Seoul hantavirus as a cause of acute kidney injury in the United Kingdom Thesis submitted in accordance with the requirements of the University of Liverpool for the degree of Doctor in Philosophy Lisa Jane Jameson April, 2015 Department of Clinical Infection, Microbiology & Immunology Institute of Global Health University of Liverpool Memorandum Apart from the help and advice acknowledged, this thesis represents the work of the author ------------------------------------------------------------ Lisa Jane Jameson Page | 1 Table of Contents Memorandum......................................................................................................... 1 Table of contents.................................................................................................... 2 Acknowledgments................................................................................................... 4 Abstract................................................................................................................... 5 List of abbreviations................................................................................................ 6 List of figures........................................................................................................... 8 List of tables............................................................................................................ 9 List of original publications...................................................................................... 10 Chapter 1................................................................................................................. 11 1.1 Classification...................................................................................................... 12 1.2 Historical review................................................................................................ 12 1.3 Virus structure and replication.......................................................................... 17 1.4 Life cycle............................................................................................................ 19 1.5 Relationship between hantaviruses and their natural hosts............................. 19 1.6 Distribution........................................................................................................ 20 1.7 Transmission...................................................................................................... 23 1.8 Hantavirus causing human disease................................................................... 24 1.9 Non-pathogenic hantaviruses............................................................................ 26 1.10 Pathogenesis................................................................................................... 27 1.11 Treatment........................................................................................................ 28 1.12 Aetiological diagnosis...................................................................................... 30 1.13 Control and prevention................................................................................... 32 1.14 Hantaviruses in the United Kingdom............................................................... 32 1.15 Rationale.......................................................................................................... 40 1.16 Seoul hantavirus.............................................................................................. 40 1.17 Aims................................................................................................................. 44 Chapter 2................................................................................................................. 45 2.1 Rodent sampling................................................................................................ 45 2.2 RNA extraction................................................................................................... 47 2.3 Cells and viruses................................................................................................ 48 2.4 Virus isolation.................................................................................................... 49 2.5 Polymerase chain reaction................................................................................ 50 2.6 Sequencing of PCR products.............................................................................. 52 2.7 Virus ultracentrifugation................................................................................... 56 2.8 Inactivation of hantaviruses by UV irradiation.................................................. 56 2.9 Fluorescent focus assay..................................................................................... 57 2.10 Immunofluorescence assays............................................................................ 57 2.11 In-house ELISA................................................................................................. 59 2.12 Sero-prevalence studies.................................................................................. 60 2.13 Human sera and saliva sampling..................................................................... 62 2.14 Data processing and statistical analysis........................................................... 62 63 Page | 2 Chapter 3................................................................................................................. 3.1 Introduction....................................................................................................... 63 3.2 Hantavirus in Yorkshire...................................................................................... 64 3.3 Investigation in Oxfordshire.............................................................................. 68 3.4 Genomic analysis of UK SEOV strains................................................................ 71 3.5 Phylogenetic analysis......................................................................................... 73 3.6 Molecular detection of hantaviral RNA in human sample................................ 77 3.7 Discussion.......................................................................................................... 78 Chapter 4................................................................................................................. 82 4.1 Introduction....................................................................................................... 82 4.2 Sero-prevalence in Yorkshire farmers and rural dwellers................................. 85 4.3 Sero-prevalence in British military personnel................................................... 91 4.4 Sero-prevalence in renal patients...................................................................... 93 4.5 Discussion.......................................................................................................... 94 Chapter 5................................................................................................................. 97 5.1 Introduction....................................................................................................... 97 5.2 Isolation of Seoul virus strain Humber.............................................................. 100 5.3 Unsuccessful isolations...................................................................................... 102 5.4 Quantification of Humber virus......................................................................... 102 5.5 UV inactivation of Humber virus....................................................................... 103 5.6 Inactivated whole virus as antigen in an ELISA.................................................. 106 5.7 Ultracentrifugation of Humber virus................................................................. 109 5.8 Discussion.......................................................................................................... 109 Chapter 6................................................................................................................. 114 6.1 Hantavirus as a confirmed human pathogen in the UK..................................... 114 6.2 Circulation of other hantavirus species............................................................. 116 6.3 Clinical definition and raised awareness........................................................... 118 6.4 Future work....................................................................................................... 119 6.5 Conclusion......................................................................................................... 120 References............................................................................................................... 121 Appendices............................................................................................................. 139 Page | 3 Acknowledgments First and foremost I would like to thank my supervisors Roger Hewson (PHE Porton), Robert Christley (University of Liverpool) and Tim Brooks (PHE Porton) for their time, expertise and support over the past three years. I would also like to thank Gail Carson (University of Oxford) and Sareen Galbraith (Leeds Metropolitan University) for their supervision in the early stages. Training was provided by key people at PHE Porton including: Babak Afrough, Barry Atkinson, Natalie Baker, Christopher Logue, Edmund Newman and Emma Rayner. In addition I would like to thank Richard Vipond and Cathy Rowe for mentoring me for the