Complete genome characterisation of a novel 26th bluetongue virus serotype from Kuwait

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.     

Identification and characterization of a novel non-structural protein of bluetongue virus

Bluetongue virus (BTV) is the causative agent of a major disease of livestock (bluetongue). For over two decades, it has been widely accepted that the 10 segments of the dsRNA genome of BTV encode for 7 structural and 3 non-structural proteins. The non-structural proteins (NS1, NS2, NS3/NS3a) play different key roles during the viral replication cycle. In this study we show that BTV expresses a fourth non-structural protein (that we designated NS4) encoded by an open reading frame in segment 9 overlapping the open reading frame encoding VP6. NS4 is 77-79 amino acid residues in length and highly conserved among several BTV serotypes/strains. NS4 was expressed early post-infection and localized in the nucleoli of BTV infected cells. By reverse genetics, we showed that NS4 is dispensable for BTV replication in vitro, both in mammalian and insect cells, and does not affect viral virulence in murine models of bluetongue infection. Interestingly, NS4 conferred a replication advantage to BTV-8, but not to BTV-1, in cells in an interferon (IFN)-induced antiviral state. However, the BTV-1 NS4 conferred a replication advantage both to a BTV-8 reassortant containing the entire segment 9 of BTV-1 and to a BTV-8 mutant with the NS4 identical to the homologous BTV-1 protein. Collectively, this study suggests that NS4 plays an important role in virus-host interaction and is one of the mechanisms played, at least by BTV-8, to counteract the antiviral response of the host. In addition, the distinct nucleolar localization of NS4, being expressed by a virus that replicates exclusively in the cytoplasm, offers new avenues to investigate the multiple roles played by the nucleolus in the biology of the cell.     

Characterising Non-Structural Protein NS4 of African Horse Sickness Virus

African horse sickness is a serious equid disease caused by the orbivirus African horse sickness virus (AHSV). The virus has ten double-stranded RNA genome segments encoding seven structural and three non-structural proteins. Recently, an additional protein was predicted to be encoded by genome segment 9 (Seg-9), which also encodes VP6, of most orbiviruses. This has since been confirmed in bluetongue virus and Great Island virus, and the non-structural protein was named NS4. In this study, in silico analysis of AHSV Seg-9 sequences revealed the existence of two main types of AHSV NS4, designated NS4-I and NS4-II, with different lengths and amino acid sequences. The AHSV NS4 coding sequences were in the +1 reading frame relative to that of VP6. Both types of AHSV NS4 were expressed in cultured mammalian cells, with sizes close to the predicted 17–20 kDa. Fluorescence microscopy of these cells revealed a dual cytoplasmic and nuclear, but not nucleolar, distribution that was very similar for NS4-I and NS4-II. Immunohistochemistry on heart, spleen, and lung tissues from AHSV-infected horses showed that NS4 occurs in microvascular endothelial cells and mononuclear phagocytes in all of these tissues, localising to the both the cytoplasm and the nucleus. Interestingly, NS4 was also detected in stellate-shaped dendritic macrophage-like cells with long cytoplasmic processes in the red pulp of the spleen. Finally, nucleic acid protection assays using bacterially expressed recombinant AHSV NS4 showed that both types of AHSV NS4 bind dsDNA, but not dsRNA. Further studies will be required to determine the exact function of AHSV NS4 during viral replication.     

Bluetongue Virus Nonstructural Protein NS3/NS3a Is Not Essential for Virus Replication

