Cell-Free Varicella-Zoster Virus in Cultured Human Melanoma Cells

Abstract

Varicella-zoster virus (VZV) has been isolated and serially propagated in a continuous cell line derived from a human malignant melanoma tumour. Human melanoma cells (HMC) have been further evaluated as a substrate for the production of cell-free virus and compare favourably with human embryo cells. Within 60 h after inoculation with VZV-infected cells, HMC monolayers incubated at 32 degrees C exhibited advanced syncytial cytopathic effect, and the overlying culture medium contained greater than 10(2) p.f.u./ml. The cell pellet from a mechanically dispersed 150 cm2 monolayer yielded 10(5) p.f.u. after sonic disruption, while the medium ('scraping medium') in which the cells had been harvested contained up to one log more infectious virus than was found in the cells from the same monolayer. When infected cells were subjected to Dounce homogenization, most of the infectivity was found in the nuclear fraction. The concentration and purification of cell-free virus were also investigated. Concentration was carried out by three methods: ultracentrifugation, dialysis against hydrophilic compounds and liquid polymer phase separation. The first two procedures caused considerable loss of biological activity, whereas precipitation with 8% polyethylene glycol resulted in a 50-fold increase in titre. Purification of cell-free virus with retention of infectivity was achieved by rate zonal centrifugation in linear potassium tartrate gradients. Infectious virus was also recovered after sedimentation in combination equilibrium-viscosity gradients of potassium tartrate and glycerol, but not after centrifugation to equilibrium in caesium chloride gradients.