MERS-coronavirus replication induces severe in vitro cytopathology and is strongly inhibited by cyclosporin A or interferon-α treatment
Identifieur interne : 001144 ( Pmc/Corpus ); précédent : 001143; suivant : 001145MERS-coronavirus replication induces severe in vitro cytopathology and is strongly inhibited by cyclosporin A or interferon-α treatment
Auteurs : Adriaan H. De Wilde ; V. Stalin Raj ; Diede Oudshoorn ; Theo M. Bestebroer ; Stefan Van Nieuwkoop ; Ronald W. A. L. Limpens ; Clara C. Posthuma ; Yvonne Van Der Meer ; Montserrat Bárcena ; Bart L. Haagmans ; Eric J. Snijder ; Bernadette G. Van Den HoogenSource :
- The Journal of General Virology [ 0022-1317 ] ; 2013.
Abstract
Coronavirus (CoV) infections are commonly associated with respiratory and enteric disease in humans and animals. The 2003 outbreak of severe acute respiratory syndrome (SARS) highlighted the potentially lethal consequences of CoV-induced disease in humans. In 2012, a novel CoV (Middle East Respiratory Syndrome coronavirus; MERS-CoV) emerged, causing 49 human cases thus far, of which 23 had a fatal outcome. In this study, we characterized MERS-CoV replication and cytotoxicity in human and monkey cell lines. Electron microscopy of infected Vero cells revealed extensive membrane rearrangements, including the formation of double-membrane vesicles and convoluted membranes, which have been implicated previously in the RNA synthesis of SARS-CoV and other CoVs. Following infection, we observed rapidly increasing viral RNA synthesis and release of high titres of infectious progeny, followed by a pronounced cytopathology. These characteristics were used to develop an assay for antiviral compound screening in 96-well format, which was used to identify cyclosporin A as an inhibitor of MERS-CoV replication in cell culture. Furthermore, MERS-CoV was found to be 50–100 times more sensitive to alpha interferon (IFN-α) treatment than SARS-CoV, an observation that may have important implications for the treatment of MERS-CoV-infected patients. MERS-CoV infection did not prevent the IFN-induced nuclear translocation of phosphorylated STAT1, in contrast to infection with SARS-CoV where this block inhibits the expression of antiviral genes. These findings highlight relevant differences between these distantly related zoonotic CoVs in terms of their interaction with and evasion of the cellular innate immune response.
Url:
DOI: 10.1099/vir.0.052910-0
PubMed: 23620378
PubMed Central: 3749523
Links to Exploration step
PMC:3749523Le document en format XML
<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">MERS-coronavirus replication induces severe <italic>in vitro</italic> cytopathology and is strongly inhibited by cyclosporin A or interferon-α treatment</title><author><name sortKey="De Wilde, Adriaan H" sort="De Wilde, Adriaan H" uniqKey="De Wilde A" first="Adriaan H." last="De Wilde">Adriaan H. De Wilde</name><affiliation><nlm:aff id="aff1">Molecular Virology Laboratory, Department of Medical Microbiology, Leiden University Medical Center, Leiden, The Netherlands</nlm:aff></affiliation></author><author><name sortKey="Raj, V Stalin" sort="Raj, V Stalin" uniqKey="Raj V" first="V. Stalin" last="Raj">V. Stalin Raj</name><affiliation><nlm:aff id="aff2">Viroscience Lab, Erasmus MC, Rotterdam, The Netherlands</nlm:aff></affiliation></author><author><name sortKey="Oudshoorn, Diede" sort="Oudshoorn, Diede" uniqKey="Oudshoorn D" first="Diede" last="Oudshoorn">Diede Oudshoorn</name><affiliation><nlm:aff id="aff1">Molecular Virology Laboratory, Department of Medical Microbiology, Leiden University Medical Center, Leiden, The Netherlands</nlm:aff></affiliation></author><author><name sortKey="Bestebroer, Theo M" sort="Bestebroer, Theo M" uniqKey="Bestebroer T" first="Theo M." last="Bestebroer">Theo M. Bestebroer</name><affiliation><nlm:aff id="aff2">Viroscience Lab, Erasmus MC, Rotterdam, The Netherlands</nlm:aff></affiliation></author><author><name sortKey="Van Nieuwkoop, Stefan" sort="Van Nieuwkoop, Stefan" uniqKey="Van Nieuwkoop S" first="Stefan" last="Van Nieuwkoop">Stefan Van Nieuwkoop</name><affiliation><nlm:aff id="aff2">Viroscience Lab, Erasmus MC, Rotterdam, The Netherlands</nlm:aff></affiliation></author><author><name sortKey="Limpens, Ronald W A L" sort="Limpens, Ronald W A L" uniqKey="Limpens R" first="Ronald W. A. L." last="Limpens">Ronald W. A. L. Limpens</name><affiliation><nlm:aff id="aff3">Section Electron Microscopy, Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, The Netherlands</nlm:aff></affiliation></author><author><name sortKey="Posthuma, Clara C" sort="Posthuma, Clara C" uniqKey="Posthuma C" first="Clara C." last="Posthuma">Clara C. Posthuma</name><affiliation><nlm:aff id="aff1">Molecular Virology Laboratory, Department of Medical Microbiology, Leiden University Medical Center, Leiden, The Netherlands</nlm:aff></affiliation></author><author><name sortKey="Van Der Meer, Yvonne" sort="Van Der Meer, Yvonne" uniqKey="Van Der Meer Y" first="Yvonne" last="Van Der Meer">Yvonne Van Der Meer</name><affiliation><nlm:aff id="aff1">Molecular Virology Laboratory, Department of Medical Microbiology, Leiden University Medical Center, Leiden, The Netherlands</nlm:aff></affiliation></author><author><name sortKey="Barcena, Montserrat" sort="Barcena, Montserrat" uniqKey="Barcena M" first="Montserrat" last="Bárcena">Montserrat Bárcena</name><affiliation><nlm:aff id="aff3">Section Electron Microscopy, Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, The Netherlands</nlm:aff></affiliation></author><author><name sortKey="Haagmans, Bart L" sort="Haagmans, Bart L" uniqKey="Haagmans B" first="Bart L." last="Haagmans">Bart L. Haagmans</name><affiliation><nlm:aff id="aff2">Viroscience Lab, Erasmus MC, Rotterdam, The Netherlands</nlm:aff></affiliation></author><author><name sortKey="Snijder, Eric J" sort="Snijder, Eric J" uniqKey="Snijder E" first="Eric J." last="Snijder">Eric J. Snijder</name><affiliation><nlm:aff id="aff1">Molecular Virology Laboratory, Department of Medical Microbiology, Leiden University Medical Center, Leiden, The Netherlands</nlm:aff></affiliation></author><author><name sortKey="Van Den Hoogen, Bernadette G" sort="Van Den Hoogen, Bernadette G" uniqKey="Van Den Hoogen B" first="Bernadette G." last="Van Den Hoogen">Bernadette G. Van Den Hoogen</name><affiliation><nlm:aff id="aff2">Viroscience Lab, Erasmus MC, Rotterdam, The Netherlands</nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">23620378</idno><idno type="pmc">3749523</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3749523</idno><idno type="RBID">PMC:3749523</idno><idno type="doi">10.1099/vir.0.052910-0</idno><date when="2013">2013</date><idno type="wicri:Area/Pmc/Corpus">001144</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">001144</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">MERS-coronavirus replication induces severe <italic>in vitro</italic> cytopathology and is strongly inhibited by cyclosporin A or interferon-α treatment</title><author><name sortKey="De Wilde, Adriaan H" sort="De Wilde, Adriaan H" uniqKey="De Wilde A" first="Adriaan H." last="De Wilde">Adriaan H. De Wilde</name><affiliation><nlm:aff id="aff1">Molecular Virology Laboratory, Department of Medical Microbiology, Leiden University Medical Center, Leiden, The Netherlands</nlm:aff></affiliation></author><author><name sortKey="Raj, V Stalin" sort="Raj, V Stalin" uniqKey="Raj V" first="V. Stalin" last="Raj">V. Stalin Raj</name><affiliation><nlm:aff id="aff2">Viroscience Lab, Erasmus MC, Rotterdam, The Netherlands</nlm:aff></affiliation></author><author><name sortKey="Oudshoorn, Diede" sort="Oudshoorn, Diede" uniqKey="Oudshoorn D" first="Diede" last="Oudshoorn">Diede Oudshoorn</name><affiliation><nlm:aff id="aff1">Molecular Virology Laboratory, Department of Medical Microbiology, Leiden University Medical Center, Leiden, The Netherlands</nlm:aff></affiliation></author><author><name sortKey="Bestebroer, Theo M" sort="Bestebroer, Theo M" uniqKey="Bestebroer T" first="Theo M." last="Bestebroer">Theo M. Bestebroer</name><affiliation><nlm:aff id="aff2">Viroscience Lab, Erasmus MC, Rotterdam, The Netherlands</nlm:aff></affiliation></author><author><name sortKey="Van Nieuwkoop, Stefan" sort="Van Nieuwkoop, Stefan" uniqKey="Van Nieuwkoop S" first="Stefan" last="Van Nieuwkoop">Stefan Van Nieuwkoop</name><affiliation><nlm:aff id="aff2">Viroscience Lab, Erasmus MC, Rotterdam, The Netherlands</nlm:aff></affiliation></author><author><name sortKey="Limpens, Ronald W A L" sort="Limpens, Ronald W A L" uniqKey="Limpens R" first="Ronald W. A. L." last="Limpens">Ronald W. A. L. Limpens</name><affiliation><nlm:aff id="aff3">Section Electron Microscopy, Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, The Netherlands</nlm:aff></affiliation></author><author><name sortKey="Posthuma, Clara C" sort="Posthuma, Clara C" uniqKey="Posthuma C" first="Clara C." last="Posthuma">Clara C. Posthuma</name><affiliation><nlm:aff id="aff1">Molecular Virology Laboratory, Department of Medical Microbiology, Leiden University Medical Center, Leiden, The Netherlands</nlm:aff></affiliation></author><author><name sortKey="Van Der Meer, Yvonne" sort="Van Der Meer, Yvonne" uniqKey="Van Der Meer Y" first="Yvonne" last="Van Der Meer">Yvonne Van Der Meer</name><affiliation><nlm:aff id="aff1">Molecular Virology Laboratory, Department of Medical Microbiology, Leiden University Medical Center, Leiden, The Netherlands</nlm:aff></affiliation></author><author><name sortKey="Barcena, Montserrat" sort="Barcena, Montserrat" uniqKey="Barcena M" first="Montserrat" last="Bárcena">Montserrat Bárcena</name><affiliation><nlm:aff id="aff3">Section Electron Microscopy, Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, The Netherlands</nlm:aff></affiliation></author><author><name sortKey="Haagmans, Bart L" sort="Haagmans, Bart L" uniqKey="Haagmans B" first="Bart L." last="Haagmans">Bart L. Haagmans</name><affiliation><nlm:aff id="aff2">Viroscience Lab, Erasmus MC, Rotterdam, The Netherlands</nlm:aff></affiliation></author><author><name sortKey="Snijder, Eric J" sort="Snijder, Eric J" uniqKey="Snijder E" first="Eric J." last="Snijder">Eric J. Snijder</name><affiliation><nlm:aff id="aff1">Molecular Virology Laboratory, Department of Medical Microbiology, Leiden University Medical Center, Leiden, The Netherlands</nlm:aff></affiliation></author><author><name sortKey="Van Den Hoogen, Bernadette G" sort="Van Den Hoogen, Bernadette G" uniqKey="Van Den Hoogen B" first="Bernadette G." last="Van Den Hoogen">Bernadette G. Van Den Hoogen</name><affiliation><nlm:aff id="aff2">Viroscience Lab, Erasmus MC, Rotterdam, The Netherlands</nlm:aff></affiliation></author></analytic><series><title level="j">The Journal of General Virology</title><idno type="ISSN">0022-1317</idno><idno type="eISSN">1465-2099</idno><imprint><date when="2013">2013</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>Coronavirus (CoV) infections are commonly associated with respiratory and enteric disease in humans and animals. The 2003 outbreak of severe acute respiratory syndrome (SARS) highlighted the potentially lethal consequences of CoV-induced disease in humans. In 2012, a novel CoV (Middle East Respiratory Syndrome coronavirus; MERS-CoV) emerged, causing 49 human cases thus far, of which 23 had a fatal outcome. In this study, we characterized MERS-CoV replication and cytotoxicity in human and monkey cell lines. Electron microscopy of infected Vero cells revealed extensive membrane rearrangements, including the formation of double-membrane vesicles and convoluted membranes, which have been implicated previously in the RNA synthesis of SARS-CoV and other CoVs. Following infection, we observed rapidly increasing viral RNA synthesis and release of high titres of infectious progeny, followed by a pronounced cytopathology. These characteristics were used to develop an assay for antiviral compound screening in 96-well format, which was used to identify cyclosporin A as an inhibitor of MERS-CoV replication in cell culture. Furthermore, MERS-CoV was found to be 50–100 times more sensitive to alpha interferon (IFN-α) treatment than SARS-CoV, an observation that may have important implications for the treatment of MERS-CoV-infected patients. MERS-CoV infection did not prevent the IFN-induced nuclear translocation of phosphorylated STAT1, in contrast to infection with SARS-CoV where this block inhibits the expression of antiviral genes. These findings highlight relevant differences between these distantly related zoonotic CoVs in terms of their interaction with and evasion of the cellular innate immune response.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Belouzard, S" uniqKey="Belouzard S">S. Belouzard</name></author><author><name sortKey="Millet, J K" uniqKey="Millet J">J. K. Millet</name></author><author><name sortKey="Licitra, B N" uniqKey="Licitra B">B. N. Licitra</name></author><author><name sortKey="Whittaker, G R" uniqKey="Whittaker G">G. R. Whittaker</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Bergman, S J" uniqKey="Bergman S">S. J. Bergman</name></author><author><name sortKey="Ferguson, M C" uniqKey="Ferguson M">M. C. Ferguson</name></author><author><name sortKey="Santanello, C" uniqKey="Santanello C">C. Santanello</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Brockway, S M" uniqKey="Brockway S">S. M. Brockway</name></author><author><name sortKey="Clay, C T" uniqKey="Clay C">C. T. Clay</name></author><author><name sortKey="Lu, X T" uniqKey="Lu X">X. T. Lu</name></author><author><name sortKey="Denison, M R" uniqKey="Denison M">M. R. Denison</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Cameron, M J" uniqKey="Cameron M">M. J. Cameron</name></author><author><name sortKey="Kelvin, A A" uniqKey="Kelvin A">A. A. Kelvin</name></author><author><name sortKey="Leon, A J" uniqKey="Leon A">A. J. Leon</name></author><author><name sortKey="Cameron, C M" uniqKey="Cameron C">C. M. Cameron</name></author><author><name sortKey="Ran, L" uniqKey="Ran L">L. Ran</name></author><author><name sortKey="Xu, L" uniqKey="Xu L">L. Xu</name></author><author><name sortKey="Chu, Y K" uniqKey="Chu Y">Y. K. Chu</name></author><author><name sortKey="Danesh, A" uniqKey="Danesh A">A. Danesh</name></author><author><name sortKey="Fang, Y" uniqKey="Fang Y">Y. Fang</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Cervantes Barragan, L" uniqKey="Cervantes Barragan L">L. Cervantes-Barragan</name></author><author><name sortKey="Zust, R" uniqKey="Zust R">R. Züst</name></author><author><name sortKey="Weber, F" uniqKey="Weber F">F. Weber</name></author><author><name sortKey="Spiegel, M" uniqKey="Spiegel M">M. Spiegel</name></author><author><name sortKey="Lang, K S" uniqKey="Lang K">K. S. Lang</name></author><author><name sortKey="Akira, S" uniqKey="Akira S">S. Akira</name></author><author><name sortKey="Thiel, V" uniqKey="Thiel V">V. Thiel</name></author><author><name sortKey="Ludewig, B" uniqKey="Ludewig B">B. Ludewig</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="De Groot, R J" uniqKey="De Groot R">R. J. de Groot</name></author><author><name sortKey="Cowley, J A" uniqKey="Cowley J">J. A. Cowley</name></author><author><name sortKey="Enjuanes, L" uniqKey="Enjuanes L">L. Enjuanes</name></author><author><name sortKey="Faaberg, K S" uniqKey="Faaberg K">K. S. Faaberg</name></author><author><name sortKey="Perlman, S" uniqKey="Perlman S">S. Perlman</name></author><author><name sortKey="Rottier, P J" uniqKey="Rottier P">P. J. Rottier</name></author><author><name sortKey="Snijder, E J" uniqKey="Snijder E">E. J. Snijder</name></author><author><name sortKey="Ziebuhr, J" uniqKey="Ziebuhr J">J. Ziebuhr</name></author><author><name sortKey="Gorbalenya, A E" uniqKey="Gorbalenya A">A. E. Gorbalenya</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="De Groot, R J" uniqKey="De Groot R">R. J. de Groot</name></author><author><name sortKey="Baker, S C" uniqKey="Baker S">S. C. Baker</name></author><author><name sortKey="Baric, R S" uniqKey="Baric R">R. S. Baric</name></author><author><name sortKey="Brown, C S" uniqKey="Brown C">C. S. Brown</name></author><author><name sortKey="Drosten, C" uniqKey="Drosten C">C. Drosten</name></author><author><name sortKey="Enjuanes, L" uniqKey="Enjuanes L">L. Enjuanes</name></author><author><name sortKey="Fouchier, R A" uniqKey="Fouchier R">R. A. Fouchier</name></author><author><name sortKey="Galiano, M" uniqKey="Galiano M">M. Galiano</name></author><author><name sortKey="Gorbalenya, A E" uniqKey="Gorbalenya A">A. E. Gorbalenya</name></author><author><name sortKey="Other Authors" uniqKey="Other Authors">other authors</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="De Wilde, A H" uniqKey="De Wilde A">A. H. de Wilde</name></author><author><name sortKey="Zevenhoven Dobbe, J C" uniqKey="Zevenhoven Dobbe J">J. C. Zevenhoven-Dobbe</name></author><author><name sortKey="Van Der Meer, Y" uniqKey="Van Der Meer Y">Y. van der Meer</name></author><author><name sortKey="Thiel, V" uniqKey="Thiel V">V. Thiel</name></author><author><name sortKey="Narayanan, K" uniqKey="Narayanan K">K. Narayanan</name></author><author><name sortKey="Makino, S" uniqKey="Makino S">S. Makino</name></author><author><name sortKey="Snijder, E J" uniqKey="Snijder E">E. J. Snijder</name></author><author><name sortKey="Van Hemert, M J" uniqKey="Van Hemert M">M. J. van Hemert</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Drosten, C" uniqKey="Drosten C">C. Drosten</name></author><author><name sortKey="Gunther, S" uniqKey="Gunther S">S. Günther</name></author><author><name sortKey="Preiser, W" uniqKey="Preiser W">W. Preiser</name></author><author><name sortKey="Van Der Werf, S" uniqKey="Van Der Werf S">S. van der Werf</name></author><author><name sortKey="Brodt, H R" uniqKey="Brodt H">H. R. Brodt</name></author><author><name sortKey="Becker, S" uniqKey="Becker S">S. Becker</name></author><author><name sortKey="Rabenau, H" uniqKey="Rabenau H">H. Rabenau</name></author><author><name sortKey="Panning, M" uniqKey="Panning M">M. Panning</name></author><author><name sortKey="Kolesnikova, L" uniqKey="Kolesnikova L">L. Kolesnikova</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Fouchier, R A" uniqKey="Fouchier R">R. A. Fouchier</name></author><author><name sortKey="Hartwig, N G" uniqKey="Hartwig N">N. G. Hartwig</name></author><author><name sortKey="Bestebroer, T M" uniqKey="Bestebroer T">T. M. Bestebroer</name></author><author><name sortKey="Niemeyer, B" uniqKey="Niemeyer B">B. Niemeyer</name></author><author><name sortKey="De Jong, J C" uniqKey="De Jong J">J. C. de Jong</name></author><author><name sortKey="Simon, J H" uniqKey="Simon J">J. H. Simon</name></author><author><name sortKey="Osterhaus, A D" uniqKey="Osterhaus A">A. D. Osterhaus</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Frieman, M" uniqKey="Frieman M">M. Frieman</name></author><author><name sortKey="Yount, B" uniqKey="Yount B">B. Yount</name></author><author><name sortKey="Heise, M" uniqKey="Heise M">M. Heise</name></author><author><name sortKey="Kopecky Bromberg, S A" uniqKey="Kopecky Bromberg S">S. A. Kopecky-Bromberg</name></author><author><name sortKey="Palese, P" uniqKey="Palese P">P. Palese</name></author><author><name sortKey="Baric, R S" uniqKey="Baric R">R. S. Baric</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Garlinghouse, L E" uniqKey="Garlinghouse L">L. E. Garlinghouse</name></author><author><name sortKey="Smith, A L" uniqKey="Smith A">A. L. Smith</name></author><author><name sortKey="Holford, T" uniqKey="Holford T">T. Holford</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Gorbalenya, A E" uniqKey="Gorbalenya A">A. E. Gorbalenya</name></author><author><name sortKey="Enjuanes, L" uniqKey="Enjuanes L">L. Enjuanes</name></author><author><name sortKey="Ziebuhr, J" uniqKey="Ziebuhr J">J. Ziebuhr</name></author><author><name sortKey="Snijder, E J" uniqKey="Snijder E">E. J. Snijder</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Gosert, R" uniqKey="Gosert R">R. Gosert</name></author><author><name sortKey="Kanjanahaluethai, A" uniqKey="Kanjanahaluethai A">A. Kanjanahaluethai</name></author><author><name sortKey="Egger, D" uniqKey="Egger D">D. Egger</name></author><author><name sortKey="Bienz, K" uniqKey="Bienz K">K. Bienz</name></author><author><name sortKey="Baker, S C" uniqKey="Baker S">S. C. Baker</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Haagmans, B L" uniqKey="Haagmans B">B. L. Haagmans</name></author><author><name sortKey="Kuiken, T" uniqKey="Kuiken T">T. Kuiken</name></author><author><name sortKey="Martina, B