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Updated: Sep 25, 2025
Detection of Low Copy Number Integrated Viral DNA Formed by In Vitro Hepatitis B Infection
Published on: November 7, 2018
Mutational spectrum of hepatitis C virus in patients with chronic hepatitis C determined by single molecule real-time
Fumiyasu Nakamura1, Haruhiko Takeda1, Yoshihide Ueda2,3
1Department of Gastroenterology and Hepatology, Graduate School of Medicine, Kyoto University, Kyoto, Japan.
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.
Insights
Hepatitis C virus (HCV) resistance-associated substitutions (RASs) emerge through both transition and transversion mutations. Understanding these mutation patterns is key to improving direct-acting antiviral (DAA) therapy for chronic HCV infection.
Area of Science:
- Virology
- Genetics
- Molecular Biology
Background:
- Direct-acting antiviral (DAA) treatment for hepatitis C virus (HCV) is challenged by the emergence of drug-resistant strains.
- Mutations in the HCV genome lead to resistance-associated substitutions (RASs), compromising treatment efficacy.
- Understanding the specific mutation patterns driving RAS emergence is crucial for optimizing antiviral strategies.
Purpose of the Study:
- To investigate the mutational spectrum and substitution patterns of HCV-RNA during direct-acting antiviral (DAA) treatment.
- To compare RAS emergence in patients with chronic HCV infection (non-transplant and post-liver transplant) with in vitro HCV models.
- To elucidate the role of viral polymerase fidelity in shaping HCV mutational biases.
Main Methods:
- High-accuracy single-molecule real-time long-read sequencing was employed.
- HCV-RNA was analyzed from two HCV replicon cell lines and serum samples from eight patients with unsuccessful DAA treatment.
- Mutational biases and substitution patterns leading to RASs were characterized.
Main Results:
- Dominant transition substitutions (A>G, U>C) were observed in HCV-RNA under DAA pressure, consistent across cell lines and patient groups.
- These mutational biases persisted even after DAA treatment cessation.
- RASs emerged through both transition (e.g., NS5A-Y93) and transversion (e.g., NS3-D168, NS5A-L31) substitutions.
- HCV-NS5B polymerase fidelity contributes to a mutational bias favoring transitions.
Conclusions:
- The HCV NS5B polymerase exhibits fidelity leading to a mutational bias, primarily transition substitutions.
- While this bias influences overall mutation patterns, RASs emerge via both transition and transversion mutations.
- DAA treatment pressure selects for and promotes the proliferation of RAS-harboring HCV clones.
