key: cord-339720-d1stzy8w authors: Zhao, Yuan; Wang, Junbin; Kuang, Dexuan; Xu, Jingwen; Yang, Mengli; Ma, Chunxia; Zhao, Siwen; Li, Jingmei; Long, Haiting; Ding, Kaiyun; Gao, Jiahong; Liu, Jiansheng; Wang, Haixuan; Li, Haiyan; Yang, Yun; Yu, Wenhai; Yang, Jing; Zheng, Yinqiu; Wu, Daoju; Lu, Shuaiyao; Liu, Hongqi; Peng, Xiaozhong title: Susceptibility of tree shrew to SARS-CoV-2 infection date: 2020-04-30 journal: bioRxiv DOI: 10.1101/2020.04.30.029736 sha: doc_id: 339720 cord_uid: d1stzy8w Since SARS-CoV-2 became a pandemic event in the world, it has not only caused huge economic losses, but also a serious threat to global public health. Many scientific questions about SARS-CoV-2 and COVID-19 were raised and urgently need to be answered, including the susceptibility of animals to SARS-CoV-2 infection. Here we tested whether tree shrew, an emerging experimental animal domesticated from wild animal, is susceptible to SARS-CoV-2 infection. No clinical signs were observed in SARS-CoV-2 inoculated tree shrews during this experiment except the increasing body temperature (above 39° C) particular in female animals during infection. Low levels of virus shedding and replication in tissues occurred in all three age groups, each of which showed his own characteristics. Histopathological examine revealed that pulmonary abnormalities were mild but the main changes although slight lesions were also observed in other tissues. In summary, tree shrew is not susceptible to SARS-CoV-2 infection and may not be a suitable animal for COVID-19 related researches. The manifestation caused by SARS-CoV-2 infection is known as COVID-19. Since the first case of SARS-CoV-2 infection reported in Wuhan, China, it has been about five months. It causes a pandemic with more than three million confirmed cases and nearly 230 thousand deaths in addition to huge economic losses to the world 1 . Nevertheless, it is widely considered to be controllable according to the experiences from China and other countries. However, there are still some critical aspects that need to be further investigated in patients, such as cytokine storm, immunopathogenic damages, tropism of SARS-CoV-2, and other sources of SARS-CoV-2 infection besides bat and pangolin that are regarded as the origin of SARS-CoV-2 [2] [3] [4] . From these points of view, studies of animals become essentially important. In fact, several animal models of COVID-19 have been recently reported in murine 5 , hamster 6 , ferret 7 , and non-human primate [8] [9] [10] [11] , which recapitulate COVID-19 from different aspects. In terms of susceptibility to SARS-CoV-2, in addition to these experimental animals, domestic animals and pets are also investigated 12 . Cats, as a popular pet, could be an important source of SARS-CoV-2 infection due to their close relationship with human beings. The tree shrew, also known as Tupaia belangeris, is genetically demonstrated to be close to primates [13] [14] [15] . Therefore, it is being developed to be an experimental animal that could be an alternative to primates in biomedical research due to its unique characteristics 16 . In fact, tree shrew has been used for several animal models of virus infections, including hepatitis B 17 , influenza virus [18] [19] [20] , and Zika virus 21 . However, Tupaia model of high pathogenic viruses has not been reported yet, including SARS-CoV-2. Several reports show that SARS-CoV-2 may originate from wild animals [2] [3] [4] . Replication of SARS-CoV-2 in tree shrews is still unknown. In this study, in order to determine the possibility of tree shrew as a COVID-19 model, we tested the susceptibility of tree shrew to SARS-CoV-2 infection. We found that SARS-CoV-2 had limited replication and shedding in tree shrew, and cause mild histopathology, but no typical symptom is observed in infected tree shrew as reported in COVID-19 patients. Tree shew, Tupaia belangeri chinensis, were bred in the institute of medical biology, Chinese Academy of Medical Sciences. After quarantine and before infection, animals were transferred to ABSL-3 facility and housed in the isolated ventilation cages, with 12-hour light and 12-hour dark. All animal procedures were approved by the Institutional Animal Care and Use Committee of Institute Procedures in this study are outlined in Figure 1 . Total 24 tree shrews were used in this