Skip to main content
Log in

Sequence analysis of hepatitis B virus DNA in immunologically negative infection

  • Original Papers
  • Published:
Archives of Virology Aims and scope Submit manuscript

Summary

It was previously demonstrated that the serum of some patients without immunological evidence of HBV infection contains the virus. Here we demonstrated by sequence analysis that the serum of such a patient contained a mixed HBV population. In comparison with HBV genomes of different genotypes twenty-two nucleotide variations were found in all clones sequenced in parallel. One nucleotide variation was identified within the enhancer I. Twelve of the twenty-two nucleotide variations caused altogether fifteen changes of amino acid sequence in known or predicted viral proteins. The proteins of the P open reading frame, which are most important for viral replication, were affected by nine amino acid substitutions. Three amino acid substitutions concerned the product of the X gene, a transcriptional transactivator of various viral and cellular promoters. Three mutations were only observed in some of the clones. One point mutation affected the direct repeats of the enhancer II. It occurred together with an 8 bp-deletion involving the C promoter region and the X gene. The third mutation was a single insertion, causing a fusion of the X and C gene. One or several of the identified mutations could be responsible for the diminished rate of replication and consequently for the low-titred, immunologically negative HBV infection.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Similar content being viewed by others

References

  1. Bartenschlager R, Schaller H (1988 a) The aminoterminal domain of the hepadnaviral P-gene encodes the terminal protein (genome-linked protein) believed to prime reverse transcription. EMBO J 7: 4185–4192

    Google Scholar 

  2. Bartenschlager R, Schaller H (1988 b) The P gene product of hepatitis B virus is required as a structural component for genomic RNA encapsidation. J Virol 64: 5324–5332

    Google Scholar 

  3. Ben-Levy R, Faktor O, Berger I, Shaul Y (1989) Cellular factors that interact with the hepatitis B virus enhancer. Mol Cell Biol 9: 1804–11809

    Google Scholar 

  4. Bhatnager PK, Papas E, Blum HE, Milich DR, Nitecki D, Karels MJ, Vyas GN (1982) Immune response to synthetic peptide analogues of HBsAg for ‘a’ determinant. Proc Natl Acad Sci USA 78: 4400–4404

    Google Scholar 

  5. Bichko V, Pushko P, Dreilina D, Pumpen P, Gren E (1985) Subtype ayw variant of hepatitis B virus. DNA primary structure analysis. FEBS Lett 185: 208–212

    Google Scholar 

  6. Blum HE, Zhang Z-S, Galun E, von Weizsäcker F, Garner B, Liang TJ, Wands JR (1992) Hepatitis B virus X protein is not central to the viral life cycle in vitro. J Virol 66: 1223–1227

    Google Scholar 

  7. Bonino F, Brunetto MR, Rizetto M, Will H (1991) Hepatitis B virus unable to secrete antigen. Gastroenterology 100: 1138–1141

    Google Scholar 

  8. Choo QL, Kuo G, Weiner AJ, Overby LR, Bradley DW, Houghton M (1989) Isolation of a cDNA-clone derived from a bloodborne non-A, non-B viral hepatitis genome. Science 244: 359–362

    Google Scholar 

  9. Faruqi AF, Roychoudhury S, Greenberg R, Israel J, Shih C (1991) Replication-defective missense mutations within the terminal protein and spacer/intron regions of the polymerase gene of human hepatitis B virus. Virology 183: 764–768

    Google Scholar 

  10. Figus A, Blum HE, Vyas GN, Virgillis SD, Cao A, Lippi M, Lai E, Balestrieri A (1984) Hepatitis B viral nucleotide sequences in non-A, non-B hepatitis B virus-related chronic liver disease. Hepatology 4: 364–368

    Google Scholar 

  11. Galibert F, Mandart E, Fitoussi F, Tiollais P, Charnay P (1979) Nucleotide sequence of the hepatitis B virus genome (subtype ayw) cloned inE. coli. Nature 281: 646–650

    Google Scholar 

  12. Hirsch RC, Lavine JE, Chang L-J, Varmus HE, Ganem D (1990) Polymerase gene products of hepatitis B viruses are required for genomic RNA packaging as well as for reverse transcription. Nature 344: 552–555

    Google Scholar 

  13. Honigwachs J, Faktor O, Dikstein K, Shaul Y, Laub O (1989) Liver-specific expression of hepatitis B virus is determined by the combined action of the core gene promotor and the enhancer. J Virol 63: 919–924

    Google Scholar 

  14. Horwich AL, Furtak K, Pugh J, Summers J (1990) Synthesis of hepadnavirus particles that contain replication-defective duck hepatitis B virus genome in cultured HuH7 cells. J Virol 64: 642–650

