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The spike (S) protein, a critical mediator of viral entry, undergoes extensive N-linked glycosylation. To elucidate the functional significance of these post-translational modifications, we employed a reverse genetics system to generate 19 recombinant PEDV strains with single-site mutations at predicted N-glycosylation sites. In vitro experiments revealed that mutations at residues N118, N216, N726, N1232, and N1249 significantly attenuated viral replication and reduced plaque size. Our data demonstrated that these mutations impaired viral attachment and internalization. Importantly, in vivo pathogenicity assays in piglets indicated that the N1232Q and N1249Q mutants presented minimal faecal viral shedding and no clinical symptoms, suggesting their potential as live attenuated vaccine candidates. These findings underscore the critical role of S protein glycosylation in PEDV infectivity and virulence, providing a molecular basis for rational vaccine design.<\/jats:p>","DOI":"10.1186\/s13567-025-01606-9","type":"journal-article","created":{"date-parts":[[2025,8,29]],"date-time":"2025-08-29T10:39:33Z","timestamp":1756463973000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":11,"title":["N-glycosylation of the PEDV spike protein modulates viral replication and pathogenicity"],"prefix":"10.1186","volume":"56","author":[{"given":"Huixin","family":"Zhu","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhaoyang","family":"Feng","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Meng","family":"Sun","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sinuo","family":"Zhang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhen","family":"Yang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Juan","family":"Bai","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ping","family":"Jiang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Gongguan","family":"Liu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xing","family":"Liu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4264-3306","authenticated-orcid":false,"given":"Xianwei","family":"Wang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2025,8,29]]},"reference":[{"key":"1606_CR1","doi-asserted-by":"publisher","first-page":"2048","DOI":"10.3201\/eid1912.121088","volume":"19","author":"J Wang","year":"2013","unstructured":"Wang J, Zhao P, Guo L, Liu Y, Du Y, Ren S, Li J, Zhang Y, Fan Y, Huang B, Liu S, Wu J (2013) Porcine epidemic diarrhea virus variants with high pathogenicity, China. 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All experimental protocols for animals were conducted following the National Guidelines for Housing and Care of Laboratory Animals (China) and performed in accordance with Nanjing Agricultural University (NAU) institutional regulations after approval by the Institutional Animal Care and Ethics Committee of NAU (SYXK(SU)2017-0007).","order":2,"name":"Ethics","group":{"name":"EthicsHeading","label":"Ethics approval and consent to participate"}},{"value":"The authors declare that they have no competing interests.","order":3,"name":"Ethics","group":{"name":"EthicsHeading","label":"Competing interests"}}],"article-number":"172"}}