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Indian Journal of Biotechnology (IJBT) >
IJBT Vol.09 [2010] >
IJBT Vol.09(2) [April 2010] >
| Title: | Differentiation of Indian isolates of bluetongue virus serotype 1 from Australian and African isolates based on analysis of vp5 gene |
| Authors: | Manjunatha, B N
Prasad, Minakshi
Maan, S
Prasad, G |
| Keywords: | Bluetongue
BTV-1
cloning
RE analysis
RT-PCR
sequencing
vp5 gene |
| Issue Date: | Apr-2010 |
| Publisher: | CSIR |
| Abstract: |
Bluetongue
virus (BTV), prototype species of genus Orbivirus,
belongs to the family Reoviridae.
It is a non-enveloped, double shelled virus with ten segmented dsRNA genome. RNA segment
6 encodes an outer capsid serotype specific virus protein VP5. A pair of primers (forward
207-229 bp & reverse 1284-1304) was designed from the published BTV-1
segment 6 sequences to specifically amplify vp5 gene from Indian isolates of
BTV. These primers specifically amplified PCR product of 1098 bp from cell
culture adapted isolates of BTV-1 (Hisar isolate-BTV-1H, Avikanagar isolate-BTV-1A
and Sirsa isolate-BTV-1S3), but did not give any
amplification with BTV-9 and BTV-23, indicating serotype specificity. vp5
coding sequences amplified from Indian BTV-1 isolates were cloned into pPCR
ScriptTM Amp SK
(+) vector and transformed into XL10-Gold® Kan ultracompetent Escherichia coli cells. The positive clones selected by blue–white
screening and colony touch PCR were sequenced. The sequence analysis of the vp5
gene (253-1255 bp) revealed that Indian isolates of BTV-1 showed 89-91.1%
nucleotide identity with Australian isolates of BTV-1, whereas it showed only
77-79.7% similarity with the BTV-1 African isolates. All three Indian isolates
shared 99.4% nucleotide sequence similarity amongst themselves. Comparison of
the deduced amino acid sequences revealed that the Indian BTV-1 isolates shared 96.7-98.8% and 94.9-95.8% amino acid similarity with Australian and
African BTV-1 isolates, respectively. In silico restriction enzyme (RE) profile analysis of vp5 gene
sequences showed that Indian isolates of BTV-1 can be differentiated from other
BTV-1 isolates from South Africa and Australia using TaqI and BsmI restriction
endonucleases.
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| Page(s): | 117-125 |
| ISSN: | 0975-0967 (Online); 0972-5849 (Print) |
| Source: | IJBT Vol.09(2) [April 2010]
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