Bluetongue Virus Serotype Research Articles

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

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

Seroprevalence and Potential Risk Factors of Bluetongue Virus Infection in Domestic Cattle and Goats in Guangxi Province, Southern China.

Bluetongue is one of the most important vector-borne viral diseases that can lead to significant economic losses as a result of reduction of productivity and even death in some susceptible ruminants. However, epidemiological information on bluetongue virus (BTV) infection in cattle and goats is scarce in China. To determine the seropositive rate and risk factors of BTV infection in cattle and goats in Guangxi province, a subtropical region in Southern China, a total of 548 cattle serum samples and 6567 goat serum samples collected from 13 cities across Guangxi province during 2003-2015 were analyzed and found that the seroprevalence is 44.5% (244/548) in cattle and 28.0% (1837/6567) in goats and the main BTV serotypes are BTV-1, -2, -4, and -8. Climatic zone, age, and species are found to be the likely risk factors for BTV infection. To our knowledge, this is the first large-scale serological survey for BTV infection in domestic cattle and goats in Guangxi province, Southern China.

Development of a Digital RT-PCR Method for Absolute Quantification of Bluetongue Virus in Field Samples.

Bluetongue (BT) is a major Office International des Epizooties (OIE)-listed disease of wild and domestic ruminants caused by several serotypes of Bluetongue virus (BTV), a virus with a segmented dsRNA genome belonging to the family Reoviridae, genus Orbivirus. BTV is transmitted through the bites of Culicoides midges. The aim of this study was to develop a new method for quantification of BTV Seg-10 by droplet digital RT-PCR (RTdd-PCR), using nucleic acids purified from complex matrices such as blood, tissues, and midges, that notoriously contain strong PCR inhibitors. First, RTdd-PCR was optimized by using RNAs purified from serially 10-fold dilutions of a BTV-1 isolate (105.43TCID50/ml up to 10−0.57 TCID50/ml) and from the same dilutions spiked into fresh ovine EDTA-blood and spleen homogenate. The method showed a good degree of linearity (R2 ≥ 0.995). The limit of detection (LoD) and the limit of quantification (LoQ) established were 10−0.67TCID50/ml (0.72 copies/μl) and 100.03TCID50/ml (3.05 copies/μl) of BTV-1, respectively. Second, the newly developed test was compared, using the same set of biological samples, to the quantitative RT-PCR (RT-qPCR) detecting Seg-10 assay widely used for the molecular diagnosis of BTV from field samples. Results showed a difference mean of 0.30 log between the two assays with these samples (p < 0.05). Anyway, the analysis of correlation demonstrated that both assays provided similar measurements with a very close agreement between the systems.

Ecological Dynamics Impacting Bluetongue Virus Transmission in North America.

Bluetongue virus (BTV) is an arbovirus transmitted to domestic and wild ruminants by certain species of Culicoides midges. The disease resulting from infection with BTV is economically important and can influence international trade and movement of livestock, the economics of livestock production, and animal welfare. Recent changes in the epidemiology of Culicoides-transmitted viruses, notably the emergence of exotic BTV genotypes in Europe, have demonstrated the devastating economic consequences of BTV epizootics and the complex nature of transmission across host-vector landscapes. Incursions of novel BTV serotypes into historically enzootic countries or regions, including the southeastern United States (US), Israel, Australia, and South America, have also occurred, suggesting diverse pathways for the transmission of these viruses. The abundance of BTV strains and multiple reassortant viruses circulating in Europe and the US in recent years demonstrates considerable genetic diversity of BTV strains and implies a history of reassortment events within the respective regions. While a great deal of emphasis is rightly placed on understanding the epidemiology and emergence of BTV beyond its natural ecosystem, the ecological contexts in which BTV maintains an enzootic cycle may also be of great significance. This review focuses on describing our current knowledge of ecological factors driving BTV transmission in North America. Information presented in this review can help inform future studies that may elucidate factors that are relevant to longstanding and emerging challenges associated with prevention of this disease.

