A plasmid-launched reverse genetics system for the human coronavirus HCoV-OC43
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Authors
Somerville, Samuel
Smith, Professor Everett Clinton
Issue Date
2023-04-28
Type
Other
Language
en_US
Keywords
Scholarship Sewanee 2023 , University of the South , Coronavirus , Reverse-Genetics , Transformation-Associated Recombination
Alternative Title
Abstract
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. HCoV-OC43 is an attractive model for studying HCoV replication, as it is related to both epidemic and pandemic HCoVs but generally causes only cold-like illness. However, the use of HCoV-OC43 as a model system to study HCoVs is complicated by the lack of reagent availability, in particular robust genetics systems. We recently established a system to generate recombinant viruses for the murine coronavirus mouse hepatitis virus, strain A59 (MHV-A59). Using this technical knowledge, a similar yeast-derived transformation-associated recombination (TAR) system was generated for HCoV-OC43. Using commercially available purified HCoV-OC43, we used reverse transcription (RT) to generate overlapping viral dsDNA genomic fragments. All fragments were assembled in the correct order and the resultant full-length dsDNA genome was synthesized without RT-generated mutations. Following transfection of the assembled plasmid, both viral cytopathic effect and RT-PCR on purified viral RNA indicate that HCoV-OC43 was recovered successfully. Development of this tractable system will enable many mechanistic studies regarding HCoV-OC43 replication which will enhance our understanding of CoV biology.
Description
Citation
Publisher
University of the South