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.

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.

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.

Evidence for the circulation of equine encephalosis virus in Israel since 2001.

Equine encephalosis virus (EEV) distribution was thought to be limited to southern Africa until 2008 when we reported EEV in Israel. It was then assumed that the clinical presentation resembled the initial incursion in Israel. To investigate further we conducted a retrospective analysis of equine sera, which had been collected for diagnosis of other suspected diseases, via serum neutralisation test. The data demonstrated that EEV was circulating as early as 2001 with incidence ranging from 20-100% for time period 2001-2008. As the symptoms of EEV can be similar to other equine notifiable diseases this is a significant finding which highlights the need for vigilance and education to accurately diagnose new and emerging diseases.

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.

Serotype specific primers and gel-based RT-PCR assays for 'typing' African horse sickness virus: identification of strains from Africa.

African horse sickness is a devastating, transboundary animal disease, that is 'listed' by the Office International des Epizooties (OIE). Although attenuated, inactivated and subunit vaccines have been developed for African horse sickness virus (AHSV), these are serotype-specific and their effective deployment therefore relies on rapid and reliable identification of virus type. AHSV serotype is controlled by the specificity of interactions between neutralising antibodies, and components of the outer-capsid, particularly protein VP2 (encoded by AHSV genome segment 2 (Seg-2)). We report the development and evaluation of novel gel based reverse transcription-PCR (RT-PCR) assays targeting AHSV Seg-2, which can be used to very significantly increase the speed and reliability of detection and identification (compared to virus neutralisation tests) of the nine serotypes of AHSV. Primer sets were designed targeting regions of Seg-2 that are conserved between strains within each of the AHSV serotype (types 1 to 9). These assays were evaluated using multiple AHSV strains from the orbivirus reference collection at IAH (www.reoviridae.org/dsRNA_virus_proteins/ReoID/AHSV-isolates.htm). In each case the Seg-2 primers showed a high level of specificity and failed to cross-amplify the most closely related heterologous AHSV types, or other related orbiviruses (such as bluetongue virus (BTV), or equine encephalosis virus (EEV)). The assays are rapid and sensitive, and can be used to detect and type viral RNA in blood, tissue samples, or cultivated viral suspensions within 24 h. They were used to identify AHSV strains from recent outbreaks in sub-Saharan African countries. These methods also generate cDNAs suitable for sequencing and phylogenetic analyses of Seg-2, identifying distinct virus lineages within each virus-type and helping to identify strain movements/origins. The RT-PCR methods described here provide a robust and versatile tool for rapid and specific detection and identification of AHSV serotypes 1 to 9.

A competitive ELISA for the detection of group-specific antibody to equine encephalosis virus.

A polyclonal antibody-based, group-specific, competitive ELISA (C-ELISA) for the detection of antibodies to equine encephalosis virus (EEV) was developed. The assay measures the competition between a specific guinea pig antiserum and a test serum, for a pre-titrated EEV antigen. The C-ELISA detected antibodies to the seven known EEV serotypes. Reference antisera raised against other arboviruses did not cross react with EEV antigen. Negative sera from horses in the United Kingdom were used to establish the baseline for a negative population. Negative and positive populations of South African horses, selected on the basis of virus neutralisation were assayed subsequently. Optimal test parameters, where sensitivity≅specificity≅100%, were calculated by two-graph receiver operator characteristic (TG-ROC) analysis to be at a cut-off value of 29.5% inhibition. Results show the EEV C-ELISA described to be sensitive, specific and reliable. Used in conjunction with ELISAs available for African horse sickness virus (AHSV), differential serological diagnosis between EEV and AHSV can be achieved.

Genetic and phylogenetic analysis of the outer-coat proteins VP2 and VP5 of epizootic haemorrhagic disease virus (EHDV): comparison of genetic and serological data to characterise the EHDV serogroup.

The outer-coat proteins, VP2 and VP5, of epizootic haemorrhagic disease virus (EHDV) are important for host cell binding during the initiation of infection. They are also known to determine virus serotype. This study presents a complete genetic and phylogenetic analysis of these proteins (and the genes that code for them) to allow comparison of the selective pressures acting on each and the correlation of genetic sequence data with serotype. Accession numbers, gene and protein sizes, ORF positions, G+C contents, terminal hexanucleotides, start and stop codons and phylogenetic relationships are all presented. The results show that VP2 is highly variable, is under great pressure to adapt and can be correlated with serotype. While also variable, VP5 appears to be under less adaptive pressure than VP2 but still shows some correlation with serotype. Seven serotypes of EHDV have been defined in this study, although the results do show that some serotypes are extremely closely related--and highlight the benefit of using both molecular and serologic analyses. Analysis of the terminal hexanucleotides showed that the 5' terminus is under greater purifying selection than the 3'. Evidence is also presented that both segments 2 and 6 (coding for VP2 and VP5 respectively) have grown via gene duplication and subsequent mutation.

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.

