Studies on the formation of hyperplastic focus by variola virus in human cell cultures: I. In vitro quantitation of variola virus by focus counting in HeLa and FL cell cultures

Abstract

Conditions under which variola virus, both major and minor, can be quantitated by counting hyperplastic foci in HeLa and FL cell cultures were studied. Foci were countable as red spots of 0.3–0.5 mm diameter when infected cultures were stained with neutral red at 4–6 days after inoculation. Focus-forming activity was inactivated by antiserum against vaccinia virus, but was insensitive to antibody after an initial adsorption period. Some physicochemical treatments inactivated focus-forming activity in parallel with loss of pock-forming activity. It was shown by density gradient centrifugation in sucrose that the peak of pock-forming and focus-forming activities coincided. Fluorescent antibody studies revealed that all cells in a focus contained specific viral antigen. These results are considered to substantiate that foci are formed by variola virus.