Performance evaluation of in vitro diagnostic kits for hepatitis B virus infection using the regional reference panel of Japan.

BACKGROUND: Hepatitis B virus (HBV) infection is a global public health concern. Precise and sensitive detection of viral markers, including HBV DNA and HBs antigen (Ag), is essential to determine HBV infection. METHODS: The sensitivities and specificities of 5 HBV DNA and 14 HBsAg kits were evaluated using World Health Organization International Standards (WHO IS) and the Regional Reference Panel (RRP) consisting of 64 HBsAg-negative and 80 HBsAg-positive specimens. RESULTS: All 5 HBV DNA kits detected HBV DNA in the WHO IS at a concentration of 10 IU/mL. The sensitivity and specificity to the RRP were 98.8-100% and 96.9-100%, respectively. HBV DNA titers were well correlated among the 5 kits regardless of HBV genotype. However, discordance of the HBV DNA titer was found in 5 specimens measured by CAP/CTM HBV v2.0. Among 12 automated HBsAg kits, the minimum detectable concentrations in the WHO IS varied from 0.01 to 0.1 IU/mL. Two lateral flow assays were positive for WHO IS concentrations greater than or equal to 1.0 and 0.1 IU/mL, respectively. When analyzed by the RRP, 12 automated kits exhibited a sensitivity of 98.8-100%, and 2 lateral flow assays showed sensitivities of 93.8% and 100%. The specificities of HBsAg kits were 100%. In the quantification of HBsAg, some kits showed a poor correlation of measurements with each other and showed up to a 1.7-fold difference in the regression coefficient of HBsAg titers. There were variations in the correlations of measurements among HBsAg kits when analyzed by genotype. CONCLUSIONS: Five HBV DNA kits showed sufficient sensitivity and specificity to determine HBV infection. HBV DNA titers were compatible with each other irrespective of HBV genotypes. HBsAg kits had enough sensitivity and specificity to screen for HBV infection. One of the lateral flow assays had a nearly equivalent sensitivity to that of the automated HBsAg kit. HBsAg titers quantified by the evaluated kits were not compatible across the kits. Genotype-dependent amino acid variations might affect the quantification of HBsAg titers.

N-Terminal PreS1 Sequence Regulates Efficient Infection of Cell-Culture-Generated Hepatitis B Virus.

BACKGROUND AND AIMS: An efficient cell-culture system for hepatitis B virus (HBV) is indispensable for research on viral characteristics and antiviral reagents. Currently, for the HBV infection assay in cell culture, viruses derived from HBV genome-integrated cell lines of HepG2.2.15 or HepAD-38 are commonly used. However, these viruses are not suitable for the evaluation of polymorphism-dependent viral characteristics or resistant mutations against antiviral reagents. HBV obtained by the transient transfection of the ordinary HBV molecular clone has limited infection efficiencies in cell culture. APPROACH AND RESULTS: We found that an 11-amino-acid deletion (d11) in the preS1 region enhances the infectivity of cell-culture-generated HBV (HBVcc) to sodium taurocholate cotransporting polypeptide-transduced HepG2 (HepG2/NTCP) cells. Infection of HBVcc derived from a d11-introduced genotype C strain (GTC-d11) was ~10-fold more efficient than infection of wild-type GTC (GTC-wt), and the number of infected cells was comparable between GTC-d11- and HepG2.2.15-derived viruses when inoculated with the same genome equivalents. A time-dependent increase in pregenomic RNA and efficient synthesis of covalently closed circular DNA were detected after infection with the GTC-d11 virus. The involvement of d11 in the HBV large surface protein in the enhanced infectivity was confirmed by an HBV reporter virus and hepatitis D virus infection system. The binding step of the GTC-d11 virus onto the cell surface was responsible for this efficient infection. CONCLUSIONS: This system provides a powerful tool for studying the infection and propagation of HBV in cell culture and also for developing the antiviral strategy against HBV infection.

Emergence of Hepatitis C Virus Genotype 2c Infection Among Human Immunodeficiency Virus-Infected Men Who Have Sex With Men in Tokyo, Japan.

ABSTRACT: We report on hepatitis C virus genotype 2c infection in 12 human immunodeficiency virus-infected men who have sex with men in Tokyo, Japan. The uncommon strains from the 12 patients were genetically clustered; they suggested an emerging outbreak in this population at high risk of sexually transmitted infections.

