Characterization of anti-MERS-CoV antibodies against various recombinant structural antigens of MERS-CoV in an imported case in China
Identifieur interne : 000C27 ( Pmc/Corpus ); précédent : 000C26; suivant : 000C28Characterization of anti-MERS-CoV antibodies against various recombinant structural antigens of MERS-CoV in an imported case in China
Auteurs : Wenling Wang ; Huijuan Wang ; Yao Deng ; Tie Song ; Jiaming Lan ; Guizhen Wu ; Changwen Ke ; Wenjie TanSource :
- Emerging Microbes & Infections [ 2222-1751 ] ; 2019.
Abstract
The first imported case of Middle East respiratory syndrome (MERS) in China recently
occurred, allowing for the characterization of antibody titers in a series of the
patient’s sera using the following methods based on recombinant viral structural antigens:
inactivated MERS coronavirus (MERS-CoV) enzyme-linked immunosorbent assay (ELISA),
recombinant MERS-CoV spike (S, or fragments of S) ELISA, nucleoprotein (NP) ELISA and MERS
S pseudovirus particle-based neutralization test (ppNT). A longitudinal profile of the
infection showed that seroconversion detected by ELISAs based on the recombinant
extracellular domain, S, S1 and receptor-binding domain (RBD) antigens occurred as early
as neutralizing antibodies were detected by the ppNT and earlier than antibodies were
detected by the inactivated MERS-CoV and N-terminal domain (NTD) ELISAs. Antibodies
detected by the NP ELISA occurred last. Strong correlations were found between the S1, RBD
and NP ELISAs and the inactivated MERS-CoV ELISA. The S and RBD ELISAs were highly
correlated with the commercial S1 ELISA. The S ELISA strongly correlated with the ppNT,
although the MERS-CoV, S1, NTD and RBD ELISAs were also significantly correlated with the
ppNT (
Url:
DOI: 10.1038/emi.2016.114
PubMed: 27826140
PubMed Central: 5148018
Links to Exploration step
PMC:5148018Le document en format XML
<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Characterization of anti-MERS-CoV antibodies against various recombinant
structural antigens of MERS-CoV in an imported case in China</title>
<author><name sortKey="Wang, Wenling" sort="Wang, Wenling" uniqKey="Wang W" first="Wenling" last="Wang">Wenling Wang</name>
<affiliation><nlm:aff id="aff1"><institution>Key Laboratory of Medical Virology, Ministry of Health, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention</institution>
, Beijing 102206,<country>China</country>
</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Wang, Huijuan" sort="Wang, Huijuan" uniqKey="Wang H" first="Huijuan" last="Wang">Huijuan Wang</name>
<affiliation><nlm:aff id="aff1"><institution>Key Laboratory of Medical Virology, Ministry of Health, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention</institution>
, Beijing 102206,<country>China</country>
</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Deng, Yao" sort="Deng, Yao" uniqKey="Deng Y" first="Yao" last="Deng">Yao Deng</name>
<affiliation><nlm:aff id="aff1"><institution>Key Laboratory of Medical Virology, Ministry of Health, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention</institution>
, Beijing 102206,<country>China</country>
</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Song, Tie" sort="Song, Tie" uniqKey="Song T" first="Tie" last="Song">Tie Song</name>
<affiliation><nlm:aff id="aff2"><institution>Guangdong Provincial Center for Disease Control and Prevention</institution>
, Guangzhou 511430, Guangdong Province,<country>China</country>
</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Lan, Jiaming" sort="Lan, Jiaming" uniqKey="Lan J" first="Jiaming" last="Lan">Jiaming Lan</name>
<affiliation><nlm:aff id="aff1"><institution>Key Laboratory of Medical Virology, Ministry of Health, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention</institution>
, Beijing 102206,<country>China</country>
</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Wu, Guizhen" sort="Wu, Guizhen" uniqKey="Wu G" first="Guizhen" last="Wu">Guizhen Wu</name>
<affiliation><nlm:aff id="aff1"><institution>Key Laboratory of Medical Virology, Ministry of Health, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention</institution>
, Beijing 102206,<country>China</country>
</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Ke, Changwen" sort="Ke, Changwen" uniqKey="Ke C" first="Changwen" last="Ke">Changwen Ke</name>
<affiliation><nlm:aff id="aff2"><institution>Guangdong Provincial Center for Disease Control and Prevention</institution>
, Guangzhou 511430, Guangdong Province,<country>China</country>
</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Tan, Wenjie" sort="Tan, Wenjie" uniqKey="Tan W" first="Wenjie" last="Tan">Wenjie Tan</name>
<affiliation><nlm:aff id="aff1"><institution>Key Laboratory of Medical Virology, Ministry of Health, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention</institution>
, Beijing 102206,<country>China</country>
</nlm:aff>
</affiliation>
</author>
</titleStmt>
<publicationStmt><idno type="wicri:source">PMC</idno>
<idno type="pmid">27826140</idno>
<idno type="pmc">5148018</idno>
<idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5148018</idno>
<idno type="RBID">PMC:5148018</idno>
<idno type="doi">10.1038/emi.2016.114</idno>
<date when="2019">2019</date>
<idno type="wicri:Area/Pmc/Corpus">000C27</idno>
<idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000C27</idno>
</publicationStmt>
<sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Characterization of anti-MERS-CoV antibodies against various recombinant
structural antigens of MERS-CoV in an imported case in China</title>
<author><name sortKey="Wang, Wenling" sort="Wang, Wenling" uniqKey="Wang W" first="Wenling" last="Wang">Wenling Wang</name>
<affiliation><nlm:aff id="aff1"><institution>Key Laboratory of Medical Virology, Ministry of Health, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention</institution>
, Beijing 102206,<country>China</country>
</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Wang, Huijuan" sort="Wang, Huijuan" uniqKey="Wang H" first="Huijuan" last="Wang">Huijuan Wang</name>
<affiliation><nlm:aff id="aff1"><institution>Key Laboratory of Medical Virology, Ministry of Health, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention</institution>
, Beijing 102206,<country>China</country>
</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Deng, Yao" sort="Deng, Yao" uniqKey="Deng Y" first="Yao" last="Deng">Yao Deng</name>
<affiliation><nlm:aff id="aff1"><institution>Key Laboratory of Medical Virology, Ministry of Health, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention</institution>
, Beijing 102206,<country>China</country>
</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Song, Tie" sort="Song, Tie" uniqKey="Song T" first="Tie" last="Song">Tie Song</name>
<affiliation><nlm:aff id="aff2"><institution>Guangdong Provincial Center for Disease Control and Prevention</institution>
, Guangzhou 511430, Guangdong Province,<country>China</country>
</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Lan, Jiaming" sort="Lan, Jiaming" uniqKey="Lan J" first="Jiaming" last="Lan">Jiaming Lan</name>
<affiliation><nlm:aff id="aff1"><institution>Key Laboratory of Medical Virology, Ministry of Health, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention</institution>
, Beijing 102206,<country>China</country>
