1887

Abstract

To investigate the viral replication cycle and genomic heterogeneity of hepatitis C virus (HCV), we established an HCV cultivation system by using a primary hepatocyte culture from patients with chronic hepatitis C. Liver tissue was obtained by needle biopsy or surgery, then hepatocytes were isolated by collagenase digestion. After several weeks, we determined the HCV RNA titre of the cultured cells and supernatant by a competitive polymerase chain reaction (PCR) method. A significant amount of HCV RNA was observed in the cells and supernatant during cultivation. Negative-strand RNA, regarded as a marker of viral replication, could be detected by a strand-specific reverse transcription PCR method and the HCV core protein could be detected by immunofluorescence microscopy. Many HCV particles released into the supernatant were infectious. In addition, we compared the nucleotide sequences in the E2/NS1 region of pre- and post-cultivation hepatocytes for 8 weeks. At the beginning of the culture period, three major HCV types containing two subtypes were isolated. Following cultivation, the same types were isolated from the cultured hepatocytes in the same ratio as prior to cultivation. We could detect the same clones in this patient’s serum, but we observed genetic variability over a 6 month interval. One clone detected throughout the 6 month period mutated extensively in the hypervariable region. These results indicated that HCV can replicate in cultured hepatocytes, and that infectious virions are released into the supernatant. This cultivation system should facilitate the study of HCV genomic heterogeneity, infection and replication.

Loading

Article metrics loading...

/content/journal/jgv/10.1099/0022-1317-77-5-1043
1996-05-01
2024-03-29
Loading full text...

Full text loading...

/deliver/fulltext/jgv/77/5/JV0770051043.html?itemId=/content/journal/jgv/10.1099/0022-1317-77-5-1043&mimeType=html&fmt=ahah

