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

Summary

The faint plaques formed with HEP Flury strain of rabies virus in chick embryo cells by the original method were thought ascribable to acid production from virus-infected cells coupled by the weak buffering action of the agar overlay medium. When the overlay medium contained appropriate concentrations of alkalies, clear plaques could be observed. The optimal conditions were (i) incorporation of 0.02% NaOH, 0.0025m Tris-HCl buffer of pH 8.2 and 2% calf serum in the base overlay medium, and (ii) neutral red staining after 7 days' incubation at 35‡ C. Under these conditions the plaque size was approximately 2 mm in diameter and autointerference caused by higher multiplicity of infection showed a diminishing trend. Mouse passaged NOPM strain also formed fairly clear plaques when agarose was substituted for agar, the plaque titer being almost equal to its mouse LD titer. That these plaques were formed by infection of the virus was testified by a neutralization test using a standard antiserum prepared with rabbit passaged CVS strain. Adsorption of rabies virus to chick embryo cells was found to proceed slowly, the maximal adsorption requiring 4 hours or longer. The adsorption efficiency was equal between 35‡ and 37‡C.