Monitoring and Pathogenesis of Flavivirus Infections in Wild Birds and Domestic Poultry in Germany

University of Veterinary Medicine Hannover

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 minute ml milliliter

ND50 neutralization dose 50 nm nanometer qRT-PCR quantitative real-time reverse transcriptase polymerase chain reaction

RNA ribonucleic acid

ROC receiver operating characteristic spp. species

TBEV tick-borne encephalitis virus

TCID50 tissue culture infection dose 50

U/L Unit per liter

USUV Usutu virus

VNT virus neutralization test

WNND West Nile neuroinvasive disease

WNV West Nile virus

List of Figures

Figure 1. Quantitative real-time RT-PCR (qRT-PCR) results of the blood and swab samples of the infected ducks (D 01 - D 19) in copies/µl ……………………….……………………… 26

Figure 2. Antibody response of the infected ducks against TBEV, by virus neutralization test and ELISA .………………………………………………………………………………….. 27

Figure 3. Histopathology and immunohistochemistry of TBEV infected ducks ……………. 29

Figure 4. Results of quantitative real-time RT-PCR (qRT-PCR) of the blood and swab samples of the intramuscularly and subcutaneously infected chickens in copies/µl …………………. 49

Figure 5. Results of quantitative real-time polymerase chain reactions (qRT-PCR) of the organ samples of the intramuscularly (i.m.) and subcutaneously (s.c.) infected chickens in copies/µl .………………………………………………………………………………………………. 50

Figure 6. Course of antibody responses of the intramuscularly (i.m.) and subcutaneously inoculated (s.c.) chickens against TBEV monitored by virus neutralization test (depicted in log titers) ………………………………….………………………………………………… 51

List of Tables

Table 1. Results of the tissue samples by quantitative real-time RT-PCR (qRT-PCR), titration on PK15 cells and immunohistochemistry (IHC) .…………………………….……………. 30

Supplemental Table 1: Overview of the histopathological results obtained in TBEV infected ducks. Immunohistochemical results are shown for brain samples only …………………… 32

Chapter 1: Introduction

West Nile virus (WNV), Usutu virus (USUV) and tick-borne encephalitis virus (TBEV) belong to the family Flaviviridae, genus Flavivirus. The family of the Flaviviridae includes three more genera, the Pestiviruses, Hepaciviruses and Pegiviruses (Simmonds et al., 2017). While WNV and USUV belong to the Japanese encephalitis virus (JEV) serocomplex along with viruses like JEV, Murray Valley encephalitis virus and St. Louis encephalitis virus, TBEV is part of the tick-borne encephalitis virus serocomplex (Barrows et al., 2018; Mansfield et al., 2011).

Flaviviruses are small, enveloped viruses with an icosahedral symmetry and a diameter of 40 – 60 nm containing a single stranded positive-sense RNA genome of approximately 11 kb with a 5´cap structure, but no 3´poly A tail (Brinton, 2013; Simmonds et al., 2017). The genome consists of a unique open reading frame (ORF) flanked by two untranslated regions (UTRs) at the 5´ and 3´ end (Heinz and Stiasny, 2010). The ORF is translated into one long polyprotein, which is processed co- and post-translationally into structural proteins (C, prM, E) and nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) (Barrows et al., 2018; Lindenbach and Rice, 2003; Simmonds et al., 2017). The capsid protein (C) forms the capsid, in which the RNA is embedded. The envelope (E) protein and the pre-membrane (prM) protein are necessary for virus attachment, entry and membrane fusion. Furthermore, the envelope protein is the main target for neutralizing antibodies (Beasley and Barrett, 2002). The nonstructural proteins have multiple functions and for instance are involved in virus replication and immune evasion (Chancey et al., 2015).

WNV circulates in an enzootic cycle between ornithophilic mosquitoes as vectors and avian host species (Troupin and Colpitts, 2016). In several mosquito species efficient transmission has been demonstrated experimentally, nevertheless, mosquitoes of the Culex pipiens complex are considered to be the primary vectors for WNV (Ciota, 2017; Farajollahi et al., 2011; Fros et al., 2015a). In most birds an infection remains inapparent, however, highly susceptible birds such as owls, birds of prey and several passerines (like jays, crows and sparrows) can develop a neurological disease and the infection may end fatally (Komar, 2003; Perez-Ramirez et al., 2014). Not all infected bird species are able to transmit the virus to feeding mosquitoes,

primarily depending on the level of their viremia (Perez-Ramirez et al., 2014). When clinical symptoms occur, they manifest in neurological signs, like ataxia, paralysis, torticollis, opisthotonus and incoordination, but also in nonspecific symptoms like lethargy, ruffled plumage and weight loss (Perez-Ramirez et al., 2014; van der Meulen et al., 2005). Transmission to other vertebrate species like humans and horses via bridge vectors (mosquitoes feeding on both, mammals and birds) is also possible. However, humans and horses do not develop a viremia which is sufficiently high to infect feeding mosquitoes and they therefore represent dead end hosts (Troupin and Colpitts, 2016). In humans, the majority of infections remain inapparent (80%) or manifest in mild flu-like symptoms (20%). Less than 1% develop West Nile neuroinvasive disease (WNND), associated with meningitis, encephalitis or meningoencephalitis (David and Abraham, 2016; Kramer et al., 2008; Petersen et al., 2013). In addition to infections via mosquito bites, infections via blood transfusions, organ transplantations, breastfeeding and most likely intrauterine infections are possible (Saxena et al., 2017; Sejvar, 2016). Similarly, only up to 10% of the equines develop clinical disease with neurological symptoms (Angenvoort et al., 2013). Typical symptoms are ataxia, paresis, paralysis, recumbency and fever. Besides humans and horses, many other animal species are susceptible to a WNV infection, such as for example cats, dogs, sheep, hamsters, but also alligators and some frog species (van der Meulen et al., 2005). At least seven different WNV genetic lineages can be distinguished, of which only lineage 1 and 2 cause human disease (Zannoli and Sambri, 2019). WNV was first isolated in 1937 from the blood of a woman suffering from a febrile illness in the West Nile district of Uganda and now occurs on almost every continent of the world, except Antarctica (Papa, 2017; Smithburn et al., 1940). In Europe, neutralizing antibodies against WNV were first detected in humans from Albania, however, no viral RNA was detected in these patients (Bárdoš et al., 1959). The first outbreak in Europe associated with WNND and death in humans and horses occurred in the Camargue region in Southern France (1962/1963) (Murgue et al., 2002). Since the 1990s, outbreaks have occurred in several European countries, such as Romania, Russia, France, Italy and Hungary (Bakonyi et al., 2013; Calistri et al., 2010; David and Abraham, 2016; Platonov et al., 2001; Tsai et al., 1998). Major outbreaks took place in Romania in 1996 with over 390 humans infected and in Russia with over 800 human cases

(Platonov et al., 2001; Tsai et al., 1998). In 1999, WNV was introduced into the United States of America, with the first outbreak in the city of New York and surrounding areas (Lanciotti et al., 1999). The virus resembled a WNV isolate previously detected in geese in Israel (Banet- Noach et al., 2003; Bin et al., 2001). Over the next years, WNV spread over the whole continent, causing neurological disease among humans and horses and a high mortality in birds, especially of the zoological order Passeriformes (Komar, 2003; Roehrig, 2013). During the last two decades, WNV has spread further throughout Europe with cases reported in numerous other countries (such as Algeria, Bulgaria, Croatia, Greece, Hungary, Italy, Kosovo, Montenegro, the former Yugoslav republic of Macedonia, Russia, Serbia, Spain, Portugal, and Austria) (Chancey et al., 2015; David and Abraham, 2016). Starting from the Mediterranean countries, a steady northward expansion was observed. Despite virus circulation in close neighboring countries of Germany such as France, Austria and Czech Republic for several years, WNV was first detected in Germany in 2018 (Aberle et al., 2018; Bahuon et al., 2016; Rudolf et al., 2014; Ziegler et al., 2019). Altogether 12 birds, including Eurasian Blackbirds (Turdus merula), Northern Goshawks (Accipiter gentilis) and several owl species, and furthermore two horses in Eastern and Southeastern Germany were affected (Ziegler et al., 2019). In 2019, WNV was detected in Germany (Saxony-Anhalt) again in a Snowy Owl (Bubo scandiacus) already in the beginning of July. In general, WNV cases reappeared in the same geographical regions as in the previous year, however, at much higher numbers (Friedrich-Loeffler-Institut, 2019). The high number of WNV infected birds and several horses in Eastern Germany in 2019 show that WNV was able to overwinter in mosquitoes and has established in a local mosquito/bird cycle.

USUV, a flavivirus closely related to WNV was first isolated from a Culex neavei in South Africa in 1959 (McIntosh, 1985). The transmission cycle resembles that of WNV, with birds as virus reservoir and amplifying hosts, and ornithophilic mosquitoes as vectors (Ashraf et al., 2015). Mosquitoes of the Culex genus, especially Culex pipiens, are considered to be the main vector for USUV in Europe, but the virus was also detected in mosquitoes of several other genera (Cle et al., 2019). In experimental studies Culex pipiens were at higher temperatures (28 °C) more vector competent for USUV than for WNV (Fros et al., 2015b). Infections in birds usually are inapparent, but can be fatal for highly susceptible bird species such as Eurasian Blackbirds and Great Grey Owls (Strix nebulosa). Clinical symptoms resemble those of a WNV

infection and are dominated by neurological signs apart from non-specific symptoms like apathy, ruffled feathers, weight loss and missing flight behavior. USUV was considered to have a low zoonotic potential. However, a series of human USUV infections were reported in Africa and several European countries, albeit mainly in immunocompromised persons (Grottola et al., 2017; Nikolay et al., 2011; Vilibic-Cavlek et al., 2014). Different clinical manifestations such as fever, headache, rash, but also severe disease associated with neurological symptoms have been described (Roesch et al., 2019). Just recently, a patient showing facial paralysis associated with an acute USUV infection had been reported in France, representing probably an atypical neurological presentation (Simonin et al., 2018). Furthermore, USUV specific RNA was also detected among healthy, asymptomatic blood donors posing a risk for recipients of blood donations (Allering et al., 2012; Bakonyi et al., 2017b; Cadar et al., 2017b; Carletti et al., 2019; Zaaijer et al., 2019). In other vertebrates, such as equids, dogs, deer, wild boar and tree squirrels, neutralizing antibodies against USUV have been detected, suggesting a natural USUV infection (Barbic et al., 2013; Ben Hassine et al., 2014; Durand et al., 2016; Escribano-Romero et al., 2015; Garcia-Bocanegra et al., 2016; Romeo et al., 2018). Furthermore, USUV-RNA was found in bats and rodents (Cadar et al., 2014; Diagne et al., 2019). Eight different USUV lineages can be distinguished to date: Africa 1 – 3 and Europe 1 – 5 (Gaibani and Rossini, 2017). In Germany, currently the USUV lineages Africa 2, Africa 3, Europe 2, Europe 3 and Europe 5 are circulating. Retrospectively, in Europe USUV emerged for the first time in Italy in 1996 (Weissenböck et al., 2013). In 2001, the first large outbreak, affecting predominantly Eurasian Blackbirds and Great Grey Owls, occurred in Austria (Weissenböck et al., 2003; Weissenböck et al., 2002). In the following years the virus was detected in different European countries, such as Hungary, Switzerland, Spain, Belgium, Czech Republic, France and Croatia (Gaibani and Rossini, 2017). In Germany, USUV was isolated for the first time from a pool of Culex pipiens pipiens mosquitoes in Southwestern Germany in 2010 (Jöst et al., 2011). During the next two years, USUV caused a massive die-off among Eurasian blackbirds around the Upper Rhine Valley (Becker et al., 2012). Since 2013, the number of USUV positive birds has decreased, and has remained limited to Southwestern Germany, however, with sporadic detections in Bonn (2014) and Berlin (2015) (Cadar et al., 2015; Ziegler et al., 2016; Ziegler et al., 2015). In 2016, the virus reemerged causing large outbreaks in North Rhine-Westphalia and around Leipzig and

Halle (Cadar et al., 2017a; Sieg et al., 2017). Not only in Germany, but also in the close neighboring countries such as the Netherlands and Belgium large epidemics were registered during the year 2016 (Garigliany et al., 2017; Rijks et al., 2016). In 2017, the spread towards Northern Germany continued, where USUV was detected for the first time in Hamburg, Bremen and Hannover. In the following year (2018), the largest USUV outbreak in Germany with a high mortality rate among birds associated with the death of several hundred Eurasian Blackbirds and other bird species took place. In the same year, the virus was for the first time detected in all federal states of Germany (NABU, 2019).

Another important flavivirus is the TBE virus, which in contrast to the two other viruses described above, is predominantly transmitted by ticks. TBEV is circulating in so-called “natural foci”, between ticks as vectors and small mammals as hosts and reservoirs (Dobler et al., 2011). The virus is not found evenly distributed in the tick population, unlike other tick-borne pathogens such as Borrelia burgdorferi s.l., but is distributed in a patchy pattern (Zeman, 1997). The size of the natural foci can vary from a few square meters up to several square kilometers (Lindquist and Vapalahti, 2008). Once a tick is infected, the virus can persist from one stage of development to the next stage, so that ticks can excrete the virus during their whole lifetime (trans-stadial transmission). As the life cycle of Ixodes ticks can take up to six years, TBEV can circulate in the same area for a long period (Michelitsch et al., 2019). However, the transmission rates between the different tick stages are presumably not as high as assumed (Slovák et al., 2014). Trans-ovarial transmission, from an infected tick to its eggs, most probably does not play a significant role in the transmission cycle (Danielová et al., 2002). Each tick stage targets different animal species. Whereas larvae and nymphs are primarily found on smaller animals, including birds, adults mainly target large animals (Černý, 1975). Another mechanism of virus transmission is the so- called co-feeding, where infected and non-infected ticks feed in close proximity on the same host (Labuda et al., 1993). Successful virus transmission via co-feeding is also possible if the host already has antibodies against TBEV (Randolph, 2011). Reservoir hosts for TBEV predominantly are rodents such as the yellow-necked mouse (Apodemus flavicollis) and the bank vole (Myodes glareolus), but also insectivores like hedgehogs may serve as hosts (Dobler et al., 2012; Michelitsch et al., 2019; Schönbächler et

al., 2019). Antibodies against TBEV were found in various other wild animals such as foxes, wild boars and deer, but no clinical symptoms were observed (Dobler et al., 2012). These species are susceptible but most likely do not play a role in the transmission cycle (Klaus et al., 2016b). However, wild animals are considered to play a role in the passive transport of infected ticks (Dobler et al., 2011). Clinical manifestation has been reported in several animal species such as horses, dogs, sheep, goats, mouflons and monkeys (Bagó et al., 2002; Böhm et al., 2017; Klaus et al., 2013; Süss et al., 2008; Weissenböck et al., 1998; Zindel and Wyler, 1983). TBEV-RNA and neutralizing antibodies against TBEV were occasionally detected in various bird species in the past (Ernek, 1975; Ernek et al., 1975). Furthermore, infection studies with different bird species were conducted about fifty years ago. Most of the challenged birds were not susceptible to an infection. Chickens and ducks, however, developed a viremia and/ or seroconversion (Ernek, 1962; Ernek et al., 1969a; Ernek et al., 1969b; Streissle, 1958; van Tongeren, 1983). Nevertheless, the possible role of avian species in the transmission cycle of TBEV is still unclear. Humans are accidental hosts and do not contribute to the maintenance of TBEV in nature. TBEV is primarily transmitted by tick bite. Additionally, an alimentary infection via raw, unpasteurized milk is possible. Sporadic outbreaks of foodborne TBEV have been reported from several countries, also just recently from Germany (Brockmann et al., 2018; Dorko et al., 2018; Holzmann et al., 2009; Paulsen et al., 2019). The fatality rate can be as high as 20-40% after infection with the Far Eastern subtype, but is in general lower than 2% for the Western European subtype (Beauté et al., 2018). The majority of infections (70-98%) remain inapparent (Ruzek et al., 2019). In case of a biphasic course of disease, the initial phase manifests in febrile illness, headache, body pain with non-specific symptoms (Bogovic and Strle, 2015). In some cases, after a symptomless period a second phase with neurological symptoms follows: 50% develop meningitis, 40% meningoencephalitis, and 10% meningoencephalomyelitis (Riccardi et al., 2019). Several vaccines for humans are available for a protection against TBE (Smit and Postma, 2015). TBEV is endemic in wide parts of Asia and Europe, with more than 10,000 human cases reported every year (Lindquist and Vapalahti, 2008). However, the number of cases most likely is higher, as TBEV is not a notifiable disease in all European countries. In Europe, highest incidences are reported from the Czech Republic, Lithuania, Latvia and Estonia (Beauté et al.,

