IN-VITRO ESTABLISHMENT OF LYTIC AND NONPRODUCTIVE INFECTION BY HERPES-SIMPLEX VIRUS TYPE-1 IN 3-DIMENSIONAL KERATINOCYTE CULTURE

The F strain of herpes simplex virus type 1 (HSV-1) was tested for its ability to produce lytic or nonproductive infection in squamous epith elial cells cultured in a three-dimensional organotypic tissue culture . For the tissue culture, we used HaCat cells (immortalized skin kerat inocytes) and normal fibroblasts derived from the skin. The cultures w ere infected with HSV-1 (5 PFU) either when the epithelial cells had g rown as a monolayer with a confluence of 80% on the collagen fibroblas t gel or 30 min after lifting of the epithelial cells into the air-liq uid interface. The cultures were collected 1 week after inoculation. T ypical cytopathic effects of HSV infection (ballooning and reticular d egeneration with multinucleate giant cells) were seen only in those cu ltures in which the epithelial cells were infected before lifting. The presence of HSV was confirmed by DNA and RNA in situ hybridization an d PCR. No morphological changes were found in cultures infected after lifting into the air-liquid interface. No infectious virus was recover ed either from cells or culture supernatant. However, these cultures w ere positive for HSV DNA on PCR and showed expression of the LAT gene by in situ hybridization and Northern blot (RNA) hybridization. The pr esent results indicate that both nonproductive and lytic HSV infection can be produced in vitro and the outcome of the infection depends on the time of viral inoculation in relation to epithelial maturation.