Bluetongue Research Articles

Bluetongue virus capsid protein VP5 perforates membranes at low endosomal pH during viral entry.

Bluetongue virus (BTV) is a non-enveloped virus and causes substantial morbidity and mortality in ruminants such as sheep. Fashioning a receptor-binding protein (VP2) and a membrane penetration protein (VP5) on the surface, BTV releases its genome-containing core (VP3 and VP7) into the host cell cytosol after perforation of the endosomal membrane. Unlike enveloped ones, the entry mechanisms of non-enveloped viruses into host cells remain poorly understood. Here we applied single-particle cryo-electron microscopy, cryo-electron tomography and structure-guided functional assays to characterize intermediate states of BTV cell entry in endosomes. Four structures of BTV at the resolution range of 3.4-3.9 Å show the different stages of structural rearrangement of capsid proteins on exposure to low pH, including conformational changes of VP5, stepwise detachment of VP2 and a small shift of VP7. In detail, sensing of the low-pH condition by the VP5 anchor domain triggers three major VP5 actions: projecting the hidden dagger domain, converting a surface loop to a protonated β-hairpin that anchors VP5 to the core and stepwise refolding of the unfurling domains into a six-helix stalk. Cryo-electron tomography structures of BTV interacting with liposomes show a length decrease of the VP5 stalk from 19.5 to 15.5 nm after its insertion into the membrane. Our structures, functional assays and structure-guided mutagenesis experiments combined indicate that this stalk, along with dagger domain and the WHXL motif, creates a single pore through the endosomal membrane that enables the viral core to enter the cytosol. Our study unveils the detailed mechanisms of BTV membrane penetration and showcases general methods to study cell entry of other non-enveloped viruses.

Identification and Genetic Characterization of Viral Pathogens in Ruminant Gestation Abnormalities, Israel, 2015–2019

Infectious agents including viruses are important abortifacients and can cause fetal abnormalities in livestock animals. Here, samples that had been collected in Israel from aborted or malformed ruminant fetuses between 2015 and 2019 were investigated for the presence of the following viruses: the reoviruses bluetongue virus (BTV) and epizootic hemorrhagic disease virus (EHDV), the flaviviruses bovine viral diarrhea virus (BVDV) and border disease virus (BDV), the peribunyaviruses Shuni virus (SHUV) and Akabane virus (AKAV), bovine herpesvirus type 1 (BoHV-1) and bovine ephemeral fever virus (BEFV). Domestic (cattle, sheep, goat) and wild/zoo ruminants were included in the study. The presence of viral nucleic acid or antigen could be confirmed in 21.8 % of abnormal pregnancies (213 out of 976 investigated cases), with peribunyaviruses, reoviruses and pestiviruses being the most prevalent. At least four different BTV serotypes were involved in abnormal courses of pregnancy in Israel. The subtyping of pestiviruses revealed the presence of two BDV and several distinct BVDV type 1 strains. The peribunyaviruses AKAV and SHUV were identified annually throughout the study period, however, variation in the extent of virus circulation could be observed between the years. In 2018, AKAV even represented the most detected pathogen in cases of small domestic ruminant gestation abnormalities. In conclusion, it was shown that various viruses are involved in abnormal courses of pregnancy in ruminants in Israel.

Sialic Acid Binding Sites in VP2 of Bluetongue Virus and Their Use during Virus Entry.

