Annotation: Human cells of neuronal origin represent an excellent tool for the investigation of neuropathogenesis of TBE. The maturation, replication process of tick-borne encephalitis virus (TBEV) and ultrastructural changes induced by infection in the neuroblasts cell line (UKF-NB-4) was studied by electron microscopy. I compared electron microscopical aspects (appearance) of TEM images of neuroblasts cells prepared by (1) conventional chemical fixation, resin-embedding and sectionig; (2) rapid freezing of cell monolayers at high pressure and sectioning of freeze substituted samples. The most interesting fact, however, is that vitrification preserves the cell in close to native state, whereas chemical fixation and dehydration can not take place without extensive intra- and intermolecular cross-linking and aggregation. The appearance of the cytoplasm and nucleoplasm of neu-roblasts cells were different in conditions (1) and (2). The excellent ultrastructure of the cytoplasmic and nuclear membranes and organels of neuroblasts cells processed by (2) confirmed the potentional of the method for preservation of cellular fine structures. The infection of neuroblastoma cells was associated with number of major morphological changes, including proliferation of membranes of the rough endoplasmic reticulum and rearrangement of cytoskeletal structures. The viral particles were located mainly in the cisterna´s of ER but also in the cytoplasm. In the cytoplasm I observed virions in the asso-ciations with microtubules and neurosecretory dense core vesicles. The transport of viral particles inside of the transport vesicles was obsereved from ER to Golgi apparatus. Free nucleocapsids were not confirmed. The observed pattern corresponded to both trans and cis type of maturation. The TBEV-infected neuroblasts cells exhibited either apoptotic or necrotic morphological changes. I observed the apoptotic signs (condensation, margination and fragmentation of chromatin in nucleus) and other alterations, such as disorganisation of cytoplasm, presence of the vacuoles and high density of cytoplasm. This report also de-scribes scanning electron microscope study of the surface features of neuroblasts cells. We observed virus-mediated cytopathic effect. The cells infected with TBEV were rounded with rough and rugged topography.

Le ré-encodage aléatoire des codons à grande échelle est une nouvelle méthode d'atténuation virale qui consiste en l'insertion d'un grand nombre de mutations synonymes, individuellement peu délétères, de façon aléatoire dans une ou plusieurs régions codantes d'un virus. Cette approche permet de diminuer de façon significative et modulable le fitness réplicatif des virus in cellulo et in vivo, ainsi que la pathogénicité du virus chez la souris, tout en induisant une protection immunitaire spécifique et efficace lors d'une nouvelle infection par le virus sauvage. Les virus ré-encodés présentent également une grande stabilité et une absence de réversion ce qui en font des candidats vaccins très prometteurs en termes d'efficacité et de fiabilité pour la conception de candidats vaccins vivants atténués contre une grande variété de virus à ARN. La combinaison du ré-encodage aléatoire et d'une nouvelle méthode de génétique inverse permettant de générer de nouveaux virus en quelques jours: ISA (Amplicon Subgenomique Infectieux), est une approche prometteuse qui pourrait aider au développement de vaccins vivants atténués de nouvelle génération en un temps record. / Large-scale random codon re-encoding is a new method of viral attenuation consisting in the insertion of a high number of slightly deleterious synonymous mutations, randomly, in one or several coding regions of a virus. This approach significantly reduces the replicative fitness of re-encoded viruses in cellulo and in vivo, as viral pathogenicity, while inducing a specific and effective immune response in mice against a new infection with wild-type viruses. Re-encoded viruses also present a high stability and an absence of reversion, making them promising vaccine candidates in term of reliability and efficiency for the conception of new vaccine candidates against a wide variety of RNA viruses. Combination of random re-encoding with a new method of revers genetics allowing to generate new viruses in days : ISA (Infectious Subgenomic Amplicons) would be very helpful to develop new-generation vaccine candidates.