</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Wu, Guizhen" sort="Wu, Guizhen" uniqKey="Wu G" first="Guizhen" last="Wu">Guizhen Wu</name>
<affiliation><nlm:aff id="aff1"><institution>Key Laboratory of Medical Virology, Ministry of Health, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention</institution>
, Beijing 102206,<country>China</country>
</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Ke, Changwen" sort="Ke, Changwen" uniqKey="Ke C" first="Changwen" last="Ke">Changwen Ke</name>
<affiliation><nlm:aff id="aff2"><institution>Guangdong Provincial Center for Disease Control and Prevention</institution>
, Guangzhou 511430, Guangdong Province,<country>China</country>
</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Tan, Wenjie" sort="Tan, Wenjie" uniqKey="Tan W" first="Wenjie" last="Tan">Wenjie Tan</name>
<affiliation><nlm:aff id="aff1"><institution>Key Laboratory of Medical Virology, Ministry of Health, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention</institution>
, Beijing 102206,<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>The first imported case of Middle East respiratory syndrome (MERS) in China recently
occurred, allowing for the characterization of antibody titers in a series of the
patient’s sera using the following methods based on recombinant viral structural antigens:
inactivated MERS coronavirus (MERS-CoV) enzyme-linked immunosorbent assay (ELISA),
recombinant MERS-CoV spike (S, or fragments of S) ELISA, nucleoprotein (NP) ELISA and MERS
S pseudovirus particle-based neutralization test (ppNT). A longitudinal profile of the
infection showed that seroconversion detected by ELISAs based on the recombinant
extracellular domain, S, S1 and receptor-binding domain (RBD) antigens occurred as early
as neutralizing antibodies were detected by the ppNT and earlier than antibodies were
detected by the inactivated MERS-CoV and N-terminal domain (NTD) ELISAs. Antibodies
detected by the NP ELISA occurred last. Strong correlations were found between the S1, RBD
and NP ELISAs and the inactivated MERS-CoV ELISA. The S and RBD ELISAs were highly
correlated with the commercial S1 ELISA. The S ELISA strongly correlated with the ppNT,
although the MERS-CoV, S1, NTD and RBD ELISAs were also significantly correlated with the
ppNT (<italic>P</italic>
<0.001).</p>
</div>
</front>
<back><div1 type="bibliography"><listBibl><biblStruct></biblStruct>
<biblStruct><analytic><author><name sortKey="Da X0a Guan, W" uniqKey="Da X0a Guan W">W Da
Guan</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Park, Sw" uniqKey="Park S">SW Park</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Wernery, U" uniqKey="Wernery U">U Wernery</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="Park, Wb" uniqKey="Park W">WB Park</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Wu, J" uniqKey="Wu J">J Wu</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Lu, G" uniqKey="Lu G">G Lu</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Wang, Yq" uniqKey="Wang Y">YQ Wang</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Lan, J" uniqKey="Lan J">J Lan</name>
</author>
</analytic>
</biblStruct>
<biblStruct><analytic><author><name sortKey="Zhao, J" uniqKey="Zhao J">J Zhao</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="Li, G" uniqKey="Li G">G Li</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="Min, Ck" uniqKey="Min C">CK Min</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">27826140</article-id>
<article-id pub-id-type="pmc">5148018</article-id>
<article-id pub-id-type="publisher-id">12040281</article-id>
<article-id pub-id-type="doi">10.1038/emi.2016.114</article-id>
<article-categories><subj-group subj-group-type="heading"><subject>Original Articles</subject>
</subj-group>
</article-categories>
<title-group><article-title>Characterization of anti-MERS-CoV antibodies against various recombinant
structural antigens of MERS-CoV in an imported case in China</article-title>
</title-group>
<contrib-group><contrib contrib-type="author"><name><surname>Wang</surname>
<given-names>Wenling</given-names>
</name>
<xref ref-type="aff" rid="aff1"><sup>1</sup>
</xref>
<xref ref-type="corresp" rid="AN0001"><sup>*</sup>
</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Wang</surname>
<given-names>Huijuan</given-names>
</name>
<xref ref-type="aff" rid="aff1"><sup>1</sup>
</xref>
<xref ref-type="corresp" rid="AN0001"><sup>*</sup>
</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Deng</surname>
<given-names>Yao</given-names>
</name>
<xref ref-type="aff" rid="aff1"><sup>1</sup>
</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Song</surname>
<given-names>Tie</given-names>
</name>
<xref ref-type="aff" rid="aff2"><sup>2</sup>
</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Lan</surname>
<given-names>Jiaming</given-names>
</name>
<xref ref-type="aff" rid="aff1"><sup>1</sup>
</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Wu</surname>
<given-names>Guizhen</given-names>
</name>
<xref ref-type="aff" rid="aff1"><sup>1</sup>
</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Ke</surname>
<given-names>Changwen</given-names>
</name>
<xref ref-type="aff" rid="aff2"><sup>2</sup>
</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Tan</surname>
<given-names>Wenjie</given-names>
</name>
<xref ref-type="aff" rid="aff1"><sup>1</sup>
</xref>
<xref ref-type="corresp" rid="AN0002"></xref>
</contrib>
<aff id="aff1"><label>1</label>
<institution>Key Laboratory of Medical Virology, Ministry of Health, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention</institution>
, Beijing 102206,<country>China</country>
</aff>
<aff id="aff2"><label>2</label>
<institution>Guangdong Provincial Center for Disease Control and Prevention</institution>
, Guangzhou 511430, Guangdong Province,<country>China</country>
</aff>
</contrib-group>
<author-notes><corresp id="AN0001"><label>*</label>
These authors contributed equally to this
work.</corresp>
<corresp id="AN0002">WJ Tan E-mail: <email>tanwj28@163.com</email>
</corresp>
</author-notes>
<pub-date pub-type="collection"><year>2016</year>
</pub-date>
<pub-date pub-type="epub"><day>25</day>
<month>1</month>
<year>2019</year>
</pub-date>
<volume>5</volume>
<issue>1</issue>
<fpage seq="71">1</fpage>
<lpage>12</lpage>
<history><date date-type="received"><day>14</day>
<month>6</month>
<year>2016</year>
</date>
<date date-type="rev-recd"><day>09</day>
<month>8</month>
<year>2016</year>
</date>
<date date-type="accepted"><day>04</day>
<month>9</month>
<year>2016</year>
</date>
</history>
<permissions><copyright-statement>© 2016 The Author(s)</copyright-statement>
<copyright-year>2016</copyright-year>
<copyright-holder>The Author(s)</copyright-holder>
<license license-type="open-access" xlink:href="https://creativecommons.org/licenses/by/4.0/"><license-p><bold>Open Access</bold>
This work is licensed under a Creative Commons
Attribution 4.0 International 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 permission from the license holder to reproduce the
material. To view a copy of this license, visit <ext-link ext-link-type="uri" xlink:href="https://creativecommons.org/licenses/by/4.0/">https://creativecommons.org/licenses/by/4.0/</ext-link>
</license-p>
</license>
</permissions>
