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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Synthetic Reconstruction of Zoonotic and Early Human Severe Acute Respiratory Syndrome Coronavirus Isolates That Produce Fatal Disease in Aged Mice<xref ref-type="fn" rid="fn1">▿</xref> </title><author><name sortKey="Rockx, Barry" sort="Rockx, Barry" uniqKey="Rockx B" first="Barry" last="Rockx">Barry Rockx</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Sheahan, Timothy" sort="Sheahan, Timothy" uniqKey="Sheahan T" first="Timothy" last="Sheahan">Timothy Sheahan</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Donaldson, Eric" sort="Donaldson, Eric" uniqKey="Donaldson E" first="Eric" last="Donaldson">Eric Donaldson</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Harkema, Jack" sort="Harkema, Jack" uniqKey="Harkema J" first="Jack" last="Harkema">Jack Harkema</name><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="Heise, Mark" sort="Heise, Mark" uniqKey="Heise M" first="Mark" last="Heise">Mark Heise</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Pickles, Raymond" sort="Pickles, Raymond" uniqKey="Pickles R" first="Raymond" last="Pickles">Raymond Pickles</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Cameron, Mark" sort="Cameron, Mark" uniqKey="Cameron M" first="Mark" last="Cameron">Mark Cameron</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Kelvin, David" sort="Kelvin, David" uniqKey="Kelvin D" first="David" last="Kelvin">David Kelvin</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><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">17507479</idno><idno type="pmc">1933338</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1933338</idno><idno type="RBID">PMC:1933338</idno><idno type="doi">10.1128/JVI.00505-07</idno><date when="2007">2007</date><idno type="wicri:Area/Pmc/Corpus">000717</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000717</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Synthetic Reconstruction of Zoonotic and Early Human Severe Acute Respiratory Syndrome Coronavirus Isolates That Produce Fatal Disease in Aged Mice<xref ref-type="fn" rid="fn1">▿</xref> </title><author><name sortKey="Rockx, Barry" sort="Rockx, Barry" uniqKey="Rockx B" first="Barry" last="Rockx">Barry Rockx</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Sheahan, Timothy" sort="Sheahan, Timothy" uniqKey="Sheahan T" first="Timothy" last="Sheahan">Timothy Sheahan</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Donaldson, Eric" sort="Donaldson, Eric" uniqKey="Donaldson E" first="Eric" last="Donaldson">Eric Donaldson</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Harkema, Jack" sort="Harkema, Jack" uniqKey="Harkema J" first="Jack" last="Harkema">Jack Harkema</name><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="Heise, Mark" sort="Heise, Mark" uniqKey="Heise M" first="Mark" last="Heise">Mark Heise</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Pickles, Raymond" sort="Pickles, Raymond" uniqKey="Pickles R" first="Raymond" last="Pickles">Raymond Pickles</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Cameron, Mark" sort="Cameron, Mark" uniqKey="Cameron M" first="Mark" last="Cameron">Mark Cameron</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Kelvin, David" sort="Kelvin, David" uniqKey="Kelvin D" first="David" last="Kelvin">David Kelvin</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><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation><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="2007">2007</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>The severe acute respiratory syndrome (SARS) epidemic was characterized by high mortality rates in the elderly. The molecular mechanisms that govern enhanced susceptibility of elderly populations are not known, and robust animal models are needed that recapitulate the increased pathogenic phenotype noted with increasing age. Using synthetic biology and reverse genetics, we describe the construction of a panel of isogenic SARS coronavirus (SARS-CoV) strains bearing variant spike glycoproteins that are representative of zoonotic strains found in palm civets and raccoon dogs, as well as isolates spanning the early, middle, and late phases of the SARS-CoV epidemic. The recombinant viruses replicated efficiently in cell culture and demonstrated variable sensitivities to neutralization with antibodies. The human but not the zoonotic variants replicated efficiently in human airway epithelial cultures, supporting earlier hypotheses that zoonotic isolates are less pathogenic in humans but can evolve into highly pathogenic strains. All viruses replicated efficiently, but none produced clinical disease or death in young animals. In contrast, severe clinical disease, diffuse alveolar damage, hyaline membrane formation, alveolitis, and death were noted in 12-month-old mice inoculated with the palm civet HC/SZ/61/03 strain or early-human-phase GZ02 variants but not with related middle- and late-phase epidemic or raccoon dog strains. This panel of SARS-CoV recombinants bearing zoonotic and human epidemic spike glycoproteins will provide heterologous challenge models for testing vaccine efficacy against zoonotic reintroductions as well as provide the appropriate model system for elucidating the complex virus-host interactions that contribute to more-severe and fatal SARS-CoV disease and acute respiratory distress in the elderly.