Abstract
Duck viral hepatitis, caused by Duck Hepatitis A Virus Type 3 (DHAV-3), remains a major threat to young ducklings. Although DHAV-3 has circulated in China since the 1999s, the complete genomic architecture, exact virulence parameters, and evolutionary distance between early Yunnan isolates and current field strains have remained undefined. This study investigated six DHAV-3 strains isolated in Yunnan Province, China, between 2004 and 2006, to elucidate their genetic and biological characteristics. Full-genome sequencing and phylogenetic analysis revealed >99.5% nucleotide and >99.6% amino acid identity among the strains, suggesting a common ancestral origin. In vivo challenge assays showed rapid onset of clinical signs and >90% mortality in ducklings within 36 h post-inoculation. Embryonic deaths began at 24 h post-infection and peaked by 90 h. Viral replication was efficient in DEF, DEK, Vero, and BHK-21 cells, but absent in chicken fibroblasts (DF-1). Comparative genomic analysis between the YN/LR/2005 strain and recent field isolates (2022-2024) revealed substantial nucleotide divergence in structural regions, with 32 unique amino acid substitutions-all five located in the immunodominant VP1 region that may influence viral antigenicity and host interaction-alongside changes in N-glycosylation sites and alterations in protein secondary structure. Histopathological examination confirmed characteristic hepatic lesions. These findings demonstrate that while DHAV-3 has undergone genetic evolution, it retains high virulence, underscoring the need for ongoing molecular surveillance and supporting future vaccine and diagnostic development.