    Google Scholar 

  15. Hu KQ, Siddiqui A (1991) Regulation of the hepatitis B virus gene expression by the enhancer element I. Virology 181: 721–726

    Google Scholar 

  16. Kim SH, Hong SP, Kim SK, Lee WS, Rho HM (1992) Replication of a mutant hepatitis B virus with a fused X-C reading frame in hepatoma cells. J Gen Virol 73: 2421–2424

    Google Scholar 

  17. Kobayashi M, Koike K (1984) Complete nucleotide sequence of hepatitis B virus DNA of subtype adr and its conserved gene organization. Gene 30: 227–232

    Google Scholar 

  18. Kuo G, Choo QL, Alter HJ, Gitnick GL, Redeker AG, Purcell RH, Miyamura T, Dienstag JL, Alter MJ, Stevens CE, Tegtmeier GE, Bonino F, Colombo M, Lee WS, Kuo C, Berger K, Shuster JR, Overby LR, Bradley DW, Houghton M (1989) An assay for circulating antibodies to a major etiologic virus of human non-A, non-B hepatitis. Science 244: 362–364

    Google Scholar 

  19. Lai ME, Mazzoleni AP, Balestrieri A, Melis A, Porru A (1992) Sequence analysis of HBV genomes isolated from patients with HBsAg-negative chronic liver disease (unpublished, Accession no. [EMBL data library] X 65259)

  20. Liang TJ, Blum HE, Wands JR (1990) Characterization and biological properties of a hepatitis B virus isolated from a patient without hepatitis B virus serologic markers. Hepatology 12: 204–212

    Google Scholar 

  21. Lo SJ, You L-R, Lee Y-HW (1990) The hepatitis B virus X-C-fusion protein is unlikely to be produced by the mechanism of ribosomal frameshifting. Virology 178: 584–587

    Google Scholar 

  22. López-Cabrera M, Letovsky J, Hu K-Q, Siddiqui A (1990) Multiple liver-specific factors bind to the hepatitis B virus core/pregenomic promoter: trans-activation and repression by CCAAT/enhancer binding protein. Proc Natl Acad Sci USA 87: 5069–5073

    Google Scholar 

  23. López-Cabrera M, Letovsky J, Hu K-QW, Siddiqui A (1991) Transcriptional factor C/EBP binds to and transactivates the enhancer element II of the hepatitis B virus. Virology 183: 825–829

    Google Scholar 

  24. Nakamura T, Tokino T, Nagaya T, Matsubara K (1988) Microdeletion associated with the integration process of hepatitis B virus DNA. Nucleic Acids Res 16: 4865–4873

    Google Scholar 

  25. Norder H, Hammas B, Löfdahl S, Couroucé A-M, Magnius LO (1992) Comparison of the amino acid sequences of nine different serotypes of hepatitis B surface antigen and genomic classification of the corresponding hepatitis B virus strains. J Gen Virol 73: 1201–1208

    Google Scholar 

  26. Norder H, Couroucé A-M, Magnius LO (1992) Molecular basis of hepatitis B virus serotype variations within the four major subtypes. J Gen Virol 73: 3141–3145

    Google Scholar 

  27. Okamoto H, Imai M, Shimozaki M, Hoshi Y, Iizuka H, Gotanda T, Tsuda F, Miyakawa Y, Mayumi M (1986) Nucleotide sequence of a cloned hepatitis B virus genome, subtype ayr: comparison with genomes of the other three subtypes. J Gen Virol 67: 2305–2314

    Google Scholar 

  28. Okamoto H, Tsuda F, Sakugawa H, Sastrosoewignjo RI, Mitsunobu I, Miyakawa Y, Mayumi M (1988) Typing hepatitis B virus by homology in nucleotide sequence: comparison of surface antigen subtypes. J Gen Virol 69: 2575–2583

    Google Scholar 

  29. Ono Y, Onda H, Sasada R, Igarashi K, Sugino Y, Nishioka K (1983) The complete nucleotide sequences of the cloned hepatitis B virus DNA; subtype adr and adw. Nucleic Acids Res 11: 1747–1757

    Google Scholar 

  30. Preisler-Adams S, Schlayer H-J, Peters T, Korp R, Rasenack J (1993) Complete nucleotide sequence of a hepatitis B virus, subtype adw 2, and identification of three types of C open reading frame. Nucleic Acids Res 21: 2258

    Google Scholar 

  31. Radzivill G, Tucker W, Schaller H (1990) Mutational analysis of the hepatitis B virus P gene product: domain structure and RNaseH activity. J Virol 64: 613–620

    Google Scholar 

  32. Renbao G, Meijin C, Lueping S, Suwen Q, Zaiping L (1987) The complete nucleotide sequence of the cloned DNA of hepatitis B virus subtype adr in pADR-1. Sci Sin 30: 507–521

