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                <text>Vaccine efficacy in senescent mice challenged with recombinant SARS-CoV bearing epidemic and zoonotic spike variants.</text>
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                <text>Damon Deming, Timothy Sheahan, Mark Heise, Boyd Yount, Nancy Davis, Amy Sims, Mehul Suthar, Jack Harkema, Alan Whitmore, Raymond Pickles, Ande West, Eric Donaldson, Kristopher Curtis, Robert Johnston, Ralph Baric</text>
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                <text>In 2003, severe acute respiratory syndrome coronavirus (SARS-CoV) was identified as the etiological agent of severe acute respiratory syndrome, a disease characterized by severe pneumonia that sometimes results in death. SARS-CoV is a zoonotic virus that crossed the species barrier, most likely originating from bats or from other species including civets, raccoon dogs, domestic cats, swine, and rodents. A SARS-CoV vaccine should confer long-term protection, especially in vulnerable senescent populations, against both the 2003 epidemic strains and zoonotic strains that may yet emerge from animal reservoirs. We report the comprehensive investigation of SARS vaccine efficacy in young and senescent mice following homologous and heterologous challenge.Using Venezuelan equine encephalitis virus replicon particles (VRP) expressing the 2003 epidemic Urbani SARS-CoV strain spike (S) glycoprotein (VRP-S) or the nucleocapsid (N) protein from the same strain (VRP-N), we demonstrate that VRP-S, but not VRP-N vaccines provide complete short- and long-term protection against homologous strain challenge in young and senescent mice. To test VRP vaccine efficacy against a heterologous SARS-CoV, we used phylogenetic analyses, synthetic biology, and reverse genetics to construct a chimeric virus (icGDO3-S) encoding a synthetic S glycoprotein gene of the most genetically divergent human strain, GDO3, which clusters among the zoonotic SARS-CoV. icGD03-S replicated efficiently in human airway epithelial cells and in the lungs of young and senescent mice, and was highly resistant to neutralization with antisera directed against the Urbani strain. Although VRP-S vaccines provided complete short-term protection against heterologous icGD03-S challenge in young mice, only limited protection was seen in vaccinated senescent animals. VRP-N vaccines not only failed to protect from homologous or heterologous challenge, but resulted in enhanced immunopathology with eosinophilic infiltrates within the lungs of SARS-CoV-challenged mice. VRP-N-induced pathology presented at day 4, peaked around day 7, and persisted through day 14, and was likely mediated by cellular immune responses.This study identifies gaps and challenges in vaccine design for controlling future SARS-CoV zoonosis, especially in vulnerable elderly populations. The availability of a SARS-CoV virus bearing heterologous S glycoproteins provides a robust challenge inoculum for evaluating vaccine efficacy against zoonotic strains, the most likely source of future outbreaks.</text>
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                <text>DOI: 10.1371/journal.pmed.0030525</text>
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                <text>DOI: 10.1371/journal.pmed.0030001</text>
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                <text>Konstantin Pervushin, Edward Tan, Krupakar Parthasarathy, Xin Lin, Fengli Jiang, Dejie Yu, Ardcharaporn Vararattanavech, Tuck Wah Soong, Ding Xiang Liu, Jaume Torres</text>
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                <text>The envelope (E) protein from coronaviruses is a small polypeptide that contains at least one alpha-helical transmembrane domain. Absence, or inactivation, of E protein results in attenuated viruses, due to alterations in either virion morphology or tropism. Apart from its morphogenetic properties, protein E has been reported to have membrane permeabilizing activity. Further, the drug hexamethylene amiloride (HMA), but not amiloride, inhibited in vitro ion channel activity of some synthetic coronavirus E proteins, and also viral replication. We have previously shown for the coronavirus species responsible for severe acute respiratory syndrome (SARS-CoV) that the transmembrane domain of E protein (ETM) forms pentameric alpha-helical bundles that are likely responsible for the observed channel activity. Herein, using solution NMR in dodecylphosphatidylcholine micelles and energy minimization, we have obtained a model of this channel which features regular alpha-helices that form a pentameric left-handed parallel bundle. The drug HMA was found to bind inside the lumen of the channel, at both the C-terminal and the N-terminal openings, and, in contrast to amiloride, induced additional chemical shifts in ETM. Full length SARS-CoV E displayed channel activity when transiently expressed in human embryonic kidney 293 (HEK-293) cells in a whole-cell patch clamp set-up. This activity was significantly reduced by hexamethylene amiloride (HMA), but not by amiloride. The channel structure presented herein provides a possible rationale for inhibition, and a platform for future structure-based drug design of this potential pharmacological target.</text>
