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AIM: To develop a fast, low-cost diagnostic strategy to identify single point mutations in highly variable genomes such as hepatitis C virus (HCV). METHODS: In patients with HCV infection, resistance-associated amino acid substitutions within the viral quasispecies prior to therapy can confer decreased susceptibility to direct-acting antiviral agents and lead to treatment failure and virological relapse. One such naturally occurring mutation is the Q80K substitution in the HCV-NS3 protease gene, which confers resistance to PI inhibitors, particularly simeprevir. Low-cost, highly sensitive techniques enabling routine detection of these single point mutations would be useful to identify patients at a risk of treatment failure. LightCycler methods, based on real-time PCR with sequence-specific probe hybridization, have been implemented in most diagnostic laboratories. However, this technique cannot identify single point mutations in highly variable genetic environments, such as the HCV genome. To circumvent this problem, we developed a new method to homogenize all nucleotides present in a region except the point mutation of interest. RESULTS: Using nucleotide-specific probes Q, K, and R substitutions at position 80 were clearly identified at a sensitivity of 10% (mutations present at a frequency of at least 10% were detected). The technique was successfully applied to identify the Q80K substitution in 240 HCV G1 serum samples, with performance comparable to that of direct Sanger sequencing, the current standard procedure for this purpose. The new method was then validated in a Catalonian population of 202 HCV G1-infected individuals. Q80K was detected in 14.6% of G1a patients and 0% of G1b in our setting. CONCLUSION: A fast, low-cost diagnostic strategy based on real-time PCR and fluorescence resonance energy transfer probe melting curve analysis has been successfully developed to identify single point mutations in highly variable genomes such as hepatitis C virus. This technique can be adapted to detect any single point mutation in highly variable genomes. Sí

Background & Aims Occult HBV infection (OBI) is defined by the presence of HBV DNA in the liver and/or serum and negative HBsAg testing. Since the implementation of highly sensitive HBV DNA screening, OBI is also detected in healthy blood donors. The aims of this study were to investigate HBV-specific immune responses and genetic variability in donors with OBI, established by HBV DNA in serum. Methods HBV-specific T-cell responses to HBV antigens were tested in 34 OBI donors by IFN-γ ELISpot, cytometric bead array, and intracellular cytokine staining. As comparison populations, 36 inactive HBV carriers, 22 donors with spontaneously resolved HBV infection, 24 vaccinated donors, and 25 seronegative donors were also included. Surface, pre-S, and pre-c/core genes from 44 genotype D isolates (24 OBI and 20 HBsAg-positive) were sequenced. Results The immune response of OBI donors to the 3 HBV antigens was 29–41%, similar to the response in subjects with resolved HBV infection and higher than that in HBsAg-positive subjects. On sequence analysis, OBI donors presented a higher HBsAg mutation rate than HBsAg-positive subjects. Mutations were clustered in the major hydrophilic region of HBsAg, and no stop codons or relevant mutations that could affect antigen formation or detection were observed. Conclusions Our results suggest that immune response can suppress viral replication to low levels and HBsAg expression to undetectable levels in OBI blood donors. Relevant mutations were not found in the genomic HBsAg coding region. Hence, the fact that HBsAg was not detected in OBI is likely due to low HBsAg production, rather than to a failure of laboratory reagents.

Hepatitis C virus (HCV)-specific T cell responses are essential for HCV control, and chronic infection is characterized by functionally altered antigen-specific T cells. It has been proposed that the early inactivation of specific CD4(+) T cell responses may be involved in establishment of HCV persistence. We have investigated whether HCV-specific CD4(+) T cells dysfunction can be reversed in vitro. Nonstructural protein 3 (NS3) and core-specific CD4(+) T cells from eight chronically infected and eight spontaneously resolved HCV individuals were selected through transient CD154 (CD40 ligand) expression, and their functional profile (IFN-γ, IL-2, TNF-α, IL-10 and IL-4 production by enzyme-linked immunospot assay, cytometric bead array and intracellular cytokine staining, and proliferation by carboxy-fluorescein diacetate succinimidyl ester dilution assay) was determined both ex vivo and after in vitro expansion of sorted CD154-expressing cells in the absence of specific antigen in IL-7/IL-15-supplemented medium. Ex vivo bulk CD4(+) T cells from chronic patients expressed CD154 in most cases, albeit at lower frequencies than those of resolved patients (0.11%vs 0.41%; P = 0.01), when stimulated with NS3, but not core, although they had a markedly impaired capacity to produce IL-2 and IFN-γ. Antigen-free in vitro expansion of NS3-specific CD154(+) cells from chronic patients restored IFN-γ and IL-2 production and proliferation to levels similar to those of patients with spontaneously resolved infection. Hence, NS3-specific CD4(+) T cell response can be rescued in most chronic HCV patients by in vitro expansion in the absence of HCV-specific antigen. These results might provide a rationale for adoptive immunotherapy.

Injection of dendritic cells (DCs) presenting viral proteins constitutes a promising approach to stimulate T cell immunity against hepatitis C virus (HCV). Here we describe a strategy to enhance antigen loading and immunostimulatory functions of DCs useful in the preparation of therapeutic vaccines. Incubation of murine DCs with CFm40L, an adapter molecule containing the coxsackie-adenovirus receptor fused to the ecto-domain of murine CD40L-induced DC maturation, produced high amounts of interleukin-12 and up-regulation of molecules associated with T helper 1 responses. Accordingly, targeting of an adenovirus encoding HCV NS3 protein (AdNS3) to DCs with CFm40L strongly enhanced NS3 presentation in vitro, activating interferon-γ–producing T cells. Moreover, immunization of mice with these DCs promoted strong CD4 and CD8 T cell responses against HCV NS3. CFh40L, a similar adapter molecule containing human CD40L, enhanced transduction and maturation of human monocyte-derived DCs. Comparison of DCs transduced with AdNS3 and CFh40L from patients with chronic HCV infection and healthy donors revealed similar maturation levels. More importantly, DCs from the patients induced NS3-specific responses when transduced with AdNS3 and CFh40L but not with AdNS3 alone. Conclusion: DCs transduced with AdNS3 and the adapter molecule CFm/h40L exhibit enhanced immunostimulatory functions, induce robust anti-HCV NS3 immunity in animals, and can induce antiviral immune responses in subjects with chronic HCV infection. This strategy may serve as therapeutic vaccination for patients with chronic hepatitis C. (HEPATOLOGY 2011;)

BACKGROUND: Blood donors are routinely screened for hepatitis C virus (HCV) infection. Some show weak anti-HCV responses, often restricted to a single antigen on confirmatory immunoblot (recombinant immunoblot assay [RIBA]) testing. The aim of this study was to investigate the extent to which such RIBA-indeterminate donors had previously been exposed to HCV. STUDY DESIGN AND METHODS: T-cell responses to HCV recombinant proteins (core, NS3, and NS3 helicase) were analyzed using an interferon-γ (IFN-γ) enzyme-linked immunospot (ELISpot) assay and quantification of cytokines in culture supernatants in 27 RIBA-indeterminate donors, 60 RIBA-confirmed donors (48 with and 12 without HCV RNA), and 30 RIBA-negative donors. RESULTS: HCV-specific T-cell responses were identified in 13 (48%) RIBA-indeterminate donors, 33 (55%) RIBA-confirmed donors, and 4 (13%) RIBA-negative controls (p = 0.008 and p

Background. To study the feasibility of a response-guided therapy for chronic hepatitis C virus (HCV) infection in patients coinfected with human immunodeficiency virus (HIV) in a tertiary care hospital. Methods. Treatment duration was individualized on the basis of week 4 and week 12 virologic response. Sixty patients were enrolled and received pegylated interferon alfa-2b (1.5 μg/kg per week) plus weight-based ribavirin (800-1400 mg/day). Patients who achieved a rapid virologic response, defined as viral load

The epidemic of hepatitis C virus (HCV) infection in Europe is continuously evolving and epidemiological parameters (prevalence, incidence, disease transmission patterns and genotype distribution) have changed substantially during the last 15 years. Four main factors contribute to such changes: increased blood transfusion safety, improvement of healthcare conditions, continuous expansion of intravenous drug use and immigration to Europe from endemic areas. As a result, intravenous drug use has become the main risk factor for HCV transmission, prevalent infections have increased and genotype distribution has changed and diversified. Hence, prevalence data from studies conducted a decade ago may not be useful to estimate the current and future burden of HCV infection and additional epidemiological studies should be conducted, as well as new preventive strategies implemented to control the silent epidemic. This review summarizes recently published data on the epidemiology of HCV infection in Europe focusing on the factors currently shaping the epidemic.

