Puumala Viruses by One-Step Real-Time RT-PCR

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hantavirů, z toho v souvislosti s klinickými případy Konce ´ a ´ hantavirového RNA genomu jsou rodově HPS . Více než klinických případů HPS bylo hlá specifické, vysoce konzervované a reverzně komplemen šeno v USA od data objevení syndromu , a četné další tární a jsou tedy schopné tvořit struktury ve tvaru držadla případy z Jižní Ameriky. pánvice znak čeledi Bunyaviridae. Tyto struktury fungují jako promotor a hrají úlohu při regulaci virové replikace a transkripce . Segmentovaný genom relativně snad CHARAKTERISTIKA HANTAVIRŮ (MORFOLOGIE, no podléhá reasortaci a také homologní rekombinaci, což STRUKTURA, GENOM A ZPŮSOB PŘENOSU) jsou klíčové procesy určující jejich bohatou genetickou Morfologie hantavirů je typická pro členy čeledi diverzitu a odrážející se v evoluci hantavirů . Bunyaviridae : jsou to obalené, většinou sférické nebo Na rozdíl od hlodavců s celoživotní perzistentní infekcí ovoidní částice s průměrem nm. Mohou ale tvořit člověk nehraje zásadní roli při cirkulaci hantavirů v pří i protáhlé útvary, které se u jiných bunyavirů tak často rodním ohnisku nákazy, jde o tzv. konečného hostitele nevyskytují. Na povrchu mají charakteristický šachov deadend host, který se nakazí náhodně při vstupu do nicový vzor složený ze čtvercových, přibližně x nm ohniska po kontaktu s infikovaným hlodavcem. Nákaza velkých morfologických podjednotek. Viriony obsahují se přenáší aerosolem obsahujícím infikované exkrety husté jádro s ribonukleoproteinovými strukturami a po hlodavců sliny, moč, trus. Ačkoliv inhalační cesta pře vrchové glykoproteiny uložené v dvojvrstvém lipidovém nosu hantavirů je klíčová, byla popsána i onemocnění po obalu , . Virové částice jsou inaktivovány teplem kousnutí infikovaným hlodavcem. U viru Andes je navíc min/ °C, kyselým pH, detergenty, organickými zvažován interhumánní přenos , , . rozpouštědly a UV zářením . Genomy hantavirů sestávají ze tří jednořetězcových RNA segmentů, které jsou převážně negativní polarity . GEOGRAFICKÉ ROZŠÍŘENÍ HANTAVIRŮ Velký L, „large“ segment je přibližně nukleo Hlavní geografické rozdělení hantavirů je na hanta tidů dlouhý , kóduje protein L, který slouží jako viry Starého a Nového světa. Hantaviry Starého světa RNAdependentníRNApolymeráza , . Střední M, se vyskytují v mnoha oblastech Evropy a Asie a me „medium“ segment o velikosti nukleotidů kóduje zi nejdůležitější patogenní zástupce patří hantaviry glykoproteinový prekurzor , který se posttranslač Hantaan, Puumala, DobravaBelgrade a Seoul , . ně štěpí na dva glykoproteiny, G kD a G kD. Viry Saaremaa, Kurkino, DobravaBelgrade a Sochi, Proteiny G a G fungují jako antigeny rozpoznávané které podle katalogu ICTV International Committee on neutralizačními protilátkami . Glykoproteiny mají Taxonomy of Viruses byly původně deklarovány jako od N konec vystavený na povrchu virionu a C konec ukot lišné viry, jsou nyní na základě konsenzu expertů řazeny vený v membráně a pravděpodobně se účastní interakce pouze jako čtyři odlišné genotypy viru DobravaBelgrade viru s hostitelskou buňkou při její infekci . Malý . Hantaviry Starého světa jsou přenášeny hlodavci S, „small“ segment kóduje nukleokapsidový protein N z podčeledi Murinae řád Rodentia, podřád Myomorpha, čeleď a je až nukleotidů dlouhý . V rámci zástup Muridae . Hantavirus Puumala je členem skupiny ců hantavirů se délka a sekvence S segmentu podstatně hantavirů Starého světa, které jsou přenášeny hlodavci nemění, což naznačuje, že hraje důležitou funkční úlo podčeledi Arvicolinae řád Rodentia, podřád Myomorpha, hu . N protein se syntetizuje v rané fázi infekce čeleď Cricetidae. Hantaviry Nového světa se vyskytují a jde o nejhojnější virový protein, který hraje klíčovou v Severní a Jižní Americe a jejich hostiteli jsou křečci roli v některých důležitých procesech životního cyklu podčeledí Neotominae a Sigmodontinae řád Rodentia, podřád viru: zamezuje např. přístupu nukleáz hostitelské buňky Myomorpha, nadčeleď Muroidea, čeleď Cricetidae , . k virové RNA a podílí se na replikaci viru a sestavování Přehled významných hantavirů Starého i Nového světa virionů . Pravděpodobně interaguje s hostitelskou včetně patogenity, hlodavčích hostitelů a geografického buňkou a ovlivňuje imunitní odpověď na infekci . rozšíření je uveden v tabulkách a .

Tabuľka 1. Přehled patogenních hantavirů Starého světa, včetně rezervoárů a geografického rozšíření [21] Table 1. List of Old World hantaviruses pathogenic for human, including reservoirs and geographic distribution [21] Název viru Onemocnění Rezervoár Geografické rozšíření Hantaan HFRS Apodemus agrarius Čína, Jižní Korea, Rusko, Taiwan Seoul HFRS Rattus norvegicus, R. rattus celosvětové Dobrava- Apodemus flavicollis, A. agrarius, HFRS evropská část Ruska, Estonsko, střední a jihovýchodní Evropa (Balkán) Belgrade A. sylvaticus Puumala HFRS (NE) Myodes glareolus Evropa (včetně západního Ruska) Tula HFRS Microtus arvalis Evropa (včetně Ruska) Thailand HFRS Bandicota indica, B. savile jihovýchodní Asie: Srí Lanka, Indie, jih Číny, Laos, Taiwan, Thajsko, Vietnam Amur HFRS Apodemus peninsulae Rusko, severovýchod Číny, východní ostrovy Sachalin a Hokkaidó Soochong HFRS Apodemus peninsulae Jižní Korea Sangassou * Holomyscus simus centrální Afrika

