Histopathologic Changes and SARS–CoV-2 Immunostaining in the Lung of a Patient With COVID-19
Identifieur interne : 000802 ( Pmc/Corpus ); précédent : 000801; suivant : 000803Histopathologic Changes and SARS–CoV-2 Immunostaining in the Lung of a Patient With COVID-19
Auteurs : Huilan Zhang ; Peng Zhou ; Yanqiu Wei ; Huihui Yue ; Yi Wang ; Ming Hu ; Shu Zhang ; Tanze Cao ; Chengqing Yang ; Ming Li ; Guangyun Guo ; Xianxiang Chen ; Ying Chen ; Mei Lei ; Huiguo Liu ; Jianping Zhao ; Peng Peng ; Cong-Yi Wang ; Ronghui DuSource :
- Annals of Internal Medicine [ 0003-4819 ] ; 2020.
Url:
DOI: 10.7326/M20-0533
PubMed: 32163542
PubMed Central: 7081173
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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Histopathologic Changes and SARS–CoV-2 Immunostaining in the Lung of a Patient With COVID-19</title><author><name sortKey="Zhang, Huilan" sort="Zhang, Huilan" uniqKey="Zhang H" first="Huilan" last="Zhang">Huilan Zhang</name><affiliation><nlm:aff>NONE</nlm:aff></affiliation></author><author><name sortKey="Zhou, Peng" sort="Zhou, Peng" uniqKey="Zhou P" first="Peng" last="Zhou">Peng Zhou</name><affiliation><nlm:aff>NONE</nlm:aff></affiliation></author><author><name sortKey="Wei, Yanqiu" sort="Wei, Yanqiu" uniqKey="Wei Y" first="Yanqiu" last="Wei">Yanqiu Wei</name><affiliation><nlm:aff>NONE</nlm:aff></affiliation></author><author><name sortKey="Yue, Huihui" sort="Yue, Huihui" uniqKey="Yue H" first="Huihui" last="Yue">Huihui Yue</name><affiliation><nlm:aff>NONE</nlm:aff></affiliation></author><author><name sortKey="Wang, Yi" sort="Wang, Yi" uniqKey="Wang Y" first="Yi" last="Wang">Yi Wang</name><affiliation><nlm:aff>NONE</nlm:aff></affiliation></author><author><name sortKey="Hu, Ming" sort="Hu, Ming" uniqKey="Hu M" first="Ming" last="Hu">Ming Hu</name><affiliation><nlm:aff>NONE</nlm:aff></affiliation></author><author><name sortKey="Zhang, Shu" sort="Zhang, Shu" uniqKey="Zhang S" first="Shu" last="Zhang">Shu Zhang</name><affiliation><nlm:aff>NONE</nlm:aff></affiliation></author><author><name sortKey="Cao, Tanze" sort="Cao, Tanze" uniqKey="Cao T" first="Tanze" last="Cao">Tanze Cao</name><affiliation><nlm:aff>NONE</nlm:aff></affiliation></author><author><name sortKey="Yang, Chengqing" sort="Yang, Chengqing" uniqKey="Yang C" first="Chengqing" last="Yang">Chengqing Yang</name><affiliation><nlm:aff>NONE</nlm:aff></affiliation></author><author><name sortKey="Li, Ming" sort="Li, Ming" uniqKey="Li M" first="Ming" last="Li">Ming Li</name><affiliation><nlm:aff>NONE</nlm:aff></affiliation></author><author><name sortKey="Guo, Guangyun" sort="Guo, Guangyun" uniqKey="Guo G" first="Guangyun" last="Guo">Guangyun Guo</name><affiliation><nlm:aff>NONE</nlm:aff></affiliation></author><author><name sortKey="Chen, Xianxiang" sort="Chen, Xianxiang" uniqKey="Chen X" first="Xianxiang" last="Chen">Xianxiang Chen</name><affiliation><nlm:aff>NONE</nlm:aff></affiliation></author><author><name sortKey="Chen, Ying" sort="Chen, Ying" uniqKey="Chen Y" first="Ying" last="Chen">Ying Chen</name><affiliation><nlm:aff>NONE</nlm:aff></affiliation></author><author><name sortKey="Lei, Mei" sort="Lei, Mei" uniqKey="Lei M" first="Mei" last="Lei">Mei Lei</name><affiliation><nlm:aff>NONE</nlm:aff></affiliation></author><author><name sortKey="Liu, Huiguo" sort="Liu, Huiguo" uniqKey="Liu H" first="Huiguo" last="Liu">Huiguo Liu</name><affiliation><nlm:aff>NONE</nlm:aff></affiliation></author><author><name sortKey="Zhao, Jianping" sort="Zhao, Jianping" uniqKey="Zhao J" first="Jianping" last="Zhao">Jianping Zhao</name><affiliation><nlm:aff>NONE</nlm:aff></affiliation></author><author><name 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wicri:corpus="PMC">000802</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Histopathologic