Middle East Respiratory Syndrome Coronavirus Efficiently Infects Human Primary T Lymphocytes and Activates the Extrinsic and Intrinsic Apoptosis Pathways
Identifieur interne : 000E23 ( Pmc/Corpus ); précédent : 000E22; suivant : 000E24Middle East Respiratory Syndrome Coronavirus Efficiently Infects Human Primary T Lymphocytes and Activates the Extrinsic and Intrinsic Apoptosis Pathways
Auteurs : Hin Chu ; Jie Zhou ; Bosco Ho-Yin Wong ; Cun Li ; Jasper Fuk-Woo Chan ; Zhong-Shan Cheng ; Dong Yang ; Dong Wang ; Andrew Chak-Yiu Lee ; Chuangen Li ; Man-Lung Yeung ; Jian-Piao Cai ; Ivy Hau-Yee Chan ; Wai-Kuen Ho ; Kelvin Kai-Wang To ; Bo-Jian Zheng ; Yanfeng Yao ; Chuan Qin ; Kwok-Yung YuenSource :
- The Journal of Infectious Diseases [ 0022-1899 ] ; 2015.
Abstract
Middle East respiratory syndrome (MERS) is associated with a mortality rate of >35%. We previously showed that MERS coronavirus (MERS-CoV) could infect human macrophages and dendritic cells and induce cytokine dysregulation. Here, we further investigated the interplay between human primary T cells and MERS-CoV in disease pathogenesis. Importantly, our results suggested that MERS-CoV efficiently infected T cells from the peripheral blood and from human lymphoid organs, including the spleen and the tonsil. We further demonstrated that MERS-CoV infection induced apoptosis in T cells, which involved the activation of both the extrinsic and intrinsic apoptosis pathways. Remarkably, immunostaining of spleen sections from MERS-CoV–infected common marmosets demonstrated the presence of viral nucleoprotein in their CD3+ T cells. Overall, our results suggested that the unusual capacity of MERS-CoV to infect T cells and induce apoptosis might partly contribute to the high pathogenicity of the virus.
Url:
DOI: 10.1093/infdis/jiv380
PubMed: 26203058
PubMed Central: 7107330
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PMC:7107330Le document en format XML
<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Middle East Respiratory Syndrome Coronavirus Efficiently Infects Human Primary T Lymphocytes and Activates the Extrinsic and Intrinsic Apoptosis Pathways</title><author><name sortKey="Chu, Hin" sort="Chu, Hin" uniqKey="Chu H" first="Hin" last="Chu">Hin Chu</name><affiliation><nlm:aff id="af1"><addr-line>State Key Laboratory of Emerging Infectious Diseases</addr-line></nlm:aff></affiliation><affiliation><nlm:aff id="af2"><addr-line>Department of Microbiology</addr-line></nlm:aff></affiliation><affiliation><nlm:aff id="af3"><addr-line>Research Centre of Infection and Immunology</addr-line></nlm:aff></affiliation><affiliation><nlm:aff id="af4"><addr-line>Carol Yu Centre for Infection</addr-line></nlm:aff></affiliation></author><author><name sortKey="Zhou, Jie" sort="Zhou, Jie" uniqKey="Zhou J" first="Jie" last="Zhou">Jie Zhou</name><affiliation><nlm:aff id="af1"><addr-line>State Key Laboratory of Emerging Infectious Diseases</addr-line></nlm:aff></affiliation><affiliation><nlm:aff id="af2"><addr-line>Department of Microbiology</addr-line></nlm:aff></affiliation><affiliation><nlm:aff id="af3"><addr-line>Research Centre of Infection and Immunology</addr-line></nlm:aff></affiliation><affiliation><nlm:aff id="af4"><addr-line>Carol Yu Centre for Infection</addr-line></nlm:aff></affiliation></author><author><name sortKey="Wong, Bosco Ho Yin" sort="Wong, Bosco Ho Yin" uniqKey="Wong B" first="Bosco Ho-Yin" last="Wong">Bosco Ho-Yin Wong</name><affiliation><nlm:aff id="af2"><addr-line>Department of Microbiology</addr-line></nlm:aff></affiliation></author><author><name sortKey="Li, Cun" sort="Li, Cun" uniqKey="Li C" first="Cun" last="Li">Cun Li</name><affiliation><nlm:aff id="af2"><addr-line>Department of Microbiology</addr-line></nlm:aff></affiliation></author><author><name sortKey="Chan, Jasper Fuk Woo" sort="Chan, Jasper Fuk Woo" uniqKey="Chan J" first="Jasper Fuk-Woo" last="Chan">Jasper Fuk-Woo Chan</name><affiliation><nlm:aff id="af1"><addr-line>State Key Laboratory of Emerging Infectious Diseases</addr-line></nlm:aff></affiliation><affiliation><nlm:aff id="af2"><addr-line>Department of Microbiology</addr-line></nlm:aff></affiliation><affiliation><nlm:aff id="af3"><addr-line>Research Centre of Infection and Immunology</addr-line></nlm:aff></affiliation><affiliation><nlm:aff id="af4"><addr-line>Carol Yu Centre for Infection</addr-line></nlm:aff></affiliation></author><author><name sortKey="Cheng, Zhong Shan" sort="Cheng, Zhong Shan" uniqKey="Cheng Z" first="Zhong-Shan" last="Cheng">Zhong-Shan Cheng</name><affiliation><nlm:aff id="af2"><addr-line>Department of Microbiology</addr-line></nlm:aff></affiliation></author><author><name sortKey="Yang, Dong" sort="Yang, Dong" uniqKey="Yang D" first="Dong" last="Yang">Dong Yang</name><affiliation><nlm:aff id="af2"><addr-line>Department of Microbiology</addr-line></nlm:aff></affiliation></author><author><name sortKey="Wang, Dong" sort="Wang, Dong" uniqKey="Wang D" first="Dong" last="Wang">Dong Wang</name><affiliation><nlm:aff id="af2"><addr-line>Department of Microbiology</addr-line></nlm:aff></affiliation></author><author><name sortKey="Lee, Andrew Chak Yiu" sort="Lee, Andrew Chak Yiu" uniqKey="Lee A" first="Andrew Chak-Yiu" last="Lee">Andrew Chak-Yiu Lee</name><affiliation><nlm:aff id="af2"><addr-line>Department of Microbiology</addr-line></nlm:aff></affiliation></author><author><name sortKey="Li, Chuangen" sort="Li, Chuangen" uniqKey="Li C" first="Chuangen" last="Li">Chuangen Li</name><affiliation><nlm:aff id="af2"><addr-line>Department of Microbiology</addr-line></nlm:aff></affiliation></author><author><name sortKey="Yeung, Man Lung" sort="Yeung, Man Lung" uniqKey="Yeung M" first="Man-Lung" last="Yeung">Man-Lung Yeung</name><affiliation><nlm:aff id="af1"><addr-line>State Key Laboratory of Emerging Infectious Diseases</addr-line></nlm:aff></affiliation><affiliation><nlm:aff id="af2"><addr-line>Department of Microbiology</addr-line></nlm:aff></affiliation><affiliation><nlm:aff id="af3"><addr-line>Research Centre of Infection and Immunology</addr-line></nlm:aff></affiliation><affiliation><nlm:aff id="af4"><addr-line>Carol Yu Centre for Infection</addr-line></nlm:aff></affiliation></author><author><name sortKey="Cai, Jian Piao" sort="Cai, Jian Piao" uniqKey="Cai J" first="Jian-Piao" last="Cai">Jian-Piao Cai</name><affiliation><nlm:aff id="af2"><addr-line>Department of Microbiology</addr-line></nlm:aff></affiliation></author><author><name sortKey="Chan, Ivy Hau Yee" sort="Chan, Ivy Hau Yee" uniqKey="Chan I" first="Ivy Hau-Yee" last="Chan">Ivy Hau-Yee Chan</name><affiliation><nlm:aff id="af5"><addr-line>Department of Surgery, University of Hong Kong</addr-line>,<institution>Hong Kong Special Administrative Region</institution></nlm:aff></affiliation></author><author><name sortKey="Ho, Wai Kuen" sort="Ho, Wai Kuen" uniqKey="Ho W" first="Wai-Kuen" last="Ho">Wai-Kuen Ho</name><affiliation><nlm:aff id="af5"><addr-line>Department of Surgery, University of Hong Kong</addr-line>,<institution>Hong Kong Special Administrative Region</institution></nlm:aff></affiliation></author><author><name sortKey="To, Kelvin Kai Wang" sort="To, Kelvin Kai Wang" uniqKey="To K" first="Kelvin Kai-Wang" last="To">Kelvin Kai-Wang To</name><affiliation><nlm:aff id="af1"><addr-line>State Key Laboratory of Emerging Infectious Diseases</addr-line></nlm:aff></affiliation><affiliation><nlm:aff id="af2"><addr-line>Department of Microbiology</addr-line></nlm:aff></affiliation><affiliation><nlm:aff id="af3"><addr-line>Research Centre of Infection and Immunology</addr-line></nlm:aff></affiliation><affiliation><nlm:aff id="af4"><addr-line>Carol Yu Centre for Infection</addr-line></nlm:aff></affiliation></author><author><name sortKey="Zheng, Bo Jian" sort="Zheng, Bo Jian" uniqKey="Zheng B" first="Bo-Jian" last="Zheng">Bo-Jian Zheng</name><affiliation><nlm:aff id="af1"><addr-line>State Key Laboratory of Emerging Infectious Diseases</addr-line></nlm:aff></affiliation><affiliation><nlm:aff id="af2"><addr-line>Department of Microbiology</addr-line></nlm:aff></affiliation><affiliation><nlm:aff id="af3"><addr-line>Research Centre of Infection and Immunology</addr-line></nlm:aff></affiliation><affiliation><nlm:aff id="af4"><addr-line>Carol Yu Centre for Infection</addr-line></nlm:aff></affiliation></author><author><name sortKey="Yao, Yanfeng" sort="Yao, Yanfeng" uniqKey="Yao