Evaluation of a mouse model for the West Nile virus group for the purpose of determining viral pathotypes.

West Nile virus (WNV; family Flaviviridae; genus Flavivirus) group members are an important cause of viral meningoencephalitis in some areas of the world. They exhibit marked variation in pathogenicity, with some viral lineages (such as those from North America) causing high prevalence of severe neurological disease, whilst others (such as Australian Kunjin virus) rarely cause disease. The aim of this study was to characterize WNV disease in a mouse model and to elucidate the pathogenetic features that distinguish disease variation. Tenfold dilutions of five WNV strains (New York 1999, MRM16 and three horse isolates of WNV-Kunjin: Boort and two isolates from the 2011 Australian outbreak) were inoculated into mice by the intraperitoneal route. All isolates induced meningoencephalitis in different proportions of infected mice. WNVNY99 was the most pathogenic, the three horse isolates were of intermediate pathogenicity and WNVKUNV-MRM16 was the least, causing mostly asymptomatic disease with seroconversion. Infectivity, but not pathogenicity, was related to challenge dose. Using cluster analysis of the recorded clinical signs, histopathological lesions and antigen distribution scores, the cases could be classified into groups corresponding to disease severity. Metrics that were important in determining pathotype included neurological signs (paralysis and seizures), meningoencephalitis, brain antigen scores and replication in extra-neural tissues. Whereas all mice infected with WNVNY99 had extra-neural antigen, those infected with the WNV-Kunjin viruses only occasionally had antigen outside the nervous system. We conclude that the mouse model could be a useful tool for the assessment of pathotype for WNVs.

Development and preliminary validation of a MERS-CoV ELISA for serological testing of camels and alpacas.

This study describes the development and preliminary validation of a new serological assay using MERS-CoV S1 protein in an indirect enzyme-linked immunosorbent assay (ELISA) format. This assay has the advantage of being able to test MERS-CoV serum samples in a PC2 laboratory without the need for a high-level biocontainment laboratory (PC3 or PC4), which requires highly trained and skilled staff and a high level of resources and equipment. Furthermore, this MERS-CoV S1 ELISA enables a larger number of samples to be tested quickly, with results obtained in approximately five hours. The MERS-CoV S1 ELISA demonstrated high analytical specificity, with no cross-reactivity observed in serum of animals infected with other viruses, including different coronaviruses. We tested 166 positive and 40 negative camel serum samples and have estimated the diagnostic sensitivity (DSe) to be 99.4% (95% CI: 96.7 - 100.0%) and diagnostic specificity (DSp) to be 100% (95% CI: 97.2%-100.0%) relative to the assigned serology results (ppNT and VNT) using a S/P ratio cut-off value of >0.58. The findings of this study showed that our MERS-CoV S1 ELISA was more sensitive than the commercial EUROIMMUN ELISA (Se 99.4% vs 84.9%) and comparable to the ppNT assay, and therefore could be used as a diagnostic aid in countries in the Middle East where MERS-CoV is endemic in dromedary camels. The assay reagents and protocol were easily adapted and transferred from an Australian laboratory to a laboratory in the University of Hong Kong. Thus, the results described here show that the MERS-CoV S1 ELISA represents a cheap, rapid, robust, and reliable assay to support surveillance of MERS-CoV in camels in endemic regions.

Comprehensive mapping of West Nile virus (WNV)- and Japanese encephalitis virus serocomplex-specific linear B-cell epitopes from WNV non-structural protein 1.