    Google Scholar 

  33. Repp R, Keller C, Borkhardt A, Csecke A, Schaefer S, Gerlich WH, Lampert F (1992) Detection of a hepatitis B virus variant with a truncated X gene and enhancer II. Arch Virol 125: 299–304

    Google Scholar 

  34. Rossner MT (1992) Review: hepatitis B virus X-gene product: a promiscuous transcriptional activator. J Med Virol 36: 101–117

    Google Scholar 

  35. Saiki RK, Gelfund DH, Stoffel S, Scharf SJ, Higuchi R, Horn GT, Mullis KB, Erlich HA (1988) Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239: 487–491

    Google Scholar 

  36. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory, New York

    Google Scholar 

  37. Santantonio T, Pontisso P, Milella M, Chemello L, Luchena N, Pastore G (1989) Detection of hepatitis B virus DNA in serum by spot hybridization technique: sensitivity and specificity of radiolabeled and biotin-labeled probes. Res Clin Lab 20: 29–35

    Google Scholar 

  38. Sastrosoewignjo RJ, Omi S, Okamoto H, Mayumi M, Rustam M, Sujudi X (1987) The complete nucleotide sequence of HBV DNA clone of subtype adw. ICMR Ann 7: 51–60

    Google Scholar 

  39. Schlayer H-J, Peters T, Preisler S, Berthold H, Gerok W, Rasenack J (1992) Cause and frequency of posttransfusion hepatitis after open-heart surgery. Clin Invest 70: 579–584

    Google Scholar 

  40. Schödel F, Moriartry AM, Peterson DL, Zheng J, Hughes JL, Will H, Leturcq DJ, McGee JS, Milich DR (1992) The position of heterologous epitopes inserted in hepatitis B virus core particles determined their immunogenicity. J Virol 66: 106–114

    Google Scholar 

  41. Seeger C, Ganem D, Varmus HE (1986) Biochemical and genetic evidence for the hepatitis B virus replication strategy. Science 232: 477–484

    Google Scholar 

  42. Summers J, Mason W (1982) Replication of the genome of a hepatitis B-like virus by reverse transcription of an RNA intermediate. Cell 29: 403–415

    Google Scholar 

  43. Thiers V, Nakajima E, Kremsdorf D, Mack D, Schellekens H, Driss F, Goudeau A, Wands J, Sninsky J, Tiollais P, Bréchot C (1988) Sequences in blood samples negative for HBV serologic markers. Lancet 2: 1273–1276

    Google Scholar 

  44. Tindall KR, Kungel TA (1988) Fidelity of DNA synthesis by the Thermus aquaticus DNA polymerase. Biochemistry 27: 6008–6013

    Google Scholar 

  45. Tiollais P, Pourcel C, Dejean A (1985) The hepatitis B virus. Nature 317: 489–495

    Google Scholar 

  46. Tong SP, Li J, Vitvitsky L, Trépo C (1990) Active hepatitis B replication in the presence of anti-Hbe is associated with viral variants containing an inactive pre C region. Virology 176: 596–603

    Google Scholar 

  47. Valenzuela P, Quiroga M, Zaldivar J, Gray P, Rutter W (1980) The nucleotide sequence of the hepatitis B viral genome and the identification of the major viral genes. In: Fields B, Jaenisch R, Fox C (eds) Animal virus genetics. Academic Press, New York, pp 57–70

    Google Scholar 

  48. Walter E, Blum HE, Offensperger WB, Zeschnigk C, Offensperger S, Gerok W (1987) Spot-blot hybridization assay for the detection of hepatitis B virus DNA in serum: factors determining its sensitivity and specificity. Hepatology 7: 557–562

    Google Scholar 

  49. Wang GH, Seeger C (1992) The reverse transcriptase of hepatitis B virus acts as a protein primer for viral DNA synthesis. Cell 71: 663–670

    Google Scholar 

  50. Will H, Reiser W, Weimer T, Pfaff E, Bucher M, Sprengel R, Cattaneo R, Schaller H (1987) Replication strategy of human hepatitis B virus. J Virol 61: 909–911

    Google Scholar 

  51. Zhou D-X, Yen TSB (1990) Differential regulation of the hepatitis B virus surface gene promotors by a second viral enhancer. J Biol Chem 265: 20731–20734

    Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Rights and permissions

Reprints and permissions

About this article

Cite this article

Preisler-Adams, S., Schlayer, H.J., Peters, T. et al. Sequence analysis of hepatitis B virus DNA in immunologically negative infection. Archives of Virology 133, 385–396 (1993). https://doi.org/10.1007/BF01313777

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF01313777

Keywords

Navigation