Infection and pathogenesis of canine, equine, and human influenza viruses in canine tracheas

Abstract

Influenza A viruses (IAVs) can jump species barriers and occasionally cause epidemics, epizootics, pandemics, and panzootics. Characterizing the infection dynamics at the target tissues of natural hosts is central to understanding the mechanisms that control host range, tropism, and virulence. Canine influenza virus (CIV; H3N8) originated after the transfer of an equine influenza virus (EIV) into dogs. Thus, comparing CIV and EIV isolates provides an opportunity to study the determinants of influenza virus emergence. Here we characterize the replication of canine, equine, and human IAVs in the trachea of the dog, a species to which humans are heavily exposed. We define a phenotype of infection for CIV, which is characterized by high levels of virus replication and extensive tissue damage. CIV was compared to evolutionarily distinct EIVs, and the early EIV isolates showed an impaired ability to infect dog tracheas, while EIVs that circulated near the time of CIV emergence exhibited a CIV-like infection phenotype. Inoculating dog tracheas with various human IAVs (hIAVs) showed that they infected the tracheal epithelium with various efficiencies depending on the virus tested. Finally, we show that reassortant viruses carrying gene segments of CIV and hIAV are viable and that addition of the hemagglutinin (HA) and neuraminidase (NA) of CIV to the 2009 human pandemic virus results in a virus that replicates at high levels and causes significant lesions. This provides important insights into the role of evolution on viral emergence and on the role of HA and NA as determinants of pathogenicity.

Importance: Influenza A viruses (IAVs) have entered new host species in recent history, sometimes with devastating consequences. Canine influenza virus (CIV) H3N8 originated from a direct transfer of an equine influenza virus (EIV) in the early 2000s. We studied the infection patterns of IAVs that circulate in dogs or to which dogs are commonly exposed and showed that CIV emergence was likely caused by an adaptive driver, as evolutionarily distinct EIVs display distinct infection phenotypes. We also showed that many human viruses can infect dog tracheas and that reassortment with CIV results in viable viruses. Finally, we showed that the hemagglutinin and neuraminidase of CIV act as virulence factors. Our findings have significant implications because they show that dogs might act as "mixing vessels" in which novel viruses with pandemic potential could emerge and also provide experimental evidence supporting the role of viral evolution in influenza virus emergence.

FIG 1

Canine tracheal explants are maintained in culture without major histophysiological changes. (A) Representative light micrographs of explant sections maintained in culture for 1, 3, and 5 days. EP, epithelium; LP, lamina propria; SM, submucosa; CA, cartilage. Black horizontal bars represent 200 μm. (B) Higher-magnification micrographs of the epithelial layer of the trachea. Black horizontal bars represent 50 μm. (C) Ciliar activity of explants maintained in culture at different time points. Bars represent averages (± SEMs) of the results of three independent experiments.

FIG 2

Infection of canine tracheal explants with CIV results in a distinct infection phenotype. (A) Histological features of dog tracheas infected with A/canine/New York/51864/2008 (CIV) and mock-infected controls at different times postinfection. Lesions are shown in sections stained with hematoxylin and eosin. Infected cells were detected by immunohistochemical staining of the NP viral protein (white and yellow arrows indicate basal and ciliated cells, respectively). Positive cells are stained brown. Apoptotic cells were detected by immunohistochemical staining of caspase 3. Positive cells are stained brown. (B) Graphical representation of the results of bead clearance assays in infected and control explants. Vertical bars represent average times (± SEMs) to clear the beads in three independent experiments. (C) Growth kinetics of CIV in tracheal explants. Vertical bars represent average titers (± SEMs) from three independent experiments. (D) Immunohistochemical detection of dividing cells (Ki-67). Nuclei of positive cells are stained brown and are indicated by arrows. Black horizontal bars represent 50 μm.

FIG 3

Infection of canine tracheal explants with a 2003 EIV isolate results in a CIV-like infection phenotype. (A) Histological features of dog tracheas infected with A/equine/South Africa/2003 (EIV/03) and mock-infected controls at different times postinfection. Lesions, infected cells, and apoptotic cells are shown as in Fig. 2. Black horizontal bars represent 50 μm. (B) Graphical representation of the results of bead clearance assays in infected and control explants. Vertical bars represent average times (± SEMs) to clear the beads in three independent experiments. (C) Growth kinetics of EIV/03 in tracheal explants. Vertical bars represent average titers (± SEMs) from three independent experiments.

