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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Angiotensin-converting enzyme 2 (ACE2) proteins of different bat species confer variable susceptibility to SARS-CoV entry</title><author><name sortKey="Hou, Yuxuan" sort="Hou, Yuxuan" uniqKey="Hou Y" first="Yuxuan" last="Hou">Yuxuan Hou</name><affiliation><nlm:aff id="Aff1"><institution-wrap><institution-id institution-id-type="GRID">grid.9227.e</institution-id><institution-id institution-id-type="ISNI">0000000119573309</institution-id><institution>State Key Laboratory of Virology, Wuhan Institute of Virology,</institution><institution>Chinese Academy of Sciences (CAS),</institution></institution-wrap>Wuhan, 430071 Hubei China</nlm:aff></affiliation></author><author><name sortKey="Peng, Cheng" sort="Peng, Cheng" uniqKey="Peng C" first="Cheng" last="Peng">Cheng Peng</name><affiliation><nlm:aff id="Aff1"><institution-wrap><institution-id institution-id-type="GRID">grid.9227.e</institution-id><institution-id institution-id-type="ISNI">0000000119573309</institution-id><institution>State Key Laboratory of Virology, Wuhan Institute of Virology,</institution><institution>Chinese Academy of Sciences (CAS),</institution></institution-wrap>Wuhan, 430071 Hubei China</nlm:aff></affiliation></author><author><name sortKey="Yu, Meng" sort="Yu, Meng" uniqKey="Yu M" first="Meng" last="Yu">Meng Yu</name><affiliation><nlm:aff id="Aff2"><institution-wrap><institution-id institution-id-type="GRID">grid.413322.5</institution-id><institution-id institution-id-type="ISNI">0000 0001 2188 8254</institution-id><institution>Australian Animal Health Laboratory,</institution><institution>Commonwealth Scientific and Industrial Research Organization Livestock Industries,</institution></institution-wrap>PO Bag 24, Geelong, VIC 3220 Australia</nlm:aff></affiliation></author><author><name sortKey="Li, Yan" sort="Li, Yan" uniqKey="Li Y" first="Yan" last="Li">Yan Li</name><affiliation><nlm:aff id="Aff1"><institution-wrap><institution-id institution-id-type="GRID">grid.9227.e</institution-id><institution-id institution-id-type="ISNI">0000000119573309</institution-id><institution>State Key Laboratory of Virology, Wuhan Institute of Virology,</institution><institution>Chinese Academy of Sciences (CAS),</institution></institution-wrap>Wuhan, 430071 Hubei China</nlm:aff></affiliation></author><author><name sortKey="Han, Zhenggang" sort="Han, Zhenggang" uniqKey="Han Z" first="Zhenggang" last="Han">Zhenggang Han</name><affiliation><nlm:aff id="Aff1"><institution-wrap><institution-id institution-id-type="GRID">grid.9227.e</institution-id><institution-id institution-id-type="ISNI">0000000119573309</institution-id><institution>State Key Laboratory of Virology, Wuhan Institute of Virology,</institution><institution>Chinese Academy of Sciences (CAS),</institution></institution-wrap>Wuhan, 430071 Hubei China</nlm:aff></affiliation></author><author><name sortKey="Li, Fang" sort="Li, Fang" uniqKey="Li F" first="Fang" last="Li">Fang Li</name><affiliation><nlm:aff id="Aff3"><institution-wrap><institution-id institution-id-type="GRID">grid.17635.36</institution-id><institution-id institution-id-type="ISNI">0000000419368657</institution-id><institution>Department of Pharmacology,</institution><institution>University of Minnesota Medical School,</institution></institution-wrap>Minneapolis, MN 55455 USA</nlm:aff></affiliation></author><author><name sortKey="Wang, Lin Fa" sort="Wang, Lin Fa" uniqKey="Wang L" first="Lin-Fa" last="Wang">Lin-Fa Wang</name><affiliation><nlm:aff id="Aff2"><institution-wrap><institution-id institution-id-type="GRID">grid.413322.5</institution-id><institution-id institution-id-type="ISNI">0000 0001 2188 8254</institution-id><institution>Australian Animal Health Laboratory,</institution><institution>Commonwealth Scientific and Industrial Research Organization Livestock Industries,</institution></institution-wrap>PO Bag 24, Geelong, VIC 3220 Australia</nlm:aff></affiliation></author><author><name sortKey="Shi, Zhengli" sort="Shi, Zhengli" uniqKey="Shi Z" first="Zhengli" last="Shi">Zhengli Shi</name><affiliation><nlm:aff id="Aff1"><institution-wrap><institution-id institution-id-type="GRID">grid.9227.e</institution-id><institution-id institution-id-type="ISNI">0000000119573309</institution-id><institution>State Key Laboratory of Virology, Wuhan Institute of Virology,</institution><institution>Chinese Academy of Sciences (CAS),</institution></institution-wrap>Wuhan, 430071 Hubei China</nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">20567988</idno><idno type="pmc">7086629</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7086629</idno><idno type="RBID">PMC:7086629</idno><idno type="doi">10.1007/s00705-010-0729-6</idno><date when="2010">2010</date><idno type="wicri:Area/Pmc/Corpus">000266</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000266</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Angiotensin-converting enzyme 2 (ACE2) proteins of different bat species confer variable susceptibility to SARS-CoV entry</title><author><name sortKey="Hou, Yuxuan" sort="Hou, Yuxuan" uniqKey="Hou Y" first="Yuxuan" last="Hou">Yuxuan Hou</name><affiliation><nlm:aff id="Aff1"><institution-wrap><institution-id institution-id-type="GRID">grid.9227.e</institution-id><institution-id institution-id-type="ISNI">0000000119573309</institution-id><institution>State Key Laboratory of Virology, Wuhan Institute of Virology,</institution><institution>Chinese Academy of Sciences (CAS),</institution></institution-wrap>Wuhan, 430071 Hubei China</nlm:aff></affiliation></author><author><name sortKey="Peng, Cheng" sort="Peng, Cheng" uniqKey="Peng C" first="Cheng" last="Peng">Cheng Peng</name><affiliation><nlm:aff id="Aff1"><institution-wrap><institution-id institution-id-type="GRID">grid.9227.e</institution-id><institution-id institution-id-type="ISNI">0000000119573309</institution-id><institution>State Key Laboratory of Virology, Wuhan Institute of Virology,</institution><institution>Chinese Academy of Sciences (CAS),</institution></institution-wrap>Wuhan, 430071 Hubei China</nlm:aff></affiliation></author><author><name sortKey="Yu, Meng" sort="Yu, Meng" uniqKey="Yu M" first="Meng" last="Yu">Meng Yu</name><affiliation><nlm:aff id="Aff2"><institution-wrap><institution-id institution-id-type="GRID">grid.413322.5</institution-id><institution-id institution-id-type="ISNI">0000 0001 2188 8254</institution-id><institution>Australian Animal Health Laboratory,</institution><institution>Commonwealth Scientific and Industrial Research Organization Livestock Industries,</institution></institution-wrap>PO Bag 24, Geelong, VIC 3220 Australia</nlm:aff></affiliation></author><author><name sortKey="Li, Yan" sort="Li, Yan" uniqKey="Li Y" first="Yan" last="Li">Yan Li</name><affiliation><nlm:aff id="Aff1"><institution-wrap><institution-id institution-id-type="GRID">grid.9227.e</institution-id><institution-id institution-id-type="ISNI">0000000119573309</institution-id><institution>State Key Laboratory of Virology, Wuhan Institute of Virology,</institution><institution>Chinese Academy of Sciences (CAS),</institution></institution-wrap>Wuhan, 430071 Hubei China</nlm:aff></affiliation></author><author><name sortKey="Han, Zhenggang" sort="Han, Zhenggang" uniqKey="Han Z" first="Zhenggang" last="Han">Zhenggang Han</name><affiliation><nlm:aff id="Aff1"><institution-wrap><institution-id institution-id-type="GRID">grid.9227.e</institution-id><institution-id institution-id-type="ISNI">0000000119573309</institution-id><institution>State Key Laboratory of Virology, Wuhan Institute of Virology,</institution><institution>Chinese Academy of Sciences (CAS),</institution></institution-wrap>Wuhan, 430071 Hubei China</nlm:aff></affiliation></author><author><name sortKey="Li, Fang" sort="Li, Fang" uniqKey="Li F" first="Fang" last="Li">Fang Li</name><affiliation><nlm:aff id="Aff3"><institution-wrap><institution-id institution-id-type="GRID">grid.17635.36</institution-id><institution-id institution-id-type="ISNI">0000000419368657</institution-id><institution>Department of Pharmacology,</institution><institution>University of Minnesota Medical School,</institution></institution-wrap>Minneapolis, MN 55455 USA</nlm:aff></affiliation></author><author><name sortKey="Wang, Lin Fa" sort="Wang, Lin Fa" uniqKey="Wang L" first="Lin-Fa" last="Wang">Lin-Fa Wang</name><affiliation><nlm:aff id="Aff2"><institution-wrap><institution-id institution-id-type="GRID">grid.413322.5</institution-id><institution-id institution-id-type="ISNI">0000 0001 2188 8254</institution-id><institution>Australian Animal Health Laboratory,</institution><institution>Commonwealth Scientific and Industrial Research Organization Livestock Industries,</institution></institution-wrap>PO Bag 24, Geelong, VIC 3220 Australia</nlm:aff></affiliation></author><author><name sortKey="Shi, Zhengli" sort="Shi, Zhengli" uniqKey="Shi Z" first="Zhengli" last="Shi">Zhengli Shi</name><affiliation><nlm:aff id="Aff1"><institution-wrap><institution-id institution-id-type="GRID">grid.9227.e</institution-id><institution-id