Abstract
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 nonstructural 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 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. 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.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Myung, J., Khalap, N., Kalkeri, G. et al., Inducible model to study negative strand RNA synthesis and assembly of hepatitis C virus from a full-length cDNA clone, J. Virol. Meth., 2001, 94: 55–67.
Wen Yumei, Modern Medicinal Microbiology (in Chinese), Shanghai: Shanghai Press, 1999, 1143–1154.
Pietschmann, T., Lohmann, V., Kaul, A. et al., Persistent and transient replication of full-length hepatitis C virus genomes in cell culture, J. Virol., 2002, 76: 4008–4021.
Bartenschlager, R., Lohmann, V., Novel cell culture systems for the hepatitis C virus, Antiviral Res., 2001, 52: 1–17.
Edward, M. M., Jag, A. G., Eric, J. M. et al., Persistent replication of hepatitis C virus replicons expressing the β-lactamase reporter in subpopulations of highly permissive Huh7 cells, J. Virol., 2003, 77(5): 2928–2935.
Seipp, S., Mueller, H. M., Pfaff, E. et al., Establishment of persistent hepatitis C virus infection and replication in vitro, J. Gen. Virol., 1997, 78: 2467–2476.
Mizuno, M., Yamada, G., Tanaka, T. et al., Virion-like structures in HeLa G. cells transfected with the full length sequence of the hepatitis C virus genome, Gastroenterology, 1995, 109: 1933–1940.
Keriln, J. B., Jane, A. M., Joseph, M. et al., Efficient replication of hepatitis C virus genotype I a RNAs in cell culture, J. Virol., 2003, 77(5): 3181–3190.
Lim, S., P., Soo, H. M., Brenner, S. et al., Inducible system in human hepatoma cell lines for hepatitis C virus production, Virology, 2002, 303: 79–99.
Dash, S., Halim, A. B., Tsuji, H. et al., Transfection of HepG2 cells with infectious hepatitis C virus genome, Am. J. Pathol., 1997, 151: 363–373.
Dash, S., Kalkeri, G., Transmission of HCV to a chimpanzee using virus particles produced in an RNA-transfected HepG2 cell culture, J. Med. Virol., 2001, 65(2): 276–281.
Li Xinqiang, Li Yan, Chinese patent, ZL 02 1 17666.3, 2004, 1-01-21.
Fuerst, T. R., Niles, E. G., Studier, F. W. et al., Eukaryotic transient-expression system based on recombinant vaccinia virus that synthesizes bacteriophage T7 RNA polymerase, Proc. Natl. Acad. Sci. USA, 1986, 83: 8122–8126.
Aizaki, H., Aoki, Y., Harada, T. et al., Full-length complementary DNA of hepatitis C virus genome from an infectious blood sample, Hepatology (Baltimore, Md), 1998, 27(2): 621–627.
Sambrook, J., Fritsch, E. F., Maniatis, T., Molecular Cloning — a Laboratory Manual, 2nd ed., New York: Cold Spring Harbor Laboratory Press, 1989, 15.3-15.108.
Kaito, M., Watanabe, S., Tsukiyama-Kohara, K. et al., Hepatitis C virus particle detected by immunoelectron microscopy study, J. Gen. Virol., 1994, 75: 1755–1760.
Li, X., Jeffers, L. J., Shao, L. et al., Identification of hepatitis C virus by immunoelectron microscopy, J. Viral Hepatitis, 1995, 2: 227–234.
Craggs, J. K., Ball, J. K., Thomson, B. J. et al., Development of a strand-specific RT-PCR based assay to detect the replicative form of hepatitis C virus RNA, J. Virol. Meth., 2001, 94: 111–120.
Lin, L., Fevery, J., Yap, S. M. et al., A novel strand-specific RT-PCR for detection of hepatitis C virus negative-strand RNA(replicate intermediate): Evidence of absence or very low level of HCV replication in peripheral blood mononuclear cells, J. Virol. Meth., 2002, 100: 97–105.
Clarke, B., Molecular virology of hepatitis C virus, J. Gen. Virol., 1997, 78: 2397–2410.
Landford, R. E., Sureau, C., Jacob, J. R. et al., Demonstration of in vitro infection of chimpanzee hepatocytes with hepatitis C virus using strand-specific RT/PCR, Virology, 1994, 202: 606–614.
Krieger, N., Lohmann, V., Bartenschlager, R. et al., Enhancement of hepatitis C virus replication by cell culture-adaptive mutations, J. Virol., 2001, 75(10): 4614–4624.
Lohmann, V., Korner, F., Dobierzewska, A. et al., Mutations in hepatitis C virus RNA conferring cell culture adaption, J. Virol., 2001, 75(3): 1437–1449.
Volker, L., Sandra, H., Ulrike, H. et al., Viral and cellular determinants of Hepatitis C virus RNA replication in cell culture, J. Virol., 2003, 77(5): 3007–3019.
Sakamoto, N., Enomoto, N., Kurosaki, M. et al., Detection and quantitation of hepatitis C virus RNA replication in the liver, J. Hepatol., 1994, 20: 593–597.
Author information
Authors and Affiliations
Corresponding authors
About this article
Cite this article
Yao, X., Guo, J., Zheng, C. et al. Establishment of an in vitro cell culture system transfected by full-length HCV cDNA genome. Chin. Sci. Bull. 49, 1358–1363 (2004). https://doi.org/10.1360/03wc0369
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1360/03wc0369