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                <text>DOI: 10.1371/journal.ppat.1000511</text>
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                <text>No therapeutics or vaccines currently exist for human coronaviruses (HCoVs). The Severe Acute Respiratory Syndrome-associated coronavirus (SARS-CoV) epidemic in 2002-2003, and the recent emergence of Middle East Respiratory Syndrome coronavirus (MERS-CoV) in April 2012, emphasize the high probability of future zoonotic HCoV emergence causing severe and lethal human disease. Additionally, the resistance of SARS-CoV to ribavirin (RBV) demonstrates the need to define new targets for inhibition of CoV replication. CoVs express a 3'-to-5' exoribonuclease in nonstructural protein 14 (nsp14-ExoN) that is required for high-fidelity replication and is conserved across the CoV family. All genetic and biochemical data support the hypothesis that nsp14-ExoN has an RNA proofreading function. Thus, we hypothesized that ExoN is responsible for CoV resistance to RNA mutagens. We demonstrate that while wild-type (ExoN+) CoVs were resistant to RBV and 5-fluorouracil (5-FU), CoVs lacking ExoN activity (ExoN-) were up to 300-fold more sensitive. While the primary antiviral activity of RBV against CoVs was not mutagenesis, ExoN- CoVs treated with 5-FU demonstrated both enhanced sensitivity during multi-cycle replication, as well as decreased specific infectivity, consistent with 5-FU functioning as a mutagen. Comparison of full-genome next-generation sequencing of 5-FU treated SARS-CoV populations revealed a 16-fold increase in the number of mutations within the ExoN- population as compared to ExoN+. Ninety percent of these mutations represented A:G and U:C transitions, consistent with 5-FU incorporation during RNA synthesis. Together our results constitute direct evidence that CoV ExoN activity provides a critical proofreading function during virus replication. Furthermore, these studies identify ExoN as the first viral protein distinct from the RdRp that determines the sensitivity of RNA viruses to mutagens. Finally, our results show the importance of ExoN as a target for inhibition, and suggest that small-molecule inhibitors of ExoN activity could be potential pan-CoV therapeutics in combination with RBV or RNA mutagens.</text>
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                <text>DOI: 10.1371/journal.ppat.1003565</text>
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                <text>Research Driven by Curiosity: The Journey from Basic Molecular Biology and Virology to Studies of Human Pathogenic Coronaviruses.</text>
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                <text>DOI: 10.1371/journal.ppat.1005023</text>
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                <text>Assessment of algorithms for inferring positional weight matrix motifs of transcription factor binding sites using protein binding microarray data.</text>
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            <description>An entity primarily responsible for making the resource</description>
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                <text>Yaron Orenstein, Chaim Linhart, Ron Shamir</text>
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                <text>The new technology of protein binding microarrays (PBMs) allows simultaneous measurement of the binding intensities of a transcription factor to tens of thousands of synthetic double-stranded DNA probes, covering all possible 10-mers. A key computational challenge is inferring the binding motif from these data. We present a systematic comparison of four methods developed specifically for reconstructing a binding site motif represented as a positional weight matrix from PBM data. The reconstructed motifs were evaluated in terms of three criteria: concordance with reference motifs from the literature and ability to predict in vivo and in vitro bindings. The evaluation encompassed over 200 transcription factors and some 300 assays. The results show a tradeoff between how the methods perform according to the different criteria, and a dichotomy of method types. Algorithms that construct motifs with low information content predict PBM probe ranking more faithfully, while methods that produce highly informative motifs match reference motifs better. Interestingly, in predicting high-affinity binding, all methods give far poorer results for in vivo assays compared to in vitro assays.</text>
