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Non-structural protein 4 (NS4) of insect-borne and tick-borne orbiviruses is encoded by genome segment 9, from a secondary open reading frame. Though a protein dispensable for bluetongue virus (BTV) replication, it has been shown to counter the interferon response in cells infected with BTV or African horse sickness virus. We further explored the functional role(s) of NS4 proteins of BTV and the tick-borne Great Island virus (GIV). We show that NS4 of BTV or GIV helps an E3L deletion mutant of vaccinia virus to replicate efficiently in interferon-treated cells, further confirming the role of NS4 as an interferon antagonist. Our results indicate that ectopically expressed NS4 of BTV localised with caspase 3 within the nucleus and was found in a protein complex with active caspase 3 in a pull-down assay. Previous studies have shown that pro-apoptotic caspases (including caspase 3) suppress type I interferon response by cleaving mediators involved in interferon signalling. Our data suggest that orbivirus NS4 plays a role in modulating the apoptotic process and/or regulating the interferon response in mammalian cells, thus acting as a virulence factor in pathogenesis.

At least 12 serotypes of 'atypical' bluetongue virus (BTV-25 to BTV-36) have been identified to date. These atypical serotypes fail to infect/replicate in Culicoides -derived cell lines and/or adult Culicoides vectors and hence can no longer be transmitted by these vectors. They appear to be horizontally transmitted from infected to in-contact ruminants, although the route(s) of infection remain to be identified. Viral genome segments 1, 2 and 3 (Seg-1, Seg2 and Seg-3) of BTV-26 were identified as involved in blocking virus replication in KC cells. We have developed Culicoides -specific expression plasmids, which we used in transfected insect cells to assess the stability of viral mRNAs and protein expression from full-length open reading frames of Seg-1, -2 and -3 of BTV-1 (a Culicoides -vectored BTV) or BTV-26. Our results indicate that the blocked replication of BTV-26 in KC cells is not due to an RNAi response, which would lead to rapid degradation of viral mRNAs. A combination of degradation/poor expression and/or modification of the proteins encoded by these segments appears to drive the failure of BTV-26 core/whole virus-particles to assemble and replicate effectively in Culicoides cells.

Bluetongue virus serotypes 1 to 24 are transmitted primarily by infected Culicoides midges, in which they also replicate. However, "atypical" BTV serotypes (BTV-25, -26, -27 and -28) have recently been identified that do not infect and replicate in adult Culicoides , or a Culicoides derived cell line (KC cells). These atypical viruses are transmitted horizontally by direct contact between infected and susceptible hosts (primarily small ruminants) causing only mild clinical signs, although the exact transmission mechanisms involved have yet to be determined. We used reverse genetics to generate a strain of BTV-1 (BTV-1 RG C7 ) which is less virulent, infecting IFNAR (-/-) mice without killing them. Reassortant viruses were also engineered, using the BTV-1 RG C7 genetic backbone, containing individual genome segments derived from BTV-26. These reassortant viruses were used to explore the genetic control of horizontal transmission (HT) in the IFNAR (-/-) mouse model. Previous studies showed that genome segments 1, 2 and 3 restrict infection of Culicoides cells, along with a minor role for segment 7. The current study demonstrates that genome segments 2, 5 and 10 of BTV-26 (coding for proteins VP2, NS1 and NS3/NS3a/NS5, respectively) are individually sufficient to promote HT.

Bluetongue (BT) is a severe and economically important disease of ruminants that is widely distributed around the world, caused by the bluetongue virus (BTV). More than 28 different BTV serotypes have been identified in serum neutralisation tests (SNT), which, along with geographic variants (topotypes) within each serotype, reflect differences in BTV outer-capsid protein VP2. VP2 is the primary target for neutralising antibodies, although the basis for cross-reactions and serological variations between and within BTV serotypes is poorly understood. Recombinant BTV VP2 proteins (rVP2) were expressed in Nicotiana benthamiana , based on sequence data for isolates of thirteen BTV serotypes (primarily from Europe), including three 'novel' serotypes (BTV-25, -26 and -27) and alternative topotypes of four serotypes. Cross-reactions within and between these viruses were explored using rabbit anti-rVP2 sera and post BTV-infection sheep reference-antisera, in I-ELISA (with rVP2 target antigens) and SNT (with reference strains of BTV-1 to -24, -26 and -27). Strong reactions were generally detected with homologous rVP2 proteins or virus strains/serotypes. The sheep antisera were largely serotype-specific in SNT, but more cross-reactive by ELISA. Rabbit antisera were more cross-reactive in SNT, and showed widespread, high titre cross-reactions against homologous and heterologous rVP2 proteins in ELISA. Results were analysed and visualised by antigenic cartography, showing closer relationships in some, but not all cases, between VP2 topotypes within the same serotype, and between serotypes belonging to the same 'VP2 nucleotype'.

