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The use of new Direct Acting Antivirals, specific of HCV, has greatly improved the HCV treatment. Most of the DAA are specific of HCV genotypes. Genotyping methods may target different regions of the HCV genome, though only the whole genome sequencing could confirm the correct genotype. The present study describes the virological investigation of a treatment failure due to the false identification of an unusual 2k/1b recombinant HCV form. It describes the sequencing methods, and the similarity analysis of the sequences to different genotype query sequences, to identify the recombination breakpoint., (© 2018 Wiley Periodicals, Inc.)

Direct-acting antiviral (DAA) drug performances depend on the viral genotype. So international recommendations give typing of the virus a prerequisite for treatment choice and patient management. Commercially available HCV genotyping kits are scarce and this analysis is often in-house using tedious PCRs and Sanger sequencing, leading to a lack of standardization. A newly commercialized HCV genotyping assay based on real-time PCR has been developed by Roche Diagnostics (Mannheim, Germany). We compared this new assay with our in-house PCRs -sequencing technique on 101 regular samples and 81 LiPA failures or low viral load samples. No genotype or 1a/1b subtype mismatch was observed. Two samples were misidentified at the subtype level without clinical impact. Three genotype 1b and two genotype 1a samples with low viral load could not be subtyped. Nevertheless, 13 (13%) samples from the regular panel and 35 (43%) from the more difficult-to-type panels failed to give results on first pass with the Roche kit. Failures were mostly associated with genotype 3 subtype a, with genotype 4 subtype non-a, or with viral loads <200 IU/mL (p = 0.0061). The workflow allowed a non-specialized technician to obtain results in less than 4 hours whereas 2 to 3 days and experienced staff were required with the in-house assay. In conclusion, the Roche cobas® HCV GT kit is easy and rapid to use and provides reliable results. The high rate of uninterpretable results particularly for low viral load samples and less frequent genotypes, and the absence of subtyping for non-genotype 1 could require sending complex samples to a specialized laboratory.

Background: Recent data have suggested that failure to achieve sustained virological response with direct-acting antiviral therapy is usually due to relapse and is primarily associated with the emergence of resistance-associated substitutions. The aim of this study was to investigate the prevalence and characterization of non-structural-5A resistance-associated substitutions in patients infected with HCV genotypes 1, 3 and 4 treated by direct-acting antiviral therapy, including anti-non-structural-5A, and to characterize the pre-existing resistance-associated substitutions in subjects treated with anti-non-structural-5A inhibitors., Methods: From January 2014 to March 2016, 2,995 patients infected with HCV genotypes 1, 3 and 4 were exposed to non-structural-5A inhibitors. Sequencing results at the time of virological failure were available for 61 patients; sequencing at baseline was available for 35 of these patients., Results: Among the 35 patients with sequencing results available at baseline, 15 had no resistance-associated substitution, 16 had only one resistance-associated substitution, and 4 had more than one resistance-associated substitution. Resistance-associated substitutions were harbored in 57% of the sequences in the non-structural-5A region. Among the 61 patients sequenced at virological failure, 50 (82%) patients presented at least one resistance-associated substitutions inducing a high level of resistance to non-structural-5A inhibitors (>10-fold resistance)., Conclusions: This pooled analysis suggests that non-structural-5A resistance-associated substitutions screening should be recommended when considering retreatment with a non-structural-5A inhibitor regimen in patients who have previously experienced failed non-structural-5A treatment.

Background: The existing literature about HCV association with, and replication in mosquitoes is extremely poor. To fill this gap, we performed cellular investigations aimed at exploring (i) the capacity of HCV E1E2 glycoproteins to bind on Aedes mosquito cells and (ii) the ability of HCV serum particles (HCVsp) to replicate in these cell lines., Methods: First, we used purified E1E2 expressing baculovirus-derived HCV pseudo particles (bacHCVpp) so we could investigate their association with mosquito cell lines from Aedes aegypti (Aag-2) and Aedes albopictus (C6/36). We initiated a series of infections of both mosquito cells (Ae aegypti and Ae albopictus) with the HCVsp (Lat strain - genotype 3) and we observed the evolution dynamics of viral populations within cells over the course of infection via next-generation sequencing (NGS) experiments., Results: Our binding assays revealed bacHCVpp an association with the mosquito cells, at comparable levels obtained with human hepatocytes (HepaRG cells) used as a control. In our infection experiments, the HCV RNA (+) were detectable by RT-PCR in the cells between 21 and 28 days post-infection (p.i.). In human hepatocytes HepaRG and Ae aegypti insect cells, NGS experiments revealed an increase of global viral diversity with a selection for a quasi-species, suggesting a structuration of the population with elimination of deleterious mutations. The evolutionary pattern in Ae albopictus insect cells is different (stability of viral diversity and polymorphism)., Conclusions: These results demonstrate for the first time that natural HCV could really replicate within Aedes mosquitoes, a discovery which may have major consequences for public health as well as in vaccine development.