Y" first="Yanfeng" last="Yao">Yanfeng Yao</name><affiliation><nlm:aff id="af6"><addr-line>Institute of Laboratory Animal Sciences</addr-line>,<institution>Chinese Academy of Medical Sciences</institution></nlm:aff></affiliation></author><author><name sortKey="Qin, Chuan" sort="Qin, Chuan" uniqKey="Qin C" first="Chuan" last="Qin">Chuan Qin</name><affiliation><nlm:aff id="af6"><addr-line>Institute of Laboratory Animal Sciences</addr-line>,<institution>Chinese Academy of Medical Sciences</institution></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="af1"><addr-line>State Key Laboratory of Emerging Infectious Diseases</addr-line></nlm:aff></affiliation><affiliation><nlm:aff id="af2"><addr-line>Department of Microbiology</addr-line></nlm:aff></affiliation><affiliation><nlm:aff id="af3"><addr-line>Research Centre of Infection and Immunology</addr-line></nlm:aff></affiliation><affiliation><nlm:aff id="af4"><addr-line>Carol Yu Centre for Infection</addr-line></nlm:aff></affiliation><affiliation><nlm:aff id="af7"><addr-line>Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases</addr-line>,<institution>Zhejiang University</institution>,<country>China</country></nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">26203058</idno><idno type="pmc">7107330</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7107330</idno><idno type="RBID">PMC:7107330</idno><idno type="doi">10.1093/infdis/jiv380</idno><date when="2015">2015</date><idno type="wicri:Area/Pmc/Corpus">000E23</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000E23</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Middle East Respiratory Syndrome Coronavirus Efficiently Infects Human Primary T Lymphocytes and Activates the Extrinsic and Intrinsic Apoptosis Pathways</title><author><name sortKey="Chu, Hin" sort="Chu, Hin" uniqKey="Chu H" first="Hin" last="Chu">Hin Chu</name><affiliation><nlm:aff id="af1"><addr-line>State Key Laboratory of Emerging Infectious Diseases</addr-line></nlm:aff></affiliation><affiliation><nlm:aff id="af2"><addr-line>Department of Microbiology</addr-line></nlm:aff></affiliation><affiliation><nlm:aff id="af3"><addr-line>Research Centre of Infection and Immunology</addr-line></nlm:aff></affiliation><affiliation><nlm:aff id="af4"><addr-line>Carol Yu Centre for Infection</addr-line></nlm:aff></affiliation></author><author><name sortKey="Zhou, Jie" sort="Zhou, Jie" uniqKey="Zhou J" first="Jie" last="Zhou">Jie Zhou</name><affiliation><nlm:aff id="af1"><addr-line>State Key Laboratory of Emerging Infectious Diseases</addr-line></nlm:aff></affiliation><affiliation><nlm:aff id="af2"><addr-line>Department of Microbiology</addr-line></nlm:aff></affiliation><affiliation><nlm:aff id="af3"><addr-line>Research Centre of Infection and Immunology</addr-line></nlm:aff></affiliation><affiliation><nlm:aff id="af4"><addr-line>Carol Yu Centre for Infection</addr-line></nlm:aff></affiliation></author><author><name sortKey="Wong, Bosco Ho Yin" sort="Wong, Bosco Ho Yin" uniqKey="Wong B" first="Bosco Ho-Yin" last="Wong">Bosco Ho-Yin Wong</name><affiliation><nlm:aff id="af2"><addr-line>Department of Microbiology</addr-line></nlm:aff></affiliation></author><author><name sortKey="Li, Cun" sort="Li, Cun" uniqKey="Li C" first="Cun" last="Li">Cun Li</name><affiliation><nlm:aff id="af2"><addr-line>Department of Microbiology</addr-line></nlm:aff></affiliation></author><author><name sortKey="Chan, Jasper Fuk Woo" sort="Chan, Jasper Fuk Woo" uniqKey="Chan J" first="Jasper Fuk-Woo" last="Chan">Jasper Fuk-Woo Chan</name><affiliation><nlm:aff id="af1"><addr-line>State Key Laboratory of Emerging Infectious Diseases</addr-line></nlm:aff></affiliation><affiliation><nlm:aff id="af2"><addr-line>Department of Microbiology</addr-line></nlm:aff></affiliation><affiliation><nlm:aff id="af3"><addr-line>Research Centre of Infection and Immunology</addr-line></nlm:aff></affiliation><affiliation><nlm:aff id="af4"><addr-line>Carol Yu Centre for Infection</addr-line></nlm:aff></affiliation></author><author><name sortKey="Cheng, Zhong Shan" sort="Cheng, Zhong Shan" uniqKey="Cheng Z" first="Zhong-Shan" last="Cheng">Zhong-Shan Cheng</name><affiliation><nlm:aff id="af2"><addr-line>Department of Microbiology</addr-line></nlm:aff></affiliation></author><author><name sortKey="Yang, Dong" sort="Yang, Dong" uniqKey="Yang D" first="Dong" last="Yang">Dong Yang</name><affiliation><nlm:aff id="af2"><addr-line>Department of Microbiology</addr-line></nlm:aff></affiliation></author><author><name sortKey="Wang, Dong" sort="Wang, Dong" uniqKey="Wang D" first="Dong" last="Wang">Dong Wang</name><affiliation><nlm:aff id="af2"><addr-line>Department of Microbiology</addr-line></nlm:aff></affiliation></author><author><name sortKey="Lee, Andrew Chak Yiu" sort="Lee, Andrew Chak Yiu" uniqKey="Lee A" first="Andrew Chak-Yiu" last="Lee">Andrew Chak-Yiu Lee</name><affiliation><nlm:aff id="af2"><addr-line>Department of Microbiology</addr-line></nlm:aff></affiliation></author><author><name sortKey="Li, Chuangen" sort="Li, Chuangen" uniqKey="Li C" first="Chuangen" last="Li">Chuangen Li</name><affiliation><nlm:aff id="af2"><addr-line>Department of Microbiology</addr-line></nlm:aff></affiliation></author><author><name sortKey="Yeung, Man Lung" sort="Yeung, Man Lung" uniqKey="Yeung M" first="Man-Lung" last="Yeung">Man-Lung Yeung</name><affiliation><nlm:aff id="af1"><addr-line>State Key Laboratory of Emerging Infectious Diseases</addr-line></nlm:aff></affiliation><affiliation><nlm:aff id="af2"><addr-line>Department of Microbiology</addr-line></nlm:aff></affiliation><affiliation><nlm:aff id="af3"><addr-line>Research Centre of Infection and Immunology</addr-line></nlm:aff></affiliation><affiliation><nlm:aff id="af4"><addr-line>Carol Yu Centre for Infection</addr-line></nlm:aff></affiliation></author><author><name sortKey="Cai, Jian Piao" sort="Cai, Jian Piao" uniqKey="Cai J" first="Jian-Piao" last="Cai">Jian-Piao Cai</name><affiliation><nlm:aff id="af2"><addr-line>Department of Microbiology</addr-line></nlm:aff></affiliation></author><author><name sortKey="Chan, Ivy Hau Yee" sort="Chan, Ivy Hau Yee" uniqKey="Chan I" first="Ivy Hau-Yee" last="Chan">Ivy Hau-Yee Chan</name><affiliation><nlm:aff id="af5"><addr-line>Department of Surgery, University of Hong Kong</addr-line>,<institution>Hong Kong Special Administrative Region</institution></nlm:aff></affiliation></author><author><name sortKey="Ho, Wai Kuen" sort="Ho, Wai Kuen" uniqKey="Ho W" first="Wai-Kuen" last="Ho">Wai-Kuen Ho</name><affiliation><nlm:aff id="af5"><addr-line>Department of Surgery, University of Hong Kong</addr-line>,<institution>Hong Kong Special Administrative Region</institution></nlm:aff></affiliation></author><author><name sortKey="To, Kelvin Kai Wang" sort="To, Kelvin Kai Wang" uniqKey="To K" first="Kelvin Kai-Wang" last="To">Kelvin Kai-Wang To</name><affiliation><nlm:aff id="af1"><addr-line>State Key Laboratory of Emerging Infectious Diseases</addr-line></nlm:aff></affiliation><affiliation><nlm:aff id="af2"><addr-line>Department of Microbiology</addr-line></nlm:aff></affiliation><affiliation><nlm:aff id="af3"><addr-line>Research Centre of Infection and Immunology</addr-line></nlm:aff></affiliation><affiliation><nlm:aff id="af4"><addr-line>Carol Yu Centre for Infection</addr-line></nlm:aff></affiliation></author><author><name sortKey="Zheng, Bo Jian" sort="Zheng, Bo Jian" uniqKey="Zheng B" first="Bo-Jian" last="Zheng">Bo-Jian Zheng</name><affiliation><nlm:aff id="af1"><addr-line>State Key Laboratory of Emerging Infectious Diseases</addr-line></nlm:aff></affiliation><affiliation><nlm:aff id="af2"><addr-line>Department of Microbiology</addr-line></nlm:aff></affiliation><affiliation><nlm:aff id="af3"><addr-line>Research Centre of Infection and Immunology</addr-line></nlm:aff></affiliation><affiliation><nlm:aff id="af4"><addr-line>Carol Yu Centre for Infection</addr-line></nlm:aff></affiliation></author><author><name sortKey="Yao, Yanfeng" sort="Yao, Yanfeng" uniqKey="Yao Y" first="Yanfeng" last="Yao">Yanfeng Yao</name><affiliation><nlm:aff id="af6"><addr-line>Institute of Laboratory Animal Sciences</addr-line>,<institution>Chinese Academy of Medical Sciences</institution></nlm:aff></affiliation></author><author><name sortKey="Qin, Chuan" sort="Qin, Chuan" uniqKey="Qin C" first="Chuan" last="Qin">Chuan