2015, 64, č. 4 EPIDEMIOLOGIE, MIKROBIOLOGIE, IMUNOLOGIE 189

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Tabulka 2. Přehled patogenních hantavirů Nového světa, včetně rezervoárů a geografického rozšíření [21]. Table 2. List of New World hantaviruses pathogenic for human, including reservoirs and geographic distribution [21]. Název viru Onemocnění Rezervoár Geografické rozšíření Sin Nombre HPS Peromyscus maniculatus USA (včetně Aljašky), Mexiko, Kanada Bayou HPS Oryzomys palustris jihozápad USA Black Creek Canal HPS Sigmodon hispidus Florida New York HPS Peromyscus leucopus USA, Kanada, Mexiko Monongahela HPS Peromyscus maniculatus západ USA, Kanada Andes HPS Oligoryzomys longicaudatus Jižní Amerika, hlavně Argentina, Chile, Brazílie Araraquara HPS Bolomys lasiurus Brazílie Juquitiba HPS Oligoryzomys nigripes Brazílie, Uruguay Laguna Negra HPS Calomys laucha Brazílie, Paraguay, sever Argentiny, Bolívie Castelo dos Sohos HPS Oligoryzomys moojeni Brazílie Anajatuba HPS Oligoryzomys fornesii Brazílie Choclo HPS Oligoryzomys fulvescens Panama Bermejo HPS Oligoryzomys chacoensis Argentina Lechiguanas HPS Oligoryzomys flavescens Argentina, jihovýchod Brazílie, Uruguay Orán HPS Oligoryzomys longicaudatus Argentina Rio Mamore HPS Oligoryzomys microtis Bolívie, Brazílie, Peru, Argentina, Paraguay Rio Mearim HPS Holochilus sciureus Brazílie

Vysvětlivky: HFRS – hantavirová horečka s renálním selháním; NE – nephropathia epidemica; HPS – hantavirový plicní syndrom. * detegovány protilátky u lidí

Explanations: HFRS – haemorrhagic fever with renal syndrome; NE – nephropathia epidemica; HPS – hantavius pulmonary syndrome * antibodies detected in humans

V poslední době bylo díky progresivním molekulárním sérologicky v České republice popsali Kobzík a Daneš technikám především sekvenování nové generace objeve u farmářů na Břeclavsku . Jednalo se o pacienty s leh no několik nových zástupců hantavirů také u hmyzožrav čí až středně těžkou formou onemocnění. Následně ců Soricomorpha − rejsků, krtků, a u letounů řád Chiroptera bylo v roce vyšetřeno lidských sér: protilát , . Dosud však není nic známo o jejich patogenním ky k hantavirům se našly jen u vyšetřených sér. potenciálu pro obratlovce včetně člověka. Nejvyšší procento pozitivity dosahovala séra ze skupi Důležitou roli pro udržování ohnisek hantavirů Starého ny zemědělců z jižní Moravy . Pejčoch a Kříž dále světa v přírodě hrají místa, kde jsou ideální podmínky zkoumali přítomnost protilátek v krevních sérech pro přemnožení hlodavců stohy slámy, seníky nebo náhodně vybraných osob starších let . Protilátky rozptýlená lidská obydlí. Hantaviry Nového světa, re některých osob reagovaly s antigenem viru Hantaan spektive hlodavci, kteří je přenášejí, upřednostňují spíše osob, séropozitivita , u jiných s antigenem viru aridní oblasti . Puumala osob, prevalence , , u některých s oběma antigeny. K podobným výsledkům došla ve své starší studii i L. Matyášová u obyvatel Prahy, Severočeského, HANTAVIRY V ČESKÉ REPUBLICE Východočeského a Západočeského kraje vyšetřených Při hloubkové rešerši domácí literatury za posledních osob, séropozitivita , . Přítomnost protilátek třicet jsme kriticky vyhodnotili asi prací zabývajících v krevním séru v populaci nasvědčuje, že organismus se hantaviry, na základě kterých analyzujeme výzkum se s virem setkal, avšak infekce mohla mít mírný nebo na našem území. asymptomatický průběh, nebo byla chybně diagnosti První česká přehledná práce o hantavirech byla uveřej kována, což je u hantaviróz bohužel běžný jev. Dále je něna dokonce až v roce . Ovšem z historického nutné zmínit, že protilátky detegované v testu ELISA hlediska pocházejí nejstarší údaje týkající se výskytu proti viru Hantaan v séru vyšetřovaných osob reagují hantavirů ve střední Evropě z roku z východního zkříženě s virem DobravaBelgrade. V posledních dvou Slovenska z oblasti Ruské Poruby, místa první epide dekádách bylo u nás publikováno několik důležitých mie HFRS v bývalém Československu v . letech . studií zabývajících se séroprevalencí hantavirových pro století, kde byl detegován antigen HFRS u hlodavců tilátek v lidské populaci. V rozsáhlé retrospektivní studii Myodes glareolus, Apodemus flavicollis a Apodemus agrarius Státního zdravotního ústavu v Praze séra získaná ze a následně i u hlodavců z Česka spolu s potvrzeným sérologických přehledů z let bylo vyšetřeno nálezem protilátek u lidí , . Vůbec první tři lidské celkem osob z jižních a jihozápadních Čech bývalé případy způsobené infekcí virem Puumala a potvrzené okresy Klatovy, Strakonice, Prachatice, Český Krumlov,

