A live, impaired-fidelity coronavirus vaccine protects in an aged, immunocompromised mouse model of lethal disease
Identifieur interne : 000444 ( Pmc/Corpus ); précédent : 000443; suivant : 000445A live, impaired-fidelity coronavirus vaccine protects in an aged, immunocompromised mouse model of lethal disease
Auteurs : Rachel L. Graham ; Michelle M. Becker ; Lance D. Eckerle ; Meagan Bolles ; Mark R. Denison ; Ralph S. BaricSource :
- Nature Medicine [ 1078-8956 ] ; 2012.
Abstract
Attenuated viruses can be highly effective vaccines. In this issue, Ralph Baric and colleagues report that inactivating mutations in the exonuclease ExoN of a mouse-adapted SARS coronavirus impair replication fidelity and cause a mutator phenotype. The resulting attenuated virus protected mice against a lethal coronavirus challenge.
The online version of this article (doi:10.1038/nm.2972) contains supplementary material, which is available to authorized users.
Url:
DOI: 10.1038/nm.2972
PubMed: 23142821
PubMed Central: 3518599
Links to Exploration step
PMC:3518599Le document en format XML
<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">A live, impaired-fidelity coronavirus vaccine protects in an aged, immunocompromised mouse model of lethal disease</title><author><name sortKey="Graham, Rachel L" sort="Graham, Rachel L" uniqKey="Graham R" first="Rachel L" last="Graham">Rachel L. Graham</name><affiliation><nlm:aff id="Aff1"><institution-wrap><institution-id institution-id-type="GRID">grid.10698.36</institution-id><institution-id institution-id-type="ISNI">0000000122483208</institution-id><institution>Department of Epidemiology,</institution><institution>University of North Carolina at Chapel Hill,</institution></institution-wrap>Chapel Hill, North Carolina USA</nlm:aff></affiliation></author><author><name sortKey="Becker, Michelle M" sort="Becker, Michelle M" uniqKey="Becker M" first="Michelle M" last="Becker">Michelle M. Becker</name><affiliation><nlm:aff id="Aff2"><institution-wrap><institution-id institution-id-type="GRID">grid.152326.1</institution-id><institution-id institution-id-type="ISNI">0000 0001 2264 7217</institution-id><institution>Department of Pediatrics,</institution><institution>Vanderbilt University,</institution></institution-wrap>Nashville, Tennessee USA</nlm:aff></affiliation></author><author><name sortKey="Eckerle, Lance D" sort="Eckerle, Lance D" uniqKey="Eckerle L" first="Lance D" last="Eckerle">Lance D. Eckerle</name><affiliation><nlm:aff id="Aff2"><institution-wrap><institution-id institution-id-type="GRID">grid.152326.1</institution-id><institution-id institution-id-type="ISNI">0000 0001 2264 7217</institution-id><institution>Department of Pediatrics,</institution><institution>Vanderbilt University,</institution></institution-wrap>Nashville, Tennessee USA</nlm:aff></affiliation></author><author><name sortKey="Bolles, Meagan" sort="Bolles, Meagan" uniqKey="Bolles M" first="Meagan" last="Bolles">Meagan Bolles</name><affiliation><nlm:aff id="Aff3"><institution-wrap><institution-id institution-id-type="GRID">grid.10698.36</institution-id><institution-id institution-id-type="ISNI">0000000122483208</institution-id><institution>Department of Microbiology and Immunology,</institution><institution>University of North Carolina at Chapel Hill,</institution></institution-wrap>Chapel Hill, North Carolina USA</nlm:aff></affiliation></author><author><name sortKey="Denison, Mark R" sort="Denison, Mark R" uniqKey="Denison M" first="Mark R" last="Denison">Mark R. Denison</name><affiliation><nlm:aff id="Aff2"><institution-wrap><institution-id institution-id-type="GRID">grid.152326.1</institution-id><institution-id institution-id-type="ISNI">0000 0001 2264 7217</institution-id><institution>Department of Pediatrics,</institution><institution>Vanderbilt University,</institution></institution-wrap>Nashville, Tennessee USA</nlm:aff></affiliation><affiliation><nlm:aff id="Aff4"><institution-wrap><institution-id institution-id-type="GRID">grid.152326.1</institution-id><institution-id institution-id-type="ISNI">0000 0001 2264 7217</institution-id><institution>Department of Pathology,</institution><institution>Microbiology and Immunology, Vanderbilt University,</institution></institution-wrap>Nashville, Tennessee USA</nlm:aff></affiliation></author><author><name sortKey="Baric, Ralph S" sort="Baric, Ralph S" uniqKey="Baric R" first="Ralph S" last="Baric">Ralph S. Baric</name><affiliation><nlm:aff id="Aff1"><institution-wrap><institution-id institution-id-type="GRID">grid.10698.36</institution-id><institution-id institution-id-type="ISNI">0000000122483208</institution-id><institution>Department of Epidemiology,</institution><institution>University of North Carolina at Chapel Hill,</institution></institution-wrap>Chapel Hill, North Carolina USA</nlm:aff></affiliation><affiliation><nlm:aff id="Aff3"><institution-wrap><institution-id institution-id-type="GRID">grid.10698.36</institution-id><institution-id institution-id-type="ISNI">0000000122483208</institution-id><institution>Department of Microbiology and Immunology,</institution><institution>University of North Carolina at Chapel Hill,</institution></institution-wrap>Chapel Hill, North Carolina USA</nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">23142821</idno><idno type="pmc">3518599</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3518599</idno><idno type="RBID">PMC:3518599</idno><idno type="doi">10.1038/nm.2972</idno><date when="2012">2012</date><idno type="wicri:Area/Pmc/Corpus">000444</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000444</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">A live, impaired-fidelity coronavirus vaccine protects in an aged, immunocompromised mouse model of lethal disease</title><author><name sortKey="Graham, Rachel L" sort="Graham, Rachel L" uniqKey="Graham R" first="Rachel L" last="Graham">Rachel L. Graham</name><affiliation><nlm:aff id="Aff1"><institution-wrap><institution-id institution-id-type="GRID">grid.10698.36</institution-id><institution-id institution-id-type="ISNI">0000000122483208</institution-id><institution>Department of Epidemiology,</institution><institution>University of North Carolina at Chapel Hill,</institution></institution-wrap>Chapel Hill, North Carolina USA</nlm:aff></affiliation></author><author><name sortKey="Becker, Michelle M" sort="Becker, Michelle M" uniqKey="Becker M" first="Michelle M" last="Becker">Michelle M. Becker</name><affiliation><nlm:aff id="Aff2"><institution-wrap><institution-id institution-id-type="GRID">grid.152326.1</institution-id><institution-id institution-id-type="ISNI">0000 0001 2264 7217</institution-id><institution>Department of Pediatrics,</institution><institution>Vanderbilt University,</institution></institution-wrap>Nashville, Tennessee USA</nlm:aff></affiliation></author><author><name sortKey="Eckerle, Lance D" sort="Eckerle, Lance D" uniqKey="Eckerle L" first="Lance D" last="Eckerle">Lance D. Eckerle</name><affiliation><nlm:aff id="Aff2"><institution-wrap><institution-id institution-id-type="GRID">grid.152326.1</institution-id><institution-id institution-id-type="ISNI">0000 0001 2264 7217</institution-id><institution>Department of Pediatrics,</institution><institution>Vanderbilt University,</institution></institution-wrap>Nashville, Tennessee USA</nlm:aff></affiliation></author><author><name sortKey="Bolles, Meagan" sort="Bolles, Meagan" uniqKey="Bolles M" first="Meagan" last="Bolles">Meagan Bolles</name><affiliation><nlm:aff id="Aff3"><institution-wrap><institution-id institution-id-type="GRID">grid.10698.36</institution-id><institution-id institution-id-type="ISNI">0000000122483208</institution-id><institution>Department of Microbiology and Immunology,</institution><institution>University of North Carolina at Chapel Hill,</institution></institution-wrap>Chapel Hill, North Carolina USA</nlm:aff></affiliation></author><author><name sortKey="Denison, Mark R" sort="Denison, Mark R" uniqKey="Denison M" first="Mark R" last="Denison">Mark R. Denison</name><affiliation><nlm:aff id="Aff2"><institution-wrap><institution-id institution-id-type="GRID">grid.152326.1</institution-id><institution-id institution-id-type="ISNI">0000 0001 2264 7217</institution-id><institution>Department of Pediatrics,</institution><institution>Vanderbilt University,</institution></institution-wrap>Nashville, Tennessee USA</nlm:aff></affiliation><affiliation><nlm:aff id="Aff4"><institution-wrap><institution-id institution-id-type="GRID">grid.152326.1</institution-id><institution-id institution-id-type="ISNI">0000 0001 2264 7217</institution-id><institution>Department of Pathology,</institution><institution>Microbiology and Immunology, Vanderbilt University,</institution></institution-wrap>Nashville, Tennessee USA</nlm:aff></affiliation></author><author><name sortKey="Baric, Ralph S" sort="Baric, Ralph S" uniqKey="Baric R" first="Ralph S" last="Baric">Ralph S. Baric</name><affiliation><nlm:aff id="Aff1"><institution-wrap><institution-id institution-id-type="GRID">grid.10698.36</institution-id><institution-id institution-id-type="ISNI">0000000122483208</institution-id><institution>Department of Epidemiology,</institution><institution>University of North Carolina at Chapel Hill,</institution></institution-wrap>Chapel Hill, North Carolina USA</nlm:aff></affiliation><affiliation><nlm:aff id="Aff3"><institution-wrap><institution-id institution-id-type="GRID">grid.10698.36</institution-id><institution-id institution-id-type="ISNI">0000000122483208</institution-id><institution>Department of Microbiology and Immunology,</institution><institution>University of North Carolina at Chapel Hill,</institution></institution-wrap>Chapel Hill, North Carolina USA</nlm:aff></affiliation></author></analytic><series><title level="j">Nature Medicine</title><idno type="ISSN">1078-8956</idno><idno type="eISSN">1546-170X</idno><imprint><date when="2012">2012</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p id="Par1">Attenuated viruses can be highly effective vaccines. In this issue, Ralph Baric and colleagues report that inactivating mutations in the exonuclease ExoN of a mouse-adapted SARS coronavirus impair replication fidelity and cause a mutator phenotype. The resulting attenuated virus protected mice against a lethal coronavirus challenge.</p><sec><title>Supplementary information</title><p>The online version of this article (doi:10.1038/nm.2972) contains supplementary material, which is available to authorized users.</p></sec></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Jones, Ke" uniqKey="Jones K">KE Jones</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Li, Y" uniqKey="Li Y">Y Li</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Morens, Dm" uniqKey="Morens D">DM Morens</name></author><author><name sortKey="Fauci, As" uniqKey="Fauci A">AS Fauci</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Sessa, R" uniqKey="Sessa R">R Sessa</name></author><author><name sortKey="Palagiano, C" uniqKey="Palagiano C">C Palagiano</name></author><author><name sortKey="Scifoni, Mg" uniqKey="Scifoni M">MG Scifoni</name></author><author><name sortKey="Di Pietro, M" uniqKey="Di Pietro M">M di Pietro</name></author><author><name sortKey="Del Piano, M" uniqKey="Del Piano M">M Del Piano</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Cheng, Vc" uniqKey="Cheng V">VC Cheng</name></author><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="Yuen, Ky" uniqKey="Yuen K">KY Yuen</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Graham, Rl" uniqKey="Graham R">RL Graham</name></author><author><name sortKey="Baric, Rs" uniqKey="Baric R">RS Baric</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Pfefferle, S" uniqKey="Pfefferle S">S Pfefferle</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Vijgen, L" uniqKey="Vijgen L">L Vijgen</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Vignuzzi, M" uniqKey="Vignuzzi M">M Vignuzzi</name></author><author><name sortKey="Wendt, E" uniqKey="Wendt E">E Wendt</name></author><author><name sortKey="Andino, R" uniqKey="Andino R">R Andino</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Drake, Jw" uniqKey="Drake J">JW Drake</name></author><author><name sortKey="Holland, Jj" uniqKey="Holland J">JJ Holland</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Holland, Jj" uniqKey="Holland J">JJ Holland</name></author><author><name sortKey="Domingo, E" uniqKey="Domingo E">E Domingo</name></author><author><name sortKey="De La Torre, Jc" uniqKey="De La Torre J">JC de la Torre</name></author><author><name sortKey="Steinhauer, Da" uniqKey="Steinhauer D">DA Steinhauer</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Vignuzzi, M" uniqKey="Vignuzzi M">M Vignuzzi</name></author><author><name sortKey="Stone, Jk" uniqKey="Stone J">JK Stone</name></author><author><name sortKey="Arnold, Jj" uniqKey="Arnold J">JJ Arnold</name></author><author><name sortKey="Cameron, Ce" uniqKey="Cameron C">CE Cameron</name></author><author><name sortKey="Andino, R" uniqKey="Andino R">R Andino</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Coffey, Ll" uniqKey="Coffey L">LL Coffey</name></author><author><name sortKey="Beeharry, Y" uniqKey="Beeharry Y">Y Beeharry</name></author><author><name sortKey="Borderia, Av" uniqKey="Borderia A">AV Borderia</name></author><author><name sortKey="Blanc, H" uniqKey="Blanc H">H Blanc</name></author><author><name sortKey="Vignuzzi, M" uniqKey="Vignuzzi M">M Vignuzzi</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Lauring, As" uniqKey="Lauring A">AS Lauring</name></author><author><name sortKey="Andino, R" uniqKey="Andino