Changes and SARS–CoV-2 Immunostaining in the Lung of a Patient With COVID-19</title><author><name sortKey="Zhang, Huilan" sort="Zhang, Huilan" uniqKey="Zhang H" first="Huilan" last="Zhang">Huilan Zhang</name><affiliation><nlm:aff>NONE</nlm:aff></affiliation></author><author><name sortKey="Zhou, Peng" sort="Zhou, Peng" uniqKey="Zhou P" first="Peng" last="Zhou">Peng Zhou</name><affiliation><nlm:aff>NONE</nlm:aff></affiliation></author><author><name sortKey="Wei, Yanqiu" sort="Wei, Yanqiu" uniqKey="Wei Y" first="Yanqiu" last="Wei">Yanqiu Wei</name><affiliation><nlm:aff>NONE</nlm:aff></affiliation></author><author><name sortKey="Yue, Huihui" sort="Yue, Huihui" uniqKey="Yue H" first="Huihui" last="Yue">Huihui Yue</name><affiliation><nlm:aff>NONE</nlm:aff></affiliation></author><author><name sortKey="Wang, Yi" sort="Wang, Yi" uniqKey="Wang Y" first="Yi" last="Wang">Yi Wang</name><affiliation><nlm:aff>NONE</nlm:aff></affiliation></author><author><name sortKey="Hu, Ming" sort="Hu, Ming" uniqKey="Hu M" first="Ming" last="Hu">Ming Hu</name><affiliation><nlm:aff>NONE</nlm:aff></affiliation></author><author><name sortKey="Zhang, Shu" sort="Zhang, Shu" uniqKey="Zhang S" first="Shu" last="Zhang">Shu Zhang</name><affiliation><nlm:aff>NONE</nlm:aff></affiliation></author><author><name sortKey="Cao, Tanze" sort="Cao, Tanze" uniqKey="Cao T" first="Tanze" last="Cao">Tanze Cao</name><affiliation><nlm:aff>NONE</nlm:aff></affiliation></author><author><name sortKey="Yang, Chengqing" sort="Yang, Chengqing" uniqKey="Yang C" first="Chengqing" last="Yang">Chengqing Yang</name><affiliation><nlm:aff>NONE</nlm:aff></affiliation></author><author><name sortKey="Li, Ming" sort="Li, Ming" uniqKey="Li M" first="Ming" last="Li">Ming Li</name><affiliation><nlm:aff>NONE</nlm:aff></affiliation></author><author><name sortKey="Guo, Guangyun" sort="Guo, Guangyun" uniqKey="Guo G" first="Guangyun" last="Guo">Guangyun Guo</name><affiliation><nlm:aff>NONE</nlm:aff></affiliation></author><author><name sortKey="Chen, Xianxiang" sort="Chen, Xianxiang" uniqKey="Chen X" first="Xianxiang" last="Chen">Xianxiang Chen</name><affiliation><nlm:aff>NONE</nlm:aff></affiliation></author><author><name sortKey="Chen, Ying" sort="Chen, Ying" uniqKey="Chen Y" first="Ying" last="Chen">Ying Chen</name><affiliation><nlm:aff>NONE</nlm:aff></affiliation></author><author><name sortKey="Lei, Mei" sort="Lei, Mei" uniqKey="Lei M" first="Mei" last="Lei">Mei Lei</name><affiliation><nlm:aff>NONE</nlm:aff></affiliation></author><author><name sortKey="Liu, Huiguo" sort="Liu, Huiguo" uniqKey="Liu H" first="Huiguo" last="Liu">Huiguo Liu</name><affiliation><nlm:aff>NONE</nlm:aff></affiliation></author><author><name sortKey="Zhao, Jianping" sort="Zhao, Jianping" uniqKey="Zhao J" first="Jianping" last="Zhao">Jianping Zhao</name><affiliation><nlm:aff>NONE</nlm:aff></affiliation></author><author><name sortKey="Peng, Peng" sort="Peng, Peng" uniqKey="Peng P" first="Peng" last="Peng">Peng Peng</name><affiliation><nlm:aff>NONE</nlm:aff></affiliation></author><author><name sortKey="Wang, Cong Yi" sort="Wang, Cong Yi" uniqKey="Wang C" first="Cong-Yi" last="Wang">Cong-Yi Wang</name><affiliation><nlm:aff>NONE</nlm:aff></affiliation></author><author><name sortKey="Du, Ronghui" sort="Du, Ronghui" uniqKey="Du R" first="Ronghui" last="Du">Ronghui Du</name><affiliation><nlm:aff>NONE</nlm:aff></affiliation></author></analytic><series><title level="j">Annals of Internal Medicine</title><idno type="ISSN">0003-4819</idno><idno type="eISSN">1539-3704</idno><imprint><date when="2020">2020</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><back><div1 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<front><journal-meta><journal-id