A Study by Fluorescence Microscopy of the Replication of Lymphogranuloma venereum Virus in HeLa Cell Monolayers

In HeLa cells infected with a single infectious unit of lymphogranuloma venereum virus and stained with acridine orange, one particle (initial body) of ribonucleic acid (RNA) about 2 μ diam. was seen by fluorescence microscopy in the cell cytoplasm after incubation for 6–8 hr. at 37°. After 11–12 hr. of incubation, an average of 2 particles/infected cell was found. Thereafter the number increased exponentially with a mean generation time of 2–2 1/4 hr. The particles then remained discrete and in a circumscribed area in the cytoplasm until 18–21 hr., when a vacuole was formed around the initial bodies. By 21–24 hr. smaller particles ranging in diameter from 1 μ to about 0.25μ, and in colour from the orange fluorescence characteristic of RNA to yellowish green, were detected amongst the initial bodies. At 33 hr., the initial bodies were almost entirely replaced by smaller particles or elementary bodies, most of which stained yellowish green and had the green fluorescence of deoxyribonucleic acid (DNA) only after treatment with ribonuclease. The elementary body of lymphogranuloma venereumum thus consists of a DNA particle surrounded by a layer containing a detectable amount of RNA. After 33 hr. the number of elementary bodies decreased; presumably because infective virus had been released, since at 40–44 hr. initial bodies appeared in previously uninfected cells. HeLa cells infected with more than one infectious unit often contained more than one focus of infection, indicating that replication is not confined to a single site in the cytoplasm.