MOLECULAR CHARACTERIZATION OF BLUETONGUE VIRUS SEROTYPE 9 ISOLATES
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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 three isolates of BTV9. The titration of the three isolates MBN,K8 and W20 of BTV9 in BHK-21 cells was done for 5 passages in BHK-21. Titres were 103.57, 103.8, 104.6, 105.33 and 107.55 TCID50/ml for MBN, 102.57, 103.4, 103.5, 104.25 and 105 TCID50/ml for K8 and 102.69, 102.99, 103.58, 104.68 and 105.64TCID50/ml for W20 isolate.
Further, one step growth curve experiments were conducted for the isolates in BHK-21 cells. All three isolates had maximum titres at 48h. The inoculum size had no appreciable effect on the harvest of the virus in the dilutions tested. RT-PCR was standardized for detection of VP7, NS1 and VP2 genes of BTV. For VP7 gene : cDNA synthesis at 420C for 45 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 , final extension cycle of 4 min at 720C were found to be suitable.For VP2 gene: cDNA synthesis at 420C for 45 min then initial denaturation at 940C for 3 min, 35 cycles of denaturation at 940C for 30s, annealing at 550C for 30s and extension at 680C for 2 min, final extension cycle of 10 min at 680C were found to be suitable.
Molecular characterization of the K8 isolate was taken up by the sequencing of NS1 (M6 segment), VP7 (segment 7) and VP2 (L2 segment) genes. The sequences were compared to the available sequences in genbank. NS1 nucleotide sequence analysis revealed that, NS1 gene sequence of BTV 9 (K8) is closely related to BTV 9 (MBN), BTV 2 and BTV 15.Further, NS1 derived amino acid analysis indicated cent percent homology between BTV9 K8 and BTV9 MBN isolates and close identity with BTV2 and BTV15. VP7 nucleotide sequence and derived amino acid analysis revealed that, BTV 9 (K8) is closely related to BTV 9 (MBN), BTV 2 and BTV 15.
VP2 nucleotide sequences, responsible for serotype specificity, segregated into 10 distinct lineages with greatest sequence similarities between serologically related serotypes BTV 9 and BTV 5.Further analysis indicated that BTV 9 (K8) was closely related to MBN, Afandou isolates of same serotype BTV 9 and BTV5 but more distantly related to other 22 serotypes of BTV.
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