A quantitative analysis of the spatial and temporal evolution patterns of the bluetongue virus outbreak in the island of Lesvos, Greece, in2014.

Bluetongue virus (BTV) causes an infectious disease called bluetongue, a vector-borne viral disease of ruminants, which has major implications and causes severe economic damage due to its effect on livestock. These economic costs are mostly ascribed to the trade restrictions imposed during the epidemic period. In August 2014, an epidemic of bluetongue occurred in the island of Lesvos, Greece. The epidemic was severe and evolved over time, lasting until December 2014. The total cases of infected farms were 490, including a total number of 136,368 small ruminants. In this paper, we describe a bluetongue virus serotype 4 (BTV-4) epidemic and utilize Bayesian epidemic models to capture the spatio-temporal spread of the disease. Our study provides important insights into the drivers of BTV transmission and has implications for designing control strategies. The results showed strong spatial autocorrelations, with BTV being more likely to spread between farms located nearby. The spatial modelling results proposed a certain spatial radius (~12km) around the onset of a similar epidemic for imposing restrictions on animal movement, which can be sufficient for the control of the disease and limit economic damage.

Bluetongue Serotype 3 in Israel 2013-2018: Clinical Manifestations of the Disease and Molecular Characterization of Israeli Strains.

In this paper, the results of the diagnostic activities on Bluetongue virus serotype 3 (BTV-3) conducted at Kimron Veterinary Institute (Beit Dagan, Israel) between 2013 and 2018 are reported. Bluetongue virus is the causative agent of bluetongue (BT), a disease of ruminants, mostly transmitted by competent Culicoides species. In Israel, BTV-3 circulation was first detected in 2013 from a sheep showing classical BT clinical signs. It was also evidenced in 2016, and, since then, it has been regularly detected in Israeli livestock. Between 2013 and 2017, BTV-3 outbreaks were limited in sheep flocks located in the southern area only. In 2018, BTV-3 was instead found in the Israeli coastal area being one of the dominant BTV serotypes isolated from symptomatic sheep, cattle and goats. In Israeli sheep, BTV-3 was able to cause BT classical clinical manifestations and fatalities, while in cattle and goats infection ranged from asymptomatic forms to death cases, depending on either general welfare of the herds or on the occurrence of viral and bacterial co-infections. Three different BTV-3 strains were identified in Israel between 2013 and 2018: ISR-2019/13 isolated in 2013, ISR-2153/16 and ISR-2262/2/16 isolated in 2016. Sequencing and phylogenetic analysis of these strains showed more than 99% identity by segment (Seg) 2, 5, 6, 7, and 8 sequences. In contrast, a wide range of diversity among these strains was exhibited in other viral gene segments, implying the occurrence of genome reassortment between these local circulating strains and those originating from Africa. The genome sequences of the BTV-3 isolated in 2017 and 2018 were most closely related to those of the ISR-2153/16 strain suggesting their common ancestor. Comparison of BTV-3 Israeli strains with those recently detected in the Mediterranean region uncovered high percentage identity (98.19–98.28%) only between Seg-2 of all Israeli strains and the BTV-3 Zarzis/TUN2016 strain. A 98.93% identity was also observed between Seg-4 sequences of ISR-2019/13 and the BTV-3 Zarzis/TUN2016 strain. This study demonstrated that BTV-3 has been circulating in the Mediterranean region at least since 2013, but, unlike the other Mediterranean strains, Israeli BTV-3 were able to cause clinical signs also in cattle.

The Comparison of Three Statistical Models for Syndromic Surveillance in Cattle Using Milk Production Data.

