RNA genome packaging and capsid assembly of bluetongue virus visualized in host cells.

Unlike those of double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), and ssRNA viruses, the mechanism of genome packaging of dsRNA viruses is poorly understood. Here, we combined the techniques of high-resolution cryoelectron microscopy (cryo-EM), cellular cryoelectron tomography (cryo-ET), and structure-guided mutagenesis to investigate genome packaging and capsid assembly of bluetongue virus (BTV), a member of the Reoviridae family of dsRNA viruses. A total of eleven assembly states of BTV capsid were captured, with resolutions up to 2.8 Å, with most visualized in the host cytoplasm. ATPase VP6 was found underneath the vertices of capsid shell protein VP3 as an RNA-harboring pentamer, facilitating RNA packaging. RNA packaging expands the VP3 shell, which then engages middle- and outer-layer proteins to generate infectious virions. These revealed "duality" characteristics of the BTV assembly mechanism reconcile previous contradictory co-assembly and core-filling models and provide insights into the mysterious RNA packaging and capsid assembly of Reoviridae members and beyond.

The effect of pH on the structure of Bluetongue virus VP5.

The unenveloped Bluetongue virus capsid comprises several structural layers, the inner two comprising a core, which assembles before addition of the outer proteins, VP2 and VP5. Two symmetric trimers of VP5 fit like pegs into two distinct pits on the core and undergo pH conformational changes in the context of the virus, associated with cell entry. Here we show that in isolation VP5 alone undergoes essentially the same changes with pH and confirm a helical transition, indicating that VP5 is a motor during cell entry. In the absence of VP5 the two pits on the core differ from each other, presumably due to the asymmetric underlying structure of VP3, the innermost capsid protein. On insertion of VP5 these pits become closely similar and remain similar at low pH whilst VP5 is present. This natural asymmetry presumably destabilises the attachment of VP5, facilitating ejection upon low pH, membrane penetration and cell entry.

Whole-transcriptome analyses of ovine lung microvascular endothelial cells infected with bluetongue virus.

Bluetongue virus (BTV) infection induces profound and intricate changes in the transcriptional profile of the host to facilitate its survival and replication. However, there have been no whole-transcriptome studies on ovine lung microvascular endothelial cells (OLMECs) infected with BTV. In this study, we comprehensively analysed the whole-transcriptome sequences of BTV-1 serotype-infected and mock-infected OLMECs and subsequently performed bioinformatics differential analysis. Our analysis revealed 1215 differentially expressed mRNA transcripts, 82 differentially expressed long noncoding RNAs (lncRNAs) transcripts, 63 differentially expressed microRNAs (miRNAs) transcripts, and 42 differentially expressed circular RNAs (circRNAs) transcripts. Annotation from Gene Ontology, enrichment from the Kyoto Encyclopedia of Genes and Genomes, and construction of endogenous competing RNA network analysis revealed that the differentially expressed RNAs primarily participated in viral sensing and signal transduction pathways, antiviral and immune responses, inflammation, and extracellular matrix (ECM)-related pathways. Furthermore, protein‒protein interaction network analysis revealed that BTV may regulate the conformation of ECM receptor proteins and change their biological activity through a series of complex mechanisms. Finally, on the basis of real-time fluorescence quantitative polymerase chain reaction results, the expression trends of the differentially expressed RNA were consistent with the whole-transcriptome sequencing data, such as downregulation of the expression of COL4A1, ITGA8, ITGB5, and TNC and upregulation of the expression of CXCL10, RNASEL, IRF3, IRF7, and IFIHI. This study provides a novel perspective for further investigations of the mechanism of the ECM in the BTV-host interactome and the pathogenesis of lung microvascular endothelial cells.

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

Bluetongue 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.

"Frozen evolution" of an RNA virus suggests accidental release as a potential cause of arbovirus re-emergence.

