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Hepatitis C virus genotype 5a subgenomic replicons for evaluation of direct-acting antiviral agents.

ABSTRACT: Hepatitis C virus (HCV) exists as six major genotypes that differ in geographical distribution, pathogenesis, and response to antiviral therapy. In vitro replication systems for all HCV genotypes except genotype 5 have been reported. In this study, we recovered genotype 5a full-length genomes from four infected voluntary blood donors in South Africa and established a G418-selectable subgenomic replicon system using one of these strains. The replicon derived from the wild-type sequence failed to replicate in Huh-7.5 cells. However, the inclusion of the S2205I amino acid substitution, a cell culture-adaptive change originally described for a genotype 1b replicon, resulted in a small number of G418-resistant cell colonies. HCV RNA replication in these cells was confirmed by quantification of viral RNA and detection of the nonstructural protein NS5A. Sequence analysis of the viral RNAs isolated from multiple independent cell clones revealed the presence of several nonsynonymous mutations, which were localized mainly in the NS3 protein. These mutations, when introduced back into the parental backbone, significantly increased colony formation. To facilitate convenient monitoring of HCV RNA replication levels, the mutant with the highest replication level was further modified to express a fusion protein of firefly luciferase and neomycin phosphotransferase. Using such replicons from genotypes 1a, 1b, 2a, 3a, 4a, and 5a, we compared the effects of various HCV inhibitors on their replication. In conclusion, we have established an in vitro replication system for HCV genotype 5a, which will be useful for the development of pan-genotype anti-HCV compounds.

SUBMITTER: Wose Kinge CN

PROVIDER: S-EPMC4135880 | biostudies-literature | 2014 Sep

REPOSITORIES: biostudies-literature

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Hepatitis C virus genotype 5a subgenomic replicons for evaluation of direct-acting antiviral agents.

Wose Kinge Constance N CN Espiritu Christine C Prabdial-Sing Nishi N Sithebe Nomathamsaqa Patricia NP Saeed Mohsan M Rice Charles M CM

Antimicrobial agents and chemotherapy 20140630 9

Hepatitis C virus (HCV) exists as six major genotypes that differ in geographical distribution, pathogenesis, and response to antiviral therapy. In vitro replication systems for all HCV genotypes except genotype 5 have been reported. In this study, we recovered genotype 5a full-length genomes from four infected voluntary blood donors in South Africa and established a G418-selectable subgenomic replicon system using one of these strains. The replicon derived from the wild-type sequence failed to ...[more]

PMID: 24982066

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Project description:ImportanceRecent reports based on the US Food and Drug Administration's voluntary Adverse Events Reporting System raised questions about the safety of direct-acting antivirals (DAAs) for treatment of the hepatitis C virus (HCV).ObjectiveTo assess the rates of adverse events in patients with HCV infection exposed to DAAs compared with those not exposed.Design, setting, and participantsA retrospective cohort study calculated unadjusted adverse event rates for exposed vs unexposed time, using claims and clinical data from 3 health systems between January 1, 2012, and December 31, 2017. Of 82 419 eligible adults, a total of 33 808 who met eligibility criteria (age, 18-88 years; HCV quantitative result or genotype from 2012 or later; continuously enrolled; naive to DAA treatment at baseline) were included. Marginal structural modeling methods were used to adjust time-to-event analyses for characteristics that are associated with both outcomes and probability of treatment.Interventions or exposuresExposure to DAAs compared with no DAA exposure.Main outcomes and measuresDeath, multiple organ failure, liver cancer, hepatic decompensation, acute-on-chronic liver event, acute myocardial infarction, ischemic or hemorrhagic stroke, arrhythmia, acute kidney failure, nonliver cancer, hepatitis B reactivation, hospitalizations, and emergency department visits.ResultsOf the 33 808 patients who met all inclusion criteria, 20 899 (61.8%) were men; mean (SD) age was 57.2 (10.6) years. In unadjusted analyses, DAA exposure was associated with significantly lower rates of death (10.7 vs 33.7 events per 1000 person-years; rate ratio [RR], 0.32, 95% CI, 0.25-0.40). Seven other unadjusted adverse clinical events ratios were below 70% and statistically significant favoring the DAA group: multiple organ failure (RR, 0.56; 95% CI, 0.44-0.72), liver cancer (RR, 0.62; 95% CI, 0.48-0.80), hepatic decompensation (RR, 0.62; 95% CI, 0.52-0.73), acute-on-chronic liver event (RR, 0.68; 95% CI, 0.56-0.84), acute myocardial infarction (RR, 0.64; 95% CI, 0.42-0.97), ischemic stroke (RR, 0.63; 95% CI, 0.42-0.95), and hemorrhagic stroke (RR, 0.47; 95% CI, 0.25-0.89); none favored the non-DAA group. In the marginal structural modeling-adjusted analysis, DAA exposure was associated with statistically significant lower odds of adverse events than non-DAA exposure for death (adjusted odds ratio [aOR], 0.42; 95% CI, 0.30-0.59), multiple organ failure (aOR, 0.67; 95% CI, 0.49-0.90), hepatic decompensation (aOR, 0.61; 95% CI, 0.49-0.76), acute-on-chronic liver event (aOR, 0.71; 95% CI, 0.56-0.91), and arrhythmia (aOR, 0.47; 95% CI, 0.25-0.88).Conclusions and relevanceDirect-acting antiviral exposure may not be associated with higher rates of any serious adverse events, including those related to liver, kidney, and cardiovascular systems. Safety concerns based on previous reports did not appear to be supported in this study with more comprehensive data and rigorous statistical methods.