West Nile virus (WNV) non-structural protein 1 (NS1) elicits protective immune responses during infection of animals. WNV NS1-specific antibody responses can provide the basis for serological diagnostic reagents, so the antigenic sites in NS1 that are targeted by host immune responses need to be identified and the conservation of these sites among the Japanese encephalitis virus (JEV) serocomplex members also needs to be defined. The present study describes the mapping of linear B-cell epitopes in WNV NS1. We screened eight NS1-specific mAbs and antisera (polyclonal antibodies; pAbs) from mice immunized with recombinant NS1 for reactivity against 35 partially overlapping peptides covering the entire WNV NS1. The screen using mAbs identified four WNV-specific (including Kunjin virus) epitopes, located at aa 21-36, 101-116, 191-206 and 261-276 in WNV NS1. However, using pAbs, only three WNV-specific epitopes were identified, located at positions 101-116, 191-206 and 231-246. Two of these epitopes (aa 21-36 and 261-276) had different reactivity with mAbs and pAbs. The knowledge and reagents generated in this study have potential applications in differential diagnostics and epitope-based marker vaccine development for WNV and viruses of the JEV serocomplex.

Identification of two linear B-cell epitopes from West Nile virus NS1 by screening a phage-displayed random peptide library.

BACKGROUND: The West Nile virus (WNV) nonstructural protein 1 (NS1) is an important antigenic protein that elicits protective antibody responses in animals and can be used for the serological diagnosis of WNV infection. Although previous work has demonstrated the vital role of WNV NS1-specific antibody responses, the specific epitopes in the NS1 have not been identified. RESULTS: The present study describes the identification of two linear B-cell epitopes in WNV NS1 through screening a phage-displayed random 12-mer peptide library with two monoclonal antibodies (mAbs) 3C7 and 4D1 that directed against the NS1. The mAbs 3C7 and 4D1 recognized phages displaying peptides with the consensus motifs LTATTEK and VVDGPETKEC, respectively. Exact sequences of both motifs were found in the NS1 ((895)LTATTEK(901) and (925)VVDGPETKEC(934)). Further identification of the displayed B cell epitopes were conducted using a set of truncated peptides expressed as MBP fusion proteins. The data indicated that (896)TATTEK(901) and (925)VVDGPETKEC(934) are minimal determinants of the linear B cell epitopes recognized by the mAbs 3C7 and 4D1, respectively. Antibodies present in the serum of WNV-positive horses recognized the minimal linear epitopes in Western blot analysis, indicating that the two peptides are antigenic in horses during infection. Furthermore, we found that the epitope recognized by 3C7 is conserved only among WNV strains, whereas the epitope recognized by 4D1 is a common motif shared among WNV and other members of Japanese encephalitis virus (JEV) serocomplex. CONCLUSIONS: We identified TATTEK and VVDGPETKEC as NS1-specific linear B-cell epitopes recognized by the mAbs 3C7 and 4D1, respectively. The knowledge and reagents generated in this study may have potential applications in differential diagnosis and the development of epitope-based marker vaccines against WNV and other viruses of JEV serocomplex.

Identification of a conserved JEV serocomplex B-cell epitope by screening a phage-display peptide library with a mAb generated against West Nile virus capsid protein.

BACKGROUND: The West Nile virus (WNV) capsid (C) protein is one of the three viral structural proteins, encapsidates the viral RNA to form the nucleocapsid, and is necessary for nuclear and nucleolar localization. The antigenic sites on C protein that are targeted by humoral immune responses have not been studied thoroughly, and well-defined B-cell epitopes on the WNV C protein have not been reported. RESULTS: In this study, we generated a WNV C protein-specific monoclonal antibody (mAb) and defined the linear epitope recognized by the mAb by screening a 12-mer peptide library using phage-display technology. The mAb, designated as 6D3, recognized the phages displaying a consensus motif consisting of the amino acid sequence KKPGGPG, which is identical to an amino acid sequence present in WNV C protein. Further fine mapping was conducted using truncated peptides expressed as MBP-fusion proteins. We found that the KKPGGPG motif is the minimal determinant of the linear epitope recognized by the mAb 6D3. Western blot (WB) analysis demonstrated that the KKPGGPG epitope could be recognized by antibodies contained in WNV- and Japanese encephalitis virus (JEV)-positive equine serum, but was not recognized by Dengue virus 1-4 (DENV1-4)-positive mice serum. Furthermore, we found that the epitope recognized by 6D3 is highly conserved among the JEV serocomplex of the Family Flaviviridae. CONCLUSION: The KKPGGPG epitope is a JEV serocomplex-specific linear B-cell epitope recognized by the 6D3 mAb generated in this study. The 6D3 mAb may serve as a novel reagent in development of diagnostic tests for JEV serocomplex infection. Further, the identification of the B-cell epitope that is highly conserved among the JEV serocomplex may support the rationale design of vaccines against viruses of the JEV serocomplex.