institution-id-type="ISNI">0000000119573309</institution-id><institution>State Key Laboratory of Virology, Wuhan Institute of Virology,</institution><institution>Chinese Academy of Sciences (CAS),</institution></institution-wrap>Wuhan, 430071 Hubei China</nlm:aff></affiliation></author></analytic><series><title level="j">Archives of Virology</title><idno type="ISSN">0304-8608</idno><idno type="eISSN">1432-8798</idno><imprint><date when="2010">2010</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>The discovery of SARS-like coronavirus in bats suggests that bats could be the natural reservoir of SARS-CoV. However, previous studies indicated the angiotensin-converting enzyme 2 (ACE2) protein, a known SARS-CoV receptor, from a horseshoe bat was unable to act as a functional receptor for SARS-CoV. Here, we extended our previous study to ACE2 molecules from seven additional bat species and tested their interactions with human SARS-CoV spike protein using both HIV-based pseudotype and live SARS-CoV infection assays. The results show that ACE2s of <italic>Myotis daubentoni</italic> and <italic>Rhinolophus sinicus</italic> support viral entry mediated by the SARS-CoV S protein, albeit with different efficiency in comparison to that of the human ACE2. Further, the alteration of several key residues either decreased or enhanced bat ACE2 receptor efficiency, as predicted from a structural modeling study of the different bat ACE2 molecules. These data suggest that <italic>M. daubentoni</italic> and <italic>R. sinicus</italic> are likely to be susceptible to SARS-CoV and may be candidates as the natural host of the SARS-CoV progenitor viruses. Furthermore, our current study also demonstrates that the genetic diversity of ACE2 among bats is greater than that observed among known SARS-CoV susceptible mammals, highlighting the possibility that there are many more uncharacterized bat species that can act as a reservoir of SARS-CoV or its progenitor viruses. This calls for continuation and expansion of field surveillance studies among different bat populations to eventually identify the true natural reservoir of SARS-CoV.</p><sec><title>Electronic supplementary material</title><p>The online version of this article (doi:10.1007/s00705-010-0729-6) contains supplementary material, which is available to authorized users.</p></sec></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Cui, J" uniqKey="Cui J">J Cui</name></author><author><name sortKey="Han, N" uniqKey="Han N">N Han</name></author><author><name sortKey="Streicker, D" uniqKey="Streicker D">D Streicker</name></author><author><name sortKey="Li, G" uniqKey="Li G">G Li</name></author><author><name sortKey="Tang, X" uniqKey="Tang X">X Tang</name></author><author><name sortKey="Shi, Z" uniqKey="Shi Z">Z Shi</name></author><author><name sortKey="Hu, Z" uniqKey="Hu Z">Z Hu</name></author><author><name sortKey="Zhao, G" uniqKey="Zhao G">G Zhao</name></author><author><name sortKey="Fontanet, A" uniqKey="Fontanet A">A Fontanet</name></author><author><name sortKey="Guan, Y" uniqKey="Guan Y">Y Guan</name></author><author><name sortKey="Wang, L" uniqKey="Wang L">L Wang</name></author><author><name sortKey="Jones, G" uniqKey="Jones G">G Jones</name></author><author><name sortKey="Field, He" uniqKey="Field H">HE Field</name></author><author><name sortKey="Daszak, P" uniqKey="Daszak P">P Daszak</name></author><author><name sortKey="Zhang, S" uniqKey="Zhang S">S Zhang</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Fenn, Td" uniqKey="Fenn T">TD Fenn</name></author><author><name sortKey="Ringe, D" uniqKey="Ringe D">D Ringe</name></author><author><name sortKey="Petsko, Ga" uniqKey="Petsko G">GA Petsko</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Jones, Ta" uniqKey="Jones T">TA Jones</name></author><author><name sortKey="Zou, Jy" uniqKey="Zou J">JY Zou</name></author><author><name sortKey="Cowan, Sw" uniqKey="Cowan S">SW Cowan</name></author><author><name sortKey="Kjeldgaard, M" uniqKey="Kjeldgaard M">M Kjeldgaard</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Ksiazek, Tg" uniqKey="Ksiazek T">TG Ksiazek</name></author><author><name sortKey="Erdman, D" uniqKey="Erdman D">D Erdman</name></author><author><name sortKey="Goldsmith, Cs" uniqKey="Goldsmith C">CS Goldsmith</name></author><author><name sortKey="Zaki, Sr" uniqKey="Zaki S">SR Zaki</name></author><author><name sortKey="Peret, T" uniqKey="Peret T">T Peret</name></author><author><name sortKey="Emery, S" uniqKey="Emery S">S Emery</name></author><author><name sortKey="Tong, S" uniqKey="Tong S">S Tong</name></author><author><name sortKey="Urbani, C" uniqKey="Urbani C">C Urbani</name></author><author><name sortKey="Comer, Ja" uniqKey="Comer J">JA Comer</name></author><author><name sortKey="Lim, W" uniqKey="Lim W">W Lim</name></author><author><name sortKey="Rollin, Pe" uniqKey="Rollin P">PE Rollin</name></author><author><name sortKey="Dowell, Sf" uniqKey="Dowell S">SF Dowell</name></author><author><name sortKey="Ling, Ae" uniqKey="Ling A">AE Ling</name></author><author><name sortKey="Humphrey, Cd" uniqKey="Humphrey C">CD Humphrey</name></author><author><name sortKey="Shieh, Wj" uniqKey="Shieh W">WJ Shieh</name></author><author><name sortKey="Guarner, J" uniqKey="Guarner J">J Guarner</name></author><author><name sortKey="Paddock, Cd" uniqKey="Paddock C">CD Paddock</name></author><author><name sortKey="Rota, P" uniqKey="Rota P">P Rota</name></author><author><name sortKey="Fields, B" uniqKey="Fields B">B Fields</name></author><author><name sortKey="Derisi, J" uniqKey="Derisi J">J DeRisi</name></author><author><name sortKey="Yang, Jy" uniqKey="Yang J">JY Yang</name></author><author><name sortKey="Cox, N" uniqKey="Cox N">N Cox</name></author><author><name sortKey="Hughes, Jm" uniqKey="Hughes J">JM Hughes</name></author><author><name sortKey="Leduc, Jw" uniqKey="Leduc J">JW LeDuc</name></author><author><name sortKey="Bellini, Wj" uniqKey="Bellini W">WJ Bellini</name></author><author><name sortKey="Anderson, Lj" uniqKey="Anderson L">LJ Anderson</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Lau, Sk" uniqKey="Lau S">SK Lau</name></author><author><name sortKey="Woo, Pc" uniqKey="Woo P">PC Woo</name></author><author><name sortKey="Li, Ks" uniqKey="Li K">KS Li</name></author><author><name sortKey="Huang, Y" uniqKey="Huang Y">Y Huang</name></author><author><name sortKey="Tsoi, Hw" uniqKey="Tsoi H">HW Tsoi</name></author><author><name sortKey="Wong, Bh" uniqKey="Wong B">BH Wong</name></author><author><name sortKey="Wong, Ss" uniqKey="Wong S">SS Wong</name></author><author><name sortKey="Leung, Sy" uniqKey="Leung S">SY Leung</name></author><author><name sortKey="Chan, Kh" uniqKey="Chan K">KH Chan</name></author><author><name sortKey="Yuen, Ky" uniqKey="Yuen K">KY Yuen</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Li, F" uniqKey="Li F">F Li</name></author><author><name sortKey="Li, W" uniqKey="Li W">W Li</name></author><author><name sortKey="Farzan, M" uniqKey="Farzan M">M Farzan</name></author><author><name sortKey="Harrison, Sc" uniqKey="Harrison S">SC Harrison</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Li, F" uniqKey="Li F">F Li</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Li, W" uniqKey="Li W">W Li</name></author><author><name sortKey="Shi, Z" uniqKey="Shi Z">Z Shi</name></author><author><name sortKey="Yu, M" uniqKey="Yu M">M Yu</name></author><author><name sortKey="Ren, W" uniqKey="Ren W">W Ren</name></author><author><name sortKey="Smith, C" uniqKey="Smith C">C Smith</name></author><author><name sortKey="Epstein, Jh" uniqKey="Epstein J">JH Epstein</name></author><author><name sortKey="Wang, H" uniqKey="Wang H">H Wang</name></author><author><name sortKey="Crameri, G" uniqKey="Crameri G">G Crameri</name></author><author><name sortKey="Hu, Z" uniqKey="Hu Z">Z Hu</name></author><author><name sortKey="Zhang, H" uniqKey="Zhang H">H Zhang</name></author><author><name sortKey="Zhang, J" uniqKey="Zhang J">J Zhang</name></author><author><name sortKey="Mceachern, J" uniqKey="Mceachern J">J McEachern</name></author><author><name sortKey="Field, H" uniqKey="Field H">H Field</name></author><author><name sortKey="Daszak, P" uniqKey="Daszak P">P Daszak</name></author><author><name sortKey="Eaton, Bt" uniqKey="Eaton B">BT Eaton</name></author><author><name sortKey="Zhang, S" uniqKey="Zhang S">S Zhang</name></author><author><name sortKey="Wang, Lf" uniqKey="Wang L">LF Wang</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Li, W" uniqKey="Li W">W Li</name></author><author><name sortKey="Zhang, C" uniqKey="Zhang C">C Zhang</name></author><author><name sortKey="Sui, J" uniqKey="Sui J">J Sui</name></author><author><name sortKey="Kuhn, Jh" uniqKey="Kuhn J">JH Kuhn</name></author><author><name sortKey="Moore, Mj" uniqKey="Moore M">MJ Moore</name></author><author><name sortKey="Luo, S" uniqKey="Luo S">S Luo</name></author><author><name sortKey="Wong, Sk" uniqKey="Wong S">SK Wong</name></author><author><name sortKey="Huang, Ic" uniqKey="Huang I">IC Huang</name></author><author><name sortKey="Xu, K" uniqKey="Xu K">K Xu</name></author><author><name sortKey="Vasilieva, N" uniqKey="Vasilieva N">N