P-83. Adaptation of fowl adenovirus serotype -4 in chicken embryo fibroblastic cell culture and its confirmation by polymerase chain reaction

Fowl adenovirus serotype-4 produces hydropericardium syndrome in poultry birds. The disease causes heavy mortality in broilers and is manifested by acute death On post mortem examination, typical hydropericardium is normally seen with enlarged livers. The disease was first reported from Pakistan and subsequently from various countries including India. The virus isolate used for this study was obtained from the Department of Veterinary Pathology in the form of infected liver homogenate recovered from a local outbreak in Himachal Pradesh. The infected liver homogenate were filtered through 0.22 μm filter and was then tested once for its viability by inoculating chicks. Liver samples thus obtained from the dead chicks that showed typical hydropericardium were collected in sterile PBS and used to prepare the virus inoculum used in our study. A 20 per cent liver homogenate was prepared in PBS incorporating antibiotics (Penicillin 1000 IU/ml and Streptomycin 1 mg/ml), centrifuged and the supernatant filtered through 0.22 μm filter. The suspension was checked for sterility by standard methods. The virus was titrated in embryonated chicken eggs to find out its EID₅₀. Chicken embryo fibroblastic cells were prepared according to standard procedures and inoculated with the virus preparation after it exhibited 90 per cent confluence and observed for 1 week. The virus could be adapted to grow in chicken embryo fibroblastic cell at the fifth passage level. At fifth passage, there was rounding of cells, aggregation and clumping and detachment of monolayer which progressed sequentially from the 2^nd day post inoculation. Complete detachment was evident after 120 days post inoculation. Subsequent passages from the eight passage onwards showed reduction of time interval and pronounced cytopathic effect starting from 24 hours. The virus was passaged up to 10^th passage level. The EID₅₀ titer when measured at 10^th passage level showed a increase in titer level. Harvested material from the 10^th passage level when inoculated again into chicks showed mortality after 24 hours post inoculation. A 0.7 kb product amplifying the hexon gene of Fowl adenovirus -4 could be amplified from the infected cell culture supernatant as well as the organs of the dead chick post inoculation thus confirming the isolate as Fowl adenovirus-4. Fowl adenovirus-4 produced cytopathic effects in chicken embryo fibroblastic cell cultures from the fifth passage onwards. The cytopathic effects observed were rounding and detachment of cells that increased in severity with subsequent passage levels. Hexon gene specific primers could amplify the virus from the infected cell supernatants as well as from different organs from chick infected with the supernatant.