Anti-viral effects of interferon-λ3 on hepatitis B virus infection in cell culture.

AIM: Interferon (IFN)-λ3 is known to have antiviral effects against various pathogens. Recently, it has been reported that the production of IFN-λ3 in colon cells after the administration of nucleotide analogs is expected to reduce hepatitis B surface antigen in chronic hepatitis B patients. Here, we aimed to prove the antiviral effects of IFN-λ3 on hepatitis B virus (HBV) by using an in vitro HBV production and infection system. METHODS: We used HepG2.2.15-derived HBV as an inoculum and the replication-competent molecular clone of HBV as a replication model. RESULTS: By administering IFN-λ3 to HepG2 cells transfected with the HBV molecular clone, the production of hepatitis B surface antigen and hepatitis B core-related antigen was reduced dose-dependently. IFN-λ3 treatment also reduced the number of HBV-positive cells and the synthesis of covalently closed circular DNA after infection of HepG2.2.15-derived HBV to sodium taurocholate cotransporting polypeptide-transduced HepG2 cells. The inhibitory effect on HBV infection by IFN-λ3 was confirmed by using a recombinant a HBV reporter virus system. To elucidate the underlying mechanisms of the anti-HBV effect of IFN-λ3, we assessed the transcription of HBV RNA and the production of core-associated HBV DNA in HBV molecular clone-transfected HepG2 cells, and found that both parameters were reduced by IFN-λ3. CONCLUSIONS: We observed that the administration of IFN-λ3 inhibits HBV infection and the production of HBV proteins at the HBV RNA transcription level. This finding provides novel insight into the treatment of chronic hepatitis B patients with the administration or induction of IFN-λ3.

Impact of resistance-associated variant dominancy on treatment in patients with HCV genotype 1b receiving daclatasvir/asunaprevir.

Sustained virological responses (SVR) by daclatasvir (DCV) and asunaprevir (ASV) therapy for genotype 1b hepatitis C virus (HCV) infected patients has been significantly affected by pre-existence of Y93 H resistance-associated variants (RAVs) in the non-structural protein 5A (NS5A) region. The aim of this study was to elucidate the dominancy of naturally occurring RAVs in viral quasispecies on treatment outcomes in patients with HCV. In total, 138 patients were prospectively selected from 152 patients treated with DCV and ASV, where evaluation of treatment outcomes at 12 weeks post-treatment was possible. Pre-treatment RAVs in the non-structural protein 3 and NS5A regions were detected by polymerase chain reaction (PCR)-Invader assays, and the ratio of Y93H RAVs in viral quasispecies was measured by quantitative PCR-Invader assay. Among 25 patients detected the Y93H RAV, the Y93H ratio was 1-25% in 5 patients, 26-75% in 7 patients, and ≥76% in 13 patients. Overall, SVR at 12 weeks after the completion of treatment (SVR12) was 91% (125/138), and those with Y93H ratios of <1%, 1-25%, 26-75%, and ≥76% were 99%, 100%, 71%, and 23%, respectively. Thus, the SVR12 decreased as the HCV Y93H ratio increased (P < 0.0001). The dominancy of pre-treatment RAVs of DCV and ASV affected its treatment outcomes, suggesting that evaluating the dominancy of HCV RAVs could be required for every other direct-acting antiviral agent treatments. J. Med. Virol. 89:99-105, 2017. © 2016 Wiley Periodicals, Inc.

Antimicrobial peptide LL-37 attenuates infection of hepatitis C virus.

