Molecular cloning and comparative sequence analyses of bluetongue virus S1 segments by selective synthesis of specific full-length DNA copies of dsRNA genes.

Using primers complementary to the conserved sequences of the 3' ends of bluetongue virus genomic dsRNA segments, full-length DNA clones of all 10 dsRNA genes from the five U.S. BTV serotypes were synthesized and amplified by a novel method (ClampR). This amounts to nearly 100,000 base pairs of dsRNA cloned as unique full-length DNA copies. This continuous one-tube procedure combined cloning of the denatured dsRNA with reverse transcriptase and the selective amplification of full-length DNA by the polymerase chain reaction. ClampR-derived clones of the genomic segment S1 of BTV-11 encoding the serogroup antigen, VP7, were sequenced and shown to be complete copy, containing a total of 1156 bp and a long open reading frame of 349 amino acids. Comparative sequence analyses of BTV-11 S1 with those of the other U.S. serotypes show that 95.2% of the nucleotides are conserved between BTV-11 and -10, while only 79.0% of the bases are identical between BTV-11 and -13. Comparison of the VP7 proteins demonstrates that 100% of the amino acids are conserved between BTV-11 and -10 and 93.7% of these residues are identical between VP7 of BTV-11 and -13. The adaptation of the polymerase chain reaction to the full-length cloning and amplification of dsRNA (ClampR) should greatly facilitate molecular studies within the Reoviridae family.