SREENIVASULU, D (Major)SATYANARAYANA CHETTY, MALAHA SINGARI, NDASAIAH, C.B2016-12-152016-12-152008-01http://krishikosh.egranth.ac.in/handle/1/90395THESESABSTRACT : Hither it became a herculean task for the researchers to develop a suitable vaccine for BTV to suit the needs of sheep farmers and to reduce the economic losses since the virus exists as multiple serotypes. Further the virus undergoes frequent molecular changes becoming major impediment in the development of vaccine. Hence, a thrush has been given in understanding the molecular epidemiology of the virus both at nucleotide and deduced amino acid sequences. The present study indicated that BHK-21 cell lines were suitable for good yield of the virus. The electrophoretic migration pattern of indigenous isolates revealed 10 discrete bands. The RT-PCR using specific primers amplified the DNA of 1804 which was specific for L2 gene segment of BTV2. Cloning of PCR product was done in pTZ57R/T vector and E-coli DH5α host. The recombinant colonies were screened by blue-white selection and confirmation of recombinant colonies with desired insert was done by colony PCR and sequencing. The sequence analysis using BLAST N search indicated that the sequence obtained was specific to BTV-2 VP2 gene corresponding the region 852-2656 (1804 bases). Multiple sequence alignments of the nucleotide and amino acid sequences with other BTV2 isolates of different regions showed divergence in the viruses isolates. The bluetongue isolates BTV2-TPT and BTV-2MBNR showed the divergence of 0.9 to 1.2% at nucleic acid and amino acid level which indicated that the occurrence of variation within the serotype though they are isolated from different areas in different periods. The study indicated the BTV2 isolates are closely related to China and Taiwan isolates with nucleotide homology of 88.1 to 93.3. Phylogenetic study indicated that these BTV2 (indigenous) isolates along with BTV2 China and BTV2 Taiwan formed one clade in monophyletic tree indicating these might have evolved from common evolutionary pathway.enBLUETONGUE VIRUS; GENE SEQUENCING; sheepThesis