A cytopathic agent was isolated from crusts of patients diagnosed as smallpox in Indonesia in 1965, using Japanese monkey kidney (JMK) tissue cultures. The isolated agent was identified as smallpox virus, based on its biological and virological properties. When formation of pocks approximately 1 mm in diameter 48-72 hours after the agent had been introduced onto the chorioallantoic membrane of fertilized chick embryos. It produced CPE in continuous JMK cell cultures, in which multinuclear giant cells were also formed. The agent is ether-resistant and non-pathogenic for 3-week-old white mice intracerebrally and for rabbits intradermally. The agent agglutinated chicken erythrocytes, and this reaction was inhibited by anti-vaccinia serum. Specific adsorption of chicken erythrocytes on the infected culture cells (hemadsorption reaction) could also be seen. The infected culture cells exhibited positive reaction against anti-vaccinia immunofluorescent globulin. The specific flurorescence was seen in the cytoplasm. Plaques were formed on JMK cell culture under agar overlay media. Bytaking advantage of this phenomenon; cell-virus adsorption time, relationship of plaque count to virus concentration, and fifty per cent plaque neutralization were studied, obtaining reproducible results.
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