A plasmid-launched, highly tractable coronavirus reverse genetics system

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Sterling, Cade
Wilson, Natalie R.
Harris, Debreiona Y.
Smith, Everett Clinton
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Scholarship Sewanee 2022 , University of the South , coronavirus , reverse genetics , transformation-associated recombination , mouse hepatitis virus , viral reverse genetics
Coronaviruses (CoVs) are a large family of single-stranded, positive-sense RNA viruses that infect a wide range of vertebrate hosts. Along with SARS-CoV-2, the etiological agent of the COVID-19 pandemic, there are six other characterized human CoVs (HCoVs). Infection by the four endemic HCoVs (e.g., -OC43, -NL63, -229E, -HKU1) are often associated with the common cold, while infection with epidemic HCoVs (e.g., SARS-CoV and MERS-CoV) can result in severe disease with high mortality. The generation of recombinant viruses is an important tool for understanding viral replication and for testing potential therapeutics; however, the systems used to engineer these viruses are often inefficient and labor intensive. Several systems to engineer recombinant CoVs have been developed over the past two decades, but none are suitable for the rapid generation of large panels of recombinant viruses due to technical constraints. Transformation-associated recombination (TAR) is a cloning technique which leverages the high rate and efficiency of homologous recombination in yeast to assemble large plasmids of foreign DNA. We leveraged TAR and recent reports of SARS-CoV-2 reverse genetics systems to engineer a plasmid-launched, TAR-assembled system. Herein, we demonstrate that a TAR-generated murine CoV (MHV-A59) has similar viral yield kinetics as wild-type MHV-A59 and provide proof-of-principle for the utility of this system to generate a panel of recombinant viruses in only a few weeks.