'za. \,'13 EVENTS OF HEPADNAVIRUS REPLICATION Ming QIAO M.B., B.S. Department of Microbiology and lmmunology University of Adelaide Adelaide South Australia A thesis submitted to the University of Adelaide in fulfilment of the requirements for the degree of Doctor of Philosophy June 1993 /rvuanàuÅ tq() + CONTENTS ABSTRACT v¡i DECLARATION x ACKNOWLEDGEM ENTS x¡ ABBREVIAT¡ONS x¡¡¡ PUBLICATIONS xvi GHAPTER 1: INTRODUCTION 1.1 HBV Epidemiology and Transmission 1 1.2 The HePadnaviridae 3 1.3 Structure of HBV Particles 5 1.4 Antigenic and Biophysical Characteristics of Hepatitis B Surface Antigen Particles 7 1.5 Hepadnavirus Genome Organisation I 1.5.1 Genome Siructure I 1.5.2ORF-S and lts Function s 1.5.3 Virus Nucleocaps¡d (ORF'C) 11 1.5.4 The DNA Polymerase Gene and lts Product 1 3 1.5.5 The ORF-X and Product 15 1.6 Transcrlption and Gontrol Elements of the HePadnavirus Genome 17 1.6.1 The 3.5kb RNA Transcript and lts Promoter 17 1.6.2The 2.1 and2.4 RNA Transcripts and Their Promoters 1e 1.6.3 The 0.7kb RNA Transcript and lts Promoter 1e 1.6.4 Transcriptional Enhancer Elements 20 1.7 Hepadnavirus Genome Replication 2'l SteP One: Formation of CCC DNA 22 il Step Two: Synthesis and Packaging of pregenomic RNA 22 Step Three: Synthesis of Minus Strand 23 Step Four: Synthesis of Plus Strand 24 1.8 Host Range and Tissue Tropism 26 1.9 The Hepadnavirus Receptor and Virus Entry 27 1.10 Serological Diagnosis of Hepatitis B 29 1.11 Pathogenesis of HBV lnfection 32 1 .1 1 .1 Hepatocyte Injury and lmmunological Mechanisms 32 1.11.2 Mechanisms of HBV Persistence 34 1.12 Aims of Thesis 36 GHAPTER 2: MATERIALS AND METHODS 2.1 Purification of Virus-Associated particles 38 2.1.1 Source of Hepadnavirus-Positive Samples 38 2.1 .2 Virus Centrifugation 39 2.1.3 CsCl Gradient Centrifugation for Pulification of DHBsAg 39 2.1.4 Column Chiomatography and Sucrose Gradient Centrifugation for Purification of HBV Virions 40 2.1.5 Virus Particle Examination by Electron Microscopy 40 2.2 Production of Antibodies 41 2.2.1 lmmunisation Procedures 41 2.2.2 Purification of lmmunoglobulin G from Antiserum 41 2.2.3 Purification of Fab Fragment 42 2.2.419G lodination 43 2.3 lmmunohistochemistry 43 2.3.1 Preparat¡on of Tissue Sections 43 2.3.2 Source of Antibodies 44 2.3.3 Adsorption of Antisera with Normal Liver Tissues 44 2.3.4 lmm unofluorescence 45 ilt 2.3.5 lmmunoblotting 45 2.4 Nucleic Acid Hybridisation 47 2.4.1 Sample Preparation 47 2.4.2 Preparation of Hybridisation Membranes 48 2.4.3 Preparation of Radiolabelled Probes 50 2.4.4 Hybridisation and Post-Hybridisation Procedures 54 2.5 Polymerase Ghain Reaction 55 2.5.1 DHBV PCR 56 2.5.2 HBV PCR 56 2.6 Detection of DHBSAg and Ant¡-DHBs by RIA 57 2.6.1 DHBsAg Detection 57 2.6.2 Anti-DHBs Detection 58 2.7 Liver Cell Membrane Preparation and Eukaryotic Cell Culture 58 2.7.1 Preparation of Hepatocyte Membranes 58 2.7 .2 Enzymatic Confirmation 59 2.7.9 Total Proteìn Quantitation 60 2.7. Media anO òufture Methods 60 2.8 Hepadnaviridae Receptor-Binding Assays 61 2.8.1 Binding Assay in Liver Membranes by Solid-Phase RIA 61 2.8.2 HBV-Receptor Binding in Monolayer Culture 62 2.8.3 lnhibition Assays 63 2.8.4 Detection of HBV lnternalisation 64 2.9 Use of Animal Model 64 2.9.1 Source of Animals and Conditions of Animal Housing 64 2.9.2 Duck lnoculation 65 2.9.3 Partial Hepatectomy 65 IV CHAPTER 3: ESTABLISHMENT OF SEROLOGICAL ASSAYS FOR DETECTION OF DUCK HEPATITIS B INFECTION 3.1 lntroduction and Aims 67 3.1.1 Natural lnfection of Duck Hepatitis B Virus 67 3.1.2 DHBV Biology: A Comparison with the Mammalian Hepadnaviruses 69 3.1.3 Use of Duck Models to Study Hepatitis B Virus 71 3.1.4 Background and Aims 72 3.2 Experimental Design 72 3.3 Results 74 3.3.1 Purificatiori.of DHBsAg 74 3.3.2 Production and Specificity of the Rabbit Anti-DHBs 76 3.3.3 Optimisation of the RIA 77 3.3.4 Calculation of Cut-Off Value 78 3.3.5 SensitivitY of the RIA 78 3.4 Discussion 79 3.4.1 Evaluation of lmmunogen (DHBsAg) 79 g.4.zTitre and Specificity of the Antisera 81 3.4.3 Evaluation of the RIA 81 CHAPTER 4: SEROLOGICAL ANALYSIS OF DUCK HEPATITIS B VIRUS INFECTION 4.1 lntroduction and Aims 82 4.2 Experimental Design and Results 83 4.2.1 Serological Examination of Neonatal and Post-Neonatal Ducks after DHBV lnfection 83 4.2.2lncubation Period and Viraemic Stage 84 4.2.3 Seroconversion Events 85 4.2.4 Patterns of Serological Responses 85 V 4.2.5 Correlation of Serological Markers: DHBV DNA and DHBSAg 86 4.3 Discussion 87 GHAPTER 5: FACTORS INFLUENCING THE AGE-RELATED