Organ distribution of severe acute respiratory syndrome (SARS) associated coronavirus (SARS‐CoV) in SARS patients: implications for pathogenesis and virus transmission pathways
Identifieur interne : 001208 ( Pmc/Corpus ); précédent : 001207; suivant : 001209Organ distribution of severe acute respiratory syndrome (SARS) associated coronavirus (SARS‐CoV) in SARS patients: implications for pathogenesis and virus transmission pathways
Auteurs : Yanqing Ding ; Li He ; Qingling Zhang ; Zhongxi Huang ; Xiaoyan Che ; Jinlin Hou ; Huijun Wang ; Hong Shen ; Liwen Qiu ; Zhuguo Li ; Jian Geng ; Junjie Cai ; Huixia Han ; Xin Li ; Wei Kang ; Desheng Weng ; Ping Liang ; Shibo JiangSource :
- The Journal of Pathology [ 0022-3417 ] ; 2004.
Abstract
We previously identified the major pathological changes in the respiratory and immune systems of patients who died of severe acute respiratory syndrome (SARS) but gained little information on the organ distribution of SARS‐associated coronavirus (SARS‐CoV). In the present study, we used a murine monoclonal antibody specific for SARS‐CoV nucleoprotein, and probes specific for a SARS‐CoV RNA polymerase gene fragment, for immunohistochemistry and
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DOI: 10.1002/path.1560
PubMed: NONE
PubMed Central: 7167761
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PMC:7167761Le document en format XML
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<author><name sortKey="Jiang, Shibo" sort="Jiang, Shibo" uniqKey="Jiang S" first="Shibo" last="Jiang">Shibo Jiang</name>
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<series><title level="j">The Journal of Pathology</title>
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<front><div type="abstract" xml:lang="en"><title>Abstract</title>
<p>We previously identified the major pathological changes in the respiratory and immune systems of patients who died of severe acute respiratory syndrome (SARS) but gained little information on the organ distribution of SARS‐associated coronavirus (SARS‐CoV). In the present study, we used a murine monoclonal antibody specific for SARS‐CoV nucleoprotein, and probes specific for a SARS‐CoV RNA polymerase gene fragment, for immunohistochemistry and <italic>in situ</italic>
hybridization, respectively, to detect SARS‐CoV systematically in tissues from patients who died of SARS. SARS‐CoV was found in lung, trachea/bronchus, stomach, small intestine, distal convoluted renal tubule, sweat gland, parathyroid, pituitary, pancreas, adrenal gland, liver and cerebrum, but was not detected in oesophagus, spleen, lymph node, bone marrow, heart, aorta, cerebellum, thyroid, testis, ovary, uterus or muscle. These results suggest that, in addition to the respiratory system, the gastrointestinal tract and other organs with detectable SARS‐CoV may also be targets of SARS‐CoV infection. The pathological changes in these organs may be caused directly by the cytopathic effect mediated by local replication of the SARS‐CoV; or indirectly as a result of systemic responses to respiratory failure or the harmful immune response induced by viral infection. In addition to viral spread through a respiratory route, SARS‐CoV in the intestinal tract, kidney and sweat glands may be excreted via faeces, urine and sweat, thereby leading to virus transmission. This study provides important information for understanding the pathogenesis of SARS‐CoV infection and sheds light on possible virus transmission pathways. This data will be useful for designing new strategies for prevention and treatment of SARS. Copyright © 2004 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.</p>
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<front><journal-meta><journal-id journal-id-type="nlm-ta">J Pathol</journal-id>
<journal-id journal-id-type="iso-abbrev">J. Pathol</journal-id>
<journal-id journal-id-type="doi">10.1002/(ISSN)1096-9896</journal-id>
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<issn pub-type="ppub">0022-3417</issn>
<issn pub-type="epub">1096-9896</issn>
<publisher><publisher-name>John Wiley & Sons, Ltd.</publisher-name>
<publisher-loc>Chichester, UK</publisher-loc>
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<article-meta><article-id pub-id-type="pmc">7167761</article-id>
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<article-id pub-id-type="publisher-id">PATH1560</article-id>
<article-categories><subj-group subj-group-type="overline"><subject>Rapid Communication</subject>
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<title-group><article-title>Organ distribution of severe acute respiratory syndrome (SARS) associated coronavirus (SARS‐CoV) in SARS patients: implications for pathogenesis and virus transmission pathways</article-title>
<alt-title alt-title-type="right-running-head">Organ distribution of SARS‐CoV</alt-title>
</title-group>
<contrib-group><contrib id="au1" contrib-type="author" corresp="yes"><name><surname>Ding</surname>
<given-names>Yanqing</given-names>
</name>
<xref ref-type="aff" rid="af1"><sup>1</sup>
</xref>
<address><email>dyq@fimmu.com</email>
</address>
</contrib>
<contrib id="au2" contrib-type="author"><name><surname>He</surname>
<given-names>Li</given-names>
</name>
<xref ref-type="aff" rid="af1"><sup>1</sup>
</xref>
</contrib>
<contrib id="au3" contrib-type="author"><name><surname>Zhang</surname>
<given-names>Qingling</given-names>
</name>
<xref ref-type="aff" rid="af1"><sup>1</sup>
</xref>
</contrib>
<contrib id="au4" contrib-type="author"><name><surname>Huang</surname>
<given-names>Zhongxi</given-names>
</name>
<xref ref-type="aff" rid="af1"><sup>1</sup>
</xref>
</contrib>
<contrib id="au5" contrib-type="author"><name><surname>Che</surname>
<given-names>Xiaoyan</given-names>
</name>
<xref ref-type="aff" rid="af2"><sup>2</sup>
</xref>
</contrib>
<contrib id="au6" contrib-type="author"><name><surname>Hou</surname>
<given-names>Jinlin</given-names>
</name>
<xref ref-type="aff" rid="af3"><sup>3</sup>
</xref>
</contrib>
<contrib id="au7" contrib-type="author"><name><surname>Wang</surname>
<given-names>Huijun</given-names>
</name>
<xref ref-type="aff" rid="af1"><sup>1</sup>
</xref>
</contrib>
<contrib id="au8" contrib-type="author"><name><surname>Shen</surname>
<given-names>Hong</given-names>
</name>
<xref ref-type="aff" rid="af1"><sup>1</sup>
</xref>
</contrib>
<contrib id="au9" contrib-type="author"><name><surname>Qiu</surname>
<given-names>Liwen</given-names>
</name>
<xref ref-type="aff" rid="af3"><sup>3</sup>
</xref>
</contrib>
<contrib id="au10" contrib-type="author"><name><surname>Li</surname>
<given-names>Zhuguo</given-names>
</name>
<xref ref-type="aff" rid="af1"><sup>1</sup>
</xref>
</contrib>
<contrib id="au11" contrib-type="author"><name><surname>Geng</surname>
<given-names>Jian</given-names>
</name>
<xref ref-type="aff" rid="af1"><sup>1</sup>
</xref>
</contrib>
