Serological Cross-Reactions between Expressed VP2 Proteins from Different Bluetongue Virus Serotypes.

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'.

The Interplay between Bluetongue Virus Infections and Adaptive Immunity.

Viral infections have long provided a platform to understand the workings of immunity. For instance, great strides towards defining basic immunology concepts, such as MHC restriction of antigen presentation or T-cell memory development and maintenance, have been achieved thanks to the study of lymphocytic choriomeningitis virus (LCMV) infections. These studies have also shaped our understanding of antiviral immunity, and in particular T-cell responses. In the present review, we discuss how bluetongue virus (BTV), an economically important arbovirus from the Reoviridae family that affects ruminants, affects adaptive immunity in the natural hosts. During the initial stages of infection, BTV triggers leucopenia in the hosts. The host then mounts an adaptive immune response that controls the disease. In this work, we discuss how BTV triggers CD8+ T-cell expansion and neutralizing antibody responses, yet in some individuals viremia remains detectable after these adaptive immune mechanisms are active. We present some unpublished data showing that BTV infection also affects other T cell populations such as CD4+ T-cells or γδ T-cells, as well as B-cell numbers in the periphery. This review also discusses how BTV evades these adaptive immune mechanisms so that it can be transmitted back to the arthropod host. Understanding the interaction of BTV with immunity could ultimately define the correlates of protection with immune mechanisms that would improve our knowledge of ruminant immunology.

Pathological and immunological characterization of bluetongue virus serotype 1 infection in type I interferons blocked immunocompetent adult mice.

Introduction: Wild-type adult mice with intact interferon (IFN) system were neither susceptible to bluetongue virus (BTV) infection nor showed signs of morbidity/mortality. Establishment of immunologically competent wild-type adult mouse model with type I IFNs blockade is necessary to assess the pathogenesis, immune responses and testing of BTV vaccines. Objectives: Present study aimed to establish and characterize BTV serotype 1 infection in immunocompetent adult mice with type I IFNs blockade at the time of infection by studying immune responses and sequential pathology. Methods: Adult mice were administered with anti-mouse IFN-α/ß receptor subunit-1 (IFNAR1) blocking antibody (Clone: MAR1-5A3) 24 h before and after BTV serotype 1 infection, and sacrificed at various time points. Sequential pathology, BTV localization by immunohistochemistry and quantification by qRT-PCR, immune cell kinetics and apoptosis by flow cytometry, and cytokines estimation by c-ELISA and qRT-PCR were studied. Results: IFNAR blocked-infected mice developed clinical signs and typical lesions of BT; whereas, isotype-infected control mice did not develop any disease. The IFNAR blocked-infected mice showed enlarged, edematous, and congested lymph nodes (LNs) and spleen, and vascular (congestion and hemorrhage) and pneumonic lesions in lungs. Histopathologically, marked lymphoid depletion with "starry-sky pattern" due to lymphocytes apoptosis was noticed in the LNs and spleen. BTV antigen was detected and quantified in lymphoid organs, lungs, and other organs at various time points. Initial leukopenia (increased CD4+/CD8+ T cells ratio) followed by leukocytosis (decreased CD4+/CD8+ T cells ratio) and significantly increased biochemical values were noticed in IFNAR blocked-infected mice. Increased apoptotic cells in PBMCs and tissues coincided with viral load and levels of different cytokines in blood, spleen and draining LNs and notably varied between time points in IFNAR blocked-infected mice. Conclusion: Present study is first to characterize BTV serotype 1 infection in immunocompetent adult mouse with type I IFNs blockade. The findings will be useful for studying pathogenesis and testing the efficacy of BTV vaccines.

A Duplex Fluorescent Microsphere Immunoassay for Detection of Bluetongue and Epizootic Hemorrhagic Disease Virus Antibodies in Cattle Sera.

