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. 1995 Sep;69(9):5437–5444. doi: 10.1128/jvi.69.9.5437-5444.1995

A novel method for efficient amplification of whole hepatitis B virus genomes permits rapid functional analysis and reveals deletion mutants in immunosuppressed patients.

S Günther 1, B C Li 1, S Miska 1, D H Krüger 1, H Meisel 1, H Will 1
PMCID: PMC189390  PMID: 7636989

Abstract

Current knowledge of hepatitis B virus (HBV) sequence heterogeneity is based mainly on sequencing of amplified subgenomic HBV fragments. Here, we describe a method which allows sensitive amplification and simplified functional analysis of full-length HBV genomes with or without prior cloning. By this method, a large number of HBV genomes were cloned from sera of six immunosuppressed kidney transplant patients. Two size classes of HBV genomes, one 3.2 kb and another about 2.0 kb in size, were found in all patients. The genome population from one serum sample was studied in detail by size analysis of subgenomic PCR fragments and sequencing. Regions with deletions and insertions were mapped in the C gene and pre-S region. Up to 100% of HBV genomes in all other immunosuppressed patients also had deletions in the C gene. Our results demonstrate the potential of the established method for the structural and functional characterization of heterogeneous populations of complete virion-encapsidated HBV DNAs and suggest that HBV genomes with C gene deletions can have a selective advantage in immunosuppressed patients.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Ackrill A. M., Naoumov N. V., Eddleston A. L., Williams R. Specific deletions in the hepatitis B virus core open reading frame in patients with chronic active hepatitis B. J Med Virol. 1993 Oct;41(2):165–169. doi: 10.1002/jmv.1890410213. [DOI] [PubMed] [Google Scholar]
  2. Barnes W. M. PCR amplification of up to 35-kb DNA with high fidelity and high yield from lambda bacteriophage templates. Proc Natl Acad Sci U S A. 1994 Mar 15;91(6):2216–2220. doi: 10.1073/pnas.91.6.2216. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Blum H. E., Galun E., Liang T. J., von Weizsäcker F., Wands J. R. Naturally occurring missense mutation in the polymerase gene terminating hepatitis B virus replication. J Virol. 1991 Apr;65(4):1836–1842. doi: 10.1128/jvi.65.4.1836-1842.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chen P. J., Chen C. R., Sung J. L., Chen D. S. Identification of a doubly spliced viral transcript joining the separated domains for putative protease and reverse transcriptase of hepatitis B virus. J Virol. 1989 Oct;63(10):4165–4171. doi: 10.1128/jvi.63.10.4165-4171.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Fernholz D., Galle P. R., Stemler M., Brunetto M., Bonino F., Will H. Infectious hepatitis B virus variant defective in pre-S2 protein expression in a chronic carrier. Virology. 1993 May;194(1):137–148. doi: 10.1006/viro.1993.1243. [DOI] [PubMed] [Google Scholar]
  6. Fouillot N., Tlouzeau S., Rossignol J. M., Jean-Jean O. Translation of the hepatitis B virus P gene by ribosomal scanning as an alternative to internal initiation. J Virol. 1993 Aug;67(8):4886–4895. doi: 10.1128/jvi.67.8.4886-4895.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Galibert F., Mandart E., Fitoussi F., Tiollais P., Charnay P. Nucleotide sequence of the hepatitis B virus genome (subtype ayw) cloned in E. coli. Nature. 1979 Oct 25;281(5733):646–650. doi: 10.1038/281646a0. [DOI] [PubMed] [Google Scholar]
  8. Günther S., Meisel H., Reip A., Miska S., Krüger D. H., Will H. Frequent and rapid emergence of mutated pre-C sequences in HBV from e-antigen positive carriers who seroconvert to anti-HBe during interferon treatment. Virology. 1992 Mar;187(1):271–279. doi: 10.1016/0042-6822(92)90315-g. [DOI] [PubMed] [Google Scholar]
  9. Hasegawa K., Huang J., Rogers S. A., Blum H. E., Liang T. J. Enhanced replication of a hepatitis B virus mutant associated with an epidemic of fulminant hepatitis. J Virol. 1994 Mar;68(3):1651–1659. doi: 10.1128/jvi.68.3.1651-1659.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Köck J., Schlicht H. J. Analysis of the earliest steps of hepadnavirus replication: genome repair after infectious entry into hepatocytes does not depend on viral polymerase activity. J Virol. 1993 Aug;67(8):4867–4874. doi: 10.1128/jvi.67.8.4867-4874.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Miska S., Will H. Hepatitis B virus C-gene variants. Arch Virol Suppl. 1993;8:155–169. doi: 10.1007/978-3-7091-9312-9_16. [DOI] [PubMed] [Google Scholar]