An increasing number of new hepatitis C virus NS3-protease inhibitors are being evaluated for the treatment of chronic hepatitis C. Treatment-induced selection of mutants conferring resistance to protease inhibitors has been shown both in vivo and in vitro. A specific mutation, A156T has been shown to confer high-level resistance to several such agents (BILN2061, VX-950, SCH446211 (SCH6) and SCH503034). Here we report the presence of the A156T mutation in close to 1% of NS3 sequences within the liver quasispecies of a chronic hepatitis C patient never treated with anti-NS3-protease inhibitors.

Sofosbuvir displays a high phenotypic barrier to resistance, and it is a component of several combination therapies for hepatitis C virus (HCV) infections. HCV fitness can be a determinant of decreased sensitivity to direct-acting antiviral agents such as telaprevir or daclatasvir, but fitness-dependent decreased drug sensitivity has not been established for drugs with a high phenotypic barrier to resistance. Low- and high-fitness HCV populations and biological clones derived from them were used to infect Huh-7.5 hepatoma cells. Sofosbuvir efficacy was analyzed by measuring virus progeny production during several passages and by selection of possible sofosbuvir resistance mutations determined by sequencing the NS5B-coding region of the resulting populations. Sofosbuvir exhibited reduced efficacy against high-fitness HCV populations, without the acquisition of sofosbuvirspecific resistance mutations. A reduced sofosbuvir efficacy, similar to that observed with the parental populations, was seen for high-fitness individual biological clones. In independently derived high-fitness HCV populations or clones passaged in the presence of sofosbuvir, M289L was selected as the only substitution in the viral polymerase NS5B. In no case was the sofosbuvir-specific resistance substitution S282T observed. High HCV fitness can lead to decreased sensitivity to sofosbuvir, without the acquisition of specific sofosbuvir resistance mutations. Thus, fitness-dependent drug sensitivity can operate with HCV inhibitors that display a high barrier to resistance. This mechanism may underlie treatment failures not associated with selection of sofosbuvirspecific resistance mutations, linked to in vivo fitness of pretreatment viral populations., MINECO | Instituto de Salud Carlos III (ISCIII) (PI13/00456). This work, including the efforts of Rafael Esteban, was funded by MINECO | Instituto de Salud Carlos III (ISCIII) (PI15/00829). This work, including the efforts of Esteban Domingo, was funded by Ministerio de Economía y Competitividad (MINECO) (BFU-2011-23604); Comunidad Autónoma de Madrid (S2013/ABI-2906).

HepatitisCvirus (HCV)is classified into seven major genotypesand67 subtypes. Recent studies haveshownthat inHCVgenotype 1-infected patients, response rates to regimens containingdirect-acting antivirals(DAAs)are subtype dependent. Currently available genotypingmethods have limited subtyping accuracy.Wehave evaluated theperformanceof adeep-sequencing-basedHCVsubtyping assay, developed for the 454/GS-Junior platform, incomparisonwith thoseof twocommercial assays (VersantHCVgenotype 2.0andAbbott Real-timeHCVGenotype II)andusingdirectNS5Bsequencing as a gold standard (direct sequencing), in 114 clinical specimenspreviously tested by first-generation hybridization assay (82 genotype 1 and32 with uninterpretable results). Phylogenetic analysis of deep-sequencing reads matched subtype 1 callingbypopulation Sanger sequencing (69%1b,31%1a) in81 specimensandidentified amixed-subtype infection (1b/3a/1a) inone sample. Similarly,amongthe 32previously indeterminate specimens, identical genotypeandsubtype results were obtained by directanddeep sequencing inall but four samples with dual infection. In contrast, both VersantHCVGenotype 2.0andAbbott Real-timeHCVGenotype II failed subtype 1 calling in13 (16%)samples eachandwere unable to identify theHCVgenotype and/or subtype inmore thanhalfof the nongenotype 1 samples.Weconcluded that deep sequencing ismore efficient forHCVsubtyping than currently available methods andallows qualitative identificationofmixed infectionsandmay bemorehelpfulwith respect toinforming treatment strategies withnewDAA-containing regimens across allHCVsubtypes

Lethal mutagenesis is an antiviral approach that consists in extinguishing a virus by an excess of mutations acquired during replication in the presence of a mutagen. Here we show that favipiravir (T-705) is a potent mutagenic agent for hepatitis C virus (HCV) during its replication in human hepatoma cells. T-705 leads to an excess of G!A and C!U transitions in the mutant spectrum of preextinction HCV populations. Infectivity decreased significantly in the presence of concentrations of T-705 which are 2- to 8-fold lower than its cytotoxic concentration 50 (CC50). Passaging the virus five times in the presence of 400 μM T-705 resulted in virus extinction. Since T-705 has undergone advanced clinical trials for approval for human use, the results open a new approach based on lethal mutagenesis to treat hepatitis C virus infections. If proven effective for HCV in vivo, this new anti-HCV agent may be useful in patient groups that fail current therapeutic regimens., BFU-2011-23604, SAF2014-52400-R, S2013/ABI-2906 (PLATESA from Comunidad de Madrid/FEDER) and Fundación R. Areces. The work in Barcelona was funded by Instituto de Salud Carlos III, grants PI13-00456 and PI15-00829, cofinanced by the European Regional Development Fund (ERDF).

The influence of glucose abnormalities on the efficacy of antiviral treatment is unknown. This study investigated whether glucose abnormalities (impaired fasting glucose and type 2 diabetes) influence the response to antiviral therapy with interferon plus ribavirin in patients with chronic hepatitis C.A total of 178 treatment-naïve patients with chronic hepatitis C treated with combination therapy were retrospectively studied. SVR was assessed after completing treatment. Fasting plasmatic glucose was measured prior to therapy.Compared with nonresponders (N = 111), patients with SVR (N = 67) had lower plasma glucose (94.1 +/- 12.7 vs 104.4 +/- 25.8 mg/dL, P= 0.001) and a lower prevalence of glucose abnormalities (24.24%vs 44.14%, P= 0.012). The SVR rate was 45.13% in patients with normoglycemia (N = 113), 28.26% in patients with impaired fasting glucose (N = 46), and 15.78% in type 2 diabetic patients (N = 19) (P0.001). Multivariate logistic regression identified genotype 1 (OR 1.55, 95% CI 1.01-2.41, P= 0.05), gamma-glutamyltranspeptidase level (OR 6.41, 95% CI 1.86-22.07, P= 0.003), and presence of glucose abnormalities (OR 2.33, 95% CI 1.04-5.20, P= 0.039) as being independently associated with the absence of an SVR. In addition, patients with glucose abnormalities (N = 65) showed a lower virological response rate when compared with a subgroup of normoglycemic patients (N = 65) matched for sex, age, and liver fibrosis (24.6%vs 44.6%, P= 0.001).Glucose abnormalities are an independent predictor of poor virological response to combined therapy in hepatitis C virus infected patients.

Background & Aims: The second slope of viral decline induced by interferon treatment has been suggested to be influenced mainly by the hepatitis C virus (HCV)-specific T-cell response; however, this hypothesis needs to be validated by results derived from experimental studies. Methods: To address this issue, the HCV-specific T-cell response of 32 genotype-1–infected patients of the 270 patients enrolled in the dynamically individualized treatment of hepatitis C infection and correlates of viral/host dynamics phase III, open-label, randomized, multicenter trial was studied in relation to viral kinetics and treatment outcome. Results: Greater proliferative responses by HCV-specific CD8 cells were found before treatment in patients with a fast viral decline and with a sustained viral response. However, no significant improvement of HCV-specific CD8 responses was observed in the first weeks of therapy in both rapid viral responder and non–rapid viral responder patients. A mild enhancement of proliferative T-cell responses and a partial restoration of the cytotoxic T-cell potential was expressed only late during treatment, likely favored by HCV clearance. Conclusions: Early restoration of an efficient T-cell response does not seem to be an essential requirement for a rapid viral decline in the first weeks of treatment. However, patients presenting a better HCV-specific CD8 cell proliferative potential at baseline are more likely to present a rapid and sustained viral response. Therefore, future treatment protocols should consider the development of strategies aimed at improving HCV-specific T-cell responses.

