Establishment Of An In Vitro Cell Culture System Transfected By Full-length HCV CDNA Genome

A new cell culture system expressing the entire HCV genome has been established in vitro. To initiate transcription of HCV RNA, HeLa cells were transfected with a recombinant plasmid( pHCV ) containing full-length HCV cDNA genome by using lipofectamine 2000, followed by infection with recombinant vaccinia virus vTF7-3 containing the T7 RNA polymerase gene. In order to identify the replication and assembly of HCV, RT-PCR , strand specific RT-PCR, Fluorescent quantitative RT-PCR, Western-blot, conventional negative contrast electron microscopy, immunoelectron microscopy were used in the experiment. The results were as follows:1, By using RT-PCR, strand specific RT-PCR we could detect the synthesis of positive and negative strand of HCV RNA in the transfected HeLa cell, which indicates that pHCV could transcript and replicate HCV genome efficiently in the transfected cells. Fluorescent quantitative RT-PCR assay revealed the titer of HCV was 107 copies/mL in our cell culture system, which was significantly higher than that of infected patients' sera and that from all reported cell culture systems. The increase of HCV genome is a dynamic change, which was initiated after transfection and peaked at 48h post transfection. Then it began to decrease step by step.2, Western-blot analysis detected the expression of HCV structure and nonstructure proteins in the transfected cells, the molecular of which is coherent with other reports.3, The observation of conventional electronmicroscopy and immunoelectron microscopy reveals that transfected HeLa cells assembled 47 nm virus-like particles, which could be recognized by anti-HCV E2 antibodies.4, In the reinfection experiment we infected the permissive cell line Huh7 with the filtrated cell lysis from HeLa cell transfected. RT-PCR and strand specific RT-PCR assay demonstrate the presense of positive and negative-strand HCV in the Huh7 cell infected. This indicates that HCV particles produced from pHCV were infectious, which could replicate efficiently in Huh7 cell and form a negative-strand RNA imtermediate.To sum up, the applicability of our novel cell culture system is wide. It could be useful in the searching for the antiviral agents , the large-scale production of vaccines and the post-genome research of HCV. Furthermore, it could also offer a cell model for studying theantiviral drug sensitivity, cell tropism, and the persistent infection of HCV.