<self-uri content-type="pdf" xlink:href="TEMI_5_12040281.pdf"></self-uri>
<abstract><p>The first imported case of Middle East respiratory syndrome (MERS) in China recently
occurred, allowing for the characterization of antibody titers in a series of the
patient’s sera using the following methods based on recombinant viral structural antigens:
inactivated MERS coronavirus (MERS-CoV) enzyme-linked immunosorbent assay (ELISA),
recombinant MERS-CoV spike (S, or fragments of S) ELISA, nucleoprotein (NP) ELISA and MERS
S pseudovirus particle-based neutralization test (ppNT). A longitudinal profile of the
infection showed that seroconversion detected by ELISAs based on the recombinant
extracellular domain, S, S1 and receptor-binding domain (RBD) antigens occurred as early
as neutralizing antibodies were detected by the ppNT and earlier than antibodies were
detected by the inactivated MERS-CoV and N-terminal domain (NTD) ELISAs. Antibodies
detected by the NP ELISA occurred last. Strong correlations were found between the S1, RBD
and NP ELISAs and the inactivated MERS-CoV ELISA. The S and RBD ELISAs were highly
correlated with the commercial S1 ELISA. The S ELISA strongly correlated with the ppNT,
although the MERS-CoV, S1, NTD and RBD ELISAs were also significantly correlated with the
ppNT (<italic>P</italic>
<0.001).</p>
</abstract>
<kwd-group kwd-group-type="author"><title>Keywords</title>
<kwd>antibody</kwd>
<kwd>enzyme-linked immunosorbent assay</kwd>
<kwd>MERS-CoV</kwd>
<kwd>patient</kwd>
<kwd>recombinant structural antigens</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="15"></ref-count>
<page-count count="6"></page-count>
</counts>
</article-meta>
</front>
<body><sec><title>Introduction</title>
<p>Middle East respiratory syndrome (MERS) is caused by MERS coronavirus (MERS-CoV), and as of
23 March 2016, it had affected 26 countries, with 1698 cases and at least 609
deaths.<sup><xref rid="bib1">1</xref>
</sup>
Sensitive serological assays independent of
the use of live MERS-CoV and biosafety level 3 laboratories are important for clinical
diagnosis and research. Recombinant antigen-based enzyme-linked immunosorbent assays
(ELISAs) are preferred for diagnosis owing to their high efficiency and reproducibility.
Importantly, such assays do not require cultivation of MERS-CoV in biosafety level 3
facilities, which are not readily available in most laboratories. Although an S1-based ELISA
Kit is commercially available and has been used in many studies,<sup><xref rid="bib2">2</xref>
, <xref rid="bib3">3</xref>
, <xref rid="bib4">4</xref>
, <xref rid="bib5">5</xref>
, <xref rid="bib6">6</xref>
</sup>
data are limited on comparing ELISAs that
detect MERS-CoV antibodies in humans based on alternative recombinant antigens, including
the extracellular domain (representative of the full-length spike protein (S)), N-terminal
domain (NTD), receptor-binding domain (RBD) and nucleoprotein (NP). We used a series of sera
from the first imported case of MERS in China to longitudinally evaluate antibody production
and to compare the antigenicity of whole inactivated MERS-CoV, S, S1, NTD, RBD and NP. The
sera were also used to investigate the correlation of ELISAs with the pseudovirus
particle-based neutralization test (ppNT).</p>
</sec>
<sec><title>Materials and Methods</title>
<sec><title>Case report and blood samples</title>
<p>The first MERS-CoV case in China was imported from the Republic of Korea and confirmed
during hospital admission at the end of May 2015. A case investigation revealed a history
of exposure to the first confirmed MERS case in South Korea when the male patient in
question had visited his father who had been admitted to the same hospital ward as that of
the Korean patient with MERS-CoV. On 20 May, while in Korea, the patient complained of
feeling unwell but had no respiratory symptoms; his temperature was 38.7 °C on 25 May.
However, 1 day later, against medical advice, he traveled from South Korea to Guangdong
province (mainland China) via Hong Kong. On 27 May 2015, the Chinese Ministry of Health
was notified of the patient’s entry into China by the World Health Organization (WHO). The
man was confirmed to be MERS-CoV-positive on 28 May and was immediately admitted to a
hospital.<sup><xref rid="bib7">7</xref>
</sup>
He was discharged from the hospital at
the end of June 2015. A series of venous blood samples were collected from the patient
after he was admitted to the hospital. Normal control sera from 40 healthy blood donors
(both sexes) were used to detect background values and calculate the cutoff values for the
methods used in this study. The blood samples were processed within 24 h of collection,
and the sera were stored at −80 °C.</p>
</sec>
<sec><title>Virus preparation</title>
<p>MERS-CoV (strain EMC/2012) was kindly provided by Professor Ron Fouchier (Erasmus Medical
Centre, Rotterdam, The Netherlands). The virus was propagated in Vero cells (ATCC,
Manassas, VA, USA) in Dulbecco’s modified Eagle medium supplemented with 2% fetal calf
serum, 100 international units/mL penicillin and 100 μg/mL streptomycin at 37 °C in 5%
CO<sub>2</sub>
. All experiments related to live MERS-CoV were performed according to the
standard operating procedures of the biosafety level-3 facility in the Chinese Center for
Disease Control and Prevention (China CDC). Quantified MERS-CoV was inactivated with 0.4%
formaldehyde for seven days. The inactivated MERS-CoV was then centrifuged at 3000 r.p.m.
for 1 min at 4 °C to collect the supernatant after confirmation that it was non-infectious
by titration in Vero cells. Subsequently, the supernatant was concentrated by
centrifugation at 24 000 r.p.m. for 2 h at 4 °C, and the pellet was collected and
dissolved in phosphate-buffered saline (PBS) overnight. The concentrated MERS-CoV was
quantified using a BCA-Based Protein Quantification Kit (Applygen Technologies Inc.,
Beijing, China) and stored at −80 °C until use. In addition, the inactivated MERS-CoV was
characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western
blotting (WB) assay.</p>
</sec>
<sec><title>Preparation of recombinant proteins</title>
<p>The recombinant His-tagged extracellular domain (amino acids 1–1297) of MERS-CoV
(HCoV-EMC/2012) spike (S) expressed in Baculovirus–Insect cells was purchased from Sino
Biological Inc. (Beijing, China). Construction of expression plasmids for the RBD (amino
acids 367–606) of MERS-CoV and the method used for subsequent expression in
Baculovirus–Insect cells were described previously.<sup><xref rid="bib8">8</xref>
</sup>
The NTD (amino acids 18–353) of S was expressed in Baculovirus–Insect cells and was
provided by the Institute of Microbiology, Chinese Academy of Sciences. Expression and
purification of NP in a prokaryotic system were performed as described
previously.<sup><xref rid="bib9">9</xref>
</sup>
Purified NTD, RBD and NP were
identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and WB.</p>
</sec>
<sec><title>Serological tests</title>
<p>ELISA plates were coated at 4 °C overnight with inactivated MERS-CoV particles or
recombinant proteins (50 ng/well) in carbonate buffer (pH 9.6). Each well of the plates
was then incubated with blocking solution consisting of 10% goat serum in PBS for two h at
37 °C. The wells were washed five times with PBS containing 0.05% Tween 20 (PBS-T).
