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The large size of the CoV genome (around 30\u2009kb) represents a major obstacle to the development of reverse genetics systems, which are invaluable for basic research and antiviral drug screening. In this study, we established a rapid and convenient method for generating reverse genetic systems for various CoVs using a bacterial artificial chromosome (BAC) vector and Gibson DNA assembly. Using this system, we constructed infectious cDNA clones of coronaviruses from three genera: human coronavirus 229E (HCoV\u2010229E) of the genus <jats:italic>Alphacoronavirus<\/jats:italic>, mouse hepatitis virus A59 (MHV\u201059) of <jats:italic>Betacoronavirus<\/jats:italic>, and porcine deltacoronavirus (PDCoV\u2010Haiti) of <jats:italic>Deltacoronavirus<\/jats:italic>. Since beta coronaviruses including severe acute respiratory syndrome coronavirus (SARS\u2010CoV), severe acute respiratory syndrome coronavirus 2 (SARS\u2010CoV\u20102), and Middle East respiratory syndrome coronavirus (MERS\u2010CoV) represent major human pathogens, we modified the infectious clone of the beta coronavirus MHV\u2010A59 by replacing its NS5a gene with a fluorescent reporter gene to create a system suitable for high\u2010throughput drug screening. Thus, this study provides a practical and cost\u2010effective approach to developing reverse genetics platforms for CoV research and antiviral drug screening.<\/jats:p>","DOI":"10.1002\/jmv.70171","type":"journal-article","created":{"date-parts":[[2025,1,17]],"date-time":"2025-01-17T10:04:09Z","timestamp":1737108249000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":4,"title":["Rapid Generation of Reverse Genetics Systems for Coronavirus Research and High\u2010Throughput Antiviral Screening Using Gibson DNA Assembly"],"prefix":"10.1002","volume":"97","author":[{"given":"Xinrong","family":"Zhou","sequence":"first","affiliation":[{"name":"State Key Laboratory of Respiratory Disease, National Clinical Research Center for Respiratory Disease, Guangzhou Institute of Respiratory Health The First Affiliated Hospital of Guangzhou Medical University Guangzhou Guangdong China"},{"name":"Department of Basic Research Guangzhou National 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