E" uniqKey="Martina B">B. E. Martina</name></author><author><name sortKey="Fouchier, R A" uniqKey="Fouchier R">R. A. Fouchier</name></author><author><name sortKey="Rimmelzwaan, G F" uniqKey="Rimmelzwaan G">G. F. Rimmelzwaan</name></author><author><name sortKey="Van Amerongen, G" uniqKey="Van Amerongen G">G. van Amerongen</name></author><author><name sortKey="Van Riel, D" uniqKey="Van Riel D">D. van Riel</name></author><author><name sortKey="De Jong, T" uniqKey="De Jong T">T. de Jong</name></author><author><name sortKey="Itamura, S" uniqKey="Itamura S">S. Itamura</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Hamre, D" uniqKey="Hamre D">D. Hamre</name></author><author><name sortKey="Procknow, J J" uniqKey="Procknow J">J. J. Procknow</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Hussain, S" uniqKey="Hussain S">S. Hussain</name></author><author><name sortKey="Perlman, S" uniqKey="Perlman S">S. Perlman</name></author><author><name sortKey="Gallagher, T M" uniqKey="Gallagher T">T. M. Gallagher</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Huynh, J" uniqKey="Huynh J">J. Huynh</name></author><author><name sortKey="Li, S" uniqKey="Li S">S. Li</name></author><author><name sortKey="Yount, B" uniqKey="Yount B">B. Yount</name></author><author><name sortKey="Smith, A" uniqKey="Smith A">A. Smith</name></author><author><name sortKey="Sturges, L" uniqKey="Sturges L">L. Sturges</name></author><author><name sortKey="Olsen, J C" uniqKey="Olsen J">J. C. Olsen</name></author><author><name sortKey="Nagel, J" uniqKey="Nagel J">J. Nagel</name></author><author><name sortKey="Johnson, J B" uniqKey="Johnson J">J. B. Johnson</name></author><author><name sortKey="Agnihothram, S" uniqKey="Agnihothram S">S. Agnihothram</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Kindler, E" uniqKey="Kindler E">E. Kindler</name></author><author><name sortKey="J Nsd Ttir, H R" uniqKey="J Nsd Ttir H">H. R. Jónsdóttir</name></author><author><name sortKey="Muth, D" uniqKey="Muth D">D. Muth</name></author><author><name sortKey="Hamming, O J" uniqKey="Hamming O">O. J. Hamming</name></author><author><name sortKey="Hartmann, R" uniqKey="Hartmann R">R. Hartmann</name></author><author><name sortKey="Rodriguez, R" uniqKey="Rodriguez R">R. Rodriguez</name></author><author><name sortKey="Geffers, R" uniqKey="Geffers R">R. Geffers</name></author><author><name sortKey="Fouchier, R A" uniqKey="Fouchier R">R. A. Fouchier</name></author><author><name sortKey="Drosten, C" uniqKey="Drosten C">C. Drosten</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Knoops, K" uniqKey="Knoops K">K. Knoops</name></author><author><name sortKey="Kikkert, M" uniqKey="Kikkert M">M. Kikkert</name></author><author><name sortKey="Worm, S H" uniqKey="Worm S">S. H. Worm</name></author><author><name sortKey="Zevenhoven Dobbe, J C" uniqKey="Zevenhoven Dobbe J">J. C. Zevenhoven-Dobbe</name></author><author><name sortKey="Van Der Meer, Y" uniqKey="Van Der Meer Y">Y. van der Meer</name></author><author><name sortKey="Koster, A J" uniqKey="Koster A">A. J. Koster</name></author><author><name sortKey="Mommaas, A M" uniqKey="Mommaas A">A. M. Mommaas</name></author><author><name sortKey="Snijder, E J" uniqKey="Snijder E">E. J. Snijder</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Knoops, K" uniqKey="Knoops K">K. Knoops</name></author><author><name sortKey="Barcena, M" uniqKey="Barcena M">M. Bárcena</name></author><author><name sortKey="Limpens, R W" uniqKey="Limpens R">R. W. Limpens</name></author><author><name sortKey="Koster, A J" uniqKey="Koster A">A. J. Koster</name></author><author><name sortKey="Mommaas, A M" uniqKey="Mommaas A">A. M. Mommaas</name></author><author><name sortKey="Snijder, E J" uniqKey="Snijder E">E. J. Snijder</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Kopecky Bromberg, S A" uniqKey="Kopecky Bromberg S">S. A. Kopecky-Bromberg</name></author><author><name sortKey="Martinez Sobrido, L" uniqKey="Martinez Sobrido L">L. Martinez-Sobrido</name></author><author><name sortKey="Palese, P" uniqKey="Palese P">P. Palese</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Ksiazek, T G" uniqKey="Ksiazek T">T. G. Ksiazek</name></author><author><name sortKey="Erdman, D" uniqKey="Erdman D">D. Erdman</name></author><author><name sortKey="Goldsmith, C S" uniqKey="Goldsmith C">C. S. Goldsmith</name></author><author><name sortKey="Zaki, S R" uniqKey="Zaki S">S. R. Zaki</name></author><author><name sortKey="Peret, T" uniqKey="Peret T">T. Peret</name></author><author><name sortKey="Emery, S" uniqKey="Emery S">S. Emery</name></author><author><name sortKey="Tong, S" uniqKey="Tong S">S. Tong</name></author><author><name sortKey="Urbani, C" uniqKey="Urbani C">C. Urbani</name></author><author><name sortKey="Comer, J A" uniqKey="Comer J">J. A. Comer</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Kuiken, T" uniqKey="Kuiken T">T. Kuiken</name></author><author><name sortKey="Fouchier, R A" uniqKey="Fouchier R">R. A. Fouchier</name></author><author><name sortKey="Schutten, M" uniqKey="Schutten M">M. Schutten</name></author><author><name sortKey="Rimmelzwaan, G F" uniqKey="Rimmelzwaan G">G. F. Rimmelzwaan</name></author><author><name sortKey="Van Amerongen, G" uniqKey="Van Amerongen G">G. van Amerongen</name></author><author><name sortKey="Van Riel, D" uniqKey="Van Riel D">D. van Riel</name></author><author><name sortKey="Laman, J D" uniqKey="Laman J">J. D. Laman</name></author><author><name sortKey="De Jong, T" uniqKey="De Jong T">T. de Jong</name></author><author><name sortKey="Van Doornum, G" uniqKey="Van Doornum G">G. van Doornum</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Lau, S K" uniqKey="Lau S">S. K. Lau</name></author><author><name sortKey="Woo, P C" uniqKey="Woo P">P. C. Woo</name></author><author><name sortKey="Li, K S" uniqKey="Li K">K. S. Li</name></author><author><name sortKey="Huang, Y" uniqKey="Huang Y">Y. Huang</name></author><author><name sortKey="Tsoi, H W" uniqKey="Tsoi H">H. W. Tsoi</name></author><author><name sortKey="Wong, B H" uniqKey="Wong B">B. H. Wong</name></author><author><name sortKey="Wong, S S" uniqKey="Wong S">S. S. Wong</name></author><author><name sortKey="Leung, S Y" uniqKey="Leung S">S. Y. Leung</name></author><author><name sortKey="Chan, K H" uniqKey="Chan K">K. H. Chan</name></author><author><name sortKey="Yuen, K Y" uniqKey="Yuen K">K. Y. Yuen</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Li, W" uniqKey="Li W">W. Li</name></author><author><name sortKey="Shi, Z" uniqKey="Shi Z">Z. Shi</name></author><author><name sortKey="Yu, M" uniqKey="Yu M">M. Yu</name></author><author><name sortKey="Ren, W" uniqKey="Ren W">W. Ren</name></author><author><name sortKey="Smith, C" uniqKey="Smith C">C. Smith</name></author><author><name sortKey="Epstein, J H" uniqKey="Epstein J">J. H. Epstein</name></author><author><name sortKey="Wang, H" uniqKey="Wang H">H. Wang</name></author><author><name sortKey="Crameri, G" uniqKey="Crameri G">G. Crameri</name></author><author><name sortKey="Hu, Z" uniqKey="Hu Z">Z. Hu</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Mcintosh, K" uniqKey="Mcintosh K">K. McIntosh</name></author><author><name sortKey="Dees, J H" uniqKey="Dees J">J. H. Dees</name></author><author><name sortKey="Becker, W B" uniqKey="Becker W">W. B. Becker</name></author><author><name sortKey="Kapikian, A Z" uniqKey="Kapikian A">A. Z. Kapikian</name></author><author><name sortKey="Chanock, R M" uniqKey="Chanock R">R. M. Chanock</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Muller, M A" uniqKey="Muller M">M. A. Müller</name></author><author><name sortKey="Raj, V S" uniqKey="Raj V">V. S. Raj</name></author><author><name sortKey="Muth, D" uniqKey="Muth D">D. Muth</name></author><author><name sortKey="Meyer, B" uniqKey="Meyer B">B. Meyer</name></author><author><name sortKey="Kallies, S" uniqKey="Kallies S">S. Kallies</name></author><author><name sortKey="Smits, S L" uniqKey="Smits S">S. L. Smits</name></author><author><name sortKey="Wollny, R" uniqKey="Wollny R">R. Wollny</name></author><author><name sortKey="Bestebroer, T M" uniqKey="Bestebroer T">T. M. Bestebroer</name></author><author><name sortKey="Specht, S" uniqKey="Specht S">S. Specht</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Nagy, P D" uniqKey="Nagy P">P. D. Nagy</name></author><author><name sortKey="Wang, R Y" uniqKey="Wang R">R. Y. Wang</name></author><author><name sortKey="Pogany, J" uniqKey="Pogany J">J. Pogany</name></author><author><name sortKey="Hafren, A" uniqKey="Hafren A">A. Hafren</name></author><author><name sortKey="Makinen, K" uniqKey="Makinen K">K. Makinen</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Paragas, J" uniqKey="Paragas J">J. Paragas</name></author><author><name sortKey="Blatt, L M" uniqKey="Blatt L">L. M. Blatt</name></author><author><name sortKey="Hartmann, C" uniqKey="Hartmann C">C. Hartmann</name></author><author><name sortKey="Huggins, J W" uniqKey="Huggins J">J. W. Huggins</name></author><author><name sortKey="Endy, T P" uniqKey="Endy T">T. P. Endy</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Perlman, S" uniqKey="Perlman S">S. Perlman</name></author><author><name sortKey="Netland, J" uniqKey="Netland J">J. Netland</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Pfefferle, S" uniqKey="Pfefferle S">S. Pfefferle</name></author><author><name sortKey="Oppong, S" uniqKey="Oppong S">S. Oppong</name></author><author><name sortKey="Drexler, J F" uniqKey="Drexler J">J. F. Drexler</name></author><author><name sortKey="Gloza Rausch, F" uniqKey="Gloza Rausch F">F. Gloza-Rausch</name></author><author><name sortKey="Ipsen, A" uniqKey="Ipsen A">A. Ipsen</name></author><author><name sortKey="Seebens, A" uniqKey="Seebens A">A. Seebens</name></author><author><name sortKey="Muller, M A" uniqKey="Muller M">M. A. Müller</name></author><author><name sortKey="Annan, A" uniqKey="Annan A">A. Annan</name></author><author><name sortKey="Vallo, P" uniqKey="Vallo P">P. Vallo</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Pfefferle, S" uniqKey="Pfefferle S">S. Pfefferle</name></author><author><name sortKey="Schopf, J" uniqKey="Schopf J">J. Schöpf</name></author><author><name sortKey="Kogl, M" uniqKey="Kogl M">M. Kögl</name></author><author><name sortKey="Friedel, C C" uniqKey="Friedel C">C. C. Friedel</name></author><author><name sortKey="Muller, M A" uniqKey="Muller M">M. A. Müller</name></author><author><name sortKey="Carbajo Lozoya, J" uniqKey="Carbajo Lozoya J">J. Carbajo-Lozoya</name></author><author><name sortKey="Stellberger, T" uniqKey="Stellberger T">T. Stellberger</name></author><author><name sortKey="Von Dall Rmi, E" uniqKey="Von Dall Rmi E">E. von Dall’Armi</name></author><author><name sortKey="Herzog, P" uniqKey="Herzog P">P. Herzog</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Raj, V S" uniqKey="Raj V">V. S. Raj</name></author><author><name sortKey="Mou, H" uniqKey="Mou H">H. Mou</name></author><author><name sortKey="Smits, S L" uniqKey="Smits S">S. L. Smits</name></author><author><name sortKey="Dekkers, D H" uniqKey="Dekkers D">D. H. Dekkers</name></author><author><name sortKey="Muller, M A" uniqKey="Muller M">M. A. Müller</name></author><author><name sortKey="Dijkman, R" uniqKey="Dijkman R">R. Dijkman</name></author><author><name sortKey="Muth, D" uniqKey="Muth D">D. Muth</name></author><author><name sortKey="Demmers, J A" uniqKey="Demmers J">J. A. Demmers</name></author><author><name sortKey="Zaki, A" uniqKey="Zaki A">A. Zaki</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Randall, R E" uniqKey="Randall R">R. E. Randall</name></author><author><name sortKey="Goodbourn, S" uniqKey="Goodbourn S">S. Goodbourn</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Ratia, K" uniqKey="Ratia K">K. Ratia</name></author><author><name sortKey="Saikatendu, K S" uniqKey="Saikatendu K">K. S. Saikatendu</name></author><author><name sortKey="Santarsiero, B D" uniqKey="Santarsiero B">B. D. Santarsiero</name></author><author><name sortKey="Barretto, N" uniqKey="Barretto N">N. Barretto</name></author><author><name sortKey="Baker, S C" uniqKey="Baker S">S. C. Baker</name></author><author><name sortKey="Stevens, R C" uniqKey="Stevens R">R. C. Stevens</name></author><author><name sortKey="Mesecar, A D" uniqKey="Mesecar A">A. D. Mesecar</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Rose, K M" uniqKey="Rose K">K. M. Rose</name></author><author><name sortKey="Elliott, R" uniqKey="Elliott R">R. Elliott</name></author><author><name sortKey="Martinez Sobrido, L" uniqKey="Martinez Sobrido L">L. Martínez-Sobrido</name></author><author><name sortKey="Garcia Sastre, A" uniqKey="Garcia Sastre A">A. García-Sastre</name></author><author><name sortKey="Weiss, S R" uniqKey="Weiss S">S. R. Weiss</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Roth Cross, J K" uniqKey="Roth Cross J">J. K. Roth-Cross</name></author><author><name sortKey="Martinez Sobrido, L" uniqKey="Martinez Sobrido L">L. Martínez-Sobrido</name></author><author><name sortKey="Scott, E P" uniqKey="Scott E">E. P. Scott</name></author><author><name sortKey="Garcia Sastre, A" uniqKey="Garcia Sastre A">A. García-Sastre</name></author><author><name sortKey="Weiss, S R" uniqKey="Weiss S">S. R. Weiss</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Sims, A C" uniqKey="Sims A">A. C. Sims</name></author><author><name sortKey="Tilton, S C" uniqKey="Tilton S">S. C. Tilton</name></author><author><name sortKey="Menachery, V D" uniqKey="Menachery V">V. D. Menachery</name></author><author><name sortKey="Gralinski, L E" uniqKey="Gralinski L">L. E. Gralinski</name></author><author><name sortKey="Sch Fer, A" uniqKey="Sch Fer A">A. Schäfer</name></author><author><name sortKey="Matzke, M M" uniqKey="Matzke M">M. M. Matzke</name></author><author><name sortKey="Webb Robertson, B J" uniqKey="Webb Robertson B">B. J. Webb-Robertson</name></author><author><name sortKey="Chang, J" uniqKey="Chang J">J. Chang</name></author><author><name sortKey="Luna, M L" uniqKey="Luna M">M. L. Luna</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Snijder, E J" uniqKey="Snijder E">E. J. Snijder</name></author><author><name sortKey="Wassenaar, A L" uniqKey="Wassenaar A">A. L. Wassenaar</name></author><author><name sortKey="Spaan, W J" uniqKey="Spaan W">W. J. Spaan</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Snijder, E J" uniqKey="Snijder E">E. J. Snijder</name></author><author><name sortKey="Bredenbeek, P J" uniqKey="Bredenbeek P">P. J. Bredenbeek</name></author><author><name sortKey="Dobbe, J C" uniqKey="Dobbe J">J. C. Dobbe</name></author><author><name sortKey="Thiel, V" uniqKey="Thiel V">V. Thiel</name></author><author><name sortKey="Ziebuhr, J" uniqKey="Ziebuhr J">J. Ziebuhr</name></author><author><name sortKey="Poon, L L" uniqKey="Poon L">L. L. Poon</name></author><author><name sortKey="Guan, Y" uniqKey="Guan Y">Y. Guan</name></author><author><name sortKey="Rozanov, M" uniqKey="Rozanov M">M. Rozanov</name></author><author><name sortKey="Spaan, W J" uniqKey="Spaan W">W. J. Spaan</name></author><author><name sortKey="Gorbalenya, A E" uniqKey="Gorbalenya A">A. E. Gorbalenya</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Snijder, E J" uniqKey="Snijder E">E. J. Snijder</name></author><author><name sortKey="Van Der Meer, Y" uniqKey="Van Der Meer Y">Y. van der Meer</name></author><author><name sortKey="Zevenhoven Dobbe, J" uniqKey="Zevenhoven Dobbe J">J. Zevenhoven-Dobbe</name></author><author><name sortKey="Onderwater, J J" uniqKey="Onderwater J">J. J. Onderwater</name></author><author><name sortKey="Van Der Meulen, J" uniqKey="Van Der Meulen J">J. van der Meulen</name></author><author><name sortKey="Koerten, H K" uniqKey="Koerten H">H. K. Koerten</name></author><author><name sortKey="Mommaas, A M" uniqKey="Mommaas A">A. M. Mommaas</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Stertz, S" uniqKey="Stertz S">S. Stertz</name></author><author><name sortKey="Reichelt, M" uniqKey="Reichelt M">M. Reichelt</name></author><author><name sortKey="Spiegel, M" uniqKey="Spiegel M">M. Spiegel</name></author><author><name sortKey="Kuri, T" uniqKey="Kuri T">T. Kuri</name></author><author><name sortKey="Martinez Sobrido, L" uniqKey="Martinez Sobrido L">L. Martínez-Sobrido</name></author><author><name sortKey="Garcia Sastre, A" uniqKey="Garcia Sastre A">A. García-Sastre</name></author><author><name sortKey="Weber, F" uniqKey="Weber F">F. Weber</name></author><author><name sortKey="Kochs, G" uniqKey="Kochs G">G. Kochs</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Taguchi, F" uniqKey="Taguchi F">F. Taguchi</name></author><author><name sortKey="Siddell, S G" uniqKey="Siddell S">S. G. Siddell</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Ulasli, M" uniqKey="Ulasli M">M. Ulasli</name></author><author><name sortKey="Verheije, M H" uniqKey="Verheije M">M. H. Verheije</name></author><author><name sortKey="De Haan, C A" uniqKey="De Haan C">C. A. de Haan</name></author><author><name sortKey="Reggiori, F" uniqKey="Reggiori F">F. Reggiori</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Van Boheemen, S" uniqKey="Van Boheemen S">S. van Boheemen</name></author><author><name sortKey="De Graaf, M" uniqKey="De Graaf M">M. de Graaf</name></author><author><name sortKey="Lauber, C" uniqKey="Lauber C">C. Lauber</name></author><author><name sortKey="Bestebroer, T M" uniqKey="Bestebroer T">T. M. Bestebroer</name></author><author><name sortKey="Raj, V S" uniqKey="Raj V">V. S. Raj</name></author><author><name sortKey="Zaki, A M" uniqKey="Zaki A">A. M. Zaki</name></author><author><name sortKey="Osterhaus, A D" uniqKey="Osterhaus A">A. D. Osterhaus</name></author><author><name sortKey="Haagmans, B L" uniqKey="Haagmans B">B. L. Haagmans</name></author><author><name sortKey="Gorbalenya, A E" uniqKey="Gorbalenya A">A. E. Gorbalenya</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Van Den Worm, S H" uniqKey="Van Den Worm S">S. H. van den Worm</name></author><author><name sortKey="Eriksson, K K" uniqKey="Eriksson K">K. K. Eriksson</name></author><author><name sortKey="Zevenhoven, J C" uniqKey="Zevenhoven J">J. C. Zevenhoven</name></author><author><name sortKey="Weber, F" uniqKey="Weber F">F. Weber</name></author><author><name sortKey="Zust, R" uniqKey="Zust R">R. Züst</name></author><author><name sortKey="Kuri, T" uniqKey="Kuri T">T. Kuri</name></author><author><name sortKey="Dijkman, R" uniqKey="Dijkman R">R. Dijkman</name></author><author><name sortKey="Chang, G" uniqKey="Chang G">G. Chang</name></author><author><name sortKey="Siddell, S G" uniqKey="Siddell S">S. G. Siddell</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Van Der Hoek, L" uniqKey="Van Der Hoek L">L. van der Hoek</name></author><author><name sortKey="Pyrc, K" uniqKey="Pyrc K">K. Pyrc</name></author><author><name sortKey="Jebbink, M F" uniqKey="Jebbink M">M. F. Jebbink</name></author><author><name sortKey="Vermeulen Oost, W" uniqKey="Vermeulen Oost W">W. Vermeulen-Oost</name></author><author><name sortKey="Berkhout, R J" uniqKey="Berkhout R">R. J. Berkhout</name></author><author><name sortKey="Wolthers, K C" uniqKey="Wolthers K">K. C. Wolthers</name></author><author><name sortKey="Wertheim Van Dillen, P M" uniqKey="Wertheim Van Dillen P">P. M. Wertheim-van Dillen</name></author><author><name sortKey="Kaandorp, J" uniqKey="Kaandorp J">J. Kaandorp</name></author><author><name sortKey="Spaargaren, J" uniqKey="Spaargaren J">J. Spaargaren</name></author><author><name sortKey="Berkhout, B" uniqKey="Berkhout B">B. Berkhout</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Van Der Meer, Y" uniqKey="Van Der Meer Y">Y. van der Meer</name></author><author><name sortKey="Van Tol, H" uniqKey="Van Tol H">H. van Tol</name></author><author><name sortKey="Locker, J K" uniqKey="Locker J">J. K. Locker</name></author><author><name sortKey="Snijder, E J" uniqKey="Snijder E">E. J. Snijder</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Van Hemert, M J" uniqKey="Van Hemert M">M. J. van Hemert</name></author><author><name sortKey="De Wilde, A H" uniqKey="De Wilde A">A. H. de Wilde</name></author><author><name sortKey="Gorbalenya, A E" uniqKey="Gorbalenya A">A. E. Gorbalenya</name></author><author><name sortKey="Snijder, E J" uniqKey="Snijder E">E. J. Snijder</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Van Hemert, M J" uniqKey="Van Hemert M">M. J. van Hemert</name></author><author><name sortKey="Van Den Worm, S H" uniqKey="Van Den Worm S">S. H. van den Worm</name></author><author><name sortKey="Knoops, K" uniqKey="Knoops K">K. Knoops</name></author><author><name sortKey="Mommaas, A M" uniqKey="Mommaas A">A. M. Mommaas</name></author><author><name