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MACHEREY NAGEL viral ribonucleic acid rna
Viral Ribonucleic Acid Rna, supplied by MACHEREY NAGEL, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 95 stars, based on 1 article reviews
Price from $9.99 to $1999.99
viral ribonucleic acid rna - by Bioz Stars, 2020-04
95/100 stars

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Related Articles

RNA Extraction:

Article Title: Anti-viral activity of culinary and medicinal mushroom extracts against dengue virus serotype 2: an in-vitro study
Article Snippet: .. Viral ribonucleic acid (RNA) was extracted from cell pellets according to the instructions provided by the Nucleospin RNA extraction kit (MACHEREY-NAGEL, Germany). .. The purity and concentration of the extracted RNA were assessed using a BioSpectrophotometer (EPPENDORF, Hamburg, Germany).

Quantitative RT-PCR:

Article Title: Anti-viral activity of culinary and medicinal mushroom extracts against dengue virus serotype 2: an in-vitro study
Article Snippet: Effect of mushroom extract on the expression levels of dengue envelope (ENV) and non-structural protein 5 (NS5) genes The inhibitory activities of the mushroom extracts in the simultaneous, attachment and penetration assays were further evaluated by RT-qPCR to study the expression levels of the dengue envelope (ENV) and non-structural protein 5 (NS5) genes. .. Viral ribonucleic acid (RNA) was extracted from cell pellets according to the instructions provided by the Nucleospin RNA extraction kit (MACHEREY-NAGEL, Germany).

Concentration Assay:

Article Title: Anti-viral activity of culinary and medicinal mushroom extracts against dengue virus serotype 2: an in-vitro study
Article Snippet: Viral ribonucleic acid (RNA) was extracted from cell pellets according to the instructions provided by the Nucleospin RNA extraction kit (MACHEREY-NAGEL, Germany). .. The purity and concentration of the extracted RNA were assessed using a BioSpectrophotometer (EPPENDORF, Hamburg, Germany).

Cell Culture:

Article Title: Anti-viral activity of culinary and medicinal mushroom extracts against dengue virus serotype 2: an in-vitro study
Article Snippet: Vero cells were grown to approximately 90% confluency, infected with DENV-2 and cultured in the presence of 2000 μg/ml MNCC of the mushroom extracts. .. Viral ribonucleic acid (RNA) was extracted from cell pellets according to the instructions provided by the Nucleospin RNA extraction kit (MACHEREY-NAGEL, Germany).

Expressing:

Article Title: Anti-viral activity of culinary and medicinal mushroom extracts against dengue virus serotype 2: an in-vitro study
Article Snippet: Paragraph title: Effect of mushroom extract on the expression levels of dengue envelope (ENV) and non-structural protein 5 (NS5) genes ... Viral ribonucleic acid (RNA) was extracted from cell pellets according to the instructions provided by the Nucleospin RNA extraction kit (MACHEREY-NAGEL, Germany).

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  • 86
    MACHEREY NAGEL real time rt pcr viral rna
    Time- and dose-dependent DENV infection of primary microvascular endothelial HMVEC-d cells and the endothelial cell line HMEC-1. HMEC-1 (A, C) and HMVEC-d cells (E) were infected with DENV-2 at a MOI 1. Viral infectivity was quantified at different times after infection by flow cytometry using an anti-DENV-2 specific antibody (A, E). The amount of viral <t>RNA</t> was determined in the supernatant of infected HMEC-1 cells by means of real-time <t>RT-PCR</t> at different times after infection (C). Alternatively, HMEC-1 (B, D) and HMVEC-d cells (F) were treated with medium only (mock) or infected with DENV at a MOI of 1, 2 or 4. Viral infectivity was quantified 24 h post infection by flow cytometry (B, F). The amount of viral RNA was determined in the supernatant of infected HMEC-1 cells at 24 h post infection by means of real-time RT-PCR (D). The means and standard deviations of three independent experiments are shown.
    Real Time Rt Pcr Viral Rna, supplied by MACHEREY NAGEL, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/real time rt pcr viral rna/product/MACHEREY NAGEL
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    real time rt pcr viral rna - by Bioz Stars, 2020-04
    86/100 stars
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    99
    MACHEREY NAGEL viral rna
    Model of the interplay between NS5A and PI4KIIIα. A: Consensus sequence (red) of the PI4KIIIα interaction site (PFIS) derived from 672 NS5A sequences of all genotypes in the Los Alamos <t>HCV</t> database ( http://hcv.lanl.gov ). Green numbers in the top line refer to the degree of conservation (rounded). Numbers on the left and right refer to the positions of the flanking amino acids within NS5A. Variations from the consensus are listed according to their frequency. A proline found in the JFH-1 PFIS is marked in blue. Variants found only once are not shown. B: NS5A (light brown) interacts with PI4KIIIα (red). This interaction regulates NS5A phosphorylation status directly or indirectly. Active kinase promotes NS5A p56 formation, a fraction of which is hyperphosphorylated resulting in p58. P56 might positively influence viral <t>RNA</t> replication either directly or by affecting the morphology of the replication sites, for which additional host factors are probably required. PI4KIIIα interaction with NS5A and NS5B is required to trigger lipid kinase activity. This leads to formation of new PI4P pools, presumably involved in membranous web morphology.
    Viral Rna, supplied by MACHEREY NAGEL, used in various techniques. Bioz Stars score: 99/100, based on 42 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/viral rna/product/MACHEREY NAGEL
    Average 99 stars, based on 42 article reviews
    Price from $9.99 to $1999.99
    viral rna - by Bioz Stars, 2020-04
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    86
    MACHEREY NAGEL qrt pcr viral rna
    Replication studies of the BHCV1 isolate. (A) Luciferase activity in the electroporated cells with the sugbenomic RNAs in both transient (4 h–4 d) and mid-term fashions (4 d–21 d). (B) HCV <t>RNA</t> in the electroporated cells at similar time points as determined by quantitative reverse-transcription real-time <t>PCR</t> (qRT-PCR). For A and B, data represent the mean values from duplicate wells, each measured in triplicate, from a representative experiment of 3 (mean ± SD; n = 6). (C) Western blot analysis of core protein in electroporated cells with full-length RNAs expressed intracellularly in short-term (4–96 h) and mid-term fashions (9–21 d). Positive and negative control cell lysates were obtained from Huh7.5 cells transfected with a core protein expression plasmid under the control of the CMV promoter (pcDNA3.1/Core) or an empty plasmid (pcDNA3.1), respectively.
    Qrt Pcr Viral Rna, supplied by MACHEREY NAGEL, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/qrt pcr viral rna/product/MACHEREY NAGEL
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    qrt pcr viral rna - by Bioz Stars, 2020-04
    86/100 stars
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    Image Search Results


