School of Biomedical Sciences The Role of Genetic Diversity in the Replication, Pathogenicity and Virulence of Murray Valley Encephalitis Virus Aziz-ur-Rahman Niazi This thesis is presented for the Degree of Doctor of Philosophy of Curtin University September 2013 Declaration To the best of my knowledge and belief, this thesis contains no material previously published by any other person except where due acknowledgment has been made. This thesis contains no material which has been accepted for the award of any other degree or diploma in any university. Signature:…………………. Date:………………………. Acknowledgement First, I am humbly grateful to The Almighty God for granting me both the ability and determination to carry out this PhD. Next, I express my sincerest gratitude to both my parents who I hold with the highest regard, for their support and affability that made the undertaking of this thesis possible. I only wish that my mother were still alive to share in its completion. My sincere thanks are extended to my wife, Sonia Mohammadi, whom I am forever indebted to for giving up her ambitions of going to university to instead raise our lovely baby daughter, Alia Saba Niazi, born at the beginning of this PhD. Sonia is now expecting our son who will be born soon after completion of this PhD. Special heartfelt thanks also go to my extended family back home whose support has provided me with additional strength and energy to complete this PhD. I would like to cordially express my thanks to my supervisor, Dr David Thomas Williams, first for his help in applying for an Australian Biosecurity Cooperative Research Centre (AB-CRC) scholarship, then for guiding me and inspiring me to be a virologist. David’s support throughout the process has been essential. I would also specially thank my co-supervisor Dr Paul Costantino for introducing me to the wonderful world of infectious diseases that eventually led me to do a PhD. I have been greatly fortunate to have had Paul as a co-supervisor whose advice throughout this thesis has made the journey more delightful. Sincere appreciation goes to my other co-supervisor Dr Beng Hooi Chua whose enthusiasm and knowledge in animal work and molecular cloning greatly enhanced the laboratory work. My thanks go to Dr Sinead Diviney whose guidance in the first two years of my PhD was fundamental to my progress. My special thanks also go to my associate supervisor, Associate Professor Cheryl Johansen from the University of Western Australia, for her support, guidance and time throughout this PhD. I also thank Professor Ricardo Mancera who took care of the administrative supervision for the second half of my PhD. i I also express my sincere thanks to Dr John Fielder from the Learning Centre, Curtin University, for his advice on my writing development. My sincere appreciation also goes to Ms Chris Kerin, Curtin University International Sponsored Student Unit, whose dedication and support made this PhD journey possible. Chris’ support at the beginning and throughout this thesis was fundamental to my progress. I also sincerely thank many fellow PhD students, including Mr Naz Hasan Huda, Ms Malini Visweswaran, Ms Julia Kohn, Ms Ganga Senarathna, Ms Gaewyn Ellison, Vishal Chaturvedi, and Tin Fei Sim, who I met and developed good friendship with, which made this PhD journey more enjoyable. I am sincerely grateful to the AB-CRC for providing me with the stipend scholarship for the duration of this PhD and to Curtin University for providing additional funds and waiving the tuition fee for the course of this thesis. I also thank the Government of the Islamic Republic of Afghanistan and the Government of Australia for permitting me to undertake this PhD project. ii CONTRIBUTORS I greatly acknowledge the following people whose contribution made this possible. Associate Professor Cheryl Johansen (Arbovirus Surveillance and Research Laboratory, University of Western Australia) for providing MVEV isolates (Chapter 2) and homogenates of mosquito pools (Chapter 3 and 4). Professor Peter McMinn (University of Sydney) for providing MVEV infectious clone (Chapter 3, 4 and 6). Dr Simone Warner (Department of Planning and Infrastructure, Victoria) and Dr Mary Carr (Primary Industries and Regions, South Australia) for providing the clinical samples (Chapter 3). Mr Adam Foord (Australian Animal Health Laboratory, Geelong, Victoria) for testing clinical samples at AAHL using the real-time RT-qPCR described in Chapter 3. Dr Glenys Chidlow (PathWest Laboratory Medicine, WA) for testing a few environmental samples at PathWest by real-time RT-qPCR (Chapter 3). Mr Ivano Broz (Australian Animal Health Laboratory, Geelong, Victoria) for providing bioinformatics support and for conducting the processing and filtering of NGS datasets, providing per-nucleotide variance data and SNP reports (Chapter 4). Professor Roy Hall (University of Queensland) for providing anti-MVEV monoclonal antibodies (Chapter 5 and 6). Dr David Williams, Dr Sinead Diviney and Dr Beng Hooi Chua for providing oligonucleotide primers for complete genome sequencing of MVEV (Chapter 6). Dr Christine