Links to Exploration step
Le document en format XML
<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">A sensitive and specific antigen detection assay for Middle East respiratory syndrome coronavirus</title><author><name sortKey="Chen, Yixin" sort="Chen, Yixin" uniqKey="Chen Y" first="Yixin" last="Chen">Yixin Chen</name><affiliation><nlm:aff id="AFF0001"><institution>State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, School of Life Sciences, Xiamen University</institution>, Xiamen 361102, Fujian Province,<country>China</country></nlm:aff></affiliation><affiliation><nlm:aff id="AFF0002"><institution>National Institute of Diagnostics and Vaccine Development in Infectious Diseases, School of Public Health, Xiamen University</institution>, Xiamen 361102, Fujian Province,<country>China</country></nlm:aff></affiliation></author><author><name sortKey="Chan, Kwok Hung" sort="Chan, Kwok Hung" uniqKey="Chan K" first="Kwok-Hung" last="Chan">Kwok-Hung Chan</name><affiliation><nlm:aff id="AFF0003"><institution>Department of Microbiology, The University of Hong Kong</institution>, Hong Kong,<country>China</country></nlm:aff></affiliation></author><author><name sortKey="Kang, Yahong" sort="Kang, Yahong" uniqKey="Kang Y" first="Yahong" last="Kang">Yahong Kang</name><affiliation><nlm:aff id="AFF0001"><institution>State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, School of Life Sciences, Xiamen University</institution>, Xiamen 361102, Fujian Province,<country>China</country></nlm:aff></affiliation><affiliation><nlm:aff id="AFF0002"><institution>National Institute of Diagnostics and Vaccine Development in Infectious Diseases, School of Public Health, Xiamen University</institution>, Xiamen 361102, Fujian Province,<country>China</country></nlm:aff></affiliation></author><author><name sortKey="Chen, Honglin" sort="Chen, Honglin" uniqKey="Chen H" first="Honglin" last="Chen">Honglin Chen</name><affiliation><nlm:aff id="AFF0003"><institution>Department of Microbiology, The University of Hong Kong</institution>, Hong Kong,<country>China</country></nlm:aff></affiliation><affiliation><nlm:aff id="AFF0004"><institution>State Key Laboratory of Emerging Infectious Diseases, The University of Hong Kong</institution>, Hong Kong,<country>China</country></nlm:aff></affiliation><affiliation><nlm:aff id="AFF0005"><institution>Research Centre of Infection and Immunology, The University of Hong Kong</institution>, Hong Kong,<country>China</country></nlm:aff></affiliation><affiliation><nlm:aff id="AFF0006"><institution>Carol Yu Centre for Infection, The University of Hong Kong</institution>, Hong Kong,<country>China</country></nlm:aff></affiliation></author><author><name sortKey="Luk, Hayes Kh" sort="Luk, Hayes Kh" uniqKey="Luk H" first="Hayes Kh" last="Luk">Hayes Kh Luk</name><affiliation><nlm:aff id="AFF0003"><institution>Department of Microbiology, The University of Hong Kong</institution>, Hong Kong,<country>China</country></nlm:aff></affiliation></author><author><name sortKey="Poon, Rosana Ws" sort="Poon, Rosana Ws" uniqKey="Poon R" first="Rosana Ws" last="Poon">Rosana Ws Poon</name><affiliation><nlm:aff id="AFF0003"><institution>Department of Microbiology, The University of Hong Kong</institution>, Hong Kong,<country>China</country></nlm:aff></affiliation></author><author><name sortKey="Chan, Jasper Fw" sort="Chan, Jasper Fw" uniqKey="Chan J" first="Jasper Fw" last="Chan">Jasper Fw Chan</name><affiliation><nlm:aff id="AFF0003"><institution>Department of Microbiology, The University of Hong Kong</institution>, Hong Kong,<country>China</country></nlm:aff></affiliation><affiliation><nlm:aff id="AFF0004"><institution>State Key Laboratory of Emerging Infectious Diseases, The University of Hong Kong</institution>, Hong Kong,<country>China</country></nlm:aff></affiliation><affiliation><nlm:aff id="AFF0005"><institution>Research Centre of Infection and Immunology, The University of Hong Kong</institution>, Hong Kong,<country>China</country></nlm:aff></affiliation><affiliation><nlm:aff id="AFF0006"><institution>Carol Yu Centre for Infection, The University of Hong Kong</institution>, Hong Kong,<country>China</country></nlm:aff></affiliation></author><author><name sortKey="Yuen, Kwok Yung" sort="Yuen, Kwok Yung" uniqKey="Yuen K" first="Kwok-Yung" last="Yuen">Kwok-Yung Yuen</name><affiliation><nlm:aff id="AFF0003"><institution>Department of Microbiology, The University of Hong Kong</institution>, Hong Kong,<country>China</country></nlm:aff></affiliation><affiliation><nlm:aff id="AFF0004"><institution>State Key Laboratory of Emerging Infectious Diseases, The University of Hong Kong</institution>, Hong Kong,<country>China</country></nlm:aff></affiliation><affiliation><nlm:aff id="AFF0005"><institution>Research Centre of Infection and Immunology, The University of Hong Kong</institution>, Hong Kong,<country>China</country></nlm:aff></affiliation><affiliation><nlm:aff id="AFF0006"><institution>Carol Yu Centre for Infection, The University of Hong Kong</institution>, Hong Kong,<country>China</country></nlm:aff></affiliation></author><author><name sortKey="Xia, Ningshao" sort="Xia, Ningshao" uniqKey="Xia N" first="Ningshao" last="Xia">Ningshao Xia</name><affiliation><nlm:aff id="AFF0001"><institution>State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, School of Life Sciences, Xiamen University</institution>, Xiamen 361102, Fujian Province,<country>China</country></nlm:aff></affiliation><affiliation><nlm:aff id="AFF0002"><institution>National Institute of Diagnostics and Vaccine Development in Infectious Diseases, School of Public Health, Xiamen University</institution>, Xiamen 361102, Fujian Province,<country>China</country></nlm:aff></affiliation></author><author><name sortKey="Lau, Susanna Kp" sort="Lau, Susanna Kp" uniqKey="Lau S" first="Susanna Kp" last="Lau">Susanna Kp Lau</name><affiliation><nlm:aff id="AFF0003"><institution>Department of Microbiology, The University of Hong Kong</institution>, Hong Kong,<country>China</country></nlm:aff></affiliation><affiliation><nlm:aff id="AFF0004"><institution>State Key Laboratory of Emerging Infectious Diseases, The University of Hong Kong</institution>, Hong Kong,<country>China</country></nlm:aff></affiliation><affiliation><nlm:aff id="AFF0005"><institution>Research Centre of Infection and Immunology, The University of Hong Kong</institution>, Hong Kong,<country>China</country></nlm:aff></affiliation><affiliation><nlm:aff id="AFF0006"><institution>Carol Yu Centre for Infection, The University of Hong Kong</institution>, Hong Kong,<country>China</country></nlm:aff></affiliation></author><author><name sortKey="Woo, Patrick Cy" sort="Woo, Patrick Cy" uniqKey="Woo P" first="Patrick Cy" last="Woo">Patrick Cy Woo</name><affiliation><nlm:aff id="AFF0003"><institution>Department of Microbiology, The University of Hong Kong</institution>, Hong Kong,<country>China</country></nlm:aff></affiliation><affiliation><nlm:aff id="AFF0004"><institution>State Key Laboratory of Emerging Infectious Diseases, The University of Hong Kong</institution>, Hong Kong,<country>China</country></nlm:aff></affiliation><affiliation><nlm:aff id="AFF0005"><institution>Research Centre of Infection and Immunology, The University of Hong Kong</institution>, Hong Kong,<country>China</country></nlm:aff></affiliation><affiliation><nlm:aff id="AFF0006"><institution>Carol Yu Centre for Infection, The University of Hong Kong</institution>, Hong Kong,<country>China</country></nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">26421268</idno><idno type="pmc">4575394</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4575394</idno><idno type="RBID">PMC:4575394</idno><idno type="doi">10.1038/emi.2015.26</idno><date when="2019">2019</date><idno type="wicri:Area/Pmc/Corpus">000C25</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000C25</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">A sensitive and specific antigen detection assay for Middle East respiratory syndrome coronavirus</title><author><name sortKey="Chen, Yixin" sort="Chen, Yixin" uniqKey="Chen Y" first="Yixin" last="Chen">Yixin Chen</name><affiliation><nlm:aff id="AFF0001"><institution>State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, School of Life Sciences, Xiamen University</institution>, Xiamen 361102, Fujian Province,<country>China</country></nlm:aff></affiliation><affiliation><nlm:aff id="AFF0002"><institution>National Institute of Diagnostics and Vaccine Development in Infectious Diseases, School of Public Health, Xiamen University</institution>, Xiamen 361102, Fujian Province,<country>China</country></nlm:aff></affiliation></author><author><name sortKey="Chan, Kwok Hung" sort="Chan, Kwok Hung" uniqKey="Chan K" first="Kwok-Hung" last="Chan">Kwok-Hung Chan</name><affiliation><nlm:aff id="AFF0003"><institution>Department of Microbiology, The University of Hong Kong</institution>, Hong Kong,<country>China</country></nlm:aff></affiliation></author><author><name sortKey="Kang, Yahong" sort="Kang, Yahong" uniqKey="Kang Y" first="Yahong" last="Kang">Yahong Kang</name><affiliation><nlm:aff id="AFF0001"><institution>State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, School of Life Sciences, Xiamen University</institution>, Xiamen 361102, Fujian Province,<country>China</country></nlm:aff></affiliation><affiliation><nlm:aff id="AFF0002"><institution>National Institute of Diagnostics and Vaccine Development in Infectious Diseases, School of Public Health, Xiamen University</institution>, Xiamen 361102, Fujian Province,<country>China</country></nlm:aff></affiliation></author><author><name sortKey="Chen, Honglin" sort="Chen, Honglin" uniqKey="Chen H" first="Honglin" last="Chen">Honglin Chen</name><affiliation><nlm:aff id="AFF0003"><institution>Department of Microbiology, The University of Hong Kong</institution>, Hong Kong,<country>China</country></nlm:aff></affiliation><affiliation><nlm:aff id="AFF0004"><institution>State Key Laboratory of Emerging Infectious Diseases, The University of Hong Kong</institution>, Hong Kong,<country>China</country></nlm:aff></affiliation><affiliation><nlm:aff id="AFF0005"><institution>Research Centre of Infection and Immunology, The University of Hong Kong</institution>, Hong Kong,<country>China</country></nlm:aff></affiliation><affiliation><nlm:aff id="AFF0006"><institution>Carol Yu Centre for Infection, The University of Hong Kong</institution>, Hong