Published online Sep 15, 2004. doi: 10.3748/wjg.v10.i18.2670
Revised: December 13, 2003
Accepted: December 29, 2003
Published online: September 15, 2004
AIM: To develop a cell culture system capable of producing high titer hepatitis C virus (HCV) stocks with recombinant vaccinia viruses as helpers.
METHODS: Two plasmids were used for the generation of recombinant 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 open reading frame (ORF) directly linked to a vaccinia late promoter in PSC59. These two plasmids were co-transfected into BHK21 cells, which were then infected with vTF7-3 recombinant vaccinia helper viruses.
RESULTS: After 5 d of incubation, approximately 3.6 × 107 copies of HCV RNA were present per milliliter of cell culture supernatant, as detected by fluorescence quantitative RT-PCR (FQ-PCR). The yield of recombinant HCV using this cell system increased 100- to 1000-fold compared to in vitro-transcribed HCV genomic RNA or selective subgenomic HCV RNA molecule method.
CONCLUSION: This cell culture system is capable of producing high titer recombinant HCV.