Qin</name><affiliation><nlm:aff id="af6"><addr-line>Institute of Laboratory Animal Sciences</addr-line>,<institution>Chinese Academy of Medical Sciences</institution></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="af1"><addr-line>State Key Laboratory of Emerging Infectious Diseases</addr-line></nlm:aff></affiliation><affiliation><nlm:aff id="af2"><addr-line>Department of Microbiology</addr-line></nlm:aff></affiliation><affiliation><nlm:aff id="af3"><addr-line>Research Centre of Infection and Immunology</addr-line></nlm:aff></affiliation><affiliation><nlm:aff id="af4"><addr-line>Carol Yu Centre for Infection</addr-line></nlm:aff></affiliation><affiliation><nlm:aff id="af7"><addr-line>Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases</addr-line>,<institution>Zhejiang University</institution>,<country>China</country></nlm:aff></affiliation></author></analytic><series><title level="j">The Journal of Infectious Diseases</title><idno type="ISSN">0022-1899</idno><idno type="eISSN">1537-6613</idno><imprint><date when="2015">2015</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><title>Abstract</title><p>Middle East respiratory syndrome (MERS) is associated with a mortality rate of >35%. We previously showed that MERS coronavirus (MERS-CoV) could infect human macrophages and dendritic cells and induce cytokine dysregulation. Here, we further investigated the interplay between human primary T cells and MERS-CoV in disease pathogenesis. Importantly, our results suggested that MERS-CoV efficiently infected T cells from the peripheral blood and from human lymphoid organs, including the spleen and the tonsil. We further demonstrated that MERS-CoV infection induced apoptosis in T cells, which involved the activation of both the extrinsic and intrinsic apoptosis pathways. Remarkably, immunostaining of spleen sections from MERS-CoV–infected common marmosets demonstrated the presence of viral nucleoprotein in their CD3<sup>+</sup> T cells. Overall, our results suggested that the unusual capacity of MERS-CoV to infect T cells and induce apoptosis might partly contribute to the high pathogenicity of the virus.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Zaki, Am" uniqKey="Zaki A">AM Zaki</name></author><author><name sortKey="Van Boheemen, S" uniqKey="Van Boheemen S">S van Boheemen</name></author><author><name sortKey="Bestebroer, Tm" uniqKey="Bestebroer T">TM Bestebroer</name></author><author><name sortKey="Osterhaus, Ad" uniqKey="Osterhaus A">AD Osterhaus</name></author><author><name sortKey="Fouchier, Ra" uniqKey="Fouchier R">RA Fouchier</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Bermingham, A" uniqKey="Bermingham A">A Bermingham</name></author><author><name sortKey="Chand, Ma" uniqKey="Chand M">MA Chand</name></author><author><name sortKey="Brown, Cs" uniqKey="Brown C">CS 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<front><journal-meta><journal-id journal-id-type="nlm-ta">J Infect Dis</journal-id><journal-id journal-id-type="iso-abbrev">J. Infect. Dis</journal-id><journal-id journal-id-type="publisher-id">jid</journal-id><journal-id journal-id-type="hwp">jinfdis</journal-id><journal-title-group><journal-title>The Journal of Infectious Diseases</journal-title></journal-title-group><issn pub-type="ppub">0022-1899</issn><issn pub-type="epub">1537-6613</issn><publisher><publisher-name>Oxford University Press</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">26203058</article-id><article-id pub-id-type="pmc">7107330</article-id><article-id pub-id-type="doi">10.1093/infdis/jiv380</article-id><article-id pub-id-type="publisher-id">jiv380</article-id><article-categories><subj-group subj-group-type="heading"><subject>Major Articles and Brief Reports</subject><subj-group subj-group-type="category-toc-heading"><subject>Viruses</subject></subj-group></subj-group><subj-group subj-group-type="category-oup-series"><subject>Editor's Choice</subject></subj-group></article-categories><title-group><article-title>Middle East Respiratory Syndrome Coronavirus Efficiently Infects Human Primary T Lymphocytes and Activates the Extrinsic and Intrinsic Apoptosis Pathways</article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Chu</surname><given-names>Hin</given-names></name><xref ref-type="aff" rid="af1">1</xref><xref ref-type="aff" rid="af2">2</xref><xref ref-type="aff" rid="af3">3</xref><xref ref-type="aff" rid="af4">4</xref><pmc-comment>kyyuen@hku.hk </pmc-comment>
<xref ref-type="corresp" rid="d206e283"></xref></contrib><contrib contrib-type="author"><name><surname>Zhou</surname><given-names>Jie</given-names></name><xref ref-type="aff" rid="af1">1</xref><xref ref-type="aff" rid="af2">2</xref><xref ref-type="aff" rid="af3">3</xref><xref ref-type="aff" rid="af4">4</xref></contrib><contrib contrib-type="author"><name><surname>Wong</surname><given-names>Bosco Ho-Yin</given-names></name><xref ref-type="aff" rid="af2">2</xref></contrib><contrib contrib-type="author"><name><surname>Li</surname><given-names>Cun</given-names></name><xref ref-type="aff" rid="af2">2</xref></contrib><contrib contrib-type="author"><name><surname>Chan</surname><given-names>Jasper Fuk-Woo</given-names></name><xref ref-type="aff" rid="af1">1</xref><xref ref-type="aff" rid="af2">2</xref><xref ref-type="aff" rid="af3">3</xref><xref ref-type="aff" rid="af4">4</xref></contrib><contrib contrib-type="author"><name><surname>Cheng</surname><given-names>Zhong-Shan</given-names></name><xref ref-type="aff" rid="af2">2</xref></contrib><contrib contrib-type="author"><name><surname>Yang</surname><given-names>Dong</given-names></name><xref ref-type="aff" rid="af2">2</xref></contrib><contrib contrib-type="author"><name><surname>Wang</surname><given-names>Dong</given-names></name><xref ref-type="aff" rid="af2">2</xref></contrib><contrib contrib-type="author"><name><surname>Lee</surname><given-names>Andrew Chak-Yiu</given-names></name><xref ref-type="aff" rid="af2">2</xref></contrib><contrib contrib-type="author"><name><surname>Li</surname><given-names>Chuangen</given-names></name><xref ref-type="aff" rid="af2">2</xref></contrib><contrib contrib-type="author"><name><surname>Yeung</surname><given-names>Man-Lung</given-names></name><xref ref-type="aff" rid="af1">1</xref><xref ref-type="aff" rid="af2">2</xref><xref ref-type="aff" rid="af3">3</xref><xref ref-type="aff" rid="af4">4</xref></contrib><contrib contrib-type="author"><name><surname>Cai</surname><given-names>Jian-Piao</given-names></name><xref ref-type="aff" rid="af2">2</xref></contrib><contrib contrib-type="author"><name><surname>Chan</surname><given-names>Ivy Hau-Yee</given-names></name><xref ref-type="aff" rid="af5">5</xref></contrib><contrib contrib-type="author"><name><surname>Ho</surname><given-names>Wai-Kuen</given-names></name><xref ref-type="aff" rid="af5">5</xref></contrib><contrib contrib-type="author"><name><surname>To</surname><given-names>Kelvin Kai-Wang</given-names></name><xref ref-type="aff" rid="af1">1</xref><xref ref-type="aff" rid="af2">2</xref><xref ref-type="aff" rid="af3">3</xref><xref ref-type="aff" rid="af4">4</xref></contrib><contrib contrib-type="author"><name><surname>Zheng</surname><given-names>Bo-Jian</given-names></name><xref ref-type="aff" rid="af1">1</xref><xref ref-type="aff" rid="af2">2</xref><xref ref-type="aff" rid="af3">3</xref><xref ref-type="aff" rid="af4">4</xref></contrib><contrib contrib-type="author"><name><surname>Yao</surname><given-names>Yanfeng</given-names></name><xref ref-type="aff" rid="af6">6</xref></contrib><contrib contrib-type="author"><name><surname>Qin</surname><given-names>Chuan</given-names></name><xref ref-type="aff" rid="af6">6</xref></contrib><contrib contrib-type="author"><name><surname>Yuen</surname><given-names>Kwok-Yung</given-names></name><xref ref-type="aff" rid="af1">1</xref><xref ref-type="aff" rid="af2">2</xref><xref ref-type="aff" rid="af3">3</xref><xref ref-type="aff" rid="af4">4</xref><xref ref-type="aff" rid="af7">7</xref></contrib></contrib-group><aff id="af1"><label>1</label><addr-line>State Key Laboratory of Emerging Infectious Diseases</addr-line></aff><aff id="af2"><label>2</label><addr-line>Department of Microbiology</addr-line></aff><aff id="af3"><label>3</label><addr-line>Research Centre of Infection and Immunology</addr-line></aff><aff id="af4"><label>4</label><addr-line>Carol Yu Centre for Infection</addr-line></aff><aff id="af5"><label>5</label><addr-line>Department of Surgery, University of Hong Kong</addr-line>,<institution>Hong Kong Special Administrative Region</institution></aff><aff id="af6"><label>6</label><addr-line>Institute of Laboratory Animal Sciences</addr-line>,<institution>Chinese Academy of Medical Sciences</institution></aff><aff id="af7"><label>7</label><addr-line>Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases</addr-line>,<institution>Zhejiang University</institution>,<country>China</country></aff><author-notes><corresp id="d206e283">Correspondence: K.