journal-id-type="nlm-ta">Ann Intern Med</journal-id><journal-id journal-id-type="iso-abbrev">Ann. Intern. Med</journal-id><journal-id journal-id-type="publisher-id">aim</journal-id><journal-title-group><journal-title>Annals of Internal Medicine</journal-title></journal-title-group><issn pub-type="ppub">0003-4819</issn><issn pub-type="epub">1539-3704</issn><publisher><publisher-name>American College of Physicians</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">32163542</article-id><article-id pub-id-type="pmc">7081173</article-id><article-id pub-id-type="doi">10.7326/M20-0533</article-id><article-id pub-id-type="publisher-id">aim-olf-M200533</article-id><article-categories><subj-group subj-group-type="heading"><subject>Letters: Observations</subject></subj-group></article-categories><title-group><article-title>Histopathologic Changes and SARS–CoV-2 Immunostaining in the Lung of a Patient With COVID-19</article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Zhang</surname><given-names>Huilan</given-names></name><degrees>PhD*</degrees><xref ref-type="aff" rid="aff1"></xref></contrib><contrib contrib-type="author"><name><surname>Zhou</surname><given-names>Peng</given-names></name><degrees>PhD*</degrees><xref ref-type="aff" rid="aff2"></xref></contrib><contrib contrib-type="author"><name><surname>Wei</surname><given-names>Yanqiu</given-names></name><degrees>MD*</degrees><xref ref-type="aff" rid="aff1"></xref></contrib><contrib contrib-type="author"><name><surname>Yue</surname><given-names>Huihui</given-names></name><degrees>MD*</degrees><xref ref-type="aff" rid="aff1"></xref></contrib><contrib contrib-type="author"><name><surname>Wang</surname><given-names>Yi</given-names></name><degrees>PhD*</degrees><xref ref-type="aff" rid="aff1"></xref></contrib><contrib contrib-type="author"><name><surname>Hu</surname><given-names>Ming</given-names></name><degrees>MD*</degrees><xref ref-type="aff" rid="aff3"></xref></contrib><contrib contrib-type="author"><name><surname>Zhang</surname><given-names>Shu</given-names></name><degrees>PhD</degrees><xref ref-type="aff" rid="aff1"></xref></contrib><contrib contrib-type="author"><name><surname>Cao</surname><given-names>Tanze</given-names></name><degrees>MD</degrees><xref ref-type="aff" rid="aff4"></xref></contrib><contrib contrib-type="author"><name><surname>Yang</surname><given-names>Chengqing</given-names></name><degrees>MD</degrees><xref ref-type="aff" rid="aff3"></xref></contrib><contrib contrib-type="author"><name><surname>Li</surname><given-names>Ming</given-names></name><degrees>MD</degrees><xref ref-type="aff" rid="aff3"></xref></contrib><contrib contrib-type="author"><name><surname>Guo</surname><given-names>Guangyun</given-names></name><degrees>MD</degrees><xref ref-type="aff" rid="aff3"></xref></contrib><contrib contrib-type="author"><name><surname>Chen</surname><given-names>Xianxiang</given-names></name><degrees>MD</degrees><xref ref-type="aff" rid="aff3"></xref></contrib><contrib contrib-type="author"><name><surname>Chen</surname><given-names>Ying</given-names></name><degrees>MD</degrees><xref ref-type="aff" rid="aff2"></xref></contrib><contrib contrib-type="author"><name><surname>Lei</surname><given-names>Mei</given-names></name><degrees>MD</degrees><xref ref-type="aff" rid="aff5"></xref></contrib><contrib contrib-type="author"><name><surname>Liu</surname><given-names>Huiguo</given-names></name><degrees>PhD</degrees><xref ref-type="aff" rid="aff1"></xref></contrib><contrib contrib-type="author"><name><surname>Zhao</surname><given-names>Jianping</given-names></name><degrees>PhD</degrees><xref ref-type="aff" rid="aff1"></xref></contrib><contrib contrib-type="author"><name><surname>Peng</surname><given-names>Peng</given-names></name><degrees>MD</degrees><xref