Mammalian Scribble is a target for proteins encoded by human papilloma virus, retro- and flaviviruses. Tick-borne encephalitis virus (TBEV) is a flavivirus that have evolved distinct strategies to escape antiviral responses. Information of how flaviviruses intrude on cell integrity comes from understanding of the roles that host-factors play when they interfere with viruses. The first part of this thesis describes a novel interaction between the TBEVNS5 protein and Scribble. The importance of the interaction was demonstrated by RNAi-mediated depletion of Scribble, which prevented suppression of JAK-STAT signaling by NS5. Together, these results define Scribble as a novel target for NS5. TBEV is known to cause central nervous system disease TBE in humans that can lead to cognitive dysfunction. A unifying theme in CNS related diseases are defects in neuronal extensions. We therefore addressed the effects of TBEV expression in PC12 cell differentiation, which is characterized by extensive neurite growth. Our data show that TBEVNS5 suppresses neurite outgrowth through the Rho GTPase Rac1. These findings provide evidence that Rac1 is an indirect target of NS5 in neurite inhibition. Scribble was recently implicated in spine morphogenesis. Thus, we tested the role of Scribble in neurite elongation. Depletion of Scribble in PC12 cells, reduced neurite density but increased length of those remaining. Moreover, Scribble bound components in the Ras/ERK cascade in a growth factor dependent manner. Together, these results demonstrate that Scribble controls neurite elongation by scaffolding MAPK components. Moreover, as loss of dendritic spines, actin-rich protrusions on neurons, is a feature in cognitive dysfunction we speculate that cognitive dysfunction in TBE might involve disturbed Scribble expression by NS5. We also investigated the binding between NS1 of Influenza A virus and Scribble. The PDZ domains of Scribble are usually selective for specific C-terminal motifs in proteins. Because NS1 has a canonical PDZ motif we tested if binding to Scribble depends on this motif. We found that Scribble binds NS1; the association is dependent on the NS1 C-terminus that is recognized by PDZ3-4 of Scribble. Together, these results suggest that Scribble is a target for the H5N1 NS1 protein / At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: In press. Paper 3: Manuscript. Paper 4: Manuscript.

Tick-borne encephalitis (TBE) virus is an emerging pathogen that causes severe infections in humans. Infection risk areas are mostly defined based on the incidence of human cases, a method which does not work well in areas with sporadic TBE cases. Thus, sentinel animals may help to better estimate the existing risk. Serological tests should be thoroughly evaluated for this purpose. Here, we tested three test formats to assess the use of dogs as sentinel animals. A total of 208 dog sera from a known endemic area in Southern Germany were tested in an All-Species-ELISA and indirect immunofluorescence assays (IIFA), according to the manufacturer’s instructions. Sensitivity and specificity for both were determined in comparison to the micro-neutralization test (NT) results. Of all 208 samples, 22.1% tested positive in the micro-NT. A total of 18.3% of the samples showed characteristic fluorescence in the IIFA and were, thus, judged positive. In comparison to the micro-NT, a sensitivity of 78.3% and a specificity of 98.8% was obtained. In the ELISA, 19.2% of samples tested positive, with a sensitivity of 84.8% and a specificity of 99.4%. The ELISA is a highly specific test for TBE-antibody detection in dogs and should be well suited for acute diagnostics. However, due to deficits in sensitivity, it cannot replace the NT, at least for epidemiological studies. With even lower specificity and sensitivity, the same applies to IIFA.

No description available.