AIM: Although recent studies indicate that supplementation with vitamin D (VD) potentiates a sustained viral response by interferon-based therapy to chronic hepatitis C, detailed mechanisms are not fully defined. The production of cathelicidin, an antimicrobial peptide, has been demonstrated to be part of the VD-dependent antimicrobial pathway in innate immunity. Cathelicidin is known to directly kill or inhibit the growth of microbial pathogens including mycobacteria and viruses. METHODS: We used a hepatitis C virus (HCV) cell culture system to clarify the anti-HCV effects of the human cathelicidin, LL-37. HuH-7 cells were administrated with LL-37 and infected with cell culture-generated HCV (HCVcc). HCV propagation was estimated by measuring the level of HCV core antigen (Ag). RESULTS: Treatment with LL-37 resulted in decreased intra- and extracellular levels of HCV core Ag, suggesting inhibition of HCV propagation. To assess the effects of LL-37 on HCV replication, JFH-1 subgenomic replicon RNA-transfected cells were treated with LL-37. However, inhibition of HCV replication was not detected by this assay. To clarify the effects on HCV infection, we treated HCVcc with LL-37 and removed the antimicrobial peptide prior to use of the virus in infection. This exposure of HCVcc to LL-37 diminished the infectivity titers in a dose-dependent fashion. Iodixanol density gradient analysis revealed that the peak fraction of infectivity titer was eliminated by LL-37 treatment. CONCLUSION: The VD-associated antimicrobial peptide LL-37 attenuated the infectivity of HCV. This anti-HCV effect of LL-37 may explain the contribution of VD to the improved efficacy of interferon-based therapy.

Antiviral effect of peptoids on hepatitis B virus infection in cell culture.

Although antimicrobial peptides have been shown to inactivate viruses through disruption of their viral envelopes, clinical use of such peptides has been hampered by a number of factors, especially their enzymatically unstable structures. To overcome the shortcomings of antimicrobial peptides, peptoids (sequence-specific N-substituted glycine oligomers) mimicking antimicrobial peptides have been developed. We aimed to demonstrate the antiviral effects of antimicrobial peptoids against hepatitis B virus (HBV) in cell culture. The anti-HBV activity of antimicrobial peptoids was screened and evaluated in an infection system involving the HBV reporter virus and HepG2.2.15-derived HBV. By screening with the HBV reporter virus infection system, three (TM1, TM4, and TM19) of 12 peptoids were identified as reducing the infectivity of HBV, though they did not alter the production levels of HBs antigen in cell culture. These peptoids were not cytotoxic at the evaluated concentrations. Among these peptoids, TM19 was confirmed to reduce HBV infection most potently in a HepG2.2.15-derived HBV infection system that closely demonstrates authentic HBV infection. In cell culture, the most effective administration of TM19 was virus treatment at the infection step, but the reduction in HBV infectivity by pre-treatment or post-treatment of cells with TM19 was minimal. The disrupting effect of TM19 targeting infectious viral particles was clarified in iodixanol density gradient analysis. In conclusion, the peptoid TM19 was identified as a potent inhibitor of HBV. This peptoid prevents HBV infection by disrupting viral particles and is a candidate for a new class of anti-HBV reagents.

High levels of hepatitis B virus after the onset of disease lead to chronic infection in patients with acute hepatitis B.

BACKGROUND: Some patients with acute hepatitis B virus (HBV) infection develop chronic infection. However, the method for identifying these patients has not been established. METHODS: We followed 215 Japanese patients with acute HBV infection until the clearance of hepatitis B surface antigen (HBsAg) or the development of chronic infection. Levels of HBsAg and HBV DNA were serially monitored from the onset. RESULTS: Of the 215 patients, 113 (52.5%) possessed HBV genotype A, 26 (12.0%) genotype B, and 73 (34.0%) genotype C. Twenty-one of the 215 (9.8%) developed chronic infection, with the persistence of HBsAg for >6 months. The rate of chronicity of genotype A, B, and C was 12.4%, 3.8%, and 8.2%. Of the 21 patients, only 6 (2.8%) patients, including 5 with genotype A, failed to clear HBsAg within 12 months. Levels of HBsAg at 12 weeks and HBV DNA at 4 weeks were useful for distinguishing the patients who became chronic from those who did not (P < .001 and P < .001, respectively). Likewise, the levels of HBsAg at 12 weeks and HBV DNA at 8 weeks were useful for discriminating between the patients who lost HBsAg within 12 months and those who did not (P < .01 and P < .05, respectively). CONCLUSIONS: In acute HBV infection, clearance of HBV may happen between 6 and 12 months from the onset. Only those who fail to clear HBV within 12 months from the onset may develop chronic infection.

Neutralization of hepatitis B virus with vaccine-escape mutations by hepatitis B vaccine with large-HBs antigen.