190 EPIDEMIOLOGIE, MIKROBIOLOGIE, IMUNOLOGIE 2015, 64, č. 4

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České Budějovice, Jindřichův Hradec, Tábor a osob přenášený hrabošem polním Microtus arvalis a také nově ze Vsetínska, kdy ve vzorcích vyšetřených kitem ELISA popsaný virus Seewis, přenášený rejskem obecným Sorex byly prokázány protilátky k virům Puumala osob araneus a rejskem malým S. minutus , , . Virus a Hantaan osoba, tedy průměrná promořenost ,. Tula je nejčastěji se vyskytujícím hantavirem u našich Vyšší přítomnost IgG protilátek byla zjištěna především hlodavců, s prevalencí pohybující se kolem . v okrajových věkových skupinách, tedy u nejmladších Poprvé byl izolován na jižní Moravě z hraboše M. arvalis a nejstarších sledovaných osob . Zelená a Januška a je považován za nepatogenní , . V roce byla vyšetřili séra pacientů z Moravskoslezského regio však popsána infekce tímto virem u imunokompromito nu a zjistili prevalenci osob reagovalo proti viru vaného jedince z Ostravska . Hantaan a proti viru Puumala, osoby proti obě ma antigenům . Klinické symptomy kompatibilní s hantavirózou byly zjištěny u z těchto séropozitivních ONEMOCNĚNÍ osob. V jiné studii hemodialyzovaných pacientů ze U člověka hantaviry primárně infikují buňky endotelu západočeského regionu z celkového počtu vyšetře v plicích nebo ledvinách a také makrofágy, i když virový ných séropozitivita , vykazovalo protilátky proti antigen je přítomný i v dalších tkáních . Klinický antigenům Puumala a Hantaan, což naznačuje možnou průběh infekce se liší v závislosti na druhu viru a jeho ge souvislost mezi chronickým postižením ledvin a pří notypu jednotlivé genotypy viru DobravaBelgrade mají tomností patogenních hantavirů . Výskyt protilátek různě závažný průběh, důležitou roli hraje genetická celková séropozitivita , proti virům Hantaan je predispozice zejména HLA systém, pohlaví, věk, virová dinci, Puumala jedinci a Seoul jedinci byl zjištěn nálož anebo imunokompetence člověka, který se s virem u zdravých vojáků v letech . Vyšší výskyt setkal . Onemocnění spojené s hantaviry Starého protilátek k hantavirům u vojáků ve srovnání s běžnou světa, tj. hemoragická horečka s renálním syndromem populací lze vysvětlit častým pobytem příslušníků armá HFRS, postihuje hlavně ledviny, zatímco hantavirový dy ve volné přírodě především ve výcvikových prostorech plicní syndrom HPS, jehož původci jsou hantaviry a tedy vyšší expozicí aerosolové infekci . Nového světa, je spojený se selháním kardiopulmo Během let se podle databáze EPIDAT na na nálního systému . V současnosti přibývá důkazů šem území vyskytlo hlášených případů HFRS , poukazujících na určitou podobnost mezi těmito dvěma a jeden popsaný importovaný případ u vojáka složek syndromy. Jak HFRS tak i HPS mohou vést ke zhoršené UNPROFOR, který se nakazil při misi na Balkáně . funkci ledvin, krvácení a kardiopulmonálnímu postižení Klinická onemocnění způsobená hantaviry byla dia v důsledku zvýšené propustnosti kapilár a sníženého gnostikována v Čechách i na Moravě, a to především počtu krevních destiček trombocytopenie . na Ostravsku respektive na československém pomezí a v jižních Čechách , . V České republice byly po Hemoragická horečka s renálním syndromem HFRS, psány lidské infekce hantaviry především u dospělých, „Haemorrhagic Fever with Renal Syndrome“ ale vyskytlo se i několik případů onemocnění u dětí. HFRS je akutní horečnaté onemocnění v začátcích při V podstatě jde vždy o ojedinělé případy. V roce byly pomínající chřipku, které může vést až k závažným popsány první tři případy infekce nephropathia epidemica krvácivým stavům a k selhání ledvin . Typický průběh u dětí hospitalizovaných s intersticiální nefritidou HFRS se skládá z pěti stadií: fáze horečky, fáze hypoten . Děti nevykazovaly typické příznaky infekce virem ze, fáze oligurie, diuretická fáze a fáze rekonvalescence Puumala, kterou pravděpodobně získaly vdechnutím in . Jednotlivé fáze se dají zřetelně odlišit v průběhu těžké fikovaného aerosolu na víkendové chatě při svých opako formy onemocnění způsobeného hantaviry Hantaan vaných návštěvách. V srpnu byl hlášen případ le nebo DobravaBelgrade. tého chlapce, který se infikoval u obce Ostravice poblíž hranic se Slovenskem. Vyvinula se u něho těžká forma Infekce viry Hantaan a DobravaBelgrade HFRS způsobená virem DobravaBelgrade . Případy Viry Hantaan a DobravaBelgrade způsobují těžkou těžké formy HFRS původce opět virus DobravaBelgrade formu HFRS . Inkubační doba činí týdnů u vi byly dokumentovány u dvou imunokompetentních ru DobravaBelgrade, poté nastupuje febrilní fáze mužů po pobytu na horské chatě na slovenskočeském dní s příznaky připomínajícími chřipku vysoká pomezí. Rizikovým faktorem zde byl úklid chaty těsně horečka, zimnice, bolesti hlavy a svalů a intenzivní před návštěvou . Jediná popsaná epidemie hantaviry bolest zad , . Často se vyskytuje bolest v dutině u nás se odehrála na Prachaticku, kde bylo mezi roky břišní, žaludeční obtíže, nevolnost, zvracení, malát až popsáno případů nephropatia epidemica, nost a poruchy vizu , , . Dochází ke zvýšení způsobené virem Puumala. Pejčoch et al. zde provedli propustnosti kapilár, která vede k zarudnutí tváří, geobotanický průzkum spojený s odchytem drobných krku, objevují se petechie a krvácení do spojivek. Po hlodavců a charakterizovali vhodný biotop pro výskyt dnech vysoká horečka ustoupí a pacient se dostane hantavirů: smíšený les s predominancí buku Fagus do fáze hypotenze , která může trvat hodiny až dny sylvatica . většinou dny. Pokles tlaku bývá někdy prudký může Ve střední Evropě v současnosti cirkulují mezi hlodavci dojít až k šoku, objevuje se trombocytopenie, leuko a hmyzožravci hantaviry: virus DobravaBelgrade je cytóza, zvýšený hematokrit a je výrazná proteinurie. přenášený jak myšicí temnopásou A. agrarius genotyp S trombocytopenií souvisí krvácivé projevy po těle se Kurkino klinicky lehčí průběh infekce, tak myšicí lesní objevují hematomy, může být hematemeza, epistaxe, A. flavicolis genotyp Dobrava doprovázený těžším prů hematurie, meléna, případně i krvácení do centrálního během infekce. Dalším hantavirem je virus Puumala, nervového systému, které je mnohdy smrtelné , . přenášený norníkem rudým Myodes glareolus, virus Tula, Z laboratorních ukazatelů lze zmínit zvýšené hladiny