sortKey="Gorbalenya, A E" uniqKey="Gorbalenya A">A. E. Gorbalenya</name></author><author><name sortKey="Snijder, E J" uniqKey="Snijder E">E. J. Snijder</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Van Kasteren, P B" uniqKey="Van Kasteren P">P. B. van Kasteren</name></author><author><name sortKey="Bailey Elkin, B A" uniqKey="Bailey Elkin B">B. A. Bailey-Elkin</name></author><author><name sortKey="James, T W" uniqKey="James T">T. W. James</name></author><author><name sortKey="Ninaber, D K" uniqKey="Ninaber D">D. K. Ninaber</name></author><author><name sortKey="Beugeling, C" uniqKey="Beugeling C">C. Beugeling</name></author><author><name sortKey="Khajehpour, M" uniqKey="Khajehpour M">M. Khajehpour</name></author><author><name sortKey="Snijder, E J" uniqKey="Snijder E">E. J. Snijder</name></author><author><name sortKey="Mark, B L" uniqKey="Mark B">B. L. Mark</name></author><author><name sortKey="Kikkert, M" uniqKey="Kikkert M">M. Kikkert</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Versteeg, G A" uniqKey="Versteeg G">G. A. Versteeg</name></author><author><name sortKey="Bredenbeek, P J" uniqKey="Bredenbeek P">P. J. Bredenbeek</name></author><author><name sortKey="Van Den Worm, S H" uniqKey="Van Den Worm S">S. H. van den Worm</name></author><author><name sortKey="Spaan, W J" uniqKey="Spaan W">W. J. Spaan</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Vijaykrishna, D" uniqKey="Vijaykrishna D">D. Vijaykrishna</name></author><author><name sortKey="Smith, G J" uniqKey="Smith G">G. J. Smith</name></author><author><name sortKey="Zhang, J X" uniqKey="Zhang J">J. X. Zhang</name></author><author><name sortKey="Peiris, J S" uniqKey="Peiris J">J. S. Peiris</name></author><author><name sortKey="Chen, H" uniqKey="Chen H">H. Chen</name></author><author><name sortKey="Guan, Y" uniqKey="Guan Y">Y. Guan</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Vijgen, L" uniqKey="Vijgen L">L. Vijgen</name></author><author><name sortKey="Keyaerts, E" uniqKey="Keyaerts E">E. Keyaerts</name></author><author><name sortKey="Moes, E" uniqKey="Moes E">E. Moës</name></author><author><name sortKey="Thoelen, I" uniqKey="Thoelen I">I. Thoelen</name></author><author><name sortKey="Wollants, E" uniqKey="Wollants E">E. Wollants</name></author><author><name sortKey="Lemey, P" uniqKey="Lemey P">P. Lemey</name></author><author><name sortKey="Vandamme, A M" uniqKey="Vandamme A">A. M. Vandamme</name></author><author><name sortKey="Van Ranst, M" uniqKey="Van Ranst M">M. Van Ranst</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Weber, F" uniqKey="Weber F">F. Weber</name></author><author><name sortKey="Wagner, V" uniqKey="Wagner V">V. Wagner</name></author><author><name sortKey="Rasmussen, S B" uniqKey="Rasmussen S">S. B. Rasmussen</name></author><author><name sortKey="Hartmann, R" uniqKey="Hartmann R">R. Hartmann</name></author><author><name sortKey="Paludan, S R" uniqKey="Paludan S">S. R. Paludan</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Woo, P C" uniqKey="Woo P">P. C. Woo</name></author><author><name sortKey="Lau, S K" uniqKey="Lau S">S. K. Lau</name></author><author><name sortKey="Chu, C M" uniqKey="Chu C">C. M. Chu</name></author><author><name sortKey="Chan, K H" uniqKey="Chan K">K. H. Chan</name></author><author><name sortKey="Tsoi, H W" uniqKey="Tsoi H">H. W. Tsoi</name></author><author><name sortKey="Huang, Y" uniqKey="Huang Y">Y. Huang</name></author><author><name sortKey="Wong, B H" uniqKey="Wong B">B. H. Wong</name></author><author><name sortKey="Poon, R W" uniqKey="Poon R">R. W. Poon</name></author><author><name sortKey="Cai, J J" uniqKey="Cai J">J. J. Cai</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Woo, P C" uniqKey="Woo P">P. C. Woo</name></author><author><name sortKey="Wang, M" uniqKey="Wang M">M. Wang</name></author><author><name sortKey="Lau, S K" uniqKey="Lau S">S. K. Lau</name></author><author><name sortKey="Xu, H" uniqKey="Xu H">H. Xu</name></author><author><name sortKey="Poon, R W" uniqKey="Poon R">R. W. Poon</name></author><author><name sortKey="Guo, R" uniqKey="Guo R">R. Guo</name></author><author><name sortKey="Wong, B H" uniqKey="Wong B">B. H. Wong</name></author><author><name sortKey="Gao, K" uniqKey="Gao K">K. Gao</name></author><author><name sortKey="Tsoi, H W" uniqKey="Tsoi H">H. W. Tsoi</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Yoshikawa, T" uniqKey="Yoshikawa T">T. Yoshikawa</name></author><author><name sortKey="Hill, T E" uniqKey="Hill T">T. E. Hill</name></author><author><name sortKey="Yoshikawa, N" uniqKey="Yoshikawa N">N. Yoshikawa</name></author><author><name sortKey="Popov, V L" uniqKey="Popov V">V. L. Popov</name></author><author><name sortKey="Galindo, C L" uniqKey="Galindo C">C. L. Galindo</name></author><author><name sortKey="Garner, H R" uniqKey="Garner H">H. R. Garner</name></author><author><name sortKey="Peters, C J" uniqKey="Peters C">C. J. Peters</name></author><author><name sortKey="Tseng, C T" uniqKey="Tseng C">C. T. Tseng</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Zaki, A M" uniqKey="Zaki A">A. M. Zaki</name></author><author><name sortKey="Van Boheemen, S" uniqKey="Van Boheemen S">S. van Boheemen</name></author><author><name sortKey="Bestebroer, T M" uniqKey="Bestebroer T">T. M. Bestebroer</name></author><author><name sortKey="Osterhaus, A D" uniqKey="Osterhaus A">A. D. Osterhaus</name></author><author><name sortKey="Fouchier, R A" uniqKey="Fouchier R">R. A. Fouchier</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Zeng, F Y" uniqKey="Zeng F">F. Y. Zeng</name></author><author><name sortKey="Chan, C W" uniqKey="Chan C">C. W. Chan</name></author><author><name sortKey="Chan, M N" uniqKey="Chan M">M. N. Chan</name></author><author><name sortKey="Chen, J D" uniqKey="Chen J">J. D. Chen</name></author><author><name sortKey="Chow, K Y" uniqKey="Chow K">K. Y. Chow</name></author><author><name sortKey="Hon, C C" uniqKey="Hon C">C. C. Hon</name></author><author><name sortKey="Hui, K H" uniqKey="Hui K">K. H. Hui</name></author><author><name sortKey="Li, J" uniqKey="Li J">J. Li</name></author><author><name sortKey="Li, V Y" uniqKey="Li V">V. Y. Li</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Zheng, B" uniqKey="Zheng B">B. Zheng</name></author><author><name sortKey="He, M L" uniqKey="He M">M. L. He</name></author><author><name sortKey="Wong, K L" uniqKey="Wong K">K. L. Wong</name></author><author><name sortKey="Lum, C T" uniqKey="Lum C">C. T. Lum</name></author><author><name sortKey="Poon, L L" uniqKey="Poon L">L. L. Poon</name></author><author><name sortKey="Peng, Y" uniqKey="Peng Y">Y. Peng</name></author><author><name sortKey="Guan, Y" uniqKey="Guan Y">Y. Guan</name></author><author><name sortKey="Lin, M C" uniqKey="Lin M">M. C. Lin</name></author><author><name sortKey="Kung, H F" uniqKey="Kung H">H. F. Kung</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Zhong, Y" uniqKey="Zhong Y">Y. Zhong</name></author><author><name sortKey="Tan, Y W" uniqKey="Tan Y">Y. W. Tan</name></author><author><name sortKey="Liu, D X" uniqKey="Liu D">D. X. Liu</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Zhou, P" uniqKey="Zhou P">P. Zhou</name></author><author><name sortKey="Li, H" uniqKey="Li H">H. Li</name></author><author><name sortKey="Wang, H" uniqKey="Wang H">H. Wang</name></author><author><name sortKey="Wang, L F" uniqKey="Wang L">L. F. Wang</name></author><author><name sortKey="Shi, Z" uniqKey="Shi Z">Z. Shi</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Zust, R" uniqKey="Zust R">R. Züst</name></author><author><name sortKey="Cervantes Barragan, L" uniqKey="Cervantes Barragan L">L. Cervantes-Barragan</name></author><author><name sortKey="Habjan, M" uniqKey="Habjan M">M. Habjan</name></author><author><name sortKey="Maier, R" uniqKey="Maier R">R. Maier</name></author><author><name sortKey="Neuman, B W" uniqKey="Neuman B">B. W. Neuman</name></author><author><name sortKey="Ziebuhr, J" uniqKey="Ziebuhr J">J. Ziebuhr</name></author><author><name sortKey="Szretter, K J" uniqKey="Szretter K">K. J. Szretter</name></author><author><name sortKey="Baker, S C" uniqKey="Baker S">S. C. Baker</name></author><author><name sortKey="Barchet, W" uniqKey="Barchet W">W. Barchet</name></author></analytic></biblStruct></listBibl></div1></back></TEI><pmc article-type="research-article"><pmc-dir>properties open_access</pmc-dir>
<front><journal-meta><journal-id journal-id-type="nlm-ta">J Gen Virol</journal-id><journal-id journal-id-type="iso-abbrev">J. Gen. Virol</journal-id><journal-id journal-id-type="publisher-id">JGV</journal-id><journal-id journal-id-type="hwp">vir</journal-id><journal-title-group><journal-title>The Journal of General Virology</journal-title></journal-title-group><issn pub-type="ppub">0022-1317</issn><issn pub-type="epub">1465-2099</issn><publisher><publisher-name>Society for General Microbiology</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">23620378</article-id><article-id pub-id-type="pmc">3749523</article-id><article-id pub-id-type="publisher-id">052910</article-id><article-id pub-id-type="doi">10.1099/vir.0.052910-0</article-id><article-categories><subj-group subj-group-type="article-type"><subject>Standard</subject></subj-group><subj-group subj-group-type="heading"><subject>Animal</subject><subj-group subj-group-type="heading"><subject>RNA viruses</subject></subj-group></subj-group></article-categories><title-group><article-title>MERS-coronavirus replication induces severe <italic>in vitro</italic> cytopathology and is strongly inhibited by cyclosporin A or interferon-α treatment</article-title><alt-title alt-title-type="recto-page-foot"><ext-link ext-link-type="uri" xlink:href="http://vir.sgmjournals.org">http://vir.sgmjournals.org</ext-link></alt-title><alt-title alt-title-type="verso-running-head">A. H. de Wilde and others</alt-title><alt-title alt-title-type="recto-running-head">MERS-CoV replication and sensitivity to CsA and IFN-α</alt-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>de Wilde</surname><given-names>Adriaan H.</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref></contrib><contrib contrib-type="author"><name><surname>Raj</surname><given-names>V. Stalin</given-names></name><xref ref-type="aff" rid="aff2"><sup>2</sup></xref></contrib><contrib contrib-type="author"><name><surname>Oudshoorn</surname><given-names>Diede</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref></contrib><contrib contrib-type="author"><name><surname>Bestebroer</surname><given-names>Theo M.</given-names></name><xref ref-type="aff" rid="aff2"><sup>2</sup></xref></contrib><contrib contrib-type="author"><name><surname>van Nieuwkoop</surname><given-names>Stefan</given-names></name><xref ref-type="aff" rid="aff2"><sup>2</sup></xref></contrib><contrib contrib-type="author"><name><surname>Limpens</surname><given-names>Ronald W. A. L.</given-names></name><xref ref-type="aff" rid="aff3"><sup>3</sup></xref></contrib><contrib contrib-type="author"><name><surname>Posthuma</surname><given-names>Clara C.</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref></contrib><contrib contrib-type="author"><name><surname>van der Meer</surname><given-names>Yvonne</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref></contrib><contrib contrib-type="author"><name><surname>Bárcena</surname><given-names>Montserrat</given-names></name><xref ref-type="aff" rid="aff3"><sup>3</sup></xref></contrib><contrib contrib-type="author"><name><surname>Haagmans</surname><given-names>Bart L.</given-names></name><xref ref-type="aff" rid="aff2"><sup>2</sup></xref></contrib><contrib contrib-type="author"><name><surname>Snijder</surname><given-names>Eric J.</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref></contrib><contrib contrib-type="author" corresp="yes"><name><surname>van den Hoogen</surname><given-names>Bernadette G.</given-names></name><xref ref-type="aff" rid="aff2"><sup>2</sup></xref></contrib><aff id="aff1"><label>1</label>Molecular Virology Laboratory, Department of Medical Microbiology, Leiden University Medical Center, Leiden, The Netherlands</aff><aff id="aff2"><label>2</label>Viroscience Lab, Erasmus MC, Rotterdam, The Netherlands</aff><aff id="aff3"><label>3</label>Section Electron Microscopy, Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, The Netherlands</aff></contrib-group><author-notes><corresp id="cor1"><bold>Correspondence</bold> Eric. J. Snijder <email xlink:href="E.J.Snijder@lumc.nl">E.J.Snijder@lumc.nl</email> Bernadette G. van den Hoogen <email xlink:href="b.vandenhoogen@erasmusmc.nl">b.vandenhoogen@erasmusmc.nl</email></corresp></author-notes><pub-date pub-type="ppub"><month>8</month><year>2013</year></pub-date><pub-date pub-type="pmc-release"><month>8</month><year>2013</year></pub-date><pmc-comment> PMC Release delay is 0 months and 0 days and was based on the
<volume>94</volume><issue>Pt 8</issue><fpage>1749</fpage><lpage>1760</lpage><history><date date-type="received"><day>04</day><month>3</month><year>2013</year></date><date date-type="accepted"><day>22</day><month>4</month><year>2013</year></date></history><permissions><copyright-statement>© 2013 SGM</copyright-statement><license license-type="open-access" xlink:href="http://creativecommons.org/licenses/by/2.5/"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</license-p></license></permissions><self-uri xlink:title="pdf" xlink:type="simple" xlink:href="1749_vir052910.pdf"></self-uri><abstract><p>Coronavirus (CoV) infections are commonly associated with respiratory and enteric disease in humans and animals. The 2003 outbreak of severe acute respiratory syndrome (SARS) highlighted the potentially lethal consequences of CoV-induced disease in humans. In 2012, a novel CoV (Middle East Respiratory Syndrome coronavirus; MERS-CoV) emerged, causing 49 human cases thus far, of which 23 had a fatal outcome. In this study, we characterized MERS-CoV replication and cytotoxicity in human and monkey cell lines. Electron microscopy of infected Vero cells revealed extensive membrane rearrangements, including the formation of double-membrane vesicles and convoluted membranes, which have been implicated previously in the RNA synthesis of SARS-CoV and other CoVs. Following infection, we observed rapidly increasing viral RNA synthesis and release of high titres of infectious progeny, followed by a pronounced cytopathology. These characteristics were used to develop an assay for antiviral compound screening in 96-well format, which was used to identify cyclosporin A as an inhibitor of MERS-CoV replication in cell culture. Furthermore, MERS-CoV was found to be 50–100 times more sensitive to alpha interferon (IFN-α) treatment than SARS-CoV, an observation that may have important implications for the treatment of MERS-CoV-infected patients. MERS-CoV infection did not prevent the IFN-induced nuclear translocation of phosphorylated STAT1, in contrast to infection with SARS-CoV where this block inhibits the expression of antiviral genes. These findings highlight relevant differences between these distantly related zoonotic CoVs in terms of their interaction with and evasion of the cellular innate immune response.</p></abstract><funding-group><award-group><funding-source id="sp1">Council for Chemical Sciences (CW)</funding-source><award-id rid="sp1">700.57.301</award-id></award-group><award-group><funding-source id="sp2">Council for Earth and Life Sciences (ALW)</funding-source><award-id rid="sp2">836.10.003</award-id></award-group><award-group><funding-source id="sp3">EU-FP7-Health project SILVER</funding-source><award-id rid="sp3">260644</award-id></award-group></funding-group><custom-meta-group><custom-meta><meta-name>openaccess</meta-name><meta-value>free</meta-value></custom-meta><custom-meta><meta-name>OpenAccessEmbargo</meta-name><meta-value>0</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id="sec1-1"><title>Introduction</title><p>In June 2012, a previously unknown coronavirus was isolated from a 60-year-old Saudi Arabian patient who died from acute respiratory distress syndrome and multiple organ failure (<xref ref-type="bibr" rid="r59">Zaki <italic>et al.</italic>, 2012</xref>). Subsequently, the novel virus was isolated from several additional residents of and visitors to the Arabian Peninsula suffering from similar respiratory symptoms. In retrospect, a cluster of respiratory infections in Jordan (April 2012) was linked to the same agent, although no convincing evidence for human-to-human transmission was obtained. This was clearly different for a cluster of three UK cases in early 2013, consisting of a patient who had travelled to Saudi Arabia and two family members without recent travel history outside the UK. In the past year, various names have been used to refer to this newly identified CoV, including novel (beta)coronavirus (nCoV) and human coronavirus EMC (HCoV-EMC), but following a recent recommendation by the coronavirus study group of ICTV and other experts (<xref ref-type="bibr" rid="r1a">de Groot <italic>et al.</italic>, 2013</xref>) we will use Middle East Respiratory Syndrome coronavirus (MERS-CoV) throughout this paper. Up to May 2013, 49 confirmed MERS cases, including 23 fatalities, have been recorded (<ext-link ext-link-type="uri" xlink:href="http://www.who.int/csr/don/archive/disease/coronavirus_infections/en/">http://www.who.int/csr/don/archive/disease/coronavirus_infections/en/</ext-link>).</p><p>Coronavirus (CoV) infections are associated with respiratory and enteric disease in humans and animals. Since the 1960s, two human CoVs (HCoVs OC43 and 229E) have been known to cause mild respiratory disease (<xref ref-type="bibr" rid="r15">Hamre & Procknow, 1966</xref>; <xref ref-type="bibr" rid="r26">McIntosh <italic>et al.</italic>, 1967</xref>), but it was the 2003 outbreak of severe acute respiratory syndrome (SARS; fatality rate ~10 %) that revealed the potentially lethal consequences of CoV-induced disease in humans (<xref ref-type="bibr" rid="r8">Drosten <italic>et al.</italic>, 2003</xref>; <xref ref-type="bibr" rid="r22">Ksiazek <italic>et al.</italic>, 2003</xref>). Two years later, bats were identified as the most likely animal reservoir for this zoonotic CoV (<xref ref-type="bibr" rid="r24">Lau <italic>et al.</italic>, 2005</xref>; <xref ref-type="bibr" rid="r25">Li <italic>et al.</italic>, 2005</xref>). Subsequently, a wide variety of bat-associated CoVs was discovered (<xref ref-type="bibr" rid="r53">Vijaykrishna <italic>et al.</italic>, 2007</xref>; <xref ref-type="bibr" rid="r57">Woo <italic>et al.</italic>, 2007</xref>), and two additional human CoVs (NL63 and HKU1) (<xref ref-type="bibr" rid="r9">Fouchier <italic>et al.</italic>, 2004</xref>; <xref ref-type="bibr" rid="r47">van der Hoek <italic>et al.</italic>, 2004</xref>; <xref ref-type="bibr" rid="r56">Woo <italic>et al.</italic>, 2005</xref>) were also identified. Although the general capacity of bat CoVs to switch hosts appears to be rather restricted (<xref ref-type="bibr" rid="r27">Müller <italic>et al.</italic>, 2012</xref>), the possibility of SARS-CoV re-emergence or zoonotic transfer of other animal CoVs has remained a public health concern over the past 10 years.</p><p>CoVs are classified in four genera (alpha-, beta-, gamma- and deltacoronaviruses; <xref ref-type="bibr" rid="r6">de Groot <italic>et al.</italic>, 2012</xref>), and our previous analysis of the MERS-CoV genome (<xref ref-type="bibr" rid="r45">van Boheemen <italic>et al.</italic>, 2012</xref>) identified the newly emerging agent as a member of lineage C of the genus <italic>Betacoronavirus</italic>. Strikingly, as in the case of SARS-CoV, the closest known relatives of MERS-CoV are bat coronaviruses, such as HKU-4 and HKU-5 (<xref ref-type="bibr" rid="r45">van Boheemen <italic>et al.</italic>, 2012</xref>; <xref ref-type="bibr" rid="r57">Woo <italic>et al.</italic>, 2007</xref>). The evolutionary distance to SARS-CoV (lineage B) is considerable, a notion further supported by recent comparative studies revealing important differences in receptor usage (<xref ref-type="bibr" rid="r27">Müller <italic>et al.</italic>, 2012</xref>; <xref ref-type="bibr" rid="r33">Raj <italic>et al.</italic>, 2013</xref>).</p><p>Mammalian viruses have to cope with the host cell’s innate responses, including those triggered by activation of the type I interferon (IFN) pathway (reviewed by <xref ref-type="bibr" rid="r34">Randall & Goodbourn, 2008</xref>). CoVs, including SARS-CoV, appear to have evolved a variety of mechanisms to block or evade such antiviral responses (reviewed by <xref ref-type="bibr" rid="r30">Perlman & Netland, 2009</xref>; <xref ref-type="bibr" rid="r62">Zhong <italic>et al.