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

Department of Biology, California Lutheran University, Thousand Oaks, California, USA.
Virology Journal (Impact Factor: 2.18). 02/2006; 3(1):81. DOI: 10.1186/1743-422X-3-81
Source: PubMed


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.

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    • "We previously reported an in vitro system that can replicate HCV for extended periods of time [5]. Later reports from our laboratories included an analysis of the 5'UTR of CIMM-HCV [6] and the discovery of significant HCV variants [7]. Rare insertions and deletions have also been seen by others [8]. "
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    Virology Journal 02/2007; 4(1):106. DOI:10.1186/1743-422X-4-106 · 2.18 Impact Factor
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    • "We also noted that each of the 313 samples had at least one clone that contained an extra C in the string of C's from bases 120 to 126. Other isolates from different patients sometimes also contain an extra C in this region [11]. "
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    Virology Journal 02/2006; 3(1):82. DOI:10.1186/1743-422X-3-82 · 2.18 Impact Factor
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