Background: The emergence of new strains in RNA viruses is mainly due to mutations or intra and inter-genotype homologous recombination. Non-homologous recombinations may be deleterious and are rarely detected. In previous studies, we identified HCV-1b strains bearing two tandemly repeated V3 regions in the NS5A gene without ORF disruption. This polymorphism may be associated with an unfavorable course of liver disease and possibly involved in liver carcinogenesis. Here we aimed at characterizing the origin of these mutant strains and identifying the evolutionary mechanism on which the V3 duplication relies., Methods: Direct sequencing of the entire NS5A and E1 genes was performed on 27 mutant strains. Quasispecies analyses in consecutive samples were also performed by cloning and sequencing the NS5A gene for all mutant and wild strains. We analyzed the mutant and wild-type sequence polymorphisms using Bayesian methods to infer the evolutionary history of and the molecular mechanism leading to the duplication-like event., Results: Quasispecies were entirely composed of exclusively mutant or wild-type strains respectively. Mutant quasispecies were found to have been present since contamination and had persisted for at least 10 years. This V3 duplication-like event appears to have resulted from non-homologous recombination between HCV-1b wild-type strains around 100 years ago. The association between increased liver disease severity and these HCV-1b mutants may explain their persistence in chronically infected patients., Conclusions: These results emphasize the possible consequences of non-homologous recombination in the emergence and severity of new viral diseases.

Therapy for hepatitis C is currently undergoing a revolution. The arrival of new antiviral agents targeting viral proteins reinforces the need for a better knowledge of the viral strains infecting each patient. Hepatitis C virus (HCV) whole genome sequencing provides essential information for precise typing, study of the viral natural history or identification of resistance-associated variants. First performed with Sanger sequencing, the arrival of next-generation sequencing (NGS) has simplified the technical process and provided more detailed data on the nature and evolution of viral quasi-species. We will review the different techniques used for HCV complete genome sequencing and their applications, both before and after the apparition of NGS. The progress brought by new and future technologies will also be discussed, as well as the remaining difficulties, largely due to the genomic variability.

The pretherapeutic presence of protease inhibitor (PI) resistance-associated variants (RAVs) has not been shown to be predictive of triple-therapy outcomes in treatment-naive patients. However, they may influence the outcome in patients with less effective pegylated interferon (pegIFN)-ribavirin (RBV) backbones. Using hepatitis C virus (HCV) population sequence analysis, we retrospectively investigated the prevalence of baseline nonstructural 3 (NS3) RAVs in a multicenter cohort of poor IFN-RBV responders (i.e., prior null responders or patients with a viral load decrease of <1 log IU/ml during the pegIFN-RBV lead-in phase). The impact of the presence of these RAVs on the outcome of triple therapy was studied. Among 282 patients, the prevalances (95% confidence intervals) of baseline RAVs ranged from 5.7% (3.3% to 9.0%) to 22.0% (17.3% to 27.3%), depending to the algorithm used. Among mutations conferring a >3-fold shift in 50% inhibitory concentration (IC50) for telaprevir or boceprevir, T54S was the most frequently detected mutation (3.9%), followed by A156T, R155K (0.7%), V36M, and V55A (0.35%). Mutations were more frequently found in patients infected with genotype 1a (7.5 to 23.6%) than 1b (3.3 to 19.8%) (P = 0.03). No other sociodemographic or viroclinical characteristic was significantly associated with a higher prevalence of RAVs. No obvious effect of baseline RAVs on viral load was observed. In this cohort of poor responders to IFN-RBV, no link was found with a sustained virological response to triple therapy, regardless of the algorithm used for the detection of mutations. Based on a cross-study comparison, baseline RAVs are not more frequent in poor IFN-RBV responders than in treatment-naive patients and, even in these difficult-to-treat patients, this study demonstrates no impact on treatment outcome, arguing against resistance analysis prior to treatment., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