Orbiviruses form the largest genus of the family Reoviridae consisting of at least 23 different virus species. One of these is the bluetongue virus (BTV) and causes severe hemorrhagic disease in ruminants, and is transmitted by bites of Culicoides midges. BTV is a non-enveloped virus which is released from infected cells by cell lysis and/or a unique budding process induced by nonstructural protein NS3/NS3a encoded by genome segment 10 (Seg-10). Presence of both NS3 and NS3a is highly conserved in Culicoides borne orbiviruses which is suggesting an essential role in virus replication. We used reverse genetics to generate BTV mutants to study the function of NS3/NS3a in virus replication. Initially, BTV with small insertions in Seg-10 showed no CPE but after several passages these BTV mutants reverted to CPE phenotype comparable to wtBTV, and NS3/NS3a expression returned by repair of the ORF. These results show that there is a strong selection for functional NS3/NS3a. To abolish NS3 and/or NS3a expression, Seg-10 with one or two mutated start codons (mutAUG1, mutAUG2 and mutAUG1+2) were used to generate BTV mutants. Surprisingly, all three BTV mutants were generated and the respective AUG^Met→GCC^Ala mutations were maintained. The lack of expression of NS3, NS3a, or both proteins was confirmed by westernblot analysis and immunostaining of infected cells with NS3/NS3a Mabs. Growth of mutAUG1 and mutAUG1+2 virus in BSR cells was retarded in both insect and mammalian cells, and particularly virus release from insect cells was strongly reduced. Our findings now enable research on the role of RNA sequences of Seg-10 independent of known gene products, and on the function of NS3/NS3a proteins in both types of cells as well as in the host and insect vector.     

Identification of the Genome Segments of Bluetongue Virus Serotype 26 (Isolate KUW2010/02) that Restrict Replication in a Culicoides sonorensis Cell Line (KC Cells)

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.     

蓝舌病病毒基因节段2与6的重配导致病毒血清型与增殖特性的改变

【背景】蓝舌病病毒(Bluetongue Virus,BTV)是一种侵染反刍动物的虫媒病毒,基因重配可引起病毒的快速变异。【目的】通过我国强致病性BTV-16型毒株与弱致病性BTV-4型毒株间Seg-2与Seg-6基因节段的重配,探讨病毒基因重配与表型变异之间的关系。【方法】采用全长cDNA扩增与高通量测序获取BTV-16/V158的全基因组序列,构建病毒的真核表达质粒,通过免疫荧光与WesternBlot检测目的蛋白表达;通过RT-PCR、体外转录与细胞转染等方法建立BTV反向遗传体系并获取基因重配病毒;通过蚀斑分析、增殖曲线分析与血清中和试验,比较亲本毒株与基因重配病毒在生物学特性上的差异。【结果】获取的BTV-16/V158毒株基因组大小为19 186 bp,与中国和印度BTV-16型毒株具有最近的亲缘关系;将表达BTV VP1、VP3与NS2的真核表达质粒转染细胞,检测到目的蛋白的表达;将BTV的7种真核表达质粒与基因组ssRNA共转染BHK-21细胞,成功拯救出与亲本毒株生物学特性一致的病毒;将BTV-16/V158毒株的Seg-2与Seg-6替换为BTV-4/YTS4毒株的对应基因节段,拯救出基因重配病毒BTV-16/V158-RG (BTV-4/S2,S6);与亲本病毒相比较,基因重配病毒在BHK-21细胞上形成的蚀斑变小,增殖能力减弱,血清型由BTV-16型转化为BTV-4型。【结论】建立了我国流行BTV-16型毒株的反向遗传体系,BTVSeg-2与Seg-6的基因重配可引起病毒在细胞上增殖能力的改变与血清型改变。研究结果为BTV基因重配致病毒变异与新型基因工程疫苗的研究提供了基础。     

Bluetongue virus non-structural protein 1 is a positive regulator of viral protein synthesis