Kong,<country>China</country></nlm:aff></affiliation></author><author><name sortKey="Luk, Hayes Kh" sort="Luk, Hayes Kh" uniqKey="Luk H" first="Hayes Kh" last="Luk">Hayes Kh Luk</name><affiliation><nlm:aff id="AFF0003"><institution>Department of Microbiology, The University of Hong Kong</institution>, Hong Kong,<country>China</country></nlm:aff></affiliation></author><author><name sortKey="Poon, Rosana Ws" sort="Poon, Rosana Ws" uniqKey="Poon R" first="Rosana Ws" last="Poon">Rosana Ws Poon</name><affiliation><nlm:aff id="AFF0003"><institution>Department of Microbiology, The University of Hong Kong</institution>, Hong Kong,<country>China</country></nlm:aff></affiliation></author><author><name sortKey="Chan, Jasper Fw" sort="Chan, Jasper Fw" uniqKey="Chan J" first="Jasper Fw" last="Chan">Jasper Fw Chan</name><affiliation><nlm:aff id="AFF0003"><institution>Department of Microbiology, The University of Hong Kong</institution>, Hong Kong,<country>China</country></nlm:aff></affiliation><affiliation><nlm:aff id="AFF0004"><institution>State Key Laboratory of Emerging Infectious Diseases, The University of Hong Kong</institution>, Hong Kong,<country>China</country></nlm:aff></affiliation><affiliation><nlm:aff id="AFF0005"><institution>Research Centre of Infection and Immunology, The University of Hong Kong</institution>, Hong Kong,<country>China</country></nlm:aff></affiliation><affiliation><nlm:aff id="AFF0006"><institution>Carol Yu Centre for Infection, The University of Hong Kong</institution>, Hong Kong,<country>China</country></nlm:aff></affiliation></author><author><name sortKey="Yuen, Kwok Yung" sort="Yuen, Kwok Yung" uniqKey="Yuen K" first="Kwok-Yung" last="Yuen">Kwok-Yung Yuen</name><affiliation><nlm:aff id="AFF0003"><institution>Department of Microbiology, The University of Hong Kong</institution>, Hong Kong,<country>China</country></nlm:aff></affiliation><affiliation><nlm:aff id="AFF0004"><institution>State Key Laboratory of Emerging Infectious Diseases, The University of Hong Kong</institution>, Hong Kong,<country>China</country></nlm:aff></affiliation><affiliation><nlm:aff id="AFF0005"><institution>Research Centre of Infection and Immunology, The University of Hong Kong</institution>, Hong Kong,<country>China</country></nlm:aff></affiliation><affiliation><nlm:aff id="AFF0006"><institution>Carol Yu Centre for Infection, The University of Hong Kong</institution>, Hong Kong,<country>China</country></nlm:aff></affiliation></author><author><name sortKey="Xia, Ningshao" sort="Xia, Ningshao" uniqKey="Xia N" first="Ningshao" last="Xia">Ningshao Xia</name><affiliation><nlm:aff id="AFF0001"><institution>State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, School of Life Sciences, Xiamen University</institution>, Xiamen 361102, Fujian Province,<country>China</country></nlm:aff></affiliation><affiliation><nlm:aff id="AFF0002"><institution>National Institute of Diagnostics and Vaccine Development in Infectious Diseases, School of Public Health, Xiamen University</institution>, Xiamen 361102, Fujian Province,<country>China</country></nlm:aff></affiliation></author><author><name sortKey="Lau, Susanna Kp" sort="Lau, Susanna Kp" uniqKey="Lau S" first="Susanna Kp" last="Lau">Susanna Kp Lau</name><affiliation><nlm:aff id="AFF0003"><institution>Department of Microbiology, The University of Hong Kong</institution>, Hong Kong,<country>China</country></nlm:aff></affiliation><affiliation><nlm:aff id="AFF0004"><institution>State Key Laboratory of Emerging Infectious Diseases, The University of Hong Kong</institution>, Hong Kong,<country>China</country></nlm:aff></affiliation><affiliation><nlm:aff id="AFF0005"><institution>Research Centre of Infection and Immunology, The University of Hong Kong</institution>, Hong Kong,<country>China</country></nlm:aff></affiliation><affiliation><nlm:aff id="AFF0006"><institution>Carol Yu Centre for Infection, The University of Hong Kong</institution>, Hong Kong,<country>China</country></nlm:aff></affiliation></author><author><name sortKey="Woo, Patrick Cy" sort="Woo, Patrick Cy" uniqKey="Woo P" first="Patrick Cy" last="Woo">Patrick Cy Woo</name><affiliation><nlm:aff id="AFF0003"><institution>Department of Microbiology, The University of Hong Kong</institution>, Hong Kong,<country>China</country></nlm:aff></affiliation><affiliation><nlm:aff id="AFF0004"><institution>State Key Laboratory of Emerging Infectious Diseases, The University of Hong Kong</institution>, Hong Kong,<country>China</country></nlm:aff></affiliation><affiliation><nlm:aff id="AFF0005"><institution>Research Centre of Infection and Immunology, The University of Hong Kong</institution>, Hong Kong,<country>China</country></nlm:aff></affiliation><affiliation><nlm:aff id="AFF0006"><institution>Carol Yu Centre for Infection, The University of Hong Kong</institution>, Hong Kong,<country>China</country></nlm:aff></affiliation></author></analytic><series><title level="j">Emerging Microbes & Infections</title><idno type="eISSN">2222-1751</idno><imprint><date when="2019">2019</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>Since its emergence in 2012, more than 900 laboratory-confirmed cases of Middle East respiratory syndrome (MERS) have been reported with a fatality rate of more than 30%. However, no antigen detection assay for commercial use is available for diagnosis. In this study, the full-length nucleocapsid protein (NP) gene of MERS coronavirus (MERS-CoV) was cloned and expressed in <italic>Escherichia coli</italic>. A MERS-CoV NP capture enzyme-linked immunosorbent assay (ELISA) using two MERS-CoV-NP-specific monoclonal antibodies (MAbs) generated was developed. The ELISA was evaluated using 129 nasopharyngeal aspirates (NPAs) positive for various respiratory viruses and simulated positive NPAs by adding serial dilutions of MERS-CoV. Using a cutoff OD of 0.19, all 129 NPAs positive for respiratory viruses showed very low OD, with a specificity of 100%. For the two simulated MERS-CoV-positive NPAs with serial dilutions of live MERS-CoV, all samples with ≥10 50% tissue culture infective dose (TCID<sub>50</sub>)/0.1 mL showed positive results. For the 10 additional NPAs with 20 and 200 TCID<sub>50</sub>/0.1 mL of live MERS-CoV added, all were positive. A highly sensitive and specific MAbs-based antigen capture ELISA has been developed for MERS. This sensitive and specific antigen capture ELISA should be useful for detection of MERS-CoV in human and dromedaries and in field studies.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Chan, Jf" uniqKey="Chan J">JF Chan</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Zaki, Am" uniqKey="Zaki A">AM Zaki</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Van Boheemen, S" uniqKey="Van Boheemen S">S van Boheemen</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="De Groot, Rj" uniqKey="De Groot R">RJ de Groot</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Reusken, Cb" uniqKey="Reusken C">CB Reusken</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Haagmans, Bl" uniqKey="Haagmans B">BL Haagmans</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Briese, T" uniqKey="Briese T">T Briese</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Hemida, Mg" uniqKey="Hemida M">MG Hemida</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Woo, Pc" uniqKey="Woo P">PC Woo</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Bermingham, A" uniqKey="Bermingham A">A Bermingham</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Corman, Vm" uniqKey="Corman V">VM Corman</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Guery, B" uniqKey="Guery B">B Guery</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Lu, X" uniqKey="Lu X">X Lu</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Lau, Sk" uniqKey="Lau S">SK Lau</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Lau, Sk" uniqKey="Lau S">SK Lau</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Chan, Jf" uniqKey="Chan J">JF Chan</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Lau, Sk" uniqKey="Lau S">SK Lau</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Chan, Kh" uniqKey="Chan K">KH Chan</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Gui, X" uniqKey="Gui X">X Gui</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Gui, X" uniqKey="Gui X">X Gui</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Yuan, Q" uniqKey="Yuan Q">Q Yuan</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Lau, Sk" uniqKey="Lau S">SK Lau</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Lau, Sk" uniqKey="Lau S">SK Lau</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Lau, Sk" uniqKey="Lau S">SK Lau</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Woo, Pc" uniqKey="Woo P">PC Woo</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Woo, Pc" uniqKey="Woo P">PC Woo</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Drosten, C" uniqKey="Drosten C">C Drosten</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Kapoor, M" uniqKey="Kapoor M">M Kapoor</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Corman, Vm" uniqKey="Corman V">VM Corman</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Lau, Sk" uniqKey="Lau S">SK Lau</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Wang, Q" uniqKey="Wang Q">Q Wang</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Reusken, Cb" uniqKey="Reusken C">CB Reusken</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Meyer, B" uniqKey="Meyer B">B Meyer</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Azhar, Ei" uniqKey="Azhar E">EI Azhar</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Chu, Dk" uniqKey="Chu D">DK Chu</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Nowotny, N" uniqKey="Nowotny N">N Nowotny</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Alagaili, An" uniqKey="Alagaili A">AN Alagaili</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">Emerg Microbes Infect</journal-id><journal-id journal-id-type="iso-abbrev">Emerg Microbes Infect</journal-id><journal-id journal-id-type="publisher-id">TEMI</journal-id><journal-id journal-id-type="publisher-id">temi20</journal-id><journal-title-group><journal-title>Emerging Microbes & Infections</journal-title></journal-title-group><issn pub-type="epub">2222-1751</issn><publisher><publisher-name>Taylor & Francis</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">26421268</article-id><article-id pub-id-type="pmc">4575394</article-id><article-id