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1.  Rescue of Recent Virulent and Avirulent Field Strains of Bluetongue Virus by Reverse Genetics 
PLoS ONE  2012;7(2):e30540.
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. More recently, BTV6 and BTV11 were reported in North-Western Europe in 2008. These latter strains are closely related to live-attenuated vaccine, whereas BTV8 is virulent and can induce severe disease in ruminants, including cattle. 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 known and unknown BTV-serotypes needs a rapid response to supply effective vaccines, and research to study this phenomenon. Recently, orbivirus research achieved an important breakthrough by the establishment of reverse genetics for BTV1. Here, reverse genetics for two recent BTV strains representing virulent BTV8 and avirulent BTV6 was developed. For this purpose, extensive sequencing of full-genomes was performed, resulting in the consensus sequences of BTV8/net07 and BTV6/net08. The recovery of ‘synthetic BTV’, respectively rgBTV8 and rgBTV6, completely from T7-derived RNA transcripts was confirmed by silent mutations by which these ‘synthetic BTVs’ could be genetically distinguished from wild type BTV, respectively wtBTV6 and wtBTV8. The in vitro and in vivo properties of rgBTV6 or rgBTV8 were comparable to the properties of their parent strains. The asymptomatic or avirulent properties of rgBTV6 and the virulence of rgBTV8 were confirmed by experimental infection of sheep. Reverse genetics of the vaccine-related BTV6 provides a perfect start to develop new generations of BT-vaccines. Reverse genetics of the virulent BTV8 will accelerate research on the special features of BTV8, like transmission by species of Culicoides in a moderate climate, transplacental transmission, and pathogenesis in cattle.
PMCID: PMC3281837  PMID: 22363444
2.  A Synthetic Biology Approach for a Vaccine Platform against Known and Newly Emerging Serotypes of Bluetongue Virus 
Journal of Virology  2014;88(21):12222-12232.
Bluetongue is one of the major infectious diseases of ruminants and is caused by bluetongue virus (BTV), an arbovirus existing in nature in at least 26 distinct serotypes. Here, we describe the development of a vaccine platform for BTV. The advent of synthetic biology approaches and the development of reverse genetics systems has allowed the rapid and reliable design and production of pathogen genomes which can be subsequently manipulated for vaccine production. We describe BTV vaccines based on “synthetic” viruses in which the outer core proteins of different BTV serotypes are incorporated into a common tissue-culture-adapted backbone. As a means of validation for this approach, we selected two BTV-8 synthetic reassortants and demonstrated their ability to protect sheep against virulent BTV-8 challenge. In addition to further highlight the possibilities of genome manipulation for vaccine production, we also designed and rescued a synthetic BTV chimera containing a VP2 protein, including regions derived from both BTV-1 and BTV-8. Interestingly, while the parental viruses were neutralized only by homologous antisera, the chimeric proteins could be neutralized by both BTV-1 and BTV-8 antisera. These data suggest that neutralizing epitopes are present in different areas of the BTV VP2 and likely “bivalent” strains eliciting neutralizing antibodies for multiple strains can be obtained.
IMPORTANCE Overall, this vaccine platform can significantly reduce the time taken from the identification of new BTV strains to the development and production of new vaccines, since the viral genomes of these viruses can be entirely synthesized in vitro. In addition, these vaccines can be brought quickly into the market because they alter the approach, but not the final product, of existing commercial products.
PMCID: PMC4248921  PMID: 25142610
3.  Complete Genome Characterisation of a Novel 26th Bluetongue Virus Serotype from Kuwait 
PLoS ONE  2011;6(10):e26147.
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.
PMCID: PMC3198726  PMID: 22031822
4.  Characterization of Protection Afforded by a Bivalent Virus-Like Particle Vaccine against Bluetongue Virus Serotypes 1 and 4 in Sheep 
PLoS ONE  2011;6(10):e26666.
Bluetongue virus (BTV) is an economically important, arthropod borne, emerging pathogen in Europe, causing disease mainly in sheep and cattle. Routine vaccination for bluetongue would require the ability to distinguish between vaccinated and infected individuals (DIVA). Current vaccines are effective but are not DIVA. Virus-like particles (VLPs) are highly immunogenic structural mimics of virus particles, that only contain a subset of the proteins present in a natural infection. VLPs therefore offer the potential for the development of DIVA compatible bluetongue vaccines.
Methodology/Principal Findings
Merino sheep were vaccinated with either monovalent BTV-1 VLPs or a bivalent mixture of BTV-1 VLPs and BTV-4 VLPs, and challenged with virulent BTV-1 or BTV-4. Animals were monitored for clinical signs, antibody responses, and viral RNA. 19/20 animals vaccinated with BTV-1 VLPs either alone or in combination with BTV-4 VLPs developed neutralizing antibodies to BTV-1, and group specific antibodies to BTV VP7. The one animal that showed no detectable neutralizing antibodies, or group specific antibodies, had detectable viral RNA following challenge but did not display any clinical signs on challenge with virulent BTV-1. In contrast, all control animals' demonstrated classical clinical signs for bluetongue on challenge with the same virus. Six animals were vaccinated with bivalent vaccine and challenged with virulent BTV-4, two of these animals had detectable viral levels of viral RNA, and one of these showed clinical signs consistent with BTV infection and died.
