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Clinical Diagnosis of Hepatitis B Infection: Applications of the Polymerase Chain Reaction

  • Chapter
Diagnosis of Human Viruses by Polymerase Chain Reaction Technology

Part of the book series: Frontiers of Virology ((FRVIROLOGY,volume 1))

Summary

Hepatitis B virus (HBV) infection serves as a good example of the use of the polymerase chain reaction (PCR) for the diagnosis of viral infection. The technique is at least 104 times more sensitive than current dot blot hybridization assays for HBV DNA. PCR overcomes the disadvantage of low concentrations of HBV DNA in patients with a low level of viraemia, and permits the detection, cloning, and sequencing of HBV genomes from such patients. Use of this assay indicates that loss of HBeAg is associated with a decrease rather than complete disappearance of HBV DNA.

A number of other applications of PCR have been informative. The most interesting application of PCR has been to study the genetic heterogeneity of HBV. These investigations have identified variants of HBV by generating fragments of DNA that could be sequenced either directly or by subcloning the amplified fragments into M13 and sequencing. Critical point mutations have been detected by this means. Variants of HBV have been described which are not recognized by the classical serological reagents for HBsAg. Vaccine escape mutants, characterized by a point mutation from guanosine to adenosine, have been recognized. These mutants are not neutralized in anti-HBs-positive vaccines.

Genetic mutants have recently been described in serum of anti-HBe and HBV DNA positive patients. PCR amplification and subsequent sequencing of genomic DNA in patients with this form of chronic hepatitis B has indicated that one or more nucleotide substitutions in the pre-C region of the genome account for the absent expression of HBeAg.

Thus PCR methodology has already altered our perceptions of the natural history of chronic hepatitis B infection, and is likely to shed further light on this complex disease. The technique is proving a useful utility in the diagnosis of hepatitis B and a valuable research tool for advancing our understanding of HBV variants, vaccine escape mutants, and hepatocellular carcinoma.

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References

  • Akahane Y, Yamanaka T, Suzuki H, Sugai Y, Tsuda F, Yotsumoto S, Omi S, Okamoto H, Miyakawa Y, Mayumi M (1990) Chronic active hepatitis with hepatitis B virus DNA and antibody against e antigen in the serum. Disturbed synthesis and secretion of e antigen from hepatocytes due to a point mutation in the precore region. Gastroenterology 99:1113–1119

    CAS  Google Scholar 

  • Bonino F, Hoyer B, Nelson J, Engle R, Verme G, Gerin J (1981) Hepatitis B virus DNA in the sera of HBsAg carriers: a marker of active hepatitis B virus replication in the liver. Hepatology 1:386–391

    Article  PubMed  CAS  Google Scholar 

  • Brechot C (1990) Polymerase chain reaction. A new tool for the study of viral infections in hepatology. J Hepatol 11:124–129

    Article  PubMed  CAS  Google Scholar 

  • Brechot C, Hadchouel M, Scotto J, Degos F, Charnay P, Trepo C, Tiollais P (1981a) Detection of hepatitis B virus DNA in liver and serum: a direct appraisal of the chronic carrier state. Lancet 2:765–768

    Article  PubMed  CAS  Google Scholar 

  • Brechot C, Hadchouel M, Scotto J, Fonck M, Potet F, Vyas GN, Tiollais P (1981 b) State of hepatitis B virus DNA in hepatocytes of patients with hepatitis B surface antigen-positive and-negative liver diseases. Proc Natl Acad Sci USA 78:3906–3910

    Article  PubMed  CAS  Google Scholar 

  • Brechot C, Nalpas B, Courouce AM, Duhamel G, Callard P, Caraot F, Tiollais P, Berthelot P (1982) Evidence that hepatitis B virus has a role in liver-cell carcinoma in alcoholic liver disease. N Engl J Med 306:1384–1387

    Article  PubMed  CAS  Google Scholar 

  • Brechot C, Degos F, Lugassy C, Thiers V, Zafrani S, Franco D, Bismuth H, Trepo C, Benhamou JP, Wands J et al. (1985) Hepatitis B virus DNA in patients with chronic liver disease and negative tests for hepatitis B surface antigen. N Engl J Med 312:270–276

    Article  PubMed  CAS  Google Scholar 

  • Bressac B, Kew M, Wands J, Ozturk M (1991) Selective G to T mutations of p53 gene in hepatocellular carcinoma from southern Africa. Nature 350:429–431

