Predicted structure and domain organization of rotavirus capping enzyme and innate immune antagonist VP3.

UNLABELLED: Rotaviruses and orbiviruses are nonturreted Reoviridae members. The rotavirus VP3 protein is a multifunctional capping enzyme and antagonist of the interferon-induced cellular oligoadenylate synthetase-RNase L pathway. Despite mediating important processes, VP3 is the sole protein component of the rotavirus virion whose structure remains unknown. In the current study, we used sequence alignment and homology modeling to identify features common to nonturreted Reoviridae capping enzymes and to predict the domain organization, structure, and active sites of rotavirus VP3. Our results suggest that orbivirus and rotavirus capping enzymes share a domain arrangement similar to that of the bluetongue virus capping enzyme. Sequence alignments revealed conserved motifs and suggested that rotavirus and orbivirus capping enzymes contain a variable N-terminal domain, a central guanine-N7-methyltransferase domain that contains an additional inserted domain, and a C-terminal guanylyltransferase and RNA 5'-triphosphatase domain. Sequence conservation and homology modeling suggested that the insertion in the guanine-N7-methyltransferase domain is a ribose-2'-O-methyltransferase domain for most rotavirus species. Our analyses permitted putative identification of rotavirus VP3 active-site residues, including those that form the ribose-2'-O-methyltransferase catalytic tetrad, interact with S-adenosyl-l-methionine, and contribute to autoguanylation. Previous reports have indicated that group A rotavirus VP3 contains a C-terminal 2H-phosphodiesterase domain that can cleave 2'-5' oligoadenylates, thereby preventing RNase L activation. Our results suggest that a C-terminal phosphodiesterase domain is present in the capping enzymes from two additional rotavirus species. Together, these findings provide insight into a poorly understood area of rotavirus biology and are a springboard for future biochemical and structural studies of VP3. IMPORTANCE: Rotaviruses are an important cause of severe diarrheal disease. The rotavirus VP3 protein caps viral mRNAs and helps combat cellular innate antiviral defenses, but little is known about its structure or enzymatic mechanisms. In this study, we used sequence- and structure-based alignments with related proteins to predict the structure of VP3 and identify enzymatic domains and active sites therein. This work provides insight into the mechanisms of rotavirus transcription and evasion of host innate immune defenses. An improved understanding of these processes may aid our ability to develop rotavirus vaccines and therapeutics.

Equine encephalosis virus: evidence for circulation beyond southern Africa.

Prior to the recent outbreak of equine encephalosis in Israel in 2009, equine encephalosis virus (EEV) had only been isolated from equids in South Africa. In this study we show the first evidence for the circulation of EEV beyond South Africa in Ethiopia, Ghana and The Gambia, indicating that EEV is likely to be freely circulating and endemic in East and West Africa. Sequence analysis revealed that the EEV isolate circulating in The Gambia was closely related to an EEV isolate that was isolated from a horse from Israel during the EEV outbreak in 2009, indicating that the two viruses have a common ancestry. Interestingly horses in Morocco tested negative for EEV antibodies indicating that the Sahara desert may be acting as a geographical barrier to the spread to the virus to North African countries. This evidence for EEV circulation in countries in East and West Africa sheds light on how the virus may have reached Israel to cause the recent outbreak in 2009.

Animal diseases caused by orbiviruses, Algeria.

Antibodies against bluetongue virus were detected in cattle, sheep, goats, and camels in Algeria in 2008. Antibodies against epizootic hemorrhagic disease virus were detected in cattle, but antibodies against African horse sickness virus were not detected in horses and mules. Epizootic hemorrhagic disease in northern Africa poses a major risk for the European Union.

Isolation of Tibet Orbivirus from Culicoides jacobsoni (Diptera, Ceratopogonidae) in China.

