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Zoonotické Viry U Volně Žijících Endotermních Obratlovců

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Zoonotické Viry U Volně Žijících Endotermních Obratlovců MASARYKOVA UNIVERZITA PŘÍRODOVĚDECKÁ FAKULTA Ústav experimentální biologie Oddělení mikrobiologie a molekulární biotechnologie Zoonotické viry u volně žijících endotermních obratlovců Dizertační práce Brno 2017 Petra Straková MASARYKOVA UNIVERZITA PŘÍRODOVĚDECKÁ FAKULTA Ústav experimentální biologie Oddělení mikrobiologie a molekulární biotechnologie Zoonotické viry u volně žijících endotermních obratlovců Dizertační práce Petra Straková Školitel: prof. RNDr. Zdeněk Hubálek, DrSc. Brno 2017 Bibliografický záznam Autor: Mgr. Petra Straková Ústav biologie obratlovců AV ČR v.v.i., Brno - detašované pracoviště Valtice a Ústav experimentální biologie, PřF MU, Brno Název práce: Zoonotické viry u volně žijících endotermních obratlovců Studijní program: Biologie Studijní obor: Mikrobiologie Školitel: prof. RNDr. Zdeněk Hubálek, DrSc. Ústav biologie obratlovců AV ČR v.v.i., Brno - detašované pracoviště Valtice a Ústav experimentální biologie, PřF MU, Brno Valtice Akademický rok: 2016/2017 Počet stran: 155 + publikace Klíčová slova: emergentní zoonózy, hantaviry, flaviviry, virus západonilské horečky, virus Usutu, virus hepatitidy E, Česká republika, Evropa Bibliographic entry Author: Mgr. Petra Straková Institute of Vertebrate Biology of the Czech Academy of Sciences, Brno – laboratory Valtice, and Department of Experimental Biology, Faculty of Science, Masaryk University, Brno Title of Dissertation: Zoonotic viruses associated with free-living endotherm vertebrates Degree Programme: Biology Field of Study: Microbiology Supervisor: prof. RNDr. Zdeněk Hubálek, DrSc. Institute of Vertebrate Biology of the Czech Academy of Sciences, Brno - laboratory Valtice, and Department of Experimental Biology, Faculty of Science, Masaryk University, Brno Academic Year: 2016/2017 Number of Pages: 155 + scientific articles Keywords: emerging zoonoses, hantaviruses, flaviviruses, West Nile virus, Usutu virus, hepatitis E virus, Czech Republic, Europe © Petra Straková, Masarykova univerzita, 2017 Abstrakt Předložená dizertační práce je koncipovaná jako soubor komentovaných publikací zabývajících se zoonotickými viry u volně žijících endotermních obratlovců. Z velkého výčtu zoonotických virů vyskytujících se v Evropě byly vybrány ty, které jsou v Evropě dobře popsané, mohou způsobovat onemocnění u lidí se širokou škálou symptomů, a které byly detekované v České republice. Tato práce se věnuje konkrétně detekcí hantavirů u volně žijících hlodavců, flavivirů u volně žijícího vodního ptactva a viru hepatitidy E u divokých prasat a další lovné zvěře. Na tyto viry se stále pohlíží jako na tzv. emergentní (tj. nově se objevující) viry a v některých zemích jsou dlouhodobě monitorovány. Hlavní důvody pro tento monitoring jsou narůstající počet klinických případů u lidí i jejich rostoucí veterinární význam. Jedním z důležitých výsledků této dizertační práce byl popis prvního hantaviru detekovaného u netopýra pestrého (Nyctalus noctula) v Evropě. Dále byla provedena vůbec první studie zabývající se prevalencí hantavirů u hlodavců v Litvě, monitoring Dobrava-Belgrade hantaviru v Německu a screening hlodavců na hantaviry z České republiky. U flavivirů a viru hepatitidy E jsme sledovali přítomnost specifických protilátek u volně žijících ptáků a lovné zvěře. Z výsledků vyplývá, že studované viry cirkulují v určitých oblastech ve volné přírodě a představují možné riziko pro zdraví obyvatel. Abstract This Ph.D. thesis is compiled as a collection of commented scientific articles focusing on zoonotic viruses in free-living endotherm vertebrates. From a large number of zoonotic viruses presented in Europe, we selected those that are well described in Europe, can cause diseases in humans with a scale of different symptoms and were also detected in the Czech Republic. This thesis aimed at the detection of hantaviruses in the free-living rodents, flaviviruses in the free-living waterfowl and hepatitis E virus in wild boars and other game animals. These viruses belong to the so-called emerging viruses, and in some countries, they are monitored over a long period. The reasons for these monitoring studies are mainly increasing numbers of clinical cases and their rising veterinary importance. During our research, we detected all selected viruses in free-living animals in the Czech Republic. The most important result was the discovery of the first bat-borne hantavirus in Europe. We carried out the first prevalence study of hantaviruses in the local rodent population in Lithuania, in Germany we monitored the presence of Dobrava-Belgrade virus and we also did a screening for hantaviruses in the rodents from the Czech Republic. In flaviviruses and hepatitis E virus we focused on the