For optimal antiviral therapy, the hepatitis C virus (HCV) genotype needs to be determined, as it remains a strong predictor of sustained viral response. In this study, we assessed the number of HCV genotyping results that could not be determined using the commercially available line probe assay (LiPA) (Versant hepatitis C virus genotype 2.0 assay) in a large international panel of samples from 9,874 HCV-positive patients. In-house sequencing assays targeting the 5' untranslated region (UTR), core region, NS3 region, and NS5B region of the HCV genome and phylogenetic analyses were used to resolve these LiPA failures. Among all cases, the genotypes of 51 samples (0.52%) could not be determined with the LiPA. These undetermined results were observed more frequently among samples from non-European regions (mainly the Arabian Peninsula). The use of sequencing assays coupled with phylogenetic analysis provided reliable genotype results for 86% of the LiPA failures, which exhibited higher rates of genotypes 4, 5, and 6 than did LiPA-resolved genotypes. As expected, the 5' UTR was not sufficiently variable for clear discrimination between genotypes 1 and 6, but it also resulted in errors in classification of some genotype 3 and 4 cases using well-known Web-based BLAST programs. This study demonstrates the low frequency of genotyping failures with the Versant hepatitis C virus genotype 2.0 assay (LiPA) and also underlines the need for a complex combination of sequences and phylogenetic analyses in order to genotype these particular HCV strains correctly.

Hepatitis C virus (HCV) protease inhibitor resistance-associated substitutions are selected during triple-therapy breakthrough. This multicenter quality control study evaluated the expertise of 23 French laboratories in HCV protease inhibitor resistance genotyping. A panel of 12 well-defined blinded samples comprising two wild-type HCV strains, nine transcripts from synthetic NS3 mutant samples or from clinical strains, and one HCV RNA-negative sample was provided to the participating laboratories. The results showed that any laboratory with expertise in sequencing techniques should be able to provide reliable HCV protease inhibitor resistance genotyping. Only a 0.7% error rate was reported for the amino acid sites studied. The accuracy of substitution identification ranged from 75% to 100%, depending on the laboratory. Incorrect results were mainly related to the methodology used. The results could be improved by changing the primers and modifying the process in order to avoid cross-contamination. This study underlines the value of quality control programs for viral resistance genotyping, which is required prior to launching observational collaborative multicenter studies on HCV resistance to direct-acting antiviral agents.

Direct-acting antiviral (DAA) drug performances depend on the viral genotype. So international recommendations give typing of the virus a prerequisite for treatment choice and patient management. Commercially available HCV genotyping kits are scarce and this analysis is often in-house using tedious PCRs and Sanger sequencing, leading to a lack of standardization. A newly commercialized HCV genotyping assay based on real-time PCR has been developed by Roche Diagnostics (Mannheim, Germany). We compared this new assay with our in-house PCRs -sequencing technique on 101 regular samples and 81 LiPA failures or low viral load samples. No genotype or 1a/1b subtype mismatch was observed. Two samples were misidentified at the subtype level without clinical impact. Three genotype 1b and two genotype 1a samples with low viral load could not be subtyped. Nevertheless, 13 (13%) samples from the regular panel and 35 (43%) from the more difficult-to-type panels failed to give results on first pass with the Roche kit. Failures were mostly associated with genotype 3 subtype a, with genotype 4 subtype non-a, or with viral loads <200 IU/mL (p = 0.0061). The workflow allowed a non-specialized technician to obtain results in less than 4 hours whereas 2 to 3 days and experienced staff were required with the in-house assay. In conclusion, the Roche cobas® HCV GT kit is easy and rapid to use and provides reliable results. The high rate of uninterpretable results particularly for low viral load samples and less frequent genotypes, and the absence of subtyping for non-genotype 1 could require sending complex samples to a specialized laboratory. [ABSTRACT FROM AUTHOR]