    Article  PubMed  CAS  Google Scholar 

  • Brunetto MR, Stemler M, Bonino F, Schodel F, Oliveri F, Rizzetto M, Verme G, Will H (1990) A new hepatitis B virus strain in patients with severe anti-HBe positive chronic hepatitis B. J Hepatol 10:258–261

    Article  PubMed  CAS  Google Scholar 

  • Brunetto MR, Giarin MM, Oliveri F, Chiaberge E, Baldi M, Alfarano A, Serra A, Saracco G, Verme G, Will H, Bonino F (1991) Wild-type and e antigen-minus hepatitis B viruses and course of chronic hepatitis. Proc Natl Acad Sci USA 88:4186–4190

    Article  PubMed  CAS  Google Scholar 

  • Carman WF, Hadziyannis S, McGarvey MJ et al. (1989) Mutation preventing formation of hepatitis B e antigen in patients with chronic hepatitis B infection. Lancet 2:588–590

    Article  PubMed  CAS  Google Scholar 

  • Carman WF, Zanetti AR, Karayiannis P, Waters J, Manzillo G, Tanzi E, Zuckerman AJ, Thomas HC (1990) Vaccine-induced escape mutant of hepatitis B virus. Lancet 336:325–329

    Article  PubMed  CAS  Google Scholar 

  • Chang C, Enders G, Sprengel R, Peters N, Varmus HE, Ganem D (1987) Expression of the precore region of an avian hepatitis B virus is not required for viral replication. J Virol 61:3322–3325

    PubMed  CAS  Google Scholar 

  • Coursaget P, Yvonnet B, Bourdil C, Mevelec MN, Adamowicz P, Barres JL, Chotard J, N’Doye R, Diop-Mar I, Chiron JP (1987) HBsAG positive reactivity in man not due to hepatitis B virus. Lancet 2:1354–1358

    Article  PubMed  CAS  Google Scholar 

  • Fagan EA, Smith PM, Davison F, Williams R (1986) Fulminant hepatitis B in successive female sexual partners of two anti-HBe-positive males. Lancet 2:538–540

    Article  PubMed  CAS  Google Scholar 

  • Feray C, Zignego AL, Samuel D, Bismuth A, Reynes M, Tiollais P, Bismuth H, Brechot C (1990) Persistent hepatitis B virus infection of mononuclear blood cells without concomitant liver infection. The liver transplantation model. Transplantation 49:1155–1158

    Article  PubMed  CAS  Google Scholar 

  • Fiordalisi G, Cariani E, Mantero G, Zanetti A, Tanzi E, Chiaramonte M, Primi D (1990) High genomic variability in the pre-C region of hepatitis B virus in anti-HBe, HBV DNA-positive chronic hepatitis. J Med Virol 31:297–300

    Article  PubMed  CAS  Google Scholar 

  • Gerken G, Paterlini P, Manns M, Housset C, Terre S, Dienes H-P, Hess G, Gerlich WH, Berthelot P, Zum Büschenfelde K-HM, Brechot C (1991) Assay of hepatitis B virus DNA by polymerase chain reaction and its relationship to pre-S-and S-encoded viral surface antigens. Hepatology 13:158–166

    Article  PubMed  CAS  Google Scholar 

  • Hadziyannis SJ, Lieberman HM, Karvoimtzis GG, Shafritz DA (1983) Analysis of liver disease, nuclear HBcAg, viral replication and hepatitis B virus DNA in liver and serum of HBeAg versus anti-HBe positive carriers of hepatitis B virus. Hepatology 3:656–662

    Article  PubMed  CAS  Google Scholar 

  • Hosono S, Lee CS, Chou MJ, Yang CS, Shih CH (1991) Molecular analysis of the p53 alleles in primary hepatocellular carcinomas and cell lines. Oncogene 6:237–243

    PubMed  CAS  Google Scholar 

  • Hsu IC, Metcalf RA, Sun T, Welsh JA, Wang NJ, Harris CC (1991) Mutational hotspot in the p53 gene in human hepatocellular carcinomas. Nature 350:427–428

    Article  PubMed  CAS  Google Scholar 

  • Jackson JB (1991) Polymerase chain reaction assay for detection of hepatitis B virus. Am J Clin Pathol 95:442–444

    PubMed  CAS  Google Scholar 

  • Kaneko S, Miller RH (1990) Characterization of primers for optimal amplification of hepatitis B virus DNA in the polymerase chain reaction assay. J Virol Methods 29:225–229

    Article  PubMed  CAS  Google Scholar 

  • Kaneko S, Miller RH, Feinstone SM, Unoura M, Kobayashi K, Hattori N, Purcell RH (1989) Detection of serum hepatitis B virus DNA in patients with chronic hepatitis using the polymerase chain reaction assay. Proc Natl Acad Sci USA 86:312–316

