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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.
Page(s): 117-125
ISSN: 0975-0967 (Online); 0972-5849 (Print)
Source:IJBT Vol.09(2) [April 2010]

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