Impact of VP2 mutations on viral fitness in canine parvovirus

Canine parvovirus type 2 (CPV-2) is a highly contagious pathogen responsible for severe gastroenteritis in carnivores, particularly in dogs. Continuous antigenic evolution has resulted in the emergence of CPV-2a, CPV-2b, and CPV-2c variants, defined by amino acid substitutions in the VP2 capsid protein. In this study, genetically altered CPV-2 viruses carrying common VP2 mutations (S297A, V300G, D305Y, Y324I, N426D, N426E, and T440A) were generated to evaluate their effects on viral behavior in vitro. Mutations were individually introduced into an infectious backbone (strain 447), classified as CPV-2a based on VP2 sequence analysis, and compared with the ancestral CPV-2 backbone (strain 265). Viral dynamics were assessed using quantitative PCR and immunofluorescence assays to examine extracellular viral DNA and intracellular infection, enabling discrimination between replication and virion release. Distinct profiles were observed among mutants, particularly for N426D, N426E, and T440A, and the impact of each substitution was influenced by the genetic background. Polyclonal antibodies induced by commercial CPV-2 vaccines neutralized both the CPV-2 control and the CPV-2 N426E mutant, indicating cross-reactivity. These findings demonstrate that VP2 evolution must be interpreted through functional approaches rather than solely by variant classification and provide experimentally based information relevant for molecular surveillance and vaccination strategies.