BACKGROUND: Tibet Orbivirus (TIBOV) is a recently discovered Orbivirus known to infect cattle, Asian buffalo and goats in south-western China. It was first isolated from mosquitoes and subsequently from biting midges (Culicoides spp.) in Yunnan, China, indicating that it is an arbovirus. Little is known of its potential to cause disease, but the economic importance of related viruses promoted an investigation of potential Culicoides spp. vectors of TIBOV. METHODS: Biting midges were collected approximately once per week between May and December 2020, at a cattle farm in Wulong village, Shizong County, Yunnan Province, China. Approximately 3000 specimens of nine species were subsequently used in attempts to isolate virus, and a further 2000 specimens of six species were tested for the presence of bluetongue virus (BTV) and TIBOV using a RT-qPCR test. RESULTS: Virus isolation attempts resulted in the isolation of three viruses. One isolate from a pool of Culicoides jacobsoni was identified as TIBOV, while the other two viruses from C. orientalis and C. tainanus remain unidentified but are not BTV or TIBOV. RT-qPCR analysis did not detect BTV in any specimens, but a single pool containing five specimens of C. jacobsoni and another containing five specimens of C. tainanus produced PCR quantification cycle (Cq) values of around 28 that may indicate infection with TIBOV. CONCLUSIONS: The isolation of TIBOV from C. jacobsoni satisfies one criterion required to prove its status as a vector of this virus. This isolation is supported by a low Cq value produced from a different pool of this species in the RT-qPCR test. The low Cq value obtained from a pool of C. tainanus suggests that this species may also be able to satisfy this criterion. Both of these species are widespread throughout Asia, with C. jacobsoni extending into the Pacific region, which raises the possibility that TIBOV may be more widespread than is currently known.

Highly efficient vaccines for Bluetongue virus and a related Orbivirus based on reverse genetics.

Bluetongue virus (BTV) reverse genetics (RG), available since 2007, has allowed the dissection of the virus replication cycle, including discovery of a primary replication stage. This information has allowed the generation of Entry-Competent-Replication-Abortive (ECRA) vaccines, which enter cells and complete primary replication but fail to complete the later stage. A series of vaccine trials in sheep and cattle either with a single ECRA serotype or a cocktail of multiple ECRA serotypes have demonstrated that these vaccines provide complete protection against virulent virus challenge without cross-serotype interference. Similarly, an RG system developed for the related African Horse Sickness virus, which causes high mortality in equids has provided AHSV ECRA vaccines that are protective in horses. ECRA vaccines were incapable of productive replication in animals despite being competent for cell entry. This technology allows rapid generation of emerging Orbivirus vaccines and offers immunogenicity and safety levels that surpass attenuated or recombinant routes.

Prevalence of serotype specific antibody to equine encephalosis virus in Thoroughbred yearlings in South Africa (1999-2004).

Cohorts of yearlings were sampled over a period of 6 years in a retrospective serological survey to establish the annual prevalence of serotype specific antibody to equine encephalosis virus on Thoroughbred stud farms distributed within defined geographical regions of South Africa. Seasonal seroprevalence varied between 3.6% and 34.7%, revealing both single and multiple serotype infections in an individual yearling. During the course of this study serotypes 1 and 6 were most frequently and extensively identified while the remaining serotypes 2, 3, 4, 5 and 7 were all identified as sporadic and localized infections affecting only individual horses. This study of the seasonal prevalence of equine encephalosis virus has a corollary and serves as a useful model in the seasonal incidence of the serotypes of African horse sickness and bluetongue in regions where the respective diseases are endemic.

One after the other: A novel Bluetongue virus strain related to Toggenburg virus detected in the Piedmont region (North-western Italy), extends the panel of novel atypical BTV strains.

In this rapid communication, a novel atypical bluetongue virus (BTV) strain detected in goats in the Piedmont region (north-western Italy) is described. This strain, BTV-Z ITA2017, is most related in Seg-2/VP-2 (83.8% nt/82.7% aa) to strain TOV of BTV-25. Reactive antisera of goats positive by cELISA for BTV antibodies failed to neutralize a chimeric virus expressing the outermost protein of TOV. Infected animals displayed low levels of RNAemia and absence of clinical signs consistent with bluetongue infection, a scenario described in animals infected with atypical BTV strains.

Orbiviruses: A North American Perspective.