    Time- and dose-dependent DENV infection of primary microvascular endothelial HMVEC-d cells and the endothelial cell line HMEC-1. HMEC-1 (A, C) and HMVEC-d cells (E) were infected with DENV-2 at a MOI 1. Viral infectivity was quantified at different times after infection by flow cytometry using an anti-DENV-2 specific antibody (A, E). The amount of viral RNA was determined in the supernatant of infected HMEC-1 cells by means of real-time RT-PCR at different times after infection (C). Alternatively, HMEC-1 (B, D) and HMVEC-d cells (F) were treated with medium only (mock) or infected with DENV at a MOI of 1, 2 or 4. Viral infectivity was quantified 24 h post infection by flow cytometry (B, F). The amount of viral RNA was determined in the supernatant of infected HMEC-1 cells at 24 h post infection by means of real-time RT-PCR (D). The means and standard deviations of three independent experiments are shown.

    Journal: PLoS ONE

    Article Title: Sulfated Escherichia coli K5 Polysaccharide Derivatives Inhibit Dengue Virus Infection of Human Microvascular Endothelial Cells by Interacting with the Viral Envelope Protein E Domain III

    doi: 10.1371/journal.pone.0074035

    Figure Lengend Snippet: Time- and dose-dependent DENV infection of primary microvascular endothelial HMVEC-d cells and the endothelial cell line HMEC-1. HMEC-1 (A, C) and HMVEC-d cells (E) were infected with DENV-2 at a MOI 1. Viral infectivity was quantified at different times after infection by flow cytometry using an anti-DENV-2 specific antibody (A, E). The amount of viral RNA was determined in the supernatant of infected HMEC-1 cells by means of real-time RT-PCR at different times after infection (C). Alternatively, HMEC-1 (B, D) and HMVEC-d cells (F) were treated with medium only (mock) or infected with DENV at a MOI of 1, 2 or 4. Viral infectivity was quantified 24 h post infection by flow cytometry (B, F). The amount of viral RNA was determined in the supernatant of infected HMEC-1 cells at 24 h post infection by means of real-time RT-PCR (D). The means and standard deviations of three independent experiments are shown.

    Article Snippet: RNA extraction and real-time RT-PCR Viral RNA was extracted from 150 µl of supernatant with the Nucleospin® RNA Virus Kit (Macherey-Nagel, Düren, Germany) according to the manufacturer’s instructions.

    Techniques: Infection, Flow Cytometry, Cytometry, Quantitative RT-PCR

    Model of the interplay between NS5A and PI4KIIIα. A: Consensus sequence (red) of the PI4KIIIα interaction site (PFIS) derived from 672 NS5A sequences of all genotypes in the Los Alamos HCV database ( http://hcv.lanl.gov ). Green numbers in the top line refer to the degree of conservation (rounded). Numbers on the left and right refer to the positions of the flanking amino acids within NS5A. Variations from the consensus are listed according to their frequency. A proline found in the JFH-1 PFIS is marked in blue. Variants found only once are not shown. B: NS5A (light brown) interacts with PI4KIIIα (red). This interaction regulates NS5A phosphorylation status directly or indirectly. Active kinase promotes NS5A p56 formation, a fraction of which is hyperphosphorylated resulting in p58. P56 might positively influence viral RNA replication either directly or by affecting the morphology of the replication sites, for which additional host factors are probably required. PI4KIIIα interaction with NS5A and NS5B is required to trigger lipid kinase activity. This leads to formation of new PI4P pools, presumably involved in membranous web morphology.

