OIE Reference Laboratory Reports
Activities in 2011
Name of disease (or topic) for
which you are a designated OIE
Reference Laboratory:
Transmissible gastroenteritis virus
Address of laboratory:
Food Animal Health Research Program
Ohio Agricultural Research and Development Center
The Ohio State University
1680 Madison Avenue
Wooster, OH 44691
UNITED STATES OF AMERICA
Tel.:
330-263-3744
Fax:
330-263-3677
e-mail address:
saif.2@osu.edu
website:
Name (including Title and
Position) of Head of Laboratory
(Responsible Official):
http://www.oardc.ohio-state.edu/lsaiflab/
Y. M. Saif, DVM, PhD
Professor and Department Head, Food
Animal Health Research Program
Name (including Title and
Position) of OIE Reference
Expert:
Linda J. Saif, MS, PhD
Distinguished University Professor
Name (including Title and
Position) of writer of this report
(if different from above):
Linda J. Saif, MS, PhD
Distinguished University Professor
Annual reports of OIE Reference Centres, 2011
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Transmissible gastroenteritis
Part I: Summary of general activities related to the disease
1.
Test(s) in use/or available for the specified disease/topic at your laboratory
Test
For
Specificity
Total
ELISA
Antibody
TGEV & PRCV
10
ELISA
Antibody
Differentiate TGEV/PRCV
5
ELISA
Antigen
TGEV & PRCV
10
CCIF*
Antigen
TGEV & PRCV
20
SN (plaque reduction)
Antibody
TGEV & PRCV
15
RT-PCR and nested PCR
Viral RNA
Differentiate TGEV/PRCV
20
*CCIF – cell culture immunofluorescent test using ST cells
2.
Production and distribution of diagnostic reagents
The diagnostic reagents described in the above tests have been produced and standardized in our laboratory as
indicated in our previous publications. They have been routinely provided in varying amounts depending on
demand to diagnostic labs in the U.S., Canada, other OIE member countries and elsewhere as requested for
reference reagents. In 2006/2007 they were provided to the American Type Culture Collection (ATCC) who will
distribute them worldwide, including to be used as reference reagents for alpha- (TGEV/PRCV) and betacoronaviruses when testing for SARS or other coronaviruses in animals. In addition we are receiving many
requests for RT-PCR testing, reference TGEV RNA for RT-PCR tests and RT-PCR assay protocols as well as
requests for our actual primers for RT-PCR from countries that cannot afford to buy them. These reagents are
provided without cost but have to be paid for from Dr L.J. Saif’s research funds as no other funds are provided to
the OIE expert lab.
Type of reagent
Amount supplied nationally
(including for own use)
Amount supplied to other
countries
Control Pos/Neg sera
10ml each (ATTC)
4 aliquots of 0.5ml each
Viral RNA for TGEV/PRCV
Produced for each RT-PCV assay
As required for RT-PCR assays
Monoclonal Antibodies
10 ml
4 aliquots of 0.5ml each
Part II: Activities specifically related to the mandate
of OIE Reference Laboratories
3.
International harmonisation and standardisation of methods for diagnostic testing or the
production and testing of vaccines
Besides within the U.S. and Canada, diagnostic tests, procedures or reagents have been provided to laboratories in
Vietnam, China, Korea, South America and the Soviet Union to use for the detection and differentiation of TGEV
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Annual reports of OIE Reference Centres, 2011
Transmissible gastroenteritis
and PRCV infections. These include highly specific monoclonal antibodies and reagents prepared in germfree
animals and reference antibody positive and negative sera.
a)
Establishment and maintenance of a network with other OIE Reference Laboratories
designated for the same pathogen or disease and organisation of regular inter-laboratory
proficiency testing to ensure comparability of results
Our lab is the only OIE reference lab for TGEV.
b)
Organisation of inter-laboratory proficiency testing with laboratories other than OIE
Reference Laboratories for the same pathogens and diseases to ensure equivalence of
results
All methods have been standardized and published using known postive and negative samples and unrelated
enteric pathogens to determine sensitivity and specificity. Many of our methods have been adopted by the
other labs.
4.
Preparation and supply of international reference standards for diagnostic tests or vaccines
See Table 2.
Besides within the U.S. and Canada, diagnostic tests, procedures or reagents have been provided to laboratories in
Vietnam, China, Korea, South America and the Soviet Union to use for the detection and differentiation of TGEV
and PRCV infections. These include highly specific monoclonal antibodies and reagents prepared in germfree
animals, reference TGEV and PRCV strains and viral RNA and reference antibody positive and negative sera.
Our lab was designated as a WHO reference lab for Animal Coronaviruses in 2004 and is part of the SARS WHO
lab network. In this capacity we will supply TGEV/PRCV and bovine coronavirus strains and antisera to other
WHO reference labs and to other international labs including those of OIE member countries through the ATCC.
