1887

Abstract

Hepatitis C virus (HCV) populations consist of heterogeneous mixtures of genetically different but closely related variants defined as a ‘quasispecies’. The longitudinal fluctuation of HCV quasispecies populations in chronic hepatitis C has not been elucidated. Serial plasma samples were obtained from four patients with chronic hepatitis C (two patients without any treatment and two patients treated with interferon), and cDNA fragments containing the 5′-terminal region of the E2 gene of HCV were amplified from plasma RNA using PCR. Since conventional cloning of PCR products detects only a small part of the entire population, PCR products of each sample were separated by electrophoresis using single-strand conformation polymorphism (SSCP) analysis, which can distinguish DNA fragments of the same size as different electrophoretic bands depending on their sequence-specific conformation. Separated DNA fragments were recovered from SSCP bands in gels and their nucleotide sequences determined. SSCP electrophoresis separated PCR products into bands with different mobility. Sequence analysis of these bands confirmed that HCV populations in each patient are composed of quasispecies with different E2-hypervariable regions (HVR), which are known to contain antibody epitopes. Different patterns of variation in the HVR of quasispecies were observed in individual patients with different clinical features over time during chronic infection. Following interferon treatment, some quasispecies disappeared during the treatment and reappeared after the end of the treatment, whereas other quasispecies in the same patient remained during the treatment suggesting that the sensitivity to interferon is different among quasispecies.

Loading

Article metrics loading...

/content/journal/jgv/10.1099/0022-1317-75-6-1361
1994-06-01
2021-10-21
Loading full text...

Full text loading...

/deliver/fulltext/jgv/75/6/JV0750061361.html?itemId=/content/journal/jgv/10.1099/0022-1317-75-6-1361&mimeType=html&fmt=ahah

