Development of reverse transcriptase-polymerase chain reaction-based assays and sequencing for typing European strains of bluetongue virus and differential diagnosis of field and vaccine strains.

Bluetongue virus (BTV) is a double-stranded (ds) RNA virus, classified within the genus Orbivirus, family Reoviridae, which causes bluetongue (BT), an infectious, non-contagious disease of ruminants. The virus exists as 24 distinct serotypes, which are currently identified by virus isolation and serum neutralisation assays. The most variable outer capsid protein VP2 (encoded by genome segment 2), is the primary determinant of BTV serotype. Reverse transcriptase-polymerase chain reaction (RT-PCR) assays, based on amplification of segment 2, have been developed for identification of the five European BTV types (BTV-1, BTV-2, BTV-4, BTV-9 and BTV-16). Primer pairs were designed that are specific for each BTV serotype. The resulting RT-PCR assay was both sensitive and specific, providing BTV typing within 24 h. Perfect agreement was recorded between the RT-PCR and virus neutralisation assays. The primers for each serotype could successfully amplify the BTV isolates of that serotype from different regions and showed no cross-amplification of the most closely related BTV serotypes. RT-PCR primers were also developed for the discrimination of field and vaccine strains of BTV serotypes currently circulating in Europe. The primer pairs which could amplify field and vaccine strains of BTV-1, BTV-2, BTV-4 and BTV-9 were validated with several isolates of each serotype from various geographic origins around the world and their type specificity was again tested with the most closely related serotypes. Overall, these RT-PCR assays provide a rapid and reliable method for the identification and differentiation of field and vaccine strains of different BTV types. The primers used in this study are listed on the website of the Institute for Animal Health, Pirbright.