ABSTRACT: BACKGROUND: Bluetongue virus (BTV) is a double-stranded RNA (dsRNA) virus of the Reoviridae family, which encodes its genes in ten linear dsRNA segments. BTV mRNAs are synthesised by the viral RNA-dependent RNA polymerase (RdRp) as exact plus sense copies of the genome segments. Infection of mammalian cells with BTV rapidly replaces cellular protein synthesis with viral protein synthesis, but the regulation of viral gene expression in the Orbivirus genus has not been investigated. RESULTS: Using an mRNA reporter system based on genome segment 10 of BTV fused with GFP we identify the protein characteristic of this genus, non-structural protein 1 (NS1) as sufficient to upregulate translation. The wider applicability of this phenomenon among the viral genes is demonstrated using the untranslated regions (UTRs) of BTV genome segments flanking the quantifiable Renilla luciferase ORF in chimeric mRNAs. The UTRs of viral mRNAs are shown to be determinants of the amount of protein synthesised, with the pre-expression of NS1 increasing the quantity in each case. The increased expression induced by pre-expression of NS1 is confirmed in virus infected cells by generating a replicating virus which expresses the reporter fused with genome segment 10, using reverse genetics. Moreover, NS1-mediated upregulation of expression is restricted to mRNAs which lack the cellular 3[PRIME] poly(A) sequence identifying the 3[PRIME] end as a necessary determinant in specifically increasing the translation of viral mRNA in the presence of cellular mRNA. CONCLUSIONS: NS1 is identified as a positive regulator of viral protein synthesis. We propose a model of translational regulation where NS1 upregulates the synthesis of viral proteins, including itself, and creates a positive feedback loop of NS1 expression, which rapidly increases the expression of all the viral proteins. The efficient translation of viral reporter mRNAs among cellular mRNAs can account for the observed replacement of cellular protein synthesis with viral protein synthesis during infection.     

Synthesis of bluetongue virus (BTV) corelike particles by a recombinant baculovirus expressing the two major structural core proteins of BTV.

The L3 and M7 genes of bluetongue virus (BTV), which encode the two major core proteins of the virus (VP3 and VP7, respectively), were inserted into a baculovirus dual-expression transfer vector and a recombinant baculovirus expressing both foreign genes isolated following in vivo recombination with wild-type Autographa californica nuclear polyhedrosis virus DNA. Spodoptera frugiperda insect cells infected with the recombinant synthesized large amounts of BTV corelike particles. These particles have been shown to be similar to authentic BTV cores in terms of size, appearance, stoichiometric arrangement of VP3 to VP7 (ratio, 2:15), and the predominance of VP7 on the surface of the particles. In infected insect cells, the corelike particles were observed in paracrystalline arrays. The formation of these structures indicates that neither the BTV double-stranded viral RNA species nor the associated minor core proteins are necessary for assembly of cores in insect cells. Furthermore, the three BTV nonstructural proteins NS1, NS2, and NS3, are not required to assist or direct the formation of empty corelike particles from VP3 and VP7.     

Genomic sequences of Australian bluetongue virus prototype serotypes reveal global relationships and possible routes of entry into Australia

Bluetongue virus (BTV) is transmitted by biting midges (Culicoides spp.). It causes disease mainly in sheep and occasionally in cattle and other species. BTV has spread into northern Europe, causing disease in sheep and cattle. The introduction of new serotypes, changes in vector species, and climate change have contributed to these changes. Ten BTV serotypes have been isolated in Australia without apparent associated disease. Simplified methods for preferential isolation of double-stranded RNA (dsRNA) and template preparation enabled high-throughput sequencing of the 10 genome segments of all Australian BTV prototype serotypes. Phylogenetic analysis reinforced the Western and Eastern topotypes previously characterized but revealed unique features of several Australian BTVs. Many of the Australian BTV genome segments (Seg-) were closely related, clustering together within the Eastern topotypes. A novel Australian topotype for Seg-5 (NS1) was identified, with taxa spread across several serotypes and over time. Seg-1, -2, -3, -4, -6, -7, -9, and -10 of BTV_2_AUS_2008 were most closely related to the cognate segments of viruses from Taiwan and Asia and not other Australian viruses, supporting the conclusion that BTV_2 entered Australia recently. The Australian BTV_15_AUS_1982 prototype was revealed to be unusual among the Australian BTV isolates, with Seg-3 and -8 distantly related to other BTV sequences from all serotypes.     