2018). In Germany, most cases occur in Southern Germany in the federal states of Baden- Wurttemberg and Bavaria (Hellenbrand et al., 2019). The majority of cases happen during the peak of tick activity, from April to November (Bogovic and Strle, 2015). Originally, three different subtypes were distinguished: The Western European subtype (mainly transmitted by the tick species Ixodes ricinus) and the Siberian and Far Eastern subtypes (mostly transmitted by the tick species Ixodes persulcatus) (Valarcher et al., 2015). Two more subtypes have just recently been proposed: The Baikalian and the Himalayan subtype (Dai et al., 2018; Kovalev and Mukhacheva, 2017). Recent investigations in Finland have shown that a transmission of the Siberian subtype by Ixodes ricinus is also possible (Jääskeläinen et al., 2006; Kuivanen et al., 2018). In Germany, TBEV has also been detected just recently in Dermacentor reticulatus ticks, suggesting that this tick species most likely also contributes to the virus circulation (Chitimia-Dobler et al., 2019).

Birds, especially migratory birds, play a significant role in the dispersal of several (zoonotic) pathogens along their major flyways. On one hand, they can become infected and serve as biological carriers, on the other hand they are able to transport arthropod vectors as mechanical carriers (Hubálek, 2004; Reed et al., 2003; Sparagano et al., 2015). A memorable example is the spread of WNV throughout the United States of America within several years, with wild birds playing a substantial role. Therefore, monitoring and pathogenesis studies in wild birds are essential prerequisites for the development of early warning systems for the occurrence of and exposure to zoonotic pathogens. The aim of the presented work was to establish a nation-wide surveillance network for wild and zoo birds, in order to monitor the introduction and spread of emerging pathogens in Germany. The focus was on the molecular and serological investigation for the Flaviviruses WNV and USUV, as birds are the main virus reservoir and amplifying hosts for these viruses. The here presented data summarize the results of the wild bird monitoring from 2014 to 2018, with in total over 3,600 investigated bird samples. Translocation of Ixodes ticks is considered to be the main mechanism for the spread of TBEV to new areas. However, it is unclear whether such a translocation can occur by active transport via birds or if birds may even function as a susceptible natural virus reservoir. Therefore, chickens and ducks, as an animal model species, were infected with the TBEV strain Neudoerfl

and investigated for clinical symptoms, viremia, virus shedding, viral loads in the organs and pathology (ducks). The aim was to find out whether these birds are able to play a role in the transmission cycle of TBEV and to determine the pathogenesis of TBEV.

Chapter 2: Manuscript I

West Nile Virus and Usutu Virus Monitoring of Wild Birds in Germany

Friederike Michel1, Dominik Fischer2, Martin Eiden1, Maximilian Reuschel3, Kerstin Müller4, Monika Rinder5, Christine Fast1, Sylvia Urbaniak6, Florian Brandes7, Rebekka Schwehn8, Renke Lühken9, Martin H. Groschup1 and Ute Ziegler1,*

1 Friedrich-Loeffler Insitut (FLI), Federal Research Institute for Animal Health, Institute of Novel and Emerging Infectious Diseases, Südufer 10, D-17493 Greifswald-Insel Riems, Germany; 2 Clinic for Birds, Reptiles, Amphibians and Fish, Justus Liebig University Giessen, Frankfurter Straße 91, D-35392 Giessen, Germany; 3 Clinic for Small Mammals, Reptiles and Birds, University of Veterinary Medicine Hannover, Foundation, Bünteweg 9, D-30559 Hannover, Germany; 4 Department of Veterinary Medicine, Small Animal Clinic, Freie Universität Berlin, Oertzenweg 19 b, D-14163 Berlin, Germany; 5 Clinic for Birds, Small Mammals, Reptiles and Ornamental Fish, Centre for Clinical Veterinary Medicine, Ludwig Maximilians University Munich, Sonnenstraße 18, D-85764 Oberschleißheim, Germany; 6 Birds of Prey Rehab Center Rhineland (Greifvogelhilfe Rheinland), Roermonder Straße 34, D-41379 Brüggen, Germany; 7 Wildtier-und Artenschutzstation, Hohe Warte 1, D-31553 Sachsenhagen, Germany; 8 Seehundstation Nationalpark-Haus Norden-Norddeich, Dörper Weg 24, D-26506 Norden, Germany; 9 Bernhard-Nocht-Institute for Tropical Medicine, WHO Collaborating Centre for Arbovirus and Hemorrhagic Fever Reference and Research, Bernhardt-Nocht Straße 74, D- 20359 Hamburg, Germany; * Corresponding author

By systematically setting up a unique nation-wide wild bird surveillance network, we monitored migratory and resident birds for zoonotic arthropod-borne virus infections, such as the flaviviruses West Nile virus (WNV) and Usutu virus (USUV). More than 1900 wild bird blood samples, from 20 orders and 136 different bird species, were collected between 2014 and 2016. Samples were investigated by WNV and USUV-specific real-time polymerase chain reactions as well as by differentiating virus neutralization tests. Dead bird surveillance data, obtained from organ investigations in 2016, were also included. WNV-specific RNA was not detected, whereas four wild bird blood samples tested positive for USUV-specific RNA. Additionally, 73 USUV-positive birds were detected in the 2016 dead bird surveillance. WNV neutralizing antibodies were predominantly found in long-distance, partial and short-distance migrants, while USUV neutralizing antibodies were mainly detected in resident wild bird species, preferentially with low seroprevalences. To date, WNV-specific RNA has neither been detected in wild birds, nor in mosquitoes, thus, we conclude that WNV is not yet present in Germany. Continued wild bird and mosquito monitoring studies are essential to detect the incursion of zoonotic viruses and to allow risk assessments for zoonotic pathogens

Published 2018 in

International Journal of Environmental Research and Public Health

Volume 15, Issue 1

DOI: 10.3390/ijerph15010171

https://www.mdpi.com/1660-4601/15/1/171

Chapter 3: Manuscript II

Evidence for West Nile Virus and Usutu Virus Infections in Wild and Resident Birds in Germany, 2017 and 2018

Friederike Michel 1,2, Michael Sieg 3, Dominik Fischer 4, Markus Keller 1, Martin Eiden 1, Maximilian Reuschel 5, Volker Schmidt 6, Rebekka Schwehn 5,7, Monika Rinder 8, Sylvia Urbaniak 9, Kerstin Müller 10, Martina Schmoock 11,12, Renke Lühken 13, Patrick Wysocki 14, Christine Fast 1, Michael Lierz 4, Rüdiger Korbel 8, Thomas W.

Vahlenkamp 3, Martin H. Groschup 1,2, and Ute Ziegler 1,2,*

1 Friedrich-Loeffler Insitut (FLI), Federal Research Institute for Animal Health, Institute of Novel and Emerging Infectious Diseases, Südufer 10, D-17493 Greifswald-Insel Riems, Germany; 2 German Centre for Infection Research (DZIF); 3 Institute of Virology (Faculty of veterinary medicine), Leipzig University, An den Tierkliniken 29, D-04103 Leipzig, Germany; 4 Clinic for Birds, Reptiles, Amphibians and Fish, Justus Liebig University Giessen, Frankfurter Straße 91, D-35392 Giessen, Germany; 5 Clinic for Small Mammals, Reptiles and Birds, University of Veterinary Medicine Hannover, Foundation, Bünteweg 9, D-30559 Hannover, Germany; 6 Clinic for Birds and Reptiles (Faculty of veterinary medicine), Leipzig University, An den Tierkliniken 17, D-04103 Leipzig, Germany; 7 Seehundstation Nationalpark-Haus Norden-Norddeich, Dörper Weg 24, D-26506 Norden, Germany; 8 Clinic for Birds, Small Mammals, Reptiles and Ornamental Fish, Centre for Clinical Veterinary Medicine,Ludwig Maximilians University Munich, Sonnenstrase 18, D-85764 Oberschleißheim, Germany; 9 Birds of Prey Rehab Center Rhineland (Greifvogelhilfe Rheinland)/ Tierarztpraxis Sudhoff, Hehnerholt 105, D-41069 Mönchengladbach, Germany; 10 Department of Veterinary Medicine, Small Animal Clinic, Freie Universitat Berlin, Oertzenweg 19 b, D-14163 Berlin, Germany; 11 Wildpark Schwarze Berge, Am Wildpark 1, D-21224 Rosengarten, Germany; 12 Tiermedizin am Rothenbaum, Rothenbaumchaussee 195, D-20149 Hamburg, Germany; 13 Bernhard-Nocht-Institute for Tropical Medicine, WHO Collaborating Centre for Arbovirus and Hemorrhagic Fever Reference and Research, Bernhardt-Nocht Strase 74, D- 20359 Hamburg, Germany; 14 Friedrich-Loeffler-Institut (FLI), Federal Research Institute for Animal Health, Institute of Epidemiology, Südufer 10, D-17493 Greifswald-Insel Riems, Germany; * Corresponding author

Wild birds play an important role as reservoir hosts and vectors for zoonotic arboviruses and foster their spread. Usutu virus (USUV) has been circulating endemically in Germany since 2011, while West Nile virus (WNV) was first diagnosed in several bird species and horses in 2018. In 2017 and 2018, we screened 1709 live wild and zoo birds with real-time polymerase chain reaction and serological assays. Moreover, organ samples from bird carcasses submitted in 2017 were investigated. Overall, 57 blood samples of the live birds (2017 and 2018), and 100 organ samples of dead birds (2017) were positive for USUV-RNA, while no WNV-RNA- positive sample was found. Phylogenetic analysis revealed the first detection of USUV lineage Europe 2 in Germany and the spread of USUV lineages Europe 3 and Africa 3 towards Northern Germany. USUV antibody prevalence rates were high in Eastern Germany in both years. On the contrary, in Northern Germany, high seroprevalence rates were first detected in 2018, with the first emergence of USUV in this region. Interestingly, high WNV-specific neutralizing antibody titers were observed in resident and short-distance migratory birds in Eastern Germany in 2018, indicating the first signs of a local WNV circulation.

Published 2019 in

Viruses Volume 11, Issue 7 DOI: 10.3390/v11070674

https://www.mdpi.com/1999-4915/11/7/674

Chapter 4: Manuscript III

Role of ducks in the transmission cycle of tick-borne encephalitis virus?

Friederike Michel 1, Ute Ziegler 1, Christine Fast 1, Martin Eiden 1, Christine Klaus 2, Gerhard Dobler 3, Katrin Stiasny 4 and Martin H. Groschup 1,*

1 Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Institute of Novel and Emerging Infectious Diseases, Greifswald-Insel Riems, Südufer 10, 17493 Greifswald-Insel Riems, Germany 2 Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Institute of Bacterial Infections and Zoonoses, Naumburger Str. 96 a, 07743 Jena, Germany 3 Department of Virology and Rickettsiology, Bundeswehr Institute of Microbiology, Neuherbergstr. 11, 80937 Munich, Germany 4 Department of Virology, Medical University of Vienna, Kinderspitalgasse 15, 1090 Vienna, Austria * Corresponding author

4.1. Abstract

Tick-borne encephalitis virus (TBEV), a member of the family Flaviviridae, is the most important tick-transmitted arbovirus in Europe. It can cause severe illnesses in humans and in various animal species. The main mechanism for the spread of TBEV into new areas is considered to be the translocation of infected ticks. To find out whether ducks can function as a natural virus reservoir in addition to serving as passive transport vectors, we carried out an experimental TBEV challenge study to reveal their susceptibility and resulting pathogenesis. Nineteen ducks were inoculated subcutaneously with TBEV strain “Neudoerfl” and monitored for 21 days. Blood, oropharyngeal and cloacal swabs were collected throughout the experiment and organ samples upon necropsy at the end of the study. All samples were tested for TBEV- RNA by real-time polymerase chain reaction. TBEV specific antibodies were determined by virus neutralization test and ELISA. Organ samples were examined histopathologically and by immunohistochemistry. The inoculated ducks did not show any clinical symptoms. TBEV-specific RNA was detected in all brain samples as well as in a few blood and swab samples. Moreover, all challenged birds produced TBEV antibodies and showed a mild to severe acute to subacute necrotizing encephalitis. TBEV specific antigen was detected in the brain of 14 ducks by immunohistochemistry. The short and low viremic phases, as well as the low virus load in tissues suggest that ducks should not be considered as reservoir hosts. However, due to the high antibody levels, ducks can serve as sentinel species for the detection of natural TBEV foci.