-Y. Yuen, Carol Yu Centre for Infection, Department of Microbiology, University of Hong Kong, Queen Mary Hospital, 102 Pokfulam Rd, Pokfulam, Hong Kong Special Administrative Region, China (<email>kyyuen@hku.hk</email>).</corresp></author-notes><pub-date pub-type="ppub"><day>15</day><month>3</month><year>2016</year></pub-date><pub-date pub-type="epub" iso-8601-date="2015-07-22"><day>22</day><month>7</month><year>2015</year></pub-date><volume>213</volume><issue>6</issue><fpage>904</fpage><lpage>914</lpage><history><date date-type="received"><day>29</day><month>4</month><year>2015</year></date><date date-type="accepted"><day>1</day><month>6</month><year>2015</year></date></history><permissions><copyright-statement>© The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: <email>journals.permissions@oup.com</email>.</copyright-statement><copyright-year>2015</copyright-year><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 xlink:href="jiv380.pdf"></self-uri><related-article related-article-type="article-reference" id="d35e366" vol="213" page="877" xlink:href="26203059" ext-link-type="pubmed"></related-article><abstract><title>Abstract</title><p>Middle East respiratory syndrome (MERS) is associated with a mortality rate of >35%. We previously showed that MERS coronavirus (MERS-CoV) could infect human macrophages and dendritic cells and induce cytokine dysregulation. Here, we further investigated the interplay between human primary T cells and MERS-CoV in disease pathogenesis. Importantly, our results suggested that MERS-CoV efficiently infected T cells from the peripheral blood and from human lymphoid organs, including the spleen and the tonsil. We further demonstrated that MERS-CoV infection induced apoptosis in T cells, which involved the activation of both the extrinsic and intrinsic apoptosis pathways. Remarkably, immunostaining of spleen sections from MERS-CoV–infected common marmosets demonstrated the presence of viral nucleoprotein in their CD3<sup>+</sup> T cells. Overall, our results suggested that the unusual capacity of MERS-CoV to infect T cells and induce apoptosis might partly contribute to the high pathogenicity of the virus.</p></abstract><kwd-group><kwd>MERS-CoV</kwd><kwd>T lymphocytes</kwd><kwd>apoptosis</kwd><kwd>caspase</kwd><kwd>tonsil</kwd><kwd>spleen</kwd><kwd>marmosets</kwd></kwd-group><funding-group><award-group><funding-source><named-content content-type="funder-name">Research Grants Council of the Hong Kong Special Administrative Region</named-content></funding-source><award-id>T11/707/15</award-id></award-group><award-group><funding-source><named-content content-type="funder-name">Providence Foundation Limited</named-content></funding-source></award-group><award-group><funding-source><named-content content-type="funder-name">University of Hong Kong Small Project Funding</named-content></funding-source><award-id>201409176024</award-id></award-group><award-group><funding-source><named-content content-type="funder-name">Hong Kong Health and Medical Research Fund</named-content></funding-source><award-id>14131392</award-id></award-group><award-group><funding-source><named-content content-type="funder-name">Hong Kong Research Grants Council</named-content></funding-source><award-id>N_HKU728/14</award-id></award-group><award-group><funding-source><named-content content-type="funder-name">National Science and Technology Major Projects of Infectious Disease</named-content></funding-source><award-id>2012ZX10004501-004</award-id></award-group></funding-group></article-meta></front><body><p><related-article related-article-type="article-reference" id="RA1" vol="213" page="877" xlink:href="26203059" ext-link-type="pubmed"><bold>(See the editorial commentary by Ying, Li, and Dimitrov on pages 877–9.)</bold></related-article></p><p>Middle East respiratory syndrome coronavirus (MERS-CoV) is a novel zoonotic betacoronavirus that causes severe acute respiratory syndrome (SARS)–like disease with extrapulmonary manifestations [<xref rid="JIV380C1" ref-type="bibr">1–3</xref>]. Despite the high resemblance between MERS and SARS, the case-fatality rate of MERS is >30%, which is 3 times than that of SARS [<xref rid="JIV380C4" ref-type="bibr">4–6</xref>]. Moreover, severe lymphopenia, which is an important prognostic factor for SARS, is commonly observed in patients with MERS [<xref rid="JIV380C7" ref-type="bibr">7</xref>, <xref rid="JIV380C8" ref-type="bibr">8</xref>]. The high pathogenicity of MERS-CoV is partly explained by the MERS-CoV receptor dipeptidyl peptidase 4 (DPP4), which is abundantly found in most host cells [<xref rid="JIV380C9" ref-type="bibr">9</xref>, <xref rid="JIV380C10" ref-type="bibr">10</xref>]. In in vitro and ex vivo studies, MERS-CoV exhibits broad tissue tropism that is unparalleled by other coronaviruses [<xref rid="JIV380C11" ref-type="bibr">11–14</xref>]. Upon entry, MERS-CoV efficiently evades the host innate immune response by the viral accessory proteins 4a, 4b, and 5, by papain-like protease, and by membrane (M) protein, which are potent interferon antagonists, and by downregulating the expression of genes within the antigen presentation pathway [<xref rid="JIV380C15" ref-type="bibr">15–21</xref>]. Moreover, we have previously demonstrated that MERS-CoV causes productive infection of monocyte-derived macrophages (MDMs) and monocyte-derived dendritic cells (MoDCs), the key professional antigen-presenting cells, which may facilitate systemic virus dissemination [<xref rid="JIV380C14" ref-type="bibr">14</xref>, <xref rid="JIV380C22" ref-type="bibr">22</xref>]. Finally, MERS-CoV induces an aberrant cytokine/chemokine response that may be responsible for the cytokine dysregulation in severe MERS cases [<xref rid="JIV380C14" ref-type="bibr">14</xref>, <xref rid="JIV380C22" ref-type="bibr">22</xref>, <xref rid="JIV380C23" ref-type="bibr">23</xref>].</p><p>Intriguingly, DPP4 is recognized as an activation marker on T cells and plays pivotal roles in T-cell functions, including activation [<xref rid="JIV380C24" ref-type="bibr">24</xref>], signal transduction [<xref rid="JIV380C25" ref-type="bibr">25</xref>], and costimulation [<xref rid="JIV380C26" ref-type="bibr">26</xref>]. We postulate that the abundant expression of DPP4 on T cells may render the cells highly susceptible to MERS-CoV infection. Importantly, a recent study demonstrated that infection of DPP4-transduced T-cell–deficient mice with MERS-CoV resulted in the persistence of MERS-CoV in the lungs, while virus was cleared in control and B-cell–deficient mice [<xref rid="JIV380C27" ref-type="bibr">27</xref>]. These findings suggested that T cells might play crucial roles in the pathogenesis of MERS-CoV. In this study, we showed that MERS-CoV was capable of efficiently infecting human T cells from the peripheral blood, spleen, and tonsil. Moreover, MERS-CoV could infect the T cells in the spleen of common marmosets. Importantly, MERS-CoV infection in T cells induced massive apoptosis involving activation of both the extrinsic and intrinsic apoptosis pathways, which might be important in the pathogenesis of MERS.</p><sec sec-type="methods" id="s2"><title>MATERIALS AND METHODS</title><sec id="s2a"><title>Preparation of Peripheral Blood Mononuclear Cells (PBMCs), T Cells, and Spleen- and Tonsil- Disassociated Cells</title><p>PBMCs and T cells were prepared as preciously described [<xref rid="JIV380C28" ref-type="bibr">28</xref>]. Tonsil- and spleen-disassociated cells were obtained from individuals undergoing splenectomy and tonsillectomy; all individuals provided written consent before cells were collected. Details are presented in the <xref ref-type="supplementary-material" rid="sup1">Supplementary Materials</xref>.