Vasilieva</name></author><author><name sortKey="Murakami, A" uniqKey="Murakami A">A Murakami</name></author><author><name sortKey="He, Y" uniqKey="He Y">Y He</name></author><author><name sortKey="Marasco, Wa" uniqKey="Marasco W">WA Marasco</name></author><author><name sortKey="Guan, Y" uniqKey="Guan Y">Y Guan</name></author><author><name sortKey="Choe, H" uniqKey="Choe H">H Choe</name></author><author><name sortKey="Farzan, M" uniqKey="Farzan M">M Farzan</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Li, W" uniqKey="Li W">W Li</name></author><author><name sortKey="Wong, Sk" uniqKey="Wong S">SK Wong</name></author><author><name sortKey="Li, F" uniqKey="Li F">F Li</name></author><author><name sortKey="Kuhn, Jh" uniqKey="Kuhn J">JH Kuhn</name></author><author><name sortKey="Huang, Ic" uniqKey="Huang I">IC Huang</name></author><author><name sortKey="Choe, H" uniqKey="Choe H">H Choe</name></author><author><name sortKey="Farzan, M" uniqKey="Farzan M">M Farzan</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Peiris, Js" uniqKey="Peiris J">JS Peiris</name></author><author><name sortKey="Lai, St" uniqKey="Lai S">ST Lai</name></author><author><name sortKey="Poon, Ll" uniqKey="Poon L">LL Poon</name></author><author><name sortKey="Guan, Y" uniqKey="Guan Y">Y Guan</name></author><author><name sortKey="Yam, Ly" uniqKey="Yam L">LY Yam</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Ren, W" uniqKey="Ren W">W Ren</name></author><author><name sortKey="Li, W" uniqKey="Li W">W Li</name></author><author><name sortKey="Yu, M" uniqKey="Yu M">M Yu</name></author><author><name sortKey="Hao, P" uniqKey="Hao P">P Hao</name></author><author><name sortKey="Zhang, Y" uniqKey="Zhang Y">Y Zhang</name></author><author><name sortKey="Zhou, P" uniqKey="Zhou P">P Zhou</name></author><author><name sortKey="Zhang, S" uniqKey="Zhang S">S Zhang</name></author><author><name sortKey="Zhao, G" uniqKey="Zhao G">G Zhao</name></author><author><name sortKey="Zhong, Y" uniqKey="Zhong Y">Y Zhong</name></author><author><name sortKey="Wang, S" uniqKey="Wang S">S Wang</name></author><author><name sortKey="Wang, Lf" uniqKey="Wang L">LF Wang</name></author><author><name sortKey="Shi, Z" uniqKey="Shi Z">Z Shi</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Ren, W" uniqKey="Ren W">W Ren</name></author><author><name sortKey="Qu, X" uniqKey="Qu X">X Qu</name></author><author><name sortKey="Li, W" uniqKey="Li W">W Li</name></author><author><name sortKey="Han, Z" uniqKey="Han Z">Z Han</name></author><author><name sortKey="Yu, M" uniqKey="Yu M">M Yu</name></author><author><name sortKey="Zhou, P" uniqKey="Zhou P">P Zhou</name></author><author><name sortKey="Zhang, Sy" uniqKey="Zhang S">SY Zhang</name></author><author><name sortKey="Wang, Lf" uniqKey="Wang L">LF Wang</name></author><author><name sortKey="Deng, H" uniqKey="Deng H">H Deng</name></author><author><name sortKey="Shi, Z" uniqKey="Shi Z">Z Shi</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Tamura, K" uniqKey="Tamura K">K Tamura</name></author><author><name sortKey="Dudley, J" uniqKey="Dudley J">J Dudley</name></author><author><name sortKey="Nei, M" uniqKey="Nei M">M Nei</name></author><author><name sortKey="Kumar, S" uniqKey="Kumar S">S Kumar</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Thompson, Jd" uniqKey="Thompson J">JD Thompson</name></author><author><name sortKey="Gibson, Tj" uniqKey="Gibson T">TJ Gibson</name></author><author><name sortKey="Plewniak, F" uniqKey="Plewniak F">F Plewniak</name></author><author><name sortKey="Jeanmougin, F" uniqKey="Jeanmougin F">F Jeanmougin</name></author><author><name sortKey="Higgins, Dg" uniqKey="Higgins D">DG Higgins</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Tu, C" uniqKey="Tu C">C Tu</name></author><author><name sortKey="Crameri, G" uniqKey="Crameri G">G Crameri</name></author><author><name sortKey="Kong, X" uniqKey="Kong X">X Kong</name></author><author><name sortKey="Chen, J" uniqKey="Chen J">J Chen</name></author><author><name sortKey="Sun, Y" uniqKey="Sun Y">Y Sun</name></author><author><name sortKey="Yu, M" uniqKey="Yu M">M Yu</name></author><author><name sortKey="Xiang, H" uniqKey="Xiang H">H Xiang</name></author><author><name sortKey="Xia, X" uniqKey="Xia X">X Xia</name></author><author><name sortKey="Liu, S" uniqKey="Liu S">S Liu</name></author><author><name sortKey="Ren, T" uniqKey="Ren T">T Ren</name></author><author><name sortKey="Yu, Y" uniqKey="Yu Y">Y Yu</name></author><author><name sortKey="Eaton, Bt" uniqKey="Eaton B">BT Eaton</name></author><author><name sortKey="Xuan, H" uniqKey="Xuan H">H Xuan</name></author><author><name sortKey="Wang, Lf" uniqKey="Wang L">LF Wang</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Yu, M" uniqKey="Yu M">M Yu</name></author><author><name sortKey="Stevens, V" uniqKey="Stevens V">V Stevens</name></author><author><name sortKey="Berry, Jd" uniqKey="Berry J">JD Berry</name></author><author><name sortKey="Crameri, G" uniqKey="Crameri G">G Crameri</name></author><author><name sortKey="Mceachern, J" uniqKey="Mceachern J">J McEachern</name></author><author><name sortKey="Tu, C" uniqKey="Tu C">C Tu</name></author><author><name sortKey="Shi, Z" uniqKey="Shi Z">Z Shi</name></author><author><name sortKey="Liang, G" uniqKey="Liang G">G Liang</name></author><author><name sortKey="Weingartl, H" uniqKey="Weingartl H">H Weingartl</name></author><author><name sortKey="Cardosa, J" uniqKey="Cardosa J">J Cardosa</name></author><author><name sortKey="Eaton, Bt" uniqKey="Eaton B">BT Eaton</name></author><author><name sortKey="Wang, Lf" uniqKey="Wang L">LF Wang</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Yuan, J" uniqKey="Yuan J">J Yuan</name></author><author><name sortKey="Hon, Cc" uniqKey="Hon C">CC Hon</name></author><author><name sortKey="Li, Y" uniqKey="Li Y">Y Li</name></author><author><name sortKey="Wang, D" uniqKey="Wang D">D Wang</name></author><author><name sortKey="Xu, G" uniqKey="Xu G">G Xu</name></author><author><name sortKey="Zhang, H" uniqKey="Zhang H">H Zhang</name></author><author><name sortKey="Zhou, P" uniqKey="Zhou P">P Zhou</name></author><author><name sortKey="Poon, Ll" uniqKey="Poon L">LL Poon</name></author><author><name sortKey="Lam, Tt" uniqKey="Lam T">TT Lam</name></author><author><name sortKey="Leung, Fc" uniqKey="Leung F">FC Leung</name></author><author><name sortKey="Shi, Z" uniqKey="Shi Z">Z Shi</name></author></analytic></biblStruct></listBibl></div1></back></TEI><pmc article-type="research-article"><pmc-dir>properties open_access</pmc-dir>
<front><journal-meta><journal-id journal-id-type="nlm-ta">Arch Virol</journal-id><journal-id journal-id-type="iso-abbrev">Arch. Virol</journal-id><journal-title-group><journal-title>Archives of Virology</journal-title></journal-title-group><issn pub-type="ppub">0304-8608</issn><issn pub-type="epub">1432-8798</issn><publisher><publisher-name>Springer Vienna</publisher-name><publisher-loc>Vienna</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">20567988</article-id><article-id pub-id-type="pmc">7086629</article-id><article-id pub-id-type="publisher-id">729</article-id><article-id pub-id-type="doi">10.1007/s00705-010-0729-6</article-id><article-categories><subj-group subj-group-type="heading"><subject>Original Article</subject></subj-group></article-categories><title-group><article-title>Angiotensin-converting enzyme 2 (ACE2) proteins of different bat species confer variable susceptibility to SARS-CoV entry</article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Hou</surname><given-names>Yuxuan</given-names></name><xref ref-type="aff" rid="Aff1">1</xref></contrib><contrib contrib-type="author"><name><surname>Peng</surname><given-names>Cheng</given-names></name><xref ref-type="aff" rid="Aff1">1</xref></contrib><contrib contrib-type="author"><name><surname>Yu</surname><given-names>Meng</given-names></name><xref ref-type="aff" rid="Aff2">2</xref></contrib><contrib contrib-type="author"><name><surname>Li</surname><given-names>Yan</given-names></name><xref ref-type="aff" rid="Aff1">1</xref></contrib><contrib contrib-type="author"><name><surname>Han</surname><given-names>Zhenggang</given-names></name><xref ref-type="aff" rid="Aff1">1</xref></contrib><contrib contrib-type="author"><name><surname>Li</surname><given-names>Fang</given-names></name><xref ref-type="aff" rid="Aff3">3</xref></contrib><contrib contrib-type="author" corresp="yes"><name><surname>Wang</surname><given-names>Lin-Fa</given-names></name><address><email>linfa.wang@csiro.au</email></address><xref ref-type="aff" rid="Aff2">2</xref></contrib><contrib contrib-type="author" corresp="yes"><name><surname>Shi</surname><given-names>Zhengli</given-names></name><address><email>zlshi@wh.iov.cn</email></address><xref ref-type="aff" rid="Aff1">1</xref></contrib><aff id="Aff1"><label>1</label><institution-wrap><institution-id institution-id-type="GRID">grid.9227.e</institution-id><institution-id institution-id-type="ISNI">0000000119573309</institution-id><institution>State Key Laboratory of Virology, Wuhan Institute of Virology,</institution><institution>Chinese Academy of Sciences (CAS),</institution></institution-wrap>Wuhan, 430071 Hubei