4.2. Introduction

Tick borne encephalitis virus (TBEV) is a tick-borne Flavivirus, which is endemic in many European countries and throughout most of Asia (Mansfield et al., 2009). Originally, three different TBEV subtypes were distinguished: The Western European, the Siberian and the Far Eastern subtype (Valarcher et al., 2015). Just recently two additional subtypes have been proposed (Dai et al., 2018; Kovalev and Mukhacheva, 2017). TBEV is circulating in small, geographically defined natural foci between the tick, as vector and small mammals such as

rodents, as hosts and reservoirs (Dobler et al., 2012; Dobler et al., 2011). In Europe, TBEV is in most cases transmitted by Ixodes ricinus, whereas in Russia and Asia the most important tick vector is Ixodes persulcatus (Ruzek et al., 2019). Non-viremic transmission by co-feeding of infected and non-infected ticks in close proximity is also considered to play a substantial role in maintaining TBEV circulation (Labuda and Randolph, 1999). Moreover suitable environmental, socio-economic and climatic conditions are essential for the establishment of natural foci (Estrada-Peña and de la Fuente, 2014; Korenberg, 2009). More than 10,000 human cases are reported in Europe and Asia every year (Bogovic and Strle, 2015). Even more, the true number of cases is most probably much higher, as mild cases often remain undiagnosed and since TBEV is not in all European countries a notifiable disease (Donoso Mantke et al., 2011). For Germany the number of clinical infections fluctuates around a median of 283 human cases reported every year (RKI, 2019). High prevalence areas are Baden-Württemberg and Bavaria with most of the cases (89% in 2001 till 2018) (Hellenbrand et al., 2019). Moreover, several regions in Hesse and Thuringia and isolated districts in Saarland, Rhineland Palatine and Saxony are also TBEV risk areas. In addition, single autochthonous human TBE cases are reported from other federal states caused by the patchy pattern of TBEV natural foci that can be fairly small (Kupča et al., 2010; RKI, 2019). In most cases TBEV is transmitted by tick bites. However, alimentary infections by the consumption of unpasteurized dairy food products are also possible (Brockmann et al., 2018; Offerdahl et al., 2016). Approximately one third of the infected humans suffer from neurological symptoms, while the majority of infections manifests only in mild non-specific symptoms or is inapparent (Kaiser, 2012). Humans as well as various animal species like dogs, horses, monkeys and ruminants are accidental hosts (Klaus et al., 2016b). Infections are possible in these species, however, they probably play only a minor role in the virus transmission cycle. During the last decades a geographic expansion, characterized by an increase in the number of high risk areas and the emergence of new natural TBEV foci, was observed in Germany and in various other European countries (Riccardi et al., 2019; RKI, 2019). The way of dispersal of TBEV to previously unaffected regions and the establishment of new natural foci is not quite clear. The distribution of infected ticks by wild animals, such as rodents or deer, or even by

humans transporting infested animals is a possible option (Boelke et al., 2019). Another possibility is the transport of TBEV infected ticks by birds, which can easily cross rough terrain (Hasle, 2013). Birds are playing an important role in the spread of various arboviruses or their respective vectors, along their major flyways (Hubálek, 2004; Jourdain et al., 2007). In several European countries migratory and resident wild birds were screened for TBEV infected ticks, however the virus prevalence of infested ticks attached on birds seems to vary substantially. For instance, in Latvia 14% (Kazarina et al., 2015) of the ticks were TBEV positive, whereas the prevalence in Sweden (0.53%) (Waldenström et al., 2007) and Germany (0%) (Klaus et al., 2016a) was quite low or non-detectable. Nonetheless, considering the annual migration of billions of birds, infected ticks may still be carried by birds, even if the TBEV prevalence in them is rather low. Mainly nymphs and larvae of Ixodid ticks are feeding on birds and need only a short period (2 to 7 days) for their blood meal (Balashov, 1972). During this short time birds are flying only short distances, which speaks against the long distance transport of infected ticks (Klaus et al., 2016a). Nevertheless a spread over shorter distances, for instance from one stopover site to another is already a progression. Whether birds are also playing an active role in the transmission cycle of TBEV, apart from their function as mechanical vector, is not fully understood. The available literature regarding animal experiments dates back to the 1960s. A variety of wild and domestic bird species were tested, but most of them did not exhibit a viremia or showed a seroconversion (Ernek, 1962, 1964; Ernek et al., 1969a; Ernek et al., 1969b; Grešíková et al., 1962; Nosek et al., 1962; Řeháček et al., 1963; Streissle, 1958; van Tongeren, 1983). Ducks, however, developed a viremia and seemed to be susceptible for the investigated TBEV strains. Furthermore, the virus was isolated several times from wild ducks and neutralizing antibodies were detected, indicating the possibility of a natural infection (Ernek, 1967, 1975; Ernek et al., 1975). Mallards (Anas platyrhynchos) are partial migratory birds, depending on their geographic origin. Ducks from Northern Europe are usually migratory birds, whereas ducks from Central Europe travel over shorter distances or do not migrate (van Toor et al., 2013). A spread of infectious diseases by ducks therefore seems possible. In this study we challenged domestic ducks with the TBE strain Neudoerfl. The aim was to reveal whether ducks are susceptible for TBEV Neudoerfl and may even function as silent virus carrier.

4.3. Materials and methods

4.3.1. Ethics statement and animals

The duck infection experiments described in this publication were approved by the State Office of Agriculture, Food safety, and Fishery in Mecklenburg-Western Pomerania, Germany on the basis of national and European legislation in particular directive 2010/63/EU (Reference number 7221.3-1-075/16). Twenty Peking Ducks were purchased at the age of four weeks and monitored daily for their physical health. Pre-challenge samples were taken and examined by quantitative real-time RT-PCR (qRT-PCR) and virus neutralization test (VNT) to exclude previous infections with TBEV. To clear a Salmonella spp. infection, the ducks were treated with Enrofloxacin. The animals were challenged eventually at the age of six weeks.

4.3.2. Virus challenge

The TBEV animal experiment was carried out under biosafety level 3 conditions. Nineteen 5 ducks were infected subcutaneously (s.c.) with 10 TCID50/ml virus dilution of TBEV strain Neudoerfl (GenBank accession no. U27495) and one duck was kept as a negative control. Virus diluted in minimal essential medium (MEM) was injected subcutaneously in the knee folds of each animal (0.5 ml per side). For control purpose residual virus solution was back titrated to verify that there was no virus infectivity loss during the challenge procedure. During the infection experiments, the ducks were examined daily for clinical symptoms following a defined score sheet (scores 0 (no clinical changes) up to 3 (severe disease)). Changes in behavior, body posture, respiratory symptoms, plumage, feed intake, defecation and the nutritional condition in general were documented daily. Blood samples, oropharyngeal- and cloacal swabs were taken on days 0, 2, 4, 6, (8 and 12 just swabs), 10, 14 and 21 post infection (dpi) and the body weight was measured (additional time points 17 and 19 dpi). Blood samples were centrifuged and the blood cruor and serum aliquoted and stored at -70°C. Swab samples were placed in 2 ml MEM containing antimicrobials (Gentamicin, Amphotericin, Lincomycin, Enrofloxacin) and shaked for 30 min. The supernatant was decanted and stored at -70°C for further examination. After 21 days the ducks were euthanized and tissue samples (e.g. brain,

liver, spleen, heart, bursa cloacalis) were taken for virological investigations and for histopathology.

4.3.3. Virological analysis

Tissue samples were homogenized in 500µl MEM with antibiotics (Penicillin/ Streptomycin). Supernatants were collected after centrifugation and tested by qRT-PCR. Samples with Ct values below 30 were titrated on PK 15 cells. After seven days cell monolayers were formalin-fixed and stained with 1% crystal violet solution to reveal cythopathic effects.

4.3.4. Quantitative real-time RT-PCR (qRT-PCR)

Viral RNA of the avian blood samples was isolated from the blood cruor using the RNeasy Mini Kit (Qiagen, Hilden, Germany), according to the manufacturer’s instructions. The viral RNA of the swab and tissue sample supernatants was extracted using a Biosprint 96 (Qiagen, Hilden, Germany) and the NucleoMag VET Kit® (Macherey-Nagel, Düren, Germany). An internal control RNA (IC2 RNA) was extracted together with all samples (Hoffmann et al., 2006). Extracted RNA was eventually analzyed by a TBEV specific qRT-PCR using the protocol described by Schwaiger and Cassinotti (2003). A synthetic RNA was used for the quantification of the exact number of genome copies in the swab, blood and organ samples. Copy numbers above three copies/µl RNA were regarded as positive, one to three copies/µl were inconclusive, and less than one copy/µl RNA were negative.

4.3.5. Serology

Neutralizing antibody titers against TBEV strain “Neudoerfl” were determined by a virus neutralization test (VNT) as described before (Ziegler et al., 2013). All samples were run in duplicate and cytopathic effects were read after seven days. The neutralizing antibody titer

(neutralization dose 50% (ND50)) of a serum sample was defined as the maximum dilution which inhibited cytopathic effects in fifty percent of the wells and was calculated according to the Behrens-Kaerber method.

Serum antibody levels from 0, 6, 14 and 21 dpi were additionally investigated using a commercially available competition ELISA (Immunozym FSME IgM Kit, Progen GmbH, Germany). The samples were analyzed by the modified version published by Klaus et al. (2011) to determine total immunoglobulin. The VNT and ELISA results were compared to estimate cut-off values to distinguish positive, inconclusive, and negative ELISA results. The upper cut- off value was calculated using a receiver operating characteristic analysis (ROC analysis) with regard to the criterion “minimum ROC distance” using the software R version 3.6.0 – “Planting of a Tree” with the package OptimalCutpoints (López-Ratón et al., 2014; Metz, 1978; R, 2019). Additionally a lower cut off was determined by the mean value plus three standard deviations of the negative controls (Lardeux et al., 2016).

4.3.6. Histopathology and immunohistochemistry

Tissue samples were fixed in neutral buffered formalin (4%), embedded in paraffin and stained with hematoxylin/eosin (H&E). Brain and spleen samples as well as tissues showing histopathological alterations which might have been associated to a TBEV infection were examined by IHC. For this purpose 3µm sections were cut, deparaffinised and rehydrated. Endogenous peroxidase was blocked using 3% hydrogen peroxide/methanol followed by a proteinase K digestion step (10 mg/ml) for 15 min at 37°C to retrieve the virus antigen. The primary antibody, a polyclonal rabbit antibody against TBEV, (kindly provided by Dr. Karin Stiasny Medical University of Vienna, Austria) was used at a dilution of 1:2,000. Negative control sections were incubated only with goat serum. The slides were developed by using Rabbit Envision HRP and Diaminobenzidine (DAB) as substrate. Tissue samples were rated depending on the percentage of positive cells with scores from 0 to 3 (0 = no positive cells/negative; 1 = ˂ 1% positive cells = mildly affected; 2 = ≥ 1% and ˂ 5% positive cells = moderately affected; 3 = ≥ 5% positive cells = severely affected).

4.4. Results

None of the infected ducks showed TBEV related clinical symptoms and the animals steadily gained weight (data not shown). The clinical score was “0” in all categories during the whole

experiment. However, one duck (Duck 16) had to be euthanized on 8 dpi due to a technopathy, which was not associated with the TBEV infection.

4.4.1. TBEV detection by qRT-PCR

TBEV specific RNA was detected in few blood samples, oropharyngeal and cloacal swab samples with high Ct values, whereas the samples of the remaining infected ducks were negative (Figure 1). TBEV-RNA was detected in the swab samples on day 2 (5/19), 6 (1/19) and 8 (1/19) post infection (pi). In few blood samples TBEV-RNA was found on day 4 (2/19) and 6 (3/19) pi. Duck 10 and Duck 19 were virus positive in the blood samples on two following sampling days. The brain samples of all ducks (19/19) were tested positive for TBEV-RNA, including the brain sample of Duck 16, which was already euthanized after 8 dpi. Additionally, TBEV-RNA was detected in the spleens of few birds (4/19) (Table 1). The other organ samples tested were negative in all ducks. Brain samples with Ct values below 30 were titrated on cells, but a virus cultivation from the tissue samples on cell culture was not successful.

4.4.2. Serological results

All infected ducks seroconverted with high virus neutralizing antibody titers. Neutralizing antibodies were first detected on 6 dpi (17/19). The titers did reach their maximum on day 10 and 14 post infection and ranged then from 1:960 to 1:20,480 ND50 (Figure 2 A). The ROC analysis for the criterion “minimum ROC distance” estimated a cut-off value of 15.2079 and defined the upper cut-off to 16. All values above this cut-off were regarded TBEV positive. Additionally, a lower cut-off was calculated to define a range of inconclusive test results between the lower and the upper cut-offs. Based on the median and three deviations of the ELISA results of the negative ducks, investigated before the infection, the lower cut-off was estimated to 0.72262. Thus, ELISA results ranging between 0.72262 and 16 were regarded as inconclusive. On day 6 post infection two VNT negative ducks were inconclusive in the ELISA, while one VNT positive was inconclusive in ELISA. On day 14 and 21 post infection the ELISA results are in accordance with the VNT results: All ducks were clearly positive on these days (Figure 2 B).

4.4.3. Gross lesions, histopathology and immunohistochemistry

The gross examination of the animals revealed no specific lesions indicating a viral disease. In histopathology all ducks showed an acute lymphohistiocytic (6/19) or subacute lymphoplasmacellular (13/19) non-suppurative necrotizing encephalitis, and one animal also a meningitis. The alterations were mild (5/19) up to moderate (13/19) or severe (1/19). The cerebrum was involved in all cases, and the mesencephalon, which was not available in all ducks, was involved to a lesser degree (12/15), so was brain stem (12/19) and cerebellum (11/19). Alterations were seen in white and grey matter and consisted of perivascular lymphohistiocytic or lymphoplasmacellular cuffing (Figure 3 A, B), as well as multifocal neuronal and glial single cell necrosis, not in all cases associated with a mild glial and/or inflammatory cell reaction (Figure 3 C). These necrotic foci were mainly seen in cerebrum (18/19), less frequent in mesencephalon (3/15), brain stem (3/19) and cerebellum (2/19). Clear signs of degeneration (karyorrhexis) were also seen in perivascular and migrated inflammatory cells. Only few glia nodules were seen, mainly in cerebrum (17/19), but also in mesencephalon (7/17), cerebellum (6/17) and brain stem (4/17). Additionally, some animals revealed a mild acute non-suppurative vasculitis (7/19) and nine ducks showed a reactive astrogliosis of varying degree (Figure 3 D). By immunohistochemistry TBEV viral antigen was detected in the brain of 14 out of 19 infected ducks. All animals showed only a mild accumulation, which was confined to neurons and neuronal processes of the cerebrum (Figure 3 E, F). Only one animal revealed a staining reaction in the mesencephalon, too. The infected cells were in parts associated to histopathological alterations of the brain (i.e. perivascular cuffing, foci of necrosis), but randomly distributed positive cells were also seen. Further alterations of unknown origin were also seen in some ducks. This includes a focal acute non-suppurative vasculitis in the gut wall of one animal as well as a follicular hyperplasia of the spleen in six ducks. Viral antigen was not detectable. Additionally, in several animals lesions were seen, which were most probably due to an unrelated bacterial or parasitic etiology. Supplemental Table 1 summarizes these additional diagnosis.

