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Comparative Study
. 2022 Feb 23;96(4):e0173921.
doi: 10.1128/JVI.01739-21. Epub 2021 Dec 15.

Comparison of Experimental Middle East Respiratory Syndrome Coronavirus Infection Acquired by Three Individual Routes of Infection in the Common Marmoset

Michelle Nelson  1 , Lyn M O'Brien  1 , Carwyn Davies  1 , Emma Keyser  1 , Wendy Butcher  1 , Sophie J Smither  1 , Alejandro Nunez  2 , F Javier Salguero  3 , M Stephen Lever  1
Affiliations
Comparative Study

Comparison of Experimental Middle East Respiratory Syndrome Coronavirus Infection Acquired by Three Individual Routes of Infection in the Common Marmoset

Michelle Nelson et al. J Virol. .

Abstract

Two strains of Middle East respiratory syndrome coronavirus (MERS-CoV), England 1 and Erasmus Medical Centre/2012 (EMC/2012), were used to challenge common marmosets (Callithrix jacchus) by three routes of infection: aerosol, oral, and intranasal. Animals challenged by the intranasal and aerosol routes presented with mild, transient disease, while those challenged by the oral route presented with a subclinical immunological response. Animals challenged with MERS-CoV strain EMC/2012 by the aerosol route responded with primary and/or secondary pyrexia. Marmosets had minimal to mild multifocal interstitial pneumonia, with the greatest relative severity being observed in animals challenged by the aerosol route. Viable virus was isolated from the host in throat swabs and lung tissue. The transient disease described is consistent with a successful host response and was characterized by the upregulation of macrophage and neutrophil function observed in all animals at the time of euthanasia. IMPORTANCE Middle East respiratory syndrome is caused by a human coronavirus, MERS-CoV, similar to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Humans typically exhibit fever, cough, shortness of breath, gastrointestinal issues, and breathing difficulties, which can lead to pneumonia and/or renal complications. This emerging disease resulted in the first human lethal cases in 2012 and has a case fatality rate of approximately 36%. Consequently, there is a need for medical countermeasures and appropriate animal models for their assessment. This work has demonstrated the requirement for higher concentrations of virus to cause overt disease. Challenge by the aerosol, intranasal, and oral routes resulted in no or mild disease, but all animals had an immunological response. This shows that an appropriate early immunological response is able to control the disease.

Keywords: Callithrix jacchus; Middle East respiratory syndrome; aerosols; animal model; coronavirus; interstitial pneumonia; lung disease; macrophages; neutrophils.

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Conflict of interest statement

The authors declare no conflict of interest.

We do not have any conflicts of interest.