Bluetongue virus (BTV) causes internationally reportable hemorrhagic disease in cattle, sheep, and white-tailed deer. The closely related, and often co-circulating, epizootic hemorrhagic disease virus causes a clinically similar devastating disease in white-tailed deer, with increasing levels of disease in cattle in the past 10 years. Transmitted by Culicoides biting midges, together, they constitute constant disease threats to the livelihood of livestock owners. In cattle, serious economic impacts result from decreased animal production, but most significantly from trade regulations. For effective disease surveillance and accurate trade regulation implementation, rapid, sensitive assays that can detect exposure of cattle to BTV and/or EHDV are needed. We describe the development and validation of a duplex fluorescent microsphere immunoassay (FMIA) to simultaneously detect and differentiate antibodies to BTV and EHDV in a single bovine serum sample. Performance of the duplex FMIA for detection and differentiation of BTV and EHDV serogroup antibodies was comparable, with higher sensitivity than commercially available single-plex competitive enzyme-linked immunosorbent assays (cELISA) for detection of each virus antibody separately. The FMIA adds to the currently available diagnostic tools for hemorrhagic orbiviral diseases in cattle as a sensitive, specific assay, with the benefits of serogroup differentiation in a single serum sample, and multiplexing flexibility in a high-throughput platform.

Development of recombinant NS1-NS3 antigen based indirect ELISA for detection of bluetongue antibodies in sheep.

Bluetongue is an insect borne (Culicoides) viral disease of small ruminants. The virus blankets the globe with a wide serotypic variation, numbered from 1 to 28. In India 21 different serotypes have been reported to be circulating across the various agro-climatic zones of the country. Non-structural proteins (NSPs) of bluetongue virus have always remained ideal target for differentiation of infected from vaccinated animals. The current study is an extrapolation of our previous work where a novel fusion construct comprising of bluetongue viral segment NS1 and NS3 was successfully cloned, expressed, purified with an efficient strategy for its suitable implementation as a diagnostic antigen. In this study, the applicability of the fusion construct has been further evaluated and optimised for field applicability. The fusion construct used in an ELISA platform projected a relative diagnostic sensitivity and specificity of 98.1% and 95.5% respectively against a pre-established test panel. The rNS1-NS3 ELISA showed substantially good agreement with the commercial BTV antibody detection kit. Finally, the study brings together the diagnostic capability of two NSPs, which can be a handy tool for sero-surveillance of bluetongue.

Ovine viperin inhibits bluetongue virus replication.

Viral infections can lead to interferon production, which achieves its antiviral function primarily by activating the JAK/STAT pathway and inducing multiple interferon-stimulated genes (ISGs). Although considerable ISGs have been identified in antiviral researches, little is known about ISGs in bluetongue virus (BTV) infection. Viperin is the most highly induced ISG following BTV infection, which suggests that it may play a critical role in the anti-BTV immune response. The aim of this study was to characterize ovine Viperin (oViperin) and explore whether it can inhibit BTV replication. We cloned the coding sequences (CDS) of sheep Viperin, and the sequence analysis showed that oViperin displayed a high similarity with other species. oViperin has a leucine zipper in the N-terminal, a CxxxCxxC motif in the SAM domain, and a conservative C-terminus. We found that oViperin mRNA expression was significantly up-regulated in a time- and multiplicity of infection (MOI)-dependent manner following BTV infection. oViperin overexpression resulted in a significant inhibition in BTV replication, whereas an oViperin knockdown in MDOK cells increased BTV replication. This study shows for the first time, that oViperin has antiviral activity towards BTV infection and provides important information to research the interaction between BTV and oViperin.

Highly efficient vaccines for Bluetongue virus and a related Orbivirus based on reverse genetics.

Bluetongue virus (BTV) reverse genetics (RG), available since 2007, has allowed the dissection of the virus replication cycle, including discovery of a primary replication stage. This information has allowed the generation of Entry-Competent-Replication-Abortive (ECRA) vaccines, which enter cells and complete primary replication but fail to complete the later stage. A series of vaccine trials in sheep and cattle either with a single ECRA serotype or a cocktail of multiple ECRA serotypes have demonstrated that these vaccines provide complete protection against virulent virus challenge without cross-serotype interference. Similarly, an RG system developed for the related African Horse Sickness virus, which causes high mortality in equids has provided AHSV ECRA vaccines that are protective in horses. ECRA vaccines were incapable of productive replication in animals despite being competent for cell entry. This technology allows rapid generation of emerging Orbivirus vaccines and offers immunogenicity and safety levels that surpass attenuated or recombinant routes.

