Plaque assay of rabies virus in chick embryo cells

Abstract

It was attempted to establish a plaque assay method for rabies virus using primary chick embryo (CE) cell monolayers. Independently of our investigation, Kondo (1) found plaque formation in CE cells of a CE fibroblast-adapted line of the HEP Flury strain (2). However, according to his result, the incubation period required for the development of plaques was not constant, varying from l0 to 20 days depending upon each test. In contrast, our present results show that plaques become visible in a constant period of 6 days, and that the HEP Flury strain need not be further adapted to cultured CE fibroblasts in order to be able to form plaques in CE cells.Nine-day chick embryos were trypsinized by the method described elsewhere (3), suspended in a growth mediu~t (Earle~s balanced salt solution supplemented with 0.5% lactalbumin hydrolysate, 0.1~o yeast extract, 10% calf serum, 0.01 M Tris buffer of pH 7.2, and antibiotics) at a concentration of 2,000,000 cells per ml. and sown in 5 ml. amounts into 60 mm. petri dishes. In this procedure, it was essential to minimize the time lapse during which the cells were placed in the cold. After 24 hours' incubation at 37 ~ C, the dishes were washed once with PBS (4) and received 0.05 ml. per dish of virus inocula, which had been made by diluting a seed virus (a 20% emulsion of brains of chick embryos which had been infected at the age of 7 days with the HEP Flury strain) with 0.1 ~ yolk-saline (3). The dishes were incubated at 37 ~ C for one hour for * This study was supported in part by UoS. PHS grant AI-04248 and a grant from the World Health Organization. F i g . 1. P l a q u e s f o r m e d i n C E cells b y I~E P F l u r y s t r a i n of r a b i e s v i r u s . T r i s b u f f e r s of p i t 7.2, 7.8 a n d 8.6 w e r e i n c o r p o r a t e d i n t h e o v e r l a y m e d i a , as i n d i c a t e d . ~ C~ m" 9 6. Yoshino, K., A. Kondo, iV. Kuma, and H. Taniguchi: Infection of the oneday old fertile hen's egg with rabies virus VI. Strain differences in the egg adaptability, Arch. ges. Virusforsch. 10~ 684--697 (1960).

An improvement in the plaque assay of rabies virus in chick embryo cells

“…Multiplicity of Infection (m.o.i.) Plaque-forming units of virus were determined by the earlier standard method (1). Cells in a monolayer dish were dispersed with a 0.02% EDTA solution containing 0.05% trypsin, and the number of cells counted as described earlier (8).…”

“…Test The immune rabbit serum was prepared as described previously (1). It had been obtained by immunizing a rabbit with suspensions of CVS-strain-infected rabbit brains.…”

“…Secondly, monolayers inoculated with a high concentration of virus showed autointerference (4). Finally, only the high egg passage (HEP) Flury strain could be titrated by this method, while other strains even after many CE cell passages formed pinpoint plaques (1). It was the purpose of the present study to find a means to eliminate these drawbacks.…”

“…Introduction Quantitation of rubies virus infectivity by plaque assay in chick embryo (CE) (1,2) and BHK-21 cells (3) greatly facilitated the analyses of biological properties of this virus. Since it is possible that the continuous cell line may alter the sensitivity to virus depending upon cultural conditions, we prefer primary CE cells for the sake of experimental constancy.…”

Susceptibility of Various Cell Lines to Rabies Virus

“…The ability of some cells to support plaque formation was also tested in certain cell lines. The procedures were essentially the same as those described by Yoshino et al (1966) except that calf serum and agar in the overlay were replaced by heat-inactivated fetal calf serum and 2% Sephadex G-200 (Schneider, 1973), respectively. Infected cultures were observed for plaques for 8 days.…”