Cooper, Dr Richard Parsons and Dr Marilyn Bennet-Chambers (Curtin University) for their support in statistical analyses (Chapter 5 and 6). iii ABSTRACT Murray Valley encephalitis virus (MVEV) is the main causative agent of arboviral encephalitis in Australia. Of the four genotypes of MVEV (G1-G4), only G1 and G2 are found in Australia. G1 is dominant, while G2 is confined to Kununurra in the northeast Kimberley region of Western Australia (WA). Prior to this thesis, G2 MVEV had not been detected since 1995. In order to accurately characterise the distribution of the different MVEV genotypes in WA, nucleotide sequencing and phylogenetic analyses were performed on the partial envelope gene of all (seventy one) MVEV isolates collected from mosquitoes in northern WA between 2005 and 2009. Four G2 isolates were identified (5.6% of all isolates sequenced) from Fitzroy Crossing and Broome in the west Kimberley region. This indicates that G2 continues to circulate in WA, and beyond its previously recognised geographic range of Kununurra. Of the sixty seven G1 isolates, forty five (67.2%) belonged to sublineage G1a, and twenty two (32.8%) belonged to sublineage G1b. To further characterise the genetic diversity within these isolates, sequencing and analysis of the full-length prM-E genes and the 3ʹ- untranslated region (3ʹUTR) was performed on representative isolates. Analysis of the full-length prM-E genes indicated a similar phylogenetic relationship between all of the MVEV isolates compared to that obtained using a partial analysis of the envelope gene. Analysis of the 3ʹUTR sequences identified a unique 18-nucleotide deletion in all G2 isolates from 1991, 2006 and 2009, which may serve as a genetic marker of recent G2 strains. In order to facilitate the detection and quantification of viral RNA from all four genotypes of MVEV in the laboratory, surveillance and clinical settings, a real-time quantitative RT-PCR (RT-qPCR) was developed and validated. This assay demonstrated a high level of efficiency, sensitivity and specificity to all four MVEV genotypes and detected MVEV in infected pools of mosquito homogenates, as well as infected brains and tissues of veterinary clinical specimens. iv Next generation sequencing (NGS) was employed to characterise the depth of genetic diversity and mutation spectra in the G1 and G2 MVEV populations. The complexity of mutant spectra within each sample quasispecies was determined by calculating the percentage of sequence clones as well as nucleotides that differed from the consensus sequence. This approach has been used by many researchers to detect low-frequency mutations and variants in a viral population. NGS technology revealed that G1 samples are highly genetically diverse while G2 samples have a lower level of genetic complexity and sequence heterogeneity. The lower level of genetic diversity in G2 MVEV is associated with low abundance of this MVEV genotype in nature. The phenotypic features of representative isolates from recent G1a, G1b and G2 were characterised and compared with those of the prototype viruses – the primary ancestral viruses – of G1 (MVE-1-51) and G2 (OR156) in a single-step growth curve assay and a mouse model of pathogenesis. There was no significant difference between the replication kinetics of G1 and G2 isolates in DF1 cells compared to their prototype viruses. In contrast, recent G1 isolates demonstrated significantly lower replication kinetics in C6/36 cells compared to G1 prototype virus; whereas, one recent G2 isolate displayed a significantly higher replicative fitness than the G2 prototype virus in these cells. In an in vivo mouse infection model, all G1 isolates demonstrated a dose-dependent mortality rate, characteristic of highly virulent strains. In contrast, G2 isolates demonstrated a significant reduction in neuroinvasiveness, and increases in average survival time and 50% humane dose end point (HD50) values, indicative of attenuation. In an experimental evolution study, the basis of MVEV restricted genetic diversity (the observation that MVEV has not been subjected to a high level of genetic variation) was explored. It was hypothesised that the genetic stability of MVEV results from alternate cycling between mosquito vectors and avian hosts. Genetically homogeneous MVEV G1 and G2 clonal populations were passaged sequentially in mosquito C6/36 or avian DF1 cells or alternately between the two cell lines. Passaging G1 and G2 MVEV in mosquito cells did not result in significant differences in genetic and phenotypic characteristics. In contrast, serial passage of v G1 and G2 MVEV in avian cells and alternately between mosquito and avian cells resulted in accumulation of mutations that were associated with significant differences in replicative ability in a single-step growth curve assay and an attenuation of virulence in mice. This suggests that the restriction of MVEV genetic diversity may be a result of alternating replication between mosquitoes, in which purifying selection is strong, and not as a result of replication in avian hosts, where purifying selection is relaxed.