Background Hepatitis C virus (HCV) genotyping is crucial in clinical practise for determining the type and duration of antiviral therapy. Between 2009 and 2014, 24 (7.95%) of all HCV genotype 3 (HCV-3) cases obtained indeterminate results via the RealTime HCV Genotype II assay (Abbott) at a tertiary care center in Spain. HCV-3 is the second most common genotype worldwide. Moreover, it has been associated with a higher risk of liver disease progression and a lower response to the latest antivirals. Objective Given the clinical significance of accurately identifying HCV-3, we aimed to characterize the genetic diversity of the HCV 5′ untranslated region (5′ UTR), the target of genotyping assays, by ultradeep pyrosequencing (UDPS). Study design For the 24 indeterminate samples, the 5′ UTR-core was amplified and subjected to UDPS with the 454/GS-Junior platform (Roche). The genotype/subtype of each identified haplotype was assigned by phylogenetic analysis. For comparison, three additional samples correctly identified as HCV-3 by the real-time assay were also analyzed. Results HCV genotyping based on 5′ UTR-core UDPS was in agreement with NS5B Sanger sequencing in all cases, confirming the absence of mixed infections and recombination events. The generated 5′ UTR sequences proved the presence of one to three polymorphisms at the probe-binding site of the Abbott assay, thereby differentiating indeterminate from correctly genotyped HCV-3 samples. Conclusions The observed naturally occurring polymorphisms provide insight into regional differences observed with genotype 3, their impact on genotyping assay performance, and potential improvement and designing options.

The Wilms' tumor suppressor WT1 is a transcriptional regulator present in the fetal but not in the mature liver. Its expression and functional role in liver diseases remains unexplored. In this study, we analyzed WT1 expression by reverse-transcription polymerase chain reaction (RT-PCR) and by immunohistochemistry in normal and diseased livers. In addition, we performed in vitro studies in isolated rat hepatocytes to investigate WT1 regulation and function. We detected WT1 messenger RNA (mRNA) in 18% of normal livers, 17% of chronic hepatitis with minimal fibrosis, 49% of chronic hepatitis with bridging fibrosis, and 71% of cirrhotic livers. In cirrhosis, WT1 immunoreactivity was localized to the nucleus of hepatocytes. WT1 mRNA abundance correlated inversely with prothrombin time (P = .04) and directly with serum bilirubin (P = .002) and with the MELD score (P = .001) of disease severity. In rats, WT1 expression was present in fetal hepatocytes and in the cirrhotic liver but not in normal hepatic tissue. In vitro studies showed that isolated primary hepatocytes express WT1 when stimulated with transforming growth factor β (TGF-β) or when the cells undergo dedifferentiation in culture. Moreover, we found that WT1 down-regulates hepatocyte nuclear factor 4 (HNF-4), a factor that is essential to maintain liver function and metabolic regulation in the mature organ. Hepatic expression of HNF-4 was impaired in advanced human cirrhosis and negatively correlated with WT1 mRNA levels (P = .001). In conclusion, we show that WT1 is induced by TGF-β and down-regulates HNF-4 in liver cells. WT1 is reexpressed in the cirrhotic liver in relation to disease progression and may play a role in the development of hepatic insufficiency in cirrhosis. (Hepatology 2003;38:148-157.)

The aim of this retrospective study was to determine the predictive value of alanine aminotransferase (ALT) levels for histologic findings in patients with chronic hepatitis C virus (HCV) infection. Data on 864 HCV RNA-positive patients were collected. ALT values were obtained at the time of biopsy (before treatment), and normal ALT values were defined as normal values obtained at serial evaluations during a 6-month period. Histologic results were scored using the METAVIR system. Among all patients, 99% of those with elevated ALT levels had a score of at least F1 (positive predictive value [PPV], 99%) and 88% had a score greater than A1F1. Among patients with persistently normal ALT values, 65% had a score of at least F1 (negative predictive value [NPV], 35%) and 26% had a score greater than A1F1. The receiver operating characteristics analysis indicates that the ALT threshold for the best compromise sensitivity-specificity was about 2.25 times the upper limit of normal (ULN). In conclusion, almost all HCV RNA-positive patients with elevated ALT levels have some degree of fibrosis. However, an important proportion of patients with persistently normal ALT levels also show some histologic signs of fibrosis; the degree of fibrosis is usually mild but is sometimes more marked, and in rare cases cirrhosis may be present. In this subset of patients, the indication of liver biopsy and the potential benefit of therapy need to be further evaluated. These results suggest the need to revisit the algorithm for liver biopsy practice.

Co-infection by human immunodeficiency virus and hepatitis B and C viruses is quite common because they share similar routes of transmission. The introduction of highly active antiretroviral therapy has significantly improved the life expectancy of HIV-infected patients in the last few years. However, chronic viral hepatitis represents an emerging cause of morbidity and mortality in this population, either as a result of end-stage liver disease or as a consequence of hepatotoxicity induced by antiretroviral drugs. The main goal of the Consensus Conference was to establish specific recommendations for the management of chronic viral hepatitis B and C in HIV-infected patients. The role of orthotopic liver transplantation for co-infected individuals with end-stage liver disease was also assessed.

All Rights Reserved. HepatitisCvirus(HCV)is classified into seven major genotypesand67 subtypes. Recent studies haveshownthat inHCVgenotype 1-infected patients, response rates to regimens containingdirect-acting antivirals(DAAs)are subtype dependent. Currently available genotypingmethods have limited subtyping accuracy.Wehave evaluated theperformanceof adeep-sequencing-basedHCVsubtyping assay, developed for the 454/GS-Junior platform, in comparisonwith thoseof two commercial assays (VersantHCVgenotype 2.0andAbbott Real-timeHCVGenotype II)andusingdirectNS5Bsequencing as a gold standard (direct sequencing), in 114 clinical specimenspreviously tested by first-generation hybridization assay (82 genotype 1and32 with uninterpretable results). Phylogenetic analysis of deep-sequencing reads matched subtype 1 callingbypopulation Sanger sequencing(69%1b,31%1a) in 81 specimensandidentified amixed-subtype infection (1b/3a/1a) in one sample. Similarly,amongthe 32previously indeterminate specimens, identical genotypeandsubtype results were obtained by directanddeep sequencing in all but four samples with dual infection. In contrast, both VersantHCVGenotype 2.0andAbbott Real-timeHCVGenotype II failed subtype 1 calling in 13 (16%) samples eachandwere unable to identify theHCVgenotype and/or subtype inmore than half of the nongenotype 1 samples.Weconcluded that deep sequencing ismore efficient forHCVsubtyping than currently available methodsandallows qualitative identificationofmixed infectionsandmay bemorehelpfulwith respect to informing treatment strategies withnewDAA-containing regimens across allHCVsubtypes., This study has been supported by CDTI (Centro para el Desarrollo Tecnológico Industrial), Spanish Ministry of Economics and Competitiveness (MINECO), IDI-20110115; MINECO projects SAF 2009-10403; and also by the Spanish Ministry of Health, Instituto de Salud Carlos III (FIS) projects PI10/01505, PI12/01893, and PI13/00456. CIBERehd is funded by the Instituto de Salud Carlos III, Madrid, Spain. Work at CBMSO was supported by grant MINECO-BFU2011-23604, FIPSE, and Fundación Ramón Areces.

