SCHOOL OF BIOMEDICAL AND ALLIED HEALTH SCIENCES COLLEGE OF HEALTH SCIENCES UNIVERSITY OF GHANA HUMAN METAPNEUMOVIRUS AND RESPIRATORY SYNCYTIAL VIRUSES IN GHANAIAN CHILDREN BELOW FIVE YEARS WITH ACUTE LOWER RESPIRATORY TRACT INFECTIONS IN ACCRA, GHANA BY ANNA ABA HAYFORD (10264096) A THESIS SUBMITTED TO THE UNIVERSITY OF GHANA, LEGON IN PARTIAL FULFILLMENT OF THE REQUIREMENT FOR THE AWARD OF MASTER OF PHILOSOPHY DEGREE IN MEDICAL MICROBIOLOGY JULY 2017 DECLARATION I hereby declare that this research is a result of my own research work carried out at the Virology Department of the Noguchi Memorial Institute for Medical Research, under the supervision of Professor Theophilus Koku Adiku of the Department of Medical Microbiology, School of Biomedical and Allied Health Sciences (SBAHS) Korle Bu and Dr. John Kofi Odoom of the Noguchi Memorial Institute for Medical Research, Legon. I further declare that, any references used in this thesis that is not mine has been duly acknowledged and that this work has not been submitted to any other institution for the purposes of obtaining a degree. ……....………………………………… Date…………..…….. ANNA ABA HAYFORD (STUDENT) ….……………………………………. Date………………... PROF. THEOPHILUS KOKU ADIKU (SUPERVISOR) …………………………...…………… Date………………… DR. JOHN KOFI ODOOM (SUPERVISOR) i DEDICATION Dedicated to my beloved family. ii ACKNOWLEDGEMENT A research work of this magnitude would not have been possible without the help of many individuals. I wish to express my profound gratitude to my supervisor Professor Theophilus Adiku who encouraged me to write on this topic. The Professor, despite great pressure of work was of great help to me by taking time off his tight schedule to glance through the work at various stages and offered advice, useful criticism and suggestion and encouragement. To my external supervisor, Dr. Kofi Odoom, I owe a debt of immeasurable gratitude. He was very helpful with bioinformatics and phylogenetic analysis in this work and for agreeing to be my external supervisor. Sincerely, I cannot neglect mentioning Dr. (Mrs.) Evangeline Obodai for her immense contribution during the entire work. She assisted in availing to me the relevant laboratory reagents and was exceptionally helpful in answering my many random scientific questions. Dr. Margaret Neizer (Clinician) and Gifty Okine, both of the Princess Marie- Louise Hospital were equally very helpful with sample collection and also deserve mentioning. To Professor William Kwabena Ampofo, Head of Virology Department and Director of the Noguchi Memorial Institute for Medical Research (NMIMR), Legon, who granted me access to use the facility of the institute, I am most grateful. Without the cooperation and technical support of the following persons, this work could not have been accomplished; Dr. and Dr. (Mrs) Bonney, Prince Kofi Parbie, Naa Dedei iii Aryequaye and Christopher Abana all of the NMIMR who individually and collectively engaged in the sequencing of my samples. In addition, Dr. Gloria Adjapong of the Center for Plant Medicine Research – Mampong (CPMR), also took it upon herself to proof read the whole work and offered me very useful suggestions. I thank Elijah Paa Edu-Quansah, Yaw Larbi, Prince Asare (NMIMR) and Dr. Calys- Tagoe (Community Health, UG) for helping out with my data analysis. Finally, my appreciation goes to my husband Kwame, my children, Elorm, David and Elinam, who had to bear my long absence from home. My Mum, Charlotte for her unflinching support throughout my academic life. My prayerful in-laws, Rev and Mrs. Kafintu- Kwashie; for their prayers and supplications which preserved me throughout this academic exercise. My siblings Getty, Paapa and Ma-Adwoa; My friends Sethina, Nancy, Justice, Nana Aba, Vivian, Leonora and Diana Ahu Prah (NMIMR, Bacteriology Department) for their continuous moral support not forgetting the head and colleagues of the Department of Medical Microbiology especially Dr. Japhet Opithan, Mr Boamah, Makafui and my two wonderful Administrative Secretaries: Ms Evelyn Omane and Ms Emelia Aryettey. iv TABLE OF CONTENTS DECLARATION ................................................................................................................. i DEDICATION .................................................................................................................... ii ACKNOWLEDGEMENT ................................................................................................. iii TABLE OF CONTENTS .................................................................................................... v LIST OF FIGURES ......................................................................................................... viii LIST OF TABLES ............................................................................................................. ix LIST OF ABBREVIATIONS AND ACRONYMNS ........................................................ x ABSTRACT ....................................................................................................................... xi CHAPTER ONE ................................................................................................................. 1 1.0 INTRODUCTION ........................................................................................................ 1 1.1 BACKGROUND ....................................................................................................... 1 1.2 PROBLEM STATEMENT ....................................................................................... 