There is good evidence that BTV-1 VLPs delivered as monovalent or bivalent immunogen protect from bluetongue disease on challenge with virulent BTV-1. However, it is possible that there is some interference in protective response for BTV-4 in the bivalent BTV-1 and BTV-4 VLP vaccine. This raises the question of whether all combinations of bivalent BTV vaccines are possible, or if immunodominance of particular serotypes could interfere with vaccine efficacy.
PMCID: PMC3202233  PMID: 22046324
5.  Genetic modification of Bluetongue virus by uptake of "synthetic" genome segments 
Virology Journal  2010;7:261.
Since 1998, several serotypes of Bluetongue virus (BTV) have invaded several southern European countries. In 2006, the unknown BTV serotype 8 (BTV8/net06) unexpectedly invaded North-West Europe and has resulted in the largest BT-outbreak ever recorded. More recently, in 2008 BTV serotype 6 was reported in the Netherlands and Germany. This virus, BTV6/net08, is closely related to modified-live vaccine virus serotype 6, except for genome segment S10. This genome segment is closer related to that of vaccine virus serotype 2, and therefore BTV6/net08 is considered as a result of reassortment. Research on orbiviruses has been hampered by the lack of a genetic modification method. Recently, reverse genetics has been developed for BTV based on ten in vitro synthesized genomic RNAs. Here, we describe a targeted single-gene modification system for BTV based on the uptake of a single in vitro synthesized viral positive-stranded RNA. cDNAs corresponding to BTV8/net06 genome segments S7 and S10 were obtained by gene synthesis and cloned downstream of the T7 RNA-polymerase promoter and upstream of a unique site for a restriction enzyme at the 3'-terminus for run-off transcription. Monolayers of BSR cells were infected by BTV6/net08, and subsequently transfected with purified in vitro synthesized, capped positive-stranded S7 or S10 RNA from BTV8/net06 origin. "Synthetic" reassortants were rescued by endpoint dilutions, and identified by serotype-specific PCR-assays for segment 2, and serogroup-specific PCRs followed by restriction enzyme analysis or sequencing for S7 and S10 segments. The targeted single-gene modification system can also be used to study functions of viral proteins by uptake of mutated genome segments. This method is also useful to generate mutant orbiviruses for other serogroups of the genus Orbivirus for which reverse genetics has not been developed yet.
PMCID: PMC2958914  PMID: 20929545
6.  Rapid Generation of Replication-Deficient Monovalent and Multivalent Vaccines for Bluetongue Virus: Protection against Virulent Virus Challenge in Cattle and Sheep 
Journal of Virology  2013;87(17):9856-9864.
Since 1998, 9 of the 26 serotypes of bluetongue virus (BTV) have spread throughout Europe, and serotype 8 has suddenly emerged in northern Europe, causing considerable economic losses, direct (mortality and morbidity) but also indirect, due to restriction in animal movements. Therefore, many new types of vaccines, particularly subunit vaccines, with improved safety and efficacy for a broad range of BTV serotypes are currently being developed by different laboratories. Here we exploited a reverse genetics-based replication-deficient BTV serotype 1 (BTV-1) (disabled infectious single cycle [DISC]) strain to generate a series of DISC vaccine strains. Cattle and sheep were vaccinated with these viruses either singly or in cocktail form as a multivalent vaccine candidate. All vaccinated animals were seroconverted and developed neutralizing antibody responses to their respective serotypes. After challenge with the virulent strains at 21 days postvaccination, vaccinated animals showed neither any clinical reaction nor viremia. Further, there was no interference with protection with a multivalent preparation of six distinct DISC viruses. These data indicate that a very-rapid-response vaccine could be developed based on which serotypes are circulating in the population at the time of an outbreak.
PMCID: PMC3754119  PMID: 23824810
7.  Pathogenicity study in sheep using reverse-genetics-based reassortant bluetongue viruses 
Veterinary Microbiology  2014;174(1-2):139-147.
•Use of reverse genetics to generate reassortant BTV viruses for testing in animals.•Two structural and one non-structural proteins are involved in pathogenicity.•Molecular basis of bluetongue disease appears to be highly complex.
