Activities in 2009
Rabies
Prof. Dr Anthony R. Fooks
Veterinary Laboratories Agency (Weybridge), Rabies and Wildlife Zoonoses Group,
Department of Virology, New Haw, Addlestone, Surrey KT15 3NB, United Kingdom
Tel.: (+44-1932) 35.78.40, Fax: (+44-1932) 35.72.39
t.fooks@vla.defra.gsi.gov.uk, website: www.vla.gov.uk
Summary of general activities related to the disease
1.
2.
Test(s) in use/or available for the specified disease at your laboratory
Test
For
Specificity
Total
Fluorescent Antibody Test (FAT)
Antigen detection
RABV and other lyssaviruses
1,028
Rabies Tissue Culture Infection Test
(RTCIT)
Virus isolation
RABV and other lyssaviruses
679
Mouse Inoculation Test
Virus isolation
RABV and other lyssaviruses
58
Reverse-transcriptase Polymerase
Chain Reaction (RT-PCR)
RNA detection
RABV and other lyssaviruses
(validated for genotypes 1-7)
333
Sequencing
Genotype
differentiation
RABV and other lyssaviruses
(validated for genotypes 1-7)
2
Fluorescent Antibody Virus
Neutralisation Test (FAVN)
Antibody titration
RABV
11,665
Modified Fluorescent Antibody Virus
Neutralisation Test (mFAVN)
Antibody titration
EBLVs
192
Production and distribution of diagnostic reagents
2.1. Diagnostic samples/ viruses sent out
Collaborator
Prof Neil Hall, School of Biological
Sciences, University of Liverpool
Country
UK
Samples
Virus isolates (n=11) consisting of lyssavirus cDNA
genotypes 1 – 4; Africa 4 Lyssavirus (n=1)
Dr Thomas Muller, FLI,
Wusterhausen
Germany
Africa 2, 3, 4, Lyssaviruses (Thai, Chinese and US isolates).
Dr Christian Drosten, Institute of
Virology, University of Bonn
Medical Centre, Bonn
Germany
Non-infectious RNA from Lagos Bat Virus isolates (n=4)
Dr Christian Drosten, Institute of
Virology, University of Bonn
Medical Centre, Bonn
Germany
Virus isolates of Lagos bat virus isolates (n=3)
Dr Ed Wright, Wohl Virion Centre,
UCL, London
UK
Rabbit sera raised against Eurasian lyssaviruses using
inactivated tissue culture supernatant for IRKV, ARAV,
KHUV and WCBV
Annual reports of OIE Reference Laboratories and Collaborating Centres, 2009
1
Rabies
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
3.1. Antibody titration – proficiency testing for serological testing of blood from dogs/cats
The VLA has successfully participated in annual FAVN proficiency schemes, organised by the EU Community
reference laboratory, AFSSA, Nancy, France.
3.2. Antibody titration – proficiency testing for serological testing of blood from bats
The VLA has provided a QA proficiency scheme for the detection of neutralising antibodies to European Bat
Lyssaviruses (EBLV-1 and -2). Two European laboratories (including the VLA) participated in the proficiency scheme.
3.3. Rabies diagnosis – OIE Twining Project
The VLA has organised a proficiency scheme with the Changchun University of Agriculture and Animal Sciences
(CUAAS) to harmonise testing protocols for rabies, as part of the OIE-funded twining project.
4.
Preparation and supply of international reference standards for diagnostic tests or vaccines
Preparation of positive control dog serum is underway through close collaboration with Professor Tu of the
Changchun Veterinary Institute, Changchun, Jilin Province, China. Rabies seropositive dog serum has been
produced in China and has been sent to the VLA. The antiserum is currently being checked, titrated for
neutralising antibodies and diluted in our laboratory to produce low positive standard reagents for use in
serological tests as part of the OIE International Standards Bank.
5.