pub-id-type="publisher-id">12040307</article-id><article-id pub-id-type="doi">10.1038/emi.2015.26</article-id><article-categories><subj-group subj-group-type="heading"><subject>Original Articles</subject></subj-group></article-categories><title-group><article-title>A sensitive and specific antigen detection assay for Middle East respiratory syndrome coronavirus</article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Chen</surname><given-names>Yixin</given-names></name><xref ref-type="aff" rid="AFF0001"><sup>1</sup></xref><xref ref-type="aff" rid="AFF0002"><sup>2</sup></xref></contrib><contrib contrib-type="author"><name><surname>Chan</surname><given-names>Kwok-Hung</given-names></name><xref ref-type="aff" rid="AFF0003"><sup>3</sup></xref></contrib><contrib contrib-type="author"><name><surname>Kang</surname><given-names>Yahong</given-names></name><xref ref-type="aff" rid="AFF0001"><sup>1</sup></xref><xref ref-type="aff" rid="AFF0002"><sup>2</sup></xref></contrib><contrib contrib-type="author"><name><surname>Chen</surname><given-names>Honglin</given-names></name><xref ref-type="aff" rid="AFF0003"><sup>3</sup></xref><xref ref-type="aff" rid="AFF0004"><sup>4</sup></xref><xref ref-type="aff" rid="AFF0005"><sup>5</sup></xref><xref ref-type="aff" rid="AFF0006"><sup>6</sup></xref></contrib><contrib contrib-type="author"><name><surname>Luk</surname><given-names>Hayes KH</given-names></name><xref ref-type="aff" rid="AFF0003"><sup>3</sup></xref></contrib><contrib contrib-type="author"><name><surname>Poon</surname><given-names>Rosana WS</given-names></name><xref ref-type="aff" rid="AFF0003"><sup>3</sup></xref></contrib><contrib contrib-type="author"><name><surname>Chan</surname><given-names>Jasper FW</given-names></name><xref ref-type="aff" rid="AFF0003"><sup>3</sup></xref><xref ref-type="aff" rid="AFF0004"><sup>4</sup></xref><xref ref-type="aff" rid="AFF0005"><sup>5</sup></xref><xref ref-type="aff" rid="AFF0006"><sup>6</sup></xref></contrib><contrib contrib-type="author"><name><surname>Yuen</surname><given-names>Kwok-Yung</given-names></name><xref ref-type="aff" rid="AFF0003"><sup>3</sup></xref><xref ref-type="aff" rid="AFF0004"><sup>4</sup></xref><xref ref-type="aff" rid="AFF0005"><sup>5</sup></xref><xref ref-type="aff" rid="AFF0006"><sup>6</sup></xref></contrib><contrib contrib-type="author"><name><surname>Xia</surname><given-names>Ningshao</given-names></name><xref ref-type="aff" rid="AFF0001"><sup>1</sup></xref><xref ref-type="aff" rid="AFF0002"><sup>2</sup></xref></contrib><contrib contrib-type="author"><name><surname>Lau</surname><given-names>Susanna KP</given-names></name><xref ref-type="aff" rid="AFF0003"><sup>3</sup></xref><xref ref-type="aff" rid="AFF0004"><sup>4</sup></xref><xref ref-type="aff" rid="AFF0005"><sup>5</sup></xref><xref ref-type="aff" rid="AFF0006"><sup>6</sup></xref></contrib><contrib contrib-type="author"><name><surname>Woo</surname><given-names>Patrick CY</given-names></name><xref ref-type="aff" rid="AFF0003"><sup>3</sup></xref><xref ref-type="aff" rid="AFF0004"><sup>4</sup></xref><xref ref-type="aff" rid="AFF0005"><sup>5</sup></xref><xref ref-type="aff" rid="AFF0006"><sup>6</sup></xref><xref ref-type="corresp" rid="AN0001"></xref></contrib><aff id="AFF0001"><label>1</label><institution>State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, School of Life Sciences, Xiamen University</institution>, Xiamen 361102, Fujian Province,<country>China</country></aff><aff id="AFF0002"><label>2</label><institution>National Institute of Diagnostics and Vaccine Development in Infectious Diseases, School of Public Health, Xiamen University</institution>, Xiamen 361102, Fujian Province,<country>China</country></aff><aff id="AFF0003"><label>3</label><institution>Department of Microbiology, The University of Hong Kong</institution>, Hong Kong,<country>China</country></aff><aff id="AFF0004"><label>4</label><institution>State Key Laboratory of Emerging Infectious Diseases, The University of Hong Kong</institution>, Hong Kong,<country>China</country></aff><aff id="AFF0005"><label>5</label><institution>Research Centre of Infection and Immunology, The University of Hong Kong</institution>, Hong Kong,<country>China</country></aff><aff id="AFF0006"><label>6</label><institution>Carol Yu Centre for Infection, The University of Hong Kong</institution>, Hong Kong,<country>China</country></aff></contrib-group><author-notes><corresp id="AN0001">PCY Woo E-mail: <email>pcywoo@hku.hk</email></corresp></author-notes><pub-date pub-type="collection"><year>2015</year></pub-date><pub-date pub-type="epub"><day>25</day><month>1</month><year>2019</year></pub-date><volume>4</volume><issue>1</issue><fpage seq="11">1</fpage><lpage>5</lpage><history><date date-type="received"><day>02</day><month>2</month><year>2015</year></date><date date-type="rev-recd"><day>02</day><month>3</month><year>2015</year></date><date date-type="accepted"><day>04</day><month>3</month><year>2015</year></date></history><permissions><copyright-statement>© 2015 The Author(s)</copyright-statement><copyright-year>2015</copyright-year><copyright-holder>The Author(s)</copyright-holder><license license-type="open-access" xlink:href="http://creativecommons.org/licenses/by-nc-sa/3.0/"><license-p>This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permissing from the license holder to reproduce the material. To view a copy of this license, visit <ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by-nc-sa/3.0/">http://creativecommons.org/licenses/by-nc-sa/3.0/</ext-link></license-p></license><license><license-p>This article is made available via the PMC Open Access Subset for unrestricted re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the COVID-19 pandemic or until permissions are revoked in writing. Upon expiration of these permissions, PMC is granted a perpetual license to make this article available via PMC and Europe PMC, consistent with existing copyright protections.</license-p></license></permissions><self-uri content-type="pdf" xlink:href="TEMI_4_12040307.pdf"></self-uri><abstract id="Abs1"><p>Since its emergence in 2012, more than 900 laboratory-confirmed cases of Middle East respiratory syndrome (MERS) have been reported with a fatality rate of more than 30%. However, no antigen detection assay for commercial use is available for diagnosis. In this study, the full-length nucleocapsid protein (NP) gene of MERS coronavirus (MERS-CoV) was cloned and expressed in <italic>Escherichia coli</italic>. A MERS-CoV NP capture enzyme-linked immunosorbent assay (ELISA) using two MERS-CoV-NP-specific monoclonal antibodies (MAbs) generated was developed. The ELISA was evaluated using 129 nasopharyngeal aspirates (NPAs) positive for various respiratory viruses and simulated positive NPAs by adding serial dilutions of MERS-CoV. Using a cutoff OD of 0.19, all 129 NPAs positive for respiratory viruses showed very low OD, with a specificity of 100%. For the two simulated MERS-CoV-positive NPAs with serial dilutions of live MERS-CoV, all samples with ≥10 50% tissue culture infective dose (TCID<sub>50</sub>)/0.1 mL showed positive results. For the 10 additional NPAs with 20 and 200 TCID<sub>50</sub>/0.1 mL of live MERS-CoV added, all were positive. A highly sensitive and specific MAbs-based antigen capture ELISA has been developed for MERS. This sensitive and specific antigen capture ELISA should be useful for detection of MERS-CoV in human and dromedaries and in field studies.</p></abstract><kwd-group kwd-group-type="author"><title>Keywords</title><kwd>antigen</kwd><kwd>detection</kwd><kwd>ELISA</kwd><kwd>MERS coronavirus</kwd></kwd-group><counts><fig-count count="5"></fig-count><table-count count="0"></table-count><equation-count count="0"></equation-count><ref-count count="37"></ref-count><page-count count="5"></page-count></counts></article-meta></front><body><sec><title>Introduction</title><p>Since its emergence in 2012, Middle East respiratory syndrome (MERS) has affected more than 20 countries globally with a total of more than 900 laboratory-confirmed cases and a fatality rate of over 30%. Similar to severe acute respiratory syndrome (SARS), MERS is also caused by a coronavirus (CoV), named MERS-CoV;<sup><xref rid="bib1" ref-type="bibr">1</xref>,<xref rid="bib2" ref-type="bibr">2</xref>,<xref rid="bib3" ref-type="bibr">3</xref>,<xref rid="bib4" ref-type="bibr">4</xref></sup> but unlike the SARS epidemic which has rapidly died off after the intermediate amplification animal hosts were segregated from humans by closure of wild animal markets in southern China, the MERS epidemic has persisted for more than two years with no signs of abatement. The persistence of the epidemic may be partly due to the difficulty to control the contact between the people and dromedary camels, a major zoonotic source of MERS-CoV.<sup><xref rid="bib5" ref-type="bibr">5</xref>,<xref rid="bib6" ref-type="bibr">6</xref>,<xref rid="bib7" ref-type="bibr">7</xref>,<xref rid="bib8" ref-type="bibr">8</xref>,<xref rid="bib9" ref-type="bibr">9</xref></sup> Since there is still no effective anti-viral agent for the treatment of MERS-CoV infections, rapid and accurate laboratory diagnosis is essential for commencement of infection control measures.</p><p>Although MERS-CoV can be cultured and antibody detection methods are available, laboratory diagnosis of human MERS-CoV infections is mainly achieved by quantitative real-time RT-PCR, using the RNA-dependent RNA polymerase, the region upstream to the envelope gene or the nucleocapsid gene of MERS-CoV as the target.<sup><xref rid="bib10" ref-type="bibr">10</xref>,<xref rid="bib11" ref-type="bibr">11</xref>,<xref rid="bib12" ref-type="bibr">12</xref>,<xref rid="bib13" ref-type="bibr">13</xref></sup> However, the cost of quantitative real-time RT-PCR is still high and such expertise may not be available, particularly in the clinical microbiology laboratories in the Middle East where most of the cases were identified. Enzyme-linked immunosorbent assay (ELISA) for antigen detection offers an inexpensive and user-friendly way for laboratory diagnosis of respiratory viral infections, including SARS.<sup><xref rid="bib14" ref-type="bibr">14</xref>,<xref rid="bib15" ref-type="bibr">15</xref></sup> As for MERS, antigen detection ELISA will be useful not only for laboratory diagnosis of patients with suspected MERS but also for field studies on dromedaries as well as other suspected animal reservoirs. So far, such an antigen detection assay is not available for MERS-CoV. In this article, we report the development and evaluation of a monoclonal antibody (MAb)-based capture ELISA for MERS-CoV nucleocapsid protein (NP) detection.