4 1.3 JUSTIFICATION ...................................................................................................... 5 1.4 AIM ........................................................................................................................... 5 1.4.1 SPECIFIC OBJECTIVES................................................................................... 6 CHAPTER TWO ................................................................................................................ 7 2.0 LITERATURE REVIEW ............................................................................................. 7 2.1 HISTORY AND STRUCTURE OF RESPIRATORY SYNCYTIAL VIRUS ........ 7 2.2 MOLECULAR EPIDEMIOLOGY OF RSV ............................................................ 9 2.3 RSV TRANSMISSION........................................................................................... 11 2.4 PATHOGENESIS OF RESPIRATORY SYNCTIAL VIRUS ............................... 11 2.5 CLINICAL PRESENTATIONS ............................................................................. 12 2.6 BURDEN OF DISEASE ASSOCIATED WITH RSV ........................................... 14 2.7 LABORATORY DIAGNOSIS ............................................................................... 15 2.8 RISK FACTORS ASSOCIATED WITH RSV INFECTIONS .............................. 16 2.9 SEASONALITY ..................................................................................................... 16 2.10 TREATMENT AND MANAGEMENT OF RSV INFECTION .......................... 17 2.11 HISTORY AND STRUCTURE OF HUMAN METAPNEUMOVIRUS (HMPV) ....................................................................................................................................... 18 v 2.12 MOLECULAR EPIDEMIOLOGY OF HMPV .................................................... 20 2.13 TRANSMISSION ................................................................................................. 21 2.14 PATHOGENESIS OF HMPV .............................................................................. 22 2.15 CLINICAL PRESENTATIONS OF HMPV ........................................................ 22 2.16 SEASONALITY ................................................................................................... 23 2.17 LABORATORY DIAGNOSIS OF HMPV .......................................................... 23 2.18 DISEASE BURDEN ASSOCIATED WITH HMPV ........................................... 24 2.19 TREATMENT AND MANAGEMENT OF HMPV ............................................ 24 CHAPTER THREE .......................................................................................................... 26 3.0 METHODOLOGY ..................................................................................................... 26 3.1 STUDY DESIGN .................................................................................................... 26 3.2 STUDY SITE .......................................................................................................... 26 3.3 RECRUITMENT OF SUBJECTS .......................................................................... 27 3.4 INCLUSION CRITERIA ........................................................................................ 27 3.5 EXCLUSION CRITERIA ....................................................................................... 27 3.6 CASE DEFINITION OF ACUTE LOWER RESPIRATORY INFECTION (ALRTI) ........................................................................................................................ 27 3.7 SAMPLING SIZE ................................................................................................... 28 3.8 SAMPLING METHOD .......................................................................................... 28 3.9 TOOLS AND TECHNIQUES FOR SAMPLE AND DATA COLLECTION ....... 29 3.9.1 Questionnaire .................................................................................................... 29 3.9.2 Sample collection/Transportation /Storage ...................................................... 29 3.10 LABORATORY INVESTIGATION/ PROCEDURE .......................................... 30 3.10.1 Sample preparation (RNA extraction) ............................................................ 30 3.11 Reverse TRANSCRIPTION (RT) of viral RNA ................................................... 31 3.12 CONVENTIONAL PCR ....................................................................................... 31 3.13 GEL ELECTROPHORESIS ................................................................................. 35 3.14 PCR PRODUCT PURIFICATION AND SEQUENCING REACTION ............. 35 3.15 CYCLE SEQUENCING OF PCR PRODUCTS ................................................... 36 3.16 PURIFICATION OF SEQUENCED PRODUCTS .............................................. 37 3.17 SEQUENCE DATA ANALYSES ........................................................................ 38 3.18 DATA ANALYSIS ............................................................................................... 39 vi 3.19 ETHICAL CONSIDERATIONS .......................................................................... 39 CHAPTER FOUR ............................................................................................................. 40 4.0 RESULTS ................................................................................................................... 40 4.1 PARTICIPANTS’ DEMOGRAPHIC ..................................................................... 40 4.2 DETECTED VIRUSES AMONG STUDY PARTICIPANTS ............................... 