Evaluation of the antiviral effectiveness of ribavirin and acyclic nucleotide phosphonates to radish mosaic virus. Virus inoculation of plants with RaMV and immunological assay of the virus by ELISA. Subsequent application of antiviral agents and monitoring relative content of the virus in plants. Subsequent processing of data in tables and graphs, and then statistical evaluation.

Local DNA structures are alternative DNA conformations which can be formed aside from typical B-DNA conformation. These structures often play pivotal roles in regulation of basic biological processes, such as DNA replication, transcription or binding of specific ligands. This biological significance makes alternative DNA secondary structures a potential drug target. In this diploma thesis, local structures in genomes of viruses from Flaviviridae and Retroviridae families are analysed using bioinformatics tools. Furthermore, these structures are visualised using atomic force microscopy.

4 Summary Altogether, the six most common bee viruses which infect the honey bee (Apis mellifera) were monitored in the territory of the Czech Republic between 2006 and 2009. Parallel infections of viruses (DWV, ABPV and BQCV) in bee adults and parallel co- infection of viruses with fungal diseases caused by Nosema apis and Nosema ceranae were confirmed by PCR tests. A new sensitive method of detection of the originator of the American foulbrood (Paenibacillus larvae) from bee debris was developed for the practical use of detection of AFB disease in bee populations. Various approaches for the extraction of spores from bee debris and lyses of spores were compared. The sensitivity of PCR tests for the presence of Paenibacillus larvae in debris was compared with the classic cultivation method. The PCR method for the detection American foulbrood was further studied and developed to be more efficient. A new method, based on a matrix-like sample re-arrangement and a use of pooled samples, has been developed for testing 1000 samples in 35 PCR reactions. Another goal was to develop a robust and fast screening method for American foulbrood based on the cultivation test using paper sheets RIDA®COUNT with a specific cultivation medium, specific selection conditions for Paenibacillus larvae and chromogen visualization...

Influenza illness occures masively in human population for centuries. Influenza is an acute disorder of viral origin. Currently influenza can be found all over the world. The viruses of influenza are circulating continously in all continents not only in the human population, but in animals too. Every year the population is affected by the seasonal epidemic of different magnitude. Influenza illness pose an important medical, social and economical problem. Next to the seasonal outbreaks occuring every year the influenza pandemic can appear after several tens of years. The pandemic of influenza would mean spread all over the world and it is caused by the new variant of the influenza virus. In past the influenza pandemics caused mortality of millions of people all over the world, high economical losses and social collapse of the states in the history. That´s why the particular states prepare their national pandemic plans. The early preparedness for possible influenza pandemic elaborated in the national pandemic plans and the measures following from the plans constitute a tool for reduction of extent, impact and outcomes of pandemic. The quality of preparedness can influence the economical functions of states, medical impacts of illness and final number of the victims. The aim of the dissertation is to compare the proposed measures responding to the influenza pandemic arising from the national pandemic plans of the Czech and Slovak Republic, to ascertain the state of preparedness to the influenza pandemic following from the pandemic plan of the Czech Republic and the situation in awareness of the civil population about the influenza pandemic in the Czech Republic. The theoretical part of the dissertation is focused on the epidemiological characteristic of the influenza illness. In the sphere of interest the issue of influenza pandemics is explained in terms of their history and the next field of the dissertation is focused on the pandemic plans. The research part of the dissertation is focused on the solution to the given research questions. Two research questions were specified to accomplish the defined aim: What are the differences between the approaches and measures in the pandemic plans of the Czech and Slovak Republic? What is the knowledge and awareness of civil population about the influenza pandemic in the Czech Republic? Regarding the issue the methodology the first research question included analysis of the content of the pandemic plans of the Czech and Slovak Republic and performance of the Checklist method in order to obtain an overview on approach, procedures and standard of preparedness in the territory of both states and possibilities their reciprocal comparison. Under the second research question the awareness and knowledge of the civil population about the pandemic influenza in the Czech Republic was investigated. For acquiring the data of researching study there was used The method of quantitative research was used to acquire the data for the research survey. The form of twenty questions was created. The first part of the questionnaire was focused on the general knowledge about influenza and the second part on the influenza pandemic and related measures. The respondents chose from the answers defined in advance. There wasn´t used the random sampling of the respondents. Fifty respondents from the non-representative sample filled out the form for acquiring the basic knowledge and finding out theoretical information about the influenza pandemic. Subsequently the statistical analysis was made from the obtained data using the methods of descriptive and mathematic statistic. It follows from the analysing results that the knowledge about the influenza illness and influenza pandemic exists among the civil population which means the laic public, but they are the lower extent and do not correspond to the normal frequency distribution.