Type I hypersensitivity is induced in cattle PBMC during Bluetongue virus Taiwan isolate infection.

Bluetongue is a fatal viral disease in ruminants and has serious economic impacts on the livestock industry. Interactions between bluetongue virus (BTV) and immune cells are interesting because of the unique scenarios in each combination of animal species/breed and viral virulence/serotype. This study investigated the immune response in bovine peripheral blood mononuclear cells (PBMC) infected by the BTV2 Taiwan strain. The replication of the virus was limited in monocytes and monocyte-derived macrophages (MDM), and lymphocytes were less permissive. The cytokine mRNA of IL-4 in PBMC was expressed earlier and in greater quantities than that of innate immunity (TNFα, IL-1ß) and cell mediated immunity (CMI) (IFNγ), and the IL-4 protein was stably present in the culture medium until 72 h post-infection (hpi). Even in MDM reconstituted with autologous lymphocyte (MDM-Lymphocyte), the IL-4 still had high mRNA expression level. The level of IgE antibody also increased at 24-72 hpi, suggestive of the engagement of type I hypersensitivity in the pathogenesis. The anti-viral activity contained in the culture supernatant was transferrable to recipient infected PBMC from other cows. However, in infected MDM largely free of lymphocytes, mRNA expressions of IL-1ß, TNFα and IL-12p40 were normally expressed from 6 to 48 hpi, supporting the notion that IL-4 elaborated by lymphocytes in PBMC mediated the inhibition of both innate immunity and CMI to BTV2. The sum of responses subsequent to the early IL-4 expression likely constitutes part of the unique scenario in the current BTV2-Cow experimental combination biased toward Th2 response.

Serological survey of bluetongue virus in sheep from Minas Gerais / Inquérito sorológico do vírus da língua azul em ovinos de Minas Gerais

Bluetongue is an infectious, non-contagious disease that affects domestic and wild ruminants, caused by a virus from the Orbivirus genus, Reoviridae family, transmitted by arthropod vectors of the Culicoides genus. This paper aims to be the first serological survey of bluetongue in sheep from the Meso-regions of Campo das Vertentes and South and Southeast of Minas Gerais. Samples were collected from sheep from different properties. The serum samples were submitted to Agar Gel Immunodiffusion (AGID) and competitive Enzyme-Linked Immunosorbent Assay (cELISA). 303 serum samples were submitted to AGID and cELISA. In these samples, 164 (54.13%) were positive in the AGID technique, and 171 (56.44%) positive in the cELISA technique, with an almost perfect agreement between the techniques (kappa index = 0.887). In all visited properties, positive animals have been found in the herd. Animals acquired from properties of the studied mesoregions were more likely to be positive in IDGA and cELISA tests than animals acquired from properties in other regions of Brazil (p<0.001). These results suggest that bluetongue virus (BTV) is widespread in the mesoregions of Campo das Vertentes and South and Southeast of Minas Gerais.(AU)

A língua azul (LA) é uma doença infecciosa, não contagiosa, que acomete ruminantes domésticos e silvestres, causada por um vírus do gênero Orbivirus da família Reoviridae, transmitida por vetores artrópodes do gênero Culicoides. O presente estudo representa o primeiro trabalho a realizar um inquérito sorológico da língua azul em rebanhos ovinos nas Mesorregiões de Campo das Vertentes e Sul e Sudoeste de Minas Gerais. Foram coletadas amostras de soro de ovinos de diferentes propriedades. As amostras de soro foram submetidas aos testes de imunodifusão em gel de ágar (IDGA) e ensaio de imunoadsorção enzimática por competição (cELISA). Ao todo 303 amostras de soro foram submetidas ao IDGA e cELISA. Dessas amostras, 164 (54,13%) foram positivas na técnica de IDGA e 171 (56,44%) positivas na técnica de cELISA, havendo concordância quase perfeita entre as técnicas (índice kappa = 0,887). Em todas as propriedades visitadas, foram encontrados animais positivos no rebanho. Animais adquiridos de propriedades das Mesorregiões estudadas, tiveram mais chances de serem positivos nos testes de IDGA e cELISA do que animais adquiridos de propriedades de outras Regiões do Brasil (p<0,001). Esses resultados sugerem que o vírus da língua azul encontra-se disseminado em ovinos nas Mesorregiões de Campo das Vertentes e Sul e Sudoeste de Minas Gerais.(AU)