Aliquots of 100 μL of serially diluted sera were added to wells of the ELISA plates,
followed by further incubation for 1.5 h at 37 °C. After five washes with PBS-T, the
plates were incubated with horseradish peroxidase (HRP)-labeled goat anti-human
immunoglobulin G (IgG) or IgM antibodies for one h at 37 °C.
3,3',5,5'-tetramethylbenzidine (Sigma, St. Louis, MO, USA) was added at 100 μL/well after
five washes with PBS-T, and the wells were incubated for 5 min at room temperature. Then,
50 μL of 2 M H<sub>2</sub>
SO<sub>4</sub>
was added to each well to terminate the reaction,
and the optical density (OD) was immediately read at 450 nm. The average OD<sub>450</sub>
values for the normal controls were calculated for IgG and IgM, and the cutoff values were
determined as the average of normal controls+3 SD. The same normal control sera mixed in
equal volumes served as negative controls.</p>
<p>The S1-based ELISA Kit for detecting IgG was purchased from EUROIMMUNE (Luebeck,
Germany), and analyses were performed according to the manufacturer’s instructions. The
kit included a calibrator for defining the cutoff value recommended by the manufacturer.
Values below the cutoff were considered negative and those above were considered positive.
S1-specific IgM levels were not determined using an S1-based ELISA Kit because such
determinations were outside the range of applications of the kit.</p>
<p>Pseudovirus expressing the MERS-CoV S protein was prepared using the Env-defective and
luciferase-expressing HIV-1 genome, and the ppNT was performed as described
previously.<sup><xref rid="bib10">10</xref>
</sup>
Briefly, a lentivirus-based MERS-CoV
S pseudovirus was preincubated with serially diluted serum samples from the patients with
MERS-CoV at 37 °C for one h, and the mixtures were distributed into 96-well plates
containing monolayers of Huh7.5 cells. After 24 h of incubation, fresh medium was added,
followed by incubation for an additional 48 h. The cells were washed with PBS and then
lysed using the lysis reagent included in the Luciferase Kit (Promega, Madison, WI, USA).
Aliquots of cell lysates were transferred to 96-well Costar flat-bottomed luminometry
plates (Corning Costar, Corning, NY, USA), followed by addition of the luciferase
substrate (Promega). Relative fluorescence intensity values were immediately determined
using a Gaomax luminometer (Promega). The luciferase activity of Huh7.5-CD81 cells treated
with pseudovirus alone (reference group) was defined as 100% infection. Cells not treated
with pseudovirus were included as a background control. The results were expressed as the
percentage of infection compared with those of the control group (Huh7.5 cells treated
with only pseudovirus preparations). Fifty percent reductions in relative fluorescence
intensity were used to calculate MERS-CoV-specific neutralizing antibody (NAb) titers in
the sera. The NAb titers were defined as the highest serum dilutions that resulted in a
50% reduction in luciferase activity. NAb titers lower than 1:100 were considered
negative.</p>
</sec>
<sec><title>Statistical analysis</title>
<p>All experiments were performed at least three times. All data were analyzed using SPSS
(IBM, New York, NY, USA) and Graphpad Prism 5 (Graphpad Software Inc., La Jolla, CA, USA).
Pearson’s correlation coefficients between different assays were calculated, and
significance was defined as <italic>P</italic>
<0.05.</p>
</sec>
</sec>
<sec><title>Results</title>
<p>Inactivated MERS-CoV was characterized by WB and was shown to contain S and NP viral
components (<xref rid="SM0001">Supplementary Figure S1</xref>
). Purified
NTD, RBD and NP were found to be highly pure and antigenic by WB (<xref rid="SM0001">Supplementary Figure S1</xref>
). Normal controls, i.e., sera from 40 healthy
blood donors, were used to determine background values and calculate cutoff levels. IgG and
IgM against inactivated MERS-CoV, S, NTD, RBD and NP and IgG against S1 in the control sera
were detected by ELISA, and cutoff values were determined (<xref rid="SM0002">Supplementary Figure S2</xref>
).</p>
<p>A longitudinal profile of binding antibodies (IgG and IgM) and NAb against MERS-CoV in sera
from the first case of imported MERS-CoV in China was analyzed depending on the whole
MERS-CoV and recombinant protein ELISAs and the ppNT. <xref ref-type="fig" rid="F0001">Figures 1</xref>
and <xref ref-type="fig" rid="F0002">2</xref>
showed that the ELISAs
readily detected specific antibodies in the patient’s sera. The MERS-CoV S-based ELISA
showed the highest sensitivity, followed by the S1- and RBD-based ELISAs; the inactivated
MERS-CoV-, NTD- and NP-based ELISAs were not as sensitive as the S-, S1- and RBD-based
ELISAs. Moreover, the S-, S1- and RBD-based ELISAs showed broader measurement ranges than
those based on inactivated MERS-CoV, NTD and NP. In the ppNT assay, NAb positivity appeared
as early as anti-S antibody positivity, as determined by the ELISAs (<xref ref-type="fig" rid="F0001">Figure 1</xref>
). Anti-S antibodies were first detected by ELISA (IgM on day 6
and IgG on day 5 after admission), followed by antibodies against S1 and RBD, then
antibodies against MERS-CoV particles and NTD and, finally, antibodies against NP (by days
12–13 after admission) (<xref rid="SM0003">Supplementary Figure
S3</xref>
).</p>
<fig id="F0001" orientation="portrait" position="float"><label>Figure 1</label>