4.5. Discussion

Tick-borne encephalitis has become a growing health problem in endemic European and Asian countries with a global increase in human cases (Beauté et al., 2018; Lundkvist et al., 2011; Velay et al., 2018). Multiple different factors like the weather, environmental changes and host abundance, but also the growing awareness of the health authorities and improved diagnostics are playing a role in the increased incidence of TBEV (and its tick vector) during the last decades (Petri et al., 2010; Randolph, 2010). Furthermore an expansion of the risk areas has been observed and new natural foci/ endemic areas have emerged (Beauté et al., 2018; de Graaf et al., 2016). The background for the new appearance of natural foci is currently under extensive discussion. The possible role of birds in the spread of ticks and tick-borne pathogens, like TBEV, is not yet fully elucidated. Apart from being a possible mechanical vector for infected ticks, birds may represent a potential virus reservoir for TBEV. Infection experiments with various bird species, different TBEV strains and inoculation methods were already conducted about sixty years ago – with all limitations of then available diagnostic technologies: Infected Great tits (Parus major), House sparrows (Passer domesticus), Pheasants (Phasanius colchicus), Common buzzards (Buteo buteo) and European kestrels (Falco tinnunculus) did not develop clinical symptoms or a viremia and only occasionally neutralizing antibodies were detected (Ernek and Lichard, 1964; Grešíková et al., 1962; Nosek et al., 1962; Řeháček et al., 1963). Common coots (Fulica atra) and chickens (Gallus gallus domesticus), however, showed a viremia but no clinical symptoms (Streissle, 1958; van Tongeren and Timmers, 1961). Animal experiments with different TBEV strains were conducted on wild and domestic ducks (Anas platyrhynchos/ Anas platyrhynchos domesticus) between the 1960s and 1980s (Ernek, 1962; Ernek et al., 1969a; Ernek et al., 1969b; van Tongeren, 1983). In these experiments, ducks were infected with TBEV positive homogenized mouse brain tissue of strain Hypr or strain Graz I. In the experiments a viremia lasting over several days and seroconversion was seen. As experimentally infected ducks seemed to be susceptible to the TBEV strains Hypr and Graz I in principle, we decided to investigate their susceptibility to the TBEV strain Neudoerfl, representing the prototype of the European subtype, which was not tested before. Another reason is that mallards (Anas platyrhynchos) are partial migratory birds, enabling the transport

of infected ticks. The aim of this study was to find out whether ducks can be productively infected with TBEV strain Neudoerfl. In the here presented animal trial no clinical symptoms were observed among the infected ducks, which is in accordance with the duck experiments in the past. In comparison to the challenge study with TBEV strain Hypr and Graz I conducted by Ernek et al. and van Tongeren, where the majority of the ducks developed a viremia which was lasting over several days, TBEV-RNA was detected only sporadically in the blood of a few ducks in the present study. Differences in the neuropathogenicity of different strains within the European subtype are known: TBEV Neudoerfl has a low neuropathogenicity, whereas the neuropathogenicity after an infection with TBEV Hypr, is higher (Dobler et al., 2016). Although TBEV-RNA was detected only in a few ducks in the blood, all infected ducks of the here described animal experiment developed neutralizing antibodies. A seroconversion was detected early (on 6 dpi) in some ducks, thus it may was not possible to form a prolonged viremia, as the virus was removed too rapidly from the bloodstream. Remarkably, neutralizing antibody levels reached very high titers with up to 20,480 ND50, indicating a strong stimulation of the immune system. It is not possible to compare the antibody titers with these of the animal experiments conducted on ducks in the past, as methodological details used were different or unknown. Interestingly TBEV specific RNA was detected in the brain samples of all infected ducks, albeit it was impossible at any time point to re-isolate virus. It is possible, that viral loads in the organ samples were too low or the detected virus was not viable. The histopathological observation of a nonsuppurative encephalitis with distinct neuronal necrosis, foci of neuronophagia, gliosis and perivascular lymphohistiocytic or lymphoplasmacellular cuffing is largely in accordance to neuropathology described for mammals (Bagó et al., 2002; Böhm et al., 2017; Süss et al., 2007; Völker et al., 2017; Weissenböck et al., 1998). However, in birds the involvement of the meninges was rare. Additionally, the main target region in birds seemed to be the cerebrum, followed by mesencephalon, cerebellum and brain stem. This distribution of lesions showed some resemblance to dogs (Völker et al., 2017), with a decreasing intensity from cranial to caudal. Interestingly, several birds displayed signs of a mild vasculitis, which is a frequent observation in West Nile virus infected birds. Furthermore, a reactive astrogliosis seen in several animals

indicated the beginning glial scar formation due to a previous severe tissue damage. Even more interestingly were the distinct signs of the degeneration in glial and mononuclear inflammatory cells within the perivascular cuffs and throughout the neuropil, which in that extent cannot be seen in West Nile virus and Usutu virus infected birds. These lesions were also described by others (Böhm et al., 2017; Weissenböck et al., 1998). Additionally a distinct number of granzyme B releasing cells were detectable in TBEV infections in monkeys (Süss et al., 2008) and humans (Gelpi, 2005; Gelpi et al., 2006), indicating an, at least partly, involvement of immunopathological processes for some of the tissue damage. Therefore future studies are needed to further investigate whether these signs of karyorrhexis are due to viral or cytokine induced apoptosis or necrosis. However, viral antigen was not detected in glial cells, glial nodules or in foci of acute neuronophagia. A distinct staining reaction was only seen in neurons and neuronal processes, which were often closely associated to inflammatory processes, but never in the center of it. As described before, only a small amount of viral antigen was found, if at all (Böhm et al., 2017; Völker et al., 2017; Weissenböck et al., 1998). This is not a surprise as all birds were killed at 20 or 21 dpi (except one on 8 dpi). Flavivirus infections such as TBEV in humans and dogs and WNV in birds are rapidly cleared, hence there is only a small window for an antigen detection (Angenvoort et al., 2014; Weissenböck et al., 1998). There is a divergence between the widespread histopathological lesions throughout all regions of the brain and the locally restricted viral antigen detection only in the cerebrum (except for one detection in the mesencephalon). Such a pattern was also described in a monkey before (Süss et al., 2008). Results regarding histopathology and seroconversion were similar to historical reports (Ernek, 1962; Ernek et al., 1969a; Ernek et al., 1969b; van Tongeren, 1983). However, a high and prolonged viremia like in the previous infection studies with ducks has not been observed, which may be due to the lower virulence of the TBEV strain Neudoerfl strain. Therefore ducks do not play a role as an undetected virus reservoir in the ongoing TBEV endemic. Natural foci for TBEV are often found by tick flagging followed by molecular testing. Serosurveillance of sentinel animals (e.g. sheep and goats) is an alternative approach to identify TBEV foci (Klaus et al., 2012). According to Komar (2001) the perfect sentinel species is susceptible to the infection, with rapid seroconversion, yet not developing a clinical disease. Furthermore the sentinel should not contribute to the local pathogen transmission. Our animal

experiments show that ducks fulfill these criteria. Ducks are often kept in free-range husbandry thereby coming in contact with ticks. Monitoring the presence of neutralizing antibodies at the time of slaughter is feasible. The investigation of these ducks in addition to the monitoring of ticks could help to define the distribution/occurrence of TBEV in affected areas or (even help) to detect new natural foci.

4.6. Conclusion

The duck challenge experiments show their susceptibility to TBEV strain Neudoerfl. However, as ducks did not develop an extended viremia, they are neither a reservoir nor amplification host, hence do not play a role in the transmission cycle of this virus. However, ducks developed high antibody levels after an infection with TBEV and may therefore be used as sentinels to detect new natural foci.

Acknowledgement

We would like to thank Gesine Kreplin and Cornelia Steffen for the excellent technical assistance and the animal caretakers. This study was funded by the German Center for Infection Research (DZIF) Project Number TTU 01.801.

4.7. Figures

Figure 1. Quantitative real-time RT-PCR (qRT-PCR) results of the blood and swab samples of the infected ducks (D 01 - D 19) in copies/µl. B = Blood samples, PS = Pharyngeal Swab, CS = Cloacal Swab

B Figure 2. Antibody response of the infected ducks against TBEV, by virus neutralization test and ELISA. (A) Antibody response against TBEV, by virus neutralization test (depicted in log titers). Data of the neutralizing antibody response for all ducks are presented in a box-plot. Minimum and maximum values are represented by the respective end of the whiskers and outliers as

dots. The box includes 50% of the values of all investigated animals per day and the median is depicted as a line. (B) Total immunoglobulin detected against TBEV by ELISA in units per liter (U/L) on day 6, 14 and 21 post infection. The Cut off values are depicted as red lines: Samples with < 0.72262 U/L were regarded as negative, ≥ 0.72262 U/L and ≤ 16 U/L as inconclusive, and > 16 U/L as positive. Data of the antibody response for all ducks are presented in a box-plot. Minimum and maximum values are represented by the respective end of the whiskers and outliers as dots. The box includes 50% of the values of all investigated animals per day and the median is depicted as a line.

Figure 3. Histopathology and immunohistochemistry of TBEV infected ducks. (A) H&E, Duck E04, cerebrum, severe lymphohistiocytic perivascular cuffing, gliosis and glial/neuronal single cell necrosis in adjacent neuropil; (B) H&E, Duck E06, cerebellum, mild perivascular cuffing and glia nodule; (C) H&E, Duck E04, cerebrum, mild lymphohistiocytic perivascular cuffing with signs of degeneration as well as glial/neuronal necrosis in adjacent neuropil; (D) H&E, Duck E13, cerebrum, focal reactive astrogliosis; (E+F). Immunohistochemistry (polyclonal antibody anti TBEV), Duck E06 and E14, cerebrum, lesion associated neuronal detection of TBEV antigen; A-C: bar 50 µm, D-F: bar 20 µm

4.8. Tables

Table 1. Results of the tissue samples by quantitative real-time RT-PCR (qRT-PCR), titration on PK15 cells and immunohistochemistry (IHC).

Duck Tissue sample Ct cop/µl RNA log TCID50/ml IHC D 01 Brain 27.83 551.03 n.v.d. + D 01 Spleen N/A / n.d. - D 02 Brain 28.75 287.90 n.v.d. + D 02 Spleen N/ A / n.d. - D 03 Brain 29.16 215.91 n.v.d. + D 03 Spleen 35.66 2.27 n.d. - D 04 Brain 29.46 174.92 n.v.d. - D 04 Spleen N/A / n.d. - D 05 Brain 31.30 48.08 n.d. - D 05 Spleen N/A / n.d. - D 06 Brain 29.46 175.22 n.v.d. + D 06 Spleen 35.38 2.75 n.d. - D 07 Brain 31.63 38.34 n.d. + D 07 Spleen N/A / n.d. - D 08 Brain 30.60 79.00 n.d. + D 08 Spleen N/A / n.d. - D 09 Brain 27.65 626.33 n.v.d. + D 09 Spleen N/A / n.d. - D 10 Brain 31.16 53.33 n.d. + D 10 Spleen N/A / n.d. - D 11 Brain 33.49 10.36 n.d. - D 11 Spleen N/A / n.d. - D 12 Brain 30.97 60.72 n.d. - D 12 Spleen N/A / n.d. - D 13 Brain 32.90 15.67 n.d. - D 13 Spleen N/A / n.d. - D 14 Brain 31.23 50.59 n.d. + D 14 Spleen N/A / n.d. - D 15 Brain 32.17 26.20 n.d. + D 15 Spleen N/A / n.d. - D 16 Brain 29.13 221.28 n.v.d. + D 16 Spleen 31.11 55.04 n.d. - D 17 Brain 31.78 34.51 n.d. + D 17 Spleen N/A / n.d. -

D 18 Brain 30.70 73.52 n.v.d. + D 18 Spleen 32.99 14.71 n.d. - D 19 Brain 30.87 65.37 n.v.d. + D 19 Spleen N/A / n.d. - N/A = no Ct, n.d. = not done, n.v.d. = no virus detected; IHC (Immunohistochemistry): + = positive cells, - = negative

31 32

Supplemental Table 1: Overview of the histopathological results obtained in TBEV infected ducks. Immunohistochemical results are shown for brain samples only.

Brain Brain stem Cerebellum Mesencephalon Cerebrum Necrotizing Encephalitis Additional Findings Duck HE* IHC HE* IHC HE* IHC HE* IHC with … D01 0 0 1.5 0 na 0 1.5 1 Mild acute nonsupp. Moderate chronic active fibrinous Vasculitis, reactive suppurative peritonitis with Astrogliosis mineralization D02 1 0 0 0 na 0 1 1 -- -- D03 2 0 2 0 2 0 2 1 Reactive Astrogliosis -- D04 2 0 2 0 2 0 2 0 Mild acute nonsupp. -- Vasculitis, reactive Astrogliosis D05 1.5 0 0 0 1.5 0 2 0 -- Focal (gut) acute lymphohistiocytic Vasculitis, mild follicular hyperplasia of spleen D06 0 0 2 0 2 0 2.5 1 Reactive Astrogliosis -- D07 1 0 1 0 na 0 1 1 -- Mild follicular hyperplasia of spleen D08 1.5 0 1.5 0 na 0 1.5 1 -- Mild follicular hyperplasia of spleen, mild acute suppurative myocarditis D09 1.5 0 0 0 1.5 0.5 1.5 0 -- -- D10 0 0 0 0 0 0 1 1 -- Moderate subacute necrotizing Hepatitis and Splenitis D11 1.5 0 0 0 1.5 0 1.5 0 Mild acute nonsupp. Moderate chronic active Vasculitis suppurative Peritonitis and necrotizing Hepatitis and Cholangitis D12 0 0 0 0 1 0 1 0 Mild acute nonsupp. -- Vacultis

D13 0 0 0 0 0 0 2 0 Mild lymphohistiocytic Mild follicular hyperplasia of Meningitis, reactive spleen Astrogliosis D14 0 0 2 0 2 0 2 1 Reactive Astrogliosis -- D15 0 0 2 0 2 0 2 1 Mild acute nonsupp. Mild granulomatous pneumonia Vasculitis D16 1 0 1 0 1 0 1 1 -- Moderate acute arthritis, erosive pododermatitis D17 2 0 2 0 0 0 2 1 Reactive Astrogliosis, Mild follicular hyperplasia of mild acute nonsupp. spleen Vaculitis D18 2 0 0 0 2 0 2 1 Reactive Astrogliosis Mild follicular hyperplasia of spleen D19 GN 0 0 0 GN 0 1.5 1 Mild acute nonsupp. Focal mild acute necrotizing and Vasculitis, reactive myocarditis, mild follicular Astrogliosis hyperplasia of spleen

HE = Hematoxylin&Eosin; IHC = Immunohistochemistry for TBEV antigen (0 = no positive tissue/negative; 1 = ˂ 1% positive tissue = mildly affected; 2 = ≥ 1 % and ˂ 5 % positive tissue = moderately affected; 3 = ≥ 5 % positive tissue = severely affected); * = degree of lymphohistiocytic or lymphoplasmacellular perivascular infiltration within a necrotizing encephalitis; GN = only glial nodules; 0 = none; 1 = mild; 2 = moderate; 3 = severe; nonsupp. = non-suppurative; na = not available

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Chapter 5: Manuscript IV

Experimental infection of chickens with tick-borne encephalitis virus

Friederike Michel 1, Ute Ziegler 1, Christine Fast 1, Martin Eiden 1, Christine Klaus 2, Gerhard Dobler 3 and Martin H. Groschup 1,*

Short communication

5.1. Abstract

Tick-borne encephalitis (TBE) is an emerging arboviral zoonosis in Europe and parts of Asia causing infections of the central nervous system in humans and various animal species. In most cases the TBE virus (TBEV) is transmitted by tick bites. Small mammals are considered the main reservoir for TBEV. The question whether birds also contribute to the transmission cycle, apart from being carriers of infected ticks, is still not clear. Twenty chickens were therefore inoculated intramuscularly and another twenty chickens subcutaneously with the TBEV strain Neudoerfl. The animals were monitored daily for clinical symptoms and sampled in regular intervals. Regardless of the inoculation route, all chickens did not show clinical symptoms. TBEV-specific RNA was detected occasionally in blood and swab samples as well as in a few organ samples by TBEV-specific quantitative real-time RT- PCR. In the virus neutralization test, almost all challenged chickens produced TBEV neutralizing antibodies. Due to the short viremic phase and the low virus loads in the investigated organ samples we assume that chickens cannot function as silent carriers for TBEV and thus do not serve as an undetected virus reservoir.

