Role of seipin in lipid droplet morphology and hepatitis C virus life cycle.

Infectious hepatitis C virus (HCV) particle assembly starts at the surface of lipid droplets, cytoplasmic organelles responsible for neutral fat storage. We analysed the relationship between HCV and seipin, a protein involved in lipid droplet maturation. Although seipin overexpression did not affect the total mean volume occupied by lipid droplets nor the total triglyceride and cholesterol ester levels per cell, it caused an increase in the mean diameter of lipid droplets by 60 %, while decreasing their total number per cell. The latter two effects combined resulted in a 34 % reduction of the total outer surface area of lipid droplets per cell, with a proportional decrease in infectious viral particle production, probably due to a defect in particle assembly. These results suggest that the available outer surface of lipid droplets is a critical factor for HCV release, independent of the neutral lipid content of the cell.

Hepatitis C virus variants resistant to macrocyclic NS3-4A inhibitors subvert IFN-β induction by efficient MAVS cleavage.

BACKGROUND & AIMS: The hepatitis C virus (HCV) NS3-4A protease is essential for the HCV life cycle and a prime target of antiviral treatment strategies. Protease inhibitors, however, are limited by emergence of resistance-associated amino acid variants (RAVs). The capacity to cleave and inactivate mitochondrial antiviral-signaling protein (MAVS) in the RIG-I-signaling pathway is a cardinal feature of NS3-4A, by which HCV blocks induction of interferon-(IFN)-β, thereby promoting viral persistence. Here, we aimed to investigate the impact of NS3-4A RAVs on MAVS cleavage. METHODS: The impact of NS3-4A RAVs on MAVS cleavage was assessed using immunoblot analyses, luciferase reporter assays and molecular dynamics simulations to study the underlying molecular principles. IFN-β was quantified in serum from patients with different NS3-4A RAVs. RESULTS: We show that macrocyclic NS3-4A RAVS with substitutions at residue D168 of the protease result in an increased capacity of NS3-4A to cleave MAVS and suppress IFN-β induction compared with a comprehensive panel of RAVs and wild type HCV. Mechanistically, we show the reconstitution of a tight network of electrostatic interactions between protease and the peptide substrate that allows much stronger binding of MAVS to D168 RAVs than to the wild-type protease. Accordingly, we could show IFN-β serum levels to be lower in patients with treatment failure due to the selection of D168 variants compared to R155 RAVs. CONCLUSIONS: Our data constitutes a proof of concept that the selection of RAVs against specific classes of direct antivirals can lead to the predominance of viral variants with possibly adverse pathogenic characteristics.

Incident Hepatitis C Virus Infections in the Swiss HIV Cohort Study : changes in treatment uptake and outcomes between 1991 and 2013

Background: The hepatitis C virus (HCV) epidemic is evolving rapidly in patients infected with human immunodeficiency virus (HIV). We aimed to describe changes in treatment uptake and outcomes of incident HCV infections before and after 2006, the time-point at which major changes in HCV epidemic became apparent. Methods.  We included all adults with an incident HCV infection before June 2012 in the Swiss HIV Cohort Study, a prospective nationwide representative cohort of individuals infected with HIV. We assessed the following outcomes by time period: the proportion of patients starting an HCV therapy, the proportion of treated patients achieving a sustained virological response (SVR), and the proportion of patients with persistent HCV infection during follow-up. Results.  Of 193 patients with an HCV seroconversion, 106 were diagnosed before and 87 after January 2006. The proportion of men who have sex with men increased from 24% before to 85% after 2006 (P < .001). Hepatitis C virus treatment uptake increased from 33% before 2006 to 77% after 2006 (P < .001). Treatment was started during early infection in 22% of patients before and 91% after 2006 (P < .001). An SVR was achieved in 78% and 29% (P = .01) of patients treated during early and chronic HCV infection. The probability of having a detectable viral load 5 years after diagnosis was 0.67 (95% confidence interval [CI], 0.58-0.77) in the group diagnosed before 2006 and 0.24 (95% CI, 0.16-0.35) in the other group (P < .001). Conclusions. In recent years, increased uptake and earlier initiation of HCV therapy among patients with incident infections significantly reduced the proportion of patients with replicating HCV.

A frequent hypofunctional IRAK2 variant is associated with reduced spontaneous hepatitis C virus clearance.

UNLABELLED: Patients carrying very rare loss-of-function mutations in interleukin-1 receptor-associated kinase 4 (IRAK4), a critical signaling mediator in Toll-like receptor signaling, are severely immunodeficient, highlighting the paramount role of IRAK kinases in innate immunity. We discovered a comparatively frequent coding variant of the enigmatic human IRAK2, L392V (rs3844283), which is found homozygously in ∼15% of Caucasians, to be associated with a reduced ability to induce interferon-alpha in primary human plasmacytoid dendritic cells in response to hepatitis C virus (HCV). Cytokine production in response to purified Toll-like receptor agonists was also impaired. Additionally, rs3844283 was epidemiologically associated with a chronic course of HCV infection in two independent HCV cohorts and emerged as an independent predictor of chronic HCV disease. Mechanistically, IRAK2 L392V showed intact binding to, but impaired ubiquitination of, tumor necrosis factor receptor-associated factor 6, a vital step in signal transduction. CONCLUSION: Our study highlights IRAK2 and its genetic variants as critical factors and potentially novel biomarkers for human antiviral innate immunity. (Hepatology 2015;62:1375-1387).