ref-type="aff" rid="aff3"></xref></contrib><contrib contrib-type="author"><name><surname>Wang</surname><given-names>Cong-Yi</given-names></name><degrees>PhD</degrees><xref ref-type="aff" rid="aff1"></xref></contrib><contrib contrib-type="author"><name><surname>Du</surname><given-names>Ronghui</given-names></name><degrees>MD</degrees><xref ref-type="aff" rid="aff3"></xref></contrib><aff id="aff1"><label>1</label>Center for Biomedical Research, NHC Key Laboratory of Respiratory Disease, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China (H.Z., Y.W., H.Y., Y.W., S.Z., H.L., J.Z., C.W.)</aff><aff id="aff2"><label>2</label>CAS Key Laboratory of Special Pathogens, Wuhan Institute of Virology, Center for Biosafety Mega-Science, Chinese Academy of Sciences, Wuhan, China (P.Z., Y.C.)</aff><aff id="aff3"><label>3</label>Wuhan Pulmonary Hospital, Wuhan, China (M.H., C.Y., M.L., G.G., X.C., P.P., R.D.)</aff><aff id="aff4"><label>4</label>Pulmonary Hospital, Wuhan, China (T.C.)</aff><aff id="aff5"><label>5</label>Institution of Tuberculosis for Prevention and Cure, Wuhan Pulmonary Hospital, Wuhan, China (M.L.)</aff></contrib-group><pub-date pub-type="epub"><day>12</day><month>3</month><year>2020</year></pub-date><elocation-id>M20-0533</elocation-id><permissions><copyright-year>2020</copyright-year><copyright-holder>American College of Physicians</copyright-holder><license license-type="free"><license-p>This article is made available via the PMC Open Access Subset for unrestricted re-use for research, analyses, and text and data mining through PubMed Central. Acknowledgement of the original source shall include a notice similar to the following: “© 2020 American College of Physicians. Some rights reserved. This work permits non-commercial use, distribution, and reproduction in any medium, provided the original author and source are credited.” 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></article-meta></front><body><p><italic>Background:</italic> Although many studies have demonstrated the epidemiologic characteristics of SARS–CoV-2 disease (COVID-19), details of pathologic changes in the lung are still lacking.</p><p><italic>Objective:</italic> To describe the histopathologic changes in the lung of a patient with COVID-19.</p><p><italic>Case Report:</italic> A 72-year-old man with a history of diabetes and hypertension presented with fever and cough. His throat and pharyngeal swabs were positive for SARS–CoV-2 by day 6 after the initial symptoms. Rapidly progressive respiratory failure required endotracheal intubation and mechanical ventilation 1 week after presentation.</p><p>Lung tissue was obtained by transthoracic 14-gauge needle biopsy from the left upper anterior segment (<xref ref-type="fig" rid="f1-0533">Figure 1</xref>, <italic>A, arrow</italic>), left upper lingular segment (<xref ref-type="fig" rid="f1-0533">Figure 1</xref>, <italic>B, arrow</italic>), and left lower lobe (<xref ref-type="fig" rid="f1-0533">Figure 1</xref>, <italic>C, arrow</italic>), coinciding with ground-glass opacities on chest computed tomography (CT). Two throat swab samples were collected from the tonsils and posterior pharyngeal wall.</p><fig id="f1-0533" orientation="portrait" position="float"><label>Figure 1.</label><caption><p><bold>Computed tomographic images obtained from the patient 3 weeks after initial clinical manifestations of COVID-19 and 2 weeks before transthoracic biopsy, demonstrating ground glass–like opacifications.</bold></p><p>Pleural thickening and enlarged mediastinal lymph nodes were present. Arrows indicate the approximate locations of the subsequently obtained postmortem transthoracic needle biopsy samples. <bold>A.</bold> Left upper anterior segment. <bold>B.