    Journal: PLoS Pathogens

    Article Title: The Lipid Kinase Phosphatidylinositol-4 Kinase III Alpha Regulates the Phosphorylation Status of Hepatitis C Virus NS5A

    doi: 10.1371/journal.ppat.1003359

    Figure Lengend Snippet: Model of the interplay between NS5A and PI4KIIIα. A: Consensus sequence (red) of the PI4KIIIα interaction site (PFIS) derived from 672 NS5A sequences of all genotypes in the Los Alamos HCV database ( http://hcv.lanl.gov ). Green numbers in the top line refer to the degree of conservation (rounded). Numbers on the left and right refer to the positions of the flanking amino acids within NS5A. Variations from the consensus are listed according to their frequency. A proline found in the JFH-1 PFIS is marked in blue. Variants found only once are not shown. B: NS5A (light brown) interacts with PI4KIIIα (red). This interaction regulates NS5A phosphorylation status directly or indirectly. Active kinase promotes NS5A p56 formation, a fraction of which is hyperphosphorylated resulting in p58. P56 might positively influence viral RNA replication either directly or by affecting the morphology of the replication sites, for which additional host factors are probably required. PI4KIIIα interaction with NS5A and NS5B is required to trigger lipid kinase activity. This leads to formation of new PI4P pools, presumably involved in membranous web morphology.

    Article Snippet: To assess replication efficiency of different replicon constructs by direct measurement of viral RNA, 0.25 µg of replicon RNA was electroporated into Huh7-Lunet cells and HCV RNA was quantified by RT-PCR as described recently .In brief, viral RNA was isolated from transfected cells using the Nucleo Spin RNAII kit (Macherey-Nagel, Düren, Germany) as recommended by the manufacturer.

    Techniques: Sequencing, Derivative Assay, Activity Assay

    Mapping of the PI4KIIIα - interaction site within NS5A domain 1. A: Schematic representation of expression constructs with deletions in NS5A D1 used to identify the PI4KIIIα interaction site. HCV coding sequences are indicated by blue or grey boxes, deleted sequences by black lines. Numbers refer to amino acid positions within the HCV JFH-1 polyprotein, numbers in brackets to amino acid positions within NS5A. The EMCV-IRES is indicated by a schematic RNA secondary structure. T7 Pm, T7 promoter; AH, amphipathic helix; D1, 2, 3, subdomains of NS5A; LCS1, 2, low complexity sequences [30] . B: Huh7-Lunet T7 cells were transfected with plasmids encoding the NS3 to NS5B polyprotein of genotype 2a (JFH-1) with sub-deletions within NS5A domain 1 with or without HA-tagged PI4KIIIα (HA-PI4K), as indicated at the bottom. Newly synthesized proteins were radiolabeled and cell lysates subjected to immunoprecipitation using NS5A (lower panel) or HA-specific antibodies (upper panel). Samples were analyzed by SDS-PAGE and autoradiography and quantified by phosphoimaging. Numbers at the bottom indicate the coprecipitation efficiency of HA-PI4KIIIα with individual mutants compared to NS5A wt. Coprecipitation efficiency was normalized to the total amounts of HA-PI4K for each sample (upper panel). Note that data were not normalized to input NS5A levels due to a consistently high molar excess of NS5A compared to PI4KIIIα (data not shown). Mock: cells transfected with empty pTM vector.

    Journal: PLoS Pathogens

    Article Title: The Lipid Kinase Phosphatidylinositol-4 Kinase III Alpha Regulates the Phosphorylation Status of Hepatitis C Virus NS5A

    doi: 10.1371/journal.ppat.1003359

    Figure Lengend Snippet: Mapping of the PI4KIIIα - interaction site within NS5A domain 1. A: Schematic representation of expression constructs with deletions in NS5A D1 used to identify the PI4KIIIα interaction site. HCV coding sequences are indicated by blue or grey boxes, deleted sequences by black lines. Numbers refer to amino acid positions within the HCV JFH-1 polyprotein, numbers in brackets to amino acid positions within NS5A. The EMCV-IRES is indicated by a schematic RNA secondary structure. T7 Pm, T7 promoter; AH, amphipathic helix; D1, 2, 3, subdomains of NS5A; LCS1, 2, low complexity sequences [30] . B: Huh7-Lunet T7 cells were transfected with plasmids encoding the NS3 to NS5B polyprotein of genotype 2a (JFH-1) with sub-deletions within NS5A domain 1 with or without HA-tagged PI4KIIIα (HA-PI4K), as indicated at the bottom. Newly synthesized proteins were radiolabeled and cell lysates subjected to immunoprecipitation using NS5A (lower panel) or HA-specific antibodies (upper panel). Samples were analyzed by SDS-PAGE and autoradiography and quantified by phosphoimaging. Numbers at the bottom indicate the coprecipitation efficiency of HA-PI4KIIIα with individual mutants compared to NS5A wt. Coprecipitation efficiency was normalized to the total amounts of HA-PI4K for each sample (upper panel). Note that data were not normalized to input NS5A levels due to a consistently high molar excess of NS5A compared to PI4KIIIα (data not shown). Mock: cells transfected with empty pTM vector.

