Usutu virus (USUV) is an emerging zoonotic virus within the Flaviviridae family that can cause neurological disease in humans and wild birds. USUV is maintained in an enzootic cycle between wild birds, primarily passerine species, and ornithophilic mosquitoes, predominantly Culex spp. mosquitoes. Since its first isolation in 1959 in South Africa, USUV has spread throughout sub-Saharan Africa and Europe. Its emergence into Europe was marked by large die-offs, or epizootics, of the Eurasian blackbird (Turdus merula), as well as an increase in human cases. This dissertation sought to understand whether USUV has evolved to become more pathogenic in humans or transmissible in birds. We compared the pathogenesis of five different USUV isolates, four recent isolates: Spain 2009, Netherlands 2016, Senegal 2003, Uganda 2012, and South Africa 1959, in an interferon α/β receptor knockout (Ifnar-/-) mouse model. We observed significant mortality, high viral levels in serum and tissues in all USUV strains except for the Netherlands 2016 strain. Eighteen non-synonymous mutations were identified throughout the genome of Netherlands 2016 strain compared to the other USUV isolates. To further understand USUV infection in wild birds, we developed a physiologically relevant model of infection using juvenile chickens. In juvenile chickens, we found that the European strains were characterized by more pathogenesis and higher viral titers in tissues compared to the African strains. This work established the first viremic bird model of USUV infection. Passerine birds have been suggested to be important for USUV maintenance, however a species competent for transmission has not been identified. We first determined that wild-caught house sparrows (Passer domesticus) and Culex quinquefasciatus mosquitoes were susceptible to Netherlands 2016 and Uganda 2012 USUV strains. Following an infectious feed to assess enzootic transmission, house sparrows were able to transmit both USUV strains to Cx. quinquefasciatus mosquitoes, with the Netherlands 2016 strain being more infectious compared to the Uganda 2012 strain. The collection of these chapters provides great insights on the pathogenesis of distinct USUV strains, disease presentation in birds, and enzootic transmssion of USUV. Additionally, they indicate that USUV emergence in the United States is entirely feasible. / Ph.D. / Usutu virus (USUV) is a mosquito-borne virus that can cause neurological disease in humans and wild birds. It is in the same virus family as West Nile virus and St. Louis encephalitis virus, both of which already occur in the United States. USUV is typically maintained by spreading back and forth between wild birds, typically songbird species, and mosquitoes with an affinity for biting birds, such as Culex spp. mosquitoes. USUV was first found in South Africa in 1959 and has since spread throughout sub-Saharan Africa and Europe. Upon emergence in Europe, USUV caused massive die-offs in Eurasian blackbirds (Turdus merula) as well as an increase in human cases. My work sought to understand whether USUV has evolved to cause more disease in humans or transmissibility in wild birds. We first assessed the disease severity of African and European USUV strains in an immune compromised mouse model. We showed that African strains of USUV caused more disease and developed higher viral loads than European strains in mice that lack full immunity. To further understand USUV infection in wild birds, we developed a relevant model of infection using juvenile chickens. In juvenile chickens, we found that the European strains were characterized by more disease and higher viral loads in tissues compared to the African strains. This work established the first bird model of USUV infection where birds developed infectious virus that is detectable in blood. Finally, to identify possible reservoir hosts of USUV, we assessed USUV transmission between house sparrows (Passer domesticus) and Cx. quinquefasciatus mosquitoes. We found that house sparrows were able to transmit a European and African USUV strain to biting Cx. quinquefasciatus mosquitoes, with the European strain being more infectious to these mosquitoes. This indicates that house sparrows are competent hosts for USUV transmission and thus a potential reservoir species. The collection of these chapters provides great insights on the severity of distinct USUV strains, disease presentation in birds, and transmssion of USUV. Additionally, they indicate that USUV emergence in the United States is a very real possibility.