</p></sec><sec><title>MATERIALS AND METHODS</title><sec><title>Cloning and purification of (His)<sub>6</sub>-tagged recombinant NP (rNP) of MERS-CoV</title><p>A clinical isolate of MERS-CoV was kindly provided by RA Fouchier and colleagues.<sup><xref rid="bib2" ref-type="bibr">2</xref></sup> All work with infectious MERS-CoV was performed inside a biosafety level-2 cabinet with standard operating procedures in approved biosafety level-3 facilities.<sup><xref rid="bib16" ref-type="bibr">16</xref>,<xref rid="bib17" ref-type="bibr">17</xref>,<xref rid="bib18" ref-type="bibr">18</xref></sup> Viral culture and RNA extraction were performed as we described previously.<sup><xref rid="bib9" ref-type="bibr">9</xref>,<xref rid="bib16" ref-type="bibr">16</xref></sup> To produce a plasmid for protein purification, primers (5′-GAA TTC ATG GCA TCC CCT GCT GCA CCT-3′ and 5′-GTC GAC ATC AGT GTT AAC ATC AAT CAT-3′) were used to amplify the gene encoding the NP of MERS-CoV by RT-PCR. The sequence coding for amino acid residues 1–413 of the NP was amplified and cloned into the <italic>Eco</italic>RI and <italic>Sal</italic>I sites of expression vector pET-28a(+) (Novagen, Merck Millipore, Billerica, MA, USA) in frame and upstream of the series of six histidine residues. The rNP protein was expressed in <italic>Escherichia coli</italic> ER2566 cells (Invitrogen, Life Technologies, Grand Island, NY, USA) and purified using Ni-NTA affinity chromatograph column (Novagen, Merck Millipore, Billerica, MA, USA) according to the manufacturer’s instructions.</p></sec><sec><title>Production of anti-MERS-CoV-rNP MAbs</title><p>Anti-MERS-CoV-rNP MAbs were produced using a protocol as described previously.<sup><xref rid="bib19" ref-type="bibr">19</xref></sup> Briefly, six-week-old female BALB/c mice were immunized with 100 µg purified MERS-CoV-rNP emulsified in an equal volume of Freund’s complete adjuvant (Sigma-Aldrich, Saint Louis, MO, USA) subcutaneously. Two booster immunizations using the same antigen emulsified in incomplete Freund’s adjuvant were carried out on days 14 and 28. Blood samples were obtained from the tail veins of the immunized mice and tested for titers against purified MERS-CoV-rNP by ELISA on MERS-CoV-rNP-coated microplates. After the final booster dose, the spleens of the mice were removed, and splenocytes were fused with the mouse myeloma cell line Sp2/0-Ag-14. Culture supernatant from individual hybridoma clones was then screened against purified MERS-CoV-rNP by ELISA. After subcloning three times by limiting dilution, stable hybridoma cells were injected into the abdominal cavity of a mouse to produce ascitic fluid. The MAbs were purified first by precipitating with 50% ammonium sulfate, then with diethylaminoethyl column by high performance liquid chromatography. The concentrations of purified IgG were determined by their absorbance at OD<sub>280</sub>. All animal procedures were approved officially by the Xiamen University Institutional Committee for the Use and Care of Laboratory Animal.</p></sec><sec><title>Western blot analysis</title><p>Western blot analysis was performed according to our published protocol.<sup><xref rid="bib20" ref-type="bibr">20</xref></sup> Briefly, 1.5 µg purified (His)<sub>6</sub>-tagged rNPs of MERS-CoV, human CoV (HCoV) 229E and HCoV-OC43 respectively were subjected to 12% SDS-PAGE and were transferred to 0.22 µm nitrocellulose membranes (GE Healthcare, Little Chalfont, Buckinghamshire, UK). The membranes were blocked with 5% skim milk in Tris-buffered saline and then incubated with mouse anti-MERS-CoV-rNP immune sera at 37 °C for 60 min. Antigen–antibody interaction was detected with 1:4000 diluted horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Novex, Life Technologies, Grand Island, NY, USA) and visualized by 3,3′,5,5′-tetramethylbenzidine (TMB) liquid substrates system for membranes (Sigma Aldrich, Saint Louis, MO, USA).</p></sec><sec><title>Capture ELISA for detection of MERS-CoV NP</title><p>The capture ELISA for MERS-CoV-NP detection was developed as described previously with minor modifications.<sup><xref rid="bib21" ref-type="bibr">21</xref></sup> Microplates (Sigma Aldrich, Saint Louis, MO, USA) were pre-coated with the first anti-MERS-CoV-rNP MAb 1F6 and incubated at 37 °C overnight with a blocking reagent (phosphate-buffered saline with 2% sucrose, 0.2% casein-Na, and 2% gelatin). Inactivated MERS-CoV, HCoV-229E, HCoV-OC43, two influenza virus A strains, one influenza virus B strain and one respiratory syncytial virus (RSV) strain, and purified rNPs of MERS-CoV, HCoV-229E, and HCoV-OC43 were diluted in phosphate-buffered saline with 2% skim milk. The inactivated viral lysates were diluted to the same virus titer of 10<sup>6</sup> TCID<sub>50</sub> and further serially diluted. Specifically, 50 µL of viral lysis buffer was added to the coated well and a 50 µL of sample (fixed concentrations of purified NP or viral cell culture lysates) was then added to the wells in duplicates. The plate was shaken for 2 min and incubated at 37 °C for 30 min. After the wells were washed five times, 100 µL of the second anti-MERS-CoV-rNP MAb 7C4 conjugated with HRP was added and the plate was incubated at 37 °C for 30 min. After five washes, detection was carried out by adding of 100 µL TMB per well with incubation for 10 min followed by the addition of 50 µL 0.2 M H<sub>2</sub>SO<sub>4</sub>. The OD 450/630 nm was measured with a microplate reader. The cutoff value was set as 0.15+ mean value of 20 human nasopharyngeal aspirates (NPAs) negative for influenza A, B, and C viruses, adenovirus, RSV, parainfluenza viruses 1–4, human rhinoviruses, human metapneumovirus, HCoV-HKU1, HCoV-OC43, HCoV-229E, HCoV-NL63, and MERS-CoV.</p></sec><sec><title>Evaluation of MERS-CoV-NP capture ELISA</title><p>NPAs from 129 patients with viral respiratory tract infections (influenza A virus (<italic>n</italic> = 10), influenza B virus (<italic>n</italic> = 10), influenza C virus (<italic>n</italic> = 2), adenovirus (<italic>n</italic> = 10), RSV (<italic>n</italic> = 10), parainfluenza virus 1 (<italic>n</italic> = 5), parainfluenza virus 2 (<italic>n</italic> = 2), parainfluenza virus 3 (<italic>n</italic> = 10), parainfluenza virus 4 (<italic>n</italic> = 10), human rhinoviruses (<italic>n</italic> = 30), human metapneumovirus (<italic>n</italic> = 10), HCoV-HKU1 (<italic>n</italic> = 5), HCoV-OC43 (<italic>n</italic> = 5), HCoV-229E (<italic>n</italic> = 5), and HCoV-NL63 (<italic>n</italic> = 5)) and simulated MERS-CoV positive NPAs were used for the evaluation of the ELISA. NPAs positive for influenza C virus, parainfluenza virus 4, human rhinoviruses, human metapneumovirus, and HCoVs were determined by RT-PCR and those for the other respiratory viruses were determined by indirect immunofluorescence assay as described in our previous publications.<sup><xref rid="bib22" ref-type="bibr">22</xref>,<xref rid="bib23" ref-type="bibr">23</xref>,<xref rid="bib24" ref-type="bibr">24</xref>,<xref rid="bib25" ref-type="bibr">25</xref>,<xref rid="bib26" ref-type="bibr">26</xref></sup> MERS-CoV culture and titration were performed as we described previously.<sup><xref rid="bib16" ref-type="bibr">16</xref></sup> Simulated MERS-CoV positive NPAs were prepared by adding serial dilutions (1–500 000 TCID<sub>50</sub>/0.1 mL) of MERS-CoV to two different NPAs and 20 TCID<sub>50</sub>/0.1 mL and 200 TCID<sub>50</sub>/0.1 mL of MERS-CoV to 10 additional NPAs negative for the above mentioned respiratory viruses. The ELISA was performed as described above, using 50 µL of each NPA sample.</p></sec></sec><sec><title>RESULTS</title><sec><title>Production and characterization of MERS-CoV-rNP</title><p>The full-length NP gene of MERS-CoV was cloned and expressed in <italic>E. coli</italic> strain ER2566 with a stretch of six histidine residues at both the N and C termini. The MERS-CoV-rNP, with a molecular weight of 46 kDa, was purified by Ni-NTA affinity chromatography and was used to immunize BALB/c mice. Western blot showed that mouse anti-MERS-CoV-rNP immune sera had good reactivity with MERS-CoV and MERS-CoV-rNP, indicating that the expressed MERS-CoV-rNP has similar antigenicity to native NP of MERS-CoV (<xref rid="F0001" ref-type="fig">Figure 1</xref>). As a control, the mouse anti-MERS-CoV-rNP immune sera did not react with influenza A virus strain CA/04 (<xref rid="F0001" ref-type="fig">Figure 1</xref>). The high ELISA titer of mouse anti-MERS-CoV-rNP immune sera confirmed that the MERS-CoV-rNP has good immunogenicity in BALB/c mice, indicating that the MERS-CoV-rNP was useful for generation of anti-MERS-CoV-rNP MAbs (<xref rid="F0002" ref-type="fig">Figure 2</xref>).
<fig fig-type="figure" id="F0001" orientation="portrait" position="float"><label>Figure 1</label><p>Western blot analysis of mouse anti-MERS-CoV-rNP immune sera. Proteins loaded include: MERS-CoV-rNP, lysate of MERS-CoV (MERS-CoV), lysate of influenza A virus strain CA/04 (Flu CA/04), molecular weight marker. The arrows indicate the size of the MERS-CoV-rNP.</p><graphic xlink:href="TEMI_A_12040307_F0001_OC"></graphic></fig><fig fig-type="figure" id="F0002" orientation="portrait" position="float"><label>Figure 2</label><p>The ELISA titer of mouse anti-MERS-CoV-rNP immune sera. The MERS-CoV-rNP was coated at 100 ng/well. 1:5000 dilution of HRP conjugated Goat anti-mouse antibody was used as second antibody. Normal mouse sera were used as negative control.</p><graphic xlink:href="TEMI_A_12040307_F0002_OC"></graphic></fig></p></sec><sec><title>Generation and characterization of anti-MERS-CoV-rNP MAbs</title><p>A total of 24 MAbs were generated against MERS-CoV-rNP by ELISA. Twelve of them were confirmed to be MERS-CoV-rNP-specific MAbs by ELISA using MERS-CoV-rNP as well as HCoV-229E-rNP and HCoV-OC43-rNP (<xref rid="F0003" ref-type="fig">Figure 3</xref>). The cloning, expression, and purification procedures for HCoV-229E-rNP and HCoV-OC43-rNP were the same as described for those of MERS-CoV-rNP (data not shown). The other 12 MAbs showed different degrees of cross-reactivity among the three rNP proteins, suggesting that the NPs of different CoVs might have some common epitopes.
