K. Purohit1, M. K. Bhowmik1, S. K. Mukhopadhayay1*, H. K. Pradhan2, S. Ganguly3, A. Mondal4
1Department of Veterinary Pathology, Faculty of Veterinary & Animal Sciences, West Bengal University of Animal and Fishery Sciences, 37, K.B. Sarani, Belgachia, Kolkata – 700 037, WB, India
2High Security Animal Disease Laboratory (HSADL), Indian Veterinary Research Institute,
Bhopal – 462 021, MP, India
3AICRP-PHT (ICAR), Department of Fish Processing Technology, Faculty of Fishery Sciences, West Bengal University of Animal and Fishery Sciences, P.O. Panchasayar,
Chakgaria, Kolkata – 700 094, WB, India
4Department of Veterinary Microbiology, Faculty of Veterinary & Animal Sciences, West Bengal University of Animal and Fishery Sciences, 37, K.B. Sarani, Belgachia, Kolkata-700037, WB, India.
The present study was carried out to characterize the avian influenza virus of Indian origin by detailed molecular techniques. For this purpose, 1,423 number of tissue samples were processed for virus isolation in avian influenza virus antibody negative chicken embryos of 9-11 day old. RT-PCR was conducted from the RNA extracted from the allantoic fluid of avian influenza virus inoculated chicken embryos. A total of 515 samples were tested by RT-PCR and positive bands of specific amplification of 488 bp of HA gene were obtained by one step RT-PCR with HSAIVH9F and HSAIVH9R subtype specific primers from the three samples. A 555 bp product which included HA gene cleavage site was obtained using the primers HSAIV47F and HSAIV47R from the cDNA synthesized from the allantoic fluids of all the three isolates. The H9 subtype was also confirmed by amplifying another region of HA gene using primers H9HA 692-714 and H9HA 1011-988 which gave rise to 319 bp amplicon. The 488 bp, 555 bp and 319 bp fragments of three isolates were cloned into pGEM-T easy vector and sequenced. The results indicated species variation among sequences of H9N2 isolates.
Keywords: avian influenza virus; Indian origin; isolate; molecular techniques; RT-PCR