{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,15]],"date-time":"2026-10-15T19:42:04Z","timestamp":1792093324962,"version":"4.4.0"},"reference-count":28,"publisher":"National Academy of Sciences","issue":"27","license":[{"start":{"date-parts":[[2026,6,29]],"date-time":"2026-06-29T00:00:00Z","timestamp":1782691200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100002347","name":"Bundesministerium f\u00fcr Forschung, Technologie und Raumfahrt","doi-asserted-by":"publisher","award":["8040701710"],"award-info":[{"award-number":["8040701710"]}],"id":[{"id":"10.13039\/501100002347","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100002347","name":"Bundesministerium f\u00fcr Forschung, Technologie und Raumfahrt","doi-asserted-by":"publisher","award":["8064701703"],"award-info":[{"award-number":["8064701703"]}],"id":[{"id":"10.13039\/501100002347","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100020655","name":"EC | European Health and Digital Executive Agency","doi-asserted-by":"publisher","award":["101102733"],"award-info":[{"award-number":["101102733"]}],"id":[{"id":"10.13039\/100020655","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["www.pnas.org"],"crossmark-restriction":true},"short-container-title":["Proc. Natl. Acad. Sci. U.S.A."],"published-print":{"date-parts":[[2026,7,7]]},"abstract":"<jats:p>The origin of the polybasic furin cleavage site (FCS) of SARS-CoV-2 remains a central question in debates on the emergence of COVID-19. One hypothesis proposes a genetic relationship between the SARS-CoV-2 S1\/S2 motif RRAR and the RRVR sequence found in the mouse-adapted MERS-CoV strain MERS-MA30. Here, we combined large-scale bioinformatic analysis with experimental virology to evaluate this scenario. Analysis of over 17 million SARS-CoV-2 genomes revealed that the S:684V substitution corresponding to RRVR occurred repeatedly but only sporadically, never became phylogenetically basal, and showed limited geographic and temporal spread. Using reverse genetics, we generated SARS-CoV-2 variants encoding RRVR and demonstrated that S:684V consistently reduced viral entry efficiency and competitive fitness in multiple cell systems, including human respiratory epithelial cultures. RRVR variants did not evolve toward RRAR but instead accumulated alternative substitutions. These findings do not support an evolutionary relationship between MERS-MA30 and the SARS-CoV-2 FCS.<\/jats:p>","DOI":"10.1073\/pnas.2601806123","type":"journal-article","created":{"date-parts":[[2026,6,29]],"date-time":"2026-06-29T19:11:41Z","timestamp":1782760301000},"update-policy":"https:\/\/doi.org\/10.1073\/pnas.cm10313","source":"Crossref","is-referenced-by-count":1,"title":["Evaluation of a proposed link between the SARS-CoV-2 furin cleavage site and mouse-adapted MERS-coronavirus MA30"],"prefix":"10.1073","volume":"123","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-7730-9733","authenticated-orcid":false,"given":"Sarah M.","family":"Metzger","sequence":"first","affiliation":[{"name":"Institute of Virology, Charit\u00e9\u2014Universit\u00e4tsmedizin Berlin, Corporate Member of Freie Universit\u00e4t and Humboldt-Universit\u00e4t Berlin","place":["Berlin, Germany"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1120-9531","authenticated-orcid":false,"given":"Terry C.","family":"Jones","sequence":"additional","affiliation":[{"name":"Institute of Virology, Charit\u00e9\u2014Universit\u00e4tsmedizin Berlin, Corporate Member of Freie Universit\u00e4t and Humboldt-Universit\u00e4t Berlin","place":["Berlin, Germany"]},{"id":[{"id":"https:\/\/ror.org\/013meh722","id-type":"ROR","asserted-by":"publisher"}],"name":"Center for Pathogen Evolution, Department of Zoology, University of Cambridge","place":["Cambridge, United Kingdom"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jenny I. 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Haslam Baric referenced a 2017 molecular blueprint for COVID-19 (2024) https:\/\/jimhaslam.substack.com\/p\/baric-referenced-a-2017-molecular."},{"key":"e_1_3_4_8_2","volume-title":"Independent Assessment of The Origins OfSARS-CoV-2 from The Scientific Advisory Group for The Origins of Novel Pathogens (SAGO)","author":"World Health Organization","year":"2025","unstructured":"World Health Organization, Independent Assessment of The Origins OfSARS-CoV-2 from The Scientific Advisory Group for The Origins of Novel Pathogens (SAGO) (World Health Organization, 2025)."},{"key":"e_1_3_4_9_2","doi-asserted-by":"publisher","DOI":"10.2807\/1560-7917.ES.2017.22.13.30494"},{"key":"e_1_3_4_10_2","unstructured":"S. M. Metzger Python code from \u201cEvaluation of a proposed link between the SARS-CoV-2 furin cleavage site and mouse-adapted MERS-coronavirus MA30\u201d. Available at; https:\/\/github.com\/VirologyCharite\/PNAS-FCS-analysis. Deposited 28 May 2026."},{"key":"e_1_3_4_11_2","doi-asserted-by":"crossref","unstructured":"Livak K. J. & Schmittgen T. D. (2001). Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2\u2212\u0394\u0394CT Method. Methods 25(4) 402\u2013408. https:\/\/doi.org\/10.1006\/meth.2001.1262","DOI":"10.1006\/meth.2001.1262"},{"key":"e_1_3_4_12_2","unstructured":"S. M. Metzger Laboratory data from \u201cEvaluation of a proposed link between the SARS-CoV-2 furin cleavage site and mouse-adapted MERS-coronavirus MA30\u201d. Available at https:\/\/github.com\/VirologyCharite\/PNAS-FCS-analysis-lab-data. Deposited 28 May 2026."},{"key":"e_1_3_4_13_2","unstructured":"T. C. Jones Python to extract genotyping information from Sanger sequencing ab1 files. https:\/\/github.com\/VirologyCharite\/ab1-tool."},{"key":"e_1_3_4_14_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.celrep.2020.108630"},{"key":"e_1_3_4_15_2","unstructured":"World Health Organization COVID-19 cases World. https:\/\/data.who.int\/dashboards\/covid19\/cases. Accessed 12 October 2025."},{"key":"e_1_3_4_16_2","unstructured":"World Health Organization True extent of SARS-CoV-2 Infection through seroprevalence studies. https:\/\/www.who.int\/news\/item\/03-02-2022-true-extent-of-sars-cov-2-infection-through-seroprevalence-studies. Accessed 12 October 2025."},{"key":"e_1_3_4_17_2","unstructured":"T. C. 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