Establishment of infectious genotype 4 cell culture-derived hepatitis C virus.

To establish infectious genotype 4a (GT4a) cell culture-derived hepatitis C virus (HCVcc), we constructed full-length ED43 and 12 mutants possessing single or double mutations that increase ED43 replicon replication, and performed cell culture after RNA transfection. Sequential long-term culture of full-length ED43 RNA-transfected cells showed increased viral production in two ED43 mutants named ED43 QK/SI and TR/SI among the tested clones. These ED43 mutants possessed a common mutation, R1405G, in the NS3 helicase region and another mutation, D2413G or V2414A, in the NS5a-NS5b cleavage site. Furthermore, serial reinfection of naïve Huh7.5.1 cells accelerated peak HCV production at an earlier time point after every infection. After the fourth infection, we found a common mutation, R1405G, and six additional mutations in both ED43 QK/SI and TR/SI mutants. All seven mutations supported continuous viral production for more than 40 days in both ED43 QS-7M (QK/SI with seven mutations) and ED43 TS-7M (TR/SI with seven mutations). In addition, ED43 TS-7M did not require additional mutations for continuous virus culture up to 124 days. Both ED43 QS-7M and TS-7M were sensitive to the neutralizing E2 antibodies HCV1 and AR3A and the direct-acting antivirals, simeprevir, ledipasvir and sofosbuvir. In conclusion, we established an infectious ED43 strain containing adaptive mutations, which is important for the analysis of HCV genotype-specific pathogenesis, development of pan-genotypic agents and analysis of drug resistance.

Characterization of miR-122-independent propagation of HCV.

miR-122, a liver-specific microRNA, is one of the determinants for liver tropism of hepatitis C virus (HCV) infection. Although miR-122 is required for efficient propagation of HCV, we have previously shown that HCV replicates at a low rate in miR-122-deficient cells, suggesting that HCV-RNA is capable of propagating in an miR-122-independent manner. We herein investigated the roles of miR-122 in both the replication of HCV-RNA and the production of infectious particles by using miR-122-knockout Huh7 (Huh7-122KO) cells. A slight increase of intracellular HCV-RNA levels and infectious titers in the culture supernatants was observed in Huh7-122KO cells upon infection with HCV. Moreover, after serial passages of HCV in miR-122-knockout Huh7.5.1 cells, we obtained an adaptive mutant, HCV122KO, possessing G28A substitution in the 5'UTR of the HCV genotype 2a JFH1 genome, and this mutant may help to enhance replication complex formation, a possibility supported by polysome analysis. We also found the introduction of adaptive mutation around miR-122 binding site in the genotype 1b/2a chimeric virus, which originally had an adenine at the nucleotide position 29. HCV122KO exhibited efficient RNA replication in miR-122-knockout cells and non-hepatic cells without exogenous expression of miR-122. Competition assay revealed that the G28A mutant was dominant in the absence of miR-122, but its effects were equivalent to those of the wild type in the presence of miR-122, suggesting that the G28A mutation does not confer an advantage for propagation in miR-122-rich hepatocytes. These observations may explain the clinical finding that the positive rate of G28A mutation was higher in miR-122-deficient PBMCs than in the patient serum, which mainly included the hepatocyte-derived virus from HCV-genotype-2a patients. These results suggest that the emergence of HCV mutants that can propagate in non-hepatic cells in an miR-122-independent manner may participate in the induction of extrahepatic manifestations in chronic hepatitis C patients.

Ledipasvir and Sofosbuvir for Acute Hepatitis C Virus Monoinfection Associated with a High Risk of Acute Liver Failure.

