Modeling Abundance of Culicoides stellifer, a Candidate Orbivirus Vector, Indicates Nonrandom Hemorrhagic Disease Risk for White-Tailed Deer (Odocoileus virginianus).

(1) Background: Hemorrhagic diseases in white-tailed deer (Odocoileus virginianus) are caused by orbiviruses and have significant economic impact on the deer ranching industry in the United States. Culicoides stellifer is a suspected vector of epizootic hemorrhagic disease virus (EHDV), with recent field evidence from Florida, but its natural history is poorly understood. Studying the distribution and abundance of C. stellifer across the landscape can inform our knowledge of how virus transmission can occur locally. We may then target vector management strategies in areas where viral transmission can occur. (2) Methods: Here, we used an occupancy modeling approach to estimate abundance of adult C. stellifer females at various physiological states to determine habitat preferences. We then mapped midge abundance during the orbiviral disease transmission period (May-October) in Florida. (3) Results: We found that overall, midge abundance was positively associated with sites in closer proximity to large-animal feeders. Additionally, midges generally preferred mixed bottomland hardwood and agricultural/sand/water habitats. Female C. stellifer with different physiological states preferred different habitats. (4) Conclusions: The differences in habitat preferences between midges across states indicate that disease risk for deer is heterogeneous across this landscape. This can inform how effective vector management strategies should be implemented.

Bluetongue disease in sheep: a review / O vírus da língua azul em ovinos: uma revisão

The present review aims to show the main aspects related to bluetongue virus (BTV) infection in sheep. The bluetongue (BT) is a viral, infectious, and non-contagious disease caused by a virus (BTV) of the Orbivirus genus, transmited by a hematophagous vector of the Culicoides genus, to domestic and wild ruminants, mainly to sheep, the most susceptible species. It is caused by the association of endemic with climate conditions, with high temperatures and humidity. Economic loss is directly linked to death, abortion, weight loss, loss of milk, and meat production, and, indirectly, to the restriction on the export of animals and their by-products. The study concludes that the BTV is worldwidely spread, and probably persists due to the warm and humid climate that leads to the proliferation of Culicoides sp., being necessary to adopt measures that reduce the risk factors associated to the BTV infection.(AU)

A presente revisão objetivou apresentar os principais aspectos relacionados à infecção causada pelo vírus da língua azul em ovinos. A língua azul é uma doença viral, infecciosa e não contagiosa, causada por um vírus (BTV) do gênero Orbivírus, transmitida por meio de vetores hematófagos do gênero Culicoides a ruminantes domésticos e selvagens, principalmente aos ovinos, a espécie mais susceptível. A infecção ocorre de forma endêmica, associada a condições climáticas com elevada temperatura e umidade. As perdas econômicas estão ligadas diretamente à morte, ao abortamento, à perda de peso, à perda na produção de leite e carne, e, indiretamente, devido à restrição na exportação de animais e seus subprodutos. O estudo conclui que a língua azul está disseminada mundialmente e persiste, provavelmente, devido ao clima quente e úmido que propicia a proliferação de Culicoides sp., sendo necessário adotar medidas que diminuam os fatores de risco associados à infecção pelo vírus.(AU)

Teratogenic bluetongue and related orbivirus infections in pregnant ruminant livestock: timing and pathogen genetics are critical.

