key: cord-1033341-42wv7zl6 authors: Tang, Yan-Dong; Li, Yu-Ming; Sun, Jing; Zhang, Hong-Liang; Wang, Tong-Yun; Sun, Ming-Xia; Yang, Yue-Lin; Hu, Xiao-Liang; Zhao, Jincun; Cai, Xui-Hui title: Cell entry of SARS-CoV-2 conferred by angiotensin-converting enzyme 2 (ACE2) of different species date: 2020-06-16 journal: bioRxiv DOI: 10.1101/2020.06.15.153916 sha: 994d6fe26ffd4139910967a6895cb02e7ad8f32c doc_id: 1033341 cord_uid: 42wv7zl6 The outbreak of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses a huge threat to many countries around the world. However, where is it origin and which animals are sensitive to cross-species transmission is unclear. The interaction of virus and cell receptor is a key determinant of host range for the novel coronavirus. Angiotensin-converting enzyme 2 (ACE2) is demonstrated as the primary entry receptor for SARS-CoV-2. In this study, we evaluated the SARS-CoV-2 entry mediated by ACE2 of 11 different species of animals, and discovered that ACE2 of Rhinolophus sinicus (Chinese horseshoe bat), Felis catus (domestic cat), Canis lupus familiaris (dog), Sus scrofa (pig), Capra hircus (goat) and especially Manis javanica (Malayan pangolin) were able to render SARS-CoV-2 entry in non-susceptible cells. This is the first report that ACE2 of Pangolin could mediate SARS-CoV-2 entry which increases the presume that SARS-CoV-2 may have a pangolin origin. However, none of the ACE2 proteins from Rhinolophus ferrumequinum (greater horseshoe bat), Gallus gallus (chicken), Notechis scutatus (mainland tiger snake), Mus musculus (house mouse) rendered SARS-CoV-2 entry. Specifically, a natural isoform of Macaca mulatta (Rhesus monkey) ACE2 with a mutation of Y217N was resistance to infection, which rises the possible impact of this type of ACE2 during monkey studies of SARS-CoV-2. Overall, these results clarify that SARS-CoV-2 could engage receptors of multiple species of animals and it is a perplexed work to track SARS-CoV-2 origin and its intermediate hosts. IMPORTANCE In this study, we illustrated that SARS-CoV-2 is able to engage receptors of multiple species of animals. This indicated that it may be a perplexed work to track SARS-CoV-2 origin and discover its intermediate hosts. This feature of virus is considered to potentiate its diverse cross-species transmissibility. Of note, here is the first report that ACE2 of Pangolin could mediate SARS-CoV-2 entry which increases the possibility that SARS-CoV-2 may have a pangolin origin. And we also demonstrated that not all species of bat were sensitive to SARS-CoV-2 infection. At last, it is also important to detect the expression ratio of the Y217N ACE2 to the prototype in Rhesus monkeys to be recruited for studies on SARS-CoV-2 infection. In this study, we illustrated that SARS-CoV-2 is able to engage receptors of 2 multiple species of animals. This indicated that it may be a perplexed work to track 3 SARS-CoV-2 origin and discover its intermediate hosts. This feature of virus is 4 considered to potentiate its diverse cross-species transmissibility. Of note, here is the 5 first report that ACE2 of Pangolin could mediate SARS-CoV-2 entry which increases 6 the possibility that SARS-CoV-2 may have a pangolin origin. And we also 7 demonstrated that not all species of bat were sensitive to SARS-CoV-2 infection. At 8 last, it is also important to detect the expression ratio of the Y217N ACE2 to the 9 prototype in Rhesus monkeys to be recruited for studies on SARS-CoV-2 infection. In December 2019, a novel pneumonia, termed as COVID-19 by World Health 2 Organization (WHO) thereafter, emerged in Wuhan, China, and the causative agent 3 was soon identified as a novel coronavirus, which is termed as severe acute 4 respiratory syndrome coronavirus 2 (SARS-CoV-2) by the International Committee 5 on Taxonomy of Viruses, ICTV (1, 2). The SARS-CoV-2 outbreak