Protocol to isolate temperature-sensitive SARS-CoV-2 mutants and identify associated mutations
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Updated: Jul 27, 2025
Isolation of Fidelity Variants of RNA Viruses and Characterization of Virus Mutation Frequency
Published on: June 16, 2011
Shinya Okamura1, Akiho Yoshida1, Paola Miyazato1
1Virus Vaccine Group, BIKEN Innovative Vaccine Research Alliance Laboratories, Institute for Open and Transdisciplinary Research Initiatives, Osaka University, Suita, Osaka, Japan; The Research Foundation for Microbial Diseases of Osaka University, Suita, Osaka, Japan.
Abstract:
An inability to proliferate at high temperatures typically gives viruses an attenuated phenotype. Here, we present a protocol to obtain and isolate temperature-sensitive (TS) SARS-CoV-2 strains via 5-fluorouracile-induced mutagenesis. We describe steps for the induction of mutations in the wild-type virus and selection of TS clones. We then show how to identify the mutations associated with the TS phenotype, following forward and reverse genetics strategies. For complete details on the use and execution of this protocol, please refer to Yoshida et al. (2022).1.
