Enumeration of cell-infecting particles of Newcastle disease virus by the fluorescent antibody technique

Abstract

A procedure has been developed for the determination of the concentration of infective Newcastle disease virus (NDV) based on the enumeration of singly infected and distributed HeLa cells which are visualized by staining with fluorescent antibody. Infective virus assayed by the fluorescent cell-counting procedure is expressed in terms of cell-infecting units (CIU). Adsorption of NDV to HeLa cell monolayers reached a plateau 1 to 1.5 hours after inoculation of coverslip cultures, and 12 per cent of the infective particles inoculated failed to adsorb. The half-life of NDV in protein-free Eagle's medium at 37 degrees C. was 2.1 hours. There was a linear relationship between virus concentration and the number of infected cells. The coefficient of variation of the mean of replicate determinations of infective NDV was 8.2 per cent. The distribution of single infected HeLa cells in the monolayer corresponded to the Poisson distribution. With NDV the cell-infecting unit (CIU) determined in HeLa cells is equivalent to the plaque-forming unit in chick embryo cells and the egg infective dose. In experiments on the mechanism of dissemination of NDV in monolayer cultures of HeLa cells, NDV was found to spread from cell to cell through the extracellular milieu.