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. 2020 Nov 12;16(11):e1008949.
doi: 10.1371/journal.ppat.1008949. eCollection 2020 Nov.

Virulence and pathogenesis of SARS-CoV-2 infection in rhesus macaques: A nonhuman primate model of COVID-19 progression

Huiwen Zheng  1 , Heng Li  1 , Lei Guo  1 , Yan Liang  1 , Jing Li  1 , Xi Wang  1 , Yunguang Hu  1 , Lichun Wang  1 , Yun Liao  1 , Fengmei Yang  1 , Yanyan Li  1 , Shengtao Fan  1 , Dandan Li  1 , Pingfang Cui  1 , Qingling Wang  1 , Haijing Shi  1 , Yanli Chen  1 , Zening Yang  1 , Jinling Yang  1 , Dong Shen  1 , Wei Cun  1 , Xiaofang Zhou  2 , Xingqi Dong  3 , Yunchuan Wang  1 , Yong Chen  1 , Qing Dai  1 , Weihua Jin  1 , Zhanlong He  1 , Qihan Li  1 , Longding Liu  1
Affiliations

Virulence and pathogenesis of SARS-CoV-2 infection in rhesus macaques: A nonhuman primate model of COVID-19 progression

Huiwen Zheng et al. PLoS Pathog. .

Abstract

The COVID-19 has emerged as an epidemic, causing severe pneumonia with a high infection rate globally. To better understand the pathogenesis caused by SARS-CoV-2, we developed a rhesus macaque model to mimic natural infection via the nasal route, resulting in the SARS-CoV-2 virus shedding in the nose and stool up to 27 days. Importantly, we observed the pathological progression of marked interstitial pneumonia in the infected animals on 5-7 dpi, with virus dissemination widely occurring in the lower respiratory tract and lymph nodes, and viral RNA was consistently detected from 5 to 21 dpi. During the infection period, the kinetics response of T cells was revealed to contribute to COVID-19 progression. Our findings implied that the antiviral response of T cells was suppressed after 3 days post infection, which might be related to increases in the Treg cell population in PBMCs. Moreover, two waves of the enhanced production of cytokines (TGF-α, IL-4, IL-6, GM-CSF, IL-10, IL-15, IL-1β), chemokines (MCP-1/CCL2, IL-8/CXCL8, and MIP-1β/CCL4) were detected in lung tissue. Our data collected from this model suggested that T cell response and cytokine/chemokine changes in lung should be considered as evaluation parameters for COVID-19 treatment and vaccine development, besides of observation of virus shedding and pathological analysis.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Experimental schedule and clinical signs in rhesus macaques inoculated with SARS-CoV-2.
(A) Schedule of viral inoculation and necropsies. (B) Monitoring of the body temperature of infected animals for 27 days. (C) Animals were scored for 27 days, and their mean clinical score ±SD was calculated.
Fig 2
Fig 2. Viral shedding and viremia in rhesus macaques inoculated with SARS-CoV-2.
Oropharyngeal (A), nasal (B), and rectal (C) swab samples and blood samples (D) were collected for 27 days. Of note, 2 animals were euthanized on 3, 5 and 21 dpi, and 3 animals were euthanized on 7 and 9 dpi; thus, samples were reduced in the plots. RNA was extracted, and the viral load was determined as copies per 100 μl. The dashed line represents the detected threshold.
Fig 3
Fig 3. Viral load in tissues of rhesus macaques inoculated with SARS-CoV-2.
Necropsy tissue samples of rhesus macaques were collected on 3, 5, 7, 9 and 21 dpi. RNA was extracted, and viral load was detected by qRT-PCR as copies per 100 mg tissue in (A) lungs and tracheas; (B) LNs; (C) CNS; and (D) other tissues. Control: the archived normal tissues. Error bars represent the SD.
Fig 4
Fig 4. Histopathological changes in rhesus macaques inoculated with SARS-CoV-2.
Necropsy tissue samples of rhesus macaques were collected on 3, 5, 7, 9 and 21 dpi, stained with H&E for histopathological evaluation, or labeled with anti-N antigen antibody for IHC analysis. (A) Lung tissue samples showed mild to marked acute interstitial pneumonia demonstrated by diffuse alveolar damage and edema (black arrow), alveolar thickening (green arrow), large numbers of neutrophils, macrophages, and infiltration (blue arrow). Anti-SARS-CoV-2 antigen for IHC revealed viral N antigen in alveolar pneumocytes. (B) Trachea tissues showed damage to epithelial cells (black arrow). (C) LN samples showed enlarged germinal centers (blue arrow) in response to viral infection. IHC reveals viral N antigen within LNs. (D) Lung (E) trachea and (F) LN IHC analyses (magnification: 400×). Yellow scale bar, 100μl; Black scale bar, 50μl. Control: the archived normal tissues.
Fig 5
Fig 5. Analysis of ACE2 related to SARS-CoV-2 infection in rhesus macaques by confocal immunofluorescence assay.
Human ACE2 was detected with goat anti-human ACE2 (red) antibody followed by Alexa Fluor 647 dye-conjugated goat anti-rabbit IgG. Viral N antigen (yellow) was detected with a mouse antibody against the SARS-CoV-2 N protein followed by Alexa Flour 555 dye-conjugated goat anti-mouse IgG. Nuclei were stained with DAPI (magnification: 630×.). Control: the archived normal tissues.
Fig 6
Fig 6. Immune analysis of SARS-CoV-2-infected rhesus macaques.
(A) Lymphocyte phenotyping by T, B, and NK cells in PBMCs (upper row) and lung tissues (lower row). (B) T helper cells for IL-4, IFN-γ, IL-17 and FOXP3 expression gated in CD4+ cells from PBMCs (upper row) and lung tissues (lower row). (C)Treg cells for CD25 expression gated in CD4+CD127- cells from PBMC(upper row), and lung tissues(Lower row). Control: the archived normal lung tissues. D0: Samples from day 0(samples prior to infection)were used as controls.”*”, p<0.05; “ns”, p>0.05.
Fig 7
Fig 7. Changes of cytokines/chemokines concentrations during the SARS-COV-2 infected process.
Cytokine cytometric bead arrays were performed to measure the concentrations of 23 cytokines and chemokine. The fold change of cytokines of each samples = The concentration value of each experimental well /the concentration value of the control well. The concentration below the detection line is calculated as the concentration of limit of detection. Three multiple wells are set for each sample, and the mean±SD are finally calculated. Control: the archived normal lung tissues.
Fig 8
Fig 8. Neutralizing antibody level and ELISpot analysis of rhesus macaques infected with SARS-CoV-2.
(A) Neutralizing antibodies against SARS-CoV-2 were titered on Vero cells at 100 TCID50 virus. (B) S and N protein-specific IFN-γ+ secretion T cells were numbered on precoated PVDF plates by an ELISpot reader. Compared with controls,”*”, p<0.05; “ns”, p>0.05.
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