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Hepatitis C virus (HCV) is a major cause of chronic liver disease, frequently progressing to cirrhosis and increased risk of hepatocellular carcinoma. Current therapies are inadequate and progress in the field has been hampered by the lack of efficient HCV culture systems. By using a recently described HCV genotype 2a infectious clone that replicates and produces infectious virus in cell culture (HCVcc), we report here that HCVcc strain FL-J6͞JFH can establish long-term infections in chimpanzees and in mice containing human liver grafts. Importantly, virus recovered from these animals was highly infectious in cell culture, demonstrating efficient ex vivo culture of HCV. The improved infectivity of animal-derived HCV correlated with virions of a lower average buoyant density than HCVcc, suggesting that physical association with low-density factors influences viral infectivity. These results greatly extend the utility of the HCVcc genetic system to allow the complete in vitro and in vivo dissection of the HCV life cycle.animal model ͉ pathogenesis ͉ reverse genetics ͉ viral hepatitis A major limitation in hepatitis C virus (HCV) research has been the lack of virus culture systems. After identification of the viral genome in 1989 (1), early efforts focused on understanding the structure and function of individual viral gene products. HCV is an enveloped, positive-strand RNA virus classified in the family Flaviviridae (2). The 9.6-kb ssRNA genome encodes three structural (virion-associated) and seven nonstructural (intracellular) genes within a single ORF.The first functional cDNA clones of HCV were constructed in 1997, allowing chimpanzees to be infected after intrahepatic transfection with recombinant viral RNA (3, 4). Unfortunately, these infectious genomes failed to replicate in cell culture. By engineering HCV replicons to express a drug-selectable gene, it became possible to select for HCV RNA replication in cell culture (5). However, efficient replication required cell cultureadaptive mutations in the viral RNA (6). Moreover, only the intracellular aspects of HCV replication were modeled by these systems. For unknown reasons, cell culture-adaptive mutations can inhibit virion production in culture (T. Pietschmann and R. Bartenschlager, personal communication) and attenuate RNA infectivity in vivo (7).Recent progress in the field has come from the identification of JFH-1, a genotype 2a subgenomic replicon that does not require adaptive mutations for efficient RNA replication in culture (8). Based on this sequence, we constructed a chimeric JFH-1 genome containing the core to nonstructural protein 2 (NS2) region of HCV strain J6. This genome, FL-J6͞JFH, replicated and produced high levels of infectious virus in cell culture (HCVcc) (9), allowing us to study new aspects of the viral life cycle in tissue culture (9, 10). Similarly, full-length JFH-1 clones produced HCVcc, albeit with delayed kinetics of virus release (11, 12). HCVcc strain JFH-1 was able to transiently infect a chimpanzee, although replication le...

Two‐dimensional shear wave elastography (2D‐SWE) has proven to be efficient for the evaluation of liver fibrosis in small to moderate‐sized clinical trials. We aimed at running a larger‐scale meta‐analysis of individual data. Centers which have worked with Aixplorer ultrasound equipment were contacted to share their data. Retrospective statistical analysis used direct and paired receiver operating characteristic and area under the receiver operating characteristic curve (AUROC) analyses, accounting for random effects. Data on both 2D‐SWE and liver biopsy were available for 1,134 patients from 13 sites, as well as on successful transient elastography in 665 patients. Most patients had chronic hepatitis C (n = 379), hepatitis B (n = 400), or nonalcoholic fatty liver disease (n = 156). AUROCs of 2D‐SWE in patients with hepatitis C, hepatitis B, and nonalcoholic fatty liver disease were 86.3%, 90.6%, and 85.5% for diagnosing significant fibrosis and 92.9%, 95.5%, and 91.7% for diagnosing cirrhosis, respectively. The AUROC of 2D‐SWE was 0.022‐0.084 (95% confidence interval) larger than the AUROC of transient elastography for diagnosing significant fibrosis (P = 0.001) and 0.003‐0.034 for diagnosing cirrhosis (P = 0.022) in all patients. This difference was strongest in hepatitis B patients. Conclusion: 2D‐SWE has good to excellent performance for the noninvasive staging of liver fibrosis in patients with hepatitis B; further prospective studies are needed for head‐to‐head comparison between 2D‐SWE and other imaging modalities to establish disease‐specific appropriate cutoff points for assessment of fibrosis stage. (Hepatology 2018;67:260‐272).