<caption><p>Kinetics of the serological response in a MERS-CoV-infected patient as determined by
ELISAs for various recombinant antigens and the ppNT. Sera sampled on days 2, 3, 4, 5,
6, 8, 9, 10, 12, 13, 15, 18, 20, 27 and 28 after admission to the hospital were
retrospectively analyzed. (<bold>A</bold>
) Longitudinal profiles of IgG antibodies
against MERS-CoV, S, S1, NTD, RBD and NP in the patient who was the first to import
MERS-CoV into China; sera were determined by 1: 80 dilution in all ELISAs except by
1:101 dilution in S1 ELISA. (<bold>B</bold>
) Longitudinal profiles of IgM antibodies
against MERS-CoV, S, NTD, RBD and NP; sera were determined by 1:80 dilutioin in all
ELISAs. (<bold>C</bold>
) Longitudinal profiles of NAb against MERS-CoV. enzyme-linked
immunosorbent assay, ELISA; immunoglobulin G/M, IgG/M; Middle East respiratory syndrome
coronavirus, MERS-CoV; neutralizing antibody, NAb; nucleoprotein, NP; N-terminal domain,
NTD; optical density, OD; pseudovirus particle-based neutralization test, ppNT;
receptor-binding domain, RBD; recombinant MERS-CoV full-length spike protein, S.</p>
</caption>
<graphic xlink:href="TEMI_A_12040281_F0001_OC"></graphic>
</fig>
<fig id="F0002" orientation="portrait" position="float"><label>Figure 2</label>
<caption><p>Sensitivities of ELISAs based on different viral antigens. ELISA plates were coated
with inactivated MERS-CoV particles (<bold>A</bold>
) or purified recombinant S
(<bold>B</bold>
), S1 (<bold>C</bold>
), NTD (<bold>D</bold>
), RBD (<bold>E</bold>
) or
NP (<bold>F</bold>
) protein. Serum samples from the patient with MERS-CoV collected on
days 2 (sample 1), 15 (sample 2) and 28 (sample 3) after admission to the hospital were
serially diluted and dispensed into the wells of an ELISA plate. HRP-labeled goat
anti-human IgG (left) and IgM (right) were used as the secondary antibody, with
3,3',5,5'-tetramethylbenzidine (TMB) as the substrate. The results are expressed as the
absorbance reading at 450 nm. As the negative control had very poor ELISA responses, the
results are not shown to avoid interference with the target profiles. enzyme-linked
immunosorbent assay, ELISA; horseradish peroxidase, HRP; immunoglobulin G/M, IgG/M;
Middle East respiratory syndrome coronavirus, MERS-CoV; nucleoprotein, NP; N-terminal
domain, NTD; optical density, OD; receptor-binding domain, RBD; recombinant MERS-CoV
full-length spike protein, S.</p>
</caption>
<graphic xlink:href="TEMI_A_12040281_F0002_OC"></graphic>
</fig>
<p>The whole virus-based ELISA kit was able to detect antibodies specific for all structural
components of the virus. In the present study, correlations of the recombinant protein-based
ELISAs with that based on inactivated MERS-CoV were analyzed. The scatter plots in <xref ref-type="fig" rid="F0003">Figure 3</xref>
show excellent correlations between the
MERS-CoV ELISA and the S1, RBD and NP ELISA, with Pearson’s correlation coefficients of
0.9292–0.9488. S and NTD ELISAs were less strongly correlated with the MERS-CoV ELISA, with
Pearson’s correlation coefficients of 0.8122–0.8420. Subsequently, correlations of the S,
NTD and RBD ELISA results with those of the commercial S1 ELISA were examined. The scatter
plots in <xref ref-type="fig" rid="F0004">Figure 4</xref>
show excellent correlations
between the S1 ELISA and the S and RBD ELISAs, with Pearson’s correlation coefficients of
0.9234–0.9701, and the NTD ELISA was less strongly correlated with the S1 ELISA, with a
Pearson’s correlation coefficient of 0.8807.</p>
<fig id="F0003" orientation="portrait" position="float"><label>Figure 3</label>
<caption><p>Correlations among the inactivated virus-based and other S or N protein-based ELISA
results. The absorbance readings of the S (<bold>A</bold>
), S1 (<bold>B</bold>
), NTD
(<bold>C</bold>
), RBD (<bold>D</bold>
) and NP (<bold>E</bold>
) based IgG ELISAs
(OD<sub>450</sub>
values) were plotted against that of the virus-based IgG ELISA
(OD<sub>450</sub>
values) (data are those from <xref ref-type="fig" rid="F0001">Figure
1A</xref>
). ***<italic>P</italic>
<0.001. Enzyme-linked immunosorbent assay, ELISA;
immunoglobulin G, IgG; Middle East respiratory syndrome coronavirus, MERS-CoV;
nucleoprotein, NP; N-terminal domain, NTD; optical density, OD; receptor-binding domain,
RBD; recombinant MERS-CoV full-length spike protein, S.</p>
</caption>
<graphic xlink:href="TEMI_A_12040281_F0003_OC"></graphic>
</fig>
<fig id="F0004" orientation="portrait" position="float"><label>Figure 4</label>
<caption><p>Correlation of the Euroimmune EIA Kit for detecting anti-S1 IgG antibody of MERS-CoV
(OD<sub>450</sub>
values) with other recombinant protein-based IgG ELISAs
(OD<sub>450</sub>
values) used in this study. The absorbance readings of the S
(<bold>A</bold>
), NTD (<bold>B</bold>
) and RBD (<bold>C</bold>
) based IgG ELISAs
(OD<sub>450</sub>
values) were plotted against those of the S1-based IgG ELISA
(OD<sub>450</sub>
values) (data are those from <xref ref-type="fig" rid="F0001">Figure
1A</xref>
). ***<italic>P</italic>
<0.001. Enzyme-linked immunosorbent assay, ELISA;
immunoglobulin G, IgG; Middle East respiratory syndrome coronavirus, MERS-CoV;
nucleoprotein, NP; N-terminal domain, NTD; optical density, OD; receptor-binding domain,
RBD; recombinant MERS-CoV full-length spike protein, S.</p>
</caption>
<graphic xlink:href="TEMI_A_12040281_F0004_OC"></graphic>
</fig>
<p>The MERS S ppNT is a sensitive and specific assay used for detecting NAbs against MERS-CoV.
