Nucleotide sequence analysis of VP7 gene of Indian isolates of bluetongue virus vis-à-vis other serotypes from different parts of the world

Abstract

Bluetongue virus (BTV), a member of genus Orbivirus, a family Reoviridae, is a non-enveloped with double shelled structure and ten segmented double stranded (ds) RNA genome. The RNA segment S7 encodes an inner capsid serogroup specific viral protein VP7. To amplify coding region of VP7 gene of BTV, new primers, forward primer (18-38 bp) and reverse primer (1156-1136 bp), were designed using VP7 gene sequences available in GenBank. This primer pair successfully amplified cell culture adapted Indian isolates of BTV belonging to two different serotypes 1 and 18. The coding sequences of two Indian isolates of BTV (BTV-1H and BTV-18B) were cloned into pPCR Script-Amp SK (+) plasmid vector and transformed into XL10-Gold Kan ultracompetent E. coli cells. The positive clones selected by blue-white screening and colony touch PCR were sequenced. The sequence analysis revealed that there was 93-97% nucleotide sequence identity in VP7 gene of three different Indian serotypes of BTV. The VP7 gene sequences of Indian isolates have comparatively less sequence homology (< 80%) with American (US), and French isolates compared to South African (SA), Australian (AUS) and Chinese (PRC) isolates. In silico restriction enzyme profile analysis of VP7 gene sequences revealed that Indian isolates of BTV-1 can be differentiated from other BTV-1 isolates reported from SA, AUS and PRC using TaqI. Similarly the Indian isolates of BTV belonging to three different serotypes can be differentiated using EcoRI, Hae III and TaqI restriction enzymes.