Research and development of new procedures for diagnosis and control
5.1. Emerging technologies for the detection of rabies virus
The diagnosis of rabies is routinely based on clinical and epidemiological information, especially when exposures are
reported in rabies-endemic countries. Diagnostic tests using conventional assays that appear to be negative, even when
undertaken late in the disease and despite the clinical diagnosis, have a tendency, at times, to be unreliable. These tests
are rarely optimal and entirely dependent on the nature and quality of the sample supplied. In the course of the past three
decades, the application of molecular biology has aided in the development of tests that result in a more rapid detection
of rabies virus. These tests enable viral strain identification from clinical specimens. Currently, there are a number of
molecular tests that can be used to complement conventional tests in rabies diagnosis. Indeed the challenges in the 21st
century for the development of rabies diagnostics are not of a technical nature; these tests are available now. The
challenges in the 21st century for diagnostic test developers are two-fold: firstly, to achieve internationally accepted
validation of a test that will then lead to its acceptance by organisations globally. Secondly, the areas of the world where
such tests are needed are mainly in developing regions where financial and logistical barriers prevent their
implementation. Although developing countries with a poor healthcare infrastructure recognise that molecular-based
diagnostic assays will be unaffordable for routine use, the cost/benefit ratio should still be measured. Adoption of rapid
and affordable rabies diagnostic tests for use in developing countries highlights the importance of sharing and
transferring technology through laboratory twinning between the developed and the developing countries. Importantly
for developing countries, the benefit of molecular methods as tools is the capability for a differential diagnosis of human
diseases that present with similar clinical symptoms. Antemortem testing for human rabies is now possible using
molecular techniques. These barriers are not insurmountable and it is our expectation that if such tests are accepted and
implemented where they are most needed, they will provide substantial improvements for rabies diagnosis and
surveillance. The advent of molecular biology and new technological initiatives that combine advances in biology with
other disciplines will support the development of techniques capable of high throughput testing with a low turnaround
time for rabies diagnosis.
5.2. A robust lentiviral pseudotype neutralisation assay for in-field serosurveillance of rabies and
lyssaviruses in Africa
In a multi-institute collaboration, the feasibility of using a pseudotype based assay for lyssavirus serosurveillance
in the field in Africa was assessed. The inflexibility of existing serological techniques for detection of rabies in
surveillance constrains the benefit to be gained from many current control strategies. We analysed 304 serum
samples from Tanzanian dogs for the detection of rabies antibodies in a pseudotype assay using lentiviral vectors
2
Annual reports of OIE Reference Laboratories and Collaborating Centres, 2009
Rabies
bearing the CVS-11 envelope glycoprotein. Compared with the widely used gold standard fluorescent antibody
virus neutralisation assay, a specificity of 100% and sensitivity of 94.4% with a strong correlation of antibody
titres (r=0.915) were observed with the pseudotype assay. To increase the assay's surveillance specificity in Africa
we incorporated the envelope glycoprotein of local viruses, Lagos bat virus, Duvenhage virus or Mokola virus and
also cloned the lacZ gene to provide a reporter element. Neutralisation assays using pseudotypes bearing these
glycoproteins reveal that they provide a greater sensitivity compared to similar live virus assays and will therefore
allow a more accurate determination of the distribution of these highly pathogenic infections and the threat they
pose to human health. Importantly, the CVS-11 pseudotypes were highly stable during freeze-thaw cycles and
storage at room temperature. These results suggest the proposed pseudotype assay is a suitable option for
undertaking lyssavirus serosurveillance in areas most affected by these infections.
6.
Collection, analysis and dissemination of epizootiological data relevant to international disease
control
6.1. Analysis of vaccine-virus-associated rabies cases in red foxes (Vulpes vulpes) after oral rabies
vaccination campaigns in Germany and Austria
To eradicate rabies in foxes, almost 97 million oral rabies vaccine baits have been distributed in Germany and
Austria since 1983 and 1986, respectively. Since 2007, no terrestrial cases have been reported in either country.
The most widely used oral rabies vaccine viruses in these countries were SAD (Street Alabama Dufferin) strains,
e.g. SAD B19 (53.2%) and SAD P5/88 (44.5%). In this paper, we describe six possible vaccine-virus-associated
rabies cases in red foxes (Vulpes vulpes) detected during post-vaccination surveillance from 2001 to 2006,
involving two different vaccines and different batches. Compared to prototypic vaccine strains, full-genome
sequencing revealed between 1 and 5 single nucleotide alterations in the L gene in 5 of 6 SAD isolates, resulting in
up to two amino acid substitutions. However, experimental infection of juvenile foxes showed that those mutations
had no influence on pathogenicity. The cases described here, coming from geographically widely separated
regions, do not represent a spatial cluster. More importantly, enhanced surveillance showed that the vaccine
viruses involved did not become established in the red fox population. It seems that the number of reported
vaccine virus-associated rabies cases is determined predominantly by the intensity of surveillance after the oral
rabies vaccination campaign and not by the selection of strains.
