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

Cells. 2019 Jul 2;8(7):666. doi: 10.3390/cells8070666.

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.

Keywords: HCV; NGS; acute infection; chronic infection; nosocomial transmission; sequencing.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acute Disease
  • Adult
  • Amino Acid Substitution
  • Antiviral Agents / pharmacology
  • Antiviral Agents / therapeutic use
  • Blood Transfusion
  • Chronic Disease
  • Cross Infection / drug therapy
  • Cross Infection / transmission
  • Cross Infection / virology*
  • Drug Resistance, Viral / genetics
  • Female
  • Genotype
  • Hepacivirus / genetics*
  • Hepacivirus / pathogenicity
  • Hepatitis C / drug therapy
  • Hepatitis C / transmission
  • Hepatitis C / virology*
  • High-Throughput Nucleotide Sequencing
  • Host-Pathogen Interactions / genetics
  • Host-Pathogen Interactions / immunology
  • Humans
  • Interferons / genetics
  • Interferons / immunology
  • Male
  • Middle Aged
  • Phylogeny
  • Polymorphism, Single Nucleotide
  • Viral Nonstructural Proteins / genetics*
  • beta-Thalassemia / genetics
  • beta-Thalassemia / immunology
  • beta-Thalassemia / therapy*

Substances

  • Antiviral Agents
  • interferon-lambda, human
  • Viral Nonstructural Proteins
  • Interferons
  • NS-5 protein, hepatitis C virus