Nonstructural protein 3-4A: the Swiss army knife of hepatitis C virus.

Summary.  Hepatitis C virus (HCV) nonstructural protein 3-4A (NS3-4A) is a complex composed of NS3 and its cofactor NS4A. It harbours serine protease as well as NTPase/RNA helicase activities and is essential for viral polyprotein processing, RNA replication and virion formation. Specific inhibitors of the NS3-4A protease significantly improve sustained virological response rates in patients with chronic hepatitis C when combined with pegylated interferon-α and ribavirin. The NS3-4A protease can also target selected cellular proteins, thereby blocking innate immune pathways and modulating growth factor signalling. Hence, NS3-4A is not only an essential component of the viral replication complex and prime target for antiviral intervention but also a key player in the persistence and pathogenesis of HCV. This review provides a concise update on the biochemical and structural aspects of NS3-4A, its role in the pathogenesis of chronic hepatitis C and the clinical development of NS3-4A protease inhibitors.

Estimating the net contribution of interleukin-28B variation to spontaneous hepatitis C virus clearance.

The identification of associations between interleukin-28B (IL-28B) variants and the spontaneous clearance of hepatitis C virus (HCV) raises the issues of causality and the net contribution of host genetics to the trait. To estimate more precisely the net effect of IL-28B genetic variation on HCV clearance, we optimized genotyping and compared the host contributions in multiple- and single-source cohorts to control for viral and demographic effects. The analysis included individuals with chronic or spontaneously cleared HCV infections from a multiple-source cohort (n = 389) and a single-source cohort (n = 71). We performed detailed genotyping in the coding region of IL-28B and searched for copy number variations to identify the genetic variant or haplotype carrying the strongest association with viral clearance. This analysis was used to compare the effects of IL-28B variation in the two cohorts. Haplotypes characterized by carriage of the major alleles at IL-28B single-nucleotide polymorphisms (SNPs) were highly overrepresented in individuals with spontaneous clearance versus those with chronic HCV infections (66.1% versus 38.6%, P = 6 × 10(-9) ). The odds ratios for clearance were 2.1 [95% confidence interval (CI) = 1.6-3.0] and 3.9 (95% CI = 1.5-10.2) in the multiple- and single-source cohorts, respectively. Protective haplotypes were in perfect linkage (r(2) = 1.0) with a nonsynonymous coding variant (rs8103142). Copy number variants were not detected. We identified IL-28B haplotypes highly predictive of spontaneous HCV clearance. The high linkage disequilibrium between IL-28B SNPs indicates that association studies need to be complemented by functional experiments to identify single causal variants. The point estimate for the genetic effect was higher in the single-source cohort, which was used to effectively control for viral diversity, sex, and coinfections and, therefore, offered a precise estimate of the net host genetic contribution.

Hepatitis C Virus Nonstructural 5A Protein Inhibits Lipopolysaccharide-Mediated Apoptosis of Hepatocytes by Decreasing Expression of Toll-Like Receptor 4.

Background. Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) has been shown to modulate multiple cellular processes, including apoptosis. The aim of this study was to assess the effects of HCV NS5A on apoptosis induced by Toll-like receptor (TLR) 4 ligand, lipopolysaccharide (LPS). Methods. Apoptotic responses to TLR4 ligands and the expression of molecules involved in TLR signaling pathways in human hepatocytes were examined with or without expression of HCV NS5A. Results. HCV NS5A protected HepG2 hepatocytes against LPS-induced apoptosis, an effect linked to reduced TLR4 expression. A similar downregulation of TLR4 expression was observed in Huh-7-expressing genotype 1b and 2a. In agreement with these findings, NS5A inhibited the expression of numerous genes encoding for molecules involved in TLR4 signaling, such as CD14, MD-2, myeloid differentiation primary response gene 88, interferon regulatory factor 3, and nuclear factor-κB2. Consistent with a conferred prosurvival advantage, NS5A diminished the poly(adenosine diphosphate-ribose) polymerase cleavage and the activation of caspases 3, 7, 8, and 9 and increased the expression of anti-apoptotic molecules Bcl-2 and c-FLIP. Conclusions. HCV NS5A downregulates TLR4 signaling and LPS-induced apoptotic pathways in human hepatocytes, suggesting that disruption of TLR4-mediated apoptosis may play a role in the pathogenesis of HCV infection.

