# Molecular Characterization of an H3N2 Canine Influenza Virus Isolated from a Dog in Jiangsu, China, in 2025

## TL;DR

A new H3N2 canine influenza virus was isolated in China and found to have distinct genetic and antigenic features compared to human and avian strains.

## Contribution

The study identifies unique molecular and antigenic characteristics of a canine H3N2 isolate from China, including partial cross-reactivity with human strains.

## Key findings

- The canine H3N2 isolate has five amino acid substitutions and altered glycosylation patterns compared to human and avian strains.
- The virus shows strong binding to avian-type receptors and limited binding to human-type receptors.
- Phylogenetic analysis shows the canine strain forms an independent clade, distinct from human and avian H3N2 strains.

## Abstract

Canine influenza virus H3N2, which originally derived from avian strains, continues to evolve in dogs. In this study, we isolated an H3N2 virus from a dog in Jiangsu, China, and analyzed its antigenic and molecular characteristics. Hemagglutination inhibition assays using human antiserum indicated partial cross-reactivity with the canine virus, while no reactivity was detected with avian strains. Five amino acid substitutions and altered glycosylation patterns contributed to distinct antigenic properties in comparison with human and avian H3N2 viruses. Receptor-binding assays revealed strong affinity for avian-type a-2,3 receptors and limited binding to human-type a-2,6 receptors. These findings highlight the different genetic and antigenic characteristics of canine H3N2 virus from human and avian strains, and the necessity for sustained surveillance of canine H3N2 virus in canine populations.

To investigate the molecular characteristics of H3N2 canine influenza viruses circulating in Jiangsu, China, we isolated a H3N2 strain (A/Canine/Nanjing/CnNj01-2025) from a dog presenting with respiratory signs at the Veterinary Teaching Hospital of Nanjing Agricultural University. All eight gene segments were sequenced and compared with those of two human H3N2 strains and five avian H3N2 strains. Antigenicity and receptor-binding properties were also assessed. Phylogenetic analysis revealed that the canine isolate descended from the avian lineage and formed an independent evolutionary clade, while the human strains were more distantly related to the avian lineage. Glycosylation analysis of the HA protein revealed that the canine strain carried seven N-glycosylation sites, including a unique site at residue 97/81 (HA/H3 numbering), which serves as a molecular signature of the canine strain. Several amino-acid substitutions were identified in major antigenic sites, including D97/81N, A176/160T, N204/188D, V212/196I, and W237/222L. Analysis of internal genes showed that the canine strain harbored PB2 292T and 590S mammalian adaptation mutations, which are also present in human strains. Hemagglutination inhibition (HI) assays of the canine strain indicated moderate serologic cross-reactivity with a human H3N2 antiserum (16-fold reduction), whereas avian strains showed no cross-reactivity. Receptor-binding assays demonstrated that the virus retained predominant α-2,3 sialic acid binding, comparable to that of avian influenza viruses, and gained a modest affinity for human-type α-2,6 sialic acid receptors. Therefore, the canine H3N2 virus has undergone significant antigenic drift, developed partial serological cross-reactivity with human strains, and acquired detectable but limited binding affinity for human-type receptors. Overall, our findings suggest that the current canine H3N2 influenza virus exhibits distinct genetic and antigenic variations from human and avian strains. Continuous molecular and serological surveillance of canine influenza viruses is therefore warranted to monitor their evolutionary trends and assess the potential for cross-species transmission.