FIG 4

Infection of canine tracheal explants with early EIV isolates results in an attenuated infection phenotype. (A) Histological features of dog tracheas infected with A/equine/South Africa/2003 (EIV/03), A/equine/Miami/1963 (EIV/Miami/63), and A/equine/Uruguay/1963 (EIV/Uruguay/63) and mock-infected controls at different times postinfection. Lesions, infected cells, and apoptotic cells are shown as in Fig. 2. Black horizontal bars represent 50 μm. (B) Graphical representation of the results of bead clearance assays in infected and control explants. Vertical bars represent average times (± SEMs) to clear the beads in three independent experiments. (C) Early EIV isolates display lower replication efficiency than EIV/03. Growth kinetics of EIV/03, EIV/Miami/63, and EIV/Uruguay/63 in tracheal explants. Vertical bars represent average titers (± SEMs) from three independent experiments. Significant differences between the viruses tested are indicated with asterisks (***, P < 0.001).

FIG 5

Human influenza viruses (A/Puerto Rico/8/1934 [PR/8; H1N1] and A/Udorn/1972 [Udorn/72; H3N2]) can readily infect dog tracheas. (A) Histological features of dog tracheas infected with PR/8 and Udorn/72 and mock-infected controls at different times postinfection. Lesions, infected cells (indicated by arrows), and apoptotic cells are shown as in Fig. 2. Black horizontal bars represent 50 μm. (B) Ciliary beating is variably affected in explants infected with different human influenza viruses. Graphical representation of the results of bead clearance assays in infected and control explants. Vertical bars represent average times (± SEMs) to clear the beads in three independent experiments. (C) Human IAVs replicate at levels similar to that of CIV. Growth kinetics of PR/8 and Udorn/72 in tracheal explants. Vertical bars represent average titers (± SEMs) from three independent experiments.

FIG 6

Currently circulating human influenza viruses do not replicate in dog tracheas. (A) Histological features of dog tracheas infected with A/England/195/2009 (H1N1) and A/Perth/16/2009 (H3N2) and mock-infected controls at different times postinfection. Lesions, infected cells (indicated by arrows), and apoptotic cells are shown as in Fig. 2. Black horizontal bars represent 50 μm. (B) Ciliary beating is variably affected in explants infected with different human influenza viruses. Graphical representation of the results of bead clearance assays in infected and control explants. Vertical bars represent average times (± SEMs) to clear the beads in three independent experiments.

FIG 7

Infection phenotypes of CIV_RG and ma-Ca/04/09 in dog tracheal explants. (A) Histological features of dog tracheas infected with CIV_RG and ma-Ca/04/09 and mock-infected controls at different times postinfection. Lesions, infected cells, and apoptotic cells are shown as in Fig. 2. Black horizontal bars represent 50 μm. (B) Ciliary beating is not affected by ma-Ca/04/09. Graphical representation of the results of bead clearance assays in infected and control explants. Vertical bars represent average times (± SEMs) to clear the beads in three independent experiments. (C) CIV_RG and ma-Ca/04/09 replicate at levels similar to that of CIV. Growth kinetics of CIV_RG and ma-Ca/04/09 in tracheal explants. Vertical bars represent average titers (± SEMs) from three independent experiments.

FIG 8

Addition of HA and NA of CIV to ma-Ca/04/09 enhances virulence and viral replication in tracheal explants. (A) Histological features of dog tracheas infected with CIV_RG carrying the NS segment of ma-Ca/04/09 (CIV/NS H1N1), ma-Ca/04/09 carrying the NS segment of CIV (H1N1/NS CIV), and ma-Ca/04/09 carrying the HA and NA of CIV (HA+NA CIV/6 segments H1N1) and mock-infected controls at different times postinfection. Lesions, infected cells, and apoptotic cells are shown as in Fig. 2. Black horizontal bars represent 50 μm. (B) Ciliary beating is variably affected in dog tracheas infected with CIV/H1N1 reassortants. Graphical representation of the results of bead clearance assays in infected and control explants. Vertical bars represent average times (in minutes) (± SEMs) to clear the beads in three independent experiments. (C) A virus carrying the six internal genes of pdmH1N1 and HA and NA of CIV replicates at higher levels than CIV. Growth kinetics of reassortant viruses described above in tracheal explants. Vertical bars represent average titers (± SEMs) from three independent experiments. Significant differences between the viruses tested are indicated with asterisks (**, P < 0.01; ***, P < 0.001).

FIG 9

Addition of HA and NA of CIV to PR/8 enhances virulence and viral replication in tracheal explants. (A) Histological features of dog tracheas infected with PR8 carrying the HA and NA of CIV (HA + NA CIV/6 segments PR8) and mock-infected controls at different times postinfection. Lesions, infected cells, and apoptotic cells are shown as in Fig. 2. Black horizontal bars represent 50 μm. (B) Ciliary beating is variably affected in dog tracheas infected with the CIV/PR8 reassortant. Graphical representation of the results of bead clearance assays in infected and control explants. Vertical bars represent average times (in minutes) (± SEMs) to clear the beads in three independent experiments. (C) A reassortant virus carrying the HA and NA of CIV and the internal gene segments of PR8 replicates at higher levels than CIV. Growth kinetics of reassortant viruses described above in tracheal explants. Vertical bars represent average titers (± SEMs) from three independent experiments.