Mosquito-borne viruses, such as dengue virus (DENV), Zika virus (ZIKV), chikungunya virus (CHIKV), yellow fever virus (YFV), Japanese encephalitis virus (JEV), and West Nile virus (WNV) are major threats to global public health resulting in millions of infections and hundreds of thousands of deaths annually. The presence of these viruses and their increasing emergence/spread continues to escalate. Notably, Usutu virus (USUV; Genus: Flavivirus; Family: Flaviviridae) is one such pathogen currently causing mass die-offs of avian hosts throughout Europe. USUV is categorized in the Japanese Encephalitis virus (JEV) antigenic complex and thus shares many antigenic and pathologic characteristics with fellow members, such as JEV and WNV. Respective to human infections, USUV cases are generally asymptomatic; nonetheless, acute cases have been reported. These acute cases typically cause mild symptoms, such as fevers and rashes; however, more severe cases can result in neurologic diseases, such as encephalitis and/or meningoencephalitis. In addition to these pathologic similarities, USUV shares several ecological and geographical traits with WNV, a pathogen responsible for several outbreaks during its spread from Africa, to Europe, and eventually the United States. Currently, WNV is considered endemic in areas across the United States due to its transmission via Culex spp.; mosquitoes that are ubiquitous in the United States. These parallels suggest the possible emergence of USUV into the United States and therefore, it is imperative to broaden our knowledge of USUV and assess its potential to become a major global health concern. The overall goal of this thesis was to characterize USUV and evaluate its emergence potential in the United States by: (1) developing infectious clones of recent European and African USUV isolates as tools for characterization and analysis of USUV and (2) assessing the transmission potential of several species of North American mosquitoes. In Aim 1, we show that the aforementioned infectious clones infect and replicate similarly to their parental strains in vitro in both vertebrate and invertebrate models, as well as in transiently immunocompromised CD-1 and IFNAR-/- murine models, and thus serve as useful tools for future molecular studies focusing on USUV. Furthermore, in Aim 2, we describe the ability of field-caught (Southwest Virginia, USA) Culex spp. and Aedes spp. mosquitoes to become infected with a recent European isolate of USUV; although, we report an overall limited potential for these species to transmit this virus. Altogether, these studies form a foundation for understanding the potential emergence of USUV in the United States as well as provide necessary tools needed to aid future research on USUV emergence, transmission, and pathogenesis. / Master of Science / Usutu virus (USUV) is an emerging mosquito-borne virus that was first isolated from a mosquito in 1959 in South Africa, and since then, has become a major problem throughout Africa and Europe causing acute to severe infection in dozens of patients. Additionally, this virus is causing massive die-offs in Eurasian blackbird populations. This is particularly problematic because birds play a critical role in ecosystems as they act as forms of pest control, pollinators, and seed dispersers. Depletion of these species could lead to an imbalance and, eventually, collapse of our natural ecosystem. Additionally, there is a growing concern of USUV making its way into the United States, following a similar track of emergence to WNV's introduction in New York in 1999 and its subsequent spread throughout the states. WNV's introduction to the United States was detrimental to native bird populations and humans, and has caused tens of thousands of infections and thousands of deaths since this introduction. Research has shown USUV causes similar disease symptoms to WNV. The self-limiting illness from these viruses typically includes fever and rashes but some infections can result in more severe cases causing inflammation of the brain and surrounding areas. Like many other prominent mosquito-borne viruses, there is no specific treatment or vaccine for WNV or USUV. Because USUV is so closely related to WNV, and their similar characteristics may point towards similar emergence in the United States, it is essential to garner more information on USUV. The overall goal of this thesis was to establish a reliable tool(s) for further characterization of USUV and demonstrate the potential for USUV emergence in the United States. We first developed molecular tools, known as viral clones, that are valuable to the scientific community which allows the manipulation of USUV genetic material to perform further downstream studies. Our objective for this initial study was to create a molecular tool that would behave similarly to their natural, or "parental", virus. The results from this study suggest we have successfully produced these tools. Furthermore, we sought to determine the potential for field-caught mosquitoes from Southwest Virginia, USA to transmit a recently isolated strain of USUV. These data suggest that while these mosquitoes do have the ability to become infected with USUV, they have a limited potential to transmit this virus to animal hosts. Altogether, these studies have allowed us to expand our knowledge on USUV's potential emergence in the United States and develop powerful tools to continue this essential research.

Mosquito-borne viruses, including dengue virus (DENV), Usutu virus (USUV), West Nile virus (WNV), and Japanese encephalitis virus (JEV), are rapidly emerging, global pathogens. Though the number of people impacted by each virus varies, there have been thousands to millions of people infected. The focus of this thesis work centers around USUV and WNV; both have RNA genomes and belong to the Flaviviridae virus family. Both WNV and USUV were initially isolated in Africa and have since spread to Europe; interestingly, WNV has also spread globally and is considered endemic in the Americas. Similar to other flaviviruses, USUV and WNV are maintained in a mosquito vector-avian host transmission cycle, with spillover infection into humans. Human infections of WNV and USUV are usually asymptomatic, but in severe cases can cause neuroinvasive disease. WNV and USUV belong to the JEV serocomplex group, which indicates that antibodies produced against these viruses share a common antigen; the common antigen is hypothesized to be the envelope (E) protein on the outside of the virion. Neutralizing antibodies against both WNV and USUV have been found in birds and