The mechanisms underlying virus emergence are rarely well understood, making the appearance of outbreaks largely unpredictable. Bluetongue virus serotype 8 (BTV-8), an arthropod-borne virus of ruminants, emerged in livestock in northern Europe in 2006, spreading to most European countries by 2009 and causing losses of billions of euros. Although the outbreak was successfully controlled through vaccination by early 2010, puzzlingly, a closely related BTV-8 strain re-emerged in France in 2015, triggering a second outbreak that is still ongoing. The origin of this virus and the mechanisms underlying its re-emergence are unknown. Here, we performed phylogenetic analyses of 164 whole BTV-8 genomes sampled throughout the two outbreaks. We demonstrate consistent clock-like virus evolution during both epizootics but found negligible evolutionary change between them. We estimate that the ancestor of the second outbreak dates from the height of the first outbreak in 2008. This implies that the virus had not been replicating for multiple years prior to its re-emergence in 2015. Given the absence of any known natural mechanism that could explain BTV-8 persistence over this long period without replication, we hypothesise that the second outbreak could have been initiated by accidental exposure of livestock to frozen material contaminated with virus from approximately 2008. Our work highlights new targets for pathogen surveillance programmes in livestock and illustrates the power of genomic epidemiology to identify pathways of infectious disease emergence.

The use of path analysis to determine effects of environmental factors on the adult seasonality of Culicoides (Diptera: Ceratopogonidae) vector species in Spain.

Culicoides biting midges (Diptera: Ceratopogonidae) are the main vectors of livestock diseases such as bluetongue (BT) which mainly affect sheep and cattle. In Spain, bluetongue virus (BTV) is transmitted by several Culicoides taxa, including Culicoides imicola, Obsoletus complex, Culicoides newsteadi and Culicoides pulicaris that vary in seasonality and distribution, affecting the distribution and dynamics of BT outbreaks. Path analysis is useful for separating direct and indirect, biotic and abiotic determinants of species' population performance and is ideal for understanding the sensitivity of adult Culicoides dynamics to multiple environmental drivers. Start, end of season and length of overwintering of adult Culicoides were analysed across 329 sites in Spain sampled from 2005 to 2010 during the National Entomosurveillance Program for BTV with path analysis, to determine the direct and indirect effects of land use, climate and host factor variables. Culicoides taxa had species-specific responses to environmental variables. While the seasonality of adult C. imicola was strongly affected by topography, temperature, cover of agro-forestry and sclerophyllous vegetation, rainfall, livestock density, photoperiod in autumn and the abundance of Culicoides females, Obsoletus complex species seasonality was affected by land-use variables such as cover of natural grassland and broad-leaved forest. Culicoides female abundance was the most explanatory variable for the seasonality of C. newsteadi, while C. pulicaris showed that temperature during winter and the photoperiod in November had a strong effect on the start of the season and the length of overwinter period of this species. These results indicate that the seasonal vector-free period (SVFP) in Spain will vary between competent vector taxa and geographic locations, dependent on the different responses of each taxa to environmental conditions.

Wolbachia wAlbB inhibits bluetongue and epizootic hemorrhagic fever viruses in Culicoides midge cells.

Culicoides midges are hematophagous insects that transmit arboviruses of veterinary importance. These viruses include bluetongue virus (BTV) and epizootic hemorrhagic fever virus (EHDV). The endosymbiont Wolbachia pipientis Hertig spreads rapidly through insect host populations and has been demonstrated to inhibit viral pathogen transmission in multiple mosquito vectors. Here, we have demonstrated a replication inhibitory effect on BTV and EHDV in a Wolbachia (wAlbB strain)-infected Culicoides sonorensis Wirth and Jones W8 cell line. Viral replication was significantly reduced by day 5 for BTV and by day 2 for EHDV as detected by real-time polymerase chain reaction (RT-qPCR) of the non-structural NS3 gene of both viruses. Evaluation of innate cellular immune responses as a cause of the inhibitory effect showed responses associated with BTV but not with EHDV infection. Wolbachia density also did not play a role in the observed pathogen inhibitory effects, and an alternative hypothesis is suggested. Applications of Wolbachia-mediated pathogen interference to impact disease transmission by Culicoides midges are discussed.

