Establishment of an in vitro cell culture system transfected by full-length HCV cDNA genome

A new cell culture system expressing the entire HCV geuome has been established in vitro. To initiate transcription of HCV RNA, HeLa cells were trausfected with arecombinant plasmid containing full-length HCV cDNA geuome by using lipofectamiue 2000, followed by infection with recombinant vacciuia virus vTF7-3 containing the T7 RNA polymerase geue. Synthesis of positive-strand HCV RNA could be detected in the trausfected cells by RT-PCR. Western blot analysis revealed that HCV structural and nonstructural proteins were correctly processed. In trausfected HeLa cells 47 um virus-like particles were assembled, whichcould be recognized by auti-HCV E2 antibodies. 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. Superuataut from trausfected HeLa cells were infectious to Huh7 cells and thetiter of HCV was 106 copies/mL. Moreover, negative-strand RNA of HCV in Huh7 cells could be detected by using strand-specific RT-PCR, which demonstrated that replication of HCV occurred in the permissive cell lines.

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 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. Synthesis of positive-strand HCV RNA could be detected in the transfected cells by RT-PCR. Western blot analysis revealed that HCV structural and non structural proteins were correctly processed. In transfected HeLa cells 47 nm virus-like particles were assembled, which could be recognized by anti-HCV E2 antibodies. The liter 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. Supernatant from transfected HeLa cells were infectious to Huh7 cells and the titer of HCV was 106 copies/mL. Moreover, negative-strand RNA of HCV in Huh7 cells could be detected by using strand-specific RT-PCR, which demonstrated that replication of HCV occurred in the permissive cell lines.