References

  1. Chomczynski P., Sacchi N. 1987; Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Analytical Biochemistry 162:156–159
    [Google Scholar]
  2. Choo Q. L., Kuo G., Weiner A. J., Overby L. R., Bradley D. W., Houghton M. 1989; Isolation of a cDNA clone derived from a blood borne non-A, non-B viral hepatitis genome. Science 244:359–362
    [Google Scholar]
  3. Chuang W. L., Omata M., Ehata T., Yokosuka O., Ohto M. 1993; Concentrating missense mutations in core gene of hepatitis B virus. Digestive Diseases and Sciences 38:594–600
    [Google Scholar]
  4. Cottrell S., Bicknell D., Kaklamanis L., Bodmer W. F. 1992; Molecular analysis of APC mutations in familial adenomatous polyposis and sporadic colon carcinomas. Lancet 340:626–630
    [Google Scholar]
  5. Cristiano K., Di B. A., Hoofnagle J. H., Feinstone S. M. 1991; Hepatitis C viral RNA in serum of patients with chronic non-A, non- B hepatitis: detection by the polymerase chain reaction using multiple primer sets. Hepatology 14:51–55
    [Google Scholar]
  6. Enomoto N., Sakamoto N., Kurosaki M., Sato C., Marumo F. 1993; The hypervariable region of HCV genome changes sequentially with the progression of acute HCV infection to chronic hepatitis. Journal of Hepatology 17:415–116
    [Google Scholar]
  7. Enomoto N., Sato C., Marumo F. 1994; Hepatitis C virus remaining after interferon treatment has the variation in the hypervariable region of envelope 2 gene. Journal of Hepatology 20:252–261
    [Google Scholar]
  8. Gyllensten U. B., Erlich H. A. 1988; Generation of singlestranded DNA by the polymerase chain reaction and its application to direct sequencing of the HLA-DQA locus. Proceedings of the National Academy of Sciences U.S.A.: 857652–7656
    [Google Scholar]
  9. Holland J. J., De La Torre J. C., Steinhauer D. A. 1992; RNA virus populations as quasispecies. Current Topics in Microbiology and Immunology 176:1–20
    [Google Scholar]
  10. Kanai K., Kako M., Okamoto H. 1992; HCV genotypes in chronic hepatitis C and response to interferon. Lancet 339:1543
    [Google Scholar]
  11. Kato N., Hijikata M., Ootsuyama Y., Nakagawa M., Ohkoshi S., Sugimura T., Shimotohno 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 U.S.A.: 879524–9528
    [Google Scholar]
  12. Kato N., Ootsuyama Y., Tanaka T., Nakagawa M., Muraiso K., Ohkoshi S., Hijikata M., Shimotohno K. 1992a; Marked sequence diversity in the putative envelope proteins of hepatitis C virus. Virus Research 22:107–123
    [Google Scholar]
  13. Kato N., Ootsuyama Y., Ohkoshi S., Nakazawa T., Sekiya H., Hijikata M., Shimotohno K. 1992b; Characterization of hypervariable regions in the putative envelope protein of hepatitis C virus. Biochemical and Biophysical Research Communication 189:119–127
    [Google Scholar]
  14. Kurosaki M., Enomoto N., Marumo F., Sato C. 1993; Rapid sequence evolution of the hypervariable region of hepatitis C virus during the course of chronic hepatitis. Hepatology 18:1293–1299
    [Google Scholar]
  15. Martell M., Esteban J.I., Quer J., Genesca J., Weiner A., Esteban R., Guardia J., Gomez J. 1992; Hepatitis C virus (HCV) circulates as a population of different but closely related genomes: quasispecies nature of HCV genome distribution. Journal of Virology 66:3225–3229
    [Google Scholar]
  16. Murakawa K., Esumi M., Kato T., Kambara H., Shikata T. 1992; Heterogeneity within the nonstructural protein 5-encoding region of hepatitis C viruses from a single patient. Gene 117:229–232
    [Google Scholar]
  17. 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 U.S.A.: 883392–3396
    [Google Scholar]
  18. Okada S., Akahane Y., Suzuki H., Okamoto H., Mishiro S. 1992; The degree of variability in the amino terminal region of the E2/NS1 protein of hepatitis C virus correlates with responsiveness to interferon therapy in viremic patients. Hepatology 16:619–624
    [Google Scholar]
  19. Okamoto H., Okada S., Sugiyama Y., Tanaka T., Sugai Y., Akahane Y., Machida A., Mishiro S., Yoshizawa H., Miyakawa Y. 1990; Detection of hepatitis C virus RNA by a two- stage polymerase chain reaction with two pairs of primers deduced from the 5ʹ-non-coding region. Japanese Journal of Experimental Medicine 60:215–222
    [Google Scholar]
  20. Okamoto H., Sugiyama Y., Okada S., Kurai K., Akahane Y., Sugai Y., Tanaka T., Sato K., Tsuda F., Miyakawa Y., Mayumi M. 1992a; Typing hepatitis C virus by polymerase chain reaction with type-specific primers: application to clinical surveys and tracing infectious sources. Journal of General Virology 73:673–679
    [Google Scholar]
  21. Okamoto H., Kojima M., Okada S., Yoshizawa H., Iizuka H., Tanaka T., Muchmore E. E., Peterson D. A., Ito Y., Mishiro S. 1992b; Genetic drift of hepatitis C virus during an 8·2-year infection in a chimpanzee: variability and stability. Virology 190:894–899
    [Google Scholar]
  22. Orita M., Suzuki Y., Sekiya T., Hayashi K. 1989; Rapid and sensitive detection of point mutations and DNA polymorphisms using the polymerase chain reaction. Genomics 5:874–879
    [Google Scholar]
  23. Oshima M., Tsuchiya M., Yagasaki M., Orita T., Hasegawa M., Tomonoh K., Kojima T., Hirata Y., Yamamoto O., Sho Y., Maeda E., Arima T. 1991; cDNA clones of Japanese hepatitis C virus genomes derived from a single patient show sequence heterogeneity. Journal of General Virology 72:2805–2809
    [Google Scholar]
  24. Saiki R. K., Gelfand D. H., Stoffel S., Scharf S. J., Higuchi R., Horn T., Mullis K. B., Erlich H. A. 1988; Primer directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239:487–491
    [Google Scholar]
  25. Steinhauer D. A., Holland J. J. 1987; Rapid evolution of RNA viruses. Annual Review of Microbiology 41:409–433
    [Google Scholar]
  26. Takada N., Takase S., Enomoto N., Takada A., Date T. 1992; Clinical backgrounds of the patients having different types of hepatitis C virus genomes. Journal of Hepatology 14:35–40
    [Google Scholar]
  27. Taniguchi S., Okamoto M., Sakamoto M., Kojima M., Tsuda F., Tanaka T., Munekata E., Muchmore E. E., Peterson D. A., Mishiro S. 1993; A structurally 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. 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 180:842–884
    [Google Scholar]
  29. Weiner A. J., Geysen H. M., Christopherson C., Hall J. E., Mason T. J., Saracco G., Bonino F., Crawford K., Marion 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 U.S.A.: 893468–3472
    [Google Scholar]
  30. Yap E.-P.H., Mcgee J. O. 1992a; Nonisotopic SSCP detection in PCR products by ethidium bromide staining. Trends in Genetics 8:49
    [Google Scholar]
  31. Yap E.-P. H., Lo Y.-M. D., Cooper K., Fleming K. A., Mcgee J. O. 1992b; Exclusion of false-positive PCR viral diagnosis by single-strand conformation polymorphism. Lancet 340:736
    [Google Scholar]
  32. Yoshioka K., Kakumu S., Wakita T., Ishikawa T., Itoh Y., Takayanagi M., Higashi Y., Shibata M., Morishita T. 1992; Detection of hepatitis C virus by polymerase chain reaction to interferon-alpha therapy: relationship to genotypes of hepatitis C virus. Hepatology 16:293–299
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/0022-1317-75-6-1361
Loading
/content/journal/jgv/10.1099/0022-1317-75-6-1361
Loading

Data & Media loading...

Most cited this month Most Cited RSS feed

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