key: cord-273645-czh3zfb3 authors: Lu, Shuaiyao; Zhao, Yuan; Yu, Wenhai; Yang, Yun; Gao, Jiahong; Wang, Junbin; Kuang, Dexuan; Yang, Mengli; Yang, Jing; Ma, Chunxia; Xu, Jingwen; Qian, Xingli; Li, Haiyan; Zhao, Siwen; Li, Jingmei; Wang, Haixuan; Long, Haiting; Zhou, Jingxian; Luo, Fangyu; Ding, Kaiyun; Wu, Daoju; Zhang, Yong; Dong, Yinliang; Liu, Yuqin; Zheng, Yingqiu; Lin, Xiaochen; Jiao, Li; Zheng, Huanying; Dai, Qing; Sun, Qiangmin; Hu, Yunzhang; Ke, Changwen; Liu, Hongqi; Peng, Xiaozhong title: Comparison of SARS-CoV-2 infections among 3 species of non-human primates date: 2020-07-17 journal: bioRxiv DOI: 10.1101/2020.04.08.031807 sha: doc_id: 273645 cord_uid: czh3zfb3 COVID-19, caused by SARS-CoV-2 infection, has recently been announced as a pandemic all over the world. Plenty of diagnostic, preventive and therapeutic knowledges have been enriched from clinical studies since December 2019. However, animal models, particularly non-human primate models, are urgently needed for critical questions that could not be answered in clinical patients, evaluations of anti-viral drugs and vaccines. In this study, two families of non-human primates, Old world monkeys (12 Macaca mulatta, 6 Macaca fascicularis) and New world monkeys (6 Callithrix jacchus), were experimentally inoculated with SARS-CoV-2. Clinical signs were recorded. Samples were collected for analysis of viral shedding, viremia and histopathological examination. Increased body temperature was observed in 100% (12/12) M. mulatta, 33.3% (2/6) M. fascicularis and none (0/6) of C. jacchus post inoculation of SARS-CoV-2. All of M. mulatta and M. fascicularis showed chest radiographic abnormality. Viral genomes were detected in nasal swabs, throat swabs, anal swabs and blood from all 3 species of monkeys. Viral shedding from upper respiratory samples reached the peak between day 6 and day 8 post inoculation. From necropsied M. mulatta and M. fascicularis, the tissues showing virus positive were mainly lung, weasand, bronchus and spleen. No viral genome was seen in any of tissues from 2 necropsied C. jacchus. Severe gross lesions and histopathological changes were observed in lung, heart and stomach of SARS-CoV-2 infected animals. In summary, we have established a NHP model for COVID-19, which could be used to evaluate drugs and vaccines, and investigate viral pathogenesis. M. mulatta is the most susceptible to SARS-CoV-2 infection, followed by M. fascicularis and C. jacchus. One Sentence Summary M. mulatta is the most susceptible to SARS-CoV-2 infection as compared to M. fascicularis and C. jacchus. are going to answer with the animal models. About 16 years ago, the nonhuman primate models recapitulated several important aspects of SARS 8-11 . These models made great contributions to investigation of SARS pathogenesis, evaluation of antiviral drugs and vaccine 12 . is different from SARS in some aspects, pathogens for these two diseases share some characters, such as ACE2 receptor. Here, to establish the COVID-19 model, two families including 3 species of non-human primates, which are widely used for animal models with their own advantages and disadvantages, were experimentally infected with SARS-CoV-2, followed by comparisons of clinical symptoms, hematology, biochemical indexes, immunology and histopathology among 3 species. We found that both Given that host factors may be involved in viral pathogenesis, we designed an experiment in the present study to investigate whether host genetics, age and gender affect SARS-CoV-2 infection in non-human primates ( Figure 1 ). Two families of non-human primates, old world monkeys (12 M. mulatta, 6 M. fascicularis) and new world monkeys (6 C. jacchus), were chosen for this experiment after screening and randomly grouped based on species, age and gender ( Figure 1 ). Animals in large size (4 adults and 4 old M. mulatta, 6 M. fascicularis) were inoculated with 4.75x10 6 pfu of SARS-CoV-2 via 3 routines (intratreachuslly 4.0ml, intranasally 0.5ml and intra 0.25ml). Half dosage of the viruses was given to 4 young M. mulatta via the same 3 routines. Six C. jacchus were inoculated with 1.0x10 6 pfu intranasally. Animals were monitored daily and sampled at the indicated time points before and after viral inoculation ( Figure 1 ). Increased body temperature (BT) (above 38 ℃ ) was continuously