Monoclonal antibodies (MAbs) specific for the N and S TGEV proteins have been produced and common or
unique epitopes on the S protein of various strains of TGEV and PRCV defined. MAbs have been provided to
various researchers and diagnostic labs noted above. TGEV strains (virulent and attenuated) and PRCV strains, ST
cells and antisera have also been provided. We also have S or N gene clones as needed for research or diagnostic
assays. A baculovirus expressed TGEV S protein worked well to replace TGEV for coating plates for ELISA
antibody tests and could be tested commercially. An RT-PCR assay for TGEV/PRCV detection and differentiation
has been developed and a pan-coronavirus RT-PCV assay to detect other or new coronavirus strains has also been
devised.
5.
Research and development of new procedures for diagnosis and control
We continue to test samples from other species and from wildlife (summarized in 6) and sequence these
coronaviruses to establish their genetic relatedness to TGEV to monitor for newly emerging coronaviruses related
to TGEV and potential cross-species infections. The procedures related to TGEV/PRCV diagnosis and control are
reviewed in the Coronaviruses chapter in the new edition of Diseases of Swine to be published in 2012.
In addition we have collaborated with a colleague at the FDA to test for coronaviruses on foods (salads) and their
stability on foods to assess if they could be foodborne as was suggested for SARS.
6.
Collection, analysis and dissemination of epizootiological data relevant to international disease
control
Comparative genomic sequence analysis demonstated that SARS-like coronaviruses (CoVs) isolated from palm
civets are genetically most closely related to SARS-CoV strains infecting humans during initial outbreaks. Palm
civets are carnivores from the suborder Fissipedia, together with raccoon dogs, dogs, cats, raccoons, hyenas,
mongooses, bears, skunks, ferrets (Mustela putorius) and mink (Mustela vison). Cats, ferrets and palm civets were
all susceptible to experimental infection with SARS-CoV, and additionally a mink lung cell line (Mv1Lu) was
Annual reports of OIE Reference Centres, 2011
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Transmissible gastroenteritis
permissive to SARS-CoV, expressing a functional ACE2 receptor for viral entry. A CoV previously associated
with catarrhal gastroenteritis in mink (Mustela vison) was identified by electron microscopy in mink feces from fur
farms in the US in 1998. Initially we used our pancorona and genus-specific RT-PCR assays and demonstrated
that the newly characterized mink CoVs (MCoVs) are alphacoronaviruses (alpha-CoVs). Subsequently, we
generated full-genomic sequences and confirmed that phylogenetically, MCoVs belong to alpha-CoVs with the
closest relatedness to the recently identified but only partially sequenced ferret enteric coronaviruses (FRECV).
Molecular data presented in our study provides the first genetic evidence for a new coronavirus associated with
epizootic catarrhal gastroenteritis outbreaks in mink and demonstrates that MCoVs possess high genomic
variability and relatively low overall nucleotide sequence identities (91.7%) between contemporary strains
suggesting that they are under strong selective pressure. Additionally, new MCoVs appear to be phylogenetically
distant from human (229E and NL63) and other alpha-CoVs (TGEV) and yet they do not belong to alpha-CoV 1
species. We propose that with the FRECV, they may comprise a new species within alpha-CoVs genus (alphaCoV 2).
According to our new sequence information, and together with previous studies, our data demonstrate higher
genetic diversity among CoVs from carnivores. Due to their roles in the food chain, carnivores harboring CoVs
may serve as virus reservoirs and contribute to the evolution and emergence of new CoV strains with zoonotic or
interspecies transmission potential. Thus careful monitoring of CoVs circulating in carnivores is warranted to
detect emergence of new strains.
7.
Maintenance of a system of quality assurance, biosafety and biosecurity relevant to the
pathogen and the disease concerned
TGEV/PRCV are classified as Biosafely Label 2 infectious agents. All University and NIH mandated protocols for
handling and storing BSL 2 agents and training of personnel are strictly adhered to by our laboratory. Copies of
these completed and approved biosafety protocols are maintained by our lab and the University. The same
biosafety practices apply to our shipping of infectious agents including the mandate for training (by exam) of the
lab technicians who ship such samples.
8.
Provision of consultant expertise to OIE or to OIE Member Countries
We have provided expertise to APHIS and NVSL, USDA, and FDA labs in the U.S. and to Canadian labs
concerning issues, samples and RT-PCR assays for testing of swine or food samples for TGEV and PRCV or
antibodies for export of TGEV-free swine.
We have provided similar information on RT-PCR or antigen or antibody testing for TGEV/PRCV to the
Veterinary Diagnostic Services in Canada and to the Veterinary Diagnostics Labs in Vietnam, China and South
America. We have provided TGEV and PRCV strains or viral RNA to researchers.
