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Domain I of the 3\u2032-untranslated region (UTR) is highly structured and shaped by host-driven selection. Mammalian MBFVs usually have two functional stem\u2013loops (SLs), while avian MBFVs possess two extra SLs (SLI and SLIII), whose functions remain unclear. Using Tembusu virus (TMUV) and Japanese encephalitis virus (JEV), we mutated Domain I and characterized recombinant viruses. TMUV mutants with only one SL showed attenuated replication, with efficiency dependent on SL identity. Duplicating SLII boosted TMUV replication in mammalian and avian cells but not in mosquito cells, indicating that repeated SLs contribute to host-specific proliferation. Deleting SLI and\/or SLIII impaired TMUV replication in avian cells but not mammalian cells, and dual deletion significantly reduced virulence in ducklings but had limited impact in mice. Replacement of JEV SLI\/SLIII with homologous elements derived from a duck-associated strain improved its proliferation in avian cells. Mechanistically, SLI\/SLIII regulate TMUV replication in avian cells by interacting with the host factor CELF2, which specifically facilitates viral replication in avian cells. Collectively, SLI and SLIII are important determinants of avian host proliferation in MBFVs, improving understanding of their multi-host ecology and transmission.<\/jats:p>","DOI":"10.1093\/nar\/gkag791","type":"journal-article","created":{"date-parts":[[2026,7,31]],"date-time":"2026-07-31T11:45:47Z","timestamp":1785498347000},"source":"Crossref","is-referenced-by-count":0,"title":["Duplication of specialized\n                    <i>cis<\/i>\n                    -acting elements SLI and SLIII in the viral RNA 3\u2032UTR enables mosquito-borne orthoflavivirus adaptation to avian hosts"],"prefix":"10.1093","volume":"54","author":[{"given":"Li","family":"Mao","sequence":"first","affiliation":[{"name":"Institute of Veterinary Medicine and Immunology, Sichuan Agricultural University , Chengdu, Sichuan 611130 ,","place":["China"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yu","family":"He","sequence":"additional","affiliation":[{"name":"Institute of Veterinary Medicine 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