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ThesisItem Open Access PARTIAL SEQUENCING AND CHARACTERIZATION OF VP2 GENOME OF BLUETONGUE VIRUS SEROTYPE-2(SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA, 2006-09) BALUMAHENDIRAN, M; SREENIVASULU, D(MAJOR); SATYANARAYANA CHETTY, M; SRILATHA, Ch.ABSTRACT: Occurrence of multiple serotypes and presence of antigenic diversity with in serotypes of BTV is the major constraint for developing a protective vaccine. Hence, there is a need to identify and characterize the circulating BTV serotypes in the endemic region. In this direction, a modest study was taken up to amplify, sequence and analyse the phylogenetic relationship of Indian isolate of BTV serotype-2. A pair of primers specific to BTV-2 was designed using VP2 sequences available in Genbank to ampllfy 1797-2932 bp region. Partial amplification of 1135 bp product of VP2 gene of BTV-2 was obtained by RT-PCR and cloning of the amplified product was carried out using pTZ 57R/T plasmid vector. The positive clones were confirmed by colony lysis, PCR, restriction analysis and sequencing. A sequence of 1074 nucleotides were read from the chromatogram. The sequence analysis using BLAST N search showed that the GC content of the gene was 43 percent. Comparison of nucleotide and amino and sequence over the region 1781-2854bp (1074 bases) revealed that Indian isolate BTV-2 was having 66- 86 and 67-86 percent identity respectively with BTV-2 isolates of different regions. Whereas, 55-62 and 3548 percent identity of nucleotide and amino acid respectively with other BTV serotypes from different regions. The multiple alignment of nucleotide and amino acid sequence over the proposed region of VM revealed that there were conserved bases among BTV-2 isolates at position 2170-2223 and 754-767aa respectively. Like wise conserved bases among different serotypes at position 2828-2849 and 916-933aa respectively. The alignment of 1074 bases of VP2 gene of Indian isolate BTV-2 showed 86 percent homology with BTV Taiwan isolate and indicate its closest relationship. Restriction map analysis over the same proposed region revealed that a single conserved SalI site (at 2380bp) was present in both Indian and Taiwan isolate of BTV-2 and confirms the genetic relationship among both isolates. Phylogenetic study also revealed that Indian isolate of BTV-2 more closely related to Taiwan isolate BTVs. Thus, the study of VM sequence comparison revealed that BTV-2 of the same serotypes but from different geographical region were closely related at the nucleotide and amino acid levels and both isolates might have evolved from a common evolutionary pathway. The present study enlightened further approach of developing a recombinant vaccine by expressing the VP2 gene in suitable expression system, so as to develop a suitable desired recombinant vaccine which is the need of hour to help the sheep rearing farmers.