</bold> Left upper lingular segment. <bold>C.</bold> Left lower lobe.</p></caption><graphic xlink:href="aim-olf-M200533-M200533ff1"></graphic></fig><p>Biopsy lung sections were analyzed with hematoxylin–eosin staining, and immunostaining for SARS–CoV-2 was conducted as reported elsewhere (<xref rid="r1-0533" ref-type="bibr">1</xref>). Throat swabs were assessed for SARS–CoV-2 by using real-time reverse transcriptase polymerase chain reaction assays (<xref rid="r2-0533" ref-type="bibr">2</xref>).</p><p>The CT scans revealed patchy bilateral ground glass–like opacifications (<xref ref-type="fig" rid="f1-0533">Figure 1</xref><bold>A-C</bold>, <italic>arrows</italic>). Despite antiviral therapies, respiratory and hemodynamic instability continued and the patient died 3 weeks after diagnosis. Permission for postmortem transthoracic needle biopsy, but not autopsy, was obtained from the patient's family.</p><p>Histopathologic examination of lung biopsy tissues revealed diffuse alveolar damage, organizing phase. Denuded alveolar lining cells (<xref ref-type="fig" rid="f2-0533">Figure 2</xref>, <italic>A-1, arrow 1</italic>), with reactive type II pneumocyte hyperplasia, were noted (<xref ref-type="fig" rid="f2-0533">Figure 2</xref>, <italic>A-1, arrow 2</italic>). Intra-alveolar fibrinous exudates were present (<xref ref-type="fig" rid="f2-0533">Figure 2</xref>, <italic>A-2, arrow 3</italic>), along with loose interstitial fibrosis and chronic inflammatory infiltrates (<xref ref-type="fig" rid="f2-0533">Figure 2</xref>, <italic>A-2, arrow 4</italic>). Intra-alveolar loose fibrous plugs of organizing pneumonia were noted (<xref ref-type="fig" rid="f2-0533">Figure 2</xref>, <italic>A-3, arrow 5</italic>), with presence of intra-alveolar organizing fibrin seen in most foci (<xref ref-type="fig" rid="f2-0533">Figure 2</xref>, <italic>A-4, arrow 6</italic>).</p><fig id="f2-0533" orientation="portrait" position="float"><label>Figure 2.</label><caption><p><bold>Histopathologic examination of lung biopsy tissues and immunostaining from a patient who died of COVID-19 (×100 magnification).</bold></p><p><bold>A.</bold> Histopathologic examination revealing diffuse alveolar damage, organizing phase <bold>(A-1)</bold>; denudation of alveolar lining cells (<italic>arrow 1</italic>), with presence of reactive type II pneumocyte hyperplasia (<italic>arrow 2</italic>) <bold>(A-2)</bold>; intra-alveolar fibrinous exudates (<italic>arrow 3</italic>) and interstitial loose fibrosis with chronic inflammatory infiltrates (<italic>arrow 4</italic>) <bold>(A-3)</bold>; and intra-alveolar loose fibrous plugs (<italic>arrow 5</italic>) <bold>(A-4)</bold>. In most foci, intra-alveolar organizing fibrin is seen (<italic>arrow 6</italic>). <bold>B.</bold> Immunostaining of SARS–CoV-2 in lung sections. Images were taken under light and fluorescent conditions, respectively (f×100 magnification). Merged images were also generated. Blue arrows indicate interstitial areas between the alveoli, and green arrows indicate injured epithelial cells desquamated into the alveolar
spaces. The dashed black lines indicate the blood vessel. Immunostaining of SARS–CoV-2 was done by using a rabbit polyclonal antibody (made in house, 1:100) against the Rp3 NP protein, which is highly conserved between SARS-CoV and SARS–CoV-2, followed by probing with a Cy3-conjugated goat antirabbit IgG (1:50, Abcam, ab6939). <bold>C.</bold> Positive and negative controls for immunostaining. For the positive control, the Huh7 cells were infected with SARS–CoV-2 at multiplicity of infection of 0.5 for 48 hours. After extensive washes, the cells were then fixed with 2.5% (wt/vol) glutaraldehyde. The infected cells were stained in red, and nuclei were stained with DAPI (Beyotime, Wuhan, China) in blue. For the negative control, biopsy lung sections derived from a patient with HIV who died of fungal infection were stained in parallel with lung sections from the patient with COVID-19 as above.