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transamináz, sérového kreatininu nad µmol/l, vážného průběhu HPS je zvýšená permeabilita plicních laktátdehydrogenázy, urey, Creaktivního proteinu či kapilár, která může vyvolat hypoxémii až rychlý rozvoj Ddimerů. Většinou kolem osmého dne se krevní tlak těžkého respiračního selhání s kardiogenním šokem normalizuje, ale onemocnění pokračuje oligurgickou a selháním srdečním , . Nejvíc onemocnění HPS fází, v trvání dnů . Zhoršuje se funkce ledvin, je způsobeno virem Sin Nombre s jasnými klinickými což vede k oligurii až anurii. Asi polovina úmrtí na příznaky a minimálními projevy mimo dutinu hrudní stává právě v této fázi. Krvácivé projevy souvisejí ne . Naopak případy, u kterých nastane porucha funkce jen s extrémní trombocytopenií, ale také se zvýšenou ledvin, bývají často spojené s infekcí viry Bayou nebo propustností kapilár a rozvojem DIC diseminované Black Creek Canal . intravaskulární koagulace. DIC s šokem a multiorgá Klinický průběh HPS je rozdělen do fází s odchylkami novým selháním také bývá nejčastější příčinou úmrtí v závažnosti a výskytu mezi pacienty: fáze horečky, , . Průběh této fáze může zhoršit krvácení do kardiopulmonální fáze, fáze diuretická a zotavovací gastrointestinálního traktu nebo hemoptýza. Pacienti, fáze . Inkubační doba bývá dní a počáteční pří kteří přežijí oligurickou fázi, se dostanou do diuretic znaky zahrnují náhlý vzestup teploty, bolesti hlavy, ké fáze, která může trvat několik týdnů a nástup této svalů a žaludeční obtíže nauzea, zvracení , . fáze znamená pro pacienta pozitivní prognózu , , Objevuje se také trombocytopenie. Febrilní stadium trvá . Ledviny totiž začínají regenerovat, produkce moči přibližně dní a postupně se přidává kašel, dušnost, stoupá až na několik litrů denně litrů za den, pocit sevřeného hrudníku, tachykardie a hypotenze, a pokud ztráty tekutin nejsou nahrazeny, pacienti jsou jež jsou prvními příznaky nástupu kardiopulmonální ohroženi dehydratací . Rekonvalescence je konečnou fáze, u které se rozvíjí těžké respirační selhání , . fází onemocnění a obvykle trvá týdny až měsíce . Často dojde ke kardiogennímu šoku, laktátové acidó Letalita na infekci způsobenou těmito viry se pohybuje ze a srdeční arytmii . Právě prvních hodin od do , . po nástupu šoku je pro přežití pacienta kritických. Nezbytná bývá plicní ventilace včetně monitorování Infekce virem Seoul a podpory základních životních funkcí, přesto letalita Onemocnění způsobené virem Seoul se liší od průběhu může dosáhnout až , . Pacienti, kteří toto předchozí infekce závažností a tím, že může postihnout období přežijí, se dostávají do diuretické fáze, ve které i játra . U pacienta nelze s jistotou rozlišit pět typic dochází ke zvýšení produkce moče a stav se postupně kých fází pro HFRS . Výzkum ukázal, že onemocnění se zlepšuje . Poté nastává zdlouhavá rekonvalescence, vyskytuje převážně u letých mužů stejně jako provázená zvýšenou slabostí a únavou. u infekcí způsobených jinými hantaviry a mezi hlavní příznaky patří zvracení, horečka, bolesti svalů a bři cha, postižení spojivek a petechie. Na rozdíl od infekcí DIAGNOSTIKA HANTAVIRÓZ způsobených ostatními hantaviry bývá často přítomna Prvotní příznaky infekce hantaviry nejsou specifické, hepatitida. Letalita je pod , . a jejich diagnostika bývá složitá. V diagnostické praxi se nejčastěji využívá kombinace klinických a sérologických Infekce virem Puumala nálezů, někdy doplněné o molekulární analýzu . Virus Puumala způsobuje mírnou formu HFRS, kte rá se nazývá „nephropathia epidemica“ NE . Podobá se Přímá diagnostika HFRS, ale symptomy tohoto onemocnění jsou mírnější. Na přímou izolaci hantavirů lze použít buňky Vero Jednotlivá stadia nejsou výrazně odlišena a ledviny jsou E, ve kterých viry replikují bez tvorby cytopatického relativně málo postižené. Při infekci virem Puumala se efektu. Hantaviry také infikují linie buněk Huh a A běžně objevují příznaky jako kašel, přítomnost plicního získané z lidského karcinomu jater a plic . Obecným infiltrátu a snížená funkce plic . Tyto příznaky jsou problémem při izolačních pokusech bývá skutečnost, ale většinou málo závažné a často bývají přehlédnuty. že virémie je u člověka poměrně krátkodobá. Poslední Typicky se u pacientů vyskytuje horečka, bolesti svalů, slovinská studie však překvapivě udává průměrnou dél břicha a zad. V případě postižení ledvin se okolo třetího ku virémie u člověka dní pro virus DobravaBelgrade nebo čtvrtého dne objeví oligurie až anurie. Dialýzu a dní pro virus Puumala . Izolační pokusy jsou vyžaduje méně než pacientů. Hemoragické příznaky navíc pracné, časově náročné a pro personál rizikové petechie, hematurie nebo meléna se vyskytnou asi u . Molekulární metody založené na amplifikaci nuk případů , . Často se také objeví mlhavé vidění, leových kyselin viru, jako jsou polymerázová řetězová akutní myopie a popsán byl i glaukom , . Během reakce s využitím reverzní transkriptázy RTPCR nebo druhého týdne onemocnění se pacienti začínají zotavo realtime RTPCR, které jsou vysoce citlivé, specifické vat, ale plná úzdrava vyžaduje vždy delší dobu. Letalita a rychlé, umožňují druhovou identifikaci hantavirů po je v rozmezí od , Finsko po , Ural. Závažný sekvenaci vybraných úseků genomu a dají se použít klinický průběh infekce virem Puumala je silně vázán pro vyšetření ještě před rozvojem onemocnění. Virový na přítomnost haplotypu HLAB , zatímco u lehkých materiál RNA pro tyto testy bývá purifikován z krve průběhů je to haplotyp HLAB. nebo slin pacienta během akutní fáze infekce, případně z autopsie vzorků infikovaných tkání nejlépe ve vire Hantavirový plicní syndrom HPS, „Hantavirus mické fázi onemocnění . Úspěšnost molekulární Pulmonary Syndrome“, také označovaný HCPS, identifikace také závisí na výběru vhodných primerů „Hantavirus CardioPulmonary Syndrome“ primery a sondy se v případě Realtime PCR vážou HPS představuje mnohem závažnější onemocnění než na vysoce konzervativní oblasti v rámci L segmentu HFRS letalita dosahuje . Primární příčinou hantavirového genomu nebo vysoce homologní oblasti

