Evaluating angiotensin-converting enzyme 2-mediated SARS-CoV-2 entry across species

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic represents a global threat, and the interaction between virus angiotensin-converting enzyme (ACE2), primary entry receptor for SARS-CoV-2, is key determinant of range hosts that can be infected by virus. However, mechanisms underpinning ACE2-mediated viral across species remains unclear. Using infection assay, we evaluated SARS-CoV-2 mediated ACE2 11 different animal species. We discovered Rhinolophus sinicus (Chinese rufous horseshoe bat), Felis catus (domestic cat), Canis lupus familiaris (dog), Sus scrofa (wild pig), Capra hircus (goat), Manis javanica (Malayan pangolin) facilitated into nonsusceptible cells. Moreover, pangolin also entry, adding credence to hypothesis may have originated from pangolins. proteins ferrumequinum (greater Gallus gallus (red junglefowl), Notechis scutatus (mainland tiger snake), or Mus musculus (house mouse) did not facilitate entry. In addition, natural isoform protein Macaca mulatta (rhesus monkey) with Y217N mutation was resistant infection, highlighting possible impact this on studies in rhesus monkeys. further demonstrated Y217 residue critical ability mediate Overall, these results clarify use receptors multiple show tracking reservoirs intermediate complex. December 2019, novel pneumonia, termed disease 2019 (COVID-19) World Health Organization (WHO), emerged Wuhan, China, causative agent soon identified as coronavirus, which named International Committee Taxonomy Viruses (ICTV) (1Zhou P. Yang X.L. Wang X.G. Hu B. Zhang L. W. Si H.R. Zhu Y. Li Huang C.L. Chen H.D. J. Luo Guo H. Jiang R.D. et al.A pneumonia outbreak associated new probable bat origin.Nature. 2020; 579: 270-273Crossref PubMed Scopus (12018) Google Scholar, 2Wu F. Zhao S. Yu Y.M. Song Z.G. Tao Z.W. Tian J.H. Pei Y.Y. Yuan M.L. Y.L. Dai F.H. Liu Q.M. human China.Nature. 265-269Crossref (6145) Scholar). has been speculatively seafood market where sales include various wild animals (3Lu R. X. Niu Wu N. Bi Ma Zhan T. al.Genomic characterisation epidemiology coronavirus: Implications origins binding.Lancet. 395: 565-574Abstract Full Text PDF (6910) Bats are recognized potential reservoir 3Lu recent indicated pangolins considered (4Lam T.T. Shum M.H. H.C. Tong Y.G. Ni X.B. Liao Y.S. Wei Cheung W.Y. W.J. L.F. Leung G.M. Holmes E.C. Guan Identifying related coronaviruses Malayan pangolins.Nature. 583: 282-285Crossref (956) 5Zhang Q. Z. Probable origin COVID-19 outbreak.Curr. Biol. 30: 1346-1351.e2Abstract (819) Discovering evaluating their cross-species transmissibility scientifically very important. Unfortunately, know little about this. A study revealed ferrets cats sensitive infection; however, showed no clinical symptoms (6Shi Wen Zhong G. C. He Shuai Sun Liang Cui al.Susceptibility ferrets, cats, dogs, other domesticated SARS-coronavirus 2.Science. 368: 1016-1020Crossref (1026) Whether exist candidate model should explored. viruses host range. resembles (SARS-CoV), both (ACE2) cell 7Li Moore M.J. Vasilieva Sui Wong S.K. Berne M.A. Somasundaran M. Sullivan J.L. Luzuriaga K. Greenough T.C. Choe Farzan Angiotensin-converting functional SARS coronavirus.Nature. 2003; 426: 450-454Crossref (4001) 8Hoffmann Kleine-Weber Schroeder Kruger Herrler Erichsen Schiergens T.S. N.H. Nitsche A. Muller Drosten Pohlmann Depends TMPRSS2 blocked clinically proven protease Inhibitor.Cell. 181: 271-280.e8Abstract (10840) 9Wan Shang Graham Baric R.S. Receptor recognition Wuhan: An analysis based Decade-Long structural coronavirus.J. Virol. 94: e00127-20Crossref (2738) When retrace virus, susceptibility conferred speculated preferentially investigated (10Li Kuhn I.C. Animal Insight ACE2-S-protein interactions.J. 2006; 80: 4211-4219Crossref (216) 11Ren Qu Han Zhou S.Y. Deng Shi Difference usage (SARS) SARS-like origin.J. 2008; 82: 1899-1907Crossref (108) Before clarifying horseshow bat) SARS-CoV, scientists first provided SARS-CoV. They found responsible SARS-CoV subsequently confirmed 12Ge X.Y. Chmura A.A. Epstein Mazet J.K. Peng Y.J. C.M. Tan al.Isolation characterization uses receptor.Nature. 2013; 503: 535-538Crossref (1110) 13Hou confer variable entry.Arch. 2010; 155: 1563-1569Crossref (57) Middle East (MERS-CoV) having because MERS-CoV two MERS-CoV-related bats could utilize dipeptidyl peptidase 4 (DPP4) (14Raj V.S. Mou Smits S.L. Dekkers D.H. Dijkman Muth D. Demmers J.A. Zaki Fouchier R.A. Thiel V. Rottier P.J. Osterhaus A.D. Bosch B.J. al.Dipeptidyl emerging coronavirus-EMC.Nature. 495: 251-254Crossref (1404) 15Lau S.K.P. Fan R.Y.Y. Luk H.K.H. Fung K.S.M. E.Y.M. Ahmed S.S. Chan J.F.W. Kok R.K.H. K.H. Wernery U. Yuen K.Y. Woo P.C.Y. Replication MERS cells offers insights ancestral origins.Emerging microbes & infections. 