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                <text>Regulation of programmed ribosomal frameshifting by co-translational refolding RNA hairpins.</text>
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                <text>Che-Pei Cho, Szu-Chieh Lin, Ming-Yuan Chou, Hsiu-Ting Hsu, Kung-Yao Chang</text>
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                <text>RNA structures are unwound for decoding. In the process, they can pause the elongating ribosome for regulation. An example is the stimulation of -1 programmed ribosomal frameshifting, leading to 3' direction slippage of the reading-frame during elongation, by specific pseudoknot stimulators downstream of the frameshifting site. By investigating a recently identified regulatory element upstream of the SARS coronavirus (SARS-CoV) -1 frameshifting site, it is shown that a minimal functional element with hairpin forming potential is sufficient to down-regulate-1 frameshifting activity. Mutagenesis to disrupt or restore base pairs in the potential hairpin stem reveals that base-pair formation is required for-1 frameshifting attenuation in vitro and in 293T cells. The attenuation efficiency of a hairpin is determined by its stability and proximity to the frameshifting site; however, it is insensitive to E site sequence variation. Additionally, using a dual luciferase assay, it can be shown that a hairpin stimulated +1 frameshifting when placed upstream of a +1 shifty site in yeast. The investigations indicate that the hairpin is indeed a cis-acting programmed reading-frame switch modulator. This result provides insight into mechanisms governing-1 frameshifting stimulation and attenuation. Since the upstream hairpin is unwound (by a marching ribosome) before the downstream stimulator, this study's findings suggest a new mode of translational regulation that is mediated by the reformed stem of a ribosomal unwound RNA hairpin during elongation.</text>
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                <text>DOI: 10.1371/journal.pone.0062283</text>
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                <text>Dissecting virus entry: replication-independent analysis of virus binding, internalization, and penetration using minimal complementation of β-galactosidase.</text>
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                <text>Christine Burkard, Louis-Marie Bloyet, Oliver Wicht, Frank J van Kuppeveld, Peter J.M. Rottier, Cornelis A.M. De Haan, Berend-Jan Bosch</text>
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                <text>Studies of viral entry into host cells often rely on the detection of post-entry parameters, such as viral replication or the expression of a reporter gene, rather than on measuring entry per se. The lack of assays to easily detect the different steps of entry severely hampers the analysis of this key process in virus infection. Here we describe novel, highly adaptable viral entry assays making use of minimal complementation of the E. coli β-galactosidase in mammalian cells. Enzyme activity is reconstituted when a small intravirion peptide (α-peptide) is complementing the inactive mutant form ΔM15 of β-galactosidase. The method allows to dissect and to independently detect binding, internalization, and fusion of viruses during host cell entry. Here we use it to confirm and extend current knowledge on the entry process of two enveloped viruses: vesicular stomatitis virus (VSV) and murine hepatitis coronavirus (MHV).</text>
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                <text>DOI: 10.1371/journal.pone.0101762</text>
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                <text>PLoS ONE</text>
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                  <text>Dominio científico: Coronavirus</text>
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                <text>Profiling of substrate specificity of SARS-CoV 3CL.</text>
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                <text>Chi-Pang Chuck, Lin-Tat Chong, Chao Chen, Hak-Fun Chow, David Chi-Cheong Wan, Kam-Bo Wong</text>
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                <text>BACKGROUND: The 3C-like protease (3CL(pro)) of severe acute respiratory syndrome-coronavirus is required for autoprocessing of the polyprotein, and is a potential target for treating coronaviral infection. METHODOLOGY/PRINCIPAL FINDINGS: To obtain a thorough understanding of substrate specificity of the protease, a substrate library of 198 variants was created by performing saturation mutagenesis on the autocleavage sequence at P5 to P3' positions. The substrate sequences were inserted between cyan and yellow fluorescent proteins so that the cleavage rates were monitored by in vitro fluorescence resonance energy transfer. The relative cleavage rate for different substrate sequences was correlated with various structural properties. P5 and P3 positions prefer residues with high β-sheet propensity; P4 prefers small hydrophobic residues; P2 prefers hydrophobic residues without β-branch. Gln is the best residue at P1 position, but observable cleavage can be detected with His and Met substitutions. P1' position prefers small residues, while P2' and P3' positions have no strong preference on residue substitutions. Noteworthy, solvent exposed sites such as P5, P3 and P3' positions favour positively charged residues over negatively charged one, suggesting that electrostatic interactions may play a role in catalysis. A super-active substrate, which combined the preferred residues at P5 to P1 positions, was found to have 2.8 fold higher activity than the wild-type sequence. CONCLUSIONS/SIGNIFICANCE: Our results demonstrated a strong structure-activity relationship between the 3CL(pro) and its substrate. The substrate specificity profiled in this study may provide insights into a rational design of peptidomimetic inhibitors.</text>
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                <text>2010</text>
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                <text>DOI: 10.1371/journal.pone.0013197</text>
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                <text>PLoS ONE</text>
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                <text>Public Library of Science (PLoS)</text>
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                  <text>Dominio científico: Coronavirus</text>