Although the current hepatitis B (HB) vaccine comprising small-HBs antigen (Ag) is potent and safe, attenuated prophylaxis against hepatitis B virus (HBV) with vaccine-escape mutations (VEMs) has been reported. We investigate an HB vaccine consisting of large-HBsAg that overcomes the shortcomings of the current HB vaccine. Yeast-derived large-HBsAg is immunized into rhesus macaques, and the neutralizing activities of the induced antibodies are compared with those of the current HB vaccine. Although the antibodies induced by the current HB vaccine cannot prevent HBV infection with VEMs, the large-HBsAg vaccine-induced antibodies neutralize those infections. The HBV genotypes that exhibited attenuated neutralization via these vaccines are different. Here, we show that the HB vaccine consisting of large-HBsAg is useful to compensate for the shortcomings of the current HB vaccine. The combined use of these HB vaccines may induce antibodies that can neutralize HBV strains with VEMs or multiple HBV genotypes.

Trends of hepatitis B virus genotype distribution in chronic hepatitis B patients in Japan.

BACKGROUND: Hepatitis B virus (HBV) is one of the most prevalent chronic viral infections that causes chronic hepatitis B (CHB). In Japan, genotypes B and C account for most of acute and chronic cases of hepatitis. However, previous studies showed that the prevalence of genotype A in CHB gradually increased every 5 years. Therefore, we have conducted a nationwide survey to comprehensively investigate the trends of HBV genotype distribution in CHB patients in Japan. METHODS: 4421 CHB patients were recruited between 2015 and 2016. Clinical characteristics and distribution of CHB patients among different age groups and genotypes in 2015-2016 was compared with those in 2000-2001, 2005-2006, and 2010-2011. RESULTS: The percentages of genotype A, B, C, and D were 4.0, 16.2, 79.1, and 0.7%, respectively. While the overall percentage of CHB patients with genotype A did not change in the past 5 years, CHB with genotype A increased in young adults. On the other hand, the peak distribution of CHB with genotypes B and C, two genotypes with the largest patient population, has shifted to an older age group. CONCLUSIONS: In Japan, the peak distribution for CHB with genotypes B and C advanced to an older age group while CHB with genotype A expanded in a younger age group. Given the universal HBV vaccination launch in Japan in 2016, these pre-vaccination survey data provide important baseline information for comparative studies of the impact of universal vaccination on HBV genotypes.

Performance Evaluation of In Vitro Screening and Diagnostic Kits for Hepatitis C Virus Infection.

AIM: A reliable kit with high sensitivity and specificity is indispensable for diagnosing hepatitis C virus (HCV) infection. Detection kits for anti-HCV antibodies (anti-HCV) are used for screening, and quantification kits for HCV RNA and HCV antigen (Ag) are used for the definite diagnosis of HCV infection or the evaluation of the pathological condition of and therapeutic effects in patients with chronic hepatitis C. Several kits are currently available for these purposes and are provided for clinical use in Japan. In this study, we aimed to evaluate the performance of these kits. METHODS: We used International Standards for HCV RNA and HCV Ag and a regional reference panel to evaluate the performance of thirteen anti-HCV, five HCV RNA, and two HCV Ag kits. RESULTS: All specimens in the regional reference panel were diagnosed correctly by all anti-HCV kits, although the distributions of the quantified values varied, and the ratios of titer classification were not identical across kits. All HCV RNA kits quantified the International Standard with minimum deviation and diagnosed the specimens of the reference panel correctly. The quantified values of the International Standard by two HCV Ag kits were inconsistent. HCV Ag titers of some specimens were underestimated owing to the amino acid polymorphisms in comparison with HCV RNA titers. CONCLUSIONS: The evaluation with International Standards and the regional reference panel was useful for assessing the quality of screening and diagnostic kits for HCV infection, and such quality control is essential for the clinical usage of these kits.

Amino Acid Polymorphism in Hepatitis B Virus Associated With Functional Cure.