Evaluation of two enzyme-linked immunosorbent assays for diagnosis of bluetongue virus in wild ruminants.

Bluetongue (BT) is a reportable re-emerging vector-borne disease of animal health concern. Enzyme-linked immunosorbent assays (ELISA) are frequently used in BT surveillance programs in domestic ruminants, but their diagnostic accuracy has not been evaluated for wild ruminants, which can play an important role as natural reservoirs of bluetongue virus (BTV). The aim of this study was to assess two commercial ELISAs for BT diagnosis in wild ruminants using control sera of known BTV infection status and field samples. When control sera were tested, the double recognition ELISA (DR-ELISA) showed 100 % sensitivity (Se) and specificity (Sp), while the competitive ELISA (C-ELISA) had 86.4 % Se and 97.1 % Sp. Using field samples, the selected latent-class analysis model showed 95.7 % Se and 85.9 % Sp for DR-ELISA, 58.2 % Se and 95.8 % Sp for C-ELISA and 84.2 % Se for the serum neutralization test (SNT). Our results indicate that the DR-ELISA may be a useful diagnostic method to assess BTV circulation in endemic areas, while the C-ELISA should be selected when free-areas are surveyed. The discrepancy between control and field samples point out that the inclusion of field samples is required to assess the accuracy of commercial ELISAs for the serological diagnosis of BTV in wild ruminants.

Virological, immunological and pathological findings of transplacentally transmitted bluetongue virus serotype 1 in IFNAR1-blocked mice during early and mid gestation.

Transplacental transmission (TPT) of wild-type Indian BTV-1 had never been experimentally proved. This study was first time investigated TPT of Indian BTV-1 (isolated from aborted and stillborn goat fetal spleens). The sequential pathology, virological and immune cell kinetics (CD4+, CD8+ T-lymphocytes and NK cells in spleen and PBMCs), and apoptosis in IFNAR1-blocked pregnant mice during early (infected on 1 GD) and mid (infected on 8 GD) gestation have been studied. There was higher rate of TPT during mid stage (71.43%) than early (57.14%) stage. In early stage reduced implantation sites, early embryonic deaths, abortions, and necro-haemorrhagic lesions had observed. Mid stage, congenital defects and neurological lesions in foetuses like haemorrhages, diffuse cerebral edema, necrotizing encephalitis and decreased bone size (Alizarin red staining) were noticed. BTV-1 antigen was first time demonstrable in cells of mesometrium, decidua of embryos, placenta, uterus, ovary, and brain of foetuses by immunohistochemistry and quantified by real-time qRT-PCR. BTV-inoculated mice were seroconverted by 7 and 5 dpi, and reached peak levels by 15 and 9 dpi in early and mid gestation, respectively. CD4+ and CD8+ cells were significantly decreased (increased ratio) on 7 dpi but subsequently increased on 15 dpi in early gestation. In mid gestation, increased CD8+ cells (decreased ratio) were observed. Apoptotic cells in PBMCs and tissues increased during peak viral load. This first time TPT of wild-type Indian BTV-1 deserves to be reported for implementation of control strategies. This model will be very suitable for further research into mechanisms of TPT, overwintering, and vaccination strategies.

Putative Novel Serotypes '33' and '35' in Clinically Healthy Small Ruminants in Mongolia Expand the Group of Atypical BTV.

