Isolation and identification of duck hepatitis a virus type III and analysis of VP1 protein variation in China, 2021-2023.

Since mid-2021, frequent outbreaks of duck hepatitis A virus type 3 (DHAV-3) have occurred across China's major duck-farming regions, with rising detection rates and mounting economic losses, and vaccine protection has been challenged by immune-escape variants with a potential risk of recombination. To delineate the contemporary molecular epidemiology of DHAV-3, given that VP1 determines its antigenicity, we conducted a systematic survey across major duck-farming provinces in China from 2021 to 2023. Viruses were isolated from diseased duck livers using primary duck embryo fibroblast (DEF) cultures, followed by VP1 sequencing for all isolates and whole-genome sequencing for representative strains. VP1 sequences were obtained for 23 isolates, and three complete genomes were successfully assembled. Phylogenetic analysis was then combined with VP1 three-dimensional structural modeling to characterize lineage distribution and key variation features. Phylogenetic analysis assigned 21/23 isolates (91.3 %) to subgenotype GI-a and 2/23 (8.7 %) to GI-b. Complete genome lengths varied minimally, ranging from 7 788 to 7 789 bp, with the 5' UTR and ORF regions highly conserved and only a single-base difference observed in the 3' UTR. VP1 amino acid alignment showed that mutations were predominantly concentrated at the C-terminus; structural predictions indicated extended α-helices, elongated β-sheets and random coil to α-helix transitions in this region. These findings define the epidemiological distribution and key VP1 variation signatures of DHAV-3 in China, and provide critical data to inform targeted control strategies, enhance pathogen surveillance, and support sustainable development of the duck industry.