Genetic and epidemiological characterization of Middle Point orbivirus, a novel virus isolated from sentinel cattle in northern Australia.

Middle Point orbivirus (MPOV) was isolated in 1998 from a healthy cow pastured at Beatrice Hill farm, Middle Point (formerly Coastal Plains Research Station), 50 km east of Darwin in Australia's Northern Territory. The isolate could not be identified by using conventional serological tests, and electron microscopy indicated that it belongs to the family Reoviridae, genus Orbivirus. Genetic sequencing of segments 2 and 3 revealed that this virus is related to Yunnan orbivirus, an orbivirus known only from China and not previously associated with a vertebrate host. A real-time RT-PCR test was developed to study the epidemiology of this virus in the field. Over 150 previously unidentified viruses isolated from cattle between 1994 and 2006 were positively identified as isolates of MPOV. Serology was used to demonstrate the development of antibody responses to MPOV in cattle from multiple locations across the Northern Territory.

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.

Seroprevalence of tick-borne and mosquito-borne arboviruses in European brown hares in Northern and Western Germany.

Tríbec virus (Kemerovo serogroup, genus Orbivirus), Eyach virus (genus Coltivirus), and Tahyna virus (California encephalitis serogroup, genus Bunyavirus) are arthropod-borne viruses known to occur in Germany. These viruses are also suspected to cause human disease. So far, no information is available on their geographical distribution in Germany and their natural transmission cycles. A total of 166 sera from European brown hares (Lepus europaeus) collected in seven districts of the Federal State of Schleswig-Holstein and in four districts of the Federal State of North Rhine-Westphalia was tested by plaque reduction neutralization test (PRNT) for antibodies against Tríbec virus, Eyach virus, Tahyna virus, and Central European encephalitis virus. One out of 22 sera (4.5%) collected in the district Nord-Friesland in Schleswig-Holstein was found positive (PRNT(90) 1:10) against Tríbec virus. Neither did sera from other regions of Schleswig-Holstein nor from hares from North Rhine-Westphalia react against any of the arboviruses tested. For the first time, antibodies against Tríbec virus could be found in a European brown hare in Germany. The negative serological results for Central European encephalitis virus are in line with the current knowledge of its natural distribution within Germany. The negative serological results for Tahyna virus or Eyach virus argue against an autochthonous circulation of these viruses in the regions tested.

Tick-borne Great Island Virus: (II) Impact of age-related acquired immunity on transmission in a natural seabird host.

Tick-borne pathogen transmission is dependent upon tick number per host and the physical and temporal distribution of each feeding stage. Age-related acquired immunity to tick and pathogen may also be important but has received less attention. In this study we evaluate which of these parameters has the greatest impact on Great Island Virus (GIV) transmission between Ixodes uriae ticks and common guillemots (Uria aalge). The study system is well suited to investigate age-related effects because the guillemot population is naturally divided into 2 groups, older breeding and younger pre-breeding adult birds. The physical distribution and timing of adult and nymphal tick feeding was similar for both guillemot age groups. However, breeding birds were parasitized by significantly more ticks (mainly nymphs). Calculations based on tick number predict virus prevalence should be higher in ticks that have fed on breeding rather than pre-breeding birds. However, empirical evidence indicates the reverse. Protective acquired immunity to GIV infection may be the reason why GIV prevalence is actually significantly lower in ticks that have fed on breeders. Far more breeding (74%) than pre-breeding (12%) guillemots had antibodies that neutralized 1 or more GIV strains. Estimates of the force of infection support the view that pre-breeding birds experience higher rates of virus infection than breeding birds. The results indicate age-related acquired immunity is a key factor in GIV transmission and highlight the need to consider age-related effects and host immunity when undertaking quantitative studies of tick-borne pathogen transmission.

Tick-borne Great Island Virus: (I) Identification of seabird host and evidence for co-feeding and viraemic transmission.

Great Island Virus (GIV) is an arbovirus present in the tick Ixodes uriae, a common ectoparasite of nesting seabirds. Common guillemot (Uria aalge) and black-legged kittiwake (Rissa tridactyla) are the preferred and most abundant hosts of I. uriae on the Isle of May, Scotland. As part of a study to understand the epidemiology of GIV, the ability of guillemot and kittiwake to support tick-borne transmission of GIV was examined. GIV was present in ticks feeding in isolated guillemot colonies and guillemots had virus-specific neutralizing antibodies demonstrating previous GIV infection. By contrast, only uninfected ticks were found in colonies inhabited solely by kittiwakes. GIV was isolated from kittiwake ticks in colonies which also contained breeding guillemots but no virus-specific neutralizing antibodies were present in blood samples of kittiwake on which infected ticks were feeding. Thus guillemots are the main vertebrate hosts of GIV on the Isle of May whereas kittiwakes do not appear to be susceptible to infection. Virus infection of adult ticks feeding on guillemots was highly efficient and may involve both viraemic transmission and transmission from infected to uninfected ticks feeding together on birds that do not develop a patent viraemia.