</p></sec><sec id="s2b"><title>Infection of Common Marmosets</title><p>Approval by the institutional animal care and use committee was obtained for animal experiments [<xref rid="JIV380C29" ref-type="bibr">29</xref>]. Common marmosets were inoculated with 5 × 10<sup>6</sup> 50% tissue culture infective doses (TCID<sub>50</sub>) MERS-CoV intratracheally in 500 µL of Dulbecco's modified Eagle's medium. Organ samples were collected on necropsy as described previously [<xref rid="JIV380C29" ref-type="bibr">29–31</xref>].</p></sec><sec id="s2c"><title>Antibodies</title><p>Cell surface markers were obtained from BD Pharmingen, BioLegend, and Abcam. DPP4 antibody was obtained from R&D Systems. Immunostaining of MERS-CoV [<xref rid="JIV380C14" ref-type="bibr">14</xref>, <xref rid="JIV380C22" ref-type="bibr">22</xref>] and SARS-CoV nucleoprotein (NP) [<xref rid="JIV380C32" ref-type="bibr">32</xref>] were performed as previously described.</p></sec><sec id="s2d"><title>Virus and Infections</title><p>MERS-CoV strain EMC/2012 (passage 8) was provided by Dr Ron Fouchier (Erasmus Medical Center) [<xref rid="JIV380C1" ref-type="bibr">1</xref>, <xref rid="JIV380C33" ref-type="bibr">33</xref>]. MERS-CoV and SARS-CoV were cultured from Vero E6 cells. For infections, cells were inoculated with MERS-CoV or SARS-CoV at 2 TCID<sub>50</sub> per cell for 1 hour at 37°C.</p></sec><sec id="s2e"><title>Reverse Transcription–Quantitative Polymerase Chain Reaction (RT-qPCR)</title><p>RNA extraction and RT-qPCR were performed as previously described [<xref rid="JIV380C14" ref-type="bibr">14</xref>, <xref rid="JIV380C22" ref-type="bibr">22</xref>, <xref rid="JIV380C34" ref-type="bibr">34</xref>].</p></sec><sec id="s2f"><title>Terminal Deoxynucleotidyl Transferase dUTP Nick End Labeling (TUNEL) Assays</title><p>The TUNEL assay kit was purchased from Life Technologies. Labeling procedures were performed following protocols recommended by the manufacturer.</p></sec><sec id="s2g"><title>Active Caspase Assays</title><p>Active caspase 3 antibody was obtained from BD Pharmingen. CaspGLOW fluorescein active caspase 8 and 9 staining kits were obtained from eBioscience. Labeling was performed according to the manufacturer's protocol and is outlined in the <xref ref-type="supplementary-material" rid="sup1">Supplementary Materials</xref>.</p></sec><sec id="s2h"><title>Flow Cytometry</title><p>Immunostaining for flow cytometry was performed following standard procedures. Flow cytometry was performed using BD FACSCanto II (BD Biosciences), and data were analyzed using FlowJo vX (Tree Star).</p></sec><sec id="s2i"><title>Confocal Imaging of Marmoset Spleen Sections</title><p>MERS-CoV NP was labeled with guinea pig anti-MERS-CoV NP serum as mentioned above. CD3<sup>+</sup> T cells were identified with a mouse anti-human antibody (clone SP34-2) from BD Pharmingen. Images were acquired with a Carl Zeiss LSM 780 system.</p></sec><sec id="s2j"><title>Statistical Analysis</title><p>Statistical comparison was performed by the Student <italic>t</italic> test, using GraphPad Prism 6.</p></sec></sec><sec sec-type="results" id="s3"><title>RESULTS</title><sec id="s3a"><title>MERS-CoV Differentially Infects Subsets of Human PBMCs, and the Infectivity Correlates With Surface DPP4 Expressions</title><p>We hypothesized that T cells were potential targets of MERS-CoV infection. To test our hypothesis, we inoculated PBMCs with MERS-CoV and evaluated the proportion of MERS-CoV–infected T cells, B cells, natural killer (NK) cells, and monocytes with flow cytometry. Notably, a substantial proportion of T cells were infected by MERS-CoV. In contrast, MERS-CoV only modestly infected monocytes and NK cells, whereas it marginally infected B cells (Figure <xref ref-type="fig" rid="JIV380F1">1</xref><italic>A</italic>). Quantitatively, the average percentages (±standard deviation [SD]) of MERS-CoV NP–positive T cells, B cells, NK cells, and monocytes were 65.6% ±5.4%, 5.4% ± 2.4%, 18.5% ± 4.8%, and 13.5% ± 6.6%, respectively (Figure <xref ref-type="fig" rid="JIV380F1">1</xref><italic>A</italic>). As expected, the percentage of MERS-CoV infection was in direct accordance with the level of surface DPP4 expression on these PBMC subsets (Figure <xref ref-type="fig" rid="JIV380F1">1</xref><italic>B</italic>). Overall, we showed that MERS-CoV was capable of infecting T cells, NK cells, monocytes, and, to a less extent, B cells. The efficiency of infection highly correlated with the level of surface DPP4 expression.
</p><fig id="JIV380F1" orientation="portrait" position="float"><label>Figure 1.</label><caption><p>Middle East respiratory syndrome coronavirus (MERS-CoV) differentially infects subsets of human peripheral blood mononuclear cells (PBMCs). <italic>A</italic>, Human PBMCs were infected with MERS-CoV at 2 50% tissue culture infective doses per cell. At 24 hours after infection, infected cells were fixed with 4% paraformaldehyde (PA) and immunolabeled for detection of cell surface markers and MERS-CoV nucleoprotein (NP). The shaded curve and the solid line represent MERS-CoV NP expression from mock-infected and MERS-CoV–infected cells, respectively. The summary panel at the right represents the average percentage of infected cells from 3 different donors. <italic>B</italic>, Uninfected human PBMCs were fixed with 4% PA and immunolabeled for detection of surface DPP4 expression. The shaded curve and the solid line represented isotype and DPP4-specific staining, respectively. The summary panel at the right represents the average mean fluorescent intensity (MFI) from 3 different donors. In all panels, bars and error bars represented means and standard deviations, respectively. Statistical analyses were performed using the Student <italic>t</italic> test. *<italic>P</italic> < .001. Abbreviation: NK, natural killer.</p></caption><graphic xlink:href="jiv38001"></graphic></fig></sec><sec id="s3b"><title>MERS-CoV Efficiently Infects CD4<sup>+</sup> and CD8<sup>+</sup> T Cells and Downregulates Surface DPP4 in the Infected Cells</title><p>To further characterize MERS-CoV infection in T cells, we first challenged purified T cells with MERS-CoV and SARS-CoV and checked for NP expression. Strikingly, whereas a substantial number of T cells were infected by MERS-CoV, they were largely refractory to SARS-CoV infection (Figure <xref ref-type="fig" rid="JIV380F2">2</xref><italic>A</italic>). This differential susceptibility might be in part attributed to the discrepancy in the expression levels of DPP4 and angiotensin converting enzyme 2 in T cells (<xref ref-type="supplementary-material" rid="sup1">Supplementary Figure 1</xref>). Next, we asked whether the 2 major populations of T cells, CD4<sup>+</sup> and CD8<sup>+</sup> T cells, were equally susceptible to MERS-CoV infection. As shown in Figure <xref ref-type="fig" rid="JIV380F2">2</xref><italic>B</italic>, substantial numbers of both populations were infected by MERS-CoV. However, a significantly higher proportion of CD4<sup>+</sup> T cells (mean ± SD, 72.1% ± 9.1%) were infected, compared with CD8<sup>+</sup> cells (50.9% ± 7.7%). The degree of infection again correlated with surface DPP4 expression (Figure <xref ref-type="fig" rid="JIV380F2">2</xref><italic>C</italic>). Next, because DPP4 plays essential roles in a broad array of T-cell functions [<xref rid="JIV380C24" ref-type="bibr">24–26</xref>], we evaluated the expression level of surface and total DPP4 on CD4<sup>+</sup> or CD8<sup>+</sup> T cells after MERS-CoV infection. Remarkably, MERS-CoV significantly downregulated surface DPP4 in infected CD4<sup>+</sup> T cells by 80% and CD8<sup>+</sup> T cells by 70% (Figure <xref ref-type="fig" rid="JIV380F2">2</xref><italic>D</italic>). Changes in the total DPP4 level were less dramatic, with values of 50% and 30% for CD4<sup>+</sup> and CD8<sup>+</sup> T cells, respectively (Figure <xref ref-type="fig" rid="JIV380F2">2</xref><italic>D</italic>). In contrast to DPP4, surface or total levels of CD4 and CD8 were unaltered in infected T cells (<xref ref-type="supplementary-material" rid="sup1">Supplementary Figure 2</xref>). Together, our data demonstrated that MERS-CoV but not SARS-CoV efficiently infected T cells and significantly downregulated surface DPP4 in the infected cells.
