study, we investigated the seroprevalence of HBV
and HCV viral markers in patients with SLE who attended
a Rheumatology Outpatient Clinic at the Clinics Hospital,
Federal University of Pernambuco, Recife, Brazil. The
serum samples were tested to anti-HBc total, HBsAg,
anti-HBs and anti-HCV by third-generation enzyme
immunoassays, according to the manufacturer’s protocol.
The population was composed by 169 patients, of which
158 (93.5%) were female and 11 (6.5%) were male, with
mean age of 39±10.9 years. All patients were negative
for HBsAg. Then, the HBV infection was assessed by
positivity for anti-HBc total and its prevalence was 10.1%
(17/169), considered high when compared with Asian
studies (1-3%) and the general population of the Brazilian
Northeast (1.2%). Of the selected, 20.1% (34/169) were
anti-HBs positive isolated, it means immunity against
the virus (after infection or vaccination). The prevalence
of HCV was 1.8% (3/169), similar to prevalence in the
general Brazilian population (1.5%). It was also featured
in 79.9% (135/169) were negative for all viral markers,
suggesting their susceptibility to HBV and HCV infection.
The data presented contribute to the knowledge of these
diseases in this group of individuals. Nevertheless, it is
important to realize the detection of HBV and HCV viral
markers in patients with SLE, considering clinical and
therapeutic implications, suggesting the clinical and
laboratory follow-up of the patients.
Financial support: CNPq, PROPESQ/UFPE.
Palavras-chaves: Systemic Lupus Erythematosus,
Hepatitis B Virus, Hepatitis C Virus, Seroprevalence.
RESEARCH OF EBV-DNA IN PATIENTS WITH
SYSTEMIC LUPUS ERYTHEMATOSUS: A SERIE OF
CASES
ID: 00289-00002
Área: 05 - Virologia Humana e Saúde Pública
SOUZA,V.S.B., 2 MELO, J.H.L., 3 CAHÚ, G.G.O.M.,
SILVA, J.L.A., 5LUCENA-SILVA, N., 6KOSMINSKY, S.,
7
COÊLHO, M.R.C.D.
1. LIKA, Setor de Virologia, Laboratório de Imunopatologia
Keizo Asami, Universidade Federal de Pernambuco, Av.
Prof. Moraes Rego, S/N, Cidade Universitária, RecifePE CEP: 50670-9012. CPqAM, Depar tamento de
1
4
158
Imunologia, Centro de Pesquisas Aggeu Magalhães, Av.
Professor Moraes Rego, S/N, CDU, Recife-PE
CEP:50.670-420 Caixa Postal:74723. PPGMEDTROP,
Programa de Pós-Graduação em Medicina Tropical,
Centro de Ciências da Saúde, UFPE, Av. Prof. Moraes
Rego, S/N, Cidade Universitária, Recife-PE CEP:
50.670-4204. DCM/CCS, Departamento de Medicina
Clínica, Centro de Ciências da Saúde, UFPE, Av. Prof.
Moraes Rego, S/N, Cidade Universitária, Recife-PE CEP:
50670-9015. DFF/CCB, Departamento de Fisiologia e
Farmacologia, Centro de Ciências Biológicas, UFPE, Av.
Prof. Moraes Rego, S/N, Cidade Universitária, RecifePE CEP: 50670-901
The Epstein Barr virus (EBV) is widely distributed
worldwide, with 90% of population is infected. EBV
transmission usually occurs through oral secretions. B
lymphocytes are preferentially infected and proliferate
along with reactive T lymphocytes, resulting in increase
of lymphoid tissue. After localized lytic replication, the
infection generalizes and can be clinically manifested
as infectious mononucleosis. Increasing evidence
suggests involvement of EBV infection in immunopathogenesis of multiple autoimmune diseases, among
them is systemic lupus erythematosus (SLE). This is
characterized by heightened production of autoantibodies
that react against a variety of cellular and extracellular
constituents, like DNA and many proteins. Some key
antigens to initiate SLE show molecular mimicry with
EBV antigens, which could induce production of
autoantibodies. Molecular studies about the relationship
EBV/LES were not found in Brazil. We conducted a series
of cases to investigate the presence of EBV-DNA in SLE
patients. It was studied 23 SLE patients who attended a
Rheumatology Outpatients Clinic at the Clinics Hospital,
Federal University of Pernambuco, Recife, Brazil. The
period of study was from December 2009 to April 2010.
The PCR for EBV was performed with DNA extracted
from peripheral blood mononuclear cells (PBMC). The
studied group consisted of 22 women and one man, whose
average age was 36.4 years. There were 43.5% of nonwhites patients. It is known that SLE primarily affects
women of childbearing age and tends to be more severe
in non-white population. No patient was positive for EBVDNA, what indicate no active infection. It is small the
quantity of circulating memory B cells, where is
established EBV latent infection, what makes difficult
the virus detection at this infection stage. It is suggested
to conduct studies to better evaluate this possible
relationship by molecular biology techniques.
Financial support: CNPq.
Palavras-chaves: Systemic Lupus Erythematosus,
Epstein Barr Virus (EBV), EBV-DNA, Prevalence.
EXPRESSION OF ROTAVIRUS VP6 PROTEIN IN
ABSTRACTS
MAMMALIAN HEK293-T CELLS
ID: 00291-00001
Área: 01 - Imunobiológicos
da Silva, H.C., 2Mouta Jr., S.S., 3Leite, J.P.G., 4Moraes,
M.T.B.
1. FIOCRUZ, Fundação Oswaldo Cruz, Avenida Brasil,
4365 - Manguinhos, Rio de Janeiro - RJ, Brasil
1
Rotavirus A (RV-A), belonging to the genus Rotavirus,
family Reoviridae, is the most important pathogen
causing severe diarrhea in infants and young children,
contributing around 600,000 children deaths every year
worldwide. The major middle capsid protein, VP6,
presents conserved epitopes among the different strains
of RV-A which makes it the antigen of choice for
diagnostic assays. In addition, VP6 represents a potential
candidate for development of a vaccine against RV-A
based on viral subunit. The aim of this work was to
express VP6 of RV-A in human embryonic kidney 293-T
(HEK293-T) cells using plasmid as vector. The RV-A VP6
gene, prototype Wa, was cloned into pCR2.1-TOPO
vector and subcloned into pCI-neo mammalian expression
vector. The HEK293-T cell line was transfected with pCIneoVP6 and the cell lysates analyzed by Western
blotting. The results showed that recombinant VP6 (rVP6)
presented molecular mass similar to native VP6 and
retained the ability to oligomerize into trimers.
Furthermore, the kinetics of expression suggested the
absence of toxicity and degradation of rVP6. In order to
purify rVP6 protein from cell lysates the immunoaffinity
chromatography will be employed. The fractions collected
during purification step will be analyzed by specific ELISA
and Western blotting. This is the first time that VP6 of
human RV-A was transiently expressed in mammalian
HEK293-T cells. Our study is showing that HEK293-T
cell line represents an appropriate system to obtain rVP6
and much probably others RV-A proteins.
Financial support: Instituto Oswaldo Cruz (FIOCRUZ),
CNPq and FAPERJ.
Palavras-chaves: Cloning, Expression, HEK293-T,
Rotavirus, VP6.
DETECTION OF ANTIBODIES AGAINST rK39
ANTIGEN OF LEISHMANIA CHAGASI IN HIV
PATIENTS
ID: 00292-00001
Área: 05 - Virologia Humana e Saúde Pública
Santos, E.O., 2Pedrosa, C.M.S., 3Lemos, M.A., 4Silva,
J.L.A., 5Souza, V.S.B., 6 CAHÚ,G.G.O.M., 7COÊLHO,
M.R.C.D.
1. PPGMEDTROP, Programa de Pós-Graduação em
1
Medicina Tropical, Centro de Ciências da Saúde, UFPE,
Av. Prof. Moraes Rego, S/N, Cidade Universitária, RecifePE CEP: 50.670-4202. UNEAL, Universidade Estadual
de Alagoas, Av. Governador Luiz Cavalcante, S/N,
Arapiraca-AL CEP: 57312-2703. HEHA, Setor de
Infectologia, Hospital Escola Dr. Hélvio Auto, Rua Cônego
Fernando Lyra, S/N, Trapiche da Barra, Maceió - AL4.
LIKA, Setor de Virologia, Laboratório de Imunopatologia
Keizo Asami, Universidade Federal de Pernambuco, Av.
Prof. Moraes Rego, S/N, Cidade Universitária, Recife/
PE CEP: 50670-9015. DFF/CCB, Departamento de
Fisiologia e Farmacologia, Centro de Ciências Biológicas,
UFPE, Av. Prof. Moraes Rego, S/N - Cidade Universitária,
Recife - PE - CEP: 50670-901
The infection with human immunodeficiency virus (HIV)
is considered a challenge to public health due to the
development of opportunistic infections as a result of
immunosuppression generated by the virus. In Brazil,
there has been a phenomenon of ruralization of acquired
immunodeficiency syndrome (AIDS) and urbanization of
visceral leishmaniasis, which indicates the emergence
of this parasitosis as an important opportunistic infection
in HIV patients. The detection of anti-Leishmania
antibodies by indirect immunofluorescence assay is a
technique more applied in routine diagnosis, but has low
sensitivity in HIV/Leishmania coinfection. More specific
tests using purified or synthetic recombinants antigens
were developed. Among them, the recombinant protein
K39 (rK39), a sequence of 39 amino acids of the region
cloned kinase of Leishmania chagasi, donovani-specific
complex has been the most widely evaluated. However,
this test has been poorly evaluated in patients with HIV.
We obtained 47 samples of peripheral blood from HIV
patients who attended at University Hospital Dr. Hélvio
Auto, a reference in infectious and parasitic diseases in
the Alagoas state. A comercial immunochromatographic
test (LEISH-IT®, DiaMed) was applied for rapid detection
of antibodies against rK39 antigen, which was positive
in 8.5% (4/47) of the samples. In these patients were
not indicated spinal puncture to assess the presence of
parasites by direct examination because they did not
exhibited clinical signs of visceral leishmaniasis such
fever, hepatosplenomegaly and pancytopenia.
Considering the severity of the coinfection, this study
highlights the importance of the use of methods of easy
implementation and interpretation that will enable the rapid
and accurate diagnosis of visceral leishmaniasis in HIV
patients, allowing reduction of its morbidity and mortality.
Financial support: FAPEAL.
Palavras-chaves: HIV, Leishmania chagasi, Coinfection,
anti-rK39.
COMPARISON BETWEEN MOLECULAR METHODS
TO DETECT AND QUANTIFY HEPATITIS C VIRUS
159
ABSTRACTS
RNA
SOUZA, S.P., 2ASANO, K.M., 3Oliveira, R.N., 4HORA,
A.S., 5NOGUEIRA, J.S., 6BARROS,I.N., 7AYRES,G.R.,
8
RICHTZENHAIN, L.J., 9BRANDAO,P.E.
1. FMVZ/USP, Department of Preventive Veterinary
Medicine and Animal Health, School of Veterinary
Medicine, USP, Av Prof Dr Orlando Marques de Paiva,
87 CEP 05508270- Cidade Universitaria SP2. CRG,
Coronavirus Research Group, Av Prof Dr Orlando
Marques de Paiva, 87 CEP 05508270- Cidade
Universitaria SP3. IP, Instituto Pasteur of São Paulo,
Brazil, Av Paulista, 393 CEP 01311-000, São Paulo, SP,
Brazil
1
ID: 00293-00001
Área: 05 - Virologia Humana e Saúde Pública
Martins,PP, 2Lampe,E, 3Lewis-Ximenez, LL, 4de Sousa,
PS, 5Fernandes, CAS, 6Villar, LM
1. IOC, Instituto Oswaldo Cruz/Fiocruz, Av. Brasil,4365,
Rio de Janeiro, R.J, CEP:210409002. LACEN, Laboratório
Central Noel Nutels, Rua do Rezende,118, Centro, Rio
de Janeiro, R.J
1
Different molecular tests are available to detect and
quantify Hepatitis C virus (HCV) RNA in serum samples.
Accurate detection is essential for clinical management
and treatment of HCV patients. The aim of this study
was to evaluate methods for detection and quantification
of HCV RNA in chronic patients and healthy subjects.
Serum samples were obtained from 82 anti-HCV reactive
and 16 non reactive anti-HCV individuals and were
submitted for qualitative (RT-nested PCR, PCR-ELISA COBAS® AMPLICOR HCV Test v2.0 and TMA mediated amplification transcription) and quantitative
(quantitative PCR-ELISA - COBAS® AMPLICOR HCV
Monitor Test v2.0 and branched DNA or bDNA)
techniques for HCV RNA detection. The TMA test yielded
the highest rate (73.5%) of detection while qualitative
PCR-ELISA testing showed the lowest viral RNA
detection rate (55.1%). HCV RNA was detected by RTnested PCR in 62.2% of serum samples. When results
were compared between qualitative (RT-nested PCR vs
TMA and RT-nested vs qualitative PCR-ELISA) and
quantitative techniques, a high concordance was
observed. All anti-HCV negative samples were not
detected by any of the tests evaluated, hence the tests
showed 100% specificity. In regards to cost assessment,
qualitative tests that presented the lowest and highest
costs were RT-nested PCR and TMA, respectively. As
for the quantitative tests the lowest and highest costs
were the quantitative PCR-ELISA and bDNA,
respectively. The utility of the in house RT-nested PCR
assay for HCV diagnostics is based on its high
concordance with other commercial assays along with
its low cost.
Palavras-chaves: HCV, Diagnostic, RT-nested PCR,
Qualitative test, Quantitative test.
BOTH THE S1 AND THE S2 CODING-REGIONS OF
THE SPIKE GLYCOPROTEIN GENE OF BOVINE
CORONAVIRUS REVEALS A MARKED PHYLOGEOGRAFIC PATTERN FOR THE DISTRIBUTION OF
THE VIRUS
ID: 00294-00001
Área: 03 - Virologia Animal
160
Bovine coronavirus (BCoV) belongs to the Betacoronavirus genus, Coronaviridae family, order Nidovirales
and causes winter dysentery in adult cattle, neonatal
diarrhea, and respiratory tract infections. The disease
causes a marked decrease in milk production of dairy
cows and body condition in both calves and adults,
resulting in severe economic losses. Though largely
studied in other countries, in Brazil studies with BCoV
are relatively scarce. The aim of the study was to correlate
the molecular epidemiological study of S gene of samples
from several outbreak in Brazil. Fecal samples were
collected from 7 cows presenting diarrhea on a farm in
Sao Paulo, Minas Gerais and, Rio Grande of Sul, all of
which were previously positive to BCoV by a nestedPCR for the RdRp gene, were submitted to a RT-PCRs
to S1 (I-II nt 23653 to 24665) and S2 gene (VI-VII-VIII nt
26332 to 27693)coding-regions of the spike gene of
BCoV, for DNA sequencing, 2 amplicons resulted in
viable sequences and were submitted to genealogic
reconstruction, by the CLUSTAL/W multiple alignment
method, BioEdit program and neighbor-joining algorithm
and maximum composite likelihood (MCL) evolutionary
model implemented in Mega 4.1 with 1000 bootstrap
replicates. In addition, 61 and 58 homologous sequences
recovered from GenBank were included in the analysis
of initial and final portion of the S gene, respectively.
The analyses of the initial portion of the S gene showed
the presence of three distinct clusters, one cluster with
strains from Brazil and strains from North America, a
cluster with strains from Korea and a cluster with strains
from Italy, Germany and Korea. In both analyses only
bootstrap values above 70 were considered. Similar
results were found in the analysis of the final portion of
the S gene, the tree showed a less resolved topology,
an expected result, since the S1 portion suffers more
selective pressure that S2 portion. These analysis show
a regional signature of BCoV in different countries.
Palavras-chaves: BCov, Phylogeografic, Pattern,
Molecular, S gene.
USING MOLECULAR TOOLS TO DIAGNOSE
ABSTRACTS
CAPRINE ARTHRITIS-ENCEPHALITIS IN THE STATE
OF PERNAMBUCO
ID: 00296-00001
Área: 03 - Virologia Animal
SILVA JÚNIOR, L.C.S., 2 GOMES, A.L.V., 3 NASCIMENTO, M.C.O., 4KASSAR, T.C., 5CAMPINHO, D.S.P,
6
SANTANA, R.L., 7NASCIMENTO, S.A., 8MAIA, R.C.C.,
9
CASTRO,R.S.
1. UFRPE, Universidade Federal Rural de Pernambuco,
Rua Dom Manoel de Medeiros, s/n, Dois Irmãos, Recife
- PE2. CPqAM, Fiocruz, Centro de Pesquisa Aggeu
Magalhães, Fundação Oswaldo Cruz, Av. Professor
Moraes Rego, s/n Cidade Universitária - Recife - PE
1
The caprine arthritis-encephalitis vírus (CAEV) is an
enveloped RNA virus of the Retroviridae family and
Lentivirus genera. This virus affects goats causing clinical
or subclinical infection, with a slow and progressive
course. In the clinical form it is observed arthritis of the
anterior members, mastitis and encephalitis. The
diagnosis is generally based in the Immuno-Diffusion
Agar Gel test (IDAG), which does not permit a clear
differentiation between CAEV and another lentivirus that
affects sheeps called Maedi Visna virus (MVV).
Therefore, the main goal of this work was to develop a
PCR reaction to allow the single diagnosis of CAEV in
field samples, using primers designed to the virus gene
tat. The positive control used was obtained from the
supernatant of the caprine corneal cell culture infected
with virus from naturally infected animals from the state
of Pernambuco. The viral nucleic acid was extracted,
reverse transcribed to cDNA and quantified to be used
in the PCR studies, testing the specificity of the reaction
against the MVV cDNA, and by performing serial dilutions
of the positive control from 0,1 to 7,0 ng, where all dilutions
tested positive. The next step in this study will be to test
field samples from animals with clinical signs of caprine
arthritis-encephalitis. The results observed in this work
demonstrates that this is the first molecular diagnosis
specifically developed and optimized to detect CAEV
using the circling strain in Pernambuco, what makes it
an important diagnostic tool for this disease.
Financial Support: CNPq/MAPA.
Palavras-chaves: Caprine Arthritis-Encephalitis vírus,
Diagnosis, PCR.
QUANTITATIVE REAL TIME PCR AS A TOOL TO
DIAGNOSE CONTAGIOUS ECTHYMA VIRUS IN FIELD
SAMPLES IN THE NORTHEAST OF BRAZIL
ID: 00296-00002
Área: 03 - Virologia Animal
KASSAR, T.C., 2GOMES, A.L.V., 3CAMPINHO, D.S.P,
5
NASCIMENTO,
M.C.O.,
SANTANA,
R.L.,
6
7
NASCIMENTO, S.A., GIL, L.H.V.G., 8CASTRO,R.S.,
9
MAIA, R.C.C., 10SILVA JÚNIOR, L.C.S.
1. UFRPE, Universidade Federal Rural de Pernambuco,
Rua Dom Manoel de Medeiros, s/n, Dois Irmãos, Recife
- PE2. CPqAM, Fiocruz, Centro de Pesquisa Aggeu
Magalhães, Fundação Oswaldo Cruz, Av. Professor
Moraes Rego, s/n Cidade Universitária - Recife - PE
1
4
Contagious Ecthyma (EC) is an acute and proliferative
disease mostly affecting sheeps and goats that can also
infect human beings. It is caused by the contagious
ecthyma virus (ECV) also known as Orf virus (ORFv),
belonging to the Parapoxvirus genera and Poxviridae
family. The disease is endemic in Brazil, causing
economic losses in the herds and leading to major
discomforts of the affected animal rendering them more
susceptible to secondary bacterial infections. In many
cases, EC can be mistaken by other vesicular infections
like Foot and Mouth disease, making it necessary to
differentiated them, especially in support to the National
Foot and Mouth Disease Eradication Program (PNEFA).
In order to establish a quick, accurate and effective
diagnosis of the field samples, the quantitative real time
PCR technique is being developed in the present work
to amplify the B2L gene of the EC virus envelope. Initially,
field samples from four different animals originated from
the state of Pernambuco, presenting papular, macular
and vesicular lesions were used. The DNA from the virus
was extracted and quantified to be used in the subsequent
real time PCR reaction using SybrGreen. Three samples
were tested positive for the reaction, when evaluated
both their amplification and melting curves, and were
sequenced to determine the identity of the samples. More
tests are being developed to determine the detection
limit of the reaction in order to standardize the technique
to field samples. These results are truly encouraging in
order to establish a more powerful and state-of-the-art
technique to the diagnosis of Contagious Ecthyma from
field samples.
Financial support: CNPq /MAPA.
Palavras-chaves: Contagious Ecthyma virus, Diagnosis,
Real Time PCR.
SPATIAL AND TEMPORAL DYNAMICS OF LETHAL
CHLOROSIS OF CUCURBITS AND POPULATION
FLUCTUATION OF THRIPS VECTOR
ID: 00299-00001
Área: 06 - Virologia Vegetal
MOREIRA, A.S., 2BERGAMIN FILHO, A., 3REZENDE,
J.A.M.
1. ESALQ/USP, Escola Superior de Agricultura “Luiz de
1
161
ABSTRACTS
Queiroz”/Universidade de São Paulo, Avenida Pádua
Dias, 11 Cx 9 São Dimas - Piracicaba-SP
Cucurbits as zucchini squash (Cucurbita pepo cv.
Caserta) are affected by the lethal chlorosis disease,
caused by the tospovirus Zucchini lethal chlorosis virus
(ZLCV), which is transmitted by the thrips Frankliniella
zucchini. Our objective was to study the spatial and
temporal dynamics of the lethal chlorosis disease on
zucchini squash and monitor the thrips population. Trials
were conducted simultaneously in two experimental fields
(A and B), approximately 500 m apart, at ESALQ/USP,
between Sep./Dec. 2009, with 308 and 204 plants,
respectively. Disease incidence was evaluated every
three days, during 27 days, based on symptoms and
PTA-ELISA. Ten symptomatic and ten asymptomatic
plants were randomly chosen at each time for counting
the number of nymphs and adults of thrips present on
the upper leaf of the plants. A model of disease progress
in the time was adjusted and values of index of dispersion
(ID) and Taylor’s power law (TPL) were calculated. Thrips
population increased in the final third of the crop cycle,
which concurred with a jump on disease incidence. The
number of thrips was three times higher in the
symptomatic plants. For trial A the ID was significantly
equal to 1.0, indicating random distribution pattern of
the disease. The same pattern was found by the TPL.
For the trial B, two (3rd and 9th) out of nine evaluations
were significantly higher than 1.0 indicating aggregated
pattern of the disease, which was corroborated by the
TPL. In the temporal analysis monomolecular model was
best fit to incidence data on both trials. These results
indicate a predominance of primary infection, although
some secondary spread of ZLCV might happen during
the final third of the crop cycle.
Palavras-chaves: Cucurbita pepo, Frankliniella zucchini,
Zucchini lethal chlorosis virus.
ANALYSIS OF THE GENOME COMPOSITION OF THE
VIRUS THAT INFECTS OIL PALM DEFOLIATING
CATERPILLAR: Opsiphanes Sp
ID: 00300-00001
Área: 04 - Virologia Básica
Dias, C. S., 2Dias, R. S., 3Eller, M. R., 4Oliveira, L. L.,
Paula, S. O.
1. UFV, Universidade Federal de Viçosa, Avenida PH
Rolfs s/n Campus Universitário
1
5
Oil Palm (Elaies Jachin guineensis) is one of the most
important monocultures in Brazil, mainly in north region.
The main product extracted from this plant is this fruit
pulp – the palm oil. Demand for oil increases annually,
mainly due to the expansion and popularization of
162
biofuels. However, monocultures systems often present
problems, as recurrent diseases. One of this diseases
are the infections by lepidopteran defoliators as
Opsiphanes sp., a major pest of oil palm, and an
alternative for insecticides use is the biological control.
In this paper we analyze the genome of viral particles
purified from caterpillars with symptoms of infection
collected from oil palm plantations in Para State, Brazil.
For this, infected larvae were macerated and filtered.
The filtrate was centrifuged at 1,500 rpm for 2 minutes
and the supernatant was centrifuged at 6,000 rpm for 20
minutes. The pellet was resuspended in Milli-Q water
and subjected to sucrose gradient centrifugation (40-60%)
at 6,000 rpm for 20 minutes. The bands present in the
upper third fraction of the tube containing the infectious
agents were collected and its genetic material was
extracted by phenol-chloroform method. It was submitted
to cleavage by DNAse or RNase and its nature was
verified by electrophoresis in agarose gel 1%. After
visualization under ultra violet light, was demonstrated
that viral genome consists of a DNA strand about 23kb.
Additional tests are been developed for a complete
characterization of this virus.
Financial Support: FAPEMIG.
Palavras-chaves: Oil Palm, Defoliating caterpillar,
Opsiphanes sp.
Genotyping of pseudorabies virus by multiplex PCR
and enzymatic restriction analysis
ID: 00302-00001
Área: 03 - Virologia Animal
Fonseca Jr., A. A., 2Magalhães, C. G., 3Sales, E. B.,
D´Ambros, R., 5Heinemann, M. B., 6Leite, R. C., 7Reis.
J. K. P.
1. LANAGRO/MG, Laboratório Nacional Agropecuário,
Avenida Rômulo Joviano, s/n, Caixa Postal 50, Pedro
Leopoldo, Minas Gerais2. UFMG, Universidade Federal
de Minas Gerais, Avenida Antônio Carlos, Belo
Horizonte, Minas Gerais3. CEDISA, CEDISA, Santa
Catarina
1
4
Pseudorabies, also known as Aujeszky´s disease, is a
disease of major impact on the swine industry. It is
caused by suid herpesvirus 1 (SuHV-1) of the family
Herpesviridae, subfamily Alphaherpesvirinae. This virus
is usually genotyped by whole genome restriction using
BamHI and divided in two major genotypes I and II. The
methodology requires virus isolation and long and
complicated techniques for DNA isolation and
electrophoresis. The aim of this work is to develop a
multiplex PCR (mPCR) to amplify the genomic regions
digested by BamHI and genotype the virus by restriction
enzyme analysis. SuHV-1 genome was submitted to
WebCutter to find the regions of interest. The results
ABSTRACTS
were compared to restriction maps developed previously
by other authors. The primers were designed in
Primer3Plus. The PCR was tested in eleven isolates,
one vaccine and one attenuated strains and twenty
clinical samples (swine brain). Two multiplex PCRs
(mPCRs) using three and two primer pairs were used
the test all these samples. The mPCRs are a faster than
the methods described in the literature that require at
least 7 days to be completed and does not require any
specific instruments besides those common in any
molecular biology laboratory. Financial Support:
LANAGRO/MG, INCT-CNPq, FAPEMIG.
Palavras-chaves: Pseudorabies, Genotyping, PCR.
STRUCTURE-FUNCTION RELATIONSHIP OF
HEPATITIS C VIRUS CORE PROTEIN AND THE
UNDERSTANDING OF VIRAL CAPISID ASSEMBLY
ID: 00303-00001
Área: 04 - Virologia Básica
Souza,T.L.F., 2Braga,V.L.A., 3Ferreira,D.F., 4Peabody,
D.S., 5 Bianconi,M.L., 6 Silva,J.L., 7 Gomes,A.M.O.,
8
Oliveira,A.C.
1. UFRJ, Universidade Federal do Rio de Janeiro, Av.
Carlos Chagas Filho, 3732. UNM, University of New
Mexico, Albuquerque, NM 87131, USA
1
Hepatitis C is a worldwide public health problem. Hepatitis
C virus core protein(HCVCP) is involved in several viral
and cellular processes. This work aims a better
understanding of the viral capsid assembly in vitro and
of the structure-function relationship of the HCVCP. Using
circular dichroism and fluorescence, we show that the
HCVCP can adopt intermediate structure in different
conditions, such as in the presence of sodium dodecil
sulphate or high salt concentration. However, when
submitted to the pHs closer to the isoelectric point, the
HCVCP forms nucleocapsid-like particles(NLPs), as
verified by electron microscopy and light scattering. This
result reveals that the neutralization of basic residues is
the main factor driving the multimerization process. In
addition, we showed that nonspecific nucleic acids lead
to formation of NLPs and that this process is highly
favored by the enthalpy as verified by isothermal titration
calorimetry. HCVCP fused to GFP also was utilized and
all results were similar, indicating that the GFP does not
inhibit the process. Gel shift assays, fluorescence
polarization and fluorescence correlation spectroscopy
data indicate that the assembly is highly cooperative
since no stable intermediate was detected. These results
will shed light on the thermodynamics and structural
basis of the different functions of HCVCP, which is a
promising target to oppose the viral replication.
SUPPORT: CNPq, CAPES, FAPERJ, IMBEBB,
PRONEX, INBEB. Área: Virologia Básica Tema: HCV
Hepacivirus Arquivo: HCV-Braga.doc Apresentador:
Vanessa L de A Braga.
Palavras-chaves: Hepatitis C Virus, HCV core protein,
Viral Assembly, Virus-like particles.
MOLECULAR CHARACTERIZATION OF FOUR
ISOLATES OF Cowpea mild mottle virus.
ID: 00305-00001
Área: 06 - Virologia Vegetal
Zanardo, L. G., 2Bicalho, A. A., 3Zerbini, F. M., 4Carvalho,
C. M.
1. UFV, Universidade Federal de Viçosa, Av. P. H. Rolfs,
s/n, Campus Universitário Viçosa, Minas Gerais
1
Brazil is one of the world’s largest soybean producers.
Symptoms such as chlorosis, nerve clearing and leaf
necrosis have been found and attributed to Cowpea mild
mottle virus–CpMMV, which is causing reduced soybean
productivity and economic losses. CpMMV has a single
stranded positive-sense RNA genome and belongs to
the genus Carlavirus (family Flexiviridae). In this study,
we investigated the genetic diversity of isolates of
CpMMV by comparing viral coat protein sequences. Total
RNA was extracted, followed by RT-PCR, for the
amplification of a viral fragment of approximately 1600
bp that corresponds to the region of the coat protein and
part of the triple gene block. The product was purified
and ligated into a pGem-T EASY vector (Promega) and
cloned in E. coli DH5á. Colonies with possible
recombinant plasmids were submitted to plasmidial DNA
minipreparation by alkaline lysis and sent for sequencing.
The isolates CpMMV-Bahia, CpMMV-Goiatuba, CpMMVSorriso and CpMMV-4562-7 were sequenced and their
coat proteins were compared with all CpMMV sequences
present in GenBank. CpMMV-Bahia had the sequence
identities: CpMMV-Goiatuba (84%), CpMMV-Sorriso
(99%), CpMMV-4562-7 (99%), CpMMV_ DQ444266
(99%), CpMMV-H_ AF024628 (75%), CpMMV-M_
AF024629 (76%), CpMMV-PR_ GU191840.1 (98%),
CpMMV_ DQ885940 (84%), CpMMV_ EF635061 (98%).
CpMMV-Goiatuba had the sequence identities: CpMMVSorriso (84%), CpMMV-4562-7 (84%), CpMMV_
DQ444266 (84%), CpMMV-H_ AF024628 (76%), CpMMVM_ AF024629 (76%), CpMMV-PR_ GU191840.1 (84%),
CpMMV_ DQ885940 (99%), CpMMV_ EF635061 (85%).
CpMMV-Sorriso had the sequence identities: CpMMV4562-7 (98%), CpMMV_ DQ444266 (99%), CpMMV-H_
AF024628 (75%), CpMMV-M_ AF024629 (76%), CpMMVPR_ GU191840.1 (97%), CpMMV_ DQ885940 (83%),
CpMMV_ EF635061 (97%). The isolate CpMMV-4562-7
had the sequence identities: CpMMV_ DQ444266 (98%),
CpMMV-H_ AF024628 (76%), CpMMV-M_ AF024629
163
ABSTRACTS
(76%), CpMMV-PR_ GU191840.1 (98%), CpMMV_
DQ885940 (99%), CpMMV_ EF635061 (99%). All isolates
belong to the same species of CpMMV and the identities
between sequences varied from 75% to 99%. This work
was supported by FAPEMIG process CAG APQ- 0099209. Palavras-chaves: Characterization, Molecular,
CpMMV, diversity.
CELL GROWTH INHIBITION ACTIVITY OF
RECOMBINANT IFN ALPHA-2B GENERATES DATA
LOSS ON ANTIVIRAL ASSAY USING HEP-2C CELLS
ID: 00306-00001
Área: 01 - Imunobiológicos
considered once developing an in vitro potency
test.Financial support: FIOCRUZ/CAPES.
Palavras-chaves: Antiviral assay, Hep-2C, Interferon,
Recombinant.
MEASUREMENT OF PHOSPHORYLATED STAT1 AS
AN ALTERNATIVE ENDPOINT FOR RELATIVE
POTENCY DETERMINATION OF RECOMBINANT IFN
ALPHA USING FLOW CYTOMETRY
ID: 00306-00002
Área: 01 - Imunobiológicos
Oliveira, E.R.A., 2 Pinto, B.M.M., 3 Santos, B.A.F.,
Moura, W.C., 5Nogueira, A.C.
1. Fiocruz, Fundação Oswaldo Cruz, Av. Brasil, 4365,
Manguinhos
1
4
Oliveira, E.R.A., 2 Pinto, B.M.M., 3 Santos, B.A.F.,
Moura, W.C., 5Nogueira, A.C.
1. Fiocruz, Fundação Oswaldo Cruz, Av. Brasil 4365,
Manguinhos
1
4
Interferons (IFNs) are a group of cytokines secreted by
mammalian nucleated cells, known to exhibit antiviral,
antiproliferative and immunomodulatory activities. With
the advent of recombinant DNA technology, the
production and isolation of large amounts of IFNs became
possible. Since the recombinant IFNs are widely used in
the treatment of many diseases such as hepatitis C,
there is a considerable demand in the quality control of
INFs. We investigated whether the treatment of cells alone
with recombinat INFs may influence on the cell growth,
as measured by MTT assay, and therefore interfere with
the outcome of an antiviral assay for INFs. For this
purpose we first standardized an assay using Hep-2C
cell line and mengovirus. Moreover, we examined possible
influences of INFs in the cell-cycle of Hep-2C cell line
using propidium iodide and flow cytometry. We noticed
that the antiproliferative activity caused by IFN alpha-2b
on Hep-2C cells yielded a negative impact on final data
analysis. Proliferation assay showed a clear inhibition
effect of INF on cell growth (4.9% for 1000 IU/ml and
48% for 100 IU/ml IFN when compared to controls).
Regarding the cell cycle, an increase on cells in S + G2
phase of about 9.6 % was observed when compared to
the controls (p < 0.01) after treatment with IFN at 1000
IU/ml for 24h. Under the conditions of this study, data
analysis using parallel line model assay for ED50
determination showed a loss of linearity when considering
responses of IFN at 1000 IU/ml. The data also show a
statistically relevant (p < 0.05) reduction of cell growth
when confluent monolayers of Hep-2C cells were
stimulated with IFN at 1000 IU/ml for 24h in comparison
to controls. From the results obtained so far we conclude
that treatment with high doses of recombinat INFs might
interfere with the assay results and therefore the cell
growth and cell cycle effects of INFs on cells must be
164
The interferons (IFNs) are a large family of multi-functional
secreted cytokines involved in antiviral defense, cell
growth regulation and immune activation. Since
interferons are potent virus replication inhibitors its
recombinant preparations are used therapeutically, mainly
against virus infections, such as hepatitis C. In order to
control the potency of recombinant INFs a large variety
of assays have been developed, mostly based on the
reduction of viral cytopathic effect in susceptible cell
lines. Moreover, type I IFNs transduce intracellular signals
through the common surface receptor IFNAR1/2 leading
to the phosphorylation of signal transducers and
activators of transcription (STATs). Here, we tested the
feasibility of recobinant INF to stimulate STAT1 in a dose
dependent manner on Hep-2C cell line as an attempt to
design a new method using an alternative endpoint for
measuring recombinant INF potency. For this purpose
we developed an assay using commercially available
pSTAT1 antibody and flow cytometry. Dose-dependent
increases in pSTAT1 levels were observed in cells
stimulated with IFN alpha at doses between 31.25 and
1000 IU/ml. Using a commercially available IFN alpha
for relative potency determination we also compared the
classic cytopathic effect reduction anti-viral assay with
this novel approach of pSTAT1 determination by flow
cytometry. Data applied to the four-parameter logistic
model using Combistats software presented regression
and did not show deviations from linearity in both cases.
However, when considering pSTAT1 as an endpoint we
observed shorter 95% confidence limits of the estimated
potency. Thus, under the conditions of this study, pSTAT1
determination by flow cytometry as an alternative
endpoint to assess the potency of recombinant INF might
be a useful tool for quality control purposes. Financial
support: FIOCRUZ/CAPES.
Palavras-chaves: Cytometry, Interferon, pSTAT1,
Potency.
ABSTRACTS
EVALUATION OF DENGUE NS1 ANTIGEN TESTS
KITS FOR THE DIAGNOSIS OF ACUTE DENGUE
INFECTION.
ID: 00310-00001
Área: 05 - Virologia Humana e Saúde Pública
Silva, V.G., 2 Silva, A.M., 3 Gil, L.H.V.G., 4 Marques,
E.T.A., 5Cordeiro, M.T.
1. CPqAM-Fiocruz, Centro de Pesquisas Aggeu
Magalhães - Fiocruz, Av. Prof. Moraes Rego, s/n Campus da UFPE, Cidade Universitária, Recife, PE2.
LACEN-PE, Laboratório Central de Saúde Pública do
Estado de Pernambuco, Praça Oswaldo Cruz, s/n, Boa
Vista, Recife - Pe; CEP 50.210-000
1
Dengue virus (DENV) infection is the most common
mosquito-borne viral disease of public health significance
in most tropical and subtropical areas of the world. Rapid
diagnosis of dengue is needed for patient management,
as well as for early epidemic detection. Detection of the
dengue viral protein 1 (NS1) in the plasma/serum of
patients represents a new approach to dengue diagnosis.
The sensitivity and specificity of the Dengue Platelia
NS1-Ag ELISA (BioRad) were compared with a gold
standard reference diagnostic algorithm in 307 patients.
The immunochromatography assay NS1-Ag Strip
(BioRad) was compared with a sub-population of 90
samples. Samples were collected up to seventh day after
illness onset from 222 patients with dengue infection
along with 85 samples from other febrile illnesses. The
NS1-Ag ELISA showed 72.1% sensitivity (160/222) in
confirming dengue cases and the NS1-Ag Strip assay
showed 93.2% (69/74) when compared with NS1-ELISA
positive samples. Both kits were 100% specific, being
negative in all febrile patients without evidence of recent
DENV infection. Cross reactivity with rubella cases was
not observed. The sensitivity of the NS1-Ag ELISA in
the presence or absence of IgM in test samples was
64.8% (94/100) and 75.5% (114/151), respectively
(p=0.097). The NS1-Ag ELISA was more sensitive for
primary (94%; 94/100) than secondary (54%; 66/122)
dengue infection (p=0.0001). Similar results were
observed with the NS1-Ag rapid test. The highest NS1
antigen detection was found during the first five days
after the onset of symptoms. The NS1-Ag ELISA showed
almost the same sensitivity as the RT-PCR in confirming
acute dengue cases, 72% and 69% respectively. The
combination of NS1 and IgM detection in samples
collected in the first few days of fever can increase the
overall dengue diagnostic sensitivity. This evaluation
showed that both commercial kits offer the possibility of
early and rapid DENV infection diagnosis.
Financial support: NIAID/NIH, PDTIS-FIOCRUZ.
Palavras-chaves: Dengue, DENV NS1, NS1-Ag ELISA,
NS1-Ag teste rápido.
ROTAVIRUS AND TORQUE TENO VIRUS IN FISH
CULTURED ON CONTAMINATED WATER FROM
SINOS RIVER, SOUTHERN BRAZIL
ID: 00311-00001
Área: 02 - Virologia Ambiental
BERGAMASCHI, B., 2 Silva, J.V.S., 3 Kluge,M.,
Rodrigues, M. T., 5Silva, L.B., 6Spilki,F.R., 7Roehe
1. Feevale, Universidade Feevale, RS-239, numero 2755,
Novo Hamburgo RS2. UFRGS, Universidade Federal do
Rio Grande do Sul, Av. Paulo Gama 110, Porto Alegre/
RS
1
4
The Sinos River watershed is a major source of water
for agriculture, domestic and industrial uses for the
metropolitan region of Porto Alegre (RS, Brazil). The
domestic, industrial and agricultural sewage, as well as
waste from urban and rural areas may contain toxic,
genotoxic and mutagenic substances, as well as bacterial
and viral agents. In order to check the presence of
Rotavirus (RV) and Torque teno virus (TTV) and its
eaccumulation on the tissues of fish exposed to Sinos
River water, 10L samples were aseptically collected from
the river, in five different locations, from the source to
the delta. Conventional methods were performed to
diagnose the level of coliform contamination and chemical
composition of water samples. Piava fishes (Leporinus
obtusidens) were purchased from a local aquaculture farm
and cultured on these same water samples for 72 hours.
After, animals were euthanized and hepatopancreas was
collected, submitted to viral RNA/DNA extraction and,
after synthesis of cDNA, tissue samples were assayed
for the presence of Rotavirus and Torque teno virus
genomes by polymerase chain reaction. RV was present
in the tissues of fishes exposed to 3 out of five points,
being only found in water samples from on of these points,
thus showing that fish may be also a source for
concentration of viral agents in polluted waters. TTV was
not found on the tissues of those fishes, as well it was
not detected in water samples.
Palavras-chaves: Detection, Polymerase Chain
Reaction, Rotavirus, Torque Teno Virus.
CONSTRUCTION OF A MODIFIED VACCINIA
ANKARA VIRAL VECTOR EXPRESSING THE
LUCIFERASE GENE
ID: 00312-00001
Área: 01 - Imunobiológicos
1
DOS SANTOS, V.C, 2QUINAN, B.R., 3Coelho, F.M.,
165
ABSTRACTS
PINHO, T.M.G., 5da Silva, A.M., 6da FONSECA, F.G.
1. UFMG, Universidade Federal de MInas Gerais, Av.
Antônio Carlos, 6627 - Pampulha - Belo Horizonte - MG
CEP 31270-901
4
The use of viral vectors as tools for vaccination has
been highlighted in the last few years. The Modified
Vaccinia Ankara virus (MVA) is an attractive vector
because it is capable of generating humoral and cellular
immunities not only in immunocompetent inidivuals, but
also in the immunosupressed ones. This virus was
generated after around 570 passages in chicken embryo
fibroblasts (CEFs), which resulted in the loss of
approximately 40kb of its genome, including genes
responsible for replication in mammalian cells and for
immune response evasion. Because of that, new viral
particles are not built, yet proteins encoded in the
genome, including eventual recombinants, are expressed
in great quantities in the cytoplasm of the infected cell.
However, such overexpresion can cause the
accumulation of misfolded polupeptides mainly in the
endoplasmic reticulum (ER), and therefore, not capable
of generating an ideal immune response. The objective
of this project is to construct recombinant MVAs
expressing the luciferase gene as tool to evaluate a
possible stress of the ER as a consequence of the
overproduction of misfolded and unfolded proteins. Also
we aim to establish if the stress routes diminish the
synthesis and consequent presentation of the
recombinant proteins by the infected cell. To achieve
success, transfer plasmids encoding the luciferase gene,
with or without a downstream HAAS signal peptide,
flanked by homologous MVA sequences were built and
transfected in CEFs already infected with wild-type MVA.
In such circumstances, homologous recombination
naturally occurred and we generated MVAs virus
expressing the luciferase gene. These recombinant
viruses were selected using the visual effect of GFP
after successively round plaque purifications. Now that
the virus are built they will be used to evaluate molecular
markers of ER stress, a phenomenon characterized by
uncontrolled production of unfolded recombinant proteins.
Instituição de fomento: CAPES, CNPq, FAPEMIG.
Palavras-chaves: Luciferase, MVA, Retículo
Endoplasmático, Vacinas.
IMMUNIZATION OF BALB/C MICE WITH VIRAL
VECTOR MODIFIED VACCINIA ANKARA
EXPRESSING PROTEIN E OF DENGUE VIRUS
SEROTYPE 1
ID: 00312-00002
Área: 01 - Imunobiológicos
1
QUINAN, B.R., 2DOS SANTOS, V.C., 3Coelho, F.M.,
166
PINHO, T.M.G., 5ROCHA, E.S.O., 6OLIVEIRA, J.G., 7DA
FONSECA, F.G.
1. UFMG, Universidade Federal de Minas Gerais, Avenida
Antônio Carlos, 6627, Campus Pampulha, CEP: 31270901 Belo Horizonte MG2. CPqRR, Fundação Oswaldo
Cruz Centro de Pesquisas René Rachou, . Av. Augusto
de Lima, 1715, Barro Preto, CEP: 39100-002. Belo
Horizonte, MG
4
Dengue is considered the most important human
arthropod-borne viral disease nowadays. It is estimated
that up to 100 million cases of dengue fever and about
500 000 cases of dengue hemorrhagic fever/dengue
shock syndrome occur annually, worldwide. Dengue virus
(DENV) has four serotypes, which are transmitted by
mosquitoes of the Aedes genus. Despite the high number
of cases per year and the fact that more than two fifths
of the world‘s population are at risk for dengue, vector
control is the only weapon against dengue fever. Effective
drugs for the disease treatment and vaccines are still
not available. These data demonstrate the eminent
necessity of an efficient vaccine. Among different
techniques used for vaccine development, vectors based
on recombinant viruses have shown to be attractive and
powerful strategies. The Vaccinia Ankara Modified virus
(MVA) is one of the best vectors for vaccine
development. A recombinant MVA expressing the E
protein from Dengue virus serotype 1 was constructed.
For this purpose, shuttle plasmids containing the E cDNA
under control of a vaccinia virus promoter, a GFP marker
gene, and flanking portions of nonessential MVA DNA
were constructed. Chicken embryo fibroblasts were
transfected with the plasmids and infected with MVA. In
such circumstances, homologous recombination
between the plasmid and the MVA genome occur
naturally. After that, the recombinant MVAs were selected
by successive rounds of plaque purification using the
visual aid of GFP expression. Recombinant viruses was
propagated, purified and used to immunize BALB/c mice
to evaluate the efficiency in inducing immune response.
Serum samples and spleen of the immunized animals
were obtained and used in ELISA and ELISPOT in order
to assess the effectiveness in inducing immune
response. Neutralizing antibody titers were found for some
of the immunized animals. Overall, it wasn‘t detected a
humoral and cellular significant response. Financial
support:FIOCRUZ-PDTIS, CAPES, CNPq, FAPEMIG.
Palavras-chaves: Dengue, Imunização, MVA, Vacina.
