{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T09:40:22Z","timestamp":1785404422629,"version":"3.56.0"},"reference-count":48,"publisher":"American Society for Microbiology","issue":"6","license":[{"start":{"date-parts":[[2026,6,10]],"date-time":"2026-06-10T00:00:00Z","timestamp":1781049600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2026,6,10]],"date-time":"2026-06-10T00:00:00Z","timestamp":1781049600000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/journals.asm.org\/non-commercial-tdm-license"}],"funder":[{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"publisher","award":["R01AI185617"],"award-info":[{"award-number":["R01AI185617"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"publisher","award":["R21AI180667"],"award-info":[{"award-number":["R21AI180667"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"publisher","award":["P01AI175399"],"award-info":[{"award-number":["P01AI175399"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"publisher","award":["R01AI093848"],"award-info":[{"award-number":["R01AI093848"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"publisher","award":["U19AI42733"],"award-info":[{"award-number":["U19AI42733"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"publisher","award":["T32AI165391"],"award-info":[{"award-number":["T32AI165391"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100016616","name":"Cystic Fibrosis Research","doi-asserted-by":"publisher","award":["MCCOY17R2 and PEEPLE25XX0"],"award-info":[{"award-number":["MCCOY17R2 and PEEPLE25XX0"]}],"id":[{"id":"10.13039\/100016616","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["journals.asm.org"],"crossmark-restriction":true},"short-container-title":["mBio"],"published-print":{"date-parts":[[2026,6,10]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                  <jats:sec>\n                    <jats:title\/>\n                    <jats:p>\n                      Since the COVID-19 pandemic, several reverse genetics platforms for SARS-CoV-2 have been established. In general, a plasmid-based reverse genetics system is stable and easy to manipulate, distribute, and store. However, traditional methods for the assembly of a large viral genome in a plasmid rely on natural and artificially engineered restriction sites, which are inefficient, time-consuming, labor-intensive, and frequently not successful. Here, we developed a yeast-based homologous recombination system that allows the assembly of the SARS-CoV-2 genome as a cDNA in a bacterial artificial chromosome (BAC) plasmid in a single step. The entire protocol from cDNA construction to virus rescue is simple, rapid, accurate, highly efficient, and can be completed in 2 weeks. Using this system, we have quickly generated recombinant SARS-CoV-2 (rSARS-CoV-2) WA1, Omicron BA.2.86, and Omicron JN.1 viruses expressing mCherry, green fluorescent protein (GFP), and NanoLuc luciferase (Nluc) reporters. Insertion of these reporter genes does not significantly alter the replication of SARS-CoV-2 in cell culture. We also compared the replication kinetics of rSARS-CoV-2-WA1, BA.2.86, and JN.1 reporter viruses in\n                      <jats:italic toggle=\"yes\">ex vivo<\/jats:italic>\n                      primary human nasal epithelial (HNE) and human bronchial epithelial (HBE) cultures. Omicron BA.2.86 replicated and spread more efficiently than JN.1, which spread much faster than SARS-CoV-2 WA1 in these cultures. In summary, we have developed a highly efficient yeast-based recombinant system for the construction of infectious cDNA clones of SARS-CoV-2, enabling rapid genetic manipulation of SARS-CoV-2. In addition, the reporter viruses generated in this study will be useful for monitoring SARS-CoV-2 infection\n                      <jats:italic toggle=\"yes\">in vitro<\/jats:italic>\n                      and\n                      <jats:italic toggle=\"yes\">in vivo<\/jats:italic>\n                      .\n                    <\/jats:p>\n                    <jats:sec>\n                      <jats:title>IMPORTANCE<\/jats:title>\n                      <jats:p>\n                        Reverse genetics systems are an essential tool for probing the biology of viruses, testing antivirals, and developing live-attenuated vaccines. However, it has been a challenge to generate a rapid reverse genetics system for coronaviruses. Here, we developed a rapid, highly efficient reverse genetics system for SARS-CoV-2 that uses yeast homologous recombination. In this procedure, overlapping DNA fragments encompassing the entire SARS-CoV-2 and BAC plasmid fragments containing a yeast replication origin were mixed and transformed into yeast cells to assemble infectious cDNA clones in a single step. This system has enabled us to rapidly generate nine SARS-CoV-2 viruses: WA1, Omicron BA.2.86, and JN.1 viruses each expressing one of three reporters for tracking virus infection\n                        <jats:italic toggle=\"yes\">in vitro<\/jats:italic>\n                        and\n                        <jats:italic toggle=\"yes\">in vivo<\/jats:italic>\n                        . This method is easy, convenient, and highly efficient, generating infectious cDNA clones within 2 weeks. This system could readily be adapted to construct infectious cDNA clones for other large RNA viruses.\n                      <\/jats:p>\n                    <\/jats:sec>\n                  <\/jats:sec>","DOI":"10.1128\/mbio.00122-26","type":"journal-article","created":{"date-parts":[[2026,5,11]],"date-time":"2026-05-11T13:02:46Z","timestamp":1778504566000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":0,"title":["A rapid yeast-based reverse genetics system reveals SARS-CoV-2 Omicron BA.2.86 variant spreads faster than Omicron JN.1 variant in primary human nasal and bronchial epithelial airway cultures"],"prefix":"10.1128","volume":"17","author":[{"given":"Jiayu","family":"Xu","sequence":"first","affiliation":[{"name":"Department of Veterinary Biosciences, The Ohio State University","place":["Columbus, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Michelle","family":"Chamblee","sequence":"additional","affiliation":[{"name":"Department of Veterinary Biosciences, The Ohio State University","place":["Columbus, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Cheng Chih","family":"Hsu","sequence":"additional","affiliation":[{"name":"Department of Veterinary Biosciences, The Ohio State University","place":["Columbus, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Fei","family":"Jiang","sequence":"additional","affiliation":[{"name":"Department of Veterinary Biosciences, The Ohio State University","place":["Columbus, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Phylip","family":"Chen","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/003rfsp33","id-type":"ROR","asserted-by":"publisher"}],"name":"Center for Microbe and Immunity Research, Abigail Wexner Research Institute, 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