Isolation of Temperature Sensitive Mutants of Tanapox virus
Tanapox virus (TPV) is a member of the Poxviridae family. Because it causes transient limiting disease, the biogenesis of TPV is unclear. Use of temperature sensitive (ts mutants), unknown gene functions and the biogenesis can be determined. Such knowledge would be useful in engineering a safe and effective TPV oncolytic virus. In addition, ts mutants might be able to serve as an additional mechanism in case an oncolytic virus mutated again. In this experiment, our objective is to isolate a number of temperature sensitive (ts) mutants with a permissive temperature of 32.5 °C and a restrictive temperature of 37°C using nitrosoguanidine (N-G) as a mutagen. We used 100X-concentrated TPV-GFP with N-G and diluted it 10-fold. Infections of TPV-GFP in OMK cells were effective, and dilutions of10-7 from 100X concentration of mutated TPV-GFP produced the ideal amount of isolated plaques. None of the 108 plaques was ts, and the majority of the first 23 plaques grew better at the restrictive temperature. More plaques need to be isolated to obtain a pool of ts TPV mutants. Once obtained, these mutants would be useful in upcoming experiments, such as creating a second block for a TPV oncolytic virus and determining the biogenesis of TPV.
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