The Immunofluorescent Focus Technique in Studying the Replication of Cytomegalovirus

Abstract

A quantitative assay for cytomegalovirus involving counts of immunofluorescent foci of antigen-containing cells was developed. Counts were made 6 to 7 days after infection. The number of foci seen decreased with decreasing concentrations of virus and a linear relationship was obtained over the countable range. Comparable virus titers were obtained in parallel immunofluorescent focus and tube titrations, but the tube titration was slow and required about 30 days of observation. The focus method was used to study virus adsorption and replication in embryonic human lung fibroblasts. Adsorption proceded rapidly at 37°C; 90% of the virus adsorbed within 1 hr. Adsorption proceeded more slowly as the temperature was decreased. At 37°C production of virus antigen and maturation of virus required 4 days after inoculation of the cells, although cellular changes could be observed earlier if the cultures were fixed and stained. The infection subsequently spread to adjacent cells and increasing numbers of cells comprised the infected foci seen.