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. 2016 Mar;186(3):630-8.
doi: 10.1016/j.ajpath.2015.10.025. Epub 2015 Dec 24.

An Acute Immune Response to Middle East Respiratory Syndrome Coronavirus Replication Contributes to Viral Pathogenicity

Laura J Baseler  1 , Darryl Falzarano  2 , Dana P Scott  3 , Rebecca Rosenke  3 , Tina Thomas  2 , Vincent J Munster  2 , Heinz Feldmann  2 , Emmie de Wit  4
Affiliations

An Acute Immune Response to Middle East Respiratory Syndrome Coronavirus Replication Contributes to Viral Pathogenicity

Laura J Baseler et al. Am J Pathol. 2016 Mar.

Abstract

Middle East respiratory syndrome coronavirus (MERS-CoV) was first identified in a human with severe pneumonia in 2012. Since then, infections have been detected in >1500 individuals, with disease severity ranging from asymptomatic to severe, fatal pneumonia. To elucidate the pathogenesis of this virus and investigate mechanisms underlying disease severity variation in the absence of autopsy data, a rhesus macaque and common marmoset model of MERS-CoV disease were analyzed. Rhesus macaques developed mild disease, and common marmosets exhibited moderate to severe, potentially lethal, disease. Both nonhuman primate species exhibited respiratory clinical signs after inoculation, which were more severe and of longer duration in the marmosets, and developed bronchointerstitial pneumonia. In marmosets, the pneumonia was more extensive, with development of severe airway lesions. Quantitative analysis showed significantly higher levels of pulmonary neutrophil infiltration and higher amounts of pulmonary viral antigen in marmosets. Pulmonary expression of the MERS-CoV receptor, dipeptidyl peptidase 4, was similar in marmosets and macaques. These results suggest that increased virus replication and the local immune response to MERS-CoV infection likely play a role in pulmonary pathology severity. Together, the rhesus macaque and common marmoset models of MERS-CoV span the wide range of disease severity reported in MERS-CoV-infected humans, which will aid in investigating MERS-CoV disease pathogenesis.

