A Study by Fluorescence Microscopy of the Replication of Inclusion Blennorrhoea Virus in HeLa Cell Monolayers

Abstract

SUMMARYThe infectious agent of inclusion blennorrhoea was seen by fluorescence microscopy as a particle of deoxyribonucleic acid (DNA) 0.2 p diam.These infectious particles and particles inactivated at 4" and 56" were adsorbed by HeLa cell monolayers. The inactivated particles were visible for at least 6 hr., distributed at random on the cells, while the infectious particles rapidly disappeared. Multiple infections of the cells readily occurred and in cells so infected, a cluster of ribonucleic acid (RNA) particles was first observed at 3 hr. adjacent to the cell nucleus. Since the number of RNA particles, each of which contained a DNA core, was equal to the number of infectious particles adsorbed and since singly infected cells contained only one RNA particle, the infectious unit is almost certainly a single DNA particle. The RNA particle increased in size without dividing and the surrounding cell cytoplasm gradually dissolved to form the inclusion body. DNA particles which were protected from deoxyribonuclease by a layer containing RNA were first observed in the inclusions at 21 hr. and an average of one infective progeny was detected in each infected cell at 22-23 hr. indicating that infectivity was associated with the DNA particles. RNA, DNA and infectious DNA particles reached their maxima between 27-30 hr., 39-42 hr. and 34-38 hr.respectively and decreased thereafter. Infectious DNA particles were released from the inclusions. The total number of DNA particles/inclusion always exceeded the number of infectious DNA particles/inclusion.Glycogen was not detected in the inclusions suggesting that it may not be essential for the formation of virus.