Evidence for lineage-associated differences in Tembusu virus pathogenicity in avian hosts

Tembusu virus (TMUV) is a significant pathogen for waterfowl, yet the pathogenic differences among its evolutionary clusters remain unclear. This study systematically compared the pathogenic potential of TMUV isolates from key evolutionary branches (Clusters 2.1.1, 2.1.2, 2.2, 3.1 and 3.2) in both ducklings and chicks. Pathogenicity was assessed through in vitro replication kinetics, clinical disease, mortality, histopathology, viremia, and tissue viral loads. The results revealed host- and strain-specific pathogenicity. Duck-origin Cluster 2.1 strains were the most virulent in ducklings, while the chicken-origin Cluster 3 strain showed moderate pathogenicity. Notably, the Cluster 2.2 strain (MC) exhibited the highest replication efficiency in duck embryo fibroblasts (DEFs) but the lowest pathogenicity in vivo, indicating a disconnect between in vitro growth and in vivo virulence. All strains were weakly pathogenic in chicks. Furthermore, non-mosquito-borne transmission was demonstrated. This study elucidates the association between TMUV pathogenicity and its genetic evolution, underscores the critical role of host factors, and provides crucial insights for understanding its adaptive evolution and control.