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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Persistent Replication of Severe Acute Respiratory Syndrome Coronavirus in Human Tubular Kidney Cells Selects for Adaptive Mutations in the Membrane Protein<xref ref-type="fn" rid="fn2">▿</xref> </title><author><name sortKey="Pacciarini, Filippo" sort="Pacciarini, Filippo" uniqKey="Pacciarini F" first="Filippo" last="Pacciarini">Filippo Pacciarini</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Ghezzi, Silvia" sort="Ghezzi, Silvia" uniqKey="Ghezzi S" first="Silvia" last="Ghezzi">Silvia Ghezzi</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Canducci, Filippo" sort="Canducci, Filippo" uniqKey="Canducci F" first="Filippo" last="Canducci">Filippo Canducci</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Sims, Amy" sort="Sims, Amy" uniqKey="Sims A" first="Amy" last="Sims">Amy Sims</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Sampaolo, Michela" sort="Sampaolo, Michela" uniqKey="Sampaolo M" first="Michela" last="Sampaolo">Michela Sampaolo</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Ferioli, Elena" sort="Ferioli, Elena" uniqKey="Ferioli E" first="Elena" last="Ferioli">Elena Ferioli</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Clementi, Massimo" sort="Clementi, Massimo" uniqKey="Clementi M" first="Massimo" last="Clementi">Massimo Clementi</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Poli, Guido" sort="Poli, Guido" uniqKey="Poli G" first="Guido" last="Poli">Guido Poli</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Conaldi, Pier Giulio" sort="Conaldi, Pier Giulio" uniqKey="Conaldi P" first="Pier Giulio" last="Conaldi">Pier Giulio Conaldi</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Baric, Ralph" sort="Baric, Ralph" uniqKey="Baric R" first="Ralph" last="Baric">Ralph Baric</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Vicenzi, Elisa" sort="Vicenzi, Elisa" uniqKey="Vicenzi E" first="Elisa" last="Vicenzi">Elisa Vicenzi</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">18367528</idno><idno type="pmc">2395189</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2395189</idno><idno type="RBID">PMC:2395189</idno><idno type="doi">10.1128/JVI.00096-08</idno><date when="2008">2008</date><idno type="wicri:Area/Pmc/Corpus">000664</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000664</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Persistent Replication of Severe Acute Respiratory Syndrome Coronavirus in Human Tubular Kidney Cells Selects for Adaptive Mutations in the Membrane Protein<xref ref-type="fn" rid="fn2">▿</xref> </title><author><name sortKey="Pacciarini, Filippo" sort="Pacciarini, Filippo" uniqKey="Pacciarini F" first="Filippo" last="Pacciarini">Filippo Pacciarini</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Ghezzi, Silvia" sort="Ghezzi, Silvia" uniqKey="Ghezzi S" first="Silvia" last="Ghezzi">Silvia Ghezzi</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Canducci, Filippo" sort="Canducci, Filippo" uniqKey="Canducci F" first="Filippo" last="Canducci">Filippo Canducci</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Sims, Amy" sort="Sims, Amy" uniqKey="Sims A" first="Amy" last="Sims">Amy Sims</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Sampaolo, Michela" sort="Sampaolo, Michela" uniqKey="Sampaolo M" first="Michela" last="Sampaolo">Michela Sampaolo</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Ferioli, Elena" sort="Ferioli, Elena" uniqKey="Ferioli E" first="Elena" last="Ferioli">Elena Ferioli</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Clementi, Massimo" sort="Clementi, Massimo" uniqKey="Clementi M" first="Massimo" last="Clementi">Massimo Clementi</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Poli, Guido" sort="Poli, Guido" uniqKey="Poli G" first="Guido" last="Poli">Guido Poli</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Conaldi, Pier Giulio" sort="Conaldi, Pier Giulio" uniqKey="Conaldi P" first="Pier Giulio" last="Conaldi">Pier Giulio Conaldi</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Baric, Ralph" sort="Baric, Ralph" uniqKey="Baric R" first="Ralph" last="Baric">Ralph Baric</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Vicenzi, Elisa" sort="Vicenzi, Elisa" uniqKey="Vicenzi E" first="Elisa" last="Vicenzi">Elisa Vicenzi</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author></analytic><series><title level="j">Journal of Virology</title><idno type="ISSN">0022-538X</idno><idno type="eISSN">1098-5514</idno><imprint><date when="2008">2008</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>Severe acute respiratory syndrome (SARS) is a systemic disease characterized by both lung pathology and widespread extrapulmonary virus dissemination causing