Characterization of the stop codon readthrough signal of Colorado tick fever virus segment 9 RNA

Termination codon readthrough is utilized as a mechanism of expression of a growing number of viral and cellular proteins, but in many cases the mRNA signals that promote readthrough are poorly characterized. Here, we investigated the readthrough signal of Colorado tick fever virus (CTFV) segment 9 RNA (Seg-9). CTFV is the type-species of the genus Coltivirus within the family Reoviridae and is a tick-borne, double-stranded, segmented RNA virus. Seg-9 encodes a 36-kDa protein VP9, and by readthrough of a UGA stop codon, a 65-kDa product, VP9'. Using a reporter system, we defined the minimal sequence requirements for readthrough and confirmed activity in both mammalian and insect cell-free translation systems, and in transfected mammalian cells. Mutational analysis revealed that readthrough was UGA specific, and that the local sequence context around the UGA influenced readthrough efficiency. Readthrough was also dependent upon a stable RNA stem-loop structure beginning eight bases downstream from the UGA codon. Mutational analysis of this stem-loop revealed a requirement for the stem region but not for substructures identified within the loop. Unexpectedly, we were unable to detect a ribosomal pause during translation of the CTFV signal, suggesting that the mechanism of readthrough, at least at this site, is unlikely to be dependent upon RNA secondary-structure induced ribosomal pausing at the recoded stop codon.     

Isolates of Liao ning virus from wild-caught mosquitoes in the Xinjiang province of China in 2005

Liao ning virus (LNV) is related to Banna virus, a known human-pathogen present in south-east Asia. Both viruses belong to the genus Seadornavirus, family Reoviridae. LNV causes lethal haemorrhage in experimentally infected mice. Twenty seven isolates of LNV were made from mosquitoes collected in different locations within the Xinjiang province of north-western China during 2005. These mosquitoes were caught in the accommodation of human patients with febrile manifestations, or in animal barns where sheep represent the main livestock species. The regions where LNV was isolated are affected by seasonal encephalitis, but are free of Japanese encephalitis (JE). Genome segment 10 (Seg-10) (encoding cell-attachment and serotype-determining protein VP10) and Seg-12 (encoding non-structural protein VP12) were sequenced for multiple LNV isolates. Phylogenetic analyses showed a less homogenous Seg-10 gene pool, as compared to segment 12. However, all of these isolates appear to belong to LNV type-1. These data suggest a relatively recent introduction of LNV into Xinjiang province, with substitution rates for LNV Seg-10 and Seg-12, respectively, of 2.29×10(-4) and 1.57×10(-4) substitutions/nt/year. These substitution rates are similar to those estimated for other dsRNA viruses. Our data indicate that the history of LNV is characterized by a lack of demographic fluctuations. However, a decline in the LNV population in the late 1980s-early 1990s, was indicated by data for both Seg-10 and Seg-12. Data also suggest a beginning of an expansion in the late 1990s as inferred from Seg-12 skyline plot.     

Bluetongue virus RNA binding protein NS2 is a modulator of viral replication and assembly

Background  

Bluetongue virus (BTV) particles consist of seven structural proteins that are organized into two capsids. In addition, BTV also encodes three non-structural (NS) proteins of which protein 2 (NS2) is the RNA binding protein and is also the major component of virus encoded inclusion bodies (VIBs), which are believed to be virus assembly sites. To investigate the contribution of NS2 in virus replication and assembly we have constructed inducible mammalian cell lines expressing full-length NS2. In addition, truncated NS2 fragments were also generated in an attempt to create dominant negative mutants for NS2 function.     

Bluetongue Viruses Based on Modified-Live Vaccine Serotype 6 with Exchanged Outer Shell Proteins Confer Full Protection in Sheep against Virulent BTV8