<fig fig-type="figure" id="F0003" orientation="portrait" position="float"><label>Figure 3</label><p>Reactivity of anti-MERS-CoV-rNP MAbs in ELISA. The MERS-CoV-rNP was coated at 100 ng/well. 1:5000 dilution of HRP-conjugated goat anti-mouse antibody was used as the secondary antibody. Normal mouse sera were used as negative control.</p><graphic xlink:href="TEMI_A_12040307_F0003_OC"></graphic></fig></p></sec><sec><title>Development of capture ELISA for detection of MERS-CoV NP</title><p>To establish a capture ELISA for MERS-CoV NP detection, two anti-MERS-CoV-rNP-specific MAbs (1F6 and 7C4) were selected. MAb 1F6 was coated onto the microplate and MAb 7C4 was conjugated with HRP. This ELISA showed a high sensitivity, with detection limit to MERS-CoV-rNP of less than 1 ng/mL; and a high specificity, with no detection for the HCoV-OC43-rNP and HCoV-229E-rNP (<xref rid="F0004" ref-type="fig">Figure 4A</xref>). The high sensitivity was further confirmed with a detection limit of 10<sup>3</sup> TCID<sub>50</sub> of inactivated viral lysate of MERS-CoV culture and the high specificity was further confirmed with no detection for inactivated viral lysates of two HCoVs (HCoV-OC43 and HCoV-229E), two influenza A virus strains (CA/4/2009(H1N1) and Brisbane/10/2007(H3N2)), one influenza B virus strain (Florida/04/2006), and one RSV strain (<xref rid="F0004" ref-type="fig">Figure 4B</xref>).
<fig fig-type="figure" id="F0004" orientation="portrait" position="float"><label>Figure 4</label><p>Detection limit of anti-MERS-CoV-rNP-specific MAbs-based capture ELISA for MERS-CoV-NP. (<bold>A</bold>) Detection of MERS-CoV-rNP, HCoV-OC43-rNP, and HCoV-229E-rNP. (<bold>B</bold>) Detection of inactivated cell lysates of MERS-CoV, HCoV-OC43, HCoV-229E, influenza A virus CA/4/2009(H1N1), influenza A virus Brisbane/10/2007(H3N2), influenza B virus Florida/04/2006, and RSV cultures.</p><graphic xlink:href="TEMI_A_12040307_F0004_OC"></graphic></fig></p></sec><sec><title>Evaluation of MERS-CoV-NP capture ELISA</title><p>The mean value of 20 human NPAs negative for influenza A, B, and C viruses, adenovirus, RSV, parainfluenza viruses 1–4, human rhinoviruses, human metapneumovirus, HCoV-HKU1, HCoV-OC43, HCoV-229E, HCoV-NL63, and MERS-CoV was 0.04. Using a cutoff of 0.19 (0.15 + 0.04), all 129 NPAs positive for various viral respiratory tract infections were negative, with mean OD ± SD of 0.067 ± 0.022 (<xref rid="F0005" ref-type="fig">Figure 5</xref>). Therefore, the specificity of the capture ELISA for NPA is 100%. For the two simulated MERS-CoV positive NPAs with serial dilutions (1–500 000 TCID<sub>50</sub>/0.1 mL) of live MERS-CoV added, all samples with ≥10 TCID<sub>50</sub>/0.1 mL showed positive results (<xref rid="F0005" ref-type="fig">Figure 5</xref>). As for the 10 additional NPAs with 20 TCID<sub>50</sub>/0.1 mL and 200 TCID<sub>50</sub>/0.1 mL of live MERS-CoV added, all showed positive results (<xref rid="F0005" ref-type="fig">Figure 5</xref>).
<fig fig-type="figure" id="F0005" orientation="portrait" position="float"><label>Figure 5</label><p>Evaluation of the MERS-CoV-NP capture ELISA using 129 NPAs positive for various viral respiratory tract infections, two simulated MERS-CoV-positive NPAs spiked with serially diluted MERS-CoV cell lysates and 10 simulated MERS-CoV-positive NPAs with 20 TCID<sub>50</sub>/0.1 mL and 200 TCID<sub>50</sub>/0.1 mL of MERS-CoV. The dotted line represents the corresponding cutoff OD<sub>450</sub> value at 0.19.</p><graphic xlink:href="TEMI_A_12040307_F0005_OC"></graphic></fig></p></sec></sec><sec><title>DISCUSSION</title><p>We report the development of a MAbs-based antigen capture ELISA for MERS-CoV-NP. Our previous experience on SARS-CoV has shown that SARS-CoV-NP detection in SARS patients is highly feasible using an antigen capture ELISA-based method.<sup><xref rid="bib14" ref-type="bibr">14</xref>,<xref rid="bib15" ref-type="bibr">15</xref></sup> Since NP is also an important, highly immunogenic and abundantly expressed structural protein in MERS-CoV, its detection using similar principles should also be feasible. The NP protein is a more preferable target than the S protein because the NP protein is present in much higher amount than the S protein. In this study, using hyperimmune sera obtained from mice immunized with three doses of MERS-CoV-rNP, we confirmed that MERS-CoV-NP is abundantly present in inactivated MERS-CoV cell cultures (<xref rid="F0002" ref-type="fig">Figure 2</xref>). The antigen capture ELISA was designed using two different MAbs (1F6 and 7C4) with high and specific antibody titers against MERS-CoV-rNP (<xref rid="F0003" ref-type="fig">Figure 3</xref>). As half of the 24 MAbs generated showed different degrees of cross-reactivity against HCoV-229E and/or HCoV-OC43, suggesting the possibility of common epitopes across the different CoVs, this MAbs-based ELISA should be much more specific than a polyclonal antibodies-based ELISA using hyperimmune sera generated from two different kinds of animals immunized with MERS-CoV-rNP. In addition to the high specificity, we also observed that MERS-CoV-rNP and MERS-CoV-NP can be detected by the present assay with a protein and inactivated viral concentrations of as low as 1 ng/mL and 10<sup>3</sup> TCID<sub>50</sub>/mL respectively (<xref rid="F0004" ref-type="fig">Figure 4</xref>).</p><p>Evaluation of the diagnostic test using control and simulated clinical specimens showed that the present ELISA is highly sensitive and specific for MERS-CoV. Using more than 120 NPAs positive with various respiratory viruses, we showed that the antigen capture ELISA is highly specific. Most importantly, NPAs positive for the four human CoVs all showed very low OD values by the present MERS-CoV antigen detection assay, as the MERS-CoV-NP only shares 18.1%, 19.1%, 30.5%, and 30.5% amino acid identities with those of HCoV-229E (alphaCoV), HCoV-NL63 (alphaCoV), HCoV-OC43 (betaCoV), and HCoV-HKU1 (betaCoV) respectively. As there have been no imported MERS cases in Hong Kong so far and we do not have access to any MERS patient samples, we used simulated NPAs spiked with live MERS-CoV for evaluation. Since the assay is able to detect simulated NPA samples with viral load as low as 10 TCID<sub>50</sub>/0.1 mL of live MERS-CoV, this diagnostic tool should be suitable for early diagnosis of MERS, since the viral loads in NPAs of patients during acute infection are usually around 10<sup>6</sup> copies/mL.<sup><xref rid="bib27" ref-type="bibr">27</xref>,<xref rid="bib28" ref-type="bibr">28</xref></sup></p><p>The present MERS-CoV-NP capture ELISA is potentially useful for detecting MERS-CoV in dromedary camel samples and screening other animal specimens for MERS-CoV. Although the ancestral origin of MERS-CoV is believed to be from bats,<sup><xref rid="bib29" ref-type="bibr">29</xref>,<xref rid="bib30" ref-type="bibr">30</xref>,<xref rid="bib31" ref-type="bibr">31</xref></sup> dromedaries are likely to be the immediate animal reservoir for transmission of MERS-CoV to human. High titers of antibodies to MERS-CoV have been detected in more than 95% of adult dromedaries in the Middle East,<sup><xref rid="bib5" ref-type="bibr">5</xref>,<xref rid="bib9" ref-type="bibr">9</xref>,<xref rid="bib32" ref-type="bibr">32</xref>,<xref rid="bib33" ref-type="bibr">33</xref></sup> and MERS-CoV have also been found from the nasal samples of a small proportion of dromedaries.<sup><xref rid="bib8" ref-type="bibr">8</xref>,<xref rid="bib34" ref-type="bibr">34</xref>,<xref rid="bib35" ref-type="bibr">35</xref>,<xref rid="bib36" ref-type="bibr">36</xref></sup> As MERS-CoV was also detected in the nasal samples in dromedaries with viral loads of around 10<sup>4</sup> to 10<sup>6</sup> copies/mL,<sup><xref rid="bib37" ref-type="bibr">37</xref></sup> the present ELISA should be readily useful for detecting MERS-CoV in dromedaries. To prevent MERS infections as well as in cases of MERS outbreaks, it may be necessary to screen and isolate dromedaries that are actively shedding MERS-CoV. Since the present ELISA captures MERS-CoV-NP antigen, it should be able to detect MERS-CoV regardless of the source of the specimen, and should be extremely useful for assessing viral shedding patterns in dromedaries as well as searching for other animal sources of MERS-CoV.</p><p>This antigen detection ELISA is suitable for clinical use and field studies in the Middle East where most cases of MERS have occurred. So far, most patients with MERS either resided in or had recent travel histories to the Middle East. Therefore, a MERS diagnostic test developed should be able to be used in clinical microbiology laboratories in the Middle East. As for the animal reservoirs, the infected dromedaries also live in the Middle East as well as North Africa. Compared to quantitative real-time RT-PCR, the consumable cost of the present antigen capture ELISA is lower. It also does not require expertise on molecular technology and the expensive set-up for molecular studies, which may not be present in most clinical microbiology laboratories in the Middle East. For example, about 15 million people, including more than two million pilgrims, visit Mecca in Saudi Arabia every year. In particular, a high concentration of visitors is expected during the Hajj period. A user-friendly laboratory test would be necessary for handling such a large number of exposed people as well as potential outbreaks because of mass gathering. Therefore, the present ELISA is likely to be more suitable than quantitative real-time RT-PCR for the use in laboratories in the Middle East and North Africa, where resources and expertise could be limited. As for animals, this test would be extremely useful for tracking the transmission patterns of MERS-CoV among the dromedaries in the same farm as well as across different countries. Further refinement of the test and the development of a rapid antigen detection kit for on-the-spot diagnosis are warranted.</p></sec></body><back><sec><title>Acknowledgements</title><p> We thank Dr Wing-Man Ko, Secretary for Food and Health and Dr Constance Chan, Director of Department of Health, HKSAR, The People’s Republic of China for their continuous support. We also thank RA Fouchier (Department of Viroscience, Erasmus Medical Center, Rotterdam, the Netherlands) and colleagues for providing the MERS-CoV. This work is partly supported by grants from the National 863 Program of China (2012AA02A307); Hong Kong, Macao, and Taiwan Science and Technology Cooperation Special Project of China (2012DFH30020); the HKSAR Health and Medical Research Fund; Seed Funding for Theme-based Research Scheme and Strategic Research Theme Fund, The University of Hong Kong; Theme-based Research Scheme, Research Grant Council Grant, University Grant Council; and Consultancy Service for Enhancing Laboratory Surveillance of Emerging Infectious Disease for the HKSAR Department of Health.