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A Tissue Culture Assay for Variola Virus Based upon the Enumeration of Hyperplastic Foci

From the U. S. Army Chemical Corps Biological Laboratories, Fort Detrick, Frederick, Maryland

Abstract

The observation of hyperplastic foci in monolayer cultures of HeLa cells infected with variola virus led to the development of a tissue culture assay for this virus. The specificity of the assay was demonstrated by the fact that heat inactivation or exposure to immune serum reduced the capacity of the virus to produce the areas of hyperplasia. The proliferative lesions can be counted as early as the second day of infection; formation of secondary foci has never been detected. A linear relationship has been demonstrated between the numbers of foci formed and the concentrations of variola virus present in the inocula. The precision of the assay was established with a series of parallel titrations of a standard suspension of variola virus both in tissue culture and on the chorioallantoic membranes of embryonated chicken eggs. Counts derived from the tissue culture assay were four times higher and showed consistently less variation than those obtained by the pock method. The new tissue culture assay is simple to perform and economical of time and equipment.

Footnotes

¹ The material presented is based upon a dissertation submitted in partial fulfillment of the requirements for the Ph.D. degree at The George Washington University, Washington, D. C.