Congenital infections of domestic animals with viruses in several families, including Bunyaviridae, Flaviridae, Parvoviridae, and Reoviridae, are the cause of naturally occurring teratogenic central nervous system and/or musculoskeletal defects (arthrogryposis) in domestic animals. Congenital infections of ruminant livestock with bluetongue virus (BTV) and some related members of the genus Orbivirus (family Reoviridae) have clearly shown the critical role of gestational age at infection in determining outcome. Specifically, fetuses infected prior to mid-gestation that survive congenital BTV infection are born with cavitating central nervous system defects that range from severe hydranencephaly to cerebral cysts (porencephaly). Generally, the younger the fetus (in terms of gestational age) at infection, the more severe the teratogenic lesion at birth. Age-dependent virus infection and destruction of neuronal and/or glial cell precursors that populate the developing central nervous system are responsible for these naturally occurring virus-induced congenital defects of animals, thus lesions are most severe when progenitor cells are infected prior to their normal migration during embryogenesis. Whereas congenital infection is characteristic of certain BTV strains, notably live-attenuated (modified-live) vaccine viruses that have been passaged in embryonating eggs, transplacental transmission is not characteristic of many field strains of the virus and much remains to be determined regarding the genetic determinants of transplacental transmission of individual virus strains.

Association of vectors and environmental conditions during the emergence of Peruvian horse sickness orbivirus and Yunnan orbivirus in northern Peru.

Since 1983, cases of diseased donkeys and horses with symptoms similar to those produced by alphaviruses were identified in two departments in northern Peru; however serological testing ruled out the presence of those viruses and attempts to isolate an agent were also unproductive. In 1997, also in northern Peru, two new orbiviruses were discovered, each recognized as a causative agent of neurological diseases in livestock and domestic animals and, at the same time, mosquitoes were found to be infected with these viruses. Peruvian horse sickness virus (PHSV) was isolated from pools of culicid mosquitoes, Aedes serratus and Psorophora ferox, and Yunnan virus (YUOV) was isolated from Aedes scapularis in the subtropical jungle (upper jungle) located on the slope between the east side of the Andes and the Amazonian basin in the Department of San Martín. Both viruses later were recovered from mosquitoes collected above the slope between the west side of the Andes and the coast (Department of Piura) in humid subtropical areas associated with the Piura River basin. In this region, PHSV was isolated from Anopheles albimanus and YUOV was isolated from Ae. scapularis. We discuss the ecology of vector mosquitoes during the outbreaks in the areas where these mosquitoes were found.

A Review of Knowledge Gaps and Tools for Orbivirus Research.

Although recognized as causing emerging and re-emerging disease outbreaks worldwide since the late 1800 s, there has been growing interest in the United States and Europe in recent years in orbiviruses, their insect vectors, and the diseases they cause in domestic livestock and wildlife. This is due, in part, to the emergence of bluetongue (BT) in northern Europe in 2006-2007 resulting in a devastating outbreak, as well as severe BT outbreaks in sheep and epizootic hemorrhagic disease (EHD) outbreaks in deer and cattle in the United States. Of notable concern is the isolation of as many as 10 new BT virus (BTV) serotypes in the United States since 1999 and their associated unknowns, such as route of introduction, virulence to mammals, and indigenous competent vectors. This review, based on a gap analysis workshop composed of international experts on orbiviruses conducted in 2013, gives a global perspective of current basic virological understanding of orbiviruses, with particular attention to BTV and the closely related epizootic hemorrhagic disease virus (EHDV), and identifies a multitude of basic virology research gaps, critical for predicting and preventing outbreaks.

[Molecular-genetic characterization of the Okhotskiy virus (OKHV) and Aniva virus (ANIV) (Orbivirus, Reoviridae) isolated from the ticks Ixodes (Ceratixodes) uriae White, 1852 in high latitudes of the Northern Eurasia].

Molecular-genetic characteristics of the Okhotskiy virus (OKHV) and Aniva virus (ANIV) were studied (ID GenBank KF981623-32). These viruses are distributed over the shelf and Island areas in the high latitudes in the Okhotsk, Bering, and Barents seas and linked with nesting colonies of Alcidae seabirds and their obligatory parasites, the Ixodes uriae (Ixodidae) ticks. OKHV and ANIV are observed to be independent species within the limits of the Great Island virus (GIV) group of the Orbivirus genus of the Reoviridae family. The majority of the genes of OKHV and ANIV have high homology (VP1 - 96%, T2 - 99%, VP7 (T13) - 98%, NS1 - 94%, NS2 - 98%, NS3 - 72%, VP6 - 93%). Nevertheless, the envelope proteins containing the main specific antigenic determinants (VP2 and VP5) of OKHV and ANIV are sufficiently different (62% and 68% homology for amino acid sequences, respectively).