BACKGROUND & AIMS: To provide an adequate treatment strategy for chronic hepatitis B, it is essential to know which patients are expected to have a good prognosis and which patients do not require therapeutic intervention. Previously, we identified the substitution of isoleucine to leucine at amino acid 97 (I97L) in the hepatitis B core region as a key predictor among patients with stable hepatitis. In this study, we attempted to identify the point at which I97L affects the hepatitis B virus (HBV) life cycle and to elucidate the underlying mechanisms governing the stabilization of hepatitis. METHODS: To confirm the clinical features of I97L, we used a cohort of hepatitis B e antigen-negative patients with chronic hepatitis B infected with HBV-I97 wild-type (wt) or HBV-I97L. The effects of I97L on viral characteristics were evaluated by in vitro HBV production and infection systems with the HBV reporter virus and cell culture-generated HBV. RESULTS: The ratios of reduction in hepatitis B surface antigen and HBV DNA were higher in patients with HBV-I97L than in those with HBV-I97wt. HBV-I97L exhibited lower infectivity than HBV-I97wt in both infection systems with reporter HBV and cell culture-generated HBV. HBV-I97L virions exhibiting low infectivity primarily contained a single-stranded HBV genome. The lower efficiency of cccDNA synthesis was demonstrated after infection of HBV-I97L or transfection of the molecular clone of HBV-I97L. CONCLUSIONS: The I97L substitution reduces the level of cccDNA through the generation of immature virions with single-stranded genomes. This I97L-associated low efficiency of cccDNA synthesis may be involved in the stabilization of hepatitis.

Efficacy and safety of glecaprevir/pibrentasvir in patients with hepatitis C virus infection aged ≥75 years.

AIM: Opportunities to treat older patients with hepatitis C virus infection have increased. We investigated the efficacy and safety of glecaprevir/pibrentasvir in patients with HCV infection aged ≥75 years. METHODS: We retrospectively evaluated 131 patients with hepatitis C virus infection treated with glecaprevir/pibrentasvir at nine institutions in Japan. The patients were divided into two groups according to their age: the elderly group (n = 43, aged ≥75 years) and younger group (n = 88, aged <75 years). We compared the clinical characteristics, virologic response and adverse events between the two groups. The predictive factors for adverse events were also assessed. RESULTS: The presence of cirrhosis (27.9%), a history of hepatocellular carcinoma (23.3%) and comorbidities (88.4%) were more frequently observed in the elderly group than in the younger group. Six (14.0%) patients in the elderly group and 19 (21.6%) in the younger group dropped out before the sustained virologic response 12 assessment. In the intention-to-treat population, 86.0% in the elderly group and 78.4% in the younger group achieved sustained virologic response 12 (P = 0.30). In the modified intention-to-treat population, all patients achieved sustained virologic response 12. A total of 27.5% of patients experienced adverse events. The most frequently observed adverse events was pruritus, and was significantly associated with female sex, the presence of hemodialysis and serum albumin at baseline <4.0 g/dL. CONCLUSION: Glecaprevir/pibrentasvir therapy was effective and well tolerated, even in elderly patients with hepatitis C virus infection aged ≥75 years. Geriatr Gerontol Int 2020; ••: ••-••.

Short-term prolongation of pegylated interferon and ribavirin therapy for genotype 1b chronic hepatitis C patients with early viral response.

AIM: We tailored extended treatments using pegylated interferon (PEG IFN) and ribavirin (RBV) to viral responses after initiation of therapy and investigated the efficacy and safety of its therapy for chronic hepatitis C (CHC) patients. METHODS: Eighty-two genotype 1b CHC patients were enrolled in the present study. All patients received PEG IFN-alpha-2b and weight-based RBV therapy. We defined a viral response in which serum HCV-RNA is undetectable at week 4 as rapid viral response (RVR), detectable at week 4 and undetectable by week 12 as early viral response (EVR), and detectable at week 12 and undetectable by week 24 as late viral response (LVR). We set the treatment duration depending on viral response; 48 weeks for RVR patients and 72 weeks for LVR. Furthermore, EVR patients received a short-term extension of treatment duration to 52-60 weeks. We prospectively investigated sustained viral response (SVR) rates of these groups. RESULTS: Overall SVR rate for the total patient group was 57.3%. SVR rates of the RVR, EVR and LVR patients were 100%, 80.5% and 40.0%, respectively. Nine patients could not complete this treatment protocol. Baseline platelet count and mutation in the interferon sensitivity-determining region of NS5A were significant independent predictors of SVR, and amino acid substitution of the core region was a significant independent predictor of non-viral response by multivariate logistic regression analyses. CONCLUSION: The results indicate that short-treatment extension of PEG IFN plus RBV treatment protocols in EVR patients can improve overall SVR rates.