ABSTRACTBluetongue virus (BTV), a member of the Orbivirus genus, is transmitted by biting midges (gnats, Culicoides sp.) and is one of the most widespread animal pathogens, causing serious outbreaks in domestic animals, particularly in sheep, with high economic impact. The non-enveloped BTV particle is a double-capsid structure of seven proteins and a genome of 10 double-stranded RNA segments. Although the outermost spike-like VP2 acts as the attachment protein during BTV entry, no specific host receptor has been identified for BTV. Recent high-resolution cryo-electron (cryoEM) structures and biological data have suggested that VP2 may interact with sialic acids (SAs). To confirm this, we have generated protein-based nanoparticles displaying multivalent VP2 and used them to probe glycan arrays. The data show that VP2 binds α2,3-linked SA with high affinity but also binds α2,6-linked SA. Further, Maackia amurensis lectin II (MAL II) and Sambucus nigra lectin (SNA), which specifically bind α2,3-linked and α2,6-linked SAs, respectively, inhibited BTV infection and virus growth in susceptible sheep cells while SNA alone inhibited virus growth in Culicoides-derived cells. A combination of hydrogen deuterium exchange mass spectrometry and site-directed mutagenesis allowed the identification of the specific SA binding residues of VP2. This study provides direct evidence that sialic acids act as key receptor for BTV and that the outer capsid protein VP2 specifically binds SA during BTV entry in both mammalian and insect cells.IMPORTANCE To date no receptor has been assigned for non-enveloped bluetongue virus. To determine if the outermost spike-like VP2 protein is responsible for host cell attachment via interaction with sialic acids, we first generated a protein-based VP2-nanoparticle, for the multivalent presentation of recombinant VP2 protein. Using nanoparticles displaying VP2 to probe a glycan array, we identified that VP2 binds both α2,3-linked and α2,6-linked sialic acids. Lectin inhibitors targeting both linkages of sialic acids showed strong inhibition to BTV infection and progeny virus production in mammalian cells; however the inhibition was only seen with the lectin targeting α2,6-linked sialic acid in insect vector cells. In addition, we identified the VP2 sialic acid binding sites in the exposed tip domain. Our data provides direct evidence that sialic acids act as key receptors for BTV attachment and entry in to both mammalian and insect cells.

Determining the effects of wind-aided midge movement on the outbreak and coexistence of multiple bluetongue virus serotypes in patchy environments

Bluetongue virus (BTV) has 27 serotypes with some of them coexisting in different environments which make its control difficult. Wind-aided midge movement is a known mechanism in the spread of BTV. However, its effects on the dynamics of multiple BTV serotypes are not clear. Ordinary differential equation (ODE) and continuous-time Markov chain (CTMC) models for two BTV serotypes in an environment divided into two patches depending on the risk of infection are formulated and analysed. By approximating the CTMC model with a multitype branching process, an estimate for the probability of a major outbreak of two BTV serotypes is obtained. It is shown that without movement a major outbreak occurs in the high-risk patch, but with cattle or midge movement it occurs in both patches. When a major outbreak occurs, numerical simulations of the ODE model illustrate possible coexistence in both patches if the patches are connected by midge or cattle movement. Sensitivity analysis, based on the Latin hypercube sampling method, identified midge mortality and biting rates as being the most important in determining the magnitude of the probability of a major outbreak. These results indicate the significance of wind-aided midge movement on the outbreak and coexistence of multiple BTV serotypes in patchy environments.

Profiling of RNA Viruses in Biting Midges (Ceratopogonidae) and Related Diptera from Kenya Using Metagenomics and Metabarcoding Analysis.

ABSTRACTVector-borne diseases (VBDs) cause enormous health burden worldwide, as they account for more than 17% of all infectious diseases and over 700,000 deaths each year. A significant number of these VBDs are caused by RNA virus pathogens. Here, we used metagenomics and metabarcoding analysis to characterize RNA viruses and their insect hosts among biting midges from Kenya. We identified a total of 15 phylogenetically distinct insect-specific viruses. These viruses fall into six families, with one virus falling in the recently proposed negevirus taxon. The six virus families include Partitiviridae, Iflaviridae, Tombusviridae, Solemoviridae, Totiviridae, and Chuviridae. In addition, we identified many insect species that were possibly associated with the identified viruses. Ceratopogonidae was the most common family of midges identified. Others included Chironomidae and Cecidomyiidae. Our findings reveal a diverse RNA virome among Kenyan midges that includes previously unknown viruses. Further, metabarcoding analysis based on COI (cytochrome c oxidase subunit 1 mitochondrial gene) barcodes reveal a diverse array of midge species among the insects used in the study. Successful application of metagenomics and metabarcoding methods to characterize RNA viruses and their insect hosts in this study highlights a possible simultaneous application of these two methods as cost-effective approaches to virus surveillance and host characterization.IMPORTANCE The majority of the viruses that currently cause diseases in humans and animals are RNA viruses, and more specifically arthropod-transmitted viruses. They cause diseases such as dengue, West Nile infection, bluetongue disease, Schmallenberg disease, and yellow fever, among others. Several sequencing investigations have shown us that a diverse array of RNA viruses among insect vectors remain unknown. Some of these could be ancient lineages that could aid in comprehensive studies on RNA virus evolution. Such studies may provide us with insights into the evolution of the currently pathogenic viruses. Here, we applied metagenomics to field-collected midges and we managed to characterize several RNA viruses, where we recovered complete and nearly complete genomes of these viruses. We also characterized the insect host species that are associated with these viruses. These results add to the currently known diversity of RNA viruses among biting midges as well as their associated insect hosts.