The viral life cycle of the hepatitis C virus (HCV) has been studied mainly using different in vitro cell culture models. Studies using pseudoviral particles (HCVpp) and more recently cell culture-derived virus (HCVcc) suggest that at least three host cell molecules are important for HCV entry in vitro: the tetraspanin CD81, the scavenger receptor class B member I, and the tight junction protein Claudin-1. Whether these receptors are equally important for an in vivo infection remains to be demonstrated. We show that CD81 is indispensable for an authentic in vivo HCV infection. Prophylactic treatment with anti-CD81 antibodies completely protected human liver-uPA-SCID mice from a subsequent challenge with HCV consensus strains of different genotypes. Administration of anti-CD81 antibodies after viral challenge had no effect. M ore than 170 million people worldwide are infected with the hepatitis C virus (HCV). The lack of an effective HCV vaccine and the high cost and considerable side effects of the current standard therapies have a major impact on global public health. HCV is now a leading cause of liver cirrhosis, hepatocellular carcinoma, and liver transplantation. 1 The precise mechanism by which HCV attaches to and enters host cells remains unclear. Pileri et al. 2 identified the tetraspanin CD81 (TAPA-1) as a putative receptor for HCV. This 21-kDa surface molecule, composed of four transmembrane and two extracellular domains, efficiently bound recombinantly produced E2, one of the two viral envelope proteins. Experiments using pseudoviral particles (HCVpp) containing the HCV envelope proteins [3][4][5][6] or the recently developed infectious HCV cell culture system 7-11 have shown that CD81 expression on the target cell is essential for HCV infection of transformed hepatocytes in vitro.Despite the evidence provided by numerous in vitro experiments, definitive proof of the role of CD81 in HCV infection in vivo is missing. It is well known that cell lines are not necessarily representative for the tissue or organ of origin. Moreover, other molecules, such as scavenger receptor class B member I, 12,13 C-type lectins L-SIGN and DC-SIGN, 14-17 low-density lipoprotein receptor, 18,19 glycosaminoglycans, 20,21 and more recently, Claudin-1 22 and lipoprotein lipase, 23 have been identified as putative HCV receptors or coreceptors. In addition, all these experiments have been performed using HCVpp or cellculture-derived virus (HCVcc), which have different characteristics compared with natural HCV particles. 24 More recently, Molina et al. 25 reported that infection of primary hepatocyte cultures with serum-derived HCV could be inhibited by using anti-CD81 antibodies or via transduction of small interfering RNA that inhibited CD81 membrane expression.We therefore investigated whether HCV infection could be prevented in vivo by administering anti-CD81 mAbs to human liver-uPA-SCID mice. uPA-SCID mice are immunodeficient mice that suffer from a severe, transgene-induced liver disease. 26 These animals can be successful...

