Respiratory disease and virus shedding in rhesus macaques inoculated with SARS-CoV-2

Abstract

An outbreak of a novel coronavirus, now named SARS-CoV-2, causing respiratory disease and a ~2% case fatality rate started in Wuhan, China in December 2019. Following unprecedented rapid global spread, the World Health Organization declared COVID-19 a pandemic on March 11, 2020. Although data on disease in humans are emerging at a steady pace, certain aspects of the pathogenesis of SARS-CoV-2 can only be studied in detail in animal models, where repeated sampling and tissue collection is possible. Here, we show that SARS-CoV-2 causes respiratory disease in infected rhesus macaques, with disease lasting 8-16 days. Pulmonary infiltrates, a hallmark of human disease, were visible in lung radiographs of all animals. High viral loads were detected in swabs from the nose and throat of all animals as well as in bronchoalveolar lavages; in one animal we observed prolonged rectal shedding. Taken together, the rhesus macaque recapitulates moderate disease observed in the majority of human cases. The establishment of the rhesus macaque as a model of COVID-19 will increase our understanding of the pathogenesis of this disease and will aid development and testing of medical countermeasures.

Figure 1.. Rhesus macaques infected with SARS-CoV-2 develop respiratory disease.

Eight adult rhesus macaques were inoculated with SARS-CoV-2 isolate nCoV-WA1–2020. After inoculation, animals were observed for disease signs and scored according to a pre-established clinical scoring sheet (a). On days when clinical exams were performed, body weight (b), and body temperature (c) were measured. Respiration rate was measured, and breathing pattern was recorded, with irregular respiration patterns indicated in red (d). Ventro-dorsal and lateral radiographs were taken on clinical exam days and scored for the presence of pulmonary infiltrates by a clinical veterinarian according to a standard scoring system (0: normal; 1: mild interstitial pulmonary infiltrates; 2: moderate pulmonary infiltrates perhaps with partial cardiac border effacement and small areas of pulmonary consolidation; 3: severe interstitial infiltrates, large areas of pulmonary consolidation, alveolar patterns and air bronchograms). Individual lobes were scored and scores per animal per day were totaled and displayed (e). Grey: data derived from animals euthanized on 3 dpi; black: data derived from animals euthanized on 21 dpi. Identical symbols have been used to denote identical animals throughout the figures in this manuscript.

Figure 2.. Pulmonary infiltrates in a rhesus macaque after inoculation.

Radiographs show the progression of pulmonary infiltrates throughout the study in a single animal. Of note, this animal is denoted with a black triangle throughout the manuscript. Circles indicate areas of mild to moderate pulmonary infiltrates. A marker ® indicates right side of the animal.

Figure 3.. Viral loads in respiratory samples and bodily fluids.

Eight adult rhesus macaques were inoculated with SARS-CoV-2 isolate nCoV-WA1–2020. After inoculation, clinical exams were performed during which nose, throat, rectal and urogenital swabs were collected; viral loads in these samples were determined by qRT-PCR (a). On 1, 3, and 5 dpi, bronchoalveolar lavages were performed on the 4 animals remaining in the study through 21 dpi; viral loads and virus titers were determined in these samples. Viral loads were also determined in blood collected during clinical exams (c) and in urine collected at necropsy on 3 and 21 (d). Grey: data derived from animals euthanized on 3 dpi; black: data derived from animals euthanized on 21 dpi; red: virus was isolated from these samples. Identical symbols have been used to denote identical animals throughout the figures in this manuscript.

Figure 4.. Pathological changes in rhesus macaques infected with SARS-CoV-2.

Eight adult rhesus macaques were inoculated with SARS-CoV-2 isolate nCoV-WA1–2020. Four animals were euthanized on 3 dpi, and 4 animals on 21 dpi. Upon gross examination, lungs showed focal areas of hilar consolidation and hyperemia (circles) on 3 dpi (a) and multifocal, random consolidation and hyperemia (circles) on 21 dpi (b). At necropsy, the percentage of the area of the lungs affected by gross lesions was estimated by a board-certified veterinary pathologist (c), and lung weight to bodyweight ratio was calculated as a measure for pulmonary edema. (d). The dotted line represents baseline ratio calculated from an in-house collection of rhesus macaque lung and bodyweights from animals with grossly normal lungs. Histological analysis was performed on tissues collected at 3 dpi (e-i). (e) Pulmonary vessels are surrounded by moderate numbers of inflammatory cells (arrows) Magnification 100x. (f) Alveoli are filled with small to moderate numbers of inflammatory cells (asterisks). Adjacent alveolar interstitium (arrows) is thickened by edema, fibrin and inflammatory cells. Magnification 400x. (g) SARS-CoV-2 antigen is detected by immunohistochemistry in type I pneumocytes. Magnification 400x. (h) Pulmonary vessels are bounded by inflammatory cells (arrowhead) and hyaline membranes (arrows) line alveolar spaces. Magnification 100x. (i) Hyaline membranes line alveoli (arrows). Magnification 400x. (j) SARS-CoV-2 antigen is detected by immunohistochemistry in type I pneumocytes (asterisk) and type II pneumocytes (arrow) as well as alveolar macrophages (arrowheads). Magnification 400x. (k) SARS-CoV-2 antigen is detected by immunohistochemistry in mediastinal lymph node. Magnification 400x. (l) SARS-CoV-2 antigen is detected by immunohistochemistry in macrophages and lymphocytes in the lamina propria of the cecum. Magnification 400x.

Figure 5.. Ultrastructural analysis of lungs of rhesus macaques infected with SARS-CoV-2.

Lung tissue collected on 3 dpi was analyzed by transmission electron microscopy. The alveolar interstitium is expanded by edema (E), fibrin (F) and mononuclear (M) inflammatory cells (a). Normal collagen fibers (c) and multiple virions (arrowheads) line type I pneumocytes (arrows). Boxes in (a) indicate areas enlarged in (b-d). Scale bar in (a) represents 2μm, scale bars in (b-e) represent 0.2 μm.

Figure 6.. Viral loads in tissues collected from rhesus macaques infected with SARS-CoV-2.

Four adult rhesus macaques were inoculated with SARS-CoV-2 isolate nCoV-WA1–2020 and euthanized on 3 dpi. Thirty-seven tissues were collected at necropsy and analyzed for the presence of viral RNA by qRT-PCR. Tissues are grouped by lung lobes (a), with red symbols indicating tissues from which virus could be isolated in Vero E6 cells; other tissues from the respiratory tract (b); lymphoid tissues (c); gastrointestinal tissues (d); the central nervous system (e) and remaining tissues (f). Blue symbols in b-e indicate that viral mRNA was also detected in these tissues. Identical symbols have been used to denote identical animals throughout the figures in this manuscript. R: right; L: left; LN: lymph node.