High Security Animal Disease Laboratory, Indian Veterinary Research Institute, Anand Nagar, Bhopal-462021.
Abstracts of the papers presented at the 16th Annual Convention and International Symposium of Indian Virological Society on “Management of Vector-Borne Viruses” at International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), Patancheru-502324, Hyderabad, India, February 7–10, 2006.
Bovine viral diarrhea virus (BVDV) belongs to the Pestivirus genus within Flaviviridae family. It causes a variety of respiratory, digestive and reproductive disorders including the fatal mucosal disease in ruminants worldwide. The NS3 gene of BVDV codes for a nonstructural protein (p80), which is highly conserved among the pestiviruses, and it was targeted for development of diagnostic assay. Besides, Indian BVDV isolates are yet to be characterized in NS3 gene region. Here we report genetic analysis of NS3 gene (C- terminus 2/3rd sequence) of selected Indian BVDV isolates and expression of NS3 recombinant protein in Escherichia coli for use in BVDV diagnostics. The NS3 gene sequence analysis of Indian BVDV isolates and reference pestiviruses showed that the nucleotide percent identity varied between 69.5% and 95.2%, where as the amino acid percent identity varied between 85.9% and 98.7% showing high degree of conservation. On the basis of NS3 gene phylogeny Indian isolates were grouped into BVDV-1b. The Walker helicase motifs II, III and VI were found in Indian isolates similar to Hepatitis C virus of humans. The NS3 gene (1.152 kb) of one of the Indian isolates was amplified by RT-PCR, cloned, sequenced and subsequently inserted into Not I site of pTriEx-2-Neo expression vector and transformed into origami strain of E. coli. A recombinant protein of 50 kDa was expressed as cytoplasmic inclusion bodies. The recombinant protein was purified to homogeneity by affinity chromatography and renatured by dialysis. The protein showed distinct antigenic properties when analyzed by immunoblotting using cattle hyper-immune serum against BVDV. The ability of the recombinant protein to detect anti BVDV specific antibodies was examined in ELISA and could distinguish the positive and negative samples. Taken together, our studies showed that Indian isolates belong to BVDV-1b in NS3 gene region and the expressed recombinant NS3 protein can be used for development of immuno assay for the detection of BVDV antibodies.
