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

SARS-CoV-2 replication and distributions

Five rhesus macaques between the ages of 3 and 5 years were inoculated with 1 × 10⁶ 50% tissue-culture infectious doses (TCID₅₀) of SARS-CoV-2 via three routes: ocular conjunctival inoculation (CJ-1 and CJ-2), intragastric inoculation (IG-1 and IG-2), and intratracheal inoculation (IT-1). IT-1 was used to compare the distribution and pathogenesis of the virus after entering the host via different routes (Fig. 1). The low number of non-human primates and the single tested dose is a limitation of the study and that therefore the risk of ocular exposure in comparison to other transmission routes of infection cannot be assessed.

We observed the macaques daily for clinical signs. There was no significant change in body weight (Fig. 2a) or temperature (Fig. 2b) in any of the inoculated macaques. Routine specimens, including nasal and throat swabs, were collected at 0, 1, 3, 5, and 7 days post-inoculation (dpi). Additionally, to explore the potential excretory routes of SARS-CoV-2 in the host, conjunctival and anal swabs were also gathered. Specifically, a continuous detectable viral load was observed in nasal and throat swabs collected from CJ- and IT-inoculated animals from 1 to 7 dpi. In contrast, the virus was not detected in the swabs collected from the IG-inoculated macaques. Notably, only with infection via the conjunctival route could the viral load be detected in conjunctival swabs (average, approximately 4.33 log₁₀ RNA copies/mL) collected on 1 dpi, after which the viral load became undetectable. Furthermore, only with infection via the intratracheal route could the viral load be continuously detected in anal swabs collected from 1 to 7 dpi, with the viral load peaking at approximately 6.76 log₁₀ RNA copies/mL on 5 dpi, revealing the distinct excretory pathways of the host after inoculation via different routes (Fig. 2c).

To determine the distribution of virus and evaluate histological lesions, CJ-1, IT-1, and IG-1 were euthanized and necropsied at 7 dpi. For CJ-1, the viral load was primarily distributed in the nasolacrimal and ocular system (1.01–3.93 log₁₀ RNA copies/mL), including the lacrimal gland, optic nerve, and conjunctiva; nose (1.03–6.63 log₁₀ RNA copies/mL), including the nasal mucosa, nasal turbinate, and nostril; pharynx (4.57–5.60 log₁₀ RNA copies/mL), including the epiglottis and soft palate; oral cavity, including the cheek pouch and parotid gland; and other tissues, including the lower left lobe of the lung (4.6 log₁₀ RNA copies/mL), tonsil, inguinal and pararectal lymph nodes, stomach, duodenum, ileum, and caecum (Fig. 1d). In contrast, for IT-1, the distribution of the virus was somewhat different because viral replication was high in the different lobes of the lung (4.22–7.81 log₁₀ RNA copies/mL), and the viral load was also widely detected in the nasal septum (4.69 log₁₀ RNA copies/mL); trachea (6.61 log₁₀ RNA copies/mL); lymph organs and tissues (3.39–5.17 log₁₀ RNA copies/mL), including the mandibular lymph nodes, tonsils, and pulmonary lymph nodes; and some segments of the alimentary tract (1.11–4.93 log₁₀ RNA copies/mL), including the duodenum, ileum, colon, and caecum (Fig. 2d). There was no detectable viral load in the tissues collected from IG-1 (Supplementary Table 1). Furthermore, to evaluate the infectious virus titre in the lung at 7 dpi, virus collected from different lobes of the lungs of CJ-1 and IT-1 was inoculated onto Vero E6 cells for virus isolation. For CJ-1, SARS-CoV-2 was isolated from only the left lower lobe of the lung (2.67 log₁₀ TCID₅₀/mL). For IT-1, the virus was isolated from more lobes of the lung (2.00–5.67 log₁₀ TCID₅₀/mL), most of which showed higher virus titres than those in CJ-1. These were the left lower lobe, right lower lobe, left middle lobe, right middle lobe, right accessory lobe, left upper lobe, and right upper lobe of the lung (Fig. 2e). The distinct distributions of the virus inoculated by different routes were consistent with the anatomical structure, suggesting that the distribution of SARS-CoV-2 in the host is associated with the infection route. Furthermore, the virus was detectable in different segments of the alimentary tract and some lymphatic tissues in animals inoculated via both routes, revealing that these tissues may play important roles in the spread of the virus within the host⁸. Furthermore, specific IgG antibody against SARS-CoV-2 was detectable in CJ-2 at 14 and 21 dpi but not before infection, demonstrating that the animal had been infected with SARS-CoV-2 (Fig. 2f).