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Ssegmentu a náloži virové RNA ve vzorku , . infekcí . Intravenózně podávaný ribavirin má an Pro rozlišení různých kmenů hantavirů lze také využít tivirovou aktivitu proti hantavirům způsobujících plakredukční neutralizační test PRNT. HFRS v časných fázích, ale jeho podávání zůstává stále na experimentální úrovni. Například u pacientů Nepřímá diagnostika v Číně se prokázala snížená úmrtnost v případě, kdy Z imunologických metod se pro diagnostiku hantaviro byl ribavirin podáván během prvních dní po nástupu vých infekcí využívají ELISA enzymelinked immunosor prvotních příznaků , . Dokáže zabránit i progresi bent assays, imunofluorescenční test IF, a imunoblo onemocnění do oligurické fáze a zpomaluje selhávání tové nebo imunohistochemické testy . V současnosti funkce ledvin. Jeho použití je omezené především na jsou vyvinuté vysoce citlivé a specifické IgM a IgG ELISA těžké a rychle rozpoznané formy onemocnění, např. testy např. diagnostické sety firem Progen, Focus nebo během epidemie , . Bohužel zůstává neúčinný Euroimmun. Jako diagnostické antigeny se využívají při léčbě HPS, kde vhodné antivirotikum je třeba teprve přirozené nebo rekombinantní N proteiny viru, které jsou vyvinout . významné svou vlastností vyvolávat brzkou a dlouhotrva Imunoterapie je jedna z dalších perspektiv pro léčbu han jící imunitní odpověď. Problémem však může být nízká tavirových infekcí. Podání lidských neutralizačních pro senzitivita či zkřížená reaktivita , , . Metoda IF tilátek během akutní fáze HPS by mohlo vést k poklesu je populární metodou díky jednoduchosti provedení, ne virémie a podpořit rekonvalescenci. Studie na hlodavcích výhodou tohoto testu jsou však problémy se specificitou ukázala, že pasivní transfer neutralizačních monoklonál testu ovlivněnou zkříženými reakcemi protilátek proti ních protilátek nebo polyklonálního séra dokáže ochránit různým zástupcům hantavirů, a také nezkušenost uži zvířata před infekcí . Imunoterapie se jeví být slibnou vatelů při mikroskopickém hodnocení . Nedávno byl metodou, avšak na léčbu hantavirových infekcí by bylo vyvinut vysoce specifický a citlivý imunofluorescenční zapotřebí udržovat přiměřené koncentrace protilátek po test navržený na podkladě biočipu, který dokáže dete dostatečně dlouhou dobu, což se dosud nedaří. V sou govat protilátky ke všem známým hantavirům Starého časné době se testují také speciální imunoterapeutika, a Nového světa . Imunoblotové testy využívají také která by inhibovala zvýšenou propustnost kapilár, jež je rekombinantní antigen a patří mezi specifické a cit důsledkem hantavirové infekce . livé metody, které slouží ke konfirmaci pozitivního ELISA testu např. sety Mikrogen. Imunohistochemické metody umožňují obarvení hantavirových antigenů ve VAKCINACE vzorcích tkání, a stávají se tak ideálním nástrojem pro V Asii se využívají inaktivované monovalentní vakcíny retrospektivní diagnózu infekcí hantaviry v konzervo např. Hantavax v Koreji byť s krátkodobou protektivní vaných biopsiích . Pro detekci akutních infekcí viry odpovědí a nutností častého dávkování , . Žádná DobravaBelgrade, Hantaan a Puumala byl nedávno vy z nich se však neosvědčila pro používání v Evropě či vinut pětiminutový imunochromatografický IgM test . USA neexistuje zkřížená protektivita. Je potřeba vyvi Neutralizační test se využívá i na detekci neutralizačních nout takovou vakcínu, která by byla bezpečná, účinná protilátek vyvolaných infekcí a je díky své vysoké specifi a multivalentní, zároveň však adaptovaná na místní citě a senzitivitě považován za zlatý standard sérologické podmínky . Zatím byly vyrobeny pouze kandidátní diagnostiky , . Test je však časově náročný trvá rekombinantní vakcíny na podkladě replikačních kom dny až týdny a ne vždy je dostupný živý antigen. Kvůli ponent viru vakcinie nebo rekombinantních bakterií, aerosolům, které mohou vznikat během laboratorních které kódují jeden nebo více virových proteinů. V Evropě procedur se izolační, molekulární a sérologické meto byla vyvinuta kandidátní vakcína proti viru Puumala, dy s živým materiálem provádějí pouze v laboratořích která obsahuje proteinové komplexy s mnoha žádanými s úrovní zabezpečení BSL nebo BSL . vlastnostmi, jako imunogeny prezentující N protein viru, a která vyvolává silnou imunitní odpověď , . V současnosti se v USA pracuje na vakcíně, která využívá LÉČBA M segment virů Hantaan a Puumala . Specifickou Aktuálně proti hantavirovým nákazám nemáme k dis imunitní odpověď dokáží vyvolat i hantavirové protei pozici žádnou specifickou terapii . Léčba je proto ny získané biotechnologickou cestou z transgenních symptomatická a podpůrná. U těžkých případů je ne rostlin . Jako užitečný nástroj pro vývoj vakcín se zbytné umístit pacienta na jednotku intenzivní péče, ukázaly např. také kvasinky Sacharomyces cerevisiae po kde je zajištěno monitorování základních životních skytující vysoký výtěžek nukleokapsidových proteinů funkcí, korekce příjmu a výdeje tekutin, minerální hantaviru . ho metabolismu a acidobazické rovnováhy , , . Taktéž zavedení umělé plicní ventilace anebo mimotělní okysličování v případě HPS a hemodialýza PREVENCE u HFRS jsou u kritických nemocných nezbytné . Možnosti léčby hantaviróz jsou nedostatečné, proto Velmi důležitá je schopnost lékařů rozpoznat počáteční je velmi důležitá prevence vzniku onemocnění . příznaky hantavirové infekce. V diferenciální diagnos Nejefektivnějším způsobem prevence infekcí způsobe tice hantaviróz je nejdůležitější myslet zejména na ných hantaviry nebo jinými hlodavci přenosnými viry leptospirózu, při plicním postižení na řadu infekcí, např. arenavirus lymfocytární choriomeningitidy je které mohou způsobit postižení dolních cest dýchacích potlačení kontaktu s hlodavci a jejich exkrety sliny, a obdobně i u renálního postižení přichází v úvahu také moč, trus. Člověk by se měl vyhýbat místům, kde žijí řada možností . V současné době nemáme žádné početné populace volně žijících synantropních hlodav účinné antivirové preparáty pro léčbu hantavirových ců a dochází k hromadění jejich exkretů např. staré

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budovy, sklepy, půdy. Je důležité aktivně omezovat dočasně, protože příroda a přírodní ohniska se vyvíjejí počet jejich úkrytů a zdrojů potravy v okolí lidských a mění dál, i když velmi pomalu“ . obydlí. Dále je vhodné uzavřít vstupy do domů a budov a zamezit přístup těmto živočichům, popř. vydenzifi Poděkování kovat již potenciálně kontaminované úseky , , . Autoři děkují Evropské komisi za podporu projektu . rám Prevence ve formě osvěty by také měla směřovat k rizi cového programu EU grant FP EDENext http:// kovým skupinám, kterým hrozí vystavení volně žijícím www.edenext.eu, který se zabývá biologií a kontrolou hlodavcům pracovníci v lesnictví a zemědělství, zoolo infekcí přenášených vektory v Evropě. Publikace je v rámci gové, vojáci, trampové stejně jako imunokompromito tohoto programu katalogizována jako EDENext. Věcný vaným jedincům těhotné ženy, staří lidé, onkologičtí obsah publikace podléhá zodpovědnosti autorů a nere pacienti, pacienti po transplantaci, chronicky nemocní flektuje nutně pohled Evropské komise. Dále děkujeme lidé. Žádoucí by bylo vyšetřovat osoby s akutním či Operačnímu programu Ministerstva školství Vzdělání pro chronickým postižením ledvin na možnou souvislost konkurenceschopnost projektu CEB CZ../../. s výskytem hantavirového onemocnění, mj. za účelem a projektu Specifického výzkumu MU MUNI/A// lokalizace přírodních ohnisek nákazy. a MUNI/A//.