HUMAN RHINOVIRUS OCCURRENCE AMONG DAY
CARE CHILDREN IN SAO PAULO CITY
ID: 00313-00001
Área: 05 - Virologia Humana e Saúde Pública
ABSTRACTS
Watanabe ASA, 2Peniche L, 3Carraro E, 4Kamikawa J,
Leal E, 6Granato C, 7Bellei
1. UNIFESP, Universidade Federal de São Paulo, Rua
Pedro de Toledo, 781 15° andar - Vila Clementino2. UFPA,
Universidade Federal do Pará, Rua Augusto Corrêa, 01 Guamá - Pará
1
5
Human rhinovirus (HRV) is associated with upper and
lower respiratory tract infections and is recently pointed
as responsible for wheezing exacerbations. The recently
identified rhinovirus C specie has been associated with
bronchiolitis, wheezing, and asthma exacerbations often
resulting in hospitalization. Most of the studies have
examined patients from asthma or hospital-based
populations which may limit the knowledge of HRV C full
spectrum of outpatients clinical outcomes. The aim of
this study was to assess HRV species occurrence among
children´s samples from a hospital employee´s day-care
center collected in 2008. One hundred twenty nasal
aspirates were collected from March to December 2008
of children up to 12 years (median age 4 years) in Sao
Paulo Hospital/ UNIFESP. RT-PCR targets part of the
5’NCR, the entire VP4 gene and the 5’ part of the VP2
gene. Test sensitivity detected 10-3,25 TCID50 of HRV
39. Sequencing was accomplished using the same
primers of PCR and according to the manufacturer’s kits
instructions. Multiple sequences were aligned using
Clustal X software. The multiple-sequence alignment was
subjected to phylogenetic analyses using TOPALi
software. Rhinovirus-RNA was detected in 46.7% of
samples (56/120) and 51.8% from positive cases
presented wheezing. Phylogeny analysis identified 12
(21.4%) HRV A, 6 (10.7%) HRV B and 33 (58.9%) HRV
C. Rhinovirus C was found in 28.7% (33/115) of samples
and distribution according wheezing and no wheezing
cases was 58.6% for HRV C, 27.6% for HRV A, 10.3%
for HRV B and 59.3% HRV C, 14.8% HRV A and 11.1%
HRV B respectively. Median age of positive cases was
3 years (0.16-10) and for HRV C positive samples also
(0.41-9 years). Statistical analysis showed no relation
between wheezing and HRV C infection but a tendency
of infection in age up to five was observed. Infections
caused by HRV C are very frequent but seems not to be
related to severity as wheezing.
Palavras-chaves: Human Rhinovirus, Sequencing,
Wheezing, children.
COMPARISON BETWEEN SPECIES VERSUS FAMILY
SPECIFIC PCRs FOR DETECTION OF RHINOVIRUS
INFECTING RESPIRATORY SAMPLES
ID: 00314-00001
Área: 05 - Virologia Humana e Saúde Pública
1
SILVA, E.R.M., 2 Watanabe, A.S.A., 3 Carraro, E.,
Perosa, A.H.S, 5Granato, C.F.H, 6Bellei, N.C.J.
1. UNIFESP, Universidade Federal de São Paulo, Rua
Pedro de Toledo 781, 15º andar - Vl. Clementino CEP:
04039-0432
4
The human rhinoviruses (HRVs) are the major cause of
common cold. HRVs were recently reclassified, within
Enterovirus genus (HEV), into the Picornaviridae family.
They share many common features, including a
messenger-sense RNA genome and partial nucleotide
sequence identity. The aim of this study was to elucidate
discordant results between two different HRV detection
strategies using a third protocol. The first strategy was a
family (picornavirus) RT-PCR-hybridization with another
hybridization for enterovirus (rhinovirus by exclusion),
the second a RT-PCR-sequencing for rhinovirus and third
was a duplex semi-nested-RT-PCR that differentiates
HRV from HEV. Discordant samples were tested with
the third protocol. Adults with acute respiratory infection
(n = 290) attended in Hospital Sao Paulo – Federal
University of Sao Paulo (2001 – 2003), had samples
tested by the first protocol - RT-PCR-hybridization: 103
were positive to picornavirus with 8 positive for HEV in
hybridization so that 95 were considered positive to HRV.
All 290 samples were then tested by RT-PCR-sequencing:
77 were positive to HRV. After the first two strategies a
concordance of 83% was obtained: 61 positive results
and 180 negative results. The 49 discordant samples
were then evaluated by the third protocol: 5 were
rhinovirus positive, 2 were enterovirus positive and 42
were negative. The 42 negative ones were: 9 concordant
(negative) with RT-PCR-hybridization and 33 concordant
(negative) on RT-PCR-sequencing. These results probably
indicate that picornavirus protocol although higher
sensitivity was less specific than the others (rhinovirus)
protocols. The semi-nested protocol assessed in the
present study seems to be less sensitive and was not
useful to differentiate HRV from HEV. Sequencing assay
using different gene approach would address the best
strategy to confirm rhinovirus and enterovirus infections.
Palavras-chaves: Picornavirus, Rhinovirus, Enterovirus.
DETECTION OF HUMAN METAPNEUMOVIRUS IN
PAEDIATRIC OUTPATIENTS, WITH SUSPECTED
PANDEMIC INFLUENZA INFECTION.
ID: 00315-00001
Área: 05 - Virologia Humana e Saúde Pública
Sinohara, J., 2Carraro, E., 3Watanabe, A.S.A., 4Granato,
C.F.H., 5Bellei, N.C.J.
1. UNIFESP, Universidade Federal de São Paulo, Rua
Pedro de Toledo, 781
1
Acute viral respiratory infections are a leading cause of
167
ABSTRACTS
morbidity and mortality in children Worldwide. In 2001,
human metapneumovirus (hMPV) was first described by
Netherlands researchers and since then seems to play
an important role in respiratory tract infections especially
in children under five years of age. hMPV infection among
community children is not well known in Brazil. The aim
of this study was to determine the occurrence of unique
hMPV infection in 71 respiratory samples collected
between June and September 2009 from pediatric
patients, with suspected pandemic influenza 2009 H1N1
infection at a health care for employees´ children of São
Paulo Hospital. Samples were also screened before for
the following respiratory viruses: rhinovirus, influenza A
besides influenza A H1N1 2009. Patient´s age ranged
from 0.3 to 11 years (median 2.5 years). Viral RNA was
isolated using the QIAamp® Viral RNA kit (QIAGEN)
according manufacturer´s instructions. Detection of the
hMPV was performed by RT-PCR of the fusion (F) gene.
We also analyzed the clinical and epidemiological profile
of infections caused by hMPV. Among the 71 samples
analyzed from 64 patients, only one was positive (1.41%).
This case occurred on August during the peak of
pandemic H1N1 wave. This sample was collected from
a female child (4 years) on the 4th day of symptoms
onset. Child and mother referred previous seasonal
influenza vaccination Symptoms related by parents
included fever, coryza, cough, wheezing, dyspnea.
Despite the low incidence found among pediatric
outpatients these data suggest the need to diagnose
other viral infections that cocirculate with influenza and
may be neglected by physicians as causes of severe
respiratory disease even during a pandemic period.
Palavras-chaves: Human Metapneumovirus, Paediatric
Outpatients, RT-PCR.
OCCURRENCE OF INFLUENZA IN HOSPITALIZED
PATIENTS WITH SUSPECT INFLUENZA A H1N1
PANDEMIC INFECTION AT HOSPITAL SAO PAULO/
UNIFESP IN 2010.
ID: 00316-00001
Área: 05 - Virologia Humana e Saúde Pública
A H1N1 2009 infection. Nasal swab were collected from
94 patients (median age: 2.5 years; ranged: 0-85 years)
from January to August. Tests for influenza A and B were
performed by an in house RT-PCR and for confirmation
of influenza A H1N1 2009 by real time RT-PCR (CDC
protocol). The highest number of admissions 11.7% (11/
94) occurred during the epidemiological week 23 (06-12/
06). Most hospitalizations occurred before the end of
the vaccination program against pandemic influenza. Only
3 samples (3.2%) were positive for influenza A and 2
(2.1%) for influenza B. The male/female ratio was 1.35.
The majority of suspected samples 64.9% (61) are from
children under 12 years (median age: 2.0 years, range:
0-7 years), as the positive ones 60% (3). The positive
cases of influenza A were hospitalized during the
epidemiological weeks 16 (18-24/04), 17 (25/04-01/05)
and 18 (02-08/05), and all of them were confirmed as
infected with influenza A/California/04/2009 (H1N1) virus,
by CDC protocol. The positive cases of influenza B were
hospitalized during the epidemiological weeks 23 (0612/06) and 33 (08-14/08). The 2009 influenza A/H1N1
virus is still circulating in Sao Paulo on the second wave
of pandemic, but in low activity. The great number of
negative samples may suggest others respiratory viruses
and/or bacterial infections cocirculating in the same
period. Vaccination program against influenza A H1N1
2009 may have been responsible for reductions on
admissions and infections in 2010. Financial support:
CAPES
Palavras-chaves: Influenza, RT-PCR, influenza A H1N1
2009.
DETECTION OF HERPES SIMPLEX TYPE 1, 2 AND
VARICELLA ZOSTER VIRUS IN CORNEAL
SCRAPINGS FROM PATIENTS WITH INFECTIOUS
KERATITIS BY REAL-TIME POLYMERASE CHAIN
REACTION
ID: 00317-00001
Área: 05 - Virologia Humana e Saúde Pública
PELEGRINI, A., 2Watanabe, A.S.A., 3Bispo, P.J.M.,
NASCIMENTO, H., 5 VIEIRA, A.C.C., 6 YU, M.C.,
7
PIGNATARI, A.C.C., 8GRANATO, C.F.H., 9Lima, A.L.H.
1. UNIFESP, Laboratório de Virologia, Depto de Medicina,
Universidade Federal de São Paulo, Rua Pedro de Toledo,
781, 15º andar, Vila Clementino, SP2. UNIFESP,
Laboratório Especial de Microbiologia Clínica, Depto de
Medicina, Universidade Federal de São Paulo, Rua
Leandro Dupret, 188, Vila Clementino, SP3. UNIFESP,
Departamento de Oftalmologia, Universidade Federal de
São Paulo, Rua Borges Lagoa, 368, Vila Clementino, São
Paulo
1
4
Melchior, T.B., 2 Guatura, S.B., 3 Camargo, C.N.,
Cabeça, T.K., 5Watanabe, A.S.A., 6Granato, C.F.H.,
7
Bellei, N.C.J.
1. UNIFESP, Universidade Federal de São Paulo, Rua
Pedro de Toledo, 781 15ºandar frente
1
4
Pandemic H1N1 infected thousands of people, mostly
in the Southeast in Brazil, during 2009 first pandemic
wave. On 2010, a National Immunization Program for
different risk groups has been established from March
to June. This study identified the subtypes of influenza
viruses circulating during 2010 among patients at the
Hospital Sao Paulo/UNIFESP with suspected influenza
168
Infectious keratitis can be caused by a sor t of
ABSTRACTS
microorganisms from bacterial, fungal, viral, and parasitic
origin. Among these agents, herpes simplex viruses
types 1 (HSV-1) and 2 (HSV-2), and varicella zoster virus
(VZV) remain as important causes of blindness, making
early diagnosis and prompt initiation of appropriate
therapy essential, in order to reduce disease morbidity.
In this sense, real-time polymerase chain reaction (realtime PCR) is considered an important diagnostic method
for herpetic eye diseases because of its high sensitivity
and relatively rapid processing time. The purpose of this
study was therefore to develop a real-time PCR assay
to detect HSV-1, HSV-2 and VZV in corneal scrapings
from patients who present with clinical suspicion of
infectious keratitis. Patients admitted at Ophthalmology
Department of Sao Paulo Federal University, during the
period between May 2008 and December 2009, were
enrolled in this study, and the cases clinically diagnosed
as infectious keratitis were submitted to the sample
collection. DNA was extracted from samples using a
QIAamp DNA Mini Kit and the real-time PCR assay was
carried out with a TaqManTM universal PCR mix in the
ABIPrism® 7500. Among 63 patients eligible for testing
during the period of study, 32 (50.8%) were males and
the age was 47 years on average (8 – 93 years). In cases
with a typical herpetic ulceration, 13/15 of samples were
positive for HSV-1, corresponding to a positive predictive
value of 86.6%. On the other hand, in the cases of typical
bacterial ulceration, 9/48 (18.8%) of samples were
positive for HSV-1 and 1/48 (2.1%) were positive for VZV.
In these cases, 7/10 (70%) presented association with
severe ocular or sistemic comorbidities. We conclude
that the introduction of the real-time PCR assay in the
routine represents a valuable tool in cases of lesions of
unknown etiology and it can be useful to specialized
laboratories for a better understanding of its occurrence
in the population.
Palavras-chaves: herpes simplex type 1, herpes simplex
type 2, infectious keratitis, varicella zoster virus, realtime PCR.
DETECTION OF VIRUSES IN BOVINE FOLLICULAR FLUIDS
Maria, Avenida Roraima, 1000, Santa Maria/RS
The use of modern techniques in animal reproduction
has been one of the causes of an increasing number of
in vivo and in vitro bovine embryos produced in Brazil.
However, some of these techniques, such as artificial
insemination and in vitro fertilization, may favor
dissemination of viruses that can cause embryonic/fetal
losses or that give birth to infected animals that can
transmit these viruses, thus causing economic losses.
The aim of this study was to detect nucleic acids of
Bovine herpesviruses types 1 and 5 (BoHV-1 and BoHV5) and Bovine viral diarrhea virus (BVDV) in samples of
bovine’s follicular fluids collected from cows in an abattoir
in the state of Rio Grande do Sul, Brazil. Until now 119
samples were obtained and a recently optimized PCR
for BoHV-1 and 5 was applied in all samples. A RT-PCR
for BVDV was also optimized and applied to 50 of these
samples. Virus isolation on cultured MDBK cells was
also performed. The PCR for BoHV-1 and BoHV-5 resulted
in 102 samples positive for both viruses, 16 samples
positive only for BoHV-1 and 1 sample positive only for
BoHV-5. Thirty six samples submitted to the BVDV RTPCR were negative for BVDV RNA, and 14 were positive.
The virus isolation was negative for all samples. These
results allow us to conclude that BoHV-1 and 5 are
widespread in bovine follicular fluids analyzed until now,
while RNA of BVDV is present only in a few samples.
This study will give us an idea of the dissemination of
these viruses in the reproductive tract of cows in this
region of Brazil and motivate to perform further studies
on the impact of these viruses in bovine reproduction.
Palavras-chaves: Follicular fluids, BoHV-1, BoHV-5,
BVDV.
ROLE OF N-LINKED GLYCOSYLATION ON MAYARO
VIRUS INFECTIVITY
ID: 00319-00001
Área: 04 - Virologia Básica
CARVALHO, C.A.M., 2 PIAZZA, T., 3 SILVA, J.L.,
GOMES, A.M.O.
1. IBqM/UFRJ, Instituto de Bioquímica Médica/
Universidade Federal do Rio de Janeiro, Av. Carlos
Chagas Filho, 373 - Cid. Universitária, Rio de Janeiro,
RJ, 21941-902
1
4
ID: 00318-00001
Área: 03 - Virologia Animal
HENTGES, L.P., 2CAMPOS, F.S., 3OLIVEIRA, G.C.,
TORRES, F.D., 5 GASPERIN, B.G., 6 GONÇALVES,
P.B.D., 7FRANCO, A.C., 8RIJSEWIJK, F.A.M., 9ROEHE,
P.M.
1. UFRGS, Universidade Federal do Rio Grande do Sul,
Rua Sarmento Leite, 500, Porto Alegre/RS2. IPVDF/
FEPAGRO, Instituto de Pesquisas Veterinárias Desidério
Finamor, Estada Municipal do Conde , 6000, Eldorado
do Sul/RS3. UFSM, Universidade Federal de Santa
1
4
Mayaro virus (MAYV) is an alphavirus widespread in
South America in an endemic manner and represents
an interesting case to consider with regards to potential
for urban emergence. The viral envelope proteins mediate
cell recognition and entry and present both an Nglycosylation motif along their primary structures. These
motifs are conserved amongst other alphaviruses, which
169
ABSTRACTS
suggests that they play an important role for the virus
particle. The aim of this work was to address the role of
glycans N-linked to MAYV envelope proteins on its
infectivity, via specific cleavage by N-glycosidase F. Our
results show that digestion with N-glycosidase F
promotes a shift in the electrophoretic mobility of MAYV
envelope proteins E1 and E2, but not in the capsid protein
C. Cleavage of N-linked glycans also interfered with
MAYV infectivity. Further experiments are under course
in order to evaluate possible effects of N-deglycosylation
on MAYV structure. Our preliminary results points to
virus glycosylation as an important issue in virus biology.
Financial support: CAPES, CNPq, FAPERJ, FINEP,
INBEB and PRONEX.
Palavras-chaves: Alphavirus, Mayaro, N-glycosidase F,
N-glycosylation.
The importance of antigenemia for diagnosis of
cytomegalovirus infection: experience of a Virology
Laboratory (2000-2009).
matrix phosphoprotein pp65 of the virus, has played an
important role in the diagnosis of CMV infections, due
to its high sensitivity and positive predictive value, and
the rapidity in obtaining results. Given the profile of the
population, composed predominantly of patients with
some degree of immunosuppression, our results reinforce
the need for early diagnosis of infection, crucial for the
introduction of effective antiviral treatment and decrease
morbidity and mortality that the infection may result.
Palavras-chaves: Citomegalovirus, antigenemia,
diagnostico.
Evaluation of CMV viremia levels in bone marrow
transplanted patients: impact on clinical action and
of clinical resistance levels
ID: 00320-00003
Área: 05 - Virologia Humana e Saúde Pública
Puerari D, 2Fernandes ML, 3Parmezan SN, 4Bellei N,
Granato CFH
1. UNIFESP, Universidade Federal de São Paulo, Rua
Pedro de Toledo, 781 Vila Clementino2. FLEURY, Fleury
Medicina e Saude, Avenida General Valdomiro de Lima,
508 Jabaquara
1
5
ID: 00320-00002
Área: 05 - Virologia Humana e Saúde Pública
Parmezan SN, 2Puerari D, 3Fernandes ML, 3Bellei N,
Granato CFH
1. UNIFESP, Universidade Federal de São Paulo,
Rua‘Pedro de Toledo, 781 Vila Clementino- SP2. FLEURY,
Fleury Medicina e Saude, Avenida General Valdomiro
de lima, 508 Jabaquara
1
5
Despite advances in surveillance strategies and antiviral
drugs, infection with cytomegalovirus (CMV) continues
to pose problems for immunocompromised patients. The
antigenemia for CMV is an important marker of disease
evolution and treatment efficacy in patients. To analyze
the impact of antigenemia as a tool for diagnosis and
monitoring of active infection by cytomegalovirus (CMV),
performed in a routine Virology Laboratory over ten years.
The study included patients from a public health service,
who had clinical suspicion of CMV infection and have
been periodically investigated by the antigenemia test,
following recommendations from the literature. Laboratory
and epidemiological data were obtained from archives
of records of the laboratory in question. From January
2000 to December/2009, 21 032 tests were performed
(median year 2094), of which 8847 (42%) are from female
patients and 12 185 (57.8%) males. The positivity rate
was 16.7% (3512 tests) and in relation to the total number
of cells positivas/3x105 leukocytes, 2347 (66.8%)
showed between 10-10 cells (median 2), 964 (27, 4%)
between 11-100 cells (median 25.5) and 201 (5.7%) above
100 cells (median 197). Described in 1988, the technique
of antigenemia, which is the color of polymorphonuclear using monoclonal antibody directed against the
170
Cytomegalovirus (CMV) is among the major infectious
agents affecting bone marrow transplant patients (BMT).
The laboratory diagnosis is usually made by the research
of CMV antigen in nuclei of neutrophils, although the
diagnostic levels are not yet clear. Moreover, the
increasing use of antiviral drugs has generated increasing
level of resistance against CMV to ganciclovir. The aim
was to analyze the antigenemia assay results, verify
the medical action and the impact of the drug following
the viremic levels. We also sought to estimate levels of
resistance of CMV to ganciclovir by observing the impact
of drug use in viremic levels. We included patients seen
at Hospital São Paulo, who underwent bone marrow
transplantation and who had more than six results by
testing for CMV antigenemia assay. Clinical and
laboratory data were obtained by reading the chart and
the Clinical Virology Laboratory, which provides diagnostic
services to the Hospital. Viremia was analyzed by
immunoperoxidase reaction by analyzing 200,000 cells.
We analyzed 22 episodes of CMV infection among 16
patients. Treatment was initiated in 36.4% of episodes
where the outcome was greater than 10 cells and 36.4%
started treatment with results less than 5 cells, 9.1%,
13.6% and 9.1% of the results of 3, 4:05 cells,
respectively. Only in one episode, the treatment was
started with two cells. Regarding the effectiveness of
treatment 68.2% had good response to treatment, 9.1%
were not treated and 18.2% did not result in treatment,
and three of those died. The clinical management varied
ABSTRACTS
compared to similar results of viremia. Most situations
in which treatment was initiated, there was a fall in
antigenemia assay suggesting sensitivity to the drug.
However, considering the relatively small number of
episodes evaluated, the frequency of results suggests
that resistance indicates that other studies should be
conducted.
Palavras-chaves: Citomegalovirus, Antigenemia,
Tratamento, Resistência, Transplante de medula ossea.
group, the frequency of HBsAg was higher than in several
studies in Brazil. Anti-HCV is compatible with the national
average. Due to the serious consequences of these
chronic cases of hepatitis, the need for most effective
public health measures is suggested in combating these
illnesses in this border region. Financial
support:FAPEAM,FUAM.
Palavras-chaves: HEPATITIS B, HEPATITIS C, TRIPLE
BORDER, HBsAg, Anti-HCV.
FREQUENCE OF SEROLOGICAL MARKERS AND
RISK FACTORS FOR HEPATITIS B AND C INFECTION
IN THE TRIPLE BORDER OF THE UPPER SOLIMÕES
RIVER REGION, AMAZONAS, BRAZIL
HUMAN IMMUNODEFICIENCY VIRUS 1 AND 2
INFECTIONS IN THE TRIPLE BORDER OF THE
UPPER SOLIMÕES RIVER REGION, AMAZONAS,
BRAZIL
ID: 00321-00001
Área: 05 - Virologia Humana e Saúde Pública
ID: 00321-00002
Área: 05 - Virologia Humana e Saúde Pública
1
LETURIONDO, A.L., 2DUTRA, D.L.R., 3BENZAKEN,
A.S.
1. FUAM, Laboratório de Biologia Molecular, Fundação
Alfredo da Matta, Manaus, Brasil, Av. Codajás 24, CEP
69065-130, Cachoeirinha, Manaus-AM
LETURIONDO, A.L., 2DUTRA, D.L.R., 3BENZAKEN,
A.S.
1. FUAM, FUNDAÇÃO ALFREDO DA MATTA, AV.
CODAJAS, 24 (CEP 69065-130), CACHOEIRINHA,
MANAUS, BRASIL
Viral hepatitis is a serious public health problem affecting
billions of people globally, both hepatitis B virus (HBV)
and hepatitis C virus (HCV) are the most common causes
of liver disease worldwide, with an estimative of 78% of
Hepatocellular carcinoma cases. The objective of this
study was to collect information on behavioral,
epidemiological and laboratory data in targeted
populations. The study was based on the SASH
(Situational Analysis for Sexual Health) methodology,
mapping hot spots (bars, clubs and meeting places) to
offer invitations to hot spot goers to attend in the Health
Units. Three cities of the Amazon border with Colombia
and Peru, par ticipated in the study: Benjamin
Constant(BC), Atalaia do Norte(AN) and Tabatinga(TB).
593 blood samples were collected to ELISA for HBsAg,
anti-HCV, using kits from DiaSorin and Hepanostika HCV
Ultra. The study included 285 men and 308 women
between 14 and 64 years. The overall frequency found
for HBsAg was 6%(35/585) and anti-HCV was 0.7%(4/
585). The distribution found in the three cities for HBsAg
was 6.2%(18/290) in TB, 5.5%(11/199) in BC and 6.3%(6/
96) in AN. For anti-HCV the frequency was 0.7%(2/90)
in TB, 0.5%(1/199) in BC and 1.0%(1/96) in AN. There
was no significant association of HBsAg marker with
the level of education, age, sex, marital status, family
income, labor market, condom use, alcohol use and drug
use in the three cities involved. The four anti-HCV positive
patients (two men and two women) were aged 25-45
years, had incomplete primary education, wage income
of up to 1 MW, three reported no condoms use and one
used rarely. Although the study did not address a risk
Acquired Immune Deficiency Syndrome (AIDS) is a
pandemic responsible for the deaths of over 200 000
people in Brazil, since its inception. Every year, more
than 30 000 new cases are reported in the country. The
North region of Brazil concentrates 3.9% of all AIDS
cases notificated in the country, accumulated from 1980
to 2009. The objective of this research was to develop a
situational diagnosis of HIV 1 and 2 infection in the triple
border of the upper Solimões river region (Brazil, Colombia
and Peru), in the Brazilians cities of Tabatinga (TB),
Benjamin Constant (BC) and Atalaia do Norte (AN).
Sampling was conducted using the Situational Analysis
for Sexual Health – SASH methodo of mapping primarily
the hot spots of the “leisure circuits”, include bars and
clubs where residents (and visitors) go to “have fun “,
from the three cities. Afterwards, invitations were
distributed to the hot spots goers to attend in the Health
Units. Blood samples were collected between January
to July 2009, from 584 patients of both sexes, distributed
as follows: 289 patients from TB, 199 from BC and 96
from AN. The detection of antibodies to HIV 1 and 2 in
serum/plasma was performed by enzyme immunoassay
(ELISA), using the kit Genscreen HIV-1 / 2 Version 2
(Bio-Rad). Only four patients (0.7%), all male and age
between 25 and 39 years had HIV infection, three from
TB and one from AN. All positive patients were unmarried,
non-drug users and family income of minimum wage.
Two volunteers reported consistent condoms use in the
last year and two others stated they used rarely. Although
the study group is supposedly asymptomatic for STI
and HIV/AIDS, the frequency found of HIV infection was
1
171
ABSTRACTS
considered high compared to others studies with
vulnerable populations in Brazil. It is necessary to
improve public health policies to encourage condom use,
health screenings and sexual education. Financial
support: FAPEAM, FUAM.
Palavras-chaves: HIV1/2, TRIPLE BORDER, RISK
FACTORS, UPPER SOLIMÕES, FREQUENCE.
HIGH RISK HUMAN PAPILLOMAVIRUS INFECTIONS
IN THE TRIPLE BORDER OF THE UPPER SOLIMÕES
RIVER REGION, AMAZONAS, BRAZIL
ID: 00322-00001
Área: 05 - Virologia Humana e Saúde Pública
DUTRA, D.L.R., 2LETURIONDO, A.L., 3BENZAKEN,
A.S.
1. FUAM, LABORATÓRIO DE BIOLOGIA MOLECULAR,
FUNDAÇÃO ALFREDO DA MATTA, AV. CODAJÁS, 24
(cep 69065-130), CACHOEIRINHA, MANAUS, BRASIL2.
FUAM, GERÊNCIA DE DST, FUNDAÇÃO ALFREDO DA
MATTA, AV. CODAJÁS, 24 (cep 69065-130),
CACHOEIRINHA, MANAUS, BRASIL
1
The high risk human papillomavirus (HPV) infection is
the most important risk factor to cervical cancer and, in
Brazil, the North region is the most incident (23/100000).
The estimated number of new cases in Brazil is 18430
for the year 2010, and in the State of Amazonas are 560
according to the Instituto Nacional do Câncer - INCA.
The aim of this research was to develop a situational
diagnosis of the high risk HPV infection in the triple border
of the upper Solimões River Region(Colombia, Peru And
Brazil), in the cities of Tabatinga(TB), Benjamin
Constant(BC) and Atalaia do Norte(AN), Amazonas.
Sampling was conducted using the Situational Analysis
for Sexual Health – SASH method of mapping primarily
the hot spots of the “drinking and leisure” circuit from the
three cities to, afterwards, distribute invitations for hot
spots goers to attend at the Health Basic Units. Exfoliated
cells from the urethra and the ecto/endocervix were
obtained in a period between January and July of 2009,
from 597 male and female patients, with the use of UCM
kit (QIAGEN). The detection of the DNA of the agent
was performed by Hybrid Capture test (QIAGEN), using
a RNA probe cocktail complementary to DNA of 13 high
risk HPV types. An overall frequency of 19.4% was found
for the three sampled cities, and the frequency of positive
cases in TB (23.3% or 70/300) was significantly higher
than BC (16.6% or 33/199, p=0.07) and AN (13.3% or
13/98, p=0.03). Considering the gender, the HPV
frequency was approximately twice higher in females
(OR=1.95; p=0.0026). Adolescents presented a higher
infection risk, 30.2% compared to 18.4% among those
over 20 years old (p=0.03). Therefore, due to high rates
172
of high risk HPV infection, mainly observed in the city of
Tabatinga, it is necessary to improve public health
policies to encourage condom use and health screenings
(pap smear), especially among women and adolescents,
and thus reverse the incidence of cervical cancer in the
region.
Financial support:FAPEAM,FUAM.
Palavras-chaves: HPV, HIGH RISK, TRIPLE BORDER,
UPPER SOLIMÕES, AMAZONAS.
BOVINE ROTAVIRUS ASSOCIATED WITH DIARRHEA
IN THE TRIÂNGULO MINEIRO REGION, MINAS
GERAIS, BRAZIL
ID: 00324-00001
Área: 03 - Virologia Animal
FIGUEIREDO, E.F, 2MOURA, L.M.S, 3VILELA, E.F.,
ALVES, M.S., 5 PEREIRA, W.A.B., 6BITTAR, J.F.F.,
7
SARMENTO, R.R., 8DULGHEROFF, A.C.B., 9GOUVÊA,
V.S., 10DOMINGUES, A. L. S.
1. UFTM, Universidade Federal do Triângulo Mineiro, Av.
Frei Paulino, 30 - Bairro Abadia - Uberaba-MG2. UFRJ,
Universidade Federal do Rio de Janeiro, Av. Brigadeiro
Trompowsky, SN - C.C.S/IMPPG, Ilha do Fundão, RJ,
RJ.3. UNIUBE, Universidade de Uberaba, Av. Nenê
Sabino, 1801 Bairro Universitário, Uberaba-MG4. UFPB,
Universidade Federal da Paraíba, Cidade Universitaria
CCS - Escola Técnica de Saúde - João Pessoa-PB
1
4
Acute diarrhea is one of the most important diseases
affecting calves under one month old and has been
associated with large economic losses. Group A
rotaviruses are considered the main causative agents
of diarrhea in young calves in the world. There are no
published data on the prevalence and molecular
characterization of bovine rotavirus in the Triângulo
Mineiro region, so the aim of this study was detect and
characterize rotavirus found in faeces of calves with
diarrhea, from November 2008 to September 2009. We
collected 74 faecal samples of three cattle breeds from
five different farms. Virus detection was made by latex
agglutination (Rotavírus Tira Látex Test - BIOEASY®)
and positive samples were characterized according to
their electropherotypes after nucleic acid extraction from
stools (silica method) and genotyped by RT-PCR. The
distribution of diarrhea was uniform, without showing a
seasonal pattern. Five samples (7%) were positive for
rotavirus, detected in calves of two breeds from only
one propriety. All rotavirus positive samples showed long
electropherotypes and were genotyped as G6P[5]. Our
study emphasize the importance of rotavirus as
etiological agent of diarrhea in calves and collaborate to
understanding the molecular epidemiology of rotavirus,
essencial for the establishment of measures of prevention
ABSTRACTS
and control.
Financial support: FAPEMIG; CAPES; UFTM.
Palavras-chaves: Bovine Rotavírus, Genotyping,
Epidemiology.
RISK FACTORS AND GENOTYPING OF HEPATITIS
B VIRUS (HBV) IN PATIENTS ATTENDING STI CLINIC
AT THE FUNDAÇÃO ALFREDO DA MATTA, MANAUS,
AMAZONIA, BRAZIL
ID: 00326-00001
Área: 05 - Virologia Humana e Saúde Pública
Barbosa, T.C., 2 Dutra, D.L.R., 3 Sampaio, M.R.,
Leturiondo, A.L.
1. FUAM, Fundação Alfredo da Matta, Av. Codajás, 24 Cachoeirinha (CEP 69065-130), Manaus, Amazonas,
Brazil
1
4
Chronic Hepatitis B remains a serious public health
problem worldwide, especially in developing countries,
being the major cause of cirrhosis and hepatocellular
carcinoma. The aim of this study was to investigate the
association of risk factors with the marker total anti-HBc
using the chi-square, Fisher’s exact test and Student t
test. The detection of viral DNA was by PCR using
primers specific for S region of HBV. The sequences
generated were analyzed with bioinformatics tools, and
compared with sequences deposited in GenBank. The
sample consisted of 593 volunteer patients treated at
an STD clinic at Fundação Alfredo da Matta, ManausAM, from October 2008 to May 2009. Male participation
(62.2%) was higher than females (37.8%). Ages ranged
from 14 years to 59 years with a mean age of 29.8 years.
The variables sex, education, labor market, number of
casual partners, sexual orientation, presence of sexually
transmitted infections (STIs) and condom use showed
no significant association with the serological marker
total anti-HBc. However, the age group (40-49 years) and
marital status (stable) were significantly associated with
this marker.
Palavras-chaves: Hepatitis B, risk factors, Genotyping,
total Anti-HBc, Brazil.
RESPIRATORY VIRAL COINFECTION AMONG
HOSPITALIZED PATIENTS WITH H1N1 2009 DURING
FIRST PANDEMIC WAVE IN BRAZIL
ID: 00329-00001
Área: 05 - Virologia Humana e Saúde Pública
Camargo, C.N.,, 2Guatura, S.B.,, 3Puerari, D., 4Cabeça,
T.K., 5 Shinohara, J.S., 6 Moreira, L.P., 7 Carraro, E.,
8
Granato, C.F., 9Bellei, N.C.J.
1. UNIFESP, UNIVERSIDADE FEDERAL DE SÃO
PAULO, Rua: Pedro de Toledo , 781 15º andar
1
The 2009 H1N1 morbidity and mortality were particularly
high in Brazil during the first pandemic wave. The
frequency of viral coinfections with Infuenza A is not
clear. The purpose of this study was determine coinfection
among confirmed cases of Influenza A and other
respiratory viruses among hospitalized patients samples
collected during the first pandemic wave in a Brazilian
Sentinel Hospital. From August 19 to November 31, 2009
a total of 159 nasal/throat swab samples were collected
from patients at Sao Paulo Hospital, Sao Paulo City,
Brazil. Patient inclusion criteria according to National
Program Protocol were fever plus cough plus dyspnea
and hospitalization due to clinical suspicion of 2009 H1N1
infection. Influenza A seasonal (IAV) and H1N1 2009
detection were performed by Real Time protocol
published by CDC. Seasonal influenza virus B (IBV),
Human Rhinovirus (HRV), Human Metapneumovirus
(hMPV), Adenovirus (AdV), Human Respiratory Syncytial
Virus (HRSV) and Coronavirus (HCoV) detection was
performed as described previously. Influenza A infection
was detected in 41/159 patients (25.8%). Thirty one
specimens (19.5%) were positive for H1N1 2009 and 10
(6.3 %) were positive for seasonal IAV. Viral coinfections
with Influenza A were detected in 9/41 (21.9%) patients:
4 seasonal IAV plus AdV, 2 seasonal IVA plus HRV, 2
H1N1 plus HRSV, 2 2009 H1N1 plus hMPV and 1 triple
infection seasonal IAV plus HRV and AdV
simultaneously. Adenovirus was the most common
coinfection with Influenza A. No coinfection with IBV was
detected. 2009 H1N1 had a 4/31 rate of coinfection (13%)
and seasonal IAV had 5/10 (50%). Viral coinfections were
more common among children than adults, six of them
in children and three in adult. These data confirm that
other respiratory viruses cocirculate with influenza and
suggest further analysis of the coinfection impact on
outcome of these patients. Financial support: CAPES.
Palavras-chaves: INFLUENZA H1N1 2009,
RESPIRATORY VIRUSES, PCR.
NATURAL OCCURRENCE OF QUASISPECIES WITH
MUTATIONS OF RESISTANCE TO PROTEASE
INHIBITORS IN PATIENTS CHRONICALLY INFECTED
WITH HCV GENOTYPE 3a
ID: 00330-00001
Área: 05 - Virologia Humana e Saúde Pública
Vasconcelos-Medeiros de Sousa P.A, 2Provazzi, P.J.S,
Alves R.S, 4Queiroz, A.T.L, 5Pereira, C.A, 6Rahal, P,
7
de Carvalho-Mello, I.M.V.G.
1. IB, Instituto Butantan, Av. Vital Brasil 15002. USP,
Instituto de Biociências, USP, Rua do Matão Trav.14
nº3213. UNESP, UNESP, São José do Rio Preto, R.
Cristóvão Colombo 2265
1
3
173
ABSTRACTS
Hepatitis C virus (HCV) infection constitutes a major
health problem throughout the world and is related to
severe liver cirrhosis, end-stage liver disease and
hepatocellular carcinoma. One of the most promising
targets for antiviral therapy is nonstructural protein 3
(NS3). The N-terminal region of NS3 is a serine protease
responsible for the processing of the HCV polyprotein.
The NS3 protease is essential for viral replication.
Development of resistance to antiviral medications is
an important factor that may limit the effectiveness of
therapy for HCV infections. RNA viruses, such as HCV,
have high mutation rates and typically exist as a complex
population of genetically distinct but closely related viral
variants, commonly referred to as quasispecies. Upon
treatment, the pool of viral variants (1012 virions per
day) may allow rapid selection and outgrowth of viruses
with reduced susceptibility to an antiviral drug. The aim
of this study was to analyze the natural genetic variability
of the HCV NS3 serine protease domain in order to
identify variants carrying mutations associated with a
decreased susceptibility to protease inhibitors in patients
chronically infected with HCV. The NS3 protease domain
of the serum samples from the 17 patients infected with
hepatitis C virus genotype 3a was amplified by a nested
PCR and cloned. Around five clones from each patient
were sequenced generating 80 contigs. A A156V-S
mutation was observed in 11,76%, A39V in 5,9% and
V36L in 100%. We also observed A39G in 17%, this
mutation is not reported as a resistance mutation but
we can not ignore because it’s an important site of
mutation. These mutations were found at least in one
clone of each patient with mutation. In conclusion, the
presence of this variants associated with resistance to
protease inhibitors may interfere with the sustained
response to treatment. Individual analysis of treatment
with future inhibitors can be important to treatment
response.
Financial support: FAPESP
Palavras-chaves: HCV, Quasispecies, Mutações, NS3Protease, Inibidores de Protease.
STANDARDIZATION OF A TECHNIQUE FOR QUANTIFICATION OF NEUTRALIZING ANTIBODIES AGAINST
POLIOVIRUS USING VACCINE VIRUSES
Even after many decades of efforts to eradicate
poliomyelitis this disease is still endemic in some Asian
and African countries, representing a threat to all other
countries due to international travels. Recently new
measures to control viral stocks of poliovirus have been
proposed to the health community. Among these the use
of wild strains of polio in testing can compromise WHOs
aim of absolute poliomyelitis virus eradication. The
present study proposes to change the present
methodology for one adapted test using vaccine viruses.
For this, we used the oral vaccine Sabin and HEp-2
neutralization methodology. We tested 64 samples from
patients sera previously titrated antibodies at Fleury
Laboratory that currently uses strains of wild poliovirus,
considered the gold standard. Results of neutralization
with vaccine strains were compared and the parameters
were calculated as sensitivity, specificity, prevalence,
accuracy, positive predictive value and negative
predictive value for the three viral serotypes. Results
showed a very close approximation between the results
with the vaccine strain and the gold standard. The average
relative sensitivity was the 97,7%, specificity 83,3%,
prevalence 88,5%, accuracy 95,6%, positive predictive
value 96,5% and negative predictive value 87,6%. This
evaluation demonstrates a good performance of the
alternative diagnostic procedure, confirmed the immune
status of the patients, and provides a great alternative
to replace the wild type strain. The aim is to contribute
for eradication of poliomyelitis, minimizing risks in the
laboratory procedures and continue to perform the
serological diagnosis of polio, which nowadays has been
very important for analyzing the status of antibody
production especially in immunocompromised and
transplanted patients.
Palavras-chaves: wild poliovirus, vaccine poliovirus,
neutralization, standartization, gold standard.
SEROLOGICAL MARKERS ASSESSMENT AND
GENOTYPE CHARACTERIZATION OF HEPATITIS C
VIRUS IN PATIENTS UNDER HEMODIALYSIS
ID: 00333-00001
Área: 05 - Virologia Humana e Saúde Pública
Lemos M F, 2Compri A P, 3Spina A M M, 4Oba I T,
Moreira R C
1. IAL, Instituto Adolfo Lutz - Centro de Virologia Laboratório de Hepatites, Av Dr Arnaldo, 355 - Cerqueira
César - São Paulo - SP2. ISCMSP, Irmandade da Santa
Casa de Misericórdia de São Paulo, Rua Cesário Mota
Júnior, 112 - Vila Buarque - São Paulo - SP
1
5
ID: 00331-00001
Área: 05 - Virologia Humana e Saúde Pública
Lima, ES, 2Guatura, S, 3Watanabe, A.S.A, 4Granato,
C.F.H
1. Unifesp, Universidade Federal de São Paulo, R. Pedro
de Toledo, 781 15º andar
1
174
INTRODUCTION: Prevalence and risk of hepatitis C
transmission are higher in patients undergoing
hemodialysis than in the general population, possibly
because these patients are more exposed to risk
ABSTRACTS
situations. OBJECTIVES: To assess serological and
molecular markers and to characterize Hepatitis C virus
genotypes. CASUISTIC AND METHODS: Three blood
samples were collected with a 3-month interval from 120
volunteers of two hemodialysis centers from São Paulo
between May/2003 and July/2005. Anti-HCV was
investigated using ELISA (ETI-AB-HCVK-4, DiaSorin)
in the first samples and, in the negative ones, also in
the third blood sample. Viral RNA research was conducted
using RT-PCR in all 3 blood samples with the
AMPLICOR™ HCV Test, v2.0 ROCHE kit. Genotyping
was performed by means of sequencing methodology.
RESULTS: Anti-HCV prevalence reported in this study
was 23.3% (28/120) and seroconversion was not
observed in patients with negative serology. Out of all
the analyzed samples, HCV-RNA was detected in 27
samples [27/120 (22.5%)]; however in one of them antiHCV could not be detected. Out of the samples with
negative results for HCV-RNA, two of them had positive
serology and one of them presented a persistent
undetermined serology. HCV genotype was identified in
27 samples with detectable HCV-RNA. Genotype 1a was
the most prevalent [51.9% (14/27)], followed by genotype
1b [44.4% (12/27)] and genotype 3a [3.7% (1/27)].
CONCLUSIONS: Combination of serologic and molecular
diagnosis for HCV screening in patients under
hemodialysis may be more effective to decrease the
prevalence of this infection, which remains high. The
highest prevalence of genotype 1 in this population
corroborates the prevalence data among HCV carriers
who do not suffer from chronic renal failure. FINANCIAL
SUPPORT: FAPESP - 06/59972-8.
Palavras-chaves: GENOTYPE, HEMODIALYSIS,
HEPATITIS C.
Pevalence of HBV and HCV infections in children
and adolescents in Santos county, São Paulo state
– Brazil
ID: 00333-00002
Área: 05 - Virologia Humana e Saúde Pública
Moreira, R C, 2Ciaccia, M C C, 3Saraceni, C P, 4Spina,
A M M, 5Oba, I T, 6Lemos, M F, 7Ribeiro, J, 8Lovatti, F,
9
Santos, A P de T, 10Porta, G
1. IAL, Laboratório de Hepatites do Centro de Virologia
do Instituto Adolfo Lutz, Av Dr Arnaldo, 355 - Cerqueira
César - São Paulo - SP2. ICR – HC/ FMUSP, Instituto
da Criança – HC/ FMUSP, Av. Dr. Enéas Carvalho de
Aguiar, 647 - São Paulo - SP
1
Background: Viral hepatitis is a major public health
concern worldwide, including Brazil where population
studies are scarce. The objective of this study was to
know the prevalence of hepatitis B and C in children and
adolescents in Santos county, São Paulo state, Brazil.
Methods: We evaluated 4,677 children and adolescents
from nursery schools, elementary schools and high
schools from Santos county. Serum samples were
collected in filter paper, eluted in PBS and screened for
HBsAg, total anti-HBc and anti-HCV, employing
commercial kits (DiaSorin™, Saluggia,Italy). Results: The
prevalence of HBV and HCV infections was respectively:
anti-HBc 0.13% and anti-HCV 0.02%. One child was
identified as a chronic carrier presenting HBsAg reagent.
Conclusions: The prevalence of anti-HBc was low, which
means that the national vaccination program is effective.
Now the challenge is to improve the HCV control
measures even though there is no vaccine to date. Blood
bank and drug users’ control is the mechanism to keep
this infection in the communities under control. Financial
Support: FAPESP - 06/59972-8.
Palavras-chaves: Hepatitis B, Hepatitis C, Prevalence,
Santos, Brazil.
COMPARISON OF POLYMERASE CHAIN REACTION
AND DIRECT FLUORESCENT ASSAY TESTING FOR
RESPIRATORY SYNCYTIAL VIRUS IN BONE
MARROW TRANSPLANTED PATIENTS
ID: 00334-00001
Área: 05 - Virologia Humana e Saúde Pública
Moreira, L.P., 2 Watanabe, A.S.A., 3 Silva, E.R.M.,
Guatura, S.B., 5Carraro, E., 6Granato, C.F.H., 7Bellei,
N.C.J.
1. UNIFESP, Universidade Federal de São Paulo, Rua
Pedro de Toledo, 781, Vila Clementino - SP
1
4
Respiratory Syncytial Virus (RSV) is an important cause
of morbidity and mortality in children under 2 years old
and also immunosuppressed adults. In the
immunosupressed the progression from upper to lower
respiratory tract infection occurs rapidly and in patients
with this evolution the mortality rate has been reported
to be higher than 50%. RSV is highly contagious and the
utilization of isolation precautions for suspected cases
is required to reduce the spread of disease and the
occurrence of nosocomial infections. Rapid and sensitive
detection tests are essential for adequate clinical
management and infection control measures.
Nasopharyngeal aspirates were collected from bone
marrow transplanted patients presenting acute respiratory
illness at the Sao Paulo Hospital and tested by direct
immunofluorescence assay (IF), using specific
monoclonal antibodies, and a Reverse Transcription
followed by Polymerase Chain Reaction (RT-PCR) to
amplify the conserved region of the F protein gene. A
total of 110 samples collected between March and
December 2008 were analyzed during the study. Patients’
175
ABSTRACTS
median age was 48 years (range 5- 78) and 3 days median
time from symptoms onset. Overall 19 (17.3%) samples
were positive for RSV by at least one technique. IF was
positive in 12 (10.9%) and RT-PCR in 14 (12.7%) with a
concordance of 87.3% between both tests. Among the
19 positive samples, 7 (36.8%) were positive by the two
techniques used, 5 (26.3%) only in IF and 7 (36.8%)
were positive just by RT-PCR. We conclude that RT-PCR
was just a little more sensitive than IF to detect RSV in
bone marrow transplanted patients. The exclusive IF
positive samples can be explained by a possible RNA
degradation of the stocked samples or due the sample
concentration during IF process. Comparative studies
of techniques are very important for physicians’ choice
of best method for a rapid therapeutic intervention and
control nosocomial infections.
Financial Support: CNPq
Palavras-chaves: Immunofluorescence assay, RT-PCR,
Respiratory Syncytial Virus.
