High Security Animal Disease Laboratory, Indian Veterinary Research Institute, Anand Nagar-462021.
Abstracts of Research Papers Presented during the National Symposium of Indian Virological Society at Unit of Plant Virology, Division of Plant Pathology, Indian Agricultural Research Institute, New Delhi-110 012, October 14–1.
Bovine viral diarrhoea (BVD) is one of the most economically important infectious diseases in cattle worldwide and the causative agent, BVD virus (BVDV) is a pestivirus in the family Flaviviridae. Though prevalence of BVDV infection in Indian cattle has been established recently by us, the information on use of multiplex PCR for diagnosis and characterization of BVDV isolates at genomic level is lacking and O.I.E. also recommends its use in diagnosis and genotyping of BVDV. Moreover, presence of antibodies may interfere with detection of BVDV antigen by pestivirus antigen capture ELISA (PACE) and immunoflourescence test is time consuming besides being cumbersome. In this study, we report development of a multiplex polymerase chain reaction (PCR) assay using primers of highly conserved 5’-untranslated region (UTR) and structural glycoprotein (E1-E2) region responsible for eliciting virus neutralizing antibodies. The multiplex PCR generated two different well discernible amplicons (288 bp and 784 bp) in a single tube when thirteen Indian BVDV isolates were tested. DNA products of three isolates, one each from eastern, western and northern India were extracted from gel, purified and subjected to restriction enzyme analysis. A single PstI site was found in 5’ UTR amplicons and one HinfI and two DdeI sites were found in the E1–E2 amplicons. Further, these amplicons were found to be specific by determination of nucleotide sequences. Phylogenetic trees were constructed for the determined sequences and reference pestivirus sequences obtained from NCBI database. The sequence analysis of both the regions grouped them into BVDV 1b genotype. The study demonstrated that multiplex PCR can be used for diagnosis and genetic characterization of BVDV isolates which has implications for epidemiology and control of BVDV infections in India.
