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Whole Final 1 July 2018 Automatisch Gespeichert Faculty of Agricultural Sciences University of Hohenheim Institute of Animal Science (460) Department of Livestock Infectiology and Environmental Hygiene (460e) Establishment of reverse transcriptase polymerase chain reaction methods for the detection of newly described RNA viruses in reptiles: picornaviruses in tortoises, reptarenaviruses in snakes, and sunshinevirus in snakes. Dissertation Submitted in fulfilment of the requirements for the degree “Doktor der Agrarwissenschaften” (Dr. sc. Agrar./ Ph.D. in Agricultural Sciences) to the Faculty of Agricultural Sciences Presented by Tara H. Aqrawi B.Sc. biol., M.Sc. agr. Stuttgart 2018 Supervisor: PD Dr. Rachel E. Marschang, Dip ECZM (herpetology), FTÄ Mikrobiologie, ZB Reptilien Laboklin GmbH & Co. KG Steubenstr. 4 97688 Bad Kissingen, Germany This dissertation has been submitted in fulfilment of the requirements for the degree “Doktor der Agrarwissenschaften” (Dr. sc. Agrar. / Ph.D. in Agricultural Sciences) to the Faculty of Agricultural Sciences Date of oral exam: 18 th July 2018 Examination committee: PD Dr. Rachel E. Marschang PD Dr. Petra Kölle Prof. Dr. Ludwig Hölzle Vice-Dean and Head of the examination committee: Prof. Dr. Andrea Knierim Publications Part of the research work described in this doctoral thesis has been published in the following papers: • Marschang RE, Ihász K, Kugler R, Lengyel G, Fehér E, Marton S, Bányai K, Aqrawi T, Farkas SL. 2016: Development of a consensus reverse transcription PCR assay for the specific detection of tortoise picornaviruses. Journal of Veterinary Diagnostic Investigation, 28(3) 309 –314. • Aqrawi T, Stöhr AC, Knauf-Witzens T, Krengel A, Heckers KO, Marschang RE. 2015: Identification of snake arenaviruses in live boas and pythons in a zoo in Germany. Tierarztlische Praxis Ausg K, Kleintiere/ Heimtiere, 43(4):239-47 • Marschang RE, Stöhr AC, Aqrawi T, Hyndman TH, Plenz B, Blahak S, Pees M. 2013: First detection of Sunshine virus in pythons ( Python regius ) in Europe. In: Proceedings of the Association of Reptilian and Amphibian Veterinarians Conference. p.15 .Association of Reptilian and Amphibian Veterinarians ARAV. Table of Contents 1. Introduction ..................................................................................................... 3 2. Literature review ............................................................................................. 6 2.1 RNA viruses in reptiles ....................................................................................................... 6 2.2 Picornaviruses ................................................................................................................ 8 2.2.1 General introduction to picornaviruses ...................................................................... 8 2.2.2 Classification and replication .................................................................................... 9 2.2.3 Picornaviruses in tortoises...................................................................................... 10 2.2.4 Genome organization of picornaviruses .................................................................. 13 2.3 Arenaviruses ................................................................................................................ 14 2.3.1 General introduction of arenaviruses ...................................................................... 14 2.3.2 Classification of family Arenaviridae ....................................................................... 15 2.3.3 Genome organization of arenaviruses .................................................................... 17 2.3.4 Inclusion body disease (IBD) in snakes: History, clinical signs and hosts ................ 17 2.3.5 Arenaviruses in snakes .......................................................................................... 20 2.3.6 Diagnosis of arenaviruses and IBD in snakes ......................................................... 22 2.4 Sunshinevirus ............................................................................................................... 23 2.4.1 Clinical signs and virus diagnosis ........................................................................... 24 3. Aims of the study .......................................................................................... 26 4. Materials and methods ................................................................................. 27 4.1 Laboratory equipments and utensils ................................................................................. 27 4.2 Cell lines used in experimental trials ................................................................................. 29 4.3 Chemicals, reagents and solutions ................................................................................... 29 4.3.1 Cell culture and virus isolation ................................................................................... 29 4.3.2 Viral RNA extraction .................................................................................................. 32 4.3.3 Reverse transcription polymerase chain reaction (RT-PCR) ....................................... 34 4.3.4 Gel electrophoresis .................................................................................................... 36 4.3.5 Gel extraction ............................................................................................................ 36 4.4 Samples and isolates ....................................................................................................... 36 4.4.1 Samples for picornavirus studies ............................................................................... 36 4.4.2 Samples for arenavirus studies .................................................................................. 39 4.4.3 Samples for sunshinevirus studies ............................................................................. 40 4.4.4 Processing of diagnostic samples .............................................................................. 40 4.5 Subculture and preparation of tissue culture dishes for virus isolation ............................... 41 4.5.1 Virus isolation ............................................................................................................ 41 4.6 Extraction of viral RNA ..................................................................................................... 42 4.7 Reverse transcription – polymerase chain reaction ........................................................... 43 4.7.1 RT-PCR protocol used to identify picornaviruses from the isolates ............................. 43 4.7.1.1 Primers ............................................................................................................... 44 4.7.2 RT-PCR protocol used for the detection of arenaviruses ............................................ 46 4.7.3 RT-PCR protocol used for the detection of sunshinevirus ........................................... 47 4.7.4 RT-PCR positive controls ........................................................................................... 49 4.8 Gel electrophoresis .......................................................................................................... 49 4.9 Gel extraction ................................................................................................................... 50 4.10 Sequencing .................................................................................................................... 51 4.11 Analyses of sequences .................................................................................................. 51 5. Results ........................................................................................................... 53 5.1 Picornavirus detection ...................................................................................................... 53 5.1.1 Cell culture and RT-PCR trials ................................................................................... 53 5.1.1.1 picornavirus (previously known as virus “X” ) isolates .......................................... 53 5.1.1.2 Diagnostic samples ............................................................................................. 54 5.1.2 Sequence analysis .................................................................................................... 55 5.2 Arenavirus screening in snakes ........................................................................................ 58 5.2.1 Sequence analysis .................................................................................................... 59 5.3 Detection of sunshinevirus in snakes ................................................................................ 62 6. Discussion and conclusion .......................................................................... 66 6.1 Picornaviruses in tortoises ............................................................................................... 66 6.2 Arenaviruses in snakes .................................................................................................... 70 6.3 Sunshinevirus in snakes................................................................................................... 73 7. Summary ....................................................................................................... 76 8. Zusammenfassung ........................................................................................79 9. References ....................................................................................................
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