key: cord-0831946-9t6hp6ti authors: Zhai, Chongkai; Wang, Mingda; Chung, Hea-Jong; Hassan, Md. Mehedi; Lee, Seungkoo; Kim, Hyeon-Jin; Hong, Seong-Tshool title: Phodopus roborovskii SH101 as a systemic infection model of SARS-CoV-2 date: 2021-03-11 journal: bioRxiv DOI: 10.1101/2021.03.10.434891 sha: f114b3b801871dab8355905d3d3be68884908201 doc_id: 831946 cord_uid: 9t6hp6ti Severe acute respiratory syndrome CoV-2 (SARS-CoV-2) is currently causing a worldwide threat with its unusually high transmission rates and rapid evolution into diverse strains. Unlike typical respiratory viruses, SARS-CoV-2 frequently causes systemic infection by breaking the boundaries of the respiratory systems. The development of animal models recapitulating the clinical manifestations of COVID-19 is of utmost importance not only for the development of vaccines and antivirals but also for understanding the pathogenesis. However, there has not been developed an animal model for systemic infection of SARS-CoV-2 representing most aspects of the clinical manifestations of COVID-19 with systemic symptoms. Here we report that a hamster strain of Phodopus roborovskii SH101, a laboratory inbred hamster strain of P. roborovskii, displayed most symptoms of systemic infection upon SARS-CoV-2 infection as in the case of the human counterpart, unlike current COVID-19 animal models. P. roborovskii SH101 post-infection of SARS-CoV-2 represented most clinical symptoms of COVID-19 such as snuffling, dyspnea, cough, labored breathing, hunched posture, progressive weight loss, and ruffled fur, in addition to high fever following shaking chills. Histological examinations also revealed a serious right-predominated pneumonia as well as slight organ damages in the brain and liver, manifesting systemic COVID-19 cases. Considering the merit of a small animal as well as its clinical manifestations of SARS-CoV-2 infection in human, this hamster model seems to provide an ideal tool to investigate COVID-19. Author summary Although the current animal models supported SARS-CoV-2 replication and displayed varying degrees of illness after SARS-CoV-2 infection, the infections of SARS-CoV-2 were mainly limited to the respiratory systems of these animals, including hACE2 transgenic mice, hamsters, ferrets, fruit bats, guinea pigs, African green monkey, Rhesus macaques, and Cynomolgus macaques. While these animal models can be a modest model for the respiratory infection, there is a clear limit for use them in the study of COVID-19 that also displays multiple systemic symptoms. Therefore, the development of an animal model recapitulating COVID-19-specific symptoms such as the right-predominated pneumonia would be the utmost need to overcome the imminent threat posed by COVID-19. We identified a very interesting hamster strain, Phodopus roborovskii SH101, which mimics almost all aspects of the clinical manifestations of COVID-19 upon SARS-CoV-2 infection. Unlike the current animal models, SARS-CoV-2-infected P. roborovskii SH101 not only displayed the symptoms of respiratory infection but also clinical manifestations specific to human COVID-19 such as high fever following shaking chills, serious right-predominated pneumonia, and minor organ damages in the brain and liver. manifestations of COVID-19 with systemic symptoms. Here we report that a hamster strain of 23 Phodopus roborovskii SH101, a laboratory inbred hamster strain of P. roborovskii, displayed most 24 symptoms of systemic infection upon SARS-CoV-2 infection as in the case of the human 25 counterpart, unlike current COVID-19 animal models. P. roborovskii SH101 post-infection of 26 SARS-CoV-2 represented most clinical symptoms of COVID-19 such as snuffling, dyspnea, 27 cough, labored breathing, hunched posture, progressive weight loss, and ruffled fur, in addition to 28 high fever following shaking chills. Histological examinations also revealed a serious right-29 predominated pneumonia as well as slight organ damages in the brain and liver, manifesting 30 systemic COVID-19 cases. Considering the merit of a small animal as well as its clinical 31 manifestations of SARS-CoV-2 infection in human, this hamster model seems to provide an ideal 32 tool to investigate COVID-19. The emergence of COVID-19 upon the infection of SARS-CoV-2 in 2019 poses a mounting threat 50 to the world. In COVID-19, the infection of SARS-CoV-2 in the respiratory system causes fever, 51 shaking chills, headache, and fatigue followed by respiratory symptoms such as cough, sneeze, 52 sore throat, chest pain, atypical pneumonia, etc [1, 2] . The clinical features of COVID-19, however, 53 are not limited to the consequences of a respiratory infection [3] . Unlike a typical respiratory 54 infection, SARS-CoV-2 infects the organs other than the respiratory system from the beginning or 55 subsequent to respiratory infection, leading to diarrhea, loss of sense of smell or taste, 56 neuroinflammation manifested as encephalitis, meningitis, acute cerebrovascular disease, and 57 Guillain Barré Syndrome (GBS), multisystem inflammatory syndrome, etc [4−6]. More 58 interestingly, atypical pneumonia by SARS-CoV-2 is observed with heavy predominance in the 59 4 right lung [7] . The right-over-left predominance in pneumonia is one of the hallmarks of COVID-60 19 disease [8, 9] . As the consequences of these systemic infection, patients recovered from severe 61 or even mild COVID-19 are frequently suffering from fatigue, heart palpitations, changes in lung 62 function, muscle weakness, memory loss, concentration, brain fog, depression, anxiety, etc 63 [10−12]. 