Since 1998, Bluetongue virus (BTV)-serotypes 1, 2, 4, 9, and 16 have invaded European countries around the Mediterranean Basin. In 2006, a huge BT outbreak started after incursion of BTV serotype 8 (BTV8) in North-Western Europe. IN 2008, BTV6 and BTV11 were reported in the Netherlands and Germany, and in Belgium, respectively. In addition, Toggenburg orbivirus (TOV) was detected in 2008 in Swiss goats, which was recognized as a new serotype of BTV (BTV25). The (re-)emergency of BTV serotypes needs a rapid response to supply effective vaccines. Reverse genetics has been developed for BTV1 and more recently also for BTV6. This latter strain, BTV6/net08, is closely related to live-attenuated vaccine for serotype 6 as determined by full genome sequencing. Here, we used this strain as backbone and exchanged segment 2 and 6, respectively Seg-2 (VP2) and Seg-6 (VP5), for those of BTV serotype 1 and 8 using reverse genetics. These so-called ‘serotyped’ vaccine viruses, as mono-serotype and multi-serotype vaccine, were compared for their protective capacity in sheep. In general, all vaccinated animals developed a neutralizing antibody response against their respective serotype. After challenge at three weeks post vaccination with cell-passaged, virulent BTV8/net07 (BTV8/net07/e1/bhkp3) the vaccinated animals showed nearly no clinical reaction. Even more, challenge virus could not be detected, and seroconversion or boostering after challenge was negligible. These data demonstrate that all sheep were protected from a challenge with BTV8/net07, since sheep of the control group showed viremia, seroconversion and clinical signs that are specific for Bluetongue. The high level of cross-protection is discussed.     

Evolution and Phylogenetic Analysis of Full-Length VP3 Genes of Eastern Mediterranean Bluetongue Virus Isolates

Bluetongue virus (BTV) is the ‘type’ species of the genus Orbivirus within the family Reoviridae. The BTV genome is composed of ten linear segments of double-stranded RNA (dsRNA), each of which codes for one of ten distinct viral proteins. Previous phylogenetic comparisons have evaluated variations in genome segment 3 (Seg-3) nucleotide sequence as way to identify the geographical origin (different topotypes) of BTV isolates. The full-length nucleotide sequence of genome Seg-3 was determined for thirty BTV isolates recovered in the eastern Mediterranean region, the Balkans and other geographic areas (Spain, India, Malaysia and Africa). These data were compared, based on molecular variability, positive-selection-analysis and maximum-likelihood phylogenetic reconstructions (using appropriate substitution models) to 24 previously published sequences, revealing their evolutionary relationships. These analyses indicate that negative selection is a major force in the evolution of BTV, restricting nucleotide variability, reducing the evolutionary rate of Seg-3 and potentially of other regions of the BTV genome. Phylogenetic analysis of the BTV-4 strains isolated over a relatively long time interval (1979–2000), in a single geographic area (Greece), showed a low level of nucleotide diversity, indicating that the virus can circulate almost unchanged for many years. These analyses also show that the recent incursions into south-eastern Europe were caused by BTV strains belonging to two different major-lineages: representing an ‘eastern’ (BTV-9, -16 and -1) and a ‘western’ (BTV-4) group/topotype. Epidemiological and phylogenetic analyses indicate that these viruses originated from a geographic area to the east and southeast of Greece (including Cyprus and the Middle East), which appears to represent an important ecological niche for the virus that is likely to represent a continuing source of future BTV incursions into Europe.     

Multiserotype protection elicited by a combinatorial prime-boost vaccination strategy against bluetongue virus

Bluetongue virus (BTV) belongs to the genus Orbivirus within the family Reoviridae. The development of vector-based vaccines expressing conserved protective antigens results in increased immune activation and could reduce the number of multiserotype vaccinations required, therefore providing a cost-effective product. Recent recombinant DNA technology has allowed the development of novel strategies to develop marker and safe vaccines against BTV. We have now engineered naked DNAs and recombinant modified vaccinia virus Ankara (rMVA) expressing VP2, VP7 and NS1 proteins from BTV-4. IFNAR((-/-)) mice inoculated with DNA/rMVA-VP2,-VP7-NS1 in an heterologous prime boost vaccination strategy generated significant levels of antibodies specific of VP2, VP7, and NS1, including those with neutralizing activity against BTV-4. In addition, vaccination stimulated specific CD8(+) T cell responses against these three BTV proteins. Importantly, the vaccine combination expressing NS1, VP2 and VP7 proteins of BTV-4, elicited sterile protection against a lethal dose of homologous BTV-4 infection. Remarkably, the vaccine induced cross-protection against lethal doses of heterologous BTV-8 and BTV-1 suggesting that the DNA/rMVA-VP2,-VP7,-NS1 marker vaccine is a promising multiserotype vaccine against BTV.