Here we examined the relationship between the ppNT and various ELISAs. The scatter plots in
<xref ref-type="fig" rid="F0005">Figure 5</xref>
show that only the S ELISA had a strong
correlation with the ppNT, with a Pearson’s correlation coefficient of 0.9319. The MERS-CoV,
S1, NTD and RBD ELISAs were less strongly correlated with the MERS S ppNT, with Pearson’s
correlation coefficients of 0.71–0.8439.</p>
<fig id="F0005" orientation="portrait" position="float"><label>Figure 5</label>
<caption><p>Correlations among the ELISAs (IgG) (OD<sub>450</sub>
values) and the MERS-CoV ppNT
assay (neutralizing antibody titer) used in this study. The absorbance readings of the
inactivated MERS-CoV (<bold>A</bold>
), S (<bold>B</bold>
), S1 (<bold>C</bold>
), NTD
(<bold>D</bold>
) and RBD (<bold>E</bold>
) based IgG ELISAs (OD<sub>450</sub>
values)
(data are those from <xref ref-type="fig" rid="F0001">Figure 1A</xref>
) were plotted
against neutralizing antibody titers (data are those from <xref ref-type="fig" rid="F0001">Figure 1C</xref>
). *<italic>P</italic>
<0.05;
**<italic>P</italic>
<0.01; ***<italic>P</italic>
<0.001. Antibody, Ab;
enzyme-linked immunosorbent assay, ELISA; immunoglobulin G, IgG; Middle East respiratory
syndrome coronavirus, MERS-CoV; nucleoprotein, NP; N-terminal domain, NTD; optical
density, OD; pseudovirus particle-based neutralization test, ppNT; receptor-binding
domain, RBD; recombinant MERS-CoV full-length spike protein, S.</p>
</caption>
<graphic xlink:href="TEMI_A_12040281_F0005_OC"></graphic>
</fig>
</sec>
<sec><title>Discussion</title>
<p>To the best of our knowledge, this is the first comprehensive investigation of the
anti-MERS-CoV antibody profile in sera from a MERS patient based on a multiplex analysis of
antibodies against both the S protein and NP. The longitudinal profiles of IgG and IgM
antibodies in the sera from this case, the first to be imported into China, showed that
seroconversion of antibodies detected by the S, S1 and RBD ELISAs occurred earlier than that
detected by the inactivated MERS-CoV ELISA. The results shown in <xref ref-type="fig" rid="F0001">Figures 1</xref>
and <xref ref-type="fig" rid="F0002">2</xref>
further
illustrate that the S, S1 and RBD ELISAs are more sensitive than the whole MERS-CoV ELISA in
detecting specific antibodies. In a previous severe acute respiratory syndrome coronavirus
(SARS-CoV) study, an S protein-based ELISA also appeared to be more sensitive than a
virus-based Huada ELISA for detecting SARS-CoV-specific antibodies.<sup><xref rid="bib11">11</xref>
</sup>
The NP of SARS-CoV is an immunodominant antigen that is used to detect
SARS-CoV infection.<sup><xref rid="bib11">11</xref>
, <xref rid="bib12">12</xref>
</sup>
In
the present study, our NP-based ELISA illustrated the benefit of using recombinant NP for
detecting MERS-CoV infection. However, seroconversion of NP-specific antibodies occurred
later than seroconversion of antibodies against several S proteins and inactivated MERS-CoV.
Woo <italic>et al.</italic>
<sup><xref rid="bib12">12</xref>
</sup>
evaluated the longitudinal
profile of IgG and IgM antibodies against SARS-CoV NP in patients with pneumonia due to
SARS-CoV. The study showed that the levels of the two antibodies increased to detectable
levels by the third week of illness, which was approximately 1 week later than that observed
in another study by Lie <italic>et al.</italic>
,<sup><xref rid="bib13">13</xref>
</sup>
based
on a whole SARS-CoV ELISA. The 1-week delay in seroconversion of NP-specific antibodies in
the study by Woo <italic>et al.</italic>
<sup><xref rid="bib12">12</xref>
</sup>
compared with
the results of Li <italic>et al.</italic>
<sup><xref rid="bib13">13</xref>
</sup>
may reflect
reduced sensitivity of NP-based ELISAs based on inactivated virus particles for detecting
SARS-CoV infection at an early stage of illness, although clinical treatment may also
interfere with the appearance of antibodies. The results of the two studies confirmed the
characteristics of NP-specific antibodies in different contexts. Woo <italic>et
al.</italic>
<sup><xref rid="bib12">12</xref>
</sup>
reported that IgM antibodies against
SARS-CoV NP increased to detectable levels later than IgG antibodies, in contrast to the
phenomena described for most other pathogens. We found that IgM antibodies against some
antigens were detectable at the same time or later than IgG in the present study, which may
have been due to the earlier development of IgG than IgM antibodies or the different
sensitivities of the Ig class-specific ELISAs.<sup><xref rid="bib12">12</xref>
</sup>
</p>
<p>The S1-based ELISA Kit has the advantages of easy operation and convenience, and it has
been validated and applied in many studies.<sup><xref rid="bib2">2</xref>
, <xref rid="bib3">3</xref>
, <xref rid="bib4">4</xref>
, <xref rid="bib5">5</xref>
, <xref rid="bib6">6</xref>
, <xref rid="bib14">14</xref>
, <xref rid="bib15">15</xref>
</sup>
In the present
study, gradual increases in S1-specific serum IgG antibody levels were observed in the first
patient to import MERS-CoV into China, as was the case for most MERS-CoV patients in other
studies performed in South Korea<sup><xref rid="bib6">6</xref>
, <xref rid="bib15">15</xref>
</sup>
and the Kingdom of Saudi Arabia.<sup><xref rid="bib14">14</xref>
</sup>
Seroconversion of the S1-specific IgG antibody was observed 8 days after admission (16 days
after the onset of illness), which was consistent with previous findings.<sup><xref rid="bib6">6</xref>
</sup>
Moreover, the S1 ELISA maintained an excellent correlation
with inactivated MERS-CoV and S ELISAs (<italic>P</italic>
<0.001). In the present study,
the recombinant RBD (which is the main epitope of S1) was as sensitive as S1 for detecting
MERS-CoV infection, and there was an excellent correlation between the RBD ELISA and S1
ELISA, with a Pearson’s correlation coefficient of 0.9701. Therefore, RBD can be developed
as an alternative antigen for ELISA kits to detect MERS-CoV infection.</p>
<p>The MERS-spike ppNT assay is preferred owing to its lack of a requirement for biosafety
level 3 facilities, and it has been used extensively and validated for seroepidemiology in
humans, with a good correlation between the MERS ppNT and PRNT90.<sup><xref rid="bib3">3</xref>
</sup>
The present study confirmed the sensitivity of the ppNT when it was used
to detect NAb at an early stage of illness. Moreover, the ppNT assay showed an excellent
correlation with the S ELISA, although the latter is a binding assay while the former is a
functional neutralizing assay. The MERS-CoV, S1, NTD and RBD ELISAs (OD<sub>450</sub>
) had
acceptable but lower Pearson’s correlation coefficients (0.71–0.8439) when compared with the
ppNT titer. We speculate that this is because there are multiple components irrelevant to
neutralization in inactivated MERS-CoV and fewer epitopes related to neutralization in
recombinant S1, NTD and RBD.</p>
<p>Our study has certain limitations. We studied only one patient who was followed for only
one month after admission to the hospital. Although only one MERS-CoV case has been reported
in China, additional cases with longer follow-ups are needed to comprehensively understand
