Primer sequences for H3N2 canine influenza virus full-length amplification, polymerase activity assay, and rescue

Influenza A viruses in animal reservoirs repeatedly cross species barriers
to infect humans. Once an animal-borne virus with novel antigenicity
acquired the efficient human-to-human transmissibility, it will become
epidemic in the population. Dogs are the closest animal companions to
humans, and canine respiratory tract expresses both SAα2,3-(avian type)
and α2,6-Gal (human type) receptors. However, the role of dogs in the
ecology of influenza viruses is unclear. H3N2 avian influenza viruses were
transmitted to dogs around 2006 and have formed stable lineages. The
long-term epidemic of avian-origin H3N2 virus in canines offers the best
models to investigate the effect of dogs on the evolution of influenza
viruses. Here, we carried out a systematic and comparative identification
of the biological characteristics of H3N2 canine influenza viruses (CIVs)
isolated in worldwide over 10 years. We found that during the adaptation
of H3N2 CIVs to dogs, H3N2 CIVs became to recognize the human-like
SAα2,6-Gal receptor, gradually increased HA acid stability and replication
ability in human airway epithelial cells, and acquired a 100% transmission
rate via respiratory droplet in ferret model, which were essential
hallmarks of being adapted to humans. We also identified that the
frequency of substitutions related to human adaptation has gradually
increased in H3N2 CIVs and determined four cumulative molecular changes
responsible for the increased airborne transmission ability in ferrets.
Our results suggested that canines may serve as an intermediate for the
adaptation of avian influenza virus to humans. Continuous surveillance
coordinated with risk assessment for CIVs is necessary.