IMMUNOGENICITY AND SEROLOGICAL APPLICATIONS OF FLAVIVIRUS ED III PROTEINS AND MULTIPLEX RT-PCR FOR DETECTING NOVEL SOUTHERN AFRICAN VIRUSES Lehlohonolo Mathengtheng Thesis submitted in fulfillment of the requirements for the degree Ph.D Virology in the Department of Medical Microbiology and Virology, Faculty of Health Sciences, University of the Free State, Bloemfontein Promotor: Prof Felicity Burt, Department of Medical Microbiology and Virology, Faculty of Health Sciences, University of the Free State, Bloemfontein January 2015 Table of contents Table of contents ................................................................................................................................................. 2 Declaration ............................................................................................................................................................ i Acknowledgements .............................................................................................................................................. ii Financial Support ................................................................................................................................................ iii Lehlohonolo Mathengtheng, An Obituary ........................................................................................................... v Publications and presentations........................................................................................................................... vii List of figures ........................................................................................................................................................ ix List of tables ........................................................................................................................................................ xii List of abbreviations ........................................................................................................................................... xiii Abstract ............................................................................................................................................................ xviii LITERATURE REVIEW ........................................................................................................................................ 1 1.1. Introduction and history ....................................................................................................................... 1 1.2. Classification and molecular characteristics of flaviviruses .................................................................. 1 1.3. Transmission and epidemiology of flaviviruses .................................................................................... 3 1.3.1. Tick-borne flaviviruses ....................................................................................................................... 3 1.3.2. Mosquito-borne flaviviruses .............................................................................................................. 7 1.4. West Nile virus and Wesselsbron virus in South Africa. ....................................................................... 9 1.5. Other flaviviruses previously identified in South Africa...................................................................... 11 1.6. Prevention and treatment of flavivirus infections .............................................................................. 14 1.7. The WNV-E protein as a diagnostic agent and candidate vaccine. ..................................................... 15 1.8. Laboratory diagnosis of flavivirus infection. ....................................................................................... 17 1.9. Influence of vertebrate hosts on emerging viruses ............................................................................ 18 1.9.1. Hantaviruses .................................................................................................................................... 19 1.9.2. Hantaviruses in Africa ...................................................................................................................... 19 Problem identification, aim and objectives of the study. .......................................................................... 23 CHAPTER 2 ......................................................................................................................................................... 26 THE PREPARATION OF RECOMBINANT FLAVIVIRUS ENVELOPE DOMAIN III (EDIII) PROTEINS USING A BACTERIAL EXPRESSION SYSTEM. .................................................................................................................. 26 2.1 Introduction ......................................................................................................................................... 26 2.2. Aim ...................................................................................................................................................... 27 2.3. Materials and methods ....................................................................................................................... 27 2.3.1. Selection of viruses .......................................................................................................................... 27 2.3.2. Sequence alignment and phylogenetic analyses ............................................................................. 28 2.3.3. Extraction of RNA ............................................................................................................................. 28 2.3.4. Amplification of EDIII genes ............................................................................................................. 28 2.3.5. Purification of PCR products ............................................................................................................ 29 2.3.6. Cloning of amplicons and transformation of cells ........................................................................... 29 2.3.7. Colony PCR assay for confirmation of positive transformants ........................................................ 30 2.3.8. Plasmid purification ......................................................................................................................... 31 2.3.9. Double digestions............................................................................................................................. 31 2.3.10. Determination of DNA concentrations .......................................................................................... 31 2.3.11. DNA sequencing of positive constructs ......................................................................................... 31 2.3.12. Protein expression and SDS-PAGE analysis .................................................................................... 32 2.3.13. Protein solubility analyses ............................................................................................................. 33 2.3.14. Purification of over-expressed proteins and refolding of denatured proteins.............................. 34 2.3.15. Protein concentration determination ............................................................................................ 35 2.3.16. Characterization of proteins by Western blot analysis .................................................................. 35 2.4. RESULTS ............................................................................................................................................... 35 2.4.1. Amino acid alignments and phylogenetic tree ................................................................................ 35 2.4.2. One step RT-PCR for amplification of the genes encoding the EDIII protein of flaviviruses ........... 36 2.4.3. T/A cloning of the EDIII amplicons of LGTV, WNV and WESSV into pGEM®-T- Easy vector ............ 37 2.4.4. Cloning of the EDIII genes of LGTV, WNV and WESSV into pQE-80L and pCOLD-TF expression vectors. ....................................................................................................................................................... 38 2.4.5. Sequence data to confirm the identity of inserts ............................................................................ 41 2.4.6. SDS-PAGE analyses for expressed proteins ..................................................................................... 42 2.4.7. Protein purification and determination of protein concentration .................................................. 49 2.4.8. Western blot analysis of recombinant proteins .............................................................................. 51 2.5. SUMMARY ........................................................................................................................................... 52 CHAPTER 3 ......................................................................................................................................................... 53 DEVELOPMENT AND USE OF SEROLOGICAL ASSAYS TO DETERMINE THE PRESENCE OF FLAVIVIRUSES IN THE FREE STATE PROVINCE, SOUTH AFRICA. ................................................................................................. 53 3.1. Introduction ........................................................................................................................................ 53 3.2. Aim .....................................................................................................................................................