Trichomonas vaginalis is a causative agent of the most common non-viral sexually transmitted disease with approximately 275 mil new cases annually. Virulence of this parasitic depends on at least four factors: cell shape transformation, cytoadherence, secretion of cysteine proteases, and presence of endosymbionts. Over the past decades, extracellular vesicles appeared being another important player in the host-parasite interaction. It was discovered that T. vaginalis is one of the protists that can shed the extracellular vesicles such as exosomes and ectosomes. These vesicles are possibly involved in host-parasite communications, however limited information is available about their function. To investigate a possible role of exosomes in T. vaginalis virulence, we first selected suitable strain, which is free of endosymbionts (TV 17-2MI). Next we prepared six clones of TV 17-2MI strain to test whether the strain is homogenous concerning the virulence, or there are differences in virulence among individual cells. Mouse intraperitoneal virulence tests revealed that the clones displayed significant differences in virulence level, particularly in abscess formation and mortality of infected animals. Thus, for the first time we demonstrated heterogeneity of cells derived from a single T. vaginalis strain...

The aim of this study was to define and examin the capabilities of the low voltage transmission electron microscope LVEM 5 in the detection and identification of viruses, taking into consideration various contrasting agents, and a comparison to data from HV TEM.

1 Abstract The diploma thesis deals with the educational topic Viruses at a higher level of grammar schools. The theoretical part deals with the inclusion of the topic during school year, analyses available textbooks and other teaching materials, focuses on the theoretical side of the activation methods, and suggests teaching units. In the teaching units it mainly uses activation methods and methods targeting on critical thinking. In the practical part of the proposed teaching unit verifies in teaching, and evaluates their success in terms of the degree of remembering pupils.

Next generation sequencing is a modern method applied in plant virology for sensitive detection of previously characterized and novel pathogens without any preceding knowledge of them. In this study three novel and two already described viruses were detected by de novo assembly of Illumina single-end reads ( Hi-Seq 2500 system) from total poly(A) enriched RNA of diseased red clover (Trifolium pratense) and indicator plant (Nicotiana occidentalis 37B). The complete genomic sequence of novel Red clover carlavirus A (RCCA) was determined from Illumina reads, 5´, 3´ RACE, cloning, RT-PCR and Sanger sequencing. The presence of RCCV was also confirmed in mechanically inoculated tobacco plant.

Plasmacytoid dendritic cells (pDC) represent innate immune cells capable to detect viruses in their endosomal environment via Toll-like receptors (TLRs). Viral nuclear acid recognition leads to the massive production of type I interferon (IFN I) and induction of the antiviral state in uninfected cells. Crosslinking of the surface regulatory receptors, such as BDCA-2, with monoclonal antibodies or with some viruses leads to the activation of MEK1/2- ERK signaling pathway and inhibition of IFN I production in pDC. In this study, the role of MEK1/2 kinase has been highlighted. Its inhibition reversed the inhibitory effect of BDCA-2 crosslinking and its direct activation with PMA led to the inhibition of IFN-α production. Yet an unclear role of pDC in sensing of BK polyomavirus virus (BKV) responsible for kidney transplant rejection was investigated as a major topic of this thesis. Experiments with the pDC cell line Gen2.2 and HRPTEC primary cell line showed that pDCs were not able to detect BKV particles, however, exposure of activated Gen2.2 cells to BKV inoculum dramatically upregulated production of IFN-α. Most importantly, coculture of Gen2.2 cells with BKV- infected HRPTEC cells resulted in IFN-α and TNF-α production, which was prevented by Bafilomycin. These results suggest that BKV-infected...

Plasmacytoid dendritic cells (pDC) are a highly specialized subset of immune cells that sense viral nucleic acids by endosomal toll-like receptors 7 and 9 (TLR7/9). Activation of TLR7/9 leads to the production of type I interferons (IFN-I). Moreover, pDC contribute to the antiviral response by presenting viral antigens to T lymphocytes and link innate and adaptive immunity. pDC need to be properly regulated in order to limit excessive production of IFN-I that is associated with autoimmune diseases. Therefore, pDC possess a battery of regulatory receptors (RR) that limit TLR7/9-mediated cytokine production. This thesis focuses on the mechanism of RR-mediated inhibition of IFN-I production in pDC and explores interactions between pDC and two enveloped viruses, that possess the ability to hijack RR in pDC: hepatitis B virus (HBV) and human immunodeficiency virus (HIV). We showed, that MEK-ERK signaling pathway plays an active role in RR-mediated inhibition of IFN-I in pDC. Our results indicate that in line with other studies of our group, pharmacological targeting of MEK1/2-ERK signaling could be a strategy to re-establish immunogenic activity of pDC. Then, we investigated whether antiretroviral therapy (ART) in a cohort of 21 treatment-naive chronic HIV-infected patients has restored the number and...