6.2. Phylogenetic analysis of rabies viruses from Sudan provides evidence of a viral clade with a unique
molecular signature
Rabies is endemic in Sudan and remains a continual threat to public health as transmission to humans is
principally dog-mediated. Additionally, large-scale losses of livestock occur each year causing economic and
social dilemmas. In this study, we analysed a cohort of 143 rabies viruses circulating in Sudan collected from 10
different animal species between 1992 and 2006. Partial nucleoprotein sequence data (400 bp) were obtained and
compared to available sequence data of African classical rabies virus (RABV) isolates. The Sudanese sequences
formed a discrete cluster within the Africa 1a group, including a small number of sequences that clustered with
sequences from Ethiopian RABV. These latter sequences share an Aspartic Acid at position 106 (Asp(106)) with
all other Africa 1a group members, in contrast to the remaining Sudanese strains, which encode Glutamic Acid at
this position (Glu(106)). Furthermore, when representatives of other African and European lineages were aligned,
Glu(106) is unique to Sudan, which supports the concept of a single distinct virus strain circulating in Sudan. The
high sequence identity in all Sudanese isolates studied, demonstrates the presence of a single rabies virus biotype
for which the principal reservoir is the domestic dog.
6.3. European bat lyssavirus in a Daubenton’s bat from Scotland
We have recently isolated European bat lyssavirus (EBLV) type-2 from a Daubenton’s bat (Myotis daubentonii) in
West Lothian, Scotland. EBLV-2 has been isolated multiple times throughout England since 1996, but this is the
first isolation of live virus from a bat in Scotland, seven years after the death of a Scottish bat conservation worker
from infection with EBLV-2 in 2002. This finding re-iterates the importance of public education regarding bat
lyssavirus infections throughout the UK, and the importance of gaining a better understanding of bat lyssavirus
dynamics in their natural hosts. In late August 2009, an adult female Daubenton’s bat was observed at an historic
site in Linlithgow, West Lothian. Two days later the bat had not moved and when inspected was found dead.
Lyssavirus antigen was detected in a brain smear by the fluorescent antibody test (FAT). On the same day the
lyssavirus was confirmed as EBLV-2 by differential real-time TaqMan PCR, and 2 days after receipt of the
sample, the presence of live virus in the brain had been confirmed using a tissue culture inoculation test. Part of
the viral genome was amplified using a hemi-nested reverse transcriptase PCR and sequenced, showing a close
genetic relationship to previous UK EBLV-2 isolates. Therefore, although this finding of live virus in a Scottish
Annual reports of OIE Reference Laboratories and Collaborating Centres, 2009
3
Rabies
Daubenton’s bat confirms the public health risks suggested by serosurveillance and viral RNA in bat saliva, the
risks remain small.
6.4. Rabies in foxes, Aegean region, Turkey
At the end of 1990s fox (Vulpes vulpes) rabies became endemic after a sustained spill-over infection from dogs
(Canis lupus familiaris) in the Aegean Region, Turkey. In the last decade (1998 – 2007), a total of 1,225 animal
rabies cases were reported from this area. Only 27% of all cases were dogs, with foxes being responsible for 13%
of all rabies cases. Most cases were diagnosed in cattle (n=605). The number of reported rabies cases decreased
dramatically after mass cattle vaccination campaigns were organized. Presently, oral rabies vaccination campaigns
targeted at the fox population are undertaken to control this outbreak.
6.5. Targeted surveillance for European bat lyssaviruses in English bats
In 2003-2006, targeted (active) surveillance for European bat lyssaviruses (EBLVs) was undertaken throughout
England, focussing on species most likely to host these viruses, Myotis daubentonii and Eptesicus serotinus. Blood
was sampled for the detection of EBLV-specific neutralising antibodies and oropharyngeal swabs were taken for
the detection of viral RNA or infectious virus in saliva. Between 2003 and 2006, 273 E. serotinus and 363 M.
daubentonii blood samples were tested by the EBLV-1 or EBLV-2 specific modified fluorescent antibody
neutralization test (mFAVN). The EBLV-2 antibody prevalence estimate was 1.0-4.1% (95% CI, mean = 2.2%)
for M. Daubentonii. EBLV-1 specific antibodies were detected only in a single E. serotinus. Other non-target
species (n = 5) were sampled in small numbers (n = 24), with no EBLV specific antibody detected. No viral RNA
or live virus was detected in any of the oropharyngeal swabs analysed. Host RNA was detected from 83% of the
oropharyngeal swabs analysed (total swabs 2003-2006: n = 766). These data unequivocally prove that EBLV-2 is
present in M. daubentonii in England. In contrast, there is insufficient evidence to suggest that EBLV-1 is present
in E. serotinus in England, although further research is warranted.
