Studies of the Epidemiology of Human Herpesvirus 6 Julie Dawn Fox Submitted for the degree of Doctor of Philosophy University College London ProQuest Number: 10797776 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a com plete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest ProQuest 10797776 Published by ProQuest LLC(2018). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States C ode Microform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106- 1346 Acknowledgements. I would like to thank Prof. R. S. Tedder and Prof. J. R. Pattison for giving me the opportunity to study for a PhD within the Department of Medical Microbiology and for supervision and helpful discussion during the course of this project. I would also like to thank other members of the Virology Section for help and encouragement. Thanks are due to Dr M. Contreras (North London Blood Transfusion Centre, London), Dr H. Kangro (St Bartholomew's Hospital, London), Dr H. Holzel (Hospital for Sick Children, London), Dr T. Stammers (The Church Lane Practice, London), Dr M. Hambling (Public Health Laboratory, Leeds) Dr W. Irving (University Hospital, Nottingham) Dr H. Steel (St George's Hospital, London), Dr E. Larson (Clinical Research Centre, London) and Dr P. Venables (The Kennedy Institute of Rheumatology, London) for supplying sera and clinical information. Dr R. Honess (National Institute for Medical Research, London), who sadly died last year, supplied HHV6 control plasmids and gave encouragement and advice on molecular techniques. 2 Abstract A new human herpesvirus was isolated in 1986 from patients with various lymphoproliferative disorders and was designated human herpesvirus 6 (HHV6). This thesis describes work undertaken to define the epidemiology of HHV6 infection in order to determine any association of HHV6 infection with human disease. The close relationship of HHV6 with human cytomegalovirus and the interaction of HHV6 with other viruses are discussed. Work on the epidemiology and pathogenic significance of HHV6 depends on having sensitive and specific assays for antibody detection. The development of assays for the detection of HHV6 specific IgM and IgG antibody and the use of these assays to establish antibody prevalence levels in normal and patient population groups are described. Most adults were found to be HHV6 antibody positive and the majority of HHV6 infections in adults were due to reactivation or reinfection. Children were found to have been infected with HHV6 during the first year of life and the clinical consequences of primary HHV6 infection was investigated. HHV6 antibody prevalence and titre in adult patient groups was compared with controls and the results are discussed in the context of other published serological reports. In situ hybridization and immunohistochemical staining were used to determine the in vivo tissue tropism of HHV6 and to investigate any potential disease association of HHV6 infection. Amplification of HHV6 DNA by the polymerase chain reaction was used for the detection of HHV6 DNA in saliva samples and peripheral blood mononuclear cells from healthy individuals. A large proportion of salivary gland tissues and saliva samples was found to be HHV6 DNA positive, which may have implications for transmission of the virus. The results of a prospective study to investigate possible reactivation of HHV6 during pregnancy are presented. Samples were taken from pregnant women at various times during the antenatal and early postnatal period to determine any change in HHV6 antibody profile, HHV6 DNA in peripheral blood samples and shedding of HHV6 in saliva. The postulate is discussed that pregnant women reactivate HHV6 which results in increased salival shedding of the virus during the early postnatal period and thus transmission to their newborn offspring. 3 Contents Title page .................................................................................................................... 1 Acknowledgements ................................................................................................... 2 Abstract.......................................................................................................................3 Contents ......................................................................................................................4 List of Tables ............................................................................................................10 List of Figures ..........................................................................................................13 Layout of thesis ........................................................................................................17 Abbreviations ..........................................................................................................18 Section 1: General Introduction la. TheHerpesviridae family:........................................................................20-22 la. 1. herpesvirus D N A ..................................................................20 la. 2. herpes vims proteins ............................................................. 20 la. 3. replication of herpesviruses ................................................ 21 la. 4. classification of herpesviruses ............................................ 21 lb. Biological properties of the human herpesviruses: .................................22-30 lb. 1. herpes simplex vims (HSV) ..............................................23 lb. 2. varicella-zoster vims (VZV) ..............................................24 lb. 3. Epstein-Barr vims (EBV) ....................................................25 lb. 4. human cytomegalovims (CMV) .........................................27 lb. 5. human herpesvirus 6 (HHV6).............................................28 lc. Aims of this study and plan of experimental w ork .........................................31 Section 2: Materials and methods 2a. Tissue culture and HHV6 propagation ............................................................ 32 2b. Indirect immunofluorescence (IF): .......................................................... 34-36 2b. 1. preparation of slides and s e ra ............................................ 34 2b. 2. detection of anti-HHV6 IgG .............................................. 36 2b. 3. detection of anti-HHV6 IgM ............................................. 36 2c. Anti-complementary immunofluorescence (ACIF) for detection of anti-HHV6 IgG ...................................................................... 36 2d. Competitive radioimmunoassay (RIA) for detection of anti-HHV6: .......................................................................................... 37-40 2d. 1. globulin preparation .............................................................37 2d. 2. antigen preparation .............................................................. 38 4 2d. 3. preparation of 125I-labelled Ig G ........................................ 38 2d. 4. detection of anti-HHV6 .......................................................39 2e. Complement fixation test for detection of anti-HSV, anti-VZV and anti-CMV: ...................................................................,40-41 2e. 1. preparation of sensitized sheep red blood cells .................40 2e. 2. performance of the test .........................................................40 2f. Indirect IF for detection of anti-EBV VC A IgG ............................................. 41 2g. Solid-phase enzyme immunoassay for detection of anti-CMV IgM ............42 2h.In situ hybridization for detection of HHV6 DNA: ................................42-46 2h. 1. preparation of biotin labelled probes by nick translation ....................................................................42 2h. 2. reproducibility of the nick translation reaction .................43 2h. 3. pre-treatment of cells, slides and tissues ........................... 44 2h. 4. hybridization of biotinylated probes to target DNA . .45 2h. 5. detection of biotinylated probes ......................................... 45 2i. Production of monoclonal antibodies against HHV6:.............................46-50 2i. 1. animals and immunisation protocol ....................................46 2i. 2. culture of myeloma cells ......................................................47 2i. 3. preparation of feeder cells ................................................... 47 2i. 4. preparation of spleen cells ................................................... 47 2i. 5. cell fusion ..............................................................................48 2i. 6. culture of hybrid cells .......................................................... 48 2i. 7. detection of anti-HHV6 secreting hybridomas by indirect I F .......................................................................... 48 2i. 8. cloning by limiting dilution ................................................. 49 2i. 9. culture of hybrid clones ....................................................... 49 2j. Immunohistochemical staining for detection of HHV6- specific antigens: ...........................................................