</p></sec><ref-list><ref id="bib1"><label>1</label><mixed-citation publication-type="journal"><person-group person-group-type="author"><name name-style="western"><surname>Chan</surname><given-names>JF</given-names></name></person-group>, <person-group person-group-type="author"><name name-style="western"><surname>Lau</surname><given-names>SK</given-names></name></person-group>, <person-group person-group-type="author"><name name-style="western"><surname>To</surname><given-names>KK</given-names></name></person-group>, <person-group person-group-type="author"><name name-style="western"><surname>Cheng</surname><given-names>VC</given-names></name></person-group>, <person-group person-group-type="author"><name name-style="western"><surname>Woo</surname><given-names>PC</given-names></name></person-group>, <person-group person-group-type="author"><name name-style="western"><surname>Yuen</surname><given-names>KY</given-names></name></person-group><article-title>Middle East respiratory syndrome coronavirus: another zoonotic betacoronavirus causing severe disease</article-title>. <source>Clin Microbiol Rev</source><year>2015</year>; <volume>28</volume>: <fpage>465</fpage>–<lpage>522</lpage>.<pub-id pub-id-type="pmid">25810418</pub-id></mixed-citation></ref><ref id="bib2"><label>2</label><mixed-citation publication-type="journal"><person-group person-group-type="author"><name name-style="western"><surname>Zaki</surname><given-names>AM</given-names></name></person-group>, <person-group person-group-type="author"><name name-style="western"><surname>van Boheemen</surname><given-names>S</given-names></name></person-group>, <person-group person-group-type="author"><name name-style="western"><surname>Bestebroer</surname><given-names>TM</given-names></name></person-group>, <person-group person-group-type="author"><name name-style="western"><surname>Osterhaus</surname><given-names>AD</given-names></name></person-group>, <person-group person-group-type="author"><name name-style="western"><surname>Fouchier</surname><given-names>RA</given-names></name></person-group><article-title>Isolation of a novel coronavirus from a man with pneumonia in Saudi Arabia</article-title>. <source>N Engl J Med</source><year>2012</year>; <volume>367</volume>: <fpage>1814</fpage>–<lpage>1820</lpage>.<pub-id pub-id-type="pmid">23075143</pub-id></mixed-citation></ref><ref id="bib3"><label>3</label><mixed-citation publication-type="journal"><person-group person-group-type="author"><name name-style="western"><surname>van Boheemen</surname><given-names>S</given-names></name></person-group>, <person-group person-group-type="author"><name name-style="western"><surname>de Graaf</surname><given-names>M</given-names></name></person-group>, <person-group person-group-type="author"><name name-style="western"><surname>Lauber</surname><given-names>C</given-names></name></person-group> et al.<article-title>Genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans</article-title>. <source>MBio</source><year>2012</year>; <volume>3</volume>: pii: <fpage>e00473</fpage>–<lpage>12</lpage>.<pub-id pub-id-type="pmid">23170002</pub-id></mixed-citation></ref><ref id="bib4"><label>4</label><mixed-citation publication-type="journal"><person-group person-group-type="author"><name name-style="western"><surname>de Groot</surname><given-names>RJ</given-names></name></person-group>, <person-group person-group-type="author"><name name-style="western"><surname>Baker</surname><given-names>SC</given-names></name></person-group>, <person-group person-group-type="author"><name name-style="western"><surname>Baric</surname><given-names>RS</given-names></name></person-group> et al.<article-title>Middle East respiratory syndrome coronavirus (MERS-CoV): announcement of the Coronavirus Study Group</article-title>. <source>J Virol</source><year>2013</year>; <volume>87</volume>: <fpage>7790</fpage>–<lpage>7792</lpage>.<pub-id pub-id-type="pmid">23678167</pub-id></mixed-citation></ref><ref id="bib5"><label>5</label><mixed-citation publication-type="journal"><person-group person-group-type="author"><name name-style="western"><surname>Reusken</surname><given-names>CB</given-names></name></person-group>, <person-group person-group-type="author"><name name-style="western"><surname>Haagmans</surname><given-names>BL</given-names></name></person-group>, <person-group person-group-type="author"><name name-style="western"><surname>Muller</surname><given-names>MA</given-names></name></person-group> et al.<article-title>Middle East respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study</article-title>. <source>Lancet Infect Dis</source><year>2013</year>; <volume>13</volume>: <fpage>859</fpage>–<lpage>866</lpage>.<pub-id pub-id-type="pmid">23933067</pub-id></mixed-citation></ref><ref id="bib6"><label>6</label><mixed-citation publication-type="journal"><person-group person-group-type="author"><name name-style="western"><surname>Haagmans</surname><given-names>BL</given-names></name></person-group>, <person-group person-group-type="author"><name name-style="western"><surname>Al Dhahiry</surname><given-names>SH</given-names></name></person-group>, <person-group person-group-type="author"><name name-style="western"><surname>Reusken</surname><given-names>CB</given-names></name></person-group> et al.<article-title>Middle East respiratory syndrome coronavirus in dromedary camels: an outbreak investigation</article-title>. <source>Lancet Infect Dis</source><year>2013</year>; <volume>14</volume>: <fpage>140</fpage>–<lpage>145</lpage>.<pub-id pub-id-type="pmid">24355866</pub-id></mixed-citation></ref><ref id="bib7"><label>7</label><mixed-citation publication-type="journal"><person-group person-group-type="author"><name name-style="western"><surname>Briese</surname><given-names>T</given-names></name></person-group>, <person-group person-group-type="author"><name name-style="western"><surname>Mishra</surname><given-names>N</given-names></name></person-group>, <person-group person-group-type="author"><name name-style="western"><surname>Jain</surname><given-names>K</given-names></name></person-group> et al.<article-title>Middle East respiratory syndrome coronavirus quasispecies that include homologues of human isolates revealed through whole-genome analysis and virus cultured from dromedary camels in Saudi Arabia</article-title>. <source>MBio</source><year>2014</year>; <volume>5</volume>: <fpage>e01146</fpage>–<lpage>e01114</lpage>.<pub-id pub-id-type="pmid">24781747</pub-id></mixed-citation></ref><ref id="bib8"><label>8</label><mixed-citation publication-type="journal"><person-group person-group-type="author"><name name-style="western"><surname>Hemida</surname><given-names>MG</given-names></name></person-group>, <person-group person-group-type="author"><name name-style="western"><surname>Chu</surname><given-names>DK</given-names></name></person-group>, <person-group person-group-type="author"><name name-style="western"><surname>Poon</surname><given-names>LL</given-names></name></person-group> et al.<article-title>MERS coronavirus in dromedary camel herd, Saudi Arabia</article-title>. <source>Emerg Infect Dis</source><year>2014</year>; <volume>20</volume>: <fpage>1231</fpage>–<lpage>1234</lpage>.<pub-id pub-id-type="pmid">24964193</pub-id></mixed-citation></ref><ref id="bib9"><label>9</label><mixed-citation publication-type="journal"><person-group person-group-type="author"><name name-style="western"><surname>Woo</surname><given-names>PC</given-names></name></person-group>, <person-group person-group-type="author"><name name-style="western"><surname>Lau</surname><given-names>SK</given-names></name></person-group>, <person-group person-group-type="author"><name name-style="western"><surname>Wernery</surname><given-names>U</given-names></name></person-group> et al.<article-title>Novel betacoronavirus in dromedaries of the Middle East, 2013</article-title>. <source>Emerg Infect Dis</source><year>2014</year>; <volume>20</volume>: <fpage>560</fpage>–<lpage>572</lpage>.<pub-id pub-id-type="pmid">24655427</pub-id></mixed-citation></ref><ref id="bib10"><label>10</label><mixed-citation publication-type="journal"><person-group person-group-type="author"><name name-style="western"><surname>Bermingham</surname><given-names>A</given-names></name></person-group>, <person-group person-group-type="author"><name name-style="western"><surname>Chand</surname><given-names>MA</given-names></name></person-group>, <person-group person-group-type="author"><name name-style="western"><surname>Brown</surname><given-names>CS</given-names></name></person-group> et al.<article-title>Severe respiratory illness caused by a novel coronavirus, in a patient transferred to the United Kingdom from the Middle East, September 2012</article-title>. <source>Euro Surveill</source><year>2012</year>; <volume>17</volume>: <fpage>20290</fpage>.<pub-id pub-id-type="pmid">23078800</pub-id></mixed-citation></ref><ref id="bib11"><label>11</label><mixed-citation publication-type="journal"><person-group person-group-type="author"><name name-style="western"><surname>Corman</surname><given-names>VM</given-names></name></person-group>, <person-group person-group-type="author"><name name-style="western"><surname>Eckerle</surname><given-names>I</given-names></name></person-group>, <person-group person-group-type="author"><name name-style="western"><surname>Bleicker</surname><given-names>T</given-names></name></person-group> et al.<article-title>Detection of a novel human coronavirus by real-time reverse-transcription polymerase chain reaction</article-title>. <source>Euro Surveill</source><year>2012</year>; <volume>17</volume>: pii: <fpage>20285</fpage>.<pub-id pub-id-type="pmid">23041020</pub-id></mixed-citation></ref><ref id="bib12"><label>12</label><mixed-citation publication-type="journal"><person-group person-group-type="author"><name name-style="western"><surname>Guery</surname><given-names>B</given-names></name></person-group>, <person-group person-group-type="author"><name name-style="western"><surname>Poissy</surname><given-names>J</given-names></name></person-group>, <person-group person-group-type="author"><name name-style="western"><surname>el Mansouf</surname><given-names>L</given-names></name></person-group> et al.<article-title>Clinical features and viral diagnosis of two cases of infection with Middle East Respiratory Syndrome coronavirus: a report of nosocomial transmission</article-title>. <source>Lancet</source><year>2013</year>; <volume>381</volume>: <fpage>2265</fpage>–<lpage>2272</lpage>.