6.6. Genetic analysis of four human rabies cases reported in Turkey between 2002 and 2006
Rabies remains endemic in many regions of Turkey. As a consequence, humans are at risk of this fatal disease
through encounters with rabid animals. The present study describes four recent cases of rabies in humans.
Subsequent phylogenetic analysis of the rabies virus isolates obtained from each case demonstrates the distinct
geographical distribution of rabies virus variants within Turkey. The study suggests that rabies virus translocation
has occurred across Turkey and might be the source of the emergence of a genetically similar variant in the Golan
Heights region on the Israeli/Syrian border in 2004.
6.7. Quantitative risk assessment to compare the risk of rabies entering the United Kingdom from Turkey
via quarantine, the Pet Travel Scheme and the EU Pet Importation Policy
Rabies was eradicated from the United Kingdom (UK) in 1922 through strict controls of dog movement and
investigation of every incidence of disease. Amendments were made to the UK quarantine laws and the Pet Travel
Scheme (PETS) was subsequently introduced in 2000 for animals entering the UK from qualifying listed
countries. Consequently, companion animals can enter the UK without quarantine if they meet the specific
requirements of PETS. European Regulation 998/2003 on the non-commercial movement of pet animals initiated
the European Union Pet Importation Policy (EUPIP) in July 2004. The conditions of movement indicated in this
legislation vary with that currently instigated in a number of countries and these, including the UK, have
derogations in effect until 2010. The introduction of EUPIP will harmonise the movement of pet animals within
the EU (EUPIPlisted) but raises the possibility of domestic animals entering the UK from a non-EU state where
rabies is endemic (EUPIPunlisted). A quantitative risk assessment model has been developed to estimate the risk of
rabies entering the UK via companion animals from Turkey, if companion animals are permitted to enter through
PETS or EUPIP compared to quarantine. Specifically, the risk was assessed by estimating the probability of rabies
entering the UK per year and the number of years between rabies entry for each scheme. The model identified that
the probability of rabies entering the UK via the three schemes is highly dependent on compliance. If 100%
compliance is assumed, PETS and EUPIP unlisted (at the current level of importation) presents a lower risk than
quarantine, i.e the number of years between rabies entry is more than 170,721 years for PETS and 60,163 years for
EUPIPunlisted compared to 41,851 years for quarantine (with 95% certainty). If less than 100% compliance is
assumed, PETS and EUPIPunlisted (at the current level of importation) presents a higher risk. In addition, EUPIPlisted
and EUPIPunlisted (at an increased level of importation) present a higher risk than quarantine or PETS at 100%
compliance and at an uncertain level of compliance.
6.8. Rabies epidemiology and control in Turkey
Turkey is the only country in Europe where urban dog-mediated rabies persists. Control measures in recent decades
have reduced the burden of rabies to relatively low levels but foci of disease still persist, particularly in urban areas.
4
Annual reports of OIE Reference Laboratories and Collaborating Centres, 2009
Rabies
Occasional human cases result from this persistence although the source of these appears to be both dog and wildlife
reservoirs. This review considers the current state of rabies in Turkey including current control measures, the varying
epidemiology of the disease throughout this country and the prospects for rabies elimination.
6.9. Repeated detection of European bat lyssavirus type 2 in dead bats found at a single roost site in the UK
In August 2007, European bat lyssavirus type 2 (EBLV-2) was isolated from a Daubenton's bat found at Stokesay
Castle. In September 2008, another bat from the same vicinity of Stokesay Castle also tested positive for EBLV-2. This
is the first occurrence of repeated detection of EBLV-2 from a single site. Here, we report the detection of low levels of
viral RNA in various bat organs by qRT-PCR and detection of viral antigen by immunohistochemistry. We also report
sequence data from both cases and compare data with those derived from other EBLV-2 isolations in the UK.
6.10. Human case of rabies Belfast (UK) ex. South Africa
A 37-year-old woman was admitted to hospital and over the next 5-days developed a progressive encephalitis. Nuchal
skin biopsy, analysed using a Rabies TaqMan© PCR, demonstrated rabies virus RNA. She had a history in keeping with
exposure to rabies whilst in South Africa, but had not received pre- or post-exposure prophylaxis. She was treated with a
therapeutic coma according to the “Milwaukee protocol”, which failed to prevent the death of the patient. Rabies virus
was isolated from CSF and saliva, and rabies antibody was demonstrated in serum (from day 11 onwards) and
cerebrospinal fluid (day 13 onwards). She died on day-35 of hospitalization. Autopsy specimens demonstrated the
presence of rabies antigen, viral RNA, and viable rabies virus in the central nervous system.
