Aim:The presence of resistance-associated variants (RAV) may attenuate the efficacy of direct-acting antivirals (DAA) in combination therapy for hepatitis C. The aim of this study was to characterize the NS3 and NS5A regions of hepatitis C virus (HCV) in naturally occurring RAV. Methods:The NS3 and NS5A regions of HCV were amplified by nested polymerase chain reaction and their nucleotide sequences were determined by direct sequencing in 493 genotype 1b patients naive to DAA-based therapies. The effect of baseline RAV on response to pegylated interferon and ribavirin therapy was analyzed in 65 patients after stratification by interleukin (IL)-28B genotype.Results: The incidence of RAV was 7.9% in NS3 (V36I/L, 1.2%; T54S, 2.8%; Q80K/R, 3.0%; A156S, 0.2%; and D168E/T, 2.4%) and 20.2% in NS5A (L31I/M, 2.2%; and Y93H, 19.0%). The incidence in interferon experienced and naive patients was similar. The incidence of Y93H in NS5A was significantly higher in the IL-28B TT genotype (rs8099917) than non-TT (27.1% vs 9.5%, P < 0.001). The virological response to pegylated interferon plus ribavirin therapy was not affected by the presence of RAV in IL-28B TT genotype.Conclusion: RAV, especially Y93H in the NS5A region, were highly prevalent in DAA naive patients with genotype 1b HCV in Japan and were linked to IL-28B TT genotype. Interferonbased therapy could be an alternative for patients with RAV because these variants did not attenuate the response to that therapy. The analysis of RAV may impact the selection of the optimal treatment strategy.

Recently, microdomains of organelle membranes rich in sphingomyelin and cholesterol (called "lipid rafts") have been considered to act as a scaffold for the hepatitis C virus (HCV) replication complex. Using the HCV cell culture system, we investigated the effect of myriocin, a sphingomyelin synthesis inhibitor, on HCV replication. We also investigated the combined effect of myriocin with interferon (IFN) and myriocin with simvastatin. Myriocin suppressed replication of both a genotype 1b subgenomic HCV replicon (Huh7/Rep-Feo) and genotype 2a infectious HCV (JFH-1 HCV) in a dose-dependent manner (for subgenomic HCV-1b, maximum of 79% at 1000 nmol/L; for genomic HCV-2a, maximum of 40% at 1000 nmol/L). Combination treatment with myriocin and IFN or myriocin and simvastatin attenuated HCV RNA replication synergistically in Huh7/Rep-Feo cells. Our data demonstrate that the sphingomyelin synthesis inhibitor strongly suppresses replication of both the subgenomic HCV-1b replicon and the JFH-1 strain of genotype 2a infectious HCV, indicating that lipid metabolism could be a novel target for HCV therapy.

Viral infections activate cellular expression of type I interferons (IFNs). These responses are partly triggered by RIG-I and mediated by Cardif, TBK1, IKKe and IRF-3. This study analysed the mechanisms of dsRNA-induced IFN responses in various cell lines that supported subgenomic hepatitis C virus (HCV) replication. Transfection of dsRNA into Huh7, HeLa and HEK293 cells induced an IFN expression response as shown by IRF-3 dimerization, whilst these responses were abolished in corresponding cell lines that expressed HCV replicons. Similarly, RIG-I-dependent activation of the IFN-stimulated response element (ISRE) was significantly suppressed by cells expressing the HCV replicon and restored in replicon-eliminated cells. Overexpression analyses of individual HCV non-structural proteins revealed that NS4B, as well as NS34A, significantly inhibited RIG-I-triggered ISRE activation. Taken together, HCV replication and protein expression substantially blocked the dsRNA-triggered, RIG-I-mediated IFN expression response and this blockade was partly mediated by HCV NS4B, as well as NS34A. These mechanisms may contribute to the clinical persistence of HCV infection and could constitute a novel antiviral therapeutic target. INTRODUCTIONType I interferon (IFN) plays a central role in eliminating virus, not only following clinical therapeutic application but also as a cellular immune response (Samuel, 2001;Taniguchi & Takaoka, 2002). Hepatitis C virus (HCV) infection is characterized by persistence and replication of the virus in the liver, despite an intact host immune system (Alter, 1997). Indeed, even after administration of the currently most potent IFN reagents, as many as half of the patients are refractory to the treatment and fail to eradicate the virus (Fried et al., 2002). These features have led to speculation that HCV escapes from or attenuates the host antiviral response . Cellular antiviral responses are primarily mediated by IFN and IFN-stimulated genes (ISGs), including 2,5-oligoadenylate synthetase, dsRNA-dependent protein kinase R (PKR) and MxA proteins, as well as by as yet uncharacterized genes Stark et al., 1998). A study of experimental chimpanzee HCV infection has shown that various cytokines and chemokines are induced in the liver during the course of acute HCV infection and its clearance, and that a considerable proportion of the genes is induced by type I IFN (Bigger et al., 2001).Control of expression of ISGs is mediated by binding of type I IFNs to their receptors. Following receptor binding, STAT1 and STAT2 are phosphorylated to form ISGF-3, which translocates to the nucleus and binds the IFN-stimulated response element (ISRE), located in the promoter/enhancer region of ISGs, and activates transcription of ISGs (Samuel, 3These authors contributed equally to this work. Plasmid pcDNA3.1 (Invitrogen) was used as an empty vector for mock transfection. pRL-CMV (Promega), which expressed the Renilla luciferase protein, was used for correction of transfection efficiency.Cell culture. HCV strain JFH1-...

Background and Aim:The association between liver fibrosis, fatty liver, and cardiovascular disease (CVD) risk is unknown. Hence, this study aimed to investigate the association of liver fibrosis and fatty liver with CVD risk independent of already known CVD risk comorbidities. Methods: This is a prospective study registered with the University Hospital Medical Information Network clinical trial registry (UMIN000036175). Liver fibrosis was assessed by serum fibrosis markers including FIB-4, nonalcoholic fatty liver disease fibrosis score (NFS), and Wisteria floribunda agglutinin-positive Mac-2 binding protein (WFA + -M2BP), whereas fatty liver was diagnosed by ultrasonography. CVD risk was evaluated using the Framingham risk score (FRS), and a high CVD risk was defined as an FRS ≥ 20%. Results: A total of 3512 subjects were enrolled, and high CVD risk (FRS ≥ 20%) was observed in 17.5%. Advanced fibrosis (FIB-4 ≥ 2.67, NFS ≥ 0.675, and WFA + -M2BP ≥ 1.0) and the presence of fatty liver were significantly associated with high CVD risk independent of diabetes mellitus, dyslipidemia, and hypertension. When subjects were stratified by liver fibrosis and fatty liver, subjects with advanced fibrosis and fatty liver have the highest odds for high CVD risk (odds ratio [OR]: 5.90-35.6), followed by subjects with advanced fibrosis and without fatty liver (OR: 2.53-9.62) using subjects without advanced fibrosis and fatty liver as a reference. Conclusions: Liver fibrosis and fatty liver were associated with CVD risk independent of already known CVD risk comorbidities. The assessment of liver fibrosis and fatty liver may be useful to identify high CVD risk subjects. collection. NT analyzed data. MK and NI clinically revised the manuscript and supervised the study. NI, MK, and NI obtained funding. All authors read and approved the final version of the manuscript.