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Zoonoses and Public Health

ORIGINAL ARTICLE Reservoir-Driven Heterogeneous Distribution of Recorded Human Puumala virus Cases in South-West Germany S. Drewes1, H. Turni2, U. M. Rosenfeld1, A. Obiegala3, P. Strakova1,4,5, C. Imholt6, E. Glatthaar7, K. Dressel8, M. Pfeffer3, J. Jacob6, C. Wagner-Wiening9 and R. G. Ulrich1

1 Friedrich-Loefﬂer-Institut, Institute for Novel and Emerging Infectious Diseases, Greifswald – Insel Riems, Germany 2 Stauss & Turni Gutachterburo,€ Tubingen,€ Germany 3 Veterinarmedizinische€ Fakultat,€ Institut fur€ Tierhygiene und Offentliches€ Veterinarwesen,€ University Leipzig, Leipzig, Germany 4 Institute of Vertebrate Biology v.v.i., Academy of Sciences, Masaryk University, Brno, Czech Republic 5 Department of Experimental Biology, Faculty of Science, Masaryk University, Brno, Czech Republic 6 Julius Kuhn-Institute,€ Federal Research Centre for Cultivated Plants, Institute for Plant Protection in Horticulture and Forests, Vertebrate Research, Munster,€ Germany 7 Forstzoologisches Institut, Arbeitsbereich Wildtierokologie€ und Wildtiermanagement, Universitat€ Freiburg, Freiburg, Germany 8 sine-Institut gGmbH, Munich, Germany 9 Landesgesundheitsamt Baden-Wurttemberg,€ Referat 95 – Epidemiologie und Gesundheitsberichterstattung, Sachgebietsleitung: Infektionsepidemiologische Meldesysteme (SG4), Stuttgart, Germany

Impacts • The heterogeneous geographical distribution of recorded hantavirus disease cases is related to the abundance of Puumala virus (PUUV)-infected bank voles. • The effect of beech mast on human incidences is higher in districts with high proportional coverage of beech/oak woodland. • Local early warning modules for PUUV infections should include the prevalence of PUUV in the host which requires multiannual bank vole monitoring as well as information about mast and beech/oak woodland cover.

Keywords: Summary Hantavirus; endemic region; incidence; bank vole; prevalence; Germany Endemic regions for Puumala virus (PUUV) are located in the most affected federal state Baden-Wuerttemberg, South-West Germany, where high numbers Correspondence: of notified human hantavirus disease cases have been occurring for a long time. Rainer G. Ulrich. Friedrich-Loeffler-Institut, The distribution of human cases in Baden-Wuerttemberg is, however, heteroge- Institute for Novel and Emerging Infectious neous, with a high number of cases recorded during 2012 in four districts (H dis- Diseases, 17493 Greifswald – Insel Riems, tricts) but a low number or even no cases recorded in four other districts (L Germany. Tel.: +49 38351 7 1159; Fax: +49 38351 7 1192; districts). Bank vole monitoring during 2012, following a beech (Fagus sylvatica) E-mail: rainer.ulrich@fli.de mast year, resulted in the trapping of 499 bank voles, the host of PUUV. Analyses indicated PUUV prevalences of 7–50% (serological) and 1.8–27.5% (molecular) Received for publication January 14, 2016 in seven of eight districts, but an absence of PUUV in one L district. The PUUV prevalence differed significantly between bank voles in H and L districts. In the doi: 10.1111/zph.12319 following year 2013, 161 bank voles were trapped, with reduced bank vole abundance in almost all investigated districts except one. In 2013, no PUUV infections were detected in voles from seven of eight districts. In conclusion, the linear modelling approach indicated that the heterogeneous distribution of human PUUV cases in South-West Germany was caused by different factors including the abundance of PUUV RNA-positive bank voles, as well as by the interaction of beech mast and the proportional coverage of beech and oak (Quercus spec.) forest per district. These results can aid developing local public health risk management measures and early warning models.

et al., 2012; Faber et al., 2013; Ali et al., 2015; Castel et al., Introduction 2015). Recent studies in the district Osnabruck,€ Lower Sax- Puumala virus (PUUV), genus Hantavirus, family Bunya- ony, North-West Germany, and in Konnevesi, Central Fin- viridae, is causing the vast majority of human hantavirus land, confirmed oscillations in the prevalence of PUUV disease cases in Germany and surrounding countries infections in the bank vole populations, but also a long- (Ulrich et al., 2004; Heyman et al., 2011). Nephropathia term presence of selected PUUV strains (Razzauti et al., epidemica (NE), the hantavirus disease caused by PUUV, is 2013; Weber de Melo et al., 2015). usually characterized by a mild to moderate course with The disease incidence varied between the federal states of abrupt onset of fever, headache, myalgia, chills, conjunc- Germany, with the highest total number of cases recorded tivitis, abdominal pain, nausea, vomiting and in severe in Baden-Wuerttemberg (n = 5351), South-West Germany cases with microhaemorrhages and renal failure (Brum- (Pilaski et al., 1991; Zoller€ et al., 1995; Boone et al., 2012; mer-Korvenkontio et al., 1980; Kruger€ et al., 2013). Mild Robert Koch-Institute: SurvStat@RKI 2.0, https://surv-stat. and unspecific symptoms result in underreporting of rki.de, accessed: 21 April 2016). During the years 2007, human cases (Brummer-Korvenkontio et al., 1999; Cle- 2010 and 2012, much larger numbers of human hantavirus ment et al., 2007; Klempa et al., 2013). The reservoir host disease cases were notified with a simultaneous increase in of PUUV, the bank vole Myodes glareolus, is present in Baden-Wuerttemberg and other regions of Germany (Hof- almost entire Europe, but human cases of PUUV infection mann et al., 2008; Ettinger et al., 2012). However, even are heterogeneously distributed there (Linard et al., 2007; within this highly endemic federal state, a heterogeneous Boone et al., 2012; Ali et al., 2014; Clement et al., 2014; distribution of human PUUV cases has been observed Castel et al., 2015). Further, bank vole populations erupt (Fig. 1, Table 1). occasionally resulting in a massive increase in abundance in The objective of this study was to test potential associa- some years. Such outbreaks seem to be driven by mast of tions of bank vole abundance and serological and molec- beech (Fagus sylvatica) and oak (Quercus spec.) in Germany ular PUUV detection in bank voles with the reported and other Central European countries; predator-driven frequency of human infections and beech mast intensity processes matter in northern boreal Europe (Hansson, as well as beech/oak forest occurrence per district for dis- 1985; Hanski et al., 1991; Tersago et al., 2009; Clement tricts with a historically high number of recorded human et al., 2010; Imholt et al., 2015; Reil et al., 2015). cases (H) and for additional districts with no or low A recent study identified certain landscape and environ- numbers of recorded human cases (L) in Baden- mental factors, such as forest connectivity, winter and sum- Wuerttemberg. mer temperatures and soil water content, as drivers for the spatial distribution of NE cases in Europe (Zeimes et al., Materials and Methods 2015). Climate conditions might not only influence the virus viability and transmission directly, but indirectly by Bank voles were trapped at four H (H1, district influencing the behaviour of the human population at risk Stuttgart – site Busnau;€ H2, Tubingen€ – Mossingen;€ H3, for infection. Further, the awareness of the physicians has Goppingen€ – Geislingen; H4, Heidenheim – Steinheim) been discussed as a reason for apparent disease absence or and four L (L1, Schw€abisch Hall – Crailsheim; L2, underreporting. Thus, the detection of a novel PUUV strain Emmendingen – Kenzingen; L3, Freiburg im Breisgau – in the western part of Thuringia during the outbreak in Landwasser; L4, Waldshut – Stuhlingen)€ locations in 2012 2010 indicated that the virus may have been present but and 2013 using live traps (Fig. 1). Sites were characterized unrecognized, suggesting low awareness of physicians due by deciduous broad-leaved forest dominated by beech. Fifty to a very low number of disease cases before the 2010 out- traps were set at each site in suitable habitats for bank voles break (Faber et al., 2013). in order to maximize trapping success. Traps were baited Human PUUV infections in Germany are known since with a mix of nuts, sunflower seed, apples and mealworms the 1980s (Pilaski et al., 1991). Since the introduction of and emptied every 12 h for 36 h covering two nights and the Protection against Infection Act in 2001, in total 10 164 one day. Hay was provided for bedding and a cotton wool human hantavirus disease cases were recorded in Germany pad soaked with water for moisture. Trapping was con- (Robert Koch-Institute: SurvStat@RKI 2.0, https://survstat. ducted three times per year (May/June; July/August; rki.de, accessed: 21 April 2016). A large number of molecu- September/October). Rodent individuals were caught alive, lar epidemiological studies in bank vole reservoirs and their sex and weight determined, killed through cervical patients resulted in the demonstration of a high level of dislocation and immediately frozen and stored at À20°C PUUV sequence divergence (Pilaski et al., 1994; Heiske for subsequent laboratory analysis. Data are stated as bank et al., 1999; Asikainen et al., 2000; Essbauer et al., 2006, voles trapped per 100 trap nights (TN) reflecting maximum 2007; Schilling et al., 2007; Hofmann et al., 2008; Ettinger population abundance.