the MERS-CoV-derived antibody profile and to verify the specificity and sensitivity of
MERS-CoV- and recombinant protein-based ELISAs. Fortunately, a series of serum samples was
collected from this case. Additionally, a commercial S1-based ELISA Kit was available and
was adopted as the reference test. We found that the ELISAs of various recombinant
structural proteins, including S, S1, NTD, RBD and NP, as well as the ppNT assay, can be
used for MERS-CoV diagnosis and seroepidemiology. The S, S1 and RBD ELISAs were more
sensitive than other ELISA formats, especially the S1 and RBD ELISAs were advantageous
because the recombinant S1 and RBD are smaller than the full-length S and easier to express
and purify.</p>
<p>In conclusion, an RBD ELISA may be an alternative to an S1 ELISA for determining infection
at an early stage and in patients with poor serological responses to MERS-CoV. In addition,
an S ELISA may be an useful alternative to the MERS S ppNT, which was shown to be a reliable
surrogate of neutralization activity, and may be useful for large-scale seroepidemiological
studies of MERS-CoV infection.</p>
</sec>
<sec sec-type="supplementary-material"><title>Supplementary Material</title>
<supplementary-material content-type="local-data" id="SM0001"><caption><title>Supplementary Figure S1</title>
</caption>
<media xlink:href="TEMI_A_12040281_SM0001.pdf" mimetype="application" mime-subtype="pdf" orientation="portrait" id="d37e176" position="anchor"></media>
</supplementary-material>
<supplementary-material content-type="local-data" id="SM0002"><caption><title>Supplementary Figure S2</title>
</caption>
<media xlink:href="TEMI_A_12040281_SM0002.pdf" mimetype="application" mime-subtype="pdf" orientation="portrait" id="d37e181" position="anchor"></media>
</supplementary-material>
<supplementary-material content-type="local-data" id="SM0003"><caption><title>Supplementary Figure S3</title>
</caption>
<media xlink:href="TEMI_A_12040281_SM0003.pdf" mimetype="application" mime-subtype="pdf" orientation="portrait" id="d37e186" position="anchor"></media>
</supplementary-material>
</sec>
</body>
<back><sec><title>Acknowledgements</title>
<p>This work was supported by grants from the Megaproject for Infectious Disease Research of
China (2014ZX10004001-002), the National Basic Research Program of China (973 Program)
(2011CB504704) and the National Key Plan for Scientific Research and Development of China
(2016YFD0500301). The funders had no role in designing the study, collecting or analyzing
the data, deciding to publish or preparing the manuscript. We thank Dr Bart L Haagmans and
Dr Ron A M Fouchier (Erasmus MC, Rotterdam, The Netherlands) for providing MERS-CoV (isolate
hCoV-EMC/2012). We also thank Professor Gorge F Gao and Professor Jinghua Yan (CAS Key
Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese
Academy of Sciences) for providing the recombinant RBD and NTD proteins.</p>
</sec>
<ref-list><ref id="bib1"><label>1</label>
<mixed-citation publication-type="book"><person-group person-group-type="author"><collab>World Health Organization (WHO)</collab>
</person-group>
<source><italic>Middle
East Respiratory Syndrome Coronavirus (MERS-CoV) – Saudi Arabia, Disease Outbreak
News, 23 March 2016</italic>
</source>
.<publisher-name>Geneva, Switzerland:
WHO</publisher-name>
<year>2016</year>
Available from:<ext-link ext-link-type="uri" xlink:href="http://www.who.int/csr/don/23-march-2016-mers-saudi-arabia/en/">http://www.who.int/csr/don/23-march-2016-mers-saudi-arabia/en/</ext-link>
(accessed 12
April 2016).</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>Da
Guan</surname>
<given-names>W</given-names>
</name>
</person-group>
,<person-group person-group-type="author"><name name-style="western"><surname>Mok</surname>
<given-names>CK</given-names>
</name>
</person-group>
,<person-group person-group-type="author"><name name-style="western"><surname>Chen</surname>
<given-names>ZL</given-names>
</name>
</person-group>
<italic>et
al</italic>
<article-title>Characteristics of traveler with Middle East respiratory
syndrome, China</article-title>
.<year>2015</year>
<source><italic>Emerg Infect
Dis</italic>
</source>
<year>2015</year>
;
<volume>21</volume>
:<fpage>2278</fpage>
–<lpage>2280</lpage>
.<pub-id pub-id-type="pmid">26583433</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>Park</surname>
<given-names>SW</given-names>
</name>
</person-group>
,<person-group person-group-type="author"><name name-style="western"><surname>Perera</surname>
<given-names>RA</given-names>
</name>
</person-group>
,<person-group person-group-type="author"><name name-style="western"><surname>Choe</surname>
<given-names>PG</given-names>
</name>
</person-group>
<italic>et
al</italic>
<article-title>Comparison of serological assays in human Middle East
respiratory syndrome (MERS)-coronavirus infection</article-title>
.<source><italic>Euro
Surveill</italic>
</source>
<year>2015</year>
;
<volume>20</volume>
:<fpage>41</fpage>
.</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>Wernery</surname>
<given-names>U</given-names>
</name>
</person-group>
,<person-group person-group-type="author"><name name-style="western"><surname>El
Rasoul</surname>
<given-names>IH</given-names>
</name>
</person-group>
,<person-group person-group-type="author"><name name-style="western"><surname>Wong</surname>
<given-names>EY</given-names>
</name>
</person-group>
<italic>et
al</italic>
<article-title>A phylogenetically distinct Middle East respiratory syndrome
coronavirus detected in a dromedary calf from a closed dairy herd in Dubai with rising
seroprevalence with age</article-title>
.<source><italic>Emerg Microbes
Infect</italic>
</source>
<year>2015</year>
;
<volume>4</volume>
:<fpage>e74</fpage>
.<pub-id pub-id-type="pmid">26632876</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>Drosten</surname>
<given-names>C</given-names>
</name>
</person-group>
,<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>Müller</surname>
<given-names>MA</given-names>
</name>
</person-group>
<italic>et
al</italic>
<article-title>Transmission of MERS-coronavirus in household
contacts</article-title>
.<source><italic>N Engl J
Med</italic>
</source>
<year>2014</year>
;
<volume>371</volume>
:<fpage>828</fpage>
–<lpage>835</lpage>
.<pub-id pub-id-type="pmid">25162889</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>Park</surname>
<given-names>WB</given-names>
</name>
</person-group>
,<person-group person-group-type="author"><name name-style="western"><surname>Perera</surname>
<given-names>RA</given-names>
</name>
</person-group>
,<person-group person-group-type="author"><name name-style="western"><surname>Choe</surname>
<given-names>PG</given-names>
</name>
</person-group>
<italic>et
al</italic>
<article-title>Kinetics of serologic responses to MERS coronavirus infection
in humans, South Korea</article-title>
.<source><italic>Emerg Infect
Dis</italic>
</source>
<year>2015</year>
;
<volume>21</volume>
:<fpage>2186</fpage>
–<lpage>2189</lpage>
.<pub-id pub-id-type="pmid">26583829</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>Wu</surname>
<given-names>J</given-names>
</name>
</person-group>