5.2. Introduction

Tick borne encephalitis virus (TBEV) is a member of the family Flaviviridae and originally was divided into three subtypes: the Western European subtype which is mainly transmitted by the tick species Ixodes ricinus, and the Far Eastern- and Siberian subtypes which are primarily associated with Ixodes persulcatus (Valarcher et al., 2015). Recently two additional subtypes have been suggested (Dai et al., 2018; Kovalev and Mukhacheva, 2017). The virus circulates in small so-called natural foci between the tick vector and the amplifying/reservoir host. Reservoir hosts are small mammals, like rodents and insectivores, but also the tick itself is considered to be a virus reservoir (Dobler et al., 2012; Michelitsch et al., 2019). The natural TBEV foci occur in a patchy distribution and their size can vary from few square meters up to several square kilometers (Lindquist and Vapalahti, 2008).

Tick-borne encephalitis is endemic in Central Europe, the Baltic region, Russia and eastern Asia, with about 10,000 cases registered every year (Michelitsch et al., 2019). Over the last decades the endemic regions were expanding, and an increased number of human cases was reported, most likely due to improved diagnostics, increasing awareness, but also due to socio- economic, ecological and climatic factors (Randolph, 2010; Riccardi et al., 2019). Recently, new endemic foci were found in France and the Netherlands (de Graaf et al., 2016; Jahfari et al., 2017; Velay et al., 2018). TBEV is mostly transmitted by tick bites, but an alimentary infection via the ingestion of raw, unpasteurized milk or dairy products from sheep, goats or cows is also possible (Beauté et al., 2018). The majority of the infected humans seroconverts without showing a clinical disease or presents non-specific symptoms. Only in a small percentage a neurological disease manifests (Kaiser, 2012; Ruzek et al., 2019). TBEV infections are also described in various animal species such as dogs, horses, monkeys and ruminants (Klaus et al., 2016b). However, these species, including humans, are considered as accidental hosts. The main mechanism of the spread of TBEV to new areas and for the establishment of new natural foci seems to be the transport of infected ticks in until then not affected areas. Different ways of dispersal are currently under discussion. A transport of TBEV-infected ticks by their animal hosts such as rodents or deer, or even by humans transporting infested animals is a plausible option (Boelke et al., 2019). Birds are common hosts for immature ticks, but feed only for a short period of time so that a long distance transport is unlikely (Balashov, 1972; Hasle, 2013; Klaus et al., 2016a). However, the transport over shorter distances, such as from one stopover site to another during bird migration, may be possible. Whether birds are also playing a role in the transmission cycle of TBEV is not yet fully resolved. The available literature regarding animal experiments with birds dates back to the 1960s. In this study we therefore infected chickens with TBEV strain Neudoerfl to reveal whether they show clinical symptoms, viremia or even virus shedding.

5.3. Materials and methods

5.3.1. Animals and experimental design

One day old White Leghorn and New Hampshire chickens, randomly distributed, were raised at the Friedrich-Loeffler-Institut. The chickens were monitored daily for their physical health prior to the animal experiment. All chickens were tested negative for TBEV by quantitative real-time RT-PCR (qRT-PCR) and in the virus neutralization test (VNT). The chickens were challenged at the age of six weeks with the TBEV strain Neudoerfl 5 (GenBank accession no. U27495) and 10 tissue culture infectious doses (TCID50)/ml. Twenty chickens were inoculated respectively either intramuscularly (i.m.) in the breast muscle or subcutaneously (s.c.) in the knee folds (0.5 ml virus solution per site). Following infection, chickens were examined daily for clinical anomalies and evaluated according a defined score sheet: 0 (no clinical changes) to 3 (high - grade deviation from the physiological condition). Furthermore, blood samples, oropharyngeal- and cloacal swabs were taken on 2, 4, 6, (8 and 12 just swabs), 10, 14, 21 days post infection (dpi). Weight control was performed on each sampling day and additionally on 17 and 19 dpi. Blood, swab and organ samples were processed as described before (Michel et al. in preparation). On day 21 post infection, the chickens were euthanized and the organ samples (brain, liver, spleen, heart, caecal tonsils) were collected for virological investigations. The caecal tonsil was taken as additional organ only from the chickens inoculated subcutaneously. The TBEV challenge study was carried out under biosafety level 3 conditions. The infection experiments described in this publication were approved by the State Office of Agriculture, Food safety, and Fishery in Mecklenburg-Western Pomerania, Germany on the basis of national and European legislation in particular directive 2010/63/EU.

5.3.2. Quantitative real-time RT-PCR (qRT-PCR)

Viral RNA was extracted from the blood cruor using the RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. RNA of the swab and tissue sample supernatants was extracted using a Biosprint 96 (Qiagen, Hilden, Germany) and the NucleoMag VET Kit® (Macherey-Nagel, Düren, Germany). As an internal control, IC2 RNA

was extracted with all samples (Hoffmann et al., 2006). Extracted RNA was subsequently analyzed by a TBEV specific qRT-PCR (Schwaiger and Cassinotti, 2003). A synthetic RNA was used for quantification.

5.3.3. Serological investigations

Neutralizing antibody titers against TBEV “Neudoerfl” were determined by virus neutralization test (VNT) (Ziegler et al., 2013). Cytopathic effects were evaluated after seven days and cell monolayers were fixed with neutral buffered formalin and stained with crystal violet. The neutralizing antibody titer (neutralization dose 50% (ND50)) of a serum sample was defined as the maximum dilution which inhibited cytopathic effects in fifty percent of the wells. It was calculated according to the Behrens–Kaerber method.

5.4. Results

Neither the intramuscularly nor the subcutaneously infected chickens showed clinical symptoms and steadily gained weight (data not shown). The clinical score was “0” for all chickens during the whole experiment.

5.4.1. Detection of viral RNA by qRT-PCR

In the intramuscularly infected group TBEV specific RNA was found only in one blood sample on 2 dpi and in one cloacal swab sample (6 dpi) in two different chickens, whereas the samples of the other infected chickens remained negative during the whole experiment (Figure 4. A). The blood samples of the subcutaneously infected chickens were tested positive for TBEV- RNA on 4 dpi (n=4) and 6 dpi (n=2). One chicken (C 22) had a viremia over two following sampling days. TBEV specific RNA was detected sporadically, on the days 2, 8 and 10 post infection, in the swab samples (Figure 4. B). Organ samples were also only sporadically positive, however, high Ct values and low copy numbers. TBEV-RNA was detected in heart, liver and brain of a few intramuscularly infected chickens (Figure 5. A). Among the 20 chickens inoculated subcutaneously TBEV-RNA was found in the brain and the caecal tonsil (Figure 5. B).

5.4.2. Serology

All 20 chickens of the intramuscular infection experiment seroconverted and neutralizing antibodies were initially detected on 4 dpi. The titers reached their maximum on day 10 and

14 post infection and ranged then between ND50 1:320 to 1:20,480 (Figure 6. A) Of the chickens infected subcutaneously, 19 chickens showed a seroconversion, however, one chicken (C 32) did not seroconvert until 21 dpi. Neutralizing antibodies were first detected on day four post infection. The peak of the antibody levels was also on 10 and 14 dpi with the neutralizing antibody titers ranging between ND50 1:15 to 1: 15,360 on these days (Figure 6. B).

5.5. Discussion

Tick-borne encephalitis is an emerging health threat and can cause severe disease. The incidence of TBEV infections is constantly rising. In Germany, on average of 283 cases are reported yearly, with a maximum of 583 cases in 2018 (RKI, 2019). High risk areas are Baden- Wurttemberg, Bavaria, as most cases are registered there, but also several areas in Hesse and Thuringia and some districts in Saarland, Rhineland Palatine and Saxony. During the last years an expansion of the high risk areas, with a steady north-eastern and north-western spread was observed (Hellenbrand et al., 2019). In 2018, the district “Emsland” was declared risk area, representing the northernmost location in Germany (Boelke et al., 2019; RKI, 2019). The spreading tendency of TBEV was not seen only in Germany but also in other European countries and is influenced by several factors. Better knowledge of possible virus reservoirs may lead to a better understanding of the development and spread of these natural foci (Michelitsch et al., 2019). As already mentioned above the transport of TBEV infected ticks feeding on birds seems possible, however, only over limited distances. Apart from being a possible mechanical vector for infected ticks, birds may represent a potential virus reservoir for TBEV. Infection experiments with various bird species, different TBEV strains and inoculation methods were conducted in the past. In a chicken experiment described by Streissle (1958), 8 to 10 days old chickens were infected subcutaneously with the supernatant of homogenized brain tissue of the tick-borne encephalitis strain Graz I, belonging to the Western European

subtype. The infected chickens did not show a clinical disease, but all chickens developed a viremia lasting four to five days. Virus was detected in the blood already on day two post infection. A seroconversion or virus shedding of the infected chickens was not evaluated. In the here described study we infected chickens intramuscularly and subcutaneously with the TBEV strain Neudoerfl, which was not tested in chickens before. Furthermore, the few isolates characterized in Germany showed most often the highest homology to the strain Neudoerfl representing the prototype of the Western European subtype (Süss, 2011). In contrast to the animal experiment conducted by Streissle (1958) we were not able to detect a viremia lasting over several days. Virus was detected sporadically in the blood, particularly of subcutaneously infected chickens. Virus shedding was observed in a few chickens and exclusively in the cloacal swabs. The best explanation for these different findings is that chickens infected by Streissle (1958) were only few days old and had only an immature immune system, whereas chickens in our animal experiment were over 6 weeks old, with a fully developed immune system (Davison et al., 2008). An age dependent variation in susceptibility was also shown in different birds species infected with West-Nile virus, i.e. an increased duration and higher levels of viremia in nestlings and juveniles as compared to adults (Perez-Ramirez et al., 2014). Furthermore, differences in the findings could be related to the virus strains used. We infected the chickens with TBEV strain Neudoerfl, while Streissle (1958) used a variant of the strain Graz I. Both virus strains belong to the Western European subtype. The virus strain Graz I is not characterized in regards to its pathogenicity. The virulence of different strains within the same subtype can differ and for instance is considered to be lower in strain Neudoerfl and higher in strain Hypr (Dobler et al., 2016). All infected chickens seroconverted, except for one. Neutralizing antibodies were detected early (on 4 dpi), and the chickens developed quite high neutralizing antibody titers. In one chicken of the subcutaneously infected group, no seroconversion was detected until day 21 post infection. An explanation for this could be that the animal was a “low responder”, with a delayed and reduced production of neutralizing antibodies. This phenomena has also been observed both in repeatedly immunized goats as in humans (Klaus et al., 2010). However, as

all other birds seroconverted strongly, chickens kept in free-range husbandry can be used as sentinels for the detection of new natural TBEV foci.

5.6. Conclusion

Chickens are no suitable hosts for TBEV because they do not develop a long lasting viremia sufficient to infect feeding ticks. Therefore they most likely play no role in the TBEV transmission cycle of this virus or as virus reservoir. As chickens produce high neutralizing antibody titers within a short period of time after an infection with TBEV, they can be used as sentinel species in order to detect new local natural foci.

Acknowledgement

The authors wish to thank Cornelia Steffen and René Schöttner for their excellent technical assistance. Additionally we would like to thank the animal caretakers for their support within the experiments. This study was funded by the German Center for Infection Research (DZIF) Project Number TTU 01.801.

5.7. Figures

Figure 4. Results of quantitative real-time polymerase chain reactions (qRT-PCR) of the blood and swab samples of the intramuscularly and subcutaneously infected chickens in copies/ µl. B = Blood samples, PS = Pharyngeal Swab, CS = Cloacal Swab A qRT-PCR results of the chickens inoculated intramuscularly B qRT-PCR results of the chickens inoculated subcutaneously

Figure 5. Results of quantitative real-time polymerase chain reactions (qRT-PCR) of the organ samples of the intramuscularly (i.m.) and subcutaneously (s.c.) infected chickens in copies/µl. C = chicken A qRT-PCR results of the organ samples on 21 dpi (i.m. inoculation) B qRT-PCR results of the organ samples on 21 dpi (s.c. inoculation)

4,5

4,0

3,5

3,0

2,5

log VNT titer 2,0

1,5

1,0

0,5 0 2 4 6 10 14 21 A dpi

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4,0

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Figure 6. Course of antibody responses of the intramuscularly (i.m.) and subcutaneously inoculated (s.c.) chickens against TBEV monitored by virus neutralization test (depicted in log titers). Data of the neutralizing antibody responses for all chickens are presented in a box- plot. Minimum and maximum values are represented by the respective whiskers, and outliers

as dots. The box includes 50% of the values of all investigated animals per day and the median is depicted as a line. A Course of antibody response against TBEV by virus neutralization test (i.m. inoculation) B Course of antibody response against TBEV by virus neutralization test (s.c. inoculation)

5.8. References

Balashov, Y., 1972. A translation of "Bloodsucking ticks (Ixodoidea)—vectors of diseases of man and animals". Miscellaneous Publications of the Entomological Society of America 8, 159-376.

Beauté, J., Spiteri, G., Warns-Petit, E., Zeller, H., 2018. Tick-borne encephalitis in Europe, 2012 to 2016. Euro Surveillance 23.