Fig. 1. Hantavirus incidences in districts with high incidences (H) and low incidences (L) of Baden-Wuerttemberg, South-West Germany, during 2012 and 2013, and location of the bank vole trapping sites H1 (Stuttgart), H2 (Tubingen),€ H3 (Goppingen)€ and H4 (Heidenheim), and L1 (Schwabisch€ Hall), L2 (Emmendingen), L3 (Freiburg) and L4 (Waldshut). Background maps: Nicole Neumann, Friedrich-Loefﬂer-Institut; human PUUV incidences: Robert Koch-Institute: SurvStat@RKI 2.0, https://survstat.rki.de, accessed: 21 April 2016.

Table 1. Incidences of recorded human Puumala virus cases per 100 000 inhabitants in eight districts of Baden-Wuerttemberg from 2001 to 2015

Year

District 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015

SK Stuttgart (H1) 1.02 3.57 1.19 1.19 1.69 0.84 5.19 2.00 1.50 22.26 0.82 19.73 0.00 0.83 1.32 LK Tubingen€ (H2) 0.47 3.29 0.93 2.78 1.39 0.00 13.33 0.00 1.36 11.75 1.35 40.02 0.00 6.93 10.62 LK Goppingen€ (H3) 1.16 3.09 1.16 3.09 2.33 0.78 32.84 0.78 2.76 17.42 5.56 56.09 0.40 4.42 14.47 LK Heidenheim (H4) 0.00 10.95 0.00 4.42 1.48 0.75 90.67 2.26 3.03 16.78 3.82 58.77 1.56 1.56 6.25 LK Schwabisch€ Hall (L1) 0.00 0.00 0.53 1.59 0.53 0.00 3.17 1.06 1.06 5.84 1.06 7.49 1.07 2.66 2.13 LK Emmendingen (L2) 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.64 0.00 0.00 0.00 SK Freiburg (L3) 0.00 0.00 0.47 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.46 0.00 0.00 0.00 LK Waldshut (L4) 0.00 0.00 0.00 0.00 0.00 0.00 0.60 0.60 0.00 0.00 0.00 0.00 0.00 0.00 0.00

LK, rural district; SK, urban district. Source: Robert Koch-Institute: SurvStat@RKI 2.0, https://survstat.rki.de, accessed: 21 April 2016.

Dissection and sample collection as well as morphologi- determine the serological reactivity following a previously cal and molecular species determination for shrews, Apode- published decision tree (Mertens et al., 2009). mus spp. and Microtus spp. followed standard protocols For molecular detection of PUUV, RNA was isolated â (Angermann and Hackethal, 1999; Parson et al., 2000; from lung tissue by QIAzol Lysis Reagent (Qiagen, Hil- Schlegel et al., 2012; Nainys et al., 2015). Three age groups den, Germany) treatment following an in-house protocol of bank voles were defined based on the weight: <15, 15– (Schmidt et al., 2016). Reverse transcription polymerase 19.5 and >19.5 g (Bajer et al., 2002). chain reaction (RT-PCR) using S segment-specific primers PUUV-reactive antibodies were detected in thoracic Pu342F and Pu1102R followed a previously described stan- cavity lavage by IgG ELISA using a recombinant nucleocap- dard protocol (Essbauer et al., 2006). Amplification prod- sid protein of PUUV strain Bavaria (Mertens et al., 2011). ucts of the expected size of ca. 700 bp were sequenced by â ELISA-positive or equivocal sera were tested again to the usage of BigDye Terminator version 1.1 Cycle

Sequencing Kit (Applied Biosystems, Darmstadt, Ger- many). – Fisher’s exact test with a type I error a of 0.05 and associated odds ratios were used to test the independence of 1 13 compared prevalences between H and L districts, season Waldshut (L4) and individual weight. To analyse determining factors for human infection, two backward multiple linear regression – models were developed. For both models, the ln-transformed number of human cases standardized to 100 000 inhabitants (PUUV incidence) was the dependent variable 1 (Robert Koch-Institute: SurvStat@RKI 2.0, https://survstat. Freiburg (L3) rki.de). The ﬁrst model was designed to test which predic- tor was correlated best to human PUUV cases in the outbreak year of 2012 in all eight districts. The mean bank vole abundance index, the antibody prevalence determined –––––– by ELISA and the RNA prevalence detected using RT-PCR –– were used as potential predictors. ELISA results were cor- Emmendingen (L2) rected for the potential inﬂuence of maternal antibodies in young voles. Individuals below the cut-off value of 16 g (Kallio et al., 2010) that carried antibodies but were RT-

PCR negative were treated as carrying maternal antibodies € abisch Hall and assigned to the negative ELISA category. The second 21 Schw (L1) analysis focused on potential environmental factors determining the marked differences in human PUUV incidence among districts (n = 8). Here, the ln-transformed propor- ––––––– tional cover of beech and oak forest per district and the categorical variable of beech mast in the previous year 1 1535

