Thymidine plaque autoradiography of thymidine kinase-positive and thymidine kinase-negative herpes viruses

Plaques formed by herpes simplex virus (HSV), pseudorabies virus, and varicella-zoster virus were studied by plaque autoradiography after [¹⁴C]thymidine labeling. Standard thymidine kinase-positive (TK⁺) viruses and TK^- mutants of HSV types 1 and 2 and pseudorabies virus were studied, including cell-cultured viruses and viruses isolated from animals. Autoradiography was performed with X-ray film with an exposure time of 5 days. After development of films, TK⁺ plaques showed dark rims due to isotope incorporation, whereas TK^- plaques were minimally labeled. Plaque autoradiography of stock TK^- viruses showed reversion frequencies to the TK⁺ phenotype of <10^-3. Autoradiography indicated that TK^- virus retained the TK^- phenotype after replication in vivo. In addition, it was shown that TK^- HSV could be isolated from mouse trigeminal ganglion tissue after corneal inoculation of TK^- HSV together with TK⁺ HSV. The plaque autoradiographic procedure was very useful to evaluate proportions of TK⁺ and TK^- virus present in TK⁺-TK^- virus mixtures.