Analysis of in vitro replicated human hepatitis C virus (HCV) for the determination of genotypes and quasispecies.

Dennis Revie,Nickolas Chelyapov,David Bayles,Michael O Alberti,S Zaki Salahuddin,John G Prichard,Ravi S Braich

doi:10.1186/1743-422x-3-81

Abstract

Isolation and self-replication of infectious HCV has been a difficult task. However, this is needed for the purposes of developing rational drugs and for the analysis of the natural virus. Our recent report of an in vitro system for the isolation of human HCV from infected patients and their replication in tissue culture addresses this challenge. At California Institute of Molecular Medicine several isolates of HCV, called CIMM-HCV, were grown for over three years in cell culture. This is a report of the analysis of CIMM-HCV isolates for subtypes and quasispecies using a 269 bp segment of the 5'UTR. HCV RNA from three patients and eleven CIMM-HCV were analyzed for this purpose. All isolates were essentially identical. Isolates of HCV from one patient were serially transmitted into fresh cells up to eight times and the progeny viruses from each transmission were compared to each other and also to the primary isolates from the patient's serum. Some isolates were also transmitted to different cell types, while others were cultured continuously without retransmission for over three years. We noted minor sequence changes when HCV was cultured for extended periods of time. HCV in T-cells and non-committed lymphoid cells showed a few differences when compared to isolates obtained from immortalized B-cells. These viruses maintained close similarity despite repeated transmissions and passage of time. There were no subtypes or quasispecies noted in CIMM-HCV.

Highlights

HCV infects millions of people throughout the world and is a cause of several serious diseases
In order to assess whether particular genotypes of HCV were preferentially selected in vitro, we analyzed the 5'UTR of HCV RNA representing a number of California Institute of Molecular Medicine (CIMM)-HCV (Figure 1B)
Comparison of the 5'UTR of HCV from patients' blood and CIMM-HCV RNA was purified from patients' sera or plasma and from CIMM-HCV

Summary

Introduction

HCV infects millions of people throughout the world and is a cause of several serious diseases. It has been estimated that there are over 170 million carriers of HCV worldwide [1]. The inability to culture HCV in vitro has severely limited meaningful definitive studies leading to therapeutics and vaccines. We have developed a robust in vitro system for replicating human HCV and for extended periods of time [2]. Several studies in the past have reported in vitro replication of HCV [3,4,5,6]. None of these have yet demonstrated biologically infectious HCV isolated from patient's blood, or have grown these isolates in vitro for a significant amount of time. Wakita et al [7] recently reported the development of a (page number not for citation purposes)

Methods

Results

Conclusion