Genetic characterization of the non-structural protein-3 gene of bluetongue virus serotype-2 isolate from India

Abstract

Aim: Sequence analysis and phylogenetic studies based on non-structural protein-3 (NS3) gene are important in understanding the evolution and epidemiology of bluetongue virus (BTV). This study was aimed at characterizing the NS3 gene sequence of Indian BTV serotype-2 (BTV2) to elucidate its genetic relationship to global BTV isolates.

Materials and methods: The NS3 gene of BTV2 was amplified from infected BHK-21 cell cultures, cloned and subjected to sequence analysis. The generated NS3 gene sequence was compared with the corresponding sequences of different BTV serotypes across the world, and a phylogenetic relationship was established.

Results: The NS3 gene of BTV2 showed moderate levels of variability in comparison to different BTV serotypes, with nucleotide sequence identities ranging from 81% to 98%. The region showed high sequence homology of 93-99% at amino acid level with various BTV serotypes. The PPXY/PTAP late domain motifs, glycosylation sites, hydrophobic domains, and the amino acid residues critical for virus-host interactions were conserved in NS3 protein. Phylogenetic analysis revealed that BTV isolates segregate into four topotypes and that the Indian BTV2 in subclade IA is closely related to Asian and Australian origin strains.

Conclusion: Analysis of the NS3 gene indicated that Indian BTV2 isolate is closely related to strains from Asia and Australia, suggesting a common origin of infection. Although the pattern of evolution of BTV2 isolate is different from other global isolates, the deduced amino acid sequence of NS3 protein demonstrated high molecular stability.

Keywords: bluetongue virus; non-structural protein-3 gene; phylogenetic analysis.

Figure-1

Alignment of the amino acid sequences of bluetongue virus (BTV) non-structural protein-3 (NS3) protein. Alignment of the deduced amino acid sequences (229 aa) of BTV NS3 protein. The late motif domains PPXY/PSAP, glycosylation sites (Gly I and Gly II) and hydrophobic domains (HD I and HD II) have been underlined. The unique amino acid substitutions isoleucine (I) and glycine (G) were indicated by an arrow. The conserved residues cysteine (C) and tryptophan (W) are indicated within a box.

Figure-2

Phylogenetic tree of the non-structural protein-3 nucleotide coding sequences of different bluetongue virus (BTV) isolates of the world. The evolutionary history was inferred using the neighbor-joining method using MEGA7 with bootstrap values of 500 replicates. Different lineages and sub-lineages are indicated. Our isolate, Indian BTV serotype-2 is indicated by a triangle.