Frequência de anticorpos contra o vírus da língua azul em ovinos do estado do Ceará, Brasil / Antibodies against the bluetongue virus in sheep flocks of Ceará state, Brazil

O objetivo deste trabalho foi avaliar a ocorrência de ovinos soropositivos para o vírus da línguaazul (VLA) no Estado do Ceará, Brasil, e analisar as proteínas imunogênicas das cepas virais circulantes nesses rebanhos. O teste de imunodifusão em gel de agarose (IDGA) foi utilizado para pesquisar 271 amostras de soro oriundas de 16 rebanhos. Os resultados demonstraram que 27,3% (74/271) das amostras analisadas apresentaram anticorpos contra o agente e 68,8% (11/16) das propriedades tiveram animais positivos. O immunoblotting (IB) foi utilizado para analisar as proteínas imunogênicas do VLA a partir dos soros de animais positivos no IDGA. Os soros demonstraram forte reação contra a proteína viral VP2. Para o VLA, das sete proteínas estruturais, a VP2 é a principal a estimular a resposta imune protetora. Concluiu-se que a soropositividade para a língua azul (LA) nos rebanhos ovinos estudados no Ceará é alta, apesar dos animais não apresentarem sinais clínicos, indicativo de que o vírus ocorra de forma endêmica. Além disso, a resistência à doença apresentada pelos animais pode estar relacionada com a forte reação imunológica desses à proteína VP2. Sendo assim, outros estudos são necessários para melhor esclarecer a situação epidemiológica da LA no país, através da identificação dos vetores e sorotipos virais circulantes nas diferentes regiões.

Antibodies against the bluetongue virus in sheep flocks of Ceará state, Brazil. The objective of this work was to verify the occurrence of sheep serologically positive for bluetongue virus (BTV) in the state of Ceará, Brazil, and analyze immunogenic proteins of circulating viral strains in these flocks. The agar gel immunodifusion test (AGID) was used to examine 271 serum samples from 16 herds. The results demonstrated that 27.3% (74/271) ofthe analyzed samples presented antibodies for the agent, and that 68.8% (11/16) of the propertiespresented positive animals. Immunoblotting (IB) was used to analyze the immunogenicproteins of BTV derived from AGID positive sera. Sera showed strong reaction against viral protein VP2. Of the seven BTV structural proteins, VP2 is the major protein to elicit protective immuneresponses. It was concluded that bluetongue (BT) seropositivity in sheep flocks studied in Ceará is high, despite that the animal's do not show clinical signs, indicating that it occurs in an endemic form. The animals’ resistance to the disease may be related to the strong immune response to the protein VP2. Therefore, further studies are needed to better clarify the epidemiological situation of BT in Brazilian sheep flocks, through the identification of viral vectors and serotypes circulating in different regions.

Putative risk factors for infections with Toggenburg Orbivirus in goat herds in Southern Switzerland (Canton of Ticino).

Toggenburg Orbivirus (TOV), only detected in goats, has been described as a member of the Bluetongue virus (BTV) serogroup. The transmission pathway of the virus seems different from other Bluetongue viruses (BTVs). The objective of this study was to explore risk factors, especially the influence of alpine pasture and the presence of other livestock species, for the presence of TOV infected goats on farms. Between February 2008 and September 2009, blood samples were collected and analyzed for TOV and hereupon a total of 60 goat farm owners (37 TOV-positive and 23 TOV-negative holdings) were interviewed. Additionally, goatlings were tested for TOV by rRT-PCR prior and after alpine pasture in 2009. These goatlings were positive for TOV only after the alpine pasture. The final logistic regression model included: "exposure to goats from other farms" (OR=10.12, p=0.007), "exposure of the goats to red deer" (OR=4.79, p=0.04) and "exposure to sheep from other farms" (OR=0.05, p=0.002). These variables do not implicitly include direct contact, and the findings are only vaguely indicative for a contact-driven transmission. Furthermore, it is likely that they are only associated with, and thus indicative for, an unknown risk factor associated with alpine pasture not measured in the study. The results of this screening study do not indicate iatrogenic transmission pathways as a main transmission mode and stimulate the formulation of hypotheses on the origin, the transmission pathway and other host species for TOV. Especially, the involvement of an insect vector in transmission on alpine pasture and the relevance of vertical transmission are to be clarified.

