Full length genome characterisation of Hepatitis B virus isolates at Dr George Mukhari hospital in Pretoria, South Africa. Rindidzani Edith Magobo

Transcription

1 Full length genome characterisation of Hepatitis B virus isolates at Dr George Mukhari hospital in Pretoria, South Africa. By Rindidzani Edith Magobo Submitted in fulfilment of the requirement for the degree of MASTER OF MEDICAL SCIENCE (MEDICAL VIROLOGY) In the Department of Virology School of Pathology, Faculty of Health Sciences University of Limpopo, Medunsa Campus Supervisor: Dr SG Selabe Department of Virology Medunsa Campus Pretoria SEPTEMBER 2011 i

2 DECLARATION I, Rindidzani Edith MAGOBO, hereby declare that this work, unless where acknowledged, is my own and has not been submitted at any other institution for a degree purpose It is being submitted in fulfilment of the MSc (Med) in Medical Virology, in the Department of Virology at the University of Limpopo, MEDUNSA campus. Signature of candidate.. Date ii

3 DEDICATION This dissertation is dedicated to my precious son Tanganedzani, my parents Malindi and Luvhengo Magobo, my sisters Ndivhuwo, Ňaledzani, Tshianeo, my only brother Phathutshedzo for their never ending love, support and encouragement. iii

4 ACKNOWLEDGEMENTS First and foremost I would like to give thanks to God almighty who gave strength to pull through all difficult situations I came across during my study period. I would like to express my sincere appreciation and gratitude to following people who made contribution during the course of my studies and without whose assistance this work would not have been a success: Dr SG Selabe and Professor MJ Mphahlele for their superb supervision and all the support, motivation, courage and inspiration you gave. Thanks a lot for your guidance throughout the study. To all the postgraduate students in the Department of Virology, your motivation, encouragement and assistance are highly appreciated. Mr NJ Rakgole for technical assistance; training in laboratory techniques and assistance in analysis of the results. Dr A Lukhwareni for being a good friend, brother and a mentor, and the scientific input from the beginning of this project to its very end, thanks a million times. Special thanks to Ms L Netshifhefhe for proof reading this dissertation. I also thank financial assistance from National Research Foundation (NRF), Poliomyelitis Research Foundation (PRF) and the University of Limpopo Postgraduate Funding. Mashudu Netshikweta for being a loving, supportive and understanding partner throughout the process of dissertation preparation. Finally, I would like to extend my appreciation to my family for putting their trust in me, their support, love and for giving courage; most importantly to my precious son for being the light at the end of the tunnel when things were not looking good. iv

5 ABSTRACT Introduction: Sub-Saharan Africa is a region with hepatitis B virus (HBV) hyperendemicity with more than 8% HBsAg prevalence. An estimate of two billion people has been reported to carry HBV markers. HBV was associated with about 25% of annual deaths in Africa. HBV possesses a DNA polymerase which lacks proofreading mechanism. This results in highly variabillty and genetic diversity which poses a challenge for the diagnosis and therapeutic management of HBV infection. High mutation rate of HBV also has great implications on the development of drug resistant mutations. Moreover, HBV diversity represents a challenge for the sensitivity of immunological and molecular diagnostic assays. A number of studies on HBV full length genome have been conducted particularly in developed countries. Limited studies are available in Africa and South Africa. In South Africa, few studies have been done analysing the complete genome of HBV isolates from patients with asymptomatic carriers and fulminant hepatitis B (Owideru et al, 2001a; Owideru et al, 2001b; Kimbi et al, 2004; Kramvis et al, 2002). This study was aimed at characterising the full-length genome of HBV isolates at Dr George Mukhari Hospital, Pretoria, South Africa, with a view of developing a PCR-based technology for amplification and characterisation of HBV strains with different serological profiles. The technology, if successfully developed, will contribute in understanding the molecular mechanisms resulting in various HBV variants or isolates. Methods: The study design was exploratory. Four stored serum samples collected from HBV infected patients at Dr George Mukhari hospital, Pretoria, were used to develop the molecular technology and test the hypothesis. HBV serology was previously performed targeting 5 HBV serological markers; HBsAg, anti-hbs, anti- HBc, HBeAg and anti-hbe using Elecsys version; HBV DNA quantification was done using Cobas Amplicor HBV DNA monitor assay, HBV DNA was extracted and subjected to nested PCR assay targeting HBV full length genome as two overlapping fragments: fragment A (1670 bp) and fragment B (1868 bp). The generated PCR products for both fragments were cloned into the pgem T easy vector and 2 clones were selected from each sample. The plasmids were purified using Invisorb Spin Plasmid Mini Two and the clones were recovered by PCR assay. The sample PCR products and the clone PCR products were purified and sequenced using v