Bluetongue (BT) disease, caused by the non-enveloped bluetongue virus (BTV) belonging to the Reoviridae family, is an economically important disease that affects a wide range of wild and domestic ruminants. Currently, 26 different serotypes of BTV are recognized in the world, of which BTV-8 has been found to exhibit one of the most virulent manifestations of BT disease in livestock. In recent years incursions of BTV-8 in Europe have resulted in significant morbidity and mortality not only in sheep but also in cattle. The molecular and genetic basis of BTV-8 pathogenesis is not known. To understand the genetic basis of BTV-8 pathogenicity, we generated reassortant viruses by replacing the 3 most variable genes, S2, S6 and S10 of a recent isolate of BTV-8, in different combinations into the backbone of an attenuated strain of BTV-1. The growth profiles of these reassortant viruses were then analyzed in two different ovine cell lines derived from different organs, kidney and thymus. Distinct patterns for each reassortant virus in these two cell lines were observed. To determine the pathogenicity of these reassortant viruses, groups of BTV-susceptible sheep were infected with each of these viruses. The data suggested that the clinical manifestations of these two different serotypes, BTV-1 and BTV-8, were slightly distinct and BTV-1, when comprising all 3 genome segments of BTV-8, behaved differently to BTV-1. Our results also suggested that the molecular basis of BT disease is highly complex.
PMCID: PMC4220015  PMID: 25307940
Bluetongue virus; Serotype 8; Reassortment; Non-structural protein NS3; Pathogenicity in sheep
8.  Evaluation of the Immunogenicity of an Experimental Subunit Vaccine That Allows Differentiation between Infected and Vaccinated Animals against Bluetongue Virus Serotype 8 in Cattle 
Bluetongue virus (BTV), the causative agent of bluetongue in ruminants, is an emerging virus in northern Europe. The 2006 outbreak of BTV serotype 8 (BTV-8) in Europe was marked by an unusual teratogenic effect and a high frequency of clinical signs in cattle. Conventional control strategies targeting small ruminants were therefore extended to include cattle. Since cattle were not routinely vaccinated before 2006, the immune responses to BTV have not been studied extensively in this species. With the aims of developing a subunit vaccine against BTV-8 for differentiation between infected and vaccinated animals based on viral protein 7 (VP7) antibody detection and of improving the current understanding of the immunogenicity of BTV proteins in cattle, the immune responses induced by recombinant VP2 (BTV-8) and nonstructural protein 1 (NS1) and NS2 (BTV-2) were studied. Cows were immunized twice (with a 3-week interval) with the experimental vaccine, a commercial inactivated vaccine, or a placebo. The two vaccines induced similar neutralizing antibody responses to BTV-8. Furthermore, the antibody responses detected against VP2, NS1, and NS2 were strongest in the animals immunized with the experimental vaccine, and for the first time, a serotype cross-reactive antibody response to NS2 was shown in cattle vaccinated with the commercial vaccine. The two vaccines evoked measurable T cell responses against NS1, thereby supporting a bovine cross-reactive T cell response. Finally, VP7 seroconversion was observed after vaccination with the commercial vaccine, as in natural infections, but not after vaccination with the experimental vaccine, indicating that the experimental vaccine may allow the differentiation of vaccinated animals from infected animals regardless of BTV serotype. The experimental vaccine will be further evaluated during a virulent challenge in a high-containment facility.
PMCID: PMC3754508  PMID: 23720365
9.  Vesicular stomatitis virus replicon expressing the VP2 outer capsid protein of bluetongue virus serotype 8 induces complete protection of sheep against challenge infection 
Veterinary Research  2014;45(1):64.
Bluetongue virus (BTV) is an arthropod-borne pathogen that causes an often fatal, hemorrhagic disease in ruminants. Different BTV serotypes occur throughout many temperate and tropical regions of the world. In 2006, BTV serotype 8 (BTV-8) emerged in Central and Northern Europe for the first time. Although this outbreak was eventually controlled using inactivated virus vaccines, the epidemic caused significant economic losses not only from the disease in livestock but also from trade restrictions. To date, BTV vaccines that allow simple serological discrimination of infected and vaccinated animals (DIVA) have not been approved for use in livestock. In this study, we generated recombinant RNA replicon particles based on single-cycle vesicular stomatitis virus (VSV) vectors. Immunization of sheep with infectious VSV replicon particles expressing the outer capsid VP2 protein of BTV-8 resulted in induction of BTV-8 serotype-specific neutralizing antibodies. After challenge with a virulent BTV-8 strain, the vaccinated animals neither developed signs of disease nor showed viremia. In contrast, immunization of sheep with recombinant VP5 - the second outer capsid protein of BTV - did not confer protection. Discrimination of infected from vaccinated animals was readily achieved using an ELISA for detection of antibodies against the VP7 antigen. These data indicate that VSV replicon particles potentially represent a safe and efficacious vaccine platform with which to control future outbreaks by BTV-8 or other serotypes, especially in previously non-endemic regions where discrimination between vaccinated and infected animals is crucial.
PMCID: PMC4063687  PMID: 24928313
10.  Full Genome Characterisation of Bluetongue Virus Serotype 6 from the Netherlands 2008 and Comparison to Other Field and Vaccine Strains 
PLoS ONE  2010;5(4):e10323.