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Figures

Figure 1
Figure 1
Middle East respiratory syndrome coronavirus–inoculated nonhuman primates develop bronchointerstitial pneumonia that is histologically similar in character, but is more extensive, in common marmosets. A–D: Representative sections of lung from a rhesus macaque (A and C) and common marmoset (B and D) euthanized 3 days after inoculation. A: Unaffected pulmonary tissue (asterisk) adjacent to a focus of bronchointerstitial pneumonia. B: The lung is diffusely affected by bronchointerstitial pneumonia. C and D: The microscopic features of the bronchointerstitial pneumonia are similar in rhesus macaques and common marmosets. Alveolar septa and lumina are predominantly infiltrated by neutrophils and macrophages mixed with fibrin, hemorrhage, and edema. Hematoxylin and eosin staining was used. Original magnifications: ×4 (A and B); ×40 (C and D).
Figure 2
Figure 2
A mixed population of multinucleated cells are widely scattered throughout the bronchointerstitial pneumonia in rhesus macaques (A and C) and common marmosets (B and D). A and B: Immunohistochemistry (IHC) for Iba1 in sections of lung. Most of the multinucleated cells express Iba1 (black arrows), indicating the cells are of macrophage origin. Insets: Multinucleated cells that are not macrophages (red arrows), as indicated by their lack of Iba1 expression. C and D: IHC for pan cytokeratin in sections of lung. Most of the multinucleated cells are not of epithelial origin and do not express pan cytokeratin (black arrows). Insets: Multinucleated cells expressing pan cytokeratin (red arrows), indicating the cells are of epithelial origin. Original magnification, ×40 (main images and insets).
Figure 3
Figure 3
Middle East respiratory syndrome coronavirus–inoculated common marmosets develop more severe airway lesions than rhesus macaques. A: Respiratory epithelium in a bronchus exhibits focal loss of cilia (arrow) in a macaque 3 days after inoculation (dpi). Rare inflammatory cells are present in the bronchial lumen. B: Respiratory epithelial cells in a bronchus are eroded and attenuated (arrows) in a marmoset 3 dpi. Neutrophils and foamy macrophages infiltrate the bronchial wall and mix with edema and hemorrhage in the bronchial lumen. C: Neutrophils and foamy macrophages with minimal edema, hemorrhage, and fibrin are present in the wall and lumen of a bronchiole in a macaque 3 dpi. D: A bronchiole is occluded by a mat of fibrin (asterisk) mixed with edema, hemorrhage, and degenerate leukocytes in a marmoset 4 dpi. Hematoxylin and eosin staining was used. Original magnification, ×40 (A–D).
Figure 4
Figure 4
Common marmoset lungs contain more Middle East respiratory syndrome coronavirus (MERS-CoV) antigen than rhesus macaque lungs. A and B: Immunohistochemistry (IHC) for MERS-CoV antigen (labeled brown) in sections of lung from nonhuman primates necropsied 3 days after inoculation (dpi). Lower amounts of viral antigen are present in macaques (A) than marmosets (B). At higher magnification, viral antigen is seen in pneumocytes (left insets) and in macrophages (right insets). C and D: IHC for pan cytokeratin (labeled red) and MERS-CoV antigen (labeled brown) in the lung from a rhesus macaque necropsied 3 dpi. C: Viral antigen is present in the cytoplasm of a pneumocyte (arrow), as identified by the morphology of the cell and its expression of pan cytokeratin. D: Viral antigen is shown in a macrophage (arrow), as identified by its cellular morphology and lack of pan cytokeratin expression. E: The percentage of the lung containing MERS-CoV antigen is higher in common marmosets at both 3 and 6 dpi, as determined by digital analysis using ImageScope. Statistics could not be performed for the 6 dpi data because there were only two animals per time point. F: Pulmonary viral RNA loads are significantly higher in marmosets at both 3 and 6 dpi. ∗P < 0.05, ∗∗∗∗P < 0.0001 for rhesus macaques versus common marmosets. Original magnifications: ×20 (A and B); ×40 (insets, C and D). TCID50, 50% tissue culture infectious dose.
Figure 5
Figure 5
Quantification of inflammatory cells in the lung indicates that marmosets (white bars) exhibit higher pulmonary inflammatory cell infiltration at both 3 and 6 days after inoculation (dpi) compared with rhesus macaques (black bars). A and B: Immunohistochemistry for myeloperoxidase, a marker for neutrophils, in lung sections at 3 dpi. C: The percentage of the lung infiltrated by neutrophils is significantly higher in marmosets at 3 dpi. No statistically significant differences are noted for pulmonary infiltration by T lymphocytes (D–F), B lymphocytes (G–I), or macrophages (J–L) between macaques and marmosets at 3 dpi. The difference in pulmonary infiltration by neutrophils, B lymphocytes, and macrophages in common marmosets, compared with rhesus macaques, is greater at 6 than at 3 dpi. Statistics could not be performed for the 6 dpi data because there were only two animals per time point. Insets: The results of the ImageScope positive pixel count algorithm on the 3 dpi immunohistochemically labeled lung sections. Red and orange pixels indicate detection of specific inflammatory cell markers; cells not expressing the marker of interest are shown as blue pixels. ∗∗P < 0.01 for rhesus macaques versus common marmosets. Original magnification, ×20 (main images and insets).
Figure 6
Figure 6
Dipeptidyl peptidase 4 (DPP4) is expressed by the same cell types in rhesus macaques and common marmosets in the lung. Immunohistochemistry for DPP4 on sections of lung show DPP4 is expressed by airway epithelium (arrows) and pneumocytes in macaques (A) and marmosets (B). DPP4 was labeled purple using the Discovery Purple kit; tissues were counterstained with hematoxylin. Original magnification, ×40 (A and B).
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