SREENIVASULU, D(MAJOR)SATYANARAYANA CHETTY, MALAHA SINGARI, NHIMAJA, M2018-10-232018-10-232008-11http://krishikosh.egranth.ac.in/handle/1/5810081944THESESABSTRACT: The present study was taken up to standardize reverse transcription polymerase chain reaction (RT-PCR) for typing of bluetongue virus serotypes 2,9 and 15 isolated from the native sheep of Andhra Pradesh. Bluetongue viruses 2, 9 and 15 were propagated in BHK-21 cell line. Double stranded RNA extracted from the three serotypes resolved into nine bands with similar migration pattern in ethidium bromide stained 1% agarose gel. RT-PCR for serotyping was standardized using primers specific to VP2 gene of BTV-2, 9 and 15 serotypes. cDNA was synthesized by reverse transcribing the RNA using M-MuLV RT enzyme, after denaturing the ds RNA by heating at 95°C for 5 min in the presence of 10% DMSO. The concentration of 1.75 mM MgCl2, annealing temperature of 45°C for BTV-2, 53.8°C for BTV-9 and 61 59.2°C for BTV-15 and 30 cycle amplification were found optimum for amplification of cDNA of BTV-2, 9 and 15 by PCR. RT-PCR resulted in 653 bp product of BTV-2, 1241 bp product of BTV-9 which were defined by specific primers. However non specific amplification at two different sites i.e 700 bp and 1500 bp was noticed for BTV-15. Specificity of RT-PCR was evaluated. BTV-2 and BTV-9 specific primers could amplify only BTV-2 and BTV-9 respectively where as BTV-15 type specific primers amplified not only BTV-15 but also BTV-2 and BTV-9. Primers used for serotype detection of BTV-15 reacted with RNA of BTV-2 and BTV-9 and resulted in non specific amplification. It appears that the BTV-15 specific primers employed in the present study are not suitable for typing of BTV-15. This may be because of variations between the homologous serotypes from different geographical regions. For further studies, amplified PCR products were cloned into PTZ 57 R/T vector (T/A cloning vector) and then transformed to DH5α E coli cells. The recombinant colonies were screened by blue white selection. Positive colonies were confirmed by colony touch PCR and sequence analysis. BLAST search indicated that sequence obtained from BTV-2 PCR product and BTV-9 cloned products were specific to VP2 gene of BTV-2 and BTV-9 respectively. However, 700 bp and 1500bp products of BTV-15 were identical to VP4 gene of BTV-2, 8, 10, 11, 13 & 18 and VP1 gene of BTV-2, 8 & 10 respectively, indicating the non specific amplification of BTV-15. Multiple sequence alignment of BTV-2 nucleotide and amino acid sequences with other BTV-2 isolates from different geographical regions showed homology of 72-91.71% and 88-99% respectively. Phylogenic tree of these isolates indicate that BTV-2/TPT, BTV-2/ china and BTV-2/Taiwan might have evolved from the same evolutionary pathway. Multiple sequence alignment of nucleotide and amino acid sequences of BTV-9/MBN isolate with other BTV-9 isolates from three different geographical regions showed homology of 70-98% and 76-99% respectively. Phylogenic tree 62 of these isolates indicating that BTV-9/ MBN, BTV-9 Afaundau and BTV-9 Mediterranean basin are closely related and formed a group in monophyletic tree. Where as BTV-9 SA reference strain is having divergence of 30-31% from the remaining three isolates and formed a separate group in monophyletic treeennullThesis