In mid September 2008, clinical signs of bluetongue (particularly coronitis) were observed in cows on three different farms in eastern Netherlands (Luttenberg, Heeten, and Barchem), two of which had been vaccinated with an inactivated BTV-8 vaccine (during May-June 2008). Bluetongue virus (BTV) infection was also detected on a fourth farm (Oldenzaal) in the same area while testing for export. BTV RNA was subsequently identified by real time RT-PCR targeting genome-segment (Seg-) 10, in blood samples from each farm. The virus was isolated from the Heeten sample (IAH “dsRNA virus reference collection” [dsRNA-VRC] isolate number NET2008/05) and typed as BTV-6 by RT-PCR targeting Seg-2. Sequencing confirmed the virus type, showing an identical Seg-2 sequence to that of the South African BTV-6 live-vaccine-strain. Although most of the other genome segments also showed very high levels of identity to the BTV-6 vaccine (99.7 to 100%), Seg-10 showed greatest identity (98.4%) to the BTV-2 vaccine (RSAvvv2/02), indicating that NET2008/05 had acquired a different Seg-10 by reassortment. Although Seg-7 from NET2008/05 was also most closely related to the BTV-6 vaccine (99.7/100% nt/aa identity), the Seg-7 sequence derived from the blood sample of the same animal (NET2008/06) was identical to that of the Netherlands BTV-8 (NET2006/04 and NET2007/01). This indicates that the blood contained two different Seg-7 sequences, one of which (from the BTV-6 vaccine) was selected during virus isolation in cell-culture. The predominance of the BTV-8 Seg-7 in the blood sample suggests that the virus was in the process of reassorting with the northern field strain of BTV-8. Two genome segments of the virus showed significant differences from the BTV-6 vaccine, indicating that they had been acquired by reassortment event with BTV-8, and another unknown parental-strain. However, the route by which BTV-6 and BTV-8 entered northern Europe was not established.
PMCID: PMC2859060  PMID: 20428242
11.  Expression of VP7, a Bluetongue Virus Group Specific Antigen by Viral Vectors: Analysis of the Induced Immune Responses and Evaluation of Protective Potential in Sheep 
PLoS ONE  2014;9(11):e111605.
Bluetongue virus (BTV) is an economically important Orbivirus transmitted by biting midges to domestic and wild ruminants. The need for new vaccines has been highlighted by the occurrence of repeated outbreaks caused by different BTV serotypes since 1998. The major group-reactive antigen of BTV, VP7, is conserved in the 26 serotypes described so far, and its role in the induction of protective immunity has been proposed. Viral-based vectors as antigen delivery systems display considerable promise as veterinary vaccine candidates. In this paper we have evaluated the capacity of the BTV-2 serotype VP7 core protein expressed by either a non-replicative canine adenovirus type 2 (Cav-VP7 R0) or a leporipoxvirus (SG33-VP7), to induce immune responses in sheep. Humoral responses were elicited against VP7 in almost all animals that received the recombinant vectors. Both Cav-VP7 R0 and SG33-VP7 stimulated an antigen-specific CD4+ response and Cav-VP7 R0 stimulated substantial proliferation of antigen-specific CD8+ lymphocytes. Encouraged by the results obtained with the Cav-VP7 R0 vaccine vector, immunized animals were challenged with either the homologous BTV-2 or the heterologous BTV-8 serotype and viral burden in plasma was followed by real-time RT-PCR. The immune responses triggered by Cav-VP7 R0 were insufficient to afford protective immunity against BTV infection, despite partial protection obtained against homologous challenge. This work underscores the need to further characterize the role of BTV proteins in cross-protective immunity.
PMCID: PMC4218782  PMID: 25364822
12.  Generation of Replication-Defective Virus-Based Vaccines That Confer Full Protection in Sheep against Virulent Bluetongue Virus Challenge ▿  
Journal of Virology  2011;85(19):10213-10221.
The reverse genetics technology for bluetongue virus (BTV) has been used in combination with complementing cell lines to recover defective BTV-1 mutants. To generate a potential disabled infectious single cycle (DISC) vaccine strain, we used a reverse genetics system to rescue defective virus strains with large deletions in an essential BTV gene that encodes the VP6 protein (segment S9) of the internal core. Four VP6-deficient BTV-1 mutants were generated by using a complementing cell line that provided the VP6 protein in trans. Characterization of the growth properties of mutant viruses showed that each mutant has the necessary characteristics for a potential vaccine strain: (i) viral protein expression in noncomplementing mammalian cells, (ii) no infectious virus generated in noncomplementing cells, and (iii) efficient replication in the complementing VP6 cell line. Further, a defective BTV-8 strain was made by reassorting the two RNA segments that encode the two outer capsid proteins (VP2 and VP5) of a highly pathogenic BTV-8 with the remaining eight RNA segments of one of the BTV-1 DISC viruses. The protective capabilities of BTV-1 and BTV-8 DISC viruses were assessed in sheep by challenge with specific virulent strains using several assay systems. The data obtained from these studies demonstrated that the DISC viruses are highly protective and could offer a promising alternative to the currently available attenuated and killed virus vaccines and are also compliant as DIVA (differentiating infected from vaccinated animals) vaccines.
