title: [Construction of a full-genome HCV replicon with self-cleaving double ribozyme sequences and characterization in vitro and in vivo].
abstract: OBJECTIVE
  To construct a full-genome hepatitis C virus (HCV) replicon that will allow for direct initiation of replication and generation of infectious viral particles in an in vitro and in vivo cell system.
  
  
  METHODS
  Self-cleaving ribozyme sequences were added to each side of the HCV cDNA clone JFH1 and the replication-deficient clone JFH1/GND, then inserted into the pcDNA3.1 vector downstream of the CMV promoter. The resultant recombinant plasmids, pcDNA3.1-RZ-JFH1 and pcDNA3.1-RZ-JFH1/GND, were tested for activity in vitro and in vivo by transiently transfecting into Huh7.5 cells (5 mug/100 mm culture dish) and injecting by high-pressure tail vein injection into Kunming mice (10 - 30 mug/mouse). Quantitative reverse transcription-PCR, immunofluorescence, immunohistochemistry, and serological testing were performed to determine the replication ability and assess the properties of the recombinant plasmids in the two systems.
  
  
  RESULTS
  HCV RNA (1 - 3 * 10(6) copies/ml) was detected in the supernatant of transfected Huh7.5 cells up to 16 weeks after transfection. In addition, the viral particles from the supernatant were able to infect nave Huh7.5 cells. However, only transient viremia was achieved upon tail vein injection of the plasmid, and no HCV antigen-positive cells were detected by immunohistochemistry nor HCV-specific antibodies by serological testing.
  
  
  CONCLUSIONS
  The constructed HCV replicon was capable of stable expression in cultured cells and of efficiently generating infectious viral particles in the in vitro system over a long period. However, the HCV replicon did not show infective characteristics in an in vivo mouse system. The full-length HCV replicon may represent a useful tool for in vitro study of HCV pathological mechanisms, possibly including anti-HCV drug screening.
authors.affiliation: Institute for Infectious Diseases of PLA, Southwest Hospital, Third Military Medical University, Chongqing, China.
authors.forename: Chang-long
authors.initials: CL
authors.lastname: He
authors.forename: Qing-shan
authors.initials: QS
authors.lastname: Liu
authors.forename: Yan
authors.initials: Y
authors.lastname: Guo
authors.forename: Yan
authors.initials: Y
authors.lastname: Zhu
authors.forename: Qing
authors.initials: Q
authors.lastname: Mao
authors.forename: Lin
authors.initials: L
authors.lastname: Lan
mesh_terms: D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D005822:Genetic Vectors; D016679:Genome, Viral; D016174:Hepacivirus / Q000235:genetics* / Q000502:physiology; D006801:Humans; D008297:Male; D051379:Mice; D008815:Mice, Inbred Strains; D016337:RNA, Catalytic / Q000235:genetics*; D011995:Recombination, Genetic; D012093:Replicon; D014779:Virus Replication / Q000235:genetics*
chemical_list: D016337:RNA, Catalytic