Protocol to isolate temperature-sensitive SARS-CoV-2 mutants and identify associated mutations

Shinya Okamura; Akiho Yoshida; Paola Miyazato; Mai Matsumoto; Hirotaka Ebina

doi:10.1016/j.xpro.2023.102352

Protocol to isolate temperature-sensitive SARS-CoV-2 mutants and identify associated mutations

STAR Protoc. 2023 May 15;4(2):102352. doi: 10.1016/j.xpro.2023.102352. Online ahead of print.

Authors

Shinya Okamura¹, Akiho Yoshida¹, Paola Miyazato¹, Mai Matsumoto², Hirotaka Ebina³

Affiliations

¹ Virus 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.
² The Research Foundation for Microbial Diseases of Osaka University, Suita, Osaka, Japan.
³ Virus 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; Virus Vaccine Group, BIKEN Innovative Vaccine Research Alliance Laboratories, Research institute for Microbial Diseases, Osaka University, Suita, Osaka, Japan; Virus Vaccine Group, Center for Infectious Disease Education and Research, Osaka University, Suita, Osaka, Japan. Electronic address: hebina@biken.osaka-u.ac.jp.

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.

Keywords: Microbiology; Molecular Biology.