Interactions between bluetongue virus (BTV) and its Culicoides vectors in relation to infection, replication and dissemination play a crucial role in the epidemiology of BT. In this study, temperature and strain related factors influencing replication and dissemination of BTV are investigated with a view to providing standardised tools for assessing vector competence. Initially, a Tissue Lyser based assay was developed and optimised to enable quantification of BTV infectious particles and viral RNA in Culicoides. This system was paired with a validated real-time RT-PCR assay and used to demarcate transmissible and sub-transmissible infections in orally-infected Culicoides. In comparison to virus isolation from cell culture, this technique offered considerable advantages in terms of sensitivity, specificity, repeatability, throughput and robustness. An additional approach to assessing virus dissemination by confocal observation of immunolabelled sections of infected C. sonorensis was also successfully trialled.
Quantification of infectious virus on a KC: C. sonorensis cell line highlighted differences in replication threshold temperatures across BTV strains. Certain stains were found to replicate at a temperature of 10-12°C, a lower threshold than previously defined for BTV. The use of this assay provides a rapid alternative to replication in C. sonorensis females and overcame the issue of high mortality of vectors at low temperatures. Investigation of genotype changes and related infectivity in BTV strains following passage through insect or mammalian cells highlighted a small proportion of amino acid changes in the virus although the impact of these changes during previous studies remains unclear. In addition, the use of Drosophila melanogaster as a model for investigation of BTV replication was trialled for the first time and was demonstrated to be a promising model for study of infection and dissemination. The work presented contributes standardized techniques to investigate interactions between BTV and its Culicoides vectors. Key among these are assays to confirm virus dissemination and replication of BTV on cell lines and in vectors under constant temperature regimes.