Aspects of the molecular epidemiology of rabies in Zimbabwe and South Africa

Transcription

1 Aspects of the molecular epidemiology of rabies in Zimbabwe and South Africa DOCTOR OF PHILOSOPHY (MICROBIOLOGY) FACULTY OF NATURAL AND AGRICULTURAL SCIENCES UNIVERSITY OF PRETORIA PRETORIA University of Pretoria

2 I certify that the thesis submitted to the University of Pretoria for the degree of PH. D. (Doctor of Philosophy) has not been previously submitted by me in respect ofa degree at any other University.

3 ASPECTS OF THE MOLECULAR EPIDEMIOLOGY OF RABIES IN ZIMBABWE AND SOUTH AFRICA. (genotype 1), Lagos bat virus (genotype 2), Mokola virus (genotype 3), Duvenhage virus (genotype 4), European bat lyssaviruses 1 and 2 (genotypes 5 and 6) and the

4 viruses, infect carnivores of the family Herpestidae (the yellow mongoose Cynictis penicillata and the slender mongoose Galerella sanguinea). In an endeavour to better understand the molecular epidemiology of lyssaviruses in Zimbabwe and South Africa, we analysed nucleotide sequences of the glycoprotein and the G-L intergenic region (rabies viruses) and the nucleoprotein gene (Mokola viruses). The main aim of the studies described in this thesis was to characterise lyssaviruses (genotypes I and 3) from Zimbabwe and compare them to those present in South Africa. In addition, we wanted to establish the role of the various rabies variants in rabies epizootics in the southern African subcontinent. It could be shown from this study that all the southern African canid viruses were closely related, with no general distinction between viruses from any of the canid species. Despite the general overall similarity between the canid viruses, certain phylogenetic groupings were apparent and by association with host species, geography and year of isolation, certain groups could be identified as particular epidemiological cycles. A high genetic diversity was evident amongst viverrid rabies viruses, the opposite of our observation for canid viruses. The viverrid virus groups corresponded to geographical pockets that were independent of host species. Mokola viruses from Zimbabwe were shown to be different from those from South Africa and phylogenetic relationships of these viruses were related to their geographical location of origin. This study has demonstrated the value of multinational surveillance and investigation in understanding the epidemiology of lyssaviruses in southern Africa and elsewhere in Africa. The results presented here will serve as basis for future studies on lyssaviruses in Africa and will contribute to the improved surveillance and control programs of rabies and Mokola viruses in the region.

5 Key words: molecular epidemiology / rabies / Zimbabwe / South Africa / Lyssavirus / Mokola / phylogeny / canid/ viverrid/biotype

6 My sincere gratitude to Professor Louis Nel, who introduced me to Molecular Virology and served as my promoter for my PHD studies. Professor Nel was a perfect example for scientific advancement and his enthusiasm, dedication and never failing support inspired me throughout my studies. My sincere appreciation go to the Directorate of Central Veterinary Institute (CVL) Harare, Dr Ushewokunze-Obatolu, for granting me permission to work with virus isolates from their collection. To Dr Bingham, I say thank you for assistance with virus culture and the fruitful discussions regarding rabies epidemiology in Zimbabwe. His invaluable experience with rabies epidemiology in southern Africa and his contributions made this a more exciting area for study. I would also like to thank Dr Javangwe, from the CVL, for his assistance with typing viverrid virus isolates for these genetic studies. Thanks to my employer, the University of Zimbabwe, for granting me the study leave to conduct this research at the University of Pretoria. The Agricultural Research Council, the Foundation for Research and Development for research funds and DAAD through the Ansti-Unesco Nairobi office, for their generous financial support. The University of Zimbabwe also contributed towards part of my research and travel costs through the SIDA-SAREC funds. To my friends who supported me, your friendship is greatly appreciated. In particular, I would like to thank my colleagues from the molecular virology laboratory; Jonathan Key tel, Wanda Markotter and Jeanette Jacobs for encouragement when the odds were low. Thanks to Julian Jaftha, Marinda Oosthuisen and Dr Jacques Theron for their assistance. To Fourie Joubert, many thanks for your

7 assistance with computer analysis. Finally, I would like to give special thanks to my wife Florence who encouraged me when I needed it most, to my son and daughter, Panashe and Varaidzo, of whom I sacrificed a lot of time without attending to because of the commitments to this work.

8 PAGE ACKNOWLEDGEMENTS... VI SUMMAR Y 111 LIST OF ABBREVIA nons... Xl LIST OF FIGURES... LIST OF TABLES... CHAPTER ONE INTRODUCTION AND LITERATURE REVIEW 1.1 General introduction Rabies: a short history The Rhabdoviridae family Vesiculovirus genus Lyssavirus genus '" Rabies virus genome organisation and replication Transcription, translation and replication mechanisms of rabies virus Rabies viral proteins The glycoprotein '" The nucleoprotein The matrix protein The polymerase The phosphoprotein " '" Intergenic sequences RNA quasi-species Rabies pathogenesis 14 3 Global rabies situation Europe and Asia The Americas Rabies in Africa Rabies in southern Africa Rabies control 22 5 Methods used in the study Polymerase chain reaction " '" Nucleic acid sequencing Phylogenetic methods What is molecular phylogeny? Multiple sequence alignment Distance methods Character based methods Limitations of the tree building methods Resampling methods: Bootstrapping Aims 0f the study XIV XVI

