Sensitive plaque assay and propagation of Chuzan (Kasba) virus, a Palyam serogroup orbivirus, in BHK-21 cells.

Abstract

Various factors influencing plaque formation of Chuzan virus in BHK-21 cell monolayers were studied and a practical method for plaque assay was developed. On addition of trypsin (5 micrograms/ml) and/or diethylaminoethyl (DEAE)-dextran (50 micrograms/ml) to the virus diluent as the virus adsorption medium and agar overlay medium, the number of plaques increased. When 100 micrograms/ ml DEAE-dextran was added to the diluent and overlay medium, plaques were produced in about 10-fold higher numbers than without trypsin and DEAE-dextran. Based on these results, a practical plaque assay method for Chuzan virus was established. Using this method, one-step growth of Chuzan virus was performed at an input multiplicity of 25 plaque-forming units (PFU) per cell. Cytopathic effects were first observed at 7.5 h post-inoculation (p.i.), and were complete at 12 h p.i. The titre of cell-associated virus, after gradual decline during the first 3 h of incubation, showed a rise within 4.5 h p.i. and a rise to a plateau of 10(6.3)PFU/0.2 ml at 12 h p.i. By indirect immunofluorescence, virus-specific antigen was detected in the cytoplasm of the cells at 4.5 h p.i., and all the cells fluoresced at 6 h p.i. Haemagglutination activity was first detected in infected whole cultures at 7.5 h p.i. reaching a plateau of 1:64 at 15 h p.i. Plaque formation and haemagglutination by the virus were specifically inhibited by antisera against the original and the plaque-cloned virus.