humans across Europe. In vitro cross-neutralization of WNV and USUV has been modeled experimentally and been observed in clinical settings. The neutralizing antibody response generated against WNV has been studied extensively in mouse models; however, there are few studies which examine the neutralizing antibody response generated against USUV. Whether prior WNV exposure protects against USUV disease is also unknown. The main goal of this thesis was to characterize how a primary flavivirus exposure would influence a secondary flavivirus exposure; specifically, we wanted to observe if WNV exposure would protect against USUV disease in vivo and generate a cross-neutralizing antibody response in vitro. For the WNV exposure, we used an attenuated vaccine strain of WNV that contains the WNV E gene (D2/WN-V3) developed by our collaborators. We hypothesized that treatment with D2/WN-V3 would protect against USUV infection. Two in vivo models were used: CD-1 mice and interferon alpha-beta receptor 1 deficient (Ifnar1-/-) mice. We discovered that sera from mice vaccinated with D2/WN-V3 neutralized both WNV and USUV in vitro. In the Ifnar1-/- model, we observed that vaccinated mice had higher survival rates and lower USUV viremia levels after USUV challenge. This work helps characterize the consequences of flavivirus antibody cross-neutralization in vitro and cross-protection in vivo. As the flavivirus field moves toward the goal of creating a pan-flavivirus vaccine, both cross-reactive antibodies and cross-protection need to be considered. / Master of Science / West Nile virus (WNV) and Usutu virus (USUV) are mosquito-borne viruses that were originally isolated in Africa during the 20th century. Both viruses are maintained through a transmission cycle between mosquito vectors and avian hosts. Mosquitos transfer the infectious agent (WNV or USUV) through feeding on a bird (usually a passerine species); once in the bird, the virus can replicate to high levels. Human infections of WNV and USUV from mosquitos can also occur, with symptoms ranging from mild febrile illness to severe encephalitis or meningitis. Over the past few decades, WNV and USUV have spread to Europe, most likely through infected migratory birds. Interestingly, mosquito surveillance studies in mainland Europe have found mosquitos that tested positive for both USUV and WNV. In Europe, antibodies for both viruses have been found in humans and birds, indicating a previous exposure to WNV, USUV, or both. The neutralizing antibody response is a critical immune defense against viral infections. Neutralizing antibodies bind strongly to the outside of the virion (virus particle), preventing the virion from interacting with and infecting the host cell. For WNV and USUV, one of the targets that neutralizing antibodies bind to is the outer envelope (E) protein of the virion. In clinical settings and experimental studies, cross-neutralization of WNV and USUV has been documented. During cross-neutralization, a serum sample containing neutralizing antibodies against WNV can also neutralize USUV, and vice versa. Although the neutralizing response against WNV has been characterized in humans and lab animal models such as mice, there is little research regarding the neutralizing response against USUV. Importantly, whether prior WNV exposure provides protection against USUV infection is currently unknown. The main goal of this thesis was to characterize the disease outcome and neutralizing response against USUV after a WNV exposure. For the WNV exposure, we used a vaccine strain of WNV that contains the E gene (D2/WN-V3) developed by our collaborators. We predicted that vaccinated mice would avoid USUV clinical signs of disease and generate neutralizing responses to WNV and USUV. To do this work, we used two laboratory mouse models: mice with an intact immune response system (CD-1) and mice with a stunted immune response (Ifnar1-/-). We discovered that serum from vaccinated mice did cross-neutralize WNV and USUV. In the Ifnar1-/- model, vaccinated mice had higher survival rates and lower levels of virus in blood after USUV infection compared to unvaccinated mice. Ultimately, this work highlights the importance of characterizing the immune response against similar viruses and will inform the development of human vaccines for both viruses.

Usutu virus (USUV) is an emerging mosquito-borne arbovirus within the genus Flavivirus, family Flaviviridae. Similar to the closely related West Nile virus (WNV), USUV infections are capable of causing mass mortality in wild and captive birds, especially blackbirds. In the last few years, a massive spread of USUV was present in the avian population of Germany and other European countries. To date, no specific antiviral therapies are available. Nine different approved drugs were tested for their antiviral effects on the replication of USUV in vitro in a screening assay. Ivermectin was identified as a potent inhibitor of USUV replication in three cell types from different species, such as simian Vero CCL-81, human A549 and avian TME R. A 2- to 7-log10 reduction of the viral titer in the supernatant was detected at a non-cytotoxic concentration of 5 µM ivermectin dependent on the applied cell line. IC50 values of ivermectin against USUV lineage Africa 3 was found to be 0.55 µM in Vero CCL-81, 1.94 µM in A549 and 1.38 µM in TME-R cells. The antiviral efficacy was comparable between the USUV lineages Africa 2, Africa 3 and Europe 3. These findings show that ivermectin may be a candidate for further experimental and clinical studies addressing the treatment of USUV disease, especially in captive birds.

