Isolation of temperature-sensitive conditional lethal mutants of "fixed" rabies virus

In an attempt to induce temperature-sensitive (ts) conditional lethal mutants of rabies virus, stocks of a plaque-purified substrain of strain CVS fixed rabies virus were subjected to mutagenesis by HNO(2), 5-fluorouracil, or 5-azacytidine. It was necessary to prepare virus stocks from clones of mutagenized virus selected at random and to test subsequently each stock for possible ts characteristics by measuring its relative capacity for growth at permissive (33 C) and nonpermissive (40.5 C) temperatures. Five ts mutants were detected in tests of 161 clones of mutagenized virus. Each of the mutants exhibited a remarkably low incidence of reversion and little demonstrable "leakiness." One of the five ts mutants (ts2), which formed formed very small plaques, and another (ts1), which formed plaques of only slightly reduced size, were further characterized. Virus ts1 was more thermostable at 40.5 C than the parental virus, but the ts2 mutant was unchanged in this respect. The ts1 virus exhibited normal pathogenicity for mice, but ts2 virus caused a very irregular death pattern. Both deaths and survivors immune to rabies virus challenge were noted in all groups of mice inoculated with ts2 virus, regardless of the virus dose.