PMCID: PMC3196398  PMID: 21795358
13.  An Equine Herpesvirus Type 1 (EHV-1) Expressing VP2 and VP5 of Serotype 8 Bluetongue Virus (BTV-8) Induces Protection in a Murine Infection Model 
PLoS ONE  2012;7(4):e34425.
Bluetongue virus (BTV) can infect most species of domestic and wild ruminants causing substantial morbidity and mortality and, consequently, high economic losses. In 2006, an epizootic of BTV serotype 8 (BTV-8) started in northern Europe that caused significant disease in cattle and sheep before comprehensive vaccination was introduced two years later. Here, we evaluate the potential of equine herpesvirus type 1 (EHV-1), an alphaherpesvirus, as a novel vectored DIVA (differentiating infected from vaccinated animals) vaccine expressing VP2 of BTV-8 alone or in combination with VP5. The EHV-1 recombinant viruses stably expressed the transgenes and grew with kinetics that were identical to those of parental virus in vitro. After immunization of mice, a BTV-8-specific neutralizing antibody response was elicited. In a challenge experiment using a lethal dose of BTV-8, 100% of interferon-receptor-deficient (IFNAR−/−) mice vaccinated with the recombinant EHV-1 carrying both VP2 and VP5, but not VP2 alone, survived. VP7 was not included in the vectored vaccines and was successfully used as a DIVA marker. In summary, we show that EHV-1 expressing BTV-8 VP2 and VP5 is capable of eliciting a protective immune response that is distinguishable from that after infection and as such may be an alternative for BTV vaccination strategies in which DIVA compatibility is of importance.
PMCID: PMC3325243  PMID: 22511939
14.  Protection of Spanish Ibex (Capra pyrenaica) against Bluetongue Virus Serotypes 1 and 8 in a Subclinical Experimental Infection 
PLoS ONE  2012;7(5):e36380.
Many wild ruminants such as Spanish ibex (Capra pyrenaica) are susceptible to Bluetongue virus (BTV) infection, which causes disease mainly in domestic sheep and cattle. Outbreaks involving either BTV serotypes 1 (BTV-1) and 8 (BTV-8) are currently challenging Europe. Inclusion of wildlife vaccination among BTV control measures should be considered in certain species. In the present study, four out of fifteen seronegative Spanish ibexes were immunized with a single dose of inactivated vaccine against BTV-1, four against BTV-8 and seven ibexes were non vaccinated controls. Seven ibexes (four vaccinated and three controls) were inoculated with each BTV serotype. Antibody and IFN-gamma responses were evaluated until 28 days after inoculation (dpi). The vaccinated ibexes showed significant (P<0.05) neutralizing antibody levels after vaccination compared to non vaccinated ibexes. The non vaccinated ibexes remained seronegative until challenge and showed neutralizing antibodies from 7 dpi. BTV RNA was detected in the blood of non vaccinated ibexes from 2 to the end of the study (28 dpi) and in target tissue samples obtained at necropsy (8 and 28 dpi). BTV-1 was successfully isolated on cell culture from blood and target tissues of non vaccinated ibexes. Clinical signs were unapparent and no gross lesions were found at necropsy. Our results show for the first time that Spanish ibex is susceptible and asymptomatic to BTV infection and also that a single dose of vaccine prevents viraemia against BTV-1 and BTV-8 replication.
PMCID: PMC3364256  PMID: 22666321
15.  Transplacental transmission of field and rescued strains of BTV-2 and BTV-8 in experimentally infected sheep 
Veterinary Research  2013;44(1):75.
Transplacental transmission of bluetongue virus has been shown previously for the North European strain of serotype 8 (BTV-8) and for tissue culture or chicken egg-adapted vaccine strains but not for field strains of other serotypes. In this study, pregnant ewes (6 per group) were inoculated with either field or rescued strains of BTV-2 and BTV-8 in order to determine the ability of these viruses to cross the placental barrier. The field BTV-2 and BTV-8 strains was passaged once in Culicoides KC cells and once in mammalian cells. All virus inoculated sheep became infected and seroconverted against the different BTV strains used in this study. BTV RNA was detectable in the blood of all but two ewes for over 28 days but infectious virus could only be detected in the blood for a much shorter period. Interestingly, transplacental transmission of BTV-2 (both field and rescued strains) was demonstrated at high efficiency (6 out of 13 lambs born to BTV-2 infected ewes) while only 1 lamb of 12 born to BTV-8 infected ewes showed evidence of in utero infection. In addition, evidence for horizontal transmission of BTV-2 between ewes was observed. As expected, the parental BTV-2 and BTV-8 viruses and the viruses rescued by reverse genetics showed very similar properties to each other. This study showed, for the first time, that transplacental transmission of BTV-2, which had been minimally passaged in cell culture, can occur; hence such transmission might be more frequent than previously thought.