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.

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.

Characterization of Minaçu virus (Reoviridae: Orbivirus) and pathological changes in experimentally infected newborn mice.

Minaçu virus was isolated from Ochlerotatus scapularis (Diptera: Culicidae) in Minaçu, Goiás State, Brazil, in 1996. In attempting characterization of virus serological (hemagluttination inhibition, HI; indirect immunofluorescence assay, IFA), physicochemical [test for deoxycholate acid (DCA) sensitivity; polyacrylamide gel electrophoresis (PAGE)] tests and ultrastructural studies were made. Virus was also assayed in suckling mice after intracerebral inoculation of 0.02 ml and in VERO and C6/36 cells with 0.1 ml of viral suspension containing 10(5) LD50/ml. Inoculated and control systems were observed daily. Every 24 h, one control and two inoculated animals were killed for tissue testing, including histopathological changes by haematoxylin and eosin (HE)-stained sections, which were semi-quantified. Research into viral antigen in the tissues of mice [central nervous system (CNS), liver, heart, lungs, spleen and kidneys] was carried out by the immunohistochemical technique using the peroxidase system. The virus only replicated in VERO cells, with antigen positive by IFA. Positive complement fixation tests were only obtained using antiserum of Minaçu virus. Minaçu virus is DCA resistant; haemagglutinating activity was negative. By electronic microscopy non-enveloped virus particles were 75 nm in diameter. PAGE analysis showed Minaçu virus genome profile with 10 RNA segments. Infected, non-killed animals died 7 days after inoculation. Tissue lesions were observed in all organs, except the lungs. Intense lesions were observed in the CNS and the heart, where neurone and cardiocyte necroses, respectively, were noted. The liver, spleen and kidneys had moderate tissue changes. Viral antigens were more abundant in the CNS and the heart, and absent in the lungs. In conclusion, Minaçu virus belongs to the family Reoviridae, genus Orbivirus.

[Arboviruses and epizootic viruses]. / Les arbovirus et les virus épizootiques.

Some viral diseases are transmitted to human by arthropods (arboviroses), or by animals (zoonoses). Among more than 500 arboviruses and epizootic viruses that are classified into seven families, only a few are responsible for zoonoses or cause severe human diseases. Infected patients may show an acute disease associated with different symptoms, ranging from high fever to encephalitis, pulmonary distress, and haemorrhages. Some diseases show one or more of these symptoms and the factors responsible for severe outcomes, either linked to the virus, or to the host, or to the vector, remain poorly understood. Arboviroses and zoonoses are emerging or re-emerging diseases that need a multidisciplinary effort to control the propagation of the infectious agent and the pathogenesis in infected patients. Some viruses could be used for bioterrorism attacks. In virology, studies on the interactions of the viruses with their vectors and vertebrate hosts and on the pathophysiology of the infections will allow a better prevention of these diseases.

Transmission patterns of African horse sickness and equine encephalosis viruses in South African donkeys.