Between 2015 and 2018, we identified the presence of three so-far-unknown Bluetongue virus (BTV) strains (BTV-MNG1/2018, BTV-MNG2/2016, and BTV-MNG3/2016) circulating in clinical healthy sheep and goats in Mongolia. Virus isolation from EDTA blood samples of BTV-MNG1/2018 and BTV-MNG3/2016 was successful on the mammalian cell line BSR using blood collected from surveillance. After experimental inoculation of goats with BTV-MNG2/2016 positive blood as inoculum, we observed viraemia in one goat and with the EDTA blood of the experimental inoculation, the propagation of BTV-MNG2/2016 in cell culture was successful on mammalian cell line BSR as well. However, virus isolation experiments for BTV-MNG2/2016 on KC cells were unsuccessful. Furthermore, we generated the complete coding sequence of all three novel Mongolian strains. For atypical BTV, serotyping via the traditional serum neutralization assay is not trivial. We therefore sorted the 'putative novel atypical serotypes' according to their segment-2 sequence identities and their time point of sampling. Hence, the BTV-MNG1/2018 isolate forms the 'putative novel atypical serotype' 33, the BTV-MNG3/2016 the 'putative novel atypical serotype' 35, whereas the BTV-MNG2/2016 strain belongs to the same putative novel atypical serotype '30' as BTV-XJ1407 from China.

A comparison of different vaccination schemes used in sheep combining inactivated bluetongue vaccines against serotypes 4 and 8.

Eight different vaccination schemes using four commercially available inactivated Bluetongue vaccines against serotypes 4 and 8 in three different combinations (setting 1-3) were tested under field conditions for their ability to generate a measurable immune response in sheep. Animals of setting 1 (groups A-D) were simultaneously vaccinated using either individual injections at different locations (groups A & D) or double injection by a twin-syringe (groups B & C). For both application methods, a one-shot vaccination (groups C & D) was compared to a boosted vaccination (groups A & B). Sheep of setting 2 (groups E-G) were vaccinated in an alternating, boosted pattern at fortnightly intervals starting with serotype 4 (groups E & F) or vice versa (group G). Group H of setting 3 was vaccinated simultaneously and vaccines were injected individually as a one-shot application. Each group consisted of 30 sheep. The immunogenic response was tested in all sheep (n = 240) by ELISA (IDScreen®Bluetongue Competition), while serum neutralisation tests were performed in five to six sheep from each group (n = 45). All vaccine combinations were well tolerated by all sheep. Of all vaccines and schemes described, the simultaneous double injected boosted vaccination of setting 1 (group B) yielded the highest median serotype-specific titres 26 weeks after the first vaccination (afv) and 100% seropositive animals (ELISA) one year afv. In setting 1, there were no relevant significant differences in the immunogenic response between simultaneously applied vaccines at different sites or at the same injection site. Importantly, a one-shot vaccination induced comparable immunogenicity to a boosted injection half a year afv. Low serotype-specific neutralising antibody levels were detected in settings 2 and 3 and are attributed to diverse factors which may have influenced the measured immunogenicity.

BTV antibody longevity in cattle five to eight years post BTV-8 vaccination.

The Bluetongue virus serotype -8 (BTV-8) epizootic in Germany (2006-2008) was successfully eradicated, essentially by the massive application of commercially available inactivated BTV-8 vaccines. While a six-year antibody longevity of BTV antibodies post BTV-8 vaccination in cattle has been described previously, our study investigated the BTV-8-vaccine antibodies in cattle for up to eight years. In total, 157 bovine serum samples were analysed for the presence of group-specific BTV antibodies in both a commercial cELISA, and a BTV-8- specific serum neutralization test. A robust number of cattle were seropositive for group- and serotype-specific neutralising antibodies for five or more years. In selected animals, born and vaccinated in 2009 or later, the presence of BTV antibodies for up to eight years post BTV-8 vaccination could be confirmed. Our data also show, that booster vaccination prolonged the antibody longevity of vaccine-induced antibodies and the number of serologically positive cattle.

Evidence of bluetongue virus circulation in farmed and free-ranging cervids from the Republic of Korea: A retrospective cross-sectional study.