R">R Andino</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Lauring, As" uniqKey="Lauring A">AS Lauring</name></author><author><name sortKey="Jones, Jo" uniqKey="Jones J">JO Jones</name></author><author><name sortKey="Andino, R" uniqKey="Andino R">R Andino</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Eckerle, Ld" uniqKey="Eckerle L">LD Eckerle</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Snijder, Ej" uniqKey="Snijder E">EJ Snijder</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Nga, Pt" uniqKey="Nga P">PT Nga</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Minskaia, E" uniqKey="Minskaia E">E Minskaia</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Eckerle, Ld" uniqKey="Eckerle L">LD Eckerle</name></author><author><name sortKey="Lu, X" uniqKey="Lu X">X Lu</name></author><author><name sortKey="Sperry, Sm" uniqKey="Sperry S">SM Sperry</name></author><author><name sortKey="Choi, L" uniqKey="Choi L">L Choi</name></author><author><name sortKey="Denison, Mr" uniqKey="Denison M">MR Denison</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Denison, Mr" uniqKey="Denison M">MR Denison</name></author><author><name sortKey="Graham, Rl" uniqKey="Graham R">RL Graham</name></author><author><name sortKey="Donaldson, Ef" uniqKey="Donaldson E">EF Donaldson</name></author><author><name sortKey="Eckerle, Ld" uniqKey="Eckerle L">LD Eckerle</name></author><author><name sortKey="Baric, Rs" uniqKey="Baric R">RS Baric</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Frieman, Mb" uniqKey="Frieman M">MB Frieman</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Roberts, A" uniqKey="Roberts A">A Roberts</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Sheahan, T" uniqKey="Sheahan T">T Sheahan</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Frieman, M" uniqKey="Frieman M">M Frieman</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Sheahan, T" uniqKey="Sheahan T">T Sheahan</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Zhao, J" uniqKey="Zhao J">J Zhao</name></author><author><name sortKey="Van Rooijen, N" uniqKey="Van Rooijen N">N Van Rooijen</name></author><author><name sortKey="Perlman, S" uniqKey="Perlman S">S Perlman</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Bolles, M" uniqKey="Bolles M">M Bolles</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Deming, D" uniqKey="Deming D">D Deming</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Subbarao, K" uniqKey="Subbarao K">K Subbarao</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Kew, O" uniqKey="Kew O">O Kew</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Johnson, Aa" uniqKey="Johnson A">AA Johnson</name></author><author><name sortKey="Johnson, Ka" uniqKey="Johnson K">KA Johnson</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Johnson, Aa" uniqKey="Johnson A">AA Johnson</name></author><author><name sortKey="Johnson, Ka" uniqKey="Johnson K">KA Johnson</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Donlin, Mj" uniqKey="Donlin M">MJ Donlin</name></author><author><name sortKey="Patel, Ss" uniqKey="Patel S">SS Patel</name></author><author><name sortKey="Johnson, Ka" uniqKey="Johnson K">KA Johnson</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Patel, Ss" uniqKey="Patel S">SS Patel</name></author><author><name sortKey="Wong, I" uniqKey="Wong I">I Wong</name></author><author><name sortKey="Johnson, Ka" uniqKey="Johnson K">KA Johnson</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Wong, I" uniqKey="Wong I">I Wong</name></author><author><name sortKey="Patel, Ss" uniqKey="Patel S">SS Patel</name></author><author><name sortKey="Johnson, Ka" uniqKey="Johnson K">KA Johnson</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Smits, Sl" uniqKey="Smits S">SL Smits</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Yount, B" uniqKey="Yount B">B Yount</name></author><author><name sortKey="Roberts, Rs" uniqKey="Roberts R">RS Roberts</name></author><author><name sortKey="Lindesmith, L" uniqKey="Lindesmith L">L Lindesmith</name></author><author><name sortKey="Baric, Rs" uniqKey="Baric R">RS Baric</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Yount, B" uniqKey="Yount B">B Yount</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Graham, Rl" uniqKey="Graham R">RL Graham</name></author><author><name sortKey="Sims, Ac" uniqKey="Sims A">AC Sims</name></author><author><name sortKey="Brockway, Sm" uniqKey="Brockway S">SM Brockway</name></author><author><name sortKey="Baric, Rs" uniqKey="Baric R">RS Baric</name></author><author><name sortKey="Denison, Mr" uniqKey="Denison M">MR Denison</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Becker, Mm" uniqKey="Becker M">MM Becker</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Chey, S" uniqKey="Chey S">S Chey</name></author><author><name sortKey="Claus, C" uniqKey="Claus C">C Claus</name></author><author><name sortKey="Liebert, Ug" uniqKey="Liebert U">UG Liebert</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">Nat Med</journal-id><journal-id journal-id-type="iso-abbrev">Nat. Med</journal-id><journal-title-group><journal-title>Nature Medicine</journal-title></journal-title-group><issn pub-type="ppub">1078-8956</issn><issn pub-type="epub">1546-170X</issn><publisher><publisher-name>Nature Publishing Group US</publisher-name><publisher-loc>New York</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">23142821</article-id><article-id pub-id-type="pmc">3518599</article-id><article-id pub-id-type="publisher-id">BFnm2972</article-id><article-id pub-id-type="doi">10.1038/nm.2972</article-id><article-categories><subj-group subj-group-type="heading"><subject>Article</subject></subj-group></article-categories><title-group><article-title>A live, impaired-fidelity coronavirus vaccine protects in an aged, immunocompromised mouse model of lethal disease</article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Graham</surname><given-names>Rachel L</given-names></name><xref ref-type="aff" rid="Aff1">1</xref></contrib><contrib contrib-type="author"><name><surname>Becker</surname><given-names>Michelle M</given-names></name><xref ref-type="aff" rid="Aff2">2</xref></contrib><contrib contrib-type="author"><name><surname>Eckerle</surname><given-names>Lance D</given-names></name><xref ref-type="aff" rid="Aff2">2</xref></contrib><contrib contrib-type="author"><name><surname>Bolles</surname><given-names>Meagan</given-names></name><xref ref-type="aff" rid="Aff3">3</xref></contrib><contrib contrib-type="author"><name><surname>Denison</surname><given-names>Mark R</given-names></name><xref ref-type="aff" rid="Aff2">2</xref><xref ref-type="aff" rid="Aff4">4</xref></contrib><contrib contrib-type="author" corresp="yes"><name><surname>Baric</surname><given-names>Ralph S</given-names></name><address><email>rbaric@email.unc.edu</email></address><xref ref-type="aff" rid="Aff1">1</xref><xref ref-type="aff" rid="Aff3">3</xref></contrib><aff id="Aff1"><label>1</label><institution-wrap><institution-id institution-id-type="GRID">grid.10698.36</institution-id><institution-id institution-id-type="ISNI">0000000122483208</institution-id><institution>Department of Epidemiology,</institution><institution>University of North Carolina at Chapel Hill,</institution></institution-wrap>Chapel Hill, North Carolina USA</aff><aff id="Aff2"><label>2</label><institution-wrap><institution-id institution-id-type="GRID">grid.152326.1</institution-id><institution-id institution-id-type="ISNI">0000 0001 2264 7217</institution-id><institution>Department of Pediatrics,</institution><institution>Vanderbilt University,</institution></institution-wrap>Nashville, Tennessee USA</aff><aff id="Aff3"><label>3</label><institution-wrap><institution-id institution-id-type="GRID">grid.10698.36</institution-id><institution-id institution-id-type="ISNI">0000000122483208</institution-id><institution>Department of Microbiology and Immunology,</institution><institution>University of North Carolina at Chapel Hill,</institution></institution-wrap>Chapel Hill, North Carolina USA</aff><aff id="Aff4"><label>4</label><institution-wrap><institution-id institution-id-type="GRID">grid.152326.1</institution-id><institution-id institution-id-type="ISNI">0000 0001 2264 7217</institution-id><institution>Department of Pathology,</institution><institution>Microbiology and Immunology, Vanderbilt University,</institution></institution-wrap>Nashville, Tennessee USA</aff></contrib-group><pub-date pub-type="epub"><day>11</day><month>11</month><year>2012</year></pub-date><pub-date pub-type="ppub"><year>2012</year></pub-date><volume>18</volume><issue>12</issue><fpage>1820</fpage><lpage>1826</lpage><history><date date-type="received"><day>9</day><month>8</month><year>2012</year></date><date date-type="accepted"><day>14</day><month>9</month><year>2012</year></date></history><permissions><copyright-statement>© Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved. 2012</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" abstract-type="web-summary"><p id="Par1">Attenuated viruses can be highly effective vaccines. In this issue, Ralph Baric and colleagues report that inactivating mutations in the exonuclease ExoN of a mouse-adapted SARS coronavirus impair replication fidelity and cause a mutator phenotype. The resulting attenuated virus protected mice against a lethal coronavirus challenge.</p><sec><title>Supplementary information</title><p>The online version of this article (doi:10.1038/nm.2972) contains supplementary material, which is available to authorized users.</p></sec></abstract><abstract id="Abs2"><p id="Par2">Live, attenuated RNA virus vaccines are efficacious but subject to reversion to virulence. Among RNA viruses, replication fidelity is recognized as a key determinant of virulence and escape from antiviral therapy; increased fidelity is attenuating for some viruses. Coronavirus (CoV) replication fidelity is approximately 20-fold greater than that of other RNA viruses and is mediated by a 3′→5′ exonuclease (ExoN) activity that probably functions in RNA proofreading. In this study we demonstrate that engineered inactivation of severe acute respiratory syndrome (SARS)-CoV ExoN activity results in a stable mutator phenotype with profoundly decreased fidelity <italic>in vivo</italic> and attenuation of pathogenesis in young, aged and immunocompromised mice. The ExoN inactivation genotype and mutator phenotype are stable and do not revert to virulence, even after serial passage or long-term persistent infection <italic>in vivo</italic>. ExoN inactivation has potential for broad applications in the stable attenuation of CoVs and, perhaps, other RNA viruses.</p><sec><title>Supplementary information</title><p>The online version of this article (doi:10.1038/nm.2972) contains supplementary material, which is available to authorized users.</p></sec></abstract><kwd-group kwd-group-type="npg-subject"><title>Subject terms</title><kwd>Live attenuated vaccines</kwd><kwd>Virus-host interactions</kwd><kwd>Pathogenesis</kwd><kwd>Viral infection</kwd></kwd-group><custom-meta-group><custom-meta><meta-name>issue-copyright-statement</meta-name><meta-value>© The Author(s), under exclusive licence to Springer Nature America, Inc. 2012</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id="Sec1"><title>Main</title><p id="Par3">Of the approximately 335 emerging infectious diseases that were identified between 1940 and 2004, 60.3% originated in wildlife<sup><xref ref-type="bibr" rid="CR1">1</xref></sup>. From past pandemics, it is clear that highly pathogenic zoonoses are major threats to global human health, economic stability and national security<sup><xref ref-type="bibr" rid="CR1">1</xref>,<xref ref-type="bibr" rid="CR2">2</xref>,<xref ref-type="bibr" rid="CR3">3</xref>,<xref ref-type="bibr" rid="CR4">4</xref></sup>. SARS-CoV and swine influenza virus CA/04/09 H1N1 have caused substantial human morbidity and mortality in the 21st century. Similar to influenza, CoVs have a strong history of host shifting and cross-species transmission<sup><xref ref-type="bibr" rid="CR5">5</xref>,<xref ref-type="bibr" rid="CR6">6</xref></sup>. In addition to the emergence of SARS-CoV in 2002, which caused 50% mortality in aged populations, several other human CoVs, such as HCoV-NL63, HCoV-OC43 and HCoV-229E, probably emerged from animal reservoirs within the past 200 years<sup><xref ref-type="bibr" rid="CR7">7</xref>,<xref ref-type="bibr" rid="CR8">8</xref></sup>. The sudden emergence of new respiratory viral pathogens from animals underscores the need for new, broadly applicable vaccine strategies that rapidly and rationally attenuate emerging zoonoses, especially to protect vulnerable populations in future outbreaks.</p><p id="Par4">Vaccines have a long history of success in reducing viral disease burdens. Live, attenuated viruses are ideal vaccine candidates, as they elicit balanced innate and adaptive lifelong protective immune responses with low production and delivery costs<sup><xref ref-type="bibr" rid="CR9">9</xref></sup>. Unfortunately, broadly applicable strategies for the rational design of live, attenuated virus vaccines have remained elusive, and vaccines attenuated by chemical treatment or passage can revert to virulence, resulting in disease outbreaks in unvaccinated and immunocompromised populations<sup><xref ref-type="bibr" rid="CR9">9</xref></sup>. Moreover, the precise mechanism of attenuation often remains unclear; thus, the stability of the attenuation cannot be clearly evaluated or assured.