Statin derivatives can inhibit the replication of a range of viruses, including hepatitis C virus (HCV, Hepacivirus ), dengue virus ( Flavivirus ), African swine fever virus ( Asfarviridae ) and poliovirus ( Picornaviridae ). We assess the antiviral effect of fluvastatin in cells infected with orbiviruses (bluetongue virus (BTV) and Great Island virus (GIV)). The synthesis of orbivirus outer-capsid protein VP2 (detected by confocal immunofluorescence imaging) was used to assess levels of virus replication, showing a reduction in fluvastatin-treated cells. A reduction in virus titres of ~1.7 log (98%) in fluvastatin-treated cells was detected by a plaque assay. We have previously identified a fourth non-structural protein (NS4) of BTV and GIV, showing that it interacts with lipid droplets in infected cells. Fluvastatin, which inhibits 3-hydroxy 3-methyl glutaryl CoA reductase in the mevalonic acid pathway, disrupts these NS4 interactions. These findings highlight the role of the lipid pathways in orbivirus replication and suggest a greater role for the membrane-enveloped orbivirus particles than previously recognised. Chemical intermediates of the mevalonic acid pathway were used to assess their potential to rescue orbivirus replication. Pre-treatment of IFNAR (-/-) mice with fluvastatin promoted their survival upon challenge with live BTV, although only limited protection was observed.

Bluetongue virus (BTV) is the type species of genus Orbivirus within family Reoviridae. Bluetongue virus is transmitted between its ruminant hosts by the bite of Culicoides spp. midges. Severe BT cases are characterized by symptoms including hemorrhagic fever, particularly in sheep, loss of productivity, and death. To date, 27 BTV serotypes have been documented. These include novel isolates of atypical BTV, which have been almost fully characterized using deep sequencing technologies and do not rely on Culicoides vectors for their transmission among hosts. Due to its high economic impact, BT is an Office International des Epizooties (OIE) listed disease that is strictly controlled in international commercial exchanges. During the 20th century, BTV has been endemic in subtropical regions. In the last 15 years, novel strains of nine "typical" BTV serotypes (1, 2, 4, 6, 8, 9, 11, 14, and 16) invaded Europe, some of which caused disease in naive sheep and unexpectedly in bovine herds (particularly serotype 8). Over the past few years, three novel "atypical" serotypes (25-27) were characterized during sequencing studies of animal samples from Switzerland, Kuwait, and France, respectively. Classical serotype-specific inactivated vaccines, although expensive, were very successful in controlling outbreaks as shown with the northern European BTV-8 outbreak which started in the summer of 2006. Technological jumps in deep sequencing methodologies made rapid full characterizations of BTV genome from isolates/tissues feasible. Next-generation sequencing (NGS) approaches are powerful tools to study the variability of BTV genomes on a fine scale. This paper provides information on how NGS impacted our knowledge of the BTV genome., (© 2017 Elsevier Inc. All rights reserved.)

