Advances in Animal and Veterinary Sciences

Research Article
Adv. Anim. Vet. Sci. 2 (2S): 23 - 26. Special Issue-2 (Advances in Diagnosis and Control of Infectious Diseases of Animals)
http://dx.doi.org/10.14737/journal.aavs/2014/2.2s.23.26
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Arnab Ghosh1, Narender Singh Maan2, Aman Kumar1, Kanisht Batra1, Mahendra Singh1, Sunayna1, Sushila Maan1*
1Department of Animal Biotechnology, College of Veterinary Sciences, Lala Lajpat Rai University of Veterinary and Animal Sciences, Hisar, 125 004, Haryana, India; 2Department of Animal Nutrition, Resource faculty Department of Animal Biotechnology, College of Veterinary Sciences, Lala Lajpat Rai University of Veterinary and Animal Sciences, Hisar, 125 004, Haryana, India
*Corresponding author: sushilamaan105@gmail.com

ABSTRACT
Bluetongue (BT) is an infectious but non–contagious disease of domestic and wild ruminants and is transmitted via the bite of infected Culicoides midges. Bluetongue virus (BTV), the prototype species of the genus Orbivirus (family Reoviridae) contains double–stranded (ds) RNA genome. The dsRNA genome is arranged as ten linear segments which are packaged as exactly one copy of each within the icosahedral protein capsid of the virus particle. The genome segments encode 7 structural proteins (VP1–VP7) and four non–structural proteins (NS1–NS4). BTV exists as 26 distinct serotypes, as well as different topotypes and also has the potential to exchange genome segments. Any serotype of BTV has the potential to cause disease in ruminants with high rate of mortality and significant economic losses. For the genetic analysis of these viruses, an alternative method to already published Full–Length Amplification of cDNAs’ (FLAC) technique (Maan et al., 2007), has been developed for the amplification of genome segment 2 of BTVs. In this method genome segment 2 of BTV–9 (~2.9kb) was amplified in four different overlapping fragments using four pairs of amplification primers. Each fragment was then sequenced using at least four sequencing primers. Sequences from all four fragments were then assembled to generate a contiguous sequence. This amplification and sequencing strategy is particularly useful when the initial amount of RNA in the sample is too low (e.g. tissue or blood samples from field) to perform FLAC and/or cloning. Using this approach, sequence data can be obtained from a new sample within few hours.

Key Words: Bluetongue virus, Genome, FLAC, Overlapping fragments, Sequencing, Reassortment