</p><p id="Par5">RNA viruses encode RNA-dependent RNA polymerases that lack efficient proofreading capabilities; the resulting high error rates, which range from 10<sup>−3</sup> to 10<sup>−5</sup> mutations per site per round of replication, render RNA viruses highly vulnerable to lethal mutagenesis using chemical agents<sup><xref ref-type="bibr" rid="CR10">10</xref>,<xref ref-type="bibr" rid="CR11">11</xref></sup>. High mutation rates generate considerable genomic diversity, allowing RNA viruses to rapidly adapt to changing environmental conditions and hosts<sup><xref ref-type="bibr" rid="CR12">12</xref></sup>. Increased replication fidelity has been shown to reduce the virulence of poliovirus and chikungunya virus<sup><xref ref-type="bibr" rid="CR12">12</xref>,<xref ref-type="bibr" rid="CR13">13</xref>,<xref ref-type="bibr" rid="CR14">14</xref></sup> and has been proposed as a strategy for live, attenuated virus design<sup><xref ref-type="bibr" rid="CR15">15</xref></sup>. CoVs encode the largest known RNA virus genomes (26–32 kb), exceeding the theoretical limits of viable RNA genome size<sup><xref ref-type="bibr" rid="CR11">11</xref></sup>. Mutation rates are lower in CoVs than in other RNA viruses, approaching 2 × 10<sup>−6</sup> mutations per site per round of replication<sup><xref ref-type="bibr" rid="CR16">16</xref></sup>. Nsp14, encoded in the viral replicase gene, contains a 3′→5′ exoRNase (ExoN) of the DEDDh exonuclease superfamily<sup><xref ref-type="bibr" rid="CR17">17</xref></sup>. In addition to the CoVs, ExoN homologs are present in the members of the Nidovirales order, whose genomes are >20 kb, but are not present in the smaller arteriviruses (with genomes of 12–16 kb), suggesting that the ExoN had a crucial role in genome expansion<sup><xref ref-type="bibr" rid="CR16">16</xref>,<xref ref-type="bibr" rid="CR18">18</xref></sup>. <italic>In vitro</italic>, 3′→5′ exoRNase activity has been demonstrated for recombinant SARS-CoV nsp14 (ref. <xref ref-type="bibr" rid="CR19">19</xref>). We have engineered and recovered viable ExoN inactivation mutants from mouse hepatitis virus (MHV-ExoN) and SARS-CoV (Urbani background, SARS-ExoN). Both MHV-ExoN and SARS-ExoN inactivations are maintained stably for more than ten passages <italic>in vitro</italic> and have 15- to 20-fold increased mutation frequencies compared to wild-type MHV and SARS-CoV<sup><xref ref-type="bibr" rid="CR16">16</xref>,<xref ref-type="bibr" rid="CR20">20</xref></sup>. Thus, ExoN has a crucial role in CoV RNA genome replication fidelity <italic>in vitro</italic>, probably by directly mediating or stimulating proofreading, a function previously unknown among RNA viruses<sup><xref ref-type="bibr" rid="CR21">21</xref></sup>.</p><p id="Par6">In this study, we used the stable, low-fidelity mutator phenotype of the SARS-CoV ExoN mutants to determine whether decreased replication fidelity could be used as a rational design strategy for a live, attenuated vaccine with broad potential applications to other viruses<sup><xref ref-type="bibr" rid="CR16">16</xref>,<xref ref-type="bibr" rid="CR20">20</xref>,<xref ref-type="bibr" rid="CR21">21</xref></sup>. We evaluated (i) the impact of the inactivation of an RNA-proofreading exonuclease and the resultant mutator phenotype on CoV replication, fitness and pathogenesis; (ii) virus stability after passage or persistence <italic>in vivo</italic>; and (iii) the efficacy of using a decreased-fidelity mutant as a vaccine. Further, we assessed the potential for generating stably attenuated, reversion-resistant, immunogenic strains of known and newly identified CoVs to be used as vaccines in both immunocompetent and immunocompromised populations.</p></sec><sec id="Sec2"><title>Results</title><sec id="Sec3"><title>The mutator phenotype and decreased fitness of MA-ExoN</title><p id="Par7">We engineered nsp14 ExoN inactivation mutations into the background of the virulent mouse-adapted SARS-CoV (MAwt), yielding MA-ExoN (<xref rid="Fig1" ref-type="fig">Fig. 1a,b</xref>). We compared MAwt, which causes increased mortality and acute respiratory distress in young and aged mouse models<sup><xref ref-type="bibr" rid="CR22">22</xref>,<xref ref-type="bibr" rid="CR23">23</xref>,<xref ref-type="bibr" rid="CR24">24</xref></sup>, and MA-ExoN in <italic>in vitro</italic> growth experiments (multiplicity of infection (MOI) = 0.1 PFU per cell). MA-ExoN showed a stable growth defect of less than 1 log (<xref rid="Fig1" ref-type="fig">Fig. 1c</xref>). When placed in direct competition, MA-ExoN was clearly less fit than MAwt over successive rounds of infection (<xref rid="MOESM2" ref-type="media">Supplementary Fig. 1a,b</xref>). At 6 h after infection (p.i.), MA-ExoN genome RNA levels were roughly equivalent to those of MAwt and were lower than those of MAwt at 12 h p.i.; by 24 h p.i., MA-ExoN genome RNA levels were approximately 10% of those of MAwt (<xref rid="MOESM2" ref-type="media">Supplementary Fig. 1c</xref>). Thus, the data suggest that MA-ExoN is able to initiate and establish replication efficiently through times of peak RNA synthesis (0–6 h) but has impaired accumulation, which manifests late in one round of infection and is amplified over multiple rounds. These results are consistent with accumulating defects resulting from a markedly increased mutation rate (see the Discussion section).<fig id="Fig1"><label>Figure 1</label><caption><title>The nsp14 ExoN mutator virus in a virulent mouse-adapted SARS-CoV isogenic background.</title><p>(<bold>a</bold>) Genome organization, with the locations of the nsp14 coding sequence (black rectangle) and the mouse-adapted mutations (triangles) shown. ORF1a/b, ORF1a and ORF1b. Structural proteins are labeled as follows: S, spike; E, envelope; M, membrane; N, nucleocapsid. (<bold>b</bold>) Nsp14 ExoN motifs, DEDD domain residues (underlined) and alanine substitutions (D90A and E92A) in motif I recovered in wild-type SARS-CoV and MAwt backgrounds. (<bold>c</bold>) Growth analysis (MOI = 0.1 PFU per cell) of wild-type SARS-CoV, S-ExoN, MAwt and MA-ExoN on Vero cells. Error bars, s.d. (<bold>d</bold>) Mutation frequency from complete genome sequencing of plaque isolates of MAwt and MA-ExoN (<italic>n</italic> = 5 for both) at passage 3. The increase in mean mutation frequency (horizontal lines) in MA-ExoN compared to MAwt (11.5×) is indicated. *<italic>P</italic> < 0.01 (Mann-Whitney nonparametric test for independent samples). (<bold>e</bold>) The mutations identified with complete genome sequencing across five clones from each group. Filled circles, nonsynonymous mutations; open circles, synonymous mutations; black, noncoding mutations; red, mutations present in more than one clone; blue, mutations present in only one clone. Mouse-adapted mutations are shown as triangles on the genome schematic and were present in all sequenced genomes.</p></caption><graphic xlink:href="41591_2012_Article_BFnm2972_Fig1_HTML" id="d29e481"></graphic></fig></p><p id="Par8">We then sequenced RNA from multiple MAwt and MA-ExoN plaques. Both the MAwt background and engineered ExoN mutations were present in all sequenced MA-ExoN clones. Additionally, MA-ExoN accumulated 14-fold more unique mutations and had a mean 11.5-fold greater mutation frequency compared to MAwt (<italic>P</italic> <0.01) (<xref rid="Fig1" ref-type="fig">Fig. 1d,e</xref>). These results confirm that the growth and replication fidelity impairments of the nsp14 ExoN mutator phenotype are present in MA-ExoN and are indistinguishable from those in SARS-ExoN during replication in culture.</p></sec><sec id="Sec4"><title>MA-ExoN is attenuated <italic>in vivo</italic></title><p id="Par9">To assess MA-ExoN virulence, we infected young (10-week-old) and aged (14-month-old) female BALB/c mice with MA-ExoN or MAwt (<xref rid="Fig2" ref-type="fig">Fig. 2</xref>). Young mice infected with MAwt showed dose-dependent weight loss and recovery (<xref rid="Fig2" ref-type="fig">Fig. 2a</xref>), though they had no observable dose-dependent differences in lung titers or clearance after day 4 p.i. (<xref rid="Fig2" ref-type="fig">Fig. 2b</xref>). In contrast, young mice infected with MA-ExoN showed no signs of clinical disease and had high but not dose-dependent lung titers that were rapidly cleared by day 4 p.i. (<xref rid="Fig2" ref-type="fig">Fig. 2a,b</xref>). We then compared MA-ExoN and MAwt infection in aged, immunosenescent mice<sup><xref ref-type="bibr" rid="CR25">25</xref></sup>. Mice infected with either virus experienced dose-dependent weight loss (<xref rid="Fig2" ref-type="fig">Fig. 2c</xref>); however, although lung titers were equivalent across all doses of MA-ExoN and MAwt on day 2 p.i., mice infected with MA-ExoN cleared the virus independent of inoculation dose, whereas mice infected with MAwt had begun to clear the virus from higher-titer infections more efficiently at day 4 p.i. than from lower-titer infections (<xref rid="Fig2" ref-type="fig">Fig. 2d</xref>). Additionally, whereas aged mice infected with MA-ExoN had no mortality, MAwt-infected mice had dose-dependent mortality (<xref rid="Fig2" ref-type="fig">Fig. 2e</xref>). As described previously<sup><xref ref-type="bibr" rid="CR23">23</xref></sup>, we found little if any virus in other organs. These experiments demonstrate that MA-ExoN is attenuated in both young and aged diseased mice compared to virulent MAwt and that disease symptoms, when present, are less pronounced in MA-ExoN infections than MAwt infections.<fig id="Fig2"><label>Figure 2</label><caption><title>Weight loss and lung titer in BALB/c mice.</title><p>(<bold>a</bold>–<bold>e</bold>) MA-ExoN and MAwt infections of 10-week-old (<bold>a</bold>,<bold>b</bold>) and 12- to 14-month-old (<bold>c</bold>–<bold>e</bold>) female BALB/c mice. (<bold>a</bold>,<bold>c</bold>) Weight loss. Dark shapes and solid lines, MA-ExoN; white shapes and dashed lines, MAwt; diamonds, 10<sup>2</sup> PFU; squares, 10<sup>3</sup> PFU; triangles, 10<sup>4</sup> PFU. Error bars indicate s.d. (<bold>b</bold>,<bold>d</bold>) Lung titers. The titer for each mouse lung is indicated by an open circle; the mean titer of all mice at each time point is indicated by a horizontal bar. Days p.i. are indicated on the <italic>x</italic> axes; 2log, 3log and 4log indicate the MOIs of each virus (10<sup>2</sup>, 10<sup>3</sup> or 10<sup>4</sup> PFU, respectively). (<bold>e</bold>) Survival within aged mouse groups, calculated as the percentage of surviving mice compared to the total number of mice remaining on each day of the experiment. Line weights and symbols are as in <bold>a</bold> and <bold>c</bold>.</p></caption><graphic xlink:href="41591_2012_Article_BFnm2972_Fig2_HTML" id="d29e600"></graphic></fig></p><p id="Par10">A potential concern with live, attenuated vaccines is the chance that they could revert to virulence <italic>in vivo</italic>, particularly in immunocompromised individuals. Therefore, we assessed whether MA-ExoN was attenuated in immunocompromised mice. We used MAwt and MA-ExoN to infect young <italic>Rag</italic><sup>−/−</sup> (recombination activating gene), severe combined immunodeficiency (SCID) and <italic>Stat1</italic><sup>−/−</sup> (signal transducer and activator of transcription 1) mice, as well as background controls (C57BL/6, BALB/c and 129 mice, respectively). All MA-ExoN–infected mice had significantly less weight loss than MAwt-infected mice (<italic>P</italic> < 0.05; <xref rid="Fig3" ref-type="fig">Fig. 3a–c</xref> and <xref rid="MOESM2" ref-type="media">Supplementary Table 1</xref>). Only <italic>Stat1</italic><sup>−/−</sup> mice had any notable weight loss (∼15%) as a result of MA-ExoN infection; however, these mice did not pass experimental morbidity thresholds (<xref rid="Fig3" ref-type="fig">Fig. 3c</xref>). In contrast, all MAwt-infected <italic>Stat1</italic><sup>−/−</sup> mice died or were moribund by day 9 p.i., but MAwt infection was not lethal in C57BL/6 or 129 control mice (<xref rid="Fig3" ref-type="fig">Fig. 3a,c</xref>), as has been previously reported<sup><xref ref-type="bibr" rid="CR22">22</xref>,<xref ref-type="bibr" rid="CR26">26</xref></sup>. <italic>Rag</italic><sup>−/−</sup> and SCID mice maintained detectable amounts of MAwt and MA-ExoN virus for 14 d (<italic>Rag</italic><sup>−/−</sup>) or 60 d (SCID) beyond the background controls (<xref rid="Fig3" ref-type="fig">Fig. 3d,e</xref>) but showed no signs of illness over the course of the experiment despite a lack of viral clearance, expanding earlier reports from our laboratory that MAwt does not clear from <italic>Rag</italic><sup>−/−</sup> mice<sup><xref ref-type="bibr" rid="CR26">26</xref></sup>. The rapid clearance of MA-ExoN infection from <italic>Stat1</italic><sup>−/−</sup> mice (<xref rid="Fig3" ref-type="fig">Fig. 3f</xref>) further supports the hypothesis that clearance of SARS-CoV infection is dependent on both B and T cells<sup><xref ref-type="bibr" rid="CR27">27</xref></sup>.<fig id="Fig3"><label>Figure 3</label><caption><title>Weight loss and lung titer in young immunocompromised mice.</title><p>(<bold>a</bold>–<bold>c</bold>) MA-ExoN and MAwt infections of 10-week-old <italic>Rag</italic><sup>−/−</sup> (<bold>a</bold>), SCID (<bold>b</bold>) and <italic>Stat1</italic><sup>−/−</sup> (<bold>c</bold>) mice. C57BL/6 (<bold>a</bold>), BALB/c (<bold>b</bold>) and 129 (<bold>c</bold>) mice were included as background controls. Weight loss is shown. Dark shapes and solid lines, immunocompromised mice; white shapes and dashed lines, background control mice; circles, MA-ExoN; squares, MAwt. Error bars indicate s.d. (<bold>d</bold>–<bold>f</bold>) Lung titers for <italic>Rag</italic><sup>−/−</sup> (<bold>d</bold>), SCID (<bold>e</bold>) and <italic>Stat1</italic><sup>−/−</sup> (<bold>f</bold>) mice and background controls. The titer of each mouse lung is indicated by an open circle; the mean of all titers at each time point is indicated by a horizontal bar. Days p.i. are indicated on the <italic>x</italic> axes.