West Nile virus (WNV), Saint Louis encephalitis virus (SLEV), and Usutu virus (USUV) are emerging mosquito-borne flaviviruses. These viruses are phylogenetically closely related and belong to the Japanese encephalitis serocomplex group. Similar to other flaviviruses, these viruses are enveloped, with genomes comprising positive-sense, single-stranded RNA approximately 11 kb in length. Upon translation, a single polyprotein is produced, consisting of three structural and seven non-structural proteins. These proteins function in virus binding to the cell membrane, entry into cells, replication, immune evasion, and the production of new virus progeny. Typically, these viruses are maintained in a sylvatic cycle involving avian hosts, such as passerine birds, and mosquitoes. However, they can accidentally spill over to humans through mosquito bites or wildlife exposure. Although humans generally remain asymptomatic and do not support sufficient viral replication for transmission, they can develop febrile disease and, in some cases, severe neuroinvasive diseases, especially among the elderly or immunocompromised individuals. Due to their co-circulation in the same geographical areas and sharing similar hosts and vectors, individuals in Italy and Germany have been detected as seropositive for WNV and USUV, while seropositivity for WNV and SLEV has been observed in the Americas. Viruses in the Japanese encephalitis virus serocomplex group exhibit significant antigenic similarity. The envelope protein alone contains 12 distinct epitopes and at least three highly conserved epitopes among the JEV serocomplex. Consequently, infection with one member of the JEV serocomplex group, such as WNV, induces WNV-specific antibodies and heterotypic antibodies that can cross-neutralize other members of the JEV serocomplex group, such as USUV and SLEV. Therefore, cross-reactive epitopes can protect against heterologous virus challenges to varying extents, depending on the accessibility of the antibodies to the epitopes. Prior infection with WNV or its envelope domain III (EDIII) or non-structural protein 1 (NS1) protected mice from lethal JEV challenges. Vaccination against WNV protected mice from lethal USUV challenges, and vice versa. Immunity to JEV or SLEV protected hamsters from lethal WNV challenges. Although human sera immune to WNV cross-neutralized USUV and SLEV in vitro during serodiagnosis, the actual mechanism of cross-protection among WNV, USUV, and SLEV remains poorly characterized. Therefore, this study aims to understand the mechanism of cross-protection. Specifically, this research investigated whether human plasma immune to WNV could cross-protect mice from encephalitis caused by SLEV or USUV. Initially, WNV-specific human convalescent plasma and mouse WNV convalescent serum (as a positive control) neutralized WNV and cross-neutralized USUV and SLEV in vitro in a neutralization test. Subsequently, immunocompetent mice were intraperitoneally injected with human WNV convalescent plasma, human normal plasma, mouse WNV convalescent serum, or mouse normal serum the day before being challenged with WNV, SLEV, or USUV via footpad injection. We found that human WNV convalescent plasma provided mice with strong protection against neuroinvasive encephalitis caused by WNV. Additionally, human WNV convalescent plasma reduced the viremia titers of SLEV and USUV for several days during acute infection. Human WNV convalescent serum also demonstrated a trend towards protecting mice from SLEV-induced encephalitis, as evidenced by lower SLEV titers in the brain and histopathology scores. These findings will aid in decoding the mechanisms of cross-protection among the JEV serovars, developing therapeutic strategies against WNV, SLEV, and USUV, and anticipating potential disease outcomes, especially in regions where multiple viruses of the JEV serocomplex are endemic. / Master of Science / West Nile virus (WNV), Saint Louis encephalitis virus (SLEV), and Usutu virus (USUV) are emerging flaviviruses transmitted by mosquito bites, primarily among perching birds. However, mosquitoes can also transmit these viruses to animals and humans, especially in regions where these viruses are prevalent. The immune system, which defends against pathogens and other diseases, usually combats these viruses effectively, preventing most people from developing symptoms. The immune system has two main branches: the innate immune system, which confers immediate defense, and the adaptive immune system that includes antibodies and certain long-lasting memory cells, that can fight off infections years after the initial exposure to the same or similar disease-causing agents. Occasionally, the immune system fails to fight these viruses, particularly in the elderly or those with chronic diseases, leading to fever or severe brain inflammation called encephalitis. Currently, WNV and SLEV are circulating in the Americas, while WNV and USUV are present in European countries. Due to similar transmission methods, infection patterns, and geographical overlap, individuals might be sequentially infected with WNV and USUV in Europe, and WNV and SLEV in the Americas in their lifetime. These viruses also share common antigens, which can induce similar immune responses. Therefore, the immune response to one virus might protect against another with similar antigens. It has been reported that the immune response induced by WNV can protect against encephalitis caused by USUV or SLEV. However, it remains unclear whether this cross-protection is mediated by antibodies or a certain type of immune cells called T cells. This study investigates whether antibodies induced by WNV infection can protect against SLEV or USUV in a mouse model. Plasma, the part of blood containing antibodies, is referred to as convalescent plasma when collected after an individual has recovered from an infection or disease. Human WNV convalescent plasma was tested against SLEV and USUV using a plaque reduction neutralization test to determine the antibodies’ ability to prevent viral infection in a laboratory setting. Human WNV convalescent plasma effectively prevented SLEV and USUV from infecting cells. We then developed a mouse model that could be infected with SLEV or USUV and mimic human disease. Groups of mice were systematically transferred with human WNV convalescent plasma, human normal plasma, mouse WNV convalescent serum, or mouse normal serum one day before the infection with WNV, SLEV, or USUV. Disease conditions, such as weight loss, reduced movement, hunchback, fur loss, and occasional paralysis, were monitored until the infected mice were humanely euthanized. After euthanasia, the brains of the mice were collected to measure viral load and examine signs of encephalitis. We observed asymptomatic disease outcomes reflecting natural human infection. Both human and mouse WNV convalescent samples reduced viral load in the blood for a period in both SLEV and USUV-challenged groups. Mice treated with human WNV convalescent plasma showed a trend of lower SLEV in their brains. Additionally, mice treated with mouse WNV convalescent serum had lower SLEV titers in their brains compared to those treated with mouse normal serum. Overall, these findings suggest that human WNV convalescent plasma provides some crossprotection against SLEV- and USUV-induced diseases. Understanding the mechanism of crossprotection is crucial for developing therapeutics against these viruses and predicting disease outcomes in areas where multiple viruses of the Japanese encephalitis virus serocomplex are prevalent.