    Article Snippet: To assess replication efficiency of different replicon constructs by direct measurement of viral RNA, 0.25 µg of replicon RNA was electroporated into Huh7-Lunet cells and HCV RNA was quantified by RT-PCR as described recently .In brief, viral RNA was isolated from transfected cells using the Nucleo Spin RNAII kit (Macherey-Nagel, Düren, Germany) as recommended by the manufacturer.

    Techniques: Expressing, Construct, Transfection, Synthesized, Immunoprecipitation, SDS Page, Autoradiography, Plasmid Preparation

    Reduced PI4KIIIα interaction and RNA replication deficiency correlate with relative increase of NS5A hyperphosphorylation. A: Huh7-Lunet T7 cells were transfected with plasmids encoding the NS3 to NS5B polyprotein of genotype 2a (JFH-1) containing a wt sequence or triple alanine mutants as indicated or with empty plasmid (mock). Newly synthesized proteins were radiolabeled and cell lysates subjected to immunoprecipitation using NS5A specific antibodies. Immunocomplexes were analyzed by SDS-PAGE and autoradiography. B: Quantitative analysis of the NS5A p58/p56 ratio. Bands corresponding to NS5A p58 and p56, respectively, as shown in panel A were individually quantified by phosphoimaging to obtain a p58/p56 ratio. Error bars indicate mean values +/− SD of two independent experiments analyzed in duplicates. Significance was compared to the wt polyprotein and calculated by a paired t-test. *, p

    Journal: PLoS Pathogens

    Article Title: The Lipid Kinase Phosphatidylinositol-4 Kinase III Alpha Regulates the Phosphorylation Status of Hepatitis C Virus NS5A

    doi: 10.1371/journal.ppat.1003359

    Figure Lengend Snippet: Reduced PI4KIIIα interaction and RNA replication deficiency correlate with relative increase of NS5A hyperphosphorylation. A: Huh7-Lunet T7 cells were transfected with plasmids encoding the NS3 to NS5B polyprotein of genotype 2a (JFH-1) containing a wt sequence or triple alanine mutants as indicated or with empty plasmid (mock). Newly synthesized proteins were radiolabeled and cell lysates subjected to immunoprecipitation using NS5A specific antibodies. Immunocomplexes were analyzed by SDS-PAGE and autoradiography. B: Quantitative analysis of the NS5A p58/p56 ratio. Bands corresponding to NS5A p58 and p56, respectively, as shown in panel A were individually quantified by phosphoimaging to obtain a p58/p56 ratio. Error bars indicate mean values +/− SD of two independent experiments analyzed in duplicates. Significance was compared to the wt polyprotein and calculated by a paired t-test. *, p

    Article Snippet: To assess replication efficiency of different replicon constructs by direct measurement of viral RNA, 0.25 µg of replicon RNA was electroporated into Huh7-Lunet cells and HCV RNA was quantified by RT-PCR as described recently .In brief, viral RNA was isolated from transfected cells using the Nucleo Spin RNAII kit (Macherey-Nagel, Düren, Germany) as recommended by the manufacturer.

    Techniques: Transfection, Sequencing, Plasmid Preparation, Synthesized, Immunoprecipitation, SDS Page, Autoradiography

    New PI4P pools emerge as a consequence of HCV RNA replication. A: Scheme of the transcomplementation experiments shown in panel B–D: Huh7-Lunet cells constitutively expressing HCV NS3 to NS5A (I) or NS5A (II), respectively, were transfected with reporter replicons containing luciferase and eGFP genes as indicated to analyze for conditions rescuing RNA replication. B: Wiltype (wt-eGFP), repHIT (repHIT-eGFP) and ΔGDD reporter replicons of genotype 2a (JFH-1) were transfected into Huh7-Lunet cell lines constitutively expressing NS3-5A or NS5A of genotype 1b or 2a, as indicated. RNA replication of replicons was determined by measuring luciferase activity in cell lysates at 24 h, 48 h and 72 h post transfection relative to 4 h to normalize for transfection efficiency. Diagrams show mean values +/−SD of a representative of two experiments performed in duplicates. C. Immunofluorescence analysis of the experiment shown in panel B at 48 h post electroporation. GFP (green) or PI4P (red), respectively, was detected with specific antibodies and DAPI was used to stain nuclei (blue). D. Quantitation of intracellular PI4P levels by measuring PI4P fluorescence intensity using ImageJ analysis (IntDen read-out) on cells as shown in panel C. Data represent mean arbitrary units (AU) +/− SD of 35 GFP positive cells analyzed per condition. In case of ΔGDD, cells were randomly chosen due to the lack of GFP signals. Significance was calculated by a paired students t-test. ***, p