</p></caption><graphic xlink:href="41591_2012_Article_BFnm2972_Fig3_HTML" id="d29e761"></graphic></fig></p></sec><sec id="Sec5"><title>Mutation accumulation during persistent <italic>in vivo</italic> infection</title><p id="Par11">Infection with both MAwt and MA-ExoN persisted for at least 60 d in SCID mice (<xref rid="Fig3" ref-type="fig">Fig. 3e</xref>), potentially allowing for the most longitudinal cycles of replication and lowest immune barriers to the emergence of mutations conferring increased fitness, reversion to virulence and fidelity-compensating changes of any of the groups examined. To test this, we sequenced viral genomes from viral plaques grown from SCID mouse lung homogenates at 30 d p.i. (<xref rid="Fig4" ref-type="fig">Fig. 4</xref> and <xref rid="MOESM2" ref-type="media">Supplementary Table 2</xref>). We identified a total of 14 consensus mutations (∼100,000 nt) for MAwt, with 3 mutations shared in two or three genomes, resulting in 11 distinct mutations (4 synonymous and 7 nonsynonymous). For MA-ExoN, the engineered inactivation mutations were maintained. In contrast to MAwt, MA-ExoN plaques contained a total of 91 mutations (89 distinct: 32 synonymous and 57 nonsynonymous), corresponding to a 9.6-fold higher total mutation accumulation compared to MAwt.<fig id="Fig4"><label>Figure 4</label><caption><title>Mutation accumulation in infected SCID mice at 30 d.p.i.</title><p>The SARS-CoV genome is depicted at the top. The nsp14 ExoN coding region is denoted by a purple box, with the inactivating amino acid changes indicated above the schematic. The receptor-binding domain (RBD) is denoted by a black box. Individual SCID mouse genome sequences are represented by black horizontal lines. Dashed lines separate the nonstructural protein sequences in ORF1 and downstream ORFs. Mutations are indicated by lollipop shapes colored as follows: blue, synonymous, unique to one sequence; light blue, synonymous, present in three sequences; red, nonsynonymous, unique to one sequence; green, synonymous, present in two sequences; purple, nsp14 ExoN inactivation mutations. Mutations that alter the size of an ORF are indicated by a red Δ (deletion) or a red S (stop codon). Genome sizes, ORF and nonstructural protein boundaries and mutation marker placements are approximate.</p></caption><graphic xlink:href="41591_2012_Article_BFnm2972_Fig4_HTML" id="d29e789"></graphic></fig></p><p id="Par12">We compared mutation accumulations across two separate regions (open reading frame 1a (ORF1a), nucleotides 493–8603, and ORF1b, nucleotides 12915–16520; <xref rid="MOESM2" ref-type="media">Supplementary Figs. 2</xref> and <xref rid="MOESM2" ref-type="media">3</xref> and <xref rid="MOESM2" ref-type="media">Supplementary Table 2</xref>) for statistical determinations. Mutation accumulations were significantly higher in MA-ExoN–infected mice for both regions (<italic>P</italic> < 0.01). Additionally, there was a mean 18.3-fold increased accumulation for MA-ExoN across the ORF1a region. When we normalized the accumulations of mutations per 10 kb, MA-ExoN mutation accumulations in the ORF1a compared to the ORF1b region were not significantly different (<italic>P</italic> = 0.340) but remained significantly increased compared to the accumulation in MAwt (<italic>P</italic> < 0.001 for both ORF1a and ORF1b). We identified no new mutations in any of the three MA-ExoN plaques, suggesting no obligatory or consistent pattern of adaptation or mutational bias. The most prevalent mutation identified (C16999M) was also present in viral stocks as a polymorphism; however, its frequency in the viral population remained stable (∼40%) both <italic>in vitro</italic> and <italic>in vivo</italic> and in experiments with both BALB/c and SCID mice (<xref rid="MOESM2" ref-type="media">Supplementary Tables 2</xref> and <xref rid="MOESM2" ref-type="media">3</xref> and data not shown). The results from persistent ExoN infection over 30 d were consistent with the results from passage of SARS-ExoN virus in culture: the ratios of accumulation of unique mutations (MA-ExoN:MAwt) during replication in SCID mice ranged from ∼9.6:1 to 18.3:1, which is similar to the ratio measured between SARS-ExoN and SARS-CoV in culture<sup><xref ref-type="bibr" rid="CR16">16</xref></sup>.</p></sec><sec id="Sec6"><title>MA-ExoN resists reversion to virulence <italic>in vivo</italic></title><p id="Par13">To test the resistance to reversion to virulence during passage <italic>in vivo</italic>, we subjected MAwt and MA-ExoN to both short and long serial passages (24 h and 72 h per passage, respectively) in aged BALB/c mice (<xref rid="Fig5" ref-type="fig">Fig. 5</xref>). In both groups, viral titer remained stable from passage to passage (<xref rid="Fig5" ref-type="fig">Fig. 5a,b</xref>); additionally, viral plaque phenotypes were preserved, and the ExoN-inactivating mutations and amino acid substitutions were maintained (data not shown). In contrast, whereas MAwt titer remained stable over the 24-h serial passage, mice inoculated with lung homogenates from the 72-h passage died by day 3 in passage 2 (<xref rid="Fig5" ref-type="fig">Fig. 5b</xref>). Although MA-ExoN titer remained stable over the 72-h passage, mouse weights did not decrease during infection after passage 1 (<xref rid="Fig5" ref-type="fig">Fig. 5c</xref>). Notably, there was no evidence of a gain of virulence with serial passage in the MA-ExoN pathogenesis model: when aged mice were infected with lung homogenates from each of the final 24-h and 72-h passages, mice infected with MA-ExoN passages lost no or little weight, and MAwt-infected mice became moribund (<xref rid="Fig5" ref-type="fig">Fig. 5d</xref>).<fig id="Fig5"><label>Figure 5</label><caption><title>Virulence of passaged MA-ExoN and MAwt viruses.</title><p>(<bold>a</bold>–<bold>c</bold>) MA-ExoN and MAwt serial infections of 12-month-old female BALB/c mice. Passages (P) are indicated on the <italic>x</italic> axes. (<bold>a</bold>) Serial passage after 24 h. (<bold>b</bold>,<bold>c</bold>) Serial passage after 72 h. (<bold>a</bold>,<bold>b</bold>) Lung titers are shown, with average titers indicated by bars. In the 72-h passage, MAwt-infected mice died by day 3 of passage 2; thus, the viruses were not passaged further. (<bold>c</bold>) Weight loss in the mice infected in the 72-h passage is shown. Error bars indicate s.d. (<bold>d</bold>) MA-ExoN (24-h passage 8 and 72-h passage 3) and MAwt (passage 1) infections of 12-month-old female BALB/c mice. Weight loss is shown. Error bars indicate s.d. (<bold>e</bold>) Infections of 10-week-old female BALB/c mice with MA-ExoN and MAwt population viruses isolated from SCID mice at 30 d p.i.; weight loss is shown. Dashed line, mock (PBS) inoculation; dark shapes and solid lines, MA-ExoN; white shapes and solid lines, MAwt. For each virus, lungs from three separate mice were harvested (mouse 1–mouse 3), and viruses were subsequently inoculated without plaque purification (population); circles, mouse 1 populations (pop 1); squares, mouse 2 populations (pop 2); triangles, mouse 3 populations (pop 3). (<bold>f</bold>) Weight loss in 11-month-old female BALB/c mice infected with MA-ExoN 2.1 (with C16999A) or MA-ExoN 3.1 (without C16999A) plaque isolates from SCID mice after 30 d of infection (SCID30). Squares, SCID30 MA-ExoN 2.1; circles, SCID30 MA-ExoN 3.1. Error bars indicate s.d.</p></caption><graphic xlink:href="41591_2012_Article_BFnm2972_Fig5_HTML" id="d29e901"></graphic></fig></p><p id="Par14">To test whether persistent MA-ExoN infection could result in phenotypic reversion to virulence, we used viruses harvested from SCID mice at day 30 p.i. to infect young BALB/c mice (<xref rid="Fig5" ref-type="fig">Fig. 5e</xref>). MAwt-infected mice showed signs of morbidity (weight loss, hunched posture and ruffled fur) but recovered. In contrast, MA-ExoN–infected mice showed no clinical signs of illness, as in the initial infection (<xref rid="Fig2" ref-type="fig">Fig. 2a</xref>). Additionally, plaques containing different mutational subsets were identically attenuated after re-infection of BALB/c mice (<xref rid="Fig5" ref-type="fig">Fig. 5f</xref>). These results demonstrate that after 30 d of persistent infection, the ExoN mutator phenotype did not revert to virulence despite the greatly increased mutation rate and population diversity.</p></sec><sec id="Sec7"><title>MA-ExoN vaccination protects mice from lethal challenge</title><p id="Par15">Aged mice mount poor productive immune responses to SARS-CoV vaccines and remain highly susceptible to severe disease and lethal infection<sup><xref ref-type="bibr" rid="CR24">24</xref>,<xref ref-type="bibr" rid="CR28">28</xref></sup>, thus representing the most sensitive means to measure vaccine efficacy against lethal SARS-CoV infection. To test the efficacy of the MA-ExoN mutant as a possible vaccine against lethal challenge, we vaccinated aged BALB/c mice with MA-ExoN and allowed them to recover from infection. We then challenged the mice with a lethal dose of MAwt (<xref rid="Fig6" ref-type="fig">Fig. 6</xref>). Mock-vaccinated mice succumbed to MAwt challenge by day 3 p.i. and had high lung titers (<xref rid="Fig6" ref-type="fig">Fig. 6a,b</xref>); however, mice vaccinated with MA-ExoN were protected from illness (<xref rid="Fig6" ref-type="fig">Fig. 6a</xref>). Further, in contrast to other vaccine platforms<sup><xref ref-type="bibr" rid="CR24">24</xref>,<xref ref-type="bibr" rid="CR28">28</xref>,<xref ref-type="bibr" rid="CR29">29</xref></sup>, MA-ExoN–vaccinated mice had no detectable lung titers 2 d after challenge (<xref rid="Fig6" ref-type="fig">Fig. 6b</xref>).<fig id="Fig6"><label>Figure 6</label><caption><title>MA-ExoN vaccination protects from lethal challenge.</title><p>(<bold>a</bold>,<bold>b</bold>) Low-passage MA-ExoN and mock (PBS) vaccinations of 12-month-old female BALB/c mice followed by lethal challenge with MAwt. (<bold>a</bold>) Weight loss in challenged mice. Dark circles, 10<sup>4</sup> PFU MA-ExoN vaccination; white circles, 10<sup>2.5</sup> PFU MA-ExoN vaccination; dark triangles, PBS vaccination. Error bars indicate s.d. (<bold>b</bold>) Lung titers at day 2 after challenge. ExoN was given at a 2.5log (10<sup>2.5</sup> PFU) vaccination. The mean titer is indicated by a bar in each group. (<bold>c</bold>) Fifty-percent plaque reduction neutralization titer (PRNT<sub>50</sub>) assay using sera from PBS-vaccinated and MA-ExoN–vaccinated mice to neutralize MAwt. Reciprocal dilutions capable of effecting 50% plaque reduction are shown by circles; mean reciprocal dilutions are indicated by a bar for each group. The limit of detection for each assay, if given, is indicated by a dashed line.</p></caption><graphic xlink:href="41591_2012_Article_BFnm2972_Fig6_HTML" id="d29e986"></graphic></fig></p><p id="Par16">Additionally, mice vaccinated with MA-ExoN generated high amounts of neutralizing antibodies (mean, 1:311 ± 37.5 reciprocal 50% neutralization titer) (<xref rid="Fig6" ref-type="fig">Fig. 6c</xref>). The minimal neutralizing titers for protection against SARS-CoV infection in mice have been reported as 1:25–1:49 (ref. <xref ref-type="bibr" rid="CR30">30</xref>). Thus, even with a single vaccination, MA-ExoN provided complete protection against lethal challenge in a susceptible, immunosenescent mouse model of lethal SARS-CoV infection. To our knowledge, the MA-ExoN virus is the first approach to SARS-CoV immunization that fully protects against clinical disease and viral replication in an aged mouse model<sup><xref ref-type="bibr" rid="CR24">24</xref>,<xref ref-type="bibr" rid="CR28">28</xref>,<xref ref-type="bibr" rid="CR29">29</xref></sup>.</p></sec></sec><sec id="Sec8"><title>Discussion</title><p id="Par17">Live, attenuated vaccines have substantially reduced the global disease burden associated with viral infections, including, for example, those of measles, mumps, rubella, polio, yellow fever and chickenpox<sup><xref ref-type="bibr" rid="CR9">9</xref>,<xref ref-type="bibr" rid="CR15">15</xref></sup>. However, live, attenuated vaccines carry several risks, including primary or secondary reversion to a virulent phenotype, as in the case of poliovirus<sup><xref ref-type="bibr" rid="CR9">9</xref></sup>. In fact, attenuated phenotypes encounter natural selective pressures for reversion that can cause outbreaks of disease in unvaccinated populations<sup><xref ref-type="bibr" rid="CR31">31</xref></sup>.</p><p id="Par18">RNA virus replication fidelity has evolved to balance genome diversity and stability; therefore, inactivating an enzyme, such as nsp14-ExoN, that is responsible for high-fidelity replication could theoretically drive the virus toward instability, deleterious mutational diversity and decreased fitness in complex environments, which we observed here <italic>in vitro</italic>. It is possible that nsp14-ExoN may have other functions in viral RNA synthesis<sup><xref ref-type="bibr" rid="CR21">21</xref></sup>; however, global impairment of viral RNA synthesis alone cannot explain the <italic>in vitro</italic> results and the attenuating phenotype <italic>in vivo</italic>.