African horse sickness (AHS) and equine encephalosis (EE) viruses are endemic to southern Africa. AHS virus causes severe epidemics when introduced to naive equine populations, resulting in severe restrictions on the movement of equines between AHS-positive and negative countries. Recent zoning of South Africa has created an AHS-free zone to facilitate equine movement, but the transmission dynamics of these viruses are not fully understood. Here, we present further analyses of serosurveys of donkeys in South Africa conducted in 1983-5 and in 1993-5. Age-prevalence data are used to derive estimates of the force of infection, A. For both viruses, A was highest in the northeastern part of the country and declined towards the southwest. In most of the country, EE virus had a higher transmission rate than AHS. The force of infection increased for EE virus between 1985 and 1993, but decreased for AHS virus. Both viruses showed high levels of variation in transmission between districts within the same province, particularly in areas of intermediate transmission. These data emphasize the focal nature of these viruses, and indicate areas where further data will assist in understanding the geographical variation in transmission.

Experimental reproduction of viral chorioretinitis in kangaroos.

OBJECTIVE: To investigate whether preparations containing Wallal and/or Warrego viruses could cause disease when inoculated subcutaneously into captive kangaroos. DESIGN AND PROCEDURE: Four groups of two kangaroos, seronegative to both Wallal and Warrego virus, were each inoculated with wild Wallal virus, cultured Wallal virus, wild Warrego virus, or wild Warrego virus followed by wild Wallal virus after 3 weeks. A single uninoculated animal served as a control. Animals were monitored weekly under anaesthesia, examined ophthalmoscopically (including fundic photography), and samples collected for haematological and serum biochemical analysis, virus isolation, PCR and serological examination for antibodies against Wallal and Warrego viruses. Animals inoculated with cultured Wallal virus were killed at week 10, and remaining kangaroos were reinoculated with cultured Wallal virus at week 12. RESULTS: Virus was isolated from the blood of two kangaroos 2 weeks after inoculation with Wallal virus preparations, and from a third kangaroo 2 weeks after reinoculation. By 3 weeks after inoculation, all kangaroos given Wallal virus preparations had seroconverted to Wallal virus and one had seroconverted to Warrego virus. Fundic changes were detected in the three viraemic kangaroos 4 or more weeks after inoculation, and lesions were present in the eye and brain typical of those seen in field cases of chorioretinitis. No other kangaroos had lesions. Wallal virus was identified by PCR and immunohistochemical analysis in the retina of one affected animal and orbivirus-like particles were seen by electron microscopy in the remains of retinal cells. CONCLUSION: The condition of chorioretinitis was reproduced in three of eight kangaroos by inoculation with preparations containing Wallal virus.

Encephalopathy in suckling mice infected with Kasba (Chuzan) virus.

Kasba (Chuzan) virus (an orbivirus), strain K-47, produced encephalopathy with severe necrosis in suckling mice inoculated intracerebrally. On day 3 after inoculation with 10(3)TCID50, the mice showed severe focal encephalomalacia and meningitis. On day 4, necrosis had spread to the midbrain, cerebellum and spinal cord. From one day after inoculation, virus was recovered from the brain and the titre rose over the next 3 days. Immunohistochemical examination demonstrated viral antigens in the cytoplasm of both degenerate and intact neurons, and ependymal cells in or around necrotic lesions. The study indicated that the virus has an affinity for immature nerve cells in the brains of suckling mice and causes primary encephalomalacia. Since the lesions resembled those of the hydranencephaly-cerebellar hypoplasia syndrome in calves (Chuzan disease), the system described should prove useful in studies on pathogenesis.

Sensitive plaque assay and propagation of Chuzan (Kasba) virus, a Palyam serogroup orbivirus, in BHK-21 cells.