multiple organ injuries. In this regard, renal dysfunction is an ominous sign in patients with SARS. Indeed, clusters of SARS coronavirus (SARS-CoV) particles have been detected in the cytoplasm of renal tubular epithelial cells in postmortem studies, explaining the presence of infectious virus in the urine of SARS patients. In order to investigate the potential SARS-CoV kidney tropism, we have evaluated the susceptibility of human renal cells of tubular and glomerular origin to in vitro SARS-CoV infection. Immortalized cultures of differentiated proximal tubular epithelial cells (PTEC), glomerular mesangial cells (MC), and glomerular epithelial cells (podocytes) were found to express the SARS-CoV receptor angiotensin-converting enzyme 2 on their surface. Productive infection, however, occurred only in PTEC but not in glomerular cells. A transient infection with poor virus production was observed in MC, whereas podocytes were not permissive to SARS-CoV infection. In contrast to the cytopathic infection of the Vero E6 cell line, SARS-CoV did not cause overt cytopathic effects in PTEC or MC. Of interest, PTEC, but not MC, maintained stable levels of SARS-CoV production in serial subcultures, suggesting a persistent state of infection. In this regard, a SARS-CoV variant with increased replication capacity in PTEC was selected after four serial subculture passages. This SARS-CoV variant acquired a single nonconservative amino acid change from glutamic acid (E) to alanine (A) at position 11 in the viral membrane (M) protein. The E11A point mutation was sufficient for enhanced SARS-CoV replication and persistence in PTEC when introduced in a SARS-CoV recombinant infectious clone. These findings indicate that human PTEC may represent a site of SARS-CoV productive and persistent replication favoring the emergence of viral variants with increased replication capacity, at least in these kidney cells.</p></div></front></TEI><pmc article-type="research-article"><pmc-comment>The publisher of this article does not allow downloading of the full text in XML form.</pmc-comment>
<front><journal-meta><journal-id journal-id-type="nlm-ta">J Virol</journal-id><journal-id journal-id-type="publisher-id">jvi</journal-id><journal-title>Journal of Virology</journal-title><issn pub-type="ppub">0022-538X</issn><issn pub-type="epub">1098-5514</issn><publisher><publisher-name>American Society for Microbiology (ASM)</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">18367528</article-id><article-id pub-id-type="pmc">2395189</article-id><article-id pub-id-type="publisher-id">0096-08</article-id><article-id pub-id-type="doi">10.1128/JVI.00096-08</article-id><article-categories><subj-group subj-group-type="heading"><subject>Virus-Cell Interactions</subject></subj-group></article-categories><title-group><article-title>Persistent Replication of Severe Acute Respiratory Syndrome Coronavirus in Human Tubular Kidney Cells Selects for Adaptive Mutations in the Membrane Protein<xref ref-type="fn" rid="fn2">▿</xref> </article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Pacciarini</surname><given-names>Filippo</given-names></name><xref ref-type="aff" rid="aff1">1</xref><xref ref-type="fn" rid="fn1">†</xref></contrib><contrib contrib-type="author"><name><surname>Ghezzi</surname><given-names>Silvia</given-names></name><xref ref-type="aff" rid="aff1">1</xref><xref ref-type="fn" rid="fn1">†</xref></contrib><contrib contrib-type="author"><name><surname>Canducci</surname><given-names>Filippo</given-names></name><xref ref-type="aff" rid="aff1">2</xref><xref ref-type="aff" rid="aff1">6</xref></contrib><contrib contrib-type="author"><name><surname>Sims</surname><given-names>Amy</given-names></name><xref ref-type="aff" rid="aff1">3</xref></contrib><contrib contrib-type="author"><name><surname>Sampaolo</surname><given-names>Michela</given-names></name><xref ref-type="aff" rid="aff1">2</xref><xref ref-type="aff" rid="aff1">6</xref></contrib><contrib contrib-type="author"><name><surname>Ferioli</surname><given-names>Elena</given-names></name><xref ref-type="aff" rid="aff1">5</xref></contrib><contrib contrib-type="author"><name><surname>Clementi</surname><given-names>Massimo</given-names></name><xref ref-type="aff" rid="aff1">2</xref><xref ref-type="aff" rid="aff1">6</xref></contrib><contrib contrib-type="author"><name><surname>Poli</surname><given-names>Guido</given-names></name><xref ref-type="aff" rid="aff1">4</xref><xref ref-type="aff" rid="aff1">6</xref></contrib><contrib contrib-type="author"><name><surname>Conaldi</surname><given-names>Pier Giulio</given-names></name><xref ref-type="aff" rid="aff1">7</xref></contrib><contrib contrib-type="author"><name><surname>Baric</surname><given-names>Ralph</given-names></name><xref ref-type="aff" rid="aff1">3</xref></contrib><contrib