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.
Wu
Ying-Song
YS
Institute of Tropical Medicine, First Military Medical University, Guangzhou 510515, Guangdong Province, China.
Feng
Yu
Y
Dong
Wen-Qi
WQ
Zhang
Yan-Ming
YM
Li
Ming
M
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