© 2014, American Society for Microbiology. Passage of hepatitis C virus (HCV) in human hepatoma cells resulted in populations that displayed partial resistance to alpha interferon (IFN-α), telaprevir, daclatasvir, cyclosporine, and ribavirin, despite no prior exposure to these drugs. Mutant spectrum analyses and kinetics of virus production in the absence and presence of drugs indicate that resistance is not due to the presence of drug resistance mutations in the mutant spectrum of the initial or passaged populations but to increased replicative fitness acquired during passage. Fitness increases did not alter host factors that lead to shutoff of general host cell protein synthesis and preferential translation of HCV RNA. The results imply that viral replicative fitness is a mechanism of multidrug resistance in HCV., Ministerio de Ciencia e Innovación, Fundación Ramon Areces, and Marie Curie International outgoing fellowship 219570. CIBERehd (Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas) is funded by Instituto de Salud Carlos III. N.M.B. is supported by a JAE-DOC contract from Consejo Superior de Investigaciones Científicas (CSIC) and J.S. by a Juan de la Cierva contract from CSIC

To investigate the influence of IL28B polymorphism in occult hepatitis B infection (OBI) and whether IL28B genetic variants are associated with hepatitis B virus (HBV)-specific T-cell responses.The rs12979860 IL28B genotype was determined in 34 OBI blood donors, 22 spontaneous HBV resolvers, 36 inactive HBV carriers and 25 seronegative donors. T-cell responses to HBV recombinant proteins were assessed by interferon-γ enzyme-linked immunospot assay.The frequency of the IL28B CC genotype among OBI patients was similar to that of inactive carriers [41 vs. 39%, respectively, p = 0.961; odds ratio (OR) = 1.10; 95% confidence interval (CI) = 0.42-2.86; p = 0.845]. The IL28B CC genotype was found more frequently in spontaneous resolvers, although the differences were not significant (45 vs. 39%, spontaneous resolvers and inactive carriers, respectively; p = 0.828; OR = 1.31; 95% CI = 0.45-3.83; p = 0.622). HBV-specific T-cell responses were detected in OBIs, and significantly stronger T-cell responses towards hepatitis B envelope antigen were observed in those with the IL28B CC genotype. In spontaneous resolvers and inactive carriers, IL28B CC did not correlate with the magnitude of T-cell responses.In OBI donors, IL28B CC correlates with the intensity of HBV-specific T-cell responses. In this study, IL28B CC is not statistically associated with OBI or with HBV clearance, but a larger number of cases is needed before completely ruling out its role in HBV infection.

In previous cross-sectional studies, we demonstrated that, in most patients with chronic hepatitis C, the composition and complexity of the circulating hepatitis C virus (HCV) population do not coincide with those of the virus replicating in the liver. In the subgroup of patients with similar complexities in both compartments, the ratio of quasispecies complexity in the liver to that in serum (liver/serum complexity ratio) of paired samples correlated with disease stage. In the present study we investigated the dynamic behavior of viral population parameters in consecutive paired liver and serum samples, obtained 3 to 6 years apart, from four chronic hepatitis C patients with persistently normal transaminases and stable liver histology. We sequenced 359 clones of a genomic fragment encompassing the E2(p7)-NS2 junction, in two consecutive liver-serum sample pairs from the four patients and in four intermediate serum samples from one of the patients. The results show that the liver/serum complexity ratio is not stable but rather fluctuates widely over time. Hence, the liver/serum complexity ratio does not identify a particular group of patients but a particular state of the infecting quasispecies. Phylogenetic analysis and signature mutation patterns showed that virtually all circulating sequences originated from sequences present in the liver specimens. The overall behavior of the circulating viral quasispecies appears to originate from changes in the relative replication kinetics of the large mutant spectrum present in the infected liver.

The quasispecies nature of the hepatitis C virus (HCV) is thought to play a central role in maintaining and modulating viral replication. Several studies have tried to unravel, through the parameters that characterize HCV circulating quasispecies, prognostic markers of the disease. In a previous work we demonstrated that the parameters of circulating viral quasispecies do not always reflect those of the intrahepatic virus. Here, we have analyzed paired serum and liver quasispecies from 39 genotype 1b-infected patients with different degrees of liver damage, ranging from minimal changes to cirrhosis. Viral level was quantified by real-time reverse transcription-PCR, and viral heterogeneity was characterized through the cloning and sequencing of 540 HCV variants of a genomic fragment encompassing the E2-NS2 junction. Although in 95% of patients, serum and liver consensus HCV amino acid sequences were identical, quasispecies complexity varied considerably between the viruses isolated from each compartment. Patients with HCV quasispecies in serum more complex (26%) than, less complex (28%) than, or similarly complex (41%) to those in liver were found. Among the last, a significant correlation between fibrosis and all the parameters that measure the viral amino acid complexity was found. Correlation between fibrosis and serum viral load was found as well ( R = 0.7). With regard to the origin of the differences in quasispecies complexity between serum and liver populations, sequence analysis argued against extrahepatic replication as a quantitatively important contributing factor and supported the idea of a differential effect or different selective forces on the virus depending on whether it is circulating in serum or replicating in the liver.

SummaryHepatitis C virus (HCV) infected hemophiliacs respond at low rate to interferon (IFN) monotherapy.To assess efficacy of IFN and RBV in HIV negative hemophiliacs with chronic hepatitis C and identify early predictive factors of response.Twenty naive patients were treated with interferon and RBV for twelve months. Response was assessed by both serial ALT and HCV RNA levels.Normalization of ALT with clearance of HCV RNA occurred in seven (35%) patients. Age and age at infection were the only features associated with a higher likelihood of response. In all responders the viral load had decreased by at least one log within two months of starting treatment.Combination of interferon and ribavirin is well tolerated by hemophiliacs who achieve similar sustained response rates to non-hemophiliacs. Quantitative assessment of viral load at two months of treatment is a useful method to identify non-responders at an early stage.

We describe a rapid and reproducible method for assessment of the hepatitis C virus (HCV) load in serum samples. The method combines Taqman technology (Roche) and the ABI Prism 7700 (Perkin Elmer) real-time sequence detection system. We have optimized a single-tube reverse transcription-PCR (RT-PCR) that contains a dual-labeled fluorogenic probe to quantify the 5′ noncoding region (5′ NCR) of HCV. The probe contains a fluorescent reporter at the 5′ end and a fluorescent quencher at the 3′ end. The use of such a probe combined with the 5′-3′ nuclease activity of Taq polymerase allows direct quantitation of the PCR product by the detection of a fluorescent reporter released in the course of the exponential phase of the PCR. For accurate quantitation of the number of copies of HCV in samples containing unknown quantities, we have used serial dilutions of a synthetic 5′ NCR RNA standard of HCV that was previously quantified with an isotopic tracer. The method has a 5-log dynamic range (10 3 to 10 7 ). The coefficient of regression of the standard curve was, on average, 0.98. The intra-assay and the interassay coefficients of variation of the threshold cycle were 1% and 6.2%, respectively. Seventy-nine RNA samples from the sera of infected patients were quantified by this method. Comparison of the results with those obtained by other quantitation methods (the Quantiplex 2.0 branched-DNA assay and the Superquant assay from the National Genetics Institute) revealed a significant correlation with all of the results. The mean values were also statistically comparable. In conclusion, the high sensitivity, simplicity, and reproducibility of the real-time HCV RNA quantitation which allows the screening of large numbers of samples, combined with its wide dynamic range, make this method especially suitable for monitoring of the viral load during therapy and tailoring of treatment schedules.

To study the subtype prevalence and the phylogenetic relatedness of hepatitis C virus (HCV) sequences obtained from the Argentine general population, a large cohort of individuals was analyzed.Healthy Argentinian volunteers (n = 6251) from 12 provinces representing all geographical regions of the country were studied. All parents or legal guardians of individuals younger than 18 years provided informed written consent for participation. The corresponding written permission from all municipal authorities was obtained from each city or town where subjects were to be included. HCV RNA reverse transcription-polymerase chain reaction products were sequenced and phylogenetically analyzed. The 5' untranslated region (5'UTR) was used for RNA detection and initial genotype classification. The NS5B polymerase region, encompassing nt 8262-8610, was used for subtyping.An unexpectedly low prevalence of HCV infection in the general population (0.32%) was observed. Our data contrasted with previous studies that reported rates ranging from 1.5% to 2.5%, mainly performed in selected populations of blood donors or vulnerable groups. The latter values are in keeping with the prevalence reported by the 2007 Argentinian HCV Consensus (approximately 2%). HCV subtypes were distributed as follows: 1a (25%), 1b (25%), 2c (25%), 3a (5%), and 2j (5%). Two isolates ascribed either to genotype 1 (5%) or to genotype 3 (5%) by 5'UTR phylogenetic analysis could not be subtyped. Subtype 1a sequences comprised a highly homogeneous population and clustered with United States sequences. Genotype 1b sequences represented a heterogeneous population, suggesting that this genotype might have been introduced from different sources. Most subtype 2c sequences clustered close to the 2c reported from Italy and Southern France.HCV has a low prevalence of 0.32% in the studied general population of Argentina. The pattern of HCV introduction and transmission in Argentina appears to be a consequence of multiple events and different for each subtype.

