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.

Bovine rotavirus (BRV) poses a major threat to the global cattle industry, driving significant morbidity and mortality in young calves. In Yunnan Province, China, BRV is the primary cause of neonatal calf diarrhea (NCD), yet the molecular epidemiology of circulating strains remains poorly understood. This study aimed to investigate the molecular characteristics of bovine rotavirus strains associated with a severe outbreak of the NCD on a local farm. Fecal samples were collected from 396 calves and screened for BRV by RT-PCR targeting the VP6 gene. Positive samples were subjected to virus isolation in MA104 cells, followed by whole-genome sequencing, phylogenetic analysis, and pathogenicity assessment in suckling mice. Of 396 samples, 85 tested positive for BRV, corresponding to an animal-level prevalence of 21.5% (95% CI: 17.5–25.8%), with four fatalities recorded. A strain designated as BRV-YN1-2021 was successfully isolated, exhibiting characteristic cytopathic effects, specific immunofluorescence, and typical rotavirus morphology by electron microscopy. Genomic analysis revealed the constellation G8-P[1]-I2-R2-C2-M2-A3-N2-T6-E2-H3, identified as genotype G8P[1]. BLAST analysis showed that four genomic segments shared the highest identity with deer rotavirus strains, five with human rotavirus strains, and two with bovine rotavirus strains. Phylogenetic analysis demonstrated close relationships with US deer strains, Japanese bovine strains, and human strains circulating in China. Experimental infection in suckling mice induced diarrhea and significant intestinal histopathology, degeneration of villous epithelial cells, goblet cell hyperplasia, and inflammatory infiltration. This study reports the first isolation of a G8P[1] bovine rotavirus from a diarrhea outbreak in Chinese cattle. The multi-host genetic composition provides evidence of interspecies reassortment events, highlighting the zoonotic potential of BRV and emphasizing the need for continuous molecular surveillance to inform effective control strategies.