Two vector-borne infections have emerged and spread throughout the north-western part of Europe in the last decade: Bluetongue virus serotype-8 (BTV-8) and the Schmallenberg virus (SBV). The objective of the current study was to compare three statistical methods when applied in a syndromic surveillance context for the early detection of emerging diseases in cattle in the Netherlands. Since BTV-8 and SBV both have a negative effect on milk production in dairy cattle, routinely collected bulk milk recordings were used to compare the three statistical methods in their potential to detect drops in milk production during a period of seven years in which BTV-8 and SBV emerged. A Cusum algorithm, Bayesian disease mapping model, and spatiotemporal cluster analysis using the space-time scan statistic were performed and their performance in terms of sensitivity and specificity was compared. Spatiotemporal cluster analysis performed best for early detection of SBV in cattle in the Netherlands with a relative sensitivity of 71% compared to clinical surveillance and 100% specificity in a year without major disease outbreaks. Sensitivity to detect BTV-8 was low for all methods. However, many alerts of reduced milk production were generated several weeks before the week in which first clinical suspicions were reported. It cannot be excluded that these alerts represent the actual first signs of BTV-8 infections in cattle in the Netherlands thus leading to an underestimation of the sensitivity of the syndromic surveillance methods relative to the clinical surveillance in place.

Identification and Characterization of Bluetongue Virus Serotype 14 in Russia.

This paper reports a case of bluetongue virus (BTV) infection in the Smolensk and Kaluga regions of Russia in 2011–2012. The virus was initially detected in heifers transferred in Russia from Germany through Poland and Belarus in 2011. On day 27 of quarantine, RNA and infectious viruses of BTV were detected in four heifers, but five were serologically positive. However, on day 3 before shipment, all heifers were seronegative and PCR-negative for BTV. Thus, a few animals from this consignment were viremic without any evident subclinical infection. Based on Seg-2 (VP2 gene) and Seg-5 (NS1 gene) sequencing, the recovered virus had 99.86–100% nucleotide identity with BTV-14-like viruses such as the vaccine BTV-14 strain RSArrrr/BTV 14 and the BTV-14 isolates detected in Lithuania and Poland in 2012. Subsequently, BTV-14 was also reported in local animals in two regions of Russia. During the monitoring survey, 1623 local animals within a 300-km radius were tested, of which 471 tested positive by ELISA and 183 by PCR for BTV-14 RNA. No other serotypes were identified in either imported or aboriginal animals within that radius. The Culicoides midges trapped at the site of the outbreak in May 2012 tested positive for the BTV-14 genome, indicating that the possible mechanism of spread most likely occurs via vector bites. However, further investigation is required to confirm this hypothesis, which would provide an improved understanding of the circulation and overwintering of BTV in northern latitudes.

Deciphering type-specific neutralizing antibodies to bluetongue virus in goats of Andaman and Nicobar Islands, India.

The presence of antibodies to bluetongue virus (BTV) and the viral antigen is reported recently from the Andaman and Nicobar Islands, a group of islands at the juncture of the Bay of Bengal and the Andaman Sea. A retrospective study was conducted to investigate the presence of neutralizing antibodies to different BTV serotypes in the seroconverted goats of the Islands. Thirty six samples out of 186 serum samples tested were selected on the basis of high antibody titre as predicted in an indirect ELISA. Each of the selected serum samples was used for neutralization of six BTV serotypes (BTV-1, BTV-2, BTV-9, BTV-10, BTV-16 and BTV-23), the most commonly reported serotypes in India. Out of 36 serum samples used in the neutralization study, neutralizing antibodies could be determined in 15 samples. The neutralizing antibodies to BTV-10 were found in more number of the serum samples followed by BTV-1, BTV-2 and BTV-23 and BTV-9 and BTV-16. Many of the serum samples could neutralize more than one BTV serotypes indicating possible widespread superinfections by multiple BTV serotypes in goats in the Islands. Majority of the serum samples used in the neutralization study could not neutralize any of the six BTV serotypes commonly reported from India indicating possible circulation of other BTV serotypes yet to confirm. The present study reveals circulation of multiple BTV serotypes in Andaman and Nicobar Islands where there was no such report available earlier. The findings are laudable as the baseline information for further investigations to identify and characterize the virus and competent vectors and for implementing appropriate suitable control strategies for bluetongue in the Islands and the nearby territories.