Background & Aims Hepatitis C virus (HCV) transmission from a chronic patient to a susceptible individual is a good opportunity to study viral and host factors that may influence the natural course of hepatitis C infection towards either spontaneous recovery or chronicity. To compare a documented case of a bottleneck event in the sexual transmission of HCV from a chronically infected patient to a recipient host that cleared infection. Methods Host genetic components such as Class I and II HLA and IL28B polymorphism (rs12979860 SNPs) were identified by direct sequencing and LightMix analysis, respectively. Deep nucleotide sequence analysis of quasispecies complexity was performed using massive pyrosequencing platform (454 GS-FLX), and the CD4 specific immune response was characterized by ELISPOT. Results and Conclusions Sequencing analysis and CD4 response highlighted several NS3-helicase domains in which an interplay between amino acid variability and CD4 immune response might have contributed either to chronicity in the donor patient or to viral clearance in the receptor (newly infected) patient.

In the course of a study conducted in 1992 through 1994 of the efficacy of screening blood donors for antibodies to hepatitis C virus (HCV), we found that two patients had acquired hepatitis C after cardiac surgery, with the transmission apparently unrelated to blood transfusions. Because their surgeon had chronic hepatitis C, we sought to determine whether he was transmitting the virus to his patients.Of 222 of the surgeon's patients who participated in studies of post-transfusion hepatitis between 1988 and 1994, 6 contracted postoperative hepatitis C, despite the use of only seronegative blood for transfusions. All six patients had undergone valve-replacement surgery. Analyses were performed to compare nucleotide sequences encompassing the hypervariable region at the junction between the coding regions for envelope glycoproteins E1 and E2 in the surgeon, the patients, and 10 controls infected with the same HCV genotype.The surgeon and five of the six patients with hepatitis C unrelated to transfusion were infected with HCV genotype 3; the sixth patient had genotype 1 and was considered to have been infected from another source. Thirteen other patients of the surgeon had transfusion-associated hepatitis C and were also infected with genotype 1. The average net genetic distance between the sequences from the five patients with HCV genotype 3 and those from the surgeon was 2.1 percent (range, 1.1 to 2.5 percent; P0.001), as compared with an average distance of 7.6 percent (range, 6.1 to 8.3 percent) between the sequences from the patients and those from the controls. The results of phylogenetic-tree analysis indicated a common epidemiologic origin of the viruses from the surgeon and the five patients.Our findings provide evidence that a cardiac surgeon with chronic hepatitis C may have transmitted HCV to five of his patients during open-heart surgery.

We have investigated the reliability and reproducibility of HCV viral quasispecies quantification by ultra-deep pyrosequencing (UDPS) methods. Our study has been divided in two parts. First of all, by UDPS sequencing of clone mixes samples we have established the global noise level of UDPS and fine tuned a data treatment workflow previously optimized for HBV sequence analysis. Secondly, we have studied the reproducibility of the methodology by comparing 5 amplicons from two patient samples on three massive sequencing platforms (FLX+, FLX and Junior) after applying the error filters developed from the clonal/control study. After noise filtering the UDPS results, the three replicates showed the same 12 polymorphic sites above 0.7%, with a mean CV of 4.86%. Two polymorphic sites below 0.6% were identified by two replicates and one replicate respectively. A total of 25, 23 and 26 haplotypes were detected by GS-Junior, GS-FLX and GSFLX+. The observed CVs for the normalized Shannon entropy (Sn), the mutation frequency (Mf), and the nucleotidic diversity (Pi) were 1.46%, 3.96% and 3.78%. The mean absolute difference in the two patients (5 amplicons each), in the GS-FLX and GS-FLX+, were 1.46%, 3.96% and 3.78% for Sn, Mf and Pi. No false polymorphic site was observed above 0.5%. Our results indicate that UDPS is an optimal alternative to molecular cloning for quantitative study of HCV viral quasispecies populations, both in complexity and composition. We propose an UDPS data treatment workflow for amplicons from the RNA viral quasispecies which, at a sequencing depth of at least 10,000 reads per strand, enables to obtain sequences and frequencies of consensus haplotypes above 0.5% abundance with no erroneous mutations, with high confidence, resistant mutants as minor variants at the level of 1%, with high confidence that variants are not missed, and highly confident measures of quasispecies complexity., This study has been supported by CDTI (Centro para el Desarrollo Tecnológico Industrial), Spanish Ministry of Economics and Competitiveness (MINECO), IDI-20110115. It has also been supported by MINECO projects SAF 2009-10403, and also by the Spanish Ministry of Health Instituto de Salud Carlos III (FIS) projects PI10/01505. CIBERehd is funded by the Instituto de Salud Carlos III, Madrid. Work at CBMSO was supported by grant BFU2011-23604, FIPSE and Fundación Ramon Areces

Lethal mutagenesis, or virus extinction produced by enhanced mutation rates, is under investigation as an antiviral strategy that aims at counteracting the adaptive capacity of viral quasispecies, and avoiding selection of antiviral-escape mutants. To explore lethal mutagenesis of hepatitis C virus (HCV), it is important to establish whether ribavirin, the purine nucleoside analogue used in anti-HCV therapy, acts as a mutagenic agent during virus replication in cell culture. Here we report the effect of ribavirin during serial passages of HCV in human hepatoma Huh-7.5 cells, regarding viral progeny production and complexity of mutant spectra. Ribavirin produced an increase of mutant spectrum complexity and of the transition types associated with ribavirin mutagenesis, resulting in HCV extinction. Ribavirin-mediated depletion of intracellular GTP was not the major contributory factor to mutagenesis since mycophenolic acid evoked a similar decrease in GTP without an increase in mutant spectrum complexity. The intracellular concentration of the other nucleoside-triphosphates was elevated as a result of ribavirin treatment. Mycophenolic acid extinguished HCV without an intervening mutagenic activity. Ribavirin-mediated, but not mycophenolic acid-mediated, extinction of HCV occurred via a decrease of specific infectivity, a feature typical of lethal mutagenesis. We discuss some possibilities to explain disparate results on ribavirin mutagenesis of HCV. © 2013 Ortega-Prieto et al., BFU 2011-23604, SAF2009-10403; PI 10/01505 and ref. IDI-20110115 CDTI (Centro para el Desarrollo Tecnologico Industrial) from Ministerio de Ciencia e Innovacion; P09-CVI-5428 and P10-CVI-6561 from Junta de Andalucıa; Fundacion Ramon Areces; CIBERehd

Routine screening of blood donors for anti-hepatitis C virus (HCV) has been implemented in most developed countries. However, the independent efficacy of such screening has not been established in a controlled, prospective study. We tracked 478 patients transfused with anti-HCV-negative blood by first-generation enzymelinked immunoassay (EIA) between July 1989 and May 1990 and compared the incidence of transfusion-associated hepatitis and HCV infections with that found among 280 patients transfused with blood unscreened for anti-HCV during the immediately preceding year. Of the 280 patients who had received transfusions before donors were screened for anti-HCV, 27 (9.6%) developed posttransfusion hepatitis and 1 additional patient seroconverted to anti-HCV without evidence of hepatitis, for a risk of posttransfusion HCV infection of 10.7% (28 of 262 recipients seronegative for anti-HCV before transfusion). Of the 478 patients transfused after July 1989 with blood screened for anti-HCV, only 9 (1.9%) developed posttransfusion hepatitis for a risk reduction of 80%. Seven of the 9 residual cases of hepatitis were caused by HCV (7 of 456 recipients seronegative before transfusion or 1.5%) for a risk reduction of transfusion-associated HCV infection of 86%. In retrospect, an anti-HCV positive donor was detected by second-generation immunoassay in 4 (57%) of the 7 HCV cases from the study cohort and in 19 of the 23 (83%) cases from whom all donor samples were available for testing in the historical cohort. No additional infectious donors were detected by third-generation immunoassay or serum HCV-RNA by polymerase chain reaction. Implementation of donor screening for anti-HCV with a first-generation immunoassay appeared to be independently associated with an 80% and 86% reduction in the risks of transfusion-associated hepatitis and HCV infection, respectively. Screening of donors with second-generation immunoassays might have further reduced the risk by 57%. An additional 0.4% may be at risk of developing non-A, non-B, non-C hepatitis.

