Tropism of SARS-CoV-2, SARS-CoV, and Influenza Virus in Canine Tissue Explants

Abstract

Background: Human spillovers of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to dogs and the emergence of a highly contagious avian-origin H3N2 canine influenza virus have raised concerns on the role of dogs in the spread of SARS-CoV-2 and their susceptibility to existing human and avian influenza viruses, which might result in further reassortment.

Methods: We systematically studied the replication kinetics of SARS-CoV-2, SARS-CoV, influenza A viruses of H1, H3, H5, H7, and H9 subtypes, and influenza B viruses of Yamagata-like and Victoria-like lineages in ex vivo canine nasal cavity, soft palate, trachea, and lung tissue explant cultures and examined ACE2 and sialic acid (SA) receptor distribution in these tissues.

Results: There was limited productive replication of SARS-CoV-2 in canine nasal cavity and SARS-CoV in canine nasal cavity, soft palate, and lung, with unexpectedly high ACE2 levels in canine nasal cavity and soft palate. Canine tissues were susceptible to a wide range of human and avian influenza viruses, which matched with the abundance of both human and avian SA receptors.

Conclusions: Existence of suitable receptors and tropism for the same tissue foster virus adaptation and reassortment. Continuous surveillance in dog populations should be conducted given the many chances for spillover during outbreaks.

Keywords: COVID-19; SARS-CoV; SARS-CoV-2; dogs; ex vivo; explants; influenza.

Figure 1.

Replication kinetics of SARS-CoV-2 and SARS-CoV in canine tissue explants. Explants were infected with approximately 1 × 10⁶ PFU/mL virus. Virus titers in the culture supernatant at 1, 24, 48, 72, and 96 hpi were determined by TCID₅₀ assay with a detection limit of 1.5 log TCID₅₀/mL, denoted by the dotted lines. Each column shows the replication kinetics per virus strain. Each row displays the results per explant system. Each line color represents data from an individual dog in a single replicate. Experiments were done using tissues from at least 3 different dogs. Abbreviations: hpi, hours postinfection; L, lung; NC, nasal cavity; PFU, plaque-forming unit; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; SP, soft palate; T, trachea; TCID₅₀, 50% tissue culture infection dose.

Figure 2.

Angiotensin-converting enzyme 2 (ACE2) receptor distribution in canine respiratory and soft palate tissues. Immunohistochemical staining for ACE2 (brown) in 10% formalin-fixed paraffin-embedded canine nasal cavity, soft palate, trachea, and lung (bronchioles and alveolus) tissue explants. Scale bars = 50 μm.

Figure 3.

Replication kinetics of IAVs and IBVs in canine tissue explants. A, Explants were infected with 1 × 10⁶ PFU/mL virus. Virus titers in the culture supernatant at 1, 24, 48, and 72 hpi were determined by TCID₅₀ assay with a detection limit of 1.5 log TCID₅₀/mL, denoted by the dotted lines. Each column shows the replication kinetics per virus strain. Each row displays the results per explant system. Each line color represents data from an individual dog in a single replicate. Experiments were done using tissues from at least 3 different dogs. B, AUC above the TCID₅₀ detection limit was calculated from the virus titers at 24 to 72 hpi (mean ± SD). Statistical significance between AUC values of canine H3N2 compared to other viruses in each explant system was analyzed using 1-way ANOVA with Bonferroni posttests. *P ≤ .05, **P ≤ .01, ***P ≤ .001, ****P ≤ .0001. Abbreviations: ANOVA, analysis of variance; AUC, area under the replication kinetic curve; hpi, hours postinfection; IAV, influenza A virus; IBV, influenza B virus; L, lung; NC, nasal cavity; PFU, plaque-forming unit; SP, soft palate; T, trachea; TCID₅₀, 50% tissue culture infection dose.

Figure 4.

SA receptor distribution in canine respiratory and soft palate tissues. Binding (pinkish red) of SNA specific towards α2,6-galactose linked SA, MAAI preferentially towards N-linked or core 2 O-linked glycans containing SAα2,3-Galβ1,4GlcNAc and non-SA glycans containing SO₄-3-Galβ1,4GlcNAc, and MAAII preferentially towards O-linked glycans containing SAα2,3-Galβ1,3GalNAc and non-SA glycans containing SO₄-3-Galβ, in canine nasal cavity, soft palate, trachea, and lung (bronchioles and alveolus) tissue explants. Scale bars = 50 μm. Abbreviations: MAAI, Maackia amurensis lectin I; MAAII, Maackia amurensis lectin II; SA, sialic acid; SNA, Sambucus nigra lectin.