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.

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.