Duck Tembusu virus (DTMUV), an emergent flavivirus, causes domestic waterfowls to suffer from severe egg-drop syndrome and fatal encephalitis, greatly threatens duck production globally. Like other mosquito-borne flaviviruses, the envelope (E) protein of all DTMUV strains was N-glycosylated at the amino acid position 154. Thus far, the biological roles of DTMUV E glycosylation have remained largely unexplored. Herein, we demonstrated the key roles of E glycosylation in the replication and pathogenicity of DTMUV in ducks...