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                <text>Infectious Bronchitis Virus as a Vector for the Expression of Heterologous Genes.</text>
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                <text>Kirsten Bentley, María Armesto, Paul Britton</text>
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                <text>The avian coronavirus infectious bronchitis virus (IBV) is the causative agent of the respiratory disease infectious bronchitis of domestic fowl, and is controlled by routine vaccination. To explore the potential use of IBV as a vaccine vector a reverse genetics system was utilised to generate infectious recombinant IBVs (rIBVs) expressing the reporter genes enhanced green fluorescent protein (eGFP) or humanised Renilla luciferase (hRluc). Infectious rIBVs were obtained following the replacement of Gene 5 or the intergenic region (IR) with eGFP or hRluc, or the replacement of ORFs 3a and 3b with hRluc. The replacement of Gene 5 with an IBV codon-optimised version of the hRluc gene also resulted in successful rescue of infectious rIBV. Reporter gene expression was confirmed by fluorescence microscopy, or luciferase activity assays, for all successfully rescued rIBVs following infection of primary chick kidney (CK) cells. The genetic stability of rIBVs was analysed by serial passage on CK cells. Recombinant IBV stability varied depending on the genome region being replaced, with the reporter genes maintained up to at least passage 8 (P8) following replacement of Gene 5, P7 for replacement of the IR and P5 for replacement of ORFs 3a and 3b. Codon-optimisation of the hRluc gene, when replacing Gene 5, resulted in an increase in genome stability, with hRluc expression stable up to P10 compared to P8 for standard hRluc. Repeated passaging of rIBVs expressing hRluc at an MOI of 0.01 demonstrated an increase in stability, with hRluc expression stable up to at least P12 following the replacement of Gene 5. This study has demonstrated that heterologous genes can be incorporated into, and expressed from a range of IBV genome locations and that replacement of accessory Gene 5 offers a promising target for realising the potential of IBV as a vaccine vector for other avian pathogens.</text>
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                <text>DOI: 10.1371/journal.pone.0067875</text>
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                  <text>Dominio científico: Coronavirus</text>
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                <text>The effects of sampling on the efficiency and accuracy of k-mer indexes: Theoretical and empirical comparisons using the human genome.</text>
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                <text>Meznah Almutairy, Eric Torng</text>
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                <text>One of the most common ways to search a sequence database for sequences that are similar to a query sequence is to use a k-mer index such as BLAST. A big problem with k-mer indexes is the space required to store the lists of all occurrences of all k-mers in the database. One method for reducing the space needed, and also query time, is sampling where only some k-mer occurrences are stored. Most previous work uses hard sampling, in which enough k-mer occurrences are retained so that all similar sequences are guaranteed to be found. In contrast, we study soft sampling, which further reduces the number of stored k-mer occurrences at a cost of decreasing query accuracy. We focus on finding highly similar local alignments (HSLA) over nucleotide sequences, an operation that is fundamental to biological applications such as cDNA sequence mapping. For our comparison, we use the NCBI BLAST tool with the human genome and human ESTs. When identifying HSLAs, we find that soft sampling significantly reduces both index size and query time with relatively small losses in query accuracy. For the human genome and HSLAs of length at least 100 bp, soft sampling reduces index size 4-10 times more than hard sampling and processes queries 2.3-6.8 times faster, while still achieving retention rates of at least 96.6%. When we apply soft sampling to the problem of mapping ESTs against the genome, we map more than 98% of ESTs perfectly while reducing the index size by a factor of 4 and query time by 23.3%. These results demonstrate that soft sampling is a simple but effective strategy for performing efficient searches for HSLAs. We also provide a new model for sampling with BLAST that predicts empirical retention rates with reasonable accuracy by modeling two key problem factors.</text>
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                <text>DOI: 10.1371/journal.pone.0179046</text>
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                <text>Sequence characteristics define trade-offs between on-target and genome-wide off-target hybridization of oligoprobes.</text>
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                <text>Olga V Matveeva, Aleksey Y Ogurtsov, Nafisa N Nazipova, Svetlana A Shabalina</text>