</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</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">17507479</article-id><article-id pub-id-type="pmc">1933338</article-id><article-id pub-id-type="publisher-id">0505-07</article-id><article-id pub-id-type="doi">10.1128/JVI.00505-07</article-id><article-categories><subj-group subj-group-type="heading"><subject>Pathogenesis and Immunity</subject></subj-group></article-categories><title-group><article-title>Synthetic Reconstruction of Zoonotic and Early Human Severe Acute Respiratory Syndrome Coronavirus Isolates That Produce Fatal Disease in Aged Mice<xref ref-type="fn" rid="fn1">▿</xref> </article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Rockx</surname><given-names>Barry</given-names></name><xref ref-type="aff" rid="aff1">1</xref></contrib><contrib contrib-type="author"><name><surname>Sheahan</surname><given-names>Timothy</given-names></name><xref ref-type="aff" rid="aff1">2</xref></contrib><contrib contrib-type="author"><name><surname>Donaldson</surname><given-names>Eric</given-names></name><xref ref-type="aff" rid="aff1">2</xref></contrib><contrib contrib-type="author"><name><surname>Harkema</surname><given-names>Jack</given-names></name><xref ref-type="aff" rid="aff1">4</xref></contrib><contrib contrib-type="author"><name><surname>Sims</surname><given-names>Amy</given-names></name><xref ref-type="aff" rid="aff1">1</xref></contrib><contrib contrib-type="author"><name><surname>Heise</surname><given-names>Mark</given-names></name><xref ref-type="aff" rid="aff1">2</xref><xref ref-type="aff" rid="aff1">3</xref></contrib><contrib contrib-type="author"><name><surname>Pickles</surname><given-names>Raymond</given-names></name><xref ref-type="aff" rid="aff1">2</xref><xref ref-type="aff" rid="aff1">5</xref></contrib><contrib contrib-type="author"><name><surname>Cameron</surname><given-names>Mark</given-names></name><xref ref-type="aff" rid="aff1">6</xref></contrib><contrib contrib-type="author"><name><surname>Kelvin</surname><given-names>David</given-names></name><xref ref-type="aff" rid="aff1">6</xref></contrib><contrib contrib-type="author"><name><surname>Baric</surname><given-names>Ralph</given-names></name><xref ref-type="aff" rid="aff1">1</xref><xref ref-type="aff" rid="aff1">2</xref><xref ref-type="aff" rid="aff1">3</xref><xref ref-type="corresp" rid="cor1">*</xref></contrib></contrib-group><aff id="aff1">Department of Epidemiology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina,<label>1</label> Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina,<label>2</label> Carolina Vaccine Institute, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina,<label>3</label> Department of Pathobiology and Diagnostic Investigation, College of Veterinary Medicine, Michigan State University, East Lansing, Michigan,<label>4</label> Cystic Fibrosis/Pulmonary Research and Treatment Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina,<label>5</label> University Health Network, University of Toronto, Toronto, Ontario, Canada<label>6</label></aff><author-notes><fn id="cor1"><label>*</label><p>Corresponding author. Mailing address: Department of Epidemiology, 2107 McGavran-Greenberg, CB#7435, University of North Carolina, Chapel Hill, NC 27699-7435. Phone: (919) 966-3895. Fax: (919) 966-0584. E-mail: <email>rbaric@email.unc.edu</email></p></fn></author-notes><pub-date pub-type="ppub"><month>7</month><year>2007</year></pub-date><pub-date pub-type="epub"><day>16</day><month>5</month><year>2007</year></pub-date><volume>81</volume><issue>14</issue><fpage>7410</fpage><lpage>7423</lpage><history><date date-type="received"><day>9</day><month>3</month><year>2007</year></date><date date-type="accepted"><day>3</day><month>5</month><year>2007</year></date></history><copyright-statement>Copyright © 2007, American Society for Microbiology</copyright-statement><copyright-year>2007</copyright-year><self-uri xlink:title="pdf" xlink:href="zjv01407007410.pdf"></self-uri><abstract><p>The severe acute respiratory syndrome (SARS) epidemic was characterized by high mortality rates in the elderly. The molecular mechanisms that govern enhanced susceptibility of elderly populations are not known, and robust animal models are needed that recapitulate the increased pathogenic phenotype noted with increasing age. Using synthetic biology and reverse genetics, we describe the construction of a panel of isogenic SARS coronavirus (SARS-CoV) strains bearing variant spike glycoproteins that are representative of zoonotic strains found