A Calcium Sensor Discovered in Bluetongue Virus Nonstructural Protein 2 Is Critical for Virus Replication.

Many viruses use specific viral proteins to bind calcium ions (Ca2+) for stability or to modify host cell pathways; however, to date, no Ca2+ binding protein has been reported in bluetongue virus (BTV), the causative agent of bluetongue disease in livestock. Here, using a comprehensive bioinformatics screening, we identified a putative EF-hand-like Ca2+ binding motif in the carboxyl terminal region of BTV nonstructural phosphoprotein 2 (NS2). Subsequently, using a recombinant NS2, we demonstrated that NS2 binds Ca2+ efficiently and that Ca2+ binding was perturbed when the Asp and Glu residues in the motif were substituted by alanine. Using circular dichroism analysis, we found that Ca2+ binding by NS2 triggered a helix-to-coil secondary structure transition. Further, cryo-electron microscopy in the presence of Ca2+ revealed that NS2 forms helical oligomers which, when aligned with the N-terminal domain crystal structure, suggest an N-terminal domain that wraps around the C-terminal domain in the oligomer. Further, an in vitro kinase assay demonstrated that Ca2+ enhanced the phosphorylation of NS2 significantly. Importantly, mutations introduced at the Ca2+ binding site in the viral genome by reverse genetics failed to allow recovery of viable virus, and the NS2 phosphorylation level and assembly of viral inclusion bodies (VIBs) were reduced. Together, our data suggest that NS2 is a dedicated Ca2+ binding protein and that calcium sensing acts as a trigger for VIB assembly, which in turn facilitates virus replication and assembly.IMPORTANCE After entering the host cells, viruses use cellular host factors to ensure a successful virus replication process. For replication in infected cells, members of the Reoviridae family form inclusion body-like structures known as viral inclusion bodies (VIB) or viral factories. Bluetongue virus (BTV) forms VIBs in infected cells through nonstructural protein 2 (NS2), a phosphoprotein. An important regulatory factor critical for VIB formation is phosphorylation of NS2. In our study, we discovered a characteristic calcium-binding EF-hand-like motif in NS2 and found that the calcium binding preferentially affects phosphorylation level of the NS2 and has a role in regulating VIB assembly.

Virus-induced autophagic degradation of STAT2 as a mechanism for interferon signaling blockade.

The mammalian interferon (IFN) signaling pathway is a primary component of the innate antiviral response, and viral pathogens have evolved multiple mechanisms to antagonize this pathway and to facilitate infection. Bluetongue virus (BTV), an orbivirus of the Reoviridae family, is transmitted by midges to ruminants and causes a disease that produces important economic losses and restriction to animal trade and is of compulsory notification to the World Organization for Animal Health (OIE). Here, we show that BTV interferes with IFN-I and IFN-II responses in two ways, by blocking STAT1 phosphorylation and by degrading STAT2. BTV-NS3 protein, which is involved in virion egress, interacts with STAT2, and induces its degradation by an autophagy-dependent mechanism. This STAT2 degradative process requires the recruitment of an E3-Ub-ligase to NS3 as well as NS3 K63 polyubiquitination. Taken together, our study identifies a new mechanism by which a virus degrades STAT2 for IFN signaling blockade, highlighting the diversity of mechanisms employed by viruses to subvert the IFN response.

Hsp90 Chaperones Bluetongue Virus Proteins and Prevents Proteasomal Degradation.