Einleitung Die FSME ist die medizinisch bedeutsamste zeckenübertragene Krankheit in Europa und Asien. In Europa erkranken jährlich mehrere tausend Menschen an der FSME. Zur Seroprävalenz der FSME liegen wenige Daten vor, da seit dem Aufkommen der FSME-Impfung in den 80er Jahren in Deutschland eine Unterscheidung von Impf- und Infektionsantikörpern mittels konventioneller serologischer Methoden nicht mehr möglich war. Die Entwicklung eines neuen ELISAs im Jahr 2020, der auf dem Nichtstrukturprotein 1 (NS1) basiert, macht diese Unterscheidung möglich. Ziele der Arbeit Die Ziele der durchgeführten Studie waren die Bestimmung von epidemiologischen Zielgrößen wie Seroprävalenz, Infektions- und Immunitätsrate sowie das Generieren neuer Daten zum Manifestationsindex im endemischen Landkreis Ortenaukreis in Baden-Württemberg. Das Vorliegen historischer Daten zur Seroprävalenz, die vor Einführung der Impfung im Landkreis Ortenaukreis erhoben wurden, schaffte zudem die interessante Möglichkeit, die Häufigkeit der FSME-Infektionen in einem endemischen Gebiet über einige Jahrzehnte zu vergleichen. Des Weiteren wurden die Blutproben auf neutralisierende Antikörper hin untersucht und die serologische Immunitätsrate der Proben mit vom Robert Koch-Institut bereitgestellten Zahlen zum Impfschutz in der Bevölkerung verglichen. Material und Methoden Insgesamt wurden 2220 Blutspenderestproben, die größtenteils aus dem Landkreis Ortenaukreis in Baden-Württemberg stammen, beprobt. Die FSME-Meldezahlen für das untersuchte Gebiet wurden vom RKI bereitgestellt. Zuerst wurden alle Blutproben mit einem IgG-ELISA auf IgG-Antikörper gegen die FSME getestet. Alle in diesem ELISA positiven Proben wurden im weiteren Verlauf mit dem neuen NS1-ELISA auf IgG-Antikörper gegen das NS1-Protein getestet. Alle im IgG-ELISA positiven und im NS1-ELISA negativen Proben wurden mittels SNT auf das Vorhandensein neutralisierender Antikörper überprüft. Der IIFT wurde zur Überprüfung von kreuzreaktiven Antikörpern verwendet. Die auf Daten der Krankenversicherungen basierende Durchimpfungsrate für den Ortenaukreis wurde vom Robert Koch-Institut bereitgestellt. Ergebnisse Von den 2220 Proben wiesen 57 % (1257/2220) IgG-Antikörper gegen die FSME auf. 125 der 2220 Proben, also 5,6 %, wurden mithilfe des NS1-ELISAs positiv auf eine vorangegangene FSME-Infektion getestet. Bei wenigen Proben wurden kreuzreagierende Antikörper gefunden, die durch andere Flaviviren induziert wurden (7/2220). Es konnte ein Manifestationsindex von ca. 2 % ermittelt werden, sowie eine hohe Anzahl an stillen Infektionen von über 250 pro 100.000 Einwohnern pro Jahr. Für ca. 55 % (1150/2104) der Proben wurde ein neutralisierender Antikörpertiter gegen das FSME-Virus ermittelt. Schlussfolgerung In den dieser Arbeit zugrunde liegenden Studien wurden neue Daten zu FSME-Inzidenz und Manifestationsindex in einem endemischen Gebiet generiert. Nach Vergleich mit Daten aus dem Jahr 1986 ist festzustellen, dass die Ergebnisse für einen etwa siebenfachen Anstieg der FSME-Infektionsraten im untersuchten Gebiet trotz der Verfügbarkeit eines hochwirksamen Impfstoffs sprechen. Die serologische Immunitätsrate ist signifikant höher als die vom Robert Koch-Institut für den Ortenaukreis erhobenen Zahlen zum Impfschutz (ca. 20 %), was für eine längere Haltbarkeit der von der Impfung induzierten Antikörper sprechen könnte, als ursprünglich angenommen. Diese Arbeit verdeutlicht außerdem den Nutzen von Seroprävalenzstudien zur Ergänzung Inzidenz-basierter Risikobewertung von Gebieten, in denen das FSME-Virus endemisch ist.:1 Einleitung......................................................................................1 2 Literaturübersicht.........................................................................2 2.1 Frühsommer-Meningoenzephalitis-Virus (FSME-Virus)................2 2.1.1 Virologie................................................................................2 2.1.2 Virusreplikation.....................................................................3 2.1.3 Epidemiologie in Deutschland..............................................4 2.1.4 Übertragungszyklus des FSME-Virus...................................6 2.1.5 Infektion und Krankheitsverlauf............................................8 2.1.6 Impfung.................................................................................9 2.1.7 Diagnostik............................................................................10 2.1.8 Serologische Methoden.......................................................11 3 Publikationen...............................................................................14 3.1 Eigenanteil Publikation 1..............................................................14 3.1.1 Publikation 1........................................................................16 3.2 Eigenanteil Publikation 2..............................................................25 3.2.1 Publikation 2........................................................................26 4 Diskussion....................................................................................36 5 Zusammenfassung.......................................................................44 6 Summary......................................................................................46 7 Referenzen...................................................................................48 7.1 Literatur........................................................................................48 7.2 Abbildungsverzeichnis.................................................................59 