A 72-year-old Japanese man was referred to our hospital with yellow discoloration of the sclera and liver dysfunction. He was diagnosed with acute hepatitis C virus (HCV) infection on the basis of HCV-RNA positivity and anti-HCV seroconversion. A transjugular liver biopsy confirmed submassive hepatic necrosis. Five days after admission, no flapping tremor was observed, and the prothrombin time-international normalized ratio (PT-INR) and total bilirubin level showed increases of 1.70 and 17.8 mg/dL, respectively. The Model for End-Stage Liver Disease score was determined to be 25, and the risk of acute liver failure (ALF) was estimated to be 48% according to the Japan Hepatic Encephalopathy Prediction Model. Considering that rapid HCV clearance and temporary suppression of the immune response would prevent ALF, we prescribed oral ledipasvir (LDV) 90 mg and sofosbuvir (SOF) 400 mg for 12 weeks and intravenously injected methylprednisolone 1 g for 3 days. His PT-INR promptly improved, although the total bilirubin level increased to 30.3 mg/dL. Plasma bilirubin absorption was performed three times, and the total bilirubin level gradually decreased. HCV-RNA was still detectable at six weeks after the start of LDV/SOF therapy and finally undetectable at eight weeks. There were no adverse events associated with LDV/SOF. The patient was discharged 73 days after admission. A sustained virological response was achieved at 12 and 24 weeks after treatment. The findings from this case suggest that LDV/SOF therapy can be a promising option for acute HCV monoinfection associated with a high risk of ALF.

Trans-encapsidation of hepatitis C virus subgenomic replicon RNA with viral structure proteins.

A trans-packaging system for hepatitis C virus (HCV) subgenomic replicon RNAs was developed. HCV subgenomic replicon was efficiently encapsidated by the HCV structural proteins that were stably expressed in trans under the control of a mammalian promoter. Infectious HCV-like particles (HCV-LPs), established a single-round infection, were produced and released into culture medium in titers of up to 10(3) focus forming units/ml. Expression of NS2 protein with structural proteins (core, E1, E2, and p7) was shown to be critical for the infectivity of HCV-LPs. Anti-CD81 treatment decreased the number of infected cells, suggesting that HCV-LPs infected cells in a CD81-dependent manner. The packaging cell line should be useful both for the production of single-round infectious HCV-LPs to elucidate the mechanisms of HCV assembly, particle formation and infection to host cells, and for the development of HCV replicon-based vaccines.

Dynamic behavior of hepatitis C virus quasispecies in a long-term culture of the three-dimensional radial-flow bioreactor system.

Hepatitis C virus (HCV) exists in infected individuals as quasispecies, usually consisting of a dominant viral isolate and a variable mixture of related, yet genetically distinct, variants. A prior HCV infection system was developed using human hepatocellular carcinoma cells cultured in the three-dimensional radial-flow bioreactor (RFB), in which the cells retain morphological appearance and their differentiated hepatocyte functions for an extended period of time. This report studies the selection and alteration of the viral quasispecies in the RFB system inoculated with pooled serum derived from HCV carriers. Monitoring the viral RNA and core protein in the culture supernatants, together with nucleotide sequencing of hypervariable region 1 of the HCV genome, demonstrated that (1) the virus production intermittently fluctuated in the cultures, (2) the viral genetic diversity was markedly reduced 3 days post-infection (p.i.), and (3) dominant species changed on days 19-33p.i., suggesting that the virus populations can be selected according to susceptibility to the viral infection and replication. A therapeutic effect of interferon-alpha also demonstrated the inhibition of HCV expression. Thus, this HCV infection model in the RFB system should be useful for investigating the dynamic behavior of HCV quasispecies in cultured cells and evaluating anti-HCV compounds.

Transcriptomic comparison of human hepatoma Huh-7 cell clones with different hepatitis C virus replication efficiencies.

Hepatitis C virus (HCV) infection represents a major public health problem throughout the world. The establishment of viral replicons has enhanced our understanding of the mechanism underlying HCV replication. However, the specific virus-host cell interactions involved in HCV RNA replication are not well understood. In the present study, we isolated several human hepatoma Huh-7-derived subclones with a range of HCV RNA replication efficiencies by end-point dilution. Of these, the clones HuhTe4 and HuhTe6 were observed to proliferate at the same rate; however, HuhTe6 supported a significantly greater degree of viral RNA replication. Using cDNA microarray analysis, a total of 36 genes (0.4%) demonstrated variable expression, with a >or=2-fold difference in expression noted between HuhTe4 and HuhTe6. Among genes that are implicated in a variety of functional categories, a subset of these differentially-expressed genes has a role in signal transduction and cell communication, including thioredoxin-interacting protein, Rab6B, sorting nexin 16 and UDP-galactose:ceramide glycosyltransferase. The genes identified in this study should be examined further to determine their roles in HCV RNA replication. The Huh-7 subclones identified in this study provide a tool for identifying novel host factors involved in viral replication.

