Seroepidemiology and primary infections of the recently detected Merkel cell polyomavirus and trichodysplasia-spinulosa associated polyomavirus Tingting Chen Haartman Institute Department of Virology University of Helsinki Helsinki, Finland ACADEMIC DISSERTATION To be presented with the permission of the Medical Faculty of the University of Helsinki, for public examination in the Lecture Hall 2, Haartman Institute, Haartmaninkatu 3, on December 5th, 2013, at 12 noon Helsinki 2013 SUPERVISED BY: Professor Klaus Hedman Department of Virology Haartman Institute University of Helsinki Helsinki, Finland & Docent Maria Söderlund-Venermo Department of Virology Haartman Institute University of Helsinki Helsinki, Finland REVIEWED BY: Professor Timo Vesikari University of Tampere Medical School Vaccine Research Center Tampere, Finland & Docent Thedi Ziegler University of Turku Research Center for Child Psychiatry Institute of Clinical Medicine Turku, Finland OPPONENT: Associate Professor Mariet Feltkamp Department of Medical Microbiology Leiden University Medical Center Leiden, The Netherlands ISBN 978-952-10-9546-7 (paperback) ISBN 978-952-10-9547-4 (PDF) Helsinki 2013 CONTENTS ABSTRACT .................................................................................................................... 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 3 RESULTS ..................................................................................................................... 37 1. MCPyV and TSPyV serodiagnostics and infections ................................................. 37 1.1 Virus-like particles (I, II) ................................................................................... 37 1.2 Antibody responses (I-III) ................................................................................. 37 1.3 Seroprevalences (I-III) ...................................................................................... 39 1.4 Asymptomatic MCPyV and TSPyV primary infections among children (III) ..... 40 2. Biotin IgM antibodies.............................................................................................. 42 2.1 Observation of false MCPyV IgM positivity due to biotin antibody ................... 42 2.2 Seroprevalence of biotin IgM ............................................................................ 42 2.3 The effect of biotin antibody on various IgM-antibody assays ........................... 42 2.4 Characteristics of Biotin IgM ............................................................................ 43 DISCUSSION ............................................................................................................... 50 1. MCPyV and TSPyV serodiagnostic assays (I-III) .................................................... 50 1.1 Generation of MCPyV and TSPyV VLPs .......................................................... 50 1.2. Serodiagnostic tools for MCPyV and TSPyV ................................................... 50 2. MCPyV and TSPyV infections (I-III) ....................................................................... 52 2.1 MCPyV and TSPyV antibody responses and seroprevalence ............................. 52 2.2 Antigenic cross-reactivity between different HPyVs .......................................... 53 2.3 Asymptomatic MCPyV and TSPyV primary infections in childhood ................. 54 3. Biotin IgM antibodies (IV) ...................................................................................... 55 3.1 The effect of biotin IgM on biotinylation-associated EIAs ................................. 55 3.2 Potential origins of biotin IgM........................................................................... 56 3.3 Possible clinical biomedical effects of biotin IgM.............................................. 56 CONCLUSIONS AND FUTURE PROSPECTIVES .................................................. 57 ACKNOWLEDGEMENTS .......................................................................................... 59 REFERENCES ............................................................................................................. 60 4 ABSTRACT Two newly found polyomaviruses are related to human skin diseases; Merkel cell polyomavirus (MCPyV), identified in Merkel cell carcinomas (MCC) is believed to be an etiologic factor in the pathogenesis of MCC; trichodysplasia spinulosa-associated polyomavirus (TSPyV), discovered in TS lesions, has an apparently causative role in this rare skin disease. To investigate their seroprevalence and possible clinical significance, we have developed for these two polyomaviruses comprehensive serodiagnostic methods and studied the occurrence of antibodies to these viruses, the quality of immune responses, diagnostic criteria, and possible disease associations. In addition, the thesis aims at searching for factors that cause false results in biotinylation-based antiviral IgM enzyme immunoassays (EIAs). For MCPyV and TSPyV, we developed EIAs for virus-specific IgG, IgM and IgG avidity, by using the major capsid protein