</p><p id="Par19">It is not possible to fully separate defects caused by increased mutation load from those resulting from the replication defect observed, and both probably contribute to the phenotype. However, our results are consistent with the hypothesis that both stable and evolving defects resulting from the mutator phenotype have irrevocably attenuated MA-ExoN. These defects could include (i) mutations that impair or terminate translation, replication and transcription; (ii) mutations that impair or abolish protein functions; or (iii) changes in RNA polymerase processivity in the presence of an inactivated proofreading exonuclease. These combined impairments may be manifested with CoV ExoN mutants, as the high number of accumulated mutations per genome is unprecedented among viral mutator strains. Indeed, the phenotype observed with CoV ExoN inactivation is similar to those reported for other polymerase complexes with inactivated exonucleases, such as human mitochondrial DNA polymerase γ (pol γ)<sup><xref ref-type="bibr" rid="CR32">32</xref>,<xref ref-type="bibr" rid="CR33">33</xref></sup> and bacteriophage T7 DNA polymerase<sup><xref ref-type="bibr" rid="CR34">34</xref>,<xref ref-type="bibr" rid="CR35">35</xref>,<xref ref-type="bibr" rid="CR36">36</xref></sup>. In pol γ studies, the loss of proofreading was associated with impaired polymerase activity in a manner that was probably causal, impossible to uncouple and characterized by decreased speed, increased template dissociation and restricted access of nucleotides to the polymerase active site<sup><xref ref-type="bibr" rid="CR32">32</xref>,<xref ref-type="bibr" rid="CR33">33</xref></sup>. For CoVs, the high levels of iterative amplification of both genomic and subgenomic RNA would further accelerate these deleterious processes by providing aberrant templates. The loss of ExoN proofreading would continuously generate new potentially attenuating alleles, and defective genomes and would reduce both genome fitness and the risks for primary and secondary reversions to virulence.</p><p id="Par20">In this study, we demonstrate that MA-ExoN is attenuated in mice and that the mutant clears rapidly in the presence of an adaptive immune response. Although our <italic>in vivo</italic> experiments with MAwt recapitulated many of the phenotypes observed in aged and immunocompromised human populations, additional testing of MA-ExoN as a vaccine in primates will be necessary to further confirm its stability <italic>in vivo</italic><sup><xref ref-type="bibr" rid="CR37">37</xref></sup>. Experiments in SCID mice with persistent MA-ExoN infection verified the accumulation of mutations across the genome without evidence for the selection of either phenotypic virulence-enhancing alleles or primary genotypic reversion. Viruses harvested after passage remained avirulent, supporting the conclusions that (i) selection for virulence is not occurring; (ii) selection is being outcompeted by the gradual accumulation of attenuating mutations in individual genomes or the population mutational swarm; or (iii) the mutant is unable to generate or select for either fast-growing or slow-clearing viruses that are also more virulent. Not surprisingly, we identified a limited number of polymorphisms in the MA-ExoN virus stock that we used in subsequent experiments. Viruses with and without these mutations were fully attenuated <italic>in vivo</italic>, and the mutations were also maintained in lungs in the same frequencies as in the virus stock, suggesting that they were not selected against during passage <italic>in vivo</italic>. Thus, no single mutation or polymorphism could be clearly linked to viral attenuation except for the ExoN inactivation.</p><p id="Par21">Notably, we have shown that MA-ExoN vaccinations are completely protective against replication and lethal challenge in aged BALB/c mice, the SARS-CoV mouse pathogenesis model that captures most of the severe clinical disease outcomes in human infections. Additionally, neutralization titers were equivalent or superior to those reported in studies of two-dose alphavirus replicon S glycoprotein vaccines and killed vaccines containing alum, with the additional advantage of protecting against virus replication and clinical disease<sup><xref ref-type="bibr" rid="CR24">24</xref>,<xref ref-type="bibr" rid="CR28">28</xref>,<xref ref-type="bibr" rid="CR29">29</xref></sup>.</p><p id="Par22">Live, attenuated vaccines must have two characteristics, aside from the capacity to elicit a protective immune response: resistance to primary reversion and stable attenuation at secondary sites. We have demonstrated that MA-ExoN has both of these characteristics. In all circumstances, the engineered inactivation mutations were maintained, indicating that exonuclease activity is not crucial for the virus life cycle and that the 4-nt, 2–amino acid change presents a substantial barrier to primary reversion; further, the passage experiments suggest that the virus lacks redundant or complementing mechanisms to fully restore the loss of ExoN activity. In addition, MA-ExoN harvested from persistently infected SCID mice retained an attenuated phenotype when re-inoculated into young BALB/c mice, suggesting that persistence does not select for virulence. Future studies are necessary to address whether additional modifications could enhance and stabilize the attenuated phenotype by reducing the likelihood of gain of function by homologous recombination, such as introduction of the ExoN inactivation in a background with rewired transcriptional regulatory sequences<sup><xref ref-type="bibr" rid="CR38">38</xref></sup>, which could increase resistance to reversion.</p><p id="Par23">The inactivation of putative viral proofreading components in the pursuit of a stable vaccine constitutes a paradigm that has high potential to be broadly applicable to those members of the Nidovirales order with an exonuclease activity. In a time when metagenomics studies inform us of the likelihood of future viral emergence events—viruses that have the potential to afflict the human population much as SARS-CoV did in 2002–2003—the design and ready implementation of an attenuation strategy that can be rapidly applied to any emerging CoV potentially represents a major advance in the preservation of public health. These data should also encourage the pursuit of fidelity-impairing mutations in the replicase proteins of other RNA viruses as potential targets or the use of CoVs as vaccine vectors for rational vaccine design.</p></sec><sec id="Sec9"><title>Methods</title><sec id="Sec10"><title>Construction of SARS D plasmid with ExoN and mouse-adapted mutations.</title><p id="Par24">A SARS-CoV D plasmid was constructed by restriction digestion and ligation of existing SARS D–ExoNI<sup><xref ref-type="bibr" rid="CR16">16</xref></sup> and SARS D mouse-adapted<sup><xref ref-type="bibr" rid="CR23">23</xref></sup> plasmids. Briefly, both plasmids were restriction digested with BstB I and Xba I enzymes. After treatment of the digested SARS D–ExoNI plasmid with Antarctic Phosphatase (New England BioLabs, Ipswitch, MA, USA), fragments were isolated, purified and ligated together using T4 DNA Ligase overnight at 4 °C as described previously<sup><xref ref-type="bibr" rid="CR39">39</xref></sup>. Colonies were screened for proper insert size by restriction digestion and electrophoresis, and the presence of the appropriate mutations was verified by sequencing.</p></sec><sec id="Sec11"><title>Generation of SARS-CoV MA-ExoN mutant virus.</title><p id="Par25">Virus containing the ExoNI inactivation and mouse-adapted mutations within the viral coding sequence was produced using the infectious complementary DNA (cDNA) assembly strategy for SARS-CoV as previously described<sup><xref ref-type="bibr" rid="CR39">39</xref>,<xref ref-type="bibr" rid="CR40">40</xref></sup>. ExoN viruses were kept at low passage (one passage past virus rescue, P1) to minimize the accumulation of mutations in cell culture. For this study, an equivalently low-passaged MAwt virus was used for comparison.</p></sec><sec id="Sec12"><title><italic>In vitro</italic> passage series and viral growth and plaque assays.</title><p id="Par26">MAwt and MA-ExoN viruses were grown in Vero cells at MOI = 0.1 PFU per cell in all <italic>in vitro</italic> experiments, with the exception of the genome RNA quantification experiment, which was performed at MOI = 3 PFU per cell. Passage series and growth experiments were performed, and viral titers were determined as previously described<sup><xref ref-type="bibr" rid="CR16">16</xref></sup>.</p></sec><sec id="Sec13"><title>Competition assay.</title><p id="Par27">Vero cells were plated at 10<sup>6</sup> cells per well in six-well plates. Cells were infected at an MOI of 0.1 with combinations of MAwt and MA-ExoN at 1:1, 1:10 and 1:100 ratios favoring either MA-ExoN or MAwt and were incubated for 24 h at 37 °C. After 24 h, 100-μl aliquots of each supernatant were passed to fresh six-well plates of Vero cells for five total successive passages, and infected monolayers were harvested in TRIzol (Invitrogen, Grand Island, NY, USA). After passages were complete, RNA was purified according to the manufacturer's protocol, and first-strand cDNA was generated as described<sup><xref ref-type="bibr" rid="CR41">41</xref></sup>. PCR products were produced using the primers S32F and S34R (<xref rid="MOESM2" ref-type="media">Supplementary Table 4</xref>). Once the presence of single-band PCR products was verified by agarose gel electrophoresis and the yields were calculated by spectrometry, 100 ng of each product was restriction digested using BsrF I, which cuts in the nsp14-ExoN engineered mutation site but not in the corresponding MAwt sequence. Digested products were resolved on a 1.7% agarose gel, and normalized relative percentages of MAwt and ExoN-MA digested products were calculated using ImageJ (<ext-link ext-link-type="uri" xlink:href="http://rsbweb.nih.gov/ij/">http://rsbweb.nih.gov/ij/</ext-link>).</p></sec><sec id="Sec14"><title>Quantification of genome RNA.</title><p id="Par28">Vero cells were infected with either MAwt or MA-ExoN at MOI = 3 PFU per cell. At 6, 12 and 24 h p.i., RNA was harvested in TRIzol and isolated according to the manufacturer's protocol. First-strand cDNA was generated as described above, and real-time PCR was performed assessing for genome RNA using the primers 5′-AGCCAACCAACCTCGATCTCTTGT-3′ (forward) and 5′-TGACACCAAGAACAAGGCTCTCCA-3′ (reverse). cDNA was normalized using the GAPDH primers 5′-TGCACCACCAACTGCTTAGC-3′ (forward) and 5′-GGCATGGACTGTGGTCATGAG-3′ (reverse)<sup><xref ref-type="bibr" rid="CR42">42</xref></sup>. Normalized results were then compared as ratios of MA-ExoN to MAwt genomes using the ΔΔCt method.</p></sec><sec id="Sec15"><title>Infection of mice with SARS-CoV MAwt and MA-ExoN.</title><p id="Par29">All experimental protocols involving mice were reviewed and approved by the institutional animal care and use committee at the University of North Carolina, Chapel Hill. The following groups of mice were used: 10-week-old female BALB/c (Charles River Laboratories, Wilmington, MA, USA), 14-month-old female BALB/c (Harlan Laboratories, Indianapolis, IN, USA), 10-week-old female <italic>Stat1</italic><sup>−/−</sup> (Taconic Farms, Hudson, NY, USA; stock 002045-M-F), 10-week-old female 129S6/SvEvTac (Taconic; stock 129SVE-F), 10-week-old female <italic>Rag</italic><sup>−/−</sup> (Jackson Labs, Bar Harbor, ME, USA; stock 002216), 10-week-old female C57BL/6 (Jackson; stock 00064) and 10-week-old female SCID (Jackson; stock 001803). Mice were lightly anesthetized and infected intranasally with varying doses (10<sup>2</sup>–10<sup>4</sup> PFU, depending on the experiment) of SARS-CoV MAwt or SARS-CoV MA-ExoN. Mice were weighed daily, and on certain days specified in each experiment, a subset of mice in each group was euthanized, and their lungs were harvested for virus titer. Mice that dropped below 70% of their initial mass or were moribund were euthanized before their scheduled time points. Serial passages were inoculated as above for passage 1; subsequent passages were inoculated with 50 μl of clarified lung homogenate (lungs were homogenized in 1 ml of PBS) from the previous passage. All experiments used <italic>n</italic> = 5 mice per virus per dosage per condition (if applicable) per time point, with the exception of experiments using immunocompromised mice, in which <italic>n</italic> = 3.</p></sec><sec id="Sec16"><title>Determination of virus titer in infected mouse lungs.</title><p id="Par30">Lungs harvested for virus titer were weighed and homogenized in 1.0 ml PBS at 6,000 r.p.m. for 60 s in a MagnaLyser (Roche, Basel, Switzerland). Virus titers were determined by plaque assay on Vero cells as previously described<sup><xref ref-type="bibr" rid="CR39">39</xref></sup>.</p></sec><sec id="Sec17"><title>Determination of viral neutralization antibody titers in mouse sera.</title><p id="Par31">Mouse sera were heat inactivated for 30 min at 55 °C and then serially diluted to 1:100, 1:200, 1:400, 1:800 and 1:1,600 in PBS to a volume of 125 μl. Next, 125 μl of PBS containing low-concentration MAwt (40 PFU) or high-concentration MAwt (240 PFU) was added to each serum dilution. The virus-serum mixtures were incubated at 37 °C for 30 min. After incubation, virus titers of the mixtures were determined by plaque assay as described<sup><xref ref-type="bibr" rid="CR39">39</xref></sup>. We then calculated the PRNT<sub>50</sub> values, the serum dilutions at which plaque formation was reduced by 50% relative to that of virus stock not treated with serum.</p></sec><sec id="Sec18"><title>Viral genome sequencing.