Phylogenetic Analysis Of Bluetongue Viruses In China And S10 Gene Expression Of The Virus

Bluetongue virus(BTV) is the causative agent of bluetongue disease in domestic ruminants(sheep, cattle).It is a member of the Orbivirus genus (Reoviridae family). In this study, S10 gene sequences of 1 attenuated vaccine strain and 25 Chinese field isolates of BTV were determined and The results have revealed that all 26 510 gene segments have 822 nucleotides in length with two inrame initiation codons (nucleotides 20 to 22 and 59 to 61) and a common termination codon (nucleotides 707 to 709), which encodes two proteins (NS3 and NS3A). Nucleotide difference in the sequence of all Sl0 gene were from zero to 107 indicating identities between lOO%86. 4%. Comparison of the predicted NS3/NS3A protein encoded by the 810 gene showed little variation between the various strains (zero to 10 amino acid difference or l00%-5.6% identity). Phylogenetic analysis of the SlO gene of above strains sequenced and 16 other strains from Genebank, segregated the Chinese viruses into a monophyletic group distinct from US viruses; Nucleotide identity was 85% between China Group and US group. The various Chinese isolates segregated into two phyletic subgroups based on S10 gene sequences. The clustering of viruses was dependent of geographical origin, and independent of host species of isolation , serotype & year of isolation. By analyzing the amino acid structure of NS3, it was found that the NS3 of all the BTV strains were 229 amino acids long, in which there were 28-2 strongly basic amino acids, 24-5 strongly acidic amino acids, 79-2 hydrophobic amino acids and 56-0 polar amino acids, and that the isoelectric point of NS3 is 8. 7?. 4. With the prediction results of the transmembrane secondary structure of NS3, it revealed that the topological or alpha helical structure of NS3 from each strain was identical, with its N?and C-terminal inside the cell membrane, and that there were two transmembrane helices, each comprising of 14?0 amino acids.The position of the NS3 outside the cell membrane was from 139 to 163 amino acid. It was predicted that the third structure of NS3 possibly had three forms. A portion of the L2 gene from 15 field strain isolates and 1 attenuated vaccine strain of Chinese BTV serotype 1, corresponding to nucleotide 854?806 was amplified via RT-PCR, cloned and sequenced. Phylogenetic analysis was done on the amplified region of L2 gene of Chinese strains of BTV serotype 1. The L2 gene sequences segregated into two distinct monophyletic groups(A and B).Group A included Y863 strain isolated in 1979, Group B includes 1 attenuated vaccine strain and 14 virus strains isolated after 1996. Strains of group B formed five lineage, vaccine strain and prototype strains of BTV were divided into different lineage, These results showed that L2 sequences of Chinese BTV strains of serotype 1 had genetic variation in procession of natural evolution and embryo culture. Based on the sequences of BTV RNA segment L2 and S10 , the pairs of oligodeoxy nucleotide primers for establishing the BTV serogroup?& serotype-specific RT-PCR assay were designed and synthesized. The RT-PCR assay to determinate serogroups could detect all Chinese BTV strains of different serotypes, without cross-reaction with other members of Orbivirus. The RT-PCR assay to determinate serotypes for BTV-1 co...