SEQUENCE ANALYSIS OF VP7 (GENOME SEGMENT 9) OF THE EPIDEMIC G2 ROTAVIRUS STRAINS OVER A 25- YEAR PERIOD (1984-2009) AT DR GEORGE MUKHARI HOSPITAL BY Lekgolo Lorens MAAKE 2012 i SEQUENCE ANALYSIS OF VP7 (GENOME SEGMENT 9) OF THE EPIDEMIC G2 ROTAVIRUS STRAINS OVER A 25- YEAR PERIOD (1984-2009) AT DR GEORGE MUKHARI HOSPITAL By Lekgolo Lorens MAAKE A dissertation in fulfilment of the requirements for the degree of MSc Med (Medical Virology) Submitted In the Department of Virology School of Pathology and Pre-clinical Sciences University of Limpopo (Medunsa Campus) Supervisor Dr Mapaseka L. SEHERI; PhD University of Limpopo (Medunsa Campus) Co-supervisor Dr Selokela G. SELABE; PhD University of Limpopo (Medunsa Campus) i DECLARATION I, Lekgolo Lorens MAAKE, hereby declare that the work presented in this dissertation is original and does not incorporate any material previously submitted for purpose of degree to any other institution. This dissertation is being submitted in fulfilment for the requirements of the degree of Master of Medical Science (Medical Virology), in the Department of Virology, School of Pathology and Pre-Clinical Sciences, Faculty of Health Sciences, at the Medunsa Campus of the University of Limpopo. Works of other investigators where cited and have been duly acknowledged. Signature of student Date i DEDICATION I dedicate this dissertation to my parents Joas and Betty Maake who raised me this far, my brother Sunnyboy Maake who frequently supported me financially, my three sisters, Julia, Ruth and Refentshe whom I always miss, and finally my talkative young brother Petro. ii ACKNOWLEDGEMENTS I would like to thank the almighty God who gave me life, strength and potential to do this study. I would also wish to convey my sincere gratitude to several people and organizations for their support, motivation, advice, comments, criticisms and contributions towards the development of this study. In particular, I would like to thank: Dr LM Seheri for being a superb supervisor, sacrificing her time and for her dedication, willingness and editorial support. Dr SG Selabe for editorial support, motivation and inspiration. Mrs Ina Peenze for SOPs, QLPs, technical support, inspiration and motivation The secretaries in the Department of Virology who always updated me about all the activities and programmes of the department. Prof MJ Mphahlele for all the support, motivation, courage, inspiration and skills that he gave me throughout this project. It is nice to learn from you Prof, more especially the loyalty, tireless efforts and editorial part. Mr Nare Rakgole and Mr Harry Ngobeni for technical support and assistance in analysis of the results. Mr Martin Nyaga for being an academic friend and for training in laboratory techniques. Mrs Gugu Maphalala, Miss Leah Nemarude, Miss Lufuno Netshifhefhe and Miss Relebogile Mapuroma for their inspiration and motivation. Mr Muxe Muzeze, Miss Tsakane Sondlane, Miss Rudzani Malabi, and Miss Tebogo Seleise who have being my true friends from undergraduate degree. The MRC-DPRU and the HHRU staff members at large. Finally I would like to thank the National Research Foundation and the Poliomyelitis Research Foundation for financial assistance. iii TABLE OF CONTENTS DECLARATION...................................................................................................................... I DEDICATION......................................................................................................................... II ACKNOWLEDGEMENTS ................................................................................................. III TABLE OF CONTENTS ..................................................................................................... IV ABSTRACT ........................................................................................................................ VIII LIST OF ABBREVIATIONS .............................................................................................. XI LIST OF NUCLEOTIDES ................................................................................................ XIX LIST OF AMINO ACIDS ................................................................................................... XX LIST OF TABLES ............................................................................................................. XXI LIST OF FIGURES .......................................................................................................... XXII CHAPTER 1: EXPERIMENTAL PROPOSAL ................................................................... 1 1.1 Study problem .................................................................................................................... 1 1.2 Aim ...................................................................................................................................... 2 1.3 Objectives............................................................................................................................ 2 Primary objective ................................................................................................................... 2 Secondary objectives ............................................................................................................. 2 1.4 Expected significance of the study .................................................................................... 3 CHAPTER 2: LITERATURE REVIEW .............................................................................. 4 2.1 Introduction ........................................................................................................................ 4 2.2 History ................................................................................................................................. 4 2.3 Virology and classification ................................................................................................ 5 2.4 Diversity of rotaviruses.................................................................................................... 10 2.4.1 Point mutations ........................................................................................................... 10 2.4.2 Reassortment ............................................................................................................... 11 2.4.3 Rearrangement ............................................................................................................ 11 2.4.4 Interspecies transmission ............................................................................................ 12 2.5 Prevalence of rotavirus genotypes .................................................................................. 13 2.6 Prevalence of G2 worldwide ........................................................................................... 14 2.7 Viral proteins .................................................................................................................... 16 2.7.1 Structural proteins ....................................................................................................... 16 iv 2.7.2 Non-structural proteins ............................................................................................... 19 2.8 Transmission .................................................................................................................... 23 2.9 Pathogenesis...................................................................................................................... 24 2.10 Replication ...................................................................................................................... 27 2.11 Signs and symptoms ....................................................................................................... 28 2.12 Epidemiology .................................................................................................................. 29 2.13 Rotavirus Seasonality .................................................................................................... 30 2.14 Diagnosis ......................................................................................................................... 30 2.15 Therapy and prognosis .................................................................................................. 31 2.16 Immunity ........................................................................................................................ 32 2.17 Vaccines .......................................................................................................................... 33 CHAPTER 3: MATERIALS AND METHODS ................................................................. 36 3.1 Ethical Consideration ...................................................................................................... 36 3.2 Study Site .......................................................................................................................... 36 3.3 Study design ...................................................................................................................... 36 3.4 Sampling and Study population ..................................................................................... 37 3.5 Laboratory Methods ........................................................................................................ 39 3.5.1 Extraction of dsRNA from stool for PAGE ................................................................ 39 3.5.2 Polyacrylamide gel electrophoresis (PAGE) .............................................................