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Study of exosomes in polyomavirus infection
Hyka, Lukáš ; Šroller, Vojtěch (advisor) ; Saláková, Martina (referee)
Exosomes are extracellular vesicles of endosomal origin. It was thought, that exosomes are used by cells only as carriers for cellular waste, but it was found out, that exosomes serve in the cellular communication and have a role in viral infections. Exosomes are exploited by viruses for example for the transport of viral protein or viral RNA/DNA. One of the viruses, where the mechanism of exploitation is unknown (if any exists) is murine polyomavirus. Murine polyomavirus belongs to the family Polyomaviridae, to which other human viruses belong for example, JC virus or virus of Merkel cell carcinoma. Murine polyomavirus codes for small, large and middle T antigen and three capsid proteins. Middle T antigen is known to bind to cellular membranes. Exosomes are membrane derived structures, so we investigated a possible transfer of middle T antigen. To this goal the successful isolation of exosomes and their characterization was necessary. Exosomes were isolated by ultracentrifugation and further purified by the density gradient OptiPrep. Exosomes were characterized by electron microscopy, NanoSight and by protein exosomal markers. These markers are for example Alix and flotillin-1. The cells were transfected in order to produce middle T antigen. It was shown, that exosomes isolated from these cells...
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The role of BK polyomavirus in human cancer
Cirbusová, Adéla ; Saláková, Martina (advisor) ; Šroller, Vojtěch (referee)
BK polyomavirus is small uncoated DNA virus which is ubiquitous in human population. In imunosupresed individuals can cause severe diseases, specifically BK polyomavirus associated nephropathy (BKVaN) and hemoragic cystitidis. Except that, it is presumed that BKV may be responsible for some cases of human cancer. Some features of BKV could support this idea. There are three encoded oncoproteins in BKV genome. BKV is oncogenic in rodents, where induces multiple types of tumors and it can transform cell lines as well. Moreover, BKV DNA was found in many types of human cancer. All these facts suggest a possible role of BKV in human cancer. Bladder carcinoma in pacients after transplatation and prostate adenocarcinoma are the most likely candidates to link with BKV participation. There is no complete evidence though. Therefore, future studies are necessary to proof or even exclude BKV as a possible cause of human cancer. key words: BKV, cancer, LTag, prostate, bladder
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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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Viruses in the pathogenesis of coeliac disease
Chudá, Kateřina ; Cinek, Ondřej (advisor) ; Šroller, Vojtěch (referee)
Celiac disease is a chronic inflammatory disorder affecting the small bowel. It develops in genetically susceptible individuals upon yet unknown environmental stimuli. Environmental triggers such as infections, dietary change or other "hits" are clearly required for disease development, as only a tiny fraction of genetically susceptible subjects develops celiac disease upon gluten exposure. This thesis aims to summarize the current evidence on viruses in the pathogenesis of celiac disease regarding their relevance in population or their involvement in immune processes leading to celiac disease. Rotavirus, orthoreovirus, adenovirus, astrovirus, respiratory syncytial virus, hepatitis viruses and herpesviruses are discussed. In addition, prospective cohort studies are presented that investigate environmental triggers of type 1 diabetes and celiac disease, two diseases sharing genetic predispositions. Keywords: celiac disease, orthoreovirus, rotavirus, adenovirus, astrovirus, respiratory syncytial virus, hepatitis C virus, hepatitis B virus, prospective cohort study
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Experimental and clinically used vaccines based on vaccinia virus
Pilná, Hana ; Mělková, Zora (advisor) ; Šroller, Vojtěch (referee)
Vaccinia virus (VACV) is an enveloped DNA virus belonging in the Orthopoxviridae genus. It is a laboratory virus in which the natural host and exact origin remain unclear. However it is of great significance for human kind. First of all, different VACV strains were used for preparation of vaccines used in the smallpox eradication campaign. Even today a significant effort is made to prepare more efficient and safer vaccines against smallpox, namely because of still remaining concerns that variola virus - causative agent of smallpox - could be misused as a biological weapon. Advances in genetic engineering allowed use of VACV for additional purposes, namely as a vaccination and expression vector. VACV enables insertion of large pieces of foreign DNA into its genome and expression of this DNA in a host. Furthermore VACV replicates exclusively in a cytoplasm, decreasing a risk of incorporation of the viral DNA into the host genome. These and other features make VACV an ideal candidate as a vector for preparation of recombinant vaccines against various infectious and oncological diseases. This thesis provides a summary of both clinically used and experimental vaccines derived from VACV. Powered by TCPDF (www.tcpdf.org)
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Characterization of immune responses to human polyomavirus MCPyV.
