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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    • "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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    ABSTRACT: We recently reported the isolation and in vitro replication of hepatitis C virus. These isolates were termed CIMM-HCV and analyzed to establish genotypes and subtypes, which are reported elsewhere. During this analysis, an HCV isolated from a patient was discovered that had large deletions in the 5'UTR. 57% of the HCV RNA found in this patient's sera had 113 or 116 bp deletions. Sequence data showed that domains IIIa to IIIc were missing. Previous studies have suggested that these domains may be important for translation. In vitro replicated HCV from this patient did not contain these deletions, however, it contained a 148 bp deletion in the 5'UTR. Whereas the patient HCV lacked domains IIIa through IIIc, the isolate lacked domains IIIa through IIId. HCV from this patient continues to produce large deletions in vitro, suggesting that the deletion may not be important for the assembly or replication of the virus. This is the first report describing these large deletions.
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