has been 6 speculatively associated with a seafood market where sales also various land wild 7 animals (3). Bats are recognized as a potential natural reservoir for SARS-CoV-2 (1, 8 3). However, recently studies indicated that pangolins were also considered as 9 possible natural hosts of this coronavirus (4, 5) . Discovering the potential 10 intermediate animal hosts of SARS-CoV-2 and evaluating their possible cross-species 11 transmissibility will be scientifically very important. Unfortunately, we know little 12 about this. Currently, there are no suitable animal models for SARS-CoV-2 infection. A recent study revealed that ferrets and cats were sensitive to SARS-CoV-2 infection, 14 however, these animals showed no clinical symptoms (6) . Whether there exist other 15 animal(s) as a candidate SARS-CoV-2 infection model should be further explored. The interaction between receptor and virus is a key determinant of the host range. It has been demonstrated that SARS-CoV-2 resembles SARS-CoV, which uses 18 angiotensin-converting enzyme 2 (ACE2) as the primary cell entry receptor (1, 7-9). When we retrace the origin of coronavirus, the cell susceptibility to viruses conferred 20 by receptors of speculated animals is a preferential consideration (10, 11). Before 21 clarifying that Rhinolophus sinicus is the natural reservoir of SARS-CoV, scientists 22 first evaluated the susceptibility provided by ACE2 from different bat species to 1 SARS-CoV. They found that the ACE2 of Rhinolophus sinicus was responsible for the 2 susceptibility to SARS-CoV and subsequently confirmed that Rhinolophus sinicus 3 was the natural reservoir of SARS-CoV (10, 12, 13). The Middle East respiratory 4 syndrome coronavirus (MERS-CoV) was also recognized has a bat origin due to that 5 both the MERS-CoV and two MERS-CoV-related viruses from bats could utilize 6 human or bat dipeptidyl peptidase 4 (DPP4) for cell entry (14-16). 7 Therefore, in this study, we systemically evaluated the ability of SARS-CoV-2 to 8 infect two types of non-susceptible cells utilizing ACE2 proteins from nine different 9 species of animals and the human being to determine its possible origin and further to 10 explore its potentiate cross-species transmission. Our findings provide evidence that 11 SARS-CoV-2 was able to engage broad receptors of different species, which poses a 12 huge challenge to search the animal origin of SARS-CoV-2 for the control and 13 prevention in future. To investigate which animal's ACE2 could render SARS-CoV-2 entry, we Mus musculus (house mouse) and Macaca mulatta (Rhesus monkey) and Homo 22 sapiens (human). Synthesized DNA fragments were then sub-cloned into the 1 pCAGGS-HA vector for the expression in eukaryotic cells. The origins and GenBank 2 accession numbers of these ACE2 molecules were listed in the Table. We firstly 3 compared the nucleotide sequences of ACE2 coding regions of these animals to that 4 of human. The sequence similarities of these ACE2 cDNAs were exhibited in the 5 Table. Among these sequences, the ACE2 of Rhesus monkey was most close to 6 human, and in contrast, the ACE2 of snake was the farthest. It has been reported that 7 two virus-binding hotspots, Lys31 and Lys353 in hACE2, were critical for 8 SARS-CoV infection (17, 18) . In this study, we found that the Lys31 was not 9 conserved in ACE2 of all the 11 animal species observed in this study. However, the 10 Lys353 was conserved in all the 10 animal species except mouse (Table) . Next, we tested whether ACE2 of the nine animal species were able to render 12 SARS-CoV-2 entry to non-susceptible HEK293T cell lines. Different ACE2s could be 13 expressed and presented in the surface of HEK293T cells by IFA ( Figure 1 ). 