Sensitive plaque assay and propagation of Chuzan (kasba) virus, a palyam serogroup orbivirus, in BHK-21 cells

N. Hirano, T. Tawara, R. Nomura, A. Imai, K. Ono, R. Yamaguchi

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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 μg/ml) and/or diethylaminoethyl (DEAE)-dextran (50 μg/ml) to the virus diluent as the virus adsorption medium and agar overlay medium, the number of plaques increased. When 100 μg/ 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 106 3PFU/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.

Original languageEnglish
Pages (from-to)333-342
Number of pages10
JournalJournal of Veterinary Medicine, Series B
Issue number6
Publication statusPublished - 08-1996

All Science Journal Classification (ASJC) codes

  • Medicine(all)


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