7.
Provision of consultant expertise to OIE or to OIE Members
Institute
8.
Consultancy project
Country
The University of Texas Medical
Branch, Galveston, Texas
Development of a Ferret Model for Rabies Treatment
[NIH-NIAID-NO1-AI-30065 Part C (28)]
USA
Changchun University of Agriculture
and Animal Sciences (CUAAS),
OIE Twinning project
China
Turkish Veterinary Services, Ankara
Technical Assistance for Control of Rabies Disease’
EU funded Project Contract N°: TR.503.06/100
Turkey
European Food Standards Agency,
Parma
EFSA/ZONOSES/2008/1 - rabies and Q fever
Ghana Veterinary Medicine
Association, Accra,
Cambridge Infectious Disease Consortium, UK / The
Wellcome Trust, UK
UK/Ghana
World Health Organization, Geneva,
Expert Consultation on Rabies
Switzerland
World Health Organization, Geneva /
Bill and Melinda Gates Foundation,
Seattle,
Creating a "one medicine" paradigm shift in human
rabies prevention through dog rabies control and
eventual elimination
Switzerland/
USA
OIE World Organisation for Animal
Health, Paris,
Scientific Commission for Animal Diseases
Italy
France
Provision of scientific and technical training to personnel from other OIE Members
8.1 Training personnel from other countries in rabies diagnostic techniques
Name
Institute
Country
Amir Horowitz
London School of Hygiene and Tropical Medicine, London
Shoufeng Zhang
Changchun Veterinary Research Institute, Changchun
China
Josefina Carolina
Aznar Lopez
Institute de Salud Carlos II, Madrid
Spain
Robert Wollny
Institute of Virology, Bonn
Annual reports of OIE Reference Laboratories and Collaborating Centres, 2009
United Kingdom
Germany
5
Rabies
Name
Institute
Emilie Oudot
University of Liverpool, Liverpool
Vasiliki Christodoulou
9.
Veterinary Services, Limassol
Country
United Kingdom
Cyprus
Provision of diagnostic testing facilities to other OIE Members
9.1. Diagnostic samples / virus isolates were received from a number of sources
Collaborator
Country
Samples
Dr Katie Hampson,
University of Glasgow,
UK
UK /
Tanzania
Brain samples from dog / goat / gorilla / wildcat / genet/ jackal / honey
badger / mongoose / hyena / civet / serval / from Tanzania (n=66).
Human CSF (1)
Anne-Marie Stewart,
Ethiopian Wolf
Conservation
Programme
Ethiopia
Brain samples (6); Heart (2); Kidney (2); Liver (2); Lung (2); Small
intestine (3); Lymph node (3); Salivary Gland (1) from Ethiopian Wolf
Dr Thomas Muller,
FLI, Wusterhausen
Germany
RNA RABV (genotype 1) samples from Foxes in Poland (6); Dog sample
from Sudan (1); Human samples from Chile (2); Fox sample from
Germany (1); Fox sample from Russia (1) and an unknown sample from
Canada (1).
RNA sample (genotype 3) Mokola Virus (1)
Dr Andrew Alhassan,
Veterinary Services
Directorate, Accra
Ghana
Dog Brain samples from Ghana (n=96).
Dr Charles Rupprecht,
CDC Atlanta, Clifton
Rd, Atlanta
USA
Rabbit sera raised against emerging lyssaviruses (WCBV, ARAV, IRKV,
KHUV)
Helen Ambrose, HPA
London,
Dr S. Duthie, Biobest,
Edinburgh
UK
Serum (human) for FAVN testing in encephalitis study (n=5).
Scotland
Saliva swab (bat) in RNA Later for PCR & Screening (n=1).
Amir Horowitz,
London School of
Hygiene and Tropical
Medicine, London
UK
Human serum samples for FAVN testing (n=91).
Dr Martin Schweiger,
HPA Leeds
UK
Serum (human) samples for FAVN testing (n=24).
Dr Ron Behrens,
London School of
Hygiene and Tropical
Medicine, London
UK
Serum (human) samples for FAVN testing (n=14).
Germany
Fox brain samples (n=10); serum samples (n=34).
UK / Kenya
Serum (dog) samples for FAVN testing (n=166).
