21 research outputs found

    Productive replication of serum-derived HCV in Huh-7.5/EG(4A/4B)GLuc cells.

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    <p>(A&B) <i>Gaussia</i> activity and intracellular HCV RNA analysis in Huh-7.5/EG(4A/4B)GLuc cells inoculated with sera derived from patients 20 and 35 at the indicated time points. Results represent the mean values from duplicate wells, measured in duplicate (for both <i>Gaussia</i> and RNA), from a representative experiment of 3 (mean ± SD; n = 4) (C) Intracellular core expression in Huh-7.5/EG(4A/4B)GLuc cells inoculated with the indicated patient sera, JC1 virus or HCV negative Serum (HCV neg.) as negative control, as detected by core protein IF. 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 100x. (D) Core protein released in the supernatant from Huh-7.5/EG(4A/4B)GLuc cells infected by the indicated patient sera, JC1 virus or HCV neg. serum as negative control, 120 h post inoculation.</p

    A <em>Gaussia</em> Luciferase Cell-Based System to Assess the Infection of Cell Culture- and Serum-Derived Hepatitis C Virus

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    <div><p>Robust replication of hepatitis C virus (HCV) in cell culture occurs only with the JFH-1 (genotype 2a) recombinant genome. The aim of this study was to develop a system for HCV infection quantification analysis and apply it for the selection of patient sera that may contain cell culture infectious viruses, particularly of the most clinically important genotype 1. Initially, a hepatoma cell line (designated Huh-7.5/EG(4A/4B)GLuc) was generated that stably expressed the enhanced green fluorescent protein (EGFP) fused in-frame to the secreted <em>Gaussia</em> luciferase via a recognition sequence of the viral NS3/4A protease. Upon HCV infection, NS3/4A cleaved at its signal and the <em>Gaussia</em> was secreted to the culture medium, thus facilitating the infection quantification. The Huh-7.5/EG(4A/4B)GLuc cell line provided a rapid and highly sensitive quantification of HCV infection in cell culture using JFH-1-derived viruses. Furthermore, the Huh-7.5/EG(4A/4B)GLuc cells were also shown to be a suitable host for the discovery of anti-HCV inhibitors by using known compounds that target distinct stages of the HCV life cycle; the Ĺą-factor of this assay ranged from 0.72 to 0.75. Additionally, eighty-six sera derived from HCV genotype 1b infected liver transplant recipients were screened for their <em>in vitro</em> infection and replication potential. Approximately 12% of the sera contained <em>in vitro</em> replication-competent viruses, as deduced by the <em>Gaussia</em> signal, real time quantitative PCR, immunofluorescence and capsid protein secretion. We conclude that the Huh-7.5/EG(4A/4B)GLuc cell line is an excellent system not only for the screening of <em>in vitro</em> replication-competent serum-derived viruses, but also for the subsequent cloning of recombinant isolates. Additionally, it can be utilized for high-throughput screening of antiviral compounds.</p> </div

    Schematic of dual-function reporter vectors used in the HCV NS3/4A protease activity assay.

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    <p>(A) The various recognition sites of NS3/4A protease, IPS<sub>462–540</sub>, NLS-IPS<sub>462–540</sub>, KDEL-DE<sub>4x</sub>-4A/4B and DE<sub>4x</sub>-4A/4B were inserted between the <i>EGFP</i> and the humanized <i>Gaussia luciferase</i> gene by an in-frame fusion. Arrows indicate the NS3/4A cleavage site. Expression of the reporter genes is under the human cytomegalovirus promoter (CMV) and the selection marker for the generation of the stable cell line is neomycin phosphotrasferase (Neo<sup>R</sup>). SV40, simian virus 40. (B) Transiently trasfected Huh-7.5 cells were infected with JC1 virus at an MOI 0.5 TCID<sub>50</sub>/cell or mock infected with JFH-1/ΔGDD supernatant. At 5 days post infection, the medium was harvested and <i>Gaussia</i> activity was measured. Results are expressed as the mean values from duplicate wells, measured in duplicates, from a representative experiment of 3 (mean ± SD; n = 4).*, <i>P</i><0.05.</p

    Overview of the screening strategy.

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    <p>(A) Eighty-six clinical sera were selected from patients as described in <i>Materials and Methods</i>. Patientś number rise relative to the viral load increase. Relative <i>Gaussia</i> expression scale: <i>Gaussia</i> activity by wells infected in parallel with the JC1 virus at an MOI of 0.5 TCID<sub>50</sub>/cell was set at 100 while that by JFH-1/ΔGDD and/or HCV control negative serum (HCV neg.) was set at 0. (A) Results from 3 representative experiments. Means difference between the experiments were not significant (n.s.). Patients with <i>Gaussia</i> activity ≥40% are indicated. (B) Colours were drawn according to the mean of 3 independent experiments. In each experiment duplicate infections were performed and measured in duplicate (n = 4).</p

    List of patients with infectivity ≥ 40%.

