AIM:To develop a cell culture system capable of producinghigh titer hepatitis C virus (HCV) stocks with recombinantvaccinia viruses as helpers.METHODS:Two plasmids were used for the generation ofrecombinant HCV:one containing the full-length HCV cDNAcloned between T7 promoter and T7 terminator of pOCUS-T7vector,and the other containing the HCV polyprotein openreading frame (ORF) directly linked to a vaccinia latepromoter in PSC59.These two plasmids were co-transfectedinto BHK_(21) cells,which were then infected with vTF7-3recombinant vaccinia helper viruses.RESULTS:After 5 d of incubation,approximately 3.6×10~7copies of HCV RNA were present per milliliter of cell culturesupematant,as detected by fluorescence quantitative RT-PCR(FQ-PCR).The yield of recombinant HCV using this cellsystem increased 100- to 1 000- fold compared to in vitro-transcribed HCV genomic RNA or selective subgenomic HCVRNA molecule method.CONCLUSION:This cell culture system is capable ofproducing high titer recombinant HCV. AIM: To develop a cell culture system capable of producing high titer hepatitis C virus (HCV) stocks with recombinantvaccinia viruses as helpers. METHODS: Two plasmids were used for the generation ofrecombinant HCV: one containing the full-length HCV cDNA cloned between T7 promoter and T7 terminator of pOCUS-T7 vector, and the other containing the HCV polyprotein openreading frame (ORF) directly linked to a vaccinia late promoter in PSC59. These two plasmids were co-transfected in BHK_ (21) cells, which were then infected with vTF7-3recombinant vaccinia helper viruses .RESULTS: After 5 d of incubation, approximately 3.6 × 10 ~ 7 copies of HCV RNA were present per milliliter of cell culturesupematant, as detected by fluorescence quantitative RT-PCR (FQ-PCR). The yield of recombinant HCV using this cellsystem increased 100- to 1 000-fold compared to in vitro-transcribed HCV genomic RNA or selective subgenomic HCVRNA molecule method. CONCLUSION: This cell culture system is capable of producing high titer recomb inant HCV.