Elderly age is not a negative predictive factor for virological response to therapy with pegylated interferon-α and ribavirin in chronic hepatitis C virus patients.

BACKGROUND & AIMS: Age is frequently discussed as negative host factor to achieve a sustained virological response (SVR) to antiviral therapy of chronic hepatitis C. However, elderly patients often show advanced fibrosis/cirrhosis as known negative predictive factor. The aim of this study was to assess age as an independent predictive factor during antiviral therapy. METHODS: Overall, 516 hepatitis C patients were treated with pegylated interferon-α and ribavirin, thereof 66 patients ≥60 years. We analysed the impact of host factors (age, gender, fibrosis, haemoglobin, previous hepatitis C treatment) and viral factors (genotype, viral load) on SVR per therapy course by performing a generalized estimating equations (GEE) regression modelling, a matched pair analysis and a classification tree analysis. RESULTS: Overall, SVR per therapy course was 42.9 and 26.1%, respectively, in young and elderly patients with hepatitis C virus (HCV) genotypes 1/4/6. The corresponding figures for HCV genotypes 2/3 were 74.4 and 84%. In the GEE model, age had no significant influence on achieving SVR. In matched pair analysis, SVR was not different in young and elderly patients (54.2 and 55.9% respectively; P = 0.795 in binominal test). In classification tree analysis, age was not a relevant splitting variable. CONCLUSIONS: Age is not a significant predictive factor for achieving SVR, when relevant confounders are taken into account. As life expectancy in Western Europe at age 60 is more than 20 years, it is reasonable to treat chronic hepatitis C in selected elderly patients with relevant fibrosis or cirrhosis but without major concomitant diseases, as SVR improves survival and reduces carcinogenesis.

Extended treatment of treatment-naive patients with chronic hepatitis c virus (HCV) genotype 1-infection and slow response to pegylated interferon-alfa and ribavirin: a meta-analysis

Aims: Recently, several clinical trials analyzed if extended duration of treatment with pegylated interferon-alfa and ribavirin over 48 weeks can improve sustained virologic response (SVR) rates in HCV genotype 1-infected patients with slow virologic response. Because results of these clinical trials are conflicting, we performed a metaanalysis to determine the overall impact of extended treatment compared to standard treatment on virologic response rates in treatment-naive HCV genotype 1 slow responders. Methods: Literature search was performed independently by two observers using Pub Med, EMBASE, CENTRAL and abstracts presented in English at international liver and gastroenterology meetings. Randomized controlled clinical trials (RCTs; but studies that re-analyzed data retrospectively RCTs were also allowed) were considered if they included monoinfected treatment-naive HCV genotype 1 patients and compared treatment with pegIFN-alfa 2a or 2b in combination with ribavirin for 48 weeks versus extended treatment (up to 72 weeks) in slow responders. Primary and secondary end points were SVR rates and end-of-treatment (EOT) and relapse rates, respectively. In the present meta-analysis, study endpoints were summarized with a DerSimonian-Laird estimate for binary outcome basing on a random effects model. Results: Literature search yielded seven RTCs addressing the benefit of extended treatment with pegylated interferon-alfa and ribavirin in treatment-naive HCV genotype 1 slow responders. In total, 1330 slow responders were included in our meta-analysis. We show that extended treatment duration compared to the standard of care significantly improves SVR rates in HCV genotype 1 slow responders (12.4% improvement of overall SVR rate, 95% CI 0.055- 0.193, P = 0.0005). In addition, we show that rates of viral relapse were significantly reduced by extended treatment (24.1% reduction of relapse, 95% CI −0.3332 to −0.1487, P < 0.0001), whereas no significant impact of extended treatment on EOT response rates was found. Though extended treatment was burdened with an enhanced rate of premature treatment discontinuation due to interferonalfa- and ribavirin-related side effects, the frequency of serious adverse events was not increased. Conclusions: Treatment extension in HCV genotype 1 slow responders can improve SVR rates in difficult to treat patients and should be considered in patients who need to be treated before specific antivirals will be approved.