Figures

FIG 1
FIG 1
Viral replication of MERS-CoV in BHK cells following transfection with either human or marmoset DPP4 receptor RNA. Significant differences were determined by ANOVA, with differences shown between mock-transfected cells (Lipofectamine) and human and marmoset transfected cells (**, P < 0.01; ***, P < 0.001).
FIG 2
FIG 2
Optimization of the aerosolization of MERS-CoV. (a) Comparison of SF values following the addition of 2% fetal calf serum (FCS) to the Collison and impinger fluids. (b) Comparison of the SF values at different relative humidities, where low is 34.1% ± 2.2%, medium is 52.6% ± 3.6%, and high is 85.4% ± 6.0%. (c) Particle size distribution at different relative humidities. (d) Effect of ultracentrifugation and concentration of the viral stock on SF values. Significant differences were determined by ANOVA (a and b) or a t test (d) with data transformed, Y = log(Y), to ensure normal data (ns, not significant [P > 0.05]; *, P < 0.05).
FIG 3
FIG 3
(A and B) Temperature profiles of marmosets following challenge with the original stocks of MERS-CoV strains England 1 (A) and EMC/2012 (B) by various routes of infection. (C) Temperature profile of marmosets following challenge by the aerosol route with the concentration stock of MERS-CoV strain EMC/2012.
FIG 4
FIG 4
Heat map comparing the detection of virus by a plaque assay or PCR in tissues and swabs of marmosets challenged by the aerosol route with the concentration stock of MERS-CoV strain EMC/2012. Levels of viable virus are indicated in purple, with light purple indicating samples where <20 PFU of virus were identified and dark purple indicating samples where either 1.4 × 104 or 7.7 × 104 PFU were detected. Viral RNA was identified in all samples that were positive for viable virus, with between 1.1 × 102 and 1.2 × 102 PFU equivalents detected in light purple areas and between 4.1 × 106 and 3.7 × 106 PFU equivalents detected in dark purple areas. Viral RNA was also detected in other samples; light green indicates samples where <20 PFU equivalents were detected, and dark green indicates samples where between 1.1 × 103 and 9.0 × 104 PFU equivalents were detected.
FIG 5
FIG 5
Representative histological images from the lungs of marmosets challenged by one of two strains of MERS-CoV by different routes of challenge. (A) A low-magnification hematoxylin and eosin (H&E)-stained image from animal 110W following challenge with strain EMC/2012 by the oral route shows mild thickening of alveolar septa and lymphocytic infiltration into the perivascular location (*) (above normal limits). (B) A high-magnification H&E-stained image from animal 56W following challenge with strain England 1 by the intranasal route shows expanded alveolar septa (*) infiltrated by neutrophils with mild exudation into the alveolar spaces (arrow). (C) This tissue was further characterized by Gram Twort staining and shows no evidence of bacterial colonies in areas of neutrophilic exudation. (D and E) Immunohistochemical staining with MAC387 shows an increase of cells in the alveoli (D), and immunohistochemical staining of CD3 shows scattered T lymphocytes in areas of alveolar thickening of septa (E). (F) A high-magnification H&E-stained image from animal 55W following challenge with strain EMC/2012 by the aerosol route shows lymphocytic infiltration into the perivascular location (*) (above normal limits). (G) H&E staining of the terminal bronchiole from animal 146X euthanized 2 days following challenge with the concentrated stock of MERS-CoV by the aerosol route shows exudation. (H) This sample was further characterized by immunohistochemical staining with MAC387 and shows an accumulation of cells in lesioned areas with exudation and septal infiltration. (I) Phosphotungstic acid hematoxylin (PTAH) staining was also performed on a lung section of the other animal euthanized 2 days following challenge with the concentrated stock of MERS-CoV (animal 130X) to show fibrin in the alveolar exudation.
FIG 6
FIG 6
Location and activity of the DPP4 receptor in marmosets. (A and B) Immunohistochemical staining indicates a very strong presence of the DPP4 receptor within the alveolar spaces of marmosets (A) and no expression in the nasal cavity epithelium and moderate expression in the NALT and other submucosal structures (B). (C) The virus is observed in areas of high DPP4 receptor expression following aerosol challenge with MERS-CoV strain EMC/2012 within the terminal bronchioles and alveolar spaces. (D) An aggregate of MERS-CoV-positive lymphoid cells was observed in the nasal cavity. (E) Locations of MERS-CoV antigen and levels of expression of the DPP4 receptor in the respiratory tract and lungs of marmosets.
FIG 7
FIG 7
Clinical chemistry parameters observed in marmosets before and after challenge with two strains of MERS-CoV by various routes of challenge. (A) AST (aspartate aminotransferase); (B) ALKP (alkaline phosphatase); (C) CREA (creatinine); (D) BUN (blood urea nitrogen); (E) LYM (absolute lymphocyte count); (F) MONO (absolute monocyte count). For illustrative purposes, data from the two studies are combined on a single graph. Significant differences were determined by ANOVA with data transformed, Y = log(Y), to ensure normal data (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).
FIG 8
FIG 8
Proportions and activation status of different immune cells observed in marmoset blood following challenge with MERS-CoV strain EMC/2012 by the aerosol route. (A) Proportion of neutrophils; (B) HLA-DR expression of neutrophils; (C) CD54+ expression of neutrophils; (D) CD80+ expression of neutrophils. Significant differences were determined by ANOVA with data transformed, Y = log(Y), to ensure normal data (*, P < 0.05; **, P < 0.01; ***, P < 0.001).
FIG 9
FIG 9
Proportions and activation status of different immune cells observed in marmoset lungs following challenge with MERS-CoV. (A) Number of T cells; (B) CD16+ expression of T cells; (C) proportion of neutrophils; (D) CD80+ expression of neutrophils; (E) proportion of macrophages; (F) CD40+ expression of macrophages. For illustrative purposes, levels are shown for animals challenged with MERS-CoV strain EMC/2012 by the aerosol route and euthanized on days 2 and 7. Significant differences were determined by ANOVA with data transformed, Y = log(Y), to ensure normal data (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).
FIG 10
FIG 10
Activation status of different immune cells observed in marmoset blood following challenge with either MERS-CoV strain EMC/2012 or England 1 by different routes of challenge. (A) Cytotoxic CD8+ cells; (B) NK+ T cells; (C) CD69+ expression of NK cells; (D) CD16+ expression of NK cells. For illustrative purposes, expression levels of CD16+ on NK cells are shown for animals challenged with MERS-CoV strain EMC/2012 by the aerosol route and euthanized on days 2 and 7. Significant differences were determined by ANOVA with data transformed, Y = log(Y), to ensure normal data (*, P < 0.05; **, P < 0.01; ***, P < 0.001).
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