First Isolation of Brucella canis from a breeding kennel in Italy.

Brucella canis has been isolated for the first time in Italy in a commercial breeding kennel. It was diagnosed after a deep investigation related to the onset of reproductive disorders. Animals were tested with direct and indirect techniques. The agent was first detected in two Chihuahua aborted foetuses by direct culture. Further, it was also isolated from blood samples of dogs hosted in the kennel, which also showed reaction to conventional serological tests (microplate serum agglutination test). The isolates were identified as B. canis by standard microbiological methods and a Bruce‑ladder multiplex PCR. To investigate the genomic diversity, whole genome sequencing was used, applying the core genome Multilocus Sequence Typing (cgMLST ). In a first round of serological testing performed on 598 animals, 269 (46.1%) tested positive. In the second round of laboratory testing carried out 4‑5 weeks apart, the number of serologically positive dogs was 241 out of 683 tested (35.3%), while the number of dogs positive to isolation was 68 out of 683 tested (10.0%). The PCR showed a lack of sensitivity when compared to direct isolation. The epidemiological investigation did not identify the source of the infection, given the time elapsed from the onset of abortions to the definitive diagnosis of B. canis infection in the kennel. The genomic analyses featured the strains as ST21 and, according to the cgMLST, revealed the presence of a tight cluster with a maximum diversity of four allelic differences. The observed limited genomic variation, largely within the known outbreak cut‑offs, suggests that the outbreak herein described was likely caused by a single introduction. Moreover, in a broader scale comparison using the public available genomes, we found that the closest genome, isolated in China, differed by more than 50 alleles making not possible to find out the likely origin of the outbreak. The lack of updated data on B. canis genome sequences in the public databases, together with the limited information retrieved from the epidemiological investigations on the outbreak, hampered identification of the source of B. canis infection.

Development of a rapid lateral flow immunochromatography assay for the detection of group-specific antibodies against VP7 protein of bluetongue virus in multiple species

Bluetongue virus (BTV), the causative agent of bluetongue disease infects many domestic and wild ruminants. In the present study, colloidal gold nanoparticle-based lateral flow immunochromatography assay (LFIA) was developed to detect the group-specific antibodies to BTV in serum samples of sheep, goats, cattle, and camel. The recombinant VP7 protein of BTV conjugated to colloidal gold nanoparticles (GNPs) was used as a detector reagent. Recombinant streptococcal protein G and monoclonal antibody to BTV group-specific antigen were immobilized as the test and the control line, respectively on a nitrocellulose membrane. The protein G could capture the specific antibodies to BTV present in the serum of multiple ruminant species susceptible to BTV in a common test format and could eliminate the requirement of multiple anti-species antibodies. Upon addition of serum sample, GNP-rVP7 protein-serum complex migrated laterally onto the strip via capillary action and results were analyzed based on appearance of red colour band at test and control line. Serum samples (n = 481) of sheep, goats, cattle, and camel segregated as positive and negative by the commercial competitive-ELISA (c-ELISA) kit were tested in the fabricated LFIA strips to analyze the performance of the assay. In comparison with c-ELISA, the relative diagnostic sensitivity (DSn) of 95.2% with 91.6–97.6 (95%)) confidence interval and relative diagnostic specificity (DSp) of 99.6% 97.8–100.0 (95%) confidence interval were obtained for the optimized LFIA. The agreement between the LFIA and the c-ELISA was excellent as indicated by the kappa coefficient value of 0.949 (SE = 0.0142) with 0.9219 to 0.9779 (95%) confidence interval. The recombinant protein G based LFIA is a sensitive, specific, rapid, one-step test that can be used in the field or poorly equipped laboratories for serological diagnosis and serosurveillance of bluetongue in multiple susceptible species.

A Screening for Virus Infections in Eight Herds of Semi-domesticated Eurasian Tundra Reindeer (Rangifer tarandus tarandus) in Norway, 2013-2018.