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                <text>Profiling of substrate specificities of 3C-like proteases from group 1, 2a, 2b, and 3 coronaviruses.</text>
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                <text>Chi-Pang Chuck, Hak-Fun Chow, David Chi-Cheong Wan, Kam-Bo Wong</text>
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                <text>BACKGROUND: Coronaviruses (CoVs) can be classified into alphacoronavirus (group 1), betacoronavirus (group 2), and gammacoronavirus (group 3) based on diversity of the protein sequences. Their 3C-like protease (3CL(pro)), which catalyzes the proteolytic processing of the polyproteins for viral replication, is a potential target for anti-coronaviral infection. METHODOLOGY/PRINCIPAL FINDINGS: Here, we profiled the substrate specificities of 3CL(pro) from human CoV NL63 (group 1), human CoV OC43 (group 2a), severe acute respiratory syndrome coronavirus (SARS-CoV) (group 2b) and infectious bronchitis virus (IBV) (group 3), by measuring their activity against a substrate library of 19 × 8 of variants with single substitutions at P5 to P3' positions. The results were correlated with structural properties like side chain volume, hydrophobicity, and secondary structure propensities of substituting residues. All 3CL(pro) prefer Gln at P1 position, Leu at P2 position, basic residues at P3 position, small hydrophobic residues at P4 position, and small residues at P1' and P2' positions. Despite 3CL(pro) from different groups of CoVs share many similarities in substrate specificities, differences in substrate specificities were observed at P4 positions, with IBV 3CL(pro) prefers P4-Pro and SARS-CoV 3CL(pro) prefers P4-Val. By combining the most favorable residues at P3 to P5 positions, we identified super-active substrate sequences 'VARLQ↓SGF' that can be cleaved efficiently by all 3CL(pro) with relative activity of 1.7 to 3.2, and 'VPRLQ↓SGF' that can be cleaved specifically by IBV 3CL(pro) with relative activity of 4.3. CONCLUSIONS/SIGNIFICANCE: The comprehensive substrate specificities of 3CL(pro) from each of the group 1, 2a, 2b, and 3 CoVs have been profiled in this study, which may provide insights into a rational design of broad-spectrum peptidomimetic inhibitors targeting the proteases.</text>
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                <text>2011</text>
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                <text>DOI: 10.1371/journal.pone.0027228</text>
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                <text>PLoS ONE</text>
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                <text>Up-regulation of Mcl-1 and Bak by coronavirus infection of human, avian and animal cells modulates apoptosis and viral replication.</text>
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                <text>Yanxin Zhong, Ying Liao, Shou Guo Fang, James P Tam, Ding Xiang Liu</text>
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                <text>Virus-induced apoptosis and viral mechanisms that regulate this cell death program are key issues in understanding virus-host interactions and viral pathogenesis. Like many other human and animal viruses, coronavirus infection of mammalian cells induces apoptosis. In this study, the global gene expression profiles are first determined in IBV-infected Vero cells at 24 hours post-infection by Affymetrix array, using avian coronavirus infectious bronchitis virus (IBV) as a model system. It reveals an up-regulation at the transcriptional level of both pro-apoptotic Bak and pro-survival myeloid cell leukemia-1 (Mcl-1). These results were further confirmed both in vivo and in vitro, in IBV-infected embryonated chicken eggs, chicken fibroblast cells and mammalian cells at transcriptional and translational levels, respectively. Interestingly, the onset of apoptosis occurred earlier in IBV-infected mammalian cells silenced with short interfering RNA targeting Mcl-1 (siMcl-1), and was delayed in cells silenced with siBak. IBV progeny production and release were increased in infected Mcl-1 knockdown cells compared to similarly infected control cells, while the contrary was observed in infected Bak knockdown cells. Furthermore, IBV infection-induced up-regulation of GADD153 regulated the expression of Mcl-1. Inhibition of the mitogen-activated protein/extracellular signal-regulated kinase (MEK/ERK) and phosphoinositide 3-kinase (PI3K/Akt) signaling pathways by chemical inhibitors and knockdown of GADD153 by siRNA demonstrated the involvement of ER-stress response in regulation of IBV-induced Mcl-1 expression. These results illustrate the sophisticated regulatory strategies evolved by a coronavirus to modulate both virus-induced apoptosis and viral replication during its replication cycle.</text>
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                <text>DOI: 10.1371/journal.pone.0030191</text>
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                <text>PLoS ONE</text>
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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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            <description>A name given to the resource</description>
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                <text>Sequence-dependent fluorescence of cyanine dyes on microarrays.</text>
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            <description>An entity primarily responsible for making the resource</description>
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                <text>Christy Agbavwe, Mark M. Somoza</text>
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            </elementTextContainer>
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            <description>An account of the resource</description>