9.
Provision of scientific and technical training to personnel from other OIE Member Countries
Training in diagnostic techniques or vaccine immunity testing.
Dr Anastasia Vlasova, Post-doctoral Researcher, Soviet Union
Dr Kwonil Jung, Post-doctoral Researcher, Korea
Dr Ning Chen, Visiting Scholar, China
Dr Quan Shen, Visiting Scholar, China
Mr. Zhenwen Zhang, Visiting Scholar, China
Ms. Celina Vega, Visiting Scholar, Argentina
Ms. Marina Bok, Visiting Scholar, Argentina
Dr Pierfrancesco Pinto, Visiting Scholar, Italy
Dr Joshua Amimo, Nairobi, Kenya
10. Provision of diagnostic testing facilities to other OIE Member Countries
We mainly provided diagnostic reagents (monoclonal antibodies, RT-PCR primers, hyperimmune antisera, cell
lines, virus strains, reference positive and negative sera, etc.) and test protocols to colleagues in member countries.
This is because it is difficult to import foreign animal specimens into the US for testing. However we procured a
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Annual reports of OIE Reference Centres, 2011
Transmissible gastroenteritis
USDA import permit to import fecal RNA samples from diarrheic swine in Argentina to test for possible TGE
cases. All tested samples were negative for TGEV/PRCV and other coronaviruses using TGEV/PRCV-specific
and pan-coronavirus primers, respectively. However we continue to receive requests for testing of fecal samples
from diarrheic swine for TGEV from multiple South American countries. This raises concerns for the potential
spread of TGEV or other undiagnosed diarrheal pathogens in swine in South America.
11. Organisation of international scientific meetings on behalf of OIE or other international bodies
Participated as member of organizing committee for multiple international scientific meetings. Plenary speaker at
the International Congress on Pathogens at the Human-Animal Interface (ICOPHAI), Addis Ababa,
Ethiopia, Sept 15-17, 2011 (Multiple sponsers including OIE)
12. Participation in international scientific collaborative studies
In addition to the instances cited, we are pursuing collaborative studies on diagnosis and immunity to TGEV and
PRCV and coronaviruses with Dr K.O. Cho, Dr S.Y. Kang, Korea; Dr Luis Enjuanes, Spain; Dr To-long Thanh,
Vietnam; and Dr Alexei Zaberazhny, Soviet Union; Drs Viviana Parreno and Fernandez Fernando, Argentina.
In January 2004, our lab was designated by WHO as an international reference lab for animal coronaviruses. In
this capacity we will supply TGEV/PRCV strains and antisera to other WHO reference labs and to other
international labs through the ATCC.
13. Publication and dissemination of information relevant to the work of OIE (Coronavirus related
only)
Refereed Journal Articles
1. Vlasova, A.N., Halpin, R., Wang, S., Ghedin, E., Spiro, D.J., Saif, L.J. 2011. Molecular characterization of a
new alphacoronavirus species associated with mink epizootic catarrhal gastroenteritis. J Gen Virol. 2011; 92:136979.
2. L. Mullis, L J. Saif, Y. Zhang, X. Zhang, M.S.P. Azevedo. 2012. Stability of bovine coronavirus on lettuce
surfaces under household refrigeration conditions. Food Microbiology 30: 180e186. Online Dec 13, 2011.
Book Chapters
1. Saif, L.J., M. Pensaert, K. Sestak, S.G. Yeo and K. Jung. 2012. Coronaviruses. In: Diseases of Swine. 10th Ed
(J.J. Zimmerman, et al, Ed.), Ames, IA, Blackwell Publishing (in press).
2. Saif, L.J. 2010. Reoviridae, Coronaviridae. In: Fenner’s Veterinary Virology, 4th Ed (J. MacLachlan & E.
Dubovi, Eds). Elsevier, The Netherlands.
Abstracts
1. Vlasova, A., Halpin, R., Wang, S., Ghedin, E., Spiro, D., Saif, L. 2011. Molecular characterization of a new
alphacoronavirus species associated with mink epizootic catarrhal gastroenteritis. Proc. The XIIth Nidovirus
Symposium. June 4 – 9, 2011. Acme, MI.
2. L Mullis, J Britt, C Silva, X Zhang, LJ Saif, R Steele and MSP Azevedo. 2011. Prevalence of recently
circulating coronaviruses in domestic animals and wildlife in Arkansas, USA. Am. Soc. Virology Annual Meeting,
July, 2011. Abst.
3. C Silva, L Mullis, R Owens, LJ Saif, D Taylor, L Haynes, X Zhang and MSP Azevedo. 2011. Human
respiratory coronaviruses circulating during the influenza season in Arkansas. Am. Soc. Virology Annual Meeting,
July, 2011.
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Annual reports of OIE Reference Centres, 2011
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