<contrib id="au12" contrib-type="author"><name><surname>Cai</surname>
<given-names>Junjie</given-names>
</name>
<xref ref-type="aff" rid="af1"><sup>1</sup>
</xref>
</contrib>
<contrib id="au13" contrib-type="author"><name><surname>Han</surname>
<given-names>Huixia</given-names>
</name>
<xref ref-type="aff" rid="af1"><sup>1</sup>
</xref>
</contrib>
<contrib id="au14" contrib-type="author"><name><surname>Li</surname>
<given-names>Xin</given-names>
</name>
<xref ref-type="aff" rid="af1"><sup>1</sup>
</xref>
</contrib>
<contrib id="au15" contrib-type="author"><name><surname>Kang</surname>
<given-names>Wei</given-names>
</name>
<xref ref-type="aff" rid="af1"><sup>1</sup>
</xref>
</contrib>
<contrib id="au16" contrib-type="author"><name><surname>Weng</surname>
<given-names>Desheng</given-names>
</name>
<xref ref-type="aff" rid="af1"><sup>1</sup>
</xref>
</contrib>
<contrib id="au17" contrib-type="author"><name><surname>Liang</surname>
<given-names>Ping</given-names>
</name>
<xref ref-type="aff" rid="af1"><sup>1</sup>
</xref>
</contrib>
<contrib id="au18" contrib-type="author" corresp="yes"><name><surname>Jiang</surname>
<given-names>Shibo</given-names>
</name>
<xref ref-type="aff" rid="af4"><sup>4</sup>
</xref>
<address><email>sjiang@nybloodcenter.org</email>
</address>
</contrib>
</contrib-group>
<aff id="af1"><label><sup>1</sup>
</label>
Department of Pathology, Nan Fang Hospital, First Military Medical University, Guangzhou, China</aff>
<aff id="af2"><label><sup>2</sup>
</label>
Department of Infectious Diseases, Nan Fang Hospital, First Military Medical University, Guangzhou, China</aff>
<aff id="af3"><label><sup>3</sup>
</label>
Centre Laboratory, Zhujiang Hospital, First Military Medical University, Guangzhou, China</aff>
<aff id="af4"><label><sup>4</sup>
</label>
Laboratory of Viral Immunology, Lindsley F. Kimball Research Institute, New York Blood Center, New York, USA</aff>
<author-notes><corresp id="correspondenceTo"><label>*</label>
Yanqing Ding, Department of Pathology, First Military Medical University, Guangzhou 5101515, China.<break></break>
Shibo Jiang, Laboratory of Viral Immunology, Lindsley F. Kimball Research Institute, New York Blood Center, 310 East 67th Street, New York, NY 10021, USA.<break></break>
</corresp>
</author-notes>
<pub-date pub-type="epub"><day>07</day>
<month>5</month>
<year>2004</year>
</pub-date>
<pub-date pub-type="ppub"><month>6</month>
<year>2004</year>
</pub-date>
<volume>203</volume>
<issue>2</issue>
<issue-id pub-id-type="doi">10.1002/path.v203:2</issue-id>
<fpage>622</fpage>
<lpage>630</lpage>
<history><date date-type="received"><day>14</day>
<month>1</month>
<year>2004</year>
</date>
<date date-type="rev-recd"><day>08</day>
<month>2</month>
<year>2004</year>
</date>
<date date-type="accepted"><day>12</day>
<month>2</month>
<year>2004</year>
</date>
</history>
<permissions><copyright-statement content-type="article-copyright">Copyright © 2004 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.</copyright-statement>
<license><license-p>This article is being made freely available through PubMed Central as part of the COVID-19 public health emergency response. It can be used for unrestricted research re-use and analysis in any form or by any means with acknowledgement of the original source, for the duration of the public health emergency.</license-p>
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<abstract><title>Abstract</title>
<p>We previously identified the major pathological changes in the respiratory and immune systems of patients who died of severe acute respiratory syndrome (SARS) but gained little information on the organ distribution of SARS‐associated coronavirus (SARS‐CoV). In the present study, we used a murine monoclonal antibody specific for SARS‐CoV nucleoprotein, and probes specific for a SARS‐CoV RNA polymerase gene fragment, for immunohistochemistry and <italic>in situ</italic>
hybridization, respectively, to detect SARS‐CoV systematically in tissues from patients who died of SARS. SARS‐CoV was found in lung, trachea/bronchus, stomach, small intestine, distal convoluted renal tubule, sweat gland, parathyroid, pituitary, pancreas, adrenal gland, liver and cerebrum, but was not detected in oesophagus, spleen, lymph node, bone marrow, heart, aorta, cerebellum, thyroid, testis, ovary, uterus or muscle. These results suggest that, in addition to the respiratory system, the gastrointestinal tract and other organs with detectable SARS‐CoV may also be targets of SARS‐CoV infection. The pathological changes in these organs may be caused directly by the cytopathic effect mediated by local replication of the SARS‐CoV; or indirectly as a result of systemic responses to respiratory failure or the harmful immune response induced by viral infection. In addition to viral spread through a respiratory route, SARS‐CoV in the intestinal tract, kidney and sweat glands may be excreted via faeces, urine and sweat, thereby leading to virus transmission. This study provides important information for understanding the pathogenesis of SARS‐CoV infection and sheds light on possible virus transmission pathways. This data will be useful for designing new strategies for prevention and treatment of SARS. Copyright © 2004 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.</p>
</abstract>
<kwd-group kwd-group-type="author-generated"><kwd id="kwd1">SARS</kwd>
<kwd id="kwd2">SARS‐CoV</kwd>
<kwd id="kwd3">distribution</kwd>
<kwd id="kwd4">detection</kwd>
<kwd id="kwd5">pathogenesis</kwd>
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<funding-group><award-group id="funding-0001"><funding-source>Chinese National Foundation of Natural Sciences</funding-source>
<award-id>30340015</award-id>
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<award-group id="funding-0002"><funding-source>Military Foundation of Medical Science</funding-source>
<award-id>03F016‐2</award-id>
</award-group>
<award-group id="funding-0003"><funding-source>Foundation of Natural Sciences of Guangdong Province</funding-source>
<award-id>GD2003‐80</award-id>
</award-group>
</funding-group>
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<body><sec id="sec1-1"><title>Introduction</title>
<p>The global outbreak of severe acute respiratory syndrome (SARS) in 2003 resulted in more than 8000 cases and 774 deaths <xref rid="bib1" ref-type="ref">1</xref>
. It has been demonstrated that SARS‐associated coronavirus (SARS‐CoV) is the primary causative pathogen of SARS <xref rid="bib2" ref-type="ref">2</xref>
, <xref rid="bib3" ref-type="ref">3</xref>
, <xref rid="bib4" ref-type="ref">4</xref>
, <xref rid="bib5" ref-type="ref">5</xref>
, <xref rid="bib6" ref-type="ref">6</xref>
and may originate from some wild animals, such as Himalayan palm civets <xref rid="bib7" ref-type="ref">7</xref>
