Genome Sequence of a Reassortant Strain of Bluetongue Virus Serotype 23 from Western India

Abstract

The full genome sequence (19,177 bp) of an Indian strain (IND1988/02) of bluetongue virus (BTV) serotype 23 was determined. This virus was isolated from a sheep that had been killed during a severe bluetongue outbreak that occurred in Rahuri, Maharashtra State, western India, in 1988. Phylogenetic analyses of these data demonstrate that most of the genome segments from IND1988/02 belong to the major “eastern” BTV topotype. However, genome segment 5 belongs to the major “western” BTV topotype, demonstrating that IND1988/02 is a reassortant. This may help to explain the increased virulence that was seen during this outbreak in 1988. Genome segment 5 of IND1988/02 shows >99% sequence identity with some other BTV isolates from India (e.g., BTV-3 IND2003/08), providing further evidence of the existence and circulation of reassortant strains on the subcontinent.

GENOME ANNOUNCEMENT

Bluetongue virus (BTV) is the type species of the genus Orbivirus within the family Reoviridae. There are 26 BTV serotypes that are determined by the specificity of interactions between the virus outer capsid (particularly protein VP2) with neutralizing antibodies generated during infection of the ruminant host (10). The genome of BTV is composed of 10 linear segments of double-stranded RNA (dsRNA) packaged within the three-layered protein capsid (2, 16, 20, 23). The BTV genome codes for a total of seven structural (VP1 to VP7) and four nonstructural (NS1, NS2, NS3/3a, and NS4) proteins (2–5, 7, 11, 15, 19, 21). BTV is transmitted between its ruminant hosts primarily by Culicoides biting midges, causing a severe disease known as bluetongue (BT), particularly in naïve sheep populations and some species of deer (13).

BTV is endemic between approximately 45 to 53°N and 35°S, including areas of North and South America, Africa, the Indian subcontinent, Australasia, and Asia (14, 24). Most BTV genome segments contain nucleotide sequence variations that reflect the geographic origins of the different isolates (6, 9, 18), dividing them into “eastern” and “western” groups/topotypes (9, 17). During 1988 and 1998, multiple BTV strains were isolated in India (22). These include a strain of BTV serotype 23 (BTV-23; isolate IND1988/02) from a sheep killed during a severe BT outbreak in Rahuri, Maharashtra State, in January 1988 (22).

IND1988/02 was obtained from the Orbivirus Reference Collection at the Institute for Animal Health, Pirbright (http://www.reoviridae.org/dsRNA_virus_proteins/ReoID/btv-23.htm). Viral dsRNA was extracted from infected BHK-21 cells using TRIzol reagent (1). Full-length PCR amplicons were generated for all 10 genome segments using the FLAC technique (12) and then sequenced on a 3730 capillary sequencer (Applied Biosystems). Phased primers were used to generate near-terminal sequences (12), and walking segment-specific primers were used to determine the full nucleotide sequence. The total genome of IND1988/02 is 19,177 bp; Seg-1 to Seg-10 are 3,944 bp, 2,927 bp, 2,772 bp, 1,981 bp, 1,765 bp, 1,635 bp, 1,154 bp, 1,125 bp, 1,052 bp, and 822 bp, respectively. The lengths of the structural proteins of IND1988/02 are as follows: VP1, 1,302 amino acids (aa); VP2, 957 aa; VP3, 901 aa; VP4, 644 aa; VP5, 526 aa; VP6, 330 aa; VP7, 349 aa. The lengths of the four nonstructural proteins are as follows: NS1, 552 aa; NS2, 354 aa; NS3/NS3a, 229 aa/216 aa; NS4, 77 aa.

Phylogenetic analyses of IND1988/02 show that the majority of its genome segments belong to eastern lineages. However, Seg-5/NS1 groups very closely (>98.5% nucleotide sequence identity) with several other BTV serotypes from India (BTV-2, -3, and -9), as a monophyletic group within the “major western” topotypes (8). This demonstrates the wider circulation of this western genome segment 5 and indicates that reassortment events involving eastern strains of multiple serotypes have occurred within the Indian BTV population.

These data represent the first report of a complete genome sequence of a BTV-23 reassortant (BTV-23r). They will help to support molecular epidemiology studies and the implementation of appropriate control measures for BT on the Indian subcontinent.

Nucleotide sequence accession numbers.

The nucleotide sequences of IND1988/02 were deposited in the GenBank database under accession numbers AJ631217 for Seg-6 and JQ771823 to JQ771831 for Seg-1 to Seg-5 and Seg-7 through to Seg-10, respectively.