study, and divided into three groups with consideration of gender and age, including young (6 months to 12 months), adult (2 years to 4 years) and old (5 years to 7 years) groups. Each group contains half male and half female animals. After baseline data and samples were collected right before virus inoculation, each of tree shrew was inoculated with 1 ml 10 6 pfu SARS-CoV-2 nasally (500ul/each nostril). Clinical signs were recorded daily, including behavior, drinking and eating, breathing, feces and so on. Body temperature was also monitored every other day post viral inoculation. At the same time points, nasal, throat and anal swab, and blood samples were collected. Viral genomic RNA in these samples was quantified by RT-qPCR using virus-specific primers and probes. On day 14 post viral inoculation, six animals were anesthetized, bled and necropsied. After gross lesions were recorded, tissue samples were harvested for analysis of viral loads and histopathology. RNase/DNase-free H2O was used to elute RNA from the column. 7.5 µl of each RNA was analyzed in each well of 384-well plate by one-step RT-qPCR using gene-specific primers and probe as described before 8 . Tissue samples of heart, liver, spleen, lung, kidney, weasand, stomach, small intestine, rectum, pancreas, brain, spinal cord, uterus, penis, testis, cecum were harvested and fixed in 10% neutral buffered formalin. Paraffin-embedded tissues were cut into 5 µm of sections, followed by haematoxylin and eosin (H&E) staining. Slides were scanned with 3DHISTECH and inspected by the experienced pathologist using the manufacture provided software CaseViewer. During the period of this study, we couldn't observe any other clinical sign besides change of body temperature. After SARS-COV-2 inoculation, body temperature of tree shrew was monitored every other day. Among three ages of virus-inoculated tree shrews, all young tree shrews except TS30 showed increasing body temperature. There are 5 young tree shrews (2 males, 3 females) with peak body temperature on 6 or 8-day post inoculation (dpi), followed by gradual decline. The peaks of body temperatures in two female (TS31, TS34) and one male (TS28) young tree shrews were above 39 ( Figure 1A ). In addition, among old tree shrews, one male (TS1) and three female (TS8, TS10 and TS12) had the peak body temperature (>39 ) on 4 or 6 dpi. However, only one adult female (TS20) showed the peak body temperature (39.2 ) on 6 dpi ( Figure 1B & C) . These results indicated that young/old tree shrews and female tree shrews showed higher sensitivity to SARS-CoV-2 infection, as compared to adult and male tree shrews. On 6 dpi, we could detect genomic RNA of SARS-CoV-2 in 5 nasal swabs, 3 throat swabs, 2 anal swabs and one serum sample from young tree shrews, in 2 nasal swabs from adult tree shrews and in 2 nasal swabs, 1 anal swab from old tree shrew (Table 1) . Notably, four samples (nasal, throat, anal swabs and blood) collected from young tree shrew TS27 all showed virus RNA positive. The highest copy number of viral genomic RNA was 10 5.92 /ml in nasal swab from the young tree shrew TS27. On 8 dpi, there were four young, three adult and four old tree shrew with viral RNA positive in some of samples. The highest level of virus shedding was still from the young tree shrew (TS28). On 12 dpi, the young tree shrews showed the decreasing virus shedding and only the animal TS27 had RNA-positive throat swab. In contrast, increasing number of old tree shrews had detectable viral RNA in nasal, throat, and anal swabs (Table 1) . From these findings, we deduced that young tree shrew was more susceptible to SARS-CoV-2 infection than adult and old tree shrew. However, old tree shrew and adult tree shrews gave longer duration of virus shedding than young tree shrews. Moreover, we noticed that more male animals of adult and old tree shrews showed virus shedding than females. In order to determine viral load in tissue samples, we necropsied 8 animals from 3 ages of tree shrews on 14 dpi. Sixteen major tissues were collected from each animal. Viral genomic RNA was quantified as described in Methods. In three young tree shrews (TS26, TS27 and TS28), we could detect viral RNA from only lungs in TS26 and TS27, but not in any tissue from TS28, although these animal had higher number of viral genomic copy numbers at the