Magnitude of CD8 T-cell responses against hepatitis C virus and severity of hepatitis do not necessarily determine outcomes in acute hepatitis C virus infection.

Aim: We investigated the relationship between the magnitude of comprehensive hepatitis C virus (HCV)-specific CD8(+) T-cell responses and the clinical course of acute HCV infection. Methods: Six consecutive patients with acute HCV infection were studied. Analysis of HCV-specific CD8(+) T-cell responses was performed using an interferon-gamma-based enzyme-linked immunospot assay using peripheral CD8(+) T-cells, monocytes and 297 20-mer synthetic peptides overlapping by 10 residues and spanning the entire HCV sequence of genotype 1b. Results: Five patients presented detectable HCV-specific CD8(+) T-cell responses against a single and different peptide, whereas 1 patient showed responses against three different peptides. Neither the magnitude of HCV-specific CD8(+) T-cell responses nor the severity of hepatitis predicts the outcome of acute hepatitis. The maximum number of HCV-specific CD8(+) T-cells correlated with maximum serum alanine aminotransferase level during the course (r = 0.841, P = 0.036). Conclusions: HCV-specific CD8(+) T-cell responses were detectable in all 6 patients with acute HCV infection, and 6 novel HCV-specific CTL epitopes were identified. Acute HCV infection can resolve with detectable HCV-specific CD8(+) T-cell responses, but without development of antibody against HCV.

Little evidence that hepatitis C virus leads to a higher risk of mortality in the absence of cirrhosis and excess alcohol intake: the Swiss Hepatitis C Cohort Study.

The objective of this study was to describe the all-cause mortality of participants in the Swiss Hepatitis C Cohort compared to the Swiss general population. Patients with hepatitis C virus (HCV) infection attending secondary and tertiary care centres in Switzerland. One thousand six hundred and forty-five patients with HCV infection were followed up for a mean of over 2 years. We calculated all-cause standardized mortality ratios (SMR) and 95% confidence intervals (CI) using age, sex and calendar year-specific Swiss all-cause mortality rates. Multivariable Poisson regression was used to model the variability of SMR by cirrhotic status, HCV genotype, infection with hepatitis B virus or HIV, injection drug use and alcohol intake. Sixty-one deaths were recorded out of 1645 participants. The crude all-cause SMR was 4.5 (95% CI: 3.5-5.8). Patients co-infected with HIV had a crude SMR of 20 (95% CI: 11.1-36.1). The SMR of 1.1 (95% CI: 0.63-2.03) for patients who were not cirrhotic, not infected with HBV or HIV, did not inject drugs, were not heavy alcohol consumers (<or=40 g/day) and were not genotype 3, indicated no strong evidence of excess mortality. We found little evidence of excess mortality in hepatitis C infected patients who were not cirrhotic, in the absence of selected risk factors. Our findings emphasize the importance of providing appropriate preventive advice, such as counselling to avoid alcohol intake, in those infected with HCV.

Management of hepatitis C virus (HCV) infection in drug substitution programs.