China</aff><aff id="Aff2"><label>2</label><institution-wrap><institution-id institution-id-type="GRID">grid.413322.5</institution-id><institution-id institution-id-type="ISNI">0000 0001 2188 8254</institution-id><institution>Australian Animal Health Laboratory,</institution><institution>Commonwealth Scientific and Industrial Research Organization Livestock Industries,</institution></institution-wrap>PO Bag 24, Geelong, VIC 3220 Australia</aff><aff id="Aff3"><label>3</label><institution-wrap><institution-id institution-id-type="GRID">grid.17635.36</institution-id><institution-id institution-id-type="ISNI">0000000419368657</institution-id><institution>Department of Pharmacology,</institution><institution>University of Minnesota Medical School,</institution></institution-wrap>Minneapolis, MN 55455 USA</aff></contrib-group><pub-date pub-type="epub"><day>22</day><month>6</month><year>2010</year></pub-date><pub-date pub-type="ppub"><year>2010</year></pub-date><volume>155</volume><issue>10</issue><fpage>1563</fpage><lpage>1569</lpage><history><date date-type="received"><day>21</day><month>4</month><year>2010</year></date><date date-type="accepted"><day>12</day><month>6</month><year>2010</year></date></history><permissions><copyright-statement>© Springer-Verlag 2010</copyright-statement><license><license-p>This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic.</license-p></license></permissions><abstract id="Abs1"><p>The discovery of SARS-like coronavirus in bats suggests that bats could be the natural reservoir of SARS-CoV. However, previous studies indicated the angiotensin-converting enzyme 2 (ACE2) protein, a known SARS-CoV receptor, from a horseshoe bat was unable to act as a functional receptor for SARS-CoV. Here, we extended our previous study to ACE2 molecules from seven additional bat species and tested their interactions with human SARS-CoV spike protein using both HIV-based pseudotype and live SARS-CoV infection assays. The results show that ACE2s of <italic>Myotis daubentoni</italic> and <italic>Rhinolophus sinicus</italic> support viral entry mediated by the SARS-CoV S protein, albeit with different efficiency in comparison to that of the human ACE2. Further, the alteration of several key residues either decreased or enhanced bat ACE2 receptor efficiency, as predicted from a structural modeling study of the different bat ACE2 molecules. These data suggest that <italic>M. daubentoni</italic> and <italic>R. sinicus</italic> are likely to be susceptible to SARS-CoV and may be candidates as the natural host of the SARS-CoV progenitor viruses. Furthermore, our current study also demonstrates that the genetic diversity of ACE2 among bats is greater than that observed among known SARS-CoV susceptible mammals, highlighting the possibility that there are many more uncharacterized bat species that can act as a reservoir of SARS-CoV or its progenitor viruses. This calls for continuation and expansion of field surveillance studies among different bat populations to eventually identify the true natural reservoir of SARS-CoV.</p><sec><title>Electronic supplementary material</title><p>The online version of this article (doi:10.1007/s00705-010-0729-6) contains supplementary material, which is available to authorized users.</p></sec></abstract><kwd-group xml:lang="en"><title>Keywords</title><kwd>Salt Bridge</kwd><kwd>Severe Acute Respiratory Syndrome</kwd><kwd>ACE2 Gene</kwd><kwd>Pseudotype Virus</kwd><kwd>Severe Acute Respiratory Syndrome Coronavirus</kwd></kwd-group><custom-meta-group><custom-meta><meta-name>issue-copyright-statement</meta-name><meta-value>© Springer-Verlag 2010</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id="Sec1" sec-type="introduction"><title>Introduction</title><p>Severe acute respiratory syndrome coronavirus (SARS-CoV) is the aetiological agent responsible for the SARS outbreaks during 2002–2003, which had a huge global impact on public health, travel and the world economy [<xref ref-type="bibr" rid="CR4">4</xref>, <xref ref-type="bibr" rid="CR11">11</xref>]. The host range of SARS-CoV is largely determined by the specific and high-affinity interactions between a defined receptor-binding domain (RBD) on the SARS-CoV spike protein and its host receptor, angiontensin-converting enzyme 2 (ACE2) [<xref ref-type="bibr" rid="CR6">6</xref>, <xref ref-type="bibr" rid="CR7">7</xref>, <xref ref-type="bibr" rid="CR9">9</xref>]. It has been hypothesized that SARS-CoV was harbored in its natural reservoir, bats, and was transmitted directly or indirectly from bats to palm civets and then to humans [<xref ref-type="bibr" rid="CR10">10</xref>]. However, although the genetically related SARS-like coronavirus (SL-CoV) has been identified in horseshoe bats of the genus <italic>Rhinolophus</italic> [<xref ref-type="bibr" rid="CR5">5</xref>, <xref ref-type="bibr" rid="CR8">8</xref>, <xref ref-type="bibr" rid="CR12">12</xref>, <xref ref-type="bibr" rid="CR18">18</xref>], its spike protein was not able to use the human ACE2 (hACE2) protein as a receptor [<xref ref-type="bibr" rid="CR13">13</xref>]. Close examination of the crystal structure of human SARS-CoV RBD complexed with hACE2 suggests that truncations in the receptor-binding motif (RBM) region of SL-CoV spike protein abolish its hACE2-binding ability [<xref ref-type="bibr" rid="CR7">7</xref>, <xref ref-type="bibr" rid="CR10">10</xref>], and hence the SL-CoV found recently in horseshoe bats is unlikely to be the direct ancestor of human SARS-CoV. Also, it has been shown that the human SARS-CoV spike protein and its closely related civet SARS-CoV spike protein were not able to use a horseshoe bat (<italic>R. pearsoni</italic>) ACE2 as a receptor [<xref ref-type="bibr" rid="CR13">13</xref>], highlighting a critical missing link in the bat-to-civet/human transmission chain of SARS-CoV.</p><p>There are at least three plausible scenarios to explain the origin of SARS-CoV. First, some unknown intermediate hosts were responsible for the adaptation and transmission of SARS-CoV from bats to civets or humans. This is the most popular theory of SARS-CoV transmission at the present time [<xref ref-type="bibr" rid="CR10">10</xref>]. Second, there is an SL-CoV with a very close relationship to the outbreak SARS-CoV strains in a non-bat animal host that is capable of direct transmission from reservoir host to human or civet. Third, ACE2 from yet to be identified bat species may function as an efficient receptor, and these bats could be the direct reservoir of human or civet SARS-CoV. Unraveling which scenario is most likely to have occurred during the 2002–2003 SARS epidemic is critical for our understanding of the dynamics of the outbreak and will play a key role in helping us to prevent future outbreaks. To this end, we have extended our studies to include ACE2 molecules from different bat species and examined their interaction with the human SARS-CoV spike protein. Our results show that there is great genetic diversity among bat ACE2 molecules, especially at the key residues known to be important for interacting with the viral spike protein, and that ACE2s of <italic>Myotis daubentoni</italic> and <italic>Rhinolophus sinicus</italic> from Hubei province can support viral entry.</p></sec><sec id="Sec2" sec-type="materials|methods"><title>Materials and methods</title><sec id="Sec3"><title>Cell lines and antibodies</title><p>HeLa cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (Gibco, USA). Rabbit polyclonal antibodies against ACE2 of <italic>R. pearsoni</italic> (RpACE2) were generated using <italic>R. pearsoni</italic> ACE2 protein expressed in <italic>Escherichia coli</italic> at the Wuhan Institute of Virology following standard procedures.</p></sec><sec id="Sec4"><title>Bat sample collection and identification</title><p>Bats were sampled from their natural habitats in Hubei, Guangxi, Guizhou, Henan and Yunnan provinces in China as described previously [<xref ref-type="bibr" rid="CR8">8</xref>]. Bat identification was initially determined in the field by morphology and later confirmed in the laboratory by sequencing the mitochondrial cytochrome b gene from samples of blood cells or rectal tissue as described previously [<xref ref-type="bibr" rid="CR1">1</xref>].</p></sec><sec id="Sec5"><title>Bat ACE2 amplification and cloning</title><p>Total RNA was extracted from bat rectal tissue using TRIzol Reagent (Invitrogen, USA) and treating with RNase-free DNase I at 37°C for 30 min. First-strand cDNA was synthesized from total RNA by reverse transcription with random hexamers. Full-length bat ACE2 fragments were amplified using the forward primer bAF2 (5′-CTTGGTACCATGTCAGGCTCTTYCTGG-3′) and the reverse primer bAR2 (5′-CCGCTCGAGCTAAAAB[G/T/C]GAV[G/A/C]GTCTGAACATCATC-3′). The PCR mixture (25 μL) contained 0.5 μL cDNA, 1.5 mM MgCl<sub>2</sub> and 0.2 μM of each primer, and the fragments were amplified using the following parameters: 95°C for 5 min, 35 cycles of 94°C for 30 s, 55°C for 45 s and 68°C for 3 min, with a final elongation step at 68°C for 10 min. All bat ACE2s were cloned into pCDNA3.1 with <italic>Kpn</italic>I and <italic>Xho</italic>I, and this was verified by sequencing.