Mutational spectrum of hepatitis C virus in patients with chronic hepatitis C determined by single molecule real-time sequencing

Abstract

The emergence of hepatitis C virus (HCV) with resistance-associated substitution (RAS), produced by mutations in the HCV genome, is a major problem in direct acting antivirals (DAA) treatment. This study aimed to clarify the mutational spectrum in HCV-RNA and the substitution pattern for the emergence of RASs in patients with chronic HCV infection. HCV-RNA from two HCV replicon cell lines and the serum HCV-RNA of four non-liver transplant and four post-liver transplant patients with unsuccessful DAA treatment were analyzed using high-accuracy single-molecule real-time long-read sequencing. Transition substitutions, especially A>G and U>C, occurred prominently under DAAs in both non-transplant and post-transplant patients, with a mutational bias identical to that occurring in HCV replicon cell lines during 10-year culturing. These mutational biases were reproduced in natural courses after DAA treatment. RASs emerged via both transition and transversion substitutions. NS3-D168 and NS5A-L31 RASs resulted from transversion mutations, while NS5A-Y93 RASs was caused by transition substitutions. The fidelity of the RNA-dependent RNA polymerase, HCV-NS5B, produces mutational bias in the HCV genome, characterized by dominant transition mutations, notably A>G and U>C substitutions. However, RASs are acquired by both transition and transversion substitutions, and the RASs-positive HCV clones are selected and proliferated under DAA treatment pressure.

Figure 1

Phylogenetic analyses and mutational spectra in two HCV replicon cell lines during 10 years’ culture. (A, B) Phylogenetic analyses in HCV replicon cell line 1 (A) and 2 (B) during 10 years’ culture. Green dots denote the original HCV clones, which were originally transfected in the cell lines. Blue dots denote a subset of HCV clones at year 0, when the replicon cell lines established. Red dots demonstrate a subset of HCV clones at year 10 after 10 years’ culture from establishment. (C, D) Mutational spectra occurred during 10 years in the HCV replicon cell line 1 (C) and 2 (D) from year 0 to year 10 (left panels), compared between original clone and clones at year 0 (middle panels), and between original clone and clones at year 10 (right panels).

Figure 2

Clinical courses and phylogenetic analyses of four post-liver transplant (LT) patients during serial anti-HCV treatments by interferon-based therapy with DAA and interferon-free DAA therapy. Change of serum HCV RNA levels and HCV clones with resistance-associated substitutions at NS3 D168, NS5A L31, L32, and Y93 in four LT patients (left panels). Phylogenetic analyses of four LT patients at pre-simeprevir, interferon, and ribavirin therapy (SMV/IFN/RBV) (blue dots), post- SMV/IFN/RBV (orange dots), and pre- daclatasvir, and asunaprevir therapy (DCV/ASV) (black dots) (middle panels). Phylogenetic analyses of four LT patients at pre-DCV/ASV (black dots), post-DCV/ASV (red dots), and 18 months (M) post-DCV/ASV (green dots) (right panels).

Figure 3

Mutational spectra of patients during DAA treatment, interferon-based treatment, and natural courses after DAA treatment. Base substitution rates during DAA treatment in four non-liver transplant (Non-LT) patients (A) and four post-liver transplant (LT) patients (B); and during simeprevir, interferon and ribavirin therapy (C), and 18 months’ natural course after DAA treatment (D) in four post-liver transplant patients.

Figure 4

Mutational spectra of HCV genome in patients with chronic HCV infection. Rates of transition substitutions to total substitutions (A), and A>G and U>C transitions to total substitutions (B) during direct-acting antiviral therapy (DAA) in four non-liver transplant (Non-LT) patients, and during DAA, interferon (IFN)-based therapy, and natural course after DAA treatment in four post-liver transplant (LT) patients.

Figure 5

Mutational patterns for emergence of resistance-associated substitutions from Pre daclatasvir and asunaprevir (DCV/ASV) to Post DCV/ASV. Transition and transversion substitutions at NS3 D168 and NS5A L31 and Y93 in non-LT patients #1–4 and in LT patients #1–4 are shown in each pie chart. Mutations from Pre DCV/ASV to Post DCV/ASV were identified in each patient based on the phylogenetic analysis. The nucleotide sequences of an HCV clone at Pre DCV/ASV with the nearest clones of genetic distance from the HCV clones at Post DCV/ASV was determined as the reference sequence in each case, and all the sequences of HCV clones at Post DCV/ASV were compared with each reference sequence, where the base substitutions were identified as mutations acquired from Pre DCV/ASV to Post DCV/ASV. The frequencies of transition and transversion substitutions in each locus are shown. LT, liver transplant.