2018; 7: 209Crossref (24) 16Yang Du Tang HKU4 provide insight bat-to-human transmission coronavirus.Proc. Natl. Acad. Sci. 2014; 111: 12516-12521Crossref (190) Therefore, study, systemically infect HEK293T utilizing nine humans determine its explore transmission. Our findings evidence able engage species, poses large challenge control prevention future. Furthermore, monkey; RhACE2) mutation, amino acid To investigate synthesized full-length cDNA fragments well humans. These were pangolin), mouse), monkey), Homo sapiens (human). Synthesized then subcloned pCAGGS-HA vector expression eukaryotic GenBank accession numbers molecules listed Table 1. compared nucleotide sequence coding region similarities cDNAs exhibited Among sequences, RhACE2 most similar (hACE2), contrast, mainland snake least (Fig. 1A). It reported virus-binding hotspots, K31 K353 hACE2, (17Li Structural major barriers palm civets infections.J. 6984-6991Crossref (131) 18Wu Wilken Geraghty R.J. Mechanisms adaptation Chem. 2012; 287: 8904-8911Abstract (168) conserved all observed study. ten except mouse (Table 1).Table 1Nucleotide similarity ACE-2 ACE-2ACE-2 originLength (bp)Similarity (%)Position 31Position 353GenBank numberHomo (Human)2418100KKAB046569.1Rhinolophus (Greater bat)241886.2DKAB297479.1Rhinolophus bat)241885.5EKGQ262791.1Macaca (Rhesus monkey)aNOTE: here contains Y217N.241896.6KKNM_001135696.1Sus (Pig)241884.5KKNM_001123070.1Canis (Dog)241587KKNM_001165260.1Capra (Goat)241585.5KKKF921008.1Felis (Cat)241886.8KKAY957464.1Gallus (Chicken)242768.1EKMK560199.1Manis pangolin)241886.5KKXM_017,650,257.1Notechis (Mainland snake)248766.5QKXM_026674969Mus (Mouse)241885.2NHNM_001130513.1a NOTE: Y217N. Open table tab Next, tested whether HEK 293T lines. Different expressed 1, B C). Plasmids expressing hACE2 applied positive negative controls respectively. ratio varied according 1C). As expected, supported whereas One previous 17 years ago affinis (intermediate (12Ge allowed synthesize due absence sequence. test SARS-CoV-2. Interestingly, support but 1C), suggesting infection. replicate shed pigs, chickens, ducks replicated fairly effectively cat Although chickens molecular role avidity resistance. data dogs pigs cats. There debate regarding if 4Lam mediates pangolins) conferring Finally, snake) previously predicted (19Ji Zai Cross-species newly 2019-nCoV.J. Med. 92: 433-440Crossref (567) Snakes Old world monkeys (M. fascicularis (crab-eating macaque)) used experimental models (20Lu Gao Kuang Xu Qian al.Comparison infections among 3 non-human primates.bioRxiv. Surprisingly, our expected By investigating monkey sequence, cloned contained variations (R192G Y217N) 2A). reverted N217Y (wild-type ACE2) RhACE2, had 2B). noticed (data shown), suggests confirm hypothesis, constructed completely Potential asparagine (N)-linked glycosylation influence binding; when analyzed N217 N-linked site (N-X-T/S motif). an N217Q This blocking residue. dramatically alters reduces efficiency (21Chen Tu Qin Rhesus angiotensin converting supports Chinese macaques.Virology. 381: 89-97Crossref (20) current illustrated corresponding wild-type 2C). RBD probe examine receptor-binding using IFA. performed binding assay potently bound RBD, 3A); Y217N, Y217Q almost lost bind 3A). Receptor-binding quantified western blotting 3B). Since conformation recombinant reflect native within entire envelope glycoprotein, more relevant systems S (spike) protein, obtained result 3, C D). above significantly reduced spike protein. position 217 influenced activities disrupting homology-based modeling analyze effects substitutions at 217. generated crystal structure RBD/ACE2 complex (22Lan Ge Shan Structure domain 581: 215-220Crossref (3109) 23Shang Ye Wan Aihara Geng Auerbach basis SARS-CoV-2.Nature. 221-224Crossref (2100) located RBD; furthermore, seemed 4). activities. enter cells, need interact surface; therefore, possibility fails change surface localization induced mutation. transfected plasmids. Forty-eight hours later, detached plate stained FITC-labeled anti-ACE2 antibody. strongly FITC, mutants weakly 5A). difference transfection efficacy, permeabilized Triton X-100 ACE2. labeled FITC high level 5B).This failed mainly localization. (S) features lysosomal proteases tropism (24Zheng Lysosomal tropism.J. e01504-18Crossref (38) date, RaTG13 Yunnan Province exhibits highest fact, contrast genetically homologous great genetic diversity (13Hou number isolated serves bat-origin SARS-CoV-like suggest receptor-conferred important before Recently, Coronaviruses homology pangolins), source emergence Similarly, increases (25Yinghui G.H. Ji Ren Gong Ju Hong Cai Lan Xie al.Functional Orthologs Reveals Broad SARS-CoV-2.bioRxiv. 26Zhao Szabla Zheng J.T. Junop Zeng Lin Differential SARS-CoV-2.J. 94 (e00940-20)Crossref (81) poorly