Virus biographies. II. Growth of herpes simplex virus in tissue culture

Abstract Monolayer cultures of the human epidermoid cancer cell line HEp 2 were infected with the HF strain of herpes simplex virus (herpes virus hominis) by using an inoculum of approximately 1 infectious unit per cell. At 1, 2, 4, and 7 days thereafter the cultures were studied for number of remaining cells, infectivity of cells and culture medium, cytopathic changes, viral antigen (fluorescent antibody technique) and viral morphology and development (electron microscopy). At day 1 (16 hours) infectivity of the culture medium had decreased from the initial level and about 1% of the cells were infected. There was no obvious change in number of cells and the only evidence of cell damage was a probable reduction of cytoplasmic RNA (as judged by acridine orange staining) and margination of the chromatin in a few nuclei. A very few cells had viral antigen sharply concentrated at the nuclear membrane or distributed irregularly and diffusely throughout the nucleus. Rarely a cell contained scattered intranuclear single-membraned virus particles or dense particles resembling viral nucleoids with no membrane. At day 2 about 15% of cells contained infective virus and there was a concordant increase in antigen and virus particles and nuclear DNA, but there was still no major evidence of cell destruction. By day 4 cell destruction was apparent on routine microscopy and progression of the infectious process was also evident by the fluorescent antibody and electron microscopic studies, but the characteristic changes of the herpes virus infection were most frequent in the day 7 study when a majority of the cells which remained on the glass were infected. Intranuclear inclusions were not seen by either optical or electron microscopy. The most characteristic location of viral antigen was at the nuclear membrane as either a fine line or a thickened band encircling part or all of the nucleus. Antigen was often present within the nucleoplasm also. On days 1 and 2 this intranuclear antigen was usually distributed irregularly and diffusely but at 4 and 7 days it also occurred as distinct sharply outlined particles. The usual intranuclear virus particle consisted of a dense spherical nucleoid with a maximum diameter of 60 mμ, surrounded by a single membrane of about 100 mμ in diameter, but empty particles and naked nucleoids were numerous, and double-membraned particles of about 160 mμ in diameter were occasionally seen within the nucleus in advanced infections. Virus particles were never found in crystalline arrays. They were most numerous near or in contact with the inner lamella of the nuclear membrane or between the inner and outer lamellae. There was striking thickening and progressive evagination of the inner lamella at points of contact with virus particles, and it seems clear from these morphologic alterations that this altered inner lamella of the inner nuclear membrane becomes the second membrane of the virus particle. Extreme proliferation and plication of the nuclear membrane was common, such that the perinuclear region became a complex mixture of virus particles and membranous projections. This often produced structures resembling pods of peas, with the membrane enclosing a row of single- or double-membraned virus particles. Virus particles in the cytoplasm were almost always surrounded, either individually or as a group, with a thin third membrane, thus resulting in triple membrane particles with a total diameter of 185 to 200 mμ or a cluster of double membrane particles in a thin-walled sac. The results of this study are compared with previously published studies of herpes simplex and other viruses of the herpes group.