RSV-SPECIFIC TOTAL IgG, IgG1 AND IgG3
RESPONSE IN YOUNG CHILDREN: ANTIBODY
OCURRENCE, LEVELS AND AVIDITY
ID: 00335-00001
Área: 05 - Virologia Humana e Saúde Pública
FREITAS, G.R.O., 2SILVA, D.A.O., 3YOKOSAWA, J.,
CARNEIRO, B.M., 5 PAULA, N.T., 6 COSTA, L.F.,
7
OLIVEIRA, T.F.M., 8MINEO, J.R., 9QUEIRÓZ, D.A.O.
1. UFU, Universidade Federal de Uberlândia, Av. Pará,
1720, Bloco 4C, Campus Umuarama, 38400-902,
Uberlândia, MG, Brazil
1
4
Respiratory syncytial virus (RSV) is well recognized as
a major pathogen accounting for acute respiratory
disease (ARD) in infants and young children, causing
mainly bronchiolitis and pneumonia. Although RSV has
been intensively studied since the 60s, several aspects
of the immune response to infection still remain unclear
to date. In an attempt to better characterize the humoral
immune response during infection, this study aimed to
investigate the occurrence, levels and avidity of total
IgG, IgG1 and IgG3 antibodies to RSV by indirect and
avidity ELISA in serum samples from children d”5 years
old with RSV-ARD (cases) and without ARD (controls).
Also, a possible association between antibody avidity
and illness severity was examined. The occurrence and
levels of RSV-specific IgG were age dependent, with
infants.
Palavras-chaves: acute respiratory disease, avidity
assay, IgG antibody subclasses, respiratory syncytial
virus.
DETECTION AND GENOTYPIC ANALYSIS OF BK
176
POLYOMAVIRUS IN KIDNEY TRANSPLANT
RECIPIENTS
ID: 00336-00001
Área: 05 - Virologia Humana e Saúde Pública
Zalona, A.C.J., 2Zalis, M.G., 3Varella, R.B.
1. UFF, Universidade Federal Fluminense, Rua Prof.
Ernani Pires de Melo, 101, MIP, Laboratório de Virologia2.
UFRJ, Universidade Federal do Rio de Janeiro, HUCFF,
UFRJ, Laboratório de Infectologia e Parasitologia
Molecular
1
Introduction: BK polyomavirus (BKV) is highly prevalent
in the world population and is the major causative agent
of polyomavirus associated nephropathy (PVAN) in
kidney transplant recipients. Our goal was to determine
the prevalence of BKV infection and characterize the
subtypes of BKV in urine samples of renal transplant
recipients, correlating the presence of the virus with
clinical and laboratory parameters. Methods: The
presence of BKV infection was investigated by urine
cytology in the search of BKV-induced decoy cells and
tested by a qualitative semi-nested polymerase chain
reaction (PCR) in urine samples. The viral subtype was
determined by sequencing. Results: Of the 105 patients
recruited for this study, 51 (48.6%) were positive for BKV
by the technique of semi-nested PCR and 33 (31.4%)
by cytology in urine sample. There were no significant
associations between variables such as gender, donor
type, creatinine level, time of transplantation,
immunosuppressive regimen and positivity for BKV. We
characterized BKV subtypes Ib1, Ia and II in 34 (32.4%),
15 (14.3%) and 2 (1.9%) samples, respectively.
Conclusions: The prevalence of BKV infection in urine
samples determined by qualitative PCR test used in this
study was similar to those described in other populations
of renal transplant recipients. The subgroup Ib1 prevailed
among the population studied, although it is not the most
common subtype worldwide. This is the first report in South
America to characterize BKV in kidney transplant patients.
Palavras-chaves: BK virus, PCR, genotype, transplant,
kidney.
TWO NOVEL BEGOMOVIRUSES INFECTING
Macroptilium lathyroides IN THE STATE OF
ALAGOAS, BRAZIL
ID: 00337-00001
Área: 06 - Virologia Vegetal
Silva, S.J.C., 2Castillo-Urquiza, G.P., 3Lima, J.C., 4PioRibeiro, G., 5Lima, G.S.A., 6Assunção, I.P., 7Zerbini, F.M.
1. UFV, Universidade Federal de Viçosa, Avenida Peter
Henry Rolfs, s/n 36570-000 VIÇOSA - MG2. UFRPE,
Universidade Federal Rural de Pernambuco, AV. Dom
1
ABSTRACTS
Manoel de Medeiros s/n, Dois Irmãos - CEP: 52171-900
- Recife/PE3. UFAL, Universidade Federal de Alagoas,
Av. Lourival Melo Mota, s/n, Tabuleiro do Martins - Maceió
- AL, CEP: 57072-970
The genus Begomovirus (family Geminiviridae) includes
viruses with a circular, single- stranded DNA genome
encapsidated in twinned icosahedral particles. They are
vectored by the whitefly Bemisia tabaci and cause
serious diseases in several economically important
crops. Begomoviruses are also associated with a wide
range of weed plants, which in some cases act as
inoculum reservoirs for cultivated plants. Here, we report
the detection of two novel species associated with the
leguminous weed Macroptilium lathyroides. A total of 20
samples of M. lathyroides plants were collected in
Alagoas state, Brazil. Total DNA was extracted from each
leaf sample, and full-length viral genomes were
successfully amplified from all 20 samples using the
DNA polymerase from phage phi29, indicating that they
were all infected by a begomovirus. The amplified DNA
was cleaved with BamHI and HindIII, ligated to plasmid
vectors and cloned in E. coli DH5a. Ten clones were
obtained and completely sequenced. Sequences were
compared to those of previously characterized
begomovirus species, and the ICTV-established 89%
DNA-A identity threshold was used to determined
taxonomic placements. This analysis indicated that 6
clones corresponded to the DNA-A of typical New World,
bipartite begomoviruses. Three clones corresponded to
isolates of Bean golden mosaic virus, displaying 89-90%
identity with a BGMV isolate from common bean. These
identity values indicate that these three isolates comprise
a distinct strain of BGMV, named BGMV-Ml. Two clones
corresponded to a novel species named Macroptilium
yellow spot virus (MaYSV). One clone corresponded to
another novel species named Macroptilium yellow net
virus (MaYNV). In a phylogenetic tree, both novel species
clustered with other Brazilian begomoviruses, indicating
their indigenous origin. Financial support: FAPEMIG,
INCT em Interação Planta-Praga.
Palavras-chaves: Begomovirus, Macroptilium
lathyroides, Diversity.
THE BEGOMOVIRUS Tomato yellow spot virus
(ToYSV) INFECTS Arabidopsis thaliana
SYSTEMICALLY
ID: 00340-00001
Área: 06 - Virologia Vegetal
Gonçalves, A.B., 2Silva, F.N., 3Rocha, C.S., 4Antunes,
T.F.S, 5Zerbini, F.M.
2. UFV, Universidade Federal de Viçosa, Av. PH Rolfs s/
n Centro, Viçosa/MG 36570-000
1
The family Geminiviridae is divided into four genera
(Mastrevirus, Curtovirus, Begomovirus and Topocuvirus)
according to the type of insect vector, host range,
genome organization and phylogeny. Viruses classified
in the genus Begomovirus have a genome consisting of
one or two molecules of circular, single-stranded DNA,
each with approximately 2,600 nucleotides in length. They
are transmitted by the whitefly Bemisia tabaci
(Homoptera: Aleyrodidae) and infect dicot species.
Begomovirus diseases are a major factor limiting crop
yields in tropical and subtropical regions. Tomato yellow
spot virus (ToYSV) is a virus that causes severe
symptoms in tomato and tobacco. The model plant
Arabidopsis thaliana is widely used in studies of the
interaction between viruses and plants due to its short
life cycle, small genome size, and ease of transformation
and mutagenic analysis. The objective of this study was
to determine whether ToYSV infects A. thaliana. A.
thaliana plants were biolistically inoculated with plasmid
DNA corresponding to 1,5 copies of the DNA-A and DNAB of ToYSV or Cabbage leaf curl virus (CalCuV), a
crucifer-infecting begomovirus used as a positive control.
Inoculated plants were visually examined for the
presence and severity of symptoms. The first symptoms
appeared 20 days after inoculation (dai) and consisted
of epinasty and mild yellowing of the leaves. Infection
by ToYSV and CalCuV was confirmed by PCR in noninoculated, systemically-infected leaves at 14 and 28
dai. These results confirm that ToYSV and A. thaliana
can be used as a model system to study the interaction
between plant and begomovirus factors and in the
understanding of the viral infection cycle.
Financial support: FAPEMIG, INCT em Interação
Planta-Praga.
Palavras-chaves: ToYSV, Arabidopsis thaliana,
Interação.
MOLECULAR CHARACTERIZATION OF INFLUENZA
VIRUSES IN SOUTH, SOUTHEAST AND NORTHEAST
BRAZIL, 2010.
ID: 00342-00001
Área: 05 - Virologia Humana e Saúde Pública
MACHADO, DBB, 2 Bor n , PS, 3 SANTOS, AV,
ANDRADE, T, 5LIMA, M, 6SIQUEIRA, MM, 7OLIVEIRA,
MLA, 8MOTA, FC
1. IOC/FIOCRUZ, Instituto Oswaldo Cruz/FIOCRUZ, Av.
Brasil, 4365 - Manguinhos - Rio de Janeiro - RJ - Brasil
- HPP - sala B105
1
4
Influenza viruses belong to the Orthomyxoviridae family,
presenting a segmented RNA single strain genome and
a unique and intricate replication system among the
known viruses. These viruses are great etiological agents
of acute respiratory illness worldwide. Due to the large
177
ABSTRACTS
genomic variability, some strains becoming potentially
epidemic, overcoming the barrier of circulating antibodies
in the community. Influenza type A is a ubiquitous virus,
and its subtypes are responsible for all pandemics
described, such as the recent caused by the triple
recombinant A/H1N1 pandemic (H1N1 pdm). Rapid
detection and characterization of suspicious samples is
a key point for surveillance of these viruses. In order to
characterize circulating viruses in Brazil, nasopharyngeal
samples from three distinct Brazilian Regions (NorthEast, South-East and South) were assayed by real time
RT-PCR to detect influenza A (subtypes A/H1pdm, A/
H1 and A/H3) and influenza B viruses. Real time RTPCR results showed 13,8% of positivity for influenza A
subtype A/H1pdm, 4,8% of influenza A subtype A/H3
and 2,3% for influenza B. The phylogenetic analysis
demonstrated a good correlation between influenza A/
H1pdm and the A/H3 circulating samples and those
included in South Hemisphere vaccine.
Palavras-chaves: Influenza, Pandemic, Filogenetic
analyses.
GarV-D to detect four species of allexivirus. Potyvirus
were detected only in two samples cultivated since 2006.
Allexivirus were also found in five samples from
Guarapuava in acclimatization phase (three samples
positive for GarV-A, one sample for GarV-C and one
sample for GarV-D). The remaining samples were negative
for all viruses indicating that thermotherapy + meristem
tip culture was efficient to eliminate the viruses.
Financial support: PROAP/CAPES.
Palavras-chaves: Allexivirus, Carlavirus, Potyvirus, RTPCR.
GENETIC STRUCTURE OF A POPULATION OF THE
BEGOMOVIRUS Bean golden mosaic virus (BGMV)
THAT INFECTS LIMA BEAN (Phaseolus lunatus L.)
IN THE STATE OF ALAGOAS, BRAZIL
ID: 00345-00001
Área: 06 - Virologia Vegetal
Ramos-Sobrinho, R., 2Silva, S.J.C., 3Tavares, S.S.,
Zerbini, F.M., 5Lima, G.S.A., 6Assunção, I.P.
1. UFV, Universidade Federal de Viçosa, Avenida Peter
Henry Rolfs, s/n, Campus Universitário, 36570-000,
VIÇOSA - MG2. UFAL, Universidade Federal de Alagoas,
Av. Lourival Melo Mota, s/n, Tabuleiro do Martins - Maceió
- AL, CEP: 57072-9703. UFRPE, Universidade Federal
Rural de Pernambuco, Rua Dom Manoel de Medeiros, s/
n, Dois Irmãos - CEP: 52171-900 - Recife/PE
1
4
EVALUATION FOR THE PRESENCE OF VIRUSES ON
GARLIC SEEDS PRODUCED BY THERMOTERAPY
PLUS MERISTEM TIP CULTURE.
ID: 00343-00001
Área: 06 - Virologia Vegetal
MITUTI, T., 2 DE MARCHI, B.R., 3 TAIOQUI, A.P.,
OLIVEIRA, M.L., 5IMAIZUMI, V.M., 6KRAUSE-SAKATE,
R., 7PAVAN, M.A.
1. UNESP / FCA, Universidade Estadual Paulista “Julio
de Mesquita Filho” - Faculdade de Ciências Agronomicas,
Rua José Barbosa de Barros, 1780
1
4
Commercial garlic (Allium sativum L.) is vegetatively
propagated by bulbs that often lead to the accumulation
of viruses, mainly a mixture belonging to the Allexivirus,
Carlavirus and Potyvirus genus. These viruses induce
yield losses during successive cultivation and one
alternative is the use of garlic virus free seeds. The
Department of Plant Protection of the UNESP/FCA has
a program to produce garlic plants free of virus
associating thermotherapy and meristem tip culture. In
this work there were evaluated the quality of 42 garlic
samples from the first year after meristem culture
cultivated under greenhouse. Also 33 samples from
Guarapuava / PR in acclimatization phase (2010) and
11 samples from the same region cultivated since 2006
under greenhouse conditions with anti-aphids screen,
were analyzed for the presence of virus. The test was
performed by RT-PCR using universal primers for
potyvirus (PV1 and WCIEN), carlavirus (SLV_GCLV 7303
and SLV_GCLV 7665) and GarV-A, GarV-B, GarV-C and
178
The lima bean (P. lunatus) is one of four cultivated
species in the genus Phaseolus. The species was
domesticated in South or Central America. Among its
most important diseases are those caused by viruses,
particularly by begomoviruses (family Geminiviridae).
Infected plants display an intense yellow or golden
mosaic. The objective of this work was to characterize
the genetic structure of begomovirus populations infecting
lima beans in the state of Alagoas, by analyzing complete
DNA-A sequences. Lima bean plants with typical
symptoms of begomovirus infection were collected in a
number of areas around the states of Alagoas and
Pernambuco during 2005. Total DNA was extracted and
the viral genomic components were amplified using phage
phi29 DNA polymerase, and then cleaved with either
BamH I or Hind III. Cleaved products were ligated to the
pBluescript KS+ plasmid vector and cloned into
Escherichia coli DH5á. Clones were submitted to cleavage
with Hae III and, according to the obtained restriction
pattern, 22 were selected for sequencing. Phylogenetic
analysis was carried out with MEGA 4.0 using the
Neighbour-Joining method. The genetic diversity of the
population was evaluated using the DnaSP program,
version 5. Analysis of the 22 sequences revealed that
they all belonged to a single begomovirus species, Bean
ABSTRACTS
golden mosaic virus, with 90 to 94% nucleotide sequence
identity with a BGMV isolate from soybean. All sequences
clustered with other Brazilian BGMV isolates from
common bean and soybean in the phylogenetic tree,
although the two isolates from Pernambuco are closer
to the soybean isolate than the 20 isolates from Alagoas.
Thus, the lima bean isolates clustered based on their
geographical origin. Analysis of the population indicated
a high degree of genetic variability, significantly higher
than that observed for two begomovirus populations from
tomato obtained in the Southeastern region of Brazil.
Finnancial support: FAPEMIG, INCT em Interação
Planta-Praga e FAPEAL.
Palavras-chaves: begomoviruses, genetic variability,
Phaseolus lunatus.
EXPERIMENTAL INOCULATION OF VACCINIA
VIRUS IN COWS
and GP2 re-isolated from a crust of an experimentally
infected animal. In this study, we observed that the double
need and the sandpaper were the best methods of skin
scarifcation, with better reproduction of the disease,
although the scaririfcation with the sandpaper allowed a
more widespread distribution of the lesions. Furthermore,
the inoculum GP2 re-isolated was the best in reproducing
the lesions observed in outbreaks of VB.
Palavras-chaves: bovine vaccinia, cows, experimental
infection, Vaccinia virus.
DETECTION OF VACCINIA VIRUS IN BLOOD AND
FAECES OF EXPERIMENTALLY INFECTED COWS
ID: 00347-00002
Área: 03 - Virologia Animal
Oliveira, T.M.L., 2Guedes, M.I.M.C., 3Rehfeld, I.S.,
Assis, F.L., 5Abrahão, J.S., 6Matos, A.C.D., 7Rivetti Jr.,
A.V., 8 Gerber, P.F., 9 Trindade, G.S., 10 Kroon, E.G.,
11
Lobato, Z.I.P.
1. UFMG, Universidade Federal de Minas Gerais, Av.
Antônio Carlos, 6627 CEP:31.270-901 Pampulha BH/MG
1
4
ID: 00347-00001
Área: 03 - Virologia Animal
Guedes, M.I.M.C., 2 Rehfeld, I.S., 3Oliveira, T.M.L.,
Matos, A.C.D., 5Rivetti Jr, A.V., 6gerber, P.F., 7Abrahão,
J.S., 8 Assis, F.L., 9 Trindade, G.S., 10 Kroon, E.G.,
11
Lobato, Z.I.P.
1. UFMG, Universidade Federal de Minas Gerais, Av.
Antônio Carlos, 6627 CEP: 31.270-901 Pampulha BH/
MG
1
4
Vaccinia virus (VACV), genus Orthopoxvirus, is the
causal agent of bovine vaccinia (BV), which is a zoonosis
that affects dairy cows and their calves, besides milkers
who come in contact with sick animals. Although
outbreaks of VB have been occurring in Brazil since 1999,
and the existence of several studies about the virus and
the disease, little is known about its pathogenesis. By
the studies done so far, the main route of VACV infection
in outbreaks of VB seems to be through continuity
solutions present in the teats of cows. Due to the
difficulties in monitoring natural infection by VACV and
for the further clarification of the pathogenesis of the
disease, a study was proposed using experimental
inoculation of VACV in cows. As there is a paucity of
information in the literature about the best way to
reproduce BV in cows, a pilot study was performed using
four animals. Different instruments for scarification of
the skin for subsequent inoculation were used.
Hypodermic needle (22G and 30G), double needle (used
for vaccination against fowlpox) and sandpaper were
used. Each teat was divided into cranial and caudal, and
scarification was made in a single site or across the
whole area. The virus used was the Guarani P2 (GP2),
isolated from an outbreak of VB in Minas Gerais, Brazil.
Two GP2 inocula were tested: a cell culture clone of GP2
Bovine vaccinia (BV), an zoonosis caused by Vaccinia
virus (VACV), affects dairy cattle and milkers, causing
economic, veterinary and human health impacts. By the
clinical presentation of the disease in natural infections,
it seems that BV is a localized disease, with lesions
restricted to the skin of affected individuals. But there
are no studies about the pathogenesis of the disease in
cows to asses if there is systemic spread of the virus
and if there are different ways of VACV shedding. There
are studies showing that VACV DNA and infectious virus
can be shed in faeces and urine of infected mice. Trying
to answer some of these questions, a study was
proposed using experimental inoculation of VACV in
cows. Six crossbred not-lactating cows, serologically
negative for VACV, were used. Teats were scarified using
hypodermic needle, followed by inoculation of 2 X 106
pfu of Guarani P2 (GP2) strain of VACV. Two animals
were euthanized at the 4th dpi and other two at the 9th
dpi and different tissues were collected from all animals
for further analysis. The remaining two animals were
monitored for 35 days post infection (dpi). Blood, sera,
faeces, urine and swabs (oral, nasal and vaginal) were
collected. All animals developed lesions (papules,
vesicles, ulcers) compatible with VACV infection in cattle.
Two out of the six animals presented VACV DNA in blood
samples, starting at 2nd dpi in one animal, and at the
3rd dpi in the other. Viral DNA was continuously detected
in the blood of these animals, in an intermitent way, until
the 35th dpi in both animals. VACV DNA was also
detected in fecal samples of these animals, starting at
the 3rd dpi in both animals, and lasting until the 22nd dpi
179
ABSTRACTS
in one animal, and the 35th dpi in the other, also in an
intermitent way. This study provides new evidence that
VACV can be detected in blood and faeces of infected
cows, suggesting that BV could be a systemic disease.
Financial support: FAPEMIG / CNPq / CAPES / MAPA
(LANAGRO – MG).
Palavras-chaves: bovine vaccinia, detection on blood
and faeces, experimental infection, Vaccinia virus.
HIV-1 PROVIRAL HYPERMUTATION AND DELETION
CORRELATES WITH LOW VIRAL LOAD COUNTS IN
NEWLY DIAGNOSED PATIENTS.
ID: 00349-00001
Área: 05 - Virologia Humana e Saúde Pública
Sa-Filho DJ, 2Ambar, R.F., 3Duarte, N.B., 4Comparini,
R.G., 5Gagliani, L.H., 6Caseiro, M.M.
1. UNILUS, Centro Universitário Lusíada, Rua: Oswaldo
Cruz, 179, Boqueirão, Santos - SP – Brazil, 11045-101.
1
Background: The roles of HIV hypermutation, the
introduction of excessive G-to-A substitutions, and
deletions has been suggested as an influence in the time
to AIDS development. Objective: We focused on
characterizing the pol gene sequences obtained from
newly diagnosed patients and the protective role of HIV1 deleterious mutations. Methods: We studied 33 HIV-1
sequences from newly diagnosed patients enrolled at
the Brazilian HIV/AIDS programs. Studied Population
Group: 18 male and 15 female, with mean age of 35
years, mean viral load of 4.8 Log10 copies/mL. The HIV1 pol gene sequences alignments were generated using
Clustal X with 1059 base pair. Hypermutations were
mapped more precisely using the Hypermut 2.0 program.
Results: 2 of 33 patients showed proviral HIV-1
sequences containing deleterious mutations. The
sequences alignment revealed a sample with 68 bp
deletion at reverse transcriptase gene leading to a stop
codon. At the beginning, the patient viral load was 125
copies/mL, however, after 10 months of follow-up the
viral load increased to 6534 copies/mL. The hypermutated
sequence presented four stop codons, one at the protease
gene and three at the reverse transcriptase gene. The
patient viral load increased from 630 copies/mL to 8376
copies/mL during a 12 month period of follow-up.
Conclusion: our data reveled that low viral load counts
correlate with HIV-1 deleterious mutations in newly
diagnosed patients. However, at follow-up, these cases
demonstrated continuous viremia increase, probably
caused by a viable emerging virus. This indicates that
the deleterious mutations are important but not a sole
determinant of the viremia control. This study was
supported by the Fundação Lusíada, Centro Universitário
Lusíada, Santos – SP, Brasil.
180
Palavras-chaves: AIDS, HIV, HYPERMUTATION.
BREAST CANCER AND HUMAN PAPILLOMAVIRUS (HPV) INFECTION: NO EVIDENCE OF HPV
DNA IN A GROUP OF BRAZILIAN WOMEN.
ID: 00349-00002
Área: 05 - Virologia Humana e Saúde Pública
PIVA, C.G., 2CARVALHO, L.V., 3FACCINETTO, A.C.B.,
RODRIGUES, L.M., 5 SERRA, M.J.R., 6 CASTRO,
P.M.V., 7COMPARINI, R.G., 8PENATI, T.Q., 9CASEIRO,
M.M., 10SA-FILHO, D.J.
1. UNILUS, Centro Universitário Lusíada, Rua: Oswaldo
Cruz, 179, Boqueirão, Santos - SP – Brazil, 11045-101.
1
4
Background: human papillomavirus (HPV) infection is a
necessary cause of cervical cancer, and is etiologically
associated with a subset of cancers of the anus,
oropharynx, penis, vagina, and vulva. Several studies
have proposed an association between HPV infection
and oesophageal, laryngeal, oropharyngeal, lung,
urothelial, breast and colon cancers. However, the role
of HPV in the pathogenesis of these types of cancer is
less well established. Breast cancer is one of the most
frequently diagnosed cancers in women in Brazil. Viruses
including Epstein-Barr virus, Bittner virus and human
papillomavirus have been detected in benign breast
tissues and breast tumors. It remains unclear whether
there is an association between human papillomavirus
(HPV) infection and human breast cancer. Objective: to
investigate the presence of Human Papillomavirus (HPV)
DNA in cases of invasive ductal carcinoma of the breast.
Material and Methods: twenty eight samples were
obtained from cases of invasive ductal carcinoma
diagnosed and treated at the Hospital Guilherme Álvaro
in Santos-São Paulo, Brazil in 2007. DNA was extracted
from formalin fixed and paraffin-embedded tumor tissues
using QIAamp DNA Mini Kit (Qiagen). The quality of DNA
extracted was verified by amplifying the human CCR-5
gene. Detection of HPV DNA was performed by PCR
using primers GP5 and GP6. HPV positive and negative
controls were performed. The presence of DNA was
verified by agarose gel electrophoresis. Results: the
genomic DNA from paraffin-embedded tumor tissues
presented 23 of 28 (82%) positive amplification for the
human CCR-5 gene. Breast cancers tested showed
negative results for HPV DNA. Conclusion: our results
are in agreement with some series and showed no
evidence of HPV DNA in invasive ductal carcinoma of
the breast in a group of Brazilian women. This study was
supported by the Fundação Lusíada, Centro Universitário
Lusíada, Santos – SP, Brasil.
Palavras-chaves: Breast carcinoma, HPV, Paraffinembedded tumour.
ABSTRACTS
ANALYSIS OF CCR5 DELTA 32 HETEROZYGOSIS
IN PATIENTS WITH LONG TERM HIV-1
SUPPRESSION UNDERGOING ANTIRETROVIRAL
THERAPY.
ID: 00350-00001
Área: 05 - Virologia Humana e Saúde Pública
COMPARINI, R.G., 2ZETEHAKU, A.C., 3LINS-FILHO,
A.F.P., 4NAHON, N.C., 5SANTOS, N.T., 6GAGLIANI, L.H.,
7
CASEIRO, M.M., 8SA-FILHO, D.J.
1. UNILUS, Centro Universitáro Lusíada, Rua: Oswaldo
Cruz, 179, Boqueirão, Santos - SP – Brazil, 11045-101
1
Background: Previous researches have shown that being
heterozygous for a 32 bp deletion in the CCR5 alele is
associated with the duration of time to development of
AIDS by turning the HIV-1 entry slower. Objectives: We
examined whether CCR5 delta 32 heterozygosis was
associated with antiretroviral therapy long-term HIV-1
suppression in Santos/Brazil. In addition we described
clinical course of HIV-1 infection among those individuals.
Patients and Methods: Patients under antiretroviral
therapy presenting 3 years of viral load less than 50 HIV
RNA copies per milliliter were defined as presenting longterm HIV-1 suppression. We selected randomly 83
patients for CCR5 analysis. This analysis was based on
polymerase chain reaction (PCR), using primers spanning
the CCR5 32 bp deletion. Results: We found that 7.2%
of study subjects were heterozygous CCR5/CCR5 delta
32. The ethnic group comprised Caucasians and African
descendents. Pneumonia and Candidiasis were the most
frequent opportunistic infection. The average CD4+T cells
counts in CCR5 heterozygous group was 601, 609, 719
cells per microliter in 2005, 2006 and 2007, respectively,
and CD8+T average was 1461, 1353, 1515 cells per
microliter. Conclusion: Our data demonstrated that the
frequency of CCR5 heterozygosis in the group of patients
with long-term HIV-1 suppression is similar to those found
in general Brazilian population (6.4% to 14.2%), neither
associated to a better CD4+ T cell profile. Therefore,
this suggests that HIV-1 suppression is not associated
with CCR5 heterozygous. This study was supported by
the Fundação Lusíada, Centro Universitário Lusíada,
Santos – SP, Brasil.
Palavras-chaves: antiretroviral therapy, CCR5, HIV-1.
ELITE SUPPRESSOR OF HIV-1 REPLICATION IN
SANTOS/SP, BRAZIL: CASE REPORT.
ID: 00350-00002
Área: 05 - Virologia Humana e Saúde Pública
1
COMPARINI, R.G., 2ZETEHAKU, A.C., 3LINS-FILHO,
A.F.P., 4NAHON, N.C., 5SANTOS, N.T., 6GAGLIANI, L.H.,
7
CASEIRO, M.M., 8SA-FILHO, D.J.
1. UNILUS, Centro Universitário Lusíada, Rua Oswaldo
Cruz, 179, Boqueirão, Santos - SP CEP 11045-101
Background: Rare human immunodeficiency virus-1
infected individuals, termed elite suppressors (ES),
maintain plasma virus levels of.
Palavras-chaves: AIDS, CD4+, Elite supressor, HIV, Viral
load.
SCREENING OF THE H275Y OSELTAMIVIR
RESISTANT MUTATION IN BRAZILIAN STRAINS OF
INFLUENZA A/H1N1 pdm BY PYROSEQUENCING
ASSAY.
ID: 00351-00001
Área: 05 - Virologia Humana e Saúde Pública
Santos, C.L.S., 2 Oliveira, M.I., 3 Silva, D.B.B.,
Borborema,S.E.T., 5Ferreira, J.L.P., 6Paiva, T.M.
1. IAL, Instituto Adolfo Lutz, Av. Dr. Arnaldo 355, São
Paulo,SP
1
4
A novel A/H1N1 influenza virus emerged in Mexico in
April 2009 and since then has spread worldwide.
Strategies to prevent and treat the infections caused by
the pandemic virus have been implemented. One of these
measures includes the use of neuraminidase (NA)
inhibitor Oseltamivir, which blocks the active site of the
enzyme, preventing efficient viral multiplication.
Resistance to NA inhibitors can occur by amino acid
alteration at the target site and of major impact has been
the emergence and spread of a mutation of histidine to
tyrosine at residue 275 (H275Y) (H1 numbering) in
seasonal influenza A (H1N1) viruses. Most of the currently
circulating H1N1 pdm are susceptible to Oseltamivir, but
the detection of variants of the virus harboring the
associated molecular resistance marker H275Y are a
cause of concern. During influenza pandemic period
nasopharyngeal secretions collected from patients
presenting acute respiratory illness were sent to IAL.
The presence of H275Y mutation was investigated in a
pool of samples obtained from positive cases of H1N1
pdm infection by a pyrosequencing technology. Viral RNA
was amplified by RT-PCR using the primers Uni-sw-N1B-F780 and Uni-sw-N1-B-R1273-biot developed by CDC.
Pyrosequencing reactions were performed with purified
biotinylated single-strand DNA annealed with Uni-sw-N1B-F804 sequencing primer using both the cyclic and
programmed nucleotide dispensation strategies. The
results of this investigation demonstrated that the
Brazilian strains included in the study are drug sensitive
since the pyrograms do not reveal the alteration CAT (H)
to TAT (Y) at the 275 residue. In the light of emerging
181
ABSTRACTS
oseltamivir resistance is of crucial importance the drug
resistance surveillance follow-up aiming to contribute with
physicians regarding clinical management. Financial
support: IAL/SES/SP, Ministério da Saúde, Brasil. Área:
Virologia Humana Tema FLU – Orthomixovirus Arquivo:
FLU – Paiva.rtf Apresentador: Terezinha Maria de Paiva.
Palavras-chaves: Influenza, antiviral, resistência a
antivirais, pirosequenciamento, surveillance.
development, and evaluation of antiviral drugs
susceptibility. Financial support: Instituto Adolfo Lutz/
SES/SP, Ministério da Saúde do Brasil Área: Virologia
Humana Tema: INF Influenza Arquivo: INF – Paiva.rtf
Apresentador: Terezinha Maria de Paiva.
Palavras-chaves: Influenza, Pandemia, Isolamento de
vìrus, Caracterizacao antigenica, Caracterizacao
molecular.
INVESTIGATION OF A(H1N1) v RESPONSIBLE FOR
THE FIRST INFLUENZA PANDEMIC OF 21 CENTURY
ANTIGENIC AND GENETIC ANALYSIS OF BRAZILIAN
STRAINS ISOLATED IN THE INSTITUTO ADOLFO
LUTZ
PREVALENCE OF PROTEASE AND REVERSE
TRANSCRIPTASE DRUG RESISTANCE MUTATIONS
IN DRUG NAÏVE HIV1-POSITIVE INDIVIDUALS IN
METROPOLITAN AREA OF FLORIANÓPOLIS
ID: 00351-00002
Área: 05 - Virologia Humana e Saúde Pública
ID: 00353-00002
Área: 05 - Virologia Humana e Saúde Pública
Gräf, T., 2Bello, G., 3Battisti-Neto, A., 4Ferreira, L.G.E.,
Meirelles, I.A.K., 6Grisard, E.C., 7Morgado, M.G., 8Pinto,
A. R.
1. UFSC, Universidade Federal de Santa catarina,
Campus da Trindade, Caixa postal 476 - Florianópolis,
SC - Brasil - 88040-9702. IOC/Fiocruz, Instituto Oswaldo
Cruz, Av. Brasil, 4365, Manguinhos - Rio de Janeiro - RJ
- Brasil CEP: 21040-3603. HRSJ, Hospital Regional Dr.
Homero de Miranda Gomes, Rua Adolfo Donato da Silva,
s/nº Praia Comprida, São José - SC - Brasil4. CVS,
Centro de Vigilância em Saúde, Praça Arnoldo de Souza,
38, Centro Histórico de São José, São José - SC - Brasil
1
Paiva, T.M., Santos, C.L.S., Silva, D.B.B, Benega,
M.A., 5Ishida, M.A.
1. IAL, Instituto Adolfon Lutz, Av. Dr. Arnaldo 355, CEP
0124677 9022. IAL, Instituto Adolfo Lutz, Av. Dr. Arnaldo
355, Sao Paulo, SP, Brazil
1
2
3
4
Since April 2009, human cases of respiratory infections
originated by novel swine-origin influenza A virus,
designated pandemic A (H1N1) v, have been detected
worldwide, causing immediate international concern.
Isolation and molecular epidemiology investigations were
carried out in the present study in an attempt to monitoring
the pandemic influenza A (H1N1) v in Brazil. A total of
37.200 respiratory secretions were collected from patients
who came from Southeast, Centre East, North and
Northeast regions of Brazil. Viral RNA was extracted from
the infected cells and submitted to reverse transcriptionamplification reactions with primers set designed to cover
the complete segments of the HA, NA and MP genes.
The amplified products were directed sequenced.
Comparative sequence analysis indicated the presence
of point mutations in the HA gene of the Brazilian strains
when compared to the reference strain A/California/04/
H1N1 (2009). These alterations do not change the all
five of the known antigenic sites of the HA protein. Data
of the MP sequence analysis revealed that the strains
of this study carried the S31N mutation that confers
cross-resistance to the adamantine class of anti-influenza
drugs. Sequencing of the NA gene showed that the
neuraminidase relative drug binding pocket represented
by H275 was not altered. On the other hand a resistance
against oseltamivir was identified in a seasonal H1N1
influenza strain isolated in the southeast region of Brazil,
before pandemic period. These results emphasize the
contribution of molecular surveillance, in addition to
antigenic characterization to monitor the evolutionary
pattern of the pandemic A (H1N1) v, in order to vaccine
182
5
The introduction of antiretroviral therapy for treatment of
HIV-infected subjects significantly decreased AIDS
associated morbidity and mortality. The development of
drug resistance mutations (DRM), however, remains one
of the most serious obstacles to sustained suppression
of HIV. Since 1996 the Brazilian Ministry of Health
provides universal access to antiretroviral drugs, which
raised concerns about the emergence and spread of
resistance variants due the suboptimal compliance.
Nevertheless, studies have shown low rate of HIV-drug
resistance among drug naïve patients in several cities
of Brazil. Florianópolis, the capital city of Santa Catarina
state, has the largest amount of HIV infections in the
state and there is no data describing the prevalence of
HIV drug resistance mutations in Florianópolis. The aim
of the current report is to describe the prevalence of
DRM among HIV-infected drug naïve patients in the
metropolitan area of Florianópolis. Blood samples were
collected from 100 HIV-infected drug naïve patients
attended at public reference services (Hospital Regional
Dr. Homero de Miranda Gomes and Centro de Vigilância
em Saúde de São José). Out of the 100 blood samples,
82 HIV protease and reverse transcriptase (RT)
sequences were generated by Nested-PCR amplification
and sequencing. The mutation profile was analyzed at
ABSTRACTS
the Stanford HIV Drug Resistance Database. The
presence of at least one DRM was observed in 9.75% of
the sequences, being 2.4% to protease inhibitors, 5% to
nucleoside RT inhibitors and 3.6% to non-nucleoside RT
inhibitors drug classes. In one sequence was observed
mutations to more than one class of antiretroviral, and
three patients (3.6%) showed mutations conferring high
level resistance to at least one antiretroviral drug. The
present work shows preliminary data about primary drug
resistance in Florianópolis metropolitan area, indicating
a higher DRM prevalence than previously estimated for
Brazilian naïve patients.
Financial support: CNPq & FAPESC
Palavras-chaves: Antiretroviral drugs, Drug resistance
mutations, HIV.
CHARACTERIZATION OF HIV-1 CIRCULATING IN
METROPOLITAN AREA OF FLORIANÓPOLIS
CONFIRMS THE HIGH PREVALENCE OF SUBTYPE
C AND RECOMBINANTS BC IN SANTA CATARINA
STATE
ID: 00353-00001
Área: 05 - Virologia Humana e Saúde Pública
Gräf, T., 2Bello, G., 3Battisti-Neto, A., 4Ferreira, L.G.E,
Meirelles, I.A.K., 6Grisard, E.C., 7Morgado, M.G., 8Pinto,
A.R.
1. UFSC, Universidade Federal de Santa Catarina,
Campus da Trindade, Caixa postal 476 - Florianópolis,
SC - Brasil - 88040-9702. IOC/Fiocruz, Instituto Oswaldo
Cruz, Av. Brasil, 4365, Manguinhos - Rio de Janeiro - RJ
- Brasil CEP: 21040-3603. HRSJ, Hospital Regional
Homero de Miranda Gomes, Rua Adolfo Donato da Silva,
s/nº Praia Comprida, São José - SC - Brasil4. CVS,
Centro de Vigilância em Saúde, Praça Arnoldo de Souza,
38, Centro Histórico de São José, São José - SC - Brasil
1
5
The AIDS epidemic in southern Brazil is distinct from all
other areas in the Americas. While HIV-1 subtype B
prevails in most of the countries, with clade F and BF
recombinants having a secondary occurrence, several
studies have shown the prevalence of HIV-1 subtype C
and the recombinant form CRF31_BC in southern Brazil.
The state of Santa Catarina has one of the highest
incidences of HIV/AIDS in Brazil and Florianópolis is
the city with the highest number of cases reported within
the state. Considering the unique characteristics of HIV1 epidemic in southern Brazil and the shortage data from
metropolitan area of Florianópolis, the current report
investigates the molecular epidemic of HIV-1 in this
region. Blood samples from HIV-positive patients from
public reference services (Hospital Regional Dr. Homero
de Miranda Gomes and Centro de Vigilância em Saúde
de São José) were collected from May 2008 to February
2009. 82 out of 100 blood samples had the env and pol
genes amplified by nested-PCR and sequenced. The HIV1 sequences generated were subtyped using the
Neighbor-Joining (NJ) algorithm for constructing
phylogenetics trees and the bootscaning analysis, using
program Simplot, to identify recombination breakpoints.
The analysis performed revealed that 70% of HIV-1
sequences were subtype C, 13% were subtype B and
2% were subtype F1 or BF1 recombinants. Mosaics BC
represented 15% of the sequences and 4% of these
recombinants forms displayed CRF31_BC-like mosaic
pattern. Our results revealed a significant difference.
Palavras-chaves: HIV, Molecular Epidemiology,
Phylogeny.
ANALYSIS OF THE INFLUENCE OF PHARMACOLOGICAL INHIBITORS ON THE MULTIPLICATION CILCE OF THE YELLOW FEVER VIRUS
ID: 00354-00001
Área: 04 - Virologia Básica
Palhares, R.M., 2 Ferreira, P.C.P, 3 Kroon. E.G.,
Bonjardim, C.A.
1. GTS, Grupo de Transdução de sinais, Av. Antônio
Carlos, 6627 - Pampulha - Belo Horizonte - MG2. Lab
vírus, Laboratório de vírus, Av. Antônio Carlos, 6627 Pampulha - Belo Horizonte - MG3. ICB, Instituto de
Ciências Biológicas, Av. Antônio Carlos, 6627 - Pampulha
- Belo Horizonte - MG4. UFMG, Universidade Federal
de Minas Gerais, Av. Antônio Carlos, 6627 - Pampulha Belo Horizonte - MG
1
4
The yellow fever virus (YFV) is a small, enveloped virus,
with a single stranded and positive sense RNA genome
consisting of approximately 11Kb. The yellow fever is a
disease transmitted to humans by an insect vector and
possesses pandemics and endemics patterns, affecting
thousands of people in Africa and South America, and
travelers that had not been vaccinated, with a estimate
of 200.000 asymptomatic cases and 30.000 deaths
annually. For a successful multiplication, viruses must
be able to interact and subjugate cellular pathways of
signal transmission, manipulating the responses and
cellular functions of his host. Following this line, the
signaling pathways involving the MAPKs MKK/JNK and
MEK/ERK and the PI3K/AKT pathway, responsible for
events of proliferation, cellular differentiation and
apoptosis are extremely important. For the analysis of
the importance of those pathways in the multiplication
cycle of the YFV, experiments of growth curves in single
cycle were made in several intervals, in the presence
and in the absence of the respectives inhibitors, leading
to the observation that the inhibition of the JNK pathway
by JNK inhibitor VIII and the inhibition of the PI3K/AKT
183
ABSTRACTS
pathway by LY294002 do not correspond to a decay in
the virus multiplication, however, the inhibition of the
MEK/ERK pathway by the inhibitor UO126 leads to a
marked fall (above one logarithmic scale) in the virus
multiplication, indicating an promising target to studies
of the significance of this pathway in the cycle of YFV.
To determinate more precisely the stage of the viral
multiplication affected, experiments of microscopy of
transmission, are being conducted, as well as the
production of antibodies against non structural proteins
of the virus. Those approaches will enable us to do more
detailed analysis on the stages of the multiplication cycle
affected, giving a better understanding of the importance
of this specific pathway to the cycle of YFV.
Palavras-chaves: Yellow fever virus, virus cycle, cellular
pathways, MEK/ERK, UO126.
CYTOTOXIC AND ANTIVIRAL EFFECT OF
QUINOLONES DERIVATIVES AGAINST HERPES
SIMPLEX TYPE 1
ID: 00355-00001
Área: 05 - Virologia Humana e Saúde Pública
Oliveira, I. de B., 2Meneses, L. da C., 3Ribeiro, C. P.,
Faro, L. V., 5Almeida, J. M., 6Cunha, A. C., 7Ferreira, V.
F., 8Souza, M. C., 9Giongo, V., 10, M. C. B. V., 11Paixão, I.
C. N. de P.
1. UFF, Universidade Federal Fluminense, Rua Outeiro
de São João Batista s/ nº - Centro, Niterói, RJ - CEP:
24.020-150
24-well plates were treated for 1h with virus solutions
together with the substance 50ìM were incubated for
1,2,3 and 4h at 4°C. On cytotoxicity assay, we found
high values for CC50 in relation to the positive control,
acyclovir. On the percentage of viral inhibition assay,
substances with Cl, CH3 and Br radicals inhibited over
70% while those with NO2 and F radicals inhibited 45%
or less compared with the control virus. It can be
concluded that the substances weren’t cytotoxic to Vero
cells. Some substances inhibited above 50% viral
activity. None of the substances showed virucidal activity.
Wherefore, the substances are promising for continuation
of studies for the development of derivatives with antiviral
action.
Financial support: CNPq.
Palavras-chaves: antiviral, HSV-1, quinolones
derivatives.
CYTOTOXIC AND ANTIVIRAL ACTIVITY OF
OXOQUINOLINIC DERIVATIVES IN HERPES
SIMPLEX VIRUS TYPE 1 IN VITRO REPLICATION
ID: 00355-00002
Área: 05 - Virologia Humana e Saúde Pública
1
4
Herpes Simplex type 1 is a virus can cause recurrent
lesions mainly in the mucosa of the mouth and establish
latency in the trigeminal ganglion. Prolonged treatment
with drugs known favors the formation of resistant strains,
requiring the development of new drugs. Quinolones and
their derivatives have an important role as antiviral. The
substances used in this study are quinolones derivatives
that have a phosphonate radical at carbon 1 of the
quinolonic nucleus and replacements were made or not
at carbon 6 or 7. Vero cells were cultured in DMEM with
5% FBS and maintained at 37°C in atmosphere of 5%
CO2. The cytotoxicity assay was performed in 96-well
plates using the methodology of MTT. The substances
were added at concentrations of 12.5, 25, 50, 100, 250,
500 and 1000ìM and left for 72h. The values were
analyzed in a curve for 50% cytotoxic concentration
(CC50). The antiviral effect was assessed by the
percentage of viral inhibition. Substances 50ìM were
added 1h after the addition of viral dilution. After 20h,
cells were lysed and the title of virus supernatant was
determined by adding different dilutions on 24-well plates.
The PFU was counted by comparing wells treated with
the substances and viral control. In the virucidal assay,
184
Ribeiro, C. P., 2Oliveira, I. de B., 3Vieira, N. R. P.,
Barbosa, J. E. F., 5Ribeiro, M. S., 6Giongo, V., 7de Souza,
M. C. B. V., 8Paixão, I. C. N. de P.
1. UFF, Universidade Federal Fluminense, Rua Outeiro
de São João Batista s/ nº - Centro, Niterói, RJ - CEP:
24.020-150
1
4
Virus infections by herpes virus simplex type-1 may
cause many diseases, as cutaneous, genital infections
and encephalitis. The recurrent of latent episodes are in
closely association with immunecompetence as we see
in AIDS patients. The growing resistant strains to
aciclovir (ACV), becomes urgent to discover new
molecules with antiviral activity and low citotoxicity.
Quinolonocarboxamides, are compounds derivated from
the quinolinic system, in which a piridinic ring is found
melted into a benzenic ring, known as 4-hidroxiquinoline
or 4-oxoquinoline. To evaluate the cytotoxic and antiviral
activities of this new synthetical drugs against HSV-1,
Vero cells were incubated with crescent concentrations
of the substances for 72 hours/ 37°C / 5% CO2. After
this, CC50 and EC50 were defined by the number of
viable cells and plaque assay. Our results showed that
CC50 varies according with Fluor position. In para- the
values were lower compared with ortho position in the
benzene ring. The higher CC50 value was obtained with
addiction of phosphonates in the structure. Among the
fluor synthetical derivatives, the compound MPD-18
showed the best antiviral activity, being less citotoxicity
than acyclovir. The selective index of this substance could
ABSTRACTS
give us a promising target for antiviral drugs. However,
further assays should be carried out to determine others
in vivo toxicity and activity parameters.
Financial Support: CNPQ and PROPPi UFF.
Palavras-chaves: antiviral, HSV-1, oxoquinolinic
derivatives.
TERMOSTABILITY OF LYTIC Escherichia coli
BACTERIOPHAGES
ID: 00356-00001
Área: 04 - Virologia Básica
Fonseca, L. A. B. V., 2Dias, R. S., 3Silva, L. C. F., 4Silva,
V. D., 5Silva, L. S., 6Souza, F. O., 7Eller, M. R., 8Oliveira,
L. L., 9Paula, S. O.
1. UFV, Universidade Federal de Viçosa, Avenida PH
Rolfs s/n Campus Universitário
1
Bovine Mastitis is an infectious diseases of greatest
economic impact on dairy farming worldwide, causing
economic losses both to producer and dairy industry.