The presence and significance of hepatitis C virus infection was evaluated in 241 renal transplant recipients from our hospital. Hepatitis C virus antibodies were tested by second-generation enzyme immunoassay, followed by second- and third-generation immunoblot assays (RIBA-2 and RIBA-3); hepatitis C virus RNA was measured by nested polymerase chain reaction. Hepatitis C virus antibodies, which were detected in 46.5% of patients, were mainly present before transplantation and independently associated with the total amount of transfused blood, time in hemodialysis and duration of posttransplant follow up. Liver dysfunction (alananine aminotransferase elevation) was observed in 50% of antibody-positive recipients, and 92.5% of patients whith chronic liver disease without hepatitis B infection were infected with hepatitis C virus. Most antibody-positive patients (78.4%) tested positive by RIBA-2, but 21.6% were indeterminate; RIBA-3 was positive in 90% of these indeterminates. Hepatitis C virus RNA detection was positive in 96% of antibody-positive cases tested, in 20% of patients who were already anti-HCV negative before transplantation and also demonstrated persistence of HCV infection in all cases who, being antibody positive prior to transplantation, lost these antibodies during follow up (9% of transplanted patients). In conclusion, hepatitis C virus infection is extremely prevalent in renal transplant recipients from Spain and is the main cause of chronic liver disease in these patients. Confirmation by supplemental assays of anti-HCV antibodies is not necessary, but hepatitis C virus RNA testing is indispensable to detect those cases who lose or do not develop hepatitis C virus antibodies.

BACKGROUND The current standard for determining sustained virologic response (SVR) in patients treated for hepatitis C virus (HCV) infection is undetectable serum HCV-RNA 24 weeks after treatment. This study evaluates the value of HCV-RNA determination at 12 weeks posttreatment (W+12) to predict SVR in liver transplant (LT) patients treated with pegylated interferon and ribavirin for recurrent HCV infection. METHODS This study, performed in 2001 to 2010, included HCV-LT patients with an end-of-treatment response (undetectable serum HCV-RNA) and HCV-RNA testing at 12 and 24 weeks posttreatment (W+12/W+24). HCV-RNA was detected with a qualitative polymerase chain reaction assay (detection limit 50 IU/mL) and, when positive, measured by quantitative PCR (detection limit 600 IU/mL) up to 2006. Since 2007, a real-time PCR-based test (detection limit 15 IU/mL) has been used. The positive predictive value (PPV) was defined as the probability that SVR would occur in patients with undetectable HCV-RNA at W+12 and W+24. RESULTS Of 162 patients treated during the study period, 57 (35%) had end-of-treatment response and were included. Of these, 45 (79%) had SVR and 12 (21%) had virologic relapse. At W+12, HCV-RNA was undetectable in 45 (79%) patients, all of whom had SVR, yielding a PPV for SVR at W+12 of 100% (95% confidence interval, 75.8%-100%). CONCLUSIONS Undetectable HCV-RNA at W+12 posttreatment has a high PPV for predicting SVR. HCV-RNA testing to assess SVR at this time point seems as valid as W+24 testing and could be considered for predicting SVR in HCV-LT patients receiving treatment with pegylated interferon and ribavirin.

Background Hepatitis C decreases health related quality of life (HRQL) which is further diminished by antiviral therapy. HRQL improves after successful treatment. This trial explores the course of and factors associated with HRQL in patients given individualized or standard treatment based on early treatment response (Ditto-study). Methods The Short Form (SF)-36 Health Survey was administered at baseline (n = 192) and 24 weeks after the end of therapy (n = 128). Results At baseline HRQL was influenced by age, participating center, severity of liver disease and income. Exploring the course of HRQL (scores at follow up minus baseline), only the dimension general health increased. In this dimension patients with a relapse or sustained response differed from non-responders. Men and women differed in the dimension bodily pain. Treatment schedule did not influence the course of HRQL. Conclusions Main determinants of HRQL were severity of liver disease, age, gender, participating center and response to treatment. Our results do not exclude a more profound negative impact of individualized treatment compared to standard, possibly caused by higher doses and extended treatment duration in the individualized group. Antiviral therapy might have a more intense and more prolonged negative impact on females.

Background: Selection of amino acid substitutions associated with resistance to nucleos(t)ide-analog (NA) therapy in the hepatitis B virus (HBV) reverse transcriptase (RT) and their combination in a single viral genome complicates treatment of chronic HBV infection and may affect the overlapping surface coding region. In this study, the variability of an overlapping polymerase-surface region, critical for NA resistance, is investigated before treatment and under antiviral therapy, with assessment of NA-resistant amino acid changes simultaneously occurring in the same genome (linkage analysis) and their influence on the surface coding region. Methodology/Principal Findings: Serum samples obtained from chronic HBV-infected patients at pre-treatment and during sequential NA treatment with lamivudine, adefovir, and entecavir were analyzed by ultra-deep pyrosequencing (UDPS) using the GS-FLX platform (454 Life Sciences-Roche). The pre-treatment HBV quasispecies was not enriched with NA-resistant substitutions. The frequencies of this type of substitutions at pre-treatment did not predict the frequencies observed during lamivudine treatment. On linkage analysis of the RT region studied, NA-resistant HBV variants (except for rtA181T) were present in combinations of amino acid substitutions that increased in complexity after viral breakthrough to entecavir, at which time the combined variant rtL180M-S202G-M204V-V207I predominated. In the overlapping surface region, NA-resistant substitutions caused selection of stop codons in a significant percentage of sequences both at pre-treatment and during sequential treatment; the rtA181T substitution, related to sW172stop, predominated during treatment with lamivudine and adefovir. A highly conserved RT residue (rtL155), even more conserved than the essential residues in the RT catalytic motif YMDD, was identified in all samples. Conclusions: UDPS methodology enabled quantification of HBV quasispecies variants, even those harboring complex combinations of amino acid changes. The high percentage of potentially defective genomes, especially in the surface region, suggests effective trans-complementation of these variants. © 2012 Rodriguez-Frías et al., Centro para el Desarrollo Tecnológico Industrial; Ministry of Economy and Competitiveness

Background HCV is a major cause of morbidity and mortality in HIV-coinfected patients. Several observational studies have suggested that HCV response to pegylated interferon and ribavirin is lower in HIV-coinfected patients treated with abacavir. It has been postulated that abacavir could compete with ribavirin to be phosphorylated, leading to a reduction in the active form of the drug (triphosphorylated ribavirin). Here, we studied the effect of abacavir, tenofovir or lamivudine addition on the suppressive activity of ribavirin in an HCV RNA replicon system. Methods We used the human hepatoma HuH-7 cell clone 9B containing the HCV genotype 1b replicon I389/ NS3-3’. Cells were treated for 24 h with ribavirin (0, 10 and 50 μM) plus abacavir, tenofovir or lamivudine at doses of 0, 10 and 50 μM and HCV RNA production was quantified by real-time PCR in triplicate assays. Results were expressed as mean ±sd of the HCV RNA produced per cell (log10 IU/cell). Means were compared using the Student's t-test. Results Ribavirin treatment produced a dose-dependent suppression of HCV RNA production by the replicon system. Combination of ribavirin and interferon resulted in an additive antiviral activity. The addition of abacavir did not modify the suppressive activity of ribavirin on the replicon HCV RNA expression. Similar results were obtained when ribavirin was used in combination with tenofovir or lamivudine. Conclusions In a subgenomic HCV RNA replicon system, the antiviral effect of ribavirin was not modified by the addition of abacavir, tenofovir or lamivudine.