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                <text>Cy3 and Cy5 are among the most commonly used oligonucleotide labeling molecules. Studies of nucleic acid structure and dynamics use these dyes, and they are ubiquitous in microarray experiments. They are sensitive to their environment and have higher quantum yield when bound to DNA. The fluorescent intensity of terminal cyanine dyes is also known to be significantly dependent on the base sequence of the oligonucleotide. We have developed a very precise and high-throughput method to evaluate the sequence dependence of oligonucleotide labeling dyes using microarrays and have applied the method to Cy3 and Cy5. We used light-directed in-situ synthesis of terminally-labeled microarrays to determine the fluorescence intensity of each dye on all 1024 possible 5'-labeled 5-mers. Their intensity is sensitive to all five bases. Their fluorescence is higher with 5' guanines, and adenines in subsequent positions. Cytosine suppresses fluorescence. Intensity falls by half over the range of all 5-mers for Cy3, and two-thirds for Cy5. Labeling with 5'-biotin-streptavidin-Cy3/-Cy5 gives a completely different sequence dependence and greatly reduces fluorescence compared with direct terminal labeling.</text>
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                <text>2011</text>
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                <text>DOI: 10.1371/journal.pone.0022177</text>
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            <description>A related resource from which the described resource is derived</description>
            <elementTextContainer>
              <elementText elementTextId="1710">
                <text>PLoS ONE</text>
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            <description>An entity responsible for making the resource available</description>
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                <text>Public Library of Science (PLoS)</text>
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                <text>Science, Medicine</text>
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            <description>A language of the resource</description>
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                <text>EN</text>
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  <item itemId="185" public="1" featured="0">
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              <name>Title</name>
              <description>A name given to the resource</description>
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                  <text>Coronavirus</text>
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              <description>An account of the resource</description>
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                  <text>Dominio científico: Coronavirus</text>
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      <name>Text</name>
      <description>A resource consisting primarily of words for reading. Examples include books, letters, dissertations, poems, newspapers, articles, archives of mailing lists. Note that facsimiles or images of texts are still of the genre Text.</description>
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      <elementSet elementSetId="1">
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            <name>Title</name>
            <description>A name given to the resource</description>
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                <text>Construction and characterization of single-chain variable fragment antibody library derived from germline rearranged immunoglobulin variable genes.</text>
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            <description>An entity primarily responsible for making the resource</description>
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              <elementText elementTextId="1715">
                <text>Man Cheng, Shirley Y W Chan, Qi Zhao, Elaine Y M Chan, Shannon W. N. Au, Susanna S T Lee, Wing-tai Cheung</text>
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            <description>An account of the resource</description>
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                <text>Antibody repertoires for library construction are conventionally harvested from mRNAs of immune cells. To examine whether germline rearranged immunoglobulin (Ig) variable region genes could be used as source of antibody repertoire, an immunized phage-displayed scFv library was prepared using splenocytic genomic DNA as template. In addition, a novel frame-shifting PCR (fsPCR) step was introduced to rescue stop codon and to enhance diversity of the complementarity-determining region 3 (CDR3). The germline scFv library was initially characterized against the hapten antigen phenyloxazolone (phOx). Sequence analysis of the phOx-selective scFvs indicated that the CDRs consisted of novel as well as conserved motifs. In order to illustrate that the diversity of CDR3 was increased by the fsPCR step, a second scFv library was constructed using a single scFv clone L3G7C as a template. Despite showing similar binding characteristics towards phOx, the scFv clones that were obtained from the L3G7C-derived antibody library gave a lower non-specific binding than that of the parental L3G7C clone. To determine whether germline library represented the endogenous immune status, specific scFv clones for nucleocapsid (N) protein of SARS-associated coronavirus (SCoV) were obtained both from naïve and immunized germline scFv libraries. Both libraries yielded specific anti-N scFvs that exhibited similar binding characteristics towards recombinant N protein, except the immunized library gave a larger number of specific anti-N scFv, and clones with identical nucleotide sequences were found. In conclusion, highly diversified antibody library can be efficiently constructed using germline rearranged immunoglobulin variable genes as source of antibody repertoires and fsPCR to diversify the CDR3.</text>
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                <text>2011</text>
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            <description>An unambiguous reference to the resource within a given context</description>
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                <text>DOI: 10.1371/journal.pone.0027406</text>
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            <description>A related resource from which the described resource is derived</description>
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              <elementText elementTextId="1719">
                <text>PLoS ONE</text>
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            <description>An entity responsible for making the resource available</description>
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                <text>Public Library of Science (PLoS)</text>
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                <text>Science, Medicine</text>
