Bluetongue Virus Vaccine Research Articles

Recombinant canarypox virus vaccine co-expressing genes encoding the VP2 and VP5 outer capsid proteins of bluetongue virus induces high level protection in sheep

We describe the development and preliminary characterization of a recombinant canarypox virus vectored vaccine for protective immunization of ruminants against bluetongue virus (BTV) infection. Sheep ( n = 6) immunized with recombinant canarypox virus vector (BTV-CP) co-expressing synthetic genes encoding the two outer capsid proteins (VP2 and VP5) of BTV serotype 17 (BTV-17) developed high titers (40–160) of virus-specific neutralizing antibodies and were resistant to challenge with a field strain of BTV-17. In contrast, sheep ( n = 5) immunized with a commercial recombinant canarypox virus vector expressing the E and preM genes of West Nile virus were seronegative to BTV and developed pyrexia, lymphopenia, and extended, high-titered viremias following challenge exposure to the field strain of BTV-17. These data confirm that the BTV-CP vaccine may be useful for the protective immunization of ruminants against bluetongue, and it may avoid the problems inherent to live-attenuated (LA) BTV vaccines.

High Throughput Detection of Bluetongue Virus by a New Real-Time Fluorogenic Reverse Transcription—Polymerase Chain Reaction: Application on Clinical Samples from Current Mediterranean Outbreaks

A real-time reverse transcription-polymerase chain reaction (RT-PCR) assay was developed for the detection of bluetongue virus (BTV) in blood samples. A combination of primers specific for a highly conserved region in RNA segment 5 (based on Mediterranean BTV sequences) and a DNA probe bound to 5'-Taq nuclease-3' minor groove binder (TaqMan MGB) was used to detect a range of isolates. This real-time RT-PCR assay could detect 5.4 x 10(-3) tissue culture infectious doses (TCID50) of virus per milliliter of sample, which was comparable to our current BTV diagnostic nested RT-PCR assay. The assay detected all recent Mediterranean isolates (including serotypes 2, 4, and 16), BTV vaccine strains for serotypes 2 and 4, and 15 out of the 24 BTV reference strains available (all serotypes), but did not detect the related orbiviruses epizootic hemorrhagic disease and African horse sickness viruses. Following assay evaluation, the ability of this assay to identify BTV in recent isolates (2003, 2004) from ovine and bovine samples from an epizootic outbreak in Spain was also tested. Minor nucleotide changes (detected by sequencing viral genomes) within the probe-binding region were found to have a profound effect on virus detection. This assay has the benefits of being fast and simple, and the 96-well format enables large-scale epidemiological screening for BTV, especially when combined with a high-throughput nucleic acid extraction method.

Evaluation of antibody response and nonspecific lymphocyte blastogenesis following inoculation of a live attenuated bluetongue virus vaccine in goats

To evaluate vaccine safety, antibody response, and nonspecific lymphocyte blastogenesis following inoculation of a commercial monovalent live attenuated bluetongue virus (BTV) serotype 2 vaccine in goats. 12 nonpregnant and nonlactating Saanen goats. 6 goats were inoculated with the monovalent live attenuated BTV serotype 2 vaccine, which has been widely used in Italy during the proceding 2 years. The other 6 goats were unvaccinated and represented negative controls. Nonspecific lymphocyte blastogenesis was evaluated 14 and 7 days before and 7, 21, and 49 days after vaccination by measuring DNA synthesis in peripheral blood mononuclear cells (PBMCs) stimulated with phytohemagglutinin, concanavalin-A, and pokeweed mitogen. On the same days as lymphocyte blastogenesis, blood samples were taken to determine serum concentrations of anti-BTV antibodies. During the 7 weeks following vaccination, PBMCs obtained from vaccinated goats had a significantly decreased response to mitogens in terms of DNA synthesis, compared with PBMCs from the same goats before vaccination. Conversely during the experiment, no significant change was found in the response of the PBMCs obtained from unvaccinated goats. Starting from 21 days after vaccination, serum from vaccinated goats had anti-BTV antibodies. No anti-BTV antibodies were detected in the serum from unvaccinated goats. Inoculation of goats with the monovalent live attenuated BTV serotype 2 vaccine described herein resulted in a profound depression of nonspecific lymphocyte blastogenesis, which might compromise the resistance of vaccinated goats to pathogens.

A competitive ELISA for the detection of anti-tubule antibodies using a monoclonal antibody against bluetongue virus non-structural protein NSI

A monoclonal antibody directed against the largest bluetongue virus (BTV) non-structural protein (NS1) was used in a competitive ELISA to detect antibodies to tubules (composed of NS1) in serum samples. Anti-tubule antibodies were detected at approximately 10 days post-infection in BTV infected sheep but no such antibodies were detected in sheep injected with inactivated BTV vaccines. Tubules are also produced during the replication of other related orbiviruses, including epizootic haemorrhagic disease of deer virus (EHDV). However, antibodies to the EHDV tubules were not detected in antisera from EHDV infected animals using the BTV NS1 specific monoclonal antibody and this assay system, confirming the serogroup-specific nature of this assay. This assay may prove useful not only for confirming the inactivated nature of experimental vaccines but also for the detection of viral replication and hence measurement of protection in vaccine potency trials following virulent BTV challenge. If inactivated BTV vaccines are ever widely adopted this test could provide a valuable means of discriminating between vaccinated and infected animals.

A review of the immune response to bluetongue virus.

Summary: This review presents a series of conclusions based on immunologi­ cal studies carried out on bluetongue (BT) infections in mice, sheep and cattle over the last five years at the Animal Virus Research Institute (A VRI). These studies highlight the role of both the humoral and cellular components of the immune response and conclude that current vaccination protocols could be improved by the use of serial inoculations of monovalent bluetongue virus (BTV) vaccine preparations. In identifying areas for further research, this review pinpoints the need to investigate the possibility of immunotoleran ce in cattle following a BTV infec­ tion in utero and the importance of this in terms of import/export regulations.

Production of murine cytotoxic T lymphocytes by bluetongue virus following various immunisation procedures

The induction of bluetongue virus specific cytotoxic T lymphocytes (CTLs) in C3H mice by various live and inactivated bluetongue virus preparations was studied. Live virus preparations were shown to induce good levels of CTLs; however, inactivation of virus preparations either by beta propriolactone or glutaraldehyde induced only a low level response. The use of Freund's adjuvants and double immunisation procedures failed to improve the response of the inactivated preparations. These findings are discussed in relationship to protection from bluetongue disease with various bluetongue virus vaccines.