</italic>, 2012</xref>). For example, it was postulated that the sensing of dsRNA replication intermediates by the innate immune system is inhibited by the elaborate virus-induced membrane structures with which CoV RNA synthesis is associated (<xref ref-type="bibr" rid="r19">Knoops <italic>et al.</italic>, 2008</xref>; <xref ref-type="bibr" rid="r52">Versteeg <italic>et al.</italic>, 2007</xref>). Other evasion mechanisms have been attributed to protein functions that can be either conserved across CoVs or specific for certain CoV lineages. Proteins such as the non-structural protein nsp3 proteinase (<xref ref-type="bibr" rid="r35">Ratia <italic>et al.</italic>, 2006</xref>), the nsp16 2′-<italic>O</italic>-methyltransferase (<xref ref-type="bibr" rid="r64">Züst <italic>et al.</italic>, 2011</xref>), and the products of SARS-CoV ORFs 3b, 6 and 7a (<xref ref-type="bibr" rid="r10">Frieman <italic>et al.</italic>, 2007</xref>; <xref ref-type="bibr" rid="r16">Hussain <italic>et al.</italic>, 2008</xref>; <xref ref-type="bibr" rid="r21">Kopecky-Bromberg <italic>et al.</italic>, 2006</xref>; <xref ref-type="bibr" rid="r63">Zhou <italic>et al.</italic>, 2012</xref>), have all been described as preventing IFN induction/signalling. In particular, the SARS-CoV ORF6 protein is known to inhibit IFN-induced JAK-STAT signalling by blocking the nuclear translocation of phosphorylated STAT1 (p-STAT1), which contributes to the pathogenic potential of the virus in a mouse model (<xref ref-type="bibr" rid="r38">Sims <italic>et al.</italic>, 2013</xref>). In spite of these immune evasion strategies, treatment with type I IFNs can inhibit CoV replication <italic>in vitro</italic> (<xref ref-type="bibr" rid="r11">Garlinghouse <italic>et al.</italic>, 1984</xref>; <xref ref-type="bibr" rid="r14">Haagmans <italic>et al.</italic>, 2004</xref>; <xref ref-type="bibr" rid="r29">Paragas <italic>et al.</italic>, 2005</xref>; <xref ref-type="bibr" rid="r43">Taguchi & Siddell, 1985</xref>; <xref ref-type="bibr" rid="r61">Zheng <italic>et al.</italic>, 2004</xref>) and, for example, protects type I pneumocytes against SARS-CoV infection in macaques (<xref ref-type="bibr" rid="r14">Haagmans <italic>et al.</italic>, 2004</xref>).</p><p>Clearly, well-characterized systems for MERS-CoV replication in cell culture will be invaluable for future studies into basic virus properties and interactions with the host, including innate immune responses. Therefore, we set out to characterize the replication of MERS-CoV in different cell lines. Using this information, an assay to screen for antiviral compounds was developed, which identified cyclosporin A (CsA) as an inhibitor of MERS-CoV replication. Our first screening experiments also established that, compared with SARS-CoV, MERS-CoV replication is more sensitive to type I IFN treatment.</p></sec><sec sec-type="results" id="sec1-2"><title>Results</title><sec id="sec2-1"><title>Kinetics of MERS-CoV replication in Vero and Huh7 cells</title><p>Only a few laboratory studies on MERS-CoV replication have been reported thus far. Cells from a variety of mammalian hosts have been found to be susceptible, and infection can induce pronounced cytopathology and cell death (<xref ref-type="bibr" rid="r27">Müller <italic>et al.</italic>, 2012</xref>; <xref ref-type="bibr" rid="r59">Zaki <italic>et al.</italic>, 2012</xref>). Following entry, the CoV replicative cycle starts with translation of the positive-sense RNA genome into replicase polyproteins that are cleaved into 16 nsps (<xref ref-type="bibr" rid="r12">Gorbalenya <italic>et al.</italic>, 2006</xref>; <xref ref-type="bibr" rid="r45">van Boheemen <italic>et al.</italic>, 2012</xref>). These direct both genome replication and synthesis of the subgenomic (sg) mRNAs required to express the structural and accessory proteins. To investigate MERS-CoV replication in more detail, we used Vero and Huh7 cells to analyse viral RNA synthesis and progeny release in single-cycle infection experiments.</p><p>Hybridization analysis of the accumulation of viral RNA revealed the presence of genome RNA and seven sg transcripts, with sizes closely matching those predicted previously from the positions of conserved transcription regulatory sequences (TRSs) in the viral genome (<xref ref-type="bibr" rid="r45">van Boheemen <italic>et al.</italic>, 2012</xref>) (<xref ref-type="fig" rid="f1">Fig. 1a</xref>). The relative abundance of the various sg mRNAs was similar to what has been observed for other CoVs, with the smallest species, encoding the nucleocapsid (N) protein, being by far the most abundant transcript (<xref ref-type="fig" rid="f1">Fig. 1b</xref>). In both cell lines, viral mRNAs could be readily detected at 7 h post-infection (p.i.) and reached maximum levels around 13 h p.i. (<xref ref-type="fig" rid="f1">Fig. 1a</xref>). Viral RNA levels remained more or less constant until 24 h p.i. in Vero cells, whereas the amount isolated from Huh7 cells declined due to the more rapid development of cytopathology in this cell line between 13 and 24 h p.i. (see below). After the peak of viral RNA accumulation had been reached, the titre of virus released from MERS-CoV-infected Vero cells increased steadily from ~5×10<sup>5</sup> to ~5×10<sup>7</sup> p.f.u. ml<sup>−1</sup> (<xref ref-type="fig" rid="f1">Fig. 1c</xref>). Interestingly, the bulk of the viral progeny was released significantly earlier from Huh7 cells, although the final titre at 24 h p.i. was comparable to that obtained from Vero cells.</p><fig id="f1" fig-type="figure" position="float"><label>Fig. 1. </label><caption><p>Kinetics of MERS-CoV replication in Vero and Huh7 cells. Vero and Huh7 cells were infected with MERS-CoV (m.o.i. of 5). (a) Hybridization analysis of viral mRNAs isolated from MERS-CoV-infected cells using an oligonucleotide recognizing the viral genome and all sg mRNAs. Additional minor bands of ~3 and ~4 kb were observed (*) and may represent additional viral mRNA species that remain to be studied in more detail. However, the corresponding positions in the ORF4a/b and ORF5 coding regions do not contain a canonical core TRS sequence (AACGAA; <xref ref-type="bibr" rid="r45">van Boheemen <italic>et al.</italic>, 2012</xref>) that might provide a direct explanation for their synthesis. (b) Analysis of the relative molarities of viral genome and each of the sg mRNAs (% of total viral mRNA). mRNA sizes were calculated on the basis of the TRS positions in the viral genome sequence (<xref ref-type="bibr" rid="r45">van Boheemen <italic>et al.</italic>, 2012</xref>). Phosphorimager quantification was performed on the gel lanes with the RNA samples isolated from Vero cells at 10, 13 and 24 h p.i. (<xref ref-type="fig" rid="f1">Fig. 1a</xref>; lanes 3–5, respectively; mean±<sc>sd</sc>). (c) Release of infectious MERS-CoV progeny into the medium of infected Vero or Huh7 cells at the indicated time points, as determined by plaque assay (mean±<sc>sd</sc>; <italic>n</italic> = 4).</p></caption><graphic xlink:href="052910-f1"></graphic></fig></sec><sec id="sec2-2"><title>Antisera raised against non-structural proteins of other betacoronaviruses cross-react with MERS-CoV proteins</title><p>Despite the relatively large evolutionary distance to better-characterized CoVs, we tested a panel of antisera from our laboratory for cross-reactivity with MERS-CoV-infected cells. In contrast to a polyclonal serum recognizing the SARS-CoV N protein (data not shown), antisera against various SARS-CoV nsps (nsp3, nsp5 and nsp8; <xref ref-type="bibr" rid="r41">Snijder <italic>et al.</italic>, 2006</xref>) raised using purified recombinant proteins as antigen were found to cross-react strongly (<xref ref-type="fig" rid="f2">Fig. 2a</xref>). In addition, rabbit antisera raised against synthetic peptides (23mers) representing a small but conserved C-terminal part of SARS-CoV and MHV nsp4 strongly cross-reacted with MERS-CoV. Only small but apparently immunogenic parts of these peptides (e.g. LYQPP) are absolutely conserved between MHV and MERS-CoV nsp4 (<xref ref-type="fig" rid="f2">Fig. 2b</xref>). Conservation in other betacoronaviruses (data not shown) suggests that antisera recognizing this nsp4 region may be used for immunodetection of additional (newly emerging) CoVs.</p><fig id="f2" fig-type="figure" position="float"><label>Fig. 2. </label><caption><p>Selected rabbit antisera raised against SARS-CoV and mouse hepatitis virus (MHV) nsps cross-react with MERS-CoV proteins. (a) MERS-CoV-infected Vero cells (m.o.i. of 5) were fixed at 8 h p.i. For immunofluorescence microscopy, cells were double-labelled with a mouse mAb recognizing dsRNA (bottom row) and rabbit antisera raised against SARS-CoV nsp3, nsp4, nsp5 or nsp8, or MHV nsp4 (top row). Bar, 20 µm. (b) Sequence comparison of the C-terminal domain of nsp4 of SARS-CoV (isolate Frankfurt 1), MERS-CoV (strain EMC/2012) and MHV (strain A59). The SARS-CoV and MHV sequences correspond to the synthetic peptides used to raise rabbit anti-nsp4 sera. Residues conserved in all three viruses are highlighted in yellow, whereas residues conserved in two out of three are highlighted in grey. Amino acid numbers refer to the full-length pp1a sequence. (c) Monolayers of Vero, Vero E6, Huh7 and Calu3/2B4 cells were infected with MERS-CoV (m.o.i. of 5) and double-labelled for dsRNA (green) and nsp3 (red). Bar, 40 µm.</p></caption><graphic xlink:href="052910-f2"></graphic></fig></sec><sec id="sec2-3"><title>MERS-CoV replication structures</title><p>Subsequently, we employed a mAb recognizing dsRNA to localize intermediates in viral RNA synthesis (<xref ref-type="bibr" rid="r19">Knoops <italic>et al.</italic>, 2008</xref>; <xref ref-type="bibr" rid="r55">Weber <italic>et al.</italic>, 2006</xref>). In various cell types, the immunolabelling signals for both replicase and dsRNA localized to the perinuclear region (<xref ref-type="fig" rid="f2">Fig. 2c</xref>), where the replication structures induced by other CoVs are known to accumulate (<xref ref-type="bibr" rid="r3">Brockway <italic>et al.</italic>, 2003</xref>; <xref ref-type="bibr" rid="r13">Gosert <italic>et al.</italic>, 2002</xref>; <xref ref-type="bibr" rid="r19">Knoops <italic>et al.</italic>, 2008</xref>; <xref ref-type="bibr" rid="r41">Snijder <italic>et al.</italic>, 2006</xref>; <xref ref-type="bibr" rid="r42">Stertz <italic>et al.</italic>, 2007</xref>; <xref ref-type="bibr" rid="r44">Ulasli <italic>et al.</italic>, 2010</xref>).</p><p>We next used EM to investigate the ultrastructural and potentially cytopathic changes that MERS-CoV induces in infected cells, and focused on the membranous replication structures that support MERS-CoV RNA synthesis. The preservation of such structures, typically double-membrane vesicles (DMVs) and convoluted membranes (CMs), was found previously to be improved significantly by using protocols that include cryofixation and freeze substitution (<xref ref-type="bibr" rid="r19">Knoops <italic>et al.</italic>, 2008</xref>; <xref ref-type="bibr" rid="r41">Snijder <italic>et al.</italic>, 2006</xref>). Therefore, we applied these advanced preservation techniques, including newly developed protocols for high-pressure freezing (HPF), to MERS-CoV-infected Vero cells. Images of similarly prepared SARS-CoV-infected Vero E6 cells were included for comparison (<xref ref-type="fig" rid="f3">Fig. 3f</xref>).</p><fig id="f3" fig-type="figure" position="float"><label>Fig. 3. </label><caption><p>Membrane structures induced by MERS-CoV infection. (a–d) Electron micrographs of thin sections (100 nm) of MERS-CoV-infected Vero cells at 8 h p.i. Low magnification images of a cell containing a small cluster of DMVs (a), enlarged in (b). Some DMVs are indicated by black arrowheads and the inset displays a higher magnification of the boxed DMV in (b). Extensive membrane alterations in the perinuclear region are shown in (c), with the boxed area displayed at higher magnification in (d), where CMs (white arrows, inset) embedded in clusters of DMVs (black arrowheads) can be observed. (e, f) For comparison, (e) shows the unaltered cytoplasm of a mock-infected cell and (f) contains SARS-CoV-induced DMV (black arrowheads) as observed after HPF and freeze substitution. N, nucleus; m, mitochondria. Bars, 2 µm (a, c, e); 500 nm (b, d, f).</p></caption><graphic xlink:href="052910-f3"></graphic></fig><p>Compared with mock-infected control cells (<xref ref-type="fig" rid="f3">Fig. 3e</xref>), different degrees of distinct alterations were observed at 8 h p.i. Some cells contained relatively small DMV clusters (<xref ref-type="fig" rid="f3">Fig. 3a, b</xref>; black arrowheads and inset), whereas in others large numbers of DMVs occupied extensive areas of the perinuclear region (<xref ref-type="fig" rid="f3">Fig. 3c, d</xref>), differences that probably reflect different stages in infection progression. The diameter of MERS-CoV-induced DMVs ranged from 150 to 320 nm, comparable to what was measured previously for SARS-CoV-induced structures (<xref ref-type="bibr" rid="r19">Knoops <italic>et al.</italic>, 2008</xref>). An interesting morphological difference with our previous studies of SARS-CoV-infected cells was the presence of a dense inner DMV core, which can be attributed to technical differences in sample preparation. In terms of ultrastructural preservation, HPF is widely considered superior to the previously used plunge-freezing protocols. Also, in the case of SARS-CoV (<xref ref-type="fig" rid="f3">Fig. 3f</xref>) and the distantly related equine arteritis virus (<xref ref-type="bibr" rid="r20">Knoops <italic>et al.</italic>, 2012</xref>), a similar dense DMV core became apparent when HPF was employed. Although DMV cores are known to contain dsRNA, the implications of these ultrastructural observations remain unclear. Interestingly, CMs were always surrounded by DMV clusters and were only observed in cells that appeared to be more advanced in infection (<xref ref-type="fig" rid="f3">Fig. 3c, d</xref>; white arrows and inset). This observation strengthens the notion that DMV formation precedes the development of CMs, as postulated previously for SARS-CoV (<xref ref-type="bibr" rid="r19">Knoops <italic>et al.</italic>, 2008</xref>).</p></sec><sec id="sec2-4"><title>MERS-CoV-induced cytophatology and cell death</title><p>In cell culture, many CoVs induce severe cytopathic effect (CPE) and cell death. Infection with a number of CoVs can also induce extensive syncytium formation, due to fusion activity of the viral spike protein at neutral pH (reviewed by <xref ref-type="bibr" rid="r1">Belouzard <italic>et al.</italic>, 2012</xref>). MERS-CoV-induced cytopathology was monitored by light microscopy following low m.o.i. inoculation of monkey and human cells (<xref ref-type="fig" rid="f4">Fig. 4</xref>). In line with previous observations (<xref ref-type="bibr" rid="r59">Zaki <italic>et al.</italic>, 2012</xref>), Vero cells developed clear CPE at 2 days p.i. and detached at 3 days p.i. (<xref ref-type="fig" rid="f4">Fig. 4a</xref>). Similar observations were made for Calu3/2B4 cells (<xref ref-type="fig" rid="f4">Fig. 4b</xref>). In contrast, MERS-CoV-infected Vero E6 cells displayed only mild CPE starting at 3 days p.i and cell death was not complete after 6 days (<xref ref-type="fig" rid="f4">Fig. 4c</xref>). The development of CPE in Huh7 cells was strikingly faster compared with the three other cell lines and, following extensive syncytium formation, cells had already detached by around 17 h (<xref ref-type="fig" rid="f4">Fig. 4d</xref>). Given the low m.o.i. used and the virus replication kinetics (<xref ref-type="fig" rid="f1">Fig. 1</xref>), the syncytium formation in these only partially infected Huh7 cultures appeared to be a major factor in CPE development. Dipeptidyl peptidase-4 (DPP4) expression on Vero and Huh7 cells (<xref ref-type="bibr" rid="r33">Raj <italic>et al.</italic>, 2013</xref>) and expression levels of DPP4 on Calu3/2B4 and Vero E6 cells correlated with susceptibility to MERS-CoV (data not shown).</p><fig id="f4" fig-type="figure" position="float"><label>Fig. 4. </label><caption><p>MERS-CoV infection induces severe cytopathology in monkey and human cell lines. Monolayers of Vero (a), Calu3/2B4 (b), Vero E6 (c) and Huh7 (d) cells were infected with MERS-CoV (m.o.i. of 0.05) and analysed by light microscopy at the indicated time points. Bar, 100 µm.</p></caption><graphic xlink:href="052910-f4"></graphic></fig></sec><sec id="sec2-5"><title>Development of an assay to screen for compounds inhibiting MERS-CoV replication</title><p>The virus-induced CPE in Vero and Huh7 cells was used to develop a first assay to screen for compounds that inhibit MERS-CoV replication in cell culture. Vero cells were seeded in 96-well plates and infected at an m.o.i. of 0.005 or 0.05 (<xref ref-type="fig" rid="f5">Fig. 5a</xref>). After 2 and 3 days, CPE formation was monitored microscopically and cytotoxicity was measured using a commercial cell viability assay. Moderate CPE was observed on day 2, and by day 3 cell viability in uninfected cells had dropped below 10 % with both virus doses used (<xref ref-type="fig" rid="f5">Fig. 5a</xref>), indicating near-complete cell death. In MERS-CoV-infected Huh7 cells (<xref ref-type="fig" rid="f5">Fig. 5b</xref>), by day 1, cell viability had dropped to 79 or 24 % (after m.o.i. 0.005 or 0.05 infection, respectively), which was in line with our observations on rapid syncytium formation and CPE in this particular cell line (<xref ref-type="fig" rid="f4">Fig. 4d</xref>). By day 2, CPE was complete for both virus doses used and cells had detached (<xref ref-type="fig" rid="f5">Fig. 5b</xref>). Based on this comparison, further experiments were carried out using an m.o.i. of 0.005, and Huh7 and Vero cells were incubated for 2 or 3 days, respectively, before measuring cell viability.</p><fig id="f5" fig-type="figure" position="float"><label>Fig. 5. </label><caption><p>Development of an assay to screen for compounds inhibiting MERS-CoV replication. Vero (a, c) and Huh7 (b, d) cells in a 96-well plate format were infected at an m.o.i. of 0.005 or 0.05. Mock-infected cells (no virus) were used as a reference for unchanged cell viability (their relative viability was set at 100 %). Infected Vero cells were incubated for 2 (dark shading) or 3 (light shading) days (a) and Huh7 cells were incubated for 1 (dark shading) or 2 (light shading) days (b). (c) Vero cells were infected (dark shading) or not (light shading) with MERS-CoV (m.o.i. of 0.005) in the presence of 3 or 9 µM CsA, or 0.09 % DMSO as a solvent control. (d) Huh7 cells were infected (dark shading) or not (light shading) with MERS-CoV (m.o.i. of 0.005) in the presence of 3.75, 7.5 or 15 µM CsA, or 0.15 % DMSO. The graphs in (c) and (d) show the results of a representative experiment (mean±<sc>sd</sc>; <italic>n</italic> = 4). All experiments were repeated at least twice.</p></caption><graphic xlink:href="052910-f5"></graphic></fig><p>Previously, it was shown that replication of various CoVs, including SARS-CoV, can be inhibited by the immunosuppressive drug CsA (<xref ref-type="bibr" rid="r7">de Wilde <italic>et al.</italic>, 2011</xref>; <xref ref-type="bibr" rid="r32">Pfefferle <italic>et al.</italic>, 2011</xref>). Therefore, whilst testing whether the CPE-based assay described above could be used as an antiviral screening method, we used CsA treatment to obtain a first proof of principle. Infected Vero cells were treated with 3 or 9 µM CsA and analysed at 3 days p.i. At the concentrations used, CsA did not adversely affect the viability of mock-infected cells (<xref ref-type="fig" rid="f5">Fig. 5c</xref>). Treatment with 9 µM completely inhibited CPE and left cell viability unchanged compared with mock-infected control cells. The inhibitory effect of CsA was confirmed in Huh7 cells (<xref ref-type="fig" rid="f5">Fig. 5d</xref>), which displayed a reduced or lack of CPE upon treatment with 7.5 and 15 µM CsA, respectively. These results were corroborated by immunofluorescence microscopy analysis of CsA-treated and high m.o.i.-infected Vero and Huh7 cells, and by determining virus titres released into the medium. Both assays confirmed an almost complete block of MERS-CoV infection (data not shown). However, as reported previously for other CoVs (<xref ref-type="bibr" rid="r7">de Wilde <italic>et al.</italic>, 2011</xref>), a small fraction of MERS-CoV-infected cells appeared to be refractive to CsA treatment and supported a low level of MERS-CoV replication, even at high CsA concentrations (data not shown).