    Journal: PLoS Pathogens

    Article Title: The Lipid Kinase Phosphatidylinositol-4 Kinase III Alpha Regulates the Phosphorylation Status of Hepatitis C Virus NS5A

    doi: 10.1371/journal.ppat.1003359

    Figure Lengend Snippet: New PI4P pools emerge as a consequence of HCV RNA replication. A: Scheme of the transcomplementation experiments shown in panel B–D: Huh7-Lunet cells constitutively expressing HCV NS3 to NS5A (I) or NS5A (II), respectively, were transfected with reporter replicons containing luciferase and eGFP genes as indicated to analyze for conditions rescuing RNA replication. B: Wiltype (wt-eGFP), repHIT (repHIT-eGFP) and ΔGDD reporter replicons of genotype 2a (JFH-1) were transfected into Huh7-Lunet cell lines constitutively expressing NS3-5A or NS5A of genotype 1b or 2a, as indicated. RNA replication of replicons was determined by measuring luciferase activity in cell lysates at 24 h, 48 h and 72 h post transfection relative to 4 h to normalize for transfection efficiency. Diagrams show mean values +/−SD of a representative of two experiments performed in duplicates. C. Immunofluorescence analysis of the experiment shown in panel B at 48 h post electroporation. GFP (green) or PI4P (red), respectively, was detected with specific antibodies and DAPI was used to stain nuclei (blue). D. Quantitation of intracellular PI4P levels by measuring PI4P fluorescence intensity using ImageJ analysis (IntDen read-out) on cells as shown in panel C. Data represent mean arbitrary units (AU) +/− SD of 35 GFP positive cells analyzed per condition. In case of ΔGDD, cells were randomly chosen due to the lack of GFP signals. Significance was calculated by a paired students t-test. ***, p

    Article Snippet: To assess replication efficiency of different replicon constructs by direct measurement of viral RNA, 0.25 µg of replicon RNA was electroporated into Huh7-Lunet cells and HCV RNA was quantified by RT-PCR as described recently .In brief, viral RNA was isolated from transfected cells using the Nucleo Spin RNAII kit (Macherey-Nagel, Düren, Germany) as recommended by the manufacturer.

    Techniques: Expressing, Transfection, Luciferase, Activity Assay, Immunofluorescence, Electroporation, Staining, Quantitation Assay, Fluorescence

    NS5A but not PI4P can transcomplement replication-deficient replicons with NS5A mutations affecting PI4KIIIα interaction. A: Experimental setup of transcomplementation experiments: Huh7-Lunet cells bearing either a persistent HCV replicon (I) or constitutively expressing HCV NS3 to NS5A (II) or NS5A (III), respectively, are transfected with luciferase reporter replicons harboring the HIT triple alanine mutation to analyze for conditions rescuing RNA replication. Wt replicons and a replication deficient NS5B mutant (ΔGDD) are used for positive and negative control, respectively. B. Huh7-Lunet cells with persistent replicons (neoJFH) or constitutive expression protein of genotype 2a NS3-5A or NS5A, respectively, were subjected to immunofluorescence analysis. NS5A (red) or PI4P (green), respectively, were detected with specific antibodies and DAPI was used to stain nuclei (blue). C. Quantitation of intracellular PI4P levels by measuring PI4P fluorescence intensity using ImageJ analysis (IntDen read-out) on cells as shown in panel B and on equivalent cell lines harboring genotype 1b (Con1) replicons and proteins. PI4P levels were normalized to the mean value of naïve Huh7-Lunet cells. Data represent mean +/− SD of 35 NS5A positive cells analyzed per condition. D. Detection of NS5A and β-actin in Huh7-Lunet cells with persistent replicons or constitutive expression of NS3-5A or NS5A of genotype (Gt) 1b and 2a, respectively, by western-blot. Equal amounts of NS5A were loaded to judge variations in NS5A phosphorylation. Differences in β-actin, therefore, reflect varying NS5A expression levels. E, F: Wt (dark color), repHIT (medium color) and ΔGDD reporter replicons (light color) of either genotype 2a (panel E, JFH-1, red) or genotype 1b (panel F, Con1ET, blue) were transfected into Huh7-Lunet cell lines harboring a persistent replicon (Repl.) or constitutively expressing NS3-5A (3-5A) or NS5A (5A) of genotype 1b or 2a, as indicated. RNA replication of replicons was determined by measuring luciferase activity in cell lysates 72 h post transfection relative to 4 h to normalize for transfection efficiency. Diagrams show mean values +/−SD of at least two experiments performed in duplicates.

    Journal: PLoS Pathogens

    Article Title: The Lipid Kinase Phosphatidylinositol-4 Kinase III Alpha Regulates the Phosphorylation Status of Hepatitis C Virus NS5A

