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

Research output: Contribution to journalArticle

3 Citations (Scopus)

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 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
Volume43
Issue number6
Publication statusPublished - 01-08-1996
Externally publishedYes

Fingerprint

Palyam Virus
Palyam virus
Orbivirus
serotypes
Viruses
viruses
Dextrans
dextran
assays
Hemagglutination
Trypsin
cells
hemagglutination
trypsin
plateaus
Satellite Viruses
Indirect Fluorescent Antibody Technique
cytopathogenicity
Adsorption
Agar

All Science Journal Classification (ASJC) codes

  • Medicine(all)

Cite this

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title = "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 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.",
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Sensitive plaque assay and propagation of Chuzan (kasba) virus, a palyam serogroup orbivirus, in BHK-21 cells. / Hirano, N.; Tawara, T.; Nomura, R.; Imai, A.; Ono, K.; Yamaguchi, R.

In: Journal of Veterinary Medicine, Series B, Vol. 43, No. 6, 01.08.1996, p. 333-342.

Research output: Contribution to journalArticle

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T1 - Sensitive plaque assay and propagation of Chuzan (kasba) virus, a palyam serogroup orbivirus, in BHK-21 cells

AU - Hirano, N.

AU - Tawara, T.

AU - Nomura, R.

AU - Imai, A.

AU - Ono, K.

AU - Yamaguchi, R.

PY - 1996/8/1

Y1 - 1996/8/1

N2 - 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.

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SN - 1863-1959

IS - 6

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