key: cord-345101-h0i5o0do
authors: Koo, Bon-Sang; Oh, Hanseul; Kim, Green; Hwang, Eun-Ha; Jung, Hoyin; Lee, Youngjeon; Kang, Philyong; Park, Jae-Hak; Ryu, Choong-Min; Hong, Jung Joo
title: Transient lymphopenia and interstitial pneumonia with endotheliitis in SARS-CoV-2-infected macaques
date: 2020-08-03
journal: J Infect Dis
DOI: 10.1093/infdis/jiaa486
sha: 
doc_id: 345101
cord_uid: h0i5o0do

Using a reliable primate model is critical for developing therapeutic advances to treat humans infected with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Here, we exposed macaques to high titres of SARS-CoV-2 via combined transmission routes. We observed acute interstitial pneumonia with endotheliitis in the lungs of all infected macaques. All macaques had a significant loss of total lymphocytes during infection, which were restored over time. These data show that SARS-CoV-2 causes a coronavirus disease 2019 (COVID-19)-like disease in macaques. This new model could investigate the interaction between SARS-CoV-2 and the immune system to test therapeutic strategies.

M a n u s c r i p t 3 In March 2020, the World Health Organization classified coronavirus disease 2019 (COVID-19) as a pandemic. There is an urgent need for rapid diagnosis and development of therapeutic modalities. The absence of a reliable preclinical animal model that recapitulates patients with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection poses a major limitation to the development of improved diagnostics and therapeutics.

The immune system of non-human primates (NHPs) resembles that of humans, and the structure of the ACE2 receptor is very similar [1] . However, current studies in NHPs infected with human coronaviruses have shown inconsistent outcomes. In SARS-CoVinfected macaques, previous studies reported significant lung lesions [2, 3] , while others found oedema and inflammation in alveoli, with mild clinical findings [4, 5] . Studies reported that SARS-CoV-2 developed no severe clinical signs, but pulmonary pneumonia in 50% of cynomolgus macaques, recapitulating mild symptoms in humans [6] . However, others demonstrated that macaques showed pulmonary infiltrates and high viral loads in the lungs, suggesting moderate disease [7] . In this study, we establish a promising NHP model by addressing the following: 1) genetic and immunological variability in subspecies, 2) inoculum dose, 3) inoculum route, 4) virulent strain of virus isolated from patient, and 5) demographic background of NHPs.

A c c e p t e d M a n u s c r i p t 4

Sixteen male and female macaques, including eight healthy Cambodian-origin cynomolgus (Macaca fascicularis) and Chinese-origin rhesus macaques (Macaca mulatta), aged 3-6 years, were selected by the institutional veterinary experts based on their general health. All animals reared in indoor cages in the animal biosecurity level 3 (ABL-3) laboratory in the Korea National Primate Research Centre (KNPRC) at the Korea Research Institute of Bioscience and Biotechnology (KRIBB). Animals were anaesthetized with a combination of ketamine sodium (10 mg/kg) and tiletamine/zolazepam (5 mg/kg) for viral challenges, swabs, and blood collection. As illustrated in Supplementary Figure 1A , all animals were challenged with a total of 12.5 mL of virus (2.1 × 10 6 TCID50/mL) via intratracheal (4 mL), oral (5 mL), conjunctival (0.5 mL), intranasal (1 mL), and intravenous (2 mL) route. Both cynomolgus and rhesus macaques (n= 4 each species, two males and two females) were euthanized and necropsied at 3 days post infection (dpi). After viral challenges, all live animals were subjected to swab sampling of nasopharyngeal, oropharyngeal, conjunctival, and rectal tissues, at 0, 1, and 3 dpi.

All swab samples were collected in universal viral transport medium, centrifuged (1600 × g for 10 min), and filtered with 0.2 μm pore size syringe filters for further virus quantification. Upon necropsy, tissue samples, including respiratory, immune, intestinal, cardiovascular, and reproductive organs were grossly examined, and collected for viral detection and microscopic examination. All animal procedures were approved by the KRIBB Institutional Animal Care and Use Committee (permit no.

A c c e p t e d M a n u s c r i p t 5

A SARS-CoV-2 virus (accession no. 43326) isolated from a Korean patient was obtained from the National Culture Collection for Pathogens (Cheongju, Korea). This pathogen was passaged three times in VERO cells. The virus titre expressed as 50% tissue culture infectious doses/mL (TCID50/mL), was measured in VERO cells and determined using the Reed and Muench method. All procedures were performed in a Biosafety Cabinet class Ⅱ in the ABL-3 facility in the KNPRC at the KRIBB (permit no. KRIBB-IBC-20200206).

All tissue samples were diluted ten-fold (w/v) with sterile phosphate buffered solution (PBS, pH 7.4) and homogenized using Precellys Homogenizer (Bertin Instruments). After centrifugation, the supernatants were directly inoculated into VERO cells and incubated for 3 days at 37℃, for virus isolation to calculate the values of TCID50/mL. The viral RNA genome was extracted from the supernatant using QIAamp Viral RNA Mini Kit (Qiagen) and stored at -80℃ in the ABL-3 facility until use. RT-qPCR was performed with a primer set targeting partial regions of the ORF1b gene in the SARS-CoV-2 virus using the QIAGEN OneStep RT-PCR kit (Qiagen) as previously reported [8] . 