The molecular chaperone machinery is important for the maintenance of protein homeostasis within the cells. The principle activities of the chaperone machinery are to facilitate protein folding and organize conformationally dynamic client proteins. Prominent among the members of the chaperone family are heat shock protein 70 (Hsp70) and 90 (Hsp90). Like cellular proteins, viral proteins depend upon molecular chaperones to mediate their stabilization and folding. Bluetongue virus (BTV), which is a model system for the Reoviridae family, is a nonenveloped arbovirus that causes hemorrhagic disease in ruminants. This constitutes a significant burden upon animals of commercial significance, such as sheep and cattle. Here, for the first time, we examined the role of chaperone proteins in the viral lifecycle of BTV. Using a combination of molecular, biochemical, and microscopic techniques, we examined the function of Hsp90 and its relevance to BTV replication. We demonstrate that Hsp70, the chaperone that is commonly usurped by viral proteins, does not influence virus replication, while Hsp90 activity is important for virus replication by stabilizing BTV proteins and preventing their degradation via the ubiquitin-proteasome pathway. To our knowledge this is the first report showing the involvement of Hsp90 as a modulator of BTV infection.IMPORTANCE Protein chaperones are instrumental for maintaining protein homeostasis, enabling correct protein folding and organization; prominent members include heat shock proteins 70 and 90. Virus infections place a large burden on this homeostasis. Identifying and understanding the underlying mechanisms that facilitate Bluetongue virus replication and spread through the usurpation of host factors is of primary importance for the development of intervention strategies. Our data identify and show that heat shock protein 90, but not heat shock protein 70, stabilizes bluetongue virus proteins, safeguarding them from proteasomal degradation.

Novel Function of Bluetongue Virus NS3 Protein in Regulation of the MAPK/ERK Signaling Pathway.

Bluetongue virus (BTV) is an arbovirus transmitted by blood-feeding midges to a wide range of wild and domestic ruminants. In this report, we showed that BTV, through its nonstructural protein NS3 (BTV-NS3), is able to activate the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway, as assessed by phosphorylation levels of ERK1/2 and the translation initiation factor eukaryotic translation initiation factor 4E (eIF4E). By combining immunoprecipitation of BTV-NS3 and mass spectrometry analysis from both BTV-infected and NS3-transfected cells, we identified the serine/threonine-protein kinase B-Raf (BRAF), a crucial player in the MAPK/ERK pathway, as a new cellular interactor of BTV-NS3. BRAF silencing led to a significant decrease in the MAPK/ERK activation by BTV, supporting a model wherein BTV-NS3 interacts with BRAF to activate this signaling cascade. This positive regulation acts independently of the role of BTV-NS3 in counteracting the induction of the alpha/beta interferon response. Furthermore, the intrinsic ability of BTV-NS3 to bind BRAF and activate the MAPK/ERK pathway is conserved throughout multiple serotypes/strains but appears to be specific to BTV compared to other members of Orbivirus genus. Inhibition of MAPK/ERK pathway with U0126 reduced viral titers, suggesting that BTV manipulates this pathway for its own replication. Altogether, our data provide molecular mechanisms that unravel a new essential function of NS3 during BTV infection.IMPORTANCE Bluetongue virus (BTV) is responsible of the arthropod-borne disease bluetongue (BT) transmitted to ruminants by blood-feeding midges. In this report, we found that BTV, through its nonstructural protein NS3 (BTV-NS3), interacts with BRAF, a key component of the MAPK/ERK pathway. In response to growth factors, this pathway promotes cell survival and increases protein translation. We showed that BTV-NS3 enhances the MAPK/ERK pathway, and this activation is BRAF dependent. Treatment of MAPK/ERK pathway with the pharmacologic inhibitor U0126 impairs viral replication, suggesting that BTV manipulates this pathway for its own benefit. Our results illustrate, at the molecular level, how a single virulence factor has evolved to target a cellular function to increase its viral replication.

Dynamic network approach for the modelling of genomic sub-complexes in multi-segmented viruses.

Viruses with segmented genomes, including pathogens such as influenza virus, Rotavirus and Bluetongue virus (BTV), face the collective challenge of packaging their genetic material in terms of the correct number and types of segments. Here we develop a novel network approach to predict RNA-RNA interactions between different genomic segments. Experimental data on RNA complex formation in the multi-segmented BTV genome are used to establish proof-of-concept of this technique. In particular, we show that trans interactions between segments occur at multiple specific sites, termed segment assortment signals (SASs) that are dispersed across each segment. In order to validate the putative trans acting networks, we used various biochemical and molecular techniques which confirmed predictions of the RNA network approach. A combination of mutagenesis and reverse genetics systems revealed that the RNA-RNA interacting sites identified are indeed responsible for segment assortment and complex formation, which are essential criteria for genome packaging. This paves the way for their exploitation as novel types of drug target, either to inhibit assembly, or for designing defective interfering particles containing an incomplete set of genomic segments.

