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Cholesterol 25-hydroxylase (CH25H) is a molecular mediator of innate antiviral immunity and converts cholesterol to 25-hydroxycholesterol (25HC). Previous studies have reported that CH25H and 25HC have an antiviral effect against multiple viruses. However, the interplay between SADS-CoV infection and CH25H or 25HC is still uncertain. Here, we found that CH25H and its enzymatic product 25HC restrained SADS-CoV replication by blocking membrane fusion. Our results show that CH25H was upregulated by SADS-CoV infection in vitro and in vivo, and that it was an IFN-stimulated gene in porcine ileum epithelial cells. Moreover, CH25H and CH25H mutants lacking catalytic activity can inhibit SADS-CoV replication. Furthermore, 25HC significantly suppressed SADS-CoV infection by inhibiting virus entry. Notably, we confirmed that CH25H and 25HC blocked SADS-CoV spike protein-mediated membrane fusion. Our data provide a possible antiviral therapy against SADS-CoV and other conceivable emerging coronaviruses in the future.<\/jats:p>","DOI":"10.3390\/v15122406","type":"journal-article","created":{"date-parts":[[2023,12,11]],"date-time":"2023-12-11T06:56:00Z","timestamp":1702277760000},"page":"2406","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":8,"title":["Cholesterol 25-Hydroxylase Suppresses Swine Acute Diarrhea Syndrome Coronavirus Infection by Blocking Spike Protein-Mediated Membrane Fusion"],"prefix":"10.3390","volume":"15","author":[{"given":"Dakai","family":"Liu","sequence":"first","affiliation":[{"name":"State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Xiangfang District, Haping Road 678, Harbin 150069, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Da","family":"Shi","sequence":"additional","affiliation":[{"name":"State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Xiangfang District, Haping Road 678, Harbin 150069, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hongyan","family":"Shi","sequence":"additional","affiliation":[{"name":"State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Xiangfang District, Haping Road 678, Harbin 150069, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Liaoyuan","family":"Zhang","sequence":"additional","affiliation":[{"name":"State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Xiangfang District, Haping Road 678, Harbin 150069, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jiyu","family":"Zhang","sequence":"additional","affiliation":[{"name":"State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Xiangfang District, Haping Road 678, Harbin 150069, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Miaomiao","family":"Zeng","sequence":"additional","affiliation":[{"name":"State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Xiangfang District, Haping Road 678, Harbin 150069, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tingshuai","family":"Feng","sequence":"additional","affiliation":[{"name":"State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Xiangfang District, Haping Road 678, Harbin 150069, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiaoman","family":"Yang","sequence":"additional","affiliation":[{"name":"State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Xiangfang District, Haping Road 678, Harbin 150069, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xin","family":"Zhang","sequence":"additional","affiliation":[{"name":"State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Xiangfang District, Haping Road 678, Harbin 150069, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jianfei","family":"Chen","sequence":"additional","affiliation":[{"name":"State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Xiangfang District, Haping Road 678, Harbin 150069, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhaoyang","family":"Jing","sequence":"additional","affiliation":[{"name":"State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Xiangfang District, Haping Road 678, Harbin 150069, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhaoyang","family":"Ji","sequence":"additional","affiliation":[{"name":"State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Xiangfang District, Haping Road 678, Harbin 150069, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jialin","family":"Zhang","sequence":"additional","affiliation":[{"name":"State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Xiangfang District, Haping Road 678, Harbin 150069, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2898-8308","authenticated-orcid":false,"given":"Li","family":"Feng","sequence":"additional","affiliation":[{"name":"State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Xiangfang District, Haping Road 678, Harbin 150069, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2023,12,11]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Bonilauri, P., and Rugna, G. 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