PERSISTENCE OF INFECTIOUS HEPADNAVIRUS IN OFFSPRING BORN TO MOTHERS CONVALESCENT FROM HEPATITIS IN THE WOODCHUCK MODEL OF HEPATITIS B A thesis submitted to the School of Graduate Studies in partial fiilnllment of the requirements for the degree of Master of Science Faculty of Medicine Mernorial University of Newfoundland July 1997 St. John's National Library Bibliothèque.nationale du Canada Acquisitions and Acquisitions et Bibliographie Services setvices bibliographiques 395 Wellington Street 395. rue Weilingtm OttawaON KIAûN4 OttawaON K1AON4 Canada Canada The author has granted a non- L'auteur a accordé une licence non exclusive licence allowing the exclusive permettant à la National Library of Canada to Bibliothèque nationale du Canada de reproduce, loan, distribute or sell reproduire, prêter, distribuer ou copies of this thesis in microform, vendre des copies de cette thèse sous paper or electronic formats. la fome de microfiche/nlm, de reproduction sur papier ou sur format électronique. The author retains ownership of the L'auteur conserve la propriété du copyright in this thesis. Neither the droit d'auteur qui protège cette thèse. thesis nor substantial extracts fiom it Ni la thèse ni des extraits substantiels may be printed or otherwise de celle-ci ne doivent être imprimés reproduced without the author's ou autrement reproduits sans son permission. autorisation. Hepatitis B virus (HBV) is primarüy an hepatotropic virus, dthough evidence of virai infection in lymphoid ceils has also been observed. The virus induces life-threatening liver diseases, such as cirrhosis and hepatoceilular carcinoma and is a major public heaith problem with more than 300 million chronically infected people woddwide. It is also evident that transmission of HBV f?om infieci mothers to their babies is the most important mechanism by which the virus is rnaintained within the population. Recent findings have established the existence of a serologically undetectable persistent carrier state of HBV in apparently completely healthy individuals convalescent fiom an acute episode of hepatitis B. In these individuais, traces of HBV genomes were doaunented in semand circulating lyrnphoid cells years af€er recovery. Related hdings of the lilong hepadnavirai persistence after a transient exposure to woodchuck hepatitis virus (WHV) has been demonstrated in this laboratory in a woodchuck mode1 of hepatitis B. The ment study was undertaken to leam about the risk of hepadnavirus transmission to newbom woodchucks from mothers with cornplete serological recovery f?om s<perimentally induceci viral hepaatis and about nahiral course and molecular feahires of virus penistence in these offspring. The specific aims of this investigation were: (1) to determine whether hepadnaviral genomes cm be transmitted fiom maternai woodchucks with a past episode of acute WHV hepatitis to their offspring; (2) if in fact this vertical transmission ocairs, to ident@ reservoirs of hepadnavirus replication during long-term foUow-up of these newbom animals; (3) to characterize physicochernicai properties of molecules carrying WHV DNA in sera of these ofbpring (4) to test whether dent carriage of WHV genomes acquired derverticai transmission reflects the existence of biologically competent virus infectious to WHV-naive woodchucks, and (5) to determine whether the offSpring carrying WHV traces are susceptiile to WHV ùifection. In this work, 1 1 ofEp~gbom to 4 woodchuck mothers convalescent fkom an acute episode of Wal hepatitis were investigated. Our results have shown that serologically silent WHV carriage acquired after a self- Iirnited episode of viral hepatitis is transmîttable f?om mothers to newboms as an asymptomatic chronic infection. Importantly, al of the offspring tested carried WHV DNA through the entire follow-up, lasting for more then 3 years after birth, and remained nomactive for imrnunovirological markers of WHV infèction udess challengeci with WHV. WHV DNA and RNA specific sequences were detectable both in the liver and lymphoid cells in the majonty of the animals, although in some offspring WHV persisteci exclusively at a extrahepatic location in the lymphatic systern. Particles carrying WHV DNA in sera of offspring with WHV genomes in both the liver and lymphoid cells or the Iymphatic systern alone had physicochemical properties comparable to those of complete WHV vinons. In addition, virus containeci in oflkpring sera with or without WHV DNA expression in the liver as well as, culture supematant fiom mitogen-stimulated peripheral blood mononuclear cells were ùifectous to WHV-naive woodchucks. Finally, despite dent carriage of WHV traces, the offspring were susceptible to WHV challenge. Since there are signifiant pathobiological similarities beh~eenHBV and WHV, it is possible that a comparable situation may exist in babies bom fiom mothers with a past history of hepatitis B. There have been severai ind~dudsand organizations who 1 would iike to th& for making this project possible. Instnimental to this work has been my supervisor, Dr. Thomas Michalak, who has provided valuable support, encouragement, guidance and Wendship throughout my research. In addition, I thank my supervisory cornmittee members; Dr. David Haegert and Dr. Gary Paterno for their suggestions and advice throughout my graduate program and during the hal critique of my thesis. 