Disease is attributed to infection by several
microorganisms such as Staphylococcus aureus,
Escherichia coli and Streptococcus agalactiae. Antibiotic
is the current treatment used to control these infections,
but the emergence of resistant strains and the presence
of residues in milk have led the search for alternative
therapies. In this context, phage therapy becomes a viable
and advantageous alternative, since virus is specific to
the pathogen, and affecting only the target bacterial cell.
In addition, isolation of new phages is relatively faster
than production of new antibiotic. In this study, we
analyzed the thermostability of two phages previously
isolated from the city of Viçosa’s sewage, MG
(ufvecophage006 and ufvecophage015). Aliquots of viral
suspension were incubated for 15 minutes at different
temperatures (60, 70, 80 and 100 °C) and the title
obtained by plating assay. Plates were incubated for 18
h at 37 °C and the virus titers were measured and
compared. Ufvecophage006 had initial titers of 1.95x1010
PFU/mL After warm up to 60ºC phage does not induce
lysis, indicative of a thermosensitive phage. In contrast
the ufvecophage015 who had initial titers of 7.99x1015
PFU/mL and show a slow decrease in phage titer during
thermal treatment reducing to 2.54 x 1015 PFU/mL after
heat up to 70ºC.
Financial Support: FAPEMIG
Palavras-chaves: Bacteriophages, termostability,
escherichia coli.
DETECTION BY REVERSE TRANSCRIPTASEPOLYMERASE CHAIN REACTION OF AVIAN
INFECTIOUS
BRONCHITIS
VIRUS
AND
METAPNEUMOVIRUS FROM SOULTH BRAZI-
LIAM
ID: 00357-00001
Área: 03 - Virologia Animal
Nilzane Beltrão, 2Rodrigues; O.
1. MercoLab, Laboratório Vet. Garibaldi Ltda, RST 470,
Km 226,5 - Garibaldi/RS
1
Acute respiratory tract infections are of great importance
in the poultry industry. Avian infectious bronchitis virus
(IBV) and avian metapneumovirus (aMPV) have been
recognized as the most usual pathogens in broiler flocks
in Southern Brazil. The purpose of the present study was
to apply a molecular diagnostic test to detect IBV and
aMPV in broiler flocks reared in the State of Rio Grande
de Sul. One hundred thirty-seven avian tracheal swabs
from broiler flocks with clinical signs of respiratory
disease were collected in 2010. RNA was extracted from
the swabs using one silica-based kit and was analyzed
by an OneStep RT-PCR protocol. The RT-PCR results
showed that 70% (n=96) of the samples were positive to
one or both respiratory viruses. Furthermore, 35% (n=33)
of these flocks were infected with both IBV and aMPV,
32% (n=31) with IBV alone and 33% (n=32) with aMPV
alone. Flocks positive for both IBV and aMPV showed
higher mortality than the flocks infected only with one of
these viruses. IBV positive samples were molecularly
characterized as variant or as vaccine strain (H120)
based on a RT-PCR using specific primers. Sixty-three
(63%, n=40) of the IBV positive samples were classified
as vaccine strain H120 and 37% (n=24) were classified
as variant. These results suggest that the coinfection
with the viral agents reduced the performance of the
affected flocks and also displayed the presence of IBV
variant strains in the field. Further studies are necessary
to assess additional circulating strains, economic losses
and coinfection with other respiratory pathogens. Financial
Support: MercoLab.
Palavras-chaves: Avian respiratory virus, Avian
Infectious bronchitis virus, Metapneumovirus, RT-PCR,
Avian patology.
SEROLOGIC SURVEILLANCE FOR ARBOVIRUS IN
VILLAGE IN THE MUNICIPALITY OF INDIGENOUS
KWATINEMO ALTAMIRA-PARÁ, JANUARY TO
FEBRUARY 2010.
ID: 00361-00001
Área: 05 - Virologia Humana e Saúde Pública
MORAES,J.R.S, 2FERREIRA,M.S., 3HENRIQUES,D.F,
GAMA,E.C., 5PRAZERES,A.S.C, 6CLEBER B. SILVA,
7
VASCONCELOS,P.F.C
1
4
185
ABSTRACTS
1. IEC, INSTITUTO EVANDRO CHAGAS, BR 316;KM 7
The Brazilian native ethnic Indian Kwatinemo Asurini
village, has a population of 149 Indians and is located
on the Xingu River margins in the municipality of Altamira
in Para state. The arboviruses in general are Zoonotic
viruses maintained in the natural forest environment.
Therefore, people who maintain contact with enzootic
foci of arboviruses are most at risk of acquiring infection.
Some arboviruses are serious Public health problems,
such as the viruses .dengue (DENV), Mayaro (MAYV)
and Oropouche (OROV). Between January and February,
2010;a total of 70 human sera from people living in the
Village Kwatinemo were tested by to Haemagglutination
inhibition (HI) test against 18 different types of
arboviruses: (East Encephalitis equine virus, Western
Encephalitis equine virus, Mayaro virus, Mucambo virus,
Guaroa virus, Maguari virus, Tacaiuma virus, Yellow fever
virus wild and vaccine 17D, Dengue virus type 1, 2, 3,
and 4, Saint Louis Encephalitis virus, Rocio virus, Ilheus
virus, Caraparu virus, Oropouche virus and Catu virus).
For identification of recent infection by DENV, MAYV
and OROV it was used the IgM capture enzyme
immunoassay (MAC-ELISA). From the 70 sera tested
by HI n=57 (81.42%) showed HI antibodies to viruses of
the genera as follows: Flavivirus n=18 (31.57%),
Alphavivus n=3 (5.26%), Orthobuniavirus n=1(1,75%) and
cross-reaction to viruses belongs to the mentioned
genera n= 28 (47.35%). Monotypic reaction was
observed n=8 (14,03%) to MAYV. Analysis performed
by MAC-ELISA of 70, 38 and 4 respectively serum
samples for dengue, Mayaro and Oropouche, the specific
was detected to DENV (10%), and to MAYV (2,94%)
and all were negative to OROV. The results obtained by
the serological tests have demonstrated the active
circulation of DENV e MAYV which belongs respectively
to the genera Flavivirus and Alphavivus in the Village
Kwatinemo in 2010.
Palavras-chaves: ARBOVIRUS, FLAVIVIRUS,
ALPHAVIRUS.
ORTHOPOXVIRUS INFECTIONS IN HUMANS AND
CATTLE IN RIO DE JANEIRO BETWEEN 1999-2010.
ID: 00362-00001
Área: 05 - Virologia Humana e Saúde Pública
GONÇALVES,M.C.R., 2SCHATZMAYR, H.G, 3BARTH,
O.M.
1. FIOCRUZ / IOC, Fundação Oswaldo Cruz - Instituto
Oswaldo Cruz, Av. Brasil, 4365. Manguinhos. Pav. HPP.
sala B16
1
Vesicular Orthopoxvirus Infections caused by vaccínialike viruses, deteriorated over the past 10 years in Brazil.
186
In this project will be confirmed the presence of this virus
in 12 municipalities of the State of Rio de Janeiro. It will
be applied to materials collected from human and bovine
suspected cases, using techniques of isolation of virus
in Vero cells, PCR, electron microscopy observation,
antibody titration test by reducing plates and indirect
immunofluorescence. Clinical and epidemiological
characteristics of human and bovine cases will be
described. Clinical specimens as vesicular/pustular
liquids, crusts and human and bovine sera should be
used. It has been evaluated so far, 228 bovine and 78
human specimens. Vesicular fluid, swab and macerated
crusts collected from 19 human cases and 64 animals
were inoculated in Vero cells, tested by IFI, PCR and
electron microscopy. Up to date about 223 sera were
studied, using as antigen a vaccinia-like virus isolated
in the municipality of Cantagalo in 1999. These data
confirmed the presence of antibodies in 66,7% of human
specimens and of 68% bovine samples. Vesicular lesions
in hands, forearms and faces in humans were observed,
regional lymph edema and few hospitalizations. We will
continue to apply these technologies to new specimens
arriving to the laboratory, in order to know better the
natural history of this new zoonosis in our country.
Financial Support - CNPQ and FAPERJ Virologia
Humana.
Palavras-chaves: Orthopoxvirus, Poxvirus, Rio de
Janeiro, Brasil.
STANDARDIZATION OF DOT-ELISA FOR SEROLOGICAL DETECTION OF AVIAN METAPNEUMOVIRUS
ID: 00363-00001
Área: 03 - Virologia Animal
SARAIVA, G.L., 2 SANTOS, M.A, 3 SANTOS, M.R,
SILVA Júnior, A., 5ALMEIDA, M.R
1. UFV, UNIVERSIDADE FEDERAL DE VIÇOSA,
Avenida Peter Henry Rolfs, s/n Campus Universitário
36570-000 VIÇOSA - MG
1
4
Respiratory diseases are the major cause of economic
losses in the poultry industry, mainly by the increase in
mortality, among these diseases stand out the infection
with Avian metapneumovirus (APV). This study aimed
to standardize a dot-ELISA for serological detection of
APV. The viral sample SHS-BR-121 was previously
multiplied in culture of VERO cells, using the minimum
essential medium (MEM) supplemented with 10% fetal
bovine serum. Posteriorly, nitrocellulose disks were
sensitized with 0.70 &mug of purified antigen by the
ultracentrifugation method in sucrose gradient. After of
sensitized, the disks were blocked for 30 minutes with
200 ml of PBS-T, pH 7.6. Each nitrocellulose disk
sensitized with antigen received 200 ml of diluted serum
ABSTRACTS
(1:400) in PBS-T, pH 7.6, incubating them for an hour at
room temperature. After three washes with PBS-T, were
added 150 ml of conjugate (rabbit IgG antiIgG chicken
coupled with peroxidase), diluted 1:5,000 in PBS-T 0.05%
pH 7.6, incubating the plates with the disks for 30 minutes
at room temperature. After three washes, was added in
each disk 100 ml of the substrate (0.1% 3,3diaminobenzidine, 50 mM Tris-HCl, pH 7.6 and 0.1%
hydrogen peroxide) and incubated until the positive
control was evidenced. Finally, the reaction was stopped
by adding double distilled water and the results were
evaluated based on coloration intensity of the
nitrocellulose disks. Were used three standards for
determining the value of sensitivity: 8 serum samples
from chickens free of specific pathogens, three positive
serum samples from experimentally inoculated chickens
and 20 positive samples tested previously, by a
commercial test of ELISA. The standardized test showed
a satisfactory sensitivity of 100% of positive sera. Thus,
the test developed, is presented as a possible candidate
for serological diagnosis and monitoring of Avian
metapneumovirus.
Financial support: FAPEMIG
Palavras-chaves: STANDARDIZATION, DOT-ELISA,
SEROLOGICAL, DETECTION, AVIAN METAPNEUMOVIRUS.
PROGRESS AND SPATIAL PATTERN OF GOLDEN
MOSAIC IN FABA BEAN
ID: 00364-00001
Área: 06 - Virologia Vegetal
Silva, M. A., 2Lima, J. S., 3Silva, W. C., 4Assunção, I.
P., 5Lima, G. S. A, 6Michereff, S. J.
1. UFRPE, Universidade Federal Rural de Pernambuco,
Av. Dom Manoel de Medeiros, s/n-Dois Irmãos, CEP:
57171-900; Recife-PE2. UFAL, Universidade Federal de
Alagoas, Av. Lourival Melo Mota, s/n,Tabuleiro do Martins
- Maceió - AL, CEP: 57072-970
1
The golden mosaic, caused by ,Bean golden mosaic
virus (BGMV), causing severe losses to faba beans
growers in Brazilian Northeast Brazil. This study aimed
to analyze the temporal and spatial aspects of the
disease at two experimental plots (A and B), cultivated
with faba bean, located in Rio Largo, Alagoas. In each
plot were distributed 34 planted rows with 20 plants per
row, totaling 1.360 plants per area. The evaluation of the
incidence of golden mosaic was done by visual inspection
of all plants, at 30, 60, 90, 120 and 150 days after planting
(DAP).Golden mosaic epidemics in the two fields were
compared with respect to initial incidence (yo), maximum
incidence (ymax), estimated rate of disease progression
(RDP) and area under the disease progress curve
(AUDPC). The spatial pattern of disease was analyzed
by the techniques of mapping areas isopath, ordinary
runs, adjust the beta-binomial distribution and spatial
autocorrelation. For the analysis of progress curves,
values of yo, ymax, RDP and AUDPC were significantly
(P=0.05) higher in plot B. In both plots the aggregation
of symptomatic plants within the lines increased with
time after planting and increased incidence of disease.
In plot A there was a slight predominance of aggregation
of symptomatic plants within the lines, while plot B
predominated markedly the aggregation of diseased
plants between rows. Palavras-chaves: Phaseolus
lunatus, Begomovirus, epidemiology.
INCIDENCE AVALIATION OF POTYVIRUS AND
BADNAVIRUS IN YAM CULTURE IN ALAGOAS STATE
ID: 00364-00002
Área: 06 - Virologia Vegetal
Lima, J. S., 2Silva S. J. C., 3Tenorio, A. A. R., 4Netto, M.
S. B., 5Lima, G. S. A., 6Assunção I. P.
1. UFRPE, Universidade Federal Rural de Pernambuco,
Rua Dom Manoel de Medeiros, s/n, Dois Irmãos - CEP:
52171-900 - Recife/PE2. UFAL, Universidade federal de
Alagoas, Av. Lourival Melo Mota, s/n,Tabuleiro do Martins
- Maceió - AL, CEP: 57072-970
1
Yam culture presents a huge socioeconomics importance
to Brazil’s northeast region for being a part of the basic
diet from rural and urban populations. Yam production on
the opposite side, is affected by innumerous illnesses
that individually or in association, are responsible for
the losses on the income of the culture. The viruses
cause bigger worry, because they cause significant
losses on the quality of the tuber, what restrain the
exportations and the exchange of propagative material.
Diverse viruses have been described on this culture,
caused by members from Potyvirus and Badnavirus
genus, which are highlighted by theirs importance and
geographic distribution, occurring in simple and mixed
infections. In this study were collected plants from the
main commercial plantations from Alagoas state (Viçosa
and Arapiraca) presenting leaves with symptoms caused
by virus. For identification of virus were tested by DAS
ELISA with antibody against Potyvirus utilizing Agidia’s
kit, following the manufacturer instruction and PCR for
the detection of Badnavirus , on which are utilized
degenerated primers. Positive samples for Badnavirus
were submitted to sequencing to confirm the species
identity. From the 85 samples analyzed, in Viçosa, 56,5%
were positive for Potyvirus and 10,6% for Badnavirus, in
Arapiraca 67,0% for Potyvirus and 48,2% for Badnavirus
. From the total of 170 samples studied 19,4% presented
mixed infection . These viruses have been widely
187
ABSTRACTS
disseminated at the region.
Financial support: FAPEAL
Palavras-chaves: Yam, Badnavirus, Potyvirus.
Antônio Carlos, 6627
A safe vaccine against Dengue Virus (DENV) demands
a balanced tetravalent formulation. Until now, no
formulation was able to induce an efficient immune
response against infections caused by DENV. We
hypothesized that chimerical peptides expressing
potential immunogenic epitopes of all DENV serotypes,
could be recognized, of a trivalent and balanced way, by
serum of dengue patients and, consequently, useful to
develop safe dengue vaccine compositions. Thus, we
aimed to design, express and purify the chimerical peptide
in heterologous systems. By using bioinformatics tools,
we designed a pilot 1400bp minigene coding for a 50kD
peptide and composed by immunogenic and conserved
regions of DENV (1-4) envelope, capsid and NS1
proteins. Flanking regions were added to permit cloning
in different vectors and sequence was optimized to be
efficiently expressed Escherichia coli-based systems.
We were able to cloning the minigene in pTXB1, pQE-9
and pMAL-c5x expression vectors. The chimerical
peptide will be purified by affinity chromatography and
used in ELISA and Western-blotting assays to quantify
the peptide recognition by sera of DENV (serotypes 1, 2
and 3) infected patients and non-dengue volunteers.
Palavras-chaves: Dengue, Minigene, Peptide recognition,
Vaccine.
Rap1 is a member of the Ras family of small GTPases
that is activated by diverse extracellular stimuli in many
cell types. Either defective or excessive Rap1 activation
has been reported to contribute to malignancy via distinct
biological effects in different cell types. Aim: Evaluate
the expression of the proteins Rap1 GTPase and
p16INK4A in cervical mucous biopsies modified by
squamous intraepithelial lesion associated to the Human
papillomavirus (HPV) infection. Methods: A PCR
technique was used for detection of HPV in 150 cervical
biopsies classified as follows: normal (n=30), 42 lowgrade intraepithelial lesion (LG-SIL), 38 high-grade
intraepithelial lesions (HG-SIL) and 40 squamous cervical
cancers (SCC). Immunocytochemistry analysis of
p16INK4A and Rap1 was performed on 57 cervical
biopsies classified as: 9 normal, 22 LG-SIL, 13 HG-SIL
and 13 SCC samples. HPV DNA was detected in 49%
(73/150) of the samples. HPV DNA was detected in 59%
(25/42) of the LG-SIL; 63% (24/38) of HG-SIL samples;
55% (22/40) of the SCC samples and in 7% (2/30) of
normal samples. Overall, 77% of LG-SIL and 100% of
HG-SIL specimens exhibited a clear although weak
diffuse Rap1 immunostaining pattern. All SCC
specimens predominantely presented a high intensity
staining and a diffuse distribution of Rap1. A weak and
diffuse nuclear and cytoplasmic p16INKA staining was
observed in 50% of the LG-SIL lesions and an intense
and diffuse staining was verified in 100% of the HG-SIL
and SCC specimens. Unlike p16INK4A, Rap1 expression
was upregulated in the LG-SIL group. Both markers, Rap1
GTPase and p16INK4a, were found overexpressed in
the HG-SIL and SCC lesions. Conclusions: Our data
suggest that Rap1 GTPase is a good biomarker for
predicting the progression of HPV-related LG-SIL. A
combination of Rap1 GTPase/p16INK4A expression
seems to be useful to identify low and high grade and
also invasive lesions associated with HPV.
Palavras-chaves: cervical cancer, Human papillomavirus
(HPV), Rap1, P16INK4A.
RAP1GTPASE: A PUTATIVE BIOMARKER FOR
DIAGNOSIS AND PROGNOSIS OF CERVICAL
CANCER ASSOCIATED WITH HUMAN PAPILLOMAVIRUS INFECTION?
NOROVIRUS
AS
CAUSE
OF
SEVERE
GASTROENTERITIS AMONG CHILDREN ADMITTED
IN A PEDIATRIC HOSPITAL IN BELÉM, PARÁ,
BRAZIL
ID: 00367-00001
Área: 05 - Virologia Humana e Saúde Pública
ID: 00368-00001
Área: 05 - Virologia Humana e Saúde Pública
Figueiredo, A.C.C, 2Pascoal-Xavier, M.A, 3Oliveira,
J.G., 4Ferreira, P.C.P.
1. CPqRR/FIOCRUZ, Centro de Pesquisas Rene
Rachou/FIOCRUZ, Av. Augusto de Lima,17152. UFMG,
Universidade Federal de Minas Gerais, Av. Presidente
SIQUEIRA, J.A.M., 2CARVALHO, T.C.N., 3ARAGÃO,
G.C., 4SPADA, P.K.P., 5TEIXEIRA, D.M., 6LIMA, I.C.G,
7
OLIVEIRA, D.S., 8SANTOS, M.C., 9MASCARENHAS,
J.D.P., 10LINHARES, A.C., 11GABBAY, Y.B.
1. IEC, INSTITUTO EVANDRO CHAGAS, ROD. BR 316,
EXPRESSION AND PURIFICATION OF CHIMERICAL
PEPTIDES OF DENGUE VIRUS USING
HETEROLOGOUS SYSTEMS
ID: 00366-00002
Área: 01 - Imunobiológicos
Batista, I. C. A., 2Fonseca, D. E., 3Corrêa-Oliveira, R.,
Oliveira, J.G., 5Calzavara-Silva, C.E.
1. CPqRR/FIOCRUZ, Centro de Pesquisas Rene
Rachou/FIOCRUZ, Av. Augusto de Lima, 1715
1
4
1
188
1
ABSTRACTS
S/N, KM 07, LEVILÂNDIA, ANANINDEUA/PARÁ/
BRASIL
The norovirus (NoVs) are the leading cause of epidemic
non-bacterial outbreaks of gastroenteritis and are also
an impor tant cause of infantile sporadic acute
gastroenteritis. Epidemiological investigations
demonstrated that person-to-person contacts and
contaminated food/water are the most important ways
of transmission. The aim of this study was to determine
the frequency of the NoVs in hospitalized children with
severe gastroenteritis in Belém, Pará, Brazil. From May/
2008 to April/2009, a total of 169 fecal specimens were
collected from diarrheic children under three years old.
These samples were initially screened by the Enzyme
Immunoassay (EIA) RIDASCREEN (3rd generation) kit
following the manufacturer’s instructions. After, the
specimens were tested by reverse transcriptionpolymerase chain reaction (RT-PCR), using the primers
Mon 432/434 and Mon 431/433 to detects NoVs GI and
GII, respectively. The nucleotide sequence was
determined by direct cycle sequencing. Of the 169
samples tested, 44.4% (75/169) were positive, of which
80% (60/75) were positive for EIA and 92% (69/75) for
RT-PCR. Considering the RT-PCR as the reference
method, an agreement of 87.6% (148/169) was observed.
The genomic sequencing was done in 31.9% (22/69) of
the samples, being 95.4% (21/22) belong to GII-4d and
4.6% to GII-b. The infection was more frequent in children
aged > 6 to 12 months (51.6%-31/60). The major peak
incidence were showed in September and October/2008
(63.6%-7/11). The positivity obtained in this study
(44.4%), was higher than the ones observed in other
researches conducted in Belém: 14.6% (1992/1994),
8.9% (1998/2000) and 12.5% (2003). The sequencing
data showed that the genotype GII-4 is circulating in
Belém since 2000. The age (> 6-12 m) that showed more
prevalence of NoVs was the same observed in other
study conducted in Belém. This research demonstrated
the importance of the NoVs as responsible for severe
gastroenteritis episodes in hospitalized children in Belém,
Pará.
Palavras-chaves: children, gastroenteritis, hospital,
Norovirus.
DETECTION AND MOLECULAR CHARACTERIZATION OF ASTROVIRUS IN FECAL SPECIMENS
OF CHILDREN ADMITTED WITH DIARRHEA IN A
PEDIATRIC HOSPITAL IN BELÉM, PARÁ, BRAZIL
ID: 00368-00002
Área: 05 - Virologia Humana e Saúde Pública
CARVALHO, T.C.N., 2SIQUEIRA, J.A.M., 3ARAGÃO,
G.C., 4TEIXEIRA, D.M., 5HERNANDEZ, J.M., 6LUCENA
M.S.S., 7 OLIVEIRA, D.S., 8 SOARES, L.S.,
9
MASCARENHAS, J.D.P., 10 LINHARES, A.C.,
11
GABBAY, Y.B.
1. IEC, INSTITUTO EVANDRO CHAGAS, ROD. BR 316,
S/N, KM 7, LEVILÂNDIA, ANANINDEUA/PARÁ/BRAZIL
Astroviruses (HAstVs) belong to the Astroviridae family
and are divided into two genera: Mamastrovirus and
Avastrovirus, which infect mammals and birds,
respectively. These are round and small viruses, with 28
to 40 nm of diameter and have a peculiar star-like
appearance. HAstVs-1 is the most prevalent type
worldwide. Transmission is mainly by fecal-oral route,
but water, food, and contaminated objects and surfaces
may also play a role. The main symptom in HAstVs
disease is watery diarrhea, but vomiting, fever, anorexia
and abdominal pain can be present. This study aimed at
both detecting and molecular characterizing HAstVs from
fecal specimens of children hospitalized due to diarrhea.
Samples were collected from February/2009 to January/
2010, from children under 3 years in Belém, Pará, Brazil.
These samples were submitted to RNA extraction by
silica method, followed by reverse transcriptionpolymerase chain reaction (RT-PCR) using primers 269/
270. Positive samples were purified using the QIAquick
Gel Extraction Kit and the nucleotide sequence was
determined by direct cycle sequencing. A positivity rate
of 6.5% (10/153) was observed, with strains being
characterized as types 1 (70%), 2 (20%) and 3 (10%).
HAstVs gastroenteritis was more frequent in children aged
0-6 months (10%). The peak incidence rate of HAstVs
gastroenteritis was observed in March/2009 (20%-3/15).
The overall positivity rate in this study was lower than
that in a previous study in Belém in 2003 (14.7% - 45/
305) involving children under 3 years of age. In contrast,
our positivity rate was higher than those previously
recorded in Greece (2.3%), Taiwan (2.9%) and Nigeria (5%).
As observed in most of studies, the HAstVs-1 was
predominant with concurrent detection of type 2 and 3.
Our data provide additional evidence on the importance
of HAstVs as cause of severe gastroenteritis in Belém.
In addition, we could demonstrate which genotypes were
circulating in Belém during February/2009 to January/2010.
Palavras-chaves: Astrovirus, children, gastroenteritis, hospital.
EVIDENCE OF ERYTHROVIRUS INFECTION IN A
COHORT OF HIV-INFECTED PATIENTS ATTENDING
AT HOSPITAL UNIVERSITÁRIO ANTONIO PEDRO
(HUAP), NITEROI,RJ
ID: 00369-00001
Área: 05 - Virologia Humana e Saúde Pública
1
Pereira,RFA, 2Azevedo, KML, 3Setubal,S, 4de Lima,M,
SIQUEIRA, MMAT, 6Cubel Garcia, RCN, 7de Oliveira,
1
5
189
ABSTRACTS
SA
1. UFF, Universidade Federal Fluminense, HUAP - Rua
Marquês do Paraná, 303, 2º. AndCentro, Niterói, RJ. CEP.
24030-210.2. UNIGRANRIO, Universidade do Grnade
Rio, rua Prof. José de Souza Herdy, 1160 - Duque de
Caxias/RJ3. FIOCRUZ, Fundaçao Oswaldo Cruz, Av.
Brasil, 5000 - Manguinhos/RJ4. UFF, Universidade
Federal Fluminense, IB- Rua Professor Hernani Melo
101, São Domingos, Niterói, RJ. CEP: 24210-130
Erythrovirus infection is generally acute and self-limiting.
However in immunocompromissed individuals (HIVinfected patients) the infection is not readily cleared and
its long persistence leads to chronic anemia. In these
cases, anemia may be treated using standard intravenous
immunoglobulin infusions. Despite the availability of
treatment, laboratory diagnosis is essencial in order to
confirm those cases of anemia triggered by erytrovirus
infection. The purpose of this study was to find evidence
of erythrovirus infection in a cohort of HIV-infected
patients attending at HUAP during 2001-2008 in Niteroi,
RJ. Sera samples from 88 patients were tested for IgG
antibodies using a commercial enzyme immune-assay
(BiotrinTM, Dublin, Ireland) as also for erythrovirus DNA
by PCR using primers that amplify a 102bp fragment of
the NS1 gene. Among the 88 patients, 28 seroconverted
to IgG-positive. Two sera samples from these 29 patients,
obtained before and after seroconversion and 59 sera
from the IgG negative patients were tested by PCR.
Erythrovirus DNA was found in sera of five patients and
two of these sera were collected during the outbreak of
the disease in 2005. One of these patients evaluated
with anemia at seroconversion period but for all the others
there was not enough data to identify the occurrence of
anemia during seroconversion . Although anemia in HIVinfected patients is quite common and may be
multifatorial, the laboratorial diagnosis of erythrovirus
infection during outbreaks of the disease may be a
suitable tool and helps to elucidate these cases Finnancial
support: MCT/CNPq: 14/2008 Edital Universal (Processo
471618/2008-0) Área: Humana Tema: Parvovirus humano
Arquivo: Pereira, RFA Apresentador: Renata F A Pereira.
Palavras-chaves: diagnóstico molecular, eritrovirus, HIV.
EASTERN EQUINE ENCEPHALITIS IN NORTHEASTERN BRAZIL
ID: 00370-00001
Área: 03 - Virologia Animal
Silva, M. L. c. R., 2 Galiza, G. J. N., 3 Dantas, A.F.M.,
Oliveira, R.N., 5Iamamoto, K.., 6 Achkar, S.M., 7 RietCorrea, F.
1. UFCG/CSTR, Universidade Federal de Campina
1
4
190
Grande Centro de Saúde e Tecnologia Rural Campus de
Patos, Av. Santa Cecília, Cx. Postal 64, Patos, PB 5870000, Brasil.2. IP, Instituto Pasteur, Avenida Paulista,393
Cerqueira César 01311-000 São Paulo-SP
Outbreaks of eastern equine encephalitis observed from
May 2008 to August 2009 in the Brazilian states of
Pernambuco, Ceará, and Paraíba are reported. The
disease occurred in 93 farms affecting 229 equidae with
a case fatality rate of 80.27%. Main clinical signs were
circling, depression or hyperexcitability, ataxia, and
progressive paralysis with a clinical manifestation period
of 3-15 days. Main histologic lesions were a diffuse
lymphocytic encephalomyelitis with neuronal death,
satellitosis, neuronophagia, and hemorrhages being more
severe in the cerebral grey matter of the telencephalon,
diencephalon and mesencephalon. Some animals had
also areas of malacia in the telencephalon, thalamus
and basal nuclei. From one case, the virus was isolated
by mice inoculation and in other 13 cases was identified
as eastern equine encephalitis virus by semi-nested RTPCR. After DNA sequencing, all samples were identified
as eastern equine encephalitis through the blastn
analysis, but samples from the Ceará and Paraíba states
corresponded to the same cluster, and the sample from
Pernambuco to a different cluster.
Palavras-chaves: Brazilian semiarid, equine
encephalomyelitis, laboratory diagnosis, semi-nested RTPCR.
PARALYTIC RABIES IN SWINE
ID: 00370-00002
Área: 03 - Virologia Animal
Pessoa, C. R. M., 2Silva, M. L. C. R., 3 Gomes, A.
A.B., 4 Garcia, A.I.E., 5 Ito, F. H., 6Brandão, P. E., 7 RietCorrea, F.
1. UFCG/CSTR, Universidade Federal de Campina
Grande Centro de Saúde e Tecnologia Rural Campus de
Patos, Av. Santa Cecília, Cx. Postal 64, Patos, PB 5870000, Brasil.2. FMVZ/USP, Faculdade de Medicina
Veterinária e Zootecnia, Universidade de São Paulo, Av.
Prof Dr Orlando Marques de Paiva 87, 05508-000 Cid
Universitaria, São Paulo
1
Rabies virus was diagnosed in two pigs in the semiarid
region of Paraíba State, Brazil. Main clinical signs were
paresis and paralysis of the pelvic limbs. Samples of
tissues from the whole brain and spinal cord of a pig,
examined were negative by the direct fluorescent antibody
test and the samples of the thalamus, pons, medulla
oblongata and cervical, thoracic and lumbar medulla were
positive by mouse inoculation test. The histological
lesions of the two pigs were diffuse non-suppurative
encephalomyelitis with severe lesions in the spinal cord,
ABSTRACTS
mild to severe lesions in the brain stem, and mild or
absent lesions in the forebrain. After genetic and
phylogenetic analyses, the swine isolate was identified
as vampire bat-related rabies virus, included in a clade
very closely associated to the Desmodus rotundus bat
in Latin America. Despite the various diagnosis of rabies
in pigs this is the first report of clinical signs and
pathology of rabies in swine transmitted by vampire bats.
The existence of caves in the region favors the presence
of vampire bats and, consequently, the transmission of
the virus to other animal hosts, including wild animals,
which perpetuates the presence of virus in the Brazilian
semiarid region.
Palavras-chaves: epidemiology, vampire bats, virus
isolation, zoonotic.
SUBCLINICAL DISEASE ASSOCIATED WITH
INFECTION BY Porcine circovirus 2 (PCV-2) ON
SWINE HERD
ID: 00372-00001
Área: 03 - Virologia Animal
Simão, G. M. R, 2 Silva Júnior, A., 3 Silva, F.M.F,
Ferreira, H.C.C., 5Fausto, M.C., 6Lobato, Z.I.P, 7Fietto,
J.L.R, 8Almeida, M.R.
1. UFV, Universidade Federal de Viçosa, Av. Ph Rolfs,
s/n. Viçosa, MG.2. UFMG, Universidade Federal de
Minas Gerais, Escola de Veterinária da UFMG Av. Antônio
Carlos 6627 Caixa Postal 567, Campus d
1
Among the animals tested, 53.13% showed no
significant lesions in the lymph node, 37.5% were
classified as stage I and 9.37% in stage II lesion. Titration
of NA was variable among the pigs tested. The titles
ranged 64-2048, the latter being represented by 65.62%
of the animals. These results allowed the evaluation and
demonstration of the occurrence of subclinical disease
in the herd studied. Thus, there still remains a subject of
major research studies on the implication of this new
presentation of the disease in swine herds.
Financial support: FAPEMIG
Palavras-chaves: PCV-2, Porcine circovirus 2,
Subclinical disease.
SEROLOGIC EVIDENCE OF FLAVIVIRUS INFECTIONS IN HORSES OF BRAZIL
ID: 00373-00001
Área: 03 - Virologia Animal
SILVA, J.R., 2CHÁVEZ, J.H., 3FIGUEIREDO, L. T. M.
1. FMRP-USP, Faculdade de Medicina de Ribeirão PretoUSP, Av. Bandeirantes, 3900, Monte Alegre, Ribeirão
Preto, SP
1
4
The PCV2 is a virus belonging to the family Circoviridae
and is associated with various emerging syndromes and
related culling of pigs in the world, known PCV2
associated diseases (PCVAD). Evidence shows that this
type of viral infection has considerable viral load in
tissues associated with lymphoid depletion and may
compromise the potential production of pigs. With the
introduction of commercial vaccines against PCV2 in
the farms, forms of the disease began to be controlled.
However, no manifestation of clinical signs does not
guarantee the productive performance. Thus, the
objective of this work was to analyze the laboratory
indicators of the subclinical disease associated with
PCV2. Samples were collected from individual sera and
inguinal lymph nodes of 64 clinically healthy slaughter
pigs. Viral load in lymph nodes and serum was measured
by the polymerase chair reaction in real time and
neutralizing antibodies (NA) were titrated by viral
neutralization test. Lymph nodes were evaluated without
significant injury, stage I (mild injury), stage II (moderate
injury) or stage III (severe lesion). The viral load present
in the inguinal lymph nodes and at the 10 sera ranged
from 3.82 to 6.77 log copies PCV2/500ng DNA total and
0-2.89 log copies of PCV2/5 ìl of DNA, respectively.
Arboviruses are a serious public health problem in Brazil
and, from these, the most important are those caused
by Flavivirus. Eleven Flavivirus have been described in
Brazil including Saint Louis Encephalitis Virus (SLEV)
and Rocio Virus (ROCV) that are bird zoonotic viruses
transmitted by Culex mosquitoes. In 2006, SLEV was
detected causing the first outbreak reported in Brazil,
producing acute febrile and encephalitis cases in the
middle of a large dengue type 3 epidemic, in São José
do Rio Preto city, SP. ROCV was responsible for an
encephalitis outbreak in Vale do Ribeira region, São Paulo
state, during 1973-1980, with approximately 1000 reported
cases that produced 100 deaths. The virus has
disappeared after this outbreak. It is known that large
domestic animals such as horses are more exposed to
Culex mosquitoes than humans and probably become
infected with these arboviruses. Thus, we show here
results of a serologic survey in horses to ROCV and
SLEV. Serum samples from 753 animals from five
Brazilian States were collected. Sera were tested by an
IgG-ELISA using a purified recombinant domain III
polypeptide of SLEV envelope protein as antigen. Each
serum was tested in duplicate and also against a negative
antigen control (purified Escherichia coli extract). The
results showed that 55 of 183 (30%) animals from São
Paulo, 140/267 (52.4%) from Mato Grosso do Sul, 3/15
(20%) of Minas Gerais, 41/200 (20.5%) of Rio de Janeiro
and 32/88 (36.4%) from Paraiba, had IgG antibodies to
SLEV (35.9% of all studied animals). Besides, 144/753
(19.1%) of these 753 animals had also IgG antibodies to
191
ABSTRACTS
ROCV. The results indicate an intense circulation of
flaviviruses infecting horses in the study sites and that
human infections could also occur in the locations where
these animals live. Therefore, serosurvey in horses
proved to be an appropriate approach for surveillance of
Flavivirus infections by SLEV and ROCV.
Financial support: FAPESP
Palavras-chaves: ELISA, Flavivirus, Horses, Serology.
The Effect of Bafilomycin A1 on Sindbis Virus
Infection
ID: 00374-00001
Área: 04 - Virologia Básica
S. Hunt, 2R.Hernandez, 3D.T.Brown
1. NCSU, North Carolina State University, 128 Polk Hall
Raleigh NC
1
Bafilomycin A1 is a specific inhibitor of the vacuolarATPase, which is responsible for pH homeostasis of the
cell and for acidification of endosomes. Bafilomycin A1
has been commonly used as a method of inhibition of
infection by viruses known or suspected to follow the
path of receptor-mediated endocytosis. The exact method
of entry for Sindbis Virus, the prototype Alphavirus,
remains undecided.. To further investigate the role of the
V-ATPase in Sindbis Virus infection, the effects of
Bafilomycin A1 on the infection of BHK cells by Sindbis
Virus were studied. Bafilomycin A1 was found to block
the expression of a virus-encoded reporter gene in both
infection and transfection. The inhibitory effects of
Bafilomycin A1 were found to be reversible. The results
suggest that, in the presence of drug, virus RNA enters
the cell and is translated but that proper folding of the
product proteins requires the function of the V-ATPase.
These results underscore the difficulties encountered in
using chemical inhibitors to explore virus entry.
Palavras-chaves: Bafilomycin, BHK, Sindbis.
HEPATITIS C VIRUS IN PATIENTS WITH
TUBERCULOSIS IN CENTRAL BRAZIL
ID: 00376-00001
Área: 05 - Virologia Humana e Saúde Pública
Reis, N.R.S., 2Lopes, C.L.R., 3Teles, S.A., 4Carneiro,
M.A.S., 5Matos, M.A.D., 6Andrade, A.A., 7Marinho, T.A.,
8
Espírito-Santo, M.P., 9Lampe, E, 10Martins, R.M.B.
1. IPTSP, UFG, Instituto de Patologia Tropical e Saúde
Pública, Universidade Federal de Goiás, Caixa Postal
131, 74605-031, Goiânia-GO2. FEN, UFG, Faculdade de
Enfermagem, Universidade Federal de Goiás, Caixa
Postal 131, 74605-031, Goiânia-GO3. IOC, FIOCRUZ,
Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Av.
Brasil, 4365, Manguinhos, 21040-900, Rio de Janeiro-
RJ
Hepatitis C virus (HCV) infection is a global public health
problem. Phylogenetic analysis of full-length or partial
sequences of HCV strains has led to the identification
of seven HCV genotypes and a large number of subtypes.
Tuberculosis (TB) is still among the most life-threatening
infectious diseases, resulting in high mortality in adults.
Brazil continues to be one of the 22 countries that,
together, account for 90% of all TB cases worldwide.
However, no data are available on HCV infection in this
population in Brazil. Thus, the aim of the present study
was to investigate the HCV prevalence and genotypes/
subtypes in patients with tuberculosis in Central Brazil.
A cross-sectional survey was carried out with 402
patients with TB who were recruited at out-patient and
in-patient units in the hospital for TB treatment in Goiás
state, Central Brazil. All individuals were interviewed,
and their serum samples were tested for the presence
of antibodies to HCV. Anti-HCV positive samples were
tested for HCV RNA by PCR amplification of the 5’ NC
and NS5B regions and were genotyped using the LiPA
assay and nucleotide sequencing, respectively. The
prevalence of HCV infection was 7.5% (95% CI: 5.210.6). HCV RNA was detected in 23/30 anti-HCV positive
samples, in which genotypes 1 (n = 17) and 3 (n = 6)
were determined by LiPA. Using phylogenetic tree analysis
of the NS5B region, fourteen sequences were classified
as genotype 1, subtypes 1a (n = 12) and 1b (n = 2), and
six sequences as genotype 3, subtype 3a. In conclusion,
a high HCV infection prevalence was found among
patients with tuberculosis in Brazil, indicating that hepatitis
C may have an important impact upon TB management,
treatment and control. Finally, the HCV genotypes 1a,
1b and 3a identified in the study population are also those
that are currently predominant in Central Brazil. Financial
support: CNPq.
Palavras-chaves:
HEPATITIS
C
VIRUS,
TUBERCULOSIS, PREVALENCE, GENOTYPES.
THE PRESENCE OF THE BRAZILIAN HIV-1 SUBTYPE
B VARIANT (B’-GWGR) IS DECREASING ALONG THE
TIME
ID: 00377-00001
Área: 05 - Virologia Humana e Saúde Pública
1
192
Arruda, L.B., 2Martinez, M., 3Araújo, M. L., 4Komninakis,
S.V., 5Gonsalez, C., 6Duarte, A., 7Casseb, J.
1. IMTSP/USP, Institute of Tropical Medicine of Sao Paulo
- USP, Dr Eneas de Carvalho Aguiar Avenue, 470, São
Paulo, Brazil2. LIM56/FMSUP, Laboratory of Investigation
in Dermatology and Immunodeficiencies, Department of
Dermatology, Dr Eneas de Carvalho Aguiar Avenue, 470,
3th floor, IMTII building, Sao Paulo3. ADEE3002/FMUSP,
HIV Out-clinic, Ambulatory of Secondary Immunodeficiencies – ADEE3002, Department of Dermatology,
1
ABSTRACTS
Ho, Dr Eneas de Carvalho Aguiar Avenue, 255, Sao Paulo,
Brazil4. UNILUS, Laboratory of Molecular Biology Fundação Lusíada, R Dr Oswaldo Cruz,179, Santos, Brazil
Introdution: There are two distinct HIV-1 subtype B
variants cocirculing in Brazil. The main subtype B strain
resembles the HIV-1 isolates from USA and Europe,
showing the GPGR motif in the V3 loop crown of the
envelope protein. The Brazilan variant, named B’, has a
tryptophan replacing the proline charactering the GWGR
motif. Several studies have indicated high frequencies
of B’ variant among the Brazilian HIV-1-infected subjects
and it protective effect on disease progression. Material
and Methods: A total of 209 V3 sequences from DNA
samples of HIV-1-infected subjects under clinical
monitoring in the HIV Out-clinic ADEE3002 were
evaluated. The samples were collected in the years 2002
(n=61), 2008 (n=78) and 2010 (n=70). Results: The
frequency of GWGR variant was of 44.3% (27/61), 27%
(21/78) and 17.1% (12/70) among the sequences from
2002, 2008 and 2010, respectively, showed a significant
decrease between 2002 and 2008 (p=0.04) and 2002 and
2010 (0.001). The distribution of GPGR variant and other
amino acid changes showed an increase along the time,
but not statistically significant. Conclusion: These finding
evidence the dynamic genetic variability of the HIV-1 in
the Brazilian population and may represent an important
role in the clinical practice since the protective
characteristic of the B’ variant seems disappearing
among the Brazilian HIV-1-infected subjects.
Palavras-chaves: HIV-1, subtype B, V3 loop.
IMPACT OF ACUTE RESPIRATORY INFECTION OF
PROBABLY VIRAL ETIOLOGY ON HOSPITAL
WORKERS HEALTH CENTER SERVICE OF SAO
PAULO HOSPITAL – UNIFESP
abstracted of the medical record. ARI of probably viral
etiology was considered for the ICDs J00 to J12, J20,
J21 and J40. We analyzed 5297 medical visit charters
belonging to 2872 adult different patients and 2759 from
910 pediatric ones. HCWs most frequent reasons for
attendance were: respiratory diseases (ICD: J00-J99) 1957 (37%) and symptoms, signs and abnormal clinical
and laboratory not elsewhere classified (ICD: R00-R99) 660 (13%). Indeed, ARIs of probably viral etiology were
responsible for 1771 attendances, representing 33% of
the adult total demand. ARIs were the major reason for
attendance in each age group 20 to 29 years (41%), 30
to 39 (37%) and 40 to 65 years (29%). Among pediatric
patients, ARIs were the reason for 45.6% of the
consultations and the most common symptoms in the
patients files were cough, fever, coryza, sore throat and
nasal obstruction. A higher number of visits due to
respiratory illness occurred during winter months,
decreased in summer months although remained the first
reason of medical demand. This pilot surveillance study
assessed the impact of ARIs on emergency service from
HCWs and their children and showed the relevance of
this disease as cause of frequent medical demand.
Further studies could address cost benefit analyses of
vaccination and others interventions in this group of
patients.
Palavras-chaves: Acute respiratory infections, Health
care workers, Health care workers and children.
SURVEILLANCE OF RESPIRATORY INFECTIONS
EPISODES OF PROBABLE VIRAL ETIOLOGY IN THE
HOSPITAL SAO PAULO/UNIFESP COMPLEX
ID: 00378-00002
Área: 05 - Virologia Humana e Saúde Pública
NAKANO, M.S.L., 2RIBEIRO, R.A., 3BRANDES, P.H.R.,
WATANABE, A.S.A., 5BELLEI, N.C.J.
1. UNIFESP, Universidade Federal de São Paulo, Lab
Virologia, Rua Pedro de Toledo, 781, 15 andar, Vila
Clementino, Sao Paulo-SP
1
ID: 00378-00001
Área: 05 - Virologia Humana e Saúde Pública
RIBEIRO, R.A., 2 BRANDES, P.H.R., 3 WATANABE,
A.S.A., 4CARRARO, E., 5BELLEI, N.C.J.
1. UNIFESP, Universidade Federal de São Paulo, Lab
Virologia, Rua Pedro de Toledo, 781, 15 andar, Vila
Clementino, Sao Paulo-SP
1
Acute respiratory infections (ARIs) are the most common
disease in humans and an important cause of morbidity
and mortality worldwide. Two important risk populations
for ARIs are health care workers (HCWs) and their
children. The medical records of NASF – Hospital Workers
Health Assistance Center, of both adult and pediatric
consultations, were collected and assessed since May
4, 2009 until April 29, 2010. Information about age, sex,
date of medical consultation and the diagnose according
to International Classification of Diseases (ICD) was
4
Acute respiratory diseases (ARD) may affect people of
all ages. Respiratory viruses play a major role as etiologic
agents of this illness. The aim of this study was to
evaluate the occurrence of ARDs in health care workers
(HCW) of Sao Paulo hospital. The study consists of
interviews containing a questionnaire to obtain data of
clinical presentation. Further contact about new episodes
was accomplished to follow-up these patients. Data from
92 HCWs attended in NASF (Hospital Workers Health
Assistance Center) from September 2009 to May 2010
were collected. HCWs of different areas in the Hospital
including doctors, medical residents, nurses, cleaners,
nutritionists were interviewed. Age ranged between 21
193
ABSTRACTS
and 63 years old with average age of 34.9 years and
median of 31 years. Of them 23.9% were men and 76.1%
were women. Frequent contact with children up to 10
years old were described by 51.1% of HCWs. Flu
vaccination in 2009 was reported by 37.7%. The H1N1
vaccination in 2010 reached 63.04% of the studied
patients. Total number of episodes reported by HCWs in
the year before the present study had an average of
1.02 episodes per year per professional. After the study
this average increased because people had more
respiratory infections this year or because they reported
more episodes.