detection of specific antibodies in free living waterfowl and game animals and we confirmed their presence in our ecosystem. Presented results indicate that the studied viruses circulate in nature in selected areas and they represent a possible health threat to the human population. Poděkování Na tomto místě bych ráda poděkovala mému školiteli prof. RNDr. Zdeňku Hubálkovi, DrSc. a RNDr. Ivo Rudolfovi, Ph.D. za možnost zpracovat svou dizertační práci na detašovaném pracovišti Ústavu biologie obratlovců AV ČR, v.v.i. ve Valticích a za pomoc a pevné nervy při vzniku této práce. Mé díky patří dále Mgr. Hance Blažejové, která mi byla největší oporou ke konci mého studia, Ing. Lence Betášové, Mgr. Kristýně Venclíkové, Ph.D., Juraji Peškovi a Ladislavě Ševčíkové za výborné pracovní podmínky a příjemně strávené chvilky v kuchyňce při kávě. Za poskytnutí odchycených hlodavců a informací ohledně jejich ekologie bych chtěla poděkovat doc. RNDr. Martě Heroldové, Ph.D. z Ústavu biologie obratlovců AV ČR, v.v.i. v Brně. Dále mé poděkování patří doc. RNDr. Danielu Růžkovi, Ph.D. a MVDr. Lucii Dufkové, Ph.D. z Výzkumného ústavu veterinárního lékařství, v. v. i. v Brně za to, že jsem se mohla stát členem jejich týmu a věnovat se tématu virových infekcí netopýrů. Největší dík poputuje do Německa do týmu Dr. Rainera G. Ulricha (Institute of Novel and Emerging Infectious Diseases (INNT) ve Friedrich-Loeffler Institutu), kde jsem strávila 11 měsíců na pracovní stáži ERASMUS+. Začátky nebyly jednoduché, ale jsem velmi vděčná za příležitost a získané zkušenosti díky práci ve světově uznávaném pracovišti, kde spojení „že něco nejde,“ prostě neexistuje. Děkuji všem svým kolegům za jejich pomoc a rady, jmenovitě Stephanu Drewesovi, Stefanu Fisherovi, René Ryllovi, Maysaa Dafalla, Dörte Kaufmann a Anke Mandelkow. Za možnost provést a analyzovat data z NGS testování děkuji Dr. Dirku Höpperovi a Florianu Pfaffovi. Za to, že jsem se v Německu cítila jako doma, děkuji především Dr. Nilsu Kleyovi a jeho rodině a mým přátelům Miriam Andrada Sas, Mahmoud Naguib a Rebece König. V neposlední řadě patří velký dík mé rodině a přátelům za nekončící podporu a pochopení, a především mému příteli Kubovi za to, že mi byl po celou dobu doktorského studia oporou, se vším mi pomáhal a kriticky hodnotil všechny moje nápady. Prohlašuji, že jsem tuto dizertační práci vypracovala samostatně s využitím informačních zdrojů, které jsou v práci citovány. V Brně ………………… …………………………… Petra Straková Seznam použitých zkratek AI avian influenza (ptačí chřipka) BioEdit Biological sequence alignment Editor BLAST Basic Local Alignment Search Tool (vyhledávací nástroj sekvenčního přiložení) BSL-3 biosafety level 3 (úroveň zabezpečení) BRNV Brno virus CDC Centers for Disease Control and Prevention (Centrum pro kontrolu a prevenci nemocí), U.S.A. cDNA complementary cDNA (komplementární DNA) DEPC diethyl pyrokarbonát DNA deoxyribonucleic acid (deoxyribonukleová kyselina) DOBV Dobrava-Belgrade hantavirus ECDC European Centre for Disease Prevention and Control (Evropské centrum pro prevenci a kontrolu nemocí), Stockholm EFSA European Food Safety Authority (Evropský úřad pro bezpečnost potravin) ELISA Enzyme-Linked ImmunoSorbent Assay (enzymová imunoanalýza) EPIDAT databáze epidemiologických dat ČR, Státní zdravotní ústav FLI Friedrich-Loeffler-Institut GenBank ® genetická sekvenční databáze HFRS Hemorrhagic Fever with Renal Syndrome (hemoragická horečka s renálním syndromem) HPS Hantavirus Pulmonary Syndrome (hantavirový plicní syndrom) HTNV Hantaan virus ICTV International Committee on Taxonomy of Viruses (Mezinárodní komise pro taxonomii virů) MEGA7 Molecular Evolutionary Genetics Analysis software MERS Middle East repiratory syndrome (Blízkovýchodní respirační syndrom) MERS-CoV MERS coronavirus (MERS koronavirus) NCBI National Center for Biotechnology Information (Národní centrum pro biotechnologické informace), U.S.A. NE nephropathia epidemica NGS Next Generation Sequencing (sekvenování nové generace) PCR Polymerase Chain Reaction (polymerázová řetězová reakce) PRNT Plaque reduction neutralization test (plak redukční neutralizační test) PROMED Program for Monitoring Emerging Diseases (program pro monitorování emergentních nákaz) PUUV Puumala virus real-time RT-qPCR kvantitativní reverzně transkripční polymerázová řetězová reakce v reálném čase RNA ribonucleic acid (ribonukleová kyselina) RT-PCR reverse transcription polymerase chain reaction (reverzně transkripční polymerázová řetězová reakce) SARS Severe Acute Respiratory Syndrome (těžký akutní respirační syndrom) SARS-CoV SARS coronavirus (koronavirus SARS) SEOV Seoul virus SNV Sin Nombre virus ssRNA single stranded ribonucleic acid (jednovláknová ribonukleová kyselina) TBEV tick-borne encephalitis virus (virus klíšťové encefalitidy) TMB tetramethylbenzidin TULV Tula virus UN United Nations (Spojené národy)
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