Hepatitis C virus (HCV) infection causes chronic liver diseases and is a global public health problem. Detailed analyses of HCV have been hampered by the lack of viral culture systems. Subgenomic replicons of the JFH1 genotype 2a strain cloned from an individual with fulminant hepatitis replicate efficiently in cell culture. Here we show that the JFH1 genome replicates efficiently and supports secretion of viral particles after transfection into a human hepatoma cell line (Huh7). Particles have a density of about 1.15-1.17 g/ml and a spherical morphology with an average diameter of about 55 nm. Secreted virus is infectious for Huh7 cells and infectivity can be neutralized by CD81-specific antibodies and by immunoglobulins from chronically infected individuals. The cell culture-generated HCV is infectious for chimpanzee. This system provides a powerful tool for studying the viral life cycle and developing antiviral strategies.

No abstract

Although hepatitis C virus (HCV) is a major cause of non-A non-B hepatitis, its pathogenic role in fulminant hepatitis remains controversial. A 32-year-old man contracted hepatitis. Serum ALT concentration was reached to 6,970 IU/L, the lowest prothrombin time value was 16% and jaundice and stage II encephalopathy were developed. HCV RNA was detected in this patient by reverse transcription polymerase chain reaction in sera at the acute phase, and it was undetectable during the remission phase when anti-HCV was found. The entire genome of infected HCV was recovered, cloned, and sequenced from this patient, and compared with the clones of six other chronic hepatitis patients. Phylogenetic analysis revealed a clustering around genotype 2a and a deviation from the other 2a chronic hepatitis strains. Calculating the genetic distance in each subgenomic region revealed that the 5'untranslated region (5'UTR), core, nonstructural (NS) 3, and NS5A were severely deviated. Of 20 clones of the hypervariable region (HVR), 17 showed an identical sequence with the others showing a difference of only one amino acid. HCV was isolated from a fulminant hepatitis patient and its entire genome was recovered; a clustering around genotype 2a was observed, but the sequence deviated especially in 5'UTR, core, NS3, and NS5A; and monoclonality of the HVR sequence was found not only in the fulminant hepatitis patient but in a certain percentage of chronic hepatitis patients.

No abstract

The hepatitis C virus (HCV) genotype 2a subgenomic replicon can replicate in two human non-hepatocytederived cell lines, HeLa and 293, with in vitro-transcribed replicon RNA. Sequencing analysis revealed that mutations in HCV-derived regions were not essential for replication in these cells, as some clones displayed no mutations.Hepatitis C virus (HCV) was first identified as a causative agent of posttransfusion hepatitis in 1989 (4). The virus is considered hepatotropic and is known to cause liver diseases such as acute or chronic hepatitis, cirrhosis, and hepatocellular carcinoma (11,16,17,21). HCV has been detected not only in liver, but also in peripheral blood mononuclear cells and dendritic cells (7,12,19). However, other tissue tropisms and their regulatory factors have yet to be fully elucidated. This lack of progress in the investigations regarding the virus is primarily attributable to a lack of efficient cell culture systems and small animal models of infection. As an important step toward overcoming this disadvantage, a subgenomic HCV RNA replicon system has been developed (18). This replicon system contains the HCV internal ribosome entry site (IRES), which directs expression of the G418 selectable marker, neo r , and encephalomyocarditis virus (EMCV) IRES directs the expression of HCV nonstructural (NS) proteins NS3 to NS5B. This enabled assessment of HCV replication in cultured cells. Functional replicons have previously been reported only for genotype 1, and efficient replications of these replicons have been accomplished only in limited human hepatocyte-derived cell lines (2, 3, 9). Attempts to evaluate replication of the HCV replicon in non-hepatocyte-derived cell lines have been made previously (1,23). Some of these attempts seemed to have succeeded, but efficient replicon replication in nonhepatic cells has not been achieved by synthetic RNA transfection, and other experimental procedures have been required (1, 23). We developed a new HCV replicon system using an HCV genotype 2a clone from a patient with fulminant hepatitis (15). This replicon system provided higher colony formation efficiency and robust replication, not only in Huh7 cells but also in HepG2 and IMY-N9

