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Expression and purification of Tick-borne encephalitis virus Capsid protein
SCHMELZER, Jakob
Tick-borne encephalitis virus (TBEV) is an important pathogen of the family Flaviviridae with an emerging health concern. Virus causes severe neurological disorders to human population in Europa and Asia. Despite effective vaccine, there is no specific antiviral treatment till now. The capsid protein encapsules the viral positive-sense single-stranded RNA and is essential in packaging and assembling of new virions within the host. A deeper understanding of these interactions and their implications should lead to new antiviral strategies and drug design. This thesis deals with the expression and purification of the capsid protein without membrane anchor using Escherichia coli expression system.
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Protilátky v terapii klíšťové encefalitidy
NEDVĚDOVÁ, Lenka
Tick-borne encephalitis (TBE) is a serious live threatening disease caused by tick-borne encephalitis virus (TBEV). The therapy is based on administration of non-specific treatment with anti-inflammatory drugs or IVIg (including non-specific antibodies). Application of non-specific antibodies may present a certain level of risk - maily of developing antibody dependent enhancement. We have developed and characterized monoclonal antibodies targetting non-structural protein 1 (NS1) of TBEV in order to assess its potential in TBEtreatment and diagnostics.
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Recombinant production and characterization of protease domain from TBEV nonstructural protein NS3
VEJVODOVÁ, Kateřina
The aim of this thesis was to produce and characterize recombinant protease domain of TBEV nonstructural protein 3 (NS3). The NS3 protein of TBEV is essential in viral replication and plays an indispensable role in cleaving viral polyprotein into structural and nonstructural viral proteins, thus making it an attractive target for antiviral treatment. This thesis summarized the cloning of TBEV NS3 protease,production and purification of recombinant enzyme, biochemical and kinetic characterazion of the protease domain from TBEV NS3.
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Patogeneze a klinické aspekty infekce virem klíšťové encefalitidy
ELSTEROVÁ, Jana
This thesis contributes to knowledge about the combined impacts of the pathogenesis of the tick-borne encephalitis virus (TBEV) and the immunopathogenesis of the host on the clinical course of acute tick-borne encephalitis (TBE). The thesis further focuses on the process of TBEV neuroinvasion and the utilization of the host's immune products as potential therapeutic interventions.
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Cloning, expression and characterization of the viral proteins
Pekárek, Matěj ; Šilhán, Jan (advisor) ; Obšil, Tomáš (referee)
Over the last twenty years three new coronavirus strains have appeared in human population, that cause dangerous infections of respiratory tract. At this moment no approved drug exists, that could mitigate the infection caused by these viruses. One of the possible targets of these drugs are proteins responsible for viral genome replication. Nsp13 is primarily a helicase, that unwinds double stranded nucleic acids in the direction of 5'-3', but it also increases activity of replication and transcription complex, it participates in the synthesis of 5' cap on viral RNA and it probably mediates the backtracking of replication and transcripton complex during the process of RNA repair. On top of that nsp13 is strongly conserved among all coronaviruses and it is possible target not only for COVID-19 treatment, but also infections caused by potential future coronaviruses. Theoretical part of the thesis briefly describes the life cycle of SARS-CoV-2, functions of individual proteins of this virus, that participate in replication and transcription of RNA and it summarizes current knowledge about function of nsp13 and its potential inhibitors. In experimental part we have prepared the expression vector for nsp13, that was used for the production of nsp13. Nsp13 was then isolated and purified. (In Czech) Key...
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Structural and functional characterization of a flaviviral methyltransferase.
Todd, Matthew Zagorey ; Bouřa, Evžen (advisor) ; Honzejková, Karolína (referee)
Genus flavivirus, member of the Flaviviridae family, is a wide-spread group of viral pathogens that pose a global threat to human health. Infections result in neurological and other diseases such as encephalitis, meningitis, microcephaly or haemorrhagic fever with direct anti-viral treatment restricted to only a few members including Yellow Fever Virus (YFV) and Japanese Encephalitis Virus (JEV). The flaviviral genome consists of a positive sense single-strand RNA, translated into a long viral polyprotein cleaved by host and viral proteases into structural and non-structural proteins. Non-structural protein 5 (NS5) is a highly conserved viral protein consisting of a C-terminal RNA-dependent RNA-polymerase (RdRp) and N-terminal methyltransferase (MTase) domain. Flaviviral MTases catalyse the final step of genomic RNA cap formation, that plays a crucial role in viral translation, replication and host immune system evasion. Due to its conservative nature, the flaviviral MTase is a promising drug target. It's structural analysis is needed for the development of broad-spectrum inhibitors and anti-viral treatment. Key words Flavivirus, St. Louis Encephalitis Virus, Flaviviral methyltransferase (MTase), N-7 methylation, 2'-O methylation.
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Preparation of expression vectors for the production of ZIKA and Dengue NS3 helicase
Daňhelová, Kateřina ; Konvalinka, Jan (advisor) ; Heidingsfeld, Olga (referee)
Zika and Dengue viruses have spread, due to globalisation, to all continents which lie at least in part in the subtropic and tropic climatic zones. This spread of these viruses is a reason of an increasing number of severe diseases caused by them. New drugs, which would be effective against these infections, could be an answer to this challenge. Various viral proteins, among them also viral helicase, which is the topic of this bachelor thesis, can be a suitable drug target. The task was to prepare expression constructs for production of recombinant helicases of Zika and Dengue viruses via the suitable bacterial strain Escherichia coli. Several constructs derived from plasmid pET-16b were prepared with inserted helicase of Zika and Dengue viruses. One of them was used for the preparation of recombinant purified helicase of Zika virus, that will be used for further research. [IN CZECH] Keywords: Flavivirus, Zika virus, Dengue virus, helicase, expression, purification, enzyme activity [IN CZECH]
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Dengue vaccines
Holšteinová, Aneta ; Šroller, Vojtěch (advisor) ; Poláková, Ingrid (referee)
Since the middle of the 20th century the number of people infected by the dengue virus (DENV) has been constantly increasing. Specific antiviral drugs are not available, so the objective for eliminating transmission has become the construction of an effective and safe vaccine with an emphasis on inducing a balanced immune reaction against all of the DENV serotypes. Momenteraly, the only licenced vaccine is CYD-TDV (Chimeric Yellow Fever- Dengue, Live-Attenuated, Tetravalent Dengue Vaccine). This vaccine uses the structure of the effective and safe vaccine against the yellow fever virus (YFV) from which surface protein sequences are substituted for corresponding DENV sequences. While preclinical studies displayed promising results, complications have arised during clinical studies, thus creating limiting criteria for their use. For this reason the search for new vaccines that could ensure better safety from DENV for a wider range of people and replace CYD-TDV is ongoing. In the III. phase of the clinical studies there are two live-attenuated vaccines (TetraVax-DV and DENVax). The subunite vaccine (V180), DNA vaccine (TVDV) or inactivated vaccine (TDENV PIV) was forwarded into the I. phase. The combination of several vaccine schedules is also being used (tetravalent live-attenuated...
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Interakce viru klíšťové encefalitidy s cytoskeletem hostitelských buněk
PRANČLOVÁ, Veronika
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.
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