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Tembusu virus (TMUV), an avian mosquito-borne flavivirus, was first identified from Culex tritaeniorhynchus in 1955. To validate the effects of the 3'-untranslated region (3'UTR) in viral host-specific adaptation, we generated a set of chimeric viruses using CQW1 (duck strain) and MM 1775 (mosquito strain) as backbones with heterogeneous 3'UTRs. Compared with rMM 1775, rMM-CQ3'UTR (recombinant MM 1775 virus carrying the 3'UTR of CQW1) exhibited enhanced proliferation , with peak titers increasing by 5-fold in duck embryonic fibroblast (DEF) cells or 12-fold in baby hamster kidney (BHK-21) cells; however, the neurovirulence of rMM-CQ3'UTR was attenuated in 14-day-old Kunming mice via intracranial injection, with slower weight loss, lower mortality, and reduced viral loads.

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