</p></sec><sec id="Sec6"><title>Chimeric ACE2 construction</title><p>For samples in which full-length ACE2 amplification was unsuccessful, the N-terminal region (1–1,170 bp) was amplified using the forward primer bAF2 and the reverse primer RMR (5′-TTAGCTCCATTTCTTAGCAGGTAGG-3′). Chimeric ACE2 was constructed by combining the N-terminal region of bat ACE2 with the C-terminal portion of human ACE2 at the unique <italic>Bam</italic>HI site (1,070–1,075 bp). The chimera was subsequently cloned into pCDNA3.1 with <italic>Kpn</italic>I and <italic>Xho</italic>I and sequenced as above.</p></sec><sec id="Sec7"><title>Construction of bat ACE2 mutants</title><p>ACE2 from <italic>M. daubentoni</italic> was chosen to generate a series of ACE2 mutants using a QuikChange II Site-Directed Mutagenesis Kit (Stratagene, USA). The altered amino acid codon for each mutant is indicated as follows: I27T, N31K, K35E, and H41Y. Mutants were confirmed by sequencing.</p></sec><sec id="Sec8"><title>Sequence analysis</title><p>All bat ACE2s were submitted to GenBank (EF569964, GQ999931–GQ999938). Sequence alignment was performed using ClustalX version 1.83 [<xref ref-type="bibr" rid="CR15">15</xref>] and corrected manually. A phylogenetic tree based on amino acid (aa) sequences was constructed using the neighbor-joining (NJ) method in MEGA version 4.1. [<xref ref-type="bibr" rid="CR14">14</xref>].</p></sec><sec id="Sec9"><title>Analysis of ACE2 expression by western blotting</title><p>Lysates of HeLa cells expressing human ACE2 or bat ACE2 were separated on a 4–10% SDS-PAGE gel, followed by transfer to a polyvinylidene difluoride (PVDF) membrane using a semi-dry protein transfer apparatus (Bio-Rad, USA). The membrane was probed with a rabbit polyclonal antibody against the bat ACE2 protein (1:200) at room temperature for 1 h, followed by incubation with alkaline-phosphatase-conjugated goat anti-rabbit IgG (1:1,000) (Chemicon, Australia). The probed proteins were visualized using NBT and BCIP color development (Promega, USA).</p></sec><sec id="Sec10"><title>Pseudotype virus infection assays</title><p>An HIV-1-luciferase pseudotype virus carrying the SARS-CoV BJ01 S protein, HIV/BJ01-S, was prepared as described previously [<xref ref-type="bibr" rid="CR13">13</xref>]. HeLa cells were seeded onto 96-well plates for 18 h and then transfected with 0.2 μg recombinant plasmid containing bat or human ACE2 using 0.5 μL Lipofectamine 2000 (Invitrogen, USA) according to the manufacturer’s protocol. At 24 h post-transfection, 30 μL medium containing HIV/BJ01-S was added to each well. At 2–3 h postinfection, unadsorbed viruses were removed, and fresh medium was added. The infection was monitored by measuring luciferase activity, expressed from the reporter gene carried by the pseudovirus, using a luciferase assay kit (Promega, USA). Cells were lysed at 48 h postinfection by adding 20 μL lysis buffer provided with the kit, and 10 μL of the resulting lysates were tested for luciferase activity by the addition of 20 μL luciferase substrate in a Turner Designs TD-20/20 luminometer. Each infection experiment was conducted in triplicate, and all experiments were repeated three times.</p></sec><sec id="Sec11"><title>Live virus infection assays</title><p>Live SARS-CoV infection was carried out under BSL4 conditions at the Australian Animal Health Laboratory (AAHL) as described previously [<xref ref-type="bibr" rid="CR16">16</xref>, <xref ref-type="bibr" rid="CR17">17</xref>]. Briefly, 48 h after transfection, the time at which expression of the ACE2 receptor on the HeLa cell surface is optimal, 2 × 10<sup>6</sup> TCID<sub>50</sub> of virus was added to the cells for infection. The cells were fixed 24 h later by treatment with 100% methanol for 10 min and washed five times with PBST. The primary antibody, chicken anti-SARS-CoV S (produced against the recombinant S protein expressed in <italic>E. coli</italic> at AAHL), at a 1:500 dilution in 1% BSA/PBS, was added and incubated with the cells for 1 h at room temperature. An FITC anti-chicken conjugate (Chemicon, Australia) at 1:1,000 in 1% BSA/PBS was added after washing the cells five times and incubated with the cells for 1 h. Infection was monitored by immunofluorescent microscopic analysis.</p></sec></sec><sec id="Sec12"><title>Results and discussion</title><sec id="Sec13"><title>Cloning and expression of ACE2 genes from different bat species</title><p>ACE2 genes from seven bat species were amplified and cloned (Fig. <xref rid="Fig1" ref-type="fig">1</xref>, sFig. 1). Full-length genes were obtained from <italic>Rhinolophus ferrumequinum</italic> from Hubei province (Rf-HB), <italic>R. macrotis</italic> from Hubei province (Rm-HB), <italic>R. pearsoni</italic> from Guangxi (Rp-GX), <italic>R. pusillus</italic> from Hubei province (Rpu-HB), <italic>R. sinicus</italic> from Guangxi province (Rs-GX) and <italic>R. sinicus</italic> from Hubei province (Rs-HB). For the following bat species, amplification of the full-length coding region was not successful, and instead,the N-terminal region was cloned in frame with the C-terminal region of the human ACE2 gene to form a chimeric full-length ACE molecule: <italic>R. pearsoni</italic> from Guizhou province (Rp-GZ), <italic>Myotis</italic><italic>daubentonii</italic> bat from Yunnan province (Md-YN) and <italic>Hipposideros pratti</italic> bat from Henan province (Hp-HN). The full-length sequences of bat ACE2 are identical in size to that of hACE2 (805 aa in total). Sequence comparison showed that bat ACE2s are closely related to ACE2s of other mammals and have an aa sequence identity of 80–82% to human and civet ACE2. The aa identity of ACE2 from different bat families ranges from 78 to 84%, and within the genus <italic>Rhinolophus</italic>, the sequence identity increases to 89–98%. The major sequence variation among bat ACE2s is located in the N-terminal region, which has been identified in structural studies as the SARS-CoV-binding region [<xref ref-type="bibr" rid="CR6">6</xref>, <xref ref-type="bibr" rid="CR7">7</xref>]. A phylogenetic tree was constructed based on the sequences of bat ACE2 (sFig. 2) using the MEGA package [<xref ref-type="bibr" rid="CR14">14</xref>].<fig id="Fig1"><label>Fig. 1</label><caption><p>Sequence alignment of SARS-CoV binding regions of ACE2s from 9 bats, civet and human. The GenBank accession numbers of bat, civet and human ACE2 are as follows: human (NM021804), civet (AY881174), Rf-HB (GQ999931), Rm-HB (GQ999932), Rs-GX (GQ999933), Rp-GX (EF569964), Hp-HN (GQ999934), Rp-GZ (GQ999935), Rs-HB (GQ999936), Md-YN(GQ999937) and Rpu-HB (GQ999938). The alignment was generated using ClustalX v1.83. In <italic>black</italic> are single, fully conserved residues. In <italic>gray</italic> are strongly conserved residues. In <italic>light gray</italic> are weakly conserved residues. <italic>Asterisks</italic> indicate residues that interact directly with the receptor-binding domain of the SARS-CoV S protein</p></caption><graphic xlink:href="705_2010_729_Fig1_HTML" id="MO1"></graphic></fig></p><p>All ACE2 genes were cloned into a eukaryotic expression vector and used to transfect HeLa cells. Western blot analysis showed that all ACE2s were expressed efficiently and at very similar levels and were recognized by a rabbit anti-bat ACE2 antibody with an apparent molecular weight of approximately 100–130 kDa (Fig. <xref rid="Fig2" ref-type="fig">2</xref>c).<fig id="Fig2"><label>Fig. 2</label><caption><p>Testing of the ability of bat ACE2 proteins to mediate pseudovirus HIV/BJ01-S and live SARS- CoV infection. <bold>a</bold> HeLa cells transfected with plasmids encoding bat and human ACE2s were infected with pseudovirus HIV/BJ01-S. Infectivity was determined by measuring the activity of reporter luciferase as described in “<xref rid="Sec2" ref-type="sec">Materials and methods</xref>”. HeLa cells transfected with pcDNA3.1 and human ACE2 were used as the negative and positive controls, respectively. All tests were performed in triplicate, and the experiments were repeated three times. The <italic>error bar</italic> represents the calculated standard deviation. I27T, N31K, K35E, and H41Y are mutants of MdACE2 that were made using a QuikChange II Site-Directed Mutagenesis Kit. <bold>b</bold> SARS-CoV live virus infection using the ACE2s from bats as described in “<xref rid="Sec2" ref-type="sec">Materials and methods</xref>”. HeLa cells transfected with pcDNA3.1 and human ACE2 were used as the negative and positive controls, respectively. <bold>c</bold> Expression of bat or human ACE2. Lysates from HeLa cells transfected with plasmid expressing human or bat ACE2 were analyzed by western blot. Rabbit anti-bat ACE2 polyclonal antibody (<italic>upper panel</italic>) or β-actin monoclonal antibody (<italic>lower panel</italic>) was used as the primary antibody. <italic>Lane 1</italic> vector pcDNA3.1 control; <italic>lanes 2–10</italic> bat ACE2 from samples Rf-HB, Rm-HB, Rpu-HB, Hp-HN, Rp-HB, Rp-GZ, Rs-GX, Rs-HB and Md-YN; <italic>lanes 11–14</italic> Md-YN ACE2 mutant I27T, N31K, K35E and