Vochozková, Petra ; Šroller, Vojtěch (advisor) ; Drda Morávková, Alena (referee)
Characterization of immune responses to human polyomavirus MCPyV There have been considerable increase in number of known human polyomaviruses in recent years; currently we register twelve of them. Presumably, majority of human polyomaviruses cause lifelong persistent infection. Primary infection is usually asymptomatic and is followed by appearance of antibodies specific to polyomavirus capsid. Polyomaviruses can cause complication especially in immunocompromised people. Merkel cell polyomavirus (MCPyV) is linked to development o of Merkel cell carcinoma (MCC). Although this skin tumor is very rare, MCPyV infection is very common. Most of the human population exhibit MCPyV-specific antibodies in serum. MCPyV specific antibodies are detected in patients with MCC and their level is generally higher than in healthy individuals. MCC occurs more often in immunosuppressed individuals. It appears that protection of antibodies against tumor formation is not sufficient and the development of the tumor could be rather under the control of cellular immunity. In this study, we have prepared plasmids for production of major capsid protein VP1 and detection of antibodies specific to MCPyV capsids. Mice immunized with DNA vaccine expressing VP1 protein produced VP1 specific antibodies in serum. From insect cells infected...
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Preparation of expression vectors and virus mutants for studies of the minor structural proteins of polyomaviruses.
Cibulka, Jakub ; Forstová, Jitka (advisor) ; Šroller, Vojtěch (referee)
Polyomaviruses are small non-enveloped DNA viruses infecting birds and mammals, including human. Their capsid consists of the major capsid protein, VP1, and two minor capsid proteins, VP2 and VP3. The VP2 and VP3 proteins are supposed to have an important function in the transport of viral genome into the cell nucleus, which is a key step to facilitate viral replication. VP2 and VP3 proteins of mouse polyomavirus and SV40 have an ability to bind and disrupt cellular membranes. This feature is believed to be involved in the transport of viral genome into the nucleus. Plasmids carrying genes of the minor capsid proteins of Merkel cell polyomavirus were prepared in order to produce and visualize these proteins in mammalian cells. These proteins are known to have very unusual sequences compared to other human polyomaviruses or related mouse polyomavirus. When produced alone, the minor capsid proteins of Merkel cell polyomavirus did not significantly interact with cellular membranes, unlike the minor proteins of the mouse polyomavirus. The second goal of this work was to prepare mouse polyomavirus mutants with deletion in hydrophobic domains of VP2 and VP3 proteins. These domains are likely responsible for the mentioned membrane interactions. Prepared mutants were non-infectious. The loss of infectivity was not...
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Development of the experimental system based on Cre/loxP recombination for polyomavirus mutant production.
Hron, Tomáš ; Španielová, Hana (advisor) ; Šroller, Vojtěch (referee)
Murine polyomavirus is an important member of Polyomaviridae family offering potential applications in gene therapy and immunotherapy. Viral mutant analysis is crucial for study of the virus, however, commonly used methods of its production are laborious and give low yields. This thesis involves development of the new experimental system that can produce intact viral genome from recombinant plasmid in vivo using Cre/loxP-mediated recombination. One loxP site is unavoidably introduced into newly generated viral genome during recombination. Two variants of production plasmids generating wild type viral genome with incorporation of loxP between the poly(A) signal sites of early and late genes or into the intronic region of early genes were prepared. LoxP insertion between the poly(A) signal sites has a dramatic effect on viral gene expression and leads to complete loss of virus infectivity. Conversely, the infectious virus was obtained from the viral genome containing loxP site in the early intronic region. To ensure expression of Cre recombinase I also prepared stably transfected cell lines which can simplify the virus production. This thesis shows that newly designed system gives satisfactory yield of the virus, solves restrictions connected with commonly used methods and can be used for low infectious viral...
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Human polyomavirus isolated from Merkel cell carcinoma
Vochozková, Petra ; Šroller, Vojtěch (advisor) ; Španielová, Hana (referee)
Human polyomaviruses belong to the Polyomaviridae family. Until now, five human polyomaviruses (BK, JC, KI, WU and MCV) have been discovered. There is described the course of polyomavirus infection in the first part of the thesis. These small nonenveloped viruses penetrate into the host cell by receptor-mediated endocytosis and then travel through the ER pathway to get into the nucleus where the virus replicates and expresses viral proteins. The infection occurs during early childhood and the virus remains asymptomatic in healthy individuals. However, the virus is able to reactivate in the immunosuppressed patients and can cause some diseases. The second part of thesis is focused on the MCV. MCV genom was detected in Merkel cell carcinoma (MCC) two years ago. MCV infects the Merkel cells and its DNA integrates into host cell genome. In most cases, the MCV is detected in cancer cells using PCR. Viral sequences encoding the large and small T antigen were found in the MCC using the same method; moreover, there was expressed a significant amount of oncoproteins. These factors point out an important role of MCV in the tumor progress. The study of MCV may help to discover new approaches for the treatment of MCC and other biologically similar tumors.
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