earlier stage of SARS-CoV-2 infection. In contrast, in the adult tree shrew TS16, six tissues were RNA positive with the highest number 10 9.08 /ml in pancreas. The female adult tree shrew TS14 had viral RNA-positive uterus in addition to lung and pancreas. The other four necropsied tree shrews showed viral RNA-negative results in all 16 tissues (Table S1) . To determine the host response to SARS-CoV-2 infection, we examined sixteen tissues from each of 24 tree shrews necropsied on 14 dpi and 16 dpi. It is clinically reported that old COVID-19 patients show high morbidity and mortality, which is thought to be associated with the comorbidities 25 Firstly, more young and old tree shrews showed increasing body temperature than adult tree shrews ( Figure 1 ). Secondly, young tree shrews had more severe virus shedding at the early stage of virus infection than the other animals, and old tree shrews had a longer duration of virus shedding than others ( Table 1) . One of young male tree shrew, TS27, didn't have significant increasing body temperature, but virus shedding from nasal, throat, anal and serum was detected on 6 dpi. These results indicated the asymptomatic infection of SARS-CoV-2 in young tree shrews. Although SARS-CoV-2 infection didn't cause severe disease in all three ages of tree shrews, viral replication and mild histopathological changes were still observed in this study. Particularly, we found the severe lung abnormality of histology in one adult tree shrew, which may be caused by immunopathological responses, such as cytokine storm 28 (Table 3) . These results should further be confirmed via nucleic acid in situ hybridization, immunohistochemistry or immunofluorescent staining. In conclusion, tree shrew is not as susceptible to SARS-CoV-2 infection as the reported animal models of COVID-19, though limited replication of SARS-CoV-2 and mild histopathology was detected and observed in some tissues. In addition, commercial reagents and completely domesticated tree shrews are very limited. Therefore, tree shrew is not a suitable experimental animal for COVID-19 related studies. However, it should be very important to investigate whether wild tree shrews in nature are infected by or asymptomatic carrier of SARS-CoV-2. None declared. Total 24 tree shrews (Tupaia belangeris ) were divided into three groups (young, adult and old) according to ages. Each group included half male and half female. Each of tree shrew was inoculated with 1ml 10 6 pfu SARS-CoV-2 nasally (500ul/each) in 0 dpi. Every other day body temperature was monitored. At the same time, nasal, throat, anal and blood samples were collected for analysis of viral loads. On 14 and 16 dpi, animals were euthanatized and necropsied. Gross lesions were recorded and tissue samples were collected for further analysis. (B) On every other day as indicated in (A), body temperature of tree shrew was monitored and recorded. The software Graphpad was utilized for data processing and plotting as rainbow heat map. X represents no data collected. Body temperature beyond 39 was shown in white boxes. Young Old Adult 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Histopathological examination of affected tissues from SARS-CoV-2 infected tree shrews. On 14 and 16 dpi, tree shrews were euthanized and necropsied. Seventeen tissues were collected from each animal for histopathological analysis. From eight old tree shrews, eleven tissues with various degrade of pathological changes were representatively shown. Green arrows indicate the pathological changes described in text. Histopathology of all other tree shrews was summarized in Table 2 . Table 2 . Summary of histopathological examination of tissues from all 24 tree shrews of each age group. Sixteen tissues were collected and examined histopathogically. Six tissues showed no histopathological change. Therefore, eleven tissues were shown in this table. Each age group contained 8 tree shrews. *Number of animals with histopathological changes out of total 8 animals in each age group. Table S1 . Viral load in tissues collected from SARS-CoV-2 infected tree shrews. Density of green color is proportional to the copy number of viral genomic RNA. *animal code #Copy number of viral genomic RNA was expressed as log 10 /ml. -means undetectable. 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