in palm civets and raccoon dogs, as well as isolates spanning the early, middle, and late phases of the SARS-CoV epidemic. The recombinant viruses replicated efficiently in cell culture and demonstrated variable sensitivities to neutralization with antibodies. The human but not the zoonotic variants replicated efficiently in human airway epithelial cultures, supporting earlier hypotheses that zoonotic isolates are less pathogenic in humans but can evolve into highly pathogenic strains. All viruses replicated efficiently, but none produced clinical disease or death in young animals. In contrast, severe clinical disease, diffuse alveolar damage, hyaline membrane formation, alveolitis, and death were noted in 12-month-old mice inoculated with the palm civet HC/SZ/61/03 strain or early-human-phase GZ02 variants but not with related middle- and late-phase epidemic or raccoon dog strains. This panel of SARS-CoV recombinants bearing zoonotic and human epidemic spike glycoproteins will provide heterologous challenge models for testing vaccine efficacy against zoonotic reintroductions as well as provide the appropriate model system for elucidating the complex virus-host interactions that contribute to more-severe and fatal SARS-CoV disease and acute respiratory distress in the elderly.</p></abstract></article-meta></front><floats-wrap><fig position="float" id="f1"><label>FIG. 1.</label><caption><p>(A) Phylogenetic analysis of SARS-CoV variants isolated during the zoonotic and human phases of the epidemic. An unrooted phylogenetic gene tree of the complete genomes of 27 SARS-CoV isolates ranging from those collected from humans in early, middle, and late phases of the epidemic of 2002 of 2003 to zoonotic isolates collected in 2003 and 2004 is presented. Isolates selected as representative of spike glycoprotein sequences of zoonotic (HC/SZ/61/03 and A031G) and early (GZ02)-, middle (CUHK-W1)-, and late (Urbani)-phase human isolates are shown in shaded boxes. The black bar represents percent divergence. (B) Amino acid changes in the SARS-CoV spike glycoprotein of zoonotic and human epidemic isolates. A.A., amino acid; A031G, raccoon dog; HC/SZ/61/03, palm civet; GZ02, early human phase; CUHK-W1, middle human phase; Urbani, late human phase.</p></caption><graphic xlink:href="zjv0140793640001"></graphic></fig><fig position="float" id="f2"><label>FIG. 2.</label><caption><p>In vitro growth characteristics of recombinant SARS-CoV spike glycoprotein variants and their use of ACE-2 and cathepsin L for binding and entry. (A and B) Cultures of Vero E6 cells (A) or human airway epithelium cells (B) were infected in duplicate with the S glycoprotein variants at a high MOI of 2 or >1, respectively, as described in Materials and Methods. Virus titers at different time points were determined by a plaque assay using Vero E6 cells. (C and D) HAE cell cultures infected with the icUrbani (C) or icHC/SZ/61/03 (D) isolate were stained for icSARS-CoV nucleoprotein (green) and alpha-tubulin (red) with mouse MAbs as described in Materials and Methods, with original magnifications of ×100. Staining of HAE infected with icUrbani was representative of icCUHK-W1 and icGZ02 and of HAE infected with icHC/SZ/61/03 was representative of icA031G. (E and F) To determine the role of ACE-2 and cathepsin L in binding and entry of the different S variants, Vero E6 cells were pretreated with polyclonal antibodies directed against either human ACE-2 (E) or cathepsin L inhibitor III (F) and infected with recombinant SARS-CoV spike glycoprotein variants as described in Materials and Methods. Blocking results are expressed as the mean percentages of plaque numbers in anti-human ACE2- or cathepsin L inhibitor-treated Vero E6 cells relative to untreated cell results. + with solid black line, icUrbani; ○ with dashed black line, icCUHK-W1; × with dotted black line, icGZ02; □ with solid gray line, icHC/SZ/61/03; Δ with dotted gray line, icA031G. Error bars represent standard deviations.</p></caption><graphic xlink:href="zjv0140793640002"></graphic></fig><fig position="float" id="f3"><label>FIG. 3.</label><caption><p>Homologous and heterologous neutralization of recombinant SARS-CoV spike glycoprotein variants by mouse monoclonal antibodies and human convalescent-phase sera. Serial diluted samples of mouse monoclonal antibodies (341CD, 540CD, and 283CD) and seven convalescent human serum samples from SARS cases (Human sera) were tested for the neutralizing ability of 100 PFU of different recombinant SARS-CoV spike glycoprotein variants (icUrbani, icCUHK-W1, icGZ02, icHC/SZ/61/03, and icA031G) as described in Materials and Methods. Results are expressed as the dilution of antibody at which 50% of the viruses are neutralized. Error bars represent standard deviations.</p></caption><graphic xlink:href="zjv0140793640003"></graphic></fig><fig position="float" id="f4"><label>FIG. 4.