Orbiviruses are members of the Reoviridae family and include bluetongue virus (BTV) and epizootic hemorrhagic disease virus (EHDV). These viruses are the cause of significant regional disease outbreaks among livestock and wildlife in the United States, some of which have been characterized by significant morbidity and mortality. Competent vectors are clearly present in most regions of the globe; therefore, all segments of production livestock are at risk for serious disease outbreaks. Animals with subclinical infections also serve as reservoirs of infection and often result in significant trade restrictions. The economic and explicit impacts of BTV and EHDV infections are difficult to measure, but infections are a cause of economic loss for producers and loss of natural resources (wildlife). In response to United States Animal Health Association (USAHA) Resolution 16, the US Department of Agriculture (USDA), in collaboration with the Department of the Interior (DOI), organized a gap analysis workshop composed of international experts on Orbiviruses. The workshop participants met at the Arthropod-Borne Animal Diseases Research Unit in Manhattan, KS, May 14-16, 2013, to assess the available scientific information and status of currently available countermeasures to effectively control and mitigate the impact of an outbreak of an emerging Orbivirus with epizootic potential, with special emphasis given to BTV and EHDV. In assessing the threats, workshop participants determined that available countermeasures are somewhat effective, but several weaknesses were identified that affect their ability to prevent and control disease outbreaks effectively.

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.

A blocking ELISA for detection of antibody to a subgroup-reactive epitope of African horsesickness viral protein 7 (VP7) using a novel gamma-irradiated antigen.

A novel gamma irradiated inactivated cell culture derived African horsesickness viral (AHSV) antigen was used in a blocking ELISA (B-ELISA) for detecting antibody to a subgroup-reactive epitope of AHSV. A monoclonal antibody (MAB), class IgM, against an epitope on African horsesickness (AHS) viral protein 7 (VP7) was developed in BALBc mice and used in the B-ELISA. The MAB, designated F9H, was blocked by 69 serums from equidae with antibody to AHS, but its binding activity was not appreciably affected by 301 serums that did not contain antibodies to AHS virus. An ELISA protocol using a blocking format is described.

A rapid indirect ELISA for the serogrouping of Australian orbiviruses.

This communication describes the development and evaluation of a simple and rapid method for the classification of Australian orbiviruses into one of seven established serogroups (i.e. bluetongue, epizootic haemorrhagic disease of deer, Palyam, Eubenangee, Corriparta, Wallal, Warrego) or an 'ungrouped' category. The Australian orbivirus serogrouping ELISA (SG-ELISA) utilised a sodium deoxycholate-treated cell lysate preparation from infected BHK cells which was subsequently probed in an indirect ELISA format with polyclonal antibodies representative of each serogroup. Bound immunoglobulin was detected by the use of a recombinant streptococcal protein G-HRPO conjugate and subsequent reaction with the chromogenic substrate. All reference orbiviruses tested in the SG-ELISA were identified and were in agreement with the serogroups originally designated. Minimal inter-serogroup cross-reactions were observed. One-way cross-reactions were observed between Warrego and Mitchell River viruses.

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.

Immunohistochemical study of age-dependent brain lesions in mice infected intracerebrally with Kasba (Chuzan) virus.

When Kasba (Chuzan) virus (an orbivirus) was injected intracerebrally into 1-, 2- or 4-week-old mice, non-purulent necrotizing encephalitis developed and the mice showed nervous symptoms and became moribund. The necrotic lesions were more severe in younger animals. In 1-week-old mice, viral titres rose until 7 days post-infection, while in 2- and 4-week-old animals the titres reached a peak on day 3 and then declined gradually. Glial fibrillary acidic protein-positive astrocytes increased in the white matter, hippocampus and subpial area of the cerebral cortex of infected animals, and lectin-RCA-1-positive cells, thought to be microglial cells, increased in the necrotic lesions. The number of these glial cells increased even after viral titres had declined. In this study there were no survivors in any age group, but survival time increased with age.

Avian arboviruses of the Witless Bay seabird Sanctuary, Newfoundland, Canada.

