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

BACKGROUND: Transmission 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. METHODS: Virus 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. RESULTS: A 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. CONCLUSION: Small 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.

Bluetongue virus without NS3/NS3a expression is not virulent and protects against virulent bluetongue virus challenge.

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 non-structural 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.