9 CHAPTER TWO MOLECULAR EPIDEMIOLOGY OF CANINE RABIES IN ZIMBABWE AND SOUTH AFRICA. 2.1 Summary '" Introduction Materials and methods '" " Viruses and virus isolation Primers for RT-PCR Total RNA extraction cdna synthesis PCR Nucleotide sequencing Phylogenetic analysis Nucleotide sequence accession numbers Results '" Virus isolates, virus propagation, RNA extraction, cdna synthesis and amplification Phylogenetic analyses Discussion 64 CHAPTER THREE MOLECULAR EPIDEMIOLOGY OF VIVERRID RABIES IN ZIMBABWE AND SOUTH AFRICA. 3.1 Summary Introduction Materials and methods Rabies viruses and virus isolation Primer selection Total RNA extraction, cdna synthesis and RT-PCR Nucleic acid sequencing and phylogenetic analysis Nucleotide sequence accession numbers Results Virus isolates, RNA preparation, cdna synthesis and amplification Nucleotide sequence determination Phylogenetic analyses Discussion '" 90 CHAPTER FOUR GENOTYPIC ANALYSES OF MOKOLA VIRUSES FROM ZIMBABWE AND SOUTH AFRICA. 4.1 Summary '" Introduction Materials and methods Viruses and virus isolation Primer selection Viral RNA extraction and cdna synthesis per amplification... 99

10 4.3.5 Purification ofpcr amp Iicons and nucleic acid sequencing Phylogenetic analysis Results Mokola virus isolates, virus propagation and monoclonal antibody typing Viral RNA extraction, cdna synthesis and PCR Purification ofpcr products, nucleic acid sequencing and phylogenetic Analysis Accession numbers Discussion 105 CHAPTERS CONCLUDING REMARKS APPENDIX 1 Multiple alignment of nucleotide sequences of southern African canid viruses 141 Appendix 3: Multiple alignment of viverid rabies viruses from Zimbabwe and South Africa 171 Appendixes 4a-4d: Genetic distances of viverrid rabies viruses from Zimbabwe and South Africa Appendix S: Multiple alignment of nucleic acid sequences of Mokola viruses from Zimbabwe and South Africa Appendix 6: Genetic distances of Mokola virus nucleic acid sequences from Zimbabwe and South Africa 189

11 LIST OF ABBREVIATIONS CVS Challenge virus standard ERA Evelyn-Rokitnicki-Abelseth strain PV RNP Pasteur virus Ribonucleoprotein DNA Deoxyribonucleic acid cdna complementary DNA RNA Ribonucleic acid mrna MW SAG-2 messenger RNA Molecular weight SAD-Avirulent-Gift rabies vaccine strain SAD V-RG VNA PET Street Alabama Dufferin rabies vaccine strain Vaccinia rabies glycoprotein Virus neutralising antibodies post -exposure treatment DMI DNA-mediated immunisation I-ll I-lg ng microgram nanogram WHO ml mm pmol sp. U World Health Organisation millilitre millimolar picomoles species Units of enzyme activity

12 Ribonuclease dntp bp kb RT PCR deoxynucleotide base pair kilobases room temperature/reverse polymerase chain reaction transcription RT-PCR CNS reverse trancription polymerase chain reaction central nervous system AIDS Acquired immunodeficiency syndrome VSV vesiculostomatitis virus Mab FAT ORF NS GT monoclonal antibodies fluorescent antibody test open reading frame nonstructural genotype Phylip PAUP Phylogenetic Phylogenetic inference package inference using parsimony ML MP NJ UPGMA Maximum likelihood Maximum parsimony Neighbour joining Unweighted pair group method with arithmetic means M-MuLV RT Moloney Murine Leukemia Reverse Transcriptase EBL DNAdist European bat lyssavirus DNAdistance

13 Xlll C degrees Celsius VIZ Clustal KDa uv namely Cluster analysis kilo dalton ultraviolet CMI cell-mediated immunity V-RN GCG GDE ME Vaccinia recombinant nucleoprotein Genetics computer group Genetic data environment Minimum evolution

14 Map of southern Africa showing the routes of spread of rabies in southern Africa Map of southern Africa showing approximate enzootic regions for rabies 39 The rabies virus G-L intergenic region and the flanking domains Neighbour joining tree for the canid viruses from Zimbabwe and South Africa 56 Kitsch tree of canid viruses from Zimbabwe and South Africa 58 Map of Zimbabwe and South Africa showing the geographical location of viruses in group A 60 Map of Zimbabwe and South Africa showing the geographical location of viruses in group B Map of Zimbabwe and South Africa showing the geographical location of viruses in group C Map of southern Africa showing the geographical location of viruses in group D A neighbour-joining tree of the viverrid rabies viruses from Zimbabwe and South Africa 80 A radial tree of the viverrid rabies virus isolates from Zimbabwe and South Africa A Kitsch tree showing the relationships of the viverrid rabies viruses from Zimbabwe and South Africa 84 Map of Zimbabwe showing the geographical location of viruses in group

15 Neighbour joining tree of Mokola isolates from Zimbabwe and South Africa 102 Kitsch tree showing phylogenetic relationships of Mokola viruses from Zimbabwe and South Africa 103 Map of Zimbabwe and South Africa showing the origin of Mokola viruses used in this study. 104

16 List of canid viruses used in this study and accession numbers of their nucleotide sequences Designation, sequence and location of primers used for cdna synthesis, per and sequencing of the G-L intergenic region List of viverrid viruses used in this study and accession numbers of their corresponding nucleic acid sequences 74 List of Mokola isolates used in this study and accession numbers of their nucleotide sequences 97