    doi: 10.1371/journal.ppat.1003359

    Figure Lengend Snippet: NS5A but not PI4P can transcomplement replication-deficient replicons with NS5A mutations affecting PI4KIIIα interaction. A: Experimental setup of transcomplementation experiments: Huh7-Lunet cells bearing either a persistent HCV replicon (I) or constitutively expressing HCV NS3 to NS5A (II) or NS5A (III), respectively, are transfected with luciferase reporter replicons harboring the HIT triple alanine mutation to analyze for conditions rescuing RNA replication. Wt replicons and a replication deficient NS5B mutant (ΔGDD) are used for positive and negative control, respectively. B. Huh7-Lunet cells with persistent replicons (neoJFH) or constitutive expression protein of genotype 2a NS3-5A or NS5A, respectively, were subjected to immunofluorescence analysis. NS5A (red) or PI4P (green), respectively, were detected with specific antibodies and DAPI was used to stain nuclei (blue). C. Quantitation of intracellular PI4P levels by measuring PI4P fluorescence intensity using ImageJ analysis (IntDen read-out) on cells as shown in panel B and on equivalent cell lines harboring genotype 1b (Con1) replicons and proteins. PI4P levels were normalized to the mean value of naïve Huh7-Lunet cells. Data represent mean +/− SD of 35 NS5A positive cells analyzed per condition. D. Detection of NS5A and β-actin in Huh7-Lunet cells with persistent replicons or constitutive expression of NS3-5A or NS5A of genotype (Gt) 1b and 2a, respectively, by western-blot. Equal amounts of NS5A were loaded to judge variations in NS5A phosphorylation. Differences in β-actin, therefore, reflect varying NS5A expression levels. E, F: Wt (dark color), repHIT (medium color) and ΔGDD reporter replicons (light color) of either genotype 2a (panel E, JFH-1, red) or genotype 1b (panel F, Con1ET, blue) were transfected into Huh7-Lunet cell lines harboring a persistent replicon (Repl.) or constitutively expressing NS3-5A (3-5A) or NS5A (5A) of genotype 1b or 2a, as indicated. RNA replication of replicons was determined by measuring luciferase activity in cell lysates 72 h post transfection relative to 4 h to normalize for transfection efficiency. Diagrams show mean values +/−SD of at least two experiments performed in duplicates.

    Article Snippet: To assess replication efficiency of different replicon constructs by direct measurement of viral RNA, 0.25 µg of replicon RNA was electroporated into Huh7-Lunet cells and HCV RNA was quantified by RT-PCR as described recently .In brief, viral RNA was isolated from transfected cells using the Nucleo Spin RNAII kit (Macherey-Nagel, Düren, Germany) as recommended by the manufacturer.

    Techniques: Expressing, Transfection, Luciferase, Mutagenesis, Negative Control, Immunofluorescence, Staining, Quantitation Assay, Fluorescence, Western Blot, Activity Assay

    Impact of mutations within the PI4KIIIα binding region on HCV RNA replication and partial rescue by PI4KIIIα overexpression. A: Schematic representation of a monocistronic replicon used in this study. 5′ non-translated and 3′ non-translated regions are indicated by secondary structures. Luciferase (Luc) is connected by a cleavable ubiquitin (ubi) linker to the non-structural proteins NS3 to NS5B. C, core. B: Huh7-Lunet cells were transfected with luciferase reporter replicons bearing the indicated triple alanine substitutions. JFH-1 wt replicons (wt, green) and a mutant harboring a deletion within NS5B (ΔGDD, red) served as positive and negative controls, respectively. Replication efficiency is expressed as luciferase activity (RLU) at 24 h (light color), 48 h (medium color) and 72 h (dark color) relative to 4 h after transfection to normalize for transfection efficiency. 24 h values of mutants with delayed kinetics are highlighted by red lines. Error bars indicate mean +/− SD of a representative experiment (n = 3). C: Huh7-Lunet cells were transfected with luciferase reporter replicons as described in B. Total cellular RNA was extracted 4 h or 72 h after transfection. HCV RNA was quantified by quantitative RT-PCR and is shown as HCV copies at 72 h relative to 4 h after transfection. Error bars indicate mean +/− SD of a representative experiment (n = 6). D: Naïve Huh7-Lunet cells (control) or Huh7-Lunet cells stably overexpressing HA-tagged PI4KIIIα (HA-PI4KIIIα) were transfected with luciferase reporter replicons as described in B. Replication efficiency is expressed as luciferase activity (RLU) at 48 h relative to 4 h after transfection. Error bars indicate mean +/− SD of quadruplicate values of two independent experiments. E: Overexpression of HA-PI4KIIIα was confirmed by immunoblot of whole cell lysates of naïve Huh-7 Lunet cells (control) or HA-PI4KIIIα overexpressing cells (HA-PI4KIIIα) using antibodies directed against PI4KIIIα, HA-peptide or Calnexin.

    Journal: PLoS Pathogens

    Article Title: The Lipid Kinase Phosphatidylinositol-4 Kinase III Alpha Regulates the Phosphorylation Status of Hepatitis C Virus NS5A