Bluetongue virus (BTV) can infect most ruminant species and is usually transmitted by adult, vector-competent biting midges (Culicoides spp.). Infection with BTV can cause severe clinical signs and can be fatal, particularly in naïve sheep and some deer species. Although 24 distinct BTV serotypes were recognized for several decades, additional 'types' have recently been identified, including BTV-25 (from Switzerland), BTV-26 (from Kuwait) and BTV-27 from France (Corsica). Although BTV-25 has failed to grow in either insect or mammalian cell cultures, BTV-26 (isolate KUW2010/02), which can be transmitted horizontally between goats in the absence of vector insects, does not replicate in a Culicoides sonorensis cell line (KC cells) but can be propagated in mammalian cells (BSR cells). The BTV genome consists of ten segments of linear dsRNA. Mono-reassortant viruses were generated by reverse-genetics, each one containing a single BTV-26 genome segment in a BTV-1 genetic-background. However, attempts to recover a mono-reassortant containing genome-segment 2 (Seg-2) of BTV-26 (encoding VP2), were unsuccessful but a triple-reassortant was successfully generated containing Seg-2, Seg-6 and Seg-7 (encoding VP5 and VP7 respectively) of BTV-26. Reassortants were recovered and most replicated well in mammalian cells (BSR cells). However, mono-reassortants containing Seg-1 or Seg-3 of BTV-26 (encoding VP1, or VP3 respectively) and the triple reassortant failed to replicate, while a mono-reassortant containing Seg-7 of BTV-26 only replicated slowly in KC cells.

Since 1998 there have been significant changes in the global distribution of bluetongue virus (BTV). Ten previously exotic BTV serotypes have been detected in Europe, causing severe disease outbreaks in naïve ruminant populations. Previously exotic BTV serotypes were also identified in the USA, Israel, Australia and India. BTV is transmitted by biting midges (Culicoides spp.) and changes in the distribution of vector species, climate change, increased international travel and trade are thought to have contributed to these events. Thirteen BTV serotypes have been isolated in India since first reports of the disease in the country during 1964. Efficient methods for preparation of viral dsRNA and cDNA synthesis, have facilitated full-genome sequencing of BTV strains from the region. These studies introduce a new approach for BTV characterization, based on full-genome sequencing and phylogenetic analyses, facilitating the identification of BTV serotype, topotype and reassortant strains. Phylogenetic analyses show that most of the equivalent genome-segments of Indian BTV strains are closely related, clustering within a major eastern BTV 'topotype'. However, genome-segment 5 (Seg-5) encoding NS1, from multiple post 1982 Indian isolates, originated from a western BTV topotype. All ten genome-segments of BTV-2 isolates (IND2003/01, IND2003/02 and IND2003/03) are closely related (>99% identity) to a South African BTV-2 vaccine-strain (western topotype). Similarly BTV-10 isolates (IND2003/06; IND2005/04) show >99% identity in all genome segments, to the prototype BTV-10 (CA-8) strain from the USA. These data suggest repeated introductions of western BTV field and/or vaccine-strains into India, potentially linked to animal or vector-insect movements, or unauthorised use of 'live' South African or American BTV-vaccines in the country. The data presented will help improve nucleic acid based diagnostics for Indian serotypes/topotypes, as part of control strategies.

Bluetongue virus is the "type" species of the genus Orbivirus, family Reoviridae. Twenty four distinct bluetongue virus (BTV) serotypes have been recognized for decades, any of which is thought to be capable of causing "bluetongue" (BT), an insect-borne disease of ruminants. However, two further BTV serotypes, BTV-25 (Toggenburg orbivirus, from Switzerland) and BTV-26 (from Kuwait) have recently been identified in goats and sheep, respectively. The BTV genome is composed of ten segments of linear dsRNA, encoding 7 virus-structural proteins (VP1 to VP7) and four distinct non-structural (NS) proteins (NS1 to NS4). We report the entire BTV-26 genome sequence (isolate KUW2010/02) and comparisons to other orbiviruses. Highest identity levels were consistently detected with other BTV strains, identifying KUW2010/02 as BTV. The outer-core protein and major BTV serogroup-specific antigen "VP7" showed 98% aa sequence identity with BTV-25, indicating a common ancestry. However, higher level of variation in the nucleotide sequence of Seg-7 (81.2% identity) suggests strong conservation pressures on the protein of these two strains, and that they diverged a long time ago. Comparisons of Seg-2, encoding major outer-capsid component and cell-attachment protein "VP2" identified KUW2010/02 as 26th BTV, within a 12th Seg-2 nucleotype [nucleotype L]. Comparisons of Seg-6, encoding the smaller outer capsid protein VP5, also showed levels of nt/aa variation consistent with identification of KUW2010/02 as BTV-26 (within a 9th Seg-6 nucleotype - nucleotype I). Sequence data for Seg-2 of KUW2010/02 were used to design four sets of oligonucleotide primers for use in BTV-26, type-specific RT-PCR assays. Analyses of other more conserved genome segments placed KUW2010/02 and BTV-25/SWI2008/01 closer to each other than to other "eastern" or "western" BTV strains, but as representatives of two novel and distinct geographic groups (topotypes). Our analyses indicate that all of the BTV genome segments have evolved under strong purifying selection.

