Looking at the Molecular Target of NS5A Inhibitors throughout a Population Highly Affected with Hepatitis C Virus

Abstract

Hepatitis C virus (HCV) is associated with liver damage and an increased progression rate to cirrhosis and hepatocellular carcinoma. In Portugal, it is prevalent in vulnerable populations such as injection drug users (IDU). HCV is characterized by a high intra-host variability, and the selecting driving forces could select variants containing resistance-associated substitutions (RAS) that reduce treatment effectiveness. The main goal of this study was to analyze the sequence variation of NS5A in treatment-naïve IDU. The epidemiological and clinical status of hepatitis C were analyzed, and samples were sequenced by Sanger and Next-Generation sequencing (NGS) to assess RAS and confirm HCV subtypes. Phylogenetic classification was concordant: 1a, 52.4%; 1b, 10.7%; 3a, 20.2%; 4a, 8.3%; 4d, 7.1%; and one 2k/1b recombinant. A 1a/3a mixed infection was detected by NGS. RAS were found in 34.5% (29/84) of samples using Sanger sequencing, while in 42.9% (36/84) using NGS. In sequences from subtypes 1a and 1b, RAS K24R, M28V, Q30H/R, H58D/P/Q/R, and RAS L31M and P58S were detected, respectively. In subtype 3a, RAS A30S/T, Y93H and polymorphisms in position 62 were identified. Additionally, RAS P58L was detected in genotype 4. The strategy used for the molecular survey of baseline HCV resistance is of particular importance to achieve treatment effectiveness and contribute to the elimination of hepatitis C.

ReporType: A Flexible Bioinformatics Tool for Targeted Loci Screening and Typing of Infectious Agents

“…Hepatitis C virus (HCV) can cause both acute and chronic hepatitis, ranging in severity from a mild illness to a serious, lifelong illness, including liver cirrhosis and cancer [ 42 , 43 ]. Genotypes are traditionally defined based on the genetic variability of core, E1, and NS5B regions [ 42 , 43 ], although other polymorphic regions, such as the NS4B and NS5A genes (targets of anti-viral resistance mutations), have also proven suitable for this purpose [ 43 ].…”

“…Hepatitis C virus (HCV) can cause both acute and chronic hepatitis, ranging in severity from a mild illness to a serious, lifelong illness, including liver cirrhosis and cancer [ 42 , 43 ]. Genotypes are traditionally defined based on the genetic variability of core, E1, and NS5B regions [ 42 , 43 ], although other polymorphic regions, such as the NS4B and NS5A genes (targets of anti-viral resistance mutations), have also proven suitable for this purpose [ 43 ]. In the present study, we constructed two ABRIcate databases, one of them (“HCV_complete”) containing sequences from a fragment of the NS4B-NS5A region (n = 19), and the other (“HCV_partial”) containing only partial NS5A sequences (n = 19).…”

“…In the present study, we constructed two ABRIcate databases, one of them (“HCV_complete”) containing sequences from a fragment of the NS4B-NS5A region (n = 19), and the other (“HCV_partial”) containing only partial NS5A sequences (n = 19). This proof-of concept exercise strictly focused on testing samples analyzed in a recent article [ 43 ] conducted by the Portuguese NRL of HIV and hepatitis B and C, so it did not intend to target the whole-HCV genotype diversity. The test datasets included 83 samples with known HCV genotype, for which both genomic regions (NS4B-NS5A and partial NS5A) were provided by the NRL in raw Sanger format and respective FASTA (after manual curation) [ 43 ].…”

“…This proof-of concept exercise strictly focused on testing samples analyzed in a recent article [ 43 ] conducted by the Portuguese NRL of HIV and hepatitis B and C, so it did not intend to target the whole-HCV genotype diversity. The test datasets included 83 samples with known HCV genotype, for which both genomic regions (NS4B-NS5A and partial NS5A) were provided by the NRL in raw Sanger format and respective FASTA (after manual curation) [ 43 ]. In addition, the consensus sequences generated from amplicon-based Illumina sequencing of the same samples [ 43 ] were also screened against both “HCV_complete” and “HCV_partial” databases.…”

“…The test datasets included 83 samples with known HCV genotype, for which both genomic regions (NS4B-NS5A and partial NS5A) were provided by the NRL in raw Sanger format and respective FASTA (after manual curation) [ 43 ]. In addition, the consensus sequences generated from amplicon-based Illumina sequencing of the same samples [ 43 ] were also screened against both “HCV_complete” and “HCV_partial” databases. Of note, as the HCV databases do not cover the virus genotype diversity, they are available in an independent database folder (“databases_only_test”) in the GitHub repository.…”