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While there are no approved HEV-specific therapeutics, ribavirin and pegylated interferon, prescribed off-label, remain the current standard of care. However, ribavirin resistance and toxicity highlight the unmet clinical need to identify safer, HEV-specific antivirals. Here, we identify reactive oxygen species (ROS) promotion as a previously unrecognized host-directed antiviral mechanism against HEV, revealed through the activity of the FDA-approved drug auranofin. Auranofin, which is known to elevate intracellular ROS, displays antiviral activity against several viruses. We revealed here that auranofin exhibits robust, dose-dependent antiviral activity against two clinically relevant HEV genotypes and a ribavirin treatment failure-associated mutant. ROS inhibition reversed auranofin-mediated ROS promotion and antiviral activity, establishing a mechanistic link between ROS promotion and antiviral activity. Treatment with D-amino acid oxidase, which breaks down D-amino acids producing the ROS H\n                      <jats:sub>2<\/jats:sub>\n                      O\n                      <jats:sub>2<\/jats:sub>\n                      , exerted dose-dependent anti-HEV activity. This effect was reversed by ROS inhibition, demonstrating that ROS accumulation alone is sufficient for antiviral activity. We also revealed that ROS promotion by auranofin drives activation of antioxidant, ER stress, and interferon-stimulated gene expressions, further supporting induction of ROS-dependent antiviral signaling. Lastly, we demonstrated that combined treatment with auranofin and ribavirin exhibits synergistic antiviral activity\n                      <jats:italic toggle=\"yes\">in vitro<\/jats:italic>\n                      . These findings highlight the promotion of ROS as a previously underappreciated host-directed antiviral mechanism and support the repurposing of auranofin\u2014alone or in combination with ribavirin\u2014as a therapeutic strategy against HEV.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>IMPORTANCE<\/jats:title>\n                    <jats:p>\n                      Hepatitis E virus (HEV) lacks approved virus-specific antiviral therapies, and off-label treatments with ribavirin and pegylated interferon are limited by toxicity and emerging resistance mutants. This study identifies reactive oxygen species (ROS) promotion mediated by the FDA-approved drug auranofin and D-amino acid oxidase as an effective antiviral strategy against multiple genotypes of HEV, including two globally relevant human-associated genotypes and a ribavirin treatment failure-associated HEV mutant. The observed synergistic anti-HEV activity\n                      <jats:italic toggle=\"yes\">in vitro<\/jats:italic>\n                      for combined treatment with both auranofin and ribavirin suggests a potential clinically effective combinational therapeutic approach. ROS promotion through auranofin or other means represents an underexplored antiviral strategy with potential for broad-spectrum activity against a range of viral diseases.\n                    <\/jats:p>\n                  <\/jats:sec>","DOI":"10.1128\/jvi.01917-25","type":"journal-article","created":{"date-parts":[[2025,12,9]],"date-time":"2025-12-09T14:01:02Z","timestamp":1765288862000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":1,"title":["Reactive oxygen species promotion drives auranofin\u2019s antiviral activity against hepatitis E virus"],"prefix":"10.1128","volume":"100","author":[{"ORCID":"https:\/\/orcid.org\/0009-0009-6238-1762","authenticated-orcid":true,"given":"Kateland","family":"Tiller","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/010prmy50","id-type":"ROR","asserted-by":"publisher"}],"name":"Virginia-Maryland College of Veterinary Medicine, Department of Biomedical Sciences and Pathobiology, Virginia Polytechnic Institute and State University","place":["Blacksburg, USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"S. 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