Sequence conservation among the cognate nonstructural NS3/3A protein genes of six bluetongue viruses

Full-length cDNA copies of segment 10 genes of bluetongue virus serotypes 2, 11, 13 and 17 were synthesized by the Clamp-R method and inserted into the plasmid pUC19. The complete nucleotide sequences of these four cognate genes were sequenced and determined to be 822 nucleotides in length, smallest of the 10 genes in the bluetongue virion. These four cognate gene segments contained two in-phase and overlapping open reading frames capable of coding for two non-structural proteins of 229 and 216 amino acids with net charges of +4.5 and +5.5, respectively, at neutral pH. Comparative analyses of the predicted amino acid sequences of bluetongue virus serotypes 1, 2, 10, 11, 13 and 17 revealed (i) a high degree of sequence homology and conservation, (ii) a single conserved tryptophan located at residue 159, (iii) the presence of two conserved cysteines at residues 137 and 181 and two potential N-linked glycosylation sites at residues 63-65 and 150-152, (iv) a cluster of six prolines within a 15-amino acid region near the amino terminus, and (v) the longest 3' noncoding sequence of 113 bases among the 10 bluetongue viral genes. Phylogenetic analyses indicated that BTV-10 and -11 are very closely related and BTV-2 is the distantly related serotype of the five US bluetongue virus serotypes.