Ocular conjunctival inoculation of SARS-CoV-2 can cause mild COVID-19 in rhesus macaques

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is highly transmitted through the respiratory route, but potential extra-respiratory routes of SARS-CoV-2 transmission remain uncertain. Here we inoculated five rhesus macaques with 1 × 10⁶ TCID₅₀ of SARS-CoV-2 conjunctivally (CJ), intratracheally (IT), and intragastrically (IG). Nasal and throat swabs collected from CJ and IT had detectable viral RNA at 1-7 days post-inoculation (dpi). Viral RNA was detected in anal swabs from only the IT group at 1-7 dpi. Viral RNA was undetectable in tested swabs and tissues after intragastric inoculation. The CJ infected animal had a higher viral load in the nasolacrimal system than the IT infected animal but also showed mild interstitial pneumonia, suggesting distinct virus distributions. This study shows that infection via the conjunctival route is possible in non-human primates; further studies are necessary to compare the relative risk and pathogenesis of infection through these different routes in more detail.

Fig. 1. Graphical outline of the experimental design and sample collection.

Five male rhesus macaques (Macaca mulatta) were inoculated with 10⁶ TCID₅₀/mL SARS-CoV-2. Two rhesus macaques were inoculated via the ocular conjunctival route and named CJ-1 and CJ-2, one was inoculated via the intratracheal route and named IT-1, and two were inoculated via the intragastric route in sequence and named IG-1 and IG-2. The macaques were observed for clinical signs (body weight and temperature were tested as shown). On 0, 1, 3, 5, and 7 dpi, conjunctival, nasal, throat, and anal swabs were collected. CJ-1, IT-1, and IG-1 were euthanized and necropsied at 7 dpi. Tissues were collected to analyse the viral loads and titres. Serum was collected from all animals at 0 and 7 dpi and from CJ-2 and IG-2 at 14 and 21 dpi to examine the specific IgG antibodies against SARS-CoV-2 antigens.

Fig. 2. Clinical features, virus distributions, virus titres, and antibody detection.

Clinical signs, including body weight (a) and temperature (b), were observed. The viral loads of the conjunctival, nasal, throat, and anal swab specimens (c) from the five inoculated macaques collected at 0, 1, 3, 5, and 7 dpi were determined. The viral distribution in the majority of organs and tissues (d) and the virus titre in different lobes of the lungs (e) of CJ-1 and IT-1 collected on 7 dpi were tested and compared. The darker the blue colour is, the higher the viral load is. ELISA was used to detect specific IgG antibody against SARS-CoV-2 at 0, 7, 14, and 21 dpi (f). NC not collected.

Fig. 3. Comparison of lesions in the lungs of CJ-1 and IT-1.

Anterior–posterior and right lateral chest radiographs (a) were collected from rhesus macaques prior to SARS-CoV-2 inoculation (day 0) and at 3, 5, and 7 dpi. Areas of interstitial infiltration, indicative of pneumonia, are highlighted (red circle); obscure costophrenic angles (red arrows) and patchy lesions (blue circle) are also shown. The histopathological and immunohistochemical observations in the lungs are shown (b). Both macaques exhibited interstitial pneumonia with a thickened alveolar septum; the infiltration of inflammatory cells, mainly lymphocytes and macrophages; and some amount of exudation (red arrows) in the alveolar cavities at 7 dpi. Infection via the conjunctival route caused relatively mild pneumonia. Sequential sections were stained by H&E and subjected to IHC. Viral antigens were observed primarily in the alveolar epithelia (black arrows), along with detached degenerative cellular debris (green arrows). H&E-stained sections are shown at ×100 magnification and magnified at ×400 magnification (black frame). The IHC image shows the same field outlined in black at ×400 magnification. Black scale bar = 100 µm, red scale bar = 50 µm. Data (b) are representative of three independent experiments.

Fig. 4. The distinct viral distributions in CJ-1 and IT-1 were consistent with anatomical structure.

Organs and tissues from the nasolacrimal system and conductive system of the respiratory tract, including the lacrimal gland, nasal turbinate, nasal septum, epiglottis, soft palate, and trachea, were examined. Sequential sections were stained with H&E and subjected to IHC. Fields in the H&E images outlined in black are magnified. The IHC images show the same field outlined in black at ×400 magnification. Black scale bar = 100 µm, red scale bar = 50 µm. Data are representative of three independent experiments.