Isolation of temperature sensitive Tanapox mutants

Abstract

Because tanapox virus (TPV) causes self-limiting disease, the biogenesis of TPV is unclear. Use of temperature sensitive (ts) mutants would lead to determine unknown gene functions and TPV biogenesis, viral assembly inside host cells. In this experiment, our objective was to isolate a number of ts mutants with a permissive temperature of 32.5 ºC and a restrictive temperature of 37 ºC. We used 100x concentrated TPV-GFP with nitrosoguanidine (N-G) and diluted it 10-fold. Infections of TPV-GFP in owl monkey kidney (OMK) cells were effective, and dilutions of 10 -7 from 100x concentrated TPV-GFP produced the ideal amount of isolated plaques. None of the 108 plaques were ts mutants, and the majority of the first 23 plaques replicated better at the restrictive temperature. More plaques need to be isolated in order to obtain a pool of ts TPV mutants. Once obtained these mutants would be useful in upcoming experiments TPV biogenesis and the oncolytic potential of TPV.