1 am greatly appreciative of a Graduate Fellowship Award fiom the Canadian Liver Foundation which provided financial support durhg the course of this study. Furthemore, 1 would iike to acknowledge the Faculty of Medicine and the School of Graduate Studies for initial funding. 1was forninate to have as both coileagues and fnends the research assistants and students in the Molecular Vkology and Hepatology Research laboratory. 1 am particularly gratefil to Norma Churchill, Ingrid Pardoe and Cofieen Trelegan who have helped me in many ways throughout the course of this research project. I would like to say a speciai thanks to Chad Saunders for his constant help and understanding. Finally, 1 am indebted to my fimily; my parents, Stephen and Elsie Co£& and my sisters and brothers, especially Patricia, for their confident, unconditional support and encouragement. TABLE OF CONTENTS BUE .. ABSTRACT ......................................................... II ACKNOWLEDGEMENTS ............................................. iv TABLE OF CONTENTS ............................................... v LISTOFTABLES .................................................. 5 LIST OF FIGURES .................................................. xiv LIST OF ABBREVIATIONS .......................................... xvii CHAPTER ONE - INTRODUCTION ...................................... 1 1.1 Virai Hepatitis ............................................... 2 1 -2 Natural History of HBV Méction ................................ 4 1.3 General Characteristics of HBV ................................. 6 1.3.1 Particle Types ....................................... 6 1-3 -2 Genome Organization and Replication Strategy .............. 7 1.3.3 ProteinsofHBV .................................... 12 1 -4 The Hepadnavirus Farnily ..................................... 13 1.4.1 General Feaîures of Hepadnavimses ..................... 13 1 .4.2 Woodchuck Hepatitis Vims (WHV) ..................... 14 1.5 Viral Persistence Mer Adult Onset Infection ...................... 17 1.5 .1 The Anti-Viral Immune Response ....................... 17 1.5.2 Strategies of Virus Escape f?om Host Immune Responses ..... 20 2.5.4a WHV DNA Specific Otigonucleotide Prirners ........ 59 2.5.4b Amplification of WHV Gene Sequences by PCR ...... 63 2.5.4~PCR Controls and Assay Sensitivity ............... 64 2.5.5 Reverse Transcriptase PCR for WHV RNA Deteetion ....... 64 2.5.6 Analysis of Amplifieci WHV Gene Sequences .............. 66 2.5.6a Agarose Gel Electrophoresis ..................... 66 2.5.6b Southem Blot Anaiysis ......................... 66 2.5.6~Rezombinant WHV DNA Probe .................. 67 SPECIFIC EXPERIMENTS ............................................ 69 2.6 Characterization of Physicochemical Properties of Particles Carrying WHV DNA ..................................................... 69 2.6.1 Sedimentation Velocity and Buoyant Density Analyses ....... 69 2.6.2 Preparation of WHV DNA-Reactive Controls for Ultracentrifugation Analyses ........................... 70 2.6.2a Purification of WHV Virions ..................... 70 2.6.2b Preparation of Free WHV DNA Fragments .......... 70 2.6.3 Analysis of Sedimentation Velocity of WHV DNA in Test and ControlSarnples .................................... 71 2.6.4 Analysis of the Buoyant Density of WHV DNA in Test and Control Samples .......................................... 72 2.6.5 Examination of the Effect of DNase Digestion on WHV DNA fiom ... VUl Sucrose Fractions ...................................73 2.6.5a Preüminary Experirnent ......................... 73 2.6.5b DNase Treatment of Sucrose Fractions fkorn Test and Control Sarnples .............................. 74 2.7 Detemination of the Efféct of Mitogen Stimulation on WHV Transcription in PBMC ...................................................75 2.7.1 PBMC Stimulation with Lipopolysaccharide (LPS) and Concanavalin (Cod) ................................ 75 2.7.2 Evaluation of WHV DNA in Stirnulated PBMC,Final Celi Wash and Culture Supernatant .............................. 76 2.8 Evaluation of WHV DNA Expression in PBMC derDNase and Trypsin Tratment ................................................