Recovery of Bluetongue Virus from nucleic acid: configuration optimisation and application

Bluetongue is a viral disease of sheep (sometimes cattle and goats) that poses an economic threat to UK agriculture. Since 1998, outbreaks of Bluetongue have spread Northwards in Europe and last year the disease reached Holland. The problem of BTV is clearly growing and the use of modern genetic methods to improve vaccines as well as to understand every aspect of the replication cycle is required. A significant bottleneck however, is the inability to recover BTV from sequence defined clones. As a result, the ability to change or delete essential genes in the virus with a view to producing attenuated strains as candidate vaccines is currently impossible. This project seeks to rectify this and produce a 'reverse genetics' system for BTV. Within the last year the research team has discovered a way to recover Bluetongue virus starting from just the viral genetic material isolated from the virus. The proposed research builds on this success to test ways in which the virus can be constructed completely from cloned gene copies and then deliberately weakened to make better vaccines. The research will specifically target the strain of virus that was the cause of last year's outbreak in Holland to make new strains that can be used to protect UK livestock against this disease. If successful it may be possible to also apply the research to make better vaccines for other viruses which cause disease in human and horses.

Bluetongue virus (BTV) is an insect-vectored emerging animal pathogen with the potential to have a severe economic impact in European agriculture. BTV causes high morbidity and mortality (up to 70%) in sheep. The geographic range of BTV has extended into Europe with regularity since 1998, and BTV may now be considered endemic in Europe. In the last year there was a BTV outbreak that reached as far north as the Netherlands, thus the virus can be considered a genuine risk to UK agriculture. The central objective of the project is to develop a system for the introduction of specific mutations into the genome of BTV; a reverse genetics system Two approaches are proposed: 1. An infectious RNA approach which takes advantage of our recent finding that BTV + ve sense RNA alone is sufficient to initiate an infection (1). 2. The system in approach 1 will be used to facilitate the development of an entirely plasmid-based system that uses T7 RNA polymerase to synthesize viral +ve strands. Due to the pressing need for improved vaccines to BT disease, the project will target the production of attenuated virus that could be used as a vaccine against the virus strain that caused the 2006 outbreak in the Netherlands. This attenuated virus will be used along with strains containing nucleotide modifications and epitope tags as proof-of-principle for the system. The development of reverse genetics for BTV will be a tool that accelerates basic research in BTV molecular biology and pathogenesis. The techniques developed for BTV are likely to be transferable to other members of the Reoviridae, including African Horse Sickness virus and rotaviruses. 1. Boyce, M., and P. Roy. 2007. Recovery of infectious bluetongue virus from RNA. J Virol 81:2179-86.