Canine parvovirus (CPV) is a major pathogen causing severe gastroenteritis in dogs. Since its emergence, CPV has undergone continuous evolution, leading to the predominance of variants such as CPV-2a, CPV-2b, and CPV-2c. To characterize the genetic features and evolutionary trends of CPV-2 at a regional level, 775 fecal samples were collected from domestic and stray dogs with suspected CPV-2 infection in Shanghai between 2016 and 2025. The overall positivity rate was 23.2% (180/775); incidence was substantially higher in stray dogs (30.2%) than in domestic dogs (15.9%). Thirty-one CPV-2 strains were successfully isolated. Temporal analysis revealed a pronounced genotype shift: isolates from 2016 to 2020 were predominantly New CPV-2a, whereas CPV-2c became the dominant genotype from 2021 through 2025. Sequence analysis identified the polymorphism of VP2 gene and characteristic mutations F267Y, Y324I, N426E, Q370R and A440T in CPV-2c strains. A novel I447M mutation was detected in several isolates. Phylogenetic analysis showed that Shanghai isolates formed distinct clusters; CPV-2c strains were closely related to the Asian lineage. Structural modeling indicated that mutations at residues L87M, T101I, Y267F, A297S, G300A, Y305D, I324Y, Q370R, N426E, A440T, and I447M may alter the tertiary structure of the VP2 protein, potentially affecting antigenicity and receptor recognition. Collectively, these results demonstrate the complete genotype replacement of CPV-2 in Shanghai; CPV-2c is now predominant. Identification of the novel I447M mutation and structural analysis of key amino acid substitutions provide insight into CPV molecular evolution. These findings suggest that vaccines primarily based on older CPV-2 or CPV-2b genotypes offer suboptimal protection, highlighting the need for updated vaccine strategies targeting prevalent CPV-2c variants.

canine parvovirus; CPV-2c; genotype shift; VP2; mutation