8 Danksagung.................................................................................60 / Introduction TBE is the medically most significant tick-borne disease in Europe and Asia. In Europe, several thousand people fall ill with TBE every year. There is little data available on the seroprevalence of TBE, because the availability of TBE vaccination since the 1980s in Germany has made it no longer possible to distinguish between vaccine- and infection-induced antibodies using conventional serological methods. The development of a new ELISA in 2020 based on the non-structural protein 1 (NS1) makes this distinction possible. Objectives The study’s objectives were to determine epidemiological target points such as seroprevalence, infection and serological immunity rates and to generate new data on the manifestation index in the endemic district of Ortenaukreis in Baden-Württemberg. The availability of historical seroprevalence data collected before the introduction of TBE vaccination in the Ortenaukreis district also provided the interesting opportunity to compare the prevalence of TBE infections in an endemic area over several decades. Additionally, the blood samples were analysed for neutralising antibodies, and the serological immunity rate of the sample size was compared with numbers on the vaccination rate in the population provided by the Robert Koch Institute (RKI). Material and Methods A total of 2220 blood donor samples, most of which came from the Ortenaukreis district in Baden-Württemberg, were sampled. The RKI provided TBE reporting numbers for the region of interest. All blood samples were initially tested for IgG antibodies against TBE using an IgG ELISA. All samples positive in this ELISA were subsequently tested for IgG antibodies against the NS1 protein in the new NS1 ELISA. All samples positive in the IgG ELISA and negative in the NS1 ELISA were tested for the presence of neutralising antibodies with the SNA. The IIFA was used to check for cross-reactive antibodies. The vaccination rate for the Ortenaukreis district, based on health insurance data, was provided by the RKI. Results Of the 2220 samples, 57 % (1257/2220) showed IgG antibodies against TBE. 125 of the 2220 samples, i.e. 5.6 %, tested positive for a previous TBE infection in the NS1 ELISA. In a few samples, cross-reacting antibodies induced by other flaviviruses were found (7/2220). A manifestation index of ca. 2 % could be determined, as well as a high number of silent infections of more than 250 per 100.000 inhabitants per year. A neutralising antibody titer against the TBE virus could be determined for ca. 55 % (1150/2104) of the sample size. Conclusion In the studies on which this work is based, new TBE incidence and manifestation index data were generated for an endemic area. After comparing the data from 1986, the results indicate an approximately sevenfold increase in TBE infection rates in the studied area despite the availability of a highly effective vaccine. The serological protection rate is significantly higher than the immune protection rate numbers collected by the RKI for the Ortenaukreis (approx. 20 %), which could speak for longer durability of the antibodies induced by the vaccination than originally assumed. Furthermore, this study illustrates the use of seroprevalence studies in addition to incidence-based risk assessment of TBE endemic areas.:1 Einleitung......................................................................................1 2 Literaturübersicht.........................................................................2 2.1 Frühsommer-Meningoenzephalitis-Virus (FSME-Virus)................2 2.1.1 Virologie................................................................................2 2.1.2 Virusreplikation.....................................................................3 2.1.3 Epidemiologie in Deutschland..............................................4 2.1.4 Übertragungszyklus des FSME-Virus...................................6 2.1.5 Infektion und Krankheitsverlauf............................................8 2.1.6 Impfung.................................................................................9 2.1.7 Diagnostik............................................................................10 2.1.8 Serologische Methoden.......................................................11 3 Publikationen...............................................................................14 3.1 Eigenanteil Publikation 1..............................................................14 3.1.1 Publikation 1........................................................................16 3.2 Eigenanteil Publikation 2..............................................................25 3.2.1 Publikation 2........................................................................26 4 Diskussion....................................................................................36 5 Zusammenfassung.......................................................................44 6 Summary......................................................................................46 7 Referenzen...................................................................................48 7.1 Literatur........................................................................................48 7.2 Abbildungsverzeichnis.................................................................59 8 Danksagung.................................................................................60

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.