Isolation and genetic characterization of canine adenovirus type 2 variant from raccoon dog (Nyctereutes procynoide koresis) in Republic of Korea

Canine adenovirus type 2 (CAV-2) is a common causative agent of respiratory disease in canines. There have been no reports of CAV-2 variants isolated from raccoon dogs. This study aims to investigate the biological and genetic characteristics of a novel Korean CAV-2 variant. Madin-Darby canine kidney cells were used to isolate the CAV-2 variant from 45 fecal swab samples. Diagnostic tools such as the cytopathic effect (CPE) assay, electron microscopy, polymerase chain reaction, and immunofluorescence and hemagglutination assays were used to confirm the presence of the CAV-2 isolate. A cross-virus neutralization assay was performed to verify the novelty of this CAV variant. Genetic analysis was performed using nucleotide sequences obtained through next-generation sequencing. The isolate was confirmed to be a CAV-2 variant based on the aforementioned methods and designated CAV2232. The number of bases in the fiber and E3 genes of CAV2232 were 1,626 and 414, respectively. Phylogenetic analysis of the fiber and E3 genes confirmed that CAV2232 was classified into a different clade from the known CAV-1 and CAV-2 strains. Mice inoculated with the CAV2232 vaccine developed high virus neutralization antibody titers of 1,024 (210) against CAV2232, while mice inoculated with CAV-1 and CAV-2 vaccines had low virus neutralization antibody titers of 12.9 (23.7) and 6.5 (22.7), respectively, against CAV2232. CAV2232 isolated from wild raccoon dog feces was classified as a novel CAV-2 variant. CAV2232 may therefore be used as an antigen for new vaccine development and serological investigations.