Palavras-chaves: Respiratory Infections, Health Care
Works, Vaccination.
2004 and 2005 evolved from previous Asian strains
causing the South Korean outbreak of AHC during the
summer of 2002 (South Korea 2002). On the other hand,
the outbreak of AHC in 2009 in Pernambuco State,
originated from a reintroduction of a new CA24v strain
circulating in Asia during the year of 2007, where CA24v
is continuously in circulation since 1970. Although data
obtained by the analysis of the partial VP1 gene shows
to be consistent with those using 3C, the phylogeny
based on complete VP1 gene showed a higher temporal
resolution indicating that VP1 could be more informative
for studies of molecular epidemiology of CA24v.
Financial support: FIOCRUZ
Palavras-chaves: Acute Hemorrhagic Conjunctivitis, CA24v, Phylogenetic Analysis.
GENETIC RELATIONSHIP AMONG EPIDEMIC
STRAINS OF COXSACKIEVIRUS A24 VARIANT
ISOLATED FROM ACUTE HEMORRHAGIC
CONJUNCTIVITIS (AHC) CASES IN BRAZIL.
GENETIC DIVERSITY OF ECHOVIRUS 30 INVOLVED
IN ASEPTIC MENINGITIS CASES IN BRAZIL (1998 –
2008).
ID: 00379-00001
Área: 05 - Virologia Humana e Saúde Pública
ID: 00380-00001
Área: 05 - Virologia Humana e Saúde Pública
Tavares, F.N., 2Campos, R.M., 3Burlandy, F.M., 4Melo,
M.M.M., 5Costa, E.V., 6da Silva E.E.
1. FIOCRUZ, Laboratório de Enterovirus/ IOC, Av. Brasil,
4365, Manguinhos, CEP 21045900, RJ2. LACEN/PE,
Laboratório Central de Saúde Pública de Pernambuco,
Rua João Fernandes Vieira - Bairro Boa Vista, CEP:
50050-210 / Recife – PE
1
Acute hemorrhagic conjunctivitis (AHC) is a highly
contagious viral syndrome, self-limiting and is
characterized by sudden onset of ocular pain, swelling
of the eyelids, a foreign body sensation, epiphora
(excessive tearing), eye discharge, and photophobia.
Palpebral conjunctival follicular reaction, subconjunctival
hemorrhage, and congestion are common. The symptoms
appeared after a short incubation period of 12 to 48 hours
and the clinical signs usually disappear in 1 to 2 weeks.
Coxsackievirus A24v (CA24v) is one of major agents
causing acute hemorrhagic conjunctivitis. In Brazil, the
first reported outbreak occurred in 1987 at Pará State.
During sixteen years, the virus was not reported, until it
appeared in 2003 in Southern Brazil and spread to almost
states, causing outbreaks until 2005 in Vitória, Espírito
Santo State. More recently another outbreak occurred in
Recife, Pernambuco State, where more than 11.000
cases were reported in only one of the state public health
hospitals, in May 2009. In this study, we describe a
phylogenetic study based on 3C (549bp) and VP1 (915bp)
genes from 37 isolates of CA24v isolated from the last
five reported outbreaks of AHC occurred in Brazil (1987,
2003, 2004, 2005 and 2009). Our data suggests that
CA24v that circulated in Brazil during the years of 2003,
Aseptic meningitis is one of the most common
neurological disorders caused by Enterovirus and,
among them, Echovirus 30 (E30) is described as the
main etiological agent of many sporadic cases and
outbreaks. The application of phylogenetic analysis
allows studies of genetic diversity between viral strains.
For enteroviruses, VP1 gene is often used in these
studies, among other reasons, for its relevance in the
immune response to enteroviruses and because of its
genetic variation rates. From 302 Non-Polio Enterovirus
isolated over a period of ten years (1998 to 2008) from
Cerebrospinal Fluid (CSF) collected from aseptic
meningitis cases, 177 were identified by partial
sequencing as E30 (58,6%). Forty-eight E30 isolates
were selected for phylogenetic analysis of complete VP1
gene (876 nt). They were compared with other Brazilian
and foreign strains. Bastianni prototype strain of E30
was also included in the analysis. E30 VP1 sequences
segregated into three distinct major groups and eight
subgroups, which were tightly linked to the year of
isolation. In general, sequence divergence among E30
strains ranged from 0,2-13,8%. It was not defined a
common direct ancestor for this set of E30 strains. Isolate
36972-CE-07 was genetically related with E30 strains
1
194
DOS SANTOS, G.P.L., 2COSTA, E.V., 3TAVARES, F.N.,
COSTA, L.J., 5DA SILVA, E.E.
1. FIOCRUZ, Laboratório de Enterovírus / IOC, Av. Brasil,
4365 - Manguinhos - Rio de Janeiro, RJ – CEP: 210459002. UFRJ, Laboratório de Genética e Imunologia das
Infecções Virais - IMPPG, Av. Carlos Chagas Filho, 373
- Bl. I - Rio de Janeiro - CEP: 21944-970
4
ABSTRACTS
from Pará State (3,2-4,6% of divergence). Isolates from
Group I were genetically related with an E30 sequence
of an USA isolate from 1997 (2,4-5,7%). Two E30 isolated
in Argentina in 2007, showed close relationship to
isolates of Group III (5,8-6,0%). 36972-CE-07 strain may
descend from Pará E30 strains. One of the Group III
isolates may have originated 2007 Argentine strains.
Likewise, Group I isolates and 1997 USA isolate seems
to have a common origin. The present study corroborates
and complement existing results about E30 genetic
variability. Financial Support: CAPES; FIOCRUZ.
Palavras-chaves: Aseptic Meningitis, Echovirus 30,
Phylogenetic Analysis.
PREVALENCE OF INFECTIONS BY THE
HEPATITIS’S VIRUSES IN A RURAL LOCALITY
UNDER INFLUENCE OF A MINING PROJECT IN THE
WEST OF PARÁ, BRAZIL
ID: 00381-00001
Área: 05 - Virologia Humana e Saúde Pública
Naiara Maia, 2 Manoel Soares, 3Elisabeth Oliveira,
Ocinéa Costa, 4Ocinéa Costa, 5Renilde Alves, 6Heloisa
Nunes
1. IEC, Instituto Evandro Chagas, Rodovia BR 316 Km
7 s/nº, Levilândia, Ananindeua, Pará2. ICS/UFPA,
Instituto da Ciência e da Saúde, Universidade Federal
do Pará, Praça Camilo Salgado nº 1, Umarizal, Belem,
Pará
1
serological profile of the 19-years-old people or under
was found in 41,8% (64/153). It hasn’t been detected
any person with infection by the HCV. The absence of
HBV carriers prevented the HBeAg, anti-HBe and total
anti-HD tests to be done. The Café Torrado Community
has presented high prevalence for HAV, mild endemicity
for HBV, vaccination coverage under expectations for
HBV and low endmicity for HCV and HDV. Financial
support: Evandro Chagas Institute, Fapespa, Alcoa
Omnia Minérios.
Palavras-chaves: Endemicity, Hepatitis, Prevalence, Viral.
Host rage mutations of Sindbis virus define
alphavirus host adaptive functions.
ID: 00382-00001
Área: 04 - Virologia Básica
Hernandez, R., 2Piper, A., 3Ribeiro, M., 4Kononchik, J.,
Vancini, R., 6Hunt, S., 7Brown, D.T.
1. NCSU, North Carolina State University, 128 Polk Hall,
Raleigh NC 27695
1
5
4
Viral hepatitis’s are infectious diseases that share the
primary hepatotropism and are an important worldwide
public health problem. The emergence and the increase
of the frequency of these diseases, particularly in the
Amazon are indeed different from that observed in other
places of the world. To define the frequency of infections
markers by the viruses of hepatitis A, B, C and D in the
Café Torrado Community, Juriti, West of Para, Brazil,
currently under influence of a mining project. In November,
2009, serum samples collected in the Café Torrado
Community were submitted to analysis of serum markers
of hepatitis A (HAV), B (HBV), C (HCV) and D (HDV) by
immunoenzymatic techniques. The project was approved
by the Ethics Committee of Evandro Chagas Institute
(CAAE 0022.0.072.000-09). 232 individuals were included
in this study being 53% (123/232) female. The analysis
showed a global prevalence of 78% (181/232) for total
anti-HAV with 75% (33/44) of susceptible between 1 and
4 years of age. It hasn’t been found any HBV carrier.
This study showed a total prevalence of 2,2% (5/232)
for any HBV marker; 1,3% (3/232) had signs of previous
infection; 0,9% (2/232) had anti-HBc+ only; 35,8% (83/
232) had a compatible profile with vaccine protection,
37,9% (88/232) were susceptible to HBV and the vaccine
The broad host range of the alphaviruses, spanning phyla,
suggests that these viruses have acquired genetic
functions which have adapted them specifically to
replicate in the unrelated biochemical and genetic
environment of insects and vertebrates. We have shown
that large deletions in the transmembrane domain of the
Sindbis virus E2 membrane glycoprotein results in virus
which replicates efficiently in insect cells but not
mammalian cells( Mosquito Restricted MR mutants). We
have used random mutagenesis of the Sindbis virus
genome to produce additional mutations which restrict
the ability of the virus to replicate to the insect cell.
Products of mutagenesis where amplified in insect cells
and clones were selected which showed clear MR
phenotype. 17 MR mutants were selected for further
characterization by complementation with a well
characterized collection of Sindbis temperature sensitive
mutants. Complementation allowed us to identify the gene
in which the MR mutation resides and sequencing has
identified the changes in some of the MR mutant genes.
These results define genetic elements which are essential
for replication in the vertebrate host.
Palavras-chaves: Alphavirus, Sindbis, mutations.
NEW STRUCTURE OF DENGUE-2 VIRUS REVEALED
BY CRYO-ELECTRON MICROSCOPY AND
CHEMICAL CROSS-LINKING
ID: 00383-00001
Área: 05 - Virologia Humana e Saúde Pública
195
ABSTRACTS
Vancini, R.G., 2Paredes, A., 3Ribeiro, M., 4Ferreira, D.F.,
Hernandez, R., 6Brown, D.T.
1. NCSU, North Carolina State University, 128 Polk Hall
- Raleigh, NC2. UTHSC, University of Texas Health
Science Center, Houston, TX3. IMPPG - UFRJ,
Departamento de Microbiologia - Universidade Federal
do Rio de Janeiro, CCS - Ilha do Fundão - Rio de Janeiro,
RJ
1
5
Dengue Virus is an enveloped RNA virus that belongs to
genus Flavivirus and is considered the most important
mosquito-borne viral pathogenic agent worldwide.
Infection with Dengue virus (DENV) have a huge impact
on global health putting more than 500 million people in
tropical areas at risk of developing dengue fever.
Understanding the three dimensional structure of dengue
virus is critical to investigating its function and for the
design of agents to control the spread of this disease.
The previously proposed structure of dengue virus has
been produced in context of crystallographic work of Tick
Borne encephalitis virus and Dengue-2 E-protein. This
exercise resulted in a model 500 Å in diameter made up
of 180 copies of E protein arranged in dimers in a T=3
icosahedral geometry. In the present study we took
advantage of improved purification methods and cryoEM reconstruction to investigate the structure of Dengue
virus serotype 2 (DENV-2). In addition, the purified
particles were further stabilized by a chemical crosslinking technique. We demonstrate that our structure is
significantly larger and has a different symmetry than
previously published models, with distinct trimers of
envelope proteins protruding from the virus surface and
a triangulation number calculated to be T=13. The
resulting structure is dramatically different from those
previously published in both size and symmetry and will
contribute significantly to our understanding of the
structure, assembly and function of this important human
pathogen. Palavras-chaves: Dengue, Flavivirus,
Estrutura, Virologia Humana
RESTRICTION FRAGMENT LENGTH POLYMORPHISM AS AN USEFUL TOOL TO STUDY HIV-1
SUBTYPES AND RECOMBINANTS
the environment where it is replicating. The main
circulating viruses in Brazil belong to subtypes are B, C
and F. The co-circulation of multiple subtypes favors the
emergence of recombinant viruses formed by different
subtypes. Determination of the HIV-1 subtypes cocirculating in a target population is mainly achieved by
sequencing of sub-genomic segments followed by
phylogenetic analysis. Although sequencing is a very
reliable methodology it can be very time consuming and
expensive. In our study we sought to verify the HIV-1
subtype distribution in samples from the State of São
Paulo collected from 2006 to 2008. As opposed to
sequencing we applied a previously published method
in which subtypes can be assigned to restriction fragment
length polymorphism (RFLP) patterns. By applying this
method we could rapidly investigate the subtype
distribution in 47 samples from long standing HIV-1
infections. Our data indicated that the main circulating
virus still belong to subtype B followed by subtype F
viruses. A number of samples could not be assigned a
subtype yielding different RFLP patterns. These samples
were subsequently sequenced and analyzed by
phylogenetic methods. Sequencing analysis
demonstrated that RFLP unclassified samples were in
fact recombinant viruses. According to our results at least
36% of samples harbored subtypes B and F
recombinants. Such high recombination frequency
cer tainly allows HIV-1 to explore new genomic
compositions that may lead to better adapted viruses.
The applied RFLP method proved to still be a useful tool
when determining the HIV-1 subtype distribution and an
appropriate method to identify recombinant viruses.
Financial support: FAPESP.
Palavras-chaves: RFLP, HIV-1, subtypes, recombinants.
EXPRESSION OF AN RECOMBINANT ANTIVIRAL
PROTEIN OBTAINED FROM LONOMIA OBLIQUA
HEMOLYMPH IN A BACOLOVIRUS/CELLS Sf-9
SYSTEM
ID: 00385-00001
Área: 04 - Virologia Básica
Carmo, A. C. V., 2 Giovanni, D.N.S., 3 Corrêa, T.P.,
Martins, L.M., 5Stocco, R. C., 6Calderón, R.C., 7Veiga,
A.B.G., 8Moraes, R.H.P., 9Suazo, C.A.T., 10Mendonça,
R. Z.
1. IBU, Instituto Butantan, Av. Vital Brasil, 1500, São
Paulo2. UFSCar, Universidade Federal de São Carlos,
Rodovia Washington Luís, km 235 - SP-3103. UFCSPA,
Universidade Federal de Ciências da Saúde de Porto
Alegre, Rua Sarmento Leite, 245 Porto Alegre
1
ID: 00384-00001
Área: 05 - Virologia Humana e Saúde Pública
Nunes, E.R.M., 2Zukurov,J.P., 3Janini, M.
1. UNIFESP, Universidade Federal de São Paulo, Rua
Pedro de Toledo, 781 - 16º andar
1
The human immunodeficiency virus type 1, HIV-1 has
an important genetic diversity. The ability to diversify
the genetic information contained in the genome allows
the HIV-1 to adapt to selective pressures imposed by
196
4
The control of viral infections, especially those caused
by influenza viruses, is of great interest in Public Health.
ABSTRACTS
Several studies have shown the presence of active
principles in the hemolymph of arthropods, some of
which are of interest for the development of new
pharmacological drugs. Recently, we have demonstrated
the existence of a potent antiviral principle in the
hemolymph of Lonomia obliqua caterpillar. This purified
protein reduced virus production (TCID50 ml–1) by more
than 157 fold (from 3.3±1.25x107 to 2.1±1.5x105) to
measles virus and 61 fold to polio virus (2.8±1,08x109
to 4.58±1.42x107). Therefore, this study aims to produce
recombinant bacmids containing sequences encoding
this antiviral protein in baculovirus/Sf-9 cells system.
Total RNA from L. obliqua caterpillars was extracted with
Trizol and used in reverse transcription with oligo(d)T
primer followed by polymerase chain reactions (RT-PCR)
with specific primers for the antiviral protein, based on
the sequence deposited in GenBank database.
Restriction sites were inserted in the cDNA for ligation
in the donor plasmid pFastBac1. The recombinant
plasmid was selected in Escherichia coli DH5&alpha and
subsequently used in the transformation of Escherichia
coli DH10Bac for construction of the recombinant
bacmids. These bacmids, containing the sequence of a
protein with antiviral activity, was used for expression of
this protein in baculovírus/insect cells system. Studies
on the activity of the recombinant protein, as well as the
optimization of such an expression system for the
production of this antiviral protein in insect cells, are in
progress.
Palavras-chaves: Antiviral, Lonomia obliqua, insect cell,
protein expression.
Paulo, were grouped according to gender and age (young
and adult). Serum samples were collected and, prior to
titration, were examined for antibodies to influenza A
and B viruses by the haemagglutination inhibition (HI)
test using the corresponding antigens from the circulating
viruses in Brazil. Results and Conclusion - 20 % of cats
aged between 6 and 20 years old responded with high
antibody titers (e”640 HIU/ìL) against human influenza
type A (H1N1). Lower percentages of the animals in the
same age group, 11% and 8%, presented the same high
titers in response to human influenza types A (H3N2)
and B, respectively. In the gender group, 17 % of males
and 8% of females showed a poor antibody response
against the influenza A (H1N1) virus (titers of d”20 HIU/
ìL). Protective titres of e”40 HIU/ìL against human
influenza viruses suggest viral infection transmitted to
the domestic cats by man. In conclusion, our results
show that domestic cats, like other mammals, may play
a role in interspecies transmission and spread of the
influenza virus.
Palavras-chaves: Influenza, Cats, Host range.
AN INVESTIGATION OF THE HOST RANGE OF
HUMAN INFLUENZA VIRUSES
1
ID: 00385-00002
Área: 04 - Virologia Básica
A1Mancini, D.A.P., 2Mendonça, R.M.Z., 3Cost1, T.F.M,
4
Pinto, J.R., 5Lucas, S.R.R., 6Mendonça, R. Z., 7ManciniFilho, J.
1. IBU, Instituto Butantan, Av. Vital Brasil, 1500, São
Paulo2. USP, Faculdade de Medicina Veterinária e
Zootecnia, Universidade de São Paulo, Prof. Dr. Orlando
Marques de Paiva, 873. USP, Faculdade de Ciências
Farmacêuticas, Universidade de São Paulo, Av. Prof.
Lineu Prestes, 580
Studies on the host range of influenza viruses have been
of great importance to determine the role of animals,
once unlikely links, in the virus transmission chain.
Objective - This study aimed to investigate the circulation
of the influenza virus in cats in Brazil. Material and
Methods - Domestic cats, assisted at the clinic of the
Faculty of Veterinary Medicine at the University of São
RETROSPECTIVE STUDY OF SEROLOGY
CONGENITAL RUBELLA SYNDROME IN INFANTS
UNDER ONE YEAR REFERENCE TO INSTITUTO
EVANDRO CHAGAS - PARA, NO PRE-VACCINE
PERIOD (1989 - 1999) AND POST-VACCINE (20002005.
ID: 00386-00001
Área: 05 - Virologia Humana e Saúde Pública
Moraes, M.M, 2Silva, D.F.L, 3 Cruz, A.C.R, 4Santos,
E.C.O
1. IEC/MS, Instituto Evandro Chagas, Br 316 km 7
(levilandia)
Rubella postnatal, considered a benign childhood disease,
presents low morbidmortality but Congenital Rubella
Syndrome, which infects the newbonrs of mothers
infected during pregnancy, can cause fetal losses,
natalitymortality and a wide range of birth defects in
newborns contributing with high cost psychosocial. Fetal
infection reaches 81% in concept exposed during the
first trimester of pregnancy; in the second quarter
decreased to 67%. The objective of this study is to
evaluate by sorology the Rubella Virus (RV) in under a
year, referenced to the Evandro Chagas Institute ¨C Par¨¢,
in the period run-up to the vaccine (1989-1999) and postvaccine (2000-2005). Retrospective descriptive Study,
in the database about the humoral immune response to
specific IgM antibodies of classes for rubella virus
infected or identifying children with SRC referenced to
the Evandro Chagas Institute in the State of Par¨¢, in
the period from January 1989 to December 2005 by the
197
ABSTRACTS
ELISA method, for a total of 67 samples. Between the
age of one year with positive serology IgM, the cataract,
heart disease and heart-cataract disease association,
were more clinical manifestations that predominated in
the period run-up to the vaccine with 11.5% (6/52) 9.6%
(5/52) and 9.6% (5/52) respectively. After vaccination,
the heart disease was the most obvious manifestation,
with 26.7% (4/15) of cases.When we compare the
number of cases of SRC between periods investigated,
it was noted that there was significant difference (¦Ö2 =
15.5, p < 0.0001). After strengthening of preventive
actions through Double Viral vaccine in women of
childbearing age and the MMR Vaccine in routine
immunization programmes from 12 months of age, with
the second dose applied to four years, according to the
OPAS recommended strategy, there was a reduction in
the number of infected.
Palavras-chaves: Sindrome da rubéola congênita,
Rubéola, infecção congênita, gestante, vacinação.
GENETIC DIVERSITY OF PARTIAL S1 AND N GENE
SEQUENCES OF INFECTIOUS BRONCKITIS VIRUS
ISOLATES FROM BRAZIL
ID: 00388-00001
Área: 03 - Virologia Animal
M.F.S MONTASSIER, 2 L. BRENTANO, 3 L.J.
RICHTZENHAIN, 4C.H. OKINO, 5H.J. MONTASSIER
1. UNESP - FCAV, Universidade Estadual Paulista Campus de Jaboticabal - Depto. de Patologia Veterinária,
Rod. Prof Paulo D. Castelane, CEP-14-884-9002. CNPSA
- EMBRAPA, Centro Nacional de Pesquisa em Suínos e
Aves - EMBRAPA - Concórdia - SC, Concórdia - SC3.
FMVZ - USP, Faculdade de Medicina Veterinária e
Zootecnia da Universidade de São Paulo, Depto VPSA,
São Paulo, SP
American and Brazilian groups. Eight Brazilian IBV
isolates clustered in the American group with
Massachusetts strains, including the viruses used in live
attenuated vaccines, but two isolates clustered with
Connecticut strain or Ark DPI strain. The isolates of the
indigenous Brazilian group were further divided in 2 subgroups clustering 1988’s and 2000’s viruses, which
showed a high diversity regarding IBV strains isolated
from different countries and continents, but they were
moderately to highly related (89.8 to 99.8% of identity)
one to another and with new Brazilian variants isolated
between 2004 and 2005. The major polymorphic sites
are arranged in clusters and predominate in the regions
of S1 and N genes which code for relevant structural
and antigenic sites responsible for the expression of
important biological properties. These results high lighten
the importance of development of vaccines based on
the local strains of IBV, since they have characterized
their pathotypes and protectotypes.
Financial support: FAPESP/CNPq.
Palavras-chaves: Avian infectious bronchitis virus, Field
isolates, S1 glycoprotein, N Protein, Phylogeny.
DIVERSITY IN RELEVANT EPITOPES OF S1
GLYCOPROTEIN AND NUCLEOCAPSID (N) PROTEIN
OF BRAZILIAN VARIANTS OF INFECTIOUS
BRONCHITIS VIRUS
1
Brazil is currently one of the major poultry-producing
countries in the world and in the last few years infectious
bronchitis (IB) has become a serious problem for this
country. The control of IB in Brazil has been mainly based
on vaccination with live attenuated or inactivated
vaccines of the Massachusetts serotype. Despite this,
outbreaks of IB are still occurring in vaccinated flocks,
indicating that probably variant isolates of a different
serotype of the vaccine strain were emerging. In order to
trace the origin and evolution of avian infectious bronchitis
virus (IBV) isolates in Brazil, genomic sequencing was
used for molecular characterization of 15 IBVs isolates
between 1988 and 2000. The 5' region of the S1 gene,
codifying hypervariable regions 1 and 2, and 3' region of
the nucleocapsid gene, codifying cytotoxic T lymphocyte
epitopes, were used to construct phylogenetic trees for
analysis. Two major phylogenetic groups were identified:
198
ID: 00388-00002
Área: 03 - Virologia Animal
M.F.S MONTASSIER, 2 L. BRENTANO, 3 L.J.
RICHTZENHAIN, 4H.J. MONTASSIER
1. UNESP - FCAV, Universidade Estadual Paulista Campus de Jaboticabal - Depto. de Patologia Veterinária,
Rod. Prof Paulo D. Castelane, CEP-14-884-9002. CNPSA
- EMBRAPA, Centro Nacional de Pesquisa em Suínos e
Aves - EMBRAPA - Concórdia - SC, Concórdia - SC3.
FMVZ - USP, Faculdade de Medicina Veterinária e
Zootecnia da Universidade de São Paulo, Depto VPSA,
São Paulo, SP
1
The infectious bronchitis virus (IBV) spike glycoprotein
S1 subunit is associated with virus neutralization,
hemagglutination and cell-attachment. The N protein of
IBV is associated with the genome, playing important
role in viral replication, and immunity mediated by T
lymphocytes. These proteins have been affected in the
course of IBV evolution as a consequence of mutation
and recombination processes modeled by the host
immune-selection. Field IBV isolates were recovered
from commercial broiler or layer flocks of different regions
of Brazil, in 1988 and 2000, and were preliminarily S1genotyped as indigenous to this country. A
complementary molecular analysis, including the
ABSTRACTS
hypervariable regions 1 and 2 (HVR 1, HVR 2) of Nterminus of S1 protein and C-terminus of N protein,
identified relevant changes in key aminoacid (aa)
residues located at important B-cell or T-cell epitopes of
these proteins, when compared to reference vaccine
strain from Massachusetts serotype. Following alignment
of these newly reported S1 glycoprotein sequences with
those previously published from other IBV strains, the
more diverse regions were detected between aa residues
59 - 96, 115 – 149, which coincided with important virus
neutralizing epitopes of IBV. These aa changes led to
relevant modifications on the predictive secondary
structure, glycosylation and antigenic sites of S1
glycoprotein. The N protein sequences of these isolates
were less variable, though characteristic point mutations
were found and cause modifications in the predictive
antigenicity of this protein on the sites encompassing a
T cytotoxic epitope and B-cell epitopes. These data
demonstrate that variant IBV isolates in Brazil, despite
are continuously evolving, maintain beneficial and
important mutations on the major epitopes of S1 and N
proteins to escape from the immunity induced by
Massachusetts vaccines, so that unique variants may
persist and circulate among Brazilian poultry flocks.
Financial support: FAPESP / CNPq.
Palavras-chaves: Avian infectious bronchitis virus, S1
glycoprotein, N Protein, Variants, Epitopes.
GENERATION OF CHICKEN SINGLE CHAIN
ANTIBODY VARIABLE FRAGMENTS (SCFV) FOR
DETECTION AND DIFFERENTIATION OF AVIAN
CORONAVIRUS
ID: 00389-00001
Área: 03 - Virologia Animal
MONTASSIER H.J., 2CAETANO A.G., 3FERNANDES
C.C., 4GONÇALVES M.C.M., 5MONTASSIER M.F.S.
1. UNESP - FCAV, Universidade Estadual Paulista Faculdade de Ciências Agrárias e Veterinárais - Depto.
Patol Vet, Rod. Prof. Paulo D. Castelane, CEP-14884-900
of the individual heavy (VH) and light (VL) chain variable
gene segments followed by cloning into pCANTAB
phagemid vector. After three rounds of panning selection,
ten scFv phage display antibodies of 400 randomly picked
clones of transformed Escherichia coli, were
demonstrated to react with IBV antigens by ELISA. The
western blot analysis led to the selection of one ScFv
antibody reacting strongly with nucleocapsid (N) protein
and other reacting with subunit 1 of spike glycoprotein
(S1) of H120 strain. Additionally, the anti-S1 scFv
antibody showed a significant neutralization titre in
embryonating chicken egg test. In antigenic analysis of
different IBV strains by ELISA, the anti-N scFv antibody
was able to detect all viruses tested, while the anti-S1
could discriminate Massachusetts vaccine serotype
strains from other variants from classical reference
strains belonging to different serotypes of IBV, or even
Brazilian variant field isolates. A scFv-based indirect
immunoperoxidase (IP) procedure was also applied to
detect IBV antigens in formalin-fixed tracheal tissue
sections collected from chickens experimentally infected
with IBV. Thus, the results showed that scFv antibodies
can be advantageously used for the direct diagnosis and
antigen-typing of avian IBV strains.
Financial support: FAPESP / CNPq
Palavras-chaves: Avian infectious bronchitis virus,
Recombinant Monoclonal Antibodies, S1 glycoprotein,
Nucleoprotein, Diagnosis.
PRODUCTION AND CHARACTERIZATION OF A
RECOMBINANT N-TERMINAL FRAGMENT OF
HAEMAGGLUTININ-NEURAMINIDASE
(HN)
GLYCOPROTEIN OF NEWCASTLE DISEASE VIRUS
ID: 00389-00002
Área: 03 - Virologia Animal
1
The use of infectious bronchitis virus (IBV) serotypespecific monoclonal antibodies (MAbs) has allowed
development of efficient and practical assays such as
immunoperoxidase procedures, and different ELISA
methods for IBV direct diagnosis and serotyping.
However anti-IBV MAbs have been produced so far by
conventional hybridoma techniques, which is laborious,
and has relevant restrictions. In this study a phagedisplayed recombinant antibody library derived from
splenic mRNA of chickens immunized with H120 vaccine
strain of IBV, a member of coronavirus group 3, was
constructed as single chain variable fragments (scFv)
by overlap extension polymerase chain reaction (PCR)
GONÇALVES M.C.M., 2FERNANDES C.C., 3SILVA,
K.R., 4MONTASSIER M.F.S., 5MONTASSIER H.J.
1. UNESP - FCAV, Universidade Estadual Paulista Faculdade de Ciências Agrárias e Veterinárais - Depto.
Patol Vet, Rod. Prof. Paulo D. Castelane, CEP-14884900
1
Newcastle disease virus (NDV) is an economically
important infectious agent for broilers and layers, causing
significant losses to the poultry industry. The envelope
of NDV contains haemagglutinin-neuraminidase (HN) and
fusion (F) proteins. Besides the role of HN glycoprotein
in the interaction with cell receptors and virus spreading
to other cells, it is also the major antigenic target for the
host immune response. A number of methods, including
serological tests, have been investigated for the laboratory
diagnosis of NDV infection. The hemagglutination
inhibition (HI) test and whole-virus antigen indirect ELISA
199
ABSTRACTS
have been widely used for the detection of anti-NDV
antibodies. However both techniques have some
drawbacks with regard to sensitivity, operational
complexity, and/or cross-reactivity of subtypes for
chicken serum samples and even more restrictions for
the analysis of wild bird sera. In order to improve the
immuno-diagnosis of NDV the present study aimed to
express a set of conserved and immunogenic epitope
mapped in the amino-terminal region of HN glycoprotein
gene in E. coli. A 5’-portion of the HN glycoprotein gene
containing 1050 bp was amplified by RT-PCR from RNA
extracted from LaSota strain of NDV, and the product
was cloned into a vector appropriate for E. coli expression.
A polypeptide of approximately 50 kDa (containing polyhistidine tag) was detected in western-blot and ELISA
tests, probed with anti-histidine monoclonal antibodies
or polyclonal anti-NDV antibodies from chicken
hyperimmune serum. Thus, this recombinant fragment
showed an antigenic homology with the HN glycoprotein
from virus particles and can be used as antigen in ELISA
tests for the immuno-diagnosis of NDV, and as an
immunogen to produce anti-NDV specific antibodies for
using in competitive ELISA that is recommended assay
for the analysis of wild bird serum samples, because it
does not require species-specific anti-immunoglobulin
conjugates.
Financial support: FAPESP / CNPq
Palavras-chaves: Newcastle disease virus, Recombinant
Protein, HN glycoprotein, Antigen, ELISA.
Coxsackievirus A16 and Hand-foot-and-mouth
disease, Amapá, 2009
ID: 00390-00001
Área: 05 - Virologia Humana e Saúde Pública
Burlandy, F.M., 2Tavares, F.N., 3Oliveira, S.S., 4Costa,
A.S., 5Costa, E.V., 6da Silva, E.E.
1. FIOCRUZ, Laboratório de Enterovírus/IOC, Av. Brasil,
4365 - Manguinhos, RJ2. LACEN-AP, Laboratório Central
de Saúde Pública do Amapá, Avenida Ernestino Borges,
s/n, Macapá - AP
1
Human Enteroviruses, Picornaviridae family, can cause
a variety of diseases in humans. Although enterovirus
71 (EV71) and coxsachievirus A16 (CA16) are the main
cause of hand-foot-and-mouth disease (HFMD), the
former is the most common etiologic agent. HFMD is a
common childhood illness characterized by fever and
vesicular eruptions on hands, feet and in the mouth and
usually affects children below the age of 10, especially
those less than 5 years of age. It is generally mild and
self-limited disorder except in the case of EV71 infections,
where serious complications may occur. This study
describes the virological investigation a HFMD outbreak
200
occurred in the state of Amapa, Brazil during November
and December, 2009. Fecal specimens from 19 cases
of HFMD were clarified in the presence of chloroform
and inoculated in RD, HEp2C and L20B cells, incubated
at 36 oC and examined daily for the evidence of
cytophatic effect (CPE). A total of 5 (26,3%) specimens
were positive for virus isolation in RD cells. Isolates were
further characterized by amplification of 5’ non-coding
region (5’NCR) and molecular typing by RT-PCR and
sequencing of a portion of the VP1 gene. All of them
were molecular typed as Coxsackievirus A16. Preliminary
phylogenetic analysis showed that the CA16 isolates
from the Amapa outbreak are clustering in an independent
arm sharing >99% of identity. The results obtained point
to CA16 as the etiological agent responsible for the
HFMD outbreak occurred in the Amapa State in 2009.
Financial support: FIOCRUZ
Palavras-chaves: Coxsackievirus A16, Hand-foot-andmouth disease, Sequencing.
INCIDENCE AND PREVALENCE OF CYTOMEGALOVIROSIS IN INDIVIDUALS WITH THE HIV
DEMAND INSTITUTE OF SPONTANEOUS EVANDRO
CHAGAS-IEC/SVS/MS.
ID: 00391-00001
Área: 05 - Virologia Humana e Saúde Pública
Arruda, L.M.F, 2Silva, D.F.L, 3Moraes, M.M, 4Sagica,
F.E.S, 5Jesus, I.M
1. IEC, Instituto Evandro Chagas, Br 316 km 7 s/n
Levilândia
1
Because of the increase in the prevalence of AIDS in
the world, Cytomegalovirus infection has become a
serious public health problem in several countries, mainly
associated with serious and complex immunodeficiency
caused by HIV. The State of Pará has presented high
incidence of reported cases of AIDS, according to the
Epidemiological Bulletin from the Ministry of Health
(2009). The present study was to describe the
epidemiology of HIV positive Cytomegalovirus in patients
of spontaneous demand of IEC/SVS/MS, 2005-2009
period on the basis of the clinical-epidemiological data,
the incidence and prevalence of antibodies anti CMV
IgG/IgM. Were selected in the Section of environment/
IEC 652 individuals with HIV research for CMV by Elisa.
The epidemiological profile of demand met was: 374
(57.4%) males and age group 0-50 years, being more
frequent range 30-39 years corresponding to 212
individuals (32.5%). The prevalence of anti CMV IgG
antibodies was 99.2% and the incidence of infection
based on anti CMV IGM antibody was 1.5%. Was
observed higher prevalence of serological IgG profile (+)
IgM (-)anti CMV in asymptomatic (52.6%), being an
ABSTRACTS
PREVALENCE OF EQUINE INFECTIOUS ANEMIA IN
WORKING EQUIDAE OF CORUMBÁ, MATO GROSSO
DO SUL, BRAZIL
immunosorbent assay with a surface envelope
recombinant glycoprotein of EIA virus (ELISA rgp90). It
was found that the prevalence of seropositive was 44%
(n=418), with 2.7% (n=26) of samples displaying results
in the undetermined range of ELISA rgp90. Among the
equines, the prevalence rate was 46.7% (n=337), and
among mules, 35.6% (n=81). The results considered
undetermined within both groups were 2.5% (n=18) and
3.5% (n=8), respectively. The two donkeys were
seronegative. The prevalence in equines is significantly
greater than in mules (z=2.885; P=0.004), maybe due to
differences in susceptibility to EIA virus infection.
According to our results, the prevalence rates of EIA in
the Pantanal region remain righ. Besides the economic
losses by presumable decreased performance of the
infected equidae, EIA is also a problem regarding national
and international markets.
Financial supporte: EMBRAPA / UFMG / FUNDECT
Palavras-chaves: Agar gel immunodifusion test (AGID),
ELISA rgp90, Equine infectious anemia (EIA), Pantanal.
ID: 00393-00001
Área: 03 - Virologia Animal
EMETINE IMPACTS ON HIV REPLICATION BY RNA
PROCESSING BODIES DISRUPTION
NOGUEIRA, M.F., 2 SANTOS, C.J.S, 3 OLIVEIRA,
A.L.C., 4 MONTEZUMA, E.S, 5 JULIANO, R.S.,
6
MARQUES, D.K.S, 7REIS, J.K.P
2. UFMS - CPAN, Universidade Federal de Mato Grosso
do Sul - Campus do Pantanal, Avenida Rio Branco
nº1.270 - Vila Mamona- Corumbá, MS - CEP 79.3049023. EMBRAPA - Pantanal, Empresa Brasileira de
Pesquisa Agropecuária, Rua 21 de Setembro, 1880 - N.
Sra. de Fátima- Corumbá, MS - CEP 79320-9004. UFMG,
Universidade Federal de Minas Gerais, Av. Antônio
Carlos 6627, Campus da UFMG, Belo Horizonte, MG CEP 30123-970.
ID: 00396-00001
Área: 04 - Virologia Básica
individual asymptomatic presented the profile IgG and
IgM + serological diagnosis. Among the clinical manifestations observed were more frequent the triad: fever
(23.0%), headache (13.3%) and enfartament ganglionic
10.3% common both in AIDS and citomegalia. Symptoms such as diarrhea, dyspnea, loss of
Visual acuity, seizure, myalgia and dysphagia were
presented by patients with acute cytomegalic (1.5%, n
= 10). It was concluded that the CMV wasn’t the main
causal agent of opportunistic infection among patients
in the study. It was also observed that the young adults
age 20-29 and 30-39 years for both sexes, was the most
committed HIV and that had been previously infected
by Cytomegalovirus.
Palavras-chaves: Citomegalovirus, Aids, Prevalência,
Incidência, Sorologia.
1
Corumbá is the largest town in Mato Grosso do Sul and
95% of its territory located in the Pantanal area. Corumbá
also has the largest equidae herd of the state. Equine
infectious anemia (EIA) has been documented in
numerous diverse geographical areas, with highest
prevalence in swamp regions worldwide. In the Mato
Grosso do Sul state, this disease is considered endemic
and the majority of cases occur in the Pantanal region.
In the 1990’s, mean prevalence of EIA by the agar gel
immunodifusion test (AGID) was estimated to be 34.1%
in working horses from cattle ranches of Pantanal. The
objective of this study was to estimate the current
prevalence of EIA in working equidae in Corumbá. Blood
samples were collected from 951 animals (721 equines,
228 mules and 2 donkeys) of 42 ranches placed in the
Pantanal sub-regions of Nabileque (n=8), Paiaguás (n=20)
and Nhecolândia (n=14), from September to November,
2009. The diagnostic test used was an enzyme linked
Valadão, A.L.C., 2Peterlin, B.M., 3Tanuri, A., 4Aguiar,
R.S.
1. UFRJ, Universidade Federal do Rio de Janeiro, Av.
Brigadeiro Trompwksy CCS bloco A 2º andar sala 1212.
UCSF, University of California, San Francisco, CA 94143,
415/476-9000
1
RNA Processing Bodies (P bodies) are essential for HIV
replication. P bodies are dynamic cytoplasmic foci, where
mRNA species are stored or degraded. We observed
robust synthesis and release of virus like particles
(VLPs), but they lacked HIV genomic RNA and were not
infectious in cells treated with siRNAs against P bodies
components. In addition to RNAi approaches, we wanted
to determine if pharmacological disruption of P bodies
also inhibits HIV replication. It is well known that emetine
disrupts P bodies. Emetine is a drug clinically used in
the treatment of protozoan infection, besides its anticancer properties. TZM-BL cells were infected with HIV1NL4-3 and 20 h post-infection, cells were extensively
washed and incubated with different concentrations of
emetine. Viral particles were harvested for an additional
24 h and evaluated for p24 levels, viral infectivity and
genomic RNA incorporation. Levels of constitutive gene
expression and cells viability were also evaluated.
Immunofluorescence assays were performed in the same
cells and examined for the presence of P bodies. Results
showed that emetine did not diminish the production of
201
ABSTRACTS
VLPs, but diminished greatly their infectivity. In addition,
cells remained viable during the course of these
experiments and the emetine treatment did not affect
global RNA expression levels. In TZM-BL cells treated
with emetine, decreased viral infectivity correlates with
the disappearance of P bodies. They disappear
completely with 800 ng/mL of emetine, which corresponds
to the total loss of viral infectivity. Moreover, the genomic
RNA incorporation was reduced in VLPs from emetine
treated cells. We conclude that not only the disruption of
P bodies by specific siRNAs, but also by emetine, leads
to the release of virus like particles (VLPs) that contain
no HIV gRNA and are not infectious. This raises the
possibility to use emetine or dehydroemetine in anti-viral
therapy.
Financial Support: CAPES, CNPq, CHRP.
Palavras-chaves: HIV, Processing Bodies, Emetine, HIV
replication.
AN EXPERIMENTAL ANIMAL MODEL FOR THE
STUDY OF PATHOGENESIS OF DENGUE VIRUS
SEROTYPES 1 AND 2/ TECHNIQUE MANUAL
ID: 00397-00001
Área: 03 - Virologia Animal
Barreto-Vieira, D.F., 2Barth, O.M., 3Schatzmayr, H.G.
1. IOC/Fiocruz, Instituto Oswaldo Cruz/Fundação
Oswaldo Cruz, Av. Brasil 4365 Manguinhos Rio de
Janeiro Brasil
1
Millions of individuals are annually infected by the dengue
viruses (DENV), particularly in tropical and subtropical
regions. The mortality tax is low, but the infection can
take to a severe form characterized by hemorrhage and
shock syndrome. Already, sixty years passed since the
isolation of the DENV viruses was effective, and still no
vaccine against these viruses was developed. One
problem for the development of a candidate vaccine and
pharmacos, is the existence of some gaps referring to
pathogenesis of the Dengue Hemorrhagic Fever (DHF)
and the absence of an animal model that simulates an
infection as in human cases of the illness. Stimulated
by the necessity of an animal model to study the
pathogenesis of the DENV viruses, in 1997 was initiated
a research line to supply this gap by the Laboratory of
Morphology and Viral Morphogenesis, Department of
Virology, Instituto Oswaldo Cruz (IOC), Fundação
Oswaldo Cruz (Fiocruz). The result of ten years of
experience was presented to the scientific society
through monographs, thesis, and articles published in
national and international magazines. Part of our
experiences will be related in this manual, presenting
different techniques, and helping to elucidate some points
referring the pathogenesis of the DENV viruses. We wait
202
that this is seen and used as an instrument of orientation
for professionals interested about confrontation with this
serious problem of public health. Financial support: IOC/
Faperj/CNPq Área: Animal Básica Tema: DENV Dengue
Arquivo: DENV-Barreto-Vieira2.doc Apresentador: Debora
F. Barreto-Vieira.
Palavras-chaves: dengue virus, BALB/c mice,
ultrastructural studies, histophatology, lung.
TISSUE ALTERATIONS OF BALB/c MICE
EXPERIMENTALLY INFECTED WITH DENGUE-1
VIRUS
ID: 00397-00002
Área: 03 - Virologia Animal
Barreto-Vieira, D.F., 2Schatzmayr, H.G., 3Takiya, C.M.,
Silva, M.E.V., 5Barth, O.M.
1. IOC/FIocruz, Instituto Oswaldo Cruz/Fundação
Oswaldo Cruz, Av. Brasil 4365 Manguinhos, Rio de
Janeiro Brasil2. UFRJ, Universidade Federal do Rio de
Janeiro, Ilha do Fundão, Rio de Janeiro Brasil
1
4
Dengue, a mosquito-borne flavivirus infection in humans,
is caused by four serologically distinct viruses (DENV)
namely DENV-1, -2, -3 and -4. Dengue and its severe
manifestations, dengue haemorrhagic fever (DHF) and
dengue shock syndrome (DSS), are serious public health
problems in the tropics. There are no vaccines and there
are no animal or other models to study this human
disease. Several studies suggest that mice are a
permissive host for DENV. In the majority of these
models, the animals are immunocompromised and/or
inoculated by routes like the intracerebral, with
neuroadapted DENV. In studies with mice infected with
a neuroadapted DENV strain, damages in liver and lung
tissues can be observed. In our studies, hepatic and
pulmonar tissues of adult BALB/c mice infected
experimentally with a non-neuroadapted DENV-1 by the
intravenous and intraperitoneal routes were analyzed. The
animals were sacrified 72 hours post-infection and tissue
fragments were processed following the standard
techniques of fotonic and transmission electron
microscopy. The supernatants of the macerates of
pulmonar and hepatic tissues and the sera of the infected
mice were inoculated in the C6/36 cell line for isolation
of DENV-1. In order to demonstrate DENV particles and
antigens, these cells were processed using transmission
electron microscopy and the indirect immunofluorescence
technique. Morphological studies of the hepatic tissue
showed vacuolization and necrosis of hepatocytes,
presence of monocytes, polymorphonuclear cells and
cellular debris inside sinusoidal capillars, vascular
congestion, fibroblasts producing elastine and
inflammatory infiltrate. Dengue virus-like particles were
ABSTRACTS
observed inside cytoplasmatic vesicles of Kupffer cells.
Swelling of the interalveolar septa, presence of
erythrocytes inside alveolar spaces, inflammatory
infiltrate into the peribronchiolar space, vascular
congestion, foci of hemorrhage and presence of cellular
debris inside the bronchiolar lumen was observed in the
lung tissue. DENV-1 particles and specific dengue virus
antigen was observed in C6/36 cells inoculated with the
supernatant of spleen and lung macerates and with the
animal sera. Our results show that BALB/c mice are a
permissive host for DENV-1 replication and therefore
provide an useful model to study human dengue disease
pathogenesis. Financial support: IOC/Faperj/CNPq Área:
Animal Básica Tema: DENV Dengue Arquivo: DENVBarreto-Vieira1.doc Apresentador: Debora F. BarretoVieira.
Palavras-chaves: dengue-1 virus, BALB/c mice, lung,
liver, ultrastructural studies.
between YFV-NS5 and Ini1/hSNF5 was confirmed by
GST-pulldown
and
Co-immunoprecipitation assays. More tests will be conducted in order
to achieve a better understanding of the interaction of
these proteins. The comprehension of this interaction
and of viral replication might be useful in the future
development of drugs. FAPESP and CNPq.
Palavras-chaves: Flavivirus, Yellow fever virus, Proteinprotein interaction, NS5 protein, Ini1/hSNF5 protein.