</p><fig id="JIV380F2" orientation="portrait" position="float"><label>Figure 2.</label><caption><p>Middle East respiratory syndrome coronavirus (MERS-CoV) efficiently infects CD4<sup>+</sup> and CD8<sup>+</sup> T cells and downregulates surface dipeptidyl peptidase 4 (DPP4) in the infected cells. <italic>A</italic>, T cells were infected with MERS-CoV and severe acute respiratory syndrome CoV (SARS-CoV) at 2 50% tissue culture infective doses (TCID<sub>50</sub>) per cell. Cells were fixed at 24 hours after infection and immunolabeled for detection of expression of MERS-CoV NP or SARS-CoV NP. The shaded curve and the solid line represent virus NP expression from mock-infected and MERS/SARS-CoV–infected cells, respectively. <italic>B</italic>, T cells were infected with MERS-CoV at 2 TCID<sub>50</sub> per cell. Cells were fixed at 24 hours after infection and immunolabeled for detection of CD4 or CD8 and MERS-CoV NP expression. The shaded curve and the solid line represent NP expression from mock-infected and MERS-CoV–infected cells, respectively. <italic>C</italic>, Uninfected T cells were fixed and immunolabeled for detection of CD4 or CD8 and surface DPP4 expression. The shaded curve and the solid line represent isotype and DPP4-specific staining, respectively. <italic>D</italic>, T cells were infected with MERS-CoV at 2 TCID<sub>50</sub> per cell. Cells were fixed at 24 hours after infection and immunolabeled for detection of CD4 or CD8, DPP4, and MERS-CoV NP expression. Surface or total DPP4 was detected by labeling the cells with the DPP4 antibody before or after cell permeabilization, respectively. The shaded curve represents isotype staining of DPP4. The solid line and dotted line represent DPP4 staining from mock-infected and MERS-CoV–infected cells, respectively. The DPP4 mean fluorescence intensity (MFI) in infected cells was calculated on the basis of CD4<sup>+</sup>/MERS-CoV NP–expressing or CD8<sup>+</sup>/MERS-CoV NP–expressing double-positive T-cell data. The DPP4 MFI in mock-infected cells was calculated on the basis of CD4<sup>+</sup> or CD8<sup>+</sup> T-cell data. The summary panels at the right represent the average percentage of infected cells (<italic>A</italic> and <italic>B</italic>), MFI (<italic>C</italic>), or percentage of mock MFI (<italic>D</italic>) from 3 different donors. In all panels, bars and error bars represent means and standard deviations. Statistical analyses were performed using the Student <italic>t</italic> test. *<italic>P</italic> < .05.</p></caption><graphic xlink:href="jiv38002"></graphic></fig></sec><sec id="s3c"><title>MERS-CoV Infection of Human T Cells Is Abortive</title><p>To grasp a more comprehensive picture of the replication kinetic of MERS-CoV in T cells, we measured the genome copy number of MERS-CoV from samples harvested at different time points. In stark contrast to MDMs [<xref rid="JIV380C14" ref-type="bibr">14</xref>] and MoDCs [<xref rid="JIV380C22" ref-type="bibr">22</xref>], in which MERS-CoV infections are productive, T cells failed to support MERS-CoV replication (<xref ref-type="supplementary-material" rid="sup1">Supplementary Figure 3</xref>). The number of MERS-CoV genome copies in the cell lysates (<xref ref-type="supplementary-material" rid="sup1">Supplementary Figure 3</xref><italic>A</italic>) and supernatants (<xref ref-type="supplementary-material" rid="sup1">Supplementary Figure 3</xref><italic>B</italic>) of infected T cells remained largely steady over time. As expected, SARS-CoV similarly failed to replicate in T cells. Intriguingly, the number of intracellular MERS-CoV genome copies was 2–3 logs higher than that of SARS-CoV (<xref ref-type="supplementary-material" rid="sup1">Supplementary Figure 3</xref><italic>A</italic>). Therefore, our findings supported the notion that MERS-CoV efficiently entered T cells, although the infection was not productive, whereas SARS-CoV failed to enter T cells.</p></sec><sec id="s3d"><title>MERS-CoV but Not SARS-CoV Induces Apoptosis in T Cells</title><p>MERS-CoV is known to cause cytopathology in a number of cell lines [<xref rid="JIV380C35" ref-type="bibr">35</xref>, <xref rid="JIV380C36" ref-type="bibr">36</xref>]. To examine whether MERS-CoV infection induced cell death in T cells, we assessed TUNEL activity in MERS-CoV–infected T cells and compared the findings to those in SARS-CoV– or mock-infected T cells. TUNEL assays detect DNA fragmentation that results from apoptotic signaling cascades and identifies cells in the last phase of apoptosis [<xref rid="JIV380C37" ref-type="bibr">37</xref>]. We detected an increased number of TUNEL-positive cells in MERS-CoV–infected T cells at as early as 6 hours after infection, compared with SARS-CoV– or mock-infected T cells. At 24 and 48 hours after infection, significantly more TUNEL-positive cells were detected in MERS-CoV–infected T cells comparing with findings for the 2 other groups (Figure <xref ref-type="fig" rid="JIV380F3">3</xref><italic>A</italic>). Quantitatively, the mean percentages (±SD) of TUNEL-positive T cells upon MERS-CoV infection were 8.2% ± 2.7%, 20.0% ±7.8%, and 27.8% ± 9.7% at 6, 24, and 48 hours after infection, respectively (Figure <xref ref-type="fig" rid="JIV380F3">3</xref><italic>B</italic>). Thus, we concluded that MERS-CoV but not SARS-CoV induced apoptosis in T cells.
</p><fig id="JIV380F3" orientation="portrait" position="float"><label>Figure 3.</label><caption><p>Middle East respiratory syndrome coronavirus (MERS-CoV) induces apoptosis and substantial upregulation of active caspase 3 in infected T cells. <italic>A</italic>, T cells were infected with MERS-CoV or severe acute respiratory syndrome CoV (SARS-CoV) at 2 50% tissue culture infective doses (TCID<sub>50</sub>) per cell. Cells were fixed at the indicated time points and immunolabeled for detection of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)–positive cells. <italic>B</italic>, The average percentage of TUNEL-positive cells from 3 different donors. <italic>C</italic>, T cells were infected with MERS-CoV at 2 TCID<sub>50</sub> per cell. Cells were fixed at the indicated time points and immunolabeled for detection of MERS-CoV nucleoprotein (NP) and active caspase 3. <italic>D</italic>, The average percentage of active caspase 3–positive cells among MERS-CoV NP<sup>+</sup> cells from 3 different donors. In all panels, bars and error bars represent means and standard deviations. Statistical analyses were performed using the Student <italic>t</italic> test. *<italic>P</italic> < .01.</p></caption><graphic xlink:href="jiv38003"></graphic></fig><p>Apoptosis is a coordinated and energy-dependent process that can be triggered through the extrinsic (death receptor) or the intrinsic (mitochondrial) caspase-dependent pathways [<xref rid="JIV380C38" ref-type="bibr">38</xref>]. The 2 pathways end and converge at the execution phase, which involves the activation of execution caspases, including caspases 3, 6, and 7 [<xref rid="JIV380C39" ref-type="bibr">39</xref>]. Among them, caspase 3 is considered to be the most important executer caspase and is activated by any of the initiator caspases (caspases 2 and 8–10) [<xref rid="JIV380C38" ref-type="bibr">38</xref>]. Therefore, we evaluated the extent of caspase 3 activation in infected T cells. Remarkably, substantial active caspase 3 expression was evident in MERS-CoV–infected T cells as early as 6 hours after infection, and expression remained high over time (Figure <xref ref-type="fig" rid="JIV380F3">3</xref><italic>C</italic>). Specifically, active caspase 3 was detected at a mean frequency (±SD) of 74.9% ±6.8%, 75.3% ± 10.2%, and 63.2% ± 7.6% among MERS-CoV-NP–positive T cells at 6, 24, and 48 hours after infection, respectively (Figure <xref ref-type="fig" rid="JIV380F3">3</xref><italic>D</italic>). Caspase 3 activation was absent in mock-infected cells or in T cells that remained uninfected in the MERS-CoV–treated samples (Figure <xref ref-type="fig" rid="JIV380F3">3</xref><italic>C</italic>). In summary, we demonstrated that caspase 3 was substantially activated in MERS-CoV–infected T cells early upon infection and remained highly expressed.</p></sec><sec id="s3e"><title>Extrinsic and Intrinsic Caspase-Dependent Apoptosis Pathways Are Triggered in MERS-CoV–Infected T Cells</title><p>We then examined the degree of caspase 8 and caspase 9 activation, which represented the involvement of the extrinsic and intrinsic apoptosis pathway, respectively. Intriguingly, our results suggested that both caspase 8 and caspase 9 were activated starting as early as 1 hour after infection with MERS-CoV. The degree of caspase 8 activation in MERS-CoV–infected T cells was rather consistent over time (Figure <xref ref-type="fig" rid="JIV380F4">4</xref><italic>A</italic>), with mean levels (±SD) of 14.3% ± 2.7%, 11.4% ± 4.8%, and 15.4% ± 3.0% at 1, 6, and 24 hours after infection, respectively (Figure <xref ref-type="fig" rid="JIV380F4">4</xref><italic>B</italic>). An increasing level of caspase 9 activation was detected in MERS-CoV–infected T cells over time (Figure <xref ref-type="fig" rid="JIV380F4">4</xref><italic>C</italic>). Specifically, the mean frequencies (±SD) of MERS-CoV–infected T cells expressing active caspase 9, at 1, 6, and 24 hours after infection were 13.5% ± 4.1%, 17.6% ± 1.2%, and 31.3% ± 7.3%, respectively (Figure <xref ref-type="fig" rid="JIV380F4">4</xref><italic>D</italic>). Together, our results suggested that the extrinsic and intrinsic apoptosis pathways were triggered in MERS-CoV–infected T cells.