</title><p id="Par32">To determine the sequences of viral genomes present in SCID mouse lungs after 30 d of infection, plaques were isolated from lung samples from SCID mice at 30 d p.i. as described above. Briefly, once individual, well-resolved viral plaques were visible, they were harvested by collecting the agarose plugs above them using a 200-μl pipette tip. Each agarose plug was dropped in 0.5 ml PBS, allowed to diffuse for 24 h at 4 °C and then applied to ∼70% confluent monolayers of Vero cells in T25 flasks and incubated for 48 h at 37 °C. Infected cell monolayers were then harvested in 1 ml TRIzol. First-strand cDNA was generated as described<sup><xref ref-type="bibr" rid="CR41">41</xref></sup>. Amplicons of the viral genomes were generated as follows: for whole-genome sequencing (amplicons 1–13) and partial-genome sequencing (amplicons A–G, X and Y), the primer pairs indicated in <xref rid="MOESM2" ref-type="media">Supplementary Table 4</xref> were used in a 50-μl PCR reaction using Phusion polymerase (New England BioLabs). Five microliters of each PCR reaction were electrophoresed on agarose gels to verify the presence of correctly sized amplicons, and PCR products were purified using a Qiagen PCR Purification Kit (Qiagen, Valencia, CA, USA). Amplicons were then sequenced using the corresponding primer sets for each amplicon, as indicated in <xref rid="MOESM2" ref-type="media">Supplementary Table 4</xref>. Sequence results were analyzed using Geneious Pro 5.3.6 (Biomatters, Auckland, New Zealand) and Serial Cloner 2.1 (SerialBasics, <ext-link ext-link-type="uri" xlink:href="http://serialbasics.free.fr/Home/Home.html">http://serialbasics.free.fr/Home/Home.html</ext-link>).</p></sec><sec id="Sec19"><title>Statistical analyses.</title><p id="Par33">Statistical analyses were performed using the Mann-Whitney <italic>U</italic> test (<ext-link ext-link-type="uri" xlink:href="http://elegans.som.vcu.edu/~leon/stats/utest.html">http://elegans.som.vcu.edu/~leon/stats/utest.html</ext-link>). Significance was set at <italic>P</italic> < 0.05.</p></sec><sec id="Sec20"><title>Accession codes.</title><p id="Par34">Plaque-purified MA-ExoN isolate sequences are deposited in GenBank under the following accession codes: <ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/?term=FJ882942">FJ882942</ext-link>; <ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/?term=FJ882943">FJ882943</ext-link>; <ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/?term=FJ882945">FJ882945</ext-link>; <ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/?term=FJ882948">FJ882948</ext-link>; <ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/?term=FJ882951">FJ882951</ext-link>; <ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/?term=FJ882952">FJ882952</ext-link>; <ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/?term=FJ882953">FJ882953</ext-link>; <ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/?term=FJ882957">FJ882957</ext-link>; <ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/?term=FJ882958">FJ882958</ext-link>; <ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/?term=FJ882959">FJ882959</ext-link>; <ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/?term=FJ882961">FJ882961</ext-link>; <ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/?term=FJ882962">FJ882962</ext-link>; <ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/?term=HQ890526">HQ890526</ext-link>; <ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/?term=HQ890527">HQ890527</ext-link>; <ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/?term=HQ890528">HQ890528</ext-link>; <ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/?term=HQ890529">HQ890529</ext-link>; <ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/?term=HQ890530">HQ890530</ext-link>; <ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/?term=HQ890531">HQ890531</ext-link>; <ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/?term=HQ890532">HQ890532</ext-link>; <ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/?term=HQ890533">HQ890533</ext-link>; <ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/?term=HQ890534">HQ890534</ext-link>; <ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/?term=HQ890535">HQ890535</ext-link>; <ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/?term=HQ890536">HQ890536</ext-link>; <ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/?term=HQ890537">HQ890537</ext-link>; <ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/?term=HQ890538">HQ890538</ext-link>; <ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/?term=HQ890539">HQ890539</ext-link>; <ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/?term=HQ890540">HQ890540</ext-link>; <ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/?term=HQ890541">HQ890541</ext-link>; <ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/?term=HQ890542">HQ890542</ext-link>; <ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/?term=HQ890543">HQ890543</ext-link>; <ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/?term=HQ890544">HQ890544</ext-link>; <ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/?term=HQ890545">HQ890545</ext-link>; <ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/?term=HQ890546">HQ890546</ext-link>; <ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/?term=JF292902">JF292902</ext-link>; <ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/?term=JF292903">JF292903</ext-link>; <ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/?term=JF292904">JF292904</ext-link>; <ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/?term=JF292905">JF292905</ext-link>; <ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/?term=JF292906">JF292906</ext-link>; <ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/?term=JF292907">JF292907</ext-link>; <ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/?term=JF292908">JF292908</ext-link>; <ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/?term=JF292909">JF292909</ext-link>; <ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/?term=JF292910">JF292910</ext-link>; <ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/?term=JF292911">JF292911</ext-link>; <ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/?term=JF292912">JF292912</ext-link>; <ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/?term=JF292913">JF292913</ext-link>; <ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/?term=JF292914">JF292914</ext-link>; <ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/?term=JF292915">JF292915</ext-link>; <ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/?term=JF292916">JF292916</ext-link>; <ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/?term=JF292917">JF292917</ext-link>; <ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/?term=JF292918">JF292918</ext-link>; <ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/?term=JF292919">JF292919</ext-link>; and <ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/?term=JF292920">JF292920</ext-link>.</p></sec></sec><sec sec-type="supplementary-material"><title>Supplementary information</title><sec id="Sec21"><p><supplementary-material content-type="local-data" id="MOESM2"><media xlink:href="41591_2012_BFnm2972_MOESM2_ESM.pdf"><caption><title>Supplementary Text and Figures</title><p>Supplementary Figures 1–3 and Supplementary Tables 1–4 (PDF 450 kb)</p></caption></media></supplementary-material></p></sec></sec></body><back><ack><title>Acknowledgements</title><p>The authors thank R. Halpin, C. Town (US National Institutes of Health Microbial Genome Sequencing Contract HHSN272200900007C) and X. Lu for their assistance in sequencing <italic>in vitro</italic> isolates. This work was funded by US National Institutes of Health grants U54-AI057157 (SERCEB; R.S.B. and M.R.D.), AI075297 (R.S.B.) and 5F32AI080148 (R.L.G.).</p></ack><notes notes-type="author-contribution"><title>Author Contributions</title><p>R.L.G. designed and performed experiments, analyzed data, and wrote and edited the paper. M.M.B., L.D.E. and M.B. performed experiments, analyzed data and read the paper. M.R.D. and R.S.B. designed experiments, analyzed data, and wrote and edited the paper.</p></notes><notes notes-type="data-availability"><title>Accession codes</title><sec id="d29e1555"><title>Primary accessions</title><p><list list-type="bullet"><title>NCBI Reference Sequence</title><list-item><p id="Par37"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/?term=FJ882942">FJ882942</ext-link></p></list-item><list-item><p id="Par38"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/?term=FJ882943">FJ882943</ext-link></p></list-item><list-item><p id="Par39"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/?term=FJ882945">FJ882945</ext-link></p></list-item><list-item><p id="Par40"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/?term=FJ882948">FJ882948</ext-link></p></list-item><list-item><p id="Par41"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/?term=FJ882951">FJ882951</ext-link></p></list-item><list-item><p id="Par42"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/?term=FJ882952">FJ882952</ext-link></p></list-item><list-item><p id="Par43"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/?term=FJ882953">FJ882953</ext-link></p></list-item><list-item><p id="Par44"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/?term=FJ882957">FJ882957</ext-link></p></list-item><list-item><p id="Par45"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/?term=FJ882958">FJ882958</ext-link></p></list-item><list-item><p id="Par46"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/?term=FJ882959">FJ882959</ext-link></p></list-item><list-item><p id="Par47"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/?term=FJ882961">FJ882961</ext-link></p></list-item><list-item><p id="Par48"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/?term=FJ882962">FJ882962</ext-link></p></list-item><list-item><p id="Par49"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/?term=HQ890526">HQ890526</ext-link></p></list-item><list-item><p id="Par50"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/?term=HQ890527">HQ890527</ext-link></p></list-item><list-item><p id="Par51"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/?term=HQ890528">HQ890528</ext-link></p></list-item><list-item><p id="Par52"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/?term=HQ890529">HQ890529</ext-link></p></list-item><list-item><p id="Par53"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/?term=HQ890530">HQ890530</ext-link></p></list-item><list-item><p id="Par54"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/?term=HQ890531">HQ890531</ext-link></p></list-item><list-item><p id="Par55"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/?term=HQ890532">HQ890532</ext-link></p></list-item><list-item><p id="Par56"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/?term=HQ890533">HQ890533</ext-link></p></list-item><list-item><p id="Par57"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/?term=HQ890534">HQ890534</ext-link></p></list-item><list-item><p id="Par58"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/?term=HQ890535">HQ890535</ext-link></p></list-item><list-item><p id="Par59"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/?term=HQ890536">HQ890536</ext-link></p></list-item><list-item><p id="Par60"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/?term=HQ890537">HQ890537</ext-link></p></list-item><list-item><p id="Par61"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/?term=HQ890538">HQ890538</ext-link></p></list-item><list-item><p id="Par62"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/?term=HQ890539">HQ890539</ext-link></p></list-item><list-item><p id="Par63"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/?term=HQ890540">HQ890540</ext-link></p></list-item><list-item><p id="Par64"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/?term=HQ890541">HQ890541</ext-link></p></list-item><list-item><p id="Par65"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/?term=HQ890542">HQ890542</ext-link></p></list-item><list-item><p id="Par66"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/?term=HQ890543">HQ890543</ext-link></p></list-item><list-item><p id="Par67"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/?term=HQ890544">HQ890544</ext-link></p></list-item><list-item><p id="Par68"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/?term=HQ890545">HQ890545</ext-link></p></list-item><list-item><p id="Par69"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/?term=HQ890546">HQ890546</ext-link></p></list-item><list-item><p id="Par70"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/?term=JF292902">JF292902</ext-link></p></list-item><list-item><p id="Par71"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/?term=JF292903">JF292903</ext-link></p></list-item><list-item><p id="Par72"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/?term=JF292904">JF292904</ext-link></p></list-item><list-item><p id="Par73"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/?term=JF292905">JF292905</ext-link></p></list-item><list-item><p id="Par74"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/?term=JF292906">JF292906</ext-link></p></list-item><list-item><p id="Par75"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/?term=JF292907">JF292907</ext-link></p></list-item><list-item><p id="Par76"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/?term=JF292908">JF292908</ext-link></p></list-item><list-item><p id="Par77"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/?term=JF292909">JF292909</ext-link></p></list-item><list-item><p id="Par78"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/?term=JF292910">JF292910</ext-link></p></list-item><list-item><p id="Par79"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/?term=JF292911">JF292911</ext-link></p></list-item><list-item><p id="Par80"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/?term=JF292912">JF292912</ext-link></p></list-item><list-item><p id="Par81"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/?term=JF292913">JF292913</ext-link></p></list-item><list-item><p id="Par82"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/?term=JF292914">JF292914</ext-link></p></list-item><list-item><p id="Par83"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/?term=JF292915">JF292915</ext-link></p></list-item><list-item><p id="Par84"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/?term=JF292916">JF292916</ext-link></p></list-item><list-item><p id="Par85"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/?term=JF292917">JF292917</ext-link></p></list-item><list-item><p id="Par86"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/?term=JF292918">JF292918</ext-link></p></list-item><list-item><p id="Par87"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/?term=JF292919">JF292919</ext-link></p></list-item><list-item><p id="Par88"><ext-link ext-link-type="uri" xlink:href="http://www.ncbi.nlm.nih.gov/nuccore/?term=JF292920">JF292920</ext-link></p></list-item></list></p></sec></notes><notes notes-type="COI-statement"><title>Competing interests</title><p id="Par35">The authors declare no competing financial interests.