Zoonotic Risk, Pathogenesis, and Transmission of Avian-Origin H3N2 Canine Influenza Virus

Abstract

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 in vitro genetic compatibility and in vivo 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 in vitro, 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.IMPORTANCE 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.

Keywords: 2009 H1N1 influenza A virus; A(H1N1)pdm09; H3N2; aerosol transmission; canine influenza virus; influenza A virus; reassortment; risk assessment; viral pathogenesis; zoonosis.

FIG 1

Ribonucleoprotein complex luciferase activity and reassortant virus frequency. The luciferase activities of all 16 RNP complexes between GD06 (shaded in brown) and CA09 (shaded in purple) were compared. The ratio of Renilla luciferase to firefly luciferase (Rluc/Fluc) is expressed as the mean + standard deviation from three independent experiments, and statistical analysis was performed using a one-way ANOVA with post hoc Tukey's multiple-comparison test. The reassortant virus RNP data were frequency counts from Tables 1 and 2.

FIG 2

Viral lung titers of inoculated mice. Mice (n = 3) were intranasally inoculated with 50 μl of 10⁶ TCID₅₀/ml of virus per ml. At 4 dpi they were necropsied and lung tissues were collected for determination of virus titers. Titers are expressed as the mean ± standard deviation.

FIG 3

Growth kinetics of viruses. The growth kinetics of each virus shown were characterized in MDCK cells (A) or A549 cells (B) at an MOI of 0.001. Virus titers are expressed as the mean ± standard deviation from three independent experiments. The limit of virus detection was 10^0.699 TCID₅₀/ml. R109, reassortant 109.

FIG 4

Viral titers in nasal wash fluids from ferrets. Ferrets were inoculated with 10⁶ TCID₅₀ of the GD06 virus (A, C) or the reassortant 109 virus (E, G). Naive ferrets were exposed either by direct contact with GD06-inoculated ferrets (B) or by aerosol contact with GD06-infected ferrets (D). (F, H) The same scheme described above was followed for reassortant 109, with naive ferrets being exposed by direct contact (F) or by aerosol exposure (H). At 3, 5, 7, and 10 dpi, nasal wash fluids were collected from ferrets and titrated in MDCK cells. The end titers are expressed as the number of log₁₀ TCID₅₀ per milliliter. The limit of virus detection was 10^0.699 TCID₅₀/ml.

FIG 5

Viral titers from ferret tissues. Ferrets were inoculated with 10⁶ TCID₅₀ of GD06, reassortant 109, or PBS as a control. At 5 dpi they were humanely euthanized and nasal turbinate, tracheal, and lung tissues were collected. The tissues were homogenized and then titrated in MDCK cells. The limit of virus detection was 10^0.699 TCID₅₀/ml. No virus was detected in the lungs. Reassortant 109 replicated in nasal turbinate and tracheal tissues, whereas GD06 was detected only in nasal turbinate tissue.

FIG 6

Histopathology of ferret nasal turbinate, tracheal, and lung tissues. Ferrets were inoculated with 10⁶ TCID₅₀ of GD06 virus, reassortant 109, or PBS as a control. At 5 dpi they were euthanized and nasal turbinate, tracheal, and lung tissues were collected. One GD06-infected ferret had moderate turbinate pathology (rhinitis with moderate lamina proprial lymphoplasmacellular infiltrates and a loss of cilia or replacement of the respiratory mucosal epithelium by stratified squamous epithelium), whereas the other had minimal pathology (minimal lamina proprial lymphoplasmacellular infiltrates). Both reassortant 109-infected ferrets had moderate to severe turbinate pathology, including lymphoplasmacellular rhinitis and a loss of cilia or replacement of the normal mucosal epithelium by stratified squamous epithelium. No significant lesions were seen in the trachea of any of the ferrets. There was mild lung pathology, including peribronchitis and peribronchiolitis, in one ferret infected with reassortant 109.