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            <description>A language of the resource</description>
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                <text>EN</text>
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              <name>Title</name>
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                  <text>Coronavirus</text>
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              <name>Description</name>
              <description>An account of the resource</description>
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                  <text>Dominio científico: Coronavirus</text>
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      <name>Text</name>
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                <text>Temporal variability and social heterogeneity in disease transmission: the case of SARS in Hong Kong.</text>
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              <elementText elementTextId="1724">
                <text>Anne Cori, Pierre-Yves Boelle, Guy Thomas, Gabriel M. Leung, Alain-Jacques Valleron</text>
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            <description>An account of the resource</description>
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                <text>The extent to which self-adopted or intervention-related changes in behaviors affect the course of epidemics remains a key issue for outbreak control. This study attempted to quantify the effect of such changes on the risk of infection in different settings, i.e., the community and hospitals. The 2002-2003 severe acute respiratory syndrome (SARS) outbreak in Hong Kong, where 27% of cases were healthcare workers, was used as an example. A stochastic compartmental SEIR (susceptible-exposed-infectious-removed) model was used: the population was split into healthcare workers, hospitalized people and general population. Super spreading events (SSEs) were taken into account in the model. The temporal evolutions of the daily effective contact rates in the community and hospitals were modeled with smooth functions. Data augmentation techniques and Markov chain Monte Carlo (MCMC) methods were applied to estimate SARS epidemiological parameters. In particular, estimates of daily reproduction numbers were provided for each subpopulation. The average duration of the SARS infectious period was estimated to be 9.3 days (+/-0.3 days). The model was able to disentangle the impact of the two SSEs from background transmission rates. The effective contact rates, which were estimated on a daily basis, decreased with time, reaching zero inside hospitals. This observation suggests that public health measures and possible changes in individual behaviors effectively reduced transmission, especially in hospitals. The temporal patterns of reproduction numbers were similar for healthcare workers and the general population, indicating that on average, an infectious healthcare worker did not infect more people than any other infectious person. We provide a general method to estimate time dependence of parameters in structured epidemic models, which enables investigation of the impact of control measures and behavioral changes in different settings.</text>
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                <text>2009</text>
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              <elementText elementTextId="1727">
                <text>DOI: 10.1371/journal.pcbi.1000471</text>
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            <description>A related resource from which the described resource is derived</description>
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              <elementText elementTextId="1728">
                <text>PLoS Computational Biology</text>
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                <text>Public Library of Science (PLoS)</text>
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                <text>Biology (General)</text>
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            <description>A language of the resource</description>
            <elementTextContainer>
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                <text>EN</text>
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              <name>Title</name>
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                  <text>Coronavirus</text>
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              <description>An account of the resource</description>
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                  <text>Dominio científico: Coronavirus</text>
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                <text>Establishment, immortalisation and characterisation of pteropid bat cell lines.</text>
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                <text>Gary Crameri, Shawn Todd, Samantha Grimley, Jennifer A McEachern, Glenn A. Marsh, Craig Smith, Mary Tachedjian, Carol de Jong, Elena R Virtue, Meng Yu, Dieter Bulach, Junping Liu, Wojtek P. Michalski, Deborah Middleton, Hume E. Field, Lin-Fa Wang</text>
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            <description>An account of the resource</description>
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                <text>BACKGROUND: Bats are the suspected natural reservoir hosts for a number of new and emerging zoonotic viruses including Nipah virus, Hendra virus, severe acute respiratory syndrome coronavirus and Ebola virus. Since the discovery of SARS-like coronaviruses in Chinese horseshoe bats, attempts to isolate a SL-CoV from bats have failed and attempts to isolate other bat-borne viruses in various mammalian cell lines have been similarly unsuccessful. New stable bat cell lines are needed to help with these investigations and as tools to assist in the study of bat immunology and virus-host interactions. METHODOLOGY/FINDINGS: Black flying foxes (Pteropus alecto) were captured from the wild and transported live to the laboratory for primary cell culture preparation using a variety of different methods and culture media. Primary cells were successfully cultured from 20 different organs. Cell immortalisation can occur spontaneously, however we used a retroviral system to immortalise cells via the transfer and stable production of the Simian virus 40 Large T antigen and the human telomerase reverse transcriptase protein. Initial infection experiments with both cloned and uncloned cell lines using Hendra and Nipah viruses demonstrated varying degrees of infection efficiency between the different cell lines, although it was possible to infect cells in all tissue types. CONCLUSIONS/SIGNIFICANCE: The approaches developed and optimised in this study should be applicable to bats of other species. We are in the process of generating further cell lines from a number of different bat species using the methodology established in this study.</text>
