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emmie model custom 13
The availability of a robust disease model is essential for the development of countermeasures for Middle East respiratory syndrome coronavirus (MERS-CoV). While a rhesus macaque model of MERS-CoV has been established, the lack of uniform, severe disease in this model complicates the analysis of countermeasure studies. Modeling of the interaction between the MERS-CoV spike glycoprotein and its receptor dipeptidyl peptidase 4 predicted comparable interaction energies in common marmosets and humans. The suitability of the marmoset as a MERS-CoV model was tested by inoculation via combined intratracheal, intranasal, oral and ocular routes. Most of the marmosets developed a progressive severe pneumonia leading to euthanasia of some animals. Extensive lesions were evident in the lungs of all animals necropsied at different time points post inoculation. Some animals were also viremic; high viral loads were detected in the lungs of all infected animals, and total RNAseq demonstrated the induction of immune and inflammatory pathways. This is the first description of a severe, partially lethal, disease model of MERS-CoV, and as such will have a major impact on the ability to assess the efficacy of vaccines and treatment strategies as well as allowing more detailed pathogenesis studies.
Dipeptidyl peptidase 4 (DPP4, also known as CD26) was recently shown to be the cellular receptor for MERS-CoV [17], and the interaction between the MERS-CoV spike protein and DPP4 was subsequently determined by co-crystallography studies [18], [19]. Although DPP4 is a relatively conserved protein in general, recent data have shown that receptor specificity is likely a major factor in the species tropism of MERS-CoV [11], [20]. This suggests that mapping the spike binding region of DPP4 of a species of interest before performing experimental inoculations of animals could provide a more rational approach to identifying MERS-CoV susceptible animal models. Here, we modeled the interaction of the common marmoset DPP4 with the MERS-CoV spike protein and show that no differences exist compared to human DPP4 at the site of interaction. Subsequent inoculation of common marmosets (Callithrix jacchus) resulted in severe, even lethal, respiratory disease in inoculated animals, with widespread, coalescing bronchointerstitial pneumonia and high viral loads in the lungs of all animals.
(A) Alignment of the amino acid residues from human, common marmoset and ferret DPP4 that have been identified to interact with the receptor binding domain of the MERS-CoV spike glycoprotein. (B) Interaction model (front, back and side view) of the MERS-CoV S1 and its cognate receptor human DPP4, with amino acid differences in common marmoset DPP4 are highlighted in red.
Lungs from marmosets necropsied at 3 and 4 dpi all showed multifocal to coalescing, moderate to marked acute bronchointerstitial pneumonia (Fig. 4A,C). The pneumonia tended to be centered on small caliber and terminal bronchioles and extended into the adjacent pulmonary parenchyma. Viral antigen was exclusively associated and located throughout regions that contained pathological changes (Fig. 4B,D,F,H,J). The bronchiolar epithelium was frequently eroded, leaving attenuated bronchiolar epithelial cells. Affected bronchioles were filled with small to moderate numbers of macrophages and neutrophils, and occasionally small amounts of fibrin and edema (Fig. 4E,G). The adjacent alveolar interstitium was thickened with congestion, edema and fibrin and moderate numbers of macrophages and neutrophils (Fig. 4E, Table S4). Alveolar spaces contained moderate to marked numbers of pulmonary macrophages and neutrophils; multifocally there was pulmonary edema, fibrin, and less frequently hemorrhage (Fig. 4E). There were also rare multinucleate syncytia within alveolar spaces (Fig. 4E). At 6 dpi multifocal to coalescing areas of acute pneumonia were still visible; however, there were also extensive areas of type II pneumocyte hyperplasia (Fig. 4G) and consolidation of pulmonary fibrin resulting in multifocal hyaline membranes (Fig. 4I). These changes are consistent with a transition from acute to a more chronic reparative stage of pneumonia. Regions that were undergoing tissue remodeling showed evidence of clearing of viral antigen (Fig. 4 H). The distribution of DPP4 in marmoset lungs included type I pneumocytes (Fig. 5A) as well as bronchiolar epithelial cells and smooth muscle cells. Consistent with this location of DPP4, a two-color fluorescent staining for cytokeratin and viral antigen, as well as in situ hybridization to detect viral RNA, identified type I pneumocytes and alveolar macrophages as the primary cell type for MERS- CoV replication (Fig. 5 B,C). One of the surviving animals (CM7) had to be euthanized prior to the scheduled end of the study (48 dpi) and was found to have severe aspiration pneumonia. The lungs from the other surviving animal (CM8) appeared normal at necropsy 55 dpi. Viral antigen was not detected by IHC in either animal indicating that these animals had resolved MERS-CoV infection. All other lesions noted in the remaining tissues, such as interstitial nephritis in the kidney and the presence of giant cells in the adrenal gland, consistent with extramedullary hematopoiesis, were not considered to be clinically significant since they are typical, incidental findings in common marmosets [22].