contrib-type="author"><name><surname>Vicenzi</surname><given-names>Elisa</given-names></name><xref ref-type="aff" rid="aff1">1</xref><xref ref-type="corresp" rid="cor1">*</xref></contrib></contrib-group><aff id="aff1">Viral Pathogens and Biosafety Unit, San Raffaele Scientific Institute, Milano, Italy,<label>1</label> Laboratory of Microbiology and Virology, San Raffaele Scientific Institute, Milano, Italy,<label>2</label> University of North Carolina at Chapel Hill, Chapel Hill, North Carolina,<label>3</label> AIDS Immunopathogenesis Unit, San Raffaele Scientific Institute, Milano, Italy,<label>4</label> Department of Medicine and Public Health, University of Insubria, Varese, Italy,<label>5</label> Vita-Salute San Raffaele University, School of Medicine, Milano, Italy,<label>6</label> Laboratory of Clinical Pathology, Microbiology and Virology, Mediterranean Institute for Transplantation and Advanced Specialised Therapies, University of Pittsburgh Medical Center—Italy, Palermo, Italy<label>7</label></aff><author-notes><fn id="cor1"><label>*</label><p>Corresponding author. Mailing address: P2/P3 Laboratories, DIBIT, Via Olgettina, 58, 20132 Milano, Italy. Phone: 39-02-2643-4908. Fax: 39-02-2643-4905. E-mail: <email>vicenzi.elisa@hsr.it</email></p></fn><fn id="fn1"><label>†</label><p>F.P. and S.G. contributed equally to this work.</p></fn></author-notes><pub-date pub-type="ppub"><month>6</month><year>2008</year></pub-date><pub-date pub-type="epub"><day>26</day><month>3</month><year>2008</year></pub-date><volume>82</volume><issue>11</issue><fpage>5137</fpage><lpage>5144</lpage><history><date date-type="received"><day>15</day><month>1</month><year>2008</year></date><date date-type="accepted"><day>13</day><month>3</month><year>2008</year></date></history><permissions><copyright-statement>Copyright © 2008, American Society for Microbiology</copyright-statement></permissions><self-uri xlink:title="pdf" xlink:href="zjv01108005137.pdf"></self-uri><abstract><p>Severe acute respiratory syndrome (SARS) is a systemic disease characterized by both lung pathology and widespread extrapulmonary virus dissemination causing multiple organ injuries. In this regard, renal dysfunction is an ominous sign in patients with SARS. Indeed, clusters of SARS coronavirus (SARS-CoV) particles have been detected in the cytoplasm of renal tubular epithelial cells in postmortem studies, explaining the presence of infectious virus in the urine of SARS patients. In order to investigate the potential SARS-CoV kidney tropism, we have evaluated the susceptibility of human renal cells of tubular and glomerular origin to in vitro SARS-CoV infection. Immortalized cultures of differentiated proximal tubular epithelial cells (PTEC), glomerular mesangial cells (MC), and glomerular epithelial cells (podocytes) were found to express the SARS-CoV receptor angiotensin-converting enzyme 2 on their surface. Productive infection, however, occurred only in PTEC but not in glomerular cells. A transient infection with poor virus production was observed in MC, whereas podocytes were not permissive to SARS-CoV infection. In contrast to the cytopathic infection of the Vero E6 cell line, SARS-CoV did not cause overt cytopathic effects in PTEC or MC. Of interest, PTEC, but not MC, maintained stable levels of SARS-CoV production in serial subcultures, suggesting a persistent state of infection. In this regard, a SARS-CoV variant with increased replication capacity in PTEC was selected after four serial subculture passages. This SARS-CoV variant acquired a single nonconservative amino acid change from glutamic acid (E) to alanine (A) at position 11 in the viral membrane (M) protein. The E11A point mutation was sufficient for enhanced SARS-CoV replication and persistence in PTEC when introduced in a SARS-CoV recombinant infectious clone. These findings indicate that human PTEC may represent a site of SARS-CoV productive and persistent replication favoring the emergence of viral variants with increased replication capacity, at least in these kidney cells.</p></abstract></article-meta></front><floats-wrap><fig position="float" id="f1"><label>FIG. 1.</label><caption><p>ACE2 expression in kidney cell lines. Kidney cell lines derived from African green monkeys (Vero E6), human kidney PTEC, MC, and podocytes were analyzed for ACE2 expression by labeling the cell surface with an anti-ACE2 polyclonal Ab. The full histograms indicate cells testing positive for ACE2 expression, whereas the open histograms represent the staining with a control Ab (swine anti-goat phycoerythrin-conjugated secondary Ab). The number on the upper right indicates the percentage of ACE2<sup>+</sup> cells.</p></caption><graphic xlink:href="zjv0110805900001"></graphic></fig><fig position="float" id="f2"><label>FIG. 2.