Influenza A viruses are transmitted via the air from the nasal respiratory epithelium of ferrets

Abstract

Human influenza A viruses are known to be transmitted via the air from person to person. It is unknown from which anatomical site of the respiratory tract influenza A virus transmission occurs. Here, pairs of genetically tagged and untagged influenza A/H1N1, A/H3N2 and A/H5N1 viruses that are transmissible via the air are used to co-infect donor ferrets via the intranasal and intratracheal routes to cause an upper and lower respiratory tract infection, respectively. In all transmission cases, we observe that the viruses in the recipient ferrets are of the same genotype as the viruses inoculated intranasally, demonstrating that they are expelled from the upper respiratory tract of ferrets rather than from trachea or the lower airways. Moreover, influenza A viruses that are transmissible via the air preferentially infect ferret and human nasal respiratory epithelium. These results indicate that virus replication in the upper respiratory tract, the nasal respiratory epithelium in particular, of donors is a driver for transmission of influenza A viruses via the air.

Fig. 1. The A/H1N1 virus was transmitted from the upper respiratory tract of ferrets.

Donor ferrets 1 and 2 were inoculated intranasally with 10⁵ TCID₅₀ of the A/H1N1 virus (shown in red) and intratracheally with 10⁵ TCID₅₀ of the A/H1N1_var virus (shown in blue). Donor ferrets 3 and 4 were inoculated with the opposite placement of viruses. Recipient ferrets were added to the adjacent cage at 4 hpi. Virus titers in the nose and throat swabs were determined by TCID₅₀ assay and are indicated on the y-axis. The limit of detection of the virus titration is shown by the dotted line. The proportions of untagged (red) and tagged (blue) viruses, as determined by next-generation sequencing on the PB2 gene, are indicated by the colored bars. The gray bars correspond to samples that were not included in the next-generation sequencing. Source data are provided as a Source Data file.

Fig. 2. A/H1N1, A/H3N2 and A/H5N1_AT viruses were transmitted from the upper respiratory tract of ferrets.

Ferrets were inoculated with 10⁵ TCID₅₀ of untagged (shown in red) and tagged (shown in blue) virus pairs of A/H1N1 virus (a Donors 5–8), A/H3N2 virus (b Donors 9–12) or A/H5N1_AT virus (c Donors 13–16). Untagged and tagged viruses were inoculated either intranasally or intratracheally as indicated by the color coding in the schematic ferret representations. Untagged and tagged virus proportions at the day of virus transmission are represented by the pie charts for both donor and recipient ferrets. URT: upper respiratory tract, i.e., nasal turbinates, LRT: lower respiratory tract, i.e., combined data from two parts of the trachea and the lungs, N: nose swabs, T: throat swabs. X means that no transmission was observed. Transmission was defined by the detection of two consecutive swabs with a RT-qPCR threshold (CT-value) of 35. Source data are provided as a Source Data file.

Fig. 3. Airborne transmissible influenza A viruses infected the nasal respiratory epithelium of ferrets.

a Representative pictures of ferret nasal respiratory and nasal olfactory epithelia 2 days after intranasal inoculation with A/H1N1, A/H3N2, A/H5N1_AT or A/H5N1 viruses. Influenza A virus nucleoprotein expression was determined by immunohistochemistry (IHC) and is shown as a red stain. HE: hematoxylin-eosin stain. Scale bar 50 μm. b Percentage of epithelium that was nucleoprotein antigen-positive, as determined by IHC, were blindly assessed in the nasal respiratory epithelium (black) and nasal olfactory epithelium (light gray) of three ferrets inoculated with the respective viruses. Individual percentages are shown. Means are depicted by the horizontal lines. c Individual virus titers in the homogenized nasal turbinates (containing both nasal respiratory and olfactory epithelia) were determined by end-point titration in MDCK. Means are depicted by the horizontal lines. The limit of detection of the virus titration is shown by the dotted line. Source data are provided as a Source Data file.

Fig. 4. Airborne transmissible influenza A viruses infected the human respiratory epithelium.

Representative pictures of primary cultures of human nasal respiratory epithelium (Mucilair^TM) inoculated with A/H1N1, A/H3N2, A/H5N1_AT, A/H5N1 viruses or PBS (Mock). Influenza A virus nucleoprotein expression was determined by immunohistochemistry (IHC) and is shown as a red stain. Scale bar 100 μm.