EXPERIMENTAL INFECTION OF INFECTIOUS
BRONCHITIS VIRUS ISOLATED FROM CHICKEN AND
PIGEON
ID: 00400-00001
Área: 03 - Virologia Animal
Martini, M.C., 2Sakata, S.T., 3Silveira, F., 4Giacon, C.S.,
da Silva, L.H.A., 6 dos Santos, M.M.A.B., 7 Felippe,
P.A.N., 8Arns, C.W.
1. UNICAMP, Universidade Estadual de Campinas,
Cidade Universitária “Zeferino Vaz”; Distrito de Barão
Geraldo; 13081-970 -Campi
1
5
INI1/HSNF5 PROTEIN INTERACTS WITH YELLOW
FEVER VIRUS NS5 PROTEIN IN VITRO AND EX VIVO
ID: 00399-00001
Área: 04 - Virologia Básica
Duarte, D.V.B., 2Bronzoni, R.V.M., 3Nogueira, M.L.
1. FAMERP, Faculdade de Medicina de São José do Rio
Preto, Av. Brigadeiro Faria Lima, 5416 - Vila São Pedro 15090-000
1
Yellow Fever is a mosquito-borne hemorrhagic fever
caused by Yellow Fever Virus (YFV) that is the prototype
of the genus Flavivirus. YF is characterized by severe
hepatitis, renal failure, hemorrhage, and rapid terminal
events that lead to shock and death. The mechanism of
Flavivirus replication is not well known but includes
interactions of viral RNA with cellular and viral proteins.
The nonstructural protein 5 (NS5) is the largest and most
conserved of the Flavivirus proteins, and comprises the
methyltransferase and the RNA-dependent RNA
polymerase (RNApol) domains. NS5 is critical for many
functions, including replication, capping of RNA and hostcell gene regulation. The purpose of our study was to
identify and to characterize the interaction between NS5
and host cellular proteins. By using two-hybrid assays
in the yeast Saccharomyces cerevisiae and NS5 protein
as bait, we isolated Ini1/hSNF5. Ini1 is a component of
the SWI/SNF complex which facilitates transcription by
altering the structure of chromatin. A region of NS5
between amino acids 73 and 152, called FragA, was
identified as the minimal domain required for binding to
Ini1/hSNF5. FragA from other Flavivirus, like Dengue
types 3 and 4, as well as Saint Louis Encephalitis,
interacted with Ini1/hSNF5 in yeast two-hybrid system,
indicating that it is a conserved region. The interaction
Infectious Bronchitis Virus from Chickens (VBI)Coronavirus is a highly infectious disease and cause
economic losses in the poultry industry. This study aims
to define the pathogenesis of field samples and isolated
from Pigeon, with different phylogenetic profile from the
vaccine samples. 3 VBI chicken field isolates from main
Brazilian poultry industry and one from Pigeon
Coronavirus were chosen with different phylogenetic
profile of vaccine (H120). The samples were identified
by serial passage in embryonated eggs, by RT-PCR for
IBV and partial sequencing. For the experimental design
we divided the different samples: Positive Control_H120;
VBI field chicken; VBI field chicken; VBI field chicken
and Corona-Pigeon. Each group with 8 days of age (n=10
SPF chickens) were inoculated 200 mL of inoculum
through the eyes and nasally. The groups were kept in
isolators. The sacrifice of animals was performed every
two days post-inoculation (d.p.i.), from 2nd d.p.i until
the 10th d.p.i. We collected scrapings from the trachea,
sinus, nasal and cloacal swabs, stored in MEM and
stored in liquid nitrogen (-96°C). The material collected
was processed from the Viral RNA Extraction Kit, cDNA
kit and Polymerase Chain Reaction-PCR Kit for the
amplification of gene S. The PCR products were analyzed
by electrophoresis on 1% agarose gel and stained with
ethidium bromide and visualized on UV light. The viruses
studied were able to reproduce the disease under
experimental conditions. The birds showed evident clinical
signs of respiratory disease with apathy, prostration and
respiratory distress. After the 2nd day of the inoculation
of Corona-Pigeon, it was identified from cloacal swabs
203
ABSTRACTS
by PCR, indicating the speed of multiplication and
dissemination of the virus in vivo. The results warn us
about the short period between infection and the agent
spread among other birds. All the inoculated animals and
the positive control were able to reproduce the disease
under experimental conditions.
Palavras-chaves: Coronavirus, Chicken, Field, Pigeon,
PCR.
SEARCH OF INFECTIOUS BRONCHITIS VIRUS IN
ORNAMENTAL BIRDS OF A COMMERCIAL
HATCHERY IN MINAS GERAIS.
ID: 00400-00002
Área: 03 - Virologia Animal
Giacon, C.S., 2Silveira, F., 3Felippe, P.A.N., 4Flores, F.,
Martini, M.C., 6Armando, A.P.R.N., 7 Arns, C.W., 8da
Silva, L.H.A., 9SAKATA, S. T.
1. Unicamp, Universidade Estadual de Campinas, Cidade
Universitária “Zeferino Vaz”; 13081-970 - Campinas - SP
birdhouse.
Palavras-chaves: Coronavirus, birds, poultry, PCR, wild.
INVESTIGATION OF STRUCTURAL STABILITY OF A
VACCINE
PLATFORM
FOR
HUMAN
IMMUNODEFICIENCY VIRUS
ID: 00401-00001
Área: 04 - Virologia Básica
Barroso, S.P.C, 2 Vicente, A.C, 3 Gomes, D.C,
Peabody,D.S, 5Silva, J.L, 6Oliveira, A.C
1. UFRJ, UNIVERSIDADE FEDERAL DO RIO DE
JANEIRO, Av. Carlos Chagas Filho, 373, Cid.
Universitária- Ilha do Fundão, Rio de Janeiro2. UNM,
University of New México, Albuquerque, NM 87131
1
4
1
5
Infectious Bronchitis Virus (IBV), a Coronavirus, cause
a highly infectious and acute disease, being a major cause
of economic losses in the poultry industry, in virtually all
regions of the world. The virus replicates, not only in
respiratory tract, but also in the tissues of the digestive
tract and in other sites like: kidneys, oviduct and testicles.
The IBV is perhaps the virus that spreads quickly among
birds, and often the carrier birds can transmit the virus
until two months after the initial infection and recovered
birds remain susceptible to another infection by others
serotypes. There is evidence that IBV can infect wild
birds species, conveying it to poultry flocks. The aim of
this work was to search the IBV in ornamental birds, of
a commercial hatchery in Minas Gerais, Brazil. In this
nursery are housed 4,000 birds of 325 different species,
located in a peri-urban environment and kept in nurseries
of masonry or outdoor, allowing contact with external
birds. Samples were collected through tracheal and
cloacal swabs from 20 birds, species Dendrocygna bicolor
(fulvous), with four months of age, captive-born and with
healthy clinical aspect. The swabs were stored into
Eppendorfs tubes containing one mL of RNAlater TM,
for preservation of viral RNA, and forwarded to the
laboratory for procedures: viral RNA extraction, RT/PCR
and Nested/PCR of gene S from IBV. The samples were
compared with the virus vaccine H-120, as positive control
and a negative control. In this study, all samples were
negative for IBV. The result is justified by the fact of this
birds being young, born in captivity and not having contact
with other birds. Another important factor is that these
data demonstrate that the employed health control is
proving effective. Further studies are needed to assess
the potential epidemiological with others species of this
204
The human immunodeficiency virus (HIV) is a lentivirus,
a member of the retrovirus family. Virus-like particles
(VLPs) can be considered as dense arrays of one or
more repetitive subunits of a protein and this
characteristic confers highly advantageous properties
for their use as vaccines platforms. The platforms used
in this project are VLPs of bacteriophage MS2. Its capsid
has approximately 25 nm in diameter and is formed by
180 copies of the coat protein. It has been shown that
the single chain dimer of coat protein tolerates the
insertion of a wide variety of peptides and these are
highly immunogenic when presented in the MS2 VLP. In
this project, we evaluated the structural stability of these
VLPs for highly immunogenic peptides related to the
infectious cycle of HIV-1, by subjecting such particles
to high hydrostatic pressure (HHP) and other chemical
and physical agents. In order to do measurements, we
use light scattering, intrinsic fluorescence and circular
dichroism (CD). The results obtained so far were
performed with VLP formed by a single chain dimer of
coat protein, native coat protein and two constructions
with the Flag epitope. The center of mass deviation and
light scattering indicate that there are small changes in
the structure of VLPs with insertion of the epitope, except
for results with HHP, construction with inserts had the
highest center of mass deviation. CD measurements
indicates no change in secondary structure between
dimer and native protein, but single chain constructs with
Flag epitope had a different behavior. We are currently
conducting experiments with VLPs containing peptides
extracellular loop of CCR5 co-receptor cell and the V3
loop of gp120 of HIV-1. These peptides are described by
inducing the formation of antibodies with high potential
antiviral. Stability studies of this form of presentation of
immunogenic peptides intent to help with structural
information for the development of this vaccine platform.
Palavras-chaves: Virus-like par ticles, Bacteriophage MS2, HIV.
ABSTRACTS
ROLE PLAYED BY THE CELLULAR GENE c-fos
DURING THE REPLICATION CYCLE OF THE
ORTHOPOXVIRUS VACCINIA VIRUS
ID: 00403-00001
Área: 04 - Virologia Básica
Oliveira L.C., 2Alcântara T.C., 3Brasil B.S.A.F., 4Kroon
E.G., 5Ferreira P.C.P., 6Bonjardim C.A.
1. UFMG, Universidade Federal de Minas Gerais, Av.
Antônio Carlos, 6627 - Pampulha - Belo Horizonte - MG
- CEP: 31270-901
1
Viruses acquired the potential to intercept mimicry and
subvert signaling network to control cellular functions
aiming at creating the intracellular conditions favorable
to progeny generation and dissemination. Our group has
previously demonstrated that Vaccinia virus (VACV)
modulates and takes advantage of the activation of
MAPKs ERK1/2 and JNK1/2 signaling pathways. Here,
we describe that VACV stimulates the sustained
expression of c-Fos protein in immortalized murine
embryonic fibroblasts (MEFs) since as early as 3 up to
24 hour post-infection (hpi). Western blot analysis
revealed that c-Fos expression and its stabilization
depends exclusively on MEK1/2 pathway. On the other
hand, expression of egr-1 gene, a common target of the
MEK/ERK pathway, is not c-Fos dependent. Even though
VACV replication or viral late gene expression, as well
as EEVs release, have not been found impaired in MEFs
c-fos knockout, it was observed that both plaque size
and dissemination were diminished, under the same
conditions. Immunofluorescence and confocal
microscopy revealed a significant decrease in the number
of VACV-induced actin tails during infection in MEFs cfos-/-. Furthermore, the c-fos gene knockout leads to
defects in VACV-induced actin network dynamics, as
well as a decrease in viral A36 protein tyrosine
phosphorylation, though the transport and formation of
enveloped particles was not affected. Taken together,
our results show that c-Fos performs an important role
and contributes to the dissemination of VACV enveloped
particles. Apoio: CAPES, FAPEMIG e CNPq.
Palavras-chaves: orthopoxvirus, vaccinia virus, c-fos,
virus dissemination, virus-host interaction.
MOLECULAR IDENTIFICATION OF THE VIRUS
ISOLATED FROM MERCHANT CROPS IN
WATERMELON IN TOCANTINS, BRAZIL
ID: 00404-00001
Área: 06 - Virologia Vegetal
Santos, L.B., 2Ribeiro, F.F., 3Nascimento, I.R., 4Santos,
G.R., 5Ribeiro, S.R.R.P., 6Aguiar, R.W.S., 7Figueira, A.R.
1
1. UFT, Universidade Federal do Tocantins, Rua Badejós,
chácaras 69 e 72 Lt.07 - Zona Rural2. UFLA, Universidade
Federal de Lavras, Campus Universitário Caixa Postal
3037 Lavras - MG
The cucurbits, including watermelon [Citrullus lanatus
(Thunb) Matsum & Nakai] is subject to several diseases
caused by viruses that can substantially reduce your
productivity both quantitatively and qualitatively, and is
the most important factor in reducing the production of
watermelon in the state of the Tocantins in recent years.
Aiming to identify those viruses that generally have
similar symptoms, but may have vector control methods
and different virus isolates were collected in four regions
of the merchant watermelon plantings in the state of
Tocantins, in eight isolates from different plants
watermelon, being identified as follows: FA (Formoso do
Araguaia - TO); GR1 and GR2 (Gurupi - TO); PN1 and
PN2 (Porto Nacional - TO); LC1, LC2 and LC3 (Lagoa da
Confusão - TO). These isolates were sent to the laboratory
of Virology, Universidade Federal de Lavras (UFLA) MG for molecular identification. With the use of three
pairs of specific primers for two potyviruses being: WMV2 (Watermelon mosaic virus), PRSV-W (Papaya ringspot
virus - watermelon strain) and one for a Comovirus SqMV
(Squash mosaic virus) was performed by RT-PCR to
identify these isolates. Of the eight strains, six showed
bands for PRSV-W being: GR1, PN1, PN2, LC1, LC2
and LC3. Strain PN1 plus bands to PRSV-W has also
made a band for the SqMV showing a mixed infection.
Bands characterizing the presence of WMV-2 were not
observed in any isolate. This result demonstrates that
the state of the Tocantins virus PRSV-W is apparently
the most important crop of the watermelon.
Financial support: CNPq
Palavras-chaves: Citrullus lanatus, Vírus, Resistência, Marcador, Produção.
RISK FACTORS AND TRANSMISSION CHAIN OF
BOVINE VACCINIA IN THE DAIRY REGION OF
SERRO, MINAS GERAIS STATE, BRAZIL
ID: 00405-00001
Área: 05 - Virologia Humana e Saúde Pública
Borge, I.A, 2 Abrahão, J.S, 3 Andrade, LAO, 4 SilvaFernandes, AT, 5Bonjardim, CA, 6Ferreira P.C.P, 7Kroon,
EG, 8Trindade, GS
1. UFMG, Universidade Federal de Minas Gerais, Avenida
Antônio Carlos, 6627 Belo Horizonte, Minas Gerais
1
Since the first notification in 1999, Bovine Vaccinia (BV)
outbreaks have been reported in all five geographical
regions of Brazil. According to BV epidemiological data,
this zoonosis usually occurs in small and non205
ABSTRACTS
mechanized dairy properties, with humans and bovines
as its most affected hosts. Recent studies also
speculate on different Vaccinia virus (VACV) susceptible
species and their real roles in BV epidemiology, but
several gaps in VACV transmission cycle are still to be
investigated. Therefore, an epidemiological study
concerning bovine serosurvey, cattle handling, VACV
hosts and ecological aspects related to BV affected
properties is presently being conducted in the county of
Serro, Minas Gerais State. BV outbreaks have been
reported in this county since 2005, suggesting that VACV
still circulates in the area. Up to this date, five properties
were randomly visited and sera were collected from 20%
of milking cows. Collected sera were tested in ELISA –
IgG to detect anti-Orthopoxvirus (OPV) antibodies.
Coordinates and cattle information from each property
were obtained and ecological aspects were observed.
Unexpectedly, ELISA results demonstrated a
seropositivity of 100%. Only one property presented a
non-mechanized milking procedure, herds ranged from
68 to 243 animals and all properties did milking twice a
day. At an ecological first glance, all properties presented
areas of deforestation due to livestock and agricultural
activities, which led to problems as edge effect and
fragmentation of forests, narrowing wildlife and domestic
area contact. Ecological observations indulge the search
for different VACV hosts. Partial results suggest that VACV
circulation is no longer exclusively associated to small
non-mechanized properties. Further speculations depend
on future expeditions that will soon be made to generate
more data. After a single expedition however, the county
of Serro presented itself as a suitable area to conduce
VACV epidemiological studies.
Palavras-chaves: bovine vaccinia, ecology,
epidemiology.
Infectious Bronchitis Virus (IBV) is a Gammacoronavirus of the Coronaviridae family, which causes
respiratory, enteric and urogenital disease in chickens.
Small changes in the amino acid sequence of virus spike
protein (S) can give rise to new antigenic types. The aim
of this study was to detect IBV variants in poultry flocks
from Brazil. Twenty-two pool samples were collected from
symptomatic flocks, during 2010. Each pool contained
tissues from 3 to 5 birds and were segregated by different
organs: tracheas, lungs, kidneys, enteric contents,
spleens, chest and 5 individual tracheal swabs. Samples
were screened for the presence of IBV using a Nested
RT-PCR targeted to the 3’UTR. From these samples, 18
were positive and submitted to a new Nested RT-PCR
target to S gene resulting in six amplicons of 390bp were
then submitted to DNA sequencing and analysis.
Genealogical analysis showed a nucleotide identity range
from 86.5% to 100% among the amplified samples. Two
samples had respectively 60% and 69% nucleotide
identity with IBV/BRAZIL/2008/USP26 variant strains
(GenBank accession: FJ791272) and (GenBank
accession: FJ791269). The other four samples were 100%
identical with the Mass genotype (GenBank accession:
FJ791256), suggesting, in this last case, vaccine virus
detection. However, the variant genotype detected
grouped in an individual cluster demonstrating the
presence of a different genotype circulating in Brazil.
Therefore, the present study reinforces the circulation
of variant IBV types in Brazil and the importance of
persistent surveillance to guarantee the immunity of
flocks.
Palavras-chaves: IBV, CORONAVIRUS, SPIKE
PROTEIN.
IBV VARIANTS DETECTED IN BRAZILIAN POULTRY
FLOCKS
SLOWER INTRAHOST EVOLUTION OF V4-V6
ENVELOPE GENE AND PARAMETERS OF DISEASE
PROGRESSION
IN
BRAZILIAN
FELINE
IMMUNODEFICIENCY VIRUS
ID: 00406-00001
Área: 03 - Virologia Animal
ID: 00406-00002
Área: 03 - Virologia Animal
Santos, S., 2Brandão, P. E., 3Barros, I. N., 4Ayres, G.
R., 5Oliveira, C. P., 6Torres, C. A., 7Souza, S. O. S.,
8
Villarreal, L. Y. B., 9Richtzenhain, L. J.
1. FMVZ/ USP, FACULDADE DE MEDICINA
VETERINÁRIA E ZOOTECNIA/ UNIVERSIDADE DE
SÃO PAULO, Av. Prof. Dr. Orlando Marques de Paiva, 87
CEP 05508 270 - Cidade Universitária2. CRG,
CORONAVIRUS RESEARCH GROUP, Av. Prof. Dr.
Orlando Marques de Paiva, 87 CEP 05508 270 - Cidade
Universitária3. IVP, INTERVET/ SCHERING - PLOUGH,
Av. Sir Henry Wellcome, 335 CEP: 06714 - 050/ MOINHO
VELHO - COTIA - SP
Teixeira, B. M., 2Santos, S., 3Miyashiro, S. I., 4Passarelli,
D., 5Brandão, P. E., 6Hosie, M. J., 7Souza, S. O. S.,
8
Hagiwara, M. K.
1. FMVZ/ USP, Department of Medical Clinics, College
of Veterinary Medicine, University of São Paulo, Av. Prof.
Dr. Orlando Marques de Paiva, 87 CEP 05508 270 Cidade Universitária2. RRL/ UG, Retrovirus Research
Laboratory, Faculty of Veterinary Medicine, University of
Glasgow, Bearsden Road, Glasgow G61 1QH, United
Kingdom3. VPS/FMVZ/USP, Department of Preventive
Veterinary Medicine and Animal Health, College of
Veterinary Medicine, Uni, Av. Prof. Dr. Orlando Marques
de Paiva, 87 CEP 05508 270 - Cidade Universitária
1
206
1
ABSTRACTS
Feline immunodeficiency virus (FIV) infection in domestic
cats is associated with early robust humoral and cellular
anti-viral immune response followed by a progressive
immune suppression that results eventually in AIDS. FIV
has extensive sequence variation within the env gene
encoding the envelope glycoprotein (Env), a feature that
is typical of lentiviruses. Two viral mechanisms contribute
to the generation of variability of FIV within an individual,
the introduction of mutations into the viral genome by
the error-prone reverse transcriptase, and recombination
between pre-existing viral populations. Little is known
about Brazilian FIV strains and disease progression. The
aim of this study was to determine whether disease
progression is influenced by a viral evolution, to examine
sequence divergence of viral variants and to determine
laboratory parameters characteristic of disease
progression which allow a better description of the chronic
phase of the infection. Analysis of the V4 to V6 domains
of the FIV env gene was carried out four times over a
period of 15 months in a group of naturally infected cats.
In each sample, blood samples were analyzed for the
following: complete hematology, clinical chemistry and
serum protein electrophoresis. Unexpectedly, little
sequence variation was observed amongst viruses
circulating in samples collected at various time points
from each infected cat. Changes were observed in the
following hematological and clinical chemistry parameters
in the FIV-infected cats between the first blood sampling
and last blood sampling: packed cell volume (PCV),
hemoglobin, total white blood cells (WBC), total protein
and gamma globulin fractions. A longer follow up time
may have reveal greater intra-host viral variation as a
result of selective immune-pressure. Monitoring of
hematological and clinical chemistry parameters may
prove useful for the evaluation of disease progress.
Financial Support: CAPES
Palavras-chaves: FIV, Sequence variability, Disease
progression.
IMMUNOHISTOCHEMICAL APPROACH TO THE
PATHOGENESIS OF CLINICAL CASES OF BOVINE
HERPESVIRUS TYPE 5 INFECTIONS
Human Herpesvirus 3 (commonly known as Varicellazoster virus 1), herpes simplex viruses (HSV), and equid
Herpesvirus 1 (EHV-1) induce an intense inflammatory,
vascular and cellular response. In spite of the many
reports describing the histological lesions associated with
natural and experimental infections, the immunopathological mechanisms for the development of
neurological disorder have not been established. A total
of twenty calf brains were selected from the Veterinary
School, University of São Paulo State, Araçatuba, Brazil,
after confirmation of BoHV-5 infection by virus isolation
as well as by a molecular approach. The first part of the
study characterized the microscopic lesions associated
with the brain areas in the central nervous system (CNS)
that tested positive in a viral US9 gene hybridization
assay. The frontal cortex (Fc), parietal cortex (Pc),
thalamus (T) and mesencephalon (M) were studied.
Secondly, distinct pathogenesis mechanisms that take
place in acute cases were investigated by an
immunohistochemistry assay. This study found the frontal
cortex to be the main region where intense oxidative
stress phenomena (AOP-1) and synaptic protein
expression (SNAP-25) were closely related to
inflammatory cuffs, satellitosis and gliosis, which
represent the most frequently observed neurological
lesions. Moreover, MMP-9 expression was shown to be
localized in the leptomeninges, in the parenchyma and
around mononuclear infiltrates (p < 0.0001). These data
open a new perspective in understanding the role of the
AOP-1, MMP-9 and SNAP-25 proteins in mediating
BoHV-5 pathogenesis and the strategies of host-virus
interaction in order to invade de CNS.
Palavras-chaves: BoHV-5, immunohistochemical,
Meningoencephalitis.
DIAGNOSIS OF HUMAN HERPESVIRUS 6 AND 7
INFECTION BY THE POLYMERASE CHAIN
REACTION IN YOUNG CHILDREN WITH
EXANTHEMATIC DISEASE
ID: 00409-00001
Área: 05 - Virologia Humana e Saúde Pública
Ivna Magalhães, 2Rebeca Martins, 3Solange Oliveira,
Silvia cavalcanti
1. UFF, Universidade Federal Fluminense, Prof Ernani
Melo, 101 sala 321 - Niterói
1
ID: 00407-00001
Área: 03 - Virologia Animal
Garcia, A. F., 2Ferrari, H. F., 3Rosa, A. C. G., 4Bregano,
L. C., 5Castilho, G., 6Luvizotto, M.C.R., 7Cardoso,T. C.
2. FOA-UNESP, Faculdade de Odontologia de Araçatuba
Curso de Medicina Veterinária, Rua Clovis Pestana, 793,
Jardim Dona Amélia
4
1
Meningoencephalitis by Herpesvirus type 5 (BoHV-5) in
cattle has some features that are similar to those of
herpetic encephalitis in humans and other animal species.
The human herpesvirus 6 (HHV-6) infections are
widespread in all populations. This virus has two biological
variants: HHV-6A which is not associated with any
disease, and HHV-6B which causes exanthema subitum
in young child and like human herpesvirus 7 (HHV-7),
remains latent in host cells after primary infection. The
indirect immunofluorescence assay (IFA) for the detection
207
ABSTRACTS
of low avidity IgG is accepted as the gold standard for
diagnosing HHV-6 primary infection. However, there are
reports showing cross-reactivity between HHV-6 and
HHV-7 and this technique does not differentiate HHV-6
variants. The aim of this study was to implement a
technique of nested multiplex PCR for the diagnosis and
differentiation of infections caused by HHV-6A, 6B and
HHV-7. In this study, were included children younger than
four years old presenting rash and diagnosed with HHV6 primary infection by IFA. A hundred thirty-eight saliva
samples and 125 serum samples were selected and
separated into case group and control group according
to the results of the IFA technique. After performing the
PCR technique, we observed the frequency of viral DNA
detection. In the saliva samples from case group, 4.8%,
3.2% and 4.8% had HHV-6B, HHV-6A and HHV-7,
respectively. In the saliva samples from control group,
we observed frequency of 1.3%, 2.6% and 5.3% for the
detection of HHV-6B, HHV-6A and HHV-7, respectively.
In the serum samples from the case group, 1.7% had
HHV-6A and HHV-7, but HHV-6B could not be detected.
In the control group, the HHV-6B was not detected, but
we found a frequency of 1.5% for HHV-6A and 5.9% for
HHV-7. The evaluation of sensitivity (4.8%) and accuracy
(56%) of the PCR technique showed that this is not
adequate for the diagnosis of primary infection by HHV6B. It is interesting to notice that 25% of serum samples
with inconclusive results after IFA screening, presented
HHV-7 DNA, suggesting that the PCR can be useful for
differential diagnosis of infections caused by HHV-7.
Palavras-chaves: Herpesvirus 6, Herpesvirus 7,
exanthem subitum, PCR, serum.
IMMUNOHISTOCHEMICAL APPROACH TO DETECT
PRO-AND-ANTI-APOPTOTIC
ANTIGENS
EXPRESSION IN TH CEREBELLUM OF DOGS
NATURALLY INFECTED WITH CANINE DISTEMPER
VIRUS
ID: 00410-00002
Área: 03 - Virologia Animal
Bregano, L.C., 2Agostinho, S.D., 3Roncatti, F.T.L.B,
4
Andrade, A.L., 5 ROSA, A.C.G., 6Luvizotto, M.C.R.,
7
Cardoso, T.C.
1. IBILCE - UNESP, Instituto de Biociências, Letras e
Ciências Exatas, Rua Cristóvão Colombo, 2265 Bairro:
Jardim Nazareth CEP: 15054-000 São José do2. FOA UNESP, Faculdade de Odontologia de Araçatuba, cloves
pestana, 793, dona amelia - Araçatuba - SP
1
Canine distemper virus (CDV) is a highly contagious
pathogen and may induce multifocal demyelination in
the central nervous system of infected dogs. To enhance
cell viability and facilitate replication, viruses possess
208
multiple mechanisms to inhibit the host response.
Apoptosis serves as an innate cellular response to
infection that limits both the time and cellular machinery
available for viral replication. The present investigation
was conducted to elucidate some host-virus interactions
using the cerebellum of naturally infected dogs with CDV.
The TUNEL in situ and immunohistochemical assays
(IHC) for the expression of anti-apoptotic (p53 and BCl2) antigens and pro-apoptotic caspase-2 and -3 antigens
were performed in white matter and gray matter (granular
layer) in cerebellum of naturally infected dogs. All samples
were diagnosed as positive for CDV genome by PCR
chain reaction targeting the nucleocapsid gene. We
observed a positive correlation among p53 and BCl-2
detected in all slides from cerebellum region. The annexinV staining was observed diffusely in the molecular layer.
The TUNEL staining was intensively visualized in the
Purkinje cells. Any label could be documented for
caspases-2 and-3. In respect to viral RNA detection by
RT-PCR all positive samples were considered as being
infected by CDV (data not shown). In comparison, the
number of TUNEL and annexin-V positive cells were
positively correlated in all slides analyzed. This study
represents the first description of p53 expression in
natural cases of CDV natural occurrences. It is known
that some other viruses have the participation of p53
expression during infection, but the real function of this
mediator remains unclear in CDV disease. Thus, it is not
known the complete mechanism used by CDV to induce
or not the apoptosis. To know if apoptosis is a defense
against CDV infections or if apoptosis is a way for the
virus to spread to other cells must be investigated in
further studies.
Palavras-chaves: Immunohistochemical, canine
distemper virus, apoptosis.
DETECTION AND GENOTYPING OF EPSTEIN-BARR
VIRUS (EBV) IN CERVICAL SMEARS OF PATIENTS
COINFECTED WITH HUMAN IMMUNODEFIENCY
VIRUS (HIV)
ID: 00411-00001
Área: 05 - Virologia Humana e Saúde Pública
SANTOS, L.S., 2NOGUEIRA, F.G., 3MELGAÇO, F.G.,
OLIVEIRA, L.H.S.
1. UFF, Universidade Federal Fluminense, Rua Professor
Hernani Melo, n° 101, São Domingos - Niterói, RJ
1
4
The Epstein-Barr virus is a lymphotropic microorganism
mainly transmitted by oral route, but sexual transmission
has also been suggested. Two types of EBV (1 and 2)
have been described based on genetic differences.
Cervical cancer is associated with sexually transmitted
human papillomavirus (HPV) that infect the genital area.
ABSTRACTS
Many studies have analyzed the presence of EBV, HPV
and cervical lesions, but not yet established any
relationship between those virus and lesions in the cervix.
In our study, we investigated the presence of EpsteinBarr virus in HIV-infected women, positive or negative
for the presence of HPV. Sampling included 87 cervical
smears from HIV-infected women attended the Pathology
Cervical Service of the Hospital dos Servidores do
Estado, Rio de Janeiro. The patients aged 14-59 years.
Most of them had a low familiar income and elementary
school education. All the patients had an active sexual
life. Herpetic genital lesions were found in 23.7% of the
patients. Of the total, 68 women were positive for HPV
infection. DNA was extracted either with phenolchloroform method or by a commercial assay kit. EBV
detection and typing were performed by generic and
nested PCR respectively. Two (2.3%) of the samples were
positive for EBV, and one of them was co-infected with
HPV. This sample was infected with EBV-2, associated
with low-grade squamous intraepithelial lesion and HPV
69. The HPV negative sample, with genital herpes lesions
and normal cytology harbored EBV 1 and 2. The materials
came from women presenting CD4 count about 500 cells/
mm3. EBV detection in cervical smear from HIV positive
people was very low to establish relationship between
the presence of this virus and cytological alterations. In
spite of the small number of genital EBV infection found
in these people, the results suggest a possible sexual
transmission of Epstein-Barr virus. More studies should
be conducted. Financial support: FAPERJ, PROPP-UFF,
CNPq.
Palavras-chaves: Epstein-Barr virus, Human
immunodeficiency virus, Human papillomavirus,
Polymerase Chain Reaction.
non-insect cell lines. Despite their inability to replicate,
they can enter mammalian cells and can be engineered
to be used as expression vectors in these cells. For
this, it is necessary to express the gene of interest under
the control of a mammalian gene promoter, since
baculovirus promoters are not functional in nonpermissive cell lines. The cytomegalovirus promoter
(CMV) has been successfully used for the expression
of heterologous proteins in mammalian cell lines. Thus
several viral proteins of medical interest can be
expressed in human cells via baculovirus expression
system. This work aims to express the rabies virus
glycoprotein (RVG) in mammalian cells using recombinant
baculovirus. The envelope glycoprotein of the rabies virus
recognizes specific neural receptors and is the only viral
immunogenic protein. For recombinant virus construction,
the gene cassette containing CMV promoter and the RVG
gene was cloned into pFastBac1 vector (Invitrogen). The
recombinant virus AcCMV-RVG was obtained through
the Bac-to-Bac kit (Invitrogen) and was used to infect
insect cells. Infected cell extract was analyzed by SDSPAGE and the presence of RVG was confirmed by
western blot using an anti-rabies virus antibody. HepG2
cells, derived from human hepatoma, were transduced
with the recombinant virus, labeled with the same
antibody and analyzed by confocal microscopy. Later in
vivo tests may be done in order to analyze the
immunogenic effects of recombinant virus. In the future,
recombinant baculovirus capable of expressing RVG in
mammalian cells could be an alternative for development
of cheaper and more efficient rabies vaccine. CNPq and
FAPDF.
Palavras-chaves: Baculovirus AcMNPV, Rabies virus
glycoprotein, Mammalian cells.
CONSTRUCTION OF A VIRAL VECTOR FOR
EXPRESSION OF RABIES VIRUS GLYCOPROTEIN
IN INSECT AND MAMMALIAN CELLS
A RECOMBINANT BACULOVIRUS EXPRESSING A
SILENCING SUPPRESSOR PROTEIN (NSs) OF A
TOSPOVIRUS SUPPRESS RNAi AND ENHANCES
BIOINSECTICIDE ACTION ON INSECT CELLS AND
LARVAE
ID: 00412-00001
Área: 04 - Virologia Básica
MARTINS, G. K. M., 2BARROS, M. C. E. S., 3RIBEIRO,
B. M.
1. UnB, Departamento de Biologia Celular Instituto de
Ciências Biológicas Universidade de Brasilia, Campus
Darcy Ribeiro, Instituto de Ciências Biológicas,
Laboratorio de Virologia
1
The
Autographa
californica
multicapsid
nucleopolyhedrovirus (AcMNPV) are insect viruses and
have been widely used as biopesticides. Baculovirus is
also an important tool for the expression of heterologous
proteins in insect cells. They are considered to be safe
due to thier host specificity and inability to replicate in
ID: 00413-00001
Área: 03 - Virologia Animal
Oliveria, V. C., 2 Bar tasson, L., 3 Castro, B. M. E.,
Corrêa, J. R., 5Ribeiro, B. M., 6Resende, R. O.
1. UnB, PhD Program in Molecular Biology,
Departamento de Biologia Celular, Universidade de
Brasília, Campus Universitário Darcy Ribeiro, Asa Norte2.
Cenargen, Embrapa Recursos Genéticos e
Biotecnologia, Final W3 Norte
1
4
Tomato spotted wilt virus (TSWV), the type species of
the genus Tospovirus, encodes a nonstructural protein
(NSs) which has been shown to function as a strong
209
ABSTRACTS
suppressor of RNA silencing (RNAi) in plant cells. Since
viral RNAi suppression is conserved in various
organisms, this study aimed to explore the effect of NSsTSWV protein on RNAi pathway in permissive (BTITn5B1-4), semipermissive (UFL-AG-286) and
nonpermissive (BM-5) cell lines infected with a
recombinant baculovirus containing the TSWV NSs gene
(vAcNSs). For this, a recombinant baculovirus expressing
a green fluorescent protein (vHSP70) was used to induce
RNAi and the suppressor activity of NSs was tested.
Co-expression of NSs and GFP proteins increased 10
times GFP expression in UFL-AG-286 cells. Northern
blot analysis revealed small interfering (si)RNA isolated
from vHSGFP-infected insect cells when hybridized with
a EGFP-specific probe. In contrast, the absence of small
RNA molecules of egfp transcripts in permissive and
semipermissive cell lines indicates the suppression of
gene silencing activity mediated by NSs expression.
Confocal microscopy analysis showed that NSs
accumulated in abundance in the cytoplasm of
permissive and semipermissive cells. Interestingly, high
amounts of NSs were detected in the nuclei of
nonpermissive cells. Bioassays with third instar S.
frugiperda and A. gemmatalis larva were performed by
injection of the budded virus form (BV) of the vAcNSs
and wild type of AcMNPV viruses. The vAcNSs LT50
values were significantly lower than those for AcMNPV
on larvae of S. frugiperda [5.62 and 8.5 days with 1 BV
and 4.82 and 7.52 days with 105 BVs, respectively] and
A. gemmatalis [7.46 and 20.11 days with 1 BV and 3.20
and 7.34 days with 105 BVs, respectively]. Our data
showed that NSs protein of TSWV facilitates baculovirus
gene expression in lepidopteran cells lines and increases
the virulence of baculoviruses to their insect hosts,
probably due to the role of NSs as a RNAi suppressor.
Palavras-chaves: NSs, baculovirus, replication, insect,
cell.
OCCULT HEPATITIS B VIRUS INFECTION IN HIVINFECTED PATIENTS: EVALUATION OF
SEROLOGICAL AND MOLECULAR PARAMETERS.
ID: 00414-00001
Área: 05 - Virologia Humana e Saúde Pública
Araujo, N.M., 2Pereira, K.C., 3Mouta Jr, S.S., 4Moraes,
M.T.B., 5Kay, A., 6Arabe, J., 7Gomes, S.A.
1. FIOCRUZ, Fundação Oswaldo Cruz, Laboratório de
Virologia Molecular, Av. Brasil, 4365, Rio de Janeiro,
Brazil2. INSERM, Institut national de la santé et de la
recherche médicale - U871, 151 Cours Albert Thomas
69424, Lyon, France3. FIOCRUZ, Fundação Oswaldo
Cruz, Instituto de Pesquisa Clínica Evandro Chagas, Av.
Brasil, 4365, RJ, Brazil
Aim: To determine the prevalence of occult hepatitis B
virus (HBV) infection in a group of human
immunodeficiency virus (HIV)-infected Brazilian patients
and to investigate its association with serological and
molecular features. Methods: Sera from 144 HIV-infected
patients, negative for HBV surface antigen (HBsAg) by
commercial enzyme-linked immunosorbent assays
(ELISA), were included in this study. HBV DNA was
extracted from serum samples and the region coding for
the small HBV surface protein (S-HBsAg) was amplified
by semi-nested PCR assay. HBV nucleotide sequences
from purified PCR fragments were determined. Serum
samples from all 144 patients were retested for the
presence of HBsAg, by an in-house ELISA, using a
monoclonal anti-HBs antibody. Results: HBV-DNA was
found in 5/144 (3,5%) samples. Nucleotide sequencing
revealed that two samples were from HBV genotype A,
one belonging to subgenotype A1 and other to A2. HBsAg
amino acid mutations Q101H and F134L were detected
in one patient and may explain HBsAg negativity. Both
mutations have already been described in cases of
occult HBV infection, specially, Q101H, which displayed
up to 120-fold reduced sensitivity in HBsAg commercial
assays. Interestingly, HBsAg could be detected by a
monoclonal antibody in four samples that were negative
for HBsAg by commercial assays. Conclusions: Occult
HBV infection was observed in the study group and was
associated with mutations in HBsAg. The monoclonal
antibody used here showed a higher sensitivity to detect
HBsAg than the commercial assays, and may be an
important tool for HBsAg detection, mainly in groups
where occult HBV infection is frequent. Since occult
infection is relatively common among HIV-infected
patients, HBV molecular monitoring should be employed
for an adequate management of HBV/HIV co-infected
patients. Financial support: ANRS, CNPq.
Palavras-chaves: HBV, hepatite B oculta, HIV, mutação.
OCCULT HEPATITIS B VIRUS INFECTION IN HIVINFECTED PATIENTS: EVALUATION OF
SEROLOGICAL AND MOLECULAR PARAMETERS.
ID: 00414-00002
Área: 05 - Virologia Humana e Saúde Pública
1
210
Araujo, N.M., 2Pereira, K.C., 3Mouta Jr, S.S., 4Moraes,
M.T.B., 5Kay, A., 6Arabe, J., 7Gomes, S.A.
1. FIOCRUZ, Fundação Oswaldo Cruz, Laboratório de
Virologia Molecular, Av. Brasil, 4365, Rio de Janeiro,
Brazil2. INSERM, Institut national de la santé et de la
recherche médicale - U871, 151 Cours Albert Thomas
69424, Lyon, France3. FIOCRUZ, Fundação Oswaldo
Cruz, Instituto de Pesquisa Clínica Evandro Chagas, Av.
Brasil, 4365, RJ, Brazil
1
ABSTRACTS
Aim: To determine the prevalence of occult hepatitis B
virus (HBV) infection in a group of human
immunodeficiency virus (HIV)-infected Brazilian patients
and to investigate its association with serological and
molecular features. Methods: Sera from 144 HIV-infected
patients, negative for HBV surface antigen (HBsAg) by
commercial enzyme-linked immunosorbent assays
(ELISA), were included in this study. HBV DNA was
extracted from serum samples and the region coding for
the small HBV surface protein (S-HBsAg) was amplified
by semi-nested PCR assay. HBV nucleotide sequences
from purified PCR fragments were determined. Serum
samples from all 144 patients were retested for the
presence of HBsAg, by an in-house ELISA, using a
monoclonal anti-HBs antibody. Results: HBV-DNA was
found in 5/144 (3,5%) samples. Nucleotide sequencing
revealed that two samples were from HBV genotype A,
one belonging to subgenotype A1 and other to A2. HBsAg
amino acid mutations Q101H and F134L were detected
in one patient and may explain HBsAg negativity. Both
mutations have already been described in cases of
occult HBV infection, specially, Q101H, which displayed
up to 120-fold reduced sensitivity in HBsAg commercial
assays. Interestingly, HBsAg could be detected by a
monoclonal antibody in four samples that were negative
for HBsAg by commercial assays. Conclusions: Occult
HBV infection was observed in the study group and was
associated with mutations in HBsAg. The monoclonal
antibody used here showed a higher sensitivity to detect
HBsAg than the commercial assays, and may be an
important tool for HBsAg detection, mainly in groups
where occult HBV infection is frequent. Since occult
infection is relatively common among HIV-infected
patients, HBV molecular monitoring should be employed
for an adequate management of HBV/HIV co-infected
patients. Financial support: ANRS, CNPq.
Palavras-chaves: HBV, Hepatite B oculta, HIV, Mutação.
A POPULATION-BASED PREVALENCE STUDY OF
HEPATITIS A VIRUS USING ORAL FLUIDS IN
ISOLATED COMMUNITIES OF WETLAND SOUTHERN
MATO GROSSO.
ID: 00415-00001
Área: 05 - Virologia Humana e Saúde Pública
TOURINHO, R. S., 2Moraes, A. C., 3VILLAR, L. M.,
MURAT, P. G., 5MOUSQUER, G. J., 6MOTTA-CASTRO,
A. R. C., 7DE PAULA, V. S.
1. Fiocruz/IOC, Fundação Oswaldo Cruz/Instituto
Oswaldo Cruz, Av. Brasil 4365, Pav. Helio e Peggy Pereira
sala B2062. UFMS, Universidade Federal do Mato
Grosso do Sul, Cidade Universitária S/N- 79070900,
Campo Grande/MS
Population-based prevalence studies are an important
tool for screening of HAV infection providing important
data on the susceptible groups. However, surveillances
in isolated communities become difficulty due to the
access to these areas and the collection of blood
samples, which is the specimen conventionally used in
HAV diagnosis. This situation leads to search for
alternative fluids that are non-invasive and easier to
collect facilitating the implementation of epidemiological
studies, such as oral fluids. This study was conduct in
order to evaluate the performance of oral fluids in
prevalence studies of hepatitis A in isolated communities.
Samples were collected in wetland of southern Mato
Grosso, where none data about HAV infection are
available. The study population was composed by 225
volunteers aged 1 to 86 years old. This prevalence study
was performed using oral fluids that were previously
standardized for anti-HAV antibodies detection (98%
sensibility and specificity). Oral fluid sample was obtained
using a commercial device ChemBio. The swab was
rubbed along the teeth/gum line for 1 min, returning to
the plastic tube containing 500μL of preservative solution.
Then, oral fluid was collected by centrifugation at 1300g
for 10 min and stored at 4-8ºC. Eluates were tested in
modify commercial EIA (ImmunoCombII HAVAb) to
detect total anti-HAV antibodies after 15 days of
collection. The overall prevalence for total anti-HAV was
79.1%, corresponding to 178 reactive EIA tests out of
225 samples. The age stratified data showed a prevalence
of 47,8% between 0 and 10 years, 84% in 11-20 years
and 91,9% in subjects higher than 21 years,
demonstrating a strong trend for increasing HAV infection
rate according to growth. These results showed that oral
fluid could replace serum in HAV epidemiological studies
in isolated communities since were stable in bad
conditions of collection, storage and transportation as
demonstrated by efficiency in detecting anti-HAV
antibodies.
Palavras-chaves: Hepatitis A, Oral fluids, Prevalence.
EVALUATION OF THREE ORAL FLUID COLLECTION
DEVICES TO DETECT ANTI-HAV ANTIBODIES
AMONG NATURALLY INFECTED AND VACCINATED
VOLUNTEERS
ID: 00415-00002
Área: 05 - Virologia Humana e Saúde Pública
1
4
A1TOURINHO, R. S., 2MORAES, A. C., 3SAMPAIO, D.
V., 4CRUZ, P. B., 5DA SILVA, A. S., 6DE PAULA, V. S.
1. Fiocruz/IOC, Fundação Oswaldo Cruz/Instituto
Oswaldo Cruz, Av. Brasil 4365, Pav. Helio & Peggy
Pereira, sala B206
Pathogenesis of HAV infection is a critical point to detect
211
ABSTRACTS
antibodies among oral fluids, since immunity induced
by HAV vaccine is at least 10 times lower than resulting
from natural infection. This fact is aggravated in oral fluids
in which antibodies titers are 800 to 1,000 fold lower
than serum. Therewith, evaluation of different collection
devices will allow distinguishing immune and susceptible
individuals with greater sensitivity. This study aimed to
evaluate anti-HAV antibodies in oral fluids among
vaccinated and naturally infected individuals using three
different collection devices. For that, 90 paired serum
and oral fluid samples were collected from non-reactive
(n=35), vaccinated (n=25) and naturally infected
volunteers (n=30). Serum was collected by venipuncture,
centrifuged at 1800g/5 min, and stored at -20ºC. Oral
fluid was obtained using 3 commercial devices: Salivette,
OraSure e ChemBio. Samples ware centrifuged at 1300g/
10 min and stored at -20ºC to 4-8°C depending on
collection device. Sera and oral fluids were submitted to
commercial EIA (ImmunoCombHAVAb). Optimization
panel demonstrated that oral samples were in agreement
with all serum groups of non-reactive and naturally
infected individuals. However, in vaccinated group, there
were 2 false-negative samples collected with the OraSure
and 4 with salivette device. The oral fluid test for total
anti-HAV was 92.7% sensitive for Salivette, 96.3% for
Orasure and 100% for ChemBio. All collection devices
showed 100% of specificity. A follow-up of 5 samples
collected with ChemBio device was realize to evaluate
stability of oral fluid and it was observed that 90 days
after oral fluid collection was possible to detect anti-HAV
antibodies. These results showed that Chembio was the
oral fluid collection device which better distinguishes
between susceptible and immune individuals. This
collector can be use to facilitate the screening of age
groups to receive HAV vaccine and the implementation
of a program to control disease.
