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.

Tick-borne encephalitis virus (strain Hypr) was serially subcultured in PS cells and tick cell line IRE/CTVM19, producing four different viral variants. Biological properties of these new variants were investigated in mouse model. Possible determinants of virulence were found by full-genome sequencing. The role of glycosylation for tick-borne encephalitis virus was evaluated.

The aim of this study was to characterize the replication of tick borne encephalitis virus in mouse macrophages and investigate the influence of tick saliva derived from Ixodes ricinus on the viral replication. Moreover, the effect of interferon (the member of type I interferons) and nitric oxide on virus replication was studied.

This study was focused on vector-pathogen-host interaction. Questing ticks from field were tested for presence of Borrelia burgdorferi s.l. and host DNA. Small rodents were trapped, ticks were collected from them, infestation patterns were estimated regarding the species and stage of ticks and species, sex and body weight of the host. Ticks aquired from hosts were tested for presence of Borrellia burgdorferi s.l. and tick-borne encephalitis virus. Both results from identification of hosts and from detection of pathogens were compared to be able to investigate interactions between host, vector and pathogen.

The aim of this study was to examine the effect of tick infection with tick-borne encephalitis virus on its behaviour and development. The effect of infection on feeding performance, metamorphosis, locomotion or phototaxis was analysed. Despite the fact that we were not able to demonstrate any significant effect of infection on tick behaviour, the obtained results contribute to understanding transmission dynamics of the virus during tick life cycle including co-feeding and transovarial transmission.

This thesis is focused on the role of host cytoskeleton, primarily microtubules and microfilaments, during tick-borne encephalitis virus infection in human neuroblastoma cell line SK-N-SH and tick cell line IRE/CTVM19. The importance of cytoskeletal integrity and dynamics to the viral replication cycle were examined using specific chemical inhibitors showing the virus utilizes studied structures in both cell lines. Immunofluorescence microscopy revealed structural changes in the actin cytoskeleton during late infection in SK-N-SH cells. Moreover, differences in expression of cytoskeleton-associated genes in both cell lines were compared. Several genes with up-regulated expression in SK-N-SH cells were identified during late infection.

The proposed thesis contributes to the basic knowledge in tick (Ixodes ricinus) and tick-borne pathogens (Borrelia burgdorferi sensu lato, tick-borne encephalitis virus) ecology in particular studying the spatial distribution, host associations and its causes and consequences in Central European habitats.

The first aim of this study was to detect antiviral activities of substances isolated from natural products against tick-borne encephalitis virus in in vitro model. Resveratrol isolated from plant material and adamantane derivatives were studied in this regard. The maximum tolerated concentrations of the investigated substances were determined for the glioblastoma cell line used in the experiments using flow cytometry and subsequently. Next, the number of viral particles produced by infected cells after incubation with the studied substances was determined using plaque titration. Possible antibacterial effects of the studied materials against standard strains of bacteria Staphyloccocus aureus, Staphyloccocus epidermidis, Escherichia coli and selected strains of Borrelia burgdorferi spirochetes were examined.

We examined the influence of the host genetic background on the pathogenesis of tick-borne encephalitis. We determined virus titers in organs and serum in different time intervals post-infection for different ways of inoculation. We also stated mean survival times and antibody production in different strains of mice infected with tick-borne encephalitis virus. Moreover, differences in expression of immunologicaly important genes in brains of infected mice were compared.

Tick-borne encephalitis is a serious disease of the central nervous system. It is caused by tick-borne encephalitis virus, which is transmitted by ticks. The Czech Republic is one of the countries with the highest prevalence of this disease. Tick-borne encephalitis virus is able to replicate in several cell types. In this work we focused on macrophage line IC-21 and dendritic cells, because these cells are the first, which encounter the virus and support its spreading in the host at early stage of infection. So far there is not known any specific receptor for virus entry into cells or which signaling pathways activates. Therefore, we decided to investigate the activation of selected signaling pathways after infection with tick-borne encephalitis virus and influence of tick saliva on this activation. We employed methods of dual luciferase reporter assay, immunosandwich assay and western blot. The obtained results showed that in virus infected IC-21 cells are activated phosphatidyl-inositol pathway, NF-κB pathway, signaling molecule Erk1/2 and others. Testing of tick saliva effect revealed significantly decreased activity of NF-κB, AP-1 and CREB.