BACKGROUND: In Switzerland, intravenous drug use (IDU) accounts for 80% of newly acquired hepatitis C virus (HCV) infections. Early HCV treatment has the potential to interrupt the transmission chain and reduce morbidity/mortality due to decompensated liver cirrhosis and hepatocellular carcinoma. Nevertheless, patients in drug substitution programs are often insufficiently screened and treated. OBJECTIVE/METHODS: With the aim to improve HCV management in IDUs, we conducted a cross sectional chart review in three opioid substitution programs in St. Gallen (125 methadone and 71 heroin recipients). Results were compared with another heroin substitution program in Bern (202 patients) and SCCS/SHCS data. RESULTS: Among the methadone/heroin recipients in St. Gallen, diagnostic workup of HCV was better than expected: HCV/HIV-status was unknown in only 1% (2/196), HCV RNA was not performed in 9% (13/146) of anti-HCV-positives and the genotype missing in 15% (12/78) of HCV RNA-positives. In those without spontaneous clearance (two thirds), HCV treatment uptake was 23% (21/91) (HIV-: 29% (20/68), HIV+: 4% (1/23)), which was lower than in methadone/heroin recipients and particularly non-IDUs within the SCCS/SHCS, but higher than in the, mainly psychiatrically focussed, heroin substitution program in Bern (8%). Sustained virological response (SVR) rates were comparable in all settings (overall: 50%, genotype 1: 35-40%, genotype 3: two thirds). In St. Gallen, the median delay from the estimated date of infection (IDU start) to first diagnosis was 10 years and to treatment was another 7.5 years. CONCLUSIONS: Future efforts need to focus on earlier HCV diagnosis and improvement of treatment uptake among patients in drug substitution programs, particularly if patients are HIV-co-infected. New potent drugs might facilitate the decision to initiate treatment.

Impact of donor and recipient IL28B rs12979860 genotypes on hepatitis C virus liver graft reinfection.

BACKGROUND & AIMS: Recent studies have described a major impact of genetic variations near the IL28B gene on the natural course and outcome of antiviral therapy in chronic hepatitis C. We therefore, aimed to explore the impact of donor and recipient genotypes of these polymorphisms on hepatitis C virus (HCV) liver graft reinfection. METHODS: Donor and recipient genotypes of IL28B rs12979860C>T single nucleotide polymorphism were determined in 91 patients with HCV liver graft reinfection, 47 of whom were treated with pegylated interferon-α (PEG-IFN-α) and ribavirin. IL28B genetic polymorphisms were correlated with the natural course and treatment outcome of recurrent hepatitis C. RESULTS: Patients requiring liver transplantation due to end-stage chronic hepatitis C appeared to be selected toward the adverse genotypes rs12979860 CT/TT compared to non-transplanted HCV-infected patients (p=0.046). Patients with the donor genotype rs12979860 CC had higher peak ALT and HCV RNA serum concentrations than those with CT/TT (p=0.04 and 0.06, respectively). No association was observed between ALT/HCV RNA serum concentrations and recipient genotypes (p>0.3). More important, donor IL28B rs12979860 CC vs. CT/TT genotypes were associated with rapid, complete early, and sustained virologic response (RVR, cEVR, SVR) to treatment with PEG-IFN-α and ribavirin (p=0.003, 0.0012, 0.008, respectively), but weaker associations of recipient genotypes with RVR, cEVR, and SVR were observed as well (p=0.0046, 0.115, 0.118, respectively). CONCLUSIONS: We provide evidence for a dominant, but not exclusive impact of the donor rather than the recipient IL28B genetic background on the natural course and treatment outcome of HCV liver graft reinfection.

Low current and nadir CD4+ T-cell counts are associated with higher hepatitis C virus RNA levels in the Swiss HIV cohort study.