observed in 100% (12/12) M. mulatta, 33.3% (2/6) M. fascicularis and none (0/6) of C. jacchus post inoculation of SARS-CoV-2. BT of 7/12 M. mulatta reached the first peak on day 4-6 post inoculation (dpi) and lungs is increased and thickened, and scattered in small patches ( Figure 2B ). Overview of the chest radiograph throughout this study showed that progressive pulmonary infiltration was noted in all M. mulatta and M. fascicularis (Supplemental Figure 1 ). To know dynamics of viral replication and virus shedding, samples of nasal swabs, throat swabs, anal swabs, feces, blood and tissues were collected at the indicated time points, and SARS-CoV-2 genomes were quantitated by RT-qPCR. Swab samples collected on 2 dpi from M. mulatta and M. fascicularis showed surprisingly high levels of viral genome RNA, particularly in nasal swabs. Most of swab samples saw the second peaks of viral RNA on 6-8 dpi. In some swab samples from old world monkeys, viral RNA was still detectable on 14 dpi ( Figure 3A ). Less viral RNA were detected in throat swabs, compared to nasal swabs and anal swabs. In contrast to M. mulatta and M. fascicularis, lower levels of viral RNA were detected in swab samples from C. jacchus during two weeks post viral inoculation ( Figure 3B ). Virus shedding in feces from 8 out of 18 old world monkeys started on 6 dpi. Notably, higher levels of viral RNA were detected in feces from SXH6, HHH6, HHH11 and HHH12, from which anal swabs also gave correspondingly higher number of viral RNA. In peripheral blood, 8/18 old world monkeys and 6/6 new world monkeys had viral RNA detectable on 2 dpi. After 6 days, blood samples from nearly all old world monkeys became viral RNA-positive. On 10 dpi, viral RNA was not detectable in blood samples from almost all (23/24) of experimental monkeys ( Figure 3A ). Viral load of SARS-CoV-2 was determined in tissue samples from 8 animals necropsied on 4 dpi (HHH13), 7 dpi (HHH14), 12 dpi (HHH8), 13 dpi (SXH-1, RH2, RH5) and 15 dpi (HHH3, HHH12). RT-qPCR with SARS-CoV-2 specific primers and probes was performed to quantitate copy number of viral Figure 3D and Figure 3E ). Under TEM, no viral particle was observed in the ultrathin sections of lungs and other tissues. To determine the host response to SARS-CoV-2 infection, we firstly Pandora's box after16 years 13 , leaves us not just many mysteries but also hopes in its jar. Animal models of COVID-19, particularly non-human primate models, will give us hopes to uncover many mysteries of SARS-CoV-2 and However, animals in this study were screened prior to experiments and supposed to be no severe complications/comorbidities and relatively healthy with normal immunity. This may explain that no matter age and gender within one species, monkeys showed similar susceptibility and responses to SARS- Monitoring body temperature is one of critical steps to define COVID-19 suspected patients, which leads to the conclusion that fever is the most common in COVID-19 patients, counting for more than 85% of all inpatients 3,18,21 . Here, all SARS-CoV-2 inoculated M. mulatta had increased body temperature at some time points with a peak of 40.9 ℃ . One-third of M. fascicularis and C. jacchus had a slightly elevated body temperature. Nevertheless, this manifestation could not be defined as fever since we don't have the physiological body temperature of various species of monkeys as a reference. The other important clinical feature of COVID-19 is abnormal changes of chest radiograph from X-ray and CT 22 , which is generally considered to be direct evidence for pneumonia. Ground glass opacities (GGO) were seen in 40%-70-89.6% of COVID-19 patients, followed by consolidation (13-33.9%) and other abnormal radiographs 18, 21, 23, 24 . In this study, all animals (C. jacchus jacchus, showed severe histopathological changes in lung as pneumonia, and inflammation in liver and heart. In conclusion, the NHP model in this study simulates several important aspects of COVID-19. Using this model, we should further explore pathogenesis of SARS-CoV-2 in order to answer some critical remaining questions in clinics, such as the refractory patients, cytokine storm, antibodies against SARS-CoV-2, and so on. And this model is suitable for preclinical evaluation of anti-viral drugs and vaccines against SARS-CoV-2. All animal