Background: Previous serological screenings have indicated that Eurasian semi-domesticated tundra reindeer (Rangifer tarandus tarandus) in Finnmark, Northern Norway, are exposed to alphaherpesvirus, gammaherpesvirus and pestivirus. Alphaherpesvirus (i.e., Cervid herpesvirus 2; CvHV2) has been identified as the transmissible component of infectious keratoconjunctivitis (IKC). Limited knowledge exists on the presence and prevalence of virus infections in other herding regions in Norway, which are hosting ~67,000 semi-domesticated reindeer and have contact with other species and populations of wildlife and livestock than those present in Finnmark.Methods: Blood samples (n = 618) were obtained over five winter seasons (2013–2018), from eight different herds representing summer pasture districts in Tana, Lakselv, Tromsø, Lødingen, Hattfjelldal, Fosen, Røros, and Filefjell, distributed from North to South of the reindeer herding regions in Norway. Blood samples were investigated for specific antibodies against five viral pathogen groups, alphaherpesvirus, gammaherpesvirus (viruses in the malignant catarrhal fever group; MCFV), pestivirus, bluetongue virus (BTV), and Schmallenberg virus (SBV), by using commercial multispecies serological tests (ELISA). In addition, swab samples obtained from the nasal mucosal membrane from 486 reindeer were investigated by PCR for parapoxvirus-specific DNA.Results: Antibodies against aphaherpesvirus and MCFV were found in all eight herds, with a total prevalence of 42% (range 21–62%) and 11% (range 2–15%), respectively. Anti-Pestivirus antibodies were detected in five of eight herds, with a total prevalence of 19% (range 0–52%), with two of the herds having a particularly high seroprevalence. Antibodies against BTV or SBV were not detected in any of the animals. Parapoxvirus-specific DNA was detected in two animals representing two different herds in Finnmark.Conclusions: This study confirmed that alphaherpesvirus and MCFV are enzootic throughout the geographical reindeer herding regions in Norway, and that pestivirus is present in most of the herds, with varying seroprevalence. No exposure to BTV and SBV was evident. This study also indicated that semi-domesticated reindeer in Finnmark are exposed to parapoxvirus without disease outbreaks being reported from this region.

Pentavalent Disabled Infectious Single Animal (DISA)/DIVA Vaccine Provides Protection in Sheep and Cattle against Different Serotypes of Bluetongue Virus.

Bluetongue (BT) is a midge-borne OIE-notifiable disease of ruminants caused by the bluetongue virus (BTV). There are at least 29 BTV serotypes as determined by serum neutralization tests and genetic analyses of genome segment 2 encoding serotype immunodominant VP2 protein. Large parts of the world are endemic for multiple serotypes. The most effective control measure of BT is vaccination. Conventionally live-attenuated and inactivated BT vaccines are available but have their specific pros and cons and are not DIVA compatible. The prototype Disabled Infectious Single Animal (DISA)/DIVA vaccine based on knockout of NS3/NS3a protein of live-attenuated BTV, shortly named DISA8, fulfills all criteria for modern veterinary vaccines of sheep. Recently, DISA8 with an internal in-frame deletion of 72 amino acid codons in NS3/NS3a showed a similar ideal vaccine profile in cattle. Here, the DISA/DIVA vaccine platform was applied for other serotypes, and pentavalent DISA/DIVA vaccine for “European” serotypes 1, 2, 3, 4, 8 was studied in sheep and cattle. Protection was demonstrated for two serotypes, and neutralization Ab titers indicate protection against other included serotypes. The DISA/DIVA vaccine platform is flexible in use and generates monovalent and multivalent DISA vaccines to combat specific field situations with respect to Bluetongue.

Oviposition of Culicoides insignis (Diptera: Ceratopogonidae) under laboratory conditions with notes on the developmental life history traits of its immature stages

