Genomic and pathogenic characterization of a chicken-origin cluster 3.2 Tembusu virus isolated in China

Tembusu virus (TMUV) is an emerging flavivirus that causes encephalitis and a decrease in egg production in poultry across East and Southeast Asia. Here, we isolated and characterized a chicken-origin TMUV strain, CHN-WH, from a laying hen farm in China. Whole-genome sequencing and phylogenetic analysis based on the complete open reading frame revealed that CHN-WH belongs to cluster 3.2 and is most closely related to the mosquito-derived strain TMUV-YN2020. Comparative sequence analysis and structural modeling revealed several cluster 3.2-specific signature residues in the E protein, as well as a strain-specific M198L substitution predicted to be located in a surface-exposed region. CHN-WH replicated efficiently in BHK-21 cells, chicken embryo fibroblasts (CEFs), and duck embryo fibroblasts (DEFs). In vivo, CHN-WH caused systemic viral dissemination and neurological lesions in chicks and induced ovarian lesions accompanied by reduced egg production in laying hens. These findings indicate that CHN-WH is a pathogenic chicken-origin cluster 3.2 TMUV strain with distinct genomic and phenotypic characteristics. This study expands the current knowledge of the molecular features and pathogenic potential of the chicken-origin cluster 3.2 TMUV and provides useful information for epidemiological surveillance and vaccine development.