Hepatitis C virus (HCV) enters cells via a pH-and clathrin-dependent endocytic pathway. Scavenger receptor BI (SR-BIHepatitis C virus (HCV) is an enveloped positive-strand RNA virus and the sole member of the genus Hepacivirus, within the Flaviviridae. Approximately 170 million individuals are infected with HCV worldwide, and the majority are at risk of developing serious progressive liver disease. The principal reservoir for viral replication is believed to be hepatocytes within the liver, and until recently, minimal information was available on the mechanism(s) of HCV entry. However, the last 3 years have seen several advances that contribute to our ability to study HCV hepatotropism. First, the development of the retrovirus pseudoparticle system, in which cell entry is dependent upon the expression of HCV glycoproteins (HCVpp) (4, 20), and secondly, the ability of the JFH strain of HCV to release infectious particles in cell culture (HCVcc) (25,51,55).Early studies with a truncated soluble version(s) of HCV E2 (sE2) allowed the identification of a number of interacting cellular proteins, including the tetraspanin CD81 (16, 37), scavenger receptor class B type I (SR-BI) (43), and DC-specific ICAM-3-grabbing nonintegrin (DC-SIGN) and the related molecule DC-SIGN(R), or L-SIGN (15,18,27,40). The availability of HCVpp and infectious HCVcc has provided tools for validating these receptor candidates.CD81 is a nonglycosylated member of the tetraspanin family of proteins. Both HCVpp and HCVcc infectivities are inhibited by soluble forms of CD81 and by anti-CD81 monoclonal antibodies (MAbs), suggesting that CD81 is required for HCV infection (6,20,25). Definitive experiments showing that expression of CD81 in a CD81-negative human liver cell line, HepG2, confers infectivity support a critical role of CD81 in HCV cell entry (24,25,54,55).SR-BI is expressed within the liver, steroidogenic tissue, and macrophages and is considered to be the major receptor for high-density lipoprotein (HDL) (23). SR-BI mediates the traffic of cholesterol to and from lipoproteins by selective cholesterol uptake, cholesterol efflux, and receptor-mediated endocytosis (1,34,42,44). The SR-BI gene gives rise to at least two mRNA splice variants. The SR-BII isoform differs from SR-BI at the C terminus, which is reported to confer intracellular localization on 33,52).Experiments to validate the role of SR-BI in HCV infection have proven difficult, since all cell types studied to date express SR-BI, and small interfering RNA silencing has a modest effect on HCVpp infectivity (6,24,48). The native lipoprotein ligands have differential effects on HCV infectivity: HDL enhances infectivity, low-density (LDL) and very low-density lipoproteins (VLDL) have no effect (5, 48), and oxidized LDL abrogates infectivity (50), suggesting a complex interplay between SR-BI, lipoproteins, and HCV. Treatment of target cells

Liver and spleen SWE correlate with portal pressure and can both be used as a non-invasive method to investigate CSPH. Even though external validation is still missing, these algorithms to rule-out and rule-in CSPH using sequential SWE of liver and spleen might change the clinical practice.

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To identify immunological mechanisms that govern distinct clinical phases of a chronic hepatitis B virus (HBV) infection-immune tolerant (IT), immune active (IA), inactive carrier (IC), and hepatitis B e antigen (HBeAg)-negative (ENEG) hepatitis phases-we performed a systems biology study. Serum samples from untreated chronic HBV patients (n 5 71) were used for multiplex cytokine measurements, quantitative hepatitis B surface antigen (HBsAg), HBeAg levels, HBV genotype, and mutant analysis. Leukocytes were phenotyped using multicolor flow cytometry, and whole-blood transcriptome profiles were generated. The latter were compared with liver biopsy transcriptomes from IA (n 5 16) and IT (n 5 3) patients. HBV viral load as well as HBeAg and HBsAg levels (P < 0.001), but not leukocyte composition, differed significantly between distinct phases. Serum macrophage chemotactic protein 1, interleukin-12p40, interferon (IFN)-gammainducible protein 10, and macrophage inflammatory protein 1 beta levels were different between two or more clinical phases (P < 0.05). Comparison of blood transcriptomes identified 64 differentially expressed genes. The gene signature distinguishing IA from IT and IC patients was predominantly composed of highly up-regulated immunoglobulin-encoding genes. Modular repertoire analysis using gene sets clustered according to similar expression patterns corroborated the abundant expression of B-cell function-related genes in IA patients and pointed toward increased (ISG) transcript levels in IT patients, compared to subsequent phases. Natural killer cell activities were clustered in clinical phases with biochemical liver damage (IA and ENEG phases), whereas T-cell activities were higher in all phases, compared to IT patients. B-cell-related transcripts proved to be higher in biopsies from IA versus IT patients. Conclusion: HBV clinical phases are characterized by distinct blood gene signatures. Innate IFN and B-cell responses are highly active during the IT and IA phases, respectively. This suggests that the presumed immune tolerance in chronic HBV infections needs to be redefined. (HEPATOLOGY 2015;62:87-100) W orldwide, over 350 million people are chronically infected with the hepatitis B virus (HBV) and at increased risk of developing progressive liver fibrosis, liver failure, or hepatocellular carcinoma over the course of several decades.1,2 The natural history of a chronic HBV infection follows several