Molecular Characterization and Pathogenesis of Equine and Elephant Herpesviruses
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Aus dem Institut für Virologie des Fachbereichs Veterinärmedizin der Freien Universität Berlin Molecular characterization and pathogenesis of equine and elephant herpesviruses Inaugural-Dissertation zur Erlangung des Grades eines PhD.of Biomedical Sciences an der Freien Universität Berlin vorgelegt von Pavulraj Selvaraj Tierarzt aus Villupuram, India Berlin 2019 Journal- Nr: 4180 Gedruckt mit Genehmigung des Fachbereichs Veterinärmedizin der Freien Universität Berlin Dekan: Univ.-Prof. Dr. Jürgen Zentek Erster Gutachter: Univ.-Prof. Dr. Klaus Osterrieder Zweiter Gutachter: Prof. Dr. Benedikt Kaufer Dritter Gutachter: Univ.-Prof. Dr. Robert Klopfleisch Deskriptoren (nach CAB-Thesaurus): equid herpesvirus 1; equid herpesvirus 4; elephantid herpesvirus 1; pathogenesis; peripheral blood mononuclear cells; endothelium, outbreaks; ELISA; polymerase chain reaction Tag der Promotion: 10.12.2019 “If I have a thousand ideas and only one turns out to be good, I am satisfied”. - Alfred Nobel “Ask the right questions, and nature will open the doors to her secrets”. - C V Raman “Dream is not that you see in sleep, dream is something that does not let you sleep”. - Dr A P J Abdul kalam “The greatness of a nation and its moral progress can be judged by the way its animals are treated”. - Mahatma Gandhi “One, remember to look up at the stars and not down at your feet. Two, never give up work. Work gives you meaning and purpose and life is empty without it. Three, if you are lucky enough to find love, remember it is there and don't throw it away”. - Dr Stephen Hawking “Institute is bigger than individuals. Always work in team. Don’t fly in sky when you achieve great things, always have your feet on ground. Don’t forget where you come from”. - Dr Nitin Virmani Dedicated to…… My beloved Parents, sisters and pets IV List of contents Page List of contents ...................................................................................................................... V 2 List of figures and tables .................................................................................................... X 3 List of Abbreviations ........................................................................................................ XIII 4 Introduction ........................................................................................................................ 1 4.1 Taxonomy and phylogeny of herpesviruses .................................................................... 1 4.1.1 Classification of the order Herpesvirales ................................................................... 1 4.1.1.1 Subfamily Alphaherpesvirinae ............................................................................ 2 4.1.1.2 Subfamily Betaherpesvirinae .............................................................................. 2 4.1.1.2 Subfamily Gammaherpesvirinae ........................................................................ 3 4.1.2 Equid and elephant herpesviruses ............................................................................ 4 4.1.2.1 Classification of known equine herpesviruses, host range and disease manifestations ................................................................................................................ 4 4.1.2.2 Classification of elephant endotheliotropic herpesviruses, host and disease manifestations ................................................................................................................ 5 4.2 Important equine herpesviruses affecting equine population ........................................... 5 4.3 History of EHV ................................................................................................................ 7 4.4 Equine herpesvirus type 1 and type 4: Structure and morphology ................................... 7 4.4.1 Equine herpesvirus Structure .................................................................................... 7 4.4.2 Equine herpesvirus genome ..................................................................................... 8 4.4.3 Viral proteins............................................................................................................. 9 4.5 Epidemiology ................................................................................................................ 13 4.6 Pathogenesis and pathology of EHV ............................................................................. 15 4.6.1 Epithelial infection ................................................................................................... 16 4.6.2 Viremia, PBMC-EC interaction and endothelial infection ......................................... 17 4.7 Immune evasion strategies by EHV .............................................................................. 19 4.8 Models to study equine herpesvirus pathogenesis ........................................................ 20 4.8.1 Small animal models ............................................................................................... 20 4.8.2 In vitro models ........................................................................................................ 20 4.8.2.1 Ex vivo nasal explants ...................................................................................... 21 4.8.2.2 EREC culture ................................................................................................... 21 V 4.8.2.3 Contact assays................................................................................................. 22 4.8.2.4 Flow chamber system ...................................................................................... 23 4.8.2.5 Other promising in vitro models ........................................................................ 24 4.9 Vaccination ................................................................................................................... 25 4.10 Elephant endotheliotropic herpesvirus ......................................................................... 27 4.11 Aims of the study and project summary ....................................................................... 28 5 Materials and Methods ..................................................................................................... 31 5.1 Materials ....................................................................................................................... 31 5.1.1 Chemicals, consumables and equipments .............................................................. 31 5.1.1.1 Chemicals ........................................................................................................ 31 5.1.1.2 Consumables ................................................................................................... 33 5.1.1.3 Equipments ...................................................................................................... 34 5.1.2 Softwares ............................................................................................................... 36 5.1.3 Enzymes ................................................................................................................. 37 5.1.4 Antibodies ............................................................................................................... 37 5.1.5 Commercial molecular biology kits .......................................................................... 38 5.1.6 Antibiotics ............................................................................................................... 39 5.1.7 Bacteria, cells, viruses and plasmids ...................................................................... 39 5.1.7.1 Bacteria ............................................................................................................ 39 5.1.7.2 Cells ................................................................................................................. 39 5.1.7.3 Viruses ............................................................................................................. 40 5.1.7.4 Plasmids .......................................................................................................... 40 5.1.8 Buffers, gel and bacterial culture media .................................................................. 40 5.1.8.1 Buffers ............................................................................................................. 40 5.1.8.2 Gels ................................................................................................................. 42 5.1.8.3 Bacterial culture media ..................................................................................... 43 5.1.9 Media, cell culture supplements and composition ................................................... 44 5.1.9.1 Media and cell culture supplements ................................................................. 44 5.1.9.2 Cell culture medium composition ...................................................................... 44 5.1.10 Primers, probes and oligonucleotides ................................................................... 45 5.1.11 Quantitative (q) PCR mastermix composition and cyclic condition ........................ 47 5.2 Methods ........................................................................................................................ 47 VI 5.2.1 Cells and viruses .................................................................................................... 47 5.2.1.1 Cells ................................................................................................................