Dai, X., Shang, G., Lu, S., Yang, J., Xu, J., 2018. A new subtype of eastern tick-borne encephalitis virus discovered in Qinghai-Tibet Plateau, China. Emerging microbes & infections 7, 74.

Davison, F., Kaspers, B., Schat, K.A., 2008. Avian Immunolgy. de Graaf, J.A., Reimerink, J.H., Voorn, G.P., Bij de Vaate, E.A., de Vries, A., Rockx, B., Schuitemaker, A., Hira, V., 2016. First human case of tick-borne encephalitis virus infection acquired in the Netherlands, July 2016. Euro Surveillance 21.

Dobler, G., Bestehorn, M., Antwerpen, M., Overby-Wernstedt, A., 2016. Complete Genome Sequence of a Low-Virulence Tick-Borne Encephalitis Virus Strain. Genome Announcements 4.

Dobler, G., Gniel, D., Petermann, R., Pfeffer, M., 2012. Epidemiology and distribution of tick-borne encephalitis. Wiener medizinische Wochenschrift 162, 230-238.

Hasle, G., 2013. Transport of ixodid ticks and tick-borne pathogens by migratory birds. Frontiers in Cellular and Infection Microbiology 3, 48.

Hellenbrand, W., Kreusch, T., Böhmer, M.M., Wagner-Wiening, C., Dobler, G., Wichmann, O., Altmann, D., 2019. Epidemiology of Tick-Borne Encephalitis (TBE) in Germany, 2001-2018. Pathogens 8.

Jahfari, S., de Vries, A., Rijks, J.M., Van Gucht, S., Vennema, H., Sprong, H., Rockx, B., 2017. Tick-Borne Encephalitis Virus in Ticks and Roe Deer, the Netherlands. Emerging Infectious Diseases 23, 1028-1030.

Kaiser, R., 2012. Tick-borne encephalitis: Clinical findings and prognosis in adults. Wiener medizinische Wochenschrift 162, 239-243.

Klaus, C., Hoffmann, B., Moog, U., Schau, U., Beer, M., Süss, J., 2010. Can goats be used as sentinels for Tick-borne encephalitis (TBE) in non-endemic areas? Experimental studies and epizootiological observations. Berliner und Münchener Tierärztliche Wochenschrift 123, 441-445.

Kovalev, S.Y., Mukhacheva, T.A., 2017. Reconsidering the classification of tick-borne encephalitis virus within the Siberian subtype gives new insights into its evolutionary history. Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases 55, 159-165.

Lindquist, L., Vapalahti, O., 2008. Tick-borne encephalitis. The Lancet 371, 1861-1871.

Michelitsch, A., Wernike, K., Klaus, C., Dobler, G., Beer, M., 2019. Exploring the Reservoir Hosts of Tick-Borne Encephalitis Virus. Viruses 11.

Perez-Ramirez, E., Llorente, F., Jimenez-Clavero, M.A., 2014. Experimental infections of wild birds with West Nile virus. Viruses 6, 752-781.

Randolph, S.E., 2010. on behalf of the Eden-Tbd sub-project team: Human activities predominate in determining changing incidence of tick-borne encephalitis in Europe. Euro Surveillance 15, 24-31.

RKI, 2019. Robert Koch-Institut (RKI): FSME: Risikogebiete in Deutschland (Stand: Januar 2019) Bewertung des örtlichen Erkrankungsrisikos. Epidemiologisches Bulletin 7, 57-70.

Streissle, G., 1958. Virämie bei Küken nach subcutaner Infektion mit dem Virus der Zeckenencephalitis. Zeitschrift für Hygiene 145, 331-334.

Süss, J., 2011. Tick-borne encephalitis 2010: epidemiology, risk areas, and virus strains in Europe and Asia-an overview. Ticks and Tick-Borne Diseases 2, 2-15.

Valarcher, J.F., Hägglund, S., Juremalm, M., Blomqvist, G., Renström, L., Zohari, S., Leijon, M., Chirico, J., 2015. Tick-borne encephalitis. Rev Sci Tech Oie 34, 453-466.

Chapter 6: General discussion and conclusion

Arthropod-borne viruses (Arboviruses) circulate between vertebrate hosts and their respective arthropod vectors. They are considered to be an emerging threat to human health, as many have a zoonotic potential, i.e. can cause clinical diseases in humans. During the last decades, an increased incidence and a geographical spread of several arboviruses was observed (Weaver and Reisen, 2010). Globalization, with increased international trade and travel, plays a major role. Moreover, climate change, resulting in increased temperatures, severe droughts and extreme weather events may also be a reason for this phenomenon (Pfeffer and Dobler, 2010; Semenza and Suk, 2018). Wild birds play an important role in the transmission of several viruses. They do not only serve as hosts, but also as mechanical carriers for arthropod vectors. Every year, migratory birds travel over long distances, passing endemic regions for distinct viruses (Hubálek, 2004). Surveillance studies of vectors and hosts of zoonotic arthropod-borne viruses are essential to detect the introduction and spread of new emerging pathogens and implement public and animal health measures. In order to achieve this aim, a nationwide wild bird surveillance network was set up to monitor wild and zoo birds. Sample collectors were from all over Germany and comprised of veterinary universities or institutes, bird clinics, wild bird rescue stations, zoological gardens, as well as ornithologists. The focus of this network was the investigation for the flaviviruses WNV and USUV, as birds are the main virus reservoir for both viruses. Blood was collected from live birds in veterinary clinics and wild bird rescue centers. All blood samples were surplus from those taken initially for routine diagnostics. The samples were screened by real-time polymerase chain reaction and serological assays. Additionally, organ samples from different bird carcasses were also submitted by this network to the national reference laboratory for WNV/USUV diagnostics and selected samples from this panel were also used for this study. USUV is circulating in Germany for some time now (2010/2011), whereas WNV was first detected in Germany just recently (2018) (Becker et al., 2012; Ziegler et al., 2019). Both viruses have similar transmission cycles with birds as the main amplifying hosts and mosquitoes as vectors; consequently, co-circulation as well as overlapping transmission cycles of both viruses in the same area cannot be excluded (Nikolay, 2015).

In Manuscript I the results of the survey from 2014 to 2016 are presented, with more than 1,900 investigated blood samples from resident and migratory birds belonging to 136 different bird species and 20 zoological bird orders. In the two years 2017 and 2018, with over 1,700 wild bird blood samples, nearly as many samples were collected (Manuscript II). The majority of samples were from birds of the zoological orders Passeriformes and Accipitriformes/ Falconiformes, as bird species of these orders are known to be highly susceptible to WNV. Furthermore, all birds were categorized as resident birds (remain all-the-year in their German habitat), partial migratory birds (parts of the population stay in the German habitat and parts migrate), short-distance migratory birds (usually migrating 1,000–2,000 km and not passing through the Sahara desert), and long-distance migratory birds (usually migrating 3,000–4,000 km and/or passing the Sahara desert).

West Nile virus WNV has been circulating in the neighboring countries of Germany, such as France, Austria and the Czech Republic for years (Aberle et al., 2018; Bahuon et al., 2016; Rudolf et al., 2014). Therefore, an introduction into Germany was expected, and was just a matter of time. In both here presented monitoring studies ranging from 2014 to 2018, no WNV-specific RNA was detected in the investigated samples from live wild and zoo birds, even though the first WNV cases among horses and birds occurred in Germany during late summer and fall of 2018. These results are in accordance with the previously conducted monitoring studies, where also all collected samples were negative for WNV-RNA (Seidowski et al., 2010; Ziegler et al., 2015; Ziegler et al., 2012). Nevertheless, neutralizing antibodies against WNV were frequently detected in migratory birds and occasionally also in resident birds. Surveillance of the mosquito population also did not provide evidence for WNV circulation in Germany until 2016, despite of a proven vector competence of German mosquitoes for WNV (Jansen et al., 2019). The first WNV strain detected in Germany belonged to WNV lineage 2 and showed a close relationship to a WNV strain isolated from a mosquito from the Czech Republic in 2013. Chrono- phylogenetic analysis indicated a possible introduction into Germany already in 2016 (Ziegler et al., 2019).

The serological investigations revealed almost similar seroprevalence rates against WNV during both investigated periods, with a seroprevalence of 3.18% between 2014 and 2016, and 3.13% from 2017 to 2018. In both studies, predominantly long-distance, short-distance migratory birds and partial migrants were affected, which most likely became infected during their annual migration from Africa or Southern Europe. Furthermore, in a considerable number of birds of prey neutralizing antibodies against WNV were detected. Birds of prey represent highly susceptible bird species for a WNV infection, which may explains the increased detection of neutralizing antibodies in these birds (Busquets et al., 2012; Nemeth et al., 2006; Ziegler et al., 2013). Moreover, most birds of prey are migratory birds and most likely became infected in neighboring countries of Germany. Especially in the Czech Republic an increased mortality in birds of prey due to an infection with WNV was seen in 2017 and 2018 (Hubálek et al., 2018; Hubálek et al., 2019). Neutralizing antibodies against WNV were detected in few resident bird species, however, with very low neutralizing antibody titers (ranging between 1:10 and 1:20 ND50), even though there was no evidence of WNV circulation in Germany until 2018. The same was also observed in previous wild bird monitoring studies conducted before 2014, however, the reasons for this findings are not quite clear. A possible explanation might be that the resident birds became infected in nearby countries, where WNV already was detected, as even so-called resident birds can occasionally travel up to 50-100 km (Hubálek, 2004). In the period between 2014 and 2016, a considerable number of city pigeons showed neutralizing antibodies against WNV, which are predominantly resident birds but can also travel short distances. Furthermore, it was shown that pigeons are a suitable sentinel species for WNV and can be used as an early warning system based on regular serological investigation (Chaintoutis et al., 2014). In the Netherlands, monitoring with the focus on waterfowl was conducted and a large number of resident birds were found antibody positive for WNV. Consequently, the author assumed that WNV might already be circulating in the Netherlands (Lim et al., 2018). The same cannot be excluded for Germany, however, WNV-specific RNA was detected neither in birds nor in mosquitoes until 2018. Therefore, it would be speculative to claim on this basis that WNV was already circulating in Germany before its first detection in birds and horses in 2018. In 2018, in several birds of prey (Northern Goshawk (Accipiter gentilis), European Kestrel (Falco tinnunculus), White-tailed Sea Eagle (Haliaeetus albicilla)) and one Carrion Crow

(Corvus corone)) high neutralizing antibody titers against WNV were detected, with ND50 values ranging from 1:160 up to 1:320. These high antibody titers suggest a recent WNV infection. All birds were sampled in the regions of Leipzig and Berlin, where the first WNV- RNA positive birds had been found before in 2018. Consequently, a local virus circulation in this area was also seen in the live bird monitoring in 2018 (Manuscript II).

Usutu virus USUV, a closely related Flavivirus has been circulating in Germany since 2010/2011, causing mortality predominantly in Eurasian Blackbirds and captive Great Grey Owls (Becker et al., 2012; Jöst et al., 2011). For years, the virus occurred mainly in the region of the Upper Rhine Valley, but during the last years has spread throughout Germany (Cadar et al., 2017a; Sieg et al., 2017; Ziegler et al., 2015). In the live bird monitoring study from 2014 to 2016 USUV specific RNA was detected in only four birds originating from the regions of Dusseldorf and Giessen. In the following survey, altogether 57 birds were USUV-RNA positive in 2017 and 2018. During these two years, a massive spread of USUV correlated with high case numbers could be observed, most likely leading to the higher number of USUV positive birds than in the previous study. In addition to our live bird monitoring we received organ samples from dead birds which were submitted to the Friedrich-Loeffler-Institut (FLI) national reference laboratory for WNV by the regional veterinary laboratories of the federal states of Germany, by the German Mosquito Control Association (KABS), the Nature and Biodiversity Conservation Union (NABU), citizens and independent bird clinics, and zoological gardens. These samples were analyzed in close cooperation with the Bernhard-Nocht-Institute for Tropical Medicine in Hamburg and the Institute of Virology at the Faculty of Veterinary Medicine in Leipzig. The total number of USUV positive birds in Germany during the years 2016 and 2017 are included in this study. In both years, all investigated dead birds were negative for WNV-RNA. In 2016, in Germany in total 73 dead birds were tested positive for USUV-RNA. A spread of USUV from the original main distribution area in Southwestern Germany to the regions of Leipzig/Halle and North-Rhine Westphalia was observed. In the next year (2017), USUV- specific RNA was detected in altogether 100 birds. A further spread towards Northern Germany was seen, with first cases in Hannover, Hamburg and Bremen. The tendency of a northward

spread was also seen in the live bird monitoring with one positive Great Spotted Woodpecker from the Northern part of Lower Saxony in 2017. The most affected bird species in the live and dead bird monitoring during the time span from 2014 to 2018 was the Eurasian Blackbird. Other bird species, predominantly of the bird orders Passeriformes and Strigiformes were also occasionally tested positive for USUV-RNA. Nevertheless, Eurasian Blackbirds are considered to be the most susceptible bird species for USUV, leading to a massive reduction of the blackbird population during the first years of USUV detection in Germany (Bosch et al., 2012). Lühken et al. (2017) demonstrated a negative impact on the Blackbird population in USUV outbreak areas in comparison to areas unsuitable for USUV circulation. The reason why especially Eurasian Blackbirds are so susceptible is still unclear. Nevertheless, the seasonal conditions probably play a role. During the peak of mosquito activity, Eurasian Blackbirds are moulting, leading to a suppression of the immune system. Loss of plumage in the area of the head and ears has often been described in USUV positive birds (Bosch et al., 2012). In 2018, the largest USUV outbreak since 2011 with 1,183 confirmed USUV cases occurred in Germany (Ziegler, 2019). However, to the NABU over 10,000 cases of sick or deceased Eurasian Blackbirds were reported by concerned citizens, suggesting a much higher fatality rate (NABU, 2019). The virus spread massively throughout Germany and was detected in all federal states. The extremely hot temperatures in this year led to a shorter extrinsic incubation period, resulting in a faster transmission cycle between birds and mosquitoes. Furthermore, the infection pressure seemed to be very high in this year, leading to a broad spectrum of infected bird species, which is also reflected in our live bird monitoring of 2018. Apart from the most susceptible bird species, numerous other birds were USUV positive such as different bullfinch species (Pyrrhula pyrrhula, Pyrrhula erythaca), a European Kestrel and a Domestic Canary (Serinus canaria forma domestica). Phylogenetically eight different USUV lineages can be distinguished: Africa 1-3 and Europe 1- 5, with five of them currently circulating in Germany. Two of the four USUV-RNA positive samples from the period between 2014 and 2016, originating from Dusseldorf, could be sequenced and lineage Europe 3 was identified, which is not surprising, as it is the most prominent USUV lineage in Southwestern Germany (unpublished data). During 2017 and 2018, we sequenced USUV-positive samples from both our live and dead bird survey to determine