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    a<p>Infectivity is expressed as a percentage (% ± SD) relevant to that obtained from cells infected in parallel with JC1 viruses.</p

    Correlation between extracellular <i>Gaussia</i> activity and HCV RNA and proteins.

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    <p>Huh-7.5/EG(4A/4B)GLuc cells were infected with JC1 (A) or JFH-1 (B) viruses at an MOI of 0.5 TCID<sub>50</sub>/cell. Extracellular <i>Gaussia</i> activity and intracellular RNA were analyzed at the given time points post infection. (C) Correlation between NS3 and core proteins levels with EG(4A/4B)GLuc cleavage efficiency. Huh-7.5/EG(4A/4B)GLuc cells were infected like in (A) and at the given time points expression levels of NS3, core and EG(4A/4B)GLuc were analyzed by Western blotting by anti-NS3, anti-core and anti-GFP antibodies, respectively.</p

    TGF-β reduces the fibrinogen α-chain expression in HepG2 cells.

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    <p>Fold regulation in Fibrinogen Alpha Chain (FGA), Beta Chain (FGB) and Gamma Chain (FGG) genes regulation in HepG2 cells after 6 hours of treatment with TGF-β (10 ng/ml). Results are given as mean ± SE; ***p<0.001 vs. basal. Statistical analysis was calculated by unpaired Student’s t test. The insert shows the intensity values of the 5.9 kDa peak detected in the cellular supernatant of HepG2 cells after 48 hours of treatment with TGF-β (10 ng/ml). Results are given as mean ± SE; *p<0.05 vs. basal. Statistical analysis was calculated by Mann-Whitney U test.</p

    Lack of a 5.9 kDa Peptide C-Terminal Fragment of Fibrinogen α Chain Precedes Fibrosis Progression in Patients with Liver Disease

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    <div><p>Early detection of fibrosis progression is of major relevance for the diagnosis and management of patients with liver disease. This study was designed to find non-invasive biomarkers for fibrosis in a clinical context where this process occurs rapidly, HCV-positive patients who underwent liver transplantation (LT). We analyzed 93 LT patients with HCV recurrence, 41 non-LT patients with liver disease showing a fibrosis stage F≥1 and 9 patients without HCV recurrence who received antiviral treatment before LT, as control group. Blood obtained from 16 healthy subjects was also analyzed. Serum samples were fractionated by ion exchange chromatography and their proteomic profile was analyzed by SELDI-TOF-MS. Characterization of the peptide of interest was performed by ion chromatography and electrophoresis, followed by tandem mass spectrometry identification. Marked differences were observed between the serum proteome profile of LT patients with early fibrosis recurrence and non-recurrent LT patients. A robust peak intensity located at 5905 m/z was the distinguishing feature of non-recurrent LT patients. However, the same peak was barely detected in recurrent LT patients. Similar results were found when comparing samples of healthy subjects with those of non-LT fibrotic patients, indicating that our findings were not related to either LT or HCV infection. Using tandem mass-spectrometry, we identified the protein peak as a C-terminal fragment of the fibrinogen α chain. Cell culture experiments demonstrated that TGF-β reduces α-fibrinogen mRNA expression and 5905 m/z peak intensity in HepG2 cells, suggesting that TGF-β activity regulates the circulating levels of this protein fragment. In conclusion, we identified a 5.9 kDa C-terminal fragment of the fibrinogen α chain as an early serum biomarker of fibrogenic processes in patients with liver disease.</p></div

    Comparison of the 5.9 kDa protein peak intensity between all the groups analyzed.

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    <p>SELDI-TOF-MS intensity values of the 5.9 kDa peak of the different groups of patients studied. Intensity of the peak was markedly suppressed in all patients under an active fibrogenic process.</p

    Isolation, separation and identification of the 5.9 kDa protein peak.

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    <p>A/ Spectra obtained by SELDI-TOF-MS showing the isolated protein peak after the purification process. B/ The isolated band indicated with arrows, after running two tris-tricine gels, one stained with sypro (I) and the other with oriole (II) staining. C/ Fragment of the Fibrinogen α-Chain identified as the differential protein peak by the amino acid sequencing. The two sequences identified are shown in bold.</p
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