. Farzan's group has shown that angiotensin‐converting enzyme 2 (ACE2) is a functional receptor for SARS‐CoV <xref rid="bib8" ref-type="ref">8</xref>
. The ACE2 binding site is located in the S1 domain of SARS‐CoV spike (S) protein <xref rid="bib9" ref-type="ref">9</xref>
, <xref rid="bib10" ref-type="ref">10</xref>
, <xref rid="bib11" ref-type="ref">11</xref>
. Jiang and colleagues have demonstrated that the heptad repeat 1 and 2 regions in the S2 domain of S protein associate to form a fusion‐active oligomeric core structure and a peptide derived from the HR2 region inhibits SARS‐CoV infection <xref rid="bib12" ref-type="ref">12</xref>
.</p>
<p>The entire genome of SARS‐CoV has been sequenced and it encodes RNA polymerase, spike (S) glycoprotein, membrane (M) glycoprotein, envelope (E) glycoprotein and nucleocapsid (N) protein <xref rid="bib2" ref-type="ref">2</xref>
, <xref rid="bib3" ref-type="ref">3</xref>
, <xref rid="bib4" ref-type="ref">4</xref>
, <xref rid="bib5" ref-type="ref">5</xref>
, <xref rid="bib6" ref-type="ref">6</xref>
, <xref rid="bib13" ref-type="ref">13</xref>
. The N protein in most coronaviruses is highly conserved, immunogenic and abundantly expressed during infection <xref rid="bib14" ref-type="ref">14</xref>
. Therefore, it has been widely used as an important viral antigen in immunological and immunohistochemical assays for clinical diagnosis and laboratory detection of coronavirus infection.</p>
<p>It has been confirmed that SARS epidemics in 2003 started in the Guangdong Province of China <xref rid="bib15" ref-type="ref">15</xref>
. Recently, new SARS cases have been identified in this region <xref rid="bib16" ref-type="ref">16</xref>
. As medical research scientists working in this endemic area, we were the first systematically to analyse the autopsy tissues from patients who died of SARS in Nan Fang Hospital, Guangzhou, Guangdong and identified the major pathological changes <xref rid="bib17" ref-type="ref">17</xref>
. However, we obtained limited data on the organ and tissue distribution of the SARS‐CoV. Little information about the pathogenesis of this disease is available. This has significantly impeded the progress for designing more effective strategies for the prevention and treatment of SARS.</p>
<p>In the present study, we used immunohistochemistry and <italic>in situ</italic>
hybridization to detect SARS‐CoV in tissues from patients who died of SARS and gained insights into the pathogenesis and potential new transmission routes of SARS‐CoV.</p>
</sec>
<sec id="sec1-2"><title>Materials and methods</title>
<sec id="sec2-1"><title>Materials</title>
<p>Autopsy samples of lung, lymph node, spleen, heart, liver, kidney, adrenal gland, parathyroid gland, pituitary, bone marrow, skin, oesophagus, gastric fundus, small intestine, pancreas, thyroid, aorta, cerebrum, cerebellum, testis, ovary, uterus and striated muscle were obtained from four patients who died of SARS and four control patients who died of other diseases without SARS‐CoV infection in the Eighth People's Hospital of Guangzhou City, the Second Affiliated Hospital of Zhongshan University, and the Guangzhou Institute of Respiratory Diseases, with documented permissions from patients' family members and the Health Administration of Guangdong Province, China. The ethical issues related to this study were reviewed and approved by the Research Administration Committees of the First Military Medical University and the local hospitals.</p>
<p>All four patients (three males aged 25, 38 and 57 years, and one 62‐year‐old female) met the diagnostic criteria for SARS defined by WHO <xref rid="bib15" ref-type="ref">15</xref>
. All the patients were treated with ribavirin (an antiviral drug), and levofloxacin and doxycycline (antibacterial drugs). Only one was treated with steroids. The detailed clinical data and pathological findings have been reported previously <xref rid="bib17" ref-type="ref">17</xref>
, <xref rid="bib18" ref-type="ref">18</xref>
. These four SARS patients died on days 9, 10, 20 and 34, respectively, after onset of the disease. The four control patients were two males (32 and 52 years old) who suddenly died of dissecting aneurysm of the thoracic aorta and coronary heart disease, respectively, and two females (28 and 68 years old) who died as a result of ectopic pregnancy and cerebral haemorrhage, respectively.</p>
</sec>
<sec id="sec2-2"><title>Immunohistochemistry (IHC)</title>
<p>IHC was performed as described by Lin <italic>et al</italic>
<xref rid="bib14" ref-type="ref">14</xref>
using a mouse monoclonal antibody (mAb) directed against a recombinant SARS‐CoV N protein <xref rid="bib19" ref-type="ref">19</xref>
. Briefly, the autopsy tissue samples were fixed in 4% neutral formaldehyde, embedded in paraffin wax, sectioned at 4 µm, and incubated with 3% hydrogen peroxide to quench endogenous peroxidase activity. Microwave heat‐induced antigen retrieval in citrate buffer (pH 6.0) was required for optimal staining <xref rid="bib20" ref-type="ref">20</xref>
. Slides were incubated with the anti‐N mAb (culture supernatants at 1 : 20 dilution) in a humid chamber at 4°C overnight. After rinsing in PBS, the sections were incubated with goat anti‐mouse IgG labelled with horseradish peroxidase (HRP) (DAKO, Carpinteria, CA) at 37°C for 60 min. The slides were washed with PBS and counterstained with haematoxylin for 30 s. Antibody binding to the cells in tissues was detected with the HRP reaction kit (DAKO). The sections were then dehydrated, mounted with the Permount kit (Fisher Scientific, Pittsburgh, PA) and examined under a light microscope. SARS‐CoV infected and uninfected Vero E6 cells were used as positive and negative controls, respectively.</p>
</sec>
<sec id="sec2-3"><title>Reverse‐transcriptase polymerase chain reaction (RT‐PCR)</title>
<p>RT‐PCR for detection of the SARS‐CoV M protein gene and RNA polymerase gene fragments was established in our laboratories as previously described <xref rid="bib21" ref-type="ref">21</xref>
. Briefly, RNA was isolated from lung tissue using TRIZOL reagents (Invitrogen, Gaithersburg, MD) following the manufacturer's instructions. After this procedure, RNA was dissolved in 40 µl RNase‐free water. RT‐PCR was performed with the SuperScript One‐Step RT‐PCR kit (Invitrogen) and SARS‐CoV gene‐specific primers <xref rid="bib3" ref-type="ref">3</xref>
, ie, BNIoutS: 5′‐ATG AAT TAC CAA GTC AAT GGT TAC‐3′ and BNIoutAS: 5′‐CAT AAC CAG TCG GTA CAG CTA C‐3′ (RNA polymerase gene fragment of the SARS‐CoV strain BJ01, nt 18 134–18 323, 190 bp), and MS2: 5′‐ATG GCA GAC AAC GGT ACT ATT AC‐3′ and MA2: 5′‐CTT ACT GTA CTA GCA AAG C‐3′ (M protein gene of the SARS‐CoV strain BJ01, nt 26 379–27 045, 667 bp). RNA (1 µl) was subjected to RT‐PCR in the GeneAmp PCR System 9700 (Applied Biosystems, Framingham, MA). Initial cycling was performed at 45°C for 30 min, and 95°C for 3 min, followed by 10 cycles at 94°C for 15 s, 65–56°C (drop 1°C every cycle) for 15 s, and 72°C for 35 s. The subsequent 30 cycles were performed at 94°C for 15 s, 56°C for 15 s, and 72°C for 35 s.</p>
