1887

Abstract

In infected humans, hepatitis C virus (HCV) exists as a quasispecies typically characterized by multiple nucleotide substitutions within the second envelope gene hypervariable region 1 (HVR1). In the current study, we used heteroduplex gel shift analysis (GSA) of HVR1 sequences amplified directly from patients′ sera to define two patterns of HCV quasispecies: (i) simple quasispecies, which gave a mostly homogeneous gel shift profile with a single predominant band and (ii) complex quasispecies, which gave a gel shift profile with multiple bands. Recombinant HVR1 libraries were generated from two patients with complex HCV quasispecies (cases 1 and 2) and two patients with simple HCV quasispecies (cases 3 and 4), and 129 individual clones were analysed by either GSA, nucleotide sequencing or both techniques. In case 1 we identified a highly complex HCV quasispecies with 11 distinct HVR1 variants differing by 1–51 nucleotide changes. We found a general but not absolute correlation between GSA pattern and the number or position of nucleotide changes within HVR1. In case 2, the complex HCV quasispecies consisted of three distinct major variants; GSA of individual HVR1 clones allowed us to reconstruct the complex quasispecies pattern . In case 3, the simple quasispecies comprised 66% homogeneous clones and 33% unique minor variants differing by 1–3 nucleotides from the consensus sequence. In case 4, the simple quasispecies was 84% homogeneous, but six unique major shift variants were identified among 31 clones by GSA. In summary, HCV quasispecies can be characterized based on GSA profiles following direct PCR amplification of HVR1 sequences from patients′ serum; the GSA profiles approximate the clonal population of HCV as determined by clonal analysis. GSA of HVR1 clones showed a strong correlation with nucleotide sequencing.

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1995-07-01
2024-12-12
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References

  1. Alter M. J., Margolis H. S., Krawczynski K., Judson F. N., Mares A., Alexander W. J., Hu P. Y., Miller J. K., Gerber M. A., Sampliner R. E., Meeks E. L., Beach M. J. 1992; Natural history of community-acquired hepatitis C in the United States. New England Journal of Medicine 327:1899–1905
    [Google Scholar]
  2. Ausubel F. M., Brent R., Kingston E., Moore D. D., Seidman J. G., Smith J. A., Struhl K. 1991 Current Protocols in Molecular Biology New York: Wiley–Interscience;
    [Google Scholar]
  3. Delwart E. L., Shpaer E. G., Louwagie J., McCutchan F. E., Grez M., Rubsamen-Waigmann H., Mullins J. I. 1993; Genetic relationships determined by a DNA heteroduplex mobility assay: analysis of HIV-1 env genes. Science 262:1257–1261
    [Google Scholar]
  4. Delwart E. L., Sheppard H. W., Walker B. D., Goudsmit J., Mullins J. I. 1994; Human immunodeficiency virus type 1 evolution in vivo tracked by DNA heteroduplex mobility assays. Journal of Virology 68:6672–6683
    [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. Gretch D., Lee W., Corey L. 1992; Use of aminotransferase, hepatitis C antibody, and hepatitis C polymerase chain reaction RNA assays to establish the diagnosis of hepatitis C virus infection in a diagnostic virology laboratory. Journal of Clinical Microbiology 30:2145–2149
    [Google Scholar]
  7. Gretch D. R., Wilson J. J., Carithers R. L., Dela Rosa C., Han J. H., Corey L. 1993; Detection of hepatitis C virus RNA: comparison of one-stage polymerase chain reaction (PCR) with nested-set PCR. Journal of Clinical Microbiology 31:289–291
    [Google Scholar]
  8. Gretch D. R., Bacchi C. E., Corey L., Rosa C., Lesniewski R. R., Kowdley K., Gown A., Frank I., Perkins J. D., Carithers R. L. 1995; Persistent hepatitis C virus infection following liver transplantation: clinical and virological features. Hepatology 22: (in press)
    [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. Johnson R. J., Gretch D. R., Yamabe H., Hart J., Bacchi C. E., Hartwell P., Couser W. G., Corey L., Wener M. H., Alpers C. E., Willson R. 1993; Membranoproliferative glomerulonephritis associated with hepatitis C virus infection. New England Journal of Medicine 328:465–470
    [Google Scholar]
  11. Kato N., Ootsuyama Y., Sekiya H., Ohkoshi S., Kakazawa T., Hijikata M., Shimotohno K. 1994; Genetic drift in hypervariable region 1 of the viral genome in persistent hepatitis C virus infection. Journal of Virology 68:4776–4784
    [Google Scholar]
  12. Martell M., Esteban J. I., Quer J., Genesca J., Weiner A., Esteban R., Guraida 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]
  13. Plagemann P. G. W. 1991; Hepatitis C virus. Archives of Virology 120:165–180
    [Google Scholar]
  14. Sekiya H., Kato N., Ootsuyama Y., Nakazawa T., Yamauchi K., Shimotohno K. 1993; Genetic alterations of the putative envelope proteins encoding region of the hepatitis C virus in the progression to relapsed phase from acute hepatitis: humoral immune response to envelope glycoprotein (Gp70) of hepatitis C virus. Journal of Virology 67:3923–3930
    [Google Scholar]
  15. Simmonds P., Holmes E. C., Cha T.-A., Chan S.-W., McOmish F., Irvine B., Beall E., Yap P. L., Kolberg J., Urdea M. S. 1993; Classification of hepatitis C virus into six major genotypes and a series of subtypes by phylogenetic analysis of the NS-5 region. Journal of General Virology 74:2391–2399
    [Google Scholar]
  16. Trepo C., Habersetzer F., Bailly F., Berby F., Pichoud C., Berthillon B., Vitvitski L. 1994; Interferon therapy for hepatitis C. Antiviral Research 24:155–163
    [Google Scholar]
  17. 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 proteins. Virology 180:842–848
    [Google Scholar]
  18. Weiner A., Geyson J. 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, USA 89:3468–3472
    [Google Scholar]
  19. Weiner A. J., Thaler M. M., Crawford K., Ching K., Kansopon J., Chien D. Y., Hall J. E., Hu F., Houghton M. 1993; A unique, predominant hepatitis C virus variant found in an infant born to a mother with multiple variants. Virology 67:4365–4368
    [Google Scholar]
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