PMCID: PMC3848766  PMID: 24007601
16.  Genomic Sequences of Australian Bluetongue Virus Prototype Serotypes Reveal Global Relationships and Possible Routes of Entry into Australia 
Journal of Virology  2012;86(12):6724-6731.
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.
PMCID: PMC3393597  PMID: 22514341
17.  The Effects of Pharmacological and Lentivirus-Induced Immune Suppression on Orbivirus Pathogenesis: Assessment of Virus Burden in Blood Monocytes and Tissues by Reverse Transcription-In Situ PCR 
Journal of Virology  1998;72(7):5599-5609.
We investigated the effects of pharmacological and lentivirus-induced immunosuppression on bluetongue virus (BTV) pathogenesis as a mechanism for virus persistence and induction of clinical disease. Immunologically normal and immunosuppressed sheep were infected subcutaneously with BTV serotype 3 (BTV-3), a foreign isolate with unknown pathogenicity in North American livestock, and with North American serotype 11 (BTV-11). Erythrocyte-associated BTV RNA was detected earlier and at greater concentrations in sheep treated with immunosuppressive drugs. Similarly, viral RNA and infectious virus were detected in blood monocytes earlier and at higher frequency in immunosuppressed animals: as many as 1 in 970 monocytes revealed BTV RNA at peak viremia, compared to <1 in 105 monocytes from immunocompetent sheep. Animals infected with BTV-3 had a higher virus burden in monocytes and lesions of greater severity than those infected with BTV-11. BTV RNA was detected by in situ hybridization in vascular endothelial cells and cells of monocyte lineage, but only in tissues from immunocompromised animals, and was most abundant in animals infected with BTV-3. In contrast, reverse transcription-in situ PCR showed BTV RNA from both viral serotypes in high numbers of tissue leukocytes and vascular endothelial cells from both immunosuppressed and, to a lesser extent, immunocompetent animals. Collectively, these findings show that BTV infection is widely distributed during acute infection but replication is highly restricted in animals with normal immunity. These findings also suggest that in addition to virulence factors that define viral serotypes, immunosuppression could play a role in the natural history of orbivirus infection, allowing for higher virus burden, increased virus persistence, and greater potential for acquisition of virus by the arthropod vector.
PMCID: PMC110219  PMID: 9621018
18.  Virus and Host Factors Affecting the Clinical Outcome of Bluetongue Virus Infection 
Journal of Virology  2014;88(18):10399-10411.
Bluetongue is a major infectious disease of ruminants caused by bluetongue virus (BTV), an arbovirus transmitted by Culicoides. Here, we assessed virus and host factors influencing the clinical outcome of BTV infection using a single experimental framework. We investigated how mammalian host species, breed, age, BTV serotypes, and strains within a serotype affect the clinical course of bluetongue. Results obtained indicate that in small ruminants, there is a marked difference in the susceptibility to clinical disease induced by BTV at the host species level but less so at the breed level. No major differences in virulence were found between divergent serotypes (BTV-8 and BTV-2). However, we observed striking differences in virulence between closely related strains of the same serotype collected toward the beginning and the end of the European BTV-8 outbreak. As observed previously, differences in disease severity were also observed when animals were infected with either blood from a BTV-infected animal or from the same virus isolated in cell culture. Interestingly, with the exception of two silent mutations, full viral genome sequencing showed identical consensus sequences of the virus before and after cell culture isolation. However, deep sequencing analysis revealed a marked decrease in the genetic diversity of the viral population after passaging in mammalian cells. In contrast, passaging in Culicoides cells increased the overall number of low-frequency variants compared to virus never passaged in cell culture. Thus, Culicoides might be a source of new viral variants, and viral population diversity can be another factor influencing BTV virulence.
IMPORTANCE Bluetongue is one of the major infectious diseases of ruminants. It is caused by an arbovirus known as bluetongue virus (BTV). The clinical outcome of BTV infection is extremely variable. We show that there are clear links between the severity of bluetongue and the mammalian host species infected, while at the breed level differences were less evident. No differences were observed in the virulence of two different BTV serotypes (BTV-8 and BTV-2). In contrast, we show that the European BTV-8 strain isolated at the beginning of the bluetongue outbreak in 2006 was more virulent than a strain isolated toward the end of the outbreak. In addition, we show that there is a link between the variability of the BTV population as a whole and virulence, and our data also suggest that Culicoides cells might function as an “incubator” of viral variants.
PMCID: PMC4178883  PMID: 24991012
19.  The Spread of Bluetongue Virus Serotype 8 in Great Britain and Its Control by Vaccination 
PLoS ONE  2010;5(2):e9353.
