S-31. Cytopathic effects of fowl adenovirus serotype -4 in Vero cell culture and its confirmation by polymerase chain reaction, AGID and Dot -ELISA

Fowl adenovirus serotype-4 produces hydropericardium syndrome in poultry birds. The agent has been propagated successfully in primary chicken cell cultures which are usually established from target cells viz. chicken embryo liver cell cultures and chicken kidney cell cultures with characteristic inclusion bodies. The propagation of this virus in established cell line (Vero cell line) was attempted to find out an alternative system of propagation. 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. Viability of the isolate was checked by a passage in chicks and the harvested liver collected in PBS were then used as inoculum in the study after sterilization tests as per standard protocols. The titration of the virus isolate was done in embryonated chicken eggs to find out its EID_50. Vero cell lines obtained from NCCS, Pune were split according to standard procedures and inoculated with the virus preparation after it exhibited 90 per cent confluence and observed for a period of 1 week. Vero cell line at sixth passage level started to show cytopathic effects from 72 hours post inoculation with shrinkage and rounding of cells at a few places that progressed to more rounding and detachment and included the whole monolayer by 120 hours post inoculation. Further two passages decreased the time for the appearance of cytopathic effects from 72 hours to 48 hours and total rounding of the monolayer and more than 80 per cent sloughing by 96 hours. The virus was passaged up to 8^th passage level. The EID₅₀ titer when measured at 8^th passage level showed a slight increase in titer level. Harvested material from the 8^th passage level when again inoculated into chicks showed mortality after 72 hours post inoculation. A 700 bp product specific for the hexon gene of Fowl adenovirus-4 could be amplified from the infected cell culture supernatants as well as from the different organs of the dead chick post inoculation. The cell supernatants from the fifth passage level showed a single precipitation line in AGID against reference antiserum. Dot-ELISA showed a positive reaction from the fourth passage onwards. Vero cell line showed changes from the sixth passage level manifested by cell shrinkage, rounding of the cells and sloughing of the monolayer. A 700 bp fragment of Fowl adenovirus-4 DNA could be amplified from the infected supernatants as well from organs of chicks inoculated with the supernatant. The supernatants were also positive by Dot-ELISA and AGID.