Intranasal Infection of Ferrets with SARS-CoV-2 as a Model for Asymptomatic Human Infection

Abstract

Ferrets were experimentally inoculated with SARS-CoV-2 (severe acute respiratory syndrome (SARS)-related coronavirus 2) to assess infection dynamics and host response. During the resulting subclinical infection, viral RNA was monitored between 2 and 21 days post-inoculation (dpi), and reached a peak in the upper respiratory cavity between 4 and 6 dpi. Viral genomic sequence analysis in samples from three animals identified the Y453F nucleotide substitution relative to the inoculum. Viral RNA was also detected in environmental samples, specifically in swabs of ferret fur. Microscopy analysis revealed viral protein and RNA in upper respiratory tract tissues, notably in cells of the respiratory and olfactory mucosae of the nasal turbinates, including olfactory neuronal cells. Antibody responses to the spike and nucleoprotein were detected from 21 dpi, but virus-neutralizing activity was low. A second intranasal inoculation (re-exposure) of two ferrets after a 17-day interval did not produce re-initiation of viral RNA shedding, but did amplify the humoral response in one animal. Therefore, ferrets can be experimentally infected with SARS-CoV-2 to model human asymptomatic infection.

Keywords: SARS-CoV-2; Y453F; ferret; olfactory neuronal cells.

Figure 1

Quantification of viral RNA in clinical samples. Viral RNA quantified in (A) nasal wash and (B) throat swab samples is shown as relative equivalent units (REU) based on a viral RNA standard dilution series prepared from a stock of known titre/mL. Grey shading denotes the area below the LLoQ. Vertical lines indicate the days of inoculation and re-challenge. Viral RNA (C) was also quantified in tissues from 3, 5 and 7 days post-inoculation (dpi). Presence of infectious virus was verified by re-isolation in selected samples, as indicated by a circle or asterisk (*). Abbreviations: LLoQ, lower limit of quantification; BAL, bronchoalveolar lavage; Resp., respiratory; LN, lymph node; TB, tracheobronchial.

Figure 2

Detection of severe acute respiratory syndrome (SARS)-related coronavirus (SARS-CoV-2) in the respiratory and olfactory mucosa of infected ferrets by immunohistochemistry (IHC) and in situ hybridisation (ISH). IHC labelling detected spike antigen (a,d) in the apical cytoplasm of epithelial cells (black arrow), whereas nucleoprotein labelling (b,e) was ubiquitous throughout the cytoplasm. In the olfactory mucosa (e), nucleoprotein was present in various cell populations identified by morphology, including sustentacular cells (green arrowhead), olfactory neuronal cells (black arrowhead) and olfactory nerve fibres (red arrowhead). S gene ISH also revealed the presence of viral RNA in both the respiratory (c) and olfactory mucosa (f), and in the latter case, labelling was again identified in the sustentacular cells (green arrowhead) and olfactory neuronal cells (black arrowhead). Images taken with 400× objective.

Figure 3

Detection of viral RNA on ferret fur. Diagram delineating in red (A) the sampling location for the ferret fur along the dorsal midline and (B) the quantification of viral RNA detected on the fur of both groups of animals on the indicated days post-infection (dpi). The shading from light blue to dark blue indicates a greater amount of viral RNA detected (log₁₀ relative equivalent units (REU)/mL).

Figure 4

Ferret antibody response. Specific antibody elicited to (A) the spike (S1) glycoprotein or (B) nucleoprotein (NP) was detected in samples taken from two ferrets on 21 and on 24 dpi. Each data point represents a sample from one of eight ferrets pre- and post-inoculation. The graphs show the mean OD 450 nm values from two independent ELISA assays (which did not significantly differ). Neutralizing antibody (C) was detected in samples obtained from single ferrets on 21 and 24 dpi. The graph shows the log₂ box-and-whisker values from two independent virus-neutralizing titre (VNT) determinations.