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                <text>DOI: 10.1371/journal.pone.0046145</text>
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                <text>PLoS ONE</text>
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                  <text>Dominio científico: Coronavirus</text>
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                <text>The SARS-coronavirus-host interactome: identification of cyclophilins as target for pan-coronavirus inhibitors.</text>
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                <text>Susanne Pfefferle, Julia Schöpf, Manfred Kögl, Caroline C Friedel, Marcel A. Müller, Javier Carbajo-Lozoya, Thorsten Stellberger, Ekatarina von Dall'Armi, Petra Herzog, Stefan Kallies, Daniela Niemeyer, Vanessa Ditt, Thomas Kuri, Roland Züst, Ksenia Pumpor, Rolf Hilgenfeld, Frank Schwarz, Ralf Zimmer, Imke Steffen, Friedemann Weber, Volker Thiel, Georg Herrler, Heinz-Jürgen Thiel, Christel Schwegmann-Weßels, Stefan Pöhlmann, Jürgen Haas, Christian Drosten, Albrecht von Brunn</text>
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                <text>Coronaviruses (CoVs) are important human and animal pathogens that induce fatal respiratory, gastrointestinal and neurological disease. The outbreak of the severe acute respiratory syndrome (SARS) in 2002/2003 has demonstrated human vulnerability to (Coronavirus) CoV epidemics. Neither vaccines nor therapeutics are available against human and animal CoVs. Knowledge of host cell proteins that take part in pivotal virus-host interactions could define broad-spectrum antiviral targets. In this study, we used a systems biology approach employing a genome-wide yeast-two hybrid interaction screen to identify immunopilins (PPIA, PPIB, PPIH, PPIG, FKBP1A, FKBP1B) as interaction partners of the CoV non-structural protein 1 (Nsp1). These molecules modulate the Calcineurin/NFAT pathway that plays an important role in immune cell activation. Overexpression of NSP1 and infection with live SARS-CoV strongly increased signalling through the Calcineurin/NFAT pathway and enhanced the induction of interleukin 2, compatible with late-stage immunopathogenicity and long-term cytokine dysregulation as observed in severe SARS cases. Conversely, inhibition of cyclophilins by cyclosporine A (CspA) blocked the replication of CoVs of all genera, including SARS-CoV, human CoV-229E and -NL-63, feline CoV, as well as avian infectious bronchitis virus. Non-immunosuppressive derivatives of CspA might serve as broad-range CoV inhibitors applicable against emerging CoVs as well as ubiquitous pathogens of humans and livestock.</text>
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                <text>2011</text>
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                <text>DOI: 10.1371/journal.ppat.1002331</text>
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                <text>PLoS Pathogens</text>
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                <text>Biology (General), Immunologic diseases. Allergy</text>
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                  <text>Dominio científico: Coronavirus</text>
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                <text>Action mechanisms of lithium chloride on cell infection by transmissible gastroenteritis coronavirus.</text>
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                <text>Xiaofeng Ren, Fandan Meng, Jiechao Yin, Guangxing Li, Xunliang Li, Chao Wang, Georg Herrler</text>
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                <text>Transmissible gastroenteritis virus (TGEV) is a porcine coronavirus. Lithium chloride (LiCl) has been found to be effective against several DNA viruses, such as Herpes simplex virus and vaccinia virus. Recently, we and others have reported the inhibitory effect of LiCl on avian infectious bronchitis coronavirus (IBV) infection, an RNA virus. In the current study, the action mechanism of LiCl on cell infection by TGEV was investigated. Plaque assays and 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenyl tetrazoliumbromide (MTT) assays showed that the cell infection by TGEV was inhibited in a dose-dependent manner, when LiCl was added to virus-infected cells; the cell infection was not affected when either cells or viruses were pretreated with the drug. The inhibition of TGEV infection in vitro by LiCl was observed at different virus doses and with different cell lines. The