<pub-id pub-id-type="pmid">23727167</pub-id></mixed-citation></ref><ref id="bib13"><label>13</label><mixed-citation publication-type="journal"><person-group person-group-type="author"><name name-style="western"><surname>Lu</surname><given-names>X</given-names></name></person-group>, <person-group person-group-type="author"><name name-style="western"><surname>Whitaker</surname><given-names>B</given-names></name></person-group>, <person-group person-group-type="author"><name name-style="western"><surname>Sakthivel</surname><given-names>SK</given-names></name></person-group> et al.<article-title>Real-time reverse transcription-PCR assay panel for Middle East respiratory syndrome coronavirus</article-title>. <source>J Clin Microbiol</source><year>2014</year>; <volume>52</volume>: <fpage>67</fpage>–<lpage>75</lpage>.<pub-id pub-id-type="pmid">24153118</pub-id></mixed-citation></ref><ref id="bib14"><label>14</label><mixed-citation publication-type="journal"><person-group person-group-type="author"><name name-style="western"><surname>Lau</surname><given-names>SK</given-names></name></person-group>, <person-group person-group-type="author"><name name-style="western"><surname>Che</surname><given-names>XY</given-names></name></person-group>, <person-group person-group-type="author"><name name-style="western"><surname>Woo</surname><given-names>PC</given-names></name></person-group> et al.<article-title>SARS coronavirus detection methods</article-title>. <source>Emerg Infect Dis</source><year>2005</year>; <volume>11</volume>: <fpage>1108</fpage>–<lpage>1111</lpage>.<pub-id pub-id-type="pmid">16022791</pub-id></mixed-citation></ref><ref id="bib15"><label>15</label><mixed-citation publication-type="journal"><person-group person-group-type="author"><name name-style="western"><surname>Lau</surname><given-names>SK</given-names></name></person-group>, <person-group person-group-type="author"><name name-style="western"><surname>Woo</surname><given-names>PC</given-names></name></person-group>, <person-group person-group-type="author"><name name-style="western"><surname>Wong</surname><given-names>BH</given-names></name></person-group> et al.<article-title>Detection of severe acute respiratory syndrome (SARS) coronavirus nucleocapsid protein in SARS patients by enzyme-linked immunosorbent assay</article-title>. <source>J Clin Microbiol</source><year>2004</year>; <volume>42</volume>: <fpage>2884</fpage>–<lpage>2889</lpage>.<pub-id pub-id-type="pmid">15243033</pub-id></mixed-citation></ref><ref id="bib16"><label>16</label><mixed-citation publication-type="journal"><person-group person-group-type="author"><name name-style="western"><surname>Chan</surname><given-names>JF</given-names></name></person-group>, <person-group person-group-type="author"><name name-style="western"><surname>Chan</surname><given-names>KH</given-names></name></person-group>, <person-group person-group-type="author"><name name-style="western"><surname>Choi</surname><given-names>GK</given-names></name></person-group> et al.<article-title>Differential cell line susceptibility to the emerging novel human betacoronavirus 2c EMC/2012: implications for disease pathogenesis and clinical manifestation</article-title>. <source>J Infect Dis</source><year>2013</year>; <volume>207</volume>: <fpage>1743</fpage>–<lpage>1752</lpage>.<pub-id pub-id-type="pmid">23532101</pub-id></mixed-citation></ref><ref id="bib17"><label>17</label><mixed-citation publication-type="journal"><person-group person-group-type="author"><name name-style="western"><surname>Lau</surname><given-names>SK</given-names></name></person-group>, <person-group person-group-type="author"><name name-style="western"><surname>Lau</surname><given-names>CC</given-names></name></person-group>, <person-group person-group-type="author"><name name-style="western"><surname>Chan</surname><given-names>KH</given-names></name></person-group> et al.<article-title>Delayed induction of proinflammatory cytokines and suppression of innate antiviral response by the novel Middle East respiratory syndrome coronavirus: implications for pathogenesis and treatment</article-title>. <source>J Gen Virol</source><year>2013</year>; <volume>94</volume>: <fpage>2679</fpage>–<lpage>2690</lpage>.<pub-id pub-id-type="pmid">24077366</pub-id></mixed-citation></ref><ref id="bib18"><label>18</label><mixed-citation publication-type="journal"><person-group person-group-type="author"><name name-style="western"><surname>Chan</surname><given-names>KH</given-names></name></person-group>, <person-group person-group-type="author"><name name-style="western"><surname>Chan</surname><given-names>JF</given-names></name></person-group>, <person-group person-group-type="author"><name name-style="western"><surname>Tse</surname><given-names>H</given-names></name></person-group> et al.<article-title>Cross-reactive antibodies in convalescent SARS patients’ sera against the emerging novel human coronavirus EMC (2012) by both immunofluorescent and neutralizing antibody tests</article-title>. <source>J Infect</source><year>2013</year>; <volume>67</volume>: <fpage>130</fpage>–<lpage>140</lpage>.<pub-id pub-id-type="pmid">23583636</pub-id></mixed-citation></ref><ref id="bib19"><label>19</label><mixed-citation publication-type="journal"><person-group person-group-type="author"><name name-style="western"><surname>Gui</surname><given-names>X</given-names></name></person-group>, <person-group person-group-type="author"><name name-style="western"><surname>Ge</surname><given-names>P</given-names></name></person-group>, <person-group person-group-type="author"><name name-style="western"><surname>Wang</surname><given-names>X</given-names></name></person-group> et al.<article-title>Identification of a highly conserved and surface exposed B-cell epitope on the nucleoprotein of influenza A virus</article-title>. <source>J Med Virol</source><year>2014</year>; <volume>86</volume>: <fpage>995</fpage>–<lpage>1002</lpage>.<pub-id pub-id-type="pmid">24136709</pub-id></mixed-citation></ref><ref id="bib20"><label>20</label><mixed-citation publication-type="journal"><person-group person-group-type="author"><name name-style="western"><surname>Gui</surname><given-names>X</given-names></name></person-group>, <person-group person-group-type="author"><name name-style="western"><surname>Li</surname><given-names>R</given-names></name></person-group>, <person-group person-group-type="author"><name name-style="western"><surname>Zhang</surname><given-names>X</given-names></name></person-group> et al.<article-title>An important amino acid in nucleoprotein contributes to influenza A virus replication by interacting with polymerase PB2</article-title>. <source>Virology</source><year>2014</year>; <volume>464–465</volume>: <fpage>11</fpage>–<lpage>20</lpage>.</mixed-citation></ref><ref id="bib21"><label>21</label><mixed-citation publication-type="journal"><person-group person-group-type="author"><name name-style="western"><surname>Yuan</surname><given-names>Q</given-names></name></person-group>, <person-group person-group-type="author"><name name-style="western"><surname>Cheng</surname><given-names>XD</given-names></name></person-group>, <person-group person-group-type="author"><name name-style="western"><surname>Yang</surname><given-names>BC</given-names></name></person-group> et al.<article-title>Differential diagnosis of pandemic (H1N1) 2009 infection by detection of haemagglutinin with an enzyme-linked immunoassay</article-title>. <source>Clin Microbiol Infect</source><year>2011</year>; <volume>17</volume>: <fpage>1574</fpage>–<lpage>1580</lpage>.<pub-id pub-id-type="pmid">21054661</pub-id></mixed-citation></ref><ref id="bib22"><label>22</label><mixed-citation publication-type="journal"><person-group person-group-type="author"><name name-style="western"><surname>Lau</surname><given-names>SK</given-names></name></person-group>, <person-group person-group-type="author"><name name-style="western"><surname>To</surname><given-names>WK</given-names></name></person-group>, <person-group person-group-type="author"><name name-style="western"><surname>Tse</surname><given-names>PW</given-names></name></person-group> et al.<article-title>Human parainfluenza virus 4 outbreak and the role of diagnostic tests</article-title>. <source>J Clin Microbiol</source><year>2005</year>; <volume>43</volume>: <fpage>4515</fpage>–<lpage>4521</lpage>.<pub-id pub-id-type="pmid">16145100</pub-id></mixed-citation></ref><ref id="bib23"><label>23</label><mixed-citation publication-type="journal"><person-group person-group-type="author"><name name-style="western"><surname>Lau</surname><given-names>SK</given-names></name></person-group>, <person-group person-group-type="author"><name name-style="western"><surname>Yip</surname><given-names>CC</given-names></name></person-group>, <person-group person-group-type="author"><name name-style="western"><surname>Tsoi</surname><given-names>HW</given-names></name></person-group> et al.<article-title>Clinical features and complete genome characterization of a distinct human rhinovirus (HRV) genetic cluster, probably representing a previously undetected HRV species, HRV-C, associated with acute respiratory illness in children</article-title>. <source>J Clin Microbiol</source><year>2007</year>; <volume>45</volume>: <fpage>3655</fpage>–<lpage>3664</lpage>.<pub-id pub-id-type="pmid">17804649</pub-id></mixed-citation></ref><ref id="bib24"><label>24</label><mixed-citation publication-type="journal"><person-group person-group-type="author"><name name-style="western"><surname>Lau</surname><given-names>SK</given-names></name></person-group>, <person-group person-group-type="author"><name name-style="western"><surname>Woo</surname><given-names>PC</given-names></name></person-group>, <person-group person-group-type="author"><name name-style="western"><surname>Yip</surname><given-names>CC</given-names></name></person-group> et al.<article-title>Coronavirus HKU1 and other coronavirus infections in Hong Kong</article-title>. <source>J Clin Microbiol</source><year>2006</year>; <volume>44</volume>: <fpage>2063</fpage>–<lpage>2071</lpage>.<pub-id pub-id-type="pmid">16757599</pub-id></mixed-citation></ref><ref id="bib25"><label>25</label><mixed-citation publication-type="journal"><person-group person-group-type="author"><name name-style="western"><surname>Woo</surname><given-names>PC</given-names></name></person-group>, <person-group person-group-type="author"><name name-style="western"><surname>Lau</surname><given-names>SK</given-names></name></person-group>, <person-group person-group-type="author"><name name-style="western"><surname>Chu</surname><given-names>CM</given-names></name></person-group> et al.