Die zunehmende Ausbreitung des Usutu-Virus in Europa als Ursache für fatale Ausbruchsgeschehen innerhalb der Avifauna, insbesondere unter Sperlingsvögeln (Passeriformes) und Eulenartigen (Strigiformes), stellt in Zusammenhang mit einem neuroinvasiven sowie zoonotischen Potential ein Risiko für die Veterinär- sowie Humanmedizin dar. Trotz dieser Relevanz stehen derzeit keine zugelassenen Therapeutika gegen eine Usutu-Virus-Infektion zur Verfügung. Auf Basis indikationsfremder Substanzen mit pharmakologischer Zulassung im Sinne des drug repositioning sowie auf Grundlage der Gegenregulation viral-induzierter Modulationen des Zellmetabolismus wurde die Identifikation antiviraler Therapieoptionen gegen das Usutu-Virus in vitro angestrebt.:Abkürzungsverzeichnis Abbildungsverzeichnis Tabellenverzeichnis 1 Einleitung 2 Literaturübersicht 2.1 Das Usutu-Virus 2.1.1 Ursprung, Klassifikation und Epidemiologie 2.1.1 Transmissionszyklus und Wirtstropismus 2.1.2 Aufbau des Virions und des Genoms 2.1.3 Veterinärmedizinische Relevanz und pathologische Ausprägung 2.1.4 Humanmedizinische Bedeutung als Zoonose-Erreger 2.1.5 Vergleichende Aspekte zum West-Nil-Virus 2.2 Antivirale Präventions- und Therapieoptionen 2.2.1 Möglichkeiten und Grenzen der Immunprophylaxe 2.2.2 Pharmaka mit potentiell antiviraler Wirkung gegen Flaviviren 2.2.3 Identifizierte Substanzen gegen das Usutu-Virus 2.3 Zelluläre Systeme als antivirale Zielobjekte 2.3.1 Das Interferon-System und seine antivirale Schutzfunktion 2.3.2 Viral-induzierte Modulation des Wirtszellmetabolismus 3 Zielstellungen der Dissertation 4 Material 4.1 Zelllinien 4.2 Viruslinien 4.3 Zellkulturmedien 4.4 Pharmakologische und andere Substanzen 4.5 Chemikalien 4.6 Lösungen und Puffer 4.7 Antikörper 4.8 Reagenzien und Kit-Systeme 4.9 Enzyme und Nukleotide 4.10 Primer 4.11 Verbrauchsmaterialen 4.12 Geräte 4.13 Datenbanken und Software 5 Methodik 5.1 Zellkultivierungstechnik und PBMC-Isolation 5.2 Isolation von Usutu-Virus-Linien aus Gewebeproben in Zellkultur 5.2.1 Aufbereitung aviären Organmaterials 5.2.2 Typisierung von in Deutschland zirkulierenden Usutu-Virus-Linien (2019, 2020) 5.2.3 Virusanzucht in verschiedenen Zellkulturen zur Virusstock-Generierung 5.3 Quantifizierung des extrazellulären Virustiters 5.4 Immunfluoreszenzanalyse 5.5 Präparation pharmakologischer und anderer Substanzen 5.6 Infektionsansätze mit dem Usutu-Virus und WNV 5.7 Durchflusszytometrische Analyse 5.8 Zytotoxizitätsstudien 5.9 Extrazelluläre Fluxanalyse mittels Agilent Seahorse XF-Technologie 5.10 Statistische Analyse 6 Ergebnisse 6.1 Isolation des Usutu-Virus in Zellkultur 6.2 Replikationsdynamik des Usutu-Virus in verschiedenen Zelllinien 6.3 Pharmakologisches Screening zur antiviralen Wirksamkeit gegen das Usutu-Virus 6.4 Identifikation und Charakterisierung der antiviralen Eigenschaften von Ivermectin 6.5 Wirkung von Ivermectin gegen Linie 2 des West-Nil-Virus in einer aviären Zelllinie 6.6 Metabolischer Phänotyp Usutu-Virus-infizierter Zelllinien 6.7 Antivirale Inhibition der Glykolyse durch 2-Deoxy-D-Glukose 6.8 Einfluss von exogenem Interferon auf den Wirtszellmetabolismus unter Infektion 6.9 Auswirkung der Interferon-Rezeptor-Defizienz auf den Metabolismus unter Infektion 7 Diskussion 7.1 Typisierung und Isolation des Usutu-Virus in Zellkultur 7.2 Charakterisierung der zelltypspezifischen Permissivität und Replikationskinetik 7.3 Identifikation antiviral wirksamer Substanzen gegen das Usutu-Virus 7.4 Usutu-Virus-induzierte Modulation des Metabolismus als antiviraler Ansatz 8 Ausblick 9 Zusammenfassung 10 Summary 11 Literaturverzeichnis 12 Anhang 12.1 Zusatzmaterial 12.2 Publikation 1 12.3 Publikation 2 12.4 Publikation 3 12.5 Weitere Veröffentlichungen 13 Danksagung / The emerge of Usutu virus (USUV) in Europe as a causative agent of fatal outbreaks in avifauna, notably in Passeriformes and Strigiformes, as well as its neuroinvasive and zoonotic potential emphasize a considerable risk in veterinary and human medicine. Despite its relevance, recently, no approved drugs against USUV infections are available. The identification of antiviral therapeutic options against USUV in vitro was addressed based on the analysis of approved drugs of other medical indications in terms of drug repositioning and the counteraction of viral-induced alterations of the cellular metabolism.:Abkürzungsverzeichnis Abbildungsverzeichnis Tabellenverzeichnis 1 Einleitung 2 Literaturübersicht 2.1 Das Usutu-Virus 2.1.1 Ursprung, Klassifikation und Epidemiologie 2.1.1 Transmissionszyklus und Wirtstropismus 2.1.2 Aufbau des Virions und des Genoms 2.1.3 Veterinärmedizinische Relevanz und pathologische Ausprägung 2.1.4 Humanmedizinische Bedeutung als Zoonose-Erreger 2.1.5 Vergleichende Aspekte zum West-Nil-Virus 2.2 Antivirale Präventions- und Therapieoptionen 2.2.1 Möglichkeiten und Grenzen der Immunprophylaxe 2.2.2 Pharmaka mit potentiell antiviraler Wirkung gegen Flaviviren 2.2.3 Identifizierte Substanzen gegen das Usutu-Virus 2.3 Zelluläre Systeme als antivirale Zielobjekte 2.3.1 Das Interferon-System und seine antivirale Schutzfunktion 2.3.2 Viral-induzierte Modulation des Wirtszellmetabolismus 3 Zielstellungen der Dissertation 4 Material 4.1 Zelllinien 4.2 Viruslinien 4.3 Zellkulturmedien 4.4 Pharmakologische und andere Substanzen 4.5 Chemikalien 4.6 Lösungen und Puffer 4.7 Antikörper 4.8 Reagenzien und