</p></sec><sec id="sec2-6"><title>Enhanced sensitivity of MERS-CoV to pegylated IFN-α treatment in comparison with SARS-CoV</title><p>Type I IFNs inhibit CoV replication and can protect against infection in animal models (<xref ref-type="bibr" rid="r14">Haagmans <italic>et al.</italic>, 2004</xref>; <xref ref-type="bibr" rid="r43">Taguchi & Siddell, 1985</xref>). We therefore compared the effect of pegylated IFN-α (PEG-IFN) treatment on MERS-CoV and SARS-CoV replication <italic>in vitro</italic>. Vero cells were given PEG-IFN 4 h before low-m.o.i. infection, together with the inoculum, or 4 h after infection. At 2 days p.i., CPE was scored microscopically.</p><p>Treatment with PEG-IFN profoundly inhibited both MERS-CoV- and SARS-CoV-induced CPE and RNA levels in a dose-dependent manner (<xref ref-type="fig" rid="f6">Fig. 6</xref>). At 2 days p.i., SARS-CoV-induced CPE was reduced for all time points of PEG-IFN addition when using a dose of at least 30 ng PEG-IFN ml<sup>−1</sup> (<xref ref-type="fig" rid="f6">Fig. 6a</xref>), whereas MERS-CoV-induced CPE had already decreased using a dose of 1 ng ml<sup>−1</sup> (<xref ref-type="fig" rid="f6">Fig. 6b</xref>). For SARS-CoV, only pre-treatment with 1000 ng PEG-IFN ml<sup>−1</sup> completely prevented CPE. For MERS-CoV, complete inhibition of CPE was observed at much lower concentrations, specifically 3, 10 or 30 ng ml<sup>−1</sup>, when the drug was added to the cells before, during or after infection, respectively. Although decreased CPE was also observed in SARS-CoV-infected cultures treated with 30 ng PEG-IFN ml<sup>−1</sup>, only a 30-fold reduction in viral RNA was detected in their medium at 2 days p.i. (<xref ref-type="fig" rid="f6">Fig. 6c</xref>). For comparison, treatment of MERS-CoV-infected cells with the same PEG-IFN dose completely blocked CPE and reduced viral RNA levels in the medium by 600- to 2000-fold, depending on the timing of PEG-IFN addition (<xref ref-type="fig" rid="f6">Fig. 6d</xref>).</p><fig id="f6" fig-type="figure" position="float"><label>Fig. 6. </label><caption><p>Sensitivity of MERS-CoV and SARS-CoV to PEG-IFN. Vero cells were incubated with 0–1000 ng PEG-IFN ml<sup>−1</sup> at <italic>t</italic> = −4, <italic>t</italic> = 0 and <italic>t</italic> = 4 h p.i. Cells were infected with 100 TCID<sub>50</sub> virus per well (a, b). At 2 days p.i., cells were examined for CPE. The effect of PEG-IFN treatment on CPE induced by SARS-CoV (a) or MERS-CoV (b) is shown. CPE was scored as none (0), mild (1), moderate (2), severe (3) or complete (4). (c, d) Viral genomes in the culture medium of virus-infected cells were determined by RT-PCR. The influence of PEG-IFN treatment on the viral RNA load [genome equivalents (gen. eq.) ml<sup>−1</sup>] in the supernatants of cells infected with SARS-CoV (c) or MERS-CoV (d) is shown.</p></caption><graphic xlink:href="052910-f6"></graphic></fig><p>Our data revealed that, in the same cell line, MERS-CoV infection was 50–100 times more sensitive to PEG-IFN treatment than SARS-CoV infection. This difference may be explained by important lineage-specific genetic differences between these two zoonotic betacoronaviruses in terms of accessory protein genes encoded in the 3′ part of the genome (<xref ref-type="bibr" rid="r40">Snijder <italic>et al.</italic>, 2003</xref>; <xref ref-type="bibr" rid="r45">van Boheemen <italic>et al.</italic>, 2012</xref>). In particular, MERS-CoV does not encode a homologue of the SARS-CoV ORF6 protein, which was reported to block the IFN-induced nuclear translocation of phosphorylated transcription factor STAT1. As nuclear translocation of p-STAT1 is essential for transcriptional activation of downstream antiviral genes, the ORF6 protein makes SARS-CoV less sensitive to treatment with type I IFN (<xref ref-type="bibr" rid="r10">Frieman <italic>et al.</italic>, 2007</xref>; <xref ref-type="bibr" rid="r38">Sims <italic>et al.</italic>, 2013</xref>). IFN-induced translocation of p-STAT1 was readily observed in IFN-treated mock-infected Vero cells (<xref ref-type="fig" rid="f7">Fig. 7a–d</xref>), but not in IFN-treated SARS-CoV-infected cells (<xref ref-type="fig" rid="f7">Fig. 7e, f</xref>). In contrast, in MERS-CoV-infected and IFN-treated cultures, the translocation of p-STAT1 was detected (<xref ref-type="fig" rid="f7">Fig. 7 g, h</xref>). Together with the data on IFN sensitivity (<xref ref-type="fig" rid="f5">Fig. 5</xref>), these observations highlight important differences between SARS-CoV and MERS-CoV in terms of their interaction with the IFN signalling pathways.</p><fig id="f7" fig-type="figure" position="float"><label>Fig. 7. </label><caption><p>IFN-α induced nuclear translocation of p-STAT1 in MERS-CoV-infected Vero cells. Confocal immunofluorescence microscopy of uninfected Vero cells (a–d) and Vero cells infected (m.o.i. of 1) with SARS-CoV (e, f) or MERS-CoV (g, h). At 8 h p.i. cells were left untreated (a, b) or treated (c–h) with 1000 ng PEG-IFN ml<sup>−1</sup> for 30 min, fixed and double-labelled with antisera against SARS-CoV nsp3 (red; a–h), or p-STAT1 (green; b, d, f, h), and nuclear DNA was stained with DAPI (blue; a, c, e, g).</p></caption><graphic xlink:href="052910-f7"></graphic></fig></sec></sec><sec sec-type="discussion" id="sec1-3"><title>Discussion</title><p>Following the 2003 SARS epidemic, global CoV hunting efforts identified a wealth of previously unknown family members, in particular in bat species from several continents (<xref ref-type="bibr" rid="r6">de Groot <italic>et al.</italic>, 2012</xref>). Moreover, at least three of the four current ‘established’ human CoVs (NL63, 229E and OC43) were postulated to have originated from zoonotic reservoirs (<xref ref-type="bibr" rid="r17">Huynh <italic>et al.</italic>, 2012</xref>; <xref ref-type="bibr" rid="r31">Pfefferle <italic>et al.</italic>, 2009</xref>; <xref ref-type="bibr" rid="r54">Vijgen <italic>et al.</italic>, 2005</xref>). Recently, about a decade after the SARS outbreak, MERS-CoV was identified as the next zoonotic CoV (<xref ref-type="bibr" rid="r59">Zaki <italic>et al.</italic>, 2012</xref>) and appears to be highly pathogenic to humans: of the 49 cases confirmed thus far, 23 had a fatal outcome (<ext-link ext-link-type="uri" xlink:href="http://www.who.int/csr/don/archive/disease/coronavirus_infections/en/">http://www.who.int/csr/don/archive/disease/coronavirus_infections/en/</ext-link>). Whether zoonotic CoVs cause transient epidemics or establish a long-lasting relationship with the human host, an in-depth understanding of virus–host interactions will be required to develop effective countermeasures. In this study, we defined several basic but important parameters of MERS-CoV replication in cell culture (<xref ref-type="fig" rid="f1">Figs 1</xref>–<xref ref-type="fig" rid="f4">4</xref>). Among the tools for MERS-CoV research developed are immunoassays based on cross-reacting antisera raised against other betacoronaviruses (<xref ref-type="fig" rid="f2">Fig. 2</xref>) and a CPE-based assay that can be used to screen for antiviral effects (<xref ref-type="fig" rid="f5">Figs 5</xref> and <xref ref-type="fig" rid="f6">6</xref>).</p><p>Following the development of a high-throughput screening method for antiviral effects, proof of principle was obtained using CsA, a recently discovered inhibitor of CoV replication (<xref ref-type="bibr" rid="r7">de Wilde <italic>et al.</italic>, 2011</xref>; <xref ref-type="bibr" rid="r32">Pfefferle <italic>et al.</italic>, 2011</xref>). This drug affects the function of several members of the cellular cyclophilin family and appears to block functional interactions between viral proteins and one or multiple cyclophilin family members (<xref ref-type="bibr" rid="r28">Nagy <italic>et al.</italic>, 2011</xref>). Low-micromolar CsA concentrations blocked MERS-CoV-induced CPE in Vero and Huh7 cells (9 and 15 µM, respectively), as observed previously for other CoVs (<xref ref-type="bibr" rid="r7">de Wilde <italic>et al.</italic>, 2011</xref>; <xref ref-type="bibr" rid="r32">Pfefferle <italic>et al.</italic>, 2011</xref>). As in these previous studies (<xref ref-type="bibr" rid="r7">de Wilde <italic>et al.</italic>, 2011</xref>), a small fraction of the cells somehow remained susceptible to MERS-CoV infection, even at high CsA concentrations. Thus, virus replication could not be completely eliminated, which may ultimately lead to the development of CsA resistance in cell culture. In conclusion, these experiments established that monitoring MERS-CoV-induced CPE could be a valuable and rapid tool in screening for the potential antiviral activity of, for example, small-molecule compounds or Food and Drug Administration-approved drugs such as PEG-IFN.</p><p>Type I IFN induction, a hallmark of the early innate immune response, is counteracted by different CoV-encoded proteins. Despite these evasion strategies, IFN can be detected in sera of CoV-infected mice and humans (<xref ref-type="bibr" rid="r4">Cameron <italic>et al.</italic>, 2012</xref>; <xref ref-type="bibr" rid="r11">Garlinghouse <italic>et al.</italic>, 1984</xref>; <xref ref-type="bibr" rid="r43">Taguchi & Siddell, 1985</xref>), and CoV-infected plasmacytoid dendritic cells have been identified as a source of high IFN-α levels (<xref ref-type="bibr" rid="r5">Cervantes-Barragan <italic>et al.</italic>, 2007</xref>; <xref ref-type="bibr" rid="r37">Roth-Cross <italic>et al.</italic>, 2007</xref>). The SARS-CoV ORF6 protein, however, (partially) disrupts the downstream IFN-induced signalling in infected cells by inhibiting the nuclear translocation of p-STAT1, a critical component of both the IFN-α and IFN-γ signalling pathways (<xref ref-type="bibr" rid="r10">Frieman <italic>et al.</italic>, 2007</xref>). Although contributions from additional immune evasion mechanisms are likely, the lack of a SARS-CoV ORF6 homologue (<xref ref-type="bibr" rid="r45">van Boheemen <italic>et al.</italic>, 2012</xref>) may be a major factor in the higher sensitivity of MERS-CoV to PEG-IFN treatment, as observed in this study and other recent work (<xref ref-type="bibr" rid="r18">Kindler <italic>et al.</italic>, 2013</xref>). This was substantiated further by the finding that nuclear translocation of p-STAT1 is not blocked in MERS-CoV-infected cells (<xref ref-type="fig" rid="f7">Fig. 7</xref>), which indicates that MERS-CoV has not evolved an alternative strategy to achieve the same goal. MHV has been shown to be relatively insensitive to IFN pre-treatment; however, this virus also does not block activation and translocation of p-STAT1 but instead inhibits the induction of a subset of ISGs by IFN-α/β (<xref ref-type="bibr" rid="r36">Rose <italic>et al.</italic>, 2010</xref>). Future studies may elucidate whether MERS-CoV has evolved alternative strategies to cope with the host’s IFN response. In addition, it will be important to test whether MERS-CoV is attenuated <italic>in vivo</italic> as a result of the relative high IFN sensitivity.</p><p>PEG-IFN is a registered drug used for the treatment of chronic hepatitis B and C infections in humans (<xref ref-type="bibr" rid="r2">Bergman <italic>et al.</italic>, 2011</xref>). Several CoVs, including SARS-CoV, have been shown to be sensitive to both type I IFN treatment <italic>in vitro</italic> and PEG-IFN treatment <italic>in vivo</italic> (<xref ref-type="bibr" rid="r14">Haagmans <italic>et al.</italic>, 2004</xref>; <xref ref-type="bibr" rid="r29">Paragas <italic>et al.</italic>, 2005</xref>; <xref ref-type="bibr" rid="r61">Zheng <italic>et al.</italic>, 2004</xref>), and in this study we established a relatively high sensitivity for MERS-CoV. For example, in cynomolgus macaques, plasma levels of 1–5 ng ml<sup>−1</sup> were reached (<xref ref-type="bibr" rid="r14">Haagmans <italic>et al.</italic>, 2004</xref>), a dose that in this study significantly reduced MERS-CoV replication <italic>in vitro</italic>. The sensitivity of MERS-CoV to exogenous IFN suggests that administration of recombinant IFN merits further evaluation as a therapeutic intervention strategy if new infections with this novel virus occur.</p></sec><sec sec-type="methods" id="sec1-4"><title>Methods</title><sec id="sec2-7"><title></title><sec id="sec3-1"><title>Cells culture and virus infection.</title><p>Vero cells (ATCC CCL-81) were cultured in Eagle’s minimal essential medium (EMEM; Lonza) with 8 % FCS (PAA) and antibiotics. Huh7 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM; Lonza) containing 8 % FCS, 2 mM <sc>l</sc>-glutamine (PAA), non-essential amino acids (PAA) and antibiotics. Vero E6 and Calu3/2B4 cells were cultured as described previously (<xref ref-type="bibr" rid="r41">Snijder <italic>et al.</italic>, 2006</xref>; <xref ref-type="bibr" rid="r58">Yoshikawa <italic>et al.</italic>, 2010</xref>). Infection of Vero, Vero E6, Huh7 and Calu3/2B4 cells with MERS-CoV (strain EMC/2012; <xref ref-type="bibr" rid="r59">Zaki <italic>et al.</italic>, 2012; van Boheemen <italic>et al.</italic>, 2012</xref>) at high m.o.i. (m.o.i. of 5) was carried out in PBS containing 50 µg DEAE-dextran ml<sup>−1</sup> and 2 % FCS. Inoculations with a low dose (m.o.i. ≤ 0.05) of MERS-CoV or SARS-CoV (strain HKU-39849; <xref ref-type="bibr" rid="r60">Zeng <italic>et al.</italic>, 2003</xref>) were carried out directly in EMEM containing 2 % FCS. Virus titrations by plaque assay were performed as described previously (<xref ref-type="bibr" rid="r46">van den Worm <italic>et al.</italic>, 2012</xref>). All work with live MERS-CoV and SARS-CoV was performed inside biosafety cabinets in Biosafety Level 3 facilities at Leiden University Medical Center or Erasmus Medical Center.</p></sec><sec id="sec3-2"><title>Antibodies and drugs.</title><p>Rabbit antisera recognizing the SARS-CoV replicase subunits nsp3, nsp4, nsp5 and nsp8 have been described previously (<xref ref-type="bibr" rid="r41">Snijder <italic>et al.</italic>, 2006</xref>; <xref ref-type="bibr" rid="r50">van Hemert <italic>et al.</italic>, 2008b</xref>). Rabbit antisera recognizing the SARS-CoV nucleocapsid (N) protein and MHV nsp4 were raised as described elsewhere (<xref ref-type="bibr" rid="r39">Snijder <italic>et al.</italic>, 1994</xref>). Antigens were a full-length recombinant SARS-CoV N protein (purified from <italic>Escherichia coli</italic>) and a synthetic peptide representing the 23 C-terminal residues of MHV nsp4, respectively. p-STAT1 was detected with Alexa Fluor 488-labelled mouse anti-STAT1 (pY701; BD Biosciences), and FITC-labelled anti-mouse IgG was used to enhance the green fluorescence. Virus infection was detected using the above-mentioned anti-nsp3 sera and Alexa Fluor 594-labelled anti-rabbit IgG.</p><p>CsA (Sigma) was dissolved in DMSO and a 10 mM stock was stored in aliquots for single use at −20 °C. Peg-interferon α-2b (PEG-IFN; Pegintron) was prepared according to the manufacturer’s instruction as a 100 µg ml<sup>−1</sup> stock and stored at 4 °C.</p></sec><sec id="sec3-3"><title>Immunofluorescence microscopy.</title><p>Cells were grown on coverslips and fixed with 3 % paraformaldehyde in PBS or with 4 % formaldehyde and 70 % ethanol (p-STAT1 experiments), permeabilized with 0.1 % Triton X-100 and processed for immunofluorescence microscopy as described previously (<xref ref-type="bibr" rid="r48">van der Meer <italic>et al.</italic>, 1998</xref>). Specimens were examined with a Zeiss Axioskop 2 fluorescence microscope with an Axiocam HRc camera and Zeiss Axiovision 4.4 software or with a confocal microscope (Zeiss, LSM 700) (p-STAT1 experiments).</p></sec><sec id="sec3-4"><title>EM.</title><p>Vero cells were grown on sapphire discs and fixed at 8 h p.i. for 30 min at room temperature with 3 % paraformaldehyde and 0.25 % glutaraldehyde in 0.1 M PHEM buffer pH 6.9 [60 mM piperazide-1,4-bis (2-ethanesulfonic acid), 25 mM HEPES, 2 mM MgCl<sub>2</sub>, 10 mM EGTA] containing 50 % diluted EMEM and 1 % FCS. Cells were stored in fixative at 4 °C for 72 h and then high-pressure frozen using a Leica EM PACT2. Freeze substitution was performed in an automated system (Leica AFS2) using as freeze-substitution medium acetone containing 1 % OsO<sub>4</sub>, 0.5 % uranyl acetate and 10 % H<sub>2</sub>O. First, the samples were maintained at −90 °C for 6 h in this medium and then slowly warmed to −20 °C within 14 h, kept at −20 °C for 1 h, warmed to 0 °C at a 5 °C h<sup>−1</sup> rate and left at 0 °C for 1 h before letting the samples reach room temperature. After washing with acetone, the samples were gradually infiltrated with epoxy resin LX-112 and polymerized at 60 °C. The samples were cut into thin sections (100 nm) and counterstained with uranyl acetate and lead citrate. Imaging was performed in an FEI Tecnai12 TWIN electron microscope operating at 120 kV and equipped with an Eagle 4k cooled slow-scan charge-coupled device camera (FEI Company). The images were acquired using binning mode 2.</p></sec><sec id="sec3-5"><title>Intracellular viral RNA analysis.</title><p>Isolation of intracellular viral RNA was carried out as described previously (<xref ref-type="bibr" rid="r51">van Kasteren <italic>et al.</italic>, 2013</xref>). After drying of the gel, viral mRNAs were detected by hybridization with a <sup>32</sup>P-labelled oligonucleotide probe (5′-GCAAATCATCTAATTAGCCTAATC-3′) complementary to the 3′ end of all MERS-CoV mRNAs. Equal loading was verified in a second hybridization using a <sup>32</sup>P-labelled oligonucleotide probe (5′-GTAACCCGTTGAACCCCATT-3′) recognizing 18S rRNA (<xref ref-type="bibr" rid="r49">van Hemert <italic>et al.</italic>, 2008a</xref>). ImageQuant TL software (GE Healthcare) was used for quantification.</p></sec><sec id="sec3-6"><title>Real-time reverse transcription-PCR (RT-PCR).</title><p>RNA from 200 µl culture medium of CoV-infected cells was isolated with a MagnaPure LC Total Nucleic Acid Isolation kit (Roche) and eluted in 100 µl. RT-PCR conditions for quantifying MERS-CoV and SARS-CoV RNA and amplification parameters have been described previously (<xref ref-type="bibr" rid="r23">Kuiken <italic>et al.</italic>, 2003</xref>; <xref ref-type="bibr" rid="r33">Raj <italic>et al.</italic>, 2013</xref>). Dilutions of viral RNA isolated from MERS-CoV or SARS-CoV virus stocks with a known virus titre were used to produce a standard curve.</p></sec><sec id="sec3-7"><title>Development of a screening assay for antiviral compounds.</title><p>Huh7 or Vero cells were seeded in 96-well plates at a density of 1×10<sup>4</sup> or 2×10<sup>4</sup> cells per well, respectively. After overnight growth, cells were infected with an m.o.i. of 0.005 or 0.05. At 1–3 days after incubation, differences in cell viability caused by virus-induced CPE or by compound-specific side effects were analysed using the CellTiter 96 AQ<sub>ueous</sub> Non-Radioactive Cell Proliferation Assay (Promega), according to the manufacturer’s instructions. Absorbance at 490 nm (<italic>A</italic><sub>490</sub>) was measured using a Berthold Mithras LB 940 96-well plate reader. Infected cells were given CsA or DMSO (solvent control) prior to infection (m.o.i 0.005). Cytotoxic effects caused by CsA treatment alone were monitored in parallel plates containing mock-infected cells.</p></sec><sec id="sec3-8"><title>IFN sensitivity and p-STAT1 translocation experiments.</title><p>One day prior to infection, Vero cells were plated at a density of 10<sup>4</sup> cells per well in a 96-well plate format. At −4, 0 and 4 h p.i., cells were incubated with 0–1000 ng PEG-IFN ml<sup>−1</sup> in 250 µl. At <italic>t</italic> = 0 h, all wells were washed with PBS and infected with MERS-CoV or SARS-CoV (100 TCID<sub>50</sub> per 100 µl medium). Those cultures receiving treatment from <italic>t</italic> = −4 or <italic>t</italic> = 0 were infected in the presence of the indicated concentration PEG-IFN. After 1 h, 150 µl medium was added to the cultures of <italic>t</italic> = −4 or <italic>t</italic> = 0 cultures, and 100 µl medium was added to the untreated cultures, which at 4 h p.i. received 50 µl medium supplemented with PEG-IFN to reach a final concentration of 0–1000 ng PEG-IFN ml<sup>−1</sup>. At 48 h p.i., RNA was isolated from 50 µl cell-culture supernatant and quantified using virus-specific real-time RT-PCR assays (see above). At 48 h p.i., CPE was also scored microscopically as either none (0), mild (1), moderate (2), severe (3) or complete (4).