Chest X-ray and histopathological changes

Meanwhile, to observe the progressive pulmonary infiltration characteristic of SARS-CoV-2-related pneumonia, chest radiographs of the inoculated animals were recorded every other day after inoculation. Beginning at 3 dpi, abnormalities with varying degrees of severity appeared in the lungs. Specifically, for the CJ-inoculated animal, unlike the results collected before infection (day 0), the left upper lobe of the lung was presented opaque glass sign on 3 dpi, which then developed in the bilateral upper lobes on 5 dpi. Obscure lung markings and an opaque glass in the bilateral lobes of the lung were observed at 7 dpi. In contrast, the IT-inoculated animals developed relatively severe progressive pulmonary infiltration from 3 to 7 dpi. The right upper lobe of the lung exhibited an increased density and was obscure at 3 dpi. The right lower lobe of the lung presented obscure lung markings and lamellar ground-glass opacities at 5 dpi. Increased radiographic changes showing patchy lesions in the right upper lobe of the lung, obscure lung markings, and marked ground-glass opacities with a blurred right diaphragm were observed in the bilateral lobes of the lung at 7 dpi (Fig. 3a). Consistent with these radiographic alterations, upon microscopy examination, local lesions in the lungs of CJ-1 displayed mild interstitial pneumonia characterized by a thickened alveolar interstitium; the infiltration of inflammatory cells, primarily lymphocytes and monocytes; and a small amount of exudation in the alveolar cavities. IT-1 developed moderate and diffuse interstitial pneumonia characterized by a more thickened alveolar interstitium and more serious inflammation and exudation (Fig. 3b)⁹. Virus antigen was further confirmed by immunohistochemistry (IHC) staining with SARS-CoV-2-specific antibody. In the damaged lobes of the lungs of both CJ-1 and IT-1, SARS-CoV-2 was predominantly observed in the alveolar epithelium and exfoliated degenerative cellular debris in the alveolar cavities (Fig. 3b). Notably, the IHC results were highly consistent with the viral load detection data at 7 dpi collected from both CJ-1 and IT-1. Specifically, viral antigen was scattered in several cells in the nasolacrimal system via conjunctival inoculation but more prominent in the trachea following intratracheal inoculation (Fig. 4). Moreover, viral antigen was clearly observed in the lamina propria of the alimentary tract, suggesting that the virus can spread from the initial point of entry to gut-associated lymphoid tissue (Supplementary Fig. 1) and detected at low levels in the kidney, myocardium, and liver of IT-1 but was undetectable in CJ-1 (Supplementary Fig. 2). No substantial histopathological changes were observed in IG-1 (Supplementary Fig. 3). Briefly, the CJ-inoculated animal showed evidence of relatively mild and local interstitial pneumonia, unlike the IT-inoculated macaque, which demonstrated moderate, diffuse lesions in the lung accompanied by the increased infiltration of inflammatory cells and accumulation of exudation in the alveolar cavities.

These data demonstrate that macaques can be infected with SARS-CoV-2 via the conjunctival and intratracheal routes but not the intragastric route. Compared to the intratracheal route, with the conjunctival route, the viral load in the nasolacrimal system was higher, and the lesions in the lung were milder and local.

Ethics statement

The animal biosafety level 3 (ABSL3) facility at the Institute of Laboratory Animal Science was used to complete all the experiments with rhesus macaques. All research was performed in compliance with the Animal Welfare Act and other regulations relating to animals and experiments. The Institutional Animal Care and Use Committee of the Institute of Laboratory Animal Science, Peking Union Medical College, reviewed and authorized all the protocols in this research, including research done in animals (BLL20009).

Cells and viruses

A stock of the SARS-CoV-2 virus (SARS-CoV-2/WH-09/human/2020/CHN, accession No. MT093631.2)¹⁶ was used in this study and cultivated in Vero E6 cells maintained in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen, Carlsbad, USA) supplemented with 10% foetal bovine serum (FBS) and incubated at 37 °C and 5% CO₂ (ref. ¹⁶).

RNA extraction and quantitative RT-PCR

Total RNA was extracted from all of the collected organs^13,17 using the RNeasy Mini Kit (Qiagen, Hilden, Germany), and RT was performed with the PrimerScript RT Reagent Kit (TaKaRa, Japan) following the manufacturer’s instructions. Quantitative real-time RT-PCR was performed using the PowerUp SYBR Green Master Mix Kit (Applied Biosystems, USA), and samples were processed in duplicate with the following cycling protocol: 50 °C for 2 min; 95 °C for 2 min; 40 cycles of 95 °C for 15 s, 60 °C for 30 s, and 95 °C for 15 s; 60 °C for 1 min; and 95 °C for 45 s. The primer sequences used for RT-PCR targeted the envelope (E) gene of SARS-CoV-2 and are as follows: forward: 5′-TCGTTTCGGAAGAGACAGGT-3′, reverse: 5′-GCGCAGTAAGGATGGCTAGT-3′. The PCR products were verified by sequencing with the dideoxy method on an ABI 3730 DNA sequencer (Applied Biosystems, CA, USA). During the sequencing process, amplification was performed with specific primers. The sequences of the primers used for this process are available upon request. The obtained sequencing reads were linked with DNAMAN, and the results were compared with the MEGALIGN module in the DNAStar software package.

