GENOME ANNOUNCEMENT
Complete Genome Sequence of an Isolate of Bluetongue Virus Serotype 2, Demonstrating Circulation of a Western Topotype in Southern India Narender S. Maan,a,b Sushila Maan,a,b Marc Guimera,a Gillian Pullinger,a Karam Pal Singh,c Kyriaki Nomikou,a Manjunatha N. Belaganahalli,a and Peter P. C. Mertensa Vector-borne Diseases Programme, Institute for Animal Health, Pirbright, Surrey, United Kingdoma; College of Veterinary Sciences, LLR University of Veterinary and Animal Sciences, Hisar, Haryana, Indiab; and Pathology Laboratory, Centre for Animal Disease Research and Diagnosis, Indian Veterinary Research Institute, Izatnagar, Indiac
Bluetongue virus serotype 2 (IND2003/02) was isolated in Tiruneveli City, Tamil Nadu State, India, and is stored in the Orbivirus Reference Collection at the Institute for Animal Health, Pirbright, United Kingdom. The entire genome of this isolate was sequenced, showing that it is composed of a total of 19,203 bp (all 10 genome segments). This is the first report of the entire genome sequence of a western strain of BTV-2 isolated in India, indicating that this virus has been introduced and is circulating in the region. These data will aid in the development of diagnostics and molecular epidemiology studies of BTV-2 in the subcontinent.
B
luetongue is an important infectious disease of livestock (12) caused by arboviruses belonging to the species Bluetongue virus (BTV), genus Orbivirus, family Reoviridae, that are transmitted between their ruminant hosts by biting midges (Culicoides spp.) (3). In the last decade, repeated incursions of exotic BTV strains have occurred in Europe, the United States, and Australia (11). There are 26 known BTV serotypes, all of which have a 10segmented double-stranded RNA genome packaged within a three-layer icosahedral protein capsid (15). BTV encodes seven structural proteins (VP1 to VP7) (7, 9, 13, 17) and four nonstructural proteins (NS1, NS2, NS3/3a, and NS4) (2, 4, 5, 14, 18, 19). The BTV “serotype” is controlled by outer capsid protein VP2, encoded by genome segment 2 (Seg-2). Therefore, identification of BTV by serotype does not recognize the sequence variations that exist between isolates from different geographic regions in the remaining nine genome segments. Based on full-genome sequences, we have identified a number of distinct “topotypes” that reflect these regional genetic variations (9). The global trade in livestock has helped BTV strains to move between geographic regions; for example, both eastern and western BTV strains have been identified in Europe (6, 8). Full-genome sequencing and phylogenetic comparisons to reference strains from different geographical locations can help to determine the origins of novel outbreak strains, identify regions of topotype intermixing, and identify reassortant progeny strains. However, a lack of data concerning BTV genome sequences, epistasis, evolution, and transmission dynamics has hampered efforts to develop appropriate diagnostics and control measures. BTV is endemic in India, and eight different serotypes have been isolated there in the last decade (16). BTV-2 (IND2003/02) was isolated in Tiruneveli City, Tamil Nadu State, India, in 2003 and is stored in the Orbivirus Reference Collection at the Institute for Animal Health, Pirbright, United Kingdom (http://www .reoviridae.org/dsRNA_virus_proteins/ReoID/btv-2 .htm#IND2003/02). The virus genome RNA was purified using TRIzol from infected BHK-21 cell cultures (1). The cDNA was synthesized using the FLAC technique (10). PCR products for each of the 10 genome segments were sequenced directly by elec-
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trophoresis on an Applied Biosystems 3730 capillary DNA sequencer using a BigDye Terminator Cycle Sequencing kit. Our analyses show that the complete genome of IND2003/02 has a total length of 19,203 bp. The sizes (base pairs) of Seg-1 to Seg-10 are 3,944, 2,943, 2,772, 1,981, 1,776, 1,635, 1,153, 1,125, 1,052, and 822, respectively. The structural protein sizes (amino acids) are as follows: VP1, 1,302; VP2, 962; VP3, 901; VP4, 644; VP5, 526; VP6, 329; VP7, 349. The nonstructural protein sizes (amino acids) are as follows: NS1, 552; NS2, 354; NS3/NS3a, 229/ 216; NS4, 79. VP6 of IND2003/02w is shorter by one amino acid and NS4 is longer by two amino acids than that of other “eastern” BTV strains from India. These data will aid in the development of diagnostic assays, surveillance, identification of east-west reassortants, and control measures for BTV-2 in the region. Nucleotide sequence accession numbers. The nucleotide sequence accession numbers are AJ783905 for Seg-6 and JQ681257 to JQ681265 for Seg-1 to Seg-5 and Seg-7 to Seg-10, respectively. ACKNOWLEDGMENTS We are very grateful to Aruni Wilson, Tamil Nadu Veterinary and Animal Sciences University, Chennai, India, for sending us the virus isolate. We acknowledge funding support from the Commonwealth Commission, DEFRA, the European Commission (OrbiVac grant 245266, WildTech grant 222633-2), EMIDA grant OrbiNet K1303206, OIE, and BBSRC.
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Received 17 February 2012 Accepted 21 February 2012 Address correspondence to Peter P. C. Mertens,
[email protected]. Copyright © 2012, American Society for Microbiology. All Rights Reserved. doi:10.1128/JVI.00420-12
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3. Du Toit RM. 1944. The transmission of bluetongue and horse-sickness by Culicoides. Onderstepoort J. Vet. Sci. Anim. Ind. 19:7–16. 4. Firth AE. 2008. Bioinformatic analysis suggests that the orbivirus VP6 cistron encodes an overlapping gene. Virol. J. 5:48. 5. Huismans H, Els HJ. 1979. Characterization of the tubules associated with the replication of three different orbiviruses. Virology 92:397– 406. 6. Maan S, et al. 2009. Molecular epidemiology studies of bluetongue virus, p 135–166. In Mellor PS, Baylis M, Mertens PPC (ed), Bluetongue. Elsevier/Academic Press, London, United Kingdom. 7. Maan S, et al. 2011. Novel bluetongue virus serotype from Kuwait. Emerg. Infect. Dis. 17:886 – 889. 8. Maan S, et al. 2008. Sequence analysis of bluetongue virus serotype 8 from The Netherlands 2006 and comparison to other European strains. Virology 377:308 –318. 9. Maan S, et al. 2010. Full genome characterisation of bluetongue virus serotype 6 from The Netherlands 2008 and comparison to other field and vaccine strains. PLoS One 5:e10323. 10. Maan S, et al. 2007. Rapid cDNA synthesis and sequencing techniques for the genetic study of bluetongue and other dsRNA viruses. J. Virol. Methods 143:132–139. 11. Maclachlan NJ, Guthrie AJ. 2010. Re-emergence of bluetongue, African horse sickness, and other orbivirus diseases. Vet. Res. 41:35.
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