Bluetongue (BT) is a viral disease of ruminants transmitted by Culicoides biting midges and has the ability to spread rapidly over large distances. In the summer of 2006, BTV serotype 8 (BTV-8) emerged for the first time in northern Europe, resulting in over 2000 infected farms by the end of the year. The virus subsequently overwintered and has since spread across much of Europe, causing tens of thousands of livestock deaths. In August 2007, BTV-8 reached Great Britain (GB), threatening the large and valuable livestock industry. A voluntary vaccination scheme was launched in GB in May 2008 and, in contrast with elsewhere in Europe, there were no reported cases in GB during 2008.
Methodology/Principal Findings
Here, we use carefully parameterised mathematical models to investigate the spread of BTV in GB and its control by vaccination. In the absence of vaccination, the model predicted severe outbreaks of BTV, particularly for warmer temperatures. Vaccination was predicted to reduce the severity of epidemics, with the greatest reduction achieved for high levels (95%) of vaccine uptake. However, even at this level of uptake the model predicted some spread of BTV. The sensitivity of the predictions to vaccination parameters (time to full protection in cattle, vaccine efficacy), the shape of the transmission kernel and temperature dependence in the transmission of BTV between farms was assessed.
A combination of lower temperatures and high levels of vaccine uptake (>80%) in the previously-affected areas are likely to be the major contributing factors in the control achieved in England in 2008. However, low levels of vaccination against BTV-8 or the introduction of other serotypes could result in further, potentially severe outbreaks in future.
PMCID: PMC2825270  PMID: 20179768
20.  Reassortment between Two Serologically Unrelated Bluetongue Virus Strains Is Flexible and Can Involve any Genome Segment 
Journal of Virology  2013;87(1):543-557.
Coinfection of a cell by two different strains of a segmented virus can give rise to a “reassortant” with phenotypic characteristics that might differ from those of the parental strains. Bluetongue virus (BTV) is a double-stranded RNA (dsRNA) segmented virus and the cause of bluetongue, a major infectious disease of livestock. BTV exists as at least 26 different serotypes (BTV-1 to BTV-26). Prompted by the isolation of a field reassortant between BTV-1 and BTV-8, we systematically characterized the process of BTV reassortment. Using a reverse genetics approach, our study clearly indicates that any BTV-1 or BTV-8 genome segment can be rescued in the heterologous “backbone.” To assess phenotypic variation as a result of reassortment, we examined viral growth kinetics and plaque sizes in in vitro experiments and virulence in an experimental mouse model of bluetongue disease. The monoreassortants generated had phenotypes that were very similar to those of the parental wild-type strains both in vitro and in vivo. Using a forward genetics approach in cells coinfected with BTV-1 and BTV-8, we have shown that reassortants between BTV-1 and BTV-8 are generated very readily. After only four passages in cell culture, we could not detect wild-type BTV-1 or BTV-8 in any of 140 isolated viral plaques. In addition, most of the isolated reassortants contained heterologous VP2 and VP5 structural proteins, while only 17% had homologous VP2 and VP5 proteins. Our study has shown that reassortment in BTV is very flexible, and there is no fundamental barrier to the reassortment of any genome segment. Given the propensity of BTV to reassort, it is increasingly important to have an alternative classification system for orbiviruses.
PMCID: PMC3536370  PMID: 23097432
21.  Evolution and Phylogenetic Analysis of Full-Length VP3 Genes of Eastern Mediterranean Bluetongue Virus Isolates 
PLoS ONE  2009;4(7):e6437.
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.
PMCID: PMC2713410  PMID: 19649272
22.  Ovine and murine T cell epitopes from the non-structural protein 1 (NS1) of bluetongue virus serotype 8 (BTV-8) are shared among viral serotypes 
Veterinary Research  2014;45(1):30.
Bluetongue virus (BTV) is a non-enveloped dsRNA virus that causes a haemorrhagic disease mainly in sheep. It is an economically important Orbivirus of the Reoviridae family. In order to estimate the importance of T cell responses during BTV infection, it is essential to identify the epitopes targeted by the immune system. In the present work, we selected potential T cell epitopes (3 MHC-class II-binding and 8 MHC-class I binding peptides) for the C57BL/6 mouse strain from the BTV-8 non-structural protein NS1, using H2b-binding predictive algorithms. Peptide binding assays confirmed all MHC-class I predicted peptides bound MHC-class I molecules. The immunogenicity of these 11 predicted peptides was then determined using splenocytes from BTV-8-inoculated C57BL/6 mice. Four MHC-class I binding peptides elicited specific IFN-γ production and generated cytotoxic T lymphocytes (CTL) in BTV-8 infected mice. CTL specific for 2 of these peptides were also able to recognise target cells infected with different BTV serotypes. Similarly, using a combination of IFN-γ ELISPOT, intracellular cytokine staining and proliferation assays, two MHC-class II peptides were identified as CD4+ T cell epitopes in BTV-8 infected mice. Importantly, two peptides were also consistently immunogenic in sheep infected with BTV-8 using IFN-γ ELISPOT assays. Both of these peptides stimulated CD4+ T cells that cross-reacted with other BTV serotypes. The characterisation of these T cell epitopes can help develop vaccines protecting against a broad spectrum of BTV serotypes and differentiate infected from vaccinated animals.