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                <text>Off-target oligoprobe's interaction with partially complementary nucleotide sequences represents a problem for many bio-techniques. The goal of the study was to identify oligoprobe sequence characteristics that control the ratio between on-target and off-target hybridization. To understand the complex interplay between specific and genome-wide off-target (cross-hybridization) signals, we analyzed a database derived from genomic comparison hybridization experiments performed with an Affymetrix tiling array. The database included two types of probes with signals derived from (i) a combination of specific signal and cross-hybridization and (ii) genomic cross-hybridization only. All probes from the database were grouped into bins according to their sequence characteristics, where both hybridization signals were averaged separately. For selection of specific probes, we analyzed the following sequence characteristics: vulnerability to self-folding, nucleotide composition bias, numbers of G nucleotides and GGG-blocks, and occurrence of probe's k-mers in the human genome. Increases in bin ranges for these characteristics are simultaneously accompanied by a decrease in hybridization specificity-the ratio between specific and cross-hybridization signals. However, both averaged hybridization signals exhibit growing trends along with an increase of probes' binding energy, where the hybridization specific signal increases significantly faster in comparison to the cross-hybridization. The same trend is evident for the S function, which serves as a combined evaluation of probe binding energy and occurrence of probe's k-mers in the genome. Application of S allows extracting a larger number of specific probes, as compared to using only binding energy. Thus, we showed that high values of specific and cross-hybridization signals are not mutually exclusive for probes with high values of binding energy and S. In this study, the application of a new set of sequence characteristics allows detection of probes that are highly specific to their targets for array design and other bio-techniques that require selection of specific probes.</text>
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                <text>2018</text>
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                <text>DOI: 10.1371/journal.pone.0199162</text>
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                <text>PLoS ONE</text>
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                  <text>Dominio científico: Coronavirus</text>
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                <text>Analysis of intraviral protein-protein interactions of the SARS coronavirus ORFeome.</text>
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                <text>Albrecht von Brunn, Carola Teepe, Jeremy C. Simpson, Rainer Pepperkok, Caroline C Friedel, Ralf Zimmer, Rhonda Roberts, Ralph Baric, Jürgen Haas</text>
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                <text>The severe acute respiratory syndrome coronavirus (SARS-CoV) genome is predicted to encode 14 functional open reading frames, leading to the expression of up to 30 structural and non-structural protein products. The functions of a large number of viral ORFs are poorly understood or unknown. In order to gain more insight into functions and modes of action and interaction of the different proteins, we cloned the viral ORFeome and performed a genome-wide analysis for intraviral protein interactions and for intracellular localization. 900 pairwise interactions were tested by yeast-two-hybrid matrix analysis, and more than 65 positive non-redundant interactions, including six self interactions, were identified. About 38% of interactions were subsequently confirmed by CoIP in mammalian cells. Nsp2, nsp8 and ORF9b showed a wide range of interactions with other viral proteins. Nsp8 interacts with replicase proteins nsp2, nsp5, nsp6, nsp7, nsp8, nsp9, nsp12, nsp13 and nsp14, indicating a crucial role as a major player within the replication complex machinery. It was shown by others that nsp8 is essential for viral replication in vitro, whereas nsp2 is not. We show that also accessory protein ORF9b does not play a pivotal role for viral replication, as it can be deleted from the virus displaying normal plaque sizes and growth characteristics in Vero cells. However, it can be expected to be important for the virus-host interplay and for pathogenicity, due to its large number of interactions, by enhancing the global stability of the SARS proteome network, or play some unrealized role in regulating protein-protein interactions. The interactions identified provide valuable material for future studies.</text>
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                <text>2007</text>
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                <text>DOI: 10.1371/journal.pone.0000459</text>
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                <text>PLoS ONE</text>
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                <text>To predict the potential severity of outbreaks of infectious diseases such as SARS, HIV, TB and smallpox, a summary parameter, the basic reproduction number R(0), is generally calculated from a population-level model. R(0) specifies the average number of secondary infections caused by one infected individual during his/her entire infectious period at the start of an outbreak. R(0) is used to assess the severity of the outbreak, as well as the strength of the medical and/or behavioral interventions necessary for control. Conventionally, it is assumed that if R(0)&gt;1 the outbreak generates an epidemic, and if R(0)</text>
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                <text>DOI: 10.1371/journal.pone.0000282</text>
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                  <text>Dominio científico: Coronavirus</text>
