Detection and characterisation of bluetongue virus using the polymerase chain reaction

Abstract

Pairs of oligodeoxynucleotide primers whose sequences were based on those of RNA segment 3 that encodes the bluetongue virus serogroup-reactive protein VP3, were synthesized for three BTVs from different geographic regions of the world and for seven Australian orbiviruses. Each pair of primers was then tested for the synthesis of cDNA and in subsequent polymerase chain reactions (PCR) with all ten virus groups. All primers were serogroup-specific at low or high stringency. One pair of primers was specifically designed for its ability to serogroup a BTV isolate irrespective of its geographic origin. At either high or low stringency, this primer-pair resulted in a common and specific PCR product for each of the BTVs tested but not for the other orbiviruses. Eight pairs of primers based on RNA2 sequences (the gene segment encoding the serotype-specific protein VP2) were also synthesized for the eight Australian serotypes of BTV. Each primer-pair was serotype-specific at low or high stringency except for the BTV16A pair, which cross-reacted with BTV3A and also gave a non-specific product that differed in Mr from the authentic PCR product. Using the PCR and BTV1A RNA3-based primers, BTV1A was detected in blood samples from two sheep at 9 days post inoculation. Virus was found in the platelet, buffy-coat and packed red blood cell fractions, but not in whole blood.