Background: Combination of pegylated interferon (Peg-IFN) and ribavirin is the standard treatment for HCV infection in HIV co-infected patients. However, data available on the efficacy of this therapy in co-infected patients who failed a former interferon-based regimen are limited. Methods: We analysed the efficacy and safety of the Peg-IFN alfa-2a or alfa-2b plus ribavirin combination in a multicentre observational cohort study including 54 HCV/HIV co-infected patients who had failed to respond to or relapsed on interferon-based treatment. The primary efficacy endpoint was the proportion of patients who achieved a sustained virological response (SVR), defined as HCV RNA

Background Emerging data suggest that higher ribavirin (RBV) exposure could improve early hepatitis C virus (HCV) response. Furthermore, interindividual RBV bioavailability shows high variation, and dose-limiting haemolytic anaemia is a common adverse event. Therefore, it has been suggested that monitoring RBV serum levels could be used to drive dose modification and to optimize management of HCV-infected patients receiving combination treatment. Methods To assess the effect of RBV serum levels on HCV RNA clearance at week 4 and 12 of treatment, and to determine the correlation between RBV serum concentration and haemoglobin decrease, RBV trough levels were measured by HPLC in stored serum samples obtained from 94 HCV-HIV-coinfected patients at week 4 and 12 of treatment with peginterferon-α2b (1.5 μg/kg/weekly) plus ribavirin (800–1,200 mg/day). Results The median RBV levels increased from 1.70 μg/ml at week 4 to 1.97 μg/ml at week 12 of treatment ( P=0.001) and were independently predicted by weight-adjusted dose of RBV and co-administration of tenofovir. Haemoglobin drop was higher among patients who received zidovudine and weakly correlated with RBV level. Although RBV concentration was lower in genotype 1 or 4 HCV-infected patients who cleared the virus at treatment week 4, the ability of this parameter to discriminate between responders and non-responders at treatment week 4 and 12 was poor. Conclusion Intracellular RBV accumulation early in treatment might improve the kinetics of HCV response in difficult to treat patients. Although this hypothesis and the potential interaction between RBV and tenofovir warrant further research, our data do not support RBV serum monitoring as a tool to optimize treatment in HCV-HIV-coinfected patients.

To investigate the utility of a week-4 virological response for sustained response prediction in hepatitis C virus (HCV) genotype 3/HIV-co-infected patients treated with interferon and ribavirin for 24 weeks.Using a real-time polymerase chain reaction-based quantitative assay (COBAS AmpliPrep-COBAS-TaqMan 48; Roche Diagnostics) we retrospectively analysed samples obtained at baseline and weeks 4 and 12 from a subset of 35 HCV genotype 3-HIV co-infected patients enrolled in a randomized comparative trial of peginterferon alpha-2b versus interferon alpha-2b both in combination with ribavirin.In an intention-to-treat analysis, 78% of patients treated with peginterferon and 53% of those receiving standard interferon achieved a sustained virological response (SVR) Overall, at 4 weeks, 49% of patients had HCV RNA50 IU/ml and 63% had600 IU/ml. Of these rapid responders 88 and 86% achieved a SVR, respectively, with only one patient relapsing among end-of-treatment responders. In contrast, only 44 and 31% of patients with a week-4 HCV RNAor= 50 oror= 600 IU/ml achieved an SVR, respectively, with relapse rates of 33 and 50%, respectively. In multivariate logistic regression analysis a serum HCV RNA level below 600 IU/ml at week 4 was the strongest independent predictor of SVR (odds ratio, 11.3; 95% confidence interval, 1.7 to 75.0; P = 0.012).Monitoring early viral response may be useful to tailor the duration of treatment among patients with HCV genotype 3/HIV-co-infection. Patients whose HCV RNA falls below 600 IU/ml at 4 weeks are at low risk of relapse after 24 weeks of combination therapy.

Development of vaccination strategies against hepatitis C virus (HCV) is of paramount importance. With this aim, we tested the ability of dendritic cell-activating reagents polyinosinic-polycytidylic acid (poly(I:C)) and anti-CD40, as adjuvants to induce T-cell responses against HCV. Immunization of mice with these adjuvants induced dendritic cell maturation in vivo. Also, joint administration of poly(I:C) and anti-CD40 plus HCV antigens had a synergistic effect on the induction of anti-HCV T-cell responses. CD4 responses displayed a Th1 cytokine profile, and CD8 responses could be induced by immunization with a minimal CD8 epitope. Addition of a low amount of NS3 protein (as a source of Th epitopes) to the immunization mixture enhanced CD8 responses, whereas immunization with higher doses of NS3 induced both CD4 and CD8 responses. Surprisingly, immunization with NS3 protein but not with CD8 epitopes was able to induce CD8 responses and able to recognize cells expressing HCV antigens endogenously. Moreover, immunization with these adjuvants activated NK cells, which in turn helped to induce Th1 responses. Finally, this combined immunization protocol afforded long-lasting T-cell responses, suggesting that this strategy may prove to be useful in vaccination and/or treatment of HCV infection.

OBJECTIVES—To characterise phenotypically the minor salivary glands of patients with clinical and histological features of Sjögren's syndrome (SS) infected with hepatitis C virus (HCV). PATIENTS AND METHODS—75 consecutive patients with SS (31 primary SS, 44 secondary SS) diagnosed by preliminary European classification criteria. The presence of anti-HCV antibodies was detected by commercial third generation ELISA and by a second generation immunoblot assay. Presence of HCV genome in serum was determined by polymerase chain reaction analysis. Expression of CD3, CD4, CD8, CD20, HLA-DR, and CD25 molecules in lymphocytic and epithelial cells on minor salivary glands was detected by immunohistochemical assays. Expression of interferon γ and interleukin 4 cytokines was determined by in situ hybridisation. RESULTS—Six of 31 primary SS (19%) and one of 44 secondary SS (2%) serum samples were positive for anti-HCV by ELISA. Three samples were positive, three indeterminate, and one sample corresponding to a secondary SS patient was negative by immunoblot. The three immunoblot positive serum samples were also HCV-RNA positive by PCR assay. The study of lymphocytic cells in the diffuse infiltrate of minor salivary glands showed a predominance of the CD3 lymphocytic population. A predominance of CD4 over CD8 T cells (ratio 2:1) was observed in HCV and non- HCV infected patients. The analysis of the lymphocytic focus showed that the HCV infected patients had a predominance of CD20 positive cells. Activation molecules ( CD-25 and HLA-DR ) were expressed in HCV and non-HCV infected patients in lymphocytic and epithelial cells, however epithelial cell expression of CD25 was low in HCV infected patients. As expected, a pronounced Th1 response was observed in the lymphocytic foci of HCV patients. CONCLUSIONS—HCV infected patients may develop an autoimmune sialadenitis, similar to that described in primary SS.

Objectives: To study the viral requirements for attachment, entry and infection using natural isolates of HCV (infectious plasma of a patient infected with HCV from genotype 1b) and a cell line which has previously been shown to support HCV replication (Daudi cells). Methods: We studied attachment of HCV to Daudi cells, a human B-cell line. Quantification was done by real-time RT-PCR. Results: The best attachment levels were obtained using plasma depleted of immunocomplexes. Results of kinetics of HCV attachment to Daudi cells show that attachment is maximum at pH 7.0, and that it decreased drastically at any other pH studied (5.5, 6.0, 6.5, 7.5 and 8.0). Conclusions: Depletion of immunocomplexes and pH control during infection are important parameters to improve attachment of HCV to Daudi cells using plasma as a natural source of virus.

PMCID: PMC443556.-- Final edited version available at: http://dx.doi.org/10.1093/nar/gnh088, Many studies have tried to identify specific nucleotide sequences in the quasispecies of hepatitis C virus (HCV) that determine resistance or sensitivity to interferon (IFN) therapy, unfortunately without conclusive results. Although viral proteins represent the most evident phenotype of the virus, genomic RNA sequences determine secondary and tertiary structures which are also part of the viral phenotype and can be involved in important biological roles. In this work, a method of RNA structure analysis has been developed based on the hybridization of labelled HCV transcripts to microarrays of complementary DNA oligonucleotides. Hybridizations were carried out at non-denaturing conditions, using appropriate temperature and buffer composition to allow binding to the immobilized probes of the RNA transcript without disturbing its secondary/tertiary structural motifs. Oligonucleotides printed onto the microarray covered the entire 5' non-coding region (5'NCR), the first three-quarters of the core region, the E2–NS2 junction and the first 400 nt of the NS3 region. We document the use of this methodology to analyse the structural degree of a large region of HCV genomic RNA in two genotypes associated with different responses to IFN treatment. The results reported here show different structural degree along the genome regions analysed, and differential hybridization patterns for distinct genotypes in NS2 and NS3 HCV regions., M.M. was supported in part by grants DITTO-HCV QLK2-2000-0836 from the European Commission FP5 and Red Nacional de Gastroenterología y Hepatología (C03/02). The work in Hospital Vall d’Hebron was supported in part by grants SAF2000/0183 and PROFIT (FIT-010000-2003-51) from the Ministerio de Ciencia y Tecnología. The work at CAB was supported by the European Union, Instituto Nacional de Técnica Aerospacial, Ministerio de Ciencia y Tecnología and Comunidad de Madrid.