Economic Impact of a Bluetongue Serotype 8 Epidemic in Germany.

Background and Objectives: Germany was affected by Bluetongue virus serotype 8 (BTV-8) from 2006 to 2009 and recorded new cases since December 2018. We assessed the economic impact of the epidemic from the first cases in 2006 until 2018. Direct costs include production losses, animal deaths, and veterinary treatment. Indirect costs include surveillance, additional measures for animal export, disease control (preventive vaccination and treatment with insecticides), vector monitoring, and administration.Methodology: To estimate the financial impact of BTV-8 on different species and production types at the animal level, we performed a gross margin analysis (GMA) for dairy and beef cattle, and sheep. To estimate the impact on the national level, we used a modified framework described by Rushton et al. (1) and applied a methodology described by Bennett (2). Both the GMA and the economic model on national level were implemented in Excel and the Excel Add-in @Risk. The tools, which are widely applicable, also for other diseases, are made available here.Results: The financial impact of a BTV-8 infection at the animal level was estimated at 119–136 Euros in dairy cattle, at 27 Euros in beef cattle, and at 74 Euros in sheep. At the national level, the impact of the BTV-8 epidemic ranged between 157 and 203 million Euros (mean 180 million Euros). This figure consisted of 132 (73%) and 48 (27%) million Euros for indirect and direct costs. Indirect costs included 89 million Euros (67%) for vaccination, 18 million Euros (14%) for insecticide treatment, 15 million Euros (11%) for diagnostic testing of animals dispatched for trade, 8 million Euros (6%) for monitoring and surveillance, and 3 million Euros (2%) for administration. The highest costs were induced by a compulsory vaccination campaign in 2008 (51 million Euros; 28% of the total costs) and the disease impact on cattle in 2007 (30 million Euros; 17%).Discussion: We compare the outcome of our study with economic analyses of Bluetongue disease in other countries, and discuss the suitability of GMA and the developed tools for a wider application in veterinary economics.

Alternative methods to reduce the animal use in quality controls of inactivated BTV8 Bluetongue vaccines

The acceptance of serology data instead of challenge for market release of new batches of commercial vaccine is under evaluation by regulatory agencies in order to reduce the use of animals and costs for manufacturers. In this study two vaccines for Bluetongue virus serotype 8 were submitted to quality controls required by the European Pharmacopoeia and tested on sheep in comparison with a commercial inactivated vaccine. Body temperature, antibody titres and viraemia of vaccinated and controls sheep were recorded. In addition IL4 and IFNγ in sera and supernatant derived from in vitro stimulation of blood cells were also quantified using two commercial ELISA kit. The outer-capsid protein VP2 contained in vaccine formulations was quantified using a home-made capture-ELISA. Results obtained indicates that in-lab evaluation of cell-mediated and humoral immune response are useful parameters to predict the efficacy of BTV inactivated vaccines avoiding the challenge phase required to release new batches of vaccines with proven clinical efficacy and safety. The correlation observed between serology data and VP2 protein concentration of final product could be useful in-process control to predict if a new vaccine batch of BTV must be discarded or released to the market.

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.

Origin of Bluetongue Virus Serotype 8 Outbreak in Cyprus, September 2016.