Palavras-chaves: Hepatitis A, Oral fluids, Anti-HAV
antibodies.
artificially spiked oral fluid samples. Oral fluid specimens
were collected from 20 individuals without previous
history of HBV infection or HBV markers using two
commercial collectors: Salivette (Sarstedt, Germany)
and Chembio (Chembio Diagnostic System, USA). To
obtain oral fluid-positive HBV controls, a standard
commercial OptiQuant Acrometrix (Boston Biomedical,
USA) containing 20000000 copies/mL of HBV DNA was
used. Oral fluid pool (obtained separately from Salivette
and Chembio samples) was then submitted to serial tenfold dilutions (ranging from 2000000 to -200), tested in
parallel with a positive control (HBV DNA human positive
serum) and a negative control (negative serum and oral
fluid).DNA was extracted using QIAamp DNA Mini Kit
using 200ìL of sample volume and 150ìL of elution
volume. HBV DNA was amplified by semi-nested PCR
using primers for surface region of HBV genome. To
standardize PCR, three conditions were evaluated:
Platinum Taq DNA polymerase (5 U/ìl; Invitrogen) in a
volume of 0.1ìl (0.75 U) and 0.3ìl (1.5 U); DNA volume (2
and 5ìl); and number of amplification cycles (30 and 40
cycles). PCR products were loaded onto 1.0% agarose
gels and stained with Gel red to visualize bands of an
expected length of 1099 bp. Sensitivity was improved
when increased concentration of Platinum Taq DNA
Polimerase, and number of amplification cycles used.
HBV could be detected until 20 HBV-DNA copies/mL
using both collectors. Samples obtained with Chembio
presented nonspecific bands. HBV DNA could be
detected in artificially contaminated oral fluid using
modified PCR protocol but collector device is essential
to produce specific results. Studies using other extraction
DNA methods will be done to improve test efficiency.
Palavras-chaves: diagnosis, Hepatitis B, saliva.
EVALUATION OF ORAL FLUID AS BIOLOGICAL
SAMPLES FOR HBV DNA DETECTION.
ID: 00418-00001
Área: 01 - Imunobiológicos
ID: 00416-00001
Área: 05 - Virologia Humana e Saúde Pública
1
PORTILHO, M.M., 2MARTINS, P.P., 3MEDINA, H.C.,
LAMPE, E., 5VILLAR, L.M.
1. LAHEP - FIOCRUZ, Laboratório de Hepatites Virais Fundação Oswaldo Cruz, Rua Leopoldo Bulhões, 1480,
sala B09, Manguinhos, Rio de Janeiro, RJ
1
4
Detection of Hepatitis B virus (HBV) DNA is done in
blood samples, but in person with difficult venous access
it could be done using oral fluid. The aim of this study is
to establish a protocol for HBV DNA detection in
212
EVALUATION OF NEW IMMUNOENZYMATIC ASSAY
FOR TOTAL ANTI-HAV DETECTION USING IGY ANTIHAV
Silva, A.S., 2Vasconcelos, G.A., 3Pinto, M.A, 4De Paula,
V.S
1. Fiocruz - IOC, Laboratório de Desenvolvimento
Tecnológico em Virologia, Fundação Oswaldo Cruz - IOC,
Av. Brasil 4365 - Manguinhos, Rio de Janeiro - RJ, Brasil
Hepatitis A is an endemic disease in Brazil and Latin
America. Prevalence of this infection is related to the
low degree of hygiene and sanitation. Nowadays, due to
better sanitation conditions, the epidemiological profile
of disease is changing to older ages resulting in the
occurrence of outbreaks. Diagnostic kits for detection of
total anti-HAV generally use mammals immunoglobulin
ABSTRACTS
G (IgG) in the convalescent period of disease for
production of capture and conjugated antibodies. An
alternative to the application of mammals antibodies in
the diagnosis is the use of Immunoglobulin Y (IgY) from
birds and reptiles. The IgY has several advantages when
compared to IgG: high response against mammals
antigens, reduction of the background in imunoenzymatic
assays and it is obtained by a non-invasive method,
through the harvest of the egg yolks. The aim of this
study was to develop an immunoenzymatic assay for
total anti-HAV detection using IgY anti-HAV produced in
immunized chickens against Hepatitis A virus as capture
and conjugated. For evaluation of the immunoenzymatic
“in-house” assay with IgY anti-HAV, a panel composed
of two hundred samples was tested for total anti-HAV,
one hundred positive samples and one hundred negative
samples. The “in-house” assay showed sensibility of 88%,
specificity of 98% and efficiency of 80%. The utilization
of IgY anti-HAV in the “in-house” immunoenzymatic assay
was efficient and demonstrated a good sensitivity and
specificity. The advantages of IgY anti-HAV when
compared to IgG anti-HAV and the high sensibility and
specificity of the assay showed that IgY anti-HAV can
be use as an alternative to the IgG in immunoenzymatic
assays. Apoio Financeiro: CNPq e Fiocruz-IOC.
Palavras-chaves: IgY, Immunoenzymatic assay,
Hepatitis A, CpG.
cellular proteins and characterize it. These interactions
can be useful to design new drugs against YFV and
provide more information about the YFV replication. The
NS4B is a non conserved protein among the Flavivirus
and there is little information about the NS4B function.
The yeast two-hybrid system has been used as a genetic
tool for protein interactions detection. The method is a
model with stable, well-defined, and easy-to-manipulate
genetic system. In this study, we constructed three
different baits of NS4B and tested to toxicity to the yeast
and self-activation. All three baits showed no toxicity
and will be used in a screening. The screening will be
realized using Saccharomyces cerevisiae AH109 and
human cDNA library. Protein-protein interactions identified
by two-hybrid system will be further characterized using
GST-Pull down, co-immunoprecipitation and mass
spectrometry.
Palavras-chaves: Yellow Fever Virus, Protein-Interaction,
Flavivirus.
IDENTIFICATION OF NEW PROTEIN-PROTEIN
INTERACTION BETWEEN NS4B-YFV AND CELLULAR
PROTEINS
1
ID: 00419-00001
Área: 04 - Virologia Básica
Pacca, C.C., 2Zanfolin, J., 3Vidotto, A., 4Nogueira, M.L.
1. FAMERP, FACULDADE MEDICINA SAO JOSE RIO
PRETO, AV BRIGADEIRO F LIMA 5416
CITOTOXICITY EFFECT AND PRELIMINARY
ANTIDENGUE ASSAY OF THE SULFATED
POLYSSACHARIDES OF THE RED MARINE ALGA
Gracilaria birdiae IN C6/36 CELLS.
ID: 00420-00001
Área: 04 - Virologia Básica
Edfranck Vanderlei, 2Eloy, Y.R.G., 3Marques, M.M.M.,
Silva, A.R.A., 5Guedes, M.I.F., 6Mendonça, G.P., 7Alves,
A.W.S., 8 Lima, T.B., 9 Queiroz, I.N.L., 10 Benevides,
N.M.B.
1. UFC, Universidade Federal do Ceará, Av. Humberto
Monte S/N2. UECE, Universidade Estadual do Ceará,
Av. Paranjana, 1700
4
1
Flaviviruses are among the most important arboviruses
in the world, including yellow fever virus. Yellow Fever is
an infectious disease caused by Yellow Fever Virus (YFV),
prototype of the Flavivirus genus. The disease presents
endemic and epidemic cycles, affecting thousands of
people in tropical Africa and South America.YFV contains
a single positive-stranded RNA genome of approximately
11 Kb in length that encodes three structural proteins
(C, prM and E) and seven non-structural proteins (NS1,
NS2a, NS2b, NS3, NS4a, NS4b and NS5). The Flavivirus
genome replication occurs in the cell cytoplasm through
a replication complex involving viral and cellular proteins
and viral RNA. The current knowledge about this complex
still does not provide enough information on the biology
of the viruses, especially the YFV. The aim of this study
is identify interactions between YFV-NS4B and host
Sulfated polysaccharides (SP) are complex
macromolecules found in high concentrations in marine
algae and presents many biological and pharmaceutical
properties. The aim of this study was to examine the
cytotoxicity concentration (CC50) of the SP extracted
from red seaweed G. birdiae and to investigate the
antiviral activity against DENV-2. To evaluate the cell
viability, C6/36 cells were cultured in 96-well plates at a
density of 2×105 cells/well. After 24h of incubation, the
medium was removed and cells were treated with 200μL
of samples at different concentrations. Then, the cells
were incubated for five days, the medium was removed
and 50uL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl
tetrazolium bromide (MTT) solution (1mg/mL) was added.
The plates were reincubated for 4 h, the MTT solution
was removed and 100μL of DMSO was added. The plates
were gently shaken and read at 495nm. In addition, a
preliminary test was done to evaluate the antiviral activity.
For this, a working stock of DENV-2 was passaged 2
213
ABSTRACTS
times in C6/36 cell monolayer and the supernatant was
removed 7 days post-infection. Following, C6/36 cells
were cultured and treated with 100μL of SP (3,9-1000
ppm) and 100uL of virus suspension (negative control =
only cells; positive control = cells + virus). Five days
post-inoculation, the cultures were observed by optical
microscopy to analysis of cytopathogenic effect. The
results showed that SP in the highest concentration
(1000 ppm) presented a cell viability of 82.7% and did
not caused cytotoxicity (CC50>1000 ppm). In addition,
was showed that SP (15,62ppm) decreased the presence
of rounded different sized syncytia and inhibited the
infection process. Nevertheless, on positive control was
observed infected cells and low cell density. Thus, the
results show that the G. birdiae SP did not presented
any cytotoxicity on C6/36 cells and present antiviral effect.
Palavras-chaves: alga, Gracilaria, cytotoxicity, dengue,
virus.
ASSESSMENT OF IMMUNE RECONSTITUTION IN
PREGNANT WOMEN UNDERGOING PROPHYLAXIS
WITH ARV TO PREVENT MTCT OF HIV-1.
ID: 00421-00001
Área: 05 - Virologia Humana e Saúde Pública
transmission. Thus, ten women infected with HIV-1 and
treatment naive were evaluated prior to initiation of
HAART, in childbirth, during the suspension of HAART
and six months after delivery. With HAART, the control
of viral replication was achieved, as well as an important
addition in the levels of CD4 +T cells. Changes, such as
the reduction of immune hyper-activation, recovery of
naive T cells and central effector memory cells to their
normal levels in CD4+ and CD8+ cells subpopulations
were not observed. Therefore, although important for the
prevention of vertical transmission, which was zero in
this study, HAART prophylaxis was not able to promote
a full immune reconstitution in these patients. Financial
support: CNPQ Área: Virologia Humana Tema: HIV/Aids
Apresentador: Geane Lopes Flores.
Palavras-chaves: HIV, Immunology, pregnant.
CITOTOXICITY EFFECT AND PRELIMINARY
ANTIDENGUE ASSAY OF Aloe vera POLYSSACHARIDES IN C6/36 CELLS
ID: 00422-00001
Área: 04 - Virologia Básica
Eloy, Y.G.E, 2Vanderlei, E.S.O., 3Marques, M.M.M.,
Silva, A.R.A., 5 Guedes, M.I.F., 6 Mendonça, G.P.,
7
willamealves@rocketmail.com, 8Lima, T.B., 9Queiroz,
I.N.L., 10Benevides, N.M.B.
1. UFC, Universidade Federal do Ceará, Av. Humberto
Monte S/N2. UECE, Universidade Estadual do Ceará,
Av. Paranjana 1700
1
4
Flores, G. L., 2Pilotto, J. H., 3Giacoia Gripp, C.B.W.,
Morgado, M. G.
1. FIOCRUZ, Instituto Oswaldo Cruz, Av Brasil 4365
Manguinhos RJ
1
4
In recent years, AIDS epidemic in Brazil has shown
significant changes in its profile, characterized primarily
by its feminization. The growth of AIDS cases in women,
especially those in reproductive age, has resulted in the
increase of children acquiring AIDS through vertical
transmission. Therefore, preventing vertical transmission
of HIV-1 is a great challenge to be faced. Several studies
have shown a reduction of vertical transmission of HIV1, between zero and 2%, with appropriate use of
antiretroviral therapy during pregnancy and/or at delivery.
But this is not the only benefit of HAART towards infection
by HIV-1, since a profound decrease in the incidence of
opportunistic infections was achieved with the use of
ART and directly related to the consequent recovery of
the immune system with a clear quantitative increase of
the peripheral levels of CD4 +T cells. Other events
associated with the immunopathogenesis of HIV-1 as
the generalized process of activation and the consequent
renewal and differentiation of T lymphocytes have also
changed. In this context, the objective of this study is to
assess the immunological profile; in particular the
immune reconstitution of HIV-1 infected pregnant women
at different moments of antiretroviral therapy (HAART)
or antiretroviral prophylaxis, to prevent ver tical
214
Aloes have been used therapeutically and different
properties being ascribed to the inner, colourless, leaf
gel. Thus, the study reported here was intended to
examine the cytotoxicity concentration (CC50) of the
polysaccharides extracted from A. vera and to
investigate the antiviral activity against DENV-2. To
evaluate the cell viability, C6/36 cells were cultured in
96-well plates at a density of 2×105 cells/well. After 24h
of incubation, the medium was removed and cells were
treated with 200uL of samples at different concentrations.
Then, the cells were incubated for five days, the medium
was removed and 50uL of 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide (MTT) solution (1mg/
mL) was added. The plates were reincubated for 4 h, the
MTT solution was removed and 100μL of DMSO was
added. The plates were gently shaken and read at 495nm.
In addition, a preliminary test was done to evaluate the
antiviral activity. For this, a working stock of DENV-2
was passaged 2 times in C6/36 cell monolayer and the
supernatant was removed 7 days post-infection.
Following, C6/36 cells were cultured and treated with
100μL of A. vera polysaccharides (3,9-1000 ppm) and
100μL of virus suspension (negative control = only cells;
ABSTRACTS
positive control = cells + virus). Five days postinoculation, the cultures were observed by optical
microscopy to analysis of citopathogenic effect. The
results showed that polysaccharides in the highest
concentration (1000 ppm) presented a cell viability of
90,0% and did not caused cytotoxicity (CC50>1000 ppm).
In addition, was showed that polysaccharides at
concentration of 15,62 ppm decreased the presence of
rounded different sized syncytia and inhibited the
infection process. Nevertheless, on positive control was
observed infected cells and low cell density. Thus, the
results show that the A. vera polysaccharides did not
presented any citotoxicity on C6/36 cells and present
antiviral effect.
Palavras-chaves: Aloe vera, Dengue, DENV-2,
polysaccharides.
HIGH FREQUENCY OF HBV POLYMERASE
MUTATIONS RELATED TO VACCINE ESCAPE
AMONG BLOOD DONORS FROM SANTOS, BRAZIL
ID: 00423-00001
Área: 04 - Virologia Básica
MANTOVANI, N., 2 CARMO, E.P., 3CASTRO, D.F.,
SANTANA, L.C., 5 CÍCERO, M.F., 6 ALMEIDA, C.P.,
7
REIS, H., 8CASEIRO, M.M., 9KOMNINAKIS, S.C.V.
1. UNILUS, Centro Universitário Lusíada, Rua Oswaldo
Cruz, 179 Boqueirão - Santos2. UNIFESP, Universidade
Federal de São Paulo, Rua Pedro de Toledo, 781 Vila
Clementino – São Paulo
viral fitness and/or increase LAM resistance in the
presence of primary mutation. However, in our study it
was found in genotype A, demonstrating that there is no
relationship between genotype and mutation. In rtT128
position there was 41.2% mutations which: 85.7% was
from rtT128P and 14.6% from rtT128S. The exchange
rtT128N restores the resistant virus fitness and may
change the surface gene and form vaccine escape.
However, it is not clear whether the rtT128P and rtT128S
have this power. Despite the absence of primary
Lamivudine resistant mutations, this study verified the
presence of compensatory mutations associated with
vaccine escape related to recovery of viral fitness in the
city of Santos, Brazil. Financial Suport: Centro
Universitário Lusíada Área: Virologia Básica.
Palavras-chaves: Hepatitis, Lamivudina, Primary
Resistance.
HIGH LEVELS OF POLYMORPHISMS RELATED TO
RALTEGRAVIR
RESISTENCE
AMONG
RALTEGRAVIR NAIVE INDIVIDUALS IN BRAZIL
ID: 00423-00002
Área: 04 - Virologia Básica
1
4
Lamivudine (LAM) is a nucleoside analog inhibitors
(NRTI) that reduces HBV viral load to undetectable levels.
Treatment with LAM is safe, but the efficacy of the NRTI
is limited by the rapid emergence of drug-resistant. The
presence of LAM resistant mutations causes changes
in amino acids present in the surface antigen HBsAg
due to the overlap between the polymerase and the
surface genes. These changes may compromise the
vaccine response. Our goal were to evaluate the
transmission of LAM primary resistance and vaccine
escape in 17 serum samples from blood donors at the
Hemocenter of the Guilherme Álvaro State Hospital in
Santos, Brazil. HBV-DNA was extracted from the sera
of donors. The PCR was carried out to amplify HBV
polymerase and HBsAg genes. The direct sequencing
was performed using the Applied Biosystem BigDye ®
Terminator V3. Sequences were corrected, assembled
and mutations analyzed with Sequencher Program and
Bioedit. There was no presence of primary mutations in
the YMDD motif. However, compensatory mutations were
found: rtL80V in four samples (36.3%), rtL80F in five
(45.5%) and rtL80S in two (18.2%). The rtL80V mutation,
described only in genotypes B, C and D, is able to restore
MANTOVANI, N., 2AZEVEDO, R.G., 3CASTRO, D.F.,
DIAZ, R.S., 5KOMNINAKIS, S.C.V.
1. UNILUS, Centro Universitário Lusíada, Rua Osvaldo
Cruz, 179 Boqueirao-Santos2. UNIFESP, Universidade
Federal de São Paulo, Rua Pedro de Toledo, 781 Vila
Clementino-São Paulo
1
4
Raltegravir (RAL) is an HIV-1 integrase strand-transfer
inhibitor that has exhibited substantial efficacy and a
favorable safety profile in HIV-1infected patients. The
goal of this study was to explore the presence of natural
polymorphisms and primary mutations related to RAL
resistance among HIV-1 patients failing to multiple
antiretroviral agents. 25 plasmas from HIV-1 infected
patients with HAART failure were studied. Genetic
analysis was performed amplifying and sequencing DNA
encompassing 288 amino acids of HIV-1 integrase gene.
Genetic subtypes were analyzed using REGA HIV
Subtyping Tool. Of the 25 patients, 15 were males and
10 females. All of them are more than 18 years old and
19 patients born in Sao Paulo city. 22 patients were
infected by HIV-1 subtype B, 1 by subtype F and 2 by B/
F recombinants. No Raltegravir resistance related
mutations were observed, however we identified following
polymorphisms: V72I (44%), T97A (4%), Q146K (4%),
V151I (28%), V201I (52%), T206S (8%), I203M (12%),
S230N (4%), M154L (4%), K156N (16%) e K156R (4%).
Furthermore, we observed amino acid substitutions at
codons 163 in two patients (G163E e G163V) and 138 in
one patient (E138N). Despite the absence of RAL primary
215
ABSTRACTS
resistance mutations, we found a high frequency of
polymorphisms that were related to in vitro reduced
susceptibility to RAL. Furthermore, substitutions at
codons 163 (G163R) and 138 (E138K) are called
secondary mutations, which are capable to restore viral
fitness due to the presence of primary mutations. Further
studies are needed to determine the importance of these
polymorphisms in reducing the genetic barrier to RAL
resistance among treated individuals. Financial Suport:
FAPESP (2009/5712-3) Área: Virologia Básica.
Palavras-chaves: HIV-1, Primary Resistance, Integrase
Inhibitor.
AFRICAN SWINE FEVER VIRUS IN BRAZIL:
HISTORIC
AND
ACHIEVEMENTS
OVER
LABORATORIAL VIRUS AND ANTIBODY
SCREENING IN THE EMERGENCE PHASE
ID: 00424-00001
Área: 03 - Virologia Animal
samples, only one was HAD positive and four were IEOP
positive. From October, to December, sera were the
majority of samples in which were detected 227 immune
reagents pigs by IEOP test. In conclusion, the impressive
efficiency of implanted laboratorial techniques was
fundamental for the eradication campaign established in
1980, which assured the eradication of ASF virus in all
country after six years from the first ASF outbreak.
Palavras-chaves: African swine fever, Virus historical
research, ASFvirus and antibody screening, Laboratorial
diagnostic, ASF outbreaks.
RELATIONSHIP BETWEEN PRO-INFLAMMATORY CYTOKINE GENE EXPRESSION AND
TRACHEAL LESIONS INDUCED BY INFECTIOUS
BRONCHITIS VIRUS
ID: 00425-00001
Área: 03 - Virologia Animal
OKINO, C.H., 2GONÇALVES, M.C.M., 3FERNANDO,
F.S., 4 MONTASSIER, M.F.S., 5 TAMANINI,M.L.F.,
6
MONTASSIER,H.J.
1. FCAV - UNESP, Faculdade de Ciências Agrárias e
Veterinárias, Rod de acesso Prof Paulo Donato
Castellane, s/n, Industrial, Jaboticabal - SP2. Ammco
Pharma, Ammco Pharma Saúde Animal, R Dr Paulo
Pinto, 957, São Dimas, Piracicaba-SP
1
Freitas, T.R.P., Lyra, TMP
1. LANAGRO/MG MAPA, Laboratório Nacional
Agropecuário Ministério da Agricultura, Pecuária e
Abastecimento, avenida Rômulo Joviano, s/n2. Equalis,
Equalis- ensino equalificação superior, ww.equalis.com.br
1
2
African swine fever (ASF) is a devastating viral disease
of Suidae family in all breeds and ages. After ASF
outbreak occurred in Brasil in 1978, which caused
relevant economic and social impacts in our recent
history, was established the official laboratory for ASF
diagnosis under International rules. This work was,
therefore, carried out to revise all efforts of the laboratory
team to settle the ASF diagnosis. From June, 12th to
December 28th, 1978, 3532 pig tissue, blood and sera
were collected principally from Southeast and South
regions. Virus isolation by haemadsorption (HAD) of
erythrocytes and antibody screen (IEOP) was based on
international standards methods. The first pig samples
came from all mesoregions (MR) of Rio de Janeiro state
(RJ). Pig samples from Teresópolis city, constituted the
first isolation of ASFV out of Paracambi. ASFV was
isolated from others regions of RJ bounded each to other
by road. About the country, in the first month, 101 (56.11
%) pig samples of 180 were positive: 21 from RJ, 37
from São Paulo, 17 from Paraná and 7 from Minas Gerais.
besides other regions. The positive results were rapidly
sent to Federal government and State staff to provide
sanitary measures. Certainly, the integrated action of
the laboratory and Official Institutions contributed for ASF
outbreaks decreased in more 70% from July to August.
The detection of antibody against ASFV by IEOP test
increased from 18% in June, to 47.80 % in August, 1978.
In September, from 197 Northeast and South regions
216
Infectious bronchitis (IB) is a highly contagious respiratory
poultry disease, and it is caused by IB virus (IBV), which
initially infects the upper respiratory tract and
disseminates from this site to other tissues like urogenital and digestive tracts. Characteristic pathological
changes induced by IBV are deciliation of the ciliated
epithelia of the nose and trachea follows infection and
acute inflammatory responses in these tissues, which
evolve to inflammatory reactions in secondary bronchi,
lung, and airsacs, leading to enhanced susceptibility for
most severe bacterial infections. We studied the role of
proinflammatory cytokines (IL1&beta, IL6 and TNF) at
entry site of IBV infection to determine their relationship
with tracheal lesions (ciliostasis, histopathology and viral
replication) at different time-points after challenge (8
hours post-infection (pi), 1 day (dpi), 3dpi, 7dpi and 14dpi).
The most prominent tracheal lesions were observed since
3 dpi, increased at 7 dpi and decline at 14 dpi. IL1&beta
gene started to increase its expression at 1dpi, reaching
higher levels of expression (approximately 5 fold more
than birds of negative control samples) at 3 dpi. The IL6
expression showed a similar profile, except that it
augmented 20 fold more than the negative control
samples. TNF did not show significant differences of
expression. The increased levels of IL6 and IL1&beta
correlated positively (Spearman test) with histopathology
ABSTRACTS
and ciliostasis scores as well as viral replication on
tracheal tissues of non-immune chickens challenged with
a typical respiratory pathotype of IBV (M41 strain). These
results indicates that IL1&beta and IL6 may play a role
in IBV-induced tracheal lesions and can be related to
enhanced susceptibility for bacterial secondary infection
and alternatively, they can be used as markers to evaluate
and compare the course of infection in vaccinated and
non-immune birds.Financial suppor t: FAPESP,
CNPq,Pós-graduação em Medicina veterinária(UNESP).
Palavras-chaves: Cytokine, Infectious Bronchitis virus,
Inflammation, Interleukin 6, Interleukin 1 beta.
Replicação HIV-1 Arquivo: inibidor_hiv_poeys.rtf
Apresentador: Sandro Costa Poeys.
Palavras-chaves: hiv-1 replication inhibit, antiretroviral,
hiv therapy.
DETECTION OF BOVINE PAPILLOMAVIRUS TYPE
11 AND A PUTATIVE NEW TYPE OF BPV IN CATTLE
IN NORTHEAST REGION OF BRAZIL
ID: 00428-00001
Área: 03 - Virologia Animal
Carvalho, C. C. R., 2Batista, M. V. A., 3Reis, M. C.,
Balbino, V. Q., 5Castro, R. B., 6Freitas, A. C.
1. UFPE, Universidade Federal de Pernambuco, Av. Prof.
Moraes Rego, 1235 - Cidade Universitária, Recife - PE CEP: 50670-9012. UFRPE, Universidade Federal Rural
de Pernambuco, Rua Dom Manoel de Medeiros, s/n,
Dois Irmãos - CEP: 52171-900 - Recife/PE
1
SCREENING OF NEW COMPOUNDS THAT INHIBIT
THE REPLICATION OF HIV-1
ID: 00426-00001
Área: 05 - Virologia Humana e Saúde Pública
POEYS, S. C., 2LIMA, L. M., 3COSTA, L. J. da
1. UFRJ, Universidade Federal do Rio de Janeiro, CCS Bloco I - Cidade Universitária - Rio de Janeiro/RJ - CEP:
21.941-590
4
1
It is estimated that 44 million people are infected with
the Human Immunodeficiency Virus Type 1 (HIV-1)
worldwide. The pharmacologic therapy used to control
HIV replication consist in the administration to the patient
of a combination of at least 3 anti-retroviral drugs (Highly
Active Antiretroviral Therapy HAART), and is the only
available treatment to prevent disease progression.
However, there are several problems associated with the
long-term antiretroviral treatment, to mention the toxicity
of the drugs which leads to the low adhesion of the
patients to the treatment and the selection of resistant
and multi-resistant viral strains. Therefore, it is of great
importance to search for new drugs that could inhibit
viral replication, especially to act against new viral targets
and already existing resistant strains. The objective of
this work was to screen a library of 24 synthesized
compounds that could act against different targets during
the HIV replication cycle. The anti-viral activity of the
compounds was tested in a single-round infection in the
TZM-bl indicator cell line. Initially, cell toxicity of these
drugs was evaluated by XTT assays. 17 out of the 24
compounds tested showed no toxicity to the cells at the
highest concentration tested (100μM). From these, six
compounds demonstrated significant inhibition of HIV-1
replication (varying from 30% to 55%). The IC50 of 2 of
these compounds was calculated in 66nM, 58nM. Assays
are now being conducted to characterize the mechanism
of action of these compounds. This work demonstrates
the sensibility of one-round replication assays to screen
for new anti-retroviral drugs. Financial support: INCT/
CNPq Área: Virologia Humana Tema: Inibidores de
Papillomaviruses are DNA viruses with double-stranded
circular genome that vary from 6800-8400 base pairs.
The bovine papillomavirus (BPV) has been identified as
a cause of cancer. It has been well characterized ten
types of BPVs. Most recently, it was characterized the
BPV11, a little known virus type and its genome was
just recently deposited in GenBank, however it was not
published until now. It is very important to know the viral
types that circulate in Brazil. So, the aim of this study
was to evaluate the presence of BPVs 1-11 and identify
putative new viral types. We analyzed 36 samples of 29
animals on 16 farms located in the State of Bahia. PCRs
were performed with degenerate primers FAP59/FAP64.
Sequencing was used to distinguish the viral types. The
sample that suggested a putative new type was amplified
with “high fidelity” Taq and cloned. The clones were
sequenced and the sequences were analyzed with the
programs Pregap4 and Gap4 of Staden package and
the similarities assessed by Clustal W and BioEdit. The
most prevalent viral type (BPV2) was present in 11.1%
of lesions. BPV10 and BPV3 appeared both in 5.5% of
the samples. We also detected BPV1, BPV6 and BPV8
each in 2.7% of the samples. We also identified the
sequence of the recently described type (BPV11) in 5.5%
of the lesions. The putative new types, already described
in Southern Brazil, were also identified: BPV/BR-UEL4
was present in 11.1% of the injuries and BPV/BR-UEL3
and BPV/BR-UEL5 both found in 2.7% of the samples.
A new putative new type of BPV has been identified
showing less than 80% of identity with the closest viral
types described. The isolate was provisionally named
BPV/UFPE01 and presents 79% of identity with BPV/
BR-UEL3. Although papillomatosis has a worldwide
distribution there are no effective strategies to control or
treat this illness. The study of viral diversity and
217
ABSTRACTS
distribution is critical for effective measures for treatment
and prevention of papillomaviruses. Financial
support:CAPES.
Palavras-chaves: BPV11, Detecção, Novo Tipo,
Papilomavírus.
that bovines with papillomatosis can infect equine with
BPV leading to the development of equine sarcoid. More
studies are necessary to discover the form of viral
transmission. Financial support:CAPES.
Palavras-chaves: BPV, Detection, Blood, Equines.
DETECTION OF THE BOVINE PAPILLOMAVIRUS
TYPES 1 AND 2 AND GENE EXPRESSION IN
PERIPHERAL BLOOD OF EQUINES AND BOVINES
CREATED IN SAME COUNTRY PROPERTY IN
BRAZIL
NEW VARIANTS OF E6 AND E7 ONCOGENES OF
HUMAN PAPILLOMAVIRUS TYPE 31 IDENTIFIED IN
NORTHEASTERN
BRAZIL
AND
THEIR
RELATIONSHIP WITH PREDICTED T-CELL
EPITOPES
ID: 00428-00002
Área: 03 - Virologia Animal
ID: 00429-00001
Área: 05 - Virologia Humana e Saúde Pública
Silva, K. M. G., 2Santos, F. L., 3Vieira, R. T. A., 4Andrade,
R. L. F. S., 5Carvalho, C. C. R., 6Silva, M. A. R., 7Freitas,
A. C.
1. UFPE, Universidade Federal de Pernambuco, Av. Prof.
Moraes Rego, 1235 - Cidade Universitária, Recife - PE CEP: 50670-9012. UFPE, Universidade Federal Rural de
Pernambuco, Rua Dom Manoel de Medeiros, s/n, Dois
Irmãos - CEP: 52171-900 - Recife/PE
Chagas, B.S., 2Batista, M.V.A., 3Crovella, S., 4Freitas,
A.C.
1. UFPE, Universidade Federal de Pernambuco, Av. Prof.
Moraes Rego, 1235 - Cidade Universitária, Recife - PE CEP: 50670-901
1
Bovine Papillomavirus (BPV) is a species-specific virus
that infects cattle, however it was also described in
European buffalo that coexisted next to bovines. Strong
evidences suggest that BPV types 1 and 2 are involved
in equine sarcoid. This virus is presented in some tumors
described in equines, that do not suffer metastases.
These tumors tend to grow depending on the treatment
and on the viral types that are infecting the cells. The
objective of this study was to analyze the presence of
BPV 1 and 2 and the activity of E5 and E2 genes in
peripheral blood of equines and bovines. Of the four
evaluated horses, only one presented sarcoid confirmed
for the histopathology exam, and two bovines had
presented papillomatosis where all belonged to the same
country property. DNA and RNA extraction was carried
out in total blood. The detection of BPV 1 and 2 was
carried out using PCR with primers that amplified one
fragment of the genes E5 and E2. The expression of
these genes was verified by RT-PCR, using primers for
E5 and E2 genes of BPV-2. The expression of the E2
protein, that plays crucial role in the viral cicle regulating
the transcription, response and segregation of the viral
genome in the cells, was verified in the blood of two
animals. The expression of E5, that is the minor
oncoprotein that par ticipates in the cellular
transformation, was verified in the blood of 3 animals one of them presented one tumor. In bovines, that
belonged to the same property but did not coexist with
the horses, it was found the presence of BPV 1 and 2.
These results support the hypothesis that the blood
stream can work as reservoir and transport of BPV, and
218
1
The human papillomavirus (HPV) is a DNA virus that
infects the epithelial tissue, and the sexual contact is
the main form of their transmission. Currently, more than
120 types of HPV have been described, in addition to
subtypes and variants, based on differences in nucleotide
sequences. Advances in tumor virology has shown that
high risk HPV has an important role in the development
of cervical cancer, which is considered the second most
common malignant neoplasy among women worldwide.
In this study, we evaluated the genetic variability of
HPV31 and associated them to predicted T-cell epitopes.
To accomplish this, DNA samples from HPV31 positive
patients, coming from Recife/PE, were amplified, cloned
and sequenced with respect to genomic regions E6 and
E7. The obtained sequences were aligned with the
reference sequence deposited in a public database (NCBI)
to assess the mutations. The importance of these
mutations to the immune function of the proteins E6 and
E7 was analyzed by predicting T-cell epitopes. The
sequence analysis revealed the presence of specific
synonymous and non-synonymous variations. Some
amino acid changes in HPV31 variants, for the E6 and
E7 proteins, were mapped at sites belonging to T-cell
epitopes. These findings show that changes in E6 and
E7 mapped within the T-cell epitopes can alter the
immune recognition of cells infected with HPV, and may
have direct implications on the immune response, which
could, alone or together with other factors, clarify the
high incidence of HPV31 in Brazilian Northeastern
patients. This study contributed to expand the knowledge
about the genetic diversity of HPV and their relationship
to the immune system, being relevant to the development
of vaccines that meet national needs. Financial support:
CNPq/Facepe-PPSUS.
ABSTRACTS
Palavras-chaves: Cervical cancer, Epitopes, E6, E7,
Human papillomavirus.
EVALUATION OF CHANGES ON E6 AND E7
ONCOGENES OF HUMAN PAPILLOMAVIRUS TYPE
31 OF PATIENTS WITH CERVICAL LESION FROM
NORTHEAST REGION OF BRAZIL
EVALUATION OF GENOTYPIC RESISTANCE TO THE
NEW CLASS OF ANTIRETROVIRAL: HIV-1
INTEGRASE INHIBITORS
ID: 00430-00001
Área: 05 - Virologia Humana e Saúde Pública
CAVALCANTI, J.C., 2BATISTA, J.P.G, 3FERREIRA,
4
5
SIQUEIRA,
AFAC,
CABRAL,
G.B,
J.L.P,
6
7
RODRIGUES, R., BRIGIDO, L.F.M
1. IAL, INSTITUTO ADOLFO LUTZ, AV. DR. ARNALDO,
355 - CERQUEIRA CESAR-CEP: 01246902-SÃO
PAULO/SP-BRAZIL
1
ID: 00429-00002
Área: 05 - Virologia Humana e Saúde Pública
Chagas, B.S., 2Gurgel, A.P.A.D., 3 Batista, M.V.A.,
Crovella, S., 5Freitas, A.C.
1. UFPE, Universidade Federal de Pernambuco, Av. Prof.
Moraes Rego, 1235 - Cidade Universitária, Recife - PE CEP: 50670-901
1
4
Human Papillomavirus (HPV) has the ability to cause
clinical infection after sexual transmission. Studies
confirm the relationship between infections caused by
some HPV types and the etiology of cervical cancer,
which is considered the second leading cause of death.
E6 and E7 genes are key oncogenes to high-risk HPVs
and their products inhibit p53 and pRb, respectively,
contributing to cellular transformation. Several studies
have shown a diversity of variants of HPV types.
Differences in nucleotide sequences among HPV variants
may result in amino acid changes and can lead to different
oncogenic potential. Thus, in this study we have evaluated
the genetic variability of HPV31 concerning to
nonsynonymous nucleotide changes as well as the
physicochemical properties of amino acids in E6 and
E7 proteins. DNA positive samples of HPV31 from
Recife, State of Pernambuco, Northeastern Brazil, were
amplified, cloned and sequenced with respect to E6 and
E7 genomic regions. The obtained sequences were
aligned with the reference sequence deposited in a public
database (NCBI). The sequence analysis revealed
nonsynonymous variations, which caused changes in
amino acids with different physicochemical properties.
The amino acid substitution occurred in E6 caused
changes in the polarity and hydropathic potential, while
in E7 the amino acid substitution led to changes in side
chain. This type of variation is usually related to change
in the conformational structure of proteins, which can
lead to a functional change. The present study shows
non-conservative nucleotide changes detected in the E6
and E7 oncogenes and suggests the possibility that
structural and functional modifications on proteins can
be caused by those changes. This would impact
tumorigenesis by increasing the oncogenic potential of
HPV. Financial support: CNPq/Facepe-PPSUS.
Palavras-chaves: Amino acid, Cervical cancer, E6, E7,
Human papillomavirus.
Integrase inhibitor (INI) is considered an important
addition of the antiHIV treatment. The first INI, Raltegravir
have been recently approved for use in salvage therapy
and other drugs are currently in study. Mutations in the
codons 143/148/155 of Integrase gene (IN) have been
associated to INI resistance, second generation with
difference mutation profile are in development. The aim
of this study was to evaluate primary resistance to INI
and molecular characteristics of IN. Sequences of 53
patients not exposed for INI (30 ART and 23 naïve
patients) were enrolled. Drug susceptibility was analyzed
by Stanford Database(SDb) and Geno2pheno (G2p).
Laboratory and demographic data was analyzed by
EpiInfo. Mean age, sex, VL, TCD4 were respectively:
36yo (6-53), 64% male, 4.3log, 311cells/mm³. Clade B
in 57% and non-B in 43% (C 21%, F 11% and mosaic
11%). Major mutations were not been observed, however
SDb algorithm detected some minor mutations: V151I
11.5%, G163R 8%, S230N 6%, M154I 6%, H51R 2%
and I203M 2%. V151I were observed only in clade B and
G163R in clade F. When G2p is used, V151I and S119R
suggest reduce susceptibility to RAL and EVG
respectively. The most frequent polymorphisms were
V201I 58% and L101I 55%. Polymorphisms (means)
clade B=9 (2-15) and non-B=15 (8-21).
FINANCIAL SUPPORT CAPES
Palavras-chaves: HIV-1, ANTIRETROVIRAL THERAPY,
INTEGRASE, INTEGRASE INHIBITORS, RESCUE
THERAPY.
DETECTION OF HEPATITIS A VIRUS IN LIVER
SECTIONS USING IMMUNOGLOBULIN Y (IGY)
PRODUCED IN IMMUNIZED HENS
ID: 00431-00001
Área: 01 - Imunobiológicos
Vasconcelos, G.A., 2 Silva, A.S., 3 Kappel, L.A.,
Lanzarini, N.M., 5de Paula, V.S., 6Pinto, M.A.
1. IOC - FIOCRUZ, Instituto Oswaldo Cruz - Fundação
Oswaldo Cruz, Av. Brasil, 4365, Manguinhos, Rio de
Janeiro, RJ. CEP: 21.040-360
1
4
219
ABSTRACTS
Immunoglobulin Y (IgY) is found in birds and reptiles,
currently used by the advantage of being transported
from serum to the yolk of eggs of these animals. This
protein is purified from egg yolk of immunized birds with
a specific antigen, and the IgY easily accessible and
has bioethical character, because the animals do not
suffer any injury. Besides low cost, the amount of IgY
produced by animals is very high, corresponding ten
times the annual average production of IgG in rabbits.
Detection of Hepatitis A Virus (HAV) in liver tissue is
important in cases of acute liver failure to precisely
diagnose the cause of liver failure. Commercial anti-HAV
antibodies currently have low affinity and have high cost.
Our study aims to produce specific anti-HAV in laying
hens immunized for detection of HAV in liver. Chickens
divided into three groups were immunized with the
following schedule: Group I- commercial vaccine against
hepatitis A and CpG-ODN; Group II- HAV, incomplete
Freund’s adjuvant (IFA) and CpG-ODN; Group III- IFA
(control). The eggs were collected and purified by the
method of precipitation in polyethylene glycol. The
detection of HAV in liver were analyzed by indirect
immunofluorescence (IIF) with IgY anti-HAV was used
as primary antibody and goat IgG anti-IgY labeled with
AlexaFluor®488 as secondary antibody. We used liver
samples from monkeys experimentally infected, patient
with HAV, and patient without HAV (control). The primary
antibodies (IgY) used were purified from eggs of groups
I and II, and the antibody of the Group III was used as
control. All infected tissue sections showed specific
staining with anti-HAV IgY and no specific staining with
control IgY. The IIF of control patient has not provided
specific staining with either antibodies (anti-HAV IgY and
control IgY). These preliminary results demonstrate the
specificity of the obtained IgY, which can be useful for
diagnostic purposes, such as IIF tests for direct detection
of HAV in tissue sections.
Palavras-chaves: Immunoglobulin Y (IgY), Hepatitis A
Virus (HAV), CpG-ODN, Incomplete Freund’s Adjuvant
(IFA).
MOLECULAR CHARACTERIZATION OF CANINE
PARVOVIRUS (CPV) STRAINS DETECTED IN
VACCINATED PUPPIES
ID: 00432-00001
Área: 03 - Virologia Animal
CUBEL GARCIA, R.C.N., 2CASTRO, T.X., 3COSTA,
E.M., 4LEITE, J.P.G., 5LABARTHE, N.V.
1. UFF, Universidade Federal Fluminense, Rua Prof.
Hernani Melo 101, Niterói, RJ, CEP 24210-1302. Fiocruz,
Instituto Oswaldo Cruz, Avenida Brasil 4365, Pav. Hélio
& Peggy Pereira, 21040-360, Rio de Janeiro, RJ3. Fiocruz,
1
220
Programa de Biodiversidade e Saúde, Av. Brasil 4036,
Prédio da Expansão, sala 214, 21040-361, Rio de Janeiro,
RJ
Canine parvovirus (CPV) is still circulating in canine
population despite vaccination and seems to be
undergoing continuous evolution, with the generation of
new genetic and antigenic variants. The objective of this
study was to investigate, by partial sequencing of the
capsid VP2 gene, the CPV variants in fecal samples
collected from vaccinated puppies with enteric illness.
A total of 37 CPV-positive fecal samples by HA/HI and
PCR collected during a period of 14 years (1995-2009)
were selected. The majority of samples (28/37) were
derived from puppies up to 4 months old. Only 5/37
puppies received three doses of CPV-2 vaccine. For
sequencing analysis, two different sets of primers were
used: 555For/555Rev (4003-4585) for amplifying a 583
bp fragment which encodes the two informative AA (426
and 555) and HFor/HRev (3556-4166) for amplifying a
610 bp fragment which encodes the AA 297, 300 and
305. The sequence analysis of strains in this study
confirmed the infection with CPV field strain. From 1995
to 2006, all strains (18) were characterized as CPV-2a.
After that, both CPV-2a (n=5) and CPV-2b (n=14) were
detected. The CPV-2a strains analyzed in this study
showed a non-synonymous mutation at AA residue 297
(Ser’!Ala). A synonymous substitution at the residue 574
(A’!G substitution in nucleotide 5408) was also observed
in 15/37 samples (14 CPV-2a and one CPV-2b). Neither
nucleotide nor AA changes characteristic of CPV-2c type
were found. Our findings indicate that the cases of vaccine
failure are most likely not associated to the mutations
detected in the sequenced regions but could be related
to an incomplete vaccine protocol and the interference
of residual maternally-derived antibodies in puppies up
to four months of age. However, the monitoring of
genotyping mutations that led to new CPV strains is
essential to determinate if current vaccines will keep
providing protection against all new future variants.
Palavras-chaves: canine parvovirus, enteritis, sequence
analysis, vaccine.
SEROEPIDEMIOLOGY OF HEPATITIS B VIRUS (HBV)
IN MEN WHO HAVE SEX WITH MEN (MSM) IN
METROPOLITAN REGION OF CAMPINAS, SÃO
PAULO, BRAZIL.
ID: 00434-00001
Área: 05 - Virologia Humana e Saúde Pública
Araújo, O.R.C., 2 Savassi-Ribas, F., 3Soares, C.C.,
Gomes, S.A.
1. Fiocruz/IOC, Fiocruz/Instituto Oswaldo Cruz, Av. Brasil
4365, Manguinhos, Rio de Janeiro
1
4
ABSTRACTS
HBV infection is still a global public health problem,
although a vaccine has been available since the 80s.
Chronic infection caused by HBV affects over 350 million
people worldwide. It is estimated that one third of the
world population have been infected with the virus and
about one million of deaths occur annually as result of
this infection. Worldwide prevalence of HBV varies
according to geographical location, and there is an inverse
correlation between the degree of economic development
of the country and hepatitis B endemicity. In this study,
the epidemiology of HBV infection in a MSM population
of Campinas was evaluated. Five hundred and seventy
two serum samples collected from October 2005 to
October 2006 were analyzed. All samples were
submitted to enzyme immunoassays (ELISA) for HBV
serological markers detection (anti-HBc, HBsAg, antiHBs). HBsAg positive samples were also tested for
HBeAg and anti-HBe detection. Pre-exposure to the virus
was detected in 15% (86/572) and anti-HBs marker alone,
indicating vaccination, was found in 31.5% (180/572).
No marker was found in 53.5% (306/572), meaning
susceptibility to HBV infection. The surface antigen
HBsAg was found in 3.3% (19/572) of the studied
population. Among HBsAg positive samples, 5 were also
anti-HBs positive, 10 were anti-HBc positive and 4 showed
only HBsAg. Anti-HBc and anti-HBs were found in 9.8%
(56/572), indicating that these individuals evolved to cure
after contact with the virus. Anti-HBe and HBeAg were
found in 6 samples each and coexistence was not
detected. HBV DNA was found in 47.4% (9/19) samples.
The presence of HBeAg and anti-HBe did not interfere
with the DNA detection. The prevalence of HBsAg
prevalence in 3.3% of the population was expected, since
Brazil is considered, in general, a region of intermediate
prevalence of HBV.
Financial support: Fiocruz, PIBIC/CNPq, FAPERJ.
Palavras-chaves: Hepatitis B virus, Men who have sex
with men, Seroepidemiology.
They have 28nm of diameter, non-enveloped. These
viruses remain viable for a long period of time in sewage,
feces, water and also in hands, thus facilitating its
transmission primarily the fecal-oral route. EVs affect
mainly children 1-5 years old. This study aimed to identify
the presence of NPEV. Cell fluids positive for EV were
obtained in infected cell lines HEp-2 and RD. From these
fluids- RNA was extracted using the kit QIAamp Viral
RNA (QIAGEN). The presence of the viral genome was
demonstrated by RT-PCR, using a pair of primers 222
and 292, which anneal partly to the VP1 region a of
Poliovirus type 1. These same oligonucleotids were used
for sequencing. During 1996 to 2006, we received at IEC
538 faecal samples from DMAF cases, originated from
northern Brazil, with 78 (14.5%) turned to be positive for
NPEV. These samples were reisolated in RD and HEp-2
cell lines, resulting in 51 positive by RT-PCR. The cases
are isolated samples by were as follow: 13 isolated/16
tested from Amapá, 20/14 Amazonas, 13/11 Pará, 12/
10 Rondônia, and 03/03 Tocantins. In the state of Roraima
no positive cases during the study period and was not
able to work with the sample of Acre. 40 samples were
sequenced with, 30 identified. These were 16 Echovirus
(including 2 as Echo1, 1 Echo 3, 4 Echo 11, 2 Echo 13,
2 Echo14, 1 Echo 17, 1 Echo 19, 2 Echo 25 and, 1 Echo
33), 7 Coxsackievirus (1Cox A17, 2 Cox B5 and 4 Cox
B6), 1 Enterovirus B, 2 Enterovirus 96, 1 Enterovirus
99, 2 poliovirus type 2 and 1 type 3 (Vaccine). Isolates
were mainly obtained from children aged 1 to 5 years,
evenly distributed by gender of. With the erradication of
wild poliovirus in Brazil and the Americas. In 1991 NPEV
gains importance as a potential cause of DMAF. Data
obtained in this study improve our knowledge on the
epidemiology of NPEV in cases of DMAF the Northern
region of Brazil, end provide information on the currently
circulating strains.