</sec>
<sec id="sec2-4"><title><italic>In situ</italic>
hybridization (ISH)</title>
<p>Sense and antisense digoxigenin‐labelled SARS‐CoV RNA polymerase gene‐specific oligonucleotides (described above) were prepared through 3′ end‐labelling reactions with kits from Boehringer Mannheim (Indianapolis, IN). ISH was performed on paraffin wax‐embedded tissues with the ready‐for‐use reagents purchased from Boster Biological Technology Co Ltd (Wuhan, China) following the manufacturer's instructions. Briefly, tissue sections were dewaxed in xylol and rehydrated with gradient ethanol. Endogenous peroxidase was quenched in 3% H<sub>2</sub>
O<sub>2</sub>
. The tissues were digested with 3 mg/ml pepsin in 0.14 mol/L citric acid at 37°C for 20 min and incubated at 37°C for 3 h with prehybridization buffer containing 50% deionized formamide, 5× standard saline citrate, 2% blocking reagent, 0.1% <italic>N</italic>
‐lauroylsarcosine, 0.02% sodium dodecyl sulphate and 100 ng/ml salmon sperm DNA. The tissue sections were hybridized with digoxigenin‐labelled oligonucleotide probes (5 µg/ml) at 37°C for 18 h. The slides were rinsed in a series of graded salt solutions (2×, 0.5× and 0.2× standard saline citrate). After reaction with a digoxigenin‐labelled blocking reagent at 37°C for 30 min, the sections were exposed to biotin‐labelled mouse anti‐digoxin antibody at 37°C for 60 min, and incubated in avidin–peroxidase at 37°C for 20 min. The peroxidase conjugates were subsequently localized by using the substrate diaminobenzidine tetrahydrochloride (DAB) and counterstained with Mayer's haematoxylin.</p>
</sec>
<sec id="sec2-5"><title>Pathological examination and histochemical staining</title>
<p>Specimens were fixed with 4% neutral formaldehyde and embedded in paraffin wax. Sections (4 µm) were stained with haematoxylin and eosin (H&E) and with Macchiavello stain, and examined under a light microscope.</p>
</sec>
<sec id="sec2-6"><title>Transmission electron microscopy</title>
<p>Specimens (about 2 mm × 2 mm × 2 mm) were obtained from the autopsy tissues and fixed with 2.5% glutaraldehyde in phosphoric buffer (pH 7.4), post‐fixed with 1% osmate, dehydrated with gradient alcohol, embedded in Epon 812, double‐stained with uranium acetate and lead citromalic acid, and observed under a JEM1200 transmission electron microscope (Jeol, Tokyo, Japan).</p>
</sec>
</sec>
<sec id="sec1-3"><title>Results</title>
<p>The mouse mAb against SARS‐CoV N protein was generated using a recombinant SARS‐CoV (strain BJ01) N protein as immunogen as previously described <xref rid="bib19" ref-type="ref">19</xref>
. In an enzyme‐linked immunosorbent assay (ELISA), this mAb specifically recognized the recombinant SARS‐CoV N protein with high binding affinity (<italic>K</italic>
<sub>d</sub>
= 4 × 10<sup>−9</sup>
<sc>m</sc>
), and reacted with convalescent sera from SARS patients, but was negative to the sera of healthy blood donors <xref rid="bib22" ref-type="ref">22</xref>
. Using an immunofluorescence assay, we demonstrated that this mAb bound significantly to Vero E6 cells infected by SARS‐CoV, but did not bind to uninfected Vero E6 cells, adenovirus‐infected 293 cells or rotavirus‐infected MA104 cells (Figure <xref rid="fig1" ref-type="fig">1</xref>
). The results suggest that this mAb is specific for the SARS‐CoV N protein and can be used in IHC for detection of the distribution of SARS‐CoV in organs and tissues from SARS patients.</p>
<fig fig-type="Figure" xml:lang="en" id="fig1" orientation="portrait" position="float"><label>Figure 1</label>
<caption><p>Detection of SARS‐CoV N protein by immunofluorescence. (A) Vero E6 cells infected by SARS‐CoV (×200). (B) Uninfected Vero E6 (×200); (C) 293 cells infected with adenovirus type 5 (×400). (D) MA104 cells infected by Group A rotavirus (×200). The green colour represents the positive signal for SARS‐CoV N protein, while the negative cells without SARS‐CoV infection display a red colour</p>
</caption>
<graphic id="nlm-graphic-1" xlink:href="PATH-203-622-g001"></graphic>
</fig>
<p>Primers specific for the SARS‐CoV RNA polymerase gene fragment BNI109 (SARS‐CoV strain BJ01, nt 18 134–18 323) were developed in our laboratories as previously described <xref rid="bib21" ref-type="ref">21</xref>
. Using these primers to amplify the gene fragments from eight specimens (sputum samples, throat swabs and plasma) of established SARS cases by RT‐PCR, all the resulting gene fragments possessed 100% identity to the sequence of the corresponding RNA polymerase gene fragments of SARS‐CoV strain BJ01, confirming the specificity of these primers. The specificity of <italic>in situ</italic>
hybridization was verified by disappearance or decrease of the signal after: (i) digestion of the sections with RNase A (0.2 mg/ml) (Sigma Chemical Co, St Louis, MO) for 2 h at 37°C before ISH, and (ii) competition by adding an excess of unlabelled oligonucleotide probes. These preliminary results suggested that this ISH assay can be used for the detection of SARS‐CoV in the organs and tissues of SARS patients.</p>
<p>To confirm further the specificity of the IHC and ISH assays described above, we first tested the lung tissues of four patients who died of SARS and four patients who died of other diseases using these two assays in parallel for detection of SARS‐CoV N protein and RNA polymerase gene fragment, respectively. Our previous studies demonstrated that SARS‐CoV can be detected in certain cell types, eg pneumocytes, in the lung tissues of SARS patients using different methods, including H&E and Macchiavello's stains for observing inclusion bodies, and transmission electron microscopy for revealing corona‐like virus particles <xref rid="bib17" ref-type="ref">17</xref>
, <xref rid="bib18" ref-type="ref">18</xref>
. In the present study, inclusion bodies and corona‐like virus particles were observed in the lung tissues from all of the SARS patients (Figure <xref rid="fig2" ref-type="fig">2</xref>
A–C). However, no inclusion bodies or virus particles were identified in the lung tissues of the four control patients. The presence of SARS‐CoV in the lung tissues of SARS patients was further confirmed by RT‐PCR, since the SARS‐CoV RNA polymerase gene fragment and M protein gene were identified in lung tissues from three SARS patients (Figure <xref rid="fig2" ref-type="fig">2</xref>
D; samples from the fourth patient were tested later and showed similar results, not included in this figure). However, these SARS‐CoV RNA species were not found in lung tissues from the four control patients. These results confirm the observations by other groups that the respiratory system is the primary target for SARS‐CoV <xref rid="bib2" ref-type="ref">2</xref>
