Please use this identifier to cite or link to this item: http://nopr.niscair.res.in/handle/123456789/7788
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: <smarttagtype namespaceuri="urn:schemas-microsoft-com:office:smarttags" name="State"><smarttagtype namespaceuri="urn:schemas-microsoft-com:office:smarttags" name="City"><smarttagtype namespaceuri="urn:schemas-microsoft-com:office:smarttags" name="place"> Bluetongue virus (BTV), prototype species of genus <i style="">Orbivirus, </i>belongs to the family <i style="">Reoviridae</i>. 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 &amp; 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-1S<sub>3</sub>), 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 Script<sup>TM</sup> Amp SK (+) vector and transformed into XL10-Gold<sup>®</sup> Kan ultracompetent <i style="">Escherichia coli</i> 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. <i style="">In silico</i> 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 <i style="">Taq</i>I and <i style="">Bsm</i>I restriction endonucleases. </smarttagtype></smarttagtype></smarttagtype>
Description: 117-125
URI: http://hdl.handle.net/123456789/7788
ISSN: 0975-0967 (Online); 0972-5849 (Print)
Appears in Collections:IJBT Vol.09(2) [April 2010]

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