In September 2016, clinical signs, indicative of bluetongue, were observed in sheep in Cyprus. Bluetongue virus serotype 8 (BTV-8) was detected in sheep, indicating the first incursion of this serotype into Cyprus. Following virus propagation, Nextera XT DNA libraries were sequenced on the MiSeq instrument. Full-genome sequences were obtained for five isolates CYP2016/01-05 and the percent of nucleotide sequence (% nt) identity between them ranged from 99.92% to 99.95%, which corresponded to a few (2–5) amino acid changes. Based on the complete coding sequence, the Israeli ISR2008/13 (98.42–98.45%) was recognised as the closest relative to CYP2016/01-05. However, the phylogenetic reconstruction of CYP2016/01-05 revealed that the possibility of reassortment in several segments: 4, 7, 9 and 10. Based on the available sequencing data, the incursion BTV-8 into Cyprus most likely occurred from the neighbouring countries (e.g., Israel, Lebanon, Syria, or Jordan), where multiple BTV serotypes were co-circulating rather than from Europe (e.g., France) where a single BTV-8 serotype was dominant. Supporting this hypothesis, atmospheric dispersion modelling identified wind-transport events during July–September that could have allowed the introduction of BTV-8 infected midges from Lebanon, Syria or Israel coastlines into the Larnaca region of Cyprus.

Origin of bluetongue virus serotype 8 outbreak in Cyprus, September 2016

Origin of bluetongue virus serotype 8 outbreak in Cyprus, September 2016

Clinical cases of Bluetongue serotype 8 in calves in France in the 2018-2019 winter.

Bluetongue virus serotype 8 (BTV-8) caused an epizootic in Europe in 2006/09. Transplacental transmission of BTV-8 was demonstrated leading to abortions, congenital malformations or nervous clinical signs in newborn calves. BTV-8 re-emerged in France in 2015. Although the re-emergent strain is nearly genetically identical to the one that had circulated in 2006/2009, it has caused very few clinical cases. However, from mid-December 2018 to April 2019, cases of calves with congenital malformations or displaying nervous clinical signs occurred in some departments (French administrative unit) in mainland France. Blood samples from these animals were sent to local laboratories, and the positive ones were confirmed at the French Bluetongue reference laboratory (BT-NRL). Out of 580 samples found positive at the local laboratories, 544 were confirmed as RT-PCR BTV-8 positive. The 36 samples found positive in the local laboratories and negative in the BT-NRL were all at the limit of RT-PCR detection. Hundred eighty-eight of the confirmed samples were also tested for the presence of Schmallenberg virus (SBV) and bovine virus diarrhoea virus (BVDV) infection: 4 were found positive for BVDV and none for SBV. The main clinical signs recorded for 244 calves, for which a reporting form was completed by veterinarians, included nervous clinical signs (81%), amaurosis (72%) and decrease/ no suckling reflex (40%). Hydranencephaly and microphthalmia were reported in 19 calves out of 27 in which a necropsy was practiced after death or euthanasia. These results indicate that the re-emergent strain of BTV-8 can cross the transplacental barrier and cause congenital malformations or nervous clinical signs in calves.

Aydın Yöresindeki Sığırlarda Mavidil Enfeksiyonunun (BTV serotip 4, 9 ve 16) Serolojik Araştırılması

Bu çalışmada, Aydın ili sığırlarında mavidil enfeksiyonunun serolojik tespiti ve farklı yaş grupları arasında seropozitiflik oranlarının dağılımının belirlenmesi amaçlanmıştır. Bu amaçla, Aydın ilinde bulunan dört özel çiftlikten, yaşları 1 gün ile 9 yıl arasında değişen sığırlardan toplanan serum örnekleri kullanıldı. Sığırlar, yaşlarına göre on grupta sınıflandırıldı. Bluetongue virus (BTV) karşı spesifik antikor tespiti amacıyla mikro nötralizasyon tekniğinden faydalanıldı. Çalışmada kullanılan toplam 288 serum örneğinin yapılan laboratuvar tetkikleri neticesinde, BTV tip 4, 9 ve 16 için sırası ile %33.33 (96/288), %51.04 (147/288) ve %24.65 (71/288) seropozitiflik oranları belirlendi. BTV seropozitifliği %64.24 (185/288) olarak tespit edildi. Örneklenen tüm hayvanlarda BTV'nin bir, iki veya her üç tipi için seropozitif hayvanların sayı ve oranları sırasıyla 91 (%31.60), 59 (%20.49) ve 35 (%12.15) olarak tespit edildi. Test sonuçlarına uygulanan istatiksel sonuca dayanarak, bu üç serotipin varlığı ile yaş arasında anlamlı ilişki görülmedi. Sonuçlar, sığırların yaşlarına bakılmaksızın BTV'ye duyarlı olduklarını göstermiştir.