, <xref rid="bib3" ref-type="ref">3</xref>
, <xref rid="bib4" ref-type="ref">4</xref>
, <xref rid="bib5" ref-type="ref">5</xref>
.</p>
<fig fig-type="Figure" xml:lang="en" id="fig2" orientation="portrait" position="float"><label>Figure 2</label>
<caption><p>Detection of SARS‐CoV in lung tissues from patients who died of SARS (A–F) and from a patient who died through rupture of a thoracic aortic aneurysm (G and H). (A) Viral inclusion body in a pneumocyte (arrow; H&E, ×400). (B) Viral inclusion body in a pneumocyte (arrow; Macchiavello's stain, ×400). (C) Clusters of corona‐like viral particles in the endoplasmic reticulum of a type II alveolar epithelial cell (transmission electron microscopy; bar = 200 nm); (D) SARS‐CoV M protein gene (a, 667bp) and RNA polymerase gene fragment (b, 190bp) detected in lung tissues from three SARS cases (1–3), but not in the negative control (N), who died through rupture of a thoracic aortic aneurysm (RT‐PCR). M = marker. (E) SARS‐CoV N protein expression in pneumocytes (IHC, ×400). (F) SARS‐CoV RNA polymerase gene expression in pneumocytes (ISH, ×400); the positive cells (brown‐yellow) are indicated by arrows. (G) SARS‐CoV N protein expression (IHC, ×200). (H) SARS‐CoV RNA polymerase gene expression (ISH, ×200) is not detectable in lung tissue from the individual who died through rupture of a thoracic aortic aneurysm</p>
</caption>
<graphic id="nlm-graphic-3" xlink:href="PATH-203-622-g002"></graphic>
</fig>
<p>Since the presence of SARS‐CoV in lung tissues from these SARS patients was confirmed by the techniques described above, we used the IHC and ISH techniques to detect the SARS‐CoV N protein and RNA polymerase gene fragment, respectively, in these lung tissues. Pneumocytes in the lung tissues of SARS patients, but not those from the four control patients, contained detectable SARS‐CoV antigen and RNA. Two representative slides showing pneumocytes positive with the mAb specific for SARS‐CoV N protein and with the probes specific for SARS‐CoV RNA polymerase gene fragment are shown in Figure <xref rid="fig2" ref-type="fig">2</xref>
E and F, respectively. In contrast, none of the cells in the lung tissues of the four control patients were positive in both assays (Figure <xref rid="fig2" ref-type="fig">2</xref>
G and H). These results suggest that both IHC and ISH developed in our laboratories are specific and sensitive for the detection of SARS‐CoV in organs and tissues.</p>
<p>Using these two validated assays, we examined other organs and tissues from the patients who died of SARS and the controls. If positive cells were observed, the number of positive cells and the total number of cells in 10 high‐power views were counted under a microscope and scored as described in the footnotes to Table <xref rid="tbl1" ref-type="table">1</xref>
. As shown in Figure <xref rid="fig3" ref-type="fig">3</xref>
, SARS‐CoV antigen and RNA were found in tracheal/bronchial serous gland epithelium, gastric parietal cells, small intestinal epithelium, acidophilic cells in parathyroid and pituitary, pancreatic acinar cells, adrenal cortical and medullary cells, sweat gland cells, epithelial cells of distal convoluted renal tubules, cerebral neurones and hepatocytes near the central vein (Table <xref rid="tbl1" ref-type="table">1</xref>
, Figure <xref rid="fig3" ref-type="fig">3</xref>
, 1–11A and B). However, SARS‐CoV N protein and RNA polymerase gene fragments were not detectable in heart (Figure <xref rid="fig3" ref-type="fig">3</xref>
, 12A and B), seminiferous tubule of testis (Figure <xref rid="fig3" ref-type="fig">3</xref>
, 13A and B), thyroid, oesophagus, spleen, lymph node, bone marrow, ovary, uterus, aorta, cerebellum and muscle (Table <xref rid="tbl1" ref-type="table">1</xref>
); and in corresponding tissues of control patients, namely glandular epithelium of the gastric corpus, mucosal and glandular small intestinal epithelia, pancreatic acini, bronchial mucous glandular epithelia, cerebral cortex, pituitary, liver, adrenal gland and cutaneous sweat glands (Figure <xref rid="fig3" ref-type="fig">3</xref>
, 1–11C and D).</p>
<fig fig-type="Figure" xml:lang="en" id="fig3" orientation="portrait" position="float"><label>Figure 3</label>
<caption><p>Detection of SARS‐CoV N protein by IHC (A and C) and of SARS‐CoV RNA polymerase gene fragments by ISH (B and D) in the following samples from SARS patients (A and B; DAB; figures <xref rid="fig3" ref-type="fig">3</xref>
, 7A, 8A, 10, 13 ×200; figures <xref rid="fig1" ref-type="fig">1</xref>
, <xref rid="fig2" ref-type="fig">2</xref>
, 4–6, 8B, 9, 11, 12 ×400; 7B ×480) and from control patients (C and D; DAB; figures 1–11 × 400). (1) Bronchial serous gland epithelium; (2) gastric parietal cells; (3) small intestinal epithelium; (4) acidophilic cells of parathyroid; (5) pancreatic acinar cells; (6) acidophilic cells of pituitary; (7) adrenal cortical cells; (8) sweat gland cells; (9) cerebral neurones; (10) epithelial cells of distal convoluted renal tubule; (11) hepatocytes near the central vein; (12) myocardium; (13) seminiferous tubules of testis. The brownish‐yellow colour in cells represents positive signal for SARS‐CoV N protein in IHC, and for SARS‐CoV RNA polymerase gene fragments in ISH</p>
</caption>
<graphic id="nlm-graphic-5" xlink:href="PATH-203-622-g003"></graphic>
</fig>
<table-wrap id="tbl1" xml:lang="en" orientation="portrait" position="float"><label>Table 1</label>
<caption><p>Detection of SARS‐CoV in tissues from four SARS autopsies<xref ref-type="fn" rid="fn1">a</xref>
</p>
</caption>
<table frame="hsides" rules="groups"><col span="1"></col>
<col span="1"></col>
<col span="1"></col>
<col span="1"></col>
<col span="1"></col>
<col span="1"></col>
<col span="1"></col>
<col span="1"></col>
<col span="1"></col>
<thead valign="bottom"><tr style="border-bottom:solid 1px #000000"><th rowspan="2" align="left" valign="bottom" colspan="1"><bold>Organs and tissues</bold>
</th>
<th colspan="2" align="center" valign="bottom" rowspan="1"><bold>A1061</bold>
<xref ref-type="fn" rid="fn2">b</xref>
</th>
<th colspan="2" align="center" valign="bottom" rowspan="1"><bold>A1062</bold>
<xref ref-type="fn" rid="fn2">b</xref>
</th>
<th colspan="2" align="center" valign="bottom" rowspan="1"><bold>A1065</bold>
<xref ref-type="fn" rid="fn2">b</xref>
</th>
<th colspan="2" align="center" valign="bottom" rowspan="1"><bold>A1076</bold>
<xref ref-type="fn" rid="fn2">b</xref>
</th>
</tr>
<tr style="border-bottom:solid 1px #000000"><th align="center" valign="bottom" rowspan="1" colspan="1"><bold>IHC</bold>
</th>
<th align="center" valign="bottom" rowspan="1" colspan="1"><bold>ISH</bold>
</th>
<th align="center" valign="bottom" rowspan="1" colspan="1"><bold>IHC</bold>
</th>
<th align="center" valign="bottom" rowspan="1" colspan="1"><bold>ISH</bold>
</th>
<th align="center" valign="bottom" rowspan="1" colspan="1"><bold>IHC</bold>