6 SpectruMedix SCE2410 genetic analysis system. HBV genotyping was performed using the NCBI web-based genotyping tool. Phylogenetic analysis was done using MEGA 4 software to confirm HBV genotypes. Results: Serology results were as follows: All samples were HBsAg positive, Anti-HBs negative, anti-hbc positive and anti-hbe negative. Sample B1121 and sample 6 were HBeAg positive while samples B452 and 5 were HBeAg negative. A total of 12 PCR products were sequenced (4 study samples and 8 clones [2 clones each sample]). In total, 7 HBV full length genome sequences were deduced from this study, with 3 sequences belonging to genotype A, 2 to genotype C and 2 to genotype D. 3 HBV genotypes were detected from this study; genotype A, C and D with subgenotype A2, C1 and D1 respectively. Mutations were observed throughout the genome. In the pre-s/s open reading frame (ORF), the most significant findings were the detection of mutations within the a determinant site and major hydrophilic region (MHR). These mutations included Y161F,E164G observed in sample B1121 and B1121C1 belonging to subtype A1; 2 amino acid insertion at aa in sample 5 belonging to subtype C1. Drug resistance associated mutations were identified in the polymerase gene, these included M204T and L217R which are associated with adefovir resistance, M204T also resulted in a change from tryptophan (W) to arginine (R) at aa 196 on the overlapping surface gene on sample B452 C1. Basal core promoter (BCP) and pre core/core mutations were detected in study isolates; specifically the BCP double mutation (1762/1764) was seen in 8 isolates which belonged to subtype C1 (5) and D1 (3) and the pre-core stop codon mutations (G1896A) in 4 isolates (2 belonging to subtype C1 and the other 2 to D1). Other changes observed included a 48 nucleotides deletion in the pre-core gene, 6 nucleotides insertion in the HBx gene of all subtype D1 isolates and a 3 nucleotides deletion in subtype C1 clone. Conclusion: This study successfully optimised a PCR-based technology for the amplification and characterisation of HBV full length genome. 3 HBV genotypes were detected with subtypes A2, C1 and D1. However, the detected subtypes are rarely detected in South Africa. The detection of subtype A2 may confirm its Southern vi

7 African origin. Drug resistance associated mutations were observed in this study. These included the adefovir resistance mutation which the current study confirmed it is a naturally occurring mutation as it was detected in adefovir therapy naïve patient. The BCP and pre-core/core mutations were detected in genotype C and D isolates; however, their association with serological profile and clinical outcomes could not be deduced. Unique or novel mutations were seen in the study isolates, these included 48 nucleotides deletion in the pre core gene, 3 amino acids insertion in the RNase H and 8 amino acids deletion in the RT domain of polymerase gene. To our knowledge, these mutations have not been identified or reported in the literature. The detection of 6 nucleotide insertion in the HBx gene was reported for the first time in South African isolates. Further analysis is required to ascertain the biological significance of the unique mutations detected in this study. vii

8 Table of contents Page No. Title page...i Declaration...ii Dedication...iii Acknowledgements...iv Abstract...v Table of contents...viii List of abbreviations...xiv List of tables...xviii List of figures...xix CHAPTER ONE: INTRODUCTION AND EXPERIMENTAL PROPOSAL 1.1. Overview of hepatitis Experimental proposal Study problem Aim Specific Objectives Expected significance of the study.3 CHAPTER TWO: LITERATURE REVIEW ON HEPATITIS B VIRUS 2.1. LITERATURE REVIEW ON HEPATITIS B VIRUS HBV historical background Classification of HBV...4 viii