    Article  PubMed  CAS  Google Scholar 

  • Kaneko S, Miller RH, Di Bisceglie AM, Feinstone SM, Hoofnagle JH, Purcell RH (1990) Detection of hepatitis B virus DNA in serum by polymerase chain reaction. Gastroen-terology 99:799–804

    CAS  Google Scholar 

  • Kosaka Y, Takase K, Kojima M, Shimizu M, Inoue K, Yoshiba M, Tanaka S, Akahane Y, Okamoto H, Tsuda F et al. (1991) Fulminant hepatitis B: induction by hepatitis B virus mutants defective in the precore region and incapable of encoding e antigen. Gastroen-terology 100:1087–1094

    CAS  Google Scholar 

  • Krogsgaard K, Kryger P, Aldershvile J, Andersson P, Brechot C, Copenhagen Hepatitis Acute Programme (1985) Hepatitis B virus DNA in serum from patients with acute hepatitis B. Hepatology 5:10–30

    Article  PubMed  CAS  Google Scholar 

  • Krogsgaard K, Wantzin P, Aldershvile J, Kryger P, Andersson P, Nielsen JO (1986) Hepatitis B virus DNA in hepatitis B surface antigen-positive blood donors: relation to the hepatitis B e system and outcome in recipients. J Infect Dis 153:298–303

    Article  PubMed  CAS  Google Scholar 

  • Lampertico P, Malter JS, Colombo M, Gerber MA (1990) Detection of hepatitis B virus DNA in formalin-fixed, paraffin-embedded liver tissue by the polymerase chain reaction. Am JPathol 137:253–258

    CAS  Google Scholar 

  • Li J, Tong S, Vitvitski L, Zoulim F, Tr’po C (1990) Rapid detection and further characterization of infection with hepatitis B virus variants containing a stop codon in the distal pre-C region. J Gen Virol 71:1993–1998

    Article  PubMed  CAS  Google Scholar 

  • Liang TJ, Isselbacher KJ, Wands JR (1989) Rapid identification of low level hepatitis B-re-lated viral genome in serum. J Clin Invest 84:1367–1371

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • Liaw YF, Sheen IS, Chen TJ, Chu CM, Pao CC (1991) Incidence, determinants and significance of delayed clearance of serum HBsAg in chronic hepatitis B virus infection: a prospective study. Hepatology 13:627–631

    Article  PubMed  CAS  Google Scholar 

  • Lin HJ, Lai CL, Lau JY, Chung HT, Lauder IJ, Fong MW (1990) Evidence for intrafamilial transmission of hepatitis B virus from sequence analysis of mutant HBV DNAs in two Chinese families. Lancet 336:208–212

    Article  PubMed  CAS  Google Scholar 

  • Marcellin P, Giostra E, Martinot-Peignoux M, Loriot MA, Jaegle ML, Wolf P, Degott C, Degos F, Benhamou JP (1991) Redevelopment of hepatitis B surface antigen after renal transplantation. Gastroenterology 100:1432–1434

    PubMed  CAS  Google Scholar 

  • McMahon G, McCarthy L, Dottavio D, Ostberg L (1991) Surface antigen and polymerase gene variation in hepatitis B virus isolates from a monoclonal antibody-treated liver transplant patient. In: Hollinger BF, Lemon SM, Margolis H (eds) Viral hepatitis and liver disease. Williams and Wilkins, Baltimore, pp 219–221

    Google Scholar 

  • Milich DR, Jones JE, McLachlan A, Houghten R, Thornton GB, Hughes JL (1989) Distinction between immunogenicity and tolerogenicity among HBcAg T cell determinants. Influence of peptide-MHC interaction. J Immunol 143:3148–3156

    PubMed  CAS  Google Scholar 

  • Milich DR, Jones JE, Hughes JL, Price J, Raney AK, McLachlan A (1990) Is a function of the secreted hepatitis B e antigen to induce immunologie tolerance in utero? Proc Natl Acad Sci USA 87:6599–6603

    Article  PubMed  CAS  Google Scholar 

  • Mitsuda T, Yokota S, Mori T, Ibe M, Ookawa N, Shimizu H, Aihara Y, Yoshida N, Kosuge K, Matsuyama S (1989) Demonstration of mother-to-infant transmission of hepatitis B virus by means of polymerase chain reaction. Lancet 2:886–888