Background Approximately 40% of patients infected with hepatitis C virus (HCV) have no history of blood transfusion or other high-risk practice. Other causes should be considered. Goal The goal was to describe sexual transmission of HCV from a chronic carrier to his female partner during unprotected vaginal intercourse after removal of an intrauterine device. Study Design A heterosexual woman who was a regular blood donor acquired acute HCV infection from her sex partner, who was chronically infected. The study included an interview and phylogenetic analysis of sequences of virus obtained from the couple. Results Risk factors other than vaginal sexual relations with her partner were ruled out. Phylogenetic analysis showed that sequences from the couple clustered together in all trees generated, in comparison with local and GenBank controls. Conclusion The temporal relationship between removal of the intrauterine device and the acute hepatitis suggests that vaginal mucosal damage might have favored transmission of HCV. Barrier precautions should be suggested whenever damage of the vaginal tract has occurred.

Background/Aims : Hepatitis C has been associated with a decrease in quality of life and with neurological abnormalities. The aim of our study was to investigate the relationship between quality of life and cognitive function. Methods : Quality of life, clinical variables and neuropsychological function were evaluated in 120 patients with hepatitis C (mild chronic hepatitis, compensated cirrhosis and decompensated cirrhosis) and in healthy controls ( n =40, in each group). Results : Patients with chronic hepatitis or compensated cirrhosis showed a decrease in quality of life, in spite of unimpaired neuropsychological tests. Patients with decompensated cirrhosis exhibited a further decrease in quality of life and neuropsychological abnormalities. The decrease in quality of life was associated with the severity of liver failure, neuropsychological abnormalities and treatment with beta-blockers or diuretics. However, in the multivariable analysis, only treatment with beta-blockers or diuretics (which was limited to decompensated cirrhosis) was independently associated with quality of life. Conclusions : Hepatitis C causes a decrease in quality of life even in the absence of major cognitive impairment. The mechanisms that worsen quality of life are unknown. However, in cirrhotic outpatients with prior decompensations, treatment with beta-blockers or diuretics appears to have an important effect on quality of life.

The aim of this retrospective study was to determine the predictive value of alanine aminotransferase (ALT) levels for histologic findings in patients with chronic hepatitis C virus (HCV) infection. Data on 864 HCV RNA-positive patients were collected. ALT values were obtained at the time of biopsy (before treatment), and normal ALT values were defined as normal values obtained at serial evaluations during a 6-month period. Histologic results were scored using the METAVIR system. Among all patients, 99% of those with elevated ALT levels had a score of at least F1 (positive predictive value [PPV], 99%) and 88% had a score greater than A1F1. Among patients with persistently normal ALT values, 65% had a score of at least F1 (negative predictive value [NPV], 35%) and 26% had a score greater than A1F1. The receiver operating characteristics analysis indicates that the ALT threshold for the best compromise sensitivity-specificity was about 2.25 times the upper limit of normal (ULN). In conclusion, almost all HCV RNA-positive patients with elevated ALT levels have some degree of fibrosis. However, an important proportion of patients with persistently normal ALT levels also show some histologic signs of fibrosis; the degree of fibrosis is usually mild but is sometimes more marked, and in rare cases cirrhosis may be present. In this subset of patients, the indication of liver biopsy and the potential benefit of therapy need to be further evaluated. These results suggest the need to revisit the algorithm for liver biopsy practice.

We have found that RNase P from HeLa cells specifically and efficiently cleaves hepatitis C virus (HCV) transcripts in vitro. The evidence includes identification of the 5'-phosphate polarity of the newly generated termini at position A(2860) as well as immunological and biochemical assays. Active cleavage has been shown in five dominant sequences of HCV "quasispecies" differing at or near the position of cleavage, demonstrating that this is a general property of HCV RNA. During the analysis, a second cleavage event was found in the 3' domain of the internal ribosome entry site. We have found that HCV RNA competitively inhibits pre-tRNA cleavage by RNase P, suggesting that HCV RNA has structural similarities to tRNA. This finding sets HCV apart from other pathogens causing serious human diseases and represents the first description of human RNase P-viral RNA cleavage. Here we discuss the possible meaning of these RNase P-accessible structures built into the viral genome and their possible role in vivo. Moreover, such structures within the viral genome might be vulnerable to attack by therapeutic strategies.

We have conducted an open, prospective trial to assess the safety and efficacy of interferon alfa-2b and ribavirin in combination for the treatment of chronic hepatitis C in human immunodeficiency virus (HIV)-infected hemophiliacs. Twenty hemophiliacs coinfected with HIV and hepatitis C virus (HCV), 18 of them under highly active antiretroviral therapy (HAART), with a mean CD4+ cell count of 490 ± 176 cells/mm3 and undetectable (n = 9) or low-level HIV RNA (

To evaluate the utility of early testing for hepatitis C viremia as a predictor of treatment outcome during interferon or combination therapy.We studied 184 patients with chronic hepatitis C who received interferon and were monitored for HCV RNA. Sixty-two patients received interferon alone for 12 months and 122 patients, who were still HCV RNA positive at 2 months, received an additional 12-month course of interferon and ribavirin combination therapy.Using this strategy, sustained response occurred in a total of 34 patients (18.5%). Independent variables associated with sustained response were HCV genotype (p=0.06), viral loador = 5.1 logs/ml (p= 0.005) and negative HCV RNA at 1 month (p0.0001) in the interferon group, and female sex (p=0.04), genotype (p=0.03), viral loador = 5.5 logs/ml (p=0.01), normal ALT (p=0.001) and decline in viral loador = 1.2 logs/ml after 2 months of interferon monotherapy (p0.001) and negative viremia at 5 months of ribavirin onset (p0.0001) in the combination therapy group. Persistence of viremia at 1 month of interferon monotherapy and at 5 months of combination therapy were the strongest predictors of non-response (negative predictive value of 100% and 99%, respectively).Qualitative assessment of HCV RNA during treatment is the strongest predictor of sustained response during interferon or combination therapy for chronic hepatitis C.

Background A new virus of the Flaviviridae family, the hepatitis G virus (HGV/HGBV-C), has been identified recently. The purpose of this study was to determine the prevalence of HGV infection in healthy children, in patients with liver disease, and in human immunodeficiency virus (HIV)-1-infected patients. The role of HGV in the clinical course of chronic HCV, the response to interferon-alpha2b, and the possible implications of intravenous gamma-globulin in the transmission of the virus were also evaluated. Methods Fifty healthy children, 66 patients with a variety of liver diseases, 19 patients with acquired immune deficiency syndrome (AIDS), and various batches of commercial intravenous immunoglobulins were investigated. Viral HGV RNA (5'NCR-NS5) and anti-HGV envelope protein E2 were assayed. Results The prevalence of HGV infection was 6% in the healthy children and 42% in the liver disease group. Viremia and anti-E2 were found in 11% and 79% of patients with AIDS. Four (27%) of 15 patients with chronic HCV, receiving treatment with interferon, were coinfected by HGV and became HGV-RNA negative during therapy. One year after the end of interferon therapy, three of them were again HGV RNA positive. Conclusions The prevalence of HGV infection is high in healthy children higher in children affected with liver disease, but its potential pathologic implication is questionable, and further studies are warranted. Hepatitis G virus is sensitive to interferon therapy, although the infection often recurs after discontinuation of treatment.

GBV-C/Hepatitis G virus (GBV-C/HGV) is a newly discovered viral agent, found widely among healthy blood donors and among individuals at risk of parenterally transmitted infections. GBV-C/HGV is found frequently in coinfection with HCV. A population of 109 HCV positive patients was examined for the presence of GBV-C/HGV RNA and antibodies to E2. Of the 109 patients, 23 (21%) had serum GBV-C/HGV RNA in serum, 39 (36%) had only antibodies to E2 and 8 (7%) were positive for both markers, with an overall prevalence of 64%. Different serologic and virological patterns were observed in GBV-C/HGV exposed patients according to their infection status. Active infection was characterized by positive RT/PCR signal with primers for both the 5'UTR and NS5 genomic regions, viremia levels above 10(4) copies/mL by real time quantitative RT/PCR and absence of detectable anti-E2. In the transition phase between active infection and recovery, GBV-C/HGV RNA was only detectable by RT/PCR using primers from the 5' untranslated region and viremia levels were below 10(4) copies/ml by quantitative PCR, with or without simultaneous presence of anti-E2 antibodies. Resolved infection was characterized by absence of detectable viremia and, in most patients, by the presence of anti-E2.