the geographic distribution of the different USUV lineages. A massive spread of the USUV lineages Europe 3 and Africa 3 was observed in 2018, especially towards Northern Germany in the federal states Lower Saxony, Schleswig Holstein and Mecklenburg-Western Pomerania (Manuscript II). As mentioned above, Europe 3 was for years restricted to Southern Germany, but was first detected in the region of Leipzig and Halle in 2016, and subsequently spread all over Germany (Sieg et al., 2017). Africa 3 was introduced into Germany in 2014 and was first detected in Bonn. In Saxony and Saxony-Anhalt it was found for the first time in 2016 (Cadar et al., 2015). The lineage Africa 2 also expanded to Saxony and Saxony-Anhalt after its first detection in Berlin, and remained restricted to Eastern Germany during the past two years (Ziegler et al., 2016). Furthermore, in 2018, the USUV lineage Europe 2 was first detected in Germany, in the city of Leipzig (Manuscript II). This USUV lineage has been circulating in Austria, Hungary and Italy for several years (Bakonyi et al., 2017a; Carletti et al., 2019). Both a transboundary distribution of the different USUV lineages, but also a dispersal within Germany via migrating birds are possible explanations. The circulation of different USUV lineages in one country is not a unique phenomenon, and was observed in Austria, France and Italy as well (Carletti et al., 2019; Eiden et al., 2018; Roesch et al., 2019). Differences regarding the pathogenicity/virulence of the different USUV lineages in humans and birds are still unknown. In both investigated periods (Manuscript I and II), the majority of the USUV antibody positive birds were resident birds, but also partial migrants and some short-distance migratory birds. Neutralizing antibodies were found in a wide spectrum of different bird species, however, predominantly in birds of the order Passeriformes and frequently in Strigiformes representing the most susceptible bird species, but also in birds of prey (Accipitriformes/ Falconiformes). In the monitoring study between 2014 and 2016, the total seroprevalence for whole Germany per year was calculated, but for the years 2017 and 2018, the samples were divided into different geographic regions, to better show regional differences. Based on the total number of USUV antibody positive birds per year, an increase in seroprevalence can be observed, from 1.21% in 2014 up to 8.44% in 2018. However, it must also be considered that USUV was not as widespread in 2014 as in 2018. Looking at the different geographic regions of Northern, Eastern, and Central and Southern Germany during 2017 and 2018, variances regarding the seroprevalence were observed:

In Northern Germany, the seroprevalence increased from 1.75% in 2017 up to 9.54% in 2018. In 2018, a massive USUV outbreak resulting in numerous perished birds and most likely such high seroprevalence rates occurred around Hamburg and the North Sea. The antibody titers were also quite high in 2018 (up to 1:2,560 ND50), indicating a recent USUV infection. In Eastern Germany in both years, relatively high seroprevalence rates were found, with 21.05% in 2017 and 13.04% in 2018, and high antibody titers in both years. As USUV has been circulating in this region for several years now, this finding is not surprising. In Central and Southern Germany, seroprevalence was slightly higher in 2018. A remarkable finding is the high number of antibody positive zoo birds in 2018. Zoo birds can here be assessed as resident birds, and thus prove the ongoing USUV circulation in the investigated area. In Austria, several years after the USUV outbreak a seroprevalence in wild birds of over 50% was observed, indicating the formation of a so-called herd immunity (Meister et al., 2008). In the presented wild bird monitoring studies in Germany such a high percentage of seroreactors was not found. Nevertheless, in 2018 the so far highest USUV seroprevalence rates were registered in Germany to date. Future wild bird surveys will reveal whether the seroprevalence in the local bird populations is further increasing.

Taken together, USUV spread massively throughout Germany during 2017 and 2018, and more birds were tested positive for USUV-RNA than in the period 2014 to 2016. This finding is supported by the spread of the USUV lineages Africa 3 and Europe 3 towards Northern Germany and the first detection of Europe 2. Neutralizing antibodies against USUV were predominantly found in resident birds. The USUV seroprevalence increased during 2017 and 2018, in particular in regions where USUV had not been detected before. WNV-RNA was not found in the live bird panel from 2014 to 2018, despite the first detection among several birds and horses in Germany in 2018. Neutralizing antibodies against WNV were predominantly found in long-distance migratory birds. In 2018, however, high antibody titers indicating recent virus contacts were detected also in Eastern Germany in resident and short-distance migratory birds. These were the first signs that WNV started to circulate in Germany.

Tick-borne encephalitis virus The role of birds in the transmission cycles for the mosquito borne flaviviruses WNV and USUV is well known. However, their contribution in the transmission cycle of the tick- transmitted TBE virus is still not fully understood. Birds are common hosts for ticks, and especially migratory birds are able to carry feeding ticks. Consequently, a transport of infected ticks to previously non-infected areas and an establishment of new natural TBEV foci seems possible. Nevertheless, transport over longer distances is unlikely, as Ixodes ticks feed only for a short time (2 to 7 days) (Balashov, 1972; Klaus et al., 2016a). However, a transport of infected ticks from one stopover site to the next seems possible. In the past, TBEV was occasionally isolated from passerines, ducks, and other bird species, but no TBEV-RNA was detected in the live bird monitoring program between 2015 and 2018 (Ernek, 1962; Hubálek and Rudolf, 2012) (unpublished data). Whether birds play a role in the transmission cycle of TBEV and are a potential virus reservoir is still not fully clear. In the past, several animal experiments on various wild and domestic birds were conducted, which date back to the 1960s. Infected Great tits (Parus major), House sparrows (Passer domesticus), Pheasants (Phasanius colchicus), Common Buzzards (Buteo buteo), and European Kestrels (Falco tinnunculus) did not show any clinical signs and no viremia was detected (Ernek and Lichard, 1964; Grešíková et al., 1962; Nosek et al., 1962; Řeháček et al., 1963). Some of the buzzards, kestrels and pheasants, however, developed neutralizing antibodies after repeated inoculation. In few days old chickens (Gallus gallus domesticus) the infection was asymptomatic, but a viremia lasting several days was detected, the same applied to Common coots (Fulica atra) (Streissle, 1958; van Tongeren and Timmers, 1961). Several infection experiments were also conducted on ducks (Anas platyrhynchos/ Anas platyrhynchos domesticus) since the 1960s, with various TBEV virus strains (Graz I and Hypr) and inoculation methods (Ernek, 1962; Ernek et al., 1969a; Ernek et al., 1969b; van Tongeren, 1983). The majority of the infected ducks showed a viremia lasting over several days and developed neutralizing antibodies, regardless of the virus strain or inoculation method used. The TBEV strains detected in Germany show a close homology to the TBEV strain Neudoerfl, representing the prototype of the Western European subtype (Süss, 2011). As no experimental studies with the virus strain Neudoerfl have been conducted to date, we infected chickens and ducks, as

animal model species, with this TBEV strain. The aim was to find out whether these bird species are susceptible to TBEV and may therefore represent an undetected virus reservoir. 5,0 One group of 20 chickens was inoculated intramuscularly with a virus titer of 10 TCID50/ml and another group subcutaneously at the age of six weeks (Manuscript IV). Furthermore, 19 ducks were inoculated subcutaneously (Manuscript III). Subcutaneous inoculation was chosen for the ducks, because the previously conducted infection studies on chickens were more successful by this route and because this inoculation route most strongly resembles natural infection by an infectious tick. The birds were monitored daily for clinical symptoms and evaluated according to a clinical score sheet. Furthermore, blood, pharyngeal and cloacal swab samples were taken regularly. Twenty one days after the infection, the chickens and ducks were euthanized and organ samples were collected during necropsy. No clinical signs were observed in the chickens and ducks during the whole experiment. Furthermore, the animals were weighed in regular intervals, and steadily gained weight. In the intramuscularly infected group of chickens, TBEV-RNA was only found in one blood and one swab sample. In contrast, in the group of chickens inoculated subcutaneously, TBEV- RNA was detected sporadically in the blood samples of several chickens on days 4 and 6 post infection. The same could be observed during the duck experiment, where TBEV-specific RNA was also only detected sporadically in some of the infected ducks, but also on days 4 and 6 post infection. This stands in contrast to the findings of the experiments conducted with chickens and ducks in the past, where a viremia lasting over several days could be observed (Ernek et al., 1969b; Streissle, 1958; van Tongeren, 1983). A possible explanation may be the different virus strains used. The virus strain Graz I used in the chicken experiment and one of the duck experiments carried out in the past, has not been characterized with regard to its pathogenicity, and can barely be found in the literature. The virus strain Hypr, however, is considered to have a higher neuropathogenicity than the strain Neudoerfl (Dobler et al., 2016). Furthermore, blood samples were taken only at two-day intervals for animal welfare reasons during the first week. Therefore, it is possible that the viremia may have been missed. Moreover, the chickens infected by Streissle (1958) were only a few days old, and had an immature immune system. In contrast, the chickens in our animal experiment were six weeks old and had a further developed immune system (Davison et al., 2008). Most likely, they were less susceptible and consequently did not show a long lasting viremia. Such an age dependent

susceptibility was also seen in different bird species infected with the related flavivirus WNV. An increased duration and higher levels of viremia were seen in nestlings and juveniles in comparison to adults (Perez-Ramirez et al., 2018). Despite the lack of virus detection in the blood, all 19 infected ducks were positive for TBEV- RNA in the brain and some of the spleen samples. As the virus normally is transported to distant organs via the bloodstream, the virus most likely was detectable in the blood only for a short time, or the amount of virus particles in the blood was below the limit of detection. Virus cultivation of the TBEV-RNA positive organ samples in cell culture remained unsuccessful. It is possible that the virus load in the brain samples, with Ct values between 27 and 30, was too low. Another possibility is that 21 days after the infection the virus was no longer able to replicate. Whereas in the intramuscularly infected chickens TBEV-RNA was found occasionally in the brain, heart and liver, in the subcutaneously infected chickens viral RNA was found only in the brain and caecal tonsil (additionaly sampled in this animal experiment). As the Ct values were quite high, no virus cultivation was conducted. These results differ from those obtained in the ducks, where in all infected animals virus was detected in the brain. A possible explanation could be that chickens are less susceptible to an infection with TBEV Neudoerfl. In histopathology the ducks showed nonsuppurative encephalitis with distinct neuronal necrosis, foci of neuronophagia, gliosis and perivascular lymphohistiocytic or lymphoplasmacellular cuffing, which is in accordance with the pathology described for mammals, however, the meninges were rarely involved (Bagó et al., 2002; Böhm et al., 2017; Süss et al., 2007; Völker et al., 2017; Weissenböck et al., 1998). The main target region in birds seemed to be the cerebrum, followed by mesencephalon, cerebellum and brain stem. This distribution of lesions resembles findings in dogs (Völker et al., 2017). Furthermore, several birds showed signs of a mild vasculitis, which is also frequently seen in birds infected with WNV. Distinct signs of degeneration are seen in glial and mononuclear inflammatory cells within the perivascular cuffs, but also throughout the neuropil. Similar lesions were also described before (Weissenböck et al., 1998). In IHC stained viral antigen was only seen in neurons and neuronal processes and often close to inflammatory processes. Only low amounts of viral antigen were found in several ducks as also described for mammals. This is not surprising as all birds were euthanized 20 or 21 dpi. Flaviviruses, like TBEV in humans and

dogs as well as West Nile virus in birds are known for a rapid clearance, resulting in a small time frame for the detection of virus antigen by IHC (Angenvoort et al., 2014; Weissenböck et al., 1998). Another interesting finding is the divergence between the histopathological lesions, which were distributed throughout all regions of the brain, and the detection of viral antigens, which was, except in one case, restricted to the cerebrum. Such a pattern was described for a monkey as well (Süss et al., 2008) and in both cases the reason behind this pattern remains unclear. Unfortunately, a histopathological evaluation of the chickens was not possible. Both the subcutaneously and the intramuscularly infected chickens (Manuscript IV) seroconverted (with one exception). Neutralizing antibodies were detected early (on 4 dpi) and reached high titers. In one chicken, no seroconversion was observed during the whole experiment. An explanation for this could be that the animal was a “low responder”, with a delayed or reduced production of neutralizing antibodies. This phenomenon has also been observed in repeatedly immunized goats and humans (Klaus et al., 2010). Furthermore, there are reports about vaccine failures in humans, even when correct vaccination schedules were used (Andersson et al. 2010). All infected ducks (Manuscript III) developed neutralizing antibodies against TBEV, which were first detected on day 6 after infection. The ducks developed quite high neutralizing antibody titers with values up to 20,480 ND50. Additionally, the serum samples were investigated by the Immunozym FSME IgM kit (Progen GmbH, Germany). This ELISA kit was used in the modified version to detect total immunoglobulin and was applied for several mammalian species before (Klaus et al., 2011). Avian serum samples have never been tested to date, but the results of the ELISA shows that it can also be applied on ducks. However, it needs to be considered that the calculation of the Cut-off values is based on a fairly low number of samples.

Taken together the chickens and ducks did not show any clinical signs and a viremia and virus shedding was only sporadically observed in a few birds. Nevertheless, all chickens and ducks seroconverted, except for one chicken. In the intramuscularly and subcutaneously infected chickens, TBEV-RNA was detected occasionally in several organ samples. In contrast, in all ducks TBEV-RNA was detected in the brain and all ducks suffered from an encephalitis.

Furthermore, in 14 ducks, TBEV antigen was found by immunohistochemistry. It is unlikely that chickens and ducks do play a role as hosts in the transmission cycle of TBEV as they do not develop a high and long lasting viremia. Nevertheless, both bird species can be used as sentinel species, as they show a fast seroconversion associated with high antibody titers. The serological investigation of chickens and ducks kept in free-range housing, in addition to the monitoring of TBEV-RNA in ticks, could help to define and detect new natural foci.

