Selective assay for herpes simplex viruses expressing thymidine kinase

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Abstract
A technique for selecting herpes simples viruses expressing the viral thymidine kinase (TK+) from a population of predominantly TK- viruses was developed. This was accomplished by infecting TK- [human osteogenic sarcoma R970-5] cells and incubating the cultures under a liquid overlay medium containing methotrexate. Since the TK- cells survive in this medium for only a limited period of time, it was necessary to add fresh uninfected TK- cells 48 h after infection. The technique allowed the detection and quantitation of the TK+ virus fraction in mixtures of TK+ and TK- viruses where the TK+ fraction was present in frequencies as low as 10-5. It was also used to estimate reversion frequencies and to obtain and analyze TK+ revertants from TK- mutant strains of herpes simplex virus type 1.