key: cord-0959740-3oyivuxv
authors: Yu, Pin; Qi, Feifei; Xu, Yanfeng; Li, Fengdi; Liu, Peipei; Liu, Jiayi; Bao, Linlin; Deng, Wei; Gao, Hong; Xiang, Zhiguang; Xiao, Chong; Lv, Qi; Gong, Shuran; Liu, Jiangning; Song, Zhiqi; Qu, Yajin; Xue, Jing; Wei, Qiang; Liu, Mingya; Wang, Guanpeng; Wang, Shunyi; Yu, Haisheng; Liu, Xing; Huang, Baoying; Wang, Wenling; Zhao, Li; Wang, Huijuan; Ye, Fei; Zhou, Weimin; Zhen, Wei; Han, Jun; Wu, Guizhen; Jin, Qi; Wang, Jianwei; Tan, Wenjie; Qin, Chuan
title: Age‐related rhesus macaque models of COVID‐19
date: 2020-03-30
journal: Animal Model Exp Med
DOI: 10.1002/ame2.12108
sha: b7f053f7fe63869f0a7f4560b4209b8ff9dcadfd
doc_id: 959740
cord_uid: 3oyivuxv

BACKGROUND: Since December 2019, an outbreak of the Corona Virus disease 2019 (COVID‐19) caused by severe acute respiratory syndrome coronavirus (SARS‐CoV‐2) in Wuhan, China, has become a public health emergency of international concern. The high fatality of aged cases caused by SARS‐CoV‐2 was a need to explore the possible age‐related phenomena with non‐human primate models. METHODS: Three 3‐5 years old and two 15 years old rhesus macaques were intratracheally infected with SARS‐CoV‐2, and then analyzed by clinical signs, viral replication, chest X‐ray, histopathological changes and immune response. RESULTS: Viral replication of nasopharyngeal swabs, anal swabs and lung in old monkeys was more active than that in young monkeys for 14 days after SARS‐CoV‐2 challenge. Monkeys developed typical interstitial pneumonia characterized by thickened alveolar septum accompanied with inflammation and edema, notably, old monkeys exhibited diffuse severe interstitial pneumonia. Viral antigens were detected mainly in alveolar epithelial cells and macrophages. CONCLUSION: SARS‐CoV‐2 caused more severe interstitial pneumonia in old monkeys than that in young monkeys. Rhesus macaque models infected with SARS‐CoV‐2 provided insight into the pathogenic mechanism and facilitated the development of vaccines and therapeutics against SARS‐CoV‐2 infection.

In late December of 2019, a cluster of severe pneumonia cases caused by severe acute respiratory syndrome coronavirus (SARS-CoV-2) were reported from Wuhan, China. [1] [2] [3] It is critical to understand the pathogenicity of this virus for the prevention and treatment of Coronavirus disease 2019 (COVID-19). SARS-CoV-2 was isolated and classified as Betacoronavirus, belonging to the lineage B or subgenus Sarbecovirus, which also includes the human SARS coronavirus. 4, 5 The full virus genome of SARS-CoV-2 has about an 89% nucleotide identity with bat-SL-CoVZC45, the Spike protein has a 78% nucleotide identity with the human SARS coronavirus, and an 84% nucleotide identity with the bat-SL-CoVZC45 coronavirus. 6, 7 The SARS-infected rhesus monkey model was once established, 8 and now the SARS-CoV-2-infected rhesus monkey model can dynamically understand the pathogenicity of COVID-19.

A more comprehensive understanding of the pneumonia caused by the SARS-CoV-2 infection is essential for developing more scientific and effective prevention, diagnosis, and treatment measures. Meanwhile, the high fatality of aged cases caused by SARS-CoV-2 was a need to explore the possible age-related phenomena with non-human primate models. 9 

Professor Tan from the China Centers for Disease Control and Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum, 100 IU/mL penicillin, and 100 µg/mL streptomycin, and incubated at 37°C, 5% CO 2 . Titers for SARS-CoV-2 were resolved by a 50% tissue-culture infectious doses (TCID 50 ) assay.

Total RNA was extracted from organs as described in previous re- 

To develop a macaque animal infection model with SARS-CoV-2, three rhesus macaques (Macaca mulatta) (3-5 years old) and two 15 years old rhesus macaques were subsequently inoculated with SARS-CoV-2 by intratracheal routes containing 10 6 TCID 50 /mL. On 0, 5, 6, 7, 8, 9, 11, 13 , and 15-day post-inoculation (dpi), imageological examinations using X-rays were performed. On 3, 5, 7, 9, 11 , and 14 dpi, the nasal, throat, and anal swabs were collected in 1 mL DMEM with 50 U/mL penicillin and 50 µg/mL streptomycin. The IPTT-300 temperature probes (BMDS), which were injected interscapularly into the macaques prior to the start of the experiment, were used to monitor the temperature. Blood samples were collected before the beginning of this research and on 0, 3, 4, 6, 7, 11, and 14 dpi for hematological analysis. Lung tissues were collected at 7 dpi. All sera were collected at 3, 7 and 14 dpi to test the IgG antibodies reactive with SARS-CoV-2 antigens. For the OD450 value, more than twice higher than the negative control was regarded as positive result.