</p><p>For p-STAT1 nuclear translocation experiments, Vero cells were infected with MERS-CoV or SARS-CoV (m.o.i. of 1). At 8 h p.i., cells were treated with 1000 ng PEG-IFN ml<sup>−1</sup> for 30 min, fixed with 4 % formaldehyde and 70 % ethanol and subsequently stained for the presence of viral antigen and p-STAT1 translocation.</p></sec></sec></sec></body><back><ack><title>Acknowledgements</title><p>We are grateful to Ron Fouchier, Chris Lauber and Alexander Gorbalenya for helpful discussions, and we thank Dennis Ninaber and Corrine Beugeling for technical assistance. This research was supported in part by the <funding-source rid="sp1">Council for Chemical Sciences (CW)</funding-source>, TOP grant <award-id rid="sp1">700.57.301</award-id>, and the <funding-source rid="sp2">Council for Earth and Life Sciences (ALW)</funding-source>, MEERVOUD grant <award-id rid="sp2">836.10.003</award-id>, of the Netherlands Organization for Scientific Research (NWO), and the <funding-source rid="sp3">EU-FP7-Health project SILVER</funding-source> (grant <award-id rid="sp3">260644</award-id>).</p></ack><ref-list><title>References</title><ref id="r1"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Belouzard</surname><given-names>S.</given-names></name><name><surname>Millet</surname><given-names>J. K.</given-names></name><name><surname>Licitra</surname><given-names>B. N.</given-names></name><name><surname>Whittaker</surname><given-names>G. R.</given-names></name></person-group><bold>(</bold><year>2012</year><bold>).</bold><article-title>Mechanisms of coronavirus cell entry mediated by the viral spike protein</article-title>. <source>Viruses</source><volume>4</volume>, <fpage>1011</fpage>–<lpage>1033</lpage><pub-id pub-id-type="doi">10.3390/v4061011</pub-id><pub-id pub-id-type="pmid">22816037</pub-id></mixed-citation></ref><ref id="r2"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Bergman</surname><given-names>S. J.</given-names></name><name><surname>Ferguson</surname><given-names>M. C.</given-names></name><name><surname>Santanello</surname><given-names>C.</given-names></name></person-group><bold>(</bold><year>2011</year><bold>).</bold><article-title>Interferons as therapeutic agents for infectious diseases</article-title>. <source>Infect Dis Clin North Am</source><volume>25</volume>, <fpage>819</fpage>–<lpage>834</lpage><pub-id pub-id-type="doi">10.1016/j.idc.2011.07.008</pub-id><pub-id pub-id-type="pmid">22054758</pub-id></mixed-citation></ref><ref id="r3"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Brockway</surname><given-names>S. M.</given-names></name><name><surname>Clay</surname><given-names>C. T.</given-names></name><name><surname>Lu</surname><given-names>X. T.</given-names></name><name><surname>Denison</surname><given-names>M. R.</given-names></name></person-group><bold>(</bold><year>2003</year><bold>).</bold><article-title>Characterization of the expression, intracellular localization, and replication complex association of the putative mouse hepatitis virus RNA-dependent RNA polymerase</article-title>. <source>J Virol</source><volume>77</volume>, <fpage>10515</fpage>–<lpage>10527</lpage><pub-id pub-id-type="doi">10.1128/JVI.77.19.10515-10527.2003</pub-id><pub-id pub-id-type="pmid">12970436</pub-id></mixed-citation></ref><ref id="r4"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Cameron</surname><given-names>M. J.</given-names></name><name><surname>Kelvin</surname><given-names>A. A.</given-names></name><name><surname>Leon</surname><given-names>A. J.</given-names></name><name><surname>Cameron</surname><given-names>C. M.</given-names></name><name><surname>Ran</surname><given-names>L.</given-names></name><name><surname>Xu</surname><given-names>L.</given-names></name><name><surname>Chu</surname><given-names>Y. K.</given-names></name><name><surname>Danesh</surname><given-names>A.</given-names></name><name><surname>Fang</surname><given-names>Y.</given-names></name></person-group><bold>& other authors (</bold><year>2012</year><bold>).</bold><article-title>Lack of innate interferon responses during SARS coronavirus infection in a vaccination and reinfection ferret model</article-title>. <source>PLoS ONE</source><volume>7</volume>, <fpage>e45842</fpage><pub-id pub-id-type="doi">10.1371/journal.pone.0045842</pub-id><pub-id pub-id-type="pmid">23029269</pub-id></mixed-citation></ref><ref id="r5"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Cervantes-Barragan</surname><given-names>L.</given-names></name><name><surname>Züst</surname><given-names>R.</given-names></name><name><surname>Weber</surname><given-names>F.</given-names></name><name><surname>Spiegel</surname><given-names>M.</given-names></name><name><surname>Lang</surname><given-names>K. S.</given-names></name><name><surname>Akira</surname><given-names>S.</given-names></name><name><surname>Thiel</surname><given-names>V.</given-names></name><name><surname>Ludewig</surname><given-names>B.</given-names></name></person-group><bold>(</bold><year>2007</year><bold>).</bold><article-title>Control of coronavirus infection through plasmacytoid dendritic-cell-derived type I interferon</article-title>. <source>Blood</source><volume>109</volume>, <fpage>1131</fpage>–<lpage>1137</lpage><pub-id pub-id-type="doi">10.1182/blood-2006-05-023770</pub-id><pub-id pub-id-type="pmid">16985170</pub-id></mixed-citation></ref><ref id="r6"><mixed-citation publication-type="book"><person-group person-group-type="author"><name><surname>de Groot</surname><given-names>R. J.</given-names></name><name><surname>Cowley</surname><given-names>J. A.</given-names></name><name><surname>Enjuanes</surname><given-names>L.</given-names></name><name><surname>Faaberg</surname><given-names>K. S.</given-names></name><name><surname>Perlman</surname><given-names>S.</given-names></name><name><surname>Rottier</surname><given-names>P. J.</given-names></name><name><surname>Snijder</surname><given-names>E. J.</given-names></name><name><surname>Ziebuhr</surname><given-names>J.</given-names></name><name><surname>Gorbalenya</surname><given-names>A. E.</given-names></name></person-group><bold>(</bold><year>2012</year><bold>).</bold><article-title>Order of <italic>Nidovirales</italic></article-title>. In <source>Virus Taxonomy, the 9th Report of the International Committee on Taxonomy of Viruses</source>, pp. <fpage>785</fpage>–<lpage>795</lpage> Edited by <person-group person-group-type="editor"><name><surname>King</surname><given-names>A.</given-names></name><name><surname>Adams</surname><given-names>M.</given-names></name><name><surname>Carstens</surname><given-names>E.</given-names></name><name><surname>Lefkowitz</surname><given-names>E. J.</given-names></name></person-group><publisher-loc>Amsterdam</publisher-loc>: <publisher-name>Academic Press</publisher-name></mixed-citation></ref><ref id="r1a"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>de Groot</surname><given-names>R. J.</given-names></name><name><surname>Baker</surname><given-names>S. C.</given-names></name><name><surname>Baric</surname><given-names>R. S.</given-names></name><name><surname>Brown</surname><given-names>C. S.</given-names></name><name><surname>Drosten</surname><given-names>C.</given-names></name><name><surname>Enjuanes</surname><given-names>L.</given-names></name><name><surname>Fouchier</surname><given-names>R. A.</given-names></name><name><surname>Galiano</surname><given-names>M.</given-names></name><name><surname>Gorbalenya</surname><given-names>A. E.</given-names></name><name><surname>other authors</surname></name></person-group><bold>(</bold><year>2013</year><bold>).</bold><article-title>Middle East Respiratory Syndrome Coronavirus (MERS-CoV): Announcement of the Coronavirus Study Group</article-title>. <source>J Virol </source><volume>87</volume>, in press (<pub-id pub-id-type="doi">10.1128/JVI.01244-13</pub-id>).<pub-id pub-id-type="pmid">22816037</pub-id></mixed-citation></ref><ref id="r7"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>de Wilde</surname><given-names>A. H.</given-names></name><name><surname>Zevenhoven-Dobbe</surname><given-names>J. C.</given-names></name><name><surname>van der Meer</surname><given-names>Y.</given-names></name><name><surname>Thiel</surname><given-names>V.</given-names></name><name><surname>Narayanan</surname><given-names>K.</given-names></name><name><surname>Makino</surname><given-names>S.</given-names></name><name><surname>Snijder</surname><given-names>E. J.</given-names></name><name><surname>van Hemert</surname><given-names>M. J.</given-names></name></person-group><bold>(</bold><year>2011</year><bold>).</bold><article-title>Cyclosporin A inhibits the replication of diverse coronaviruses</article-title>. <source>J Gen Virol</source><volume>92</volume>, <fpage>2542</fpage>–<lpage>2548</lpage><pub-id pub-id-type="doi">10.1099/vir.0.034983-0</pub-id><pub-id pub-id-type="pmid">21752960</pub-id></mixed-citation></ref><ref id="r8"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Drosten</surname><given-names>C.</given-names></name><name><surname>Günther</surname><given-names>S.</given-names></name><name><surname>Preiser</surname><given-names>W.</given-names></name><name><surname>van der Werf</surname><given-names>S.</given-names></name><name><surname>Brodt</surname><given-names>H. R.</given-names></name><name><surname>Becker</surname><given-names>S.</given-names></name><name><surname>Rabenau</surname><given-names>H.</given-names></name><name><surname>Panning</surname><given-names>M.</given-names></name><name><surname>Kolesnikova</surname><given-names>L.</given-names></name></person-group><bold>& other authors (</bold><year>2003</year><bold>).</bold><article-title>Identification of a novel coronavirus in patients with severe acute respiratory syndrome</article-title>. <source>N Engl J Med</source><volume>348</volume>, <fpage>1967</fpage>–<lpage>1976</lpage><pub-id pub-id-type="doi">10.1056/NEJMoa030747</pub-id><pub-id pub-id-type="pmid">12690091</pub-id></mixed-citation></ref><ref id="r9"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Fouchier</surname><given-names>R. A.</given-names></name><name><surname>Hartwig</surname><given-names>N. G.</given-names></name><name><surname>Bestebroer</surname><given-names>T. M.</given-names></name><name><surname>Niemeyer</surname><given-names>B.</given-names></name><name><surname>de Jong</surname><given-names>J. C.</given-names></name><name><surname>Simon</surname><given-names>J. H.</given-names></name><name><surname>Osterhaus</surname><given-names>A. D.</given-names></name></person-group><bold>(</bold><year>2004</year><bold>).</bold><article-title>A previously undescribed coronavirus associated with respiratory disease in humans</article-title>. <source>Proc Natl Acad Sci U S A</source><volume>101</volume>, <fpage>6212</fpage>–<lpage>6216</lpage><pub-id pub-id-type="doi">10.1073/pnas.0400762101</pub-id><pub-id pub-id-type="pmid">15073334</pub-id></mixed-citation></ref><ref id="r10"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Frieman</surname><given-names>M.</given-names></name><name><surname>Yount</surname><given-names>B.</given-names></name><name><surname>Heise</surname><given-names>M.</given-names></name><name><surname>Kopecky-Bromberg</surname><given-names>S. A.</given-names></name><name><surname>Palese</surname><given-names>P.</given-names></name><name><surname>Baric</surname><given-names>R. S.</given-names></name></person-group><bold>(</bold><year>2007</year><bold>).</bold><article-title>Severe acute respiratory syndrome coronavirus ORF6 antagonizes STAT1 function by sequestering nuclear import factors on the rough endoplasmic reticulum/Golgi membrane</article-title>. <source>J Virol</source><volume>81</volume>, <fpage>9812</fpage>–<lpage>9824</lpage><pub-id pub-id-type="doi">10.1128/JVI.01012-07</pub-id><pub-id pub-id-type="pmid">17596301</pub-id></mixed-citation></ref><ref id="r11"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Garlinghouse</surname><given-names>L. E.</given-names><suffix>Jr</suffix></name><name><surname>Smith</surname><given-names>A. L.</given-names></name><name><surname>Holford</surname><given-names>T.</given-names></name></person-group><bold>(</bold><year>1984</year><bold>).</bold><article-title>The biological relationship of mouse hepatitis virus (MHV) strains and interferon: <italic>in vitro</italic> induction and sensitivities</article-title>. <source>Arch Virol</source><volume>82</volume>, <fpage>19</fpage>–<lpage>29</lpage><pub-id pub-id-type="doi">10.1007/BF01309365</pub-id><pub-id pub-id-type="pmid">6208879</pub-id></mixed-citation></ref><ref id="r12"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Gorbalenya</surname><given-names>A. E.</given-names></name><name><surname>Enjuanes</surname><given-names>L.</given-names></name><name><surname>Ziebuhr</surname><given-names>J.</given-names></name><name><surname>Snijder</surname><given-names>E. J.</given-names></name></person-group><bold>(</bold><year>2006</year><bold>).</bold><article-title><italic>Nidovirales</italic>: evolving the largest RNA virus genome</article-title>. <source>Virus Res</source><volume>117</volume>, <fpage>17</fpage>–<lpage>37</lpage><pub-id pub-id-type="doi">10.1016/j.virusres.2006.01.017</pub-id><pub-id pub-id-type="pmid">16503362</pub-id></mixed-citation></ref><ref id="r13"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Gosert</surname><given-names>R.</given-names></name><name><surname>Kanjanahaluethai</surname><given-names>A.</given-names></name><name><surname>Egger</surname><given-names>D.</given-names></name><name><surname>Bienz</surname><given-names>K.</given-names></name><name><surname>Baker</surname><given-names>S. C.</given-names></name></person-group><bold>(</bold><year>2002</year><bold>).</bold><article-title>RNA replication of mouse hepatitis virus takes place at double-membrane vesicles</article-title>. <source>J Virol</source><volume>76</volume>, <fpage>3697</fpage>–<lpage>3708</lpage><pub-id pub-id-type="doi">10.1128/JVI.76.8.3697-3708.2002</pub-id><pub-id pub-id-type="pmid">11907209</pub-id></mixed-citation></ref><ref id="r14"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Haagmans</surname><given-names>B. L.</given-names></name><name><surname>Kuiken</surname><given-names>T.</given-names></name><name><surname>Martina</surname><given-names>B. E.</given-names></name><name><surname>Fouchier</surname><given-names>R. A.</given-names></name><name><surname>Rimmelzwaan</surname><given-names>G. F.</given-names></name><name><surname>van Amerongen</surname><given-names>G.</given-names></name><name><surname>van Riel</surname><given-names>D.</given-names></name><name><surname>de Jong</surname><given-names>T.</given-names></name><name><surname>Itamura</surname><given-names>S.</given-names></name></person-group><bold>& other authors (</bold><year>2004</year><bold>).</bold><article-title>Pegylated interferon-α protects type 1 pneumocytes against SARS coronavirus infection in macaques</article-title>. <source>Nat Med</source><volume>10</volume>, <fpage>290</fpage>–<lpage>293</lpage><pub-id pub-id-type="doi">10.1038/nm1001</pub-id><pub-id pub-id-type="pmid">14981511</pub-id></mixed-citation></ref><ref id="r15"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Hamre</surname><given-names>D.</given-names></name><name><surname>Procknow</surname><given-names>J. J.</given-names></name></person-group><bold>(</bold><year>1966</year><bold>).</bold><article-title>A new virus isolated from the human respiratory tract</article-title>. <source>Proc Soc Exp Biol Med</source><volume>121</volume>, <fpage>190</fpage>–<lpage>193</lpage><pub-id pub-id-type="pmid">4285768</pub-id></mixed-citation></ref><ref id="r16"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Hussain</surname><given-names>S.</given-names></name><name><surname>Perlman</surname><given-names>S.</given-names></name><name><surname>Gallagher</surname><given-names>T. M.</given-names></name></person-group><bold>(</bold><year>2008</year><bold>).</bold><article-title>Severe acute respiratory syndrome coronavirus protein 6 accelerates murine hepatitis virus infections by more than one mechanism</article-title>. <source>J Virol</source><volume>82</volume>, <fpage>7212</fpage>–<lpage>7222</lpage><pub-id pub-id-type="doi">10.1128/JVI.02406-07</pub-id><pub-id pub-id-type="pmid">18448520</pub-id></mixed-citation></ref><ref id="r17"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Huynh</surname><given-names>J.</given-names></name><name><surname>Li</surname><given-names>S.</given-names></name><name><surname>Yount</surname><given-names>B.</given-names></name><name><surname>Smith</surname><given-names>A.</given-names></name><name><surname>Sturges</surname><given-names>L.</given-names></name><name><surname>Olsen</surname><given-names>J. C.</given-names></name><name><surname>Nagel</surname><given-names>J.</given-names></name><name><surname>Johnson</surname><given-names>J. B.</given-names></name><name><surname>Agnihothram</surname><given-names>S.</given-names></name></person-group><bold>& other authors (</bold><year>2012</year><bold>).</bold><article-title>Evidence supporting a zoonotic origin of human coronavirus strain NL63</article-title>. <source>J Virol</source><volume>86</volume>, <fpage>12816</fpage>–<lpage>12825</lpage><pub-id pub-id-type="doi">10.1128/JVI.00906-12</pub-id><pub-id pub-id-type="pmid">22993147</pub-id></mixed-citation></ref><ref id="r18"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Kindler</surname><given-names>E.</given-names></name><name><surname>Jónsdóttir</surname><given-names>H. R.</given-names></name><name><surname>Muth</surname><given-names>D.</given-names></name><name><surname>Hamming</surname><given-names>O. J.</given-names></name><name><surname>Hartmann</surname><given-names>R.</given-names></name><name><surname>Rodriguez</surname><given-names>R.</given-names></name><name><surname>Geffers</surname><given-names>R.</given-names></name><name><surname>Fouchier</surname><given-names>R. A.</given-names></name><name><surname>Drosten</surname><given-names>C.</given-names></name></person-group><bold>& other authors (</bold><year>2013</year><bold>).</bold><article-title>Efficient replication of the novel human betacoronavirus EMC on primary human epithelium highlights its zoonotic potential</article-title>. <source>MBio</source><volume>4</volume>, <fpage>e00611-12</fpage><pub-id pub-id-type="doi">10.1128/mBio.00611-12</pub-id><pub-id pub-id-type="pmid">23422412</pub-id></mixed-citation></ref><ref id="r19"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Knoops</surname><given-names>K.</given-names></name><name><surname>Kikkert</surname><given-names>M.</given-names></name><name><surname>Worm</surname><given-names>S. H.</given-names></name><name><surname>Zevenhoven-Dobbe</surname><given-names>J. C.</given-names></name><name><surname>van der Meer</surname><given-names>Y.</given-names></name><name><surname>Koster</surname><given-names>A. J.</given-names></name><name><surname>Mommaas</surname><given-names>A. M.</given-names></name><name><surname>Snijder</surname><given-names>E. J.</given-names></name></person-group><bold>(</bold><year>2008</year><bold>).</bold><article-title>SARS-coronavirus replication is supported by a reticulovesicular network of modified endoplasmic reticulum</article-title>. <source>PLoS Biol</source><volume>6</volume>, <fpage>e226</fpage><pub-id pub-id-type="doi">10.1371/journal.pbio.0060226</pub-id><pub-id pub-id-type="pmid">18798692</pub-id></mixed-citation></ref><ref id="r20"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Knoops</surname><given-names>K.</given-names></name><name><surname>Bárcena</surname><given-names>M.</given-names></name><name><surname>Limpens</surname><given-names>R. W.</given-names></name><name><surname>Koster</surname><given-names>A. J.</given-names></name><name><surname>Mommaas</surname><given-names>A. M.</given-names></name><name><surname>Snijder</surname><given-names>E. J.</given-names></name></person-group><bold>(</bold><year>2012</year><bold>).</bold><article-title>Ultrastructural characterization of arterivirus replication structures: reshaping the endoplasmic reticulum to accommodate viral RNA synthesis</article-title>. <source>J Virol</source><volume>86</volume>, <fpage>2474</fpage>–<lpage>2487</lpage><pub-id pub-id-type="doi">10.1128/JVI.06677-11</pub-id><pub-id pub-id-type="pmid">22190716</pub-id></mixed-citation></ref><ref id="r21"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Kopecky-Bromberg</surname><given-names>S. A.</given-names></name><name><surname>Martinez-Sobrido</surname><given-names>L.</given-names></name><name><surname>Palese</surname><given-names>P.</given-names></name></person-group><bold>(</bold><year>2006</year><bold>).