64 The mutation rate in RNA virus such as SARS-CoV-2 is dramatically high, up to a thousand 65 times higher than that of DNA virus, contributing to rapid evolution into its variants. Considering 66 the nature of RNA genome and the number of already happened cases of outbreak, the emergence 67 of new strains from furtively circulating SARS-CoV-2 even after herd immunity by vaccination 68 seems to be inevitable. In fact, the world is already observing the emergence of multiple variants 69 of SARS-CoV-2. Given the number of already spread outbreak cases, an appropriate surrogate for 70 human COVID-19 is needed to overcome the imminent threats of COVID-19 from a future 71 emergence of variant strains. Moreover, animal models could help shed light on important aspects 72 of human COVID-19 in ways that are not easily addressed or feasible in humans, such as how 73 SARS-CoV-2 causes systemic infection. 74 After the outbreak of COVID-19, various animal models for COVID-19 have been developed. 75 The current animal models, such as hACE2 transgenic mice, hamsters, ferrets, fruit bats, guinea 76 pigs, African green monkey, Rhesus macaques, and Cynomolgus macaques, supported SARS-77 CoV-2 replication and displayed varying degrees of illness when the virus was delivered into the 78 respiratory tract of these animals [13 −17] . However, the infections of SARS-CoV-2 in these 79 animals were mainly limited to the respiratory systems. Since these animal models mostly 80 recapitulated the infections of the respiratory system, each of these animals has a limited utility in 81 the study of COVID-19 [18−22] . Therefore, the development of an animal model recapitulating 82 5 systemic infection, including COVID-19-specific facet such as the right-predominated pneumonia, 83 would be the utmost need to overcome the imminent threat posed by In this work, we identified a laboratory-inbred strain of A hamster strain recapitulating COVID-19 after SARS-CoV-2 infection was identified 92 To develop an ideal animal model of COVID-19, we screened several hundred strains of mice, 93 rats, guinea pigs, and hamsters, and identified a hamster strain, Phodopus roborovskii SH101, 94 which was highly sensitive to SARS-CoV-2 infection. P. roborovskii SH101 (abbreviated as 95 SH101) is a laboratory-inbred hamster strain with prominent white patches above eyes and at the 96 base of ears (Fig 1A, B) . The average adult body weights of SH101 were about 21 g for males and 97 20 g for females, respectively. 98 As in the case of COVID-19, respiratory symptoms were immediately noticed in the SH101 99 hamsters infected with SARS-CoV-2 (Video S1~S4). The hamsters showed clear signs of 100 respiratory symptoms such as snuffling, dyspnea, cough, labored breathing, ruffled fur, and sneeze. shaking chills of human patients of COVID-19. Also, progressive and significant weight loss had 106 been observed from 2 to 4 dpi (Fig 1C, D) . All individuals were terminally ill, and the mortality 107 rate of the SH101 hamsters was 83% by 4 dpi. 108 Most interestingly, a unique uneven distribution of pneumonia was noticed immediately by 109 gross examination of the lung specimens ( Fig 1E) . The dissected lungs of the infected SH101 130 We next quantitated SARS-CoV-2 in the SH101 hamsters by quantitative RT-PCR after 131 reverse transcription (RT-qPCR). The primary organs were collected from 3 randomly assigned 132 individuals in each group at 2 and 4 dpi for analysis. High levels of viral RNA were detected in 133 the homogenates of the lung and trachea, whereas lower levels were detected in the brain, stomach, 134 intestine, liver, blood, heart, kidney, and spleen ( Fig 3E, F) with previous studies, our experiments using hACE2 transgenic mice and Syrian hamsters 175 confirmed their sensitivity to SARS-CoV-2 (Fig 5, 6 and Fig S2, S3 ) but failed to observe 176 representative symptoms of COVID-19 such as fever induction, shaking chills, the right-over-left 177 predominated pneumonia, etc. 178 The fever induction and shaking chills are the most common and primary symptoms of 179 COVID-19 that can be observed even in mild cases [26, 27] . For the first time, we were able to 180 show the cardinal symptoms of COVID-19, fever induction, shaking chills, and respiratory 181 problems, in a SARS-CoV-2-infected animal just like in the human counterpart (Fig 1 ~ 4, Fig S4 182 ~ S7 and Video S1~S4), but not in other models (Fig 5, 6) . 183 Both hACE2 transgenic mice and Syrian hamster with SARS-CoV-2 infection demonstrated 184 certain degree of organ damages, heavily damaged lungs and slightly damaged livers but no 185 detectable pathology in the brains (Fig 6A ~ D and Fig S3) . Interestingly, the viral RNA copies in 186 the lungs of SH101, the hACE2 transgenic mice, and Syrian hamsters were not much different, 6.4 187 ± 0.4 (4 dpi), 5.7 ± 0.3 (4 dpi), and 5.6 ± 0.2 (4 dpi), respectively (Fig 6E, F) (Fig S4 ~ S7) . The symptomatic severities of the young hamsters assessed by the organ 195 damages were comparable to those of adults (Fig 4 and Fig S1, S5, S6) . The virus titers of the 196 young SH101 were 7.0 ± 0.3 in the lung at 4 dpi ( Fig S7) like those of adults (Fig 3E, F) interesting COVID-19-specific facet, the right-predominated pneumonia (Fig 1E) , which are 225 uniquely observed in human COVID-19. 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