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Research
(Published
online: 25-05-2014)
16. Molecular diagnosis and adaptation of highly virulent
infectious bursal disease virus on chicken embryo fibroblast cell
- Yogender Singh, Kalpana Yadav, Vinod Kumar Singh and Rakesh
Kumar
Veterinary World, 7(5): 351-355
doi:
10.14202/vetworld.2014.351-355
Yogender Singh:
Animal Biotechnology Centre, Nanaji Deshmukh Pashu-Chikitsa Vigyan
Vishwavidyalaya, Jabalpur, Madhya Pradesh - 482004, India
Kalpana Yadav:
Animal Biotechnology Centre, Nanaji Deshmukh Pashu-Chikitsa Vigyan
Vishwavidyalaya, Jabalpur, Madhya Pradesh - 482004, India
Vinod Kumar Singh:
Division of Virology, Indian Veterinary Research Institute,
Izatnagar, Bareilly-243122, India
Rakesh Kumar:
Division of Dairy Cattle and Breeding, National Dairy
Research Institute, Karnal, Haryana-132001, India
Received: 13-03-2014, Revised: 13-04-2014, Accepted: 19-04-2014,
Published online: 25-05-2014
Corresponding author:
Yogender Singh, email: yogender_bly@yahoo.com
Abstract
Aim: To collect Infectious
bursal disease virus (IBDV) infected bursae based on postmortem
findings of clinical samples followed by molecular confirmation
and adaptation on chicken embryo fibroblast cell.
Material and Methods: Enlarged bursae were processed to
make 10% suspension in phosphate buffer saline and were used for
viral RNA isolation to carryout VP2 gene fragment amplification
using RT-PCR technique. Suspension was also used for adaptation of
IBDV on chicken embryo fibroblast cells prepared from 11 day old
chicken embryos. Infective titer of virus was calculated using
Reed and Muench method.
Results: Fifteen dead birds suspected of IBD infection were
collected from different local poultry farms of Jabalpur (Madhya
Pradesh). After post-mortem, twelve enlarged bursae sample were
used for total RNA isolation which was used for amplification of
VP2 gene. Out of twelve, eleven were found positive for VP2 gene
amplification. Virological characterization of PCR positive sample
was done on chicken embryo fibroblast cell culture and various
cytopathic effects like rounding of cells, cellular detachment and
vacuolation were shown by five IBD field isolates after 48 hours
on 4th passage. TCID50 per ml of the adapted virus on 4th passage
at 48 hours after infection was 1.46 x 107 .
Conclusion: VP2 gene amplification using RT-PCR technique
is a specific target for IBDV detection. Passaging of highly
virulent IBDV field isolates in cell culture leads to attenuation
of virus which can be exploited as a cell culture adapted vaccine
candidate.
Keywords: chicken embryo fibroblast, IBD virus, molecular
diagnosis, VP2 gene.
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