Hepatitis C Virus Awareness Among Men Who Have Sex With Men in Southwest Switzerland.

BACKGROUND: In Switzerland, the incidence of hepatitis C virus (HCV) infection in HIV-positive men who have sex with men (MSM) rose 18-fold between 1998 and 2011. We aimed to evaluate transmission risk factors, awareness, and seroprevalence of HCV among MSM in southwest Switzerland. METHODS: From 1st June 2011 to 31st August 2012, trained health care professionals invited individuals attending (1) MSM screening clinics and (2) indoor and outdoor meeting areas to complete an anonymous questionnaire. Consenting participants were rapid tested for HCV (OraQuick HCV Rapid Antibody Test). RESULTS: Of 918 MSM approached, 654 agreed to participate, most of whom (536, 82%) were enrolled via MSM screening clinics. Of 654 participants, 21 (3.2%) disclosed being HIV positive; 140 (21%) had unknown HIV status. In the preceding 12 months, 357 (55%) of 654 participants reported unprotected anal intercourse (UAI) and 321 (49%) of 654 participants reported UAI with partners of different/unknown HIV status. Not HIV serosorting was reported more frequently among HIV-positive individuals (76%, P < 0.001). Three hundred two participants (46%) were aware of HCV, awareness being higher among clinic than meeting area participants (49% vs. 33%, P = 0.04). One individual (of 654; 0.2%), with a negative HIV test result 18 months previously was newly diagnosed as being HCV positive on rapid testing. CONCLUSIONS: In this sample of predominantly HIV-negative MSM, half the participants were aware of HCV and HCV seroprevalence was low. However, high rates of UAI and of UAI without HIV serosorting were reported. Given the increasing incidence of HCV among HIV-positive men, we propose that HCV counseling should be offered to MSM regardless of HIV status, with testing offered to those at high risk.

Genomic characterization of Brazilian hepatitis C virus genotypes 1a and 1b

Parts of 5' non-coding (5' NC) and of E1 envelope regions of the hepatitis C virus (HCV) genome were amplified from sera of 26 Brazilian anti-HCV antibody-positive patients using the reverse transcription-polymerase chain reaction (RT-PCR). Fourteen samples were PCR positive with primers from the 5' NC region and 8 of them were also positive with primers from the E1 region. A genomic segment of 176 bp from the E1 region of 7 isolates was directly sequenced from PCR products. The sequences were compared with those of HCV strains isolated in other countries and the Brazilian isolates were classified by phylogenetic analysis into genotypes 1a and 1b. This could have a clinical importance since it has been shown that individuals infected with type 1 viruses are less likely to respond to treatment with interferon than individuals infected with types 2 and 3 viruses. Two quasispecies isolated from the same patient with an interval of 13 months differed by two base substitutions (1.1%). The sequence of another isolate presented a three-nucleotide deletion at codon 329

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.

In vitro-induced antibody production in chronic hepatitis C virus infection

The objectives of the present study were to assess the in vitro-induced anti-hepatitis C virus (HCV) antibody production (IVIAP) in relation to the clinical, biochemical, virologic and histologic variables of patients with HCV infection. The study included 57 patients (60% males) with HCV infection (anti-HCV and HCV-RNA positive). Alanine aminotransferase (ALT) was elevated in 89% of the patients. Mean viral load was 542,241 copies/ml and histology of the liver showed chronic hepatitis in 27/52 (52%) and cirrhosis in 11/52 (21%) patients. IVIAP levels were determined by immunoenzymatic assay at median absorbance of 0.781 at 450 nm. IVIAP was negative in 14% of the patients. When groups with IVIAP levels above and below the median were compared, high IVIAP levels were associated with the male sex, elevated ALT levels and more advanced disease stage. After logistic regression analysis, advanced histologic damage to the liver remained as the only independent variable associated with elevated IVIAP levels. Using a receiver operator characteristic curve, the best cut-off level for IVIAP was established (= 1.540), with 71% sensitivity and 94% specificity for the detection of more advanced disease stages (grades 3 and 4). These findings are consistent with the participation of immunological mechanisms in the genesis of the hepatic lesions induced by HCV and indicate that the IVIAP test may be useful as a noninvasive marker of liver damage either alone or in combination with other markers.

Molecular biology and clinical implication of hepatitis C virus

Hepatitis C virus (HCV) was first described in 1989 as the putative viral agent of non-A non-B hepatitis. It is a member of the Flaviviridae family and has been recognized as the major causative agent of chronic liver disease, including chronic active hepatitis, cirrhosis and hepatocellular carcinoma. HCV is a positive RNA virus with a genome containing approximately 9500 nucleotides. It has an open reading frame that encodes a large polyprotein of about 3000 amino acids and is characterized by extensive genetic diversity. HCV has been classified into at least 6 major genotypes with many subtypes and circulates within an infected individual as a number of closely related but distinct variants known as quasispecies. This article reviews aspects of the molecular biology of HCV and their clinical implication.

