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bProcessing bodies (P-bodies) are highly dynamic cytoplasmic granules conserved among eukaryotes. They are present under normal growth conditions and contain translationally repressed mRNAs together with proteins from the mRNA decay and microRNA (miRNA) machineries. We have previously shown that the core P-body components PatL1, LSm1, and DDX6 (Rck/p54) are required for hepatitis C virus (HCV) RNA replication; however, how HCV infection affects P-body granules and whether P-body granules per se influence the HCV life cycle remain unresolved issues. Here we show that HCV infection alters P-body composition by specifically changing the localization pattern of P-body components that are required for HCV replication. This effect was not related to an altered expression level of these components and could be reversed by inhibiting HCV replication with a polymerase inhibitor. Similar observations were obtained with a subgenomic replicon that supports only HCV translation and replication, indicating that these early steps of the HCV life cycle trigger the P-body alterations. Finally, P-body disruption by Rap55 depletion did not affect viral titers or HCV protein levels, demonstrating that the localization of PatL1, LSm1, and DDX6 in P-bodies is not required for their function on HCV. Thus, the HCV-induced changes on P-bodies are mechanistically linked to the function of specific P-body components in HCV RNA translation and replication; however, the formation of P-body granules is not required for HCV infection. Hepatitis C virus (HCV), a positive-strand RNA virus of the Flaviviridae family, is a major human pathogen. It has chronically infected around 170 million individuals worldwide and is a major cause of chronic liver disease and liver transplantation (24). Impressive advances in understanding the HCV life cycle have been achieved over the last years. These include the identification of cellular factors necessary for HCV entry into target cells, the elucidation of the viral multiplication requirements, and the description of cellular compartments necessary for particle assembly and progeny virus production (7). These advances not only increase our understanding of fundamental steps in HCV biology but also highlight the intimate relationship of virus components with cellular host factors.Components of the so-called processing bodies (P-bodies) are among the host factors required for efficient HCV multiplication (5,20,26,37,38). P-bodies are discrete and highly dynamic granules present in the cytoplasm of eukaryotic cells under normal growth conditions (18, 23). They contain translationally repressed mRNAs together with multiple proteins from the mRNA decay pathway and the microRNA (miRNA) machinery. Once in P-bodies, mRNAs can be either degraded or stored for a later return into translation (9,12,15). Experiments involving fluorescence recovery after photobleaching (FRAP) revealed that P-body components rapidly cycle in and out of these granules, indicating that there is a constant exchange of molecules with the cyt...

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Alternative strategies are required to enhance the diagnosis of silent hepatitis C virus (HCV) infections in key populations at risk. Among them, HCV prevalence and bio-behavioural data are scarce for HIV-negative men who have sex with men (MSM) and men and trans-women sex workers. We sought to describe and assess the potential benefits of a community-based one-step HCV screening and confirmatory strategy for these populations in Barcelona. The screening strategy based on a real-time RT-PCR assay for HCV-RNA detection in dried-blood spots (DBS) was validated and implemented in addition to an antibody point-of-care test in a community centre. HCV prevalence was assessed, and bio-behavioural data were collected. The molecular assay was precise, reproducible, sensitive and specific. Four HIV-negative MSM reported being currently infected (0.75% HCV self-reported prevalence). Implementation of DBS testing was easy, and acceptability was >95%, but no silent HCV case was diagnosed (N = 580). High-risk sexual practices and drug use for sex were reported frequently. HIV prevalence was 4.7% in MSM and 10% in sex workers. Self-reported prevalence of other STIs ranged from 11.3% to 36.2%. In conclusion, HCV-RNA testing in DBS showed a good performance, but the assessed one-step strategy does not seem beneficial in this setting. Although no silent HCV infections were detected, the observed high-risk behaviours and prevalence of other STIs suggest that HCV spread should be periodically monitored among these populations in Barcelona by means of behavioural surveillance, rapid antibody testing and molecular confirmation in DBS.

