Differentiation of Indian isolates of bluetongue virus serotype 1 from Australian and African isolates based on analysis of vp5 gene

Abstract

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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-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 Script^TM Amp SK (+) vector and transformed into XL10-Gold^® 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.

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