<article-title>Characterization and complete genome sequence of a novel coronavirus, coronavirus HKU1, from patients with pneumonia</article-title>. <source>J Virol</source><year>2005</year>; <volume>79</volume>: <fpage>884</fpage>–<lpage>895</lpage>.<pub-id pub-id-type="pmid">15613317</pub-id></mixed-citation></ref><ref id="bib26"><label>26</label><mixed-citation publication-type="journal"><person-group person-group-type="author"><name name-style="western"><surname>Woo</surname><given-names>PC</given-names></name></person-group>, <person-group person-group-type="author"><name name-style="western"><surname>Chiu</surname><given-names>SS</given-names></name></person-group>, <person-group person-group-type="author"><name name-style="western"><surname>Seto</surname><given-names>WH</given-names></name></person-group> et al.<article-title>Cost-effectiveness of rapid diagnosis of viral respiratory tract infections in pediatric patients</article-title>. <source>J Clin Microbiol</source><year>1997</year>; <volume>35</volume>: <fpage>1579</fpage>–<lpage>1581</lpage>.<pub-id pub-id-type="pmid">9163486</pub-id></mixed-citation></ref><ref id="bib27"><label>27</label><mixed-citation publication-type="journal"><person-group person-group-type="author"><name name-style="western"><surname>Drosten</surname><given-names>C</given-names></name></person-group>, <person-group person-group-type="author"><name name-style="western"><surname>Seilmaier</surname><given-names>M</given-names></name></person-group>, <person-group person-group-type="author"><name name-style="western"><surname>Corman</surname><given-names>VM</given-names></name></person-group> et al.<article-title>Clinical features and virological analysis of a case of Middle East respiratory syndrome coronavirus infection</article-title>. <source>Lancet Infect Dis</source><year>2013</year>; <volume>13</volume>: <fpage>745</fpage>–<lpage>751</lpage>.<pub-id pub-id-type="pmid">23782859</pub-id></mixed-citation></ref><ref id="bib28"><label>28</label><mixed-citation publication-type="journal"><person-group person-group-type="author"><name name-style="western"><surname>Kapoor</surname><given-names>M</given-names></name></person-group>, <person-group person-group-type="author"><name name-style="western"><surname>Pringle</surname><given-names>K</given-names></name></person-group>, <person-group person-group-type="author"><name name-style="western"><surname>Kumar</surname><given-names>A</given-names></name></person-group> et al.<article-title>Clinical and laboratory findings of the first imported case of middle East respiratory syndrome coronavirus to the United States</article-title>. <source>Clin Infect Dis</source><year>2014</year>; <volume>59</volume>: <fpage>1511</fpage>–<lpage>1518</lpage>.<pub-id pub-id-type="pmid">25100864</pub-id></mixed-citation></ref><ref id="bib29"><label>29</label><mixed-citation publication-type="journal"><person-group person-group-type="author"><name name-style="western"><surname>Corman</surname><given-names>VM</given-names></name></person-group>, <person-group person-group-type="author"><name name-style="western"><surname>Ithete</surname><given-names>NL</given-names></name></person-group>, <person-group person-group-type="author"><name name-style="western"><surname>Richards</surname><given-names>LR</given-names></name></person-group> et al.<article-title>Rooting the phylogenetic tree of Middle East respiratory syndrome coronavirus by characterization of a conspecific virus from an African bat</article-title>. <source>J Virol</source><year>2014</year>; <volume>88</volume>: <fpage>11297</fpage>–<lpage>11303</lpage>.<pub-id pub-id-type="pmid">25031349</pub-id></mixed-citation></ref><ref id="bib30"><label>30</label><mixed-citation publication-type="journal"><person-group person-group-type="author"><name name-style="western"><surname>Lau</surname><given-names>SK</given-names></name></person-group>, <person-group person-group-type="author"><name name-style="western"><surname>Li</surname><given-names>KS</given-names></name></person-group>, <person-group person-group-type="author"><name name-style="western"><surname>Tsang</surname><given-names>AK</given-names></name></person-group> et al.<article-title>Genetic characterization of Betacoronavirus lineage C viruses in bats reveals marked sequence divergence in the spike protein of pipistrellus bat coronavirus HKU5 in Japanese pipistrelle: implications for the origin of the novel Middle East respiratory syndrome coronavirus</article-title>. <source>J Virol</source><year>2013</year>; <volume>87</volume>: <fpage>8638</fpage>–<lpage>8650</lpage>.<pub-id pub-id-type="pmid">23720729</pub-id></mixed-citation></ref><ref id="bib31"><label>31</label><mixed-citation publication-type="journal"><person-group person-group-type="author"><name name-style="western"><surname>Wang</surname><given-names>Q</given-names></name></person-group>, <person-group person-group-type="author"><name name-style="western"><surname>Qi</surname><given-names>J</given-names></name></person-group>, <person-group person-group-type="author"><name name-style="western"><surname>Yuan</surname><given-names>Y</given-names></name></person-group> et al.<article-title>Bat origins of MERS-CoV supported by bat coronavirus HKU4 usage of human receptor CD26</article-title>. <source>Cell Host Microbe</source><year>2014</year>; <volume>16</volume>: <fpage>328</fpage>–<lpage>337</lpage>.<pub-id pub-id-type="pmid">25211075</pub-id></mixed-citation></ref><ref id="bib32"><label>32</label><mixed-citation publication-type="journal"><person-group person-group-type="author"><name name-style="western"><surname>Reusken</surname><given-names>CB</given-names></name></person-group>, <person-group person-group-type="author"><name name-style="western"><surname>Ababneh</surname><given-names>M</given-names></name></person-group>, <person-group person-group-type="author"><name name-style="western"><surname>Raj</surname><given-names>VS</given-names></name></person-group> et al.<article-title>Middle East Respiratory Syndrome coronavirus (MERS-CoV) serology in major livestock species in an affected region in Jordan, June to September 2013</article-title>. <source>Euro Surveill</source><year>2013</year>; <volume>18</volume>: <fpage>20662</fpage>.<pub-id pub-id-type="pmid">24342516</pub-id></mixed-citation></ref><ref id="bib33"><label>33</label><mixed-citation publication-type="journal"><person-group person-group-type="author"><name name-style="western"><surname>Meyer</surname><given-names>B</given-names></name></person-group>, <person-group person-group-type="author"><name name-style="western"><surname>Muller</surname><given-names>MA</given-names></name></person-group>, <person-group person-group-type="author"><name name-style="western"><surname>Corman</surname><given-names>VM</given-names></name></person-group> et al.<article-title>Antibodies against MERS coronavirus in dromedary camels, United Arab Emirates, 2003 and 2013</article-title>. <source>Emerg Infect Dis</source><year>2014</year>; <volume>20</volume>: <fpage>552</fpage>–<lpage>559</lpage>.<pub-id pub-id-type="pmid">24655412</pub-id></mixed-citation></ref><ref id="bib34"><label>34</label><mixed-citation publication-type="journal"><person-group person-group-type="author"><name name-style="western"><surname>Azhar</surname><given-names>EI</given-names></name></person-group>, <person-group person-group-type="author"><name name-style="western"><surname>El-Kafrawy</surname><given-names>SA</given-names></name></person-group>, <person-group person-group-type="author"><name name-style="western"><surname>Farraj</surname><given-names>SA</given-names></name></person-group> et al.<article-title>Evidence for camel-to-human transmission of MERS coronavirus</article-title>. <source>N Engl J Med</source><year>2014</year>; <volume>370</volume>: <fpage>2499</fpage>–<lpage>2505</lpage>.<pub-id pub-id-type="pmid">24896817</pub-id></mixed-citation></ref><ref id="bib35"><label>35</label><mixed-citation publication-type="journal"><person-group person-group-type="author"><name name-style="western"><surname>Chu</surname><given-names>DK</given-names></name></person-group>, <person-group person-group-type="author"><name name-style="western"><surname>Poon</surname><given-names>LL</given-names></name></person-group>, <person-group person-group-type="author"><name name-style="western"><surname>Gomaa</surname><given-names>MM</given-names></name></person-group> et al.<article-title>MERS coronaviruses in dromedary camels, Egypt</article-title>. <source>Emerg Infect Dis</source><year>2014</year>; <volume>20</volume>: <fpage>1049</fpage>–<lpage>1053</lpage>.<pub-id pub-id-type="pmid">24856660</pub-id></mixed-citation></ref><ref id="bib36"><label>36</label><mixed-citation publication-type="journal"><person-group person-group-type="author"><name name-style="western"><surname>Nowotny</surname><given-names>N</given-names></name></person-group>, <person-group person-group-type="author"><name name-style="western"><surname>Kolodziejek</surname><given-names>J</given-names></name></person-group><article-title>Middle East respiratory syndrome coronavirus (MERS-CoV) in dromedary camels, Oman, 2013</article-title>. <source>Euro Surveill</source><year>2014</year>; <volume>19</volume>: <fpage>20781</fpage>.<pub-id pub-id-type="pmid">24786259</pub-id></mixed-citation></ref><ref id="bib37"><label>37</label><mixed-citation publication-type="journal"><person-group person-group-type="author"><name name-style="western"><surname>Alagaili</surname><given-names>AN</given-names></name></person-group>, <person-group person-group-type="author"><name name-style="western"><surname>Briese</surname><given-names>T</given-names></name></person-group>, <person-group person-group-type="author"><name name-style="western"><surname>Mishra</surname><given-names>N</given-names></name></person-group> et al.<article-title>Middle East respiratory syndrome coronavirus infection in dromedary camels in Saudi Arabia</article-title>. <source>MBio</source><year>2014</year>; <volume>5</volume>: <fpage>e00884</fpage>–<lpage>e00814</lpage>.<pub-id pub-id-type="pmid">24570370</pub-id></mixed-citation></ref></ref-list></back></pmc></record>Pour manipuler ce document sous Unix (Dilib)
EXPLOR_STEP=$WICRI_ROOT/Sante/explor/MersV1/Data/Pmc/Corpus
HfdSelect -h $EXPLOR_STEP/biblio.hfd -nk 000C25 | SxmlIndent | more
Ou
HfdSelect -h $EXPLOR_AREA/Data/Pmc/Corpus/biblio.hfd -nk 000C25 | SxmlIndent | more
Pour mettre un lien sur cette page dans le réseau Wicri
{{Explor lien
|wiki= Sante
|area= MersV1
|flux= Pmc
|étape= Corpus
|type= RBID
|clé=
|texte=
}}
| This area was generated with Dilib version V0.6.33. |  |