</p></notes><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>Jones</surname><given-names>KE</given-names></name><etal></etal></person-group><article-title>Global trends in emerging infectious diseases</article-title><source>Nature</source><year>2008</year><volume>451</volume><fpage>990</fpage><lpage>993</lpage><pub-id pub-id-type="doi">10.1038/nature06536</pub-id><pub-id pub-id-type="pmid">18288193</pub-id></element-citation></ref><ref id="CR2"><label>2</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Li</surname><given-names>Y</given-names></name><etal></etal></person-group><article-title>On the origin of smallpox: correlating variola phylogenics with historical smallpox records</article-title><source>Proc. Natl. Acad. Sci. USA</source><year>2007</year><volume>104</volume><fpage>15787</fpage><lpage>15792</lpage><pub-id pub-id-type="doi">10.1073/pnas.0609268104</pub-id><pub-id pub-id-type="pmid">17901212</pub-id></element-citation></ref><ref id="CR3"><label>3</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Morens</surname><given-names>DM</given-names></name><name><surname>Fauci</surname><given-names>AS</given-names></name></person-group><article-title>The 1918 influenza pandemic: insights for the 21st century</article-title><source>J. Infect. Dis.</source><year>2007</year><volume>195</volume><fpage>1018</fpage><lpage>1028</lpage><pub-id pub-id-type="doi">10.1086/511989</pub-id><pub-id pub-id-type="pmid">17330793</pub-id></element-citation></ref><ref id="CR4"><label>4</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Sessa</surname><given-names>R</given-names></name><name><surname>Palagiano</surname><given-names>C</given-names></name><name><surname>Scifoni</surname><given-names>MG</given-names></name><name><surname>di Pietro</surname><given-names>M</given-names></name><name><surname>Del Piano</surname><given-names>M</given-names></name></person-group><article-title>The major epidemic infections: a gift from the Old World to the New?</article-title><source>Panminerva Med.</source><year>1999</year><volume>41</volume><fpage>78</fpage><lpage>84</lpage><pub-id pub-id-type="pmid">10230264</pub-id></element-citation></ref><ref id="CR5"><label>5</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Cheng</surname><given-names>VC</given-names></name><name><surname>Lau</surname><given-names>SK</given-names></name><name><surname>Woo</surname><given-names>PC</given-names></name><name><surname>Yuen</surname><given-names>KY</given-names></name></person-group><article-title>Severe acute respiratory syndrome coronavirus as an agent of emerging and reemerging infection</article-title><source>Clin. Microbiol. Rev.</source><year>2007</year><volume>20</volume><fpage>660</fpage><lpage>694</lpage><pub-id pub-id-type="doi">10.1128/CMR.00023-07</pub-id><pub-id pub-id-type="pmid">17934078</pub-id></element-citation></ref><ref id="CR6"><label>6</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Graham</surname><given-names>RL</given-names></name><name><surname>Baric</surname><given-names>RS</given-names></name></person-group><article-title>Recombination, reservoirs, and the modular spike: mechanisms of coronavirus cross-species transmission</article-title><source>J. Virol.</source><year>2010</year><volume>84</volume><fpage>3134</fpage><lpage>3146</lpage><pub-id pub-id-type="doi">10.1128/JVI.01394-09</pub-id><pub-id pub-id-type="pmid">19906932</pub-id></element-citation></ref><ref id="CR7"><label>7</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Pfefferle</surname><given-names>S</given-names></name><etal></etal></person-group><article-title>Distant relatives of severe acute respiratory syndrome coronavirus and close relatives of human coronavirus 229E in bats, Ghana</article-title><source>Emerg. Infect. Dis.</source><year>2009</year><volume>15</volume><fpage>1377</fpage><lpage>1384</lpage><pub-id pub-id-type="doi">10.3201/eid1509.090224</pub-id><pub-id pub-id-type="pmid">19788804</pub-id></element-citation></ref><ref id="CR8"><label>8</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Vijgen</surname><given-names>L</given-names></name><etal></etal></person-group><article-title>Complete genomic sequence of human coronavirus OC43: molecular clock analysis suggests a relatively recent zoonotic coronavirus transmission event</article-title><source>J. Virol.</source><year>2005</year><volume>79</volume><fpage>1595</fpage><lpage>1604</lpage><pub-id pub-id-type="doi">10.1128/JVI.79.3.1595-1604.2005</pub-id><pub-id pub-id-type="pmid">15650185</pub-id></element-citation></ref><ref id="CR9"><label>9</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Vignuzzi</surname><given-names>M</given-names></name><name><surname>Wendt</surname><given-names>E</given-names></name><name><surname>Andino</surname><given-names>R</given-names></name></person-group><article-title>Engineering attenuated virus vaccines by controlling replication fidelity</article-title><source>Nat. Med.</source><year>2008</year><volume>14</volume><fpage>154</fpage><lpage>161</lpage><pub-id pub-id-type="doi">10.1038/nm1726</pub-id><pub-id pub-id-type="pmid">18246077</pub-id></element-citation></ref><ref id="CR10"><label>10</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Drake</surname><given-names>JW</given-names></name><name><surname>Holland</surname><given-names>JJ</given-names></name></person-group><article-title>Mutation rates among RNA viruses</article-title><source>Proc. Natl. Acad. Sci. USA</source><year>1999</year><volume>96</volume><fpage>13910</fpage><lpage>13913</lpage><pub-id pub-id-type="doi">10.1073/pnas.96.24.13910</pub-id><pub-id pub-id-type="pmid">10570172</pub-id></element-citation></ref><ref id="CR11"><label>11</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Holland</surname><given-names>JJ</given-names></name><name><surname>Domingo</surname><given-names>E</given-names></name><name><surname>de la Torre</surname><given-names>JC</given-names></name><name><surname>Steinhauer</surname><given-names>DA</given-names></name></person-group><article-title>Mutation frequencies at defined single codon sites in vesicular stomatitis virus and poliovirus can be increased only slightly by chemical mutagenesis</article-title><source>J. Virol.</source><year>1990</year><volume>64</volume><fpage>3960</fpage><lpage>3962</lpage><pub-id pub-id-type="pmid">1695258</pub-id></element-citation></ref><ref id="CR12"><label>12</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Vignuzzi</surname><given-names>M</given-names></name><name><surname>Stone</surname><given-names>JK</given-names></name><name><surname>Arnold</surname><given-names>JJ</given-names></name><name><surname>Cameron</surname><given-names>CE</given-names></name><name><surname>Andino</surname><given-names>R</given-names></name></person-group><article-title>Quasispecies diversity determines pathogenesis through cooperative interactions in a viral population</article-title><source>Nature</source><year>2006</year><volume>439</volume><fpage>344</fpage><lpage>348</lpage><pub-id pub-id-type="doi">10.1038/nature04388</pub-id><pub-id pub-id-type="pmid">16327776</pub-id></element-citation></ref><ref id="CR13"><label>13</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Coffey</surname><given-names>LL</given-names></name><name><surname>Beeharry</surname><given-names>Y</given-names></name><name><surname>Borderia</surname><given-names>AV</given-names></name><name><surname>Blanc</surname><given-names>H</given-names></name><name><surname>Vignuzzi</surname><given-names>M</given-names></name></person-group><article-title>Arbovirus high fidelity variant loses fitness in mosquitoes and mice</article-title><source>Proc. Natl. Acad. Sci. USA</source><year>2011</year><volume>108</volume><fpage>16038</fpage><lpage>16043</lpage><pub-id pub-id-type="doi">10.1073/pnas.1111650108</pub-id><pub-id pub-id-type="pmid">21896755</pub-id></element-citation></ref><ref id="CR14"><label>14</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Lauring</surname><given-names>AS</given-names></name><name><surname>Andino</surname><given-names>R</given-names></name></person-group><article-title>Quasispecies theory and the behavior of RNA viruses</article-title><source>PLoS Pathog.</source><year>2010</year><volume>6</volume><fpage>e1001005</fpage><pub-id pub-id-type="doi">10.1371/journal.ppat.1001005</pub-id><pub-id pub-id-type="pmid">20661479</pub-id></element-citation></ref><ref id="CR15"><label>15</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Lauring</surname><given-names>AS</given-names></name><name><surname>Jones</surname><given-names>JO</given-names></name><name><surname>Andino</surname><given-names>R</given-names></name></person-group><article-title>Rationalizing the development of live attenuated virus vaccines</article-title><source>Nat. Biotechnol.</source><year>2010</year><volume>28</volume><fpage>573</fpage><lpage>579</lpage><pub-id pub-id-type="doi">10.1038/nbt.1635</pub-id><pub-id pub-id-type="pmid">20531338</pub-id></element-citation></ref><ref id="CR16"><label>16</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Eckerle</surname><given-names>LD</given-names></name><etal></etal></person-group><article-title>Infidelity of SARS-CoV nsp14-exonuclease mutant virus replication is revealed by complete genome sequencing</article-title><source>PLoS Pathog.</source><year>2010</year><volume>6</volume><fpage>e1000896</fpage><pub-id pub-id-type="doi">10.1371/journal.ppat.1000896</pub-id><pub-id pub-id-type="pmid">20463816</pub-id></element-citation></ref><ref id="CR17"><label>17</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Snijder</surname><given-names>EJ</given-names></name><etal></etal></person-group><article-title>Unique and conserved features of genome and proteome of SARS-CoV, an early split-off from the coronavirus group 2 lineage</article-title><source>J. Mol. Biol.</source><year>2003</year><volume>331</volume><fpage>991</fpage><lpage>1004</lpage><pub-id pub-id-type="doi">10.1016/S0022-2836(03)00865-9</pub-id><pub-id pub-id-type="pmid">12927536</pub-id></element-citation></ref><ref id="CR18"><label>18</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Nga</surname><given-names>PT</given-names></name><etal></etal></person-group><article-title>Discovery of the first insect nidovirus, a missing evolutionary link in the emergence of the largest RNA virus genomes</article-title><source>PLoS Pathog.</source><year>2011</year><volume>7</volume><fpage>e1002215</fpage><pub-id pub-id-type="doi">10.1371/journal.ppat.1002215</pub-id><pub-id pub-id-type="pmid">21931546</pub-id></element-citation></ref><ref id="CR19"><label>19</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Minskaia</surname><given-names>E</given-names></name><etal></etal></person-group><article-title>Discovery of an RNA virus 3′→5′ exoribonuclease that is critically involved in coronavirus RNA synthesis</article-title><source>Proc. Natl. Acad. Sci. USA</source><year>2006</year><volume>103</volume><fpage>5108</fpage><lpage>5113</lpage><pub-id pub-id-type="doi">10.1073/pnas.0508200103</pub-id><pub-id pub-id-type="pmid">16549795</pub-id></element-citation></ref><ref id="CR20"><label>20</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Eckerle</surname><given-names>LD</given-names></name><name><surname>Lu</surname><given-names>X</given-names></name><name><surname>Sperry</surname><given-names>SM</given-names></name><name><surname>Choi</surname><given-names>L</given-names></name><name><surname>Denison</surname><given-names>MR</given-names></name></person-group><article-title>High fidelity of murine hepatitis virus replication is decresed in nsp14 exoribonuclease mutants</article-title><source>J. Virol.