Palavras-chaves: Enteroviruses, Genoma Viral, Northern
Region, PCR, Sequencing.
MOLECULAR EPIDEMIOLOGY OF NON-POLIO
ENTEROVIRUSES (NPEV) ISOLATED FROM CASES
OF ACUTE FLACCID MOTOR DISABILITY IN
NORTHERN REGION OF BRAZIL, DURING 1996 - 2006.
CHARACTERIZATION OF BE AR 177325 (INHANGAPI)
AND BE AN 362159 (XIBUREMA) VIRUS STRAINS
ISOLATED IN THE NORTHERN REGION (PARÁ AND
ACRE STATES) OF BRAZIL
ID: 00435-00001
Área: 05 - Virologia Humana e Saúde Pública
ID: 00436-00001
Área: 04 - Virologia Básica
1
Alves, J. C. S., 2Alves, A. S, 3Oliveira, D.S., 4Soares,
L. S., 5Linhares, A. C., 6Wanzeller, A. L. Mm, 7Castro, C.
M. O.
1. IEC, Instituto Evandro Chagas, Secretaria de
Vigilância em Saúde, MS., Rodovia BR 316 km7 s/n
Bairro Levilândia, Ananindeua, Pará, Brazil.
1
Enteroviruses (EVs) belong to the family Picornaviridae.
The rhabdovirus strains BEAR177325 (Inhangapi) and
BEAN362159 (Xiburema) were isolated by Seção de
Arbovirologia e Febres Hemorrágicas of Evandro Chagas
Institute (SAARB/IEC) on newborn swiss mice following
Wanzeller, ALM, 2Diniz, JAP, 3Bezzera, DAM, 4Lameira,
F, 5Vieira, CMA, 6Santos, MM, 7Vasconcelos, PFC
1. IEC, Instituto Evandro Chagas, Br 316, km 07, s/n
221
ABSTRACTS
intracerebral (i.c.) inoculation, from a pool of sandfly
Lutzomyia flaviscutellata captured in the State of Pará
in 1969 and from a lot of mosquitoes Sabethes
intermedius captured in the State of Acre in 1977,
respectively. Antigens from the brains of mice infected
with these viruses reacted by the complement fixation
(CF) tests with their homologous serum samples, but
not with hyperimmune ascetic fluid of other viruses
available in the SAARB/IEC. Both viruses were included
in the family Rhabdoviridae, as antigenically non-grouped
rhabdoviruses (Karabatsos, 1985). The objective of this
study was the characterization of both viral agents at
their ultraestrutural level, aiming a future taxonomic
classification. The swiss albino mice newborn presented
susceptibility to infection by both viruses when inoculated
by via i.c.; animals death after 3 and 4 d.p.i. The viral
titer on mice was: DL/0,02 mL:10-5,15 (Inhangapi) and
to DL/0,02 mL:10-6,4 (Xiburema). Vero cells were likely
to be susceptible to two viral strains; CPE became visible
with approximately 14 d.p.i. BEAN362159 strain caused
CPE to C6/36 cells lines 4 d.p.i. Among the primary
cultures (mice astrocytes and neurons), morphological
changes of neuron were present for both virus strains
characterized respectively by the presence of refringent
bodies and destruction of the monolayer approximately
4 d.p.i. By CF test, positive result were noted for
supernatant of Vero cells and neurons infected with
Inhagapi and Xiburema viruses, infected C6/36 cells only
to Xiburema virus; negative reaction to the infected
cultures of astrocytes for both viruses; and to C6/36 when
inoculated with the virus Inhangapi. IFI have confirmed
the results obtained by CF tests, with presence of viral
antigens on positive cells.
Palavras-chaves: Characterization, Inhangapi virus,
Xiburema virus, Northern region.
of the circulating mumps virus in order to contribute with
the prevention strategy of this disease by vaccination
campaign. At present, the molecular epidemiology of
mumps virus is characterized by the co-existence of 13
different genotypes named A–M. Complications of
mumps typically involve the central nervous system.
Mumps is the second leading cause of aseptic meningitis
worldwide, and approximately 10% to 30% of patients
will have a meningitis component. During 2007 and 2008
cerebrospinal fluid collected from patients presenting
aseptic meningitis; in addition throat wash and urine, were
investigated. Viral RNA was extracted from supernatant
of Vero cell culture and the entire SH gene was amplified
by RT-PCR. The amplified products were directly
sequenced. The alignment of the SH gene amino acid
sequences of MuV strains of this study with those of
the strains representatives of all known genotypes
revealed the presence of IIL triplet and SV doubled at
positions 28-30 and 41-42, respectively. This peptide
sequence combination appears to be the signature
sequence for MuV strains of MuV genotype M, a new
lineage recently identified in Brazil. Phylogenetic analysis
confirmed that the two isolated mumps strains isolated
from cerebrospinal fluid, in this investigation, belong to
genotype M. Mumps virus molecular epidemiology follow
up in the State of São Paulo will contribute with the
surveillance and epidemiological program not only in
Brazil but also at a global level. Financial support: Instituto
Adolfo Lutz.
Palavras-chaves: ASEPTIC MENINGITS, MUMPS
VIRUS, MUMPS VIRUS OF GENOTYPE M.
MUMPS VIRUS OF GENOTYPE M ISOLATED FROM
CEREBROSPINAL FLUID OF PATIENTS
PRESENTING ASEPTIC MENINGITS
ID: 00437-00002
Área: 05 - Virologia Humana e Saúde Pública
MOLECULAR CHARACTERIZATION OF HUMAN
PARVOVIRUS B19 ASSOCIATED WITH NEUROMYELITIS
Oliveira, M.I., 2Afonso, A.M.S., 3Figueiredo, C.A, 4Curti,
S.P, 5Castrignano, S.B., 6Theobaldo, M, 7Paiva, T.M.,
8
Rasslan, Z, 9Lima, C.A., 10Marrochi, L.C.R., 11Klautau,
G.B.
1. IAL, Instituto Adolfo Lutz, Av Dr Aranldo 3552.
ISCMSP, Irmandade da Santa Casa de Misericórdia de
São Paulo, Rua Cesário Mota Júnior, 112
1
ID: 00437-00003
Área: 05 - Virologia Humana e Saúde Pública
Santos, C.L.S., 2Oliveira, M.I, 3Silva, D.B.B., 4Ishida,
M.A., 5 Benega, M.A., 6Corrêa, K.O., 7Paulino, R.S.,
8
Sasaki, N.A., 9Constantino, C.R.A., 10Paiva, T.M.
1. IAL, Instituto Adolfo Lutz, Av Dr Arnaldo 355
1
Nowadays mumps virus outbreaks are a public health
concern worldwide although considered a vaccine
preventable disease. For many years the lifelong
protection following natural Infection together with
widespread vaccination appeared to be an approach to
control the illness. On the other hand, the occurrence of
mumps cases in highly vaccinated population urge an
accurate investigation towards the molecular evolution
222
Human parvovirus B19 (HPV B19) belongs to the genus
Erythrovirus of the Parvoviridae family.HPV B19 infection
is associated with a wide range of clinical symptoms.
Genetic diversity among HPV B19 virus strains has been
reported to be very low and thus proposed three
genotypes groups. Our study reports the case of a 27year-old female patient with dizziness, cough and
expectoration, desquamative lesions in the left cervical
ABSTRACTS
region and mental confusion with meningoencephalitis.
An epidemiological investigation on the virus infection
started by collecting blood and cerebrospinal fluid (CSF)
employing serological and PCR assays. The samples
were analyzed to detect either measles, rubella, CMV,
HIV, syphilis, hepatitis, HTLV, EBV, Herpes simplex 1 e
2, and varicella zoster infection. This results showed
negatives, the sera and CSF samples was then tested
for HPV B19 infection, which was demonstrated to be
positive in CSF.The HPV B19 infection was confirmed
by both parvovirus B19-specific immunoglobulin M in
sera and PCR assay in CSF. The sequencing generated
a fragment of 423 bp, positions 1282 to 1704 in the
alignment with M13178 of the NS1 - VP1 gene.
Comparisons of the nucleotide and amino acid NS1 VP1 gene sequences of this work with those from
representative of the HBV B19 deposited in the GenBank
showed a high level of identity in genotype 1. Nucleotide
pair wise identity among strains ranged from 87.5% to
99.2%. In this study we report the isolation of B19
genotype 1 of the patient with neurological symptoms;
only a limited number of studies have been conducted
to detect this virus in CFS. This result indicates that this
virus continues to circulate in SP, Brazil. An accumulation
of similar cases is needed to elucidate the role that the
virus plays in the development of neuromyelitis. The
patient of this case report represents a rare case of B19
which might be the responsible agent for CNS infections.
Financial support: Instituto Adolfo Lutz.
Palavras-chaves: HUMAN PARVOVIRUS B19, HPV B19
ASSOCIATED, HPV B19 MOLECULAR CHARACTERIZATION.
of 21.227 biological samples were collected. Samples
were tested by MAC ELISA (enzyme immunoassay for
capture of IgM) for detection of specific IgM. A total of
8.289 (39.04%) serum samples were positive for dengue
by serology, 4.671 (56.35%) of there were females and
3,618 (43.64%) males. In 2000 5,970 samples were
tested, and 2,902 (35.01%) were positive: in 2001 we
analyzed 3,542 samples, of which 1,535 (18.52%) were
positive: in 2002 we tested 2,027 samples, 762 (9.19% )
were positive; in 2003 were analyzed 1,879 samples,
with 809 (9.76%) positive: in 2004, 1,693 samples were
tested, of which 648 (7.82%) were positive; in 2005 were
analyzed in 1,052 samples, 373 (4.50%) were positive:
in 2006 we tested 1,426 samples, 456 (5.50%) positive;
in 2007 were analyzed 2,491 samples, 579 (6.99%)
positive in 2008 and 1,147 samples were tested, of which
225 (2.71%) were positive. Finally, the highest incidence
of the disease between 2000 and 2008 occurred in
females and 2000 was the period of highest rate of
infection. Three dengue serotypes (DENV1-3) were
responsible for cases reported. Financial support: IEC/
SVS-MS.
Palavras-chaves: Dengue, Aedes aegypti, Sorologia,
MAC ELISA.
IDENTIFICATION OF CELL PROTEINS THAT
INTERACT WITH HUMAN RESPIRATORY SYNCYTIAL
VIRUS MATRIX, NUCLEO AND PHOSPO PROTEINS
ID: 00439-00001
Área: 04 - Virologia Básica
Oliveira, A.P., 2Simabuco, F.M., 3Tamura R.E., 4FarinhaArcieri L, 5Armenteros, Y.M., 6Libermann T.A., 7Zerbini,
L.F.C, 8Ventura A. M.
1. USP, Universidade de São Paulo, Cidade Universitária
Armando de Salles Oliveira2. DF/HCC, Genomics Center
and Dana Farber/Harvard Cancer Center Proteomics Core,
Boston, EUA
1
ANALYSIS OF DENGUE VIRUS INFECTION IN
BELÉM-PARÁ BETWEEN THE YEARS 2000 AND 2008
ID: 00438-00001
Área: 05 - Virologia Humana e Saúde Pública
ARAÚJO, J. B. S., 2Costa, E.S.S., 3Gama, E. C., 4Lima,
M. F., 5Rodrigues, S. G., 6Vasconcelos, P. F. C.
1. IEC, Instituto Evandro Chagas, Rodovia BR-316 km
7 s/n - Levilândia - 67030-000 - Ananindeua / Pará / Brasil
1
The Dengue is a disease caused by a Flavivirus in the
family Flaviviridae, is transmitted to humans through by
infected bites of Aedes aegypti. The clinical spectrum of
dengue virus infections ranges from asymptomatic,
through an undifferentiated viral fever syndrome, classic
dengue fever to hemorrhagic fever, which can present
with or without shock syndrome. Due to its high morbidity
and case fatality rate is considered the most important
arbovirus. The objective of this study was to examine
laboratory confirmed dengue cases that occurred in the
municipally of Belém-PA between 2000 and 2008. A total
The Human Respiratory Syncytial Virus (HRSV) matrix
(M), nucleo (N) and phospo (P) proteins are essential for
viral replication, assembly and budding. M, N and P codon
optimized genes were cloned in frame with FLAG peptide
nucleotide sequence in pcDNA3 vector. HEK-293T cells
transfection was performed with plasmid DNAs and after
48 hours the cells were washed with cold PBS, harvested
with lysis buffer and subjected to immunoprecipitation
with anti FLAG antibody conjugated with agarose beads.
Efficient expression of these proteins was demonstrated
by western-blot assays and previous mass spectrometry
data revealed several co-immunoprecipitated cell proteins.
We show in this report their identity confirmation by
repeating the pull down with anti FLAG and performing
western-blot assays with antibodies specific for each of
223
ABSTRACTS
the identified proteins. Our results indicate that N interacts
with Protein Arginin Metil-transferase (PRMT5),
Nucleofosmin (NPM) and Heat Shock Protein (Hsp70).
P interacts only with Hsp70, and M with Tropomyosin
(TPM) and NPM. Considering the intracellular localization
and functions of these proteins, the described interactions
contribute to the construction of a HRSV replication
model.
Palavras-chaves: Human Respiratory Syncytial Virus
(HRSV), Matrix protein, Nucleoprotein, Phosphoprotein,
virus-cell interaction.
ORAL IMMUNIZATION TO HEPATITIS B VACCINE:
THE PROMISING USE OF THE MESOPOROUS SBA15 SILICA ADJUVANT
ID: 00440-00001
Área: 01 - Imunobiológicos
Oliveira, DCA, 2Botosso, V.F., 3Scaramuzzi, K., 4Carvalho
L.V., 5 Tenório E.C.N., 6 Gouvea M.N., 7 Rizzi M,
8
Mussalem, J, 9Fantini M.C.A, 10Sant´Anna O.A.
1. IBut, Serviço de Virologia - Instituto Butantan, Av. Vital
Brazil, 1500, CEP - 05503-9002. IBut, Laboratório de
Imunoquímica - Instituto Butantan, Av. Vital Brasil, 15003.
Cristália Produtos Q, Cristália Produtos Químicos
Farmacêuticos, Rod. Itapira-Lindóia, Km 31,54. IFUSP,
Laboratório de Cristalografia do Instituto de Física da
Universidade de São Paulo, Rua do Matão Travessa R
Nr.187
1
The Butantan Institute initiated in 1996 the production of
the vaccine for Hepatitis B that contains particles highly
purified of the recombinant surface antigen [HBsAg]
adsorbed in Al(OH)3. It is known that 5% of the population
does not respond conveniently for the vaccination
schema. Several studies for improvement of the
immunogenicity have been developed, standing out the
use of distinct adjuvants. Mesoporous SBA-15 silica is
an inorganic material with ordered channels of uniform
hexagonal nanoestrutured pores measuring
approximately 10 nm in diameter. These particles of
silicon oxide are able to interact with atoms, ions and
molecules and due to its physicochemical properties
show great potential as vehicle/adjuvant. On the present
work we intend to avaiable the applicability of SBA-15
silica as an oral adjuvant in immunizations with the
recombinant vaccine for the rHBsAg. BALB/c mice (5/
group) received by the subcutaneous [sc] or oral route
[vo] two doses of the recombinant vaccine in SBA-15 or
in Al(OH)3 at 30 days interval. Individual serum and
pooled fecal samples of each group were periodically
collected for antibody titrations by ELISA. Animals vo
immunized with HBsAg adsorbed/encapsulated in SBA15 had increased levels of specific secretory IgA [s224
IgA] [4.5 log2] after the first immunization and had
amplified levels of specific IgG [10 log2] in the secondary
response. Animals sc immunized had undetectable sIgA and the same level of specific IgG as vo. With Al(OH)3
by subcutaneous immunizations, specific s-IgA titers
were detected only at day 7 post booster [4.5 log2] and
the levels of IgG was high [11 log2] post booster. The
overall analysis of the results indicates the promising
development of nanovaccines using SBA-15 silica as
an adjuvant to be employed in oral immunizations.
Financial support: Fundação Butantan, FAPESP,
Cristália Laboratories and CNPq. Palavras-chaves:
adjuvante, silica mesoporosa, Hepatie B, oral
immunization.
HUMAN RESPIRATORY SYNCYTIAL VIRUS WITH A
60-NUCLEOTIDE DUPLICATION IN THE G GENE
IDENTIFIED IN SÃO PAULO, BRAZIL, 2001-2009
ID: 00440-00002
Área: 05 - Virologia Humana e Saúde Pública
doNASCIMENTO, C.A., 2Oliveira, DB, 3THOMAZELLI,
L.M., 4NEGRÃO, R.C., 5CAMPOS, A.C.A., 7TENORIO,
E.C.N., 8GILIO, A.E., 10DURIGON, E.L., 10DURIGON,
G.S., 11BEREZIN, E., 11BOTOSSO, V.F.
1. IB, Instituto Butantan, Avenida Doutor Vital Brasil,
1500. Butantã - São Paulo - SP2. ICB - USP, Instituto de
Ciencias Biomédicas da Universidade de São Paulo,
Avenida Prof. Lineu Prestes - São Paulo - SP - CEP
05508-9003. HU - USP, Hospital Universitário da
Universidade de São Paulo, Av. Prof. Lineu Prestes,
2.565 . Cid. Universitária, São Paulo, SP
1
HRSV is classified into two groups, HRSVA and HRSVB,
according to their genetic and antigenic characteristics.
Both groups are classified into different genotypes that
can co-circulate during the same season, with one or
two dominant genotypes that are then replaced in
consecutive years. The emergence of isolates of HRSVB
with a 60-nt duplication in the G protein were first
described in Buenos Aires, Argentine, in 1999 and later
in several other places, spreading throughout the world,
becoming the B genotype predominant nowadays. This
virus clustered in a new genotype named BA. In order to
study the epidemiology and evolution of this new
genotype in Brazil we analyzed 4274 clinical samples
collected from children hospitalized in University Hospital
- USP and Santa Casa de Misericórdia Hospital, in São
Paulo city, during 2001 to 2009. The samples were subject
to RT-PCR followed by sequencing of the G2 region of
the G gene The 60 nt-duplication were detected in 127
samples. From 2001 to 2004 the circulation of the BA
genotype was low (only five samples). In 2005 to 2006
years the prevalence was higher, accounting for 24%
ABSTRACTS
and 12.5% of all identified samples. Within two years
the circulation declined, followed by a new peak at 2009
(26% of total samples). In these years, (2005, 2006 and
2009) the BA genotype replaced all other group B viruses.
Sequences were compared with G sequences with the
60 nt-duplication globally sampled. Preliminary
phylogenetic analysis divided Brazilian samples into five
clusters (BA-I, BAII, BAIII and BAVI and BAIV), and
almost all samples from 2005 to 2009 were clustered
together in BA-IV lineage, establishing temporal and
geographical cluster.
Palavras-chaves: Virus, HRSV, Variabilidade, Proteína
G, Genótipo BA.
TRANSMISSION OF FELINE IMMUNODEFICIENCY VIRUS FROM INFECTED QUEEN TO
KITTEN.
ID: 00442-00001
Área: 03 - Virologia Animal
Medeiros, S.O., 2Martins, A.N., 3Tanuri, A., 4Brindeiro,
R.M.
1. UFRJ, Universidade Federal do Rio de Janeiro, Av.
Brigadeiro Trompowisk s/nº, CCS, Bloco A, Sala 121 Ilha do Fundão - CEP: 2
1
Feline immunodeficiency virus (FIV) causes
immunodeficiency diseases in cats. Infectious virus is
present in saliva, and biting is the major mechanism for
spread. Vertical transmission appears to be infrequent
in nature. In experimental infection studies FIV can be
passed to kittens during the birth process or via the milk.
Here we present a case history giving evidence of mother
to kitten FIV transmission occurred in natural setting.
This work was carried out in Rio de Janeiro city. A small
colony of 20 stray cats, composed mainly by female,
was observed for five years. Most of them were trapped
in baited cages, neutered and adopted. The population
size is lowering during the years, and one untamed
female cat remained alone. This female cat was
monitored for three years. During the breeding season
two adult male cats were seen in the area. The two males,
the queen and her two kittens were captured, blood
samples were taken, and the ELISA method was used
to detect FIV-specific antibodies. We amplified and
sequenced CA, RT and V3-V4 portions of FIV isolates.
The phylogenetic analyses were performed by the
maximum likelihood method performed with MEGA 4.
The distance matrix was generated by Kimura´s twoparameter model for nucleotides. All the animals were
FIV positive and feline leukemia virus negative by ELISA.
We evaluated sequences from this group of cats and
determined their evolutionary relationships by comparison
with previously characterized isolates. The sequence data
provided evidence for queen to kitten transmission. The
variability between linked queen-kitten pairs was lower
than between unlinked cats, suggesting that
epidemiologically linked sequences were closer to each
other evolutionarily than to unlinked sequences. Our
hypothesis is that, as the animals were removed from
the original group, the reduced number of males led to
an increase in the occurrence of fights over females,
thus facilitating FIV transmission. Financial support:
CAPES.
Palavras-chaves: Lentivirus, Vertical transmission, FIV.
ROTAVIRUS AND NOROVIRUS ASSOCIED WITH
GASTROENTERITIS IN A OLDER POPULATION
ID: 00443-00001
Área: 05 - Virologia Humana e Saúde Pública
Cicconeto, 2Da Luz, 3Paesi, S.
1. UCS, Instituto de Biotecnologia da Universidade de
Caxias do Sul, Getúlio Vargas, 1130 95070-560 Caxias
do Sul, RS
1
In the world population, the diarrheas from a virus origin
are responsible for most of the half registered outbreaks
in health centers, among them the main are rotavirus
and norovirus. Rotavirus belongs to Reoviridae family, it
is a not enveloped virus which presents 11 segments of
RNA of double-stranded (dsRNA) and it is the main agent
responsible for children’s gastroenteritis, taking about
870 thousand children to death yearly. Norovirus belongs
to Caliciviridae family, it is a small virus, not enveloped
and with RNA of simple strand, it is the most expressive
in outbreaks attacking all the age levels. Both the virus
has transmission oral-fecal. Few studies with these
viruses have been realized with the elderly. The present
study analyzed the frequency of rotavirus and norovirus
in elderly from Caxias do Sul. A hundred samples were
analyzed. They were from particular laboratories of this
city in the period from 2004 to 2009. It was used the test
of agglutination in latex and electrophoresis in
polyacrylamide gel (PAGE) for rotavirus detection and
for checking of norovirus presence was realized the
reverse transcription test of polymerase chain reaction
(RT-PCR) in eight samples which were negative for
rotavirus. From the analyzed samples, it was obtained a
positivity of 5/100 (5%) for rotavirus A by the test of
agglutination in latex and 9/100 (9%) by the test EGPA.
Norovirus was found in 2/8 (25%) from the analyzed
samples. The obtained data prove the circulation of viral
agents in elder population from Caxias do Sul, which
reinforces the importance of these infection monitoring.
Financial Support: UCS Área:Virologia Humana Tema:
Gastroenterite viral Apresentador: Suelen Paesi.
Palavras-chaves: GASTROENTERITIS, NORO225
ABSTRACTS
VIRUS, OLDER, POPULATION, ROTAVIRUS.
COMPARATIVE ANALYSIS OF THE ROTAVIRUS
DIAGNOSIS BETWEEN TWO CITIES OF THE
NORTHEASTERN REGION OF RIO GRANDE DO SUL
THROUGH LATEX AND PAGE IN ELDERLY PEOPLE
ID: 00443-00002
Área: 05 - Virologia Humana e Saúde Pública
Debiazi. F., 2Trentin, B., 3Tognon, 4Paesi, S.
1. UCS, Instituto de Biotecnologia da Universidade de
Caxias do Sul, Getúluio Vargas, 1130- 95070-560 Caxias
do Sul, RS
1
Diarrhea is responsible for a high number of hospital
admissions and mortality worldwide. Over 50% of the
types of diarrhea are from viral origin, and rotavirus is
the infectious agent linked to gastroenteritis in children.
However, little is known about the outbreaks in people
who are over 60 years old. The genus Rotavirus belongs
to the Reoviridae family, and it is characterized for having
eleven double-stranded RNA segments. This
nonenveloped virus is divided in five species (A to E)
and the groups A, B and C are found in humans and
animals. The identification of this virus can be carried
out by passive agglutination (PLA) and polyacrylamide
gel electrophoresis (PAGE). The objective of this study
was to compare the results obtained by both methods,
in symptomatic and asymptomatic patients who are over
60 and live in the cities of Veranópolis and Caxias do
Sul. We tested 158 diarrhetic and nondiarrhetic fecal
samples; 158 were tested using PLA and 153 using the
PAGE method. According to the PLA method, 5(3,16%)
from the 158 samples were positive for rotavirus;
according to the PAGE method, 9(5,8%) from the 153
samples were rotavirus-positive. All the positive samples
detected by the PAGE method were from female patients
who live in Caxias do Sul and are over 60 years old.
From the 5 positive samples detected by the PLA
method, 4 were from female patients and one from a
male patient, all of them residents of Caxias do Sul and
over 60 years of age. From the samples, 4 were positive
according to both methods, which indicated that the virus
belonged to Group A. In relation to the other 5 positive
samples, one might say that the virus probably belongs
to other groups rather than A, such as B and C, which
also occur in humans and, for this reason, were not
detected by the latex method. These results indicate that
the study of viral identification must be done using the
association of PLA and PAGE methods in order to detect
the diversity of rotaviruses circulating in the elderly.
Palavras-chaves: DIAGNOSIS, ELDERLY PEOPLE,
GASTROENTERITIS, DIAGNOSIS, ROTAVIRUS.
EFFECTS OF BOTH NATURAL AND RATIONALLY
DESIGNED COMPOUNDS ON AMP PROTEIN KINASE
ACTIVATION AND HCV REPLICATION
ID: 00444-00001
Área: 05 - Virologia Humana e Saúde Pública
Jardim, A.C.G., 2Mankouri, J., 3Verow, M., 4Rahal, P.,
Harris, M.
1. IBILCE, UNESP, Instituto de Biociências, letras e
Ciências Exatas, Rua Cristovão Colombo 2265 São José
do Rio Preto/SP, Brasil.2. IMCB, Institute of Molecular
and Cellular Biology, Faculty of biological Science,
University of Leeds., Faculty of Biological Science,
University of Leeds, LS2 9JT, UK
1
5
Hepatitis C virus (HCV) infection is associated with
dysregulation of both lipid and glucose metabolism. As
well as contributing to viral replication, these
perturbations influence the pathogenesis associated with
the virus, including steatosis, insulin resistance and type2 diabetes. Adenosine 5’-monophosphate (AMP)activated protein kinase (AMPK) plays a key role in
regulation of both lipid and glucose metabolism. We have
previously shown that in cells infected with HCV, AMPK
activity is dramatically reduced. We further demonstrated
that pharmacological restoration of AMPK activity not
only abrogates the lipid accumulation observed in virusinfected cells, but also efficiently inhibits viral replication
presenting a novel target for much needed anti-HCV
therapies. Here, we have furthered this analysis
evaluating the effects of both natural and rationally
designed AMPK activators on HCV replication and
persistence. The designed compounds used in the
assays were selected from the Maybridge chemicals
library of compounds using ROCS analysis of a
compound designed to fit into the ligand binding site of
AMPK. Huh-7 cells were electroporated with luciferasebased genotype 2a (JFH-1) replicons that allow the direct
correlation of HCV replication to luciferase activity, and
the compounds assessed for their effects on both HCV
replication and cellular toxicity. We demonstrate that HCV
replication was dramatically inhibited by both natural and
rationally designed AMPK agonists that reduced both
levels of luciferase activity and HCV protein expression
assessed through western blotting for the NS5A nonstructural protein. These data are the first description of
a rationally designed kinase activator possessing antiviral activity and further clarify the recovery of AMPK
activation in HCV expressing hepatocytes as a rationale
target for anti-HCV therapies. Financial support: CAPES.
Palavras-chaves: AMP Protein kinase, Hepatitis C virus,
HCV replication, natural Inhibitors, AMPK Agonists.
ULTRASTRUCTURAL STUDIES OF BE AR 177325
226
ABSTRACTS
(INHANGAPI) AND BE AN 362159 (XIBUREMA) VIRUS
STRAINS, ISOLATED IN THE AMAZON REGION OF
BRAZIL
ID: 00445-00001
Área: 04 - Virologia Básica
Lameira, F.A.C, 2Wanzeller, A.L.M., 3Bezerra, D.A.M,
Diniz, J.A.P, 5Vasconcelos, P.F.C.
1. IEC, Instituto Evandro Chagas, Rodovia BR 316 - KM
07, S/N° - Bairro Levilândia - Ananindeua/PA
1
4
Inhangaphi and Xiburema virus strains were isolated on
newborn Swiss mice by intracerebral (i.c.) inoculation,
from pooled of sandfly Lutzomyia flaviscutellata captured
Belém Metropolitan Area, State of Pará (1969), and from
a lot of mosquitoes Sabethes intermedius captured in
Sena Madureira, State of Acre (1977), respectively. The
objective of this study was to analyze the
morphogenesis, as well as the infection process into
C6/36 cells and primary neuron cells with Xiburema and
Inhangaphi viruses. For conventional electron microscopy
infected cells were fixed for 2 h at room temperature
with 4% paraformaldehyde, 2.5% glutaraldehyde in 0.1M
cacodylate, pH 7.2, followed by post-fixation with 1%
OsO4 at 4ïC, en bloc stained with 2.5% uranyl acetate;
dehydrated through different concentrations of acetone,
and embedded in Epon. Sections were stained with uranyl
acetate and lead citrate, and then observed into a
transmission electron microcopy (TEM) (Zeiss EM 900).
Ninety-six hours after infection, TEM observations
showed viral particles which exhibited a typical bulletshape. These results suggest that these viruses have
morphologic characteristics similar to those members
of the family Rhabdoviridae. Additionally, by serology, it
was confirmed that both viruses present cross reactivity.
Finally, the hematophagous insects Lutzomyia
flaviscutellata sandfly Sabethes intermedius mosquitoes
can play a role in the maintenance cycle in nature of
Inhangapi and Xiburema viruses, respectively.
Financial Support: IEC / FUNASA Área: Virologia Básica
Tema: Estudo Ultraestrutural Apresentador: Lameira,
F.A.C.
Palavras-chaves: Inhangaphi Virus, Xiburema virus,
Ultrastructural studies, Rhabdoviridae, Amazon region.
J., 1Rodrigues, M.T., 1Frezza, R., 1Arantes, T., 1Luz, R.B.,
Spilki, F.R.
1. Feevale, Universidade Feevale, RS-239, n° 2755,
Bairro Vila Nova CEP 93525-000. Novo Hamburgo/RS.
1
Torque Teno virus is a non-enveloped virus measuring
30-32 nm in diameter, with a single-stranded, circular
DNA negative sense genome. TTV is excreted through
the fecal route and may be found in contaminated water.
The purpose of this study was to compare the results
obtained by molecular detection of TTV in surface water
collected from two affluents of the Guaíba Basin, the
Arroio Diluvio, a canalized stream located in Porto Alegre
and Rio dos Sinos, a river on metropolitan river. Cloned
TTV DNA served as positive control. Water samples were
concentrated, the extraction of viral DNA was performed
using the RTP® DNA/RNA Virus Mini Kit (Invitek)
reagent. PCR reactions were carried out using 2X PCRMix (LGC) commercial kit (25 μL), 22,5 μL of ultrapure
water, 0,5 μL of Fw1, 0,5μL of Fw2, 0,5μL of Rev and
1μL of DNA, resulting in a solution of 50 μL, using a set
of primers designed to amplify a conserved fragment
within the TTV ORF2. The reaction products were
subjected to electrophoresis on 2% agarose gels in a
TBE buffer, stained with BlueGreen Loading Dye (LGC)
and then visualized by UV light. Fourteen samples of
Diluvio’s stream and four of Rio dos Sinos were submitted
to PCR amplification. The virus presence was detected
in 4 (28,5%) samples of Diluvio stream. No positive
results were found for torque teno virus from samples
collected in Rio dos Sinos. This data shows that TTV
may be found occasionally as a contaminant of surface
water in Southern Brazil; however, TTV will be not always
present.
Financial support: CNPq; Fapergs; Feevale. Área:
Virologia Ambiental.
Palavras-chaves: Torque Teno Virus, water samples,
fecal contamination.
EFFECTIVENESS OF siRNA DIRECTED AGAINST
HUMAN RESPIRATORY SYNCYTIAL VIRUS L GENE
ID: 00460-00001
Área: 04 - Virologia Básica
Carromeu, C., 2Farinha-Arcieri, L.E., 3Moraes, C.T.P.,
Armenteros, Y.M., 5 Simabuco, F.M., 6Tamura, R.E.,
7
Durigon, E.L., 8Ventura, A.M.
1. ICB - USP, Departamento de Microbiologia, Instituto
de Ciências Biomédicas, Universidade de São Paulo,
Av. Prof. Lineu Prestes, 1374 - CEP 05508-900 - São
Paulo - SP - Brasil
1
TORQUE TENO VIRUS IN SURFACE WATERS
COLLECTED FROM DILUVIO STREAM AND RIO DOS
SINOS
ID: 00446-00001
Área: 02 - Virologia Ambiental
1
Silva, J.V.S, 1Vecchia, A.D., 1KLUGE, M., 1Comerlato,
4
The Human Respiratory Syncytial Virus (HRSV) is an
important cause of disease in the respiratory tract of
227
ABSTRACTS
infants. Despite the intense research in the field, a vaccine
or efficient treatment against HRSV is not available yet.
The ribonucleocapsid complex viral proteins (N, P, M2-1
and L) share high similarity among all strains of HRSV,
specially the L protein, responsible for all enzymatic
activities. Given the sequence identity, these genes are
suitable candidates for a broad spectrum RNA
interference therapy against diverse HRSV types. Here
we report the design of three small interfering RNAs
(siRNAs) against highly conserved regions of the HRSV
L gene and their efficacy as antiviral agents. Our results
show a blockage of virus replication and efficient silencing
of the L gene for A2 prototype and clinical isolates grown
in HEp-2 cells. Their ability to work against diverse HRSV
subtypes makes them good candidates for the
development of future anti-HRSV therapies. Financial
support: FAPESP and CNPq.
Palavras-chaves: Human Respiratory Syncytial Virus,
RNA polymerase, L protein, siRNA, HRSV.
HEPATITIS E VIRUS (HEV) EXPERIMENTAL
INFECTION STUDY IN CYNOMOLGUS MACAQUE
(MACACA FASCICULARIS)
ID: 00461-00001
Área: 05 - Virologia Humana e Saúde Pública
Carvalho, L.G., 2 Santos, D.R.L., 3 Marinho, A.M.,
4
Ramos, S., 5Cajaraville, A.C.R.A., 6 Resende, F.R.,
7
Pinto, M.A.
1. IOC/Fiocruz, Instituto Oswaldo Cruz/ Fundação
Oswaldo Cruz, Av. Brasil 4365 Manguinhos, Rio de
janeiro/RJ CEP: 21040-3602. SCPRIM/CECAL-Fiocruz,
Serviço de Criação de Primatas não humanos/Cecal,
Av. Brasil 4365 Manguinhos, Rio de Janeiro/RJ
CEP:21040-3603. UFRRJ, Laboratório de Imunologia e
Virologia Veterinária/UFRRJ, BR-465, Km 7. Seropédica/
Rio de Janeiro4. SCQA/CECAL-Fiocruz, Serviço de
Controle da Qualidade Animal/CECAL, Av. Brasil 4365
Manguinhos, Rio de Janeiro/RJ5. LTV/Bio-Fiocruz,
Laboratório de Tecnologia Virológica/Biomanguinhos, Av.
Brasil 4365 Manguinhos, Rio de Janeiro/RJ6. IOC/
Fiocruz, Biotério de Experimentação de Primatas não
Humanos/Fiocruz, Av. Brasil 4365 Manguinhos, Rio de
Janeiro/RJ CEP:21040-360
1
Several studies have demonstrated serological and
molecular evidences of hepatitis E virus (HEV) infection
in different animal species of endemic and non endemic
areas suggesting its zoonotic transmission. Besides
biochemical parameters of alanine aminotransferase ALT
quantification, serological and molecular techniques have
been applied to confirm hepatitis E acute cases. The
study aimed to develop an experimental infection in
Cynomolgus macaques to evaluate the infection course.
228
A total of twelve animals clustered into subgroups and
inoculated with different HEV genotype 3 obtained from
Brazilian and Dutch swine and human cases from
Argentina, including a negative control. Animals were
accompanied for 67 days when serum and fecal samples
were collected weekly. Samples were submitted to HEV
partial genome (HEV RNA) detection with Real time PCR
(Q-PCR) (Jothikumar et al., 2006). Serum samples were
also submitted to biochemical quantification of ALT
(enzymatic colorimetric method). A total of six animals
from ten inoculated were positive for HEV-RNA in fecal
samples, and four of them were also positive for HEVRNA in serum samples. Nevertheless, a significant raise
of ALT levels was not observed. The spread of virus in
feces and viremia characterize the infection and the
lightly alteration of ALT levels confirms imunomediated
status of the disease. The study demonstrates that HEV
circulating in commercial swine herds was able to infect
non human primates suggesting that hepatitis E acute
cases may occur in Brazil. Palavras-chaves:
Cynomolgus, Hepatite E, Infecção experimental.
ANGOLA: HIV-1 GENETIC VARIABILITY AND
INTERSUBTYPE
RECOMBINANT
FORMS
CIRCULATING IN THE NORTH
ID: 00462-00002
Área: 05 - Virologia Humana e Saúde Pública
Morais Afonso, J., 2Bello, G., 3Guimarães, M. L., 4Sojka,
M., 5Morgado, M. G.
1. IOC/FIOCRUZ, Laboratory of Aids and Molecular
Immunology, Oswaldo Cruz Institute, Oswaldo Cruz
Foundation, Manguinhos - Rio de Janeiro - RJ - Brasil
CEP: 21040-3602. CSSL, São Lucas Medical Center,
Estrada do Caxito - Kifangondo, Angola3. FM-UAN,
University Agostinho Neto - Faculty of Medicine, Av. Hoji
Ya Henda, Cidadela- Luanda, Angola. CP:116
1
Angola, a South-Western African country, presents
notably low HIV/AIDS prevalence levels (2.7%) within
the adult population when compared to other African
countries such as Namibia and Zambia, presenting 15%
and 14.3%, respectively. Nevertheless, HIV-1 within the
Angolan population shows very high and complex
genotype diversity, similar to those found in the
Democratic Republic of Congo (DRC) and Republic of
Congo, where all HIV-1 Group M clades as well as
circulating recombinant forms (CRFs) and Unique
recombinant forms (URFs) co-circulate. Therefore, there
is a need to evaluate and update HIV-1 genetic diversity
and evolution in Angola once it may pose unprecedented
challenges to diagnostic, treatment and prevention of
HIV-1. The aim of this work was to investigate the genetic
diversity of HIV-1-positive Angolan samples. Forty six
ABSTRACTS
HIV-1-positive patients were diagnosed and collected in
2008 (n= 25) and 2009 (n= 21) in either Luanda or Bengo.
Angolan samples were genotyped based on phylogenetic
and recombinant analysis from pol (PR/RT) gene. A
subset three samples presenting similar A/U/H
recombinant profile in the pol region was selected for
DNA sequencing of the gag and partial env (gp120) gene
regions. High HIV-1 genetic diversity was depicted in
the pol region, with a predominance of intersubtype
recombinant forms, CRFs (18.2%) and URFs (13.8%)
following subtypes C (17%), F (13%), A (11%), D (9%),
G and H (each 7%), J and K (each 2%) were found.
Three out of six (50%) URFs samples formed a unique
cluster (A/U/H pattern) in the RT/PR region. Further
analysis of the gag and env regions indicated a
CRF06_cpx gag/U env gp120 pattern for all three
samples. Molecular data have indicated a complex degree
of HIV-1 group M genetic variability within the Angolan
samples. Combined analysis of those three sequences
presenting the same recombinant profile in PR/RT-gagenv sequences regions suggest a pontential new HIV-1
CRF in the region. Financial suppor t: IOC/
FIOCRUZ,TWOWs,FMUAN.
Palavras-chaves: HIV-1, Angola, Genetic variability,
intersubtype recombinants.
HISTORY OF THE PLANT VIROLOGY IN BRAZIL
ID: 00463-00001
Área: 06 - Virologia Vegetal
E.W. KITAJIMA
1. ESALQ/USP, Escola Superior de Agricultura Luiz de
Queiroz, USP, CP 9, 13418-900 Piracicaba, SP, Brasil
1
Studies on plant viral diseases started around 1930’s at
the Instituto Biológico (Bitancourt, Silberschmidt) and
Instituto Agronomico de Campinas (Costa). The main
viral diseases studies were on cotton, tobacco, vegetable
crops and particular focus on citrus tristeza. Costa and
Silberschmidt started the organization of a research group
on plant viruses since 1950, which consolidated in the
1970’s. At the same time activities on plant virology
started in several parts of Brazil (UFRGS, UFPr, UFV,
UFRPe, UFCe, Unicamp, UNESP, IAA, UnB).. Graduate
programs on plant pathology, including plant virology also
began in the 1970’s (ESALQ, UnB, UFV, UFCe, UFLa,
UFRPe, UFRGS) producing new generation of plant
virologists which nucleated new research centers on plant
viruses in several centers of Embrapa (Vegetable Crops,
Biotechnology, Cerrado, Wheat, Corn and Sorghum,
Soybean, Rice and bean, Cassava and Tropical
Fruticulture, Grapevine and vine, Temperate climate
Fruticulture, Oriental Amazon) and universities (UEM,
UEL, UPF, UFAl, UFRRJ). Presently an estimate of 100
professionals and posdocs are engaged in researches
on diseases caused by plant viruses and related
pathogens, besides graduate and undergraduate
students. Many of these professionals either graduated
or have postdoctoral experience in foreign centers in the
US, Japan, Australia, Germany, France, Spain, Italy,
Portugal, the Netherlands, etc. Many cooperative
researches with invited foreign scientists were carrued
out. Publications reporting the results of these
researches are growing at steady rate with an average
of 150 new abstracts and articles yearly. At the early
years these publications were mostly made in journals
as Bragantia, Arquivos do Inst.Biológico, O Biológico,
Revista da Agricultura, etc., but after 1970, with the
advent of specialized journals as Fitopatologia Brasileira
and Summa Phytopathologica as well as in important
foreign specialized journals.
Palavras-chaves: Plant virology, Brazil, History.
AN IGM-ELISA BASED ON RECOMBINANT Dengue
virus ANTIGENS SHOWS COMPARABLE RESULTS
TO A COMMERCIAL DENGUE IGM CAPTURE ELISA
ID: 00468-00001
Área: 01 - Imunobiológicos
ROCHA, E.S.O., 2OLIVEIRA, J.G., 3 FIGUEIREDO,
L.B., 4ZEYMER, B.R., 5SANTOS, J.R., 6CECÍLIO, A.B.,
7
BONJARDIM, C.A., 8FERREIRA, P.C.P, 9KROON, E.G.
1. UFMG, Laboratório de Vírus, Universidade Federal de
Minas Gerais, Av. Antônio Carlos, 6627 - ICB bloco F4
sl 258 - Belo Horizonte, MG, Brazil2. CPqRR, Laboratório
de Imunologia Celular e Molecular, Centro de Pesquisa
Rene Rachou, Avenida Augusto de Lima, 1715 - Belo
Horizonte, MG, Brazil3. FUNED, Fundação Ezequiel Dias,
Rua Conde Pereira Carneiro, 80 - Belo Horizonte - MG
1
Dengue fever (DF) is caused by any one of the four viral
serotypes of Dengue virus (DENV) and is characterized
by an acute onset of high fever, frontal headache, retroorbital and muscular pain. The immune response during
the primary dengue infection is characterized by a rise
of IgM antibodies after the 3rd day of the onset of
symptoms and may persist as long as three months.
Several serological methods have been developed to
assess laboratory confirmation of dengue infection, which
include a large number of ELISA tests. Since no effective
commercial vaccine or treatment exist for dengue
infections, an early detection and management of severe
disease are essential to prevent death. The aim of this
study was to evaluate the effectiveness of recombinant
proteins for detection of DENV specific IgM antibodies
compared to the commercial kit Panbio Dengue IgM
capture ELISA. Recombinant proteins used as antigens
in a standardized IgM-ELISA were expressed in
229
ABSTRACTS
Escherichia coli system and purified by nickel affinity
chromatography. A panel of 53 serum samples from DF
suspected individuals residents in Belo Horizonte metro
area was provided by Fundação Ezequiel Dias. The
samples were tested by the Panbio kit and IgM-ELISA.
Forty-eight (90.6%) samples were concordant between
Panbio and IgM-ELISA, 18 positives and 30 negatives.
Since only 5 (9.4%) samples were discordant, IgMELISA sensitivity was 90.9% and specificity was 95.0%.
This study presents the IgM-ELISA as an inexpensive
and suitable kit for use in clinical laboratory. In conclusion,
our IgM-ELISA based on recombinant DENV antigens
should be potentially as useful as Panbio Kit to early
detection of dengue infections and our recombinant
antigens are reliable reagents for detection of anti-DENV
antibodies.
Financial support: CNPq, CAPES, DECIT/MS,
FAPEMIG, PRONEX-DENGUE and INCT-DENGUE.
Palavras-chaves: Dengue fever, Immunoenzymatic assay, Recombinant protein.
COMPARATIVE EVALUATION OF ENZYME-LINKED
IMMUNOSORBENT
ASSAY
BASED
ON
RECOMBINANT ANTIGENS AND AGAR GEL
IMMUNODIFFUSION FOR SERODIAGNOSIS OF
EQUINE INFECTIOUS ANEMIA
to standardize an ELISA for detection of IgG antibodies
from equine sera. The current study was designed as a
comparative evaluation of two serological methods for
detection of EIAV-specific antibodies, AGID and ELISA
based on recombinant protein. The expressed
recombinant protein was purified by nickel-chelate affinity
chromatoghaphy and we obtained a yield of 1,0mg of
gp90 per liter of induced culture. A panel of 100 brazilian
equine serum samples, 50 AGID-positive sera and 50
AGID-negative sera, were obtained and those results
associated with mean OD obtained on ELISA were
analyzed to establish the cut-off value by the ROC curve
method. The cut-off value of 0.445 showed high sensitivity
(95.7%) and high specificity (96.0%) as well as great
probability indexes for ELISA. Moreover, 96 (96%)
samples evaluated by AGID and ELISA shown
concorda