We report the evaluation of a new real-time PCR assay for hepatitis C virus (HCV) genotyping. The assay design is such that genotype 1 isolates are typed by amplification targeting the nonstructural 5b (NS5b) subgenomic region. Non-genotype 1 isolates are typed by type-specific amplicon detection in the 5 noncoding region (5NC) (method 1; HCV genotyping analyte-specific reagent assay). This method was compared with 5NC reverse hybridization (method 2; InnoLiPA HCV II) and 5NC sequencing (method 3; Trugene HCV 5NC). Two hundred ninety-five sera were tested by method 1; 223 of them were also typed by method 2 and 89 by method 3. Sequencing and phylogenetic analysis of an NS5b fragment were used to resolve discrepant results. Suspected multiple-genotype infections were confirmed by PCR cloning and pyrosequencing. Even though a 2% rate of indeterminates was obtained with method 1, concordance at the genotype level with results with methods 2 and 3 was high. Among eight discordant results, five mixed infections were confirmed. Genotype 1 subtyping efficiencies were 100%, 77%, and 74% for methods 1, 2, and 3, respectively; there were 11/101 discordants between methods 1 and 2 (method 1 was predominantly correct) and 2/34 between methods 2 and 3. Regarding genotype 2, subtyping efficiencies were 100%, 45%, and 92% by methods 1, 2, and 3, respectively; NS5b sequencing of discordants (16/17) revealed a putative new subtype within genotype 2 and that most subtype calls were not correct. Although only sequencing-based methods provide the possibility of identifying new variants, the real-time PCR method is rapid, straightforward, and simple to interpret, thus providing a good single-step alternative to more-time-consuming assays.Hepatitis C virus (HCV) is the most important cause of chronic liver disease and is the leading indication for liver transplantation (2). It is estimated that HCV infects 3% (170 million people) of the world's population (26). HCV possesses a positive-sense single-stranded RNA genome of approximately 9.5 kb, which is flanked by noncoding (NC) regions. The HCV genome encodes at least 11 proteins, which include both structural and nonstructural (NS) proteins (5, 7).HCV is known to have a high rate of genetic heterogeneity. This has allowed HCV strains to be classified into a number of genetically distinct groups, known as genotypes, subtypes, isolates, and quasispecies (5). Genome sequence heterogeneity arises due to poor fidelity of the viral polymerase during replication. Sequence variability is not evenly distributed throughout the genome. The lowest sequence variability is found in the 5ЈNC region, which is the target of choice for many molecular diagnostics assays, including genotyping tests. Nevertheless, nucleotide sequencing coupled with phylogenetic analysis of more-variable genomic regions has been recommended for HCV genotyping in consensus proposals (27). As patients infected with different genotypes respond differently to antiviral drug therapy, identification of the infecting genotype ha...

Hepatitis C virus (HCV) infection represents a major public health issue. Hepatitis C can be cured by therapy, but many infected individuals are unaware of their status. Effective HCV screening, fast diagnosis and characterization, and hepatic fibrosis staging are highly relevant for controlling transmission, treating infected patients and, consequently, avoiding end-stage liver disease. Exposure to HCV can be determined with high sensitivity and specificity with currently available third generation serology assays. Additionally, the use of point-of-care tests can increase HCV screening opportunities. However, active HCV infection must be confirmed by direct diagnosis methods. Additionally, HCV genotyping is required prior to starting any treatment. Increasingly, high-volume clinical laboratories use different types of automated platforms, which have simplified sample processing, reduced hands-on-time, minimized contamination risks and human error and ensured full traceability of results. Significant advances have also been made in the field of fibrosis stage assessment with the development of non-invasive methods, such as imaging techniques and serum-based tests. However, no single test is currently available that is able to completely replace liver biopsy. This review focuses on approved commercial tools used to diagnose HCV infection and the recommended hepatic fibrosis staging tests.

Hepatitis C virus (HCV) infection is the leading cause of chronic liver diseases. Water extracts of the leaves of the wild Egyptian artichoke (WEA) [Cynara cardunculus L. var. sylvestris (Lam.) Fiori] have been used for centuries in the Sinai Peninsula to treat hepatitis symptoms. Here we isolated and characterized six compounds from the water extracts of WEA and evaluated their HCV inhibition capacities in vitro. Importantly, two of these compounds, grosheimol and cynaropicrin, inhibited HCV with half-maximal effective concentrations (EC 50 s) in the low micromolar range. They inhibited HCV entry into target cells and were active against both cell-free infection as well as cell-cell transmission. Furthermore, the antiviral activity of both compounds was pan-genotypic as HCV genotypes 1a, 1b, 2b, 3a, 4a, 5a, 6a, and 7a were inhibited. Thus, grosheimol and cynaropicrin are promising candidates for the development of new pan-genotypic entry inhibitors of HCV infection. IMPORTANCEBecause there is no preventive HCV vaccine available today, the discovery of novel anti-HCV cell entry inhibitors could help develop preventive measures against infection. The present study describes two compounds isolated from the wild Egyptian artichoke (WEA) with respect to their structural elucidation, absolute configuration, and quantitative determination. Importantly, both compounds inhibited HCV infection in vitro. The first compound was an unknown molecule, and it was designated "grosheimol," while the second compound is the known molecule cynaropicrin. Both compounds belong to the group of sesquiterpene lactones. The mode of action of these compounds occurred during the early steps of the HCV life cycle, including cell-free and cell-cell infection inhibition. These natural compounds present promising candidates for further development into anti-HCV therapeutics. Hepatitis C virus (HCV) is an enveloped, positive-strand RNA virus classified as a separate genus (Hepacivirus) within the Flaviviridae family. It shows a high degree of genetic diversity, with 7 major circulating genotypes (1). HCV is mainly transmitted through exposure to HCV-contaminated blood. Most infections remain persistent, summing up to an estimated 150 million chronic HCV carriers worldwide (2). As persistent HCV infection frequently causes chronic hepatitis that can progress to liver cirrhosis and liver cell carcinoma, it is a major threat to human health (3, 4).Treatment options for chronically infected individuals have dramatically improved over the last few years. This has been due to the development of highly potent direct-acting antivirals (DAAs) that increased sustained response rates to over 90%, even in interferon-free combinations (5). Currently approved DAAs include NS3/4A protease inhibitors (telaprevir, boceprevir, and simeprevir), NS5A inhibitors (daclatasvir and ledipasvir), and the NS5B polymerase inhibitor sofosbuvir. Further antiviral drugs are in clinical trials and are about to be approved. Nonetheless, the rapid replication of HCV, al...