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.

Plasma hydroxy-metronidazole/ metronidazole ratio in hepatitis C virus-induced liver disease

It has been suggested that the measurement of metronidazole clearance is a sensitive method for evaluating liver function. The aim of this study was to evaluate the usefulness of plasma hydroxy-metronidazole/metronidazole ratios as indicators of dynamic liver function to detect changes resulting from the various forms of chronic hepatitis C virus (HCV) infection. A total of 139 individuals were studied: 14 healthy volunteers, 22 healthy, asymptomatic, consecutive anti-HCV-positive HCV-RNA negative subjects, 81 patients with chronic hepatitis C (49 with moderate/severe chronic hepatitis and 34 with mild hepatitis), and 20 patients with cirrhosis of the liver. HCV status was determined by the polymerase chain reaction. Plasma concentrations of metronidazole and its hydroxy-metabolite were measured by reverse-phase high-performance liquid chromatography with ultraviolet detection in a blood sample collected 10 min after the end of a metronidazole infusion. Anti-HCV-positive HCV-RNA-negative individuals demonstrated a significantly reduced capacity to metabolize intravenously infused metronidazole compared to healthy individuals (0.0478 ± 0.0044 vs 0.0742 ± 0.0232). Liver cirrhosis patients also had a reduced plasma hydroxy-metronidazole/metronidazole ratio when compared to the other groups of anti-HCV-positive individuals (0.0300 ± 0.0032 vs 0.0438 ± 0.0027 (moderate/severe chronic hepatitis) vs 0.0455 ± 0.0026 (mild chronic hepatitis) and vs 0.0478 ± 0.0044 (anti-HCV-positive, HCV-RNA-negative individuals)). These results suggest an impairment of the metronidazole metabolizing system induced by HCV infection that lasts after viral clearance. In those patients with chronic hepatitis C, this impairment is paralleled by progression of the disease to liver cirrhosis.

Hepatitis C virus seroprevalence and genotypes in patients with diffuse connective tissue diseases and spondyloarthropathies

Many extrahepatic manifestations, including rheumatic diseases, have been reported to be associated with hepatitis C virus (HCV) infection. In order to investigate the prevalence of HCV infection among patients with rheumatic diseases, in the present study we interviewed 367 patients and tested their blood samples for HCV antibodies (anti-HCV) by an enzyme-linked immunosorbent assay. Anti-HCV-reactive samples were retested for confirmation by a line immunoassay and also for HCV RNA detection by the polymerase chain reaction. HCV RNA-positive samples were genotyped by INNO-LIPA. An overall HCV infection prevalence of 1.9% (7/367) was found. Of the 7 HCV-infected patients, 4 had systemic lupus erythematosus and 3 rheumatoid arthritis, resulting in positivity rates of 2.3 and 3.4%, respectively. HCV RNA genotyping revealed the presence of subtypes 1a (57.1%), 1b (28.6%) and 3a (14.3%). The clinical course was favorable for all HCV-infected patients, except one, who died due to renal insufficiency related to lupus nephritis. These results demonstrate a low HCV infection prevalence among the population studied. In the few positive cases, we observed no adverse influence of this infection on the clinical evolution of the rheumatic disease.

Hepatitis C virus infection, cryoglobulinemia, and peripheral neuropathy: a case report

Hepatitis C virus (HCV) is essentially hepatotropic but its manifestations can extend beyond the liver. It can be associated with autoimmune diseases, such as mixed cryoglobulinemia, membranoproliferative glomerulonephritis, autoimmune thyroiditis, and lymphoproliferative disorders. The mechanisms that trigger these manifestations are not completely understood. We describe a 48-year-old man with chronic HCV infection (circulating HCV RNA and moderate hepatitis as indicated by liver biopsy), cryoglobulinemia, and sensory and motor peripheral neuropathy. The diagnosis of multineuropathy was confirmed by clinical examination and electromyographic tests. A nerve biopsy revealed an inflammatory infiltrate in the perineurial space and signs of demyelination and axonal degeneration. The patient had no improvement of neurological symptoms with the use of analgesics and neuro-modulators. He was then treated with interferon-alpha (3 million units subcutaneously, 3 times per week) and ribavirin (500 mg orally, twice a day) for 48 weeks. Six months after the end of therapy, the patient had sustained viral response (negative HCV RNA) and remission of neurological symptoms, but cryoglobulins remained positive. A review of the literature on the pathogenesis and treatment of neurological manifestations associated with HCV infection is presented. This report underscores the need for a thorough evaluation of HCV-infected patients because of the possibility of extrahepatic manifestations. Antiviral treatment with interferon and ribavirin can be effective and should be considered in patients with neurological complications associated with HCV infection.