Dr Adrian Vos, IDT
Dr Annie Cook,
London School of
Hygiene and Tropical
Medicine, London
Dr Shoufeng Zhang
Changchun Veterinary
Research Institute,
Changchun
6
China
Serum (dog) for FAVN testing (n=1).
Annual reports of OIE Reference Laboratories and Collaborating Centres, 2009
Rabies
10. Organisation of international scientific meetings on behalf of OIE or other international bodies
International Conference on Global Rabies Control, Seoul (Republic of Korea), 7-9 September 2011.
11. Participation in international scientific collaborative studies
11.1. Experimental infection of foxes with European Bat Lyssaviruses type-1 and 2
The susceptibility of foxes to European Bat Lyssaviruses type-1 and -2 was investigated. The study used silver
foxes (Vulpes vulpes) as a model. These data showed that the sensitivity of silver foxes is low by both the
intracranial and intramuscular routes. Six of 6 foxes intracranially injected with EBLV-1 and EBLV-2 died
between 8 and 282 days post-infection. Three of 21 foxes intramuscularly injected with EBLV-1 died between 14
and 24 days post-infection. None of the five foxes infected intramuscularly with EBLV-2 died. These data
suggested that the risk of a EBLV spill-over from bat to a fox is low, but with a greater probability for EBLV-1
than for EBLV-2 and that foxes seem to be able to clear the virus before it reaches the brain and cause a lethal
infection.
11.2. Detection of High Levels of European Bat Lyssavirus Type-1 Viral RNA in the Thyroid Gland of
Experimentally-Infected Eptesicus fuscus
In a multi-disciplinary study, we evaluated the susceptibility and pathology associated with an EBLV-1 infection
in Eptesicus fuscus following different routes of virus inoculation including intracranial (n=6), intramuscular
(n=14), oral (n=7) and intranasal (n=7). The presence of virus and viral RNA was detected in the thyroid gland in
bats challenged experimentally with EBLV-1, which exceeded that detected in all other extra-neural tissue. The
significance of detecting EBLV-1 in the thyroid gland of rabid bats is not well understood. We speculated that the
infection of the thyroid gland may cause subacute thyroiditis, a transient form of thyroiditis causing
hyperthyroidism, resulting in changes in adrenocortical activity that could lead to hormonal dysfunction, thereby
distinguishing the clinical presentation of rabies in the rabid host.
11.3. Experimental infection of serotine bats (Eptesicus serotinus) with European bat lyssavirus type 1a
The serotine bat (Eptesicus serotinus) accounts for the vast majority of bat rabies cases in Europe and is
considered the main reservoir for European bat lyssavirus type 1 (EBLV-1, genotype 5). However, so far the
disease has not been investigated in its native host under experimental conditions. To assess viral virulence,
dissemination and probable means of transmission, captive bats were infected experimentally with an EBLV-1a
virus isolated from a naturally infected conspecific from Germany. Twenty-nine wild caught bats were divided
into five groups and inoculated by intracranial (i.c.), intramuscular (i.m.) or subcutaneous (s.c.) injection or by
intranasal (i.n.) inoculation to mimic the various potential routes of infection. One group of bats was maintained as
uninfected controls. Mortality was highest in the i.c.-infected animals, followed by the s.c. and i.m. groups.
Incubation periods varied from 7 to 26 days depending on the route of infection. Rabies did not develop in the i.n.
group or in the negative-control group. None of the infected bats seroconverted. Viral antigen was detected in
more than 50% of the taste buds of an i.c.-infected animal. Shedding of viable virus was measured by virus
isolation in cell culture for one bat from the s.c. group at 13 and 14 days post-inoculation, i.e. 7 days before death.
In conclusion, it is postulated that s.c. inoculation, in nature caused by bites, may be an efficient way of
transmitting EBLV-1 among free-living serotine bats.