BackgroundCulicoides insignis is a confirmed vector of bluetongue virus (BTV) throughout the American tropics and a possible vector of epizootic hemorrhagic disease virus (EHDV) in Florida. Despite its importance, fundamental information on the biology and ecology of this vector species is lacking. In this study, we examined the oviposition of C. insignis under laboratory conditions, monitored the development of immature stages and attempted colonization of this species.MethodsLive C. insignis females were collected from the field using CDC-UV-LED traps, allowed to blood-feed on live chicken and given various natural substrates for oviposition in two-choice assays. The eggs deposited were transferred to 0.3% agar slants, and the hatched larvae were provided a diet of Panagrellus redivivus Linnaeus nematodes and the development of all immature stages was monitored.ResultsCulicoides insignis females exhibited an overall oviposition preference for dishes containing mud from their larval habitat as gravid females deposited a significantly higher number of eggs on these dishes (35.3 eggs/female) than on controls (17.7 eggs/female). The ovipositing females also deposited a higher percentage of eggs on substrates with habitat mud and other organically enriched muds (≥ 75.2%) compared to controls (31.0%). The larvae developed successfully to adulthood on the nematode diet, exhibiting high overall larval survival rates (85.0%). Sex ratios of the F1 generation were male biased, approximately 3:1 (male:female). Captive mating could not be induced in the F1 adults.ConclusionsMud from the larval habitat and other organically enriched muds provide strong oviposition cues to C. insignis under laboratory conditions. Further studies will be needed to identify the key biotic/abiotic factors influencing midge oviposition in the field. The agar/nematode method is effective for the rearing of C. insignis larvae. However, further studies will be needed to address the issue of male-biased sex ratios in the progeny and to examine the mating habits/cues of C. insignis in nature, which may provide clues towards inducing captive mating in the F1 adults.Graphical abstract

SEROEPIDEMIOLOGICAL SURVEY OF CAPRINE ARTHRITIS ENCEPHALITIS VIRUS AND BLUETONGUE VIRUS IN GOAT HERDS FROM PARAIBA STATE, NORTHEASTERN BRAZIL

Caprine arthritis encephalitis (CAE) and bluetongue (BT) are viral diseases affecting goats and sheep, respectively, worldwide. Both diseases are characterized by being slow and progressive, causing a variety of clinical signs and determining economic and production losses. The objective of this study was to evaluate the seroprevalence of CAE and BT and its associated factors in dairy and beef goat herds in the state of Paraíba, Northeast Brazil. In total, 6.4% and 1.8% of the animals were positive for caprine arthritis encephalitis and bluetongue, respectively, by Agarose Gel Immunodiffusion assay (AGID). No significant statistical association was found between diseases and risk factors, herd size, age, gender, and type of management in most animals evaluated. The risk factors, production system and grazing of multispecies can influence the prevalence of viral infections in goats in the state of Paraíba.

Advances in the release mechanisms of bluetongue virus

Bluetongue virus (BTV) causes Bluetongue (BT) of ruminants vectored by culicoides midges. It is also a classic model for studying the release mechanism of non-enveloped virus. This review begins with the infection and assembly of BTV, then summarizes the advances of different ways of releasing BTV. This includes BTV-induced autophagy and the release as extracellular vesicles via multivesicular bodies, BTV-induced apoptosis and the lytic release, as well as different pathways of release through budding via plasma membrane. The regulatory mechanisms of NS3 which is a key non-structural protein during the release of BTV are also discussed, providing a basis for further understanding the molecular mechanisms underpinning the infection, proliferation and release of BTV.

Modelling bluetongue risk in Kazakhstan

BackgroundBluetongue is a serious disease of ruminants caused by the bluetongue virus (BTV). BTV is transmitted by biting midges (Culicoides spp.). Serological evidence from livestock and the presence of at least one competent vector species of Culicoides suggests that transmission of BTV is possible and may have occurred in Kazakhstan.MethodsWe estimated the risk of transmission using a mathematical model of the reproduction number R0 for bluetongue. This model depends on livestock density and climatic factors which affect vector density. Data on climate and livestock numbers from the 2466 local communities were used. This, together with previously published model parameters, was used to estimate R0 for each month of the year. We plotted the results on isopleth maps of Kazakhstan using interpolation to smooth the irregular data. We also mapped the estimated proportion of the population requiring vaccination to prevent outbreaks of bluetongue.ResultsThe results suggest that transmission of bluetongue in Kazakhstan is not possible in the winter from October to March. Assuming there are vector-competent species of Culicoides endemic in Kazakhstan, then low levels of risk first appear in the south of Kazakhstan in April before spreading north and intensifying, reaching maximum levels in northern Kazakhstan in July. The risk declined in September and had disappeared by October.ConclusionThese results should aid in surveillance efforts for the detection and control of bluetongue in Kazakhstan by indicating where and when outbreaks of bluetongue are most likely to occur. The results also indicate where vaccination efforts should be focussed to prevent outbreaks of disease.Graphical abstract

DNA barcode identification and molecular detection of bluetongue virus in Culicoides biting midges (Diptera: Ceratopogonidae) from western Thailand