H41Y; <italic>lane 15</italic> human ACE2. The abbreviations of bat species are given in the main text</p></caption><graphic xlink:href="705_2010_729_Fig2_HTML" id="MO2"></graphic></fig></p></sec><sec id="Sec14"><title>Functionality of bat ACE2 as an SARS-CoV entry receptor</title><p>To examine the susceptibility of different bat ACE2 molecules to SARs-CoV entry, the HIV/BJ01-S pseudovirus system was used to infect HeLa cells transiently expressing bat ACE2 or human ACE2 genes. Among the bat ACE2s, only MdACE2 (MdACE2) and Rs-HB ACE2 demonstrated significant pseudovirus infection, as deduced from the significantly higher level of luciferase activity in comparison to background activity in the negative control (Fig. <xref rid="Fig2" ref-type="fig">2</xref>a). Although such assays are not to be viewed as an absolute quantification of receptor activity, it is nevertheless worth noting that MdACE2-mediated infection seemed to be more efficient than with Rs-HB ACE2. In the same context, it is clear that the bat ACE2s were less efficient overall than the human ACE2 in this particular assay system. The biological significance of this observation remains to be determined. The functionality of MdACE2 and Rs-HB ACE2 as SARS-CoV entry receptors was further confirmed by infection with live virus. As shown in Fig. <xref rid="Fig2" ref-type="fig">2</xref>b, both bat ACE2 proteins could clearly support SARs-CoV infection. No attempt was made to quantify infection efficiency in this study due to difficulties encountered in conducting experiments under BSL4 conditions.</p></sec><sec id="Sec15"><title>Structural modeling of bat ACE2 molecules</title><p>Homologous structural modeling of human SARS-CoV RBD complexed with MdACE2 supports MdACE2 as a receptor for human SARS-CoV S protein. The crystal structure of human SARS-CoV RBD complexed with hACE2 shows that two salt bridges at the SARS-CoV-hACE2 interface, between hACE2 Lys31 and Glu35 and between hACE2 Lys353 and hACE2 Glu38, are both buried in a hydrophobic environment and contribute critically to the SARS-CoV-hACE2 interactions (Fig. <xref rid="Fig3" ref-type="fig">3</xref>a, c) [<xref ref-type="bibr" rid="CR7">7</xref>]. Disturbance of the formation of either of these salt bridges weakens SARS-CoV-hACE2 binding. The Lys31-Glu35 salt bridge at the SARS-CoV-hACE2 interface becomes an Asn31-Lys35 hydrogen bond at the SARS-CoV-Md-YNACE2 interface (Fig. <xref rid="Fig3" ref-type="fig">3</xref>b), which possibly weakens virus-receptor binding but still is largely compatible with the virus-receptor interface. Thr27 on hACE2 supports the Lys31-Gu35 salt bridge through hydrophobic interactions with Tyr475 (Fig. <xref rid="Fig3" ref-type="fig">3</xref>a); Ile27 on MdACE2 supports the Asn31-Lys35 hydrogen bond more efficiently than Thr27 through tighter hydrophobic interactions with Tyr475 (Fig. <xref rid="Fig3" ref-type="fig">3</xref>b). Moreover, Tyr41 on hACE2 supports the Lys353-Glu38 salt bridge (Fig. <xref rid="Fig3" ref-type="fig">3</xref>c); His41 on MdACE2 supports the same salt bridge less efficiently than Tyr41 (Fig. <xref rid="Fig3" ref-type="fig">3</xref>d). Overall, MdACE2 is an efficient receptor for SARS-CoV, despite the fact that its receptor activity is lower than that of hACE2.<fig id="Fig3"><label>Fig. 3</label><caption><p>Homologous structural modeling of SARS-CoV and Md-YN ACE2 (MdACE2) interactions. <bold>a</bold> Critical salt bridge between hACE2 Lys31 and Glu35 and the hydrophobic residues surrounding it, based on the experimentally determined crystal structure of SARS-CoV RBD complexed with hACE2 (PDB 2AJF). <bold>b</bold> Homologous structural modeling of the hydrogen bond between MdACE2 Asn31 and Lys35. The modeling was done in the program O [<xref ref-type="bibr" rid="CR3">3</xref>]. <bold>c</bold> Critical salt bridge between hACE2 Lys353 and Glu38 and the hydrophobic residues surrounding it, based on the structure of SARS-CoV RBD complexed with hACE2. <bold>d</bold> Homologous structural modeling of the salt bridge between MdaACE2 Lys353 and SARS-CoV Glu38 and the hydrophobic residues surrounding it. Structural illustrations were prepared using the program Povscript [<xref ref-type="bibr" rid="CR2">2</xref>]</p></caption><graphic xlink:href="705_2010_729_Fig3_HTML" id="MO3"></graphic></fig></p><p>Compared with MdACE2, Rs-HB ACE2 contains Glu31 and Glu35, which are not compatible with each other due to their same negative charges, which disfavor virus-receptor binding. However, Rs-HB ACE2 also contains Thr27 and Tyr41, both of which support SARS-CoV entry by contributing favorably to the hydrophobic interactions at the virus-receptor interface. Thus, Rs-HB is a low-efficiency receptor for SARS-CoV. All of the other bat ACE2 molecules contain combinations of the aforementioned key residues that are completely incompatible with virus–receptor interactions. More specifically, they either contain same-charged residues at the 31 and 35 positions, which repel each other, or contain His41 and Lys27, which disfavor SARS-CoV binding (Fig. <xref rid="Fig1" ref-type="fig">1</xref>). In particular, Lys27 on some of these bat ACE2 molecules is incompatible with certain hydrophobic residues, such as Leu443 and Phe460, on SARS-CoV RBD (Fig. <xref rid="Fig3" ref-type="fig">3</xref>a, b). Therefore, these bat ACE2 molecules are not receptors for SARS-CoV.</p></sec><sec id="Sec16"><title>Site-directed mutagenesis analysis</title><p>To confirm the above homologous structural analysis, we carried out site-directed mutagenesis on MdACE2. Our results show that mutations E31K, K35E, and I27T all dramatically decrease the receptor activity of MdACE2, whereas mutation H41Y greatly increases its receptor activity (Fig. <xref rid="Fig2" ref-type="fig">2</xref>a). Therefore, our mutagenesis data further confirmed that key residues in ACE2 determine the receptor activity of MdACE2.</p><p>Our finding that <italic>M. daubentoni</italic> and <italic>R. sinicus</italic> could support SARS-CoV infection has important implications in relation to the origin of SARS-CoV. Since all lines of investigation have indicated that ACE2-binding affinity is among the important determinants for SARS-CoV host range, our data would suggest that <italic>M. Daubentonii</italic> and <italic>R. sinicus</italic> have the potential to serve as the direct reservoirs for human SARS-CoV or its highly related civet SARS-CoV. To further investigate the potential of <italic>M. Daubentonii</italic> and <italic>R. sinicus</italic> as reservoirs for SARS-CoV, more efforts will have to be directed toward widening the surveillance of bats in these families and in different geographical locations.</p><p>Another important finding of our current study is the great genetic diversity of bat ACE2 proteins, which is in contrast to the genetically homogenous hACE2 [<xref ref-type="bibr" rid="CR10">10</xref>]. Sequence variations of bat ACE2, especially in positions that are critical to SARS-CoV binding, such as residues 27, 31, 35, and 41, suggest that, in addition to the Md-YN and Rs-HB ACE2s, there may be many other bats with an ACE2 protein that makes them susceptible to SARS-CoV entry. This again highlights the need for more field surveillance and molecular characterization of different bat ACE2 proteins until the true reservoir host of SARS-CoV is identified and its spillover mechanisms and transmission pathways are fully characterized.</p></sec></sec><sec sec-type="supplementary-material"><title>Electronic supplementary material</title><sec id="SecESM1"><p>Below is the link to the electronic supplementary material.
<supplementary-material content-type="local-data" id="MOESM1"><media xlink:href="705_2010_729_MOESM1_ESM.doc"><caption><p>Supplementary material 1 (DOC 150 kb)</p></caption></media></supplementary-material></p></sec></sec></body><back><ack><p>This work was jointly funded by the State Key Program for Basic Research Grants (2005CB523004, 2010CB530100) from the Chinese Ministry of Science, Technology and the Knowledge Innovation Program Key Project administered by the Chinese Academy of Sciences (KSCX1-YW-R-07) to Z.S. and the CSIRO CEO Science Leader Award to L.-F.W. We thank Gary Crameri and Jennifer Barr for help with live virus infection studies.</p></ack><ref-list id="Bib1"><title>References</title><ref id="CR1"><label>1.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Cui</surname><given-names>J</given-names></name><name><surname>Han</surname><given-names>N</given-names></name><name><surname>Streicker</surname><given-names>D</given-names></name><name><surname>Li</surname><given-names>G</given-names></name><name><surname>Tang</surname><given-names>X</given-names></name><name><surname>Shi</surname><given-names>Z</given-names></name><name><surname>Hu</surname><given-names>Z</given-names></name><name><surname>Zhao</surname><given-names>G</given-names></name><name><surname>Fontanet</surname><given-names>A</given-names></name><name><surname>Guan</surname><given-names>Y</given-names></name><name><surname>Wang</surname><given-names>L</given-names></name><name><surname>Jones</surname><given-names>G</given-names></name><name><surname>Field</surname><given-names>HE</given-names></name><name><surname>Daszak</surname><given-names>P</given-names></name><name><surname>Zhang</surname><given-names>S</given-names></name></person-group><article-title>Evolutionary relationships between bat coronaviruses and their hosts</article-title><source>Emerg Infect Dis</source><year>2007</year><volume>13</volume><fpage>1526</fpage><lpage>1532</lpage><pub-id pub-id-type="pmid">18258002</pub-id></element-citation></ref><ref id="CR2"><label>2.