11.4. Development of a mouse monoclonal antibody cocktail for post-exposure rabies prophylaxis in
humans
As the demand for rabies post-exposure prophylaxis (PEP) treatments has increased exponentially in recent years,
the limited supply of human and equine rabies immunoglobulin (HRIG and ERIG) has failed to provide the
required passive immune component in PEP in countries where canine rabies is endemic. Replacement of HRIG
and ERIG with a potentially cheaper and efficacious alternative biological for treatment of rabies in humans,
therefore, remains a high priority. In this study, we set out to assess a mouse monoclonal antibody (MoMAb)
cocktail with the ultimate goal to develop a product at the lowest possible cost that can be used in developing
countries as a replacement for RIG in PEP. Five MoMAbs, E559.9.14, 1112-1, 62-71-3, M727-5-1, and M777-163, were selected from available panels based on stringent criteria, such as biological activity, neutralizing potency,
binding specificity, spectrum of neutralization of lyssaviruses, and history of each hybridoma. Four of these
MoMAbs recognize epitopes in antigenic site II and one recognizes an epitope in antigenic site III on the rabies
virus (RABV) glycoprotein, as determined by nucleotide sequence analysis of the glycoprotein gene of unique
MoMAb neutralization-escape mutants. The MoMAbs were produced under Good Laboratory Practice (GLP)
conditions. Unique combinations (cocktails) were prepared, using different concentrations of the MoMAbs that
Annual reports of OIE Reference Laboratories and Collaborating Centres, 2009
7
Rabies
were capable of targeting non-overlapping epitopes of antigenic sites II and III. Blind in vitro efficacy studies
showed the MoMab cocktails neutralized a broad spectrum of lyssaviruses except for lyssaviruses belonging to
phylogroups II and III. In vivo, MoMAb cocktails resulted in protection as a component of PEP that was
comparable to HRIG. In conclusion, all three novel combinations of MoMAbs were shown to have equal efficacy
to HRIG and therefore could be considered a potentially less expensive alternative biological agent for use in PEP
and prevention of rabies in humans.
12. Publication and dissemination of information relevant to the work of OIE (including list of
scientific publications, internet publishing activities, presentations at international conferences)
Publications
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
8
Zinsstag, J., Schelling, E., Bonfoh, B., Fooks, A.R., Kasymbekov, J., Waltner-Toews, D. and M. Tanner.
(2009). Towards a “One Health” Research and Application Toolbox. Veterinaria Italiana 45(1); 119-131.
Cliquet, F., Picard Meyer, E., Barrat, J., Brookes, S.M., Healy, D.M., Wasniewski, M., Litaize, E., Biarnais,
M., Johnson L. and A.R. Fooks. (2009). Experimental infection of Foxes with European bat Lyssaviruses
type-1 and 2. BMC Veterinary Research 5(1); 19.
Fooks, A.R., Johnson, N., Müller, T., Vos, A., Mansfield, K., Hicks, D., Nunez, A., Freuling, C., Neubert, L.,
Kaipf, I., Denzinger, A., Franka, R. and C.E. Rupprecht (2009). Detection of High Levels of European Bat
Lyssavirus Type-1 Viral RNA in the Thyroid Gland of Experimentally-Infected Eptesicus fuscus Bats.
Zoonoses Public Health 56; 270 – 277.
Müller, T., Bätza, H-J., Beckert, A., Bunzenthal, C., Cox, J.H., Freuling, C., Fooks, A.R., Frost, J., Geue, L.,
Hoeflechner, A., Marston, D., Neubert, A., Neubert, L., Revilla-Fernández S., Vanek, E., Vos, A., Wodak,
E., Zimmer, K. and T.C. Mettenleiter (2009). Analysis of vaccine-virus-associated rabies cases in red foxes
(Vulpes vulpes) after oral rabies vaccination campaigns in Germany and Austria. Arch. Virol. 154(7); 108191.
Marston, D.E., McElhinney, L.M., Ali, Y.H., Intisar, K.S., Ho, S.M., Freuling, C., Müller, T and A.R. Fooks.
(2009). Phylogenetic analysis of rabies virus strains from Sudan provides evidence of a viral clade with a
unique molecular signature. Virus Research 145(2); 244-50.
Freuling, C., Vos, A., Johnson, N., Kaipf, I., Denzinger, A., Neubert, L., Mansfield, K., Hicks, D., Nunez, A.,
Tordo, N., Rupprecht C. E, Fooks A.R. and Müller T. (2009). Experimental infection of Serotine bats
(Eptesicus serotinus) with European bat lyssavirus type 1a (EBLV-1a). Journal of General Virology. 90;
2493-2502.
Horton, D.L., Voller, K., Haxton, B., Johnson, N., Leech, S., Goddard, T., Wilson, C., McElhinney, L.M. and
A.R. Fooks. (2009). European bat lyssavirus in a Daubenton’s bat from Scotland. The Veterinary Record
165(13); 383-4.
