Middle East respiratory syndrome coronavirus (MERS-CoV) causes transient lower respiratory tract infection in rhesus macaques

Abstract

In 2012, a novel betacoronavirus, designated Middle East respiratory syndrome coronavirus or MERS-CoV and associated with severe respiratory disease in humans, emerged in the Arabian Peninsula. To date, 108 human cases have been reported, including cases of human-to-human transmission. The availability of an animal disease model is essential for understanding pathogenesis and developing effective countermeasures. Upon a combination of intratracheal, ocular, oral, and intranasal inoculation with 7 × 10(6) 50% tissue culture infectious dose of the MERS-CoV isolate HCoV-EMC/2012, rhesus macaques developed a transient lower respiratory tract infection. Clinical signs, virus shedding, virus replication in respiratory tissues, gene expression, and cytokine and chemokine profiles peaked early in infection and decreased over time. MERS-CoV caused a multifocal, mild to marked interstitial pneumonia, with virus replication occurring mainly in alveolar pneumocytes. This tropism of MERS-CoV for the lower respiratory tract may explain the severity of the disease observed in humans and the, up to now, limited human-to-human transmission.

Keywords: DPP4; emerging infectious disease.

Fig. 1.

Hematological changes in rhesus macaques inoculated with MERS-CoV. Total number of white blood cells (Left), neutrophils (Center), and lymphocytes (Right) were determined in blood samples obtained from animals 0, 1, 3, and 6 dpi. Of note, CoV-2, CoV-4, and CoV-6 were euthanized 3 dpi, and thus no samples are available for 6 dpi. Each line represents one animal: CoV-1 (●), CoV-2 (■), CoV-3 (▲), CoV-4 (▼), CoV-5 (♦), CoV-6 (○).

Fig. 2.

Virus shedding in rhesus macaques inoculated with MERS-CoV. Nasal swabs (A) and BAL fluid (B) were collected 1, 3, and 6 dpi. Of note, CoV-2, CoV-4, and CoV-6 were euthanized 3 dpi and thus no samples are available for 6 dpi. RNA was extracted and viral load was determined as TCID₅₀ equivalents by qRT-PCR. TCID₅₀ equivalents were extrapolated from standard curves generated by adding dilutions of RNA extracted from a HCoV-EMC/2012 stock with known virus titer in parallel to each run. Animal numbers are indicated on the x axis. Black bars indicate viral load at 1 dpi, white bars indicate viral load at 3 dpi, and gray bars indicate viral load at 6 dpi.

Fig. 3.

Viral load in respiratory tissues of rhesus macaques inoculated with MERS-CoV. Rhesus macaques were euthanized on day 3 (black bars) and day 6 (white bars) postinfection and tissue samples were collected. RNA was extracted and viral load was determined as TCID₅₀ equivalents by qRT-PCR. TCID₅₀ equivalents were extrapolated from standard curves generated by adding dilutions of RNA extracted from a HCoV-EMC/2012 stock with known virus titer in parallel to each run. Geometric mean viral loads were calculated; error bars represent SD. R, right; L, left; LN, lymph node.

Fig. 4.

Virus replication in the lungs of MERS-CoV inoculated rhesus macaques. Transmission electron microscopy was performed on lung lesion samples collected from MERS-CoV–inoculated macaques at 6 dpi. Virus particles (arrows) are visible in type I pneumocytes (Left and Right). (Scale bar, Right, 250 nm; Inset, 50 nm). B, basement membrane; E, endothelial cell; P, pneumocytes; R, red blood cell.

Fig. 5.

Histopathological changes at 3 dpi in rhesus macaques inoculated with MERS-CoV. Rhesus macaques were euthanized on day 3 postinfection and lung tissue was collected and stained with H&E. (A) Mild acute interstitial pneumonia demonstrated by mild thickening of alveolar septae by small numbers of neutrophils, macrophages, fibrin, and edema. Small numbers of inflammatory cells and fibrin also extend into alveolar spaces. (B) Marked interstitial pneumonia with bronchiolitis obliterans (arrow). Airways are filled with organizing fibrin, necrotic debris, collagen and fibroblasts. There are numerous multinucleate syncytial cells scattered throughout the alveoli (arrowhead). (C) Bronchiolitis obliterans organizing pneumonia. (D) Alveolar capillary (asterisk) bounded by degenerate, vacuolated type I pulmonary epithelium. The alveolar space contains inflammatory cells and a multinucleate syncytial cell (arrowhead). (E) ISH demonstrates viral RNA predominantly within alveolar pneumocytes. (F) Anticoronaviral IHC reveals viral antigen within alveolar pneumocytes. (Magnification: A and B, 200×; C, E, and F, 400×; D, 1,000×.)

Fig. 6.

Histopathological changes at 6 dpi in rhesus macaques inoculated with MERS-CoV. Rhesus macaques were euthanized at 6 dpi and lung tissue was collected and stained with H&E. (A) Mild acute interstitial pneumonia demonstrated by mild thickening of alveolar septae by small numbers of neutrophils, macrophages, fibrin, and edema. Small numbers of inflammatory cells and fibrin also extend into alveolar spaces. (B) Marked interstitial pneumonia with extensive type II pneumocyte hyperplasia (arrow). Alveolar spaces are flooded with edema fluid, fibrin, and inflammatory cells. (C) Type II pneumocyte hyperplasia (arrow) with alveolar flooding edema and fibrin. (D) Hyaline membranes line alveolar space (arrowhead). (E) ISH demonstrates viral RNA predominantly within alveolar pneumocytes. (F) Anticoronaviral IHC reveals viral antigen within alveolar pneumocytes. (Magnification: A and B, 200×; C–F, 400×.)

Fig. 7.

Microarray analysis of gene expression in lungs of rhesus macaques inoculated with MERS-CoV. The heatmap shows log₁₀ ratios to uninfected controls of differentially expressed genes in the right lung lower lobe and lung lesion samples as determined by Welch’s t test (P < 0.01, fold-change ≥ 2), and grouped by hierarchical clustering. A representation is shown of 173 differentially expressed genes from individual animals and sample sites at 3 dpi (A) and of 37 differentially expressed genes from individual animals and sample sites at 6 dpi (B). Functional analysis was performed on subgroups of the day 3 differentially expressed genes, and enriched functional categories identified by IPA are shown in the blue and red vertical bars to the right of the heatmap in A. Differentially expressed genes are specified in Table S2 (3 dpi) and Table S3 (6 dpi).