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Fenn</surname><given-names>TD</given-names></name><name><surname>Ringe</surname><given-names>D</given-names></name><name><surname>Petsko</surname><given-names>GA</given-names></name></person-group><article-title>POVScript+: a program for model and data visualization using persistence of vision ray-tracing</article-title><source>J Appl Crystallogr</source><year>2003</year><volume>36</volume><fpage>944</fpage><lpage>947</lpage><pub-id pub-id-type="doi">10.1107/S0021889803006721</pub-id></element-citation></ref><ref id="CR3"><label>3.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Jones</surname><given-names>TA</given-names></name><name><surname>Zou</surname><given-names>JY</given-names></name><name><surname>Cowan</surname><given-names>SW</given-names></name><name><surname>Kjeldgaard</surname><given-names>M</given-names></name></person-group><article-title>Improved methods for building protein models in electron density maps and the location of errors in these models</article-title><source>Acta Crystallogr A</source><year>1991</year><volume>47</volume><fpage>110</fpage><lpage>119</lpage><pub-id pub-id-type="doi">10.1107/S0108767390010224</pub-id><pub-id pub-id-type="pmid">2025413</pub-id></element-citation></ref><ref id="CR4"><label>4.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Ksiazek</surname><given-names>TG</given-names></name><name><surname>Erdman</surname><given-names>D</given-names></name><name><surname>Goldsmith</surname><given-names>CS</given-names></name><name><surname>Zaki</surname><given-names>SR</given-names></name><name><surname>Peret</surname><given-names>T</given-names></name><name><surname>Emery</surname><given-names>S</given-names></name><name><surname>Tong</surname><given-names>S</given-names></name><name><surname>Urbani</surname><given-names>C</given-names></name><name><surname>Comer</surname><given-names>JA</given-names></name><name><surname>Lim</surname><given-names>W</given-names></name><name><surname>Rollin</surname><given-names>PE</given-names></name><name><surname>Dowell</surname><given-names>SF</given-names></name><name><surname>Ling</surname><given-names>AE</given-names></name><name><surname>Humphrey</surname><given-names>CD</given-names></name><name><surname>Shieh</surname><given-names>WJ</given-names></name><name><surname>Guarner</surname><given-names>J</given-names></name><name><surname>Paddock</surname><given-names>CD</given-names></name><name><surname>Rota</surname><given-names>P</given-names></name><name><surname>Fields</surname><given-names>B</given-names></name><name><surname>DeRisi</surname><given-names>J</given-names></name><name><surname>Yang</surname><given-names>JY</given-names></name><name><surname>Cox</surname><given-names>N</given-names></name><name><surname>Hughes</surname><given-names>JM</given-names></name><name><surname>LeDuc</surname><given-names>JW</given-names></name><name><surname>Bellini</surname><given-names>WJ</given-names></name><name><surname>Anderson</surname><given-names>LJ</given-names></name></person-group><article-title>A novel coronavirus associated with severe acute respiratory syndrome</article-title><source>N Engl J Med</source><year>2003</year><volume>348</volume><fpage>1953</fpage><lpage>1966</lpage><pub-id pub-id-type="doi">10.1056/NEJMoa030781</pub-id><pub-id pub-id-type="pmid">12690092</pub-id></element-citation></ref><ref id="CR5"><label>5.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Lau</surname><given-names>SK</given-names></name><name><surname>Woo</surname><given-names>PC</given-names></name><name><surname>Li</surname><given-names>KS</given-names></name><name><surname>Huang</surname><given-names>Y</given-names></name><name><surname>Tsoi</surname><given-names>HW</given-names></name><name><surname>Wong</surname><given-names>BH</given-names></name><name><surname>Wong</surname><given-names>SS</given-names></name><name><surname>Leung</surname><given-names>SY</given-names></name><name><surname>Chan</surname><given-names>KH</given-names></name><name><surname>Yuen</surname><given-names>KY</given-names></name></person-group><article-title>Severe acute respiratory syndrome coronavirus-like virus in Chinese horseshoe bats</article-title><source>Proc Natl Acad Sci USA</source><year>2005</year><volume>102</volume><fpage>14040</fpage><lpage>14045</lpage><pub-id pub-id-type="doi">10.1073/pnas.0506735102</pub-id><pub-id pub-id-type="pmid">16169905</pub-id></element-citation></ref><ref id="CR6"><label>6.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Li</surname><given-names>F</given-names></name><name><surname>Li</surname><given-names>W</given-names></name><name><surname>Farzan</surname><given-names>M</given-names></name><name><surname>Harrison</surname><given-names>SC</given-names></name></person-group><article-title>Structure of SARS coronavirus spike receptor-binding domain complexed with receptor</article-title><source>Science</source><year>2005</year><volume>309</volume><fpage>1864</fpage><lpage>1868</lpage><pub-id pub-id-type="doi">10.1126/science.1116480</pub-id><pub-id pub-id-type="pmid">16166518</pub-id></element-citation></ref><ref id="CR7"><label>7.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Li</surname><given-names>F</given-names></name></person-group><article-title>Structural analysis of major species barriers between humans and palm civets for severe acute respiratory syndrome coronavirus infections</article-title><source>J Virol</source><year>2008</year><volume>82</volume><fpage>6984</fpage><lpage>6991</lpage><pub-id pub-id-type="doi">10.1128/JVI.00442-08</pub-id><pub-id pub-id-type="pmid">18448527</pub-id></element-citation></ref><ref id="CR8"><label>8.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Li</surname><given-names>W</given-names></name><name><surname>Shi</surname><given-names>Z</given-names></name><name><surname>Yu</surname><given-names>M</given-names></name><name><surname>Ren</surname><given-names>W</given-names></name><name><surname>Smith</surname><given-names>C</given-names></name><name><surname>Epstein</surname><given-names>JH</given-names></name><name><surname>Wang</surname><given-names>H</given-names></name><name><surname>Crameri</surname><given-names>G</given-names></name><name><surname>Hu</surname><given-names>Z</given-names></name><name><surname>Zhang</surname><given-names>H</given-names></name><name><surname>Zhang</surname><given-names>J</given-names></name><name><surname>McEachern</surname><given-names>J</given-names></name><name><surname>Field</surname><given-names>H</given-names></name><name><surname>Daszak</surname><given-names>P</given-names></name><name><surname>Eaton</surname><given-names>BT</given-names></name><name><surname>Zhang</surname><given-names>S</given-names></name><name><surname>Wang</surname><given-names>LF</given-names></name></person-group><article-title>Bats are natural reservoirs of SARS-like coronaviruses</article-title><source>Science</source><year>2005</year><volume>310</volume><fpage>676</fpage><lpage>679</lpage><pub-id pub-id-type="doi">10.1126/science.1118391</pub-id><pub-id pub-id-type="pmid">16195424</pub-id></element-citation></ref><ref id="CR9"><label>9.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Li</surname><given-names>W</given-names></name><name><surname>Zhang</surname><given-names>C</given-names></name><name><surname>Sui</surname><given-names>J</given-names></name><name><surname>Kuhn</surname><given-names>JH</given-names></name><name><surname>Moore</surname><given-names>MJ</given-names></name><name><surname>Luo</surname><given-names>S</given-names></name><name><surname>Wong</surname><given-names>SK</given-names></name><name><surname>Huang</surname><given-names>IC</given-names></name><name><surname>Xu</surname><given-names>K</given-names></name><name><surname>Vasilieva</surname><given-names>N</given-names></name><name><surname>Murakami</surname><given-names>A</given-names></name><name><surname>He</surname><given-names>Y</given-names></name><name><surname>Marasco</surname><given-names>WA</given-names></name><name><surname>Guan</surname><given-names>Y</given-names></name><name><surname>Choe</surname><given-names>H</given-names></name><name><surname>Farzan</surname><given-names>M</given-names></name></person-group><article-title>Receptor and viral determinants of SARS-coronavirus adaptation to human ACE2</article-title><source>EMBO J</source><year>2005</year><volume>24</volume><fpage>1634</fpage><lpage>1643</lpage><pub-id pub-id-type="doi">10.1038/sj.emboj.7600640</pub-id><pub-id pub-id-type="pmid">15791205</pub-id></element-citation></ref><ref id="CR10"><label>10.