BACKGROUND: The aim of this study was to evaluate the effect of CD4+ T-cell counts and other characteristics of HIV-infected individuals on hepatitis C virus (HCV) RNA levels. METHODS: All HIV-HCV-coinfected Swiss HIV Cohort Study participants with available HCV RNA levels and concurrent CD4+ T-cell counts before starting HCV therapy were included. Potential predictors of HCV RNA levels were assessed by multivariate censored linear regression models that adjust for censored values. RESULTS: The study included 1,031 individuals. Low current and nadir CD4+ T-cell counts were significantly associated with higher HCV RNA levels (P = 0.004 and 0.001, respectively). In individuals with current CD4+ T-cell counts < 200/microl, median HCV RNA levels (6.22 log10 IU/ml) were +0.14 and +0.24 log10 IU/ml higher than those with CD4+ T-cell counts of 200-500/microl and > 500/microl. Based on nadir CD4+ T-cell counts, median HCV RNA levels (6.12 log10 IU/ml) in individuals with < 200/microl CD4+ T-cells were +0.06 and +0.44 log10 IU/ml higher than those with nadir T-cell counts of 200-500/microl and > 500/microl. Median HCV RNA levels were also significantly associated with HCV genotype: lower values were associated with genotype 4 and higher values with genotype 2, as compared with genotype 1. Additional significant predictors of lower HCV RNA levels were female gender and HIV transmission through male homosexual contacts. In multivariate analyses, only CD4+ T-cell counts and HCV genotype remained significant predictors of HCV RNA levels. Conclusions: Higher HCV RNA levels were associated with CD4+ T-cell depletion. This finding is in line with the crucial role of CD4+ T-cells in the control of HCV infection.

Hepatitis C virus infection among Brazilian hemophiliacs: a virological, clinical and epidemiological study

We determined and analyzed risk factors of hepatitis C virus (HCV)-infected Brazilian hemophiliacs according to their virological, clinical and epidemiological characteristics. A cross-sectional and retrospective study of 469 hemophiliacs was carried out at a Brazilian blood center starting in October 1997. The prevalence of HCV infection, HCV genotypes and factors associated with HCV RNA detection was determined. The seroprevalence of anti-HCV antibodies (ELISA-3.0) was 44.6% (209/469). Virological, clinical and epidemiological assessments were completed for 162 positive patients. There were seven (4.3%) anti-HCV seroconversions between October 1992 and October 1997. During the same period, 40.8% of the positive anti-HCV hemophiliacs had abnormal alanine transaminase (ALT) levels. Plasma HCV RNA was detected by nested-RT-PCR in 116 patients (71.6%). RFLP analysis showed the following genotype distribution: HCV-1 in 98 hemophiliacs (84.5%), HCV-3 in ten (8.6%), HCV-4 in three (2.6%), HCV-2 in one (0.9%), and not typeable in four cases (3.4%). Univariate analysis indicated that older age (P = 0.017) and abnormal ALT levels (P = 0.010) were associated with HCV viremia, while the presence of inhibitor antibodies (P = 0.024) and HBsAg (P = 0.007) represented a protective factor against the presence of HCV RNA. These findings may contribute to a better understanding of the relationship between HCV infection and hemophilia.

Hepatitis C virus infection in a Brazilian population with sickle-cell anemia

Patients with sickle-cell anemia submitted to frequent blood transfusions are at risk of contamination with hepatitis C virus (HCV). Determination of HCV RNA and genotype characterization are parameters that are relevant for the treatment of the viral infection. The objective of the present study was to determine the frequency of HCV infection and the positivity for HCV RNA and to identify the HCV genotype in patients with sickle-cell anemia with a history of blood transfusion who had been treated at the Hospital of the HEMOPE Foundation. Sera from 291 patients were tested for anti-HCV antibodies by ELISA 3.0 and RIBA 3.0 Chiron and for the presence of HCV RNA by RT-PCR. HCV genotyping was performed in 19 serum samples. Forty-one of 291 patients (14.1%) were anti-HCV positive by ELISA and RIBA. Both univariate and multivariate analysis showed a greater risk of anti-HCV positivity in those who had started a transfusion regime before 1992 and received more than 10 units of blood. Thirty-four of the anti-HCV-positive patients (34/41, 82.9%) were also HCV RNA positive. Univariate analysis, used to compare HCV RNA-negative and -positive patients, did not indicate a higher risk of HCV RNA positivity for any of the variables evaluated. The genotypes identified were 1b (63%), 1a (21%) and 3a (16%). A high prevalence of HCV infection was observed in our patients with sickle-cell anemia (14.1%) compared to the population in general (3%). In the literature, the frequency of HCV infection in sickle-cell anemia ranges from 2 to 30%. The serological screening for anti-HCV at blood banks after 1992 has contributed to a better control of the dissemination of HCV infection. Because of the predominance of genotype 1, these patients belong to a group requiring special treatment, with a probable indication of new therapeutic options against HCV.