Project description:Background:The efficacy of direct-acting antivirals (DAAs) depends on the hepatitis C virus (HCV) genotype 4 (GT4) subtype which are used in the treatment of HCV. We aimed to retrospectively investigate the baseline prevalence of HCV NS5A and NS5B polymorphisms and their impact on virological outcome in GT4-infected patients treated with various DAA regimens. Patients and methods:Available plasma samples from HCV GT4-infected patients treated with different DAA regimens were analyzed at baseline and after treatment failure, where applicable. Sanger sequencing of patient-derived NS5A and NS5B regions was performed on all available samples, while ultradeep pyrosequencing (UDPS) of NS5A and NS5B regions was performed only on samples from treatment failures at different time points. Results:Sustained virological response (SVR) was achieved by 96% (48/50) of patients. Of 16 patients with baseline NS5A sequence, polymorphisms at amino acid positions associated with drug resistance were detected only at position 58: P58 (69.2%) and T58 (30.8%). Of 21 patients with baseline NS5B sequence, N142S was detected only in the two treatment failures, both with GT4d were treated with sofosbuvir (SOF)-based regimens, suggesting a potential involvement in SOF efficacy. Two patients (patient 1 [Pt1] and patient 2 [Pt2]) relapsed. In Pt1, NS5A-T56I and NS5A-Y93H/S emerged. In Pt2, NS5A-L28F emerged and a novel NS5B resistance-associated substitution (RAS), L204F, representing 1.5% of the viral population at baseline, enriched to 71% and 91.6% during and after treatment failure, respectively. UDPS of NS5B from Pt2 indicated a mixed infection of approximately 1:5, GT1a:GT4d, at baseline and GT4d during failure. Phylogenetic analysis of NS5A sequences indicated no clustering of HCV strains from patients achieving SVR vs patients who relapsed. The mean genetic distance in NS5A sequences was 5.8%, while a lower genetic distance (3.1%) was observed in NS5B sequences. Conclusion:Results from these analyses confirm the importance of UDPS in the analysis of viral quasispecies variability and the identification of novel RASs potentially associated with DAA treatment failure in HCV GT4-infected patients.

Project description:UnlabelledHepatitis C virus (HCV) is the most common cause of mixed cryoglobulinemia syndrome (MCS). The efficacy and safety of all-oral direct-acting antiviral (DAA) therapy in HCV-associated MCS (HCV-MCS) is largely unknown. The authors studied case series of patients with HCV-MCS who were treated with sofosbuvir-based regimens and historical controls treated with pegylated interferon and ribavirin in a single health care network. HCV-MCS was defined by circulating cryoglobulin associated with systemic vasculitis symptoms. Renal involvement (n = 7) was established by kidney biopsy (n = 5) or by two or more of the following clinical findings: reduced kidney function, proteinuria, or hematuria with other causes excluded (n = 2). Twelve patients received DAA therapy between December 2013 and September 2014. Median age was 61 years, 58% were male, and 50% had cirrhosis. Median baseline serum creatinine was 0.97 mg/dL (range 0.7-2.47). Four patients received rituximab concurrent with DAA therapy. Sustained virological response rate at 12 weeks (SVR12) was 83% overall. Patients with glomerulonephritis who achieved SVR12 experienced an improvement in serum creatinine and a reduction in proteinuria. Cryoglobulin levels decreased in 89% of patients, with median percent decreasing from 1.5% to 0.5% and completely disappearing in four of nine cases who had cryoglobulins measured after treatment. Serious adverse events were infrequent (17%). In contrast, the historical cohort treated with pegylated interferon and ribavirin experienced only 10% SVR12, with 100% experiencing at least one adverse event and 50% experiencing premature discontinuation due to adverse events.ConclusionSVR12 rates for sofosbuvir-based DAA regimens in HCV-MCS were 83%, significantly higher than historical controls treated with pegylated interferon and ribavirin; patients with glomerulonephritis experienced improvement in renal function, including those not concomitantly treated with immunosuppression.

Dataset Information

Hepatitis C virus genotype 5a subgenomic replicons for evaluation of direct-acting antiviral agents.

Publications

Hepatitis C virus genotype 5a subgenomic replicons for evaluation of direct-acting antiviral agents.

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