An electric homogenizer was employed to prepare tissue homogenates for 2 minutes 30 seconds in 1 mL of DMEM. The homogenates were centrifuged at 3000 rpm for 10 minutes at 4°C. The supernatant was gathered and stored at −80°C for viral titer. 

Sera from each animal were collected to detect the SARS-CoV-2 antibody through ELISA at 0, 3, 7, 11, and 14 dpi. The 96-well plates coated with 0.1 μg Spike protein from SARS-CoV-2 (Sino Biological;

Product code:40591-V08H) at 4℃ overnight were blocked by 2% BSA/PBST at room temperature for 1 hour. Sera samples were diluted at 1:100, and then were added to different wells and maintained at 37℃ for 30 minutes, followed by the goat anti-monkey antibody labeled with horseradish peroxidase (Abcam, ab112767) incubated at room temperature for 30 minutes. The reaction was built using TMB substrate and determined at 450 nm.

All the collected organs were preserved in 10% buffered formalin solution fixed, and paraffin sections (3-4 µm in thickness) were prepared according to routine practice. All the tissues sections were stained with hematoxylin and eosin (H&E). The histopathological changes of different tissues were observed under an Olympus microscope.

All the collected organs were preserved in 10% buffered formalin solution fixed, and paraffin sections (3-4 µm in thickness) were prepared routinely. An antigen retrieval kit (AR0022; Boster) was prepared the sections for 1 minute at 37°C. Three percent H 2 O 2 in methanol were used to quench endogenous peroxidases for 10 minutes. The slices were maintained at 4℃ overnight with a laboratory prepared-7D2 monoclonal antibody 11 

We report nonhuman primate models for COVID-19 infected with SARS-CoV-2 (HB-01). Three rhesus macaques (divided into younger group, YG) between 3 and 5 years old and two rhesus macaques (divided into elder group, EG) about 15 years old were inoculated with 1 × 10 6 TCID 50 of SARS-CoV-2 through intratracheal route. Clinical signs including weight loss, fever, asthenia and lethality were recorded in Table 1 , the weight was decreased obviously from 3 dpi in all the monkeys except one young monkey but without significant change of body temperature during the entire observation. Clinical signs were transient and lasted for a few days. Nasal, throat, and anal swabs were obtained from animals on 0, 3, 5, 7, 9, 11 , and 14 days post-inoculation (dpi).

Viral RNA was found in all animals from nose, pharynx, and crissum.

Virus load in the respiratory tract peaked at 3 dpi in both of YG and EG ( Figure 1A,B) . For young monkeys, the virus load in anal swabs was The percentage and counts of CD3 + CD8 + T cells, CD3 + CD4 + T cells were shown. YG (red line) and EG (blue line) were indicated in the upper right corner of each panel. All data are presented as mean ± SEM lower than that in the nasal and throat specimens ( Figure 1A) . Moreover, viral load from the anal swabs in EG was persistently detected from 3 dpi to 11 dpi, which is higher that in YG ( Figure 1B ). One monkey from each group was euthanized and necropsied at 7 dpi. Different lobes of lung were collected for detecting the viral loads. Approximate 104 Viral RNA copies/mL from the upper lung was detected in YG. As shown in Figure 1C , viral replication in EG monkeys was detected in all the lobes of lung with higher viral RNA copies (range from 104 to 107.5 copies/mL). The anterior-posterior thoracic x-rays from the young macaque showed ground-glass opacity and obscure lung markings in the upper lobe of the right lung at 7 dpi (Figure 2A ) and EG revealed no changes in the counts or percentages white blood cells ( Figure 3A ) and monocytes ( Figure 3B ), a notably higher average counts or percentage of lymphocytes was observed in YG than that in EG during the observation, but the decreased counts of lymphocytes was found both in YG and EG monkeys ( Figure 3C ). Furthermore, regarding the counts of CD3 + CD8 + T cells and CD3 + CD4 + T cells, the obvious decline in YG but not in EG was observed in Figure 3D .

Importantly, the decreased counts of lymphocytes were similar to the clinical manifestations of the SARS-CoV-2-infected-patients. 10 The specific IgG antibody against SARS-CoV-2 from the two groups were detectable at 14 dpi (Table 1) .

Collectively, SARS-CoV-2 caused more severe interstitial pneumonia in old monkeys than that in young monkeys, as demonstrated by clinical signs, viral replication, chest X-ray, histopathological changes and immune response. The age-related animal models of SARS-CoV-2

infection are meaningful for further investigation on pathogenicity and evaluation of vaccines and therapeutics. 

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