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As a major coronavirus affecting the swine industry, PEDV exhibits extensive genetic variability, which has greatly complicated disease control. Current vaccines provide suboptimal protection under field conditions. Therefore, the isolation of recently circulating strains and the establishment of a robust reverse genetics system are critical for advancing the study of emerging variants and facilitating rational vaccine development. In this study, a PEDV field strain designated PEDV-BJ-2023 was isolated from diarrheic piglets in Guizhou, China. Phylogenetic analysis based on the complete genome and spike gene classified PEDV-BJ-2023 within the emerging G2c lineage. To facilitate functional studies, a full-length infectious cDNA clone was constructed using transformation-associated recombination cloning in yeast. Furthermore, an enhanced green fluorescent protein reporter virus was generated via CRISPR\/Cas9-assisted homologous recombination by inserting an EGFP-2A cassette upstream of the nucleocapsid gene. The recombinant viruses displayed virion morphology and plaque characteristics similar to those of the parental wild-type PEDV-BJ-2023 strain, although the parental virus exhibited faster replication during the early stage of infection in vitro. In 5-day-old piglets, all three viruses caused severe diarrhea, weight loss, and intestinal lesions; however, recombinant viruses exhibited slightly reduced viral shedding and pathogenicity, with rPEDV-EGFP being the most attenuated. Notably, rPEDV-EGFP maintained stable EGFP expression over eight serial passages. This study establishes a reverse genetics platform for an emerging G2c PEDV strain and provides a stable fluorescent reporter virus, offering valuable tools for visualizing viral infection and investigating virus\u2013host interactions.<\/jats:p>","DOI":"10.3390\/v18080864","type":"journal-article","created":{"date-parts":[[2026,8,7]],"date-time":"2026-08-07T09:14:28Z","timestamp":1786094068000},"page":"864","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Isolation, Characterization and Reverse Genetic System Establishment of a Highly Virulent PEDV Strain"],"prefix":"10.3390","volume":"18","author":[{"ORCID":"https:\/\/orcid.org\/0009-0004-5327-6534","authenticated-orcid":false,"given":"Fan","family":"Zhang","sequence":"first","affiliation":[{"name":"MOA Key Laboratory of Animal Virology, Zhejiang Provincial Engineering Research Center of Animal Biological Products, Center for Veterinary Sciences, Zhejiang University, Hangzhou 310058, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Helu","family":"Liu","sequence":"additional","affiliation":[{"name":"MOA Key Laboratory of Animal Virology, Zhejiang Provincial Engineering Research Center of Animal Biological Products, Center for Veterinary Sciences, Zhejiang University, Hangzhou 310058, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Linlong","family":"Ji","sequence":"additional","affiliation":[{"name":"MOA Key Laboratory of Animal Virology, Zhejiang Provincial Engineering Research Center of Animal Biological Products, Center for Veterinary Sciences, Zhejiang University, Hangzhou 310058, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yanyang","family":"Zhou","sequence":"additional","affiliation":[{"name":"MOA Key Laboratory of Animal Virology, Zhejiang Provincial Engineering Research Center of Animal Biological Products, Center for Veterinary Sciences, Zhejiang University, Hangzhou 310058, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Heng","family":"Chen","sequence":"additional","affiliation":[{"name":"MOA Key Laboratory of Animal Virology, Zhejiang Provincial Engineering Research Center of Animal Biological Products, Center for Veterinary Sciences, Zhejiang University, Hangzhou 310058, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8852-1227","authenticated-orcid":false,"given":"Jiyong","family":"Zhou","sequence":"additional","affiliation":[{"name":"MOA Key Laboratory of Animal Virology, Zhejiang Provincial Engineering Research Center of Animal Biological Products, Center for Veterinary Sciences, Zhejiang University, Hangzhou 310058, China"},{"name":"State Key Laboratory for Diagnosis and Treatment of Severe Infectious Diseases, First Affiliated Hospital, Zhejiang University, Hangzhou 310058, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jinyan","family":"Gu","sequence":"additional","affiliation":[{"name":"MOA Key Laboratory of Animal Virology, Zhejiang Provincial Engineering Research Center of Animal Biological Products, Center for Veterinary Sciences, Zhejiang University, Hangzhou 310058, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,8,7]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"20","DOI":"10.1016\/j.virusres.2016.05.023","article-title":"Evolution, antigenicity and pathogenicity of global porcine epidemic diarrhea virus strains","volume":"226","author":"Lin","year":"2016","journal-title":"Virus Res."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"134","DOI":"10.1016\/j.tvjl.2015.02.017","article-title":"Porcine epidemic diarrhea virus infection: Etiology, epidemiology, pathogenesis and immunoprophylaxis","volume":"204","author":"Jung","year":"2015","journal-title":"Vet. 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