Sequence analysis and in vitro expression of a cDNA clone of genome segment 5 from bluetongue virus, serotype 1 from South Africa.

A full length copy of genome segment 5 of bluetongue virus serotype 1 from South Africa (BTV-1SA) was assembled from two incomplete cDNA clones. The complete nucleotide sequence was determined (1635 nucleotides in length) and an open reading frame coding for 527 amino acids was found, which was flanked by a 5' non-coding region of 25 nucleotides and a 3' non-coding region of 29 nucleotides. The cDNA clone was transferred to an Sp6 expression vector from which an RNA transcript was obtained. This transcript, when translated in vitro in a reticulocyte lysate system, produced a protein that co-migrated during electrophoresis with both protein VP5 from disrupted virus particles and VP5 translated from denatured viral dsRNA. The protein synthesized from the cDNA clone was precipitable with antisera raised against BTV-1SA virus particles and with antisera raised against a synthetic peptide, the sequence of which was obtained from the predicted amino acid sequence of BTV-1SA protein VP5. These antisera also precipitated protein VP5 translated from denatured viral dsRNA. Collectively these data indicate that the cDNA clone encodes an authentic VP5 protein product. The amino acid sequence of BTV-1SA VP5, when compared to other published sequences for VP5, contained highly conserved regions interrupted by variable domains. If two isolates of the same serotype are compared, (BTV-1SA and BTV-1AUS) only two variable regions are apparent. However, if the amino acid sequences of VP5 from two different serotypes are compared, (BTV-1SA and BTV-10), eight variable regions are detectable (two of which are in the same position as the variable regions within a serotype). The implications of these variations in the outer coat protein, VP5, are discussed.