Seroepidemiology and primary infections of the recently detected Merkel cell polyomavirus and trichodysplasia-spinulosa associated polyomavirus

................................................................................................................... 5 LIST OF ORIGINAL PUBLICATIONS ....................................................................... 7 ABBREVIATIONS ......................................................................................................... 8 INTRODUCTION .......................................................................................................... 9 REVIEW OF THE LITERATURE ............................................................................. 10 1. Overview of human polyomaviruses ........................................................................ 10 1.1 Classification and virion properties .................................................................... 10 1.2 General aspects of HPyV life cycle .................................................................... 11 1.3 General aspects of HPyV infection and diseases ................................................ 12 2. Merkel cell polyomavirus ........................................................................................ 13 2.1 Merkel cell carcinoma ....................................................................................... 13 2.2 Discovery of MCPyV ........................................................................................ 14 2.3 MCPyV genome structure and function ............................................................. 14 2.4 Infection and immunity ..................................................................................... 17 2.5 Molecular epidemiology .................................................................................... 18 2.6 Clinical aspects on MCPyV-positive and -negative MCCs ................................. 20 2.7 Association of MCPyV with pathogenesis of MCC ........................................... 21 3. Trichodysplasia-spinulosa associated polyomavirus ................................................ 23 3.1 Trichodysplasia-spinulosa disease ..................................................................... 23 3.2 Discovery of TSPyV.......................................................................................... 23 3.3 TSPyV genome ................................................................................................. 23 3.4 Epidemiology and immunity.............................................................................. 24 4. Serodiagnosis .......................................................................................................... 26 4.1 Antibody response in viral infection .................................................................. 26 4.2 EIA for antibody detection ................................................................................ 27 4.3 Interpretation of results ...................................................................................... 28 4.4 Biotinylation-based anti-viral IgM EIA ............................................................. 29 AIMS OF THE STUDY ................................................................................................ 30 MATERIALS AND METHODS .................................................................................. 31 1. Materials ................................................................................................................. 31 1.1 Patients and serum samples ............................................................................... 31 1.2 Plasmids, bacteria and cells (I, II) ...................................................................... 32 2. Methods .................................................................................................................. 32 2.1 Virus-like particle production (I, II) ................................................................... 32 2.2 Biotinylation (I, II, IV) ...................................................................................... 33 2.3 Immunoblotting (I, II, IV).................................................................................. 33 2.4 In-house antibody assays (I-IV) ......................................................................... 34 2.5 Cutoff determination (I-IV) ............................................................................... 35 2.6 Affinity determination (IV) ............................................................................... 36 2.7 Quantitative PCR (III) ....................................................................................... 36 2.8 Statistical analysis (I-IV) ................................................................................... 36