A low-passage insect-cell isolate of bluetongue virus uses a macropinocytosis-like entry pathway to infect natural target cells derived from the bovine host.

Bluetongue virus (BTV) causes an economically important disease in domestic and wildlife ruminants and is transmitted by Culicoides biting midges. In ruminants, BTV has a wide cell tropism that includes endothelial cells of vascular and lymphatic vessels as important cell targets for virus replication, and several cell types of the immune system including monocytes, macrophages and dendritic cells. Thus, cell-entry represents a particular challenge for BTV as it infects many different cell types in widely diverse vertebrate and invertebrate hosts. Improved understanding of BTV cell-entry could lead to novel antiviral approaches that can block virus transmission from cell to cell between its invertebrate and vertebrate hosts. Here, we have investigated BTV cell-entry using endothelial cells derived from the natural bovine host (BFA cells) and purified whole virus particles of a low-passage, insect-cell isolate of a virulent strain of BTV-1. Our results show that the main entry pathway for infection of BFA cells is dependent on actin and dynamin, and shares certain characteristics with macropinocytosis. The ability to use a macropinocytosis-like entry route could explain the diverse cell tropism of BTV and contribute to the efficiency of transmission between vertebrate and invertebrate hosts.

Interaction between a Unique Minor Protein and a Major Capsid Protein of Bluetongue Virus Controls Virus Infectivity.

Among the Reoviridae family of double-stranded RNA viruses, only members of the Orbivirus genus possess a unique structural protein, termed VP6, within their particles. Bluetongue virus (BTV), an important livestock pathogen, is the prototype Orbivirus BTV VP6 is an ATP-dependent RNA helicase, and it is indispensable for virus replication. In the study described in this report, we investigated how VP6 might be recruited to the virus capsid and whether the BTV structural protein VP3, which forms the internal layer of the virus capsid core, is involved in VP6 recruitment. We first demonstrated that VP6 interacts with VP3 and colocalizes with VP3 during capsid assembly. A series of VP6 mutants was then generated, and in combination with immunoprecipitation and size exclusion chromatographic analyses, we demonstrated that VP6 directly interacts with VP3 via a specific region of the C-terminal portion of VP6. Finally, using our reverse genetics system, mutant VP6 proteins were introduced into the BTV genome and interactions between VP6 and VP3 were shown in a live cell system. We demonstrate that BTV strains possessing a mutant VP6 are replication deficient in wild-type BSR cells and fail to recruit the viral replicase complex into the virus particle core. Taken together, these data suggest that the interaction between VP3 and VP6 could be important in the packaging of the viral genome and early stages of particle formation.IMPORTANCE The orbivirus bluetongue virus (BTV) is the causative agent of bluetongue disease of livestock, often causing significant economic and agricultural impacts in the livestock industry. In the study described in this report, we identified the essential region and residues of the unique orbivirus capsid protein VP6 which are responsible for its interaction with other BTV proteins and its subsequent recruitment into the virus particle. The nature and mechanism of these interactions suggest that VP6 has a key role in packaging of the BTV genome into the virus particle. As such, this is a highly significant finding, as this new understanding of BTV assembly could be exploited to design novel vaccines and antivirals against bluetongue disease.

Quantifying bluetongue vertical transmission in French cattle from surveillance data.

