Project description:Bluetongue is primarily a disease of sheep in South Africa, while cattle and goats are mostly subclinically infected. The viraemia of bluetongue virus in cattle lasts much longer than in sheep and the role of cattle in the epidemiology of bluetongue in South Africa is poorly understood. Bluetongue virus has a segmented double-stranded ribonucleic acid genome and reassortment of genomes is a common feature. The aim of the study was to investigate whether reassortment occurs between vaccine and field strains when simultaneously administered to cattle. Six cattle between the ages of 6 and 12 months were infected with five strains of modified live vaccine bluetongue virus and a virulent field isolate of bluetongue virus 4. Blood samples were subsequently collected daily from these animals from day 1 to day 39 post-inoculation. Viruses were directly isolated during viraemia from the buffy coat on Vero cells using the plaque forming unit method. Analysis of plaques indicated that no reassortants between virulent field and vaccine strains occurred and the virulent bluetongue virus 4 was identified as the predominant virus strain. However, a reassortant virus between two bluetongue virus vaccine strains was isolated from the buffy coat. Whole genome sequences from the vaccine viruses were compared to the suspected reassortant and it was found that segment 8 exchanged between the bluetongue virus 8 and bluetongue virus 9 vaccine strains. The use of the live-attenuated bluetongue virus multivalent vaccine in South Africa causes circulation of different vaccine serotypes in Culicoides spp. and susceptible hosts and cattle might provide the ideal host for reassortment to occur.
Project description:Bluetongue (BT) is a midge-borne OIE-notifiable disease of ruminants caused by the bluetongue virus (BTV). There are at least 29 BTV serotypes as determined by serum neutralization tests and genetic analyses of genome segment 2 encoding serotype immunodominant VP2 protein. Large parts of the world are endemic for multiple serotypes. The most effective control measure of BT is vaccination. Conventionally live-attenuated and inactivated BT vaccines are available but have their specific pros and cons and are not DIVA compatible. The prototype Disabled Infectious Single Animal (DISA)/DIVA vaccine based on knockout of NS3/NS3a protein of live-attenuated BTV, shortly named DISA8, fulfills all criteria for modern veterinary vaccines of sheep. Recently, DISA8 with an internal in-frame deletion of 72 amino acid codons in NS3/NS3a showed a similar ideal vaccine profile in cattle. Here, the DISA/DIVA vaccine platform was applied for other serotypes, and pentavalent DISA/DIVA vaccine for "European" serotypes 1, 2, 3, 4, 8 was studied in sheep and cattle. Protection was demonstrated for two serotypes, and neutralization Ab titers indicate protection against other included serotypes. The DISA/DIVA vaccine platform is flexible in use and generates monovalent and multivalent DISA vaccines to combat specific field situations with respect to Bluetongue.
Project description:Bluetongue (BT) is an arthropod-borne viral disease of ruminants with serious trade and socio-economic implications. Although the disease has been reported in a number of countries in sub-Saharan Africa, there is currently no information on circulating serotypes and disease distribution in Zambia. Following surveillance for BT in domestic and wild ruminants in Zambia, BT virus (BTV) nucleic acid and antibodies were detected in eight of the 10 provinces of the country. About 40% (87/215) of pooled blood samples from cattle and goats were positive for BTV nucleic acid, while one hartebeest pool (1/43) was positive among wildlife samples. Sequence analysis of segment 2 revealed presence of serotypes 3, 5, 7, 12 and 15, with five nucleotypes (B, E, F, G and J) being identified. Segment 10 phylogeny showed Zambian BTV sequences clustering with Western topotype strains from South Africa, intimating likely transboundary spread of BTV in Southern Africa. Interestingly, two Zambian viruses and one isolate from Israel formed a novel clade, which we designated as Western topotype 4. The high seroprevalence (96.2%) in cattle from Lusaka and Central provinces and co-circulation of multiple serotypes showed that BT is widespread, underscoring the need for prevention and control strategies.
Project description:BackgroundBluetongue 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 findingsMerino 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.ConclusionsThere 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.