  12. Okamoto H., Wang Y., Tanaka T., Machida A., Miyakawa Y., Mayumi M. Trans-complementation among naturally occurring deletion mutants of hepatitis B virus and integrated viral DNA for the production of viral particles with mutant genomes in hepatoma cell lines. J Gen Virol. 1993 Mar;74(Pt 3):407–414. doi: 10.1099/0022-1317-74-3-407. [DOI] [PubMed] [Google Scholar]
  13. Okamoto H., Yotsumoto S., Akahane Y., Yamanaka T., Miyazaki Y., Sugai Y., Tsuda F., Tanaka T., Miyakawa Y., Mayumi M. Hepatitis B viruses with precore region defects prevail in persistently infected hosts along with seroconversion to the antibody against e antigen. J Virol. 1990 Mar;64(3):1298–1303. doi: 10.1128/jvi.64.3.1298-1303.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Parfrey P. S., Forbes R. D., Hutchinson T. A., Kenick S., Farge D., Dauphinee W. D., Seely J. F., Guttmann R. D. The impact of renal transplantation on the course of hepatitis B liver disease. Transplantation. 1985 Jun;39(6):610–615. doi: 10.1097/00007890-198506000-00007. [DOI] [PubMed] [Google Scholar]
  15. Raimondo G., Schneider R., Stemler M., Smedile V., Rodino G., Will H. A new hepatitis B virus variant in a chronic carrier with multiple episodes of viral reactivation and acute hepatitis. Virology. 1990 Nov;179(1):64–68. doi: 10.1016/0042-6822(90)90274-u. [DOI] [PubMed] [Google Scholar]
  16. Santantonio T., Jung M. C., Miska S., Pastore G., Pape G. R., Will H. Prevalence and type of pre-C HBV mutants in anti-HBe positive carriers with chronic liver disease in a highly endemic area. Virology. 1991 Aug;183(2):840–844. doi: 10.1016/0042-6822(91)91022-9. [DOI] [PubMed] [Google Scholar]
  17. Santantonio T., Jung M. C., Schneider R., Fernholz D., Milella M., Monno L., Pastore G., Pape G. R., Will H. Hepatitis B virus genomes that cannot synthesize pre-S2 proteins occur frequently and as dominant virus populations in chronic carriers in Italy. Virology. 1992 Jun;188(2):948–952. doi: 10.1016/0042-6822(92)90559-8. [DOI] [PubMed] [Google Scholar]
  18. Takayanagi M., Kakumu S., Ishikawa T., Higashi Y., Yoshioka K., Wakita T. Comparison of envelope and precore/core variants of hepatitis B virus (HBV) during chronic HBV infection. Virology. 1993 Sep;196(1):138–145. doi: 10.1006/viro.1993.1462. [DOI] [PubMed] [Google Scholar]
  19. Terré S., Petit M. A., Bréchot C. Defective hepatitis B virus particles are generated by packaging and reverse transcription of spliced viral RNAs in vivo. J Virol. 1991 Oct;65(10):5539–5543. doi: 10.1128/jvi.65.10.5539-5543.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Tong S. P., Diot C., Gripon P., Li J., Vitvitski L., Trépo C., Guguen-Guillouzo C. In vitro replication competence of a cloned hepatitis B virus variant with a nonsense mutation in the distal pre-C region. Virology. 1991 Apr;181(2):733–737. doi: 10.1016/0042-6822(91)90908-t. [DOI] [PubMed] [Google Scholar]
  21. Ulrich P. P., Bhat R. A., Kelly I., Brunetto M. R., Bonino F., Vyas G. N. A precore-defective mutant of hepatitis B virus associated with e antigen-negative chronic liver disease. J Med Virol. 1990 Oct;32(2):109–118. doi: 10.1002/jmv.1890320208. [DOI] [PubMed] [Google Scholar]
  22. Wakita T., Kakumu S., Shibata M., Yoshioka K., Ito Y., Shinagawa T., Ishikawa T., Takayanagi M., Morishima T. Detection of pre-C and core region mutants of hepatitis B virus in chronic hepatitis B virus carriers. J Clin Invest. 1991 Dec;88(6):1793–1801. doi: 10.1172/JCI115500. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Wallace L. A., Carman W. F. Clinical implications of hepatitis B virus envelope protein variation. Int J Clin Lab Res. 1994;24(2):80–85. doi: 10.1007/BF02593904. [DOI] [PubMed] [Google Scholar]
  24. Wu H. L., Chen P. J., Tu S. J., Lin M. H., Lai M. Y., Chen D. S. Characterization and genetic analysis of alternatively spliced transcripts of hepatitis B virus in infected human liver tissues and transfected HepG2 cells. J Virol. 1991 Apr;65(4):1680–1686. doi: 10.1128/jvi.65.4.1680-1686.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]

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