Kit-Systeme 4.9 Enzyme und Nukleotide 4.10 Primer 4.11 Verbrauchsmaterialen 4.12 Geräte 4.13 Datenbanken und Software 5 Methodik 5.1 Zellkultivierungstechnik und PBMC-Isolation 5.2 Isolation von Usutu-Virus-Linien aus Gewebeproben in Zellkultur 5.2.1 Aufbereitung aviären Organmaterials 5.2.2 Typisierung von in Deutschland zirkulierenden Usutu-Virus-Linien (2019, 2020) 5.2.3 Virusanzucht in verschiedenen Zellkulturen zur Virusstock-Generierung 5.3 Quantifizierung des extrazellulären Virustiters 5.4 Immunfluoreszenzanalyse 5.5 Präparation pharmakologischer und anderer Substanzen 5.6 Infektionsansätze mit dem Usutu-Virus und WNV 5.7 Durchflusszytometrische Analyse 5.8 Zytotoxizitätsstudien 5.9 Extrazelluläre Fluxanalyse mittels Agilent Seahorse XF-Technologie 5.10 Statistische Analyse 6 Ergebnisse 6.1 Isolation des Usutu-Virus in Zellkultur 6.2 Replikationsdynamik des Usutu-Virus in verschiedenen Zelllinien 6.3 Pharmakologisches Screening zur antiviralen Wirksamkeit gegen das Usutu-Virus 6.4 Identifikation und Charakterisierung der antiviralen Eigenschaften von Ivermectin 6.5 Wirkung von Ivermectin gegen Linie 2 des West-Nil-Virus in einer aviären Zelllinie 6.6 Metabolischer Phänotyp Usutu-Virus-infizierter Zelllinien 6.7 Antivirale Inhibition der Glykolyse durch 2-Deoxy-D-Glukose 6.8 Einfluss von exogenem Interferon auf den Wirtszellmetabolismus unter Infektion 6.9 Auswirkung der Interferon-Rezeptor-Defizienz auf den Metabolismus unter Infektion 7 Diskussion 7.1 Typisierung und Isolation des Usutu-Virus in Zellkultur 7.2 Charakterisierung der zelltypspezifischen Permissivität und Replikationskinetik 7.3 Identifikation antiviral wirksamer Substanzen gegen das Usutu-Virus 7.4 Usutu-Virus-induzierte Modulation des Metabolismus als antiviraler Ansatz 8 Ausblick 9 Zusammenfassung 10 Summary 11 Literaturverzeichnis 12 Anhang 12.1 Zusatzmaterial 12.2 Publikation 1 12.3 Publikation 2 12.4 Publikation 3 12.5 Weitere Veröffentlichungen 13 Danksagung

Einleitung: Das West Nil-Virus (WNV) und das Usutu-Virus (USUV) zirkulieren seit vielen Jahren in Europa und sind auch in Deutschland endemisch geworden. Beide Viren können schwere Erkrankungen in Vögeln auslösen. Zudem kann insbesondere WNV auch zu schweren neurologischen Erkrankungen bei Menschen und Pferden führen, und unentdeckte WNV-Infektionen sind ein Risiko für die Sicherheit von Blutspenden. WNV und USUV werden von Stechmücken als biologische Vektoren übertragen, wobei sich verschiedene Mückenspezies in ihrer Vektorkompetenz unterscheiden können. Die Kenntnis über die Vektorkompetenz von Mückenspezies ist essentiell für die effiziente Überwachung und Bekämpfung dieser Viren. Weitgehend unbekannt ist jedoch, welche Auswirkungen Ko-Infektionen mit WNV und USUV auf die Vektorkompetenz von Stechmücken haben. Ziele der Untersuchungen: Ziel der ersten Studie war es, die Vektorkompetenz der bislang wenig untersuchten Mückenspezies Aedes punctor für WNV zu bestimmen. Mit der zweiten Studie sollten Mono- und simultane Ko-Infektionen mit WNV und USUV in verschiedenen Stechmückenarten (Culex pipiens Biotyp pipiens, Culex pipiens Biotyp molestus, Aedes vexans) durchgeführt werden, um die Auswirkungen von Ko-Infektionen auf die Übertragung beider Viren zu bestimmen. Tiere, Material und Methoden: Die für die Versuche verwendeten Stechmücken wurden entweder in Deutschland gesammelt oder stammten aus den Laborkolonien am Friedrich-Loeffler-Institut. Die Infektionen erfolgten oral über Blut, welches ein oder beide Viren enthielt und über Wattestäbchen angeboten wurde. Blutgesogene Weibchen wurden über einen definierten Zeitraum unter definierten Umweltbedingungen inkubiert. Nach Ablauf der Inkubationszeit wurden die noch lebenden Tiere durch das Entfernen der Beine und Flügel immobilisiert, um die Gewinnung von Speichel zu ermöglichen. Ein Teil der Speichelprobe wurde auf eine Zellkultur gegeben, um infektiöse Viruspartikel nachzuweisen. Zur Bestimmung der Infektion, Dissemination und potenzieller Übertragung wurden die Körper, die Beine und Flügel, die Speichelproben sowie der Überstand der Zellkultur mit einer quantitativen Reverse-Transkriptase-Polymerase-Kettenreaktion (RT-qPCR) auf virale RNA untersucht. Im Falle der Ko-Infektionsstudie wurden zeitgleich Mono-Infektionen durchgeführt und die Ergebnisse verglichen, um potenzielle Veränderungen in Empfänglichkeit oder Übertragung feststellen zu können. Ergebnisse: In der ersten Studie zeigte sich, dass Ae. punctor nicht vektorkompetent für WNV ist. Von insgesamt 155 untersuchten Weibchen waren nur 7 Tiere mit WNV infiziert. In den Speichelproben wurden weder infektiöse Viruspartikel noch virale RNA nachgewiesen. In der zweiten Studie konnte gezeigt werden, dass sowohl Cx. pipiens Biotyp pipiens als auch Cx. pipiens Biotyp molestus effektive Vektoren für WNV und USUV sein können. Im Gegensatz dazu waren Ae. vexans Weibchen nicht empfänglich für eine Infektion durch WNV oder USUV. In den Ko-Infektionen zeigte sich, dass die Empfänglichkeit für USUV in Cx. pipiens Biotyp pipiens verringert und in Ae. vexans erhöht war. In Cx. pipiens Biotyp molestus waren hingegen keine Unterschiede zwischen Mono- und Ko-infektionen festzustellen. Bei Cx. pipiens Biotyp molestus wurden infektiöse Partikel beider Viren in Speichelproben gefunden, was auf eine potenzielle Ko-Übertragung hindeutet. Schlussfolgerung: Aufgrund der