</label><caption><p>Kinetics of SARS-CoV replication in kidney cells. Vero E6 cells, PTEC, MC, and podocytes were infected with SARS-CoV HSR1 at an MOI of 0.1. Virus replication was measured by real-time PCR of the full genome (▴) in the supernatant of infected cells harvested every 24 h up to 4 days p.i. The quantification of subgenomic transcripts was carried out by real-time PCR on retrotranscribed cDNA obtained from infected cells every 24 h up to 4 days p.i. The nucleocapsid (N) log<sub>10</sub> copy number (▪) was normalized by 50 ng of 18S RNA measured by real-time PCR. Values represent the mean number of ORF-1b copies ± standard deviation expressed as log<sub>10</sub>/ml obtained in three independent experiments.</p></caption><graphic xlink:href="zjv0110805900002"></graphic></fig><fig position="float" id="f3"><label>FIG. 3.</label><caption><p>Kinetics of SARS-CoV growth. The supernatants from infected PTEC (▪), MC (•), and Vero E6 (▴) cells, harvested every 24 h up to 4 days p.i., were tested in a Vero E6 plaque assay to determine the levels of infectious virus. Values represent the mean PFU ± standard deviation, expressed as log<sub>10</sub>/ml obtained in three independent experiments.</p></caption><graphic xlink:href="zjv0110805900003"></graphic></fig><fig position="float" id="f4"><label>FIG. 4.</label><caption><p>Cell viability after SARS-CoV HSR1 infection of Vero E6 and human kidney cells. Cells were exposed to 4 × 10<sup>5</sup> PFU of SARS-CoV HSR1 and stained with Trypan blue dye after 72 h p.i. Photographs were taken using a digital camera connected to the light microscope at a magnification of ×40. CPE (dark) was evident in Vero E6 cells, whereas SARS-CoV-infected human kidney epithelial cells did not show decreased viability in comparison to uninfected control cell cultures.</p></caption><graphic xlink:href="zjv0110805900004"></graphic></fig><fig position="float" id="f5"><label>FIG. 5.</label><caption><p>Persistent virus production in PTEC but not in MC. Cells were infected with SARS-CoV HSR1 at an MOI of 0.1. After 3 days, cultures were split and this procedure was repeated every 3 days up to four passages. Cultivation passages are indicated with a T, and the number in subscript indicates the serial passage. At each passage, 50% of the cells were seeded in fresh medium. The SARS-CoV copy number was calculated by real-time PCR of ORF-1b in cell supernatants collected prior to cell subculture. Values represent the mean number of ORF-1b copies ± standard deviation expressed as log<sub>10</sub>/ml obtained in three independent experiments.</p></caption><graphic xlink:href="zjv0110805900005"></graphic></fig><fig position="float" id="f6"><label>FIG. 6.</label><caption><p>Proportion of viral quasispecies in the M protein following serial subculturing of PTEC. The percentage is relative to 10 clones obtained from SARS-CoV HSR1-III passaged three times in Vero E6 cells and each time point from <italic>T</italic><sub>0</sub> to <italic>T</italic><sub>4</sub>.</p></caption><graphic xlink:href="zjv0110805900006"></graphic></fig><fig position="float" id="f7"><label>FIG. 7.</label><caption><p>Kinetics of SARS-CoV HSR1 replication in PTEC and Vero E6 cells infected with the supernatant obtained from the <italic>T</italic><sub>0</sub> culture prior to cell division and the <italic>T</italic><sub>4</sub> subculture. PTEC and Vero E6 cells were infected at MOIs of 0.03 and 0.006, respectively. Both PTEC and Vero E6 cells were also incubated with a 1:1 mixture of <italic>T</italic><sub>0</sub> and <italic>T</italic><sub>4</sub> supernatants containing 5 × 10<sup>4</sup> PFU/each. Replication kinetics were measured by real-time PCR of ORF-1b in the supernatant harvested every 24 to 48 h p.i. Values represent the mean number of ORF-1b copies ± standard deviation expressed as log<sub>10</sub>/ml obtained in three independent experiments.</p></caption><graphic xlink:href="zjv0110805900007"></graphic></fig><fig position="float" id="f8"><label>FIG. 8.</label><caption><p>Kinetics of viral replication of WT and mutant infectious clone (ic) in PTEC (A) and Vero E6 cells (B). Replication kinetics were measured by real-time PCR of ORF-1b in the supernatant harvested every 24 to 48 h p.i. Values represent the mean number of ORF-1b copies ± standard deviation expressed as log<sub>10</sub>/ml obtained in two independent experiments. Infectious virus present in the PTEC supernatant 168 h p.i. (C) was determined in a Vero E6 plaque assay. Values represent the mean PFU ± standard deviation as log<sub>10</sub>/ml.</p></caption><graphic xlink:href="zjv0110805900008"></graphic></fig></floats-wrap></pmc></record>Pour manipuler ce document sous Unix (Dilib)
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