UNIVERSITA’ DEGLI STUDI DI MILANO Facoltà di Medicina Veterinaria SCUOLA DI DOTTORATO Scienze Veterinarie e dell’Allevamento DIPARTIMENTO Medicina Veterinaria TESTUDINID HERPESVIRUS 3: DETECTION AND MOLECULAR CHARACTERIZATION OF STRAINS IN ITALIAN TESTUDO SPP. Tesi di dottorato di: Dr. Marco TECILLA R10820 TUTOR: Prof.ssa Paola Roccabianca (University of Milan, Italy) CO-TUTOR: Prof. Francesco Origgi (University of Bern, Switzerland) COORDINATORE: Prof. Fulvio Gandolfi Anno Accademico 2017-2018 Index: English abstract. ....................................................................................................................... 4 Abstract in Italiano. .................................................................................................................. 6 Summary of the work. .............................................................................................................. 8 Scientific background and working hypothesis: .............................................................................. 8 Aims: .............................................................................................................................................. 10 Sample collection: ......................................................................................................................... 11 Necropsy and prospective sample collections: ............................................................................. 11 PCR: ................................................................................................................................................ 12 Host pathogen interaction immunological study: ......................................................................... 12 Virus and genomic DNA: ................................................................................................................ 12 Phage Library and DNA sequencing: ............................................................................................. 12 Fluorescence Activated Cells Sorting [FACS] Validation in VERO cells: ......................................... 15 Phagemid editing and bacterial cell transformation: .................................................................... 15 Results: .......................................................................................................................................... 16 Discussion: ..................................................................................................................................... 18 References: .................................................................................................................................... 20 Chapter 1: History of reptile medicine. .................................................................................... 25 Chapter 2: Reptile trading. ...................................................................................................... 27 Reptile trade in Italy: ..................................................................................................................... 28 References: .................................................................................................................................... 29 Chapter 3: Reptiles’ immune response to viruses. .................................................................... 30 References: .................................................................................................................................... 33 Chapter 4: Herpesviruses of chelonians. .................................................................................. 36 Introduction: .................................................................................................................................. 36 Herpesvirus of chelonians: ............................................................................................................ 36 1 Chelonid herpevirus (ChHVs): ..................................................................................................... 36 2 Emydid herpevirus (EmyHVs): .................................................................................................... 47 3 Glyptemys herpesviruses (GlyHV): ............................................................................................. 49 4 Terrapene herpesviruses (TerrHV): ............................................................................................ 50 5 Testudinid herpesvirus (TeHV): .................................................................................................. 51 Discussion: ..................................................................................................................................... 56 References: .................................................................................................................................... 57 Chapter 5: Detection of TeHVs in Italy. .................................................................................... 61 Introduction: .................................................................................................................................. 61 Material and methods: .................................................................................................................. 61 Results: .......................................................................................................................................... 73 Discussion: ..................................................................................................................................... 98 References: .................................................................................................................................. 103 Scientific communications related to the research argument: ................................................... 112 Chapter 6: The genome of Testudinid herpesvirus 3. ............................................................... 115 Introduction: ................................................................................................................................ 115 Material and Methods: ................................................................................................................ 118 Results: ........................................................................................................................................ 124 Discussion: ................................................................................................................................... 147 References: .................................................................................................................................. 152 Scientific communications related to the research argument: ................................................... 158 Chapter 7: Testudo graeca immune response against Testudinid herpesvirus 3 ....................... 159 Initial assessment .................................................................................................................. 159 Materials and methods: .............................................................................................................. 161 Results: ........................................................................................................................................ 164 Discussion: ................................................................................................................................... 167 References: .................................................................................................................................. 169 pag. 2 Scientific communications related to the research argument: ................................................... 177 New insights .......................................................................................................................... 178 Introduction: ................................................................................................................................ 178 Material and methods: ................................................................................................................ 178 Results: ........................................................................................................................................ 180 Discussion: ................................................................................................................................... 188 References: .................................................................................................................................. 189 Scientific communications related to the research argument: ................................................... 190 Conclusions ........................................................................................................................... 191 Future perspective ................................................................................................................. 195 Publications outside the PhD research project: ....................................................................... 196 Indexed publications: .................................................................................................................. 196 Non-indexed publications: .......................................................................................................... 197 pag. 3 English abstract. Herpesvirales is an ancient group of DNA viruses that infect different animal species, includ- ing reptiles. The