(yes/no) were determined for each district for the years Heidenheim (H4) 2007–2014. Values for forest cover were obtained from the federal forest census (http://www.fva-bw.de/monitoring/)

by calculating average values from the censuses in 2002 – and 2012. For the city districts H1 and L3, no forest data were available and therefore the districts closest to the € oppingen 1 trapping sites where forest data were available were chosen G (H3) (Boblingen€ and Breisgau im Hochschwarzwald, respectively). All analyses were performed in R (R Core Team, 2015) using the lm function. Model simpliﬁcation was done using drop1 and the ﬁnal model was chosen based on lowest corrected Akaike’s information criterion (Hurvich . ) and other small mammals collected at the eight trapping sites during 2012 and 2013 € ubingen T and Tsai, 1989). (H2) ––– Results ––––––– . Sorex minutus 122 108 105 90 110 38 87 76 During 2012, the number of recorded human hantavirus Myodes glareolus and 1 disease cases and incidences was higher in the districts H1 1 Stuttgart (H1) (n = 118/19.73), H2 (n = 86/40.02), H3 (n = 139/56.09) 201287 2013 2012 12 2013 80 2012 2013 15 2012 80 2013 2012 9 2013 68 2012 2013 15 2012 55 2013 2012 44 2013 32 6 40 46 57 14 and H4 (n = 75/58.77) than in the districts L1 (n = 14/ Microtus agrestis 7.49), L2 (n = 1/0.64), L3 (n = 1/0.46) and L4 (n = 0/0.00) and Sorex coronatus

(Table 1, Fig. 1). , During 2012, 499 bank voles were trapped with a range † Numbers of bank voles ( of 32–87 animals per site (Table 2). Mean trap success ran- spp. spp.* 6 15 2 11 6 9 ged from 9.5 ind./100TN in L4 to 29 ind./100TN in the city Sorex araneus Microtus arvalis † * Total Apodemus ﬂavicollis Table 2. Small mammal species Microtus district H1 (Table 3). In addition, bycatches of four Myodes glareolus Sorex

Table 3. Abundance indices and results of serological and RT-PCR investigations of bank voles trapped at the eight districts during 2012 and 2013

Mean Abundance PUUV ELISA PUUV RT-PCR

District and trapping site Year Ind./100TN SE Absolute* Per cent Absolute* Per cent

Stuttgart (H1) 2012 29.0 4.6 26/87 29.89 20/87 22.99 2013 4.0 3.6 0/12 0.00 0/12 0.00 Total 26/99 26.26 20/99 20.20

Tubingen€ (H2) 2012 26.7 10.3 19†/80 23.75 14/80 17.50 2013 5.0 1.0 0/15 0.00 0/15 0.00 Total 19/95 20.00 14/95 14.74

Goppingen€ (H3) 2012 26.7 10.2 24/80 30.00 22/80 27.50 2013 3.0 2.0 0/9 0.00 0/9 0.00 Total 24/89 26.97 22/89 24.72

Heidenheim (H4) 2012 22.7 1.5 13†/68 19.12 15/68 22.06 2013 5.0 7.0 0/15 0.00 0/15 0.00 Total 13/83 15.66 15/83 18.07

Schwabisch€ Hall (L1) 2012 18.3 17.2 13/55‡ 23.64 6/55 10.91 2013 14.7 6.7 2/44‡ 4.55 5/44 11.36 Total 15/99 15.15 11/99 11.11

Emmendingen (L2) 2012 10.7 9.7 16/32 50.00 6/32 18.75 2013 2.0 1.0 0/6 0.00 0/6 0.00 Total 16/38 42.11 6/38 15.79

Freiburg (L3) 2012 13.0 6.9 0/40 0.00 0/40 0.00 2013 15.3 7.5 0/46 0.00 0/46 0.00 Total 0/86 0.00 0/86 0.00

Waldshut (L4) 2012 19.0 2.6 4/57§ 7.02 1/57 1.75 2013 4.7 4.2 0/14 0.00 0/14 0.00 Total 4/71 5.63 1/71 1.41

Ind., individuals; TN, trap nights; SE, standard error. *Number of positive/total number of bank voles tested. †One juvenile, antibody-positive animal was excluded. ‡Total number includes one equivocal sample. §Total number includes ﬁve equivocal samples.

Microtus spp., three yellow-necked mice (Apodemus flavi- significantly higher in districts H1–H4 compared to L1–L4 collis) and 20 shrews were collected (Table 2). in IgG ELISA [P = 0.037; two tailed Fisher’s exact test Serological investigations indicated the presence of with a = 0.05, odds ratio 1.64 (1.05–2.59)] and RT-PCR PUUV infections in bank voles in seven of eight districts (P < 0.001, odds ratio 3.77 (2.02–7.04)). That year adult with prevalences of 7–50% (Table 3). RT-PCR investiga- bank voles (weight class 3, >19.5 g) showed significantly tions confirmed PUUV infections for the same seven higher prevalences compared to juvenile individuals districts with prevalences of 1.8–27.5% (Table 3). In two (weight class 1, <15.5 g) (ELISA: odds ratio 21.19 (2.92– juvenile voles (weight < 16 g), one each from sites H2 163.86), Fisher’s test P < 0.001; RT-PCR: odds ratio 6.38 and H4, exclusively PUUV-reactive antibodies, but no (1.47–27.64), Fisher’s test P < 0.008) (see Table S1 in Sup- viral RNA were detected (Table 3). Therefore, these two porting information). In addition, there was a trend that animals were regarded as harbouring maternal antibodies. RNA prevalences decreased in autumn (Season 3) com- The voles from the four H districts showed serological and pared to spring (Season 1 versus Season 3; see Table S1 in molecular prevalences of 17.5–30.0%. At the districts L1, Supporting information; RT-PCR: odds ratio 2.48 (1.28– L2 and L4 seroprevalence reached 23.6%, 50% and 7%, 4.79), Fisher’s test P = 0.007; ELISA: odds ratio 1.60 respectively, whereas the molecular prevalence ranged (0.94–2.72), Fisher’s test P = 0.08). One field vole (Micro- from 1.8% (L4), to 10.9% (L1) and 18.8% (L2). In district tus agrestis) from trapping site H1 was found to be PUUV L3, none of the bank voles was positive for PUUV. In positive in RT-PCR, but none of the yellow-necked mice 2012, the prevalence of PUUV infection in bank voles was (data not shown).

Table 4. Results of multiple linear regression analysis for the suitability antibodies in the population was not significantly corre- of methodological and environmental predictors regarding human lated with the number of human PUUV cases. Neither the PUUV cases. Categorical variable Mast [1] indicates differences com- percentage of beech and oak woodland per district nor pared to the reference category Mast [0] representing no beech mast beech mast alone predicted the existing