A comparison of different cloned bluetongue virus genome segments as probes for the detection of virus-specified RNA.

The seven largest double-stranded (ds) RNA genome segments of bluetongue virus (BTV) serotype 4 as well as genome segment 8 of BTV10 have been cloned into pBR322. The length of the cloned genes indicates that, with the exception of genome segment 1, the entire gene has been cloned in each case. A method is described for isolating different sized cDNA transcripts on alkaline sucrose gradients with very good recovery. The eight cloned genes were compared as 32P-labeled probes for the detection of dsRNA from 21 different BTV serotypes. The S1, S3, S4, S5, and S8 genes were identified as being highly conserved. Of these, S5, which codes for nonstructural protein NS1, gave the best hybridization signal with all the dsRNA isolates. All the probes hybridized significantly weaker with the dsRNA of BTV isolates from Australia and Pakistan than with dsRNA from other serotypes. Genome segment 7, which codes for the group-specific antigen P7, was not highly conserved. Even more variation was shown by genome segment 6 which codes for outer capsid polypeptide P5. S2 which codes for protein P2 is the obvious choice for a serotype-specific probe. Hybridization of this probe with dsRNA from other serotypes reflects the cross-neutralization between BTV4 and these serotypes. The hybridization results can also be used to define the relatedness of BTV4 to other serotypes. None of the probes hybridized with dsRNA from any of the other orbiviruses investigated.