Bluetongue is a vector-borne disease of ruminants with economic consequences for the livestock industry. Bluetongue virus serotype 8 (BTV-8) caused a massive outbreak in Europe in 2006/2009 and re-emerged in France in 2015. Given the unprecedented epidemiological features of this serotype in cattle, the importance of secondary routes of transmission was reconsidered and transplacental transmission of BTV-8 was demonstrated in naturally and experimentally infected cattle. Here we used surveillance data from the on-going outbreak to quantify BTV-8 vertical transmission in French cattle. We used RT-PCR pre-export tests collected from June to December 2016 on the French territory and developed a catalytic model to disentangle vertical and vector-borne transmission. A series of in silico experiments validated the ability of our framework to quantify vertical transmission provided sufficient prevalence levels. By applying our model to an area selected accordingly, we estimated a probability of vertical transmission of 56% (55.8%, 95% credible interval 41.7-70.6) in unvaccinated heifers infected late in gestation. The influence of this high probability of vertical transmission on BTV-8 spread and persistence should be further investigated.

Phosphoproteomic Analysis Reveals the Importance of Kinase Regulation During Orbivirus Infection.

Bluetongue virus (BTV) causes infections in wild and domesticated ruminants with high morbidity and mortality and is responsible for significant economic losses in both developing and developed countries. BTV serves as a model for the study of other members of the Orbivirus genus. Previously, the importance of casein kinase 2 for BTV replication was demonstrated. To identify intracellular signaling pathways and novel host-cell kinases involved during BTV infection, the phosphoproteome of BTV infected cells was analyzed. Over 1000 phosphosites were identified using mass spectrometry, which were then used to determine the corresponding kinases involved during BTV infection. This analysis yielded protein kinase A (PKA) as a novel kinase activated during BTV infection. Subsequently, the importance of PKA for BTV infection was validated using a PKA inhibitor and activator. Our data confirmed that PKA was essential for efficient viral growth. Further, we showed that PKA is also required for infection of equid cells by African horse sickness virus, another member of the Orbivirus genus. Thus, despite their preference in specific host species, orbiviruses may utilize the same host signaling pathways during their replication.

Country-wide distribution of bluetongue virus with expanding host spectrum and evidence of vector competence in Hungary.

Following the introduction of bluetongue virus type 4 (BTV-4) in 2014, country-wide monitoring of bluetongue (BT) disease was performed to see whether the virus has become enzootic in Hungary. To analyse the epizootiology of BT, over 110,000 samples collected from domestic and wild ruminants were screened for the presence of BTV RNA and virus-specific antibodies using real-time RT-PCR assay and commercial ELISA kit, respectively. During laboratory analysis, specimens collected from 333 (0.8%) cattle, 79 (2.2%) sheep, 4 (0.9%) goats, and 1 (2.3%) mouflon were found to be positive by viral RNA-detection assay. In addition, antibody to BTV was detected in 5.5% (3158/57,250) of cattle, 10.1% (517/5120) of sheep, 40% (116/290) of goat, and 5.6% (16/284) of buffalo origin samples. The majority of positive samples originated from south-western counties; however, 18 out of 19 counties reported cases or antibody prevalence in the examined animals. Genome sequencing of a representative BTV-4 strain from 2015 was also performed. When comparing this strain with the isolate BTV4-HUN2014 detected only a year earlier in Hungary, mutations at 14 sites were identified within the amplified and sequenced genome. Our findings reinforce the need for continued surveillance of BT disease in Hungary. Keywords: reoviridae; orbivirus; cattle; sheep; goat; biting midge.

Seroprevalence of bluetongue and presence of viral antigen and type-specific neutralizing antibodies in goats in Tripura, a state at Indo-Bangladesh border of northeastern India.