Biting midges of the genus Culicoides Latreille are biological vectors of bluetongue virus (BTV), a member of family Reoviridae, genus Orbivirus. About 30 species of Culicoides have been identified as competent BTV vectors worldwide. Even though high seroprevalence of BTV has been reported among livestock ruminants from western Thailand, the Culicoides species which contribute to BTV transmission remain unclear. In the present study, Culicoides were collected from eight sampling sites, located in two BTV prevalent provinces in western Thailand. Adult Culicoides were identified using wing morphology and cytochrome c oxidase subunit I (COI) mtDNA molecular marker. A total of 9,677 Culicoides specimens belonging to 7 subgenera, 3 species groups, and 23 species were identified. After comparing sequencing results with available data from GenBank, COI sequences of five species were reported for the first time from Thailand. The most abundant potential BTV vector species collected were C. peregrinus, followed by C. orientalis, C. imicola, C. oxystoma, and C. fulvus. Out of 72 Culicoides pools, 9 pools (4 from C. orientalis, 2 from C. imicola, 2 from C. oxystoma, and 1 from C. fulvus) were positive by BTV RT-PCR analyses. These results are new to Culicoides BTV vector knowledge in Thailand and will contribute to further BTV studies in this particular region.

Orbiviruses in biting midges and mosquitoes from the Zambezi region, Namibia.

The genus Orbivirus includes a variety of pathogenic viruses that are transmitted by biting midges, mosquitoes and ticks. Some of the economically most relevant orbiviruses are endemic to Namibia, like the livestock-pathogenic Bluetongue and African horse sickness viruses. Here, we assessed the diversity of orbiviruses circulating in the Zambezi region of north-eastern Namibia. A total of 10 250 biting midges and 10 206 mosquitoes were collected and screened for orbivirus infections. We identified Palyam virus (PALV) in a pool of biting midges (Culicoides sp.) sampled in the Wuparo Conservancy and three strains of Corriparta virus (CORV) in Culex sp. mosquitoes sampled in Mudumu National Park and the Mashi Conservancy. This is, to our knowledge, the first detection of PALV and CORV in Namibia. Both viruses infect vertebrates but only PALV has been reported to cause disease. PALV can cause foetal malformations and abortions in ruminants. Furthermore, a novel orbivirus, related to Kammavanpettai virus from India and Umatilla virus from North America, was discovered in biting midges (Culicoides sp.) originating from Mudumu National Park and tentatively named Mudumu virus (MUMUV). Complete genomes of PALV, CORV and MUMUV were sequenced and genetically characterized. The Namibian CORV strain showed 24.3 % nucleotide divergence in its subcore shell gene to CORV strains from Australia, indicating that African CORV variants vary widely from their Australian relatives. CORV was isolated in cell culture and replicated to high titres in mosquito and duck cells. No growth was found in rodent and primate cells. The data presented here show that diverse orbiviruses are endemic to the Zambezi region. Further studies are needed to assess their effects on wildlife and livestock.

Development of real-time RT-qPCR assays for the typing of two novel bluetongue virus genotypes derived from sheeppox vaccine

Previously, we reported the detection of two novel bluetongue virus (BTV) strains (SPvvvv/02 and SPvvvv/03), possibly representing new BTV genotypes, in a batch of sheeppox vaccine. We developed type-specific RT-qPCR assays (targeting genome segment 2) for these two new BTV strains. The limit of detection of both assays was 10 genome copies/μl and no cross-reactivity with other BTV genotypes was observed. The performance of three other BTV group-specific diagnostic assays was also tested against the putative novel genotypes. RT-qPCR assays targeting BTV segment 9 and 10 detected both strains (SPvvvv/02 and SPvvvv/03) whereas a BTV segment 1 RT-qPCR assay was unable to detect either BTV strain. The work presented here expands upon the current repertoire of RT-qPCR assays for BTV genotype determination.

RNA Origami: Packaging a Segmented Genome in Orbivirus Assembly and Replication.