</source><year>2007</year><volume>81</volume><fpage>12135</fpage><lpage>12144</lpage><pub-id pub-id-type="doi">10.1128/JVI.01296-07</pub-id><pub-id pub-id-type="pmid">17804504</pub-id></element-citation></ref><ref id="CR21"><label>21</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Denison</surname><given-names>MR</given-names></name><name><surname>Graham</surname><given-names>RL</given-names></name><name><surname>Donaldson</surname><given-names>EF</given-names></name><name><surname>Eckerle</surname><given-names>LD</given-names></name><name><surname>Baric</surname><given-names>RS</given-names></name></person-group><article-title>Coronaviruses: an RNA proofreading machine regulates replication fidelity and diversity</article-title><source>RNA Biol.</source><year>2011</year><volume>8</volume><fpage>270</fpage><lpage>279</lpage><pub-id pub-id-type="doi">10.4161/rna.8.2.15013</pub-id><pub-id pub-id-type="pmid">21593585</pub-id></element-citation></ref><ref id="CR22"><label>22</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Frieman</surname><given-names>MB</given-names></name><etal></etal></person-group><article-title>SARS-CoV pathogenesis is regulated by a STAT1 dependent but a type I, II and III interferon receptor independent mechanism</article-title><source>PLoS Pathog.</source><year>2010</year><volume>6</volume><fpage>e1000849</fpage><pub-id pub-id-type="doi">10.1371/journal.ppat.1000849</pub-id><pub-id pub-id-type="pmid">20386712</pub-id></element-citation></ref><ref id="CR23"><label>23</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Roberts</surname><given-names>A</given-names></name><etal></etal></person-group><article-title>A mouse adapted SARS coronavirus causes disease and mortality in BALB/c mice</article-title><source>PLoS Pathog.</source><year>2007</year><volume>3</volume><fpage>e5</fpage><pub-id pub-id-type="doi">10.1371/journal.ppat.0030005</pub-id><pub-id pub-id-type="pmid">17222058</pub-id></element-citation></ref><ref id="CR24"><label>24</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Sheahan</surname><given-names>T</given-names></name><etal></etal></person-group><article-title>Successful vaccination strategies that protect aged mice from lethal challenge from influenza virus and heterologous severe acute respiratory syndrome coronavirus</article-title><source>J. Virol.</source><year>2011</year><volume>85</volume><fpage>217</fpage><lpage>230</lpage><pub-id pub-id-type="doi">10.1128/JVI.01805-10</pub-id><pub-id pub-id-type="pmid">20980507</pub-id></element-citation></ref><ref id="CR25"><label>25</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Frieman</surname><given-names>M</given-names></name><etal></etal></person-group><article-title>Molecular determinants of severe acute respiratory syndrome coronavirus pathogenesis and virulence in young and aged mouse models of human disease</article-title><source>J. Virol.</source><year>2012</year><volume>86</volume><fpage>884</fpage><lpage>897</lpage><pub-id pub-id-type="doi">10.1128/JVI.05957-11</pub-id><pub-id pub-id-type="pmid">22072787</pub-id></element-citation></ref><ref id="CR26"><label>26</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Sheahan</surname><given-names>T</given-names></name><etal></etal></person-group><article-title>MyD88 is required for protection from lethal infection with a mouse-adapted SARS-CoV</article-title><source>PLoS Pathog.</source><year>2008</year><volume>4</volume><fpage>e1000240</fpage><pub-id pub-id-type="doi">10.1371/journal.ppat.1000240</pub-id><pub-id pub-id-type="pmid">19079579</pub-id></element-citation></ref><ref id="CR27"><label>27</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Zhao</surname><given-names>J</given-names></name><name><surname>Van Rooijen</surname><given-names>N</given-names></name><name><surname>Perlman</surname><given-names>S</given-names></name></person-group><article-title>Evasion by stealth: inefficient immune activation underlies poor T cell response and severe disease in SARS-CoV–infected mice</article-title><source>PLoS Pathog.</source><year>2009</year><volume>5</volume><fpage>e1000636</fpage><pub-id pub-id-type="doi">10.1371/journal.ppat.1000636</pub-id><pub-id pub-id-type="pmid">19851468</pub-id></element-citation></ref><ref id="CR28"><label>28</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Bolles</surname><given-names>M</given-names></name><etal></etal></person-group><article-title>A double-inactivated severe acute respiratory syndrome coronavirus vaccine provides incomplete protection in mice and induces increased eosinophilic proinflammatory pulmonary response upon challenge</article-title><source>J. Virol.</source><year>2011</year><volume>85</volume><fpage>12201</fpage><lpage>12215</lpage><pub-id pub-id-type="doi">10.1128/JVI.06048-11</pub-id><pub-id pub-id-type="pmid">21937658</pub-id></element-citation></ref><ref id="CR29"><label>29</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Deming</surname><given-names>D</given-names></name><etal></etal></person-group><article-title>Vaccine efficacy in senescent mice challenged with recombinant SARS-CoV bearing epideic and zoonotic spike variants</article-title><source>PLoS Med.</source><year>2006</year><volume>3</volume><fpage>e525</fpage><pub-id pub-id-type="doi">10.1371/journal.pmed.0030525</pub-id><pub-id pub-id-type="pmid">17194199</pub-id></element-citation></ref><ref id="CR30"><label>30</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Subbarao</surname><given-names>K</given-names></name><etal></etal></person-group><article-title>Prior infection and passive transfer of neutralizing antibody prevent repication of SARS coronavirus in the respiratory tract of mice</article-title><source>J. Virol.</source><year>2004</year><volume>78</volume><fpage>3572</fpage><lpage>3577</lpage><pub-id pub-id-type="doi">10.1128/JVI.78.7.3572-3577.2004</pub-id><pub-id pub-id-type="pmid">15016880</pub-id></element-citation></ref><ref id="CR31"><label>31</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Kew</surname><given-names>O</given-names></name><etal></etal></person-group><article-title>Outbreak of poliomyelitis in Hispaniola associated with circulating type 1 vaccine-derived poliovirus</article-title><source>Science</source><year>2002</year><volume>296</volume><fpage>356</fpage><lpage>359</lpage><pub-id pub-id-type="doi">10.1126/science.1068284</pub-id><pub-id pub-id-type="pmid">11896235</pub-id></element-citation></ref><ref id="CR32"><label>32</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Johnson</surname><given-names>AA</given-names></name><name><surname>Johnson</surname><given-names>KA</given-names></name></person-group><article-title>Exonuclease proofreading by human mitochondrial DNA polymerase</article-title><source>J. Biol. Chem.</source><year>2001</year><volume>276</volume><fpage>38097</fpage><lpage>38107</lpage><pub-id pub-id-type="pmid">11477094</pub-id></element-citation></ref><ref id="CR33"><label>33</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Johnson</surname><given-names>AA</given-names></name><name><surname>Johnson</surname><given-names>KA</given-names></name></person-group><article-title>Fidelity of nucleotide incorporation by human mitochondrial DNA polymerase</article-title><source>J. Biol. Chem.</source><year>2001</year><volume>276</volume><fpage>38090</fpage><lpage>38096</lpage><pub-id pub-id-type="pmid">11477093</pub-id></element-citation></ref><ref id="CR34"><label>34</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Donlin</surname><given-names>MJ</given-names></name><name><surname>Patel</surname><given-names>SS</given-names></name><name><surname>Johnson</surname><given-names>KA</given-names></name></person-group><article-title>Kinetic partitioning between the exonuclease and polymerase sites in DNA error correction</article-title><source>Biochemistry</source><year>1991</year><volume>30</volume><fpage>538</fpage><lpage>546</lpage><pub-id pub-id-type="doi">10.1021/bi00216a031</pub-id><pub-id pub-id-type="pmid">1988042</pub-id></element-citation></ref><ref id="CR35"><label>35</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Patel</surname><given-names>SS</given-names></name><name><surname>Wong</surname><given-names>I</given-names></name><name><surname>Johnson</surname><given-names>KA</given-names></name></person-group><article-title>Pre–steady-state kinetic analysis of processive DNA replication including complete characterization of an exonuclease-deficient mutant</article-title><source>Biochemistry</source><year>1991</year><volume>30</volume><fpage>511</fpage><lpage>525</lpage><pub-id pub-id-type="doi">10.1021/bi00216a029</pub-id><pub-id pub-id-type="pmid">1846298</pub-id></element-citation></ref><ref id="CR36"><label>36</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Wong</surname><given-names>I</given-names></name><name><surname>Patel</surname><given-names>SS</given-names></name><name><surname>Johnson</surname><given-names>KA</given-names></name></person-group><article-title>An induced-fit kinetic mechanism for DNA replication fidelity: direct measurement by single-turnover kinetics</article-title><source>Biochemistry</source><year>1991</year><volume>30</volume><fpage>526</fpage><lpage>537</lpage><pub-id pub-id-type="doi">10.1021/bi00216a030</pub-id><pub-id pub-id-type="pmid">1846299</pub-id></element-citation></ref><ref id="CR37"><label>37</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Smits</surname><given-names>SL</given-names></name><etal></etal></person-group><article-title>Exacerbated innate host response to SARS-CoV in aged non-human primates</article-title><source>PLoS Pathog.</source><year>2010</year><volume>6</volume><fpage>e1000756</fpage><pub-id pub-id-type="doi">10.1371/journal.ppat.1000756</pub-id><pub-id pub-id-type="pmid">20140198</pub-id></element-citation></ref><ref id="CR38"><label>38</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Yount</surname><given-names>B</given-names></name><name><surname>Roberts</surname><given-names>RS</given-names></name><name><surname>Lindesmith</surname><given-names>L</given-names></name><name><surname>Baric</surname><given-names>RS</given-names></name></person-group><article-title>Rewiring the SARS-CoV transcription circuit: engineering a recombination-resistant genome</article-title><source>Proc. Natl. Acad. Sci. USA</source><year>2006</year><volume>103</volume><fpage>12546</fpage><lpage>12551</lpage><pub-id pub-id-type="doi">10.1073/pnas.0605438103</pub-id><pub-id pub-id-type="pmid">16891412</pub-id></element-citation></ref><ref id="CR39"><label>39</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Yount</surname><given-names>B</given-names></name><etal></etal></person-group><article-title>Reverse genetics with a full length infectious cDNA of the severe acute respiratory syndrome coronavirus</article-title><source>Proc. Natl. Acad. Sci. USA</source><year>2003</year><volume>100</volume><fpage>12995</fpage><lpage>13000</lpage><pub-id pub-id-type="doi">10.1073/pnas.1735582100</pub-id><pub-id pub-id-type="pmid">14569023</pub-id></element-citation></ref><ref id="CR40"><label>40</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Graham</surname><given-names>RL</given-names></name><name><surname>Sims</surname><given-names>AC</given-names></name><name><surname>Brockway</surname><given-names>SM</given-names></name><name><surname>Baric</surname><given-names>RS</given-names></name><name><surname>Denison</surname><given-names>MR</given-names></name></person-group><article-title>The nsp2 replicase proteins of murine hepatitis virus and severe acute respiratory syndrome coronavirus are dispensable for viral replication</article-title><source>J. Virol.</source><year>2005</year><volume>79</volume><fpage>13399</fpage><lpage>13411</lpage><pub-id pub-id-type="doi">10.1128/JVI.79.21.13399-13411.2005</pub-id><pub-id pub-id-type="pmid">16227261</pub-id></element-citation></ref><ref id="CR41"><label>41</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Becker</surname><given-names>MM</given-names></name><etal></etal></person-group><article-title>Synthetic recombinant bat SARS-like coronavirus is infectious in cultured cells and in mice</article-title><source>Proc. Natl. Acad. Sci. USA</source><year>2008</year><volume>105</volume><fpage>19944</fpage><lpage>19949</lpage><pub-id pub-id-type="doi">10.1073/pnas.0808116105</pub-id><pub-id pub-id-type="pmid">19036930</pub-id></element-citation></ref><ref id="CR42"><label>42</label><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Chey</surname><given-names>S</given-names></name><name><surname>Claus</surname><given-names>C</given-names></name><name><surname>Liebert</surname><given-names>UG</given-names></name></person-group><article-title>Validation and application of normalization factors for gene expression studies in rubella virus–infected cell lines with quantitative real-time PCR</article-title><source>J. Cell. Biochem.</source><year>2010</year><volume>110</volume><fpage>118</fpage><lpage>128</lpage><pub-id pub-id-type="pmid">20217898</pub-id></element-citation></ref></ref-list></back></pmc></record>Pour manipuler ce document sous Unix (Dilib)
EXPLOR_STEP=$WICRI_ROOT/Sante/explor/SrasV1/Data/Pmc/Corpus
HfdSelect -h $EXPLOR_STEP/biblio.hfd -nk 000444 | SxmlIndent | more
Ou
HfdSelect -h $EXPLOR_AREA/Data/Pmc/Corpus/biblio.hfd -nk 000444 | SxmlIndent | more
Pour mettre un lien sur cette page dans le réseau Wicri
{{Explor lien
|wiki= Sante
|area= SrasV1
|flux= Pmc
|étape= Corpus
|type= RBID
|clé= PMC:3518599
|texte= A live, impaired-fidelity coronavirus vaccine protects in an aged, immunocompromised mouse model of lethal disease
}}
Pour générer des pages wiki
HfdIndexSelect -h $EXPLOR_AREA/Data/Pmc/Corpus/RBID.i -Sk "pubmed:23142821" \
| HfdSelect -Kh $EXPLOR_AREA/Data/Pmc/Corpus/biblio.hfd \
| NlmPubMed2Wicri -a SrasV1
| This area was generated with Dilib version V0.6.33. |  |