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The segment 10 (S10) genes of African horsesickness virus (AHSV), Palyam virus and epizootic haemorrhagic disease virus were translated in vitro in a rabbit reticulocyte lysate system. Each of the S10 genes encoded two proteins NS3 and NS3A, which were shown to be related by peptide mapping. Cloned copies of the S10 genes of two AHSV serotypes (AHSV-3 and AHSV-9) and Palyam virus were sequenced and compared to each other and to the nucleotide sequence of bluetongue virus (BTV) gene S10. Two in-phase ATG translation initiation codons reported for the S10 genes of BTV-10 and BTV-1 were conserved in the S10 genes of AHSV-3, AHSV-9 and Palyam virus, and would be able to initiate synthesis of NS3 and NS3A respectively. Comparison of the amino acid sequences of NS3 of AHSV-3 and AHSV-9 identified two areas of approximately 45 amino acids which displayed high (98%) similarity. One of these areas corresponded to the only region which displayed more than 50% amino acid similarity between NS3 of BTV, AHSV and Palyam virus. This region could represent an important structural or catalytic site of the protein. The overall amino acid similarity outside this conserved region was between 13% and 29%.

A comparison of the genes which encode non-structural protein NS3 of different orbiviruses