References

  1. Bertolini L., Iacovacci S., Ponzetto A., Gorini G., Bataglia M., Carloni G. 1993; The human bone marrow-derived B-cell line (ce) susceptible to hepatitis C virus infection. Research in Virology 144:281–285
    [Google Scholar]
  2. Brinton M. A. 1986; Replication of flaviviruses. In The Togaviridae and Flaviviridae pp 327–374 Edited by Schlesinger S., Schlesinger M. J. New York: Plenum Press;
    [Google Scholar]
  3. Choo Q.-L., Kuo G., Weiner A. J., Oberby L. R., Bradley D. W., Houghton M. 1989; Isolation of cDNA clone derived from a blood-borne non-A, non-B viral hepatitis genome. Science 244:359–362
    [Google Scholar]
  4. Choo Q.-L., Kuo G., Ralston R., Weiner A. J., Chien D., Van Nest G., Han J., Berger K., Thudium K., Kuo C., Kansopon J., McFarland J., Tabrizi A., Ching K., Moss B., Cummins L. B., Houghton M., Muchmore E. 1994; Vaccination of chimpanzees against infection by the hepatitis C virus. Proceedings of the National Academy of Sciences, USA 91:1294–1298
    [Google Scholar]
  5. Enomoto N., Kurosaki M., Tanaka Y., Marumo F., Sato C. 1994; Fluctuation of hepatitis C virus quasispecies in persistent infection and interferon treatment revealed by single-strand conformation polymorphism analysis. Journal of General Virology 75:1361–1369
    [Google Scholar]
  6. Hagiwara H., Hayashi N., Mita E., Naito M., Kasahara A., Fusamoto H., Kamada T. 1993; Quantitation of hepatitis C virus RNA in serum of asymptomatic blood donors and patients with type C chronic liver disease. Hepatology 17:545–550
    [Google Scholar]
  7. Hijikata M., Kato N., Ootsuyama Y., Nakagawa M., Ohkoshi S., Shimotohno K. 1991; Hyper variable regions in the putative glycoprotein of hepatitis C virus. Biochemical and Biophysical Reserch Communications 175:220–228
    [Google Scholar]
  8. Hijikata M., Shimizu Y. K., Kato H., Iwamoto H., Shih J. W., Alter H. J., Purcell R. H., Yoshikura H. 1993; Equilibrium centrifugation studies of hepatitis C virus: evidence for circulating immune complexes. Journal of Virology 67:1953–1958
    [Google Scholar]
  9. Iacovacci S., Sargiacomo M., Parolini I., Ponzetto A., Peschle C., Carloni G. 1993; Replication and multiplication of hepatitis C virus genome in human foetal liver cells. Research in Virology 144:275–279
    [Google Scholar]
  10. Kaito M., Watanabe S., Tsukiyama-Kohara K., Yamaguchi K., Kobayashi Y., Konishi M., Yokoi M., Ishida S., Suzuki S., Kohara M. 1994; Hepatitis C virus particle detected by immunoelectron microscopic study. Journal of General Virology 75:1755–1760
    [Google Scholar]
  11. Kaneko S., Murakami S., Unoura K., Kobayashi K. 1992; Quantitation of hepatitis C virus RNA by competitive polymerase chain reaction. Journal of Medical Virology 37:278–282
    [Google Scholar]
  12. Kato N., Hijikata M., Ootsuyama Y., Nakagawa M., Ohkoshi S., Sugimura T., Shimotono K. 1990; Molecular cloning of the human hepatitis C virus genome from Japanese patients with non-A, non-B hepatitis. Proceedings of the National Academy of Sciences, USA 87:9524–9528
    [Google Scholar]
  13. Kato N., Ootsuyama Y., Ohkoshi S., Nakazawa T., Sekiya H., Hijikata M., Shimotohno K. 1992; Characterization of hypervariable regions in the putative envelope protein of hepatitis C virus. Biochemical and Biophysical Research Communications 189:119–127
    [Google Scholar]
  14. Kimura-Kuroda J., Yasui K. 1988; Protection of mice against Japanese encephalitis virus by passive administration with monoclonal antibodies. Journal of Immunology 141:3606–3610
    [Google Scholar]
  15. Lanford R. E., Sureau C., Jacob J. R., White R., Fuerst T. R. 1994; Demonstration of in vitro infection of chimpanzee hepatocytes with hepatitis C virus using strand-specific RT/PCR. Virology 202:606–614
    [Google Scholar]
  16. Lobigs M., Dalgarno L., Schlesinger J. J., Weir R. C. 1987; Location of a neutralization determinant in the E protein of yellow fever virus (17D vaccine strain). Virology 161:474–478
    [Google Scholar]
  17. Nakatsuji Y., Matsumoto A., Tanaka E., Ogata H., Kiyosawa K. 1992; Detection of chronic hepatitis C virus infection by four diagnostic systems: first-generation and second-generation enzyme-linked immunosorbent assay, second-generation recombinant immuno-blot assay and nested polymerase chain reaction analysis. Hepatology 16:300–305
    [Google Scholar]
  18. Ogata N., Alter H. J., Miller R. H., Purcell R. H. 1991; Nucleotide sequence and mutation rate of the H strain of hepatitis C virus. Proceedings of the National Academy of Sciences, USA 88:3392–3396