Ergebnisse kann davon ausgegangen werden, dass Ae. punctor und Ae. vexans derzeit keine Rolle bei der Übertragung von WNV oder USUV spielen und daher vorerst bei Überwachungsprogrammen in Deutschland nicht berücksichtigt werden müssen. Für Cx. pipiens Mücken konnte hingegen die Rolle als Hauptvektoren für WNV und USUV durch Infektionsstudien bestätigt werden. Es konnte zudem gezeigt werden, dass die Interaktion zwischen WNV und USUV in der Stechmücke speziesabhängig variiert. Aufgrund dessen ist eine Untersuchung von Ko-Infektionen in weiteren potenziellen Vektorspezies notwendig, um ein besseres Verständnis der Interaktionen zu bekommen. Die Studie zeigt auch, dass unter Umständen auch nicht vektorkompetente Spezies durch eine Ko-Infektion eine Rolle in der Übertragung der Viren spielen könnten. In Gebieten, in denen WNV und USUV sympatrisch zirkulieren, sollte diese Erkenntnis in den Überwachungs- und Bekämpfungsstrategien berücksichtigt werden. Eine potenzielle Ko-Übertragung beider Viren wurde zwar beobachtet, trat innerhalb dieser Studie aber selten auf und scheint daher eher eine geringe Rolle spielen. / Introduction: West Nile virus (WNV) and Usutu virus (USUV) have been circulating in Europe for many years and have also become endemic in Germany. Both viruses can cause severe diseases in birds. In addition, WNV in particular can also lead to severe neurological diseases in humans and horses, and undetected WNV infections pose a risk to the safety of blood donations. Both WNV and USUV are transmitted by mosquitoes as biological vectors, whereby different mosquito species can differ in their vector competence. Knowledge of the vector competence of various mosquito species is essential for efficient surveillance and control of these viruses. What is largely unknown, however, is the impact of co-infections with WNV and USUV on the vector competence of mosquitoes. Objective: The aim of the first study was to determine the vector competence of the mosquito species Aedes punctor for WNV, which has so far been little studied. The second study aimed to perform mono- and simultaneous co-infections with WNV and USUV in different mosquito species (Culex pipiens biotype pipiens, Culex pipiens biotype molestus, Aedes vexans) in order to determine the impact of co-infections on the transmission of both viruses. Animals, material and methods: The mosquitoes used for the experiments were either collected in Germany or were taken from the laboratory colonies at the Friedrich-Loeffler-Institute. Mosquitoes were orally infected via a blood that contained one or both viruses using cotton sticks. Engorged females were incubated over a defined period of time under defined environmental conditions. At the end of the incubation period, surviving animals were immobilized by removing the legs and wings, and saliva was obtained. Part of each saliva sample was placed on cell culture to detect infectious virus particles. To determine infection, dissemination and potential transmission, the bodies, legs and wings, saliva samples and supernatant of the cell culture were analyzed for viral RNA by a quantitative reverse transcriptase polymerase chain reaction (RT-qPCR). During the co-infection study, mono-infections were carried out at the same time as the co-infections and results were compared in order to be able to determine potential changes in susceptibility or transmission. Results: In the first study, Ae. punctor showed to be not susceptible for WNV. Of a total of 155 females examined, only 7 females were found infected. Neither infectious viral particles nor viral RNA was found in saliva samples. In the second study it could be shown that Cx. pipiens biotype pipiens as well as Cx. pipiens biotype molestus can be effective vectors for WNV and USUV. In contrast, Ae. vexans was not susceptible to an infection with WNV or USUV. In Co-infections, it was shown that the susceptibility to USUV was reduced in Cx. pipiens biotype pipiens and increased in Ae. vexans. In Cx. pipiens biotype molestus, however, no differences were found between mono- and co-infections. In Cx. pipiens biotype molestus mosquitoes, infectious particles of both viruses were found in saliva samples, indicating a potential co-transmission by this species. Conclusion: Based on the results, it can be assumed that Ae. punctor and Ae. vexans do not play a role in WNV and USUV transmission and therefore currently do not need to be included in a surveillance in Germany. In contrast, the role of Cx. pipiens mosquitoes as main vectors for WNV and USUV could be confirmed by infection studies. It could also be shown that the interaction between WNV and USUV in the mosquito vector varies and is species dependent. Therefore, an investigation of co-infections in various potential vector species and even different populations is essential to get a better understanding of the interactions between both viruses within the mosquito. The study also showed that, under certain circumstances, non-vector-competent species might also play a role in the transmission of the viruses in the event of a co-infection. Thus, in areas where WNV and USUV are both endemic, surveillance and control programs should take this knowledge into account. Potential co-transmission of both viruses was observed, but was rare in this study and therefore seems to play a rather minor role.