</p></caption><graphic xlink:href="aim-olf-M200533-M200533ff2_2"></graphic></fig><p>Immunostaining of lung sections with an antibody to the Rp3 NP protein of SARS–CoV-2 revealed prominent expression on alveolar epithelial cells (<xref ref-type="fig" rid="f2-0533">Figure 2</xref>, <italic>B, top panel</italic>), including damaged, desquamated cells within the alveolar space (<xref ref-type="fig" rid="f2-0533">Figure 2</xref>, <italic>B, bottom panel, green arrows</italic>). In contrast, viral protein expression was minimally detectable on blood vessels (<xref ref-type="fig" rid="f2-0533">Figure 2</xref>, <italic>B, dashed black line</italic>) or in the interstitial areas between alveoli (<xref ref-type="fig" rid="f2-0533">Figure 2</xref>, <italic>B, bottom panel, blue arrows</italic>). Immunostaining of Huh7 cells infected with SARS–CoV and of lung sections from an HIV-positive patient who died of fungal infection served as positive and negative staining controls, respectively (<xref ref-type="fig" rid="f2-0533">Figure 2</xref>,
<italic>C</italic>).</p><p><italic>Discussion:</italic> The histopathologic changes seen on postmortem transthoracic needle biopsies from a patient with COVID-19 who had respiratory failure and radiographic bilateral ground-glass opacities are consistent with diffuse alveolar damage. Although such nonspecific findings may be seen in response to several conditions that result in respiratory failure, its demonstration in the setting of COVID-19 helps to inform the clinical course of disease.</p><p>Our study is limited by our inability to obtain larger tissue specimens. The present findings warrant further study with larger tissue samples, obtained by open or thoracoscopic lung biopsy, or autopsy, for example.</p><sec><title></title></sec></body><back><fn-group><fn id="n0-M200533"><p>This article was published at Annals.org on 12 March 2020.</p></fn></fn-group><bio id="d35e213"><p><bold>Note:</bold> Authors indicated with an asterisk (Drs. H. Zhang, P. Zhou, Y. Wei, H. Yue, Y. Wang, and M. Hu) contributed equally to this article.</p><p><bold>Disclosures:</bold> Authors have disclosed no conflicts of interest. Forms can be viewed at <uri xlink:type="simple" xlink:href="http://www.acponline.org/authors/icmje/ConflictOfInterestForms.do?msNum=M20-0533">www.acponline.org/authors/icmje/ConflictOfInterestForms.do?msNum=M20-0533</uri>.</p><p><bold>Financial Support:</bold> By the National Natural Science Foundation of China (grants 81974456 and 91749207); the Clinical Research Physician Program of Tongji Medical College, Huazhong University of Science and Technology (grant 5001540075); and the SARS-CoV-2 Pneumonia Emergency Technology Public Relations Project (grants 2020FCA009 and 2020FCA026).</p></bio><ref-list><title>References</title><ref id="r1-0533"><label> 1.</label><mixed-citation publication-type="unrecognized"><comment>Zhou P, Yang XL, Wang XG, et al. A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature. 2020. [PMID: 32015507] doi:10.1038/s41586-020-2012-7</comment></mixed-citation></ref><ref id="r2-0533"><label> 2.</label><mixed-citation publication-type="unrecognized"><comment>Corman VM, Landt O, Kaiser M, et al. Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR. Euro Surveill. 2020;25. [PMID: 31992387] doi:10.2807/1560-7917.ES.2020.25.3.2000045</comment></mixed-citation></ref></ref-list></back></pmc></record>Pour manipuler ce document sous Unix (Dilib)
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