</label><caption><p>Weight loss and lung titer results for 6-week-old female BALB/c mice infected with recombinant SARS-CoV spike glycoprotein variants icUrbani (+), icCUHK-W1 (○), icGZ02 (×), icHC/SZ/61/03 (□), and icA031G (Δ) and mock infected (diao). Mice were intranasally inoculated with 10<sup>5</sup> PFU of the SARS-CoV S variants in 50 μl PBS. (A) Body weights of infected mice were measured on a daily basis (<italic>n</italic> = 10 per group until day 2 and 5 per group until day 4). Weight changes are expressed as the mean percent changes for infected animals relative to the initial weights at day 0. (B) Lung tissues were harvested from infected mice on day 2 (black bars) and day 4 (white bars) postinfection and assayed for infectious virus as described in Materials and Methods. Tissue samples from five mice were analyzed at each time point. Error bars represent standard deviations.</p></caption><graphic xlink:href="zjv0140793640004"></graphic></fig><fig position="float" id="f5"><label>FIG. 5.</label><caption><p>Weight loss, mortality, and lung titers of 12-month-old female BALB/c mice infected with recombinant SARS-CoV spike glycoprotein variants icUrbani (+), icCUHK-W1 (○), icGZ02 (×), icHC/SZ/61/03 (□), and icA031G (Δ) and mock infected (diao). Mice were intranasally inoculated with 10<sup>5</sup> PFU of the SARS-CoV S variants in 50 μl PBS. (A and B) Body weights (A) and accumulated mortality (B) of infected mice were measured on a daily basis (<italic>n</italic> = 10 per group until day 2 and <italic>n</italic> = 5 per group until day 4). Weight changes are expressed as the mean percent changes in infected animal relative to the initial weights at day 0. (C) Lung tissues were harvested from infected mice on day 2 (black bars) and day 4 (white bars) postinfection and assayed for infectious virus as described in Materials and Methods. Tissue samples from five mice were analyzed at each time point. Error bars represent standard deviations.</p></caption><graphic xlink:href="zjv0140793640005"></graphic></fig><fig position="float" id="f6"><label>FIG. 6.</label><caption><p>Light photographs of preterminal bronchioles and terminal bronchioles (PB and TB, respectively) in the lungs of 6-week-old (A, C, and E) or 12-month-old (B, D, and F) BALB/c mice that were mock inoculated (A and B) or inoculated with recombinant SARS-CoV S glycoprotein variant isGZ02 (C and D) or icHC/SZ/61/03 (E and F) and sacrificed 4 days postinoculation. Bronchiolitis with marked attenuation or loss (arrows with closed arrowheads) of the surface epithelium (e) lining the bronchiolar lumen is present in bronchioles of 12-month-old mice infected with the viral variants (D and F) but not in the much younger, 6-week-old mice that were similarly exposed to these viral strains (C and E). Virus-induced peribronchiolar inflammation is evident in both young and old mice (dotted arrows with open arrowheads). AL, alveoli; AD, alveolar ducts; BV, blood vessels. Tissues were stained with hematoxylin and eosin. Scaling bars, 50 μm.</p></caption><graphic xlink:href="zjv0140793640006"></graphic></fig><fig position="float" id="f7"><label>FIG. 7.</label><caption><p>Light photographs of preterminal bronchioles and terminal bronchioles (PB and TB, respectively) in the lungs of 12-month-old BALB/c mice that were mock inoculated (A) or inoculated with recombinant SARS-CoV spike glycoprotein variant icUrbani (B), icGZ02 (C), icHC/SZ/61/03 (D), icCUHK-W1 (E), or icA031G (F) and sacrificed 2 days after inoculation. Acute necrotizing bronchiolitis with epithelial cell exfoliation (solid arrows with closed arrowhead) is most prominent in panels C and D, with lesions of lesser severity in the mice infected with the other S variants (B, E, and F). No epithelial (e) or inflammatory lesions are present in the bronchiole from the control mouse (A). AL, alveoli; AD, alveolar ducts; BV, blood vessels; AS, alveolar septa. Dotted arrows with open arrowheads represent peribronchiolar inflammatory cell infiltration. Tissues were stained with hematoxylin and eosin. Scaling bars, 50 μm.</p></caption><graphic xlink:href="zjv0140793640007"></graphic></fig><fig position="float" id="f8"><label>FIG. 8.</label><caption><p>Light photomicrographs of alveolar parenchyma from 12-month-old mice that were mock inoculated (A) or inoculated with recombinant SARS-CoV spike glycoprotein variant icUrbani (B), icGZ02 (C), icHC/SZ/61/03 (D), icCUHK-W1 (E), or icA031G (F) and sacrificed 4 days after inoculation. No microscopic lesions are evident in panel A, B, E, or F. Diffuse acute alveolitis with alveolar septal congestion (dotted arrows with open arrowhead) and numerous hyaline membranes in alveolar airspaces (arrows with closed arrowheads) are present in panels C and D. AL, alveoli; AD, alveolar ducts; BV, blood vessels. Tissues were stained with hematoxylin and eosin. Scaling bars, 50 μm.</p></caption><graphic xlink:href="zjv0140793640008"></graphic></fig></floats-wrap></pmc></record>Pour manipuler ce document sous Unix (Dilib)
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