Chapter 7: Summary

Monitoring and Pathogenesis of Flavivirus Infections in Wild Birds and Domestic Poultry in Germany

Friederike Michel

In the last decades several arboviruses emerged, spread and subsequently became a threat to human health due to environmental, climatic and ecological changes. Wild birds play a significant role as reservoir hosts and mechanical vectors for zoonotic pathogens and their dispersal along the major flyways. West Nile virus (WNV) is a mosquito-borne viral pathogen of global importance and can cause severe diseases in humans, horses and several bird species. In 2018, WNV was detected for the first time in birds and horses in Germany. Moreover, an epidemic spread of the closely related Usutu virus (USUV) in birds has been observed in Germany since its first introduction in 2010/2011. Both viruses are maintained in enzootic cycles between birds as virus reservoirs and mosquitoes as vectors, which makes integrated surveillance of birds and mosquitoes an urgent need. Therefore, a nation-wide wild bird surveillance network was set up in order to monitor migratory and resident birds for the zoonotic flaviviruses WNV and USUV. Sample collectors from all over Germany provided blood samples of live wild and zoo birds. Furthermore, organ samples from dead birds submitted to the Friedrich-Loeffler-Institut in 2016 and 2017 were included in this surveillance. All samples were assayed by WNV- and USUV-specific real-time PCRs and serologically by virus neutralization tests. Wild bird monitoring results from 2014 to 2018 in Germany were summarized and evaluated in the first and second manuscript of this cumulative thesis. The number of USUV-RNA positive live birds was rather small (n=4) in the time period of 2014 to 2016, whereas in the following years 2017 and 2018 more viremic cases (n=57) were detected. The expansion of USUV throughout Germany was already seen from data of the live bird sample panel. Furthermore, the locations of dead birds tested positive for USUV illustrated its geographical spread from Southern and Central Germany towards the north. Phylogenetic investigations of these virus genomes showed the presence of USUV lineages Africa 3 and Europe 3 and resulted in the first detection of Europe 2. During the entire investigation period, neutralizing antibodies

against USUV were found predominantly in resident birds, but also in partial and short-distance migratory birds. The USUV seroprevalence increased during 2017 and 2018, in particular in regions where USUV had not been detected before. WNV-RNA was not found in the live bird sample panel from 2014 until 2018, despite the first WNV cases in several bird species and in horses in Germany in 2018. During this investigation period, neutralizing antibodies against WNV were found primarily in long-distance migratory birds, indicating a possible virus contact in the winter quarters in Southern Europe or Africa. However, in 2018 high antibody titers were detected for the first time in resident and short- distance migratory birds in Eastern Germany. High antibody titers indicate a recent virus contact, most likely in Germany or neighboring countries and provide evidence for an ongoing WNV circulation in the local bird population. This has become more prominent in 2019 with the occurrence of more than 50 WNV cases in birds and numerous clinically affected horses.

Tick-borne encephalitis virus (TBEV) is another arbovirus of the genus Flavivirus. The role of birds in the transmission cycle of TBEV is not fully understood, hence the second part of the here presented study addresses this topic. For this purpose, 20 chickens were infected intramuscularly (i.m.) and 20 chickens subcutaneously (s.c.) with the TBEV strain Neudoerfl. Furthermore, 19 ducks were inoculated subcutaneously. The chickens did not show any clinical signs. Viremia or virus shedding were observed only sporadically in a few birds, however more often in the subcutaneously infected group. The results were similar for the ducks, which did not show any clinical symptoms either, and TBEV-RNA was detected only occasionally in some blood and swab samples. Nevertheless, all chickens (except one) and all ducks seroconverted. In both groups of chickens (i.m. and s.c.), TBEV-RNA was detected sporadically in different organ samples such as brain, heart, liver and the caecal tonsil. In contrast, in all ducks TBEV-RNA was detected in the brain, and in the histopathology all ducks showed an encephalitis. Furthermore, in 14 ducks, TBEV antigen was found by immunohistochemistry. As chickens and ducks did not develop a high and long-lasting viremia we conclude that they do not play a role as hosts in the transmission cycle of TBEV. Nevertheless, both bird species can be used as sentinel species as they seroconvert fast and develop high antibody levels. The serological investigation of chickens and ducks kept in free-

range housing, in addition to the monitoring of TBEV-RNA in ticks, will help to define and detect new natural foci.

The here described data emphasizes that monitoring and pathogenesis studies in wild birds can be used as early warning systems for the occurrence of zoonotic pathogens.

Chapter 8: Zusammenfassung

Zoonotische Flavivirusinfektionen: Monitoring von Wildvögeln in Deutschland und Pathogenesestudien an Wirtschaftschaftsgeflügel

Friederike Michel

Im Laufe der letzten Jahrzehnte kam es aufgrund der zunehmenden Globalisierung und des fortschreitenden Klimawandels zu einem vermehrten Auftreten und der Ausbreitung von Arboviren, in dessen Folge die Gefahr für die menschliche Gesundheit durch Übertragung dieser Viren zunahm. Wildvögel spielen in diesem Zusammenhang eine bedeutende Rolle als Reservoirwirte und/oder Vektoren im Übertragungszyklus endemischer oder neu auftretender viraler Zoonoseerreger in Zentraleuropa. Im Rahmen des weltweiten Vogelzuges können diese Erreger so über weite Strecken transportiert und damit aus Endemiegebieten in neue, bisher freie Gebiete eingeschleppt werden.

Das West-Nil-Virus (WNV) ist ein durch Stechmücken übertragenes Virus, welches schwerwiegende Erkrankungen vor allem beim Mensch und Pferd, aber auch verschiedenen Vogelarten auslösen kann. Das Virus ist weltweit verbreitet und wurde erstmals im Jahr 2018 in verschiedenen Vögeln und Pferden in Deutschland nachgewiesen. Das eng verwandte Usutu-Virus (USUV) zirkuliert bereits seit 2010/2011 in Deutschland. Während der letzten Jahre konnte eine zunehmende Ausbreitung innerhalb Deutschlands, assoziiert mit einer erhöhten Vogelsterblichkeit, beobachtet werden. Beide Viren zirkulieren zwischen Vögeln, als Virusreservoir und Mücken, als Vektoren. Monitoringstudien in Wild- und Zoovögeln, sowie der einheimischen Stechmückenpopulation geben wertvolle Hinweise über die Verbreitung neuer und neuartiger Zoonoseerreger. Vor diesem Hintergrund wurde deshalb ein deutschlandweites Wildvogelnetzwerk aufgebaut, um Zug- und Standvögel flächendeckend insbesondere auf die zoonotischen Flaviviren WNV und USUV zu untersuchen. Zu diesem Zweck wurden zum einen Blutproben von lebenden Wild- und Zoovögeln von verschiedenen ornithologisch ausgerichteten Netzwerkpartnern eingesandt. Zum anderen wurden Organproben von verendeten Vögeln, die im Rahmen des WNV Referenzlabors des Friedrich-Loeffler Instituts eingesandt wurden, in diese Studie

integriert. Alle diese Wildvogelproben wurden sowohl molekularbiologisch also auch serologisch umfangreich untersucht. Manuskript I und II fassen die Ergebnisse des deutschlandweiten Wildvogelmonitorings der Jahre 2014 bis 2018 zusammen. Im ersten Untersuchungszeitraum von 2014 bis 2016 (n=4), war die Anzahl der USUV positiven Vögel relativ gering, im Vergleich zu den Jahren 2017 und 2018 (n=57). Eine Ausbreitung des USUV nordwärts war allerdings bereits im Lebendvogelmonitoring erkennbar und wurde durch die Daten der an USUV verendeten Vögel untermauert. Besonders im Jahr 2018 kam es zu einer massiven Ausbreitung des USUV in ganz Deutschland verbunden mit einer Vielzahl verschiedener betroffener Vogelarten. Phylogenetische Analysen zeigen eine Ausbreitung der USUV Linien Afrika 3 und Europa 3, und den erstmaligen Nachweis der USUV Linie Europa 2 in Deutschland. Während des gesamten Untersuchungszeitraums wurden neutralisierende Antikörper gegen das USUV vor allem in Standvögeln nachgewiesen, aber auch in Kurz- und Teilstreckenziehern. In den Jahren 2017 und 2018 war ein deutlicher Anstieg der Seroprävalenz gegen das USUV, im Vergleich zu den vorherigen Jahren erkennbar, insbesondere in Regionen in denen das Virus erstmals nachgewiesen wurde. WNV-spezifische RNA konnte in keinem der untersuchten Vögel unseres Lebendmonitorings zwischen 2014 und 2018 nachgewiesen werden, obwohl in verendeten Vögeln und Pferden dieser Nachweis im Jahr 2018 in Deutschland gelang. Neutralisierende Antikörper gegen das WNV wurden vorrangig in Langstreckenziehern gefunden, was mit hoher Wahrscheinlichkeit auf einen Viruskontakt in den südlich gelegenen Winterquartieren hindeutet. Im Jahr 2018 konnten erstmals hohe Antikörpertiter auch in Standvögeln und Kurzstreckenziehern in Ostdeutschland nachgewiesen werden. Diese hohen Antikörper beruhen sehr wahrscheinlich auf einen kürzlich stattgefundenen Kontakt mit dem WNV in diesem Gebiet und sind Anzeichen dafür, dass eine Viruszirkulation in der einheimischen Vogelpopulation vorkommt. Untermauert werden diese ersten serologischen Hinweise aus 2018 nun durch das gehäufte Auftreten von WNV im Jahr 2019 in bereits über 50 Vögeln und bisher 15 Pferden in diesem Bereich.

Das Tick-Borne Enzephalitis Virus (TBEV) ist ein weiteres zoonotisches Arbovirus der Gattung Flavivirus. Die mögliche Rolle von Vögeln im Übertragungszyklus dieses Virus ist

noch weitgehend ungeklärt, weshalb im zweiten Teil dieser Studie jeweils 20 Hühner intramuskulär und 20 Hühner subkutan mit dem Stamm TBEV Neudoerfl infiziert wurden. Zudem wurden 19 Enten subkutan inokuliert. Die Hühner zeigten keinerlei klinische Symptome. Vereinzelt konnte in einigen Tieren eine Virämie oder Virusausscheidung nachgewiesen werden, jedoch vorrangig in der subkutan inokulierten Gruppe. Die Ergebnisse der infizierten Enten und Hühner waren nahezu identisch. Auch bei den Enten trat keine klinische Symptomatik auf und TBEV-RNA war nur sporadisch in einigen Blut- und Tupferproben nachweisbar. Eine Serokonversion konnten jedoch in allen Hühnern (bis auf einem) und allen Enten nachgewiesen werden. Postmortal gelang in den Hühnern der Nachweis von TBEV-RNA vereinzelt in Gehirn, Herz, Leber und der Zäkaltonsille. Im Gegensatz dazu waren alle infizierten Enten TBEV-RNA positiv im Gehirn und zeigten zudem eine Enzephalitis in der histopathologischen Untersuchung. Außerdem konnte in 14 Enten TBEV- Antigen mittels Immunhistochemie im Gehirn nachgewiesen werden. Da sowohl die Hühner, als auch die Enten keine hohe und langanhaltende Virämie entwickelt haben, spielen diese Vogelspezies sehr wahrscheinlich keine aktive Rolle im Übertragungszyklus des TBE Virus. Jedoch können Hühner und Enten, aufgrund ihrer schnellen Serokonversion und der hohen Antikörpertiter, als Sentineltiere genutzt werden. Serologische Untersuchungen von Wirtschaftsgeflügel in Freilandhaltung (z.B. zum Zeitpunkt der Schlachtung) könnte zusätzlich zur molekularbiologischen Untersuchung von Zecken eingesetzt werden, um neue bisher unentdeckte TBEV Naturherde zu identifizieren.

Die hier dargestellten Ergebnisse betonen, dass Monitoring- und Pathogenesestudien an Wildvögeln als Frühwarnsystem für das Auftreten zoonotischer Krankheitserreger eingesetzt werden können. Eine Fortführung des deutschlandweiten Wildvogelmonitorings ist deshalb unerlässlich.

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Chapter 10: Authors´ Contributions

Wild bird monitoring (Manuscript I + II)

Coordination and collaboration with the collectors/providers of the bird blood samples. Design of sampling strategies. Investigation of the collected samples (e.g. RNA extraction, qRT-PCR, ELISAs, differentiating VNTs of each sample for both WNV and USUV and virus isolations on tissue cultures under BSL-3 conditions). Sequencing and phylogenetic analysis of the USUV positive samples. Interpretation of the ornithological data together with the regional origin of the samples and interpretation of the molecular and serological results. Compilation and presentation of the results. Writing of the original draft of the manuscript.

Experimental infections of chickens and ducks (Manuscript III + IV)

Planning the experimental design and writing of the first draft of the application for these animal experiments according to German animal welfare legislation. Realization of the animal experiments (e.g. virus cultivation and titration, i.m. and s.c. inoculation of the chickens and ducks, checking for their health status, blood and swab sampling, euthanasia and dissection; all under BSL-3 conditions). Design of the synthetic TBEV-RNA for quantification of the genome copies. Investigation of the samples obtained (e.g. preparation of blood/swab/tissue samples for further investigations, RNA extraction, qRT-PCR, VNT, ELISA under BSL-3 conditions). Pathology support (necropsies, embedding of fixed samples for microscopy and IHC, IHC readings, histopathology was carried out by FLI pathologist). Interpretation of the data and writing the original draft of the manuscript.

Martin H. Groschup and Ute Ziegler supervised the studies scientifically.

Ute Ziegler introduced me to the serological methods (VNT, ELISA) and supported me during the animal trials.

Martin Eiden and Markus Keller taught me the molecular diagnostic methods.

Christine Fast introduced me to the IHC methodology and did H&E readings (challenge studies).

Chapter 11: Acknowledgement

First of all, I would like to thank Prof. Dr. Martin H. Groschup and Dr. Ute Ziegler for their supervision and support. I am very grateful for the opportunity to work at the Institute for Novel and Emerging Infectious Diseases at the FLI and being part of this unique and very interesting project. I would like to thank you for the assistance whenever I had questions, support during the animal trials and encouraging words, especially before forthcoming presentations. Thank you also for the help during the last weeks, when time was running short.

I address my special gratitude to the staff of the different bird clinics, rehabilitation centers and zoological gardens as part of our nation-wide wild bird surveillance network as well as the staff of the veterinary authorities and veterinary laboratories of the federal states of Germany for diligently collecting the wild bird blood samples and enabling this project. Special thanks to Dr. Dominik Fischer for the support in all ornithological questions.

Thanks for the help and support to lots of people during the animal experiments, especially Dr. Ute Ziegler, Dr. Christine Fast, René Schöttner, Gesine Kreplin and all animal caretakers.

Moreover, I would like to thank Dr. Christine Fast for giving me valuable advice regarding the interpretation of the pathological finding and the immunohistochemistry.

Furthermore, I am grateful to Dr. Martin Eiden and Dr. Markus Keller, for the support with the sequence analysis and questions regarding molecular biology and qRT-PCR.

Thank you to Cornelia Steffen and Katja Wittig for your excellent technical support and the friendly and warm atmosphere in the lab. It was a pleasure to work with you.

Special thanks also to Cora Holicki for her close friendship, but also the support during the last years and proofreading my thesis and manuscripts.

I would also like to thank a lot of people in and outside the laboratory for the support during my PhD time, in particular Arian Köhler, Annika Groth, Rebecca König, Dr. Miriam Andrada Sas, Nicole Cichon, Maren Penning, Benjamin Gutjahr, Florian Binder and René Schöttner. Thank you for the great time, friendship and motivation during my PhD studies.

Most of all I want to thank my family and Jakob Benkner who supported me all the time and have always been there for me, when I needed them. I would not have come so far without your optimism and encouragement. Thank you very much.