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Despite its increasing prevalence in Europe and reports of neuroinvasive disease in humans, the molecular and immunological determinants of USUV infection remain poorly understood, largely due to the absence of tractable experimental tools. Here, we report the generation and characterization of the first full-length fluorescent reporter USUV. Using a previously established infectious clone of the Vienna 2001 strain, we inserted the mScarlet-I gene into a permissive region of the viral genome, generating a stable and replication-competent reporter virus. In human hepatoma cells, USUV-mScarlet exhibited replication kinetics comparable to wild-type virus and maintained fluorescence over at least 10 serial passages. Application of this system identified remdesivir and interferon-alpha 2a (IFN-\u03b12a) as potent USUV inhibitors and revealed robust induction of the type I IFN response in infected mouse fibroblasts, despite overall attenuation in this cell type. In IFN signaling deficient mice, which are permissive for viral replication, USUV-mScarlet infection resulted in transient disease and reporter expression in multiple peripheral tissues. Among splenocytes, specific myeloid cell subsets were highly enriched in reporter expression, suggesting their role as targets of viral infection. Collectively, these findings demonstrate that USUV-mScarlet recapitulates wild-type infection dynamics in human hepatoma cells while enabling single-cell\/quantitative visualization and sorting of infected cells to assess viral spread and host responses. This reporter platform provides a versatile tool for studying USUV biology and will facilitate the development of targeted antivirals and vaccines against this emerging orthoflavivirus.<\/jats:p>","DOI":"10.1093\/pnasnexus\/pgag328","type":"journal-article","created":{"date-parts":[[2026,9,29]],"date-time":"2026-09-29T07:33:34Z","timestamp":1790667214000},"source":"Crossref","is-referenced-by-count":0,"title":["A versatile fluorescent reporter for Usutu virus drug screening and infection tracking"],"prefix":"10.1093","volume":"5","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-3654-163X","authenticated-orcid":false,"given":"Amy N","family":"Nelson","sequence":"first","affiliation":[{"name":"Department of Molecular Biology, Princeton University , Princeton, NJ 08544 ,","place":["USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7400-4125","authenticated-orcid":false,"given":"Aaron E","family":"Lin","sequence":"additional","affiliation":[{"name":"Department of Molecular Biology, Princeton University , Princeton, NJ 08544 ,","place":["USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Saloni","family":"Sinha","sequence":"additional","affiliation":[{"name":"Division of Gastroenterology and Hepatology, Department of Medicine, Weill Cornell Medicine , New York, NY 10065 ,","place":["USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sydney J","family":"Mullin","sequence":"additional","affiliation":[{"name":"Department of Molecular Biology, Princeton University , Princeton, NJ 08544 ,","place":["USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0000-8181-4206","authenticated-orcid":false,"given":"Jianche","family":"Liu","sequence":"additional","affiliation":[{"name":"Department of Molecular Biology, Princeton University , Princeton, NJ 08544 ,","place":["USA"]},{"name":"Computational and Systems Immunology Track, Stanford Immunology Program, Stanford University School of Medicine , Stanford, CA 94305 ,","place":["USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0001-2036-0455","authenticated-orcid":false,"given":"Alexandra G","family":"Bell","sequence":"additional","affiliation":[{"name":"Department of Molecular Biology, Princeton University , Princeton, NJ 08544 ,","place":["USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sebastian S","family":"Carver","sequence":"additional","affiliation":[{"name":"Department of Molecular Biology, Princeton University , Princeton, NJ 08544 ,","place":["USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jason","family":"Sethiadi","sequence":"additional","affiliation":[{"name":"Division of Gastroenterology and Hepatology, Department of Medicine, Weill Cornell Medicine , New York, NY 10065 ,","place":["USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0004-0955-7988","authenticated-orcid":false,"given":"Jiayu","family":"Zhang","sequence":"additional","affiliation":[{"name":"Department of Molecular Biology, Princeton University , Princeton, NJ 08544 ,","place":["USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Brigitte L","family":"Heller","sequence":"additional","affiliation":[{"name":"Department of Molecular Biology, Princeton University , Princeton, NJ 08544 ,","place":["USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8971-184X","authenticated-orcid":false,"given":"Cameron","family":"Myhrvold","sequence":"additional","affiliation":[{"name":"Department of Molecular Biology, Princeton University , Princeton, NJ 08544 ,","place":["USA"]},{"name":"Omenn-Darling Bioengineering Institute, Princeton University , Princeton, NJ 08544 ,","place":["USA"]},{"name":"Department of Chemical and Biological Engineering, Princeton University , Princeton, NJ 08544 ,","place":["USA"]},{"name":"Department of Chemistry, Princeton University , Princeton, NJ 08544 ,","place":["USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5417-5995","authenticated-orcid":false,"given":"Robert E","family":"Schwartz","sequence":"additional","affiliation":[{"name":"Division of Gastroenterology and Hepatology, Department of Medicine, Weill Cornell Medicine , New York, NY 10065 ,","place":["USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9322-7252","authenticated-orcid":false,"given":"Alexander","family":"Ploss","sequence":"additional","affiliation":[{"name":"Department of Molecular Biology, Princeton University , Princeton, NJ 08544 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