Development and validation of an IgM antibody capture ELISA for early detection of Hendra virus.

Zoonotic transmission of Hendra virus (HeV) from primary hosts (pteropid bats) to horses, and, occasionally, onward adventitious spread to humans, is associated with high mortality rates in both affected secondary species. The introduction of an effective recombinant G protein vaccine for use in horses has been a major advance for the suppression of disease risk. However, equine HeV vaccination induces neutralising antibody that is indistinguishable from a post infection immune response when using most first line serology assays (eg. VNT and some ELISAs). We have constructed and evaluated an IgM antibody capture (MAC) ELISA which employs yeast expressed HeV nucleoprotein (N). All other serology tests use the G protein which does not detect early infection and is present in the current Hendra virus vaccine and may cause ambiguity in interpretation of results. Thus, this is the first test developed using a N protein which can successfully detect a recent (primarily within the last four weeks) infection of horses with HeV and is not affected by vaccination induced antibody. Testing a limited panel (21 samples) of post infection sera, a normal serum panel (288 samples) and a post vaccination panel (163 samples), we have estimated DSe to be 100 % (95 % CI, 83.9-100.0 %) and DSp to be 98.4 % (95 % CI, 96.8-99.4 %) relative to assigned serology results (VNT, ELISA and Luminex) for the test panels. The HeV IgM MAC ELISA is intended to supplement other molecular and serology test results, with selective use, and is the only serology test which can provide an indication for recent infection which is otherwise not available.

Establishment of an immunofluorescence assay to detect IgM antibodies to Nipah virus using HeLa cells expressing recombinant nucleoprotein.

A novel indirect fluorescent antibody test (IFAT) for detection of IgM against Nipah virus (NiV) was developed using HeLa 229 cells expressing recombinant NiV nucleocapsid protein (NiV-N). The NiV IFAT was evaluated using three panels of sera: a) experimentally produced sera from NiV-N-immunized/pre-immunized macaques, b) post-infection human sera associated with a Nipah disease outbreak in the Philippines in 2014, and c) human sera from a non-exposed Malaysian population. Immunized macaque sera showed a characteristic granular staining pattern of the NiV-N expressed antigen in HeLa 229 cells, which was readily distinguished from negative-binding results of the pre-immunized macaque sera. The IgM antibody titers in sequential serum samples (n = 7) obtained from three Nipah patients correlated well with previously published results using conventional IgM capture ELISA and SNT serology. The 90 human serum samples from unexposed persons were unreactive by IFAT. The IFAT utilizing NiV-N-expressing HeLa 229 cells to detect IgM antibody in an early stage of NiV infection is an effective approach, which could be utilized readily in local laboratories to complement other capabilities in NiV-affected countries.

Identification and genomic characterization of the first isolate of bluetongue virus serotype 5 detected in Australia.