West Nile virus (WNV) infections were first detected in Germany in 2018, but information about WNV seroprevalence in horses is limited. The study’s overall goal was to gather information that would help veterinarians, horse owners, and veterinary-, and public health- authorities understand the spread of WNV in Germany and direct protective measures. For this purpose, WNV seroprevalence was determined in counties with and without previously registered WNV infections in horses, and risk factors for seropositivity were estimated. The cohort consisted of privately owned horses from nine counties in Eastern Germany. A total of 940 serum samples was tested by competitive panflavivirus ELISA (cELISA), and reactive samples were further tested by WNV IgM capture ELISA and confirmed by virus neutralization test (VNT). Information about potential risk factors was recorded by questionnaire and analyzed by logistic regression. A total of 106 serum samples showed antibodies against flaviviruses by cELISA, of which six tested positive for WNV IgM. The VNT verified a WNV infection for 54 samples (50.9%), while 35 sera neutralized tick-borne encephalitis virus (33.0%), and eight sera neutralized Usutu virus (7.5%). Hence, seroprevalence for WNV infection was 5.8% on average and was significantly higher in counties with previously registered infections (p = 0.005). The risk factor analysis showed breed type (pony), housing in counties with previously registered infections, housing type (24 h turn-out), and presence of outdoor shelter as the main significant risk factors for seropositivity. In conclusion, we estimated the extent of WNV infection in the resident horse population in Eastern Germany and showed that seroprevalence was higher in counties with previously registered equine WNV infections.

The mosquito-borne Usutu virus (USUV) is a zoonotic flavivirus and an emerging pathogen. So far therapeutical options or vaccines are not available in human and veterinary medicine. The bioenergetic profile based on extracellular flux analysis revealed an USUV infection-associated significant increase in basal and stressed glycolysis on Vero and with a tendency for basal glycolysis on the avian cell line TME-R derived from Eurasian blackbirds. On both cell lines this was accompanied by a significant drop in the metabolic potential of glycolysis. Moreover, glycolysis contributed to production of virus progeny, as inhibition of glycolysis with 2-deoxy-D-glucose reduced virus yield on Vero by one log10 step. Additionally, the increase in glycolysis observed on Vero cells after USUV infection was lost after the addition of exogenous type I interferon (IFN) b. To further explore the contribution of the IFN response pathway to the impact of USUV on cellular metabolism, USUV infection was characterized on human A549 respiratory cells with a knockout of the type I IFN receptor, either solely or together with the receptor of type III IFN. Notably, only the double knockout of types I and III IFN receptor increased permissiveness to USUV and supported viral replication together with an alteration of the glycolytic activity, namely an increase in basal glycolysis to an extent that a further increase after injection of metabolic stressors during extracellular flux analysis was not noted. This study provides evidence for glycolysis as a possible target for therapeutic intervention of USUV replication. Moreover, presented data highlight type I and type III IFN system as a determinant for human host cell permissiveness and for the infection-associated impact on glycolysis.

Expanding urbanization, climate change, and population growth contribute to increased transmission and spread of arthropod-borne viruses (arboviruses), many of which cause severe disease in humans. Pathogenic arboviruses include dengue, Zika, tick-borne encephalitis, and sindbis viruses, which together threaten more than half the global population. Thus, there is a constant need for safe, specific, and sensitive molecular tests to identify early-stage infections for accurate diagnosis and molecular epidemiological data for disease prevention and control. The study tested the biosafety of using FTA™ cards when working with pathogenic arboviruses by conducting an infectivity assay using sindbis virus. Conditions for RNA extraction and storage of arboviruses on FTA were analyzed by measuring viral RNA (vRNA) stability using a SYBR-Green, Pan-Flavi RT-qPCR method composed of degenerate primers able to detect a variety of flaviviruses. Data from a Pan-Flavi RT-qPCR study comprising of 222 clinical blood and serum samples collected from a 2018 dengue virus outbreak in Hanoi (Vietnam) was analyzed to establish applicability of FTA for molecular epidemiology and diagnosis. Results showed that sindbis virus infectivity was inhibited by FTA-adsorption. FTA-adsorbed arboviruses were extracted with the highest yield using Trizol extraction and were preserved at storage at 4-20ºC for up to 30 days. The results showed that clinical blood samples acquired higher yields of vRNA for molecular testing than serum samples and that it may be possible to perform sequencing for genomic analysis. The study suggests that FTA cards may facilitate the storage and transportation of adsorbed arboviruses for downstream molecular epidemiological and diagnostic tests.