    [Google Scholar]
  19. Okamoto H., Kurai K., Okada S. I., Yamamoto K., Iizuka H., Tanaka T., Fukuda S., Tsuda F., Mishiro S. 1992; Full-length sequence of a hepatitis C virus genome having poor homology to reported isolates: comparative study of four distinct genotypes. Virology 188:331–341
    [Google Scholar]
  20. Seki M., Honda Y. 1995; Phosphorothioate antisense oligodeoxy-nucleotides capable of inhibiting hepatitis C virus gene expression: in vitro translation assay. Journal of Biochemistry 118:1119–1204
    [Google Scholar]
  21. Shimizu Y. K., Purcell R. H., Yoshikura H. 1993; Correlation between the infectivity of hepatitis C virus in vivo and its infectivity in vitro. Proceedings of the National Academy of Sciences, USA 90:6037–6041
    [Google Scholar]
  22. Shimizu Y. K., Hijikata M., Iwamoto A. H., Alter H. J., Purcell R. H., Yoshikura H. 1994a; Neutralizing antibodies against hepatitis C virus and emergence of neutralization escape mutant viruses. Journal of Virology 68:1494–1500
    [Google Scholar]
  23. Shimizu Y. K., Yoshikura H. 1994b; Multiple infection of hepatitis C virus in cell culture and inhibition by alpha and beta interferons. Journal of Virology 68:8406–8408
    [Google Scholar]
  24. Shindo M., Di Bisceglie A. M., Akatuka T., Fong T.-L., Scaghone L., Donets M., Hoofnagle J. H., Feinstone S. M. 1994; The physical state of the negative strand of hepatitis C virus RNA in serum of patients with chronic hepatitis C. Proceedings of the National Academy of Sciences, USA 91:8719–8723
    [Google Scholar]
  25. Takehara T., Hayashi N., Mita E., Hagiwara H., Ueda K., Katayama K., Kasahara A., Husamoto H., Kamada T. 1992; Detection of the minus strand of hepatitis C virus RNA by reverse transcription and polymerase chain reaction: Implication for hepatitis C virus replication in infected tissue. Hepatology 15:387–390
    [Google Scholar]
  26. Takebe Y., Seiki Y., Fujisawa J., Hoy P., Yokota K., Arai K., Yoshida M., Arai N. 1988; SR promotor: an efficient and versatile mammalian cDNA expression system composed of the simian virus 40 early promoter and the R-U5 segment of human T- cell leukemia virus type 1 long terminal repeat. Molecular Cell Biology 8:466–472
    [Google Scholar]
  27. Taniguchi S., Okamoto H., Sakamoto M., Kojima M., Tsuda F., Tanaka T., Munekata E., Muchmore E. E., Peterson D. A., Mishiro S. 1993; A structually flexible and antigenically variable N-terminal domain of the hepatitis C virus E2/NS1 protein: implication for an escape from antibody. Virology 195:297–301
    [Google Scholar]
  28. Weiner A. J., Brauer M. J., Rosenblatt J., Richman K. H., Tung J., Crawford K., Bonino F., Saracco G., Choo Q.-L., Houghton M., Han J. H. 1991; Variable and hypervariable domains are found in the regions of HCV corresponding to the flavivirus envelope and NS1 proteins and the pestivirus envelope glycoproteins. Virology 180:842–848
    [Google Scholar]
  29. Weiner A. J., Geysen M., Christopherson C., Hall J. E., Mason T. J., Sarraco G., Bonino F., Crawford K., Marison C. D., Crawford K. A., Brunetto M., Barr P. J., Miyamura T., McHutchinson J., Houghton M. 1992; Evidence for immune selection of hepatitis C virus (HCV) putative envelope glycoprotein variants: potential role in chronic HCV infections. Proceedings of the National Academy of Sciences, USA 89:3468–3472
    [Google Scholar]
  30. Willems M., Peerlinck K., Moshage H., Deleu I., Van Den Eynde C., Vermylen J., Yap S. H. 1994; Hepatitis C virus-RNAs in plasma and in peripheral blood mononuclear cells of hemophiliacs with chronic hepatitis C: evidence for viral replication in peripheral blood mononuclear cells. Journal of Medical Virology 42:272–278
    [Google Scholar]
  31. Yoo B. J., Selby M. J., Choe J., Suh B. S., Choi S. H., Joh J. S., Nuovo G. J., Lee H., Houghton M., Han J. H. 1995; Transfection of a differentiated human hepatoma cell line (Huh7) with in vitro-transfected hepatitis C virus (HCV) RNA and establishment of a long-term culture persistently infected with HCV. Journal of Virology 69:32–38
    [Google Scholar]
  32. Zignego A. L., Macchia D., Monti M., Thiers V., Mazzetti M., Foschi M., Maggi E., Romagnani S., Gentilini P., Brechot C. 1992; Infection of peripheral mononuclear blood cells by hepatitis C virus. Journal of Hepatology 15:382–386
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/0022-1317-77-5-1043
Loading
/content/journal/jgv/10.1099/0022-1317-77-5-1043
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error