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

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-1S(3)), 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 (R) 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.