    doi: 10.1371/journal.ppat.1003359

    Figure Lengend Snippet: Impact of mutations within the PI4KIIIα binding region on HCV RNA replication and partial rescue by PI4KIIIα overexpression. A: Schematic representation of a monocistronic replicon used in this study. 5′ non-translated and 3′ non-translated regions are indicated by secondary structures. Luciferase (Luc) is connected by a cleavable ubiquitin (ubi) linker to the non-structural proteins NS3 to NS5B. C, core. B: Huh7-Lunet cells were transfected with luciferase reporter replicons bearing the indicated triple alanine substitutions. JFH-1 wt replicons (wt, green) and a mutant harboring a deletion within NS5B (ΔGDD, red) served as positive and negative controls, respectively. Replication efficiency is expressed as luciferase activity (RLU) at 24 h (light color), 48 h (medium color) and 72 h (dark color) relative to 4 h after transfection to normalize for transfection efficiency. 24 h values of mutants with delayed kinetics are highlighted by red lines. Error bars indicate mean +/− SD of a representative experiment (n = 3). C: Huh7-Lunet cells were transfected with luciferase reporter replicons as described in B. Total cellular RNA was extracted 4 h or 72 h after transfection. HCV RNA was quantified by quantitative RT-PCR and is shown as HCV copies at 72 h relative to 4 h after transfection. Error bars indicate mean +/− SD of a representative experiment (n = 6). D: Naïve Huh7-Lunet cells (control) or Huh7-Lunet cells stably overexpressing HA-tagged PI4KIIIα (HA-PI4KIIIα) were transfected with luciferase reporter replicons as described in B. Replication efficiency is expressed as luciferase activity (RLU) at 48 h relative to 4 h after transfection. Error bars indicate mean +/− SD of quadruplicate values of two independent experiments. E: Overexpression of HA-PI4KIIIα was confirmed by immunoblot of whole cell lysates of naïve Huh-7 Lunet cells (control) or HA-PI4KIIIα overexpressing cells (HA-PI4KIIIα) using antibodies directed against PI4KIIIα, HA-peptide or Calnexin.

    Article Snippet: To assess replication efficiency of different replicon constructs by direct measurement of viral RNA, 0.25 µg of replicon RNA was electroporated into Huh7-Lunet cells and HCV RNA was quantified by RT-PCR as described recently .In brief, viral RNA was isolated from transfected cells using the Nucleo Spin RNAII kit (Macherey-Nagel, Düren, Germany) as recommended by the manufacturer.

    Techniques: Binding Assay, Over Expression, Luciferase, Transfection, Mutagenesis, Activity Assay, Quantitative RT-PCR, Stable Transfection

    Viral RNA shedding detected by RRT-PCR in quail experimentally challenged with AIV. Results are expressed as inverted Ct-values and shown as means of positive individuals ± SD. Tables indicate the ratio between positive quail and total quail examined per day and sample. Ct, cycle of threshold; DPI, days post-inoculation; OS, oropharyngeal swabs; CS, cloacal swabs; FP, feather pulps. a. Quail intranasally inoculated with H7N2/LP. b. Contact quail of H7N2/LP. c . Quail intranasally inoculated with H7N1/HP. d. Contact quail of H7N1/HP. e. Quail intranasally inoculated with H5N1/HP. f. Contact quail of H5N1/HP.

    Journal: Veterinary Research

    Article Title: Pathobiology and transmission of highly and low pathogenic avian influenza viruses in European quail (Coturnix c. coturnix)

    doi: 10.1186/1297-9716-44-23

    Figure Lengend Snippet: Viral RNA shedding detected by RRT-PCR in quail experimentally challenged with AIV. Results are expressed as inverted Ct-values and shown as means of positive individuals ± SD. Tables indicate the ratio between positive quail and total quail examined per day and sample. Ct, cycle of threshold; DPI, days post-inoculation; OS, oropharyngeal swabs; CS, cloacal swabs; FP, feather pulps. a. Quail intranasally inoculated with H7N2/LP. b. Contact quail of H7N2/LP. c . Quail intranasally inoculated with H7N1/HP. d. Contact quail of H7N1/HP. e. Quail intranasally inoculated with H5N1/HP. f. Contact quail of H5N1/HP.

    Article Snippet: Viral RNA detection by RRT-PCR Viral RNA from OS, CS, FP, and drinking water samples was extracted with NucleoSpin® RNA virus kit (Macherey-Nagel, Düren, Germany) following the manufacturer’s instructions.

    Techniques: Quantitative RT-PCR

    Replication studies of the BHCV1 isolate. (A) Luciferase activity in the electroporated cells with the sugbenomic RNAs in both transient (4 h–4 d) and mid-term fashions (4 d–21 d). (B) HCV RNA in the electroporated cells at similar time points as determined by quantitative reverse-transcription real-time PCR (qRT-PCR). For A and B, data represent the mean values from duplicate wells, each measured in triplicate, from a representative experiment of 3 (mean ± SD; n = 6). (C) Western blot analysis of core protein in electroporated cells with full-length RNAs expressed intracellularly in short-term (4–96 h) and mid-term fashions (9–21 d). Positive and negative control cell lysates were obtained from Huh7.5 cells transfected with a core protein expression plasmid under the control of the CMV promoter (pcDNA3.1/Core) or an empty plasmid (pcDNA3.1), respectively.