Bluetongue virus (BTV), transmitted by midges (Culicoides sp), is distributed worldwide and causes disease in ruminants. In particular, BT can be a debilitating disease in sheep causing serious trade and socio-economic consequences at both local and global levels. Across Australia, a sentinel cattle herd surveillance program monitors the BTV activity. Prior to 2014, BTV-1, -2, -3, -7, -9, -15, -16, -20, -21 and -23 had been isolated in Australia, but no bluetongue disease has occurred in a commercial Australian flock. We routinely use a combination of serology, virus isolation, RT-PCR and next generation and conventional nucleotide sequencing technologies to detect and phylogenetically characterize incursions of novel BTV strains into Australia. Screening of Northern Territory virus isolates in 2015 revealed BTV-5, a serotype new to Australia. We derived the complete genome of this isolate and determined its phylogenetic relationship with exotic BTV-5 isolates. Gene segments 2, 6, 7 and 10 exhibited a close relationship with the South African prototype isolate RSArrrr/5. This was the first Australian isolation of a Western topotype of segment 10. Serological surveillance data highlighted the antigenic cross-reactivity between BTV-5 and BTV-9. Phylogenetic investigation of segments 2 and 6 of these serotypes confirmed their unconventional relationships within the BTV serogroup. Our results further highlighted a need for a revision of the current serologically based system for BTV strain differentiation and importantly, implied a potential for genome segments of pathogenic Western BTV strains to rapidly enter Southeast Asia. This emphasized a need for continued high-level surveillance of vectors and viruses at strategic locations in the north of Australia The expansion of routine characterization and classification of BTV to a whole genome approach is recommended, to better monitor the presence and level of establishment of novel Western topotype segments within the Australian episystem.

Assessment of a Rabies Virus Rapid Diagnostic Test for the Detection of Australian Bat Lyssavirus.

Australian bat lyssavirus (ABLV) is closely related to the classical rabies virus and has been associated with three human fatalities and two equine fatalities in Australia. ABLV infection in humans causes encephalomyelitis, resulting in fatal disease, but has no effective therapy. The virus is maintained in enzootic circulation within fruit bats (Pteropid spp.) and at least one insectivorous bat variety (Saccolaimus flaviventris). Most frequently, laboratory testing is conducted on pteropodid bat brains, either following a potential human exposure through bites, scratches and other direct contacts with bats, or as opportunistic assessment of sick or dead bats. The level of medical intervention and post-exposure prophylaxis is largely determined on laboratory testing for antigen/virus as the demonstrable infection status of the in-contact bat. This study evaluates the comparative diagnostic performance of a lateral flow test, Anigen Rabies Ag detection rapid test (RDT), in pteropodid variant of ABLV-infected bat brain tissues. The RDT demonstrated 100% agreement with the reference standard fluorescent antibody test on 43 clinical samples suggesting a potential application in rapid diagnosis of pteropodid variant of ABLV infection. A weighted Kappa value of 0.95 confirmed a high level of agreement between both tests.

A network approach for provisional assay recognition of a Hendra virus antibody ELISA: test validation with low sample numbers from infected horses.

Obtaining statistically sound numbers of sera from Hendra virus (HeV)-infected horses is problematic because affected individuals usually die or are euthanized before developing a serum antibody response. As a consequence, test validation becomes a challenge. Our approach is an extension of OIE principles for provisional recognition and included 7 validation panels tested across multiple laboratories that provided estimates for test performance characteristics. At a 0.4 S/P cutoff, 16 of 19 sera from HeV-infected horses gave positive results in the HeV soluble G, indirect ELISA (HeVsG iELISA; DSe 84.2% [95% CI: 60.4-96.6%]); 463 of 477 non-infected horse sera tested negative (DSp 97.1% [95% CI: 95.1-98.4%]). The HeVsG iELISA eliminated almost all false-positive results from the previously used HeV iELISA, with marginally decreased relative sensitivity. Assay robustness was evaluated in inter-laboratory and proficiency testing panels. The HeVsG iELISA is considered to be fit for purpose for serosurveillance and international movement of horses when virus neutralization is used for follow-up testing of positive or inconclusive serum samples.

Susceptibility of domestic dogs and cats to Australian bat lyssavirus (ABLV).

