Monitoring and Pathogenesis of Flavivirus Infections in Wild Birds and Domestic Poultry in Germany
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University of Veterinary Medicine Hannover Monitoring and Pathogenesis of Flavivirus Infections in Wild Birds and Domestic Poultry in Germany INAUGURAL – DISSERTATION in fulfillment of the requirements of the degree of Doctor of Veterinary Medicine -Doctor medicinae veterinariae- (Dr. med. vet.) submitted by Friederike Michel Böblingen Hannover 2019 Scientific supervision: Prof. Dr. Martin H. Groschup Friedrich-Loeffler-Institut Federal Research Institute for Animal Health Institute of Novel and Emerging Infectious Diseases Greifswald-Insel Riems 1st supervisor: Prof. Dr. Martin H. Groschup Friedrich-Loeffler-Institut Federal Research Institute for Animal Health Institute of Novel and Emerging Infectious Diseases Greifswald-Insel Riems 2nd supervisor: Prof. Dr. Paul Becher Institute of Virology Dept. of Infectious Diseases University of Veterinary Medicine Hannover Hannover Day of the oral examination: 30.10.2019 Sponsorship: This work was supported by the German Center for Infection Research (DZIF) To my family Table of contents Chapter 1: Introduction .............................................................................................. 1 Chapter 2: Manuscript I: West Nile Virus and Usutu Virus Monitoring of Wild Birds in Germany ........................................................................................................ 9 Chapter 3: Manuscript II: Evidence for West Nile Virus and Usutu Virus Infections in Wild and Resident Birds in Germany, 2017 and 2018 .......................................... 11 Chapter 4: Manuscript III: Role of ducks in the transmission cycle of tick-borne encephalitis virus? ..................................................................................................... 13 4.1. Abstract ............................................................................................................. 14 4.2. Introduction ...................................................................................................... 14 4.3. Materials and methods ..................................................................................... 17 4.3.1. Ethics statement and animals .......................................................................... 17 4.3.2. Virus challenge ................................................................................................ 17 4.3.3. Virological analysis ......................................................................................... 18 4.3.4. Quantitative real-time RT-PCR (qRT-PCR).................................................... 18 4.3.5. Serology ........................................................................................................... 18 4.3.6. Histopathology and immunohistochemistry .................................................... 19 4.4. Results ................................................................................................................ 19 4.4.1. TBEV detection by qRT-PCR .......................................................................... 20 4.4.2. Serological results ........................................................................................... 20 4.4.3. Gross lesions, histopathology and immunohistochemistry ............................. 21 4.5. Discussion .......................................................................................................... 22 4.6. Conclusion ......................................................................................................... 25 4.7. Figures ............................................................................................................... 26 4.8. Tables ................................................................................................................. 30 4.8. References ......................................................................................................... 34 Chapter 5: Manuscript IV: Experimental infection of chickens with tick-borne encephalitis virus ....................................................................................................... 41 5.1. Abstract ............................................................................................................. 42 5.2. Introduction ...................................................................................................... 42 5.3. Materials and methods ..................................................................................... 44 5.3.1. Animals and experimental design .................................................................... 44 5.3.2. Quantitative real-time RT-PCR (qRT-PCR).................................................... 