</bold><article-title>7a protein of severe acute respiratory syndrome coronavirus inhibits cellular protein synthesis and activates p38 mitogen-activated protein kinase</article-title>. <source>J Virol</source><volume>80</volume>, <fpage>785</fpage>–<lpage>793</lpage><pub-id pub-id-type="doi">10.1128/JVI.80.2.785-793.2006</pub-id><pub-id pub-id-type="pmid">16378980</pub-id></mixed-citation></ref><ref id="r22"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Ksiazek</surname><given-names>T. G.</given-names></name><name><surname>Erdman</surname><given-names>D.</given-names></name><name><surname>Goldsmith</surname><given-names>C. S.</given-names></name><name><surname>Zaki</surname><given-names>S. R.</given-names></name><name><surname>Peret</surname><given-names>T.</given-names></name><name><surname>Emery</surname><given-names>S.</given-names></name><name><surname>Tong</surname><given-names>S.</given-names></name><name><surname>Urbani</surname><given-names>C.</given-names></name><name><surname>Comer</surname><given-names>J. A.</given-names></name></person-group><bold>& other authors (</bold><year>2003</year><bold>).</bold><article-title>A novel coronavirus associated with severe acute respiratory syndrome</article-title>. <source>N Engl J Med</source><volume>348</volume>, <fpage>1953</fpage>–<lpage>1966</lpage><pub-id pub-id-type="doi">10.1056/NEJMoa030781</pub-id><pub-id pub-id-type="pmid">12690092</pub-id></mixed-citation></ref><ref id="r23"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Kuiken</surname><given-names>T.</given-names></name><name><surname>Fouchier</surname><given-names>R. A.</given-names></name><name><surname>Schutten</surname><given-names>M.</given-names></name><name><surname>Rimmelzwaan</surname><given-names>G. F.</given-names></name><name><surname>van Amerongen</surname><given-names>G.</given-names></name><name><surname>van Riel</surname><given-names>D.</given-names></name><name><surname>Laman</surname><given-names>J. D.</given-names></name><name><surname>de Jong</surname><given-names>T.</given-names></name><name><surname>van Doornum</surname><given-names>G.</given-names></name></person-group><bold>& other authors (</bold><year>2003</year><bold>).</bold><article-title>Newly discovered coronavirus as the primary cause of severe acute respiratory syndrome</article-title>. <source>Lancet</source><volume>362</volume>, <fpage>263</fpage>–<lpage>270</lpage><pub-id pub-id-type="doi">10.1016/S0140-6736(03)13967-0</pub-id><pub-id pub-id-type="pmid">12892955</pub-id></mixed-citation></ref><ref id="r24"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Lau</surname><given-names>S. K.</given-names></name><name><surname>Woo</surname><given-names>P. C.</given-names></name><name><surname>Li</surname><given-names>K. S.</given-names></name><name><surname>Huang</surname><given-names>Y.</given-names></name><name><surname>Tsoi</surname><given-names>H. W.</given-names></name><name><surname>Wong</surname><given-names>B. H.</given-names></name><name><surname>Wong</surname><given-names>S. S.</given-names></name><name><surname>Leung</surname><given-names>S. Y.</given-names></name><name><surname>Chan</surname><given-names>K. H.</given-names></name><name><surname>Yuen</surname><given-names>K. Y.</given-names></name></person-group><bold>(</bold><year>2005</year><bold>).</bold><article-title>Severe acute respiratory syndrome coronavirus-like virus in Chinese horseshoe bats</article-title>. <source>Proc Natl Acad Sci U S A</source><volume>102</volume>, <fpage>14040</fpage>–<lpage>14045</lpage><pub-id pub-id-type="doi">10.1073/pnas.0506735102</pub-id><pub-id pub-id-type="pmid">16169905</pub-id></mixed-citation></ref><ref id="r25"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Li</surname><given-names>W.</given-names></name><name><surname>Shi</surname><given-names>Z.</given-names></name><name><surname>Yu</surname><given-names>M.</given-names></name><name><surname>Ren</surname><given-names>W.</given-names></name><name><surname>Smith</surname><given-names>C.</given-names></name><name><surname>Epstein</surname><given-names>J. H.</given-names></name><name><surname>Wang</surname><given-names>H.</given-names></name><name><surname>Crameri</surname><given-names>G.</given-names></name><name><surname>Hu</surname><given-names>Z.</given-names></name></person-group><bold>& other authors (</bold><year>2005</year><bold>).</bold><article-title>Bats are natural reservoirs of SARS-like coronaviruses</article-title>. <source>Science</source><volume>310</volume>, <fpage>676</fpage>–<lpage>679</lpage><pub-id pub-id-type="doi">10.1126/science.1118391</pub-id><pub-id pub-id-type="pmid">16195424</pub-id></mixed-citation></ref><ref id="r26"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>McIntosh</surname><given-names>K.</given-names></name><name><surname>Dees</surname><given-names>J. H.</given-names></name><name><surname>Becker</surname><given-names>W. B.</given-names></name><name><surname>Kapikian</surname><given-names>A. Z.</given-names></name><name><surname>Chanock</surname><given-names>R. M.</given-names></name></person-group><bold>(</bold><year>1967</year><bold>).</bold><article-title>Recovery in tracheal organ cultures of novel viruses from patients with respiratory disease</article-title>. <source>Proc Natl Acad Sci U S A</source><volume>57</volume>, <fpage>933</fpage>–<lpage>940</lpage><pub-id pub-id-type="doi">10.1073/pnas.57.4.933</pub-id><pub-id pub-id-type="pmid">5231356</pub-id></mixed-citation></ref><ref id="r27"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Müller</surname><given-names>M. A.</given-names></name><name><surname>Raj</surname><given-names>V. S.</given-names></name><name><surname>Muth</surname><given-names>D.</given-names></name><name><surname>Meyer</surname><given-names>B.</given-names></name><name><surname>Kallies</surname><given-names>S.</given-names></name><name><surname>Smits</surname><given-names>S. L.</given-names></name><name><surname>Wollny</surname><given-names>R.</given-names></name><name><surname>Bestebroer</surname><given-names>T. M.</given-names></name><name><surname>Specht</surname><given-names>S.</given-names></name></person-group><bold>& other authors (</bold><year>2012</year><bold>).</bold><article-title>Human coronavirus EMC does not require the SARS-coronavirus receptor and maintains broad replicative capability in mammalian cell lines</article-title>. <source>MBio</source><volume>3</volume>, <fpage>e00515-12</fpage><pub-id pub-id-type="doi">10.1128/mBio.00515-12</pub-id><pub-id pub-id-type="pmid">23232719</pub-id></mixed-citation></ref><ref id="r28"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Nagy</surname><given-names>P. D.</given-names></name><name><surname>Wang</surname><given-names>R. Y.</given-names></name><name><surname>Pogany</surname><given-names>J.</given-names></name><name><surname>Hafren</surname><given-names>A.</given-names></name><name><surname>Makinen</surname><given-names>K.</given-names></name></person-group><bold>(</bold><year>2011</year><bold>).</bold><article-title>Emerging picture of host chaperone and cyclophilin roles in RNA virus replication</article-title>. <source>Virology</source><volume>411</volume>, <fpage>374</fpage>–<lpage>382</lpage><pub-id pub-id-type="doi">10.1016/j.virol.2010.12.061</pub-id><pub-id pub-id-type="pmid">21295323</pub-id></mixed-citation></ref><ref id="r29"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Paragas</surname><given-names>J.</given-names></name><name><surname>Blatt</surname><given-names>L. M.</given-names></name><name><surname>Hartmann</surname><given-names>C.</given-names></name><name><surname>Huggins</surname><given-names>J. W.</given-names></name><name><surname>Endy</surname><given-names>T. P.</given-names></name></person-group><bold>(</bold><year>2005</year><bold>).</bold><article-title>Interferon alfacon1 is an inhibitor of SARS-corona virus in cell-based models</article-title>. <source>Antiviral Res</source><volume>66</volume>, <fpage>99</fpage>–<lpage>102</lpage><pub-id pub-id-type="doi">10.1016/j.antiviral.2005.01.002</pub-id><pub-id pub-id-type="pmid">15911026</pub-id></mixed-citation></ref><ref id="r30"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Perlman</surname><given-names>S.</given-names></name><name><surname>Netland</surname><given-names>J.</given-names></name></person-group><bold>(</bold><year>2009</year><bold>).</bold><article-title>Coronaviruses post-SARS: update on replication and pathogenesis</article-title>. <source>Nat Rev Microbiol</source><volume>7</volume>, <fpage>439</fpage>–<lpage>450</lpage><pub-id pub-id-type="doi">10.1038/nrmicro2147</pub-id><pub-id pub-id-type="pmid">19430490</pub-id></mixed-citation></ref><ref id="r31"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Pfefferle</surname><given-names>S.</given-names></name><name><surname>Oppong</surname><given-names>S.</given-names></name><name><surname>Drexler</surname><given-names>J. F.</given-names></name><name><surname>Gloza-Rausch</surname><given-names>F.</given-names></name><name><surname>Ipsen</surname><given-names>A.</given-names></name><name><surname>Seebens</surname><given-names>A.</given-names></name><name><surname>Müller</surname><given-names>M. A.</given-names></name><name><surname>Annan</surname><given-names>A.</given-names></name><name><surname>Vallo</surname><given-names>P.</given-names></name></person-group><bold>& other authors (</bold><year>2009</year><bold>).</bold><article-title>Distant relatives of severe acute respiratory syndrome coronavirus and close relatives of human coronavirus 229E in bats, Ghana</article-title>. <source>Emerg Infect Dis</source><volume>15</volume>, <fpage>1377</fpage>–<lpage>1384</lpage><pub-id pub-id-type="doi">10.3201/eid1509.090224</pub-id><pub-id pub-id-type="pmid">19788804</pub-id></mixed-citation></ref><ref id="r32"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Pfefferle</surname><given-names>S.</given-names></name><name><surname>Schöpf</surname><given-names>J.</given-names></name><name><surname>Kögl</surname><given-names>M.</given-names></name><name><surname>Friedel</surname><given-names>C. C.</given-names></name><name><surname>Müller</surname><given-names>M. A.</given-names></name><name><surname>Carbajo-Lozoya</surname><given-names>J.</given-names></name><name><surname>Stellberger</surname><given-names>T.</given-names></name><name><surname>von Dall’Armi</surname><given-names>E.</given-names></name><name><surname>Herzog</surname><given-names>P.</given-names></name></person-group><bold>& other authors (</bold><year>2011</year><bold>).</bold><article-title>The SARS-coronavirus-host interactome: identification of cyclophilins as target for pan-coronavirus inhibitors</article-title>. <source>PLoS Pathog</source><volume>7</volume>, <fpage>e1002331</fpage><pub-id pub-id-type="doi">10.1371/journal.ppat.1002331</pub-id><pub-id pub-id-type="pmid">22046132</pub-id></mixed-citation></ref><ref id="r33"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Raj</surname><given-names>V. S.</given-names></name><name><surname>Mou</surname><given-names>H.</given-names></name><name><surname>Smits</surname><given-names>S. L.</given-names></name><name><surname>Dekkers</surname><given-names>D. H.</given-names></name><name><surname>Müller</surname><given-names>M. A.</given-names></name><name><surname>Dijkman</surname><given-names>R.</given-names></name><name><surname>Muth</surname><given-names>D.</given-names></name><name><surname>Demmers</surname><given-names>J. A.</given-names></name><name><surname>Zaki</surname><given-names>A.</given-names></name></person-group><bold>& other authors (</bold><year>2013</year><bold>).</bold><article-title>Dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-EMC</article-title>. <source>Nature</source><volume>495</volume>, <fpage>251</fpage>–<lpage>254</lpage><pub-id pub-id-type="doi">10.1038/nature12005</pub-id><pub-id pub-id-type="pmid">23486063</pub-id></mixed-citation></ref><ref id="r34"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Randall</surname><given-names>R. E.</given-names></name><name><surname>Goodbourn</surname><given-names>S.</given-names></name></person-group><bold>(</bold><year>2008</year><bold>).</bold><article-title>Interferons and viruses: an interplay between induction, signalling, antiviral responses and virus countermeasures</article-title>. <source>J Gen Virol</source><volume>89</volume>, <fpage>1</fpage>–<lpage>47</lpage><pub-id pub-id-type="doi">10.1099/vir.0.83391-0</pub-id><pub-id pub-id-type="pmid">18089727</pub-id></mixed-citation></ref><ref id="r35"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Ratia</surname><given-names>K.</given-names></name><name><surname>Saikatendu</surname><given-names>K. S.</given-names></name><name><surname>Santarsiero</surname><given-names>B. D.</given-names></name><name><surname>Barretto</surname><given-names>N.</given-names></name><name><surname>Baker</surname><given-names>S. C.</given-names></name><name><surname>Stevens</surname><given-names>R. C.</given-names></name><name><surname>Mesecar</surname><given-names>A. D.</given-names></name></person-group><bold>(</bold><year>2006</year><bold>).</bold><article-title>Severe acute respiratory syndrome coronavirus papain-like protease: structure of a viral deubiquitinating enzyme</article-title>. <source>Proc Natl Acad Sci U S A</source><volume>103</volume>, <fpage>5717</fpage>–<lpage>5722</lpage><pub-id pub-id-type="doi">10.1073/pnas.0510851103</pub-id><pub-id pub-id-type="pmid">16581910</pub-id></mixed-citation></ref><ref id="r36"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Rose</surname><given-names>K. M.</given-names></name><name><surname>Elliott</surname><given-names>R.</given-names></name><name><surname>Martínez-Sobrido</surname><given-names>L.</given-names></name><name><surname>García-Sastre</surname><given-names>A.</given-names></name><name><surname>Weiss</surname><given-names>S. R.</given-names></name></person-group><bold>(</bold><year>2010</year><bold>).</bold><article-title>Murine coronavirus delays expression of a subset of interferon-stimulated genes</article-title>. <source>J Virol</source><volume>84</volume>, <fpage>5656</fpage>–<lpage>5669</lpage><pub-id pub-id-type="doi">10.1128/JVI.00211-10</pub-id><pub-id pub-id-type="pmid">20357099</pub-id></mixed-citation></ref><ref id="r37"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Roth-Cross</surname><given-names>J. K.</given-names></name><name><surname>Martínez-Sobrido</surname><given-names>L.</given-names></name><name><surname>Scott</surname><given-names>E. P.</given-names></name><name><surname>García-Sastre</surname><given-names>A.</given-names></name><name><surname>Weiss</surname><given-names>S. R.</given-names></name></person-group><bold>(</bold><year>2007</year><bold>).</bold><article-title>Inhibition of the alpha/beta interferon response by mouse hepatitis virus at multiple levels</article-title>. <source>J Virol</source><volume>81</volume>, <fpage>7189</fpage>–<lpage>7199</lpage><pub-id pub-id-type="doi">10.1128/JVI.00013-07</pub-id><pub-id pub-id-type="pmid">17459917</pub-id></mixed-citation></ref><ref id="r38"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Sims</surname><given-names>A. C.</given-names></name><name><surname>Tilton</surname><given-names>S. C.</given-names></name><name><surname>Menachery</surname><given-names>V. D.</given-names></name><name><surname>Gralinski</surname><given-names>L. E.</given-names></name><name><surname>Schäfer</surname><given-names>A.</given-names></name><name><surname>Matzke</surname><given-names>M. M.</given-names></name><name><surname>Webb-Robertson</surname><given-names>B. J.</given-names></name><name><surname>Chang</surname><given-names>J.</given-names></name><name><surname>Luna</surname><given-names>M. L.</given-names></name></person-group><bold>& other authors (</bold><year>2013</year><bold>).</bold><article-title>Release of severe acute respiratory syndrome coronavirus nuclear import block enhances host transcription in human lung cells</article-title>. <source>J Virol</source><volume>87</volume>, <fpage>3885</fpage>–<lpage>3902</lpage><pub-id pub-id-type="doi">10.1128/JVI.02520-12</pub-id><pub-id pub-id-type="pmid">23365422</pub-id></mixed-citation></ref><ref id="r39"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Snijder</surname><given-names>E. J.</given-names></name><name><surname>Wassenaar</surname><given-names>A. L.</given-names></name><name><surname>Spaan</surname><given-names>W. J.</given-names></name></person-group><bold>(</bold><year>1994</year><bold>).</bold><article-title>Proteolytic processing of the replicase ORF1a protein of equine arteritis virus</article-title>. <source>J Virol</source><volume>68</volume>, <fpage>5755</fpage>–<lpage>5764</lpage><pub-id pub-id-type="pmid">8057457</pub-id></mixed-citation></ref><ref id="r40"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Snijder</surname><given-names>E. J.</given-names></name><name><surname>Bredenbeek</surname><given-names>P. J.</given-names></name><name><surname>Dobbe</surname><given-names>J. C.</given-names></name><name><surname>Thiel</surname><given-names>V.</given-names></name><name><surname>Ziebuhr</surname><given-names>J.</given-names></name><name><surname>Poon</surname><given-names>L. L.</given-names></name><name><surname>Guan</surname><given-names>Y.</given-names></name><name><surname>Rozanov</surname><given-names>M.</given-names></name><name><surname>Spaan</surname><given-names>W. J.</given-names></name><name><surname>Gorbalenya</surname><given-names>A. E.</given-names></name></person-group><bold>(</bold><year>2003</year><bold>).</bold><article-title>Unique and conserved features of genome and proteome of SARS-coronavirus, an early split-off from the coronavirus group 2 lineage</article-title>. <source>J Mol Biol</source><volume>331</volume>, <fpage>991</fpage>–<lpage>1004</lpage><pub-id pub-id-type="doi">10.1016/S0022-2836(03)00865-9</pub-id><pub-id pub-id-type="pmid">12927536</pub-id></mixed-citation></ref><ref id="r41"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Snijder</surname><given-names>E. J.</given-names></name><name><surname>van der Meer</surname><given-names>Y.</given-names></name><name><surname>Zevenhoven-Dobbe</surname><given-names>J.</given-names></name><name><surname>Onderwater</surname><given-names>J. J.</given-names></name><name><surname>van der Meulen</surname><given-names>J.</given-names></name><name><surname>Koerten</surname><given-names>H. K.</given-names></name><name><surname>Mommaas</surname><given-names>A. M.</given-names></name></person-group><bold>(</bold><year>2006</year><bold>).</bold><article-title>Ultrastructure and origin of membrane vesicles associated with the severe acute respiratory syndrome coronavirus replication complex</article-title>. <source>J Virol</source><volume>80</volume>, <fpage>5927</fpage>–<lpage>5940</lpage><pub-id pub-id-type="doi">10.1128/JVI.02501-05</pub-id><pub-id pub-id-type="pmid">16731931</pub-id></mixed-citation></ref><ref id="r42"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Stertz</surname><given-names>S.</given-names></name><name><surname>Reichelt</surname><given-names>M.</given-names></name><name><surname>Spiegel</surname><given-names>M.</given-names></name><name><surname>Kuri</surname><given-names>T.</given-names></name><name><surname>Martínez-Sobrido</surname><given-names>L.</given-names></name><name><surname>García-Sastre</surname><given-names>A.</given-names></name><name><surname>Weber</surname><given-names>F.</given-names></name><name><surname>Kochs</surname><given-names>G.</given-names></name></person-group><bold>(</bold><year>2007</year><bold>).</bold><article-title>The intracellular sites of early replication and budding of SARS-coronavirus</article-title>. <source>Virology</source><volume>361</volume>, <fpage>304</fpage>–<lpage>315</lpage><pub-id pub-id-type="doi">10.1016/j.virol.2006.11.027</pub-id><pub-id pub-id-type="pmid">17210170</pub-id></mixed-citation></ref><ref id="r43"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Taguchi</surname><given-names>F.</given-names></name><name><surname>Siddell</surname><given-names>S. G.</given-names></name></person-group><bold>(</bold><year>1985</year><bold>).</bold><article-title>Difference in sensitivity to interferon among mouse hepatitis viruses with high and low virulence for mice</article-title>. <source>Virology</source><volume>147</volume>, <fpage>41</fpage>–<lpage>48</lpage><pub-id pub-id-type="doi">10.1016/0042-6822(85)90225-9</pub-id><pub-id pub-id-type="pmid">2998070</pub-id></mixed-citation></ref><ref id="r44"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Ulasli</surname><given-names>M.