Chick embryo fibroblast cultures of the C/O phenotype (leukemia--free) infected with eastern equine encephalomyelitis (EEE) virus were incubated in the presence of 15 micrograms/ml N-methyl-N-nitro-N-nitrosoguanidine in the culture medium. Seven (5%) temperature-sensitive mutants were isolated from cell homogenates only in those cases where cell cultures before infection had been treated with actinomycin D. The recovered ts mutants are characterized by the marked ts- phenotype and genetic stability. The method of obtaining EEE virus ts mutants under the effect of N-methyl-N-nitro-N-nitrosoguanidine in C/O phenotype (leukemia-free) chick embryo fibroblast cultures treated before virus inoculation with actinomycin D is discussed.
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