,<person-group person-group-type="author"><name name-style="western"><surname>Yi</surname>
<given-names>L</given-names>
</name>
</person-group>
,<person-group person-group-type="author"><name name-style="western"><surname>Zou</surname>
<given-names>L</given-names>
</name>
</person-group>
<italic>et
al</italic>
<article-title>Imported case of MERS-CoV infection identified in China, May
2015: detection and lesson learned</article-title>
.<source><italic>Euro
Surveill</italic>
</source>
<year>2015</year>
; <volume>20</volume>
.</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>Lu</surname>
<given-names>G</given-names>
</name>
</person-group>
,<person-group person-group-type="author"><name name-style="western"><surname>Hu</surname>
<given-names>Y</given-names>
</name>
</person-group>
,<person-group person-group-type="author"><name name-style="western"><surname>Wang</surname>
<given-names>Q</given-names>
</name>
</person-group>
<italic>et
al</italic>
<article-title>Molecular basis of binding between novel human coronavirus
MERS-CoV and its receptor
CD26</article-title>
.<source><italic>Nature</italic>
</source>
<year>2013</year>
;
<volume>500</volume>
:<fpage>227</fpage>
–<lpage>231</lpage>
.<pub-id pub-id-type="pmid">23831647</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>Wang</surname>
<given-names>YQ</given-names>
</name>
</person-group>
,<person-group person-group-type="author"><name name-style="western"><surname>Hao</surname>
<given-names>CX</given-names>
</name>
</person-group>
,<person-group person-group-type="author"><name name-style="western"><surname>Deng</surname>
<given-names>Y</given-names>
</name>
</person-group>
<italic>et
al</italic>
<article-title>Prokaryotic expression, purification and identification of
the N protein of Middle East respiratory syndrome
coronavirus</article-title>
.<source><italic>Chin J Microbiol
Immunol</italic>
</source>
<year>2014</year>
;
<volume>34</volume>
:<fpage>764</fpage>
–<lpage>769</lpage>
.</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>Lan</surname>
<given-names>J</given-names>
</name>
</person-group>
,<person-group person-group-type="author"><name name-style="western"><surname>Yao</surname>
<given-names>Y</given-names>
</name>
</person-group>
,<person-group person-group-type="author"><name name-style="western"><surname>Deng</surname>
<given-names>Y</given-names>
</name>
</person-group>
<italic>et
al</italic>
<article-title>Recombinant receptor binding domain protein induces partial
protective immunity in rhesus macaques against Middle East respiratory syndrome
coronavirus challenge</article-title>
.<source><italic>EBio
Medicine</italic>
</source>
<year>2015</year>
;
<volume>2</volume>
:<fpage>1438</fpage>
–<lpage>1446</lpage>
.</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>Zhao</surname>
<given-names>J</given-names>
</name>
</person-group>
,<person-group person-group-type="author"><name name-style="western"><surname>Wang</surname>
<given-names>W</given-names>
</name>
</person-group>
,<person-group person-group-type="author"><name name-style="western"><surname>Wang</surname>
<given-names>W</given-names>
</name>
</person-group>
<italic>et
al</italic>
<article-title>Comparison of immunoglobulin G responses to the spike and
nucleocapsid proteins of severe acute respiratory syndrome (SARS) coronavirus in
patients with SARS</article-title>
.<source><italic>Clin Vaccine
Immunol</italic>
</source>
<year>2007</year>
;
<volume>14</volume>
:<fpage>839</fpage>
–<lpage>846</lpage>
.<pub-id pub-id-type="pmid">17475765</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>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>Wong</surname>
<given-names>BH</given-names>
</name>
</person-group>
<italic>et
al</italic>
<article-title>Longitudinal profile of immunoglobulin G (IgG), IgM, and IgA
antibodies against the severe acute respiratory syndrome (SARS) coronavirus nucleocapsid
protein in patients with pneumonia due to the SARS
coronavirus</article-title>
.<source><italic>Clin Diagn Lab
Immunol</italic>
</source>
<year>2004</year>
;
<volume>11</volume>
:<fpage>665</fpage>
–<lpage>668</lpage>
.<pub-id pub-id-type="pmid">15242938</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>Li</surname>
<given-names>G</given-names>
</name>
</person-group>
,<person-group person-group-type="author"><name name-style="western"><surname>Chen</surname>
<given-names>X</given-names>
</name>
</person-group>
,<person-group person-group-type="author"><name name-style="western"><surname>Xu</surname>
<given-names>A</given-names>
</name>
</person-group>
<article-title>Profile
of specific antibodies to the SARS-associated
coronavirus</article-title>
.<source><italic>N Engl J
Med</italic>
</source>
<year>2003</year>
;
<volume>349</volume>
:<fpage>508</fpage>
–<lpage>509</lpage>
.<pub-id pub-id-type="pmid">12890855</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>Corman</surname>
<given-names>VM</given-names>
</name>
</person-group>
,<person-group person-group-type="author"><name name-style="western"><surname>Albarrak</surname>
<given-names>AM</given-names>
</name>
</person-group>
,<person-group person-group-type="author"><name name-style="western"><surname>Omrani</surname>
<given-names>AS</given-names>
</name>
</person-group>
<italic>et
al</italic>
<article-title>Viral shedding and antibody response in 37 patients with
MERS-coronavirus infection</article-title>
.<source><italic>Clin Infect
Dis</italic>
</source>
<year>2016</year>
;
<volume>62</volume>
:<fpage>477</fpage>
–<lpage>483</lpage>
.<pub-id pub-id-type="pmid">26565003</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>Min</surname>
<given-names>CK</given-names>
</name>
</person-group>
,<person-group person-group-type="author"><name name-style="western"><surname>Cheon</surname>
<given-names>S</given-names>
</name>
</person-group>
,<person-group person-group-type="author"><name name-style="western"><surname>Ha</surname>
<given-names>NY</given-names>
</name>
</person-group>
<italic>et
al</italic>
<article-title>Comparative and kinetic analysis of viral shedding and
immunological responses in MERS patients representing a broad spectrum of disease
severity</article-title>
.<source><italic>Sci Rep</italic>
</source>
<year>2016</year>
;
<volume>6</volume>
:<fpage>25359</fpage>
.<pub-id pub-id-type="pmid">27146253</pub-id>
</mixed-citation>
</ref>
</ref-list>
<notes notes-type="ESMHint"><p>Supplementary Information for this article can be found on the <italic>Emerging Microbes
& Infections</italic>
website (<ext-link ext-link-type="uri" xlink:href="http://http://www.nature.com/emi">http://www.nature.com/emi</ext-link>
)</p>
</notes>
</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 000C27 | SxmlIndent | more
Ou
HfdSelect -h $EXPLOR_AREA/Data/Pmc/Corpus/biblio.hfd -nk 000C27 | 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= PMC:5148018 |texte= Characterization of anti-MERS-CoV antibodies against various recombinant structural antigens of MERS-CoV in an imported case in China }}
Pour générer des pages wiki
HfdIndexSelect -h $EXPLOR_AREA/Data/Pmc/Corpus/RBID.i -Sk "pubmed:27826140" \ | HfdSelect -Kh $EXPLOR_AREA/Data/Pmc/Corpus/biblio.hfd \ | NlmPubMed2Wicri -a MersV1
This area was generated with Dilib version V0.6.33. |