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                <text>2009</text>
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            <name>Identifier</name>
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              <elementText elementTextId="1736">
                <text>DOI: 10.1371/journal.pone.0008266</text>
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            </elementTextContainer>
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          <element elementId="48">
            <name>Source</name>
            <description>A related resource from which the described resource is derived</description>
            <elementTextContainer>
              <elementText elementTextId="1737">
                <text>PLoS ONE</text>
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          <element elementId="45">
            <name>Publisher</name>
            <description>An entity responsible for making the resource available</description>
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                <text>Public Library of Science (PLoS)</text>
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            <name>Coverage</name>
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                <text>Science, Medicine</text>
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                <text>EN</text>
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        <src>https://www.socictopen.socict.org/files/original/d99010200a0cebf88f039d20781a92e7.pdf</src>
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            <element elementId="50">
              <name>Title</name>
              <description>A name given to the resource</description>
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                  <text>Coronavirus</text>
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              <name>Description</name>
              <description>An account of the resource</description>
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                  <text>Dominio científico: Coronavirus</text>
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              </elementTextContainer>
            </element>
          </elementContainer>
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          <element elementId="50">
            <name>Title</name>
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                <text>Designing small universal k-mer hitting sets for improved analysis of high-throughput sequencing.</text>
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            <name>Creator</name>
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                <text>Yaron Orenstein, David Pellow, Guillaume Marçais, Ron Shamir, Carl Kingsford</text>
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                <text>With the rapidly increasing volume of deep sequencing data, more efficient algorithms and data structures are needed. Minimizers are a central recent paradigm that has improved various sequence analysis tasks, including hashing for faster read overlap detection, sparse suffix arrays for creating smaller indexes, and Bloom filters for speeding up sequence search. Here, we propose an alternative paradigm that can lead to substantial further improvement in these and other tasks. For integers k and L &gt; k, we say that a set of k-mers is a universal hitting set (UHS) if every possible L-long sequence must contain a k-mer from the set. We develop a heuristic called DOCKS to find a compact UHS, which works in two phases: The first phase is solved optimally, and for the second we propose several efficient heuristics, trading set size for speed and memory. The use of heuristics is motivated by showing the NP-hardness of a closely related problem. We show that DOCKS works well in practice and produces UHSs that are very close to a theoretical lower bound. We present results for various values of k and L and by applying them to real genomes show that UHSs indeed improve over minimizers. In particular, DOCKS uses less than 30% of the 10-mers needed to span the human genome compared to minimizers. The software and computed UHSs are freely available at github.com/Shamir-Lab/DOCKS/ and acgt.cs.tau.ac.il/docks/, respectively.</text>
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            <name>Date</name>
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                <text>2017</text>
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                <text>DOI: 10.1371/journal.pcbi.1005777</text>
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          <element elementId="48">
            <name>Source</name>
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                <text>PLoS Computational Biology</text>
              </elementText>
            </elementTextContainer>
          </element>
          <element elementId="45">
            <name>Publisher</name>
            <description>An entity responsible for making the resource available</description>
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                <text>Public Library of Science (PLoS)</text>
              </elementText>
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            <name>Coverage</name>
            <description>The spatial or temporal topic of the resource, the spatial applicability of the resource, or the jurisdiction under which the resource is relevant</description>
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                <text>Biology (General)</text>
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            <name>Language</name>
            <description>A language of the resource</description>
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                <text>EN</text>
              </elementText>
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