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Fluorescent Cell Counting as an Assay Method for Respiratory Syncytial Virus

Viral and Rickettsial Disease Laboratory, California State Department of Public Health, Berkeley, California 94704

ABSTRACT

The fluorescent cell-counting technique was applied to the enumeration of cell-infecting units of respiratory syncytial (RS) virus in human fetal diploid (HFD) cover-slip cell cultures; it was a sensitive, precise, and rapid assay method. Approximately 2 hr was required for maximal adsorption of RS virus to HFD cell monolayers. However, about 15% of the infectious virus in the inoculum remained unadsorbed; this percentage was not significantly reduced even when the adsorption period was extended to 5 hr. A linear relationship between virus concentration and the number of fluorescent cells existed over a range of 1.2 log₁₀ units. Variation of the mean of replicate determinations in a single experiment was approximately 7.5%. The distribution of single infected HFD cells on cover-slip cell cultures corresponded with the calculated frequencies of the Poisson distribution. The Chi square test for the extent of fit was calculated for several experiments, and the value of P was never less than 0.5. The addition of immune serum after virus adsorption effectively inhibited the development of detectable levels of viral antigen in secondarily infected cells.