Animal experiments

Five male rhesus macaques (Macaca mulatta) between the ages of 3 and 5 years were used in this research. All of them were negative for tuberculosis and simian immunodeficiency virus. They were inoculated with 1 × 10⁶ TCID₅₀ of SARS-CoV-2 via three routes. Two male rhesus macaques (named CJ-1 and CJ-2) were inoculated with 10⁶ TCID₅₀/mL SARS-CoV-2 via the ocular conjunctival route, one (named IT-1) was inoculated via the intratracheal route, and two (named IG-1 and IG-2) were inoculated via the intragastric route in sequence. Prior to sample collection, all animals were anaesthetised with ketamine hydrochloride (10 mg/kg). On 0, 1, 3, 5, and 7 dpi, conjunctival, nasal, throat, and anal swabs were collected and incubated in 1 mL of DMEM containing 50 µg/mL streptomycin and 50 U/mL penicillin. CJ-1, IT-1, and IG-1 were euthanized and necropsied at 7 dpi. Tissue samples were collected from the following organs to detect the viral load to analyse the distribution of the virus: the conjunctiva, lacrimal gland, optic nerve, cerebellum, cerebrum, different segments of the spinal cord, nostril, nasal turbinate, nasal mucosa, nasal septum, soft palate, cheek pouch, parotid gland, epiglottis, lingual tonsil, pharyngeal tonsil, different lobes of the lung, trachea, different lymph nodes, heart, liver, spleen, pancreas, different segments of the alimentary tract, kidney, bladder, testis, and brown adipose tissue. Serum was collected from all animals at 0 and 7 dpi and from CJ-2 and IG-2 at 14 and 21 dpi for serological detection to examine specific IgG antibodies against the SARS-CoV-2 antigen.

Preparation of homogenate supernatant

An electric homogenizer was applied to prepare tissue homogenates by incubation in 1 mL of DMEM for 2.5 min. The homogenates were centrifuged at 825 × g at 4 °C for 10 min. The supernatants were harvested and stored at −80 °C to assess the v titre. To evaluate the infectious virus titre, homogenates were prepared from different lobes of the lungs from CJ-1 and IT-1 for virus titration analysis by endpoint titration in Vero E6 cells. The virus titres of the supernatants were determined using a standard TCID₅₀ assay¹⁷.

TCID₅₀ assay

The TCID₅₀ assay was performed as following¹⁸, briefly, to measure the SARS-CoV-2 titres, 10-fold serial dilutions of the virus were used to inoculate Vero cell monolayers in DMEM containing 2% FBS, which were incubated at 37 °C for 4 days. Then, the cytopathic effect was observed, and the TCID₅₀ values were calculated by the Reed and Muench method¹⁹.

Enzyme-linked immunosorbent assay (ELISA) to detect antibodies

Serum was collected from each experimental animal at 0, 7, 14, and 21 dpi to detect SARS-CoV-2 antibodies through ELISA. Ninety-six-well plates that had been coated with 0.1 μg of the spike protein of SARS-CoV-2 (Sino Biological, 40591-V08H) and incubated at 4 °C overnight were blocked with 2% BSA/PBST at room temperature for 1 h. Serum samples were diluted 1:100, added to different wells, and maintained at 37 °C for 30 min, followed by incubation with goat anti-monkey antibody labelled with horseradish peroxidase (Abcam, ab112767, 1:10,000) at room temperature for 30 min. The absorbance at 450 nm was then determined.

Haematoxylin and eosin (H&E) staining

A ten percent buffered formalin solution was used to fix all the collected organs, and paraffin sections (3–4 µm in thickness) were prepared according to routine practice. All the tissue sections were stained with H&E. The histopathological changes in different tissues were observed under an Olympus microscope.

Immunohistochemistry

A ten percent buffered formalin solution was used to fix all the collected organs, and paraffin sections (3–4 µm in thickness) were prepared routinely as described in a previous report. Briefly, reagent from an antigen retrieval kit (Boster, AR0022) was applied to the sections at 37 °C for 1 min. A three percent solution of H₂O₂ in methanol was used to quench endogenous peroxidases for 10 min. The slices were incubated at 4 °C overnight with a laboratory-prepared 7D2 monoclonal antibody (1:200) after blocking in 1% normal goat serum. Horseradish peroxidase (HRP)-labelled goat anti-mouse IgG secondary antibody (Beijing ZSGB Biotechnology, ZDR-5307, 1:200) was added and maintained at 37 °C for 60 min. The slices were treated with 3,3′-diaminobenzidine tetrahydrochloride for visualization. The sections were counterstained with haematoxylin, dehydrated, mounted on a slide, and observed with an Olympus microscope. The sequential sections from all collected tissues were directly incubated with HRP-labelled goat anti-mouse IgG and used as the omission control for viral antigen staining. The sequential sections from all collected tissues were incubated with a recombinant anti-mouse IgG antibody [RM104] (Abcam, ab190475, 1:1000) as the negative control for the expression of viral antigen.

Statistical analysis

The collected data were analysed with GraphPad Prism 8.0 software (GraphPad Software Inc., San Diego, CA).

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.