The susceptibility of cats and dogs to Australian bat lyssavirus (ABLV; genotype VII) was investigated by intramuscular (IM) inoculation of 10(3.7)-10(5) 50% tissue culture infective doses (TCID(50)) of virus followed by observation of experimental animals for up to 3 months post-inoculation (pi). Each experiment also included positive and negative controls, animals inoculated with a bat variant of rabies virus (Eptesicus I, genotype I), or a 10% suspension of uninfected mouse brain, respectively. Each of the ABLV-inoculated cats showed occasional abnormal clinical signs, but none died. Necropsies performed at 3 months pi revealed no lesions, and no viral antigen, in the central nervous system of any cat. ABLV could not be recovered from any cats. However, rabies virus-neutralizing antibodies were detected between 4 and 14 weeks pi in the sera of all three ABLV-inoculated cats. At 2-3 weeks pi, three of the five ABLV-inoculated dogs showed very mild abnormal clinical signs that persisted for 1-2 days, after which the dogs recovered. At 3 months pi, when all dogs were necropsied, neither lesions nor ABLV antigen were detected in, and virus was not isolated from, any dog. No ABLV RNA was detected by polymerase chain reaction (PCR) in clinical or necropsy samples from the three ABLV-affected dogs. However, all ABLV-inoculated dogs seroconverted by 2 weeks pi, and serum antibody titres were higher than those observed in cats. CSF, collected at 3 months pi, was positive for rabies virus-neutralizing antibody in two ABLV-inoculated dogs.

Serological evidence for Japanese encephalitis and West Nile viruses in domestic animals of Nepal.

A regional survey was conducted in Nepal for antibody to Japanese encephalitis virus (JEV) in domestic animals. Sera from pigs, and limited numbers of ducks and horses were collected from 16 districts in 2002-2003 and subjected to three serological tests. Of 270 porcine sera tested by C-ELISA, 55% were found positive for the presence of antibodies against Japanese encephalitis virus. Additional testing for IgM antibody to JEV revealed less than 2% of C-ELISA positive sera had evidence of recent JEV infection. Plaque reduction neutralisation tests (PRNT) using JEV, Murray Valley encephalitis (MVEV) and Kunjin (KUNV) viruses implicated JEV as the flavivirus associated with the observed antibody response in most sero-positive pigs. However, eight porcine sera with predominant neutralising antibody for KUNV (an Australasian subtype of West Nile Virus) provided evidence for the circulation of West Nile virus in Nepal.

Characterisation of an Australian bat lyssavirus variant isolated from an insectivorous bat.

In 1996 a variant lyssavirus was isolated from an insectivorous bat (yellow bellied, sheath tail bat-Saccolaimus flaviventris) in Australia. The nucleocapsid protein (N), matrix protein (M), phosphoprotein (P), glycoprotein (G) and polymerase (L) genes of the Australian bat lyssavirus (ABL) insectivorous isolate were compared with that previously described from a frugivorous bat (Pteropus sp.), and showed sequence divergence at both the nucleotide and amino acid sequence level of 20% and 4-12%, respectively. Comparison of deduced protein sequences of ABL isolates from Pteropus and insectivorous bats, showed that viral isolates were homologous and varied by only a few percent. However, these viruses separated into two distinct clades; those isolated from Pteropus or those from Saccolaimus flaviventris bats, when comparisons were made at the nucleotide level. Nucleoprotein sequence comparisons also showed insectivorous isolates to be of the same putative genotype (genotype 7) as that isolated from frugivorous bats. Immediately after the isolation of ABL from an insectivorous bat, the first human case of ABL infection was identified. PCR and sequence analysis done on cerebrospinal fluid, brain and virus isolated from fresh brain tissue of this human case, was consistent with this infection originating from an insectivorous bat. Monoclonal antibody profiling studies of the virus isolated from the human brain tissues supported this conclusion. Sequence comparisons done on the nucleocapsid (N) gene of insectivorous or frugivorous bats showed no geographic associations between isolates but did delineate between the variants of ABL in Australia.

The pathobiology of two Indonesian H5N1 avian influenza viruses representing different clade 2.1 sublineages in chickens and ducks.

To determine the pathobiology of Indonesian H5N1 highly pathogenic avian influenza, two viruses representing clades 2.1.1 and 2.1.3 were inoculated into broiler chickens and Pekin ducks via the eyes, nostrils and oropharynx. In chickens, both viruses produced fulminant disease; tissue tropism was broad but predominantly endothelial and viral loads in tissues were high. Except for one case of meningoencephalitis, the infection in ducks was sub-clinical, leading only to seroconversion. In these ducks, virus and viral antigen occurred in lower amounts, mainly in the respiratory tract (airsac and sinuses), prior to day 7 after inoculation. During clinical disease, chickens shed high virus titres orally and cloacally. Ducks intermittently shed low virus titres from the oral route for up to 8 days post-inoculation. We discuss the significance of the data for understanding the pathogenesis and pathobiology of Indonesian H5N1 in chickens and ducks.

Serologic study of pig-associated viral zoonoses in Laos.

We conducted a serologic survey of four high-priority pig-associated viral zoonoses, Japanese encephalitis virus (JEV), hepatitis E virus (HEV), Nipah virus (NiV), and swine influenza virus (SIV), in Laos. We collected blood from pigs at slaughter during May 2008-January 2009 in four northern provinces. Japanese encephalitis virus hemagglutination inhibition seroprevalence was 74.7% (95% confidence interval [CI] = 71.5-77.9%), JEV IgM seroprevalence was 2.3% (95% CI = 1.2-3.2%), and HEV seroprevalence was 21.1% (95% CI = 18.1-24.0%). Antibodies to SIV were detected in 1.8% (95% CI = 0.8-2.8%) of pigs by screening enzyme-linked immunosorbent assay, and only subtype H3N2 was detected by hemagglutination inhibition in two animals with an inconclusive enzyme-linked immunosorbent assay result. No NiV antibody-positive pigs were detected. Our evidence indicates that peak JEV and HEV transmission coincides with the start of the monsoonal wet season and poses the greatest risk for human infection.

Comprehensive mapping of common immunodominant epitopes in the West Nile virus nonstructural protein 1 recognized by avian antibody responses.

West Nile virus (WNV) is a mosquito-borne flavivirus that primarily infects birds but occasionally infects humans and horses. Certain species of birds, including crows, house sparrows, geese, blue jays and ravens, are considered highly susceptible hosts to WNV. The nonstructural protein 1 (NS1) of WNV can elicit protective immune responses, including NS1-reactive antibodies, during infection of animals. The antigenicity of NS1 suggests that NS1-reactive antibodies could provide a basis for serological diagnostic reagents. To further define serological reagents for diagnostic use, the antigenic sites in NS1 that are targeted by host immune responses need to be identified and the potential diagnostic value of individual antigenic sites also needs to be defined. The present study describes comprehensive mapping of common immunodominant linear B-cell epitopes in the WNV NS1 using avian WNV NS1 antisera. We screened antisera from chickens, ducks and geese immunized with purified NS1 for reactivity against 35 partially overlapping peptides covering the entire WNV NS1. This study identified twelve, nine and six peptide epitopes recognized by chicken, duck and goose antibody responses, respectively. Three epitopes (NS1-3, 14 and 24) were recognized by antibodies elicited by immunization in all three avian species tested. We also found that NS1-3 and 24 were WNV-specific epitopes, whereas the NS1-14 epitope was conserved among the Japanese encephalitis virus (JEV) serocomplex viruses based on the reactivity of avian WNV NS1 antisera against polypeptides derived from the NS1 sequences of viruses of the JEV serocomplex. Further analysis showed that the three common polypeptide epitopes were not recognized by antibodies in Avian Influenza Virus (AIV), Newcastle Disease Virus (NDV), Duck Plague Virus (DPV) and Goose Parvovirus (GPV) antisera. The knowledge and reagents generated in this study have potential applications in differential diagnostic approaches and subunit vaccines development for WNV and other viruses of the JEV serocomplex.

Full genome sequencing and genetic characterization of Eubenangee viruses identify Pata virus as a distinct species within the genus Orbivirus.

Eubenangee virus has previously been identified as the cause of Tammar sudden death syndrome (TSDS). Eubenangee virus (EUBV), Tilligery virus (TILV), Pata virus (PATAV) and Ngoupe virus (NGOV) are currently all classified within the Eubenangee virus species of the genus Orbivirus, family Reoviridae. Full genome sequencing confirmed that EUBV and TILV (both of which are from Australia) show high levels of aa sequence identity (>92%) in the conserved polymerase VP1(Pol), sub-core VP3(T2) and outer core VP7(T13) proteins, and are therefore appropriately classified within the same virus species. However, they show much lower amino acid (aa) identity levels in their larger outer-capsid protein VP2 (<53%), consistent with membership of two different serotypes - EUBV-1 and EUBV-2 (respectively). In contrast PATAV showed significantly lower levels of aa sequence identity with either EUBV or TILV (with <71% in VP1(Pol) and VP3(T2), and <57% aa identity in VP7(T13)) consistent with membership of a distinct virus species. A proposal has therefore been sent to the Reoviridae Study Group of ICTV to recognise 'Pata virus' as a new Orbivirus species, with the PATAV isolate as serotype 1 (PATAV-1). Amongst the other orbiviruses, PATAV shows closest relationships to Epizootic Haemorrhagic Disease virus (EHDV), with 80.7%, 72.4% and 66.9% aa identity in VP3(T2), VP1(Pol), and VP7(T13) respectively. Although Ngoupe virus was not available for these studies, like PATAV it was isolated in Central Africa, and therefore seems likely to also belong to the new species, possibly as a distinct 'type'. The data presented will facilitate diagnostic assay design and the identification of additional isolates of these viruses.

Broome virus, a new fusogenic Orthoreovirus species isolated from an Australian fruit bat.

This report describes the discovery and characterization of a new fusogenic orthoreovirus, Broome virus (BroV), isolated from a little red flying-fox (Pteropus scapulatus). The BroV genome consists of 10 dsRNA segments, each having a 3' terminal pentanucleotide sequence conserved amongst all members of the genus Orthoreovirus, and a unique 5' terminal pentanucleotide sequence. The smallest genome segment is bicistronic and encodes two small nonstructural proteins, one of which is a novel fusion associated small transmembrane (FAST) protein responsible for syncytium formation, but no cell attachment protein. The low amino acid sequence identity between BroV proteins and those of other orthoreoviruses (13-50%), combined with phylogenetic analyses of structural and nonstructural proteins provide evidence to support the classification of BroV in a new sixth species group within the genus Orthoreovirus.

Peruvian horse sickness virus and Yunnan orbivirus, isolated from vertebrates and mosquitoes in Peru and Australia.

During 1997, two new viruses were isolated from outbreaks of disease that occurred in horses, donkeys, cattle and sheep in Peru. Genome characterization showed that the virus isolated from horses (with neurological disorders, 78% fatality) belongs to a new species the Peruvian horse sickness virus (PHSV), within the genus Orbivirus, family Reoviridae. This represents the first isolation of PHSV, which was subsequently also isolated during 1999, from diseased horses in the Northern Territory of Australia (Elsey virus, ELSV). Serological and molecular studies showed that PHSV and ELSV are very similar in the serotype-determining protein (99%, same serotype). The second virus (Rioja virus, RIOV) was associated with neurological signs in donkeys, cattle, sheep and dogs and was shown to be a member of the species Yunnan orbivirus (YUOV). RIOV and YUOV are also almost identical (97% amino acid identity) in the serotype-determining protein. YUOV was originally isolated from mosquitoes in China.