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. 2003 Dec;125(6):1808-17.
doi: 10.1053/j.gastro.2003.09.023.

Efficient replication of the genotype 2a hepatitis C virus subgenomic replicon

Takanobu Kato  1 , Tomoko Date, Michiko Miyamoto, Akihiro Furusaka, Katsutoshi Tokushige, Masashi Mizokami, Takaji Wakita
Affiliations

Efficient replication of the genotype 2a hepatitis C virus subgenomic replicon

Takanobu Kato et al. Gastroenterology. 2003 Dec.

Abstract

Background & aims: Although the hepatitis C virus (HCV) subgenomic replicon system has been widely used in the study of HCV, this system is available only for a few related genotypes. To develop a new replicon system, the genotype 2a clone JFH-1 was isolated from a patient with fulminant hepatitis.

Methods: A genotype 2a replicon was constructed by isolating the consensus sequence of JFH-1, transfecting G418-selectable subgenomic transcripts into Huh7 cells, and estimating the replication efficiency.

Results: The colony formation efficiency of the JFH-1 replicon was 53,200 colonies/microg RNA, significantly higher than that of the genotype 1b cell-adapted replicon, at 909 colonies/microg RNA (P < 0.05). The JFH-1 replicon RNA was transmissible to naive Huh7 cells by transfection of cellular RNA from cells containing the replicon. Sequencing of cloned replicon RNAs revealed that all but 1 had at least 1 nonsynonymous mutation. One of these mutations was shown to enhance the colony formation efficiency of the JFH-1 replicon. Furthermore, the JFH-1 replicon RNA replicated efficiently without G418 selection in a transient replication assay.

Conclusions: The genotype 2a subgenomic replicon was established in Huh7 cells and replicated efficiently with or without G418 selection. This subgenomic replicon could replicate without common amino acid mutations; however, some of the mutations found in the clones might be important in conferring higher replication phenotypes. This system provides a powerful new tool for researching HCV.

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