9 Structure of HBV particles The genome organization of HBV Pre-surface and surface gene Precore/core gene Polymerase gene HBx gene HBV genotype and subgenotypes HBV life cycle Entry and uncoating Replication of the HBV genome Assembly and Budding HBV transmission Epidemiology of HBV Distribution of HBV infection in sub-saharan Africa Burden of HBV infection in South Africa Pathogenicity of HBV Disease profile of HBV infection Acute hepatitis B infection Chronic hepatitis B virus infection Phases of chronic HBV infection Immune tolerant phase Immune clearance phase Low replication or non replication phase of HBV infection...25 ix

10 Reactivation phase Fulminant HBV infection Diagnosis of HBV Serology Liver function tests Molecular assays Prevention control and management of HBV Prevention and control of HBV Management of HBV infection HIV/HBV co-infection Effects of HIV on HBV Effects of HBV on HIV Management of HIV/HBV co-infection Genetic variability of HBV Pre-surface and surface gene mutants Basal core promoter mutants Precore mutants Core gene mutants Polymerase gene mutants HBx gene mutants Variability based on full-length genome...41 x

11 CHAPTER THREE: MATERIALS AND METHODS MATERIAL AND METHODS Ethical consideration Study design Study samples Laboratory testing HBV serological testing HBV DNA PCR assay HBV DNA extraction Optimisation of PCR assay for amplification of full length HBV genome Detection of HBV PCR products Avoiding PCR contamination HBV DNA fragments cloning Ligation of purified HBV PCR fragments into pgem -T Easy Vector Transformation of the pgem -T Easy vector clones Plasmid DNA purification Recovery of the cloned fragments Sequencing of HBV full-length genome Direct sequencing of the PCR products Nucleotide sequence recovery and editing Nucleotide sequence and amino acid multiple alignments Comparison of study sequences with the Genbank reference strains Phylogenetic tree analysis...60 xi

12 CHAPTER FOUR: OVERVIEW OF RESULTS RESULTS Overview of the Results Sequence analysis HBV NCBI genotyping tool Phylogenetic analysis Analysis of both nucleotide and amino acid sequences HBV full-length genome sequence analysis Nucleotide and amino acid sequence analysis by ORF s Pre-S/S ORF Polymerase ORF Pre-core/core gene ORF The HBX ORF CHAPTER FIVE: DISCUSSION DISCUSSION HBV genetic diversity Identification of HBV mutants within the full length genome by ORFs Pre-S/S ORF mutants affecting its binding affinity and antigenicity Polymerase ORF mutations and drug associated mutants Mutational patterns in the enhancer II and BCP regions Association of Precore/core ORF mutations with HBeAg expression and disease progression xii

13 Variability in the HBx ORF and association with HBV-related HCC Implications of the study CHAPTER SIX: CONCLUSION, LIMITATIONS AND RECOMMENDATIONS CONCLUSIONS, LIMITATIONS AND RECOMMENDATIONS Conclusions Limitations of the study Recommendations Appendix A CHAPTER SEVEN: REFERENCES List of Abbreviations µl Microliter 3TC Aa ADV AIDS ALT Anti-HBc Anti-HBe Anti-HBs AST Lamivudine Amino acid Adefovir dipivoxil Acquired immune deficiency syndrome Alanine transaminase Antibody to core antigen Antibody to hepatitis B e antigen Antibody to surface antigen Aspartate transaminase xiii

14 BCP CAESER CAH cccdna CD CDC CPH datp dctp dgtp DNA dntp DR DRC dttp Basal core promoter Canada, Australia, Europe and South African research chronic active hepatitis Covalently closed circular DNA Cluster of differentiation Centers for Disease Control and Prevention chronic persistent hepatitis deoxyadenine triphosphate Deoxycytosine triphospate Deoxyguanine triphosphate Deoxyribonucleic acid Dinucleotidephosphate Direct repeat Democratic Republic of Congo Deoxythymine triphosphate E. coli Eschericia coli EPI ER ETV FHB FTC HAART HAV HBcAg HBeAg HBsAg HBV Expanded Programme of Immunisation Endoplasmic reticulum Entecavir Fulminant hepatitis B Emtricitabine Highly active antiretroviral therapy Hepatitis A virus Hepatitis B core antigen Hepatitis B e antigen Hepatitis B surface antigen Hepatitis B virus xiv