</th>
<th align="center" valign="bottom" rowspan="1" colspan="1"><bold>ISH</bold>
</th>
<th align="center" valign="bottom" rowspan="1" colspan="1"><bold>IHC</bold>
</th>
<th align="center" valign="bottom" rowspan="1" colspan="1"><bold>ISH</bold>
</th>
</tr>
</thead>
<tbody valign="top"><tr><td align="left" valign="top" rowspan="1" colspan="1">Lung</td>
<td align="center" valign="top" rowspan="1" colspan="1">+++</td>
<td align="center" valign="top" rowspan="1" colspan="1">+++</td>
<td align="center" valign="top" rowspan="1" colspan="1">+++</td>
<td align="center" valign="top" rowspan="1" colspan="1">+++</td>
<td align="center" valign="top" rowspan="1" colspan="1">+++</td>
<td align="center" valign="top" rowspan="1" colspan="1">+++</td>
<td align="center" valign="top" rowspan="1" colspan="1">+++</td>
<td align="center" valign="top" rowspan="1" colspan="1">+++</td>
</tr>
<tr><td align="left" valign="top" rowspan="1" colspan="1">Stomach</td>
<td align="center" valign="top" rowspan="1" colspan="1">++</td>
<td align="center" valign="top" rowspan="1" colspan="1">++</td>
<td align="center" valign="top" rowspan="1" colspan="1">+</td>
<td align="center" valign="top" rowspan="1" colspan="1">+</td>
<td align="center" valign="top" rowspan="1" colspan="1">+</td>
<td align="center" valign="top" rowspan="1" colspan="1">+</td>
<td align="center" valign="top" rowspan="1" colspan="1">+</td>
<td align="center" valign="top" rowspan="1" colspan="1">+</td>
</tr>
<tr><td align="left" valign="top" rowspan="1" colspan="1">Small intestine</td>
<td align="center" valign="top" rowspan="1" colspan="1">++</td>
<td align="center" valign="top" rowspan="1" colspan="1">++</td>
<td align="center" valign="top" rowspan="1" colspan="1">+</td>
<td align="center" valign="top" rowspan="1" colspan="1">+</td>
<td align="center" valign="top" rowspan="1" colspan="1">+</td>
<td align="center" valign="top" rowspan="1" colspan="1">+</td>
<td align="center" valign="top" rowspan="1" colspan="1">+</td>
<td align="center" valign="top" rowspan="1" colspan="1">+</td>
</tr>
<tr><td align="left" valign="top" rowspan="1" colspan="1">Kidney</td>
<td align="center" valign="top" rowspan="1" colspan="1">++</td>
<td align="center" valign="top" rowspan="1" colspan="1">++</td>
<td align="center" valign="top" rowspan="1" colspan="1">++</td>
<td align="center" valign="top" rowspan="1" colspan="1">++</td>
<td align="center" valign="top" rowspan="1" colspan="1">++</td>
<td align="center" valign="top" rowspan="1" colspan="1">++</td>
<td align="center" valign="top" rowspan="1" colspan="1">++</td>
<td align="center" valign="top" rowspan="1" colspan="1">++</td>
</tr>
<tr><td align="left" valign="top" rowspan="1" colspan="1">Adrenal</td>
<td align="center" valign="top" rowspan="1" colspan="1">++</td>
<td align="center" valign="top" rowspan="1" colspan="1">++</td>
<td align="center" valign="top" rowspan="1" colspan="1">++</td>
<td align="center" valign="top" rowspan="1" colspan="1">++</td>
<td align="center" valign="top" rowspan="1" colspan="1">++</td>
<td align="center" valign="top" rowspan="1" colspan="1">++</td>
<td align="center" valign="top" rowspan="1" colspan="1">++</td>
<td align="center" valign="top" rowspan="1" colspan="1">++</td>
</tr>
<tr><td align="left" valign="top" rowspan="1" colspan="1">Skin</td>
<td align="center" valign="top" rowspan="1" colspan="1">++</td>
<td align="center" valign="top" rowspan="1" colspan="1">++</td>
<td align="center" valign="top" rowspan="1" colspan="1">++</td>
<td align="center" valign="top" rowspan="1" colspan="1">++</td>
<td align="center" valign="top" rowspan="1" colspan="1">++</td>
<td align="center" valign="top" rowspan="1" colspan="1">++</td>
<td align="center" valign="top" rowspan="1" colspan="1">++</td>
<td align="center" valign="top" rowspan="1" colspan="1">++</td>
</tr>
<tr><td align="left" valign="top" rowspan="1" colspan="1">Parathyroid</td>
<td align="center" valign="top" rowspan="1" colspan="1">++</td>
<td align="center" valign="top" rowspan="1" colspan="1">++</td>
<td align="center" valign="top" rowspan="1" colspan="1">++</td>
<td align="center" valign="top" rowspan="1" colspan="1">++</td>
<td align="center" valign="top" rowspan="1" colspan="1">++</td>
<td align="center" valign="top" rowspan="1" colspan="1">++</td>
<td align="center" valign="top" rowspan="1" colspan="1">++</td>
<td align="center" valign="top" rowspan="1" colspan="1">++</td>
</tr>
<tr><td align="left" valign="top" rowspan="1" colspan="1">Pituitary</td>
<td align="center" valign="top" rowspan="1" colspan="1">+</td>
<td align="center" valign="top" rowspan="1" colspan="1">+</td>
<td align="center" valign="top" rowspan="1" colspan="1">+</td>
<td align="center" valign="top" rowspan="1" colspan="1">+</td>
<td align="center" valign="top" rowspan="1" colspan="1">+</td>
<td align="center" valign="top" rowspan="1" colspan="1">+</td>
<td align="center" valign="top" rowspan="1" colspan="1">+</td>
<td align="center" valign="top" rowspan="1" colspan="1">+</td>
</tr>
<tr><td align="left" valign="top" rowspan="1" colspan="1">Liver</td>
<td align="center" valign="top" rowspan="1" colspan="1">+</td>
<td align="center" valign="top" rowspan="1" colspan="1">+</td>
<td align="center" valign="top" rowspan="1" colspan="1">+</td>
<td align="center" valign="top" rowspan="1" colspan="1">+</td>
<td align="center" valign="top" rowspan="1" colspan="1">+</td>
<td align="center" valign="top" rowspan="1" colspan="1">+</td>
<td align="center" valign="top" rowspan="1" colspan="1">+</td>
<td align="center" valign="top" rowspan="1" colspan="1">+</td>
</tr>
<tr><td align="left" valign="top" rowspan="1" colspan="1">Cerebrum</td>
<td align="center" valign="top" rowspan="1" colspan="1">+</td>
<td align="center" valign="top" rowspan="1" colspan="1">+</td>
<td align="center" valign="top" rowspan="1" colspan="1">+</td>
<td align="center" valign="top" rowspan="1" colspan="1">+</td>
<td align="center" valign="top" rowspan="1" colspan="1">+</td>
<td align="center" valign="top" rowspan="1" colspan="1">+</td>
<td align="center" valign="top" rowspan="1" colspan="1">+</td>
<td align="center" valign="top" rowspan="1" colspan="1">+</td>
</tr>
<tr><td align="left" valign="top" rowspan="1" colspan="1">Pancreas</td>
<td align="center" valign="top" rowspan="1" colspan="1">+</td>
<td align="center" valign="top" rowspan="1" colspan="1">+</td>
<td align="center" valign="top" rowspan="1" colspan="1">+</td>
<td align="center" valign="top" rowspan="1" colspan="1">+</td>
<td align="center" valign="top" rowspan="1" colspan="1">+</td>
<td align="center" valign="top" rowspan="1" colspan="1">+</td>
<td align="center" valign="top" rowspan="1" colspan="1">−</td>
<td align="center" valign="top" rowspan="1" colspan="1">−</td>
</tr>
<tr><td align="left" valign="top" rowspan="1" colspan="1">Oesophagus</td>
<td align="center" valign="top" rowspan="1" colspan="1">−</td>