    Journal: PLoS ONE

    Article Title: Cell Culture Replication of a Genotype 1b Hepatitis C Virus Isolate Cloned from a Patient Who Underwent Liver Transplantation

    doi: 10.1371/journal.pone.0023587

    Figure Lengend Snippet: Replication studies of the BHCV1 isolate. (A) Luciferase activity in the electroporated cells with the sugbenomic RNAs in both transient (4 h–4 d) and mid-term fashions (4 d–21 d). (B) HCV RNA in the electroporated cells at similar time points as determined by quantitative reverse-transcription real-time PCR (qRT-PCR). For A and B, data represent the mean values from duplicate wells, each measured in triplicate, from a representative experiment of 3 (mean ± SD; n = 6). (C) Western blot analysis of core protein in electroporated cells with full-length RNAs expressed intracellularly in short-term (4–96 h) and mid-term fashions (9–21 d). Positive and negative control cell lysates were obtained from Huh7.5 cells transfected with a core protein expression plasmid under the control of the CMV promoter (pcDNA3.1/Core) or an empty plasmid (pcDNA3.1), respectively.

    Article Snippet: RNA quantification by qRT-PCR Viral RNA was isolated from virus-infected or electroporated cells using the Nucleo Spin RNAII kit (Macherey-Nagel, Düren, Germany) following the manufacturer's protocol.

    Techniques: Luciferase, Activity Assay, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Negative Control, Transfection, Expressing, Plasmid Preparation

    HCV RNA and core protein detection in Huh7.5-inoculated cells. (A and B) HCV RNA was analyzed by qRT-PCR analysis at the indicated time points (A) or at 96 h post inoculation (B). Results represent the mean values from duplicate wells, measured in triplicate, from a representative experiment of 3 (mean ± SD; n = 6). (C) Cells were stained with α-core specific antibodies (clone C7-50) and α-mouse Alexa-568 antibodies (red). Cell nuclei were counterstained with DAPI (blue), magnification 100×.

    Journal: PLoS ONE

    Article Title: Cell Culture Replication of a Genotype 1b Hepatitis C Virus Isolate Cloned from a Patient Who Underwent Liver Transplantation

    doi: 10.1371/journal.pone.0023587

    Figure Lengend Snippet: HCV RNA and core protein detection in Huh7.5-inoculated cells. (A and B) HCV RNA was analyzed by qRT-PCR analysis at the indicated time points (A) or at 96 h post inoculation (B). Results represent the mean values from duplicate wells, measured in triplicate, from a representative experiment of 3 (mean ± SD; n = 6). (C) Cells were stained with α-core specific antibodies (clone C7-50) and α-mouse Alexa-568 antibodies (red). Cell nuclei were counterstained with DAPI (blue), magnification 100×.

    Article Snippet: RNA quantification by qRT-PCR Viral RNA was isolated from virus-infected or electroporated cells using the Nucleo Spin RNAII kit (Macherey-Nagel, Düren, Germany) following the manufacturer's protocol.

    Techniques: Quantitative RT-PCR, Staining

    Infectivity of the BHCV1/JFH1 chimeric virus. (A) Genetic structure of the BHCV1/JFH1 chimeric virus. Structural proteins, p7, and the first transmembrane domain of NS2 of the isolate BHCV1 (amino acids 1–844) were fused in-frame to the remaining non-structural proteins of the JFH1 isolate. Both NTRs are of JFH1 origin. (B) Schematic presentation of the experimental plan for the cell culture adaptation of the BHCV1/JFH1 chimeric virus. (C) qRT-PCR analysis of naïve Huh7.5 cells inoculated with supernatant derived from Huh7.5 cells containing the BCHV1/JFH1 virus from different passages. J6/JFH1 infection: MOI 0.2 TCID 50 /mL. Data are expressed as the mean of duplicate infections measured in triplicate (mean ± SD; n = 6). # Estimated infectivity ∼10 2 TCID 50 /mL; † Estimated infectivity ∼10 5 TCID 50 /mL.

    Journal: PLoS ONE

    Article Title: Cell Culture Replication of a Genotype 1b Hepatitis C Virus Isolate Cloned from a Patient Who Underwent Liver Transplantation

    doi: 10.1371/journal.pone.0023587

    Figure Lengend Snippet: Infectivity of the BHCV1/JFH1 chimeric virus. (A) Genetic structure of the BHCV1/JFH1 chimeric virus. Structural proteins, p7, and the first transmembrane domain of NS2 of the isolate BHCV1 (amino acids 1–844) were fused in-frame to the remaining non-structural proteins of the JFH1 isolate. Both NTRs are of JFH1 origin. (B) Schematic presentation of the experimental plan for the cell culture adaptation of the BHCV1/JFH1 chimeric virus. (C) qRT-PCR analysis of naïve Huh7.5 cells inoculated with supernatant derived from Huh7.5 cells containing the BCHV1/JFH1 virus from different passages. J6/JFH1 infection: MOI 0.2 TCID 50 /mL. Data are expressed as the mean of duplicate infections measured in triplicate (mean ± SD; n = 6). # Estimated infectivity ∼10 2 TCID 50 /mL; † Estimated infectivity ∼10 5 TCID 50 /mL.

    Article Snippet: RNA quantification by qRT-PCR Viral RNA was isolated from virus-infected or electroporated cells using the Nucleo Spin RNAII kit (Macherey-Nagel, Düren, Germany) following the manufacturer's protocol.

    Techniques: Infection, Cell Culture, Quantitative RT-PCR, Derivative Assay