Large Scale Production Of Hepatitis C Virus In An In Vitro Cell Culture System And It's Preliminary Application In Screening Of Drugs Against HCV

Ph.D. candidate Zhang Yan-ming Supervisor Li MingThe hepatitis C virus (HCV) is a major causative agent for most posttransfusion non-A, non-B hepatitis case. There are about 170 million carriers of hepatitis C virus in the world today and about 40 million in China alone. More than half of persons infected with HCV can progress to chronic infection, and of the chronically infected 10-20% will develop complications of chronic liver disease such as liver cirrorhosis and 1-5% will develop hepatocellular carcinoma. Unfortunately, only 10-20% of patients infected by HCV are respond to interferon alone, therapy of IFN- a combined with ribavirin is effective in only about 50% infected persons. The lack of tissue-culture system for HCV replication has limited the development of vaccine for HCV and new treatment.We propose a cell culture system to produce HCV in vitro. In this system, we constructed two plasmids: pT7HCV contains the HCV genomic RNA-coding region between an upstream T7 promoter and a downstream T7 terminator; pVHCV contains the HCV polyprotein open reading frame (ORF) that is linked to the vaccinia late promoter of vector. The two plasmids were co-transfected into eucaryotic cells, the cells then were infected by recombinant vaccinia virus vTF7-3 which contain a T7 RNA polymerase gene under the control of a vaccinia promoter. After cultured, recombinant HCV can be produced in the cells. We optimized the culture condition of the cells to get the highest production of recombinant HCV. Then we separate HCV by discontinuous sucrose density-gradient ultra-centrifugation. We also investigated effect of interferon and rebavirinon HCV early replication stage in this HCV culture system.Plasmid pBRT703'X(NIHJl) containing the full-length HCV genomic cDNA, was incompletely digested with HindIII and EcoR I , then HCV cDNA was reclaimed and inserted into vector pOCUS-T7 (plasmid pOCUS containing a T7 promoter and a T7 terminator) between the EcoR I and Hindlll sites, resulting insertion of the HCV sequence between the bacteriophage T7 promoter and T7 terminator sequences to create plasmid pT7HCV.Plasmid O. pMKC1A (HCV) containing the HCV polyprotein open reading frame was cut with EcoR I and Hindlll and inserted into PSC59 between the EcoR I and Stu I sites to generate plasmid pVHCV, which contained the HCV polyprotein open reading frame (ORF) linked to a vaccinia late promoter.Restriction enzyme digestion and DNA sequencing confirm the correct sequences of plasmid pT7HCV and pVHCV.Cells were then transfected with pT7HCV plus pVHCV using the LipofectaminTM2000 Reagent followed by infection with vTF7-3 vaccinia viruses. Then cells were inoculated two hours for absorption of virus. The inoculum was then removed and the cells were cultured in fresh Dulbecco's modified Eagle's medium (DMEM)with 10% fetal calf serum(FCS) in a 30 "C and 5%CO2 incubator for 2-5 days. Genomic RNA of HCV in the supernatants was detected by Reverse Transcription Polymerase Chain Reaction (RT-PCR), the titer of HCV was examined by Fluorescent Quantitative PCR (FQ-PCR), HCV proteins were tested by Indirect Immunofluorescence and Western blot analysis and the virus was observed under Immunoelectron microscopy to locate the virus in cell.As the result of experiment showed, after 96 hours of incubation in 6-well plates, 7 106 copies of HCV can be produced in the supernatants. HCV genomic RNA was amplified by RT-PCR, and structural and nonstructural proteins of HCV can be detected by immunofluorescence and Western blot. Virus-like particles of about 50nm in diameter were observedunder immuno-electron microscopy.We optimized the culture condition of HCV for the purpose of produce HCV vaccine. We selected appropriate cell line, determine quantity of plasmid for transfection and choose the best concentration of recombinant vaccinia virus to produce the most abundance HCV in supernatant. The copies of HCV genomic RNA in supernatant was detected by FQ-RT-PCR and used as guideline to determine the condition of HCV culture.Th...