Hepatitis B Virus Genotype-Dependent Vulnerability of Infected Cells to Immune Reaction in the Early Phase of Infection.

Infection with hepatitis B virus (HBV) genotype (GT)-A has been reported to predispose patients to chronic infection. To explore the immune responses in infection with different HBV genotypes and clarify the genotype-dependent pathogenicity, a system mimicking the immune reaction during the early phase of HBV infection is indispensable. To this end, we established a coculture system with the replication-competent HBV molecular clone-transfected HepG2 cells and immortalized human natural killer (NK) cells, NK-92MI. Using this system, we evaluated HBV genotype dependency in NK functions and cell death of HBV positive HepG2 cells induced by NK cells or administration of tumor necrosis factor (TNF) by use of flow cytometry. After coculture with NK cells, we found that GT-A-positive HepG2 cells exhibited lower susceptibility to NK cell-induced cell death than GT-B- or GT-C-positive HepG2 cells. The NK responses of degranulation and cytokine production were not different among transfected HBV genotypes in cocultured cells. The expression levels of death receptors in HBV-transfected HepG2 cells were not different. In GT-A-positive cells, a similar low susceptibility was detected by the external administration of TNF, although relatively higher susceptibility was observed in GT-B- and GT-C-positive cells than in GT-A-positive cells. The activation of caspase signaling was revealed to be responsible for this genotype-dependent susceptibility. In conclusion, our results indicate that the HBV genotype does not influence the NK cell function itself but rather cell vulnerability through the TNF signal pathway. This observation may explain the high chronicity rate of HBV GT-A strains even in adult infections.

Evaluation of in vitro screening and diagnostic kits for hepatitis B virus infection.

BACKGROUND: For the diagnosis of hepatitis B virus (HBV) infection, the detection and quantification of hepatitis B surface antigen (HBsAg) and HBV DNA are used. Several kits are available for this purpose, and there is a growing need for the evaluation of these kits because their performance may be affected by HBV genotype- or strain-specific polymorphisms. OBJECTIVES AND STUDY DESIGN: In this study, we used International Standards and the established regional reference panel to evaluate the performance of two HBV DNA quantitative kits, five HBsAg qualitative kits, seven HBsAg quantitative kits and three rapid immune-chromatographic tests for HBsAg. RESULTS: The quantification values of two HBV DNA quantitative kits exhibited excellent correlation. In the evaluation of HBsAg qualitative and quantitative kits, the titers of several specimens in the HBV-positive panel were below the detection limits of a few kits, and the specimens were determined as HBV-negative. Notably, the quantitative kit results exhibited low correlation values. However, when these data were analyzed for each genotype, the correlations improved. These results suggest that the HBsAg quantification data are influenced by HBV genotypes. The novel rapid immune-chromatographic test exhibited the comparable level of sensitivity to the HBsAg quantitative kits. CONCLUSIONS: We evaluated the performance of kits for the detection of HBV infection. The HBV DNA quantification data correlated with an excellent agreement, whereas the HBsAg quantification data were affected by HBV genotype. Such evaluations will be useful for estimating the quality of currently available and new HBV assay kits, and for the quality control of these kits.

[Anesthetic management of patients with tracheal bronchus].

The incidence of a tracheal bronchus supplying the right upper lobe has been reported to be in the range of 0.1 to 3%. We encountered 3 cases with the anomaly among 200 cases of lung surgery conducted over a year at our hospital. In the first case, we had difficulty in intubating the patient with a double-lumen tube. In the other two cases, the tracheal bronchus was detected by fiberoptic bronchoscopy during the lung surgery. Patients with a tracheal bronchus may be difficult or impossible to ventilate, especially when requiring unilateral lung ventilation. Therefore, the airway must be assessed carefully by fiberoptic bronchoscopy prior to unilateral lung surgery, keeping in mind the possibility of a tracheal bronchus, in order to design a safe way to secure it safely during the surgery.

Evaluation of antiviral effects of novel NS5A inhibitors in hepatitis C virus cell culture system with full-genome infectious clones.