Geographic distribution of hepatitis C virus genotypes in Brazil

Brazil is a country of continental dimension with a population of different ethnic backgrounds. Thus, a wide variation in the frequencies of hepatitis C virus (HCV) genotypes is expected to occur. To address this point, 1,688 sequential samples from chronic HCV patients were analyzed. HCV-RNA was amplified by the RT-PCR from blood samples collected from 1995 to 2000 at different laboratories located in different cities from all Brazilian States. Samples were collected in tubes containing a gel separator, centrifuged in the site of collection and sent by express mail in a refrigerated container to Laboratório Bioquímico Jardim Paulista, São Paulo, SP, Brazil. HCV- RNA was extracted from serum and submitted to RT and nested PCR using standard procedures. Nested PCR products were submitted to cycle sequencing reactions without prior purification. Sequences were analyzed for genotype determination and the following frequencies were found: 64.9% (1,095) for genotype 1, 4.6% (78) for genotype 2, 30.2% (510) for genotype 3, 0.2% (3) for genotype 4, and 0.1% (2) for genotype 5. The frequencies of HCV genotypes were statistically different among Brazilian regions (P = 0.00017). In all regions, genotype 1 was the most frequent (51.7 to 74.1%), reaching the highest value in the North; genotype 2 was more prevalent in the Center-West region (11.4%), especially in Mato Grosso State (25.8%), while genotype 3 was more common in the South (43.2%). Genotypes 4 and 5 were rarely found and only in the Southeast, in São Paulo State. The present data indicate the need for careful epidemiological surveys throughout Brazil since knowing the frequency and distribution of the genotypes would provide key information for understanding the spread of HCV.

Prevalence of hepatitis C virus (HCV) infection and HCV genotypes of hemodialysis patients in Salvador, Northeastern Brazil

Hepatitis C virus (HCV) infection has been identified as the major cause of chronic liver disease among patients on chronic hemodialysis (HD), despite the important reduction in risks obtained by testing candidate blood donors for anti-HCV antibodies and the use of recombinant erythropoietin to treat anemia. A cross-sectional study was performed to estimate the prevalence of HCV infection and genotypes among HD patients in Salvador, Northeastern Brazil. Anti-HCV seroprevalence was determined by ELISA in 1243 HD patients from all ten different dialysis centers of the city. HCV infection was confirmed by RT-PCR and genotyping was performed by restriction fragment length polymorphism. Anti-HCV seroprevalence among HD patients was 10.5% (95% CI: 8.8-12.3) (Murex anti-HCV, Abbott Murex, Chicago, IL, USA). Blood samples for qualitative HCV detection and genotyping were collected from 125/130 seropositive HD patients (96.2%). HCV-RNA was detected in 92/125 (73.6%) of the anti-HCV-positive patients. HCV genotype 1 (77.9%) was the most prevalent, followed by genotype 3 (10.5%) and genotype 2 (4.6%). Mixed infections of genotypes 1 and 3 were found in 7.0% of the total number of patients. The present results indicate a significant decrease in anti-HCV prevalence from 23.8% detected in a study carried out in 1994 to 10.5% in the present study. The HCV genotype distribution was closely similar to that observed in other hemodialysis populations in Brazil, in local candidate blood donors and in other groups at risk of transfusion-transmitted infection.