44 5.3.3. Serological investigations ............................................................................... 45 5.4. Results ................................................................................................................ 45 5.4.1. Detection of viral RNA by qRT-PCR ............................................................... 45 5.4.2. Serology ........................................................................................................... 46 5.5. Discussion .......................................................................................................... 46 5.6. Conclusion ......................................................................................................... 48 5.7. Figures ............................................................................................................... 49 5.8. References ......................................................................................................... 53 Chapter 6: General discussion and conclusion ........................................................ 56 Chapter 7: Summary ................................................................................................ 68 Chapter 8: Zusammenfassung .................................................................................. 71 Chapter 9: References .............................................................................................. 74 Chapter 10: Authors´ Contributions ........................................................................ 89 Chapter 11: Acknowledgement ............................................................................... 91 Parts of this thesis were already published in the following journals: “West Nile Virus and Usutu Virus Monitoring of Wild Birds in Germany” Michel, F., Fischer, D., Eiden, M., Reuschel, M., Müller, K., Rinder, M., Fast, C., Urbaniak, S., Brandes, F., Schwehn, R., Lühken, R., Groschup, M.H., Ziegler, U. International Journal of Environmental and Public Health (2018), Volume 15, Issue 1 “Evidence for West Nile Virus and Usutu Virus Infections in Wild and Resident Birds in Germany, 2017 and 2018” Michel, F., Sieg, M., Fischer, D., Keller, M., Eiden, M., Reuschel, M., Schmidt, V., Schwehn, R., Rinder, M., Urbaniak, S., Müller, K., Schmoock, M., Lühken, R., Wysocki, P., Fast, C., Lierz, M., Korbel, R., Vahlenkamp, T.W., Groschup, M.H., Ziegler, U. Viruses (2019), Volume 11, Issue 7 Manuscripts extracted from the doctorate project: “Role of ducks in the transmission cycle of tick-borne encephalitis virus?” Michel, F., Ziegler, U., Fast, C., Eiden, M., Klaus, C., Dobler, G., Stiasny, K., Groschup, M.H. to be submitted “Experimental infection of chickens with tick-borne encephalitis virus” Michel, F., Ziegler, U., Eiden, M., Klaus, C., Dobler, G., Groschup, M.H. to be submitted Further publications: “West Nile virus epizootic in Germany, 2018” Ziegler, U., Lühken, R., Keller, M., Cadar, D., van der Grintenc, E., Michel, F., Albrecht, K., Eiden, M., Rinder, M., Lachmann, L., Höper, D., Vina-Rodriguez, A., Gaede,W., Pohl, A., Schmidt-Chanasit, J., Groschup, M.H. Antiviral Research (2019), Volume 162, Pages 39 – 43 “Erstmalige Nachweise von West-Nil-Virus-Infektionen bei Vögeln und Pferden in Deutschland“ Ziegler, U., Keller, M., Michel, F., Eiden, M., Groschup, M.H. LABLoeffler (2018), Volume 17, Issue 2, Page 6 „Aktuelles aus dem FLI zur West-Nil-Virus-Situation“/ “News from the FLI on the West Nile virus situation” Ziegler, U., Keller, M., Michel, F., Globig, A., Denzin, N., Eiden, M., Fast, C., Gethmann, J., Bastian, M., Groschup, M.H., Conraths, F.J. Amtstierärztlicher Dienst und Lebensmittelkontrolle (2019), Volume 2, Page 78 - 81 Experimental data were also published at conferences: “Emerging zoonotic pathogens in wild birds in Germany“ Michel, F., Ziegler, U., Eiden, M., Keller, M., Dobler, G., Groschup, M.H. Junior Scientist Zoonoses Meeting 2017, 07.06.-09.06.2017, Poster presentation “West Nile virus and Usutu virus monitoring of wild birds in Germany” Michel, F., Ziegler, U., Eiden, M., Keller, M., Dobler, G., Groschup, M.H. Joint Annual Meeting of the German Society of Infectious Diseases (DGI) and the German Center for Infection Research (DZIF) 2017, 28.9.-30.09.2017, Poster presentation “Experimental infection of ducks with tick-borne encephalitis virus” Michel, F., Ziegler, U., Fast, C., Eiden, M., Keller, M., Dobler, G., Groschup, M.H. 7. FLI- Junior Scientist Symposium, 24.-26.09.2018, Poster presentation List of abbreviations µl microliter µm micrometer arbovirus arthropod-borne virus Ct value cycle threshold value DAB Diaminobenzidine dpi day post infection ELISA enzyme linked immunosorbent assay E protein envelope protein H&E hematoxylin and eosin stain IC2 RNA internal control ribonucleic acid IHC immunohistochemistry JEV Japanese encephalitis virus MEM minimal essential medium min