    Article  PubMed  CAS  Google Scholar 

  • Müller R, Gubernatis G, Farle M, Niehoff G, Klein H, Wittekind C, Tusch G, Lautz H-U, Boker K, Stängel W, Pichlmayr R (1991) Liver transplantation in HBs antigen (HBsAg) carriers. Prevention of hepatitis B virus (HBV) recurrence by passive immunization. J Hepatol 13:90–96

    Google Scholar 

  • Neurath AR, Kent SBH, Strick N et al (1985) Hepatitis B virus contains pre-S gene-encoded domains. Nature 315:154–156

    Article  PubMed  CAS  Google Scholar 

  • Okamoto H, Imai M, Tsuda F, Tanaka T, Miyakawa Y, Mayumi M (1987) Point mutation in the S gene of hepatitis B virus for a d/y or w/r subtypic change in two blood donors carrying a surface antigen of compound subtype adyr or adwr. J Virol 61:3030–3034

    PubMed  CAS  Google Scholar 

  • Okamoto H, Yotsumoto S, Akahane Y, Yamanaka T, Miyazaki Y, Sugai Y, Tsuda F, Tanaka T, Miyakawa Y, Mayumi M (1990) Hepatitis B viruses with precore region defects prevail in persistently infected hosts along with seroconversion to the antibody against e antigen. J Virol 64:1298–1303

    PubMed  CAS  Google Scholar 

  • Oren I, Hershow RC, Ben-Porath E, Krivoy N, Goldstein N, Rishpon S, Shouval D, Hadler SC, Alter MJ, Maynard JE et al. (1989) A common-source outbreak of fulminant hepatitis B in a hospital. Ann Intern Med 110:691–698

    PubMed  CAS  Google Scholar 

  • Ou J, Laub O, Rutter W (1986) Hepatitis B virus gene function: the precore region targets the core antigen to cellular membranes and causes the secretion of the e antigen. Proc Natl Acad Sci USA 83:1578–1582

    Article  PubMed  CAS  Google Scholar 

  • Peters M, Vierling J, Gershwin ME, Milich D, Chisari FV, Hoofnagle JH (1991) Immunology and the liver. Hepatology 13:977–994

    Article  PubMed  CAS  Google Scholar 

  • Raimondo G, Rodino G, Smedile V, Brancatelli S, Villari D, Longo G, Squadrito G (1990) Hepatitis B virus (HBV) markers and HBV-DNA in serum and liver tissue of patients with acute exacerbation of chronic type B hepatitis. J Hepatol 10:271–273

    Article  PubMed  CAS  Google Scholar 

  • Samuel D, Bismuth A, Mathieu D, Arulnaden J-L, Reynes M, Benhamou J-P, Brechot C, Bismuth H (1991) Passive immunoprophylaxis after liver transplantation in HBs-Agpositive patients. Lancet 337:813–815

    Article  PubMed  CAS  Google Scholar 

  • Schlicht H-J, von Brunn A, Theilmann L (1991) Antibodies in anti-HBe-positive patient sera bind to an HBe protein expressed on the cell surface of human hepatoma cells: implications for virus clearance. Hepatology 13:57–61

    PubMed  CAS  Google Scholar 

  • Schlicht H-J, Salfeld J, Schaller H (1987) The duck hepatitis B virus pre-C region encodes a signal sequence which is essential for synthesis and secretion of processed core proteins but not for virus formation. J Virol 61:3701–3709

    PubMed  CAS  Google Scholar 

  • Schlicht HJ, Esser A, Bartenschlager R, Galle P, Kuhn C, Nassal M, Niepman M, Radziwill G, Schaller H (1989) Unravelling the life cycle of hepatitis B viruses. In: Bannasch P, Keppler D, Weber G (eds) Liver cell carcinoma. Kluwer, Dordrecht, pp 79–91

    Google Scholar 

  • Scibl R, Holtke HJ, Ruger R, Meindl A, Zachau HG, Rasshofer R, Roggendorf M, Wolf H, Arnold N, Wienberg J, et al. (1990) Non-radioactive labeling and detection of nucleic acids: III. Applications of the digoxigenin system. Biol Chem Hoppe Seyler 371:939–951

    Article  Google Scholar 

  • Shafritz DA, Shouval D, Sherman HI, Hadziyannis SJ, Kew MC (1981) Integration of hepatitis B virus DNA into the genome of liver cells in chronic liver disease and hepato-cellular carcinoma. Studies in percutaneous liver biopsies and post-mortem tissue specimens. N Engl J Med 305:1067–1073