<td align="center" valign="top" rowspan="1" colspan="1">−</td>
<td align="center" valign="top" rowspan="1" colspan="1">−</td>
<td align="center" valign="top" rowspan="1" colspan="1">−</td>
<td align="center" valign="top" rowspan="1" colspan="1">−</td>
<td align="center" valign="top" rowspan="1" colspan="1">−</td>
<td align="center" valign="top" rowspan="1" colspan="1">−</td>
<td align="center" valign="top" rowspan="1" colspan="1">−</td>
</tr>
<tr><td align="left" valign="top" rowspan="1" colspan="1">Bone marrow</td>
<td align="center" valign="top" rowspan="1" colspan="1">−</td>
<td align="center" valign="top" rowspan="1" colspan="1">−</td>
<td align="center" valign="top" rowspan="1" colspan="1">−</td>
<td align="center" valign="top" rowspan="1" colspan="1">−</td>
<td align="center" valign="top" rowspan="1" colspan="1">−</td>
<td align="center" valign="top" rowspan="1" colspan="1">−</td>
<td align="center" valign="top" rowspan="1" colspan="1">−</td>
<td align="center" valign="top" rowspan="1" colspan="1">−</td>
</tr>
<tr><td align="left" valign="top" rowspan="1" colspan="1">Thyroid</td>
<td align="center" valign="top" rowspan="1" colspan="1">−</td>
<td align="center" valign="top" rowspan="1" colspan="1">−</td>
<td align="center" valign="top" rowspan="1" colspan="1">−</td>
<td align="center" valign="top" rowspan="1" colspan="1">−</td>
<td align="center" valign="top" rowspan="1" colspan="1">−</td>
<td align="center" valign="top" rowspan="1" colspan="1">−</td>
<td align="center" valign="top" rowspan="1" colspan="1">−</td>
<td align="center" valign="top" rowspan="1" colspan="1">−</td>
</tr>
<tr><td align="left" valign="top" rowspan="1" colspan="1">Spleen</td>
<td align="center" valign="top" rowspan="1" colspan="1">−</td>
<td align="center" valign="top" rowspan="1" colspan="1">−</td>
<td align="center" valign="top" rowspan="1" colspan="1">−</td>
<td align="center" valign="top" rowspan="1" colspan="1">−</td>
<td align="center" valign="top" rowspan="1" colspan="1">−</td>
<td align="center" valign="top" rowspan="1" colspan="1">−</td>
<td align="center" valign="top" rowspan="1" colspan="1">−</td>
<td align="center" valign="top" rowspan="1" colspan="1">−</td>
</tr>
<tr><td align="left" valign="top" rowspan="1" colspan="1">Lymph node</td>
<td align="center" valign="top" rowspan="1" colspan="1">−</td>
<td align="center" valign="top" rowspan="1" colspan="1">−</td>
<td align="center" valign="top" rowspan="1" colspan="1">−</td>
<td align="center" valign="top" rowspan="1" colspan="1">−</td>
<td align="center" valign="top" rowspan="1" colspan="1">−</td>
<td align="center" valign="top" rowspan="1" colspan="1">−</td>
<td align="center" valign="top" rowspan="1" colspan="1">−</td>
<td align="center" valign="top" rowspan="1" colspan="1">−</td>
</tr>
<tr><td align="left" valign="top" rowspan="1" colspan="1">Cerebellum</td>
<td align="center" valign="top" rowspan="1" colspan="1">−</td>
<td align="center" valign="top" rowspan="1" colspan="1">−</td>
<td align="center" valign="top" rowspan="1" colspan="1">−</td>
<td align="center" valign="top" rowspan="1" colspan="1">−</td>
<td align="center" valign="top" rowspan="1" colspan="1">−</td>
<td align="center" valign="top" rowspan="1" colspan="1">−</td>
<td align="center" valign="top" rowspan="1" colspan="1">−</td>
<td align="center" valign="top" rowspan="1" colspan="1">−</td>
</tr>
<tr><td align="left" valign="top" rowspan="1" colspan="1">Heart</td>
<td align="center" valign="top" rowspan="1" colspan="1">−</td>
<td align="center" valign="top" rowspan="1" colspan="1">−</td>
<td align="center" valign="top" rowspan="1" colspan="1">−</td>
<td align="center" valign="top" rowspan="1" colspan="1">−</td>
<td align="center" valign="top" rowspan="1" colspan="1">−</td>
<td align="center" valign="top" rowspan="1" colspan="1">−</td>
<td align="center" valign="top" rowspan="1" colspan="1">−</td>
<td align="center" valign="top" rowspan="1" colspan="1">−</td>
</tr>
<tr><td align="left" valign="top" rowspan="1" colspan="1">Striated muscle</td>
<td align="center" valign="top" rowspan="1" colspan="1">−</td>
<td align="center" valign="top" rowspan="1" colspan="1">−</td>
<td align="center" valign="top" rowspan="1" colspan="1">−</td>
<td align="center" valign="top" rowspan="1" colspan="1">−</td>
<td align="center" valign="top" rowspan="1" colspan="1">−</td>
<td align="center" valign="top" rowspan="1" colspan="1">−</td>
<td align="center" valign="top" rowspan="1" colspan="1">−</td>
<td align="center" valign="top" rowspan="1" colspan="1">−</td>
</tr>
<tr><td align="left" valign="top" rowspan="1" colspan="1">Testis</td>
<td align="center" valign="top" rowspan="1" colspan="1">−</td>
<td align="center" valign="top" rowspan="1" colspan="1">−</td>
<td align="center" valign="top" rowspan="1" colspan="1">−</td>
<td align="center" valign="top" rowspan="1" colspan="1">−</td>
<td align="center" valign="top" rowspan="1" colspan="1">−</td>
<td align="center" valign="top" rowspan="1" colspan="1">−</td>
<td align="center" valign="top" rowspan="1" colspan="1">−</td>
<td align="center" valign="top" rowspan="1" colspan="1">−</td>
</tr>
<tr><td align="left" valign="top" rowspan="1" colspan="1">Ovary</td>
<td align="center" valign="top" rowspan="1" colspan="1">−</td>
<td align="center" valign="top" rowspan="1" colspan="1">−</td>
<td align="center" valign="top" rowspan="1" colspan="1">−</td>
<td align="center" valign="top" rowspan="1" colspan="1">−</td>
<td align="center" valign="top" rowspan="1" colspan="1">−</td>
<td align="center" valign="top" rowspan="1" colspan="1">−</td>
<td align="center" valign="top" rowspan="1" colspan="1">−</td>
<td align="center" valign="top" rowspan="1" colspan="1">−</td>
</tr>
<tr><td align="left" valign="top" rowspan="1" colspan="1">Uterus</td>
<td align="center" valign="top" rowspan="1" colspan="1">−</td>
<td align="center" valign="top" rowspan="1" colspan="1">−</td>
<td align="center" valign="top" rowspan="1" colspan="1">−</td>
<td align="center" valign="top" rowspan="1" colspan="1">−</td>
<td align="center" valign="top" rowspan="1" colspan="1">−</td>
<td align="center" valign="top" rowspan="1" colspan="1">−</td>
<td align="center" valign="top" rowspan="1" colspan="1">−</td>
<td align="center" valign="top" rowspan="1" colspan="1">−</td>
</tr>
</tbody>
</table>
<table-wrap-foot><fn id="fn1"><label>a</label>
<p>The number of positive cells and the total number of cells specified were counted in 10 high‐power views. The percentage of positive cells was calculated. 0%, 1 to <24%, ≥25% to <49%, ≥50% to <74%, and ≥75% positive cells were recorded as ‘−’, ‘+’, ‘++’, ‘+++’, and ‘++++’, respectively. No positive signal was detected in control tissues.</p>
</fn>
<fn id="fn2"><label>b</label>
<p>Case number.</p>
</fn>
</table-wrap-foot>
<permissions><copyright-holder>John Wiley & Sons, Ltd.</copyright-holder>