Comparative Evaluation of T-Cell Immune Response to BTV Infection in Sheep Vaccinated with Pentavalent BTV Vaccine When Compared to Un-Vaccinated Animals.

Recent invasion of multiple bluetongue virus serotypes (BTV) in different regions of the world necessitates urgent development of efficient vaccine that is directed against multiple BTV serotypes. In this experimental study, cell mediated immune response and protective efficacy of binary ethylenimine (BEI) inactivated Montanide™ ISA 206 adjuvanted pentavalent (BTV-1, 2, 10, 16 and 23) vaccine was evaluated in sheep and direct challenge with homologous BTV serotypes in their respective group. Significant (P < 0.05) up-regulation of mRNA transcripts of IFN-α, IL-2, IL-6, IL-12, IFN-γ and TNF-α in PBMCs of vaccinated animals as compared to control (un-vaccinated) animals at certain time points was observed. On the other hand, there was a significant increase in mean ± SD percentage of CD8+ T cells after 7 days post challenge (DPC) but, the mean ± SD percentage of CD4+ T-cell population slightly declined at 7 DPC and enhanced after 14 DPC. Significant differences (P < 0.05) of CD8+ and CD4+T cells population was also observed between vaccinated and unvaccinated sheep. The vaccine also significantly (P < 0.05) reduced BTV RNA load in PBMCs of vaccinated animals than unvaccinated animals following challenge. There were no significant difference (P > 0.05) in cytokine induction, BTV RNA load and CD8+ and CD4+cell count among BTV-1, 2, 10, 16 and 23 serotype challenges except significant increase in mean ± SD percentage of CD8+ in BTV-2 group. These findings put forwarded that binary ethylenimine inactivated montanide adjuvanted pentavalent bluetongue vaccine has stimulated cell mediated immune response and most importantly reduced the severity of BTV-1, 2, 10, 16 and 23 infections following challenge in respective group.

Monoclonal Antibodies to the Recombinant Protein VP7 of Bluetongue Virus

Bluetongue, or catarrhal fever of sheep, is a viral vector-borne infection of ruminants, which is one of the economically significant arbovirus infections of animals. It is transmitted by blood-sucking insects of the Culicoides genus. Viral protein VP7 is a group-specific core protein of the virus, conservative for all known serotypes, and therefore represents the most suitable target for creation of diagnostic tests. To develop an immunochemical method for detecting viral infection, a panel of high-affinity monoclonal antibodies to VP7 has been obtained. The N-terminal fragment of VP7 expressed in E. coli and inactivated viral particles were used as immunogens. The resulting monoclonal antibodies are useful for detecting the virus in infected cells. As a species-specific antigen, a recombinant TrxA‑VP7_a protein has been created that contains spatial epitopes similar to those of the native viral antigen. With its help, promising antibodies were selected for diagnostics of the disease by competitive enzyme-linked immunosorbent assay, which allows the detection of specific antibodies to bluetongue virus in the sera of infected animals. Using competitive solid-phase ELISA, it has been shown that antibodies that interact most effectively with the TrxA-VP7_a protein (Bt14, Bt15, Bt18, Bt26, Bt33, Bt34, and Bt35) recognize close or overlapping VP7 epitopes. The interaction with the antigen of almost all the antibodies obtained was inhibited by reference specific sera against the bluetongue virus of 24 serotypes. The monoclonal antibody Bt14 showed maximum ability to block the interaction of VP7 with specific sera against 24 bluetongue virus serotypes. Thus, the resulting panel of monoclonal antibodies to VP7 of the bluetongue virus can be used to detect viral infection, as well as a component of kits for serological diagnosis of the disease.