Common marmosets were euthanized on day 3, 4 or 6 post inoculation and lung tissue was collected and stained with hematoxylin and eosin (H&E; panels A, C, E, G, I) or immunohistochemistry using a polyclonal α-MERS-CoV antibody (IHC; panels B, D, F, H, J). (A) Acute bronchointerstitial pneumonia centered on terminal bronchioles, with influx of inflammatory cells and thickening of alveolar septa in lung tissue collected on 3 dpi. Asterisk indicates essentially normal tissue. (B) IHC staining of sequential section of panel A reveals abundance of MERS-CoV antigen in affected areas. (C) Coalescing bronchointerstitial pneumonia inducing a diffuse lesion on 3 dpi. (D) IHC staining of sequential section of panel C reveals abundance of MERS-CoV antigen in affected areas. (E) Edema, hemorrhage and fibrin (asterisks) fill the alveolar spaces in lung tissue collected on 3 dpi. Arrowhead indicates syncytium. Inset highlights thickened alveolar interstitium with fibrin, edema and inflammatory cells. (F) IHC staining of sequential section of panel E. (G) Type II pneumocyte hyperplasia is visible on 6 dpi, as highlighted further in inset. (H) IHC staining of sequential section of panel G indicates viral antigen has been mostly cleared from remodeling tissue. (I) On 6 dpi, fibrin is consolidating into hyaline membranes (arrows). (J) IHC staining of sequential section of panel I. Magnification: A, B, C and D 4; E, F, G, H, I and J 40; inset in panel E and G 100.
Currently, the only small animal MERS-CoV challenge model available, requires transduction of animals with an adenovirus vector expressing human DPP4 [12]. Although this is a very useful model, MERS-CoV infection in this model is highly dependent on the transduction of cells and level of DPP4 expression from the adenovirus vector and thus does not necessarily reflect the natural disease process. Therefore, therapeutics indicated to inhibit MERS-CoV in in vitro studies likely need to be tested in one of the two described nonhuman primate models. As such, marmosets should likely serve as the animal of choice for future therapeutic studies where possible. Not only does the more severe, and potentially lethal disease set a higher bar for protection, it would also allow a greater differentiation to be made between disease in untreated animals versus treated animals, currently a limitation of the rhesus macaque model [7]. The marmoset model also allows the evaluation of intervention strategies at later time points as the disease process in the rhesus model is rapid and quite transient. However, late treatment that targets the virus, as with many countermeasures, is unlikely to be successful once significant lung damage has already occurred as was observed by the lack of success of very late treatment with ribavirin and interferon in human MERS-CoV cases [34]. To enable treatment of patients prior to severe lung injury, future transcriptional studies may yield early indicators of disease progression that can be used as diagnostic or prognostic tests to improve clinical management. The development of the more severe marmoset model will ensure a better pre-clinical analysis of treatments prior to proceeding to clinical trials in humans. As such, this new MERS-CoV disease model is a significant contribution to reducing the impact of MERS-CoV on global public health. 2ff7e9595c
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