Project description:Bluetongue is an economically important disease of ruminants caused by the bluetongue virus (BTV). BTV is serologically diverse, which complicates vaccination strategies. Rapid identification of the causative BTV serotypes is critical, however, real-time PCR (RT-qPCR) can be costly and time consuming to perform when the circulating serotypes are unknown. The Luminex xMAP technology is a high-throughput platform that uses fluorescent beads to detect multiple targets simultaneously. We utilized existing BTV serotyping RT-qPCR assays for BTV-1 to BTV-24 and adapted them for use with the xMAP platform. The xMAP assay specifically detected all 24 BTV serotypes when testing reference strains. In all BTV-positive samples, the sensitivity of the BTV xMAP was 87.55% whereas the sensitivity of the serotype-specific RT-qPCR was 79.85%. The BTV xMAP assay allowed for the specific detection of BTV serotypes 1-24 at a lower cost than current RT-qPCR assays. Overall, the assay provides a useful novel diagnostic tool, particularly when analyzing large sample sets. The use of the BTV xMAP assay will allow for the rapid assessment of BTV epidemiology and may inform decision-making related to control and prevention measures.
Project description:Bluetongue (BT) is a vector-borne disease of ruminants caused by Bluetongue virus (BTV). Twenty-nine different serotypes of BTV are currently reported throughout the world. The main objective of this study is the development of a subunit vaccine model that could potentially be adapted to provide broad spectrum protection against multiple BTV serotypes, which the conventional vaccines fail to address. To this end, three different BTV proteins (conserved region of viral protein [VP]2, VP5 and NS1) were expressed and purified in an Escherichia coli expression system. The immunogenicity of these proteins was tested in murine models using the MontanideTM ISA 201 VG adjuvant. BALB/c mice were immunised thrice (with individual proteins and a mixture of three proteins) at two-week intervals and were monitored until Day 40 post-infection/vaccination. Protein-specific antibodies directed against the recombinant proteins were detected by indirect enzyme-linked immunosorbent assay. Neutralising antibody (Nab) titres and cross-neutralisation against a range of BTV serotypes (BTV-1, -2, -4, -5, -9, -10, -12, -16, -21, -23 and -24) were determined by serum neutralisation test. The recombinant proteins elicited higher Nab titres compared with the inactivated vaccine group, except for BTV-1, where the inactivated vaccine group elicited higher Nab titres. Additive effect of the three proteins was not observed as the Nab titres generated with a combination of conserved VP2, VP5 and NS1 was similar to those of the individual protein groups. Whilst BTV-12 could only be neutralised by serum raised against the inactivated vaccine group, BTV-5 and -24 could not be neutralised by any of the groups tested. Our cumulative data suggest that the conserved regions of VP2 (cVP2), VP5 and NS1 could play an important part in the novel vaccine design against multiple BTV serotypes. Importantly, given that VP2 was already known to elicit a serotype-specific immune response against BT, we report, for the first time, that the conserved region of VP2 has the ability to induce cross-protective immune response.
Project description:In 2006 and 2007, sheep and cattle farms in the Netherlands were affected by an epidemic of bluetongue virus serotype 8 (BTV-8). In order to obtain insight into the within-farm spread of the virus, five affected cattle and five affected sheep farms were longitudinally monitored between early 2007 and mid or late 2008. The farms were visited between four and seven times to collect blood samples. During each visit, all animals present in the flock or herd were sampled. The samples were analysed for the presence of BTV-8 antibodies (ELISA) and BTV-8 antigen (rRT-PCR). The observed patterns of RT-PCR positives indicate a rapid within-farm virus spread during the vector season. During vector-free periods we observed a complete rRT-PCR positivity decline within a few months. During the vector season a lower bound estimate of the basic reproduction number (R0) ranges from 2.9-6.9 in the cattle herds (one herd not analysed), and from 1.3-3.2 in the sheep flocks in this study.
Project description:To determine whether transplacental transmission could explain overwintering of bluetongue virus in the United Kingdom, we studied calves born to dams naturally infected during pregnancy in 2007-08. Approximately 33% were infected transplacentally; some had compromised health. In all infected calves, viral load decreased after birth; no evidence of persistent infection was found.
Project description: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'.