Understanding how viruses with multi-segmented genomes incorporate one copy of each segment into their capsids remains an intriguing question. Here, we review our recent progress and describe the advancements made in understanding the genome packaging mechanism of a model nonenveloped virus, Bluetongue virus (BTV), with a 10-segment (S1–S10) double-strand RNA (dsRNA) genome. BTV (multiple serotypes), a member of the Orbivirus genus in the Reoviridae family, is a notable pathogen for livestock and is responsible for significant economic losses worldwide. This has enabled the creation of an extensive set of reagents and assays, including reverse genetics, cell-free RNA packaging, and bespoke bioinformatics approaches, which can be directed to address the packaging question. Our studies have shown that (i) UTRs enable the conformation of each segment necessary for the next level of RNA–RNA interaction; (ii) a specific order of intersegment interactions leads to a complex RNA network containing all the active components in sorting and packaging; (iii) networked segments are recruited into nascent assembling capsids; and (iv) select capsid proteins might be involved in the packaging process. The key features of genome packaging mechanisms for BTV and related dsRNA viruses are novel and open up new avenues of potential intervention.

Genomic Analysis Illustrated a Single Introduction and Evolution of Israeli Bluetongue Serotype 8 Virus Population 2008–2019

Outbreaks of the European Bluetongue virus (BTV) serotype 8 (BTV-8), which are characterized by activity cycles separated by years of inactivity, may be influenced by genetic changes of the virus or by herd immunity. BTV activity in Israel is characterized by similar dynamics, but differs from European countries in its vector population, environmental conditions, and lack of cattle vaccination against this serotype. Comparison of these two geographical systems and characterization of their epidemiological connection is therefore of high interest in-order to better understand the factors influencing BTV-8 evolution. BTV-8, closely related to the European strain, was introduced to Israel in 2008. It was at the center of BT outbreaks in 2010 and 2015–2016 and thereafter was lastly isolated in Israel in 2019. We performed genetic analyses of twelve BTV-8 Israeli strains isolated between 2008 and 2019 and compared them with published sequences of BTV-8 isolated in other countries. The analysis revealed a single introduction of BTV-8 into Israel and thereafter extensive occurrence of genomic drifts and multiple reassortments with local BTV strains. Comparison of the Israeli and Cypriot BTV-8 from 2015 to 2016 suggests transmission of the virus between the two countries and a separate and parallel development from European or other Israeli BTV-8 strains. The parallel development of other BTV-8 strains was demonstrated by the identification of the Israeli BTV-8 ISR-1194/1/19 strain, which exhibited common origin with reassorted Israeli BTV-8 strains from 2010 and additional reassortment of seven segments. In order to reveal the source of BTV-8 introduction into Israel we performed BEAST analysis which showed that a probable common ancestor for both European and Israeli BTV-8 presumably existed in 2003–2004. In 2019, a possible new introduction occurred in Israel, where a novel BTV-8 strain was detected, sharing ~95% identity by segments 2 and 6 with Nigerian BTV-8NIG1982/07 and European–Middle Eastern strains. The results of the study indicate that Israel and neighboring countries consist a separate environmental and evolutionary system, distinct from European ones.

Establishment of an Indirect Enzyme-linked Immunosorbent Assay for Detection of the NS4 Protein of Bluetongue Virus

Establishment of an Indirect Enzyme-linked Immunosorbent Assay for Detection of the NS4 Protein of Bluetongue Virus

Identification of a BTV-Strain-Specific Single Gene That Increases Culicoides Vector Infection Rate.

Since the 2000s, the distribution of bluetongue virus (BTV) has changed, leading to numerous epidemics and economic losses in Europe. Previously, we found a BTV-4 field strain with a higher infection rate of a Culicoides vector than a BTV-1 field strain has. We reverse-engineered parental BTV-1 and BTV-4 strains and created BTV-1/BTV-4 reassortants to elucidate the influence of individual BTV segments on BTV replication in both C. sonorensis midges and in KC cells. Substitution of segment 2 (Seg-2) with Seg-2 from the rBTV-4 significantly increased vector infection rate in reassortant BTV-14S2 (30.4%) in comparison to reverse-engineered rBTV-1 (1.0%). Replacement of Seg-2, Seg-6 and Seg-7 with those from rBTV-1 in reassortant BTV-41S2S6S7 (2.9%) decreased vector infection rate in comparison to rBTV-4 (30.2%). However, triple-reassorted BTV-14S2S6S7 only replicated to comparatively low levels (3.0%), despite containing Seg-2, Seg-6 and Seg-7 from rBTV-4, indicating that vector infection rate is influenced by interactions of multiple segments and/or host-mediated amino acid substitutions within segments. Overall, these results demonstrated that we could utilize reverse-engineered viruses to identify the genetic basis influencing BTV replication within Culicoides vectors. However, BTV replication dynamics in KC cells were not suitable for predicting the replication ability of these virus strains in Culicoides midges.