</p><fig id="JIV380F4" orientation="portrait" position="float"><label>Figure 4.</label><caption><p>The extrinsic and intrinsic caspase-dependent apoptosis pathways are activated in Middle East respiratory syndrome coronavirus (MERS-CoV)–infected T cells. T cells were infected with MERS-CoV at 2 50% tissue culture infective doses per cell. At the indicated time points, infected cells were labeled with MERS-CoV nucleoprotein (NP), as well as FITC-IETD-FMK (for caspase 8; <italic>A</italic> and <italic>B</italic>) and FITC-LEHD-FMK (for caspase 9; <italic>C</italic> and <italic>D</italic>) for 1 hour at 37°C. The average percentage of active caspase 8–positive (<italic>B</italic>) or active caspase 9–positive (<italic>D</italic>) cells among MERS-CoV NP–expressing cells from 3 different donors was illustrated. In all panels, bars and error bars represent means and standard deviations. Statistical analyses were performed using the Student <italic>t</italic> test. *<italic>P</italic> < .05.</p></caption><graphic xlink:href="jiv38004"></graphic></fig></sec><sec id="s3f"><title>MERS-CoV–Induced Caspase 3 Activation in T Cells Occurs Rapidly Upon Infection and Is Not Inhibited by UV-Inactivated MERS-CoV</title><p>To compare the extent of MERS-CoV–induced cytopathology between cell lines and primary T cells, we infected Vero E6 cells and compared the level of caspase 3 activation with that in infected T cells. Our data suggested that caspase 3 was also activated in Vero E6 cells, although the dynamic of activation was drastically different from that in T cells. Remarkably, at 6 hours after infection, only a subtle population of Vero E6 cells was infected by MERS-CoV, with no caspase 3 activation (Figure <xref ref-type="fig" rid="JIV380F5">5</xref><italic>A</italic>). Intriguingly, at 24 hours after infection, essentially all Vero E6 cells were infected, and the vast majority of the infected cells were expressing activated caspase 3 (Figure <xref ref-type="fig" rid="JIV380F5">5</xref><italic>A</italic>). On the other hand, a substantial number of T cells were infected at 6 hours after infection, with accompanying excessive caspase 3 activation. The level of infection and caspase 3 activation in T cells remained largely constant between 6 and 24 hours after infection (Figure <xref ref-type="fig" rid="JIV380F5">5</xref><italic>A</italic>). Since Vero E6 cells are known to support robust MERS-CoV replication, we speculated that caspase 3 activation in Vero E6 cells was a result of virus replication. To test this hypothesis, we inoculated Vero E6 cells and T cells with UV-inactivated MERS-CoV. The replication capacity of MERS-CoV was completely abolished with 10 minutes of UV inactivation (<xref ref-type="supplementary-material" rid="sup1">Supplementary Figure 4</xref>). Strikingly, UV inactivation completely obliterated MERS-CoV infection and caspase 3 activation in Vero E6 cells (Figure <xref ref-type="fig" rid="JIV380F5">5</xref><italic>B</italic>). In stark contrast, MERS-CoV entry and MERS-CoV–induced caspase 3 activation in T cells remained largely unaltered by UV inactivation (Figure <xref ref-type="fig" rid="JIV380F5">5</xref><italic>B</italic>). NP staining with no cell permeabilization (<xref ref-type="supplementary-material" rid="sup1">Supplementary Figure 5</xref><italic>A</italic>) or on ice before cell permeabilization (<xref ref-type="supplementary-material" rid="sup1">Supplementary Figure 5</xref><italic>B</italic>) both picked up little to no signal, suggesting the detected MERS-CoV NP signal was indeed intracellular but not from surface-attached viruses. Together, these data illustrated intrinsic differences in the dynamics of MERS-CoV infection and MERS-CoV–induced caspase 3 activation in Vero E6 cells and T cells.
</p><fig id="JIV380F5" orientation="portrait" position="float"><label>Figure 5.</label><caption><p>Middle East respiratory syndrome coronavirus (MERS-CoV)–induced caspase 3 activation in T cells occurs rapidly upon infection and is not inhibited by UV inactivation of the virus. <italic>A</italic>, T cells and Vero E6 cells were infected with MERS-CoV at 2 50% tissue culture infective doses (TCID<sub>50</sub>) per cell. Cells were fixed at the indicated time points and immunolabeled for detection of MERS-CoV nucleoprotein (NP) and active caspase 3. <italic>B</italic>, T cells and Vero E6 cells were infected with MERS-CoV at 2 TCID<sub>50</sub> per cell. In parallel, equal volumes of UV-inactivated MERS-CoV were applied to Vero E6 cells and T cells. At 24 hours after infection, cells were harvested, fixed, and immunolabeled for detection of MERS-CoV NP and active caspase 3. The illustrated result was a representative of 2 independent experiments that showed similar results.</p></caption><graphic xlink:href="jiv38005"></graphic></fig></sec><sec id="s3g"><title>T Cells in Human Lymphatic Organs, Including the Tonsil and the Spleen, Are Highly Susceptible to MERS-CoV and MERS-CoV–Induced Apoptosis</title><p>Recent pathological studies detected the presence of viral RNA in the tonsil [<xref rid="JIV380C30" ref-type="bibr">30</xref>, <xref rid="JIV380C40" ref-type="bibr">40</xref>, <xref rid="JIV380C41" ref-type="bibr">41</xref>], lymph node [<xref rid="JIV380C30" ref-type="bibr">30</xref>, <xref rid="JIV380C40" ref-type="bibr">40</xref>, <xref rid="JIV380C41" ref-type="bibr">41</xref>], and spleen [<xref rid="JIV380C30" ref-type="bibr">30</xref>] of MERS-CoV–inoculated animals. To investigate whether T cells in these lymphoid organs are as susceptible to MERS-CoV infection as those in the peripheral blood, we inoculated disassociated cells from human spleen and tonsil with MERS-CoV and estimated the percentage of infected T cells by using flow cytometry. Remarkably, CD3<sup>+</sup> T cells from the spleen and tonsil were infected to a similar extent as T cells in the peripheral blood (Figure <xref ref-type="fig" rid="JIV380F6">6</xref><italic>A</italic>). We further assessed the level of MERS-CoV–induced apoptosis in these cells. Our data suggested that the percentage of TUNEL-positive (Figure <xref ref-type="fig" rid="JIV380F6">6</xref><italic>B</italic>) and active caspase 3–positive (Figure <xref ref-type="fig" rid="JIV380F6">6</xref><italic>C</italic>) cells among MERS-CoV–infected CD3<sup>+</sup> T cells from the spleen and tonsil highly resembled that among the peripheral blood T cells. In this regard, we demonstrated that T cells in the human spleen and tonsil were highly susceptible to MERS-CoV infection and MERS-CoV–induced apoptosis.
</p><fig id="JIV380F6" orientation="portrait" position="float"><label>Figure 6.</label><caption><p>T cells in human lymphatic organs, including the spleen and the tonsil, are highly susceptible to Middle East respiratory syndrome coronavirus (MERS-CoV) and MERS-CoV–induced apoptosis. Disassociated human spleen and tonsil cells were infected with MERS-CoV at 2 50% tissue culture infective doses per cell. Cells were fixed at 24 hours after infection and assessed for MERS-CoV nucleoprotein (NP; <italic>A</italic>), terminal deoxynucleotidyl transferase dUTP nick end labeling activity (<italic>B</italic>), and active caspase 3 (<italic>C</italic>) among CD3<sup>+</sup> cells. The illustrated result was a representative of 2 independent experiments that showed similar results.</p></caption><graphic xlink:href="jiv38006"></graphic></fig></sec><sec id="s3h"><title>MERS-CoV Infects CD3<sup>+</sup> T Cells in the Spleen of Common Marmosets</title><p>Our in vitro and ex vivo data have, so far, consistently demonstrated the high susceptibility of human T cells to MERS-CoV infection. We next used the common marmoset animal model [<xref rid="JIV380C30" ref-type="bibr">30</xref>, <xref rid="JIV380C31" ref-type="bibr">31</xref>] to evaluate the involvement of T cells in MERS-CoV infection in vivo. Two male common marmosets (<italic>Callithrix jacchus</italic>; CM1 and CM2) were inoculated with 5 × 10<sup>6</sup> TCID<sub>50</sub> of MERS-CoV intratracheally. Upon necropsy of these infected animals, tissue samples were collected and analyzed for the presence of viral RNA. Notably, viral RNA was detected from the spleen tissues of both animals, with a viral gene to GAPDH ratio of 7.3 × 10<sup>4</sup> and 7.6 × 10<sup>5</sup> in CM1 and CM2, respectively (<xref ref-type="supplementary-material" rid="sup1">Supplementary Figure 6</xref>). Next, immunostaining was performed on paraformaldehyde-fixed and paraffin-embedded sections of spleen from the infected animals. Remarkably, MERS-CoV NP was readily detected in the spleen (Figure <xref ref-type="fig" rid="JIV380F7">7</xref><italic>D</italic> and <xref ref-type="fig" rid="JIV380F7">7</xref><italic>G</italic>). More importantly, MERS-CoV NP was detected in T cells, which was suggested by the colocalization between MERS-CoV NP and CD3 (Figure <xref ref-type="fig" rid="JIV380F7">7</xref><italic>D–I</italic>). As a control, no MERS-CoV NP signal was detected when preimmune serum was used in place of MERS-CoV NP antibody (Figure <xref ref-type="fig" rid="JIV380F7">7</xref><italic>A–C</italic>). Overall, we demonstrated the capacity of MERS-CoV to disseminate to the spleens of common marmosets and infect CD3<sup>+</sup> T cells therein, despite respiratory transmission of the pathogen.
