Amplification and full length sequencing of genome segment 2 and segment 6 of Indian isolates of bluetongue virus serotype 16 and 21
Abstract
One of the most important economic vector borne diseases of ruminants worldwide is caused by bluetongue (BT) virus, an orbivirus of the Reoviridae family. The virus has segmented genome and is thus prone to frequent mutations and gene reassortment leading to emergence of new genetic variants. The outer capsid proteins VP2 and VP5 are the most important proteins responsible for serotype specificity, virus neutralization and haemagglutination. Present study was undertaken to conduct full length nucleotide sequencing of vp2 and vp5 genes of two new Indian isolates VJW-64 (BTV16) and KMNO-7 (BTV21) which would lead to efficient characterization of the virus eventually helping in selection of predominant candidate vaccine strain. For the purpose of sequencing vp2 and vp5 genes were divided into several overlapping gene fragments and primer pairs specific to every such fragment were designed. Nucleotide sequence homology analysis of vp2 and vp5 genes of these two Indian isolates revealed an inter-serotypic variation of 29% to 60.1% on the basis of vp2 gene. VJW-64 (BTV16) was found to show a maximum similarity with South African reference strain of BTV3 while Indian isolate KMNO-7 (BTV21) showed a maximum similarity of 68.7% with South African reference strain of BTV14 on the basis of vp2 gene. On the basis of vp5 gene VJW-64 (BTV16) was found to show maximum similarity with South African reference strain of BTV21 while Indian isolate KMNO-7 (BTV21) showed maximum similarity with Turkey isolate of BTV16. Intra-serotypic nucleotide sequence homology analysis of vp2 gene revealed close relationship of Indian isolate VJW-64 (BTV16) with Greece and Italy isolates while vp5 gene based analysis revealed close relationship of Indian isolate VJW-64 (BTV16) with Turkey isolate and South African reference and vaccine strains. KMNO-7 (BTV21) showed nucleotide similarity of 85.3% with the South Africa reference strain of BTV21 on the basis of vp2 gene analysis, while on the basis of vp5 gene analysis KMNO-7 (BTV21) revealed more nucleotide similarity (97.6%) with Turkey isolate of BTV16 than with South African isolate of BTV21 (90.9%). In-silico restriction enzyme analysis revealed certain restriction sites which were specific to the Indian isolates of BTV16 (VJW-64) and BTV21 (KMNO-7). In this study full length vp2 and vp5 gene sequences of Indian isolates VJW-64 (BTV16) and KMNO-7 (BTV21) have been carried out for the first time. This study in itself is an important initiative towards developing a database of full length vp2 and vp5 gene sequences of Indian isolates. It paves a way for studying the protein structure and antigenicity of VP2 and VP5 proteins of these Indian isolates.