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                <text>The SARS Coronavirus 3a protein causes endoplasmic reticulum stress and induces ligand-independent downregulation of the type 1 interferon receptor.</text>
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                <text>Rinki Minakshi, Kartika Padhan, Manjusha Rani, Nabab Khan, Faizan Ahmad, Shahid Jameel</text>
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                <text>The Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) is reported to cause apoptosis of infected cells and several of its proteins including the 3a accessory protein, are pro-apoptotic. Since the 3a protein localizes to the endoplasmic reticulum (ER)-Golgi compartment, its role in causing ER stress was investigated in transiently transfected cells. Cells expressing the 3a proteins showed ER stress based on activation of genes for the ER chaperones GRP78 and GRP94. Since ER stress can cause differential modulation of the unfolded protein response (UPR), which includes the inositol-requiring enzyme 1 (IRE-1), activating transcription factor 6 (ATF6) and PKR-like ER kinase (PERK) pathways, these were individually tested in 3a-expressing cells. Only the PERK pathway was found to be activated in 3a-expressing cells based on (1) increased phosphorylation of eukaryotic initiation factor 2 alpha (eIF2alpha) and inhibitory effects of a dominant-negative form of eIF2alpha on GRP78 promoter activity, (2) increased translation of activating transcription factor 4 (ATF4) mRNA, and (3) ATF4-dependent activation of the C/EBP homologous protein (CHOP) gene promoter. Activation of PERK affects innate immunity by suppression of type 1 interferon (IFN) signaling. The 3a protein was found to induce serine phosphorylation within the IFN alpha-receptor subunit 1 (IFNAR1) degradation motif and to increase IFNAR1 ubiquitination. Confocal microscopic analysis showed increased translocation of IFNAR1 into the lysosomal compartment and flow cytometry showed reduced levels of IFNAR1 in 3a-expressing cells. These results provide further mechanistic details of the pro-apoptotic effects of the SARS-CoV 3a protein, and suggest a potential role for it in attenuating interferon responses and innate immunity.</text>
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                <text>DOI: 10.1371/journal.pone.0008342</text>
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                <text>PLoS ONE</text>
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                <text>Detection of Zika virus using reverse-transcription LAMP coupled with reverse dot blot analysis in saliva.</text>
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                <text>Maite Sabalza, Rubina Yasmin, Cheryl A Barber, Talita Castro, Daniel Malamud, Beum Jun Kim, Hui Zhu, Richard A Montagna, William R. Abrams</text>
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                <text>In recent years, there have been increasing numbers of infectious disease outbreaks that spread rapidly to population centers resulting from global travel, population vulnerabilities, environmental factors, and ecological disasters such as floods and earthquakes. Some examples of the recent outbreaks are the Ebola epidemic in West Africa, Middle East respiratory syndrome coronavirus (MERS-Co) in the Middle East, and the Zika outbreak through the Americas. We have created a generic protocol for detection of pathogen RNA and/or DNA using loop-mediated isothermal amplification (LAMP) and reverse dot-blot for detection (RDB) and processed automatically in a microfluidic device. In particular, we describe how a microfluidic assay to detect HIV viral RNA was converted to detect Zika virus (ZIKV) RNA. We first optimized the RT-LAMP assay to detect ZIKV RNA using a benchtop isothermal amplification device. Then we implemented the assay in a microfluidic device that will allow analyzing 24 samples simultaneously and automatically from sample introduction to detection by RDB technique. Preliminary data using saliva samples spiked with ZIKV showed that our diagnostic system detects ZIKV RNA in saliva. These results will be validated in further experiments with well-characterized ZIKV human specimens of saliva. The described strategy and methodology to convert the HIV diagnostic assay and platform to a ZIKV RNA detection assay provides a model that can be readily utilized for detection of the next emerging or re-emerging infectious disease.</text>
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                <text>2018</text>
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                <text>DOI: 10.1371/journal.pone.0192398</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>Coronavirus spike protein inhibits host cell translation by interaction with eIF3f.</text>
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                <text>Han Xiao, Linghui Xu, Yoshiyuki Yamada, Ding Xiang Liu</text>