14 HEK293T Plasmids expressing ACE2 of human and mice were applied as the positive 15 and negative control of the entry assay, respectively. No attempt was made to quantify 16 infection efficiency in this study due to difficulties encountered in conducting 17 experiments under BSL-3 conditions. As expected, the human ACE2 supported 18 SARS-CoV-2 entry whereas mouse ACE2 did not (Figure 1) rendered SARS-CoV-2 entry besides SARS-CoV (1). In this study, we was not able to 1 synthesize the ACE2 cDNA of Rhinolophus affinis due to the absent of its sequence. 2 Therefore, we synthesized the ACE2 cDNA of Rhinolophus sinicus and Rhinolophus 3 ferrumequinum (Greater horseshoe bat) to test whether ACE2 of other bat species was 4 responsible for the susceptibility to SARS-CoV-2. Interestingly, we found that the 5 ACE2 of Rhinolophus ferrumequinum did not support the SARS-CoV-2 entry as 6 Rhinolophus sinicus (Figure 2) , suggesting that not all species of bat were sensitive to 7 SARS-CoV-2 infection. 8 A recent study indicated that SARS-CoV-2 did not replicate and shed in dogs, 9 pigs, chickens and ducks, but fairly good in ferrets and effectively in cats (6). 10 Consistently, our study demonstrated that the cat ACE2 supported viral entry ( Figure 11 1). Although pigs, dogs and chickens were non-sensitive to SARS-CoV-2 infection, 12 we know little about its molecular mechanisms and the role of receptor avidity for the 13 resistance. Our data demonstrated that ACE2 proteins of dog and pig supported 14 SARS-CoV-2 entry as that of cat. Old world monkeys (Macaca mulatta and Macaca fascicularis) were used as 16 animal models of experimental SARS-CoV-2 infection (19) . Surprisingly, we found 17 that the ACE2 of Macaca mulatta in our study did not support the SARS-CoV-2 entry 18 as expected (Figure 1) . By investigating the monkey ACE2 sequence, we found that 19 an ACE2 isoform of Macaca mulatta, which contained two natural variations (R192G 20 and Y217N) comparing with the wild type ACE2, was cloned ( Figure 2A ). Our data 21 revealed that the ACE2 of Macaca mulatta with the Y217N mutation also fault to 22 support SARS-CoV-2 infection. When we restored Y217N mutation as wild type 1 ACE2 of Macaca mulatta, N217Y recovery the ability to support SARS-CoV-2 2 infection ( Figure 2B ). We noticed that the prototype 217Y was conserved in other 3 species of animals investigated in this study excluding Macaca mulatta (data not 4 shown), which suggests that the 217 position is a key residual for SARS-CoV-2 5 infection. 6 There is a dispute on if SARS-CoV-2 originated from bats or pangolins (1, 4, 5) . It has been demonstrated that a bat ACE2 mediated the SARS-CoV-2 entry (1). 8 However, whether ACE2 of pangolins support the virus entry was unclear. Therefore, In this study, we demonstrated that ACE2 of pig and dog rendered SARS-CoV-2 17 entry. However, a recent study reported that SARS-CoV-2 replicated poorly in dogs 18 and pigs (6) . We speculate that there exist other factors determining the host tropism However, our results showed that this Y217N-isoform of ACE2 expressed at a similar 20 level as the human ACE2 in transfected cells (data not shown). Therefore, the failure 21 of monkey ACE2 isoform to convert the cell susceptibility to SARS-CoV-2 entry is 22 not due to the poor expression of the receptor as previously speculated (28). The 1 detailed mechanism needs further investigation. It is also important to detect the 2 expression ratio of the Y217N ACE2 to the prototype in Rhesus monkeys to be 3 recruited for studies on SARS-CoV-2 infection. The full-length cDNA fragments of different species of ACE2 were synthesized at 12 either the Sangon Biotech (Shanghai, China) or TsingKe Biotech (Nanjing, China). The species and GenBank accession numbers of ACE2 sequences were listed in the 14 Table. Synthesized DNA fragments were then sub-cloned into a eukaryotic expression 15 vector pCAGGS-HA for the expression in human cell lines. 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