<license><license-p>This article is being made freely available through PubMed Central as part of the COVID-19 public health emergency response. It can be used for unrestricted research re-use and analysis in any form or by any means with acknowledgement of the original source, for the duration of the public health emergency.</license-p>
</license>
</permissions>
</table-wrap>
</sec>
<sec id="sec1-4"><title>Discussion</title>
<p>There has been a warning that SARS epidemics may recur in future <xref rid="bib23" ref-type="ref">23</xref>
; significantly, new SARS cases have recently been reported in Singapore <xref rid="bib24" ref-type="ref">24</xref>
and China <xref rid="bib16" ref-type="ref">16</xref>
. Therefore, the development of effective strategies for the protection of at‐risk populations is urgently needed. However, the pathogenesis of SARS‐CoV infection and the virus transmission pathways have not been clearly defined. The distribution of SARS‐CoV in organs and tissues in SARS patients has not been systematically determined due to the lack of antibodies and probes specific for SARS‐CoV. Recently, we developed an IHC assay, using a murine mAb specific for the SARS‐CoV N protein, and an ISH method, using probes specific for the SARS‐CoV RNA polymerase gene fragment, for <italic>in situ</italic>
detection of SARS‐CoV in tissues.</p>
<p>Consistent with our previous observations <xref rid="bib17" ref-type="ref">17</xref>
, <xref rid="bib25" ref-type="ref">25</xref>
, SARS‐CoV was predominantly detected in lung, trachea and bronchus. Strikingly, SARS‐CoV was also detected in many other organs and tissues, including stomach, small intestine, distal convoluted renal tubule, sweat gland, parathyroid, pituitary, pancreas, adrenal, liver and cerebrum (Figure <xref rid="fig3" ref-type="fig">3</xref>
and Table <xref rid="tbl1" ref-type="table">1</xref>
). However, the virus was not detected in oesophagus, thyroid, spleen, lymph node, bone marrow, testis, ovary, uterus, heart, aorta, cerebellum or muscle (Table <xref rid="tbl1" ref-type="table">1</xref>
). These results suggest that, in addition to the respiratory system, the gastrointestinal system may also be a primary target of SARS‐CoV, ie virus in contaminated food and water may enter the human body through epithelial cells covering the surface of the gastrointestinal tract, although there is no direct evidence to show that food‐borne SARS‐CoV is transmitted through the gastrointestinal system. Other organs with detectable SARS‐CoV may be secondary targets of SARS‐CoV, ie the virions in blood that are released from the primary target (eg respiratory system) may infect cells in these organs and tissues. Another possibility is that SARS‐CoV may not infect these cells, but rather binds specifically or non‐specifically to their surface, resulting in detection of SARS‐CoV antigens and RNA. The pathological changes in these organs and tissues may be caused directly by the cytopathic effect (CPE) mediated by SARS‐CoV replication in these organs; or indirectly by the systemic toxic reaction due to failure of the respiratory system, or by the harmful immune response and cytokine reaction induced by virus infection.</p>
<p>Aminopeptidase N (CD13) is a receptor for at least two human coronaviruses <xref rid="bib26" ref-type="ref">26</xref>
, <xref rid="bib27" ref-type="ref">27</xref>
and was proposed as a receptor for SARS‐CoV <xref rid="bib28" ref-type="ref">28</xref>
. It was recently demonstrated that ACE2 is a functional receptor for SARS‐CoV <xref rid="bib8" ref-type="ref">8</xref>
and a fragment in the S1 domain of the SARS‐CoV S protein is responsible for binding of the virus to ACE2 <xref rid="bib9" ref-type="ref">9</xref>
, <xref rid="bib10" ref-type="ref">10</xref>
, <xref rid="bib11" ref-type="ref">11</xref>
. Analyzing 72 human tissues by real‐time PCR, Harmer <italic>et al</italic>
<xref rid="bib29" ref-type="ref">29</xref>
demonstrated that ACE2 is expressed in human lung, gastrointestinal tract, kidney, heart and testis. Consistent with our results, SARS‐CoV was detected in lung, gastrointestinal tract and kidney, suggesting that SARS‐CoV may utilize ACE2 as a receptor to enter the cells in these organs. However, it is unclear why the cells in heart and testis cannot be infected by SARS‐CoV despite the expression of high levels of ACE2.</p>
<p>Consistent with the report of Leung <italic>et al</italic>
, who used electron microscopic and histological examination of endoscopic biopsies and post mortem material <xref rid="bib30" ref-type="ref">30</xref>
, we also detected SARS‐CoV in the epithelial cells of the alimentary tract, including stomach and small intestine (Table <xref rid="tbl1" ref-type="table">1</xref>
). This suggests that the gastrointestinal system may also be an entry route for SARS‐CoV present in food or water. Although there is no report of such transmission <xref rid="bib15" ref-type="ref">15</xref>
, caution should be exercised by the at‐risk population during the SARS‐CoV endemic season. This finding supports the hypothesis that SARS‐CoV may be released into the environment via faeces from individuals infected by SARS‐CoV. Indeed, low levels of SARS‐CoV RNA were detected in faeces from SARS patients <xref rid="bib3" ref-type="ref">3</xref>
, <xref rid="bib30" ref-type="ref">30</xref>
. Moreover, we also found SARS‐CoV in the epithelial cells of the distal convoluted renal tubule, suggesting that SARS‐CoV may also be transmitted through contaminated urine.</p>
<p>For the first time, we have demonstrated that SARS‐CoV is detectable in sweat gland cells in the skin. This suggests another route of transmission for SARS‐CoV, since this virus may be excreted in sweat and infect other people who are in direct contact with the patient's skin. Indeed, Rados suspected that SARS‐CoV could be transmitted by direct touch <xref rid="bib31" ref-type="ref">31</xref>
. If this transmission route is confirmed, new infection‐control measures should be recommended. For example, patients may have to wear gloves, disposable gowns and eye protection, in addition to N95 masks, and to avoid kissing or touching other people.</p>
</sec>
</body>
<back><ack id="sec1-ack-1"><title>Acknowledgements</title>
<p>We would like to thank the family members of the patients, the Eighth People's Hospital of Guangzhou City, the Second Affiliated Hospital of Zhongshan University, the Guangzhou Institute of Respiratory Diseases, and the Health Administration of Guangdong Province, China, for their permission to carry out the autopsies and to use the samples in this study. Sources of support were as follows: special grants from the Chinese National Foundation of Natural Sciences (No 30340015), Military Foundation of Medical Science (No 03F016‐2) and Foundation of Natural Sciences of Guangdong Province (No. GD2003‐80).</p>
</ack>
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