Bluetongue, which is caused by bluetongue virus (BTV), is a vector-borne viral disease that affects wild and domestic ruminants. Trade restrictions can have a devastating impact in areas where BTV is endemic, regardless of the incidence of clinical disease. Currently, little is known about the prevalence of BTV infection in the Republic of Korea (ROK), and limited data on the BTV situation in the ROK are available. In this study, an epidemiological survey of BTV infection in farmed and free-ranging cervids from the ROK was conducted by a countrywide retrospective cross-sectional study. In total, BTV infection was widespread in the ROK, as 74 of 790 (9.4%, 95% confidence interval = 7.5-11.6%) cervid sera samples collected from 318 herds contained antibodies to BTV. Additionally, 42 herds evaluated in this study contained BTV seropositive cervids (13.2%). Serological evidence of bluetongue virus infection was observed in 17 of 158 free-ranging cervid animals, which accounts for the prevalence rate of approximately 10.8% (17/158; 95% CI = 6.8-16.6). Neutralizing antibodies to BTV-1, -2, -4, -7, and -15 serotypes were identified and RNAs of the BTV-1, -7, and -15 serotypes were detected, indicating that BTV was circulating in the cervids in ROK. These results suggest that cervids were actively exposed to BTV in the ROK and these species might serve as an important reservoir for the transmission of BTV. This is the first report on the evidence of circulating antibodies against BTV and serotype distribution in cervids in the ROK.

Evaluation of an IGM-specific ELISA for early detection of bluetongue virus infections in domestic ruminants sera.

Competitive-ELISA (c-ELISA) is the most widely used serological test for the detection of Bluetongue virus (BTV) viral protein 7 (VP7) antibodies (Ab). However, these BTV c-ELISAs cannot to distinguish between IgG and IgM. IgM Ab are generated shortly after the primary immune response against an infectious agent, indicating a recent infection or exposure to antigens, such as after vaccination. Because the BTV genome or anti-VP7 Ab can be detected in ruminant blood months after infection, BTV diagnostic tools cannot discriminate between recent and old infections. In this study, we evaluated an IgM-capture ELISA prototype to detect ruminant anti-BTV VP7 IgM on 1,650 serum samples from cattle, sheep, or goats. Animals were BTV-naive, infected, or/and vaccinated with BTV-1, -2, -4, -8, -9, -16, or -27, and we also included 30 sera from cattle infected with the Epizootic haemorrhagic disease virus (EHDV) serotype 6. Results demonstrated that this ELISA kit is specific and can detect the presence of IgM with satisfactory diagnostic specificity and sensitivity from 1 to 5 weeks after BTV infection in domestic ruminants (for goats and cattle; for sheep, at least up to 24 days). The peak of anti-VP7 IgM was reached when the level of infectious viruses and BTV RNA in blood were the highest. The possibility of detecting BTV-RNA in IgM-positive sera allows the amplification and sequencing of the partial RNA segment 2 (encoding the serotype specific to VP2) to determine the causative BTV serotype/strain. Therefore, BTV IgM ELISA can detect the introduction of BTV (or EHDV) in an area with BTV-seropositive domestic animals regardless of their serological BTV status. This approach may also be of particular interest for retrospective epidemiological studies on frozen serum samples.

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

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

Assessment of cross-protection induced by a bluetongue virus (BTV) serotype 8 vaccine towards other BTV serotypes in experimental conditions.

