A Simple but Accurate Method for Evaluating Drug-Resistance in Infectious HCVcc System.

Jian-Rui Li,Jun-Jun Cheng,Wen-Jing Li,Jin-Hua Chen,Hu Li,Biao Dong,Zhou-Yi Wu,Chen-Chen Jiang,Jian-Dong Jiang,Meng-Hao Huang,Xiao-Qin Lv,Zong-Gen Peng

doi:10.1155/2017/1236801

Abstract

Use of direct-acting antivirals sometimes causes viral drug resistance, resulting in inefficiency in treated patients in real-world practice. Therefore, how to rapidly and accurately evaluate drug resistance is an urgent problem to be solved for rational use and development of antivirals in the future. Here, we aim to develop a new method by which we can evaluate easily but effectively whether a drug will still be efficient in the future treatment in infectious hepatitis C virus cell culture system. HCV-infected Huh7.5 cells were treated with drugs and the culture supernatants were replaced with fresh culture media containing the same drugs at 24 hours. The supernatants were harvested at 48 hours and incubated with naïve Huh7.5 cells. Intracellular HCV RNAs or proteins in the newly infected cells were extracted and analyzed at 48 hours or longer. Results showed that after being treated with telaprevir mutant viruses were easily detected which were resistant to telaprevir, while after being treated with sofosbuvir drug-resistant viruses did not emerge. In conclusion, the new method is simple and quick but accurate to evaluate whether a drug will be still efficient in the forthcoming therapeutic regimen and whether drug resistance will occur after long-term treatment with drugs.

Highlights

Hepatitis C virus (HCV) infection causes chronic hepatitis, liver fibrosis, cirrhosis, and even liver cancer, which is considered a major serious threat to people’s health worldwide [1, 2]
For selecting mutant viruses to evaluate the drug-resistant profile, HCV-positive (HCV+) Huh7.5 cells were treated with high concentration of simeprevir or sofosbuvir for 24 hours and washed and continuously incubated with fresh culture media containing the same drugs for additional 48 hours
Intracellular HCV RNAs were extracted in 48 hours and reverse-transcribed into complementary DNA (cDNA), and the full-length NS3/4A or NS5B sequence was amplified and cloned into a vector

Summary

Introduction

Hepatitis C virus (HCV) infection causes chronic hepatitis, liver fibrosis, cirrhosis, and even liver cancer, which is considered a major serious threat to people’s health worldwide [1, 2]. In the past few years, several direct-acting antiviral agents (DAAs) were approved for use in clinic, including NS3/4A protease inhibitors, NS5B polymerase inhibitors, and NS5A protein inhibitors [3,4,5,6], and the overall therapeutic effect was largely improved. Five fixed-dose combined agents were approved for clinic use, including Harvoni, Viekira Pak, Technivie, Zepatier and Epclusa, which contain NS5A inhibitor plus NS5B and/or NS3/4A inhibitor(s). Treatment with those combined agents improved the overall sustained viral response (SVR) to >90% in HCV-infected patients [11,12,13]. How to quickly evaluate whether an infection will be resistant to novel antiviral agents is a very important consideration to correctly use and develop antiviral agents in the coming future

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Results

Conclusion