Adaptive evolution of tembusu virus enhances mammalian pathogenicity and vector competence

Tembusu virus (TMUV) is an avian orthoflavivirus responsible for severe egg-drop syndrome, inflicting substantial economic losses on the poultry industry in China and Southeast Asia. TMUV exhibits zoonotic potential and has diversified into distinct phylogenetic clusters; however, the phenotypic consequences of this genetic divergence remain poorly characterized. This study systematically evaluated the mammalian pathogenicity in mice and the vector competence in Culex quinquefasciatus mosquitoes of five TMUV isolates representing major clusters (clusters 2.1.1, 2.1.2, 2.2, 3.1, and 3.2). Results showed that contemporary duck-derived isolates (clusters 2 and 3.2) demonstrated superior replication efficiency and infection rates in mosquitoes compared to the ancestral mosquito-derived isolate (MM 1775, cluster 3.1); the recently emerged chicken-derived cluster 3.2 isolate (WH2025) caused 100% mortality in the infected mice, exhibiting the highest pathogenicity. These findings indicate that TMUV evolution has selected for phenotypes with enhanced mammalian virulence and increased its fitness in mosquito vectors, highlighting a growing risk of cross-species transmission. This study provides crucial insights for risk assessment and targeted surveillance for TMUV.IMPORTANCETembusu virus (TMUV) is an emerging mosquito-borne flavivirus that has been circulating in China and Southeast Asia in recent years, causing significant economic losses to the waterfowl farming industry. In recent years, strains of TMUV cluster 3.2 have been increasingly isolated from laying hens and geese, and it has been reported that TMUV can cause mortality in mammals (dolphins). TMUV exhibits mosquito-borne transmission and potential zoonotic characteristics; however, there is a lack of systematic understanding regarding the vector transmission efficiency and changes in mammalian pathogenicity among different evolutionary clusters of TMUV. This study compares the adaptability to Culex quinquefasciatus and pathogenicity in mice of major TMUV evolutionary clusters, contributing to our understanding of TMUV's mosquito-borne transmission capacity and mammalian pathogenicity. The findings are of great significance in assessing the transmission capacity and strain risks of epidemic TMUV strains.