Vos, A., Freuling, C., Eskiimirliler, S., Ün, H., Aylan, O., Johnson, N., Gürbüz, S., Müller, W., Akkoca, N.,
Müller, T., Fooks, A.R. and H. Askaroglu. (2009). Rabies in foxes, Aegean region, Turkey. Emerging
Infectious Diseases 15(10); 1620-22.
Fooks, A.R. Weiss, R., Johnson, N., Banyard, A., Wright, E., McElhinney, L.M., Marston, D., Freuling, C.,
and T. Müller (2009). Emerging technologies for the detection of rabies virus: challenges and hopes in the
21st century. PloS Neglected Diseases 3(9); e530.
Harris, S.L., Aegerter, J., Brookes, S.M., McElhinney, L.M., Jones, G., Smith, G. and A.R. Fooks. (2009).
Targeted surveillance for European bat lyssaviruses in English bats (2003-06). Journal of Wildlife Diseases
45(4); 1030-41.
Müller, T., Dietzschold, B., Ertl, H., Fooks, AR., Freuling, C., Fehlner-Gardiner, C., Kliemt, J., Meslin, F-X.,
Franka, R., Rupprecht, C.E., Tordo, N., Wanderler, A. and Marie-Paule Kieny. (2009). Development of a
Murine Monoclonal Antibody Cocktail for Post-Exposure Rabies Prophylaxis in Humans. PloS Neglected
Diseases 3(11); e542.
Freuling C., Vos A., Johnson N., Fooks A.R. and Müller T. (2009). Bat Rabies – a Gordian knot? Berliner
Münchener Tierärztliche Wochenschrift 122(11/12); 425-33.
Wright, E., McNabb, S., Goddard, T., Horton, D.L., Lembo, T., Nel, L.H., Weiss, R.A., Cleaveland, S. and
A.R. Fooks (2009). A robust lentiviral pseudotype neutralisation assay for in-field serosurveillance of rabies
and lyssaviruses in Africa. Vaccine 27; 7178-86.
Un, H., Johnson, N., Vos, A., Muller, T., Fooks, A.R. and Aylan, O. (2009). Genetic analysis of Four Human
Rabies Cases Reported in Turkey Between 2002 and 2006. Clinical Microbiology and Infection 15(12);
1185-9.
Fooks A.R., Johnson, N. and Charles E. Rupprecht (2009). Chapter 33: Rabies. In: Vaccines for Biodefense
and Emerging and Neglected Diseases (A Barrett and L Stanberry, Eds). Elsevier Publications Ltd, pp. 605626.
Annual reports of OIE Reference Laboratories and Collaborating Centres, 2009
Rabies
16. McElhinney, L.M., Fooks, A.R. and A.D. Radford. (2009). Diagnostic tools for the detection of rabies virus.
In: Zoonoses (EJCAP Publications), pp.224-31.
17. Horton, D.L. and A.R. Fooks (2009). Infectious Diseases of the Horse (eds. T.S. Mair and R.E. Hutchinson).
In: Equine Veterinary Journal Ltd pp. 128 – 137.
18. Ramnial, V., Kosmider, R., Aylan, O., Freuling, C., Müller, T. and A.R. Fooks. (2009). Quantitative risk
assessment to compare the risk of rabies entering the United Kingdom from Turkey via quarantine, the Pet
Travel Scheme and the EU Pet Importation Policy. Epidemiology and Infection. In press.
19. Johnson, N., Un, H., Fooks, A.R., Freuling, C., Müller, T., Aylan, O. and A. Vos (2009). Rabies
epidemiology and control in Turkey: past and present. Epidemiology and Infection. In press.
20. Banyard, A.C., Johnson, N., Voller, K., Hicks, D., Nunez, A., Hartley, M. and A.R. Fooks. (2009). Repeated
detection of European bat lyssavirus type 2 in dead bats at a single roost site in the UK. Archives of Virology.
In press.
Presentations at international conferences
Conference
Country
Presentation
Humberside Zoonoses Conference
UK
Invited speaker
Ghana Veterinary Medicine Association
Ghana
Invited speaker
Rabies in the Americas
Canada
Invited speaker
International Symposium of Major Zoonoses
Korea
Invited speaker
Veterinary Vaccines
Germany
Invited speaker
13. Inscription of diagnostic kits on the OIE Register
i)
Did you participate in expert panels for the validation of candidate kits for inscription on the
OIE Register? If yes, for which kits?
No
ii)
Did you submit to the OIE candidate kits for inscription on the OIE Register? If yes, for
which kits?
None
_______________
Annual reports of OIE Reference Laboratories and Collaborating Centres, 2009
9