Nonstructural protein 5A (NS5A) inhibitors of hepatitis C virus (HCV) are known to have potent anti-viral effects; however, these inhibitors have limited activities on strains with resistant-associated substitutions or non-genotype 1 strains. To overcome these shortcomings, novel NS5A inhibitors have been developed and approved for clinical application. The aim of this study was to evaluate the anti-viral effect of novel NS5A inhibitors (derivatives of odalasvir) on HCV genotype 2 strains in a cell culture system. Chimeric JFH-1 viruses replaced with NS5A of genotypes 1 and 2 were utilized to assess the genotype-specific potencies of NS5A inhibitors. We also examined full-genome infectious clones of JFH-1, J6cc, and J8cc to confirm the effects of NS5A inhibitors on genotype 2 strains. All chimeric viruses were capable of replication at similar levels in cell culture. We examined the anti-viral effects of derivatives of the novel NS5A inhibitor and compared with the first-generation NS5A inhibitor, daclatasvir (DCV). These compounds inhibited replication of chimeric JFH-1 viruses with NS5A of genotypes 1 and 2 at low concentrations in comparison with DCV. The EC50 values of J6cc and J8cc to these compounds were more than 100-fold lower than that of DCV. By long-term culture in the presence of these compounds, we obtained highly resistant variants and identified the responsible substitutions. In conclusion, novel NS5A inhibitors displayed improved potency against HCV genotype 2 strains compared with DCV. However, the activity of these compounds was impaired by emerging resistance-associated substitutions.

Vitamin D derivatives inhibit hepatitis C virus production through the suppression of apolipoprotein.

Supplementation with vitamin D (VD) has been reported to improve the efficacy of interferon-based therapy for chronic hepatitis C. We found that 25-hydroxyvitamin D3 (25-(OH)D3), one of the metabolites of VD, has antiviral effects by inhibiting the infectious virus production of the hepatitis C virus (HCV). In this study, to clarify the underlying mechanisms of the anti-HCV effects, we searched VD derivatives that have anti-HCV effects and identified the common target molecule in the HCV life cycle by using an HCV cell culture system. After infection of Huh-7.5.1 cells with cell culture-generated HCV, VD derivatives were added to culture media, and the propagation of HCV was assessed by measuring the HCV core antigen levels in culture media and cell lysates. To determine the step in the HCV life cycle affected by these compounds, the single-cycle virus production assay was used with a CD81-negative cell line. Of the 14 structural derivatives of VD, an anti-HCV effect was detected in 9 compounds. Cell viability was not affected by these effective compounds. The 2 representative VD derivatives inhibited the infectious virus production in the single-cycle virus production assay. Treatment with these compounds and 25-(OH)D3 suppressed the expression of apolipoprotein A1 and C3, which are known to be involved in infectious virus production of HCV, and the knockdown of these apolipoproteins reduced infectious virus production. In conclusion, we identified several compounds with anti-HCV activity by screening VD derivatives. These compounds reduce the infectious virus production of HCV by suppressing the expression of apolipoproteins in host cells.

Interferon sensitivity-determining region of hepatitis C virus influences virus production and interferon signaling.

The number of amino acid substitutions in the interferon (IFN) sensitivity-determining region (ISDR) of hepatitis C virus (HCV) NS5A is a strong predictor for the outcome of IFN-based treatment. To assess the involvement of ISDR in the HCV life cycle and to clarify the molecular mechanisms influencing IFN susceptibility, we used recombinant JFH-1 viruses with NS5A of the genotype 1b Con1 strain (JFH1/5ACon1) and with NS5A ISDR containing 7 amino acid substitutions (JFH1/5ACon1/i-7mut), and compared the virus propagation and the induction of interferon-stimulated genes (ISGs). By transfecting RNAs of these strains into HuH-7-derived cells, we found that the efficiency of infectious virus production of JFH1/5ACon1/i-7mut was attenuated compared with JFH1/5ACon1. After transfecting full-length HCV RNA into HepaRG cells, the mRNA expression of ISGs was sufficiently induced by IFN treatment in JFH1/5ACon1/i-7mut-transfected but not in JFH1/5ACon1-transfected cells. These data suggested that the NS5A-mediated inhibition of ISG induction was deteriorated by amino acid substitutions in the ISDR. In conclusion, using recombinant JFH-1 viruses, we demonstrated that HCV NS5A is associated with infectious virus production and the inhibition of IFN signaling, and amino acid substitutions in the NS5A ISDR deteriorate these functions. These observations explain the strain-specific evasion of IFN signaling by HCV.