MOLECULAR CHARACTERIZATION OF BLUETONGUE SEROTYPES 2 AND 15
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Date
2010-01
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SRI VENKATESWARA VETERINARY UNIVERSITY TIRUPATI - 517 502. (A.P.) INDIA
Abstract
ABSTRACT :
The present study was taken with a view to characterize two BTV strains employed for trial vaccine. The titration of the two isolates M11 (BTV 2) and N12 (BTV 15) in BHK-21 cells was done for 5 passages. Titres were 104.55, 104.68, 106.12, 106.72 and 107.22 TCID50/ml for M11 and 102.69, 103.58, 105.64, 107.55 and 107.56 TCID50/ml for N12. Further one step growth curve experiments were conducted for the isolates in BHK-21 cells. Both the viruses had maximum titres at 48h. The inoculum size had no significant effect on the harvest in the dilutions tested. RT-PCR was standardized for detection of VP7 and NS1 genes BTV. For VP7 gene cDNA synthesis at 450C for 50 min then initial denaturation at 950C for 3 min, 30 cycles of denaturation at 950C for 20s, annealing at 390C for 60s and extension at 700C for 2 min, final extension cycle of 7 min at 700C were found to be suitable. For NS1 cDNA synthesis at 420C for 60 min then initial denaturation at 950C for 3 min, 30 cycles of denaturation at 950C for 25s, annealing at 580C for 20s and extension at 720C for 30s min, final extension cycle of 5 min at 720C were found to be suitable. Molecular characterization of the isolates was taken up by the sequencing of NS1 (M6 segment) and VP7 (segment 7). The sequences were compared to the available sequences in genbank. All NS1 nucleotide sequences segregated into 6 phylogenetic clades. Further analysis revealed that NS1 gene sequence of BTV-15 (N12) is closely related to BTV-15 (N15), BTV-15 (DQ399835). BT-2 (M11) and BTV-15 (N12) clustered together with BTV-9 BTV TPT and BTV KMTAI. All VP7 nucleotide sequences segregated into 7 phylogenetic clades. Further analysis indicated that BTV-2 (M11) was closely related to BTV-12 Brazil. BTV-15 (N12) was more closely related to BTV-15 (N15), BTV-15 (DQ399835), BTV-15 China than to BTV-15 Australia and BTV-9 (MBN).
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