Advances in Animal and Veterinary Sciences

Research Article
Adv. Anim. Vet. Sci. 3 (1S): 22 -27. Special Issue-1 (Biotechnological and molecular approaches for diagnosis, prevention and control of infectious diseases of animals)
http://dx.doi.org/10.14737/journal.aavs/2015/3.1s.22.27
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Prasad Minakshi1*, Koushlesh Ranjan2, Gaya Prasad3

1Department of Animal Biotechnology, LLR University of Veterinary and Animal Sciences, Hisar, Haryana, 125004; 2Department of Veterinary Physiology and Biochemistry, Sardar Vallabhbhai Patel University of Agriculture and Technology, Meerut, Uttar Pradesh, 250110; 3Indian Council of Agricultural Research (ICAR), New Delhi, India.

*Correspondence | Minakshi Prasad, LLR University of Veterinary and animal Sciences, Hisar, India; Email: minakshi.abt@gmail.com

Abstract
Bluetongue (BT) is a Culicoides borne infectious disease of domestic and wild ruminants. In the present study, a total of four BTV samples (GNT27/IND, MBN48/IND, MBN50/IND and VJW66/IND) of sheep origin from Andhra Pradesh state of India were inoculated to 9-11 days old chicken embryos. The BTV samples showed specific cytopathic effect (CPE) in BHK-21 cell culture. The samples also showed BTV specific characteristic migration pattern of 3:3:3:1 in RNA-Polyacrylamide gel electrophoresis. Furthermore, the ns1 gene based group specific RTPCR confirmed the samples to be BTV. Further, serotyping based on vp2 gene confirmed these samples as of BTV16 serotype. The sequence analysis of vp2 gene specific PCR products (768bp) revealed a high degree of (91-99% nucleotide and amino acid) identity within BTV isolates of this study and other BTV16 isolates from India. Further sequence analysis revealed highest nucleotide (87.7-99%) and deduced amino acid (91.3-99.5%) sequence identity with Eastern viruses from India, Japan, China, South Africa, Italy and Israel. However, it showed only 71.0-75.9% nucleotide and 81.1-87.6% deduced amino acid sequence identity with western BTV16 from Nigeria, confirmed isolates of this study as eastern topotype. The phylogenetic analysis showed a close clustering between isolates of this study and other Indian BTV16 isolates which were closely related to Japan, China and Greece isolates. The molecular analysis revealed that the isolates in the study are very close to Greece, China, Japan and other Indian isolates of BTV16.

Keywords | Bluetongue virus 16, Topotype, NS1 gene, VP2 gene, RT-PCR, Sequencing, phylogenetic analysis