Although combination therapy with interferon and ribavirin has improved the treatment for chronic hepatitis C virus (HCV) infection, the detailed anti-HCV effect of ribavirin in clinical concentrations remains uncertain. To detect the anti-HCV effect of ribavirin in lower concentrations, a sensitive and accurate assay system was developed using the reporter replicon system with an HCV genotype 2a subgenomic replicon (clone JFH-1) that exhibits robust replication in various cell lines. This reporter replicon was generated by introducing the luciferase reporter gene (instead of the neomycin resistance gene) into the subgenomic JFH-1 replicon. To assess the replication of this reporter replicon, luciferase activity was measured serially up to day 3 after transient transfection of Huh7 cells. The luciferase activity increased exponentially over the time course of the experiment. After adjustment for transfection efficiency and transfected cell viability, the impacts of interferon and ribavirin were determined. The administration of interferon and ribavirin resulted in dosedependent suppression of replicon RNA replications. The 50% inhibitory concentration of interferon and ribavirin was 1.80 IU/ml and 3.70 g/ml, respectively. In clinical concentrations, replications were reduced to 0.09% and 53.74% by interferon (100 IU/ml) and ribavirin (3 g/ml), respectively. Combination use of ribavirin and interferon enhanced the anti-HCV effect of interferon by 1.46-to 1.62-fold. In conclusion, we developed an accurate and sensitive replicon system, and the antivirus effect of interferon and ribavirin was easily detected within their clinical concentrations by this replicon system. This system will provide a powerful tool for screening new antiviral compounds against HCV.Hepatitis C virus (HCV) is a major public health problem, infecting an estimated 170 million people worldwide. HCV causes chronic liver diseases, including cirrhosis and hepatocellular carcinoma, because most patients fail to clear the virus and the persistent infection that follows (1,11,20). Current therapy for HCV-related chronic hepatitis is based on the use of interferon (IFN). However, virus clearance rates are limited to approximately 10 to 20% of cases treated with IFN only (9,23,26). Combination therapy with IFN and ribavirin improves the HCV clearance rate, although the molecular mechanism responsible for this improvement is not yet fully understood (23,25,26). However, some direct antiviral mechanisms of ribavirin have been proposed (19). One possible mechanism is the direct inhibition of HCV RNA-dependent RNA polymerase, and another possibility is the RNA mutagen effect that drives a rapidly mutating RNA virus over the threshold to "error catastrophe." The detection of these direct anti-HCV effects has been hampered by the lack of an appropriate sensitive system for evaluating HCV replication.Although HCV belongs to the Flaviviridae family and has a genome structure similar to those of the other flaviviruses (3, 27), efficient cell culture systems a...

A hepatitis C virus genotype 2a subgenomic replicon, JFH-1 replicon, was previously established using the consensus sequence of clone JFH-1 from a patient with fulminant hepatitis and, in a previous report, was indicated to replicate efficiently in Huh7. Here the replication of JFH-1 replicon was tested in HepG2, a human hepatocyte-derived cell line, and in IMY-N9, a cell line developed by fusing human hepatocytes and HepG2 cells. Following transfection with in vitro transcribed replicon RNA and selection by cultivation with G418, colonies formed in both cell lines although at efficiencies substantially lower than those of Huh7. The H2476L mutation identified in the Huh7 replicon in our previous study increased the colony formation efficiencies of the JFH-1 replicon in HepG2 and IMY-N9 cells. Higher amounts of replicon RNA were detected in IMY-N9 clones than in HepG2 clones by real time detection reverse transcription-PCR, and replicon RNA replication and viral protein expression were confirmed by Northern and Western blotting in isolated clones. Sequencing of replicon RNAs revealed that mutations found in hepatitis C virus-derived regions were not identical and that two of nine HepG2 clones and three of nine IMY-N9 clones had no or one synonymous mutation. This system with the JFH-1 replicon and three cell lines is useful not only for estimating the cellular factors affecting viral activity but also for clarifying the common gene response of the host. Hepatitis C virus (HCV),1 one of the plus-strand RNA viruses, is a principal agent in post-transfusion and sporadic acute hepatitis (1, 2). Infection with HCV leads to chronic liver diseases, including cirrhosis and hepatocellular carcinoma, because most patients fail to clear the virus and the persistent infection that follows (3-5). Although HCV belongs to the Flaviviridae family and has a genome structure similar to the other flaviviruses including yellow fever, dengue, and West Nile virus, an efficient cell culture system or small animal infection models have not yet been established (1, 6 -8). The lack of a useful system for evaluating the viral replication not only hampers the understanding of the life cycle of this virus but also prevents the development of adequate treatment for HCV infection. In an important development, a subgenomic HCV RNA replicon system containing HCV internal ribosomal entry site (IRES) driving a neomycin resistance (neo r ) gene and encephalomyocarditis virus (EMCV) IRES driving HCV nonstructural (NS) proteins NS3-NS5B has been developed (9) and has enabled the assessment of HCV replication in cultured cells. Although this represents a powerful tool in the study of HCV replication mechanisms and the search for potential antiviral agents, it was constructed with a limited HCV genotype, genotype 1, and replication has been limited to the human hepatocyte-derived cell line Huh7 (7, 10, 11).Recently we used a HCV genotype 2a clone from a patient with fulminant hepatitis to develop a new HCV replicon system, JFH-1 (12). The JFH-1 replico...

These results suggest that simultaneous stimulation of EP2 and EP4 is necessary and sufficient to elicit the effect of PGE(2)on rat primary chondrocyte differentiation.