Clinical Significance of Two Real-Time PCR Assays for Chronic Hepatitis C Patients Receiving Protease Inhibitor-Based Therapy.

The aim of this study was to determine the efficacy of two hepatitis C virus (HCV) real-time PCR assays, the COBAS AmpliPrep/COBAS TaqMan HCV test (CAP/CTM) and the Abbott RealTime HCV test (ART), for predicting the clinical outcomes of patients infected with HCV who received telaprevir (TVR)-based triple therapy or daclatasvir/asunaprevir (DCV/ASV) dual therapy. The rapid virological response rates in patients receiving TVR-based triple therapy were 92% (23/25) and 40% (10/25) for CAP/CTM and ART, respectively. The false omission rate (FOR) of ART was 93.3% (14/15), indicating that CAP/CTM could accurately predict clinical outcome in the early phase. In an independent examination of 20 patients receiving TVR-based triple therapy who developed viral breakthrough or relapse, the times to HCV disappearance by ART were longer than by CAP/CTM, whereas the times to HCV reappearance were similar. In an independent experiment of WHO standard HCV RNA serially diluted in serum containing TVR, the analytical sensitivities of CAP/CTM and ART were similar. However, cell cultures transfected with HCV and grown in medium containing TVR demonstrated that ART detected HCV RNA for a longer time than CAP/CTM. Similar results were found for 42 patients receiving DCV/ASV dual therapy. The FOR of ART was 73.3% (11/15) at week 8 after initiation of therapy, indicating that ART at week 8 could not accurately predict the clinical outcome. In conclusion, although CAP/CTM and ART detected HCV RNA with comparable analytical sensitivity, CAP/CTM might be preferable for predicting the clinical outcomes of patients receiving protease inhibitor-based therapy.

Antiviral activity of glycyrrhizin against hepatitis C virus in vitro.

Glycyrrhizin (GL) has been used in Japan to treat patients with chronic viral hepatitis, as an anti-inflammatory drug to reduce serum alanine aminotransferase levels. GL is also known to exhibit various biological activities, including anti-viral effects, but the anti-hepatitis C virus (HCV) effect of GL remains to be clarified. In this study, we demonstrated that GL treatment of HCV-infected Huh7 cells caused a reduction of infectious HCV production using cell culture-produced HCV (HCVcc). To determine the target step in the HCV lifecycle of GL, we used HCV pseudoparticles (HCVpp), replicon, and HCVcc systems. Significant suppressions of viral entry and replication steps were not observed. Interestingly, extracellular infectivity was decreased, and intracellular infectivity was increased. By immunofluorescence and electron microscopic analysis of GL treated cells, HCV core antigens and electron-dense particles had accumulated on endoplasmic reticulum attached to lipid droplet (LD), respectively, which is thought to act as platforms for HCV assembly. Furthermore, the amount of HCV core antigen in LD fraction increased. Taken together, these results suggest that GL inhibits release of infectious HCV particles. GL is known to have an inhibitory effect on phospholipase A2 (PLA2). We found that group 1B PLA2 (PLA2G1B) inhibitor also decreased HCV release, suggesting that suppression of virus release by GL treatment may be due to its inhibitory effect on PLA2G1B. Finally, we demonstrated that combination treatment with GL augmented IFN-induced reduction of virus in the HCVcc system. GL is identified as a novel anti-HCV agent that targets infectious virus particle release.

Identification of hepatitis C virus genotype 2a replicon variants with reduced susceptibility to ribavirin.

Ribavirin (RBV), a nucleoside analogue, is used in the treatment of hepatitis C virus (HCV) infection in combination with interferons. However, potential mechanisms of RBV resistance during HCV replication remain poorly understood. Serial passage of cells harboring HCV genotype 2a replicon in the presence of RBV resulted in the reduced susceptibility of the replicon to RBV. Transfection of fresh cells with RNA from RBV-resistant replicon cells demonstrated that the RBV resistance observed is largely replicon-derived. Four major amino acid substitutions: T1134S in NS3, P1969S in NS4B, V2405A in NS5A, and Y2471H in NS5B region, were identified. Site-directed mutagenesis of these mutations into the replicon indicated that Y2471H plays a role in the reduced susceptibility to RBV and leads to decrease in replication fitness. The results, in addition to analysis of sequence database, suggest that HCV variants with reduced susceptibility to RBV identified are preferential to genotype 2a.