During 2006 the first outbreak of bluetongue ever recorded in northern Europe started in Belgium and the Netherlands, spreading to Luxemburg, Germany and north-east France. The virus overwintered (2006-2007) reappearing during May-June 2007 with greatly increased severity in affected areas, spreading further into Germany and France, reaching Denmark, Switzerland, the Czech Republic and the UK. Infected animals were also imported into Poland, Italy, Spain and the UK. An initial isolate from the Netherlands (NET2006/04) was identified as BTV-8 by RT-PCR assays targeting genome segment 2. The full genome of NET2006/04 was sequenced and compared to selected European isolates, South African vaccine strains and other BTV-8 strains, indicating that it originated in sub-Saharan Africa. Although NET2006/04 showed high levels of nucleotide identity with other 'western' BTV strains, it represents a new introduction and was not derived from the BTV-8 vaccine, although its route of entry into Europe has not been established.

Bluetongue is an economically important disease of domesticated and wild ruminants caused by bluetongue virus (BTV). There are at least 36 different serotypes of BTV (the identity of which is determined by its outer-capsid protein VP2), most of which are transmitted by Culicoides biting midges. IFNAR(−/−) mice immunised with plant-expressed outer-capsid protein VP2 (rVP2) of BTV serotypes -1, -4 or -8, or the smaller outer-capsid protein rVP5 of BTV-10, or mock-immunised with PBS, were subsequently challenged with virulent strains of BTV-4 or BTV-8, or with an attenuated clone of BTV-1 (BTV-1RGC7). The mice that had received rVP2 generated a protective immune response against the homologous BTV serotype, reducing viraemia (as detected by qRT-PCR), the severity of clinical signs and mortality levels. No cross-serotype protection was observed after challenge with the heterologous BTV serotypes. However, the severity of clinical signs, viraemia and fatality levels after challenge with the attenuated strain of BTV-1 were all increased in mice immunised with rVP2 of BTV-4 and BTV-8, or with rVP5 of BTV10. The possibility is discussed that non-neutralising antibodies, reflecting serological relationships between the outer-capsid proteins of these different BTV serotypes, could lead to 'antibody-dependent enhancement of infection' (ADE). Such interactions could affect the epidemiology and emergence of different BTV strains in the field and would therefore be relevant to the design and implementation of vaccination campaigns. [ABSTRACT FROM AUTHOR]

Bioinformatic analyses have predicted that orbiviruses encode an additional, small non-structural protein (NS5) from a secondary open reading frame on genome segment 10. However, this protein has not previously been detected in infected mammalian or insect cells. NS5-specific antibodies were generated in mice and were used to identify NS5 synthesised in orbivirus-infected BSR cells or cells transfected with NS5 expression plasmids. Confocal microscopy shows that although NS5 accumulates in the nucleus, particularly in the nucleolus, which becomes disrupted, it also appears in the cell cytoplasm, co-localising with mitochondria. NS5 helps to prevent the degradation of ribosomal RNAs during infection and reduces host-cell protein synthesis However, it helps to extend cell viability by supporting viral protein synthesis and virus replication. Pulldown studies showed that NS5 binds to ssRNAs and supercoiled DNAs and demonstrates interactions with ZBP1, suggesting that it modulates host-cell responses. [ABSTRACT FROM AUTHOR]