    CAS  Google Scholar 

  • Shih L-N, Sheu J-C, Wang J-T, Huang G-T, Yang P-M, Lee H-S, Sung J-L, Wang T-H, Chen D-S (1990) Serum hepatitis B virus DNA in healthy HBsAg-negative Chinese adults evaluated by polymerase chain reaction. J Med Virol 32:257–260

    Article  PubMed  CAS  Google Scholar 

  • Stahl S, MacKay P, Magazin M, Bruce SA, Murray K (1982) Hepatitis B virus core antigen: synthesis in Escherichia coli and application in diagnosis. Proc Natl Acad Sci USA 79:1606–1610

    Article  PubMed  CAS  Google Scholar 

  • Takeda K, Akahane Y, Suzuki H, Okamoto H, Tsuda F, Miyakawa Y, Mayumi M (1990) Defects in the precore region of the HBV genome in patients with chronic hepatitis B after sustained seroconversion from HBeAg to anti-HBe induced spontaneously or with interferon therapy. Hepatology 12:1284–1289

    Article  PubMed  CAS  Google Scholar 

  • Terazawa S, Kojima M, Yamanaka T, Yotsumoto S, Okamoto H, Tsuda F, Miyakawa Y, Mayumi M (1991) Hepatitis B virus mutants with precore-region defects in two babies with fulminant hepatitis and their mothers positive for antibody to hepatitis B e antigen. Pediatr Res 29:5–9

    Article  PubMed  CAS  Google Scholar 

  • Thiers V, Nakajima E, Kremsdorf D, Mack D, Schellekens H, Driss F, Goudeau A, Wands J, Sninsky J, Tiollais P et al. (1988) Transmission of hepatitis B from hepatitis-B-seronegative subjects. Lancet 2:1273–1276

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  • Tong SP, Li JS, Vitvitski L, Trepo C (1990) Active hepatitis B virus replication in the presence of anti-HBe is associated with viral variants containing an inactive pre-C region. Virology 176:596–603

    Article  PubMed  CAS  Google Scholar 

  • Wands JR, Fujita YK, Isselbacher KJ, Degott C, Schellekens H, Dazza MC, Thiers V, Tiollais P, Brechot C (1986) Identification and transmission of hepatitis B virus-related variants. Proc Natl Acad Sci USA 83:6608–6612

    Article  PubMed  CAS  Google Scholar 

  • Wang J-T, Wang T-H, Sheu J-C, Shih L-N, Lin J-T, Chen D-S (1991) Detection of hepatitis B virus DNA by polymerase chain reaction in plasma of volunteer blood donors negative for hepatitis B surface antigen. J Infect Dis 163:397–399

    Article  PubMed  CAS  Google Scholar 

  • Yokosuka O, Omata M, Hosoda K, Tada M, Ehata T, Ohto M (1991) Detection and direct sequencing of hepatitis B virus genome by DNA amplification method. Gastroenterology 100:175–181

    PubMed  CAS  Google Scholar 

  • Zeldis JB, Lee JH, Mamish D, Finegold DJ, Sircar R, Ling Q, Knudsen PJ, Kuramoto IK, Mimms LT (1989) Direct method for detecting small quantities of hepatitis B virus DNA in serum and plasma using the polymerase chain reaction. J Clin Invest 84:1503–1508

    Article  PubMed  CAS  Google Scholar 

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Authors and Affiliations

  1. Royal Free Hospital School of Medicine, Rowland Hill Street, London, NW3 2PF, UK

    Geoffrey Dusheiko, Jianye Xu & Arie J. Zuckermann

  2. WHO Collaborating Center for Reference and Research on Viral Diseases, London, UK

    Arie J. Zuckermann

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  1. Geoffrey Dusheiko
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  2. Jianye Xu
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  3. Arie J. Zuckermann
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Editors and Affiliations

  1. Department of Molecular Virology Faculty of Medicine, Hebrew University of Jerusalem, POB 1172, Jerusalem, Israel

    Yechiel Becker

  2. Institut für Medizinische Virologie, der Universität Heidelberg, Im Neuenheimer Feld 324, W-6900, Heidelberg, Germany

    Gholamreza Darai

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Dusheiko, G., Xu, J., Zuckermann, A.J. (1992). Clinical Diagnosis of Hepatitis B Infection: Applications of the Polymerase Chain Reaction. In: Becker, Y., Darai, G. (eds) Diagnosis of Human Viruses by Polymerase Chain Reaction Technology. Frontiers of Virology, vol 1. Springer, Berlin, Heidelberg. https://doi.org/10.1007/978-3-642-84766-0_7

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  • DOI: https://doi.org/10.1007/978-3-642-84766-0_7

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