ORIGINAL ARTICLE Isolation of Bluetongue Virus Serotype 1 from Culicoides vector Captured in Livestock Farms and Sequence Analysis of the Viral Genome Segment-2 A. I. Dadawala 1 , S. K. Biswas 2 , W. Rehman 2 , K. Chand 2 , A. De 3 , B. S. Mathapati 3 , P. Kumar 1 , H. C. Chauhan 1 , B. S. Chandel 1 and B. Mondal 2 1 Department of Veterinary Microbiology, College of Veterinary science and Animal Husbandry, Sardarkrushinagar Dantiwada Agricultural University, Gujarat, India 2 Division of Virology, Indian Veterinary Research Institute, Mukteswar Campus, Uttarakhand, India 3 Project Directorate on Foot and Mouth Disease, IVRI Campus Mukteswar, Uttarakhand, India Introduction Bluetongue (BT) is an arthropod-borne viral disease pri- marily of sheep caused by the bluetongue virus (BTV). The virus can infect all ruminants, but in sheep and some species of deer the clinical signs are usually most severe. Transmission of the virus between hosts takes place by the biting insects of the genus Culicoides (Mellor, 2000), which are most abundant and active in hot and humid climates. Of more than one thousand species that occur worldwide, only 30 or so have been incriminated as potential vectors of BTV infection (Meiswinkel et al., 2008). Appropriate climatic conditions are important in the regional maintenance of BTV infection, and the virus exists throughout much of the world between latitudes 40–50°N and 35°S (Gibbs and Greiner, 1994). However, the virus recently has spread far beyond the upper limits of this traditional range in northern parts of Europe (Sae- german et al., 2008). Bluetongue is endemic in India with frequent outbreaks in sheep, and BTV antibodies are widespread in rumi- nants throughout the country. Several serotypes of BTV have been isolated from different states of India, especially from the regions where sheep population is more. BTV-1 is mostly prevalent in the north-western regions (mainly the state of Rajasthan), and virus has been isolated from sheep, goat and Culicoides (Jain et al., 1986, 1988; Prasad et al., 1994; Biswas et al., 2010). BTV-23 has been isolated from sheep from the states of Jammu and Kashmir, Utta- rakhand, Madhya Pradesh, Maharashtra and Karnataka (Mehrotra et al., 1989, 1995, 1996; Tembhurne et al., 2010), while BTV-2, BTV-9 and BTV-15 have been iso- lated from sheep in the Andhra Pradesh state (Sreenivas- ulu et al., 1999; Bommineni et al., 2008). From the Keywords: bluetongue virus serotype-1; Culicoides oxystoma; RNA; PAGE; VP2 gene Correspondence: B. Mondal. Division of Virology, Indian Veterinary Research Institute, Mukteswar Campus, Dist. Nainital 263 138, Uttarakhand, India. Tel.: +91 5942 286346; Fax: +91 5942 286347; E-mail: bimalendu.m@email.com Received for publication August 26, 2011 doi:10.1111/j.1865-1682.2011.01279.x Summary Bluetongue virus serotype-1 (BTV-1) was isolated from Culicoides oxystoma vectors captured on livestock farms in two places of Gujarat, India. The viruses were isolated on BHK-21 cells, which produced characteristic BTV-related cyto- pathic effects between 24 and 48 h post-infection. Virus antigen was demon- strated in infected cells at different passage by a BTV-specific sandwich ELISA. Further, polyacrylamide gel electrophoresis and silver staining of viral genomic RNA revealed ten double-stranded RNA segments characteristic of BTV. Sero- type of the isolates was identified by virus neutralization and PCR coupled with sequencing. The isolates were designated as SKN-7 and SKN-8 and their gen- ome segment-2 (VP2) were sequenced. Phylogenetic analyses revealed very close relationship between them although they are not identical. SKN-8 showed closer relationship with a recently isolated BTV-1 from goat. Bluetongue virus was earlier isolated from Culicoides in adjacent state more than 20 years ago, although the serotype of the virus was not determined. Transboundary and Emerging Diseases ª 2011 Blackwell Verlag GmbH • Transboundary and Emerging Diseases. 1