A vaccinia replication system for producing recombinant hepatitis C virus.
OBJECTIVE
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 3.6X10(7) 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 1 000- fold compared to in vitro- transcribed HCV genomic RNA or selective subgenomic HCV RNA molecule method.


CONCLUSIONS
This cell culture system is capable of producing high titer recombinant HCV.
Institute of Tropical Medicine, First Military Medical University, Guangzhou 510515, Guangdong Province, China.
Ying-Song
YS
Wu
Yu
Y
Feng
Wen-Qi
WQ
Dong
Yan-Ming
YM
Zhang
Ming
M
Li
D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D004274:DNA, Recombinant; D006378:Helper Viruses; D016174:Hepacivirus / Q000254:growth & development*; D007668:Kidney / Q000166:cytology; D010957:Plasmids; D014616:Vaccinia virus / Q000254:growth & development*; D014761:Viral Hepatitis Vaccines; D014773:Virology / Q000379:methods*; D014779:Virus Replication
D004274:DNA, Recombinant; D014761:Viral Hepatitis Vaccines