Two subtypes of influenza A virus (IAV), avian-origin canine influenza virus (CIV) H3N2 (CIV-H3N2) and equine-origin CIV H3N8 (CIV-H3N8), are enzootic in the canine population. Dogs have been demonstrated to seroconvert in response to diverse IAVs, and naturally occurring reassortants of CIV-H3N2 and the 2009 H1N1 pandemic virus (pdmH1N1) have been isolated. We conducted a thorough phenotypic evaluation of CIV-H3N2 in order to assess its threat to human health. Using ferret-generated antiserum, we determined that CIV-H3N2 is antigenically distinct from contemporary human H3N2 IAVs, suggesting that there may be minimal herd immunity in humans. We assessed the public health risk of CIV-H3N2 × pandemic H1N1 (pdmH1N1) reassortants by characterizing their genetic compatibility and pathogenicity and transmissibility. Using a luciferase minigenome assay, we quantified the polymerase activity of all possible 16 ribonucleoprotein (RNP) complexes (PB2, PB1, PA, NP) between CIV-H3N2 and pdmH1N1, identifying some combinations that were more active than either parental virus complex. Using reverse genetics and fixing the CIV-H3N2 hemagglutinin (HA), we found that 51 of the 127 possible reassortant viruses were viable and able to be rescued. Nineteen of these reassortant viruses had high-growth phenotypes , and 13 of these replicated in mouse lungs. A single reassortant with the NP and HA gene segments from CIV-H3N2 was selected for characterization in ferrets. The reassortant was efficiently transmitted by contact but not by the airborne route and was pathogenic in ferrets. Our results suggest that CIV-H3N2 reassortants may pose a moderate risk to public health and that the canine host should be monitored for emerging IAVs. IAV pandemics are caused by the introduction of novel viruses that are capable of efficient and sustained transmission into a human population with limited herd immunity. Dogs are a a potential mixing vessel for avian and mammalian IAVs and represent a human health concern due to their susceptibility to infection, large global population, and close physical contact with humans. Our results suggest that humans are likely to have limited preexisting immunity to CIV-H3N2 and that CIV-H3N2 × pdmH1N1 reassortants have moderate genetic compatibility and are transmissible by direct contact in ferrets. Our study contributes to the increasing evidence that surveillance of the canine population for IAVs is an important component of pandemic preparedness.

In addition to causing outbreaks in domestic poultry, subtype H7 IAVs can cause sporadic spillover infections in lower mammals and humans. In this study, we showed that SLe X expression varies among wild dabbling ducks.

Wild aquatic birds maintain a large genetically diverse pool of influenza A viruses (IAVs), which can be transmitted to lower mammals and ultimately humans. Through viral replication efficiency phenotypic analyses, only a small set of avian IAVs replicated well in the epithelial cells of swine upper respiratory tracts, and these viruses were shown to infect and cause virus shedding in pigs. Such a phenotypic trait in viral replication efficiency appears to emerge randomly and are distributed among IAVs across multiple avian species, geographic and temporal orders, and is determined not by receptor binding preference but other markers across genomic segments, such as those in the ribonucleoprotein complex. This study demonstrates that viral replication efficiency phenotypic variants exist among avian IAVs, only a few of which may result in viral shedding in pigs upon infection, providing opportunities for these viruses to become pig adapted, thus posing a higher potential risk for creating novel variants or detrimental reassortants within pig populations. IMPORTANCE Swine serve as a “mixing vessel” for generating human influenza pandemic strains. All HA subtypes of IAVs can infect swine; however, only sporadic cases of avian IAVs are reported in domestic swine. The molecular mechanisms affecting avian IAVs ability to infect swine are still not fully understood. Through phenotypic analyses, this study suggested that tissue tropisms (i.e., in swine upper respiratory tracts) of avian IAVs affect their spillovers from wild birds to pigs, and this phenotype was determined not by receptor binding preference but by other markers across genomic segments, such as those in the ribonucleoprotein complex. In addition, our results showed that such a phenotypic trait was sporadically and randomly distributed among IAVs across multiple avian species, geographic and temporal orders. This study suggested an efficient way for risk assessment of avian IAVs, such as in evaluating their potentials to be transmitted from avian to pigs.

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