NS5A Gene Analysis by Next Generation Sequencing in HCV Nosocomial Transmission Clusters of HCV Genotype 1b Infected Patients

Abstract

Background: The aim of the study was to investigate the intra-host variability through next-generation-sequencing (NGS) of the NS5A-gene in nosocomial transmission-clusters observed in two Italian hospitals among hepatitis C virus (HCV)-genotype-1b infected patients. Methods: HCV-sequencing was performed by Sanger-sequencing (NS3 + NS5A + NS5B) and by NGS (NS5A, MiSeq-Illumina) in 15 HCV-1b infected patients [five acute with onco-hematologic-disease and 10 (4/6 acute/chronic) with β-thalassemia]. Resistance-associated-substitutions (RAS) were analysed by Geno2pheno-algorithm. Nucleotide-sequence-variability (NSV, at 1%, 2%, 5%, 10% and 15% NGS-cutoffs) and Shannon entropy were estimated. Phylogenetic analysis was performed by Mega6-software and Bayesian-analysis. Results: Phylogenetic analysis showed five transmission-clusters: one involving four HCV-acute onco-hematologic-patients; one involving three HCV-chronic β-thalassemia-patients and three involving both HCV-acute and chronic β-thalassemia-patients. The NS5A-RAS Y93H was found in seven patients, distributed differently among chronic/acute patients involved in the same transmission-clusters, independently from the host-genetic IL-28-polymorphism. The intra-host NSV was higher in chronic-patients versus acute-patients, at all cutoffs analyzed (p < 0.05). Even though Shannon-entropy was higher in chronic-patients, significantly higher values were observed only in chronic β-thalassemia-patients versus acute β-thalassemia-patients (p = 0.01). Conclusions: In nosocomial HCV transmission-clusters, the intra-host HCV quasispecies divergence in patients with acute-infection was very low in comparison to that in chronic-infection. The NS5A-RAS Y93H was often transmitted and distributed differently within the same transmission-clusters, independently from the IL-28-polymorphism.

Amino acid substitutions associated with treatment failure of hepatitis C virus infection

“…We have also identified A218S in patients never subjected to SOF treatment, despite evidence that this substitution may jeopardize the access of SOF-triphosphate to the catalytic site of NS5B [37]. Bellocchi et al (2019) identified K78R and T64A in NS5A and C213S, S231N, N231S and A218S in HCV G1b-infected patients naïve to DAAs [43]. T64S in NS5A was listed as a secondary substitution accompanying P58S and Y93H in HCV G1b-infected patients who failed DCV therapy, with a resistance level of EC 50 <1 nmol/l [44].…”

Viral hepatitis in patients on hemodialysis

“…In the United States (US), HCV prevalence in HD units ranges from 5% to 44%, 3 and HD treatment is considered a risk factor for HCV transmission. A 1993‐1995 study in Greece estimated that 10% of patients seroconverted to HCV during their first 5 years of HD 20,22 . HCV genotype clustering suggests that transmission spreads within HD units 20,22 .…”

“…A 1993‐1995 study in Greece estimated that 10% of patients seroconverted to HCV during their first 5 years of HD 20,22 . HCV genotype clustering suggests that transmission spreads within HD units 20,22 . Hepatitis D and G infections are also more common among dialysis patients than in the general population.…”

Using targeted second-generation sequencing technique to guide clinical diagnosis and the effect of medication on the therapeutic effect and prognosis of respiratory tract infection in children: An observational study

“…[ 6 , 7 ] At present, there are 3 typical sequencing technologies in NGS field, including 454 from Roche Company (Switzerland), Solexa from Illumina Company (USA), and SOLID from ABI Company (USA), and sequencing technologies such as IonTorrent, Heliscope, Nanopore, SMRT, IonPGM and GeXp. [ 8 ] Among them, the 454-sequencing technology can read about 700 bp in length, and the sequencing time is 18 to 24 hours. The method adopted is pyrosequencing with an accuracy of 99.9%.…”