inhibitory effect of LiCl against TGEV infection and transcription was confirmed by RT-PCR and real-time PCR targeting viral S and 3CL-protease genes. The time-of-addition effect of the drug on TGEV infection indicated that LiCl acted on the initial and late stage of TGEV infection. The production of virus was not detected at 36 h post-infection due to the drug treatment. Moreover, immunofluorescence (IF) and flow cytometry analyses based on staining of Annexin V and propidium iodide staining of nuclei indicated that early and late cell apoptosis induced by TGEV was inhibited efficiently. The ability of LiCl to inhibit apoptosis was investigated by IF analysis of caspase-3 expression. Our data indicate that LiCl inhibits TGEV infection by exerting an anti-apoptotic effect. The inhibitory effect of LiCl was also observed with porcine epidemic diarrhea coronavirus. Together with other reports concerning the inhibitory effect of lithium salts on IBV in cell culture, our results indicate that LiCl may be a potent agent against porcine and avian coronaviruses.</text>
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                <text>DOI: 10.1371/journal.pone.0018669</text>
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                <text>PLoS ONE</text>
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        <src>https://www.socictopen.socict.org/files/original/60fa3d1b06bef633b137a8dd09dadafb.pdf</src>
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              <name>Title</name>
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                  <text>Coronavirus</text>
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                  <text>Dominio científico: Coronavirus</text>
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                <text>SARS coronavirus nsp1 protein induces template-dependent endonucleolytic cleavage of mRNAs: viral mRNAs are resistant to nsp1-induced RNA cleavage.</text>
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                <text>Cheng Huang, Kumari G Lokugamage, Janet M Rozovics, Krishna Narayanan, Bert L. Semler, Shinji Makino</text>
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                <text>SARS coronavirus (SCoV) nonstructural protein (nsp) 1, a potent inhibitor of host gene expression, possesses a unique mode of action: it binds to 40S ribosomes to inactivate their translation functions and induces host mRNA degradation. Our previous study demonstrated that nsp1 induces RNA modification near the 5'-end of a reporter mRNA having a short 5' untranslated region and RNA cleavage in the encephalomyocarditis virus internal ribosome entry site (IRES) region of a dicistronic RNA template, but not in those IRES elements from hepatitis C or cricket paralysis viruses. By using primarily cell-free, in vitro translation systems, the present study revealed that the nsp1 induced endonucleolytic RNA cleavage mainly near the 5' untranslated region of capped mRNA templates. Experiments using dicistronic mRNAs carrying different IRESes showed that nsp1 induced endonucleolytic RNA cleavage within the ribosome loading region of type I and type II picornavirus IRES elements, but not that of classical swine fever virus IRES, which is characterized as a hepatitis C virus-like IRES. The nsp1-induced RNA cleavage of template mRNAs exhibited no apparent preference for a specific nucleotide sequence at the RNA cleavage sites. Remarkably, SCoV mRNAs, which have a 5' cap structure and 3' poly A tail like those of typical host mRNAs, were not susceptible to nsp1-mediated RNA cleavage and importantly, the presence of the 5'-end leader sequence protected the SCoV mRNAs from nsp1-induced endonucleolytic RNA cleavage. The escape of viral mRNAs from nsp1-induced RNA cleavage may be an important strategy by which the virus circumvents the action of nsp1 leading to the efficient accumulation of viral mRNAs and viral proteins during infection.</text>
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                <text>2011</text>
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                <text>DOI: 10.1371/journal.ppat.1002433</text>
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                <text>PLoS Pathogens</text>
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                <text>Public Library of Science (PLoS)</text>
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                <text>Biology (General), Immunologic diseases. Allergy</text>
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            <name>Language</name>
            <description>A language of the resource</description>
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                <text>EN</text>
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