40 4.3 MOLECULAR CHARACTERIZATION............................................................... 51 4.3.1 Gel electrophoresis ........................................................................................... 51 4.3.2 Phylogenetic analysis ....................................................................................... 54 CHAPTER FIVE .............................................................................................................. 60 5.0 DISCUSSION AND CONCLUSIONS ...................................................................... 60 5.1 LIMITATIONS ....................................................................................................... 67 5.2 CONCLUSION ....................................................................................................... 68 5.3 RECOMMENDATIONS ........................................................................................ 69 REFERENCES ................................................................................................................. 70 APPENDICES .................................................................................................................. 90 APPENDIX 1 .................................................................................................................... 90 APPENDIX II ................................................................................................................... 93 APPENDIX III .................................................................................................................. 95 APPENDIX IV.................................................................................................................. 96 APPENDIX V ................................................................................................................... 97 vii LIST OF FIGURES Figure 1: Structure of RSV Source ..................................................................................... 7 Figure 2: G-Protein Structure source .................................................................................. 9 Figure 3: Structure of Human Metapneumovirus (HMPV) ............................................. 19 Figure 4: HMPV Genome. ................................................................................................ 20 Figure 5: Frequency of clinical symptoms of children with ALRTI ................................ 45 Figure 6: Monthly distribution of RSVA, RSVB and HMPV over the study period ....... 48 Figure 7: Prevalence of RSVA, RSVB and HMPV in children with ALRI ..................... 49 Figure 8: An electrophoregram (1.5% agarose gel in 1X TAE buffer) of RSV A amplification. .................................................................................................................... 51 Figure 9: An electrophoregram of amplified RSV B. ....................................................... 52 Figure 10: Gel electrophoresis of HMPV ......................................................................... 53 Figure 11: Neighbour joining trees representing phylogenetic analysis of RSV genotypes isolated in Ghana between 2015 and 2016 ....................................................................... 55 Figure 12: Graphic view of nucleotide changes between RSV-A study samples and RSV- A ON1 reference genotypes within the G.gene. ............................................................... 56 Figure 13: Amino acid changes between RSV-A study samples and RSV-A ON1 reference genotype ............................................................................................................ 57 Figure 14. Graphic view of nucleotide changes between RSV-B study samples and RSV- B BAIX reference genotypes ............................................................................................ 58 Figure 15: Amino acid changes between RSV-B study samples and RSV-B IX reference genotype. ........................................................................................................................... 59 viii LIST OF TABLES Table 1: Conventional PCR mix ....................................................................................... 32 Table 2: Conventional PCR amplification protocol.......................................................... 33 Table 3: Details of oligonucleotide used to amplify G and F Genes RSV and HMPV using conventional PCR .................................................................................................... 34 Table 4: Sequencing reaction ............................................................................................ 37 Table 5: Cycle sequencing conditions .............................................................................. 37 Table 6: Demographic characteristics of participants with respect to age groups, gender, educational status and patient category............................................................................. 41 Table 7: Association of clinical diagnosis with RSVA, RSVB and HMPV infections .... 42 Table 8: Demographic characteristics of parents’ or guardians with child’s results ........ 43 Table 9: Duration of stay in hospitals for RSVA, RSVB & HMPV patients ................... 46 Table 10: The association of RSV and HMPV infection on breastfeeding ...................... 47 Table 11: Risk factor associations with RSVA, RSVB and HMPV infections among children under five years ................................................................................................... 50 ix