Bluetongue (BT) is a notifiable multiple species transboundary viral disease of domestic and wild ruminants. Though the disease is enzootic in India, little is known of the disease burden and prevalent serotypes in Tripura, a hilly state of northeastern India sharing a vast porous border with Bangladesh. A surveillance study was conducted to understand the disease burden in goats in Tripura. Serum (n = 1240) and blood (n = 194) samples were collected during the year 2014 to 2017 from all the eight districts of Tripura. The overall prevalence of BT seroconversion was 47.58% whereas the presence of viral antigen was 20.61% at the individual level. Percent seroconversion was found more (50.47 ± 4.00, CI 41.31 to 49.47) in adult goats in comparison to the younger animals where it was 45.39 ± 2.08, CI 42.63 to 58.31. Presence of neutralizing antibodies in selected serum samples (n = 72) was investigated by serum neutralization test (SNT) against six bluetongue virus (BTV) serotypes and BTV-1 was found as most predominant (65.27%) followed by BTV-16 (26.38%), BTV-10 (20.83%), BTV-9 and 23 (13.88%), and BTV-2 (6.94%). To the best of our knowledge, this is the first study conducted in Tripura to investigate the presence of BTV antigen and type-specific neutralizing antibodies in apparently healthy goats.

The genome of the biting midge Culicoides sonorensis and gene expression analyses of vector competence for bluetongue virus.

BACKGROUND: The new genomic technologies have provided novel insights into the genetics of interactions between vectors, viruses and hosts, which are leading to advances in the control of arboviruses of medical importance. However, the development of tools and resources available for vectors of non-zoonotic arboviruses remains neglected. Biting midges of the genus Culicoides transmit some of the most important arboviruses of wildlife and livestock worldwide, with a global impact on economic productivity, health and welfare. The absence of a suitable reference genome has hindered genomic analyses to date in this important genus of vectors. In the present study, the genome of Culicoides sonorensis, a vector of bluetongue virus (BTV) in the USA, has been sequenced to provide the first reference genome for these vectors. In this study, we also report the use of the reference genome to perform initial transcriptomic analyses of vector competence for BTV. RESULTS: Our analyses reveal that the genome is 189 Mb, assembled in 7974 scaffolds. Its annotation using the transcriptomic data generated in this study and in a previous study has identified 15,612 genes. Gene expression analyses of C. sonorensis females infected with BTV performed in this study revealed 165 genes that were differentially expressed between vector competent and refractory females. Two candidate genes, glutathione S-transferase (gst) and the antiviral helicase ski2, previously recognized as involved in vector competence for BTV in C. sonorensis (gst) and repressing dsRNA virus propagation (ski2), were confirmed in this study. CONCLUSIONS: The reference genome of C. sonorensis has enabled preliminary analyses of the gene expression profiles of vector competent and refractory individuals. The genome and transcriptomes generated in this study provide suitable tools for future research on arbovirus transmission. These provide a valuable resource for these vector lineage, which diverged from other major Dipteran vector families over 200 million years ago. The genome will be a valuable source of comparative data for other important Dipteran vector families including mosquitoes (Culicidae) and sandflies (Psychodidae), and together with the transcriptomic data can yield potential targets for transgenic modification in vector control and functional studies.

Elucidating virus entry using a tetracysteine-tagged virus.

Fluorescent tags constitute an invaluable tool in facilitating a deeper understanding of the mechanistic processes governing virus-host interactions. However, when selecting a fluorescent tag for in vivo imaging of cells, a number of parameters and aspects must be considered. These include whether the tag may affect and interfere with protein conformation or localization, cell toxicity, spectral overlap, photo-stability and background. Cumulatively, these constitute challenges to be overcome. Bluetongue virus (BTV), a member of the Orbivirus genus in the Reoviridae family, is a non-enveloped virus that is comprised of two architecturally complex capsids. The outer capsid, composed of two proteins, VP2 and VP5, together facilitate BTV attachment, entry and the delivery of the transcriptionally active core in the cell cytoplasm. Previously, the significance of the endocytic pathway for BTV entry was reported, although a detailed analysis of the role of each protein during virus trafficking remained elusive due to the unavailability of a tagged virus. Described here is the successful modification, and validation, of a segmented genome belonging to a complex and large capsid virus to introduce tags for fluorescence visualization. The data generated from this approach highlighted the sequential dissociation of VP2 and VP5, driven by decreasing pH during the transition from early to late endosomes, and their retention therein as the virus particles progress along the endocytic pathway. Furthermore, the described tagging technology and methodology may prove transferable and allow for the labeling of other non-enveloped complex viruses.