</p><fig id="JIV380F7" orientation="portrait" position="float"><label>Figure 7.</label><caption><p>Middle East respiratory syndrome coronavirus (MERS-CoV) infects CD3<sup>+</sup> T cells in the spleen of infected common marmosets. Immunostaining was performed on paraformaldehyde-fixed and paraffin-embedded sections of spleen obtained on day 1 after virus challenge of animals. Colocalization between MERS-CoV nucleoprotein (NP; <italic>D</italic> and <italic>G</italic>) and CD3 (<italic>E</italic> and <italic>H</italic>) were detected in the spleen of the infected animals (arrows; <italic>D</italic>–<italic>F</italic> and <italic>G</italic>–<italic>I</italic>). CM1 and CM2 represent 2 individual common marmosets. MERS-CoV NP was not detected in the preimmune serum control (<italic>A</italic>–<italic>C</italic>). Bars represent 25 µm.</p></caption><graphic xlink:href="jiv38007"></graphic></fig></sec></sec><sec sec-type="discussion" id="s4"><title>DISCUSSION</title><p>T cells and T-cell responses play central roles in the pathogenesis of SARS [<xref rid="JIV380C42" ref-type="bibr">42</xref>, <xref rid="JIV380C43" ref-type="bibr">43</xref>]. Currently, little is known about the virus-host interaction of MERS-CoV infection in T cells. In this study, we demonstrated that human T cells were highly susceptible to MERS-CoV infection and MERS-CoV–induced apoptosis while remaining impervious to SARS-CoV. Most importantly, we showed in a common marmoset model that MERS-CoV efficiently targeted the CD3<sup>+</sup> T cells in the spleen of infected animals. Together, our results suggested that its distinct capacity to invade T cells might contribute to the high pathogenicity of MERS-CoV.</p><p>Viremia has been frequently reported in patients with SARS and those with MERS. Viral RNA was detected in the sera of SARS patients during the acute phase of infection [<xref rid="JIV380C44" ref-type="bibr">44</xref>, <xref rid="JIV380C45" ref-type="bibr">45</xref>]. MERS-CoV RNA could be detected in the blood of infected patients for a prolonged period [<xref rid="JIV380C46" ref-type="bibr">46–48</xref>]. The viremia can be attributed to circulating free viral RNA that should be transient, free virion in blood or virus in infected cells. Remarkably, our current study demonstrated that MERS-CoV but not SARS-CoV efficiently infected T cells and induced substantial apoptosis in the infected cells. This discovery indicates that the viremia in patients with SARS and those with MERS may have different clinical significance. In particular, given the capacity of MERS-CoV to invade T cells, patients with MERS who develop viremia may have a more severe outcome. Of note, viremia is not the only route for MERS-CoV to infect T cells. T cells could be attracted to the infected lung under the action of chemoattractants and therefore be infected at the primary organ of MERS-CoV infection. Alternatively, dendritic cells, which we have previously shown to support robust MERS-CoV replication [<xref rid="JIV380C22" ref-type="bibr">22</xref>], can be infected, migrate to the draining lymph nodes, and transmit the virus to naive T cells during antigen presentation. Importantly, lymphopenia is a common hematological abnormality that has been frequently reported in SARS and MERS cases [<xref rid="JIV380C7" ref-type="bibr">7</xref>, <xref rid="JIV380C49" ref-type="bibr">49</xref>]. It has been speculated that lymphopenia detected in SARS cases might be due to direct infection, tissue sequestration of lymphocytes, cytokine-mediated cell death, as well as suppression of bone marrow or thymus for T-cell generation. In this study, we demonstrated that MERS-CoV but not SARS-CoV could directly infect T cells and induce apoptosis in the infected cells. Therefore, the underlying mechanism for SARS-CoV– and MERS-CoV–induced lymphopenia could be distinct. In particular, lymphopenia in MERS but not SARS cases could be a result of direct infection of T cells and infection-induced apoptosis. In addition to apoptosis, the dramatic depletion of surface DPP4 in T cells upon MERS-CoV infection may substantially disrupt the proliferation and cellular function of T cells. The mechanism and implication of MERS-CoV–directed DPP4 depletion in T cells deserve further investigation.</p><p>Multiple genes of SARS-CoV have been implicated in caspase induction [<xref rid="JIV380C4" ref-type="bibr">4</xref>]. We showed in the current study that MERS-CoV–induced apoptosis in T cells was accompanied by the activation of caspases 3, 8, and 9. The maturation of caspases 8 and 9 suggested that the extrinsic and intrinsic apoptosis pathways were triggered by MERS-CoV infection. Currently, the exact mechanism of MERS-CoV–induced caspase upregulation is unknown. However, since UV-inactivated MERS-CoV induced caspase 3 upregulation to an extent similar to that of untreated MERS-CoV, we postulate that the detected caspase upregulation is likely attributed to the structural components of MERS-CoV, including the E, M, S, and N proteins. Nevertheless, our data demonstrated that, while approximately 70% of MERS-CoV–infected T cells were expressing active caspase 3, a considerably lower percentage of infected T cells showed signs of caspase 8 or caspase 9 activation. Although the relationship of the quantity of initiator caspases (caspases 8 and 9) and execution caspase (caspase 3) is not linear, because they are catalyzed in a cascade, it is possible that other, yet-to-be-identified pathways are also involved in caspase 3 activation and in contributing to MERS-CoV–induced apoptosis in infected T cells. Remarkably, the dynamics of MERS-CoV infection and MERS-CoV–induced apoptosis in T cells were drastically different from findings in cell lines. We demonstrated that, whereas caspase 3 induction in cell lines was gradual and replication dependent, it was abrupt and replication-independent in T cells. This discrepancy is likely due to the difference in the efficiency of MERS-CoV entry of cell lines versus primary T cells, which may in turn be a result of differential surface DPP4 expression. In this regard, if virus-induced T-cell apoptosis is a pathogenic determinant of MERS, a novel treatment regimen such as one involving anti-apoptotic agents in addition to antivirals inhibiting virus replication may be considered to improve the outcomes of patients with MERS, as in the case of VX-765 for human immunodeficiency virus infection [<xref rid="JIV380C50" ref-type="bibr">50</xref>].</p><p>Recent animal studies have identified MERS-CoV RNA in the lymphoid organs of infected animals, including common marmosets [<xref rid="JIV380C30" ref-type="bibr">30</xref>], rhesus macaques [<xref rid="JIV380C40" ref-type="bibr">40</xref>], and dromedary camels [<xref rid="JIV380C41" ref-type="bibr">41</xref>]. However, owing to the lack of reports describing samples obtained from patients with MERS during autopsy, whether the virus could invade human lymphoid organs remained unknown. To this end, we inoculated disassociated human spleen and tonsil cells with MERS-CoV. Remarkably, CD3<sup>+</sup> T cells in the spleen and tonsil were highly susceptible to MERS-CoV infection and MERS-CoV–induced apoptosis. Intriguingly, T cells from these organs appeared to demonstrate a higher degree of infection and apoptosis upon MERS-CoV challenge. This suggested that T cells at different developmental stages might be differentially affected by MERS-CoV. Finally, we examined the spleen of MERS-CoV–infected common marmosets. Importantly, our data demonstrated that MERS-CoV could disseminate to the spleens of infected animals and infect their CD3<sup>+</sup> T cells. Collectively, our ex vivo and in vivo findings suggest that MERS-CoV can invade not only T cells in the peripheral blood, but also the large repertoire of T cells in lymphoid organs, which may result in a more severe immunopathology.</p><p>In summary, our study provided in vitro, ex vivo, and in vivo evidence for MERS-CoV infection of human T cells. We demonstrated that the infection is associated with apoptosis induction, which may provide a new target for effective therapeutic interventions.</p></sec><sec sec-type="supplementary-material"><title>Supplementary Material</title><supplementary-material content-type="local-data" id="sup1"><label>Supplementary Data</label><media xlink:href="jiv380_Supplementary_Data.zip"><caption><p>Click here for additional data file.</p></caption></media></supplementary-material></sec></body><back><ack><title>Notes</title><p><bold><italic>Acknowledgments.</italic></bold> We thank Dr Birgitta Wong, Dr Jacky Lam, and the staff at the Core Facility, Li Ka Shing Faculty of Medicine, University of Hong Kong, for facilitation of the study.</p><p><bold><italic>Financial support.</italic></bold> This work was partially supported by the Research Grants Council of the Hong Kong Special Administrative Region, China (Project No. T11/707/15), the Providence Foundation Limited in memory of the late Dr Lui Hac Minh, the University of Hong Kong Small Project Funding (201409176024), the Hong Kong Health and Medical Research Fund (14131392), Hong Kong Research Grants Council (N_HKU728/14), and the National Science and Technology Major Projects of Infectious Disease (2012ZX10004501-004).</p><p><bold><italic>Potential conflicts of interest.</italic></bold> All authors: No reported conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. 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