Bluetongue is a disease in ruminants caused by the bluetongue virus (BTV), and is spread by Culicoides biting midges. Bluetongue outbreaks cause huge economic losses and death in sheep in several parts of the world. The most effective measure to control BTV is vaccination. However, both commercially available vaccines and recently developed vaccine candidates have several shortcomings. Therefore, we generated and tested next-generation vaccines for bluetongue based on the backbone of a laboratory-adapted strain of BTV-1, avirulent BTV-6 or virulent BTV-8. All vaccine candidates were serotyped with VP2 of BTV-8 and did not express NS3/NS3a nonstructural proteins, due to induced deletions in the NS3/NS3a ORF. Sheep were vaccinated once with one of these vaccine candidates and were challenged with virulent BTV-8 3 weeks after vaccination. The NS3/NS3a knockout mutation caused complete avirulence for all three BTV backbones, including for virulent BTV-8, indicating that safety is associated with the NS3/NS3a knockout phenotype. Viraemia of vaccine virus was not detected using sensitive PCR diagnostics. Apparently, the vaccine viruses replicated only locally, which will minimize spread by the insect vector. In particular, the vaccine based on the BTV-6 backbone protected against disease and prevented viraemia of challenge virus, showing the efficacy of this vaccine candidate. The lack of NS3/NS3a expression potentially enables the differentiation of infected from vaccinated animals, which is important for monitoring virus spread in vaccinated livestock. The disabled infectious single-animal vaccine for bluetongue presented here is very promising and will be the subject of future studies.

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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. IN 2008, BTV6 and BTV11 were reported in the Netherlands and Germany, and in Belgium, respectively. 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 BTV serotypes needs a rapid response to supply effective vaccines. Reverse genetics has been developed for BTV1 and more recently also for BTV6. This latter strain, BTV6/net08, is closely related to live-attenuated vaccine for serotype 6 as determined by full genome sequencing. Here, we used this strain as backbone and exchanged segment 2 and 6, respectively Seg-2 (VP2) and Seg-6 (VP5), for those of BTV serotype 1 and 8 using reverse genetics. These so-called ‘serotyped’ vaccine viruses, as mono-serotype and multi-serotype vaccine, were compared for their protective capacity in sheep. In general, all vaccinated animals developed a neutralizing antibody response against their respective serotype. After challenge at three weeks post vaccination with cell-passaged, virulent BTV8/net07 (BTV8/net07/e1/bhkp3) the vaccinated animals showed nearly no clinical reaction. Even more, challenge virus could not be detected, and seroconversion or boostering after challenge was negligible. These data demonstrate that all sheep were protected from a challenge with BTV8/net07, since sheep of the control group showed viremia, seroconversion and clinical signs that are specific for Bluetongue. The high level of cross-protection is discussed.

By using a monoclonal antibody directed against an epitope located on glycoprotein B of bovine herpesvirus 1 (BHV1), a simple, convenient blocking enzyme-linked immunosorbent assay (ELISA) which combines a high sensitivity with a low false-positive rate has been developed. The test can be performed at low variance on undiluted bovine serum samples. The epitope on glycoprotein B appears to be conserved, because it could be detected by immunostaining in all of 160 BHV1 isolates originating from 10 countries. In testing 215 anti-BHV1 antibody-negative and 179 anti-BHV1 antibody-positive serum samples, specificity and sensitivity were 0.96 and 0.99, respectively. This blocking ELISA is superior to a commercially available indirect ELISA and to the 24-h virus neutralization test in detecting low antibody levels in serum. In addition, this blocking ELISA is able to detect specific antibodies in serum as early as 7 days postinfection. To minimize any risk of introducing latent BHV1 carriers among noninfected cattle, this blocking ELISA would be, in our opinion, the test of choice.