SUSCEPTIBILITY OF DHBV INFECT¡ON OF DUCKS 5.1 lntroduction and Aims 89 5.2 Experimental Design 90 5.3 Results 91 5.3.1 Analysis of Duck Liver Membrane Preparation 91 5.g.2 Analysis oi.Purified DHBV Virions 92 5.3.3 DHBV Binding Studies 92 5.3.4 Specificity of DHBV Binding 93 5.3.5 The Effect of Hepatocyte Mitosis on DHBV Synthesis 94 5.4 Discussion 95 5.4.1 Expression of the Receptor for DHBV in Hepatocytes of Both Neonatal and Adult Ducks 95 5.4.2 lntracellular Factors and Host Susceptibility 96 5.4.3 Other Factors and Host Susceptibility 97 GHAPTER 6: INTERAGTION OF HBV WITH HOST CELLS 6.1 Background and Aims 99 6.2 Experimental Design 100 6.3 Results 101 6.3.1 HBV Binding to Human Liver Membranes and Hepatoma-Derived Cell Lines 101 6.3.2 SPecificitY AssaYs 103 6.3.3 Quantitation of HBV DNA in Cell Binding Experiments 105 6.3.4 HBV lnternalisation in HepG2 Cells 107 VI 6.4 Discussion 108 6.4. 1 HBv-Receptor lnteraction 108 6.4.2 Receptor Quantitation and the Fate of HBV 109 6.4.3 The Mode of HBV Entry into the Host Cell 110 CHAPTER 7: GONCLUDING REMARKS 7.1 Serological Events of DHBV lnfection: Significance and lmplications 112 7.2 Factors Determining Host Susceptibility 113 7.3 Hepadnavirus Entry and Early Virus-Cell lnteraction 116 7.5 Future Directions 120 REFE RENC ES 122 APPEN DIX 145 PUBLICATIONS RESULTING FROM THIS THESIS vil ABSTRACT Aspects of hepadnavirus pathogenesis and the early events of hepadnavirus replication were studied in this thesis, with particular emphasis on the examination of serological profiles of DHBV infection in neonatal and adult ducks, factors determining host susceptibility and viral persistence, and hepadnavirus binding and entry into the host cell. A radioimmunoassay was developed to detect duck hepatitis B virus surface antigen (DHBsAg) and antibody (anti-DHBs), and the results compared with those for DHBV DNA detecteO Oy dot blot hybridisation. Viraemia (DHBV DNA and/or DHBsAg) was detected in all ducks inoculated within 3 weeks post' hatch, and persistent infection developed in 93% of birds in this age group. ln contrast, only 80% and 60% of ducks inoculated 4 and 6 weeks post'hatch respectively developed viraemia, and approximatdy 7Oo/o of the viraemic ducks became carriers. Markers of viraemia were undetected in ducks inoculated I weeks post-hatch and in uninoculated controls. A tyþical anti-DHBs seroconversion developed subsequently in 2 ol 4 birds that showed transient viraemia, and antibody also developed in 3 of 7 ducks inoculated 4-8 weeks post-hatch that showed no viraemia. However, gene amplification by the polymerase chain reaction demonstrated de novo DHBV DNA in ducks from the latter group suggesting that the antibody did not result from passive vaccination. Thus, increased resistance to infection that developed with increasing age may be related to host factors. To examine the hypothesis that decreased susceptibility with increased age might be due to the loss of the receptor on the hepatocyte surface membrane in adult ducks, a receptor-binding study was performed using intact serum- V¡ii derived DHBV virions and purified liver plasma membrane from both young ducklings and adult ducks. These studies showed that ¡). DHBV was able to bind specifically to duck liver plasma membrane but not to internal membrane; ii). this binding could be inhibited by a monoclonal antibody to DHBV preS antigen, a corresponding region in hepatitis B virus that binds to human hepatocytes; and iii). there was no significant difference in the virus-binding ability between plasma membranes from ducklings and from adult ducks. Since hepatocytes in the neonatal ducks are actively dividing, in contrast to the situation in adult ducks, the effect of liver regeneration on the level of DHBV DNA replication was ihen examined by partial hepatectomy in DHBV- carrier ducks. A sharp increase was noted in the level of DHBV in the serum after partial hepatectomy suggesting that DHBV replication was enhanced in dividing hepatocytes. Thus these results indicated that the age-related difference in the susceptibility of ducks to DHBV infection is not due to the loss of the receptor but may be related to an intracellular event associated with cell division. Using a similar technique developed to examine DHBV, the HBV-receptor interaction was then examined. The study demonstrated that HBV virions bound to human liver plasma membrane and hepatoma-derived cell lines in a manner expected of a specific receptor-ligand interaction.