UNLABELLED

Tembusu virus (TMUV), an emerging avian orthoflavivirus, causes severe egg-drop syndrome and encephalitis in ducks. Although ducks are the natural host, mice serve as a valuable model for studying neuropathogenesis, as TMUV-infected mice recapitulate key neurological symptoms observed in ducks, such as paralysis and encephalitis. In the previous study, we observed that the TMUV strain CQW1 exhibited unexpectedly low neurovirulence in mice compared with earlier strains, highlighting potential genetic determinants of pathogenicity that may influence viral evolution and disease outcomes in natural hosts. In this study, we investigated the murine neurovirulence of TMUV strains from two major phylogenetic clusters (2.1 and 2.2). The Cluster 2.2 strain CHN-YC demonstrated markedly higher neurovirulence in Kunming mice than Cluster 2.1 strains (CQW1 and SCS01), with robust viral replication in the brain, pronounced histopathological damage, and elevated proinflammatory cytokine levels. Comparative genomic analysis identified seven amino acid substitutions in the E-NS1 region, with variations unique to Cluster 2.1 strains or specific to CQW1. By introducing these substitutions into CQW1 via reverse genetics, we restored high murine neurovirulence and identified the E protein substitution V487A as critical for this phenotype. Mechanistically, E-V487A enhances viral assembly, which boosts replication efficiency and . This substitution is located in the E protein transmembrane domain, a region implicated in flavivirus particle formation. Our data revealed that a naturally occurring amino acid substitution located in the transmembrane domain of the Tembusu virus E protein is responsible for its high neurovirulence in mice.

IMPORTANCE

Tembusu virus is a mosquito-borne avian orthoflavivirus, exhibiting airborne transmission. Although it primarily affects domestic fowl, TMUV demonstrates high neurovirulence in mice during laboratory studies and has been reported to spill over into humans. Recent years have seen increased genetic diversity and an expanded host range of the virus. Strains belonging to phylogenetic cluster 3 can cause severe neurological symptoms and death in mice via intranasal infection, further highlighting its risk of potential transmission to mammals. Understanding their pathogenicity and the underlying molecular basis is crucial for assessing and preventing health risks to mammals. We identified a single amino acid substitution in the TMUV E protein that critically enhances viral replication and neurovirulence in mice. The data provide insights into the molecular mechanisms of Tembusu virus pathogenesis in mammals and underscore the impact of specific genetic mutations on the viral phenotype.