Vector competence is strongly affected by a small deletion or point mutations in bluetongue virus

BackgroundTransmission of vector-borne virus by insects is a complex mechanism consisting of many different processes; viremia in the host, uptake, infection and dissemination in the vector, and delivery of virus during blood-feeding leading to infection of the susceptible host. Bluetongue virus (BTV) is the prototype vector-borne orbivirus (family Reoviridae). BTV serotypes 1–24 (typical BTVs) are transmitted by competent biting Culicoides midges and replicate in mammalian (BSR) and midge (KC) cells. Previously, we showed that genome segment 10 (S10) encoding NS3/NS3a protein is required for virus propagation in midges. BTV serotypes 25–27 (atypical BTVs) do not replicate in KC cells. Several distinct BTV26 genome segments cause this so-called ‘differential virus replication’ in vitro.MethodsVirus strains were generated using reverse genetics and their growth was examined in vitro. The midge feeding model has been developed to study infection, replication and disseminations of virus in vivo. A laboratory colony of C. sonorensis, a known competent BTV vector, was fed or injected with BTV variants and propagation in the midge was examined using PCR testing. Crossing of the midgut infection barrier was examined by separate testing of midge heads and bodies.ResultsA 100 nl blood meal containing ±105.3 TCID50/ml of BTV11 which corresponds to ±20 TCID50 infected 50% of fully engorged midges, and is named one Midge Alimentary Infective Dose (MAID50). BTV11 with a small in-frame deletion in S10 infected blood-fed midge midguts but virus release from the midgut into the haemolymph was blocked. BTV11 with S1[VP1] of BTV26 could be adapted to virus growth in KC cells, and contained mutations subdivided into ‘corrections’ of the chimeric genome constellation and mutations associated with adaptation to KC cells. In particular one amino acid mutation in outer shell protein VP2 overcomes differential virus replication in vitro and in vivo.ConclusionSmall changes in NS3/NS3a or in the outer shell protein VP2 strongly affect virus propagation in midges and thus vector competence. Therefore, spread of disease by competent Culicoides midges can strongly differ for very closely related viruses.

Characterization of bluetongue virus serotype 28

Bluetongue virus (Reoviridae; Orbivirus, BTV), which is usually transmitted by biting midges, affects wild and domestic ruminants worldwide, thereby causing an economically important disease. Recently, a putative new BTV strain was isolated from contaminated vaccine batches. In this study, we investigated the genomic and clinical characteristics of this isolate, provisionally designated BTV-28. Phylogenetic analysis of BTV-28 segment 2 (Seg-2) showed that it is related to Seg-2 from BTV serotypes 4, 10, 11, 17, 20 and 24, sharing 64%-66% identity in nucleotide sequences (nt) and 59%-62% in amino acid (aa) sequences of BTV VP2. BTV-28 Seg-6 is related to the newly reported XJ1407 BTV isolate, sharing 76.70% nt and 90.87% aa sequence identity. Seg-5 was most closely related to a South African BTV-4 strain, and all other segments showed close similarity to BTV-26. Experimental infection by injection of 6-month-old ewes caused clinical signs in all injected animals, lasting from 2 to 3days to several weeks post-infection, including high body temperature, conjunctivitis, nasal discharge and rhinitis, facial oedema, oral hyperaemia, coronitis, cough, depression and tongue cyanosis. Naïve control animals, placed together with the infected sheep, displayed clinical signs and were positive for viral RNA, but their acute disease phase was shorter than that of BTV-injected ewes. Control animals that were kept in a separated pen did not display any clinical signs and were negative for viral RNA presence throughout the experiment. Seroconversion was observed in the injected and in one of the two contact-infected animals. These findings demonstrate that BTV-28 infection of sheep can result in clinical manifestation, and the clinical signs detected in the contact animals suggest that it might be directly transmitted between the mammalian hosts.