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                <text>In response to viral infection, the expression of numerous host genes, including predominantly a number of proinflammatory cytokines and chemokines, is usually up-regulated at both transcriptional and translational levels. It was noted that in cells infected with coronavirus, transcription and translation of some of these genes were differentially induced. Drastic induction of their expression at the transcriptional level was observed in cells infected with coronavirus. However, induction of the same genes at the translational level was usually found to be minimal to moderate. To investigate the underlying mechanisms, yeast two-hybrid screen was carried out using SARS-CoV proteins as baits, revealing that a subunit of the eukaryotic initiation factor 3 (eIF3), eIF3f, may interact with the N-terminal region of the SARS-CoV spike (S) protein. This interaction was subsequently confirmed by co-immunoprecipitation and immunofluorescent staining. Meanwhile, parallel experiments confirmed that eIF3f could also interact with the S protein of another coronavirus, the avian coronavirus infectious bronchitis virus (IBV). These interactions led to the inhibition of translation of a reporter gene in both in vitro expression system and intact cells. Interestingly, IBV-infected cells stably expressing a Flag-tagged eIF3f showed much higher translation of IL-6 and IL-8, suggesting that the interaction between coronavirus S protein and eIF3f plays a functional role in controlling the expression of host genes, especially genes that are induced during coronavirus infection cycles. This study reveals a novel mechanism exploited by coronavirus to regulate viral pathogenesis.</text>
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                <text>2008</text>
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                <text>DOI: 10.1371/journal.pone.0001494</text>
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                <text>PLoS ONE</text>
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                <text>Correction: Serological Evidence of MERS-CoV Antibodies in Dromedary Camels (Camelus dromedaries) in Laikipia County, Kenya.</text>
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                <text>[This corrects the article DOI: 10.1371/journal.pone.0140125.].</text>
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                <text>DOI: 10.1371/journal.pone.0178310</text>
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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="298" public="1" featured="0">
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        <src>https://www.socictopen.socict.org/files/original/7ff685462ab8bd67e49462cbbd798edf.pdf</src>
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              <name>Title</name>
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                  <text>Coronavirus</text>
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              <name>Description</name>
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                  <text>Dominio científico: Coronavirus</text>
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                <text>Acquisition of cell-cell fusion activity by amino acid substitutions in spike protein determines the infectivity of a coronavirus in cultured cells.</text>
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                <text>Yoshiyuki Yamada, Xiao-bo Liu, Shou Guo Fang, Felicia P L Tay, Ding Xiang Liu</text>
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                <text>Coronavirus host and cell specificities are determined by specific interactions between the viral spike (S) protein and host cell receptor(s). Avian coronavirus infectious bronchitis (IBV) has been adapted to embryonated chicken eggs, primary chicken kidney (CK) cells, monkey kidney cell line Vero, and other human and animal cells. Here we report that acquisition of the cell-cell fusion activity by amino acid mutations in the S protein determines the infectivity of IBV in cultured cells. Expression of S protein derived from Vero- and CK-adapted strains showed efficient induction of membrane fusion. However, expression of S protein cloned from the third passage of IBV in chicken embryo (EP3) did not show apparent syncytia formation. By construction of chimeric S constructs and site-directed mutagenesis, a point mutation (L857-F) at amino acid position 857 in the heptad repeat 1 region of S protein was shown to be responsible for its acquisition of the cell-cell fusion activity. Furthermore, a G405-D point mutation in the S1 domain, which was acquired during further propagation of Vero-adapted IBV in Vero cells, could enhance the cell-cell fusion activity of the protein. Re-introduction of L857 back to the S gene of Vero-adapted IBV allowed recovery of variants that contain the introduced L857. However, compensatory mutations in S1 and some distant regions of S2 were required for restoration of the cell-cell fusion activity of S protein carrying L857 and for the infectivity of the recovered variants in cultured cells. This study demonstrates that acquisition of the cell-cell fusion activity in S protein determines the selection and/or adaptation of a coronavirus from chicken embryo to cultured cells of human and animal origins.</text>
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                <text>2009</text>
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            <name>Identifier</name>
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                <text>DOI: 10.1371/journal.pone.0006130</text>
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                <text>PLoS ONE</text>
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            <name>Publisher</name>
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                <text>Public Library of Science (PLoS)</text>
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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>Science, Medicine</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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