</given-names></name><name><surname>Verheije</surname><given-names>M. H.</given-names></name><name><surname>de Haan</surname><given-names>C. A.</given-names></name><name><surname>Reggiori</surname><given-names>F.</given-names></name></person-group><bold>(</bold><year>2010</year><bold>).</bold><article-title>Qualitative and quantitative ultrastructural analysis of the membrane rearrangements induced by coronavirus</article-title>. <source>Cell Microbiol</source><volume>12</volume>, <fpage>844</fpage>–<lpage>861</lpage><pub-id pub-id-type="doi">10.1111/j.1462-5822.2010.01437.x</pub-id><pub-id pub-id-type="pmid">20088951</pub-id></mixed-citation></ref><ref id="r45"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>van Boheemen</surname><given-names>S.</given-names></name><name><surname>de Graaf</surname><given-names>M.</given-names></name><name><surname>Lauber</surname><given-names>C.</given-names></name><name><surname>Bestebroer</surname><given-names>T. M.</given-names></name><name><surname>Raj</surname><given-names>V. S.</given-names></name><name><surname>Zaki</surname><given-names>A. M.</given-names></name><name><surname>Osterhaus</surname><given-names>A. D.</given-names></name><name><surname>Haagmans</surname><given-names>B. L.</given-names></name><name><surname>Gorbalenya</surname><given-names>A. E.</given-names></name></person-group><bold>& other authors (</bold><year>2012</year><bold>).</bold><article-title>Genomic characterization of a newly discovered coronavirus associated with acute respiratory distress syndrome in humans</article-title>. <source>MBio</source><volume>3</volume>, <fpage>e00473-12</fpage><pub-id pub-id-type="doi">10.1128/mBio.00473-12</pub-id><pub-id pub-id-type="pmid">23170002</pub-id></mixed-citation></ref><ref id="r46"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>van den Worm</surname><given-names>S. H.</given-names></name><name><surname>Eriksson</surname><given-names>K. K.</given-names></name><name><surname>Zevenhoven</surname><given-names>J. C.</given-names></name><name><surname>Weber</surname><given-names>F.</given-names></name><name><surname>Züst</surname><given-names>R.</given-names></name><name><surname>Kuri</surname><given-names>T.</given-names></name><name><surname>Dijkman</surname><given-names>R.</given-names></name><name><surname>Chang</surname><given-names>G.</given-names></name><name><surname>Siddell</surname><given-names>S. G.</given-names></name></person-group><bold>& other authors (</bold><year>2012</year><bold>).</bold><article-title>Reverse genetics of SARS-related coronavirus using vaccinia virus-based recombination</article-title>. <source>PLoS ONE</source><volume>7</volume>, <fpage>e32857</fpage><pub-id pub-id-type="doi">10.1371/journal.pone.0032857</pub-id><pub-id pub-id-type="pmid">22412934</pub-id></mixed-citation></ref><ref id="r47"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>van der Hoek</surname><given-names>L.</given-names></name><name><surname>Pyrc</surname><given-names>K.</given-names></name><name><surname>Jebbink</surname><given-names>M. F.</given-names></name><name><surname>Vermeulen-Oost</surname><given-names>W.</given-names></name><name><surname>Berkhout</surname><given-names>R. J.</given-names></name><name><surname>Wolthers</surname><given-names>K. C.</given-names></name><name><surname>Wertheim-van Dillen</surname><given-names>P. M.</given-names></name><name><surname>Kaandorp</surname><given-names>J.</given-names></name><name><surname>Spaargaren</surname><given-names>J.</given-names></name><name><surname>Berkhout</surname><given-names>B.</given-names></name></person-group><bold>(</bold><year>2004</year><bold>).</bold><article-title>Identification of a new human coronavirus</article-title>. <source>Nat Med</source><volume>10</volume>, <fpage>368</fpage>–<lpage>373</lpage><pub-id pub-id-type="doi">10.1038/nm1024</pub-id><pub-id pub-id-type="pmid">15034574</pub-id></mixed-citation></ref><ref id="r48"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>van der Meer</surname><given-names>Y.</given-names></name><name><surname>van Tol</surname><given-names>H.</given-names></name><name><surname>Locker</surname><given-names>J. K.</given-names></name><name><surname>Snijder</surname><given-names>E. J.</given-names></name></person-group><bold>(</bold><year>1998</year><bold>).</bold><article-title>ORF1a-encoded replicase subunits are involved in the membrane association of the arterivirus replication complex</article-title>. <source>J Virol</source><volume>72</volume>, <fpage>6689</fpage>–<lpage>6698</lpage><pub-id pub-id-type="pmid">9658116</pub-id></mixed-citation></ref><ref id="r49"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>van Hemert</surname><given-names>M. J.</given-names></name><name><surname>de Wilde</surname><given-names>A. H.</given-names></name><name><surname>Gorbalenya</surname><given-names>A. E.</given-names></name><name><surname>Snijder</surname><given-names>E. J.</given-names></name></person-group><bold>(</bold><year>2008a</year><bold>).</bold><article-title>The <italic>in vitro</italic> RNA synthesizing activity of the isolated arterivirus replication/transcription complex is dependent on a host factor</article-title>. <source>J Biol Chem</source><volume>283</volume>, <fpage>16525</fpage>–<lpage>16536</lpage><pub-id pub-id-type="doi">10.1074/jbc.M708136200</pub-id><pub-id pub-id-type="pmid">18411274</pub-id></mixed-citation></ref><ref id="r50"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>van Hemert</surname><given-names>M. J.</given-names></name><name><surname>van den Worm</surname><given-names>S. H.</given-names></name><name><surname>Knoops</surname><given-names>K.</given-names></name><name><surname>Mommaas</surname><given-names>A. M.</given-names></name><name><surname>Gorbalenya</surname><given-names>A. E.</given-names></name><name><surname>Snijder</surname><given-names>E. J.</given-names></name></person-group><bold>(</bold><year>2008b</year><bold>).</bold><article-title>SARS-coronavirus replication/transcription complexes are membrane-protected and need a host factor for activity <italic>in vitro</italic></article-title>. <source>PLoS Pathog</source><volume>4</volume>, <fpage>e1000054</fpage><pub-id pub-id-type="doi">10.1371/journal.ppat.1000054</pub-id><pub-id pub-id-type="pmid">18451981</pub-id></mixed-citation></ref><ref id="r51"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>van Kasteren</surname><given-names>P. B.</given-names></name><name><surname>Bailey-Elkin</surname><given-names>B. A.</given-names></name><name><surname>James</surname><given-names>T. W.</given-names></name><name><surname>Ninaber</surname><given-names>D. K.</given-names></name><name><surname>Beugeling</surname><given-names>C.</given-names></name><name><surname>Khajehpour</surname><given-names>M.</given-names></name><name><surname>Snijder</surname><given-names>E. J.</given-names></name><name><surname>Mark</surname><given-names>B. L.</given-names></name><name><surname>Kikkert</surname><given-names>M.</given-names></name></person-group><bold>(</bold><year>2013</year><bold>).</bold><article-title>Deubiquitinase function of arterivirus papain-like protease 2 suppresses the innate immune response in infected host cells</article-title>. <source>Proc Natl Acad Sci U S A</source><volume>110</volume>, <fpage>E838</fpage>–<lpage>E847</lpage><pub-id pub-id-type="doi">10.1073/pnas.1218464110</pub-id><pub-id pub-id-type="pmid">23401522</pub-id></mixed-citation></ref><ref id="r52"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Versteeg</surname><given-names>G. A.</given-names></name><name><surname>Bredenbeek</surname><given-names>P. J.</given-names></name><name><surname>van den Worm</surname><given-names>S. H.</given-names></name><name><surname>Spaan</surname><given-names>W. J.</given-names></name></person-group><bold>(</bold><year>2007</year><bold>).</bold><article-title>Group 2 coronaviruses prevent immediate early interferon induction by protection of viral RNA from host cell recognition</article-title>. <source>Virology</source><volume>361</volume>, <fpage>18</fpage>–<lpage>26</lpage><pub-id pub-id-type="doi">10.1016/j.virol.2007.01.020</pub-id><pub-id pub-id-type="pmid">17316733</pub-id></mixed-citation></ref><ref id="r53"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Vijaykrishna</surname><given-names>D.</given-names></name><name><surname>Smith</surname><given-names>G. J.</given-names></name><name><surname>Zhang</surname><given-names>J. X.</given-names></name><name><surname>Peiris</surname><given-names>J. S.</given-names></name><name><surname>Chen</surname><given-names>H.</given-names></name><name><surname>Guan</surname><given-names>Y.</given-names></name></person-group><bold>(</bold><year>2007</year><bold>).</bold><article-title>Evolutionary insights into the ecology of coronaviruses</article-title>. <source>J Virol</source><volume>81</volume>, <fpage>4012</fpage>–<lpage>4020</lpage><pub-id pub-id-type="doi">10.1128/JVI.02605-06</pub-id><pub-id pub-id-type="pmid">17267506</pub-id></mixed-citation></ref><ref id="r54"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Vijgen</surname><given-names>L.</given-names></name><name><surname>Keyaerts</surname><given-names>E.</given-names></name><name><surname>Moës</surname><given-names>E.</given-names></name><name><surname>Thoelen</surname><given-names>I.</given-names></name><name><surname>Wollants</surname><given-names>E.</given-names></name><name><surname>Lemey</surname><given-names>P.</given-names></name><name><surname>Vandamme</surname><given-names>A. M.</given-names></name><name><surname>Van Ranst</surname><given-names>M.</given-names></name></person-group><bold>(</bold><year>2005</year><bold>).</bold><article-title>Complete genomic sequence of human coronavirus OC43: molecular clock analysis suggests a relatively recent zoonotic coronavirus transmission event</article-title>. <source>J Virol</source><volume>79</volume>, <fpage>1595</fpage>–<lpage>1604</lpage><pub-id pub-id-type="doi">10.1128/JVI.79.3.1595-1604.2005</pub-id><pub-id pub-id-type="pmid">15650185</pub-id></mixed-citation></ref><ref id="r55"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Weber</surname><given-names>F.</given-names></name><name><surname>Wagner</surname><given-names>V.</given-names></name><name><surname>Rasmussen</surname><given-names>S. B.</given-names></name><name><surname>Hartmann</surname><given-names>R.</given-names></name><name><surname>Paludan</surname><given-names>S. R.</given-names></name></person-group><bold>(</bold><year>2006</year><bold>).</bold><article-title>Double-stranded RNA is produced by positive-strand RNA viruses and DNA viruses but not in detectable amounts by negative-strand RNA viruses</article-title>. <source>J Virol</source><volume>80</volume>, <fpage>5059</fpage>–<lpage>5064</lpage><pub-id pub-id-type="doi">10.1128/JVI.80.10.5059-5064.2006</pub-id><pub-id pub-id-type="pmid">16641297</pub-id></mixed-citation></ref><ref id="r56"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Woo</surname><given-names>P. C.</given-names></name><name><surname>Lau</surname><given-names>S. K.</given-names></name><name><surname>Chu</surname><given-names>C. M.</given-names></name><name><surname>Chan</surname><given-names>K. H.</given-names></name><name><surname>Tsoi</surname><given-names>H. W.</given-names></name><name><surname>Huang</surname><given-names>Y.</given-names></name><name><surname>Wong</surname><given-names>B. H.</given-names></name><name><surname>Poon</surname><given-names>R. W.</given-names></name><name><surname>Cai</surname><given-names>J. J.</given-names></name></person-group><bold>& other authors (</bold><year>2005</year><bold>).</bold><article-title>Characterization and complete genome sequence of a novel coronavirus, coronavirus HKU1, from patients with pneumonia</article-title>. <source>J Virol</source><volume>79</volume>, <fpage>884</fpage>–<lpage>895</lpage><pub-id pub-id-type="doi">10.1128/JVI.79.2.884-895.2005</pub-id><pub-id pub-id-type="pmid">15613317</pub-id></mixed-citation></ref><ref id="r57"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Woo</surname><given-names>P. C.</given-names></name><name><surname>Wang</surname><given-names>M.</given-names></name><name><surname>Lau</surname><given-names>S. K.</given-names></name><name><surname>Xu</surname><given-names>H.</given-names></name><name><surname>Poon</surname><given-names>R. W.</given-names></name><name><surname>Guo</surname><given-names>R.</given-names></name><name><surname>Wong</surname><given-names>B. H.</given-names></name><name><surname>Gao</surname><given-names>K.</given-names></name><name><surname>Tsoi</surname><given-names>H. W.</given-names></name></person-group><bold>& other authors (</bold><year>2007</year><bold>).</bold><article-title>Comparative analysis of twelve genomes of three novel group 2c and group 2d coronaviruses reveals unique group and subgroup features</article-title>. <source>J Virol</source><volume>81</volume>, <fpage>1574</fpage>–<lpage>1585</lpage><pub-id pub-id-type="doi">10.1128/JVI.02182-06</pub-id><pub-id pub-id-type="pmid">17121802</pub-id></mixed-citation></ref><ref id="r58"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Yoshikawa</surname><given-names>T.</given-names></name><name><surname>Hill</surname><given-names>T. E.</given-names></name><name><surname>Yoshikawa</surname><given-names>N.</given-names></name><name><surname>Popov</surname><given-names>V. L.</given-names></name><name><surname>Galindo</surname><given-names>C. L.</given-names></name><name><surname>Garner</surname><given-names>H. R.</given-names></name><name><surname>Peters</surname><given-names>C. J.</given-names></name><name><surname>Tseng</surname><given-names>C. T.</given-names></name></person-group><bold>(</bold><year>2010</year><bold>).</bold><article-title>Dynamic innate immune responses of human bronchial epithelial cells to severe acute respiratory syndrome-associated coronavirus infection</article-title>. <source>PLoS ONE</source><volume>5</volume>, <fpage>e8729</fpage><pub-id pub-id-type="doi">10.1371/journal.pone.0008729</pub-id><pub-id pub-id-type="pmid">20090954</pub-id></mixed-citation></ref><ref id="r59"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Zaki</surname><given-names>A. M.</given-names></name><name><surname>van Boheemen</surname><given-names>S.</given-names></name><name><surname>Bestebroer</surname><given-names>T. M.</given-names></name><name><surname>Osterhaus</surname><given-names>A. D.</given-names></name><name><surname>Fouchier</surname><given-names>R. A.</given-names></name></person-group><bold>(</bold><year>2012</year><bold>).</bold><article-title>Isolation of a novel coronavirus from a man with pneumonia in Saudi Arabia</article-title>. <source>N Engl J Med</source><volume>367</volume>, <fpage>1814</fpage>–<lpage>1820</lpage><pub-id pub-id-type="doi">10.1056/NEJMoa1211721</pub-id><pub-id pub-id-type="pmid">23075143</pub-id></mixed-citation></ref><ref id="r60"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Zeng</surname><given-names>F. Y.</given-names></name><name><surname>Chan</surname><given-names>C. W.</given-names></name><name><surname>Chan</surname><given-names>M. N.</given-names></name><name><surname>Chen</surname><given-names>J. D.</given-names></name><name><surname>Chow</surname><given-names>K. Y.</given-names></name><name><surname>Hon</surname><given-names>C. C.</given-names></name><name><surname>Hui</surname><given-names>K. H.</given-names></name><name><surname>Li</surname><given-names>J.</given-names></name><name><surname>Li</surname><given-names>V. Y.</given-names></name></person-group><bold>& other authors (</bold><year>2003</year><bold>).</bold><article-title>The complete genome sequence of severe acute respiratory syndrome coronavirus strain HKU-39849 (HK-39)</article-title>. <source>Exp Biol Med (Maywood)</source><volume>228</volume>, <fpage>866</fpage>–<lpage>873</lpage><pub-id pub-id-type="pmid">12876307</pub-id></mixed-citation></ref><ref id="r61"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Zheng</surname><given-names>B.</given-names></name><name><surname>He</surname><given-names>M. L.</given-names></name><name><surname>Wong</surname><given-names>K. L.</given-names></name><name><surname>Lum</surname><given-names>C. T.</given-names></name><name><surname>Poon</surname><given-names>L. L.</given-names></name><name><surname>Peng</surname><given-names>Y.</given-names></name><name><surname>Guan</surname><given-names>Y.</given-names></name><name><surname>Lin</surname><given-names>M. C.</given-names></name><name><surname>Kung</surname><given-names>H. F.</given-names></name></person-group><bold>(</bold><year>2004</year><bold>).</bold><article-title>Potent inhibition of SARS-associated coronavirus (SCOV) infection and replication by type I interferons (IFN-α/β) but not by type II interferon (IFN-γ)</article-title>. <source>J Interferon Cytokine Res</source><volume>24</volume>, <fpage>388</fpage>–<lpage>390</lpage><pub-id pub-id-type="doi">10.1089/1079990041535610</pub-id><pub-id pub-id-type="pmid">15296649</pub-id></mixed-citation></ref><ref id="r62"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Zhong</surname><given-names>Y.</given-names></name><name><surname>Tan</surname><given-names>Y. W.</given-names></name><name><surname>Liu</surname><given-names>D. X.</given-names></name></person-group><bold>(</bold><year>2012</year><bold>).</bold><article-title>Recent progress in studies of arterivirus– and coronavirus–host interactions</article-title>. <source>Viruses</source><volume>4</volume>, <fpage>980</fpage>–<lpage>1010</lpage><pub-id pub-id-type="doi">10.3390/v4060980</pub-id><pub-id pub-id-type="pmid">22816036</pub-id></mixed-citation></ref><ref id="r63"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Zhou</surname><given-names>P.</given-names></name><name><surname>Li</surname><given-names>H.</given-names></name><name><surname>Wang</surname><given-names>H.</given-names></name><name><surname>Wang</surname><given-names>L. F.</given-names></name><name><surname>Shi</surname><given-names>Z.</given-names></name></person-group><bold>(</bold><year>2012</year><bold>).</bold><article-title>Bat severe acute respiratory syndrome-like coronavirus ORF3b homologues display different interferon antagonist activities</article-title>. <source>J Gen Virol</source><volume>93</volume>, <fpage>275</fpage>–<lpage>281</lpage><pub-id pub-id-type="doi">10.1099/vir.0.033589-0</pub-id><pub-id pub-id-type="pmid">22012463</pub-id></mixed-citation></ref><ref id="r64"><mixed-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Züst</surname><given-names>R.</given-names></name><name><surname>Cervantes-Barragan</surname><given-names>L.</given-names></name><name><surname>Habjan</surname><given-names>M.</given-names></name><name><surname>Maier</surname><given-names>R.</given-names></name><name><surname>Neuman</surname><given-names>B. W.</given-names></name><name><surname>Ziebuhr</surname><given-names>J.</given-names></name><name><surname>Szretter</surname><given-names>K. J.</given-names></name><name><surname>Baker</surname><given-names>S. C.</given-names></name><name><surname>Barchet</surname><given-names>W.</given-names></name></person-group><bold>& other authors (</bold><year>2011</year><bold>).</bold><article-title>Ribose 2′-<italic>O</italic>-methylation provides a molecular signature for the distinction of self and non-self mRNA dependent on the RNA sensor Mda5</article-title>. <source>Nat Immunol</source><volume>12</volume>, <fpage>137</fpage>–<lpage>143</lpage><pub-id pub-id-type="doi">10.1038/ni.1979</pub-id><pub-id pub-id-type="pmid">21217758</pub-id></mixed-citation></ref></ref-list></back></pmc></record>Pour manipuler ce document sous Unix (Dilib)
EXPLOR_STEP=$WICRI_ROOT/Sante/explor/SrasV1/Data/Pmc/Corpus
HfdSelect -h $EXPLOR_STEP/biblio.hfd -nk 001144 | SxmlIndent | more
Ou
HfdSelect -h $EXPLOR_AREA/Data/Pmc/Corpus/biblio.hfd -nk 001144 | SxmlIndent | more
Pour mettre un lien sur cette page dans le réseau Wicri
{{Explor lien
|wiki= Sante
|area= SrasV1
|flux= Pmc
|étape= Corpus
|type= RBID
|clé= PMC:3749523
|texte= MERS-coronavirus replication induces severe in vitro cytopathology and is strongly inhibited by cyclosporin A or interferon-α treatment
}}
Pour générer des pages wiki
HfdIndexSelect -h $EXPLOR_AREA/Data/Pmc/Corpus/RBID.i -Sk "pubmed:23620378" \
| HfdSelect -Kh $EXPLOR_AREA/Data/Pmc/Corpus/biblio.hfd \
| NlmPubMed2Wicri -a SrasV1
| This area was generated with Dilib version V0.6.33. |  |