PMCID: PMC3995764  PMID: 24621015
23.  Identification and Characterization of a Novel Non-Structural Protein of Bluetongue Virus 
PLoS Pathogens  2011;7(12):e1002477.
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.
Author Summary
Bluetongue is a major infectious disease of ruminants caused by bluetongue virus (BTV), an “arbovirus” transmitted from infected to susceptible hosts by biting midges. Historically, bluetongue has been endemic almost exclusively in temperate and tropical areas of the world. However, in the last decade BTV has spread extensively in several geographical areas causing a serious burden to both animal health and the economy. BTV possesses a double-stranded RNA segmented genome. For over two decades, it has been widely accepted that the 10 segments of BTV genome encode for 7 structural and 3 non-structural proteins. In this study we discovered that BTV expresses a previously uncharacterized non-structural protein that we designated NS4. Although BTV replicates exclusively in the cytoplasm, we found NS4 to localize in the nucleoli of the infected cells. Our study shows that NS4 is not needed for viral replication both in mammalian and insect cells, and in mice. However, NS4 confers a replication advantage to BTV in cells in an antiviral state induced by interferon. In conclusion, we have elucidated a possible route by which BTV can counteract the defences of the host.
PMCID: PMC3248566  PMID: 22241985
24.  Survey of bluetongue virus infection in free-ranging wild ruminants in Switzerland 
In 2006, bluetongue virus serotype 8 (BTV-8) was detected for the first time in central Europe. Measures to control the infection in livestock were implemented in Switzerland but the question was raised whether free-ranging wildlife could be a maintenance host for BTV-8. Furthermore Toggenburg orbivirus (TOV), considered as a potential 25th BTV serotype, was detected in 2007 in domestic goats in Switzerland and wild ruminants were considered a potential source of infection. To assess prevalences of BTV-8 and TOV infections in wildlife, we conducted a serological and virological survey in red deer, roe deer, Alpine chamois and Alpine ibex between 2009 and 2011. Because samples originating from wildlife carcasses are often of poor quality, we also documented the influence of hemolysis on test results, and evaluated the usefulness of confirmatory tests.
Ten out of 1,898 animals (0.5%, 95% confidence interval 0.3-1.0%) had detectable antibodies against BTV-8 and BTV-8 RNA was found in two chamois and one roe deer (0.3%, 0.1-0.8%). Seroprevalence was highest among red deer, and the majority of positive wild animals were sampled close to areas where outbreaks had been reported in livestock. Most samples were hemolytic and the range of the optical density percentage values obtained in the screening test increased with increasing hemolysis. Confirmatory tests significantly increased specificity of the testing procedure and proved to be applicable even on poor quality samples. Nearly all samples confirmed as positive had an optical density percentage value greater than 50% in the ELISA screening.
Prevalence of BTV-8 infection was low, and none of the tested animals were positive for TOV. Currently, wild ruminants are apparently not a reservoir for these viruses in Switzerland. However, we report for the first time BTV-8 RNA in Alpine chamois. This animal was found at high altitude and far from a domestic outbreak, which suggests that the virus could spread into/through the Alps. Regarding testing procedures, hemolysis did not significantly affect test results but confirmatory tests proved to be necessary to obtain reliable prevalence estimates. The cut-off value recommended by the manufacturer for the screening test was applicable for wildlife samples.
PMCID: PMC3765105  PMID: 23941229
Bluetongue virus; Cross-sectional study; Hemolysis; Switzerland; Toggenburg orbivirus; Wildlife samples
25.  Genetic Characterization of Toggenburg Orbivirus, a New Bluetongue Virus, from Goats, Switzerland  
Emerging Infectious Diseases  2008;14(12):1855-1861.
Nucleotide sequences analysis indicates that this virus is a new serotype of bluetongue virus.
A novel bluetongue virus (BTV) termed Toggenburg orbivirus (TOV) was detected in goats from Switzerland by using real-time reverse transcription–PCR. cDNA corresponding to the complete sequence of 7 of 10 double-stranded RNA segments of the viral genome was amplified by PCR and cloned into a plasmid vector. Five clones for each genome segment were sequenced to determine a consensus sequence. BLAST analysis and dendrogram construction showed that TOV is closely related to BTV, although some genome segments are distinct from the 24 known BTV serotypes. Maximal sequence identity to any BTV ranged from 63% (segment 2) to 79% (segments 7 and 10). Because the gene encoding outer capsid protein 2 (VP2), which determines the serotype of BTV, is placed within the BTV serogroup, we propose that TOV represents an unknown 25th serotype of BTV.
PMCID: PMC2634640  PMID: 19046507
Orbivirus; bluetongue virus; goats; molecular epidemiology; genotyping; serotypes; research

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