The genus Orbivirus, family Reoviridae, includes 22 species of viruses with genomes composed of ten segments of linear dsRNA that are transmitted between their vertebrate hosts by insects or ticks, or with no identified vectors. Full-genome sequence data are available for representative isolates of the insect borne mammalian orbiviruses (including bluetongue virus), as well as a tick borne avian orbivirus (Great Island virus). However, no sequence data are as yet available for the mosquito borne avian orbiviruses. We report full-length, whole-genome sequence data for Umatilla virus (UMAV), a mosquito borne avian orbivirus from the USA, which belongs to the species Umatilla virus. Comparisons of conserved genome segments 1, 2 and 8 (Seg-1, Seg-2 and Seg-8) - encoding the polymerase-VP1, sub-core 'T2' protein and coresurface 'T13' protein, respectively, show that UMAV groups with the mosquito transmitted mammalian orbiviruses. The highest levels of sequence identity were detected between UMAV and Stretch Lagoon orbivirus (SLOV) from Australia, showing that they belong to the same virus species (with nt/aa identity of 76.04%/88.07% and 77.96%/95.36% in the polymerase and T2 genes and protein, respectively). The data presented here has assisted in identifying the SLOV as a member of the Umatilla serogroup. This sequence data reported here will also facilitate identification of new isolates, and epidemiological studies of viruses belonging to the species Umatilla virus. [ABSTRACT FROM AUTHOR]

Culicoides biting midges (Diptera: Ceratopogonidae) transmit arboviruses of veterinary or medical importance, including bluetongue virus (BTV) and Schmallenberg virus, as well as causing severe irritation to livestock and humans. Arthropod cell lines are essential laboratory research tools for the isolation and propagation of vector-borne pathogens and the investigation of host-vector-pathogen interactions. Here we report the establishment of two continuous cell lines, CNE/LULS44 and CNE/LULS47, from embryos of Culicoides nubeculosus, a midge distributed throughout the Western Palearctic region. Species origin of the cultured cells was confirmed by polymerase chain reaction (PCR) amplification and sequencing of a fragment of the cytochrome oxidase1 gene, and the absence of bacterial contamination was confirmed by bacterial 16S rRNA PCR. Both lines have been successfully cryopreserved and resuscitated. The majority of cells examined in both lines had the expected diploid chromosome number of 2n = 6. Transmission electron microscopy of CNE/LULS44 cells revealed the presence of large mitochondria within cells of a diverse population, while arrays of virus-like particles were not seen. CNE/LULS44 cells supported replication of a strain of BTV serotype 1, but not of a strain of serotype 26 which is not known to be insect-transmitted. These new cell lines will expand the scope of research on Culicoides-borne pathogens. [ABSTRACT FROM AUTHOR]

Highlights: [•] Structural capsid proteins of bluetongue virus were expressed as soluble proteins in bacteria. [•] Soluble bacterially expressed VP2 raise strong neutralising antibodies (NAbs) in Balb/c and IFNAR−/− mice. [•] Immunised IFNAR−/− are protected from lethal challenge with a homologous live BTV but not with a heterologous serotype. [•] Alternative to live or inactivated vaccines, particularly if viruses do not grow in cell culture. [•] Exclusion of antigens such as VP7 is DIVA-compatible. [Copyright &y& Elsevier]

Abstract: The genetic study of double-stranded (ds) RNA viruses by sequence analyses of full-length genome segments, or entire viral genomes, has been restricted by the technical difficulties involved in analyses of dsRNA templates. This paper describes improved methods for sequence-independent synthesis of full-length cDNA copies of dsRNA genes and associated sequencing strategies. These methods include an improved version of the ‘Single Primer Amplification Technique’ (SPAT – [Attoui, H., Billoir, F., Cantaloube, J.F., Biagini, P., de Micco, P. and de Lamballerie, X., 2000. Strategies for the sequence determination of viral dsRNA genomes. J. Virol. Methods 89, 147–158]), which is described here as ‘Full-Length Amplification of cDNAs’ (FLAC). They also include the development of direct sequencing methods (without cloning) for the resulting full-length cDNAs. These techniques, which are applicable to any viruses with segmented dsRNA genomes and conserved RNA termini, make it possible to generate sequence data rapidly from multiple isolates for molecular epidemiology studies. [Copyright &y& Elsevier]