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Despite the high virological response rates achieved with current directly acting antiviral agents (DAAs) against hepatitis C virus (HCV), around 2% to 5% of treated patients do not achieve a sustained viral response. The identification of amino acid substitutions associated with treatment failure requires analytical designs, such as subtype-specific ultradeep sequencing (UDS) methods, for HCV characterization and patient management. Using this procedure, we have identified six highly represented amino acid substitutions (HRSs) in NS5A and NS5B of HCV, which are not bona fide resistance-associated substitutions (RAS), from 220 patients who failed therapy. They were present frequently in basal and posttreatment virus of patients who failed different DAA-based therapies. Contrary to several RAS, HRSs belong to the acceptable subset of substitutions according to the PAM250 replacement matrix. Their mutant frequency, measured by the number of deep sequencing reads within the HCV quasispecies that encode the relevant substitutions, ranged between 90% and 100% in most cases. They also have limited predicted disruptive effects on the three-dimensional structures of the proteins harboring them. Possible mechanisms of HRS origin and dominance, as well as their potential predictive value for treatment response, are discussed., (Copyright © 2020 American Society for Microbiology.)

Background: Despite the high sustained virological response rates achieved with current directly-acting antiviral agents (DAAs) against hepatitis C virus (HCV), around 5-10% of treated patients do not respond to current antiviral therapies, and basal resistance to DAAs is increasingly detected among treatment-naïve infected individuals. Identification of amino acid substitutions (including those in minority variants) associated with treatment failure requires analytical designs that take into account the high diversification of HCV in more than 86 subtypes according to the ICTV website (June 2017)., Methods: The methodology has involved five sequential steps: (i) to design 280 oligonucleotide primers (some including a maximum of three degenerate positions), and of which 120 were tested to amplify NS3, NS5A-, and NS5B-coding regions in a subtype-specific manner, (ii) to define a reference sequence for each subtype, (iii) to perform experimental controls to define a cut-off value for detection of minority amino acids, (iv) to establish bioinformatics' tools to quantify amino acid replacements, and (v) to validate the procedure with patient samples., Results: A robust ultra-deep sequencing procedure to analyze HCV circulating in serum samples from patients infected with virus that belongs to the ten most prevalent subtypes worldwide: 1a, 1b, 2a, 2b, 2c, 2j, 3a, 4d, 4e, 4f has been developed. Oligonucleotide primers are subtype-specific. A cut-off value of 1% mutant frequency has been established for individual mutations and haplotypes., Conclusion: The methodological pipeline described here is adequate to characterize in-depth mutant spectra of HCV populations, and it provides a tool to understand HCV diversification and treatment failures. The pipeline can be periodically extended in the event of HCV diversification into new genotypes or subtypes, and provides a framework applicable to other RNA viral pathogens, with potential to couple detection of drug-resistant mutations with treatment planning.

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., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The allocation of liver grafts from hepatitis C virus (HCV)-positive donors in HCV-infected liver transplant (LT) recipients leads to infection with two different viral populations. In a previous study, we examined quasispecies dynamics during reinfection by clonal sequencing, which did not allow an accurate characterization of coexistence and competition events. To overcome this limitation, here we used deep-sequencing analysis of a fragment of the HCV NS5B gene in six HCV-infected LT recipients who received HCV-infected grafts. Successive expansions and contractions of quasispecies complexity were observed, evolving in all cases towards a more homogeneous population. The population that became dominant was the one displaying the highest mutant spectrum complexity. In four patients, coexistence of minority mutants, derived from the donor or the recipient, were detected. In conclusion, our study shows that, during reinfection with a different HCV strain in LT recipients, the viral population with the highest diversity always becomes dominant.

Aim: 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., Methods: 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., Results: 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., Conclusion: 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.