15 HBx HCC HCV HDV HEV HIV HLA IgG IgM kda LCR LdT LFT MHC MHR mrna Nt Hepatitis B x gene Hepatocellular carcinoma Hepatitis C virus Hepatitis D virus Hepatitis E virus Human immunodeficiency virus Human leukocyte antigen Immunoglobulin G Immunoglobulin M Kilo Dalton Ligase chain reaction Telbivudine Liver function test Major histocompatability complex Major hydrophilic region Messanger RNA Nucleotide ºC Degree celsius OHB ORF PCR pgrna Occult hepatitis B Open reading frame Polymerase chain reaction Pregenomic RNA pre-s1, Pre surface 1 pre-s2 Pre surface 2 RNA RT USA Ribonucleic acid Reverse transcriptase United States of America xv

16 WHO YMDD World health organisation Tyrosine-Methionine-Aspartate-Aspartate Nucleotides A C G T Adenine Cytosine Guanine Thymine Amino acids A R N D C E H I L K M F P S T W Alanine Arginine Asparagine Aspartic acid Cysteine Glutamic acid Histidine Isoleucine Leicine Lysine Methionine Phenylalanine Proline Serine Threonine Tryptophan xvi

17 Y V Tyrosine Valine xvii

18 List of tables Table No. Legend Page No 2.1 Geographical distribution of HBV genotypes and relationship between genotypes and serological subtypes HBV serological markers Interpretation of HBV serologic tests Summary of the study samples Sequences of HBV primers used for PCR reactions of fragment A Sequences of HBV primers used for PCR reactions of fragment B Sequences of primers used for sequencing of fragment A Sequences of primers used for sequencing of fragment B Overview of study results Summary of surface gene mutations Summary of polymerase gene mutations Summary of pre core/core gene mutations Summary of HBx gene mutations xviii

19 List of figures Figure No. Legend Page No 2.1 Schematic diagram of hepadnavirus particles Structural organization of HBV genome Schematic domains of HBV polymerase gene Schematic representation of HBV replication Global prevalence of HBV infection Characteristic of acute HBV infection with recovering HBV infection Characteristics of progression to chronic HBV infection Structure of HBsAg The location of Major drug-resistant mutations on the HBV Polymerase PCR products of fragment A using 3µl template for second round PCR assay PCR products of fragment B using 3µl template for second round PCR assay Modified schematic presentation of cloning process Modified schematic representation showing growth of recombinant plasmids on agar pgem -T Easy vector circle map Phylogenetic analysis of HBV full length genome Nucleotide alignments of HBV surface sequences for genotype A Amino acid alignments of HBV surface sequences for genotype A Nucleotide alignments of HBV surface sequences for genotype C Amino acid alignments of HBV surface sequences for genotype C...73 xix

20 4.6 Nucleotide alignments of HBV surface sequences for genotype D Amino alignments of HBV surface sequences for genotype D Nucleotide alignments of polymerase gene for genotype A Amino acid alignments of polymerase gene for genotype A Nucleotide alignments of polymerase gene for genotype C Amino acid alignments of polymerase gene for genotype C Nucleotide alignments of polymerase gene for genotype D Nucleotide alignments of polymerase gene for genotype D Multiple nucleotide alignments of pre core/core gene for genotype A Multiple amino acid alignments of pre core/core gene for genotype A Multiple nucleotide alignments of pre core/core gene for genotype C Multiple amino alignments of pre core/core gene for genotype C Multiple nucleotide alignments of pre core/core gene for genotype D Multiple amino acid alignments of pre core/core gene for genotype D Multiple nucleotide alignments of HBx gene sequences for genotype A Multiple amino acid alignments of HBx gene sequences for genotype A Multiple nucleotide alignments of HBx gene sequences for genotype C Multiple amino acid alignments of HBx gene sequences for xx

21 genotype C Multiple nucleotide alignments of HBx gene sequences for genotype D Multiple amino acid alignments of HBx gene sequences for genotyped xxi

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