Bluetongue disease is caused by bluetongue virus (BTV) and BTV serotype 8 (BTV8) caused great economic damage in Europe during the last decade. From 1998 to 2007, in addition to BTV8, Europe had to face the emergence of BTV1, 2, 4, 9, and 16, spreading in countries where the virus has never been detected before. These unprecedented outbreaks trigger the need to evaluate and compare the clinical, virological and serological features of the European BTV serotypes in the local epidemiological context. In this study groups of calves were infected with one of the following European BTV serotypes, namely BTV1, 2, 4, 9 and 16. For each tested serotype, two groups of three male Holstein calves were used: one group vaccinated against BTV8, the other non-vaccinated. Clinical signs were quantified, viral RNA was detected in blood and organs and serological relationship was assessed. Calves were euthanized 35 days post-infection and necropsied. Most of the infected animals showed mild clinical signs. A partial serological cross reactivity has been reported between BTV8 and BTV4, and between BTV1 and BTV8. BTV2 and BTV4 viral RNA only reached low levels in blood, when compared to other serotypes, whereas in vitro growth assays could not highlight significant differences. Altogether the results of this study support the hypothesis of higher adaptation of some BTV strains to specific hosts, in this case calves. Furthermore, cross-protection resulting from a prior vaccination with BTV8 was highlighted based on cross-neutralization. However, the development of neutralizing antibodies is probably not totally explaining the mild protection induced by the heterologous vaccination.

Identification of antigenic epitopes of monoclonal antibodies against the VP2 protein of the 25 serotype of bluetongue virus.

Serious outbreaks of bluetongue, an arbovirus of domestic and wild ruminants caused by bluetongue virus serotypes (BTV), have occurred around the world. More than 27 distinct serotypes are recognized throughout the world. A new virus, BTV-25 (Toggenburg orbivirus [TOV]), was first detected in Switzerland, and has not yet been found in China. VP2 is an important outer shell protein that defines BTV serotypes and is, therefore, an ideal target antigen for serotype identification. To produce a monoclonal antibody against VP2 of BTV-25, the segment 2 gene was divided into three segments, cloned into pET-28a (+) and pMAL-c5X vectors, and the protein was expressed in E. coli BL21 with different tags. Monoclonal antibodies (mAbs) were prepared by using the purified His-25A, 25B, 25C proteins as the immunogen and the purified MBP-25A, 25B, 25C proteins as the detection antigen. Twelve hybridoma cell lines stably secreting mAbs against different VP2 segments of BTV-25 were produced. The segment 2 gene was cloned into pFastBac™HT B vector and a positive recombinant plasmid pFastBac-VP2 was used to identify mAbs. The recombinant baculovirus BACV-VP2 and eukaryotic expression of protein VP2 were obtained by the recombinant bacmid BAC-VP2 transfected Sf9 insect cells; western blotting showed that only eight mAbs were reactive. Finally, we identified the epitopes of VP2 recognized by three specific mAbs (25A-2B6, 25B-2G3, 25C-4B2) using phage display technology. The linear epitopes of VP2 protein were "359LYP361", "580NT581", "620TFR622". The preparation of mAbs and identification of the epitopes provided a foundation to analyze VP2, and may assist in the serological diagnosis of BTV-25.

Evaluation of the immune response afforded by a subunit vaccine candidate against bluetongue virus in mice and sheep.

Bluetongue virus (BTV), a vector-borne pathogen, is the causative agent of bluetongue disease in ruminants. In view of the recent emergence of BTV in regions previously known to be free from the disease and/or specific serotypes or strains, optimization of the currently available vaccination strategies to control the spread of vector-borne bluetongue is crucial. The main objective of the current study was to develop a subunit vaccine candidate targeting BTV-16, a strain previously isolated in China from sheep with obvious clinical signs. To this end, five polyhistidine-tagged recombinant proteins (BTV-16 VP2, VP3, VP7, NS2 and a truncated version of VP5 (VP5-41amino acids) were expressed using the baculovirus or Escherichia coli expression system for characterization of protective activity. To determine ovine and murine immune responses to the five proteins, sheep and mice were immunized twice at 4- and 2-week intervals, respectively, with one of two different protein combinations in MontanideTM ISA201 VG adjuvant or placebo. Data from the competitive enzyme linked immunosorbent assay revealed significantly higher antibody titers in immunized than control animals. Expressed VP5 and NS2 induced a protein-specific humoral response. Interestingly, a serum neutralization test against the BTV-1 serotype showed promising cross-serotype immune response by the vaccine. Based on the collective data, we suggest that these recombinant purified proteins present promising candidates for the design of effective novel vaccines against BTV.