Respiratory syncytial virus ts mutants and nuclear immunofluorescence

Abstract

A replicated sector-plating procedure was used to isolate 35 induced temperature-sensitive (ts) mutants and 1 spontaneous ts mutant from a wild-type stock of respiratory syncytial (RS) virus cloned from recent clinical material. Seven of these mutants were ts for plaque formation at 37.degree. C and at the restrictive temperature of 39.degree. C. The wild-type strain did not differ markedly from standard laboratory strains of RS virus. It was dependent on exogenous arginine (84 .mu.g/ml) for optimal growth and was not significantly inhibited by mitomycin C (10 .mu.g/ml). It was sensitive to actinomycin D (2.5 .mu.g/ml) during the early part of the growth phase. A characteristic focal cytopathic effect was obtained in [African green monkey kidney] BS-C-1 cells. Staining of infected monolayers by indirect immunofluorescence revealed a profusion of filamentous processes extending from the plasma membrane, and a similar modification of the surface of infected cells could be visualized by scanning EM. Filament production was inhibited when certain ts mutants were incubated at 39.degree. C, confirming the virus-specific nature of the phenomenon. Thirty-four of the mutants were classified into 3 groups by immunofluorescence. Complementation was observed in mixed infection with a single mutant from each group. Nuclear and cytoplasmic immunofluorescence was detected in RS virus-infected cells using a high-titer bovine anti-bovine RS virus serum. Visualization of nuclear antigen was dependent on the inhibition of cytoplasmic fluorescence obtained when ts mutants in groups I and III were incubated at restrictive temperature.