Isolation and characterization of a red panda amdoparvovirus causing fatal respiratory disease: Identification of VP2-S447 as a key determinant of acute lung injury

Surveillance and control of wildlife-borne pathogens are essential for preventing cross-species transmission. Ongoing diversity in Aleutian virus infection among small carnivoran has expanded host range and promoted viral adaptation, leading to the emergence of previously unknown mutations as the virus spreads across geographic and species boundaries. In this study, we report the first isolation and characterization of a highly pathogenic red panda amdoparvovirus (Designated as RpAPV-Gd1) from a fatal case in a captive red panda (Ailurus fulgens) in China. Histopathological examination and in situ hybridization revealed systemic viral infection, characterized by severe acute interstitial pneumonia, multi-organ inflammation, and a marked tropism for alveolar epithelial cells. Phylogenetic analysis showed that RpAPV-Gd1 clustered closely with other red panda amdoparvovirus 2 (RpAPV-2, Amdoparvovirus carnivoran7), sharing 95.4% whole-genome identity. The virus exhibited broad in vitro cell tropism, including human cell lines (U937 and THP-1). In BALB/c mice, RpAPV-Gd1 caused 100% mortality, which was associated with cytokine storm and severe lung injury. Using reverse genetics, we identified a single amino acid substitution in the VP2 protein (S447R) as a critical determinant of pulmonary pathogenicity in the BALB/c mouse model. Viruses carrying the S447R mutation showed attenuated virulence in vivo and reduced replication efficiency in vitro. These findings further highlight that VP2 plays a key role in disease severity.