A virologic and serologic survey of arbovirus infections among seabirds and seabird ticks, Ixodes uriae, on Great Island. Witless Bay, Newfoundland, Canada, was conducted during 1971 and 1972. Kermerovo (Great Island, Bauline) and Sakhalin (Avalon) group viruses previously reported from birds and/or ticks on Great Island were prevalent among avian populations, while conclusive evidence of known nonindigenous serotypes was lacking. Circumstantial evidence-hemagglutination inhibiting antibody-of an unidentified member of the group B tick-borne encephalitis complex transmitted among marine birds of North America by I. uriae is reported. No evidence of human infections with any of these viruses was detected in a small number of biologists doing research on Great Island.

Serologic evidence for rabbit syncytium virus in eastern cottontail rabbits (Sylvilagus floridanus) in Ohio.

Thirteen of 20 eastern cottontail rabbit (Sylvilagus floridanus) sera collected near Delaware, Ohio (USA) in 1991 were positive by indirect immunofluorescent antibody test (IFAT) for antibody to rabbit syncytium virus (RSV), a Kemerovo serogroup orbivirus. In addition, two of 10 domestic bovine sera and three of 30 sheep sera collected in southeastern Ohio gave weak positive IFAT reactions to RSV.

The classification of seven serotypes of equine encephalosis virus and the prevalence of homologous antibody in horses in South Africa.

Selected isolates of equine encephalosis virus were shown to have comparable viral protein profiles and to represent seven distinct serotypes, based on cross-neutralization tests. Serotype-specific virus-neutralizing antibody in serum samples from horses confirmed the widespread occurrence of infection. The distribution and prevalence of individual serotypes however, varied considerably. Localised foci with an increased seasonal seroconversion in groups of horses to a specific serotype and the detection of an ongoing low level of infection from other serotypes within the population, confirmed the independent persistence of the viruses in a maintenance cycle. The identification of donors with antibody resulting from infection with multiple serotypes indicated a low level of cross-protection in horses to natural reinfection.

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.

Antigenic cross-reactions between tick-borne orbiviruses of the Kemerovo serogroup.

Antigenic relationships between the four subgroups of the Kemerovo serogroup of tick-borne orbiviruses were examined. Kemerovo (KEM subgroup), Broadhaven (BRD) and Wexford (WEX) [Great Island (GI) sub-group], Chenuda (CNU sub-group), and Wad Medani (WM sub-group) viruses cross-reacted in immunofluorescence tests. Complement fixation tests (CFT) revealed that KEM virus was more closely related to BRD and WEX viruses than to either CNU or WM viruses. By cross-neutralization, all the viruses were shown to be distinct; only BRD and WEX showed slight cross-reaction. In Vero cells infected with either KEM, BRD, WEX, or CNU viruses, 10 major (p1 to p10) and a variable number of minor virus-induced 35S-labelled polypeptides were detected. Comparison of the polypeptides precipitated by homologous AF revealed close similarities between BRD and WEX viruses but obvious differences between these two viruses and KEM and CNU viruses. In cross-reactions, p6 of KEM and CNU viruses, and p7 of BRD and WEX viruses were immunoprecipitated. Immunofluorescence and immunoprecipitation tests appeared to identify an inter-group specific antigen that was distinct from an intra-group specific antigen detected by CFT. The results support division of KEM-related viruses into 3 new serogroups - KEM (comprising KEM and GI subgroups), CNU and WM - and indicate that a 38 to 43kD polypeptide carries the major inter-group specific antigen.

Isolation of orbiviruses and uukuviruses from puffin ticks.

Two viruses were isolated from a pool of three female Ixodes uriae ticks found on a dead puffin (Fratercula arctica) on a beach at Arbroath, Scotland. Complement fixation tests showed that one of the viruses was an orbivirus belonging to the Kemerovo serogroup and was related to Cape Wrath virus. Cross-reactions did not occur in neutralisation tests with 4 Kemerovo group viruses previously isolated from I. uriae collected in British seabird colonies. The orbivirus was therefore named Arbroath virus. The other virus was of the Uukuniemi serogroup (family Bunyaviridae) and reacted in complement fixation and neutralisation tests with a virus isolated from I. uriae collected from a seabird colony at St Abb's Head, Scotland. Both the orbi- and the uukuviruses replicated in a tick (Rhipicephalus appendiculatus) cell line, RA-243.