For histopathological examination, all tissue samples were fixed in 10% neutral buffered formalin, embedded in paraffin, and 4-to 5-μm sections were stained with haematoxylin and eosin. For the staining of SARS-CoV-2 antigens, the immunohistochemistry assay was performed (See the Supplemental Materials for more detailed information).

Haematological evaluation was conducted using an auto-haematology analyser (Mindray BC-5000) on exam days. Whole blood samples were stained with antibodies. After using FACS TM lysing solution (BD Biosciences), blood cells were washed with PBS containing 2% foetal bovine serum. 

Group differences were determined using one-way analysis of variance (ANOVA) with Tukey's posthoc test in Prism version 8.4.2 (GraphPad Software). Significance was set at P< 0.05.

To determine whether animals had depressed behaviour during wake/sleep states after SARS-CoV-2 infection, the average mean locomotion activity (MLA) was measured by Acticals attached to collars for 3 days before and after infection. We observed decreased activity in some animals post infection (Supplementary Figure 1B) . No changes in weight and respiratory rate were observed, but most animals (13/16) showed an increase in temperature 1 dpi and returned to baseline thereafter (Supplementary Figure 1C) . Post-mortem examination of all animals at 3 dpi showed multifocal, bright red lesions in the upper, middle, and lower lobes of the lungs ( Figure 1A ). In these pulmonary lesions, acute interstitial pneumonia (thickening of alveolar wall with type-II pneumocyte hyperplasia, mononuclear and polymorphonuclear (PMN) leukocytes infiltration) was observed ( Figure 1B) and were similar to previous data in humans [9] and monkeys [6] . Additionally, endotheliitis was observed in the lungs of all animals (mononuclear and PMN leukocyte infiltrates within the intima of many vessels, oedema of vessel, and focal haemorrhage) ( Figure 1B and C) , which is in line with a recent report [10] . The viral RNA was highest in the upper respiratory swab samples and lung tissues at the earliest phase of infection, and the viral antigen was present in the lungs ( Figure 1C and D) , suggesting the predominant site of the virus. However, only low viral levels (below 10 2 ) were present in most other organs and not detected in plasma at any time point (Supplementary Figure 2) . This low viral RNA lacking replication in tissues could be detected by the presence of virus inoculated via multiple routes. Our RT-qPCR, with a primer and probe set specific for ORF1b, could not differentiate the reproducible viral RNA A c c e p t e d M a n u s c r i p t 8 from inoculated viral RNA. Without exception, all macaques had a significant decrease in total lymphocytes counts, including CD4 + and CD8 + T cells, B cells, and NK cells at 1 dpi (Figure 2A and B) . This loss was observed across naïve and memory subpopulations of T and B cells (Supplementary Figure 3) . However, our NHP model showed that these decreased values gradually recovered and returned to baseline at 7 dpi (Figure 2A and B) . RNAseq analyses revealed that genes relevant to the viral response (such as those associated with cytokine/chemokine signalling) were significantly elevated in PBMCs at 3 dpi, but no viral copies were detected ( Figure 2C and D) .

Here, we exposed NHPs to a high viral titre via combined routes, including the ocular route, nasal cavity, trachea, oral route, and blood, which have been reported as the possible routes of transmission [11] [12] [13] , regardless of it not being a physiological route. Using a high viral titre administered through combined routes, virus assays, and histopathological changes suggests that both cynomolgus and rhesus macaques are permissive to infection of SARS-CoV-2 and recapitulate COVID-19-like disease in human. During early infection, acute interstitial pneumonia with endotheliitis was observed in the lungs of all infected macaques.

Upper and lower respiratory tracts were the predominant sites of virus replication. This NHP model may also be suitable for investigating interactions between SARS-CoV-2 and the A c c e p t e d M a n u s c r i p t 9 human immune system. All macaques had a significant loss of total lymphocytes, including CD4 + and CD8 + T cells, B cells, and NK cells during early infection. Similarly, lymphopenia was reported in 83.2% of hospitalized SARS-CoV-2 patients [14] . The alteration of peripheral lymphocyte subsets seems to be correlated with severe clinical cases [15] .

Therefore, the NHP model during early infection may be used to validate the effect of immune modulators (e.g. IL-7) in combination with therapeutic approaches to improve lymphopenia. 

Receptor recognition by the novel coronavirus from Wuhan: an analysis based on decade-long structural studies of SARS coronavirus

Koch's postulates fulfilled for SARS virus

Newly discovered coronavirus as the primary cause of severe acute respiratory syndrome

Cynomolgus macaque as an animal model for severe acute respiratory syndrome

Macaque model for severe acute respiratory syndrome

Comparative pathogenesis of COVID-19, MERS, and SARS in a nonhuman primate model

Respiratory disease in rhesus macaques inoculated with SARS-CoV-2

Molecular diagnosis of a novel coronavirus (2019-nCoV) causing an outbreak of pneumonia

Pathological study of the 2019 novel coronavirus disease (COVID-19) through postmortem core biopsies

Endothelial cell infection and endotheliitis in COVID-19

Detection of SARS-CoV-2 in different types of clinical specimens

Evaluation of coronavirus in tears and conjunctival secretions of patients with SARS-CoV-2 infection

Evidence for gastrointestinal infection of SARS-CoV-2

Clinical characteristics of coronavirus disease 2019 in China

Characteristics of peripheral lymphocyte subset alteration in COVID-19 pneumonia

The authors would like to thank the staff of the Korea National Primate Research Centre for their 

The authors declare no competing financial interests.A c c e p t e d M a n u s c r i p t 11