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Li</surname><given-names>W</given-names></name><name><surname>Wong</surname><given-names>SK</given-names></name><name><surname>Li</surname><given-names>F</given-names></name><name><surname>Kuhn</surname><given-names>JH</given-names></name><name><surname>Huang</surname><given-names>IC</given-names></name><name><surname>Choe</surname><given-names>H</given-names></name><name><surname>Farzan</surname><given-names>M</given-names></name></person-group><article-title>Animal origins of the severe acute respiratory syndrome coronavirus: insight from ACE2-S-protein interactions</article-title><source>J Virol</source><year>2006</year><volume>80</volume><fpage>4211</fpage><lpage>4219</lpage><pub-id pub-id-type="doi">10.1128/JVI.80.9.4211-4219.2006</pub-id><pub-id pub-id-type="pmid">16611880</pub-id></element-citation></ref><ref id="CR11"><label>11.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Peiris</surname><given-names>JS</given-names></name><name><surname>Lai</surname><given-names>ST</given-names></name><name><surname>Poon</surname><given-names>LL</given-names></name><name><surname>Guan</surname><given-names>Y</given-names></name><name><surname>Yam</surname><given-names>LY</given-names></name></person-group><article-title>Coronavirus as a possible cause of severe acute respiratory syndrome</article-title><source>Lancet</source><year>2003</year><volume>361</volume><fpage>1319</fpage><lpage>1325</lpage><pub-id pub-id-type="doi">10.1016/S0140-6736(03)13077-2</pub-id><pub-id pub-id-type="pmid">12711465</pub-id></element-citation></ref><ref id="CR12"><label>12.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Ren</surname><given-names>W</given-names></name><name><surname>Li</surname><given-names>W</given-names></name><name><surname>Yu</surname><given-names>M</given-names></name><name><surname>Hao</surname><given-names>P</given-names></name><name><surname>Zhang</surname><given-names>Y</given-names></name><name><surname>Zhou</surname><given-names>P</given-names></name><name><surname>Zhang</surname><given-names>S</given-names></name><name><surname>Zhao</surname><given-names>G</given-names></name><name><surname>Zhong</surname><given-names>Y</given-names></name><name><surname>Wang</surname><given-names>S</given-names></name><name><surname>Wang</surname><given-names>LF</given-names></name><name><surname>Shi</surname><given-names>Z</given-names></name></person-group><article-title>Full-length genome sequences of two SARS-like coronaviruses in horseshoe bats and genetic variation analysis</article-title><source>J Gen Virol</source><year>2006</year><volume>87</volume><fpage>3355</fpage><lpage>3359</lpage><pub-id pub-id-type="doi">10.1099/vir.0.82220-0</pub-id><pub-id pub-id-type="pmid">17030870</pub-id></element-citation></ref><ref id="CR13"><label>13.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Ren</surname><given-names>W</given-names></name><name><surname>Qu</surname><given-names>X</given-names></name><name><surname>Li</surname><given-names>W</given-names></name><name><surname>Han</surname><given-names>Z</given-names></name><name><surname>Yu</surname><given-names>M</given-names></name><name><surname>Zhou</surname><given-names>P</given-names></name><name><surname>Zhang</surname><given-names>SY</given-names></name><name><surname>Wang</surname><given-names>LF</given-names></name><name><surname>Deng</surname><given-names>H</given-names></name><name><surname>Shi</surname><given-names>Z</given-names></name></person-group><article-title>Difference in receptor usage between severe acute respiratory syndrome (SARS) coronavirus and SARS-like coronavirus of bat origin</article-title><source>J Virol</source><year>2008</year><volume>82</volume><fpage>1899</fpage><lpage>1907</lpage><pub-id pub-id-type="doi">10.1128/JVI.01085-07</pub-id><pub-id pub-id-type="pmid">18077725</pub-id></element-citation></ref><ref id="CR14"><label>14.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Tamura</surname><given-names>K</given-names></name><name><surname>Dudley</surname><given-names>J</given-names></name><name><surname>Nei</surname><given-names>M</given-names></name><name><surname>Kumar</surname><given-names>S</given-names></name></person-group><article-title>MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0</article-title><source>Mol Biol Evol</source><year>2007</year><volume>24</volume><fpage>1596</fpage><lpage>1599</lpage><pub-id pub-id-type="doi">10.1093/molbev/msm092</pub-id><pub-id pub-id-type="pmid">17488738</pub-id></element-citation></ref><ref id="CR15"><label>15.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Thompson</surname><given-names>JD</given-names></name><name><surname>Gibson</surname><given-names>TJ</given-names></name><name><surname>Plewniak</surname><given-names>F</given-names></name><name><surname>Jeanmougin</surname><given-names>F</given-names></name><name><surname>Higgins</surname><given-names>DG</given-names></name></person-group><article-title>The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools</article-title><source>Nucleic Acids Res</source><year>1997</year><volume>25</volume><fpage>4876</fpage><lpage>4882</lpage><pub-id pub-id-type="doi">10.1093/nar/25.24.4876</pub-id><pub-id pub-id-type="pmid">9396791</pub-id></element-citation></ref><ref id="CR16"><label>16.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Tu</surname><given-names>C</given-names></name><name><surname>Crameri</surname><given-names>G</given-names></name><name><surname>Kong</surname><given-names>X</given-names></name><name><surname>Chen</surname><given-names>J</given-names></name><name><surname>Sun</surname><given-names>Y</given-names></name><name><surname>Yu</surname><given-names>M</given-names></name><name><surname>Xiang</surname><given-names>H</given-names></name><name><surname>Xia</surname><given-names>X</given-names></name><name><surname>Liu</surname><given-names>S</given-names></name><name><surname>Ren</surname><given-names>T</given-names></name><name><surname>Yu</surname><given-names>Y</given-names></name><name><surname>Eaton</surname><given-names>BT</given-names></name><name><surname>Xuan</surname><given-names>H</given-names></name><name><surname>Wang</surname><given-names>LF</given-names></name></person-group><article-title>Antibodies to SARS coronavirus in civets</article-title><source>Emerg Infect Dis</source><year>2004</year><volume>10</volume><fpage>2244</fpage><lpage>2248</lpage><pub-id pub-id-type="pmid">15663874</pub-id></element-citation></ref><ref id="CR17"><label>17.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Yu</surname><given-names>M</given-names></name><name><surname>Stevens</surname><given-names>V</given-names></name><name><surname>Berry</surname><given-names>JD</given-names></name><name><surname>Crameri</surname><given-names>G</given-names></name><name><surname>McEachern</surname><given-names>J</given-names></name><name><surname>Tu</surname><given-names>C</given-names></name><name><surname>Shi</surname><given-names>Z</given-names></name><name><surname>Liang</surname><given-names>G</given-names></name><name><surname>Weingartl</surname><given-names>H</given-names></name><name><surname>Cardosa</surname><given-names>J</given-names></name><name><surname>Eaton</surname><given-names>BT</given-names></name><name><surname>Wang</surname><given-names>LF</given-names></name></person-group><article-title>Determination and application of immunodominant regions of SARS coronavirus spike and nucleocapsid proteins recognized by sera from different animal species</article-title><source>J Immunol Methods</source><year>2008</year><volume>331</volume><fpage>1</fpage><lpage>12</lpage><pub-id pub-id-type="doi">10.1016/j.jim.2007.11.009</pub-id><pub-id pub-id-type="pmid">18191140</pub-id></element-citation></ref><ref id="CR18"><label>18.</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Yuan</surname><given-names>J</given-names></name><name><surname>Hon</surname><given-names>CC</given-names></name><name><surname>Li</surname><given-names>Y</given-names></name><name><surname>Wang</surname><given-names>D</given-names></name><name><surname>Xu</surname><given-names>G</given-names></name><name><surname>Zhang</surname><given-names>H</given-names></name><name><surname>Zhou</surname><given-names>P</given-names></name><name><surname>Poon</surname><given-names>LL</given-names></name><name><surname>Lam</surname><given-names>TT</given-names></name><name><surname>Leung</surname><given-names>FC</given-names></name><name><surname>Shi</surname><given-names>Z</given-names></name></person-group><article-title>Intraspecies diversity of SARS-like coronaviruses in <italic>Rhinolophus sinicus</italic> and its implications for the origin of SARS coronaviruses in humans</article-title><source>J Gen Virol</source><year>2010</year><volume>91</volume><fpage>1058</fpage><lpage>1062</lpage><pub-id pub-id-type="doi">10.1099/vir.0.016378-0</pub-id><pub-id pub-id-type="pmid">20016037</pub-id></element-citation></ref></ref-list></back></pmc></record>Pour manipuler ce document sous Unix (Dilib)
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