procedures were approved by the Institutional Animal Care and Viral stock of SARS-CoV-2 was obtained from the Center of Diseases Control, Guangdong Province China. Viruses were amplified on Vero-E6 cells and concentrated by ultrafilter system via 300kDa module (Millipore). Amplified SARS-CoV-2 were confirmed via RT-PCR, sequencing and transmission electronic microscopy, and titrated via plaque assay (10 6 pfu/ml). Three species of monkeys from two families of primates (old world monkeys and new world monkey) were used for this study. Detailed information about experimental animals was shown in Figure 1A . Animal groups and experimental schedules were outlined in Figure 1A . Referring to the NHP model of SARS, we inoculated old world monkeys with total 4.75ml of 10 6 pfu/ml SARS-CoV-2 intratracheally (4ml), intranasally (0.5ml) and on conjunctiva (0.25ml), new world monkeys with 1.0ml intranasally. Animals were daily checked for clinical sign and body temperature. At the indicated time points in Figure 1A , we anaesthetized animals with ketamine and performed the following experimental procedures. Every other day post inoculation, we took chest radiography of animals, harvested peripheral blood to prepare samples of whole blood or serum, and collected nasal, pharyngeal, and rectal swabs in 800ul Trizol LS solution (Invitrogen, US) for further analysis. Monkeys with severe signs were chosen for necropsy and pathological changes of all organs were recorded and evaluated at gross, histological and ultrastructural levels. Chest X-ray image of each anaesthetized monkey was taken with 55-75v and 8-12.5mA every other day using Mobile digital medical X-ray photography system (MobileCooper, Browiner China). Data was evaluated and scored double-blindly and independently by two radiologists. For paraffin-bedded sections, tissues were collected and fixed in 10% neutral- A novel nucleic acid hybridization technique RNAscope was performed to detect and localize viral RNA in paraffin-embedded tissue sections using SARS-CoV-2 specific probe (RNAscope® Probe-V-nCoV2019-S, ACD, Cat No. 848561, targeting the region of 21631 -23303 (NC_045512.2) of SARS-CoV-2 genome). FFPE slides were pretreated by baking, de-paraffinizing, target retrieval and protease treatment. SARS-CoV-2 specific probe was applied to the pretreated slides and hybridized with viral genome. Fluorophore was added to detect the hybridization on slides, followed by counter-staining with DAPI. Slides were scanned via 3D HISTECH system for further analysis. Levels of SARS-CoV-2-specific antibodies in serum samples were evaluated via the commercially available SARS-CoV-2 Antibody Assay Kit (ELISA) (Cat#XG100H2, China). In this kit, viral spike protein was coated in each well of 96-well plate to capture spike-specific antibodies. HRP-conjugated goat anti-human IgG (H+L) antibody was added to detect the captured spikespecific antibodies. Data was plotted via the software GraphPad. i r u s D i s c o v e r e d b y C h i n e s e S c i e n t i s t s I n v e s t i g a t i n g P n e u m o n i a O u t b r e a k . T h e w a l l s t r e e t j o u r n a l U p d a t e d J a n . 8 , 2 0 2 0 8 : 3 0 p m E T ( 2 0 2 and heart (right) fixed in 2.5% glutaraldehyde and 1% osmium tetroxide solution were for ultrastructural analysis (C). Histopathological changes were described in the text. i n o c u l a t e d w i t h S A R S - C o V - 2 . b i o R x i v , 2 0 2 0 . 2 0 0 3 . 2 0 2 1 . 0 0 1 6 2 8 ( 2 0 2 0 ) . 7 . C h a o S h a n , Y . - F . Y . , X i n g - L o u Y a n g e t a l . . I n f e c t i o n w i t h N o v e l C o r o n a v i r u s ( S A R S - C o V - 2 ) C a u HW did the animal experiments.; MY, CM, SZ, JL, YD detected viral RNA; JX did RNAscope; LJ and XL performed immunofluorescent assay Vero-E6 cells YZ did histopathological work; HZ and CK provided viral stock; QS, YH, QD, QL gave suggestions to the study. All authors have approved the submitted manuscript The authors would like to thank all staffs in National Kunming High-level Biosafety Primate Research Center for providing ABSL3-and ABSL4-related services. This study was supported by 2020YFC0841100 and 2020YFC0846400. 11.36% ↓ *Body weights (BW) of animals were measured on day 2 and day 6 post virus inoculation. Body weight change (%) was expressed as |BW on 6 dpi-BW on 2 dpi|/BW on 2 dpi.