Various factors influencing plaque formation of Chuzan virus in BHK-21 cell monolayers were studied and a practical method for plaque assay was developed. On addition of trypsin (5 micrograms/ml) and/or diethylaminoethyl (DEAE)-dextran (50 micrograms/ml) to the virus diluent as the virus adsorption medium and agar overlay medium, the number of plaques increased. When 100 micrograms/ ml DEAE-dextran was added to the diluent and overlay medium, plaques were produced in about 10-fold higher numbers than without trypsin and DEAE-dextran. Based on these results, a practical plaque assay method for Chuzan virus was established. Using this method, one-step growth of Chuzan virus was performed at an input multiplicity of 25 plaque-forming units (PFU) per cell. Cytopathic effects were first observed at 7.5 h post-inoculation (p.i.), and were complete at 12 h p.i. The titre of cell-associated virus, after gradual decline during the first 3 h of incubation, showed a rise within 4.5 h p.i. and a rise to a plateau of 10(6.3)PFU/0.2 ml at 12 h p.i. By indirect immunofluorescence, virus-specific antigen was detected in the cytoplasm of the cells at 4.5 h p.i., and all the cells fluoresced at 6 h p.i. Haemagglutination activity was first detected in infected whole cultures at 7.5 h p.i. reaching a plateau of 1:64 at 15 h p.i. Plaque formation and haemagglutination by the virus were specifically inhibited by antisera against the original and the plaque-cloned virus.

Essaouira and Kala iris: two new orbiviruses of the Kemerovo serogroup, Chenuda complex, isolated from Ornithodoros (Alectorobius) maritimus ticks in Morocco.

Essaouira and Kala Iris viruses were isolated from Ornithodoros (Alectorobius) maritimus ticks parasitizing yellow-legged gulls (Larus cachinnans) on the coast of Morocco in 1979 and 1981, respectively. Serological evidence indicates that these two viruses are new members of the Chenuda complex within the Kemerovo serogroup of the genus Orbivirus. Ecological, pathological, morphological, and physicochemical properties are compatible with these findings. The infectivity of these viruses for man and animals, including seabirds, remains unknown.

Enhanced neurovirulence of tick-borne orbiviruses resulting from genetic modulation.

The genome of orbiviruses (Reoviridae family) comprises 10 segments of double-stranded RNA. The fourth largest segment of the tick-borne Kemerovo (KEM) group orbiviruses is the genetic determinant of neurovirulence in experimentally infected mice, and segment 6 determines serotype. Reassortant viruses derived from a cross between two KEM-related viruses, Great Island (GI) and Wexford (WEX), that had the heterotypic gene combination W4G6 (segment 4 of WEX virus and segment 6 from GI virus) were nonpathogenic in mice. This apparent genetic modulation of neurovirulence may have resulted from steric interaction between the two outer capsid proteins of nonpathogenic reassortants. Further data are consistent with this hypothesis. Reassortants generated from additional KEM group viruses showed various degrees of enhanced neurovirulence in terms of their PFU/LD50 (ratio of infectivity in cell culture and in mice) and ASTmax (the average survival time at the highest virus dilution resulting in 100% mortality). Some reassortants were more pathogenic than either of their parental viruses. The results indicate that the gene determining neurovirulence dictates ASTmax, and the PFU/LD50 is a measure of the interaction between the products of the gene determining neurovirulence and that determining serotype. The nonpathogenic phenotype of a low passage isolate (St. Abb's 84-34 virus), derived from a single tick, generated neurovirulent reassortants. This result indicates that genetic modulation of KEM group viruses may occur in nature.

Identification of the major genetic determinant for neurovirulence of tick-borne orbiviruses.

Three members of the Great Island antigenic subgroup (Kemerovo serogroup) of tick-borne obiviruses produced fatal infections following intracerebral inculation of 2-day-old mice. The average survival times and PFU/LD50 ratios of mice inoculated with Wexford (WEX) virus were significantly greater than those of either Nugget (NUG) or Great Island (GI) virus. Reassortant viruses were isolated following dual infections of cell cultures with a spontaneous temperature-sensitive (ts) mutant of WEX virus, and either NUG wild-type virus or a ts mutant of GI virus. The neurovirulence for mice and derivation of the genomic RNA segments were determined for the reassortants. Analysis of this data revealed that the pathogenic phenotype of the reassortant viruses depended on the parental origin of genomic segment 4. The major genetic determinant of serotype specificity was not shown to influence neurovirulence in these investigations.