Evidence of Human adaptation for cluster 2 TMUV strain despite effective anti-viral responses

Tembusu virus (TMUV) is an emerging Orthoflavivirus with potential zoonotic importance. To date, the biological differences among TMUV strains remain poorly understood. In this study, we compared the replication of a TMUV strain belonging to the cluster 1 and one belonging to the cluster 2 in human lung epithelial cells and further examined the replication characteristics of TMUV cluster 2 strain in both human and mosquito cell lines. The TMUV cluster 2 strain demonstrated a significant higher replication efficiency than the cluster 1 strain in human cells, indicating stronger adaptation to human-derived cells. Infection of human cells with the cluster 2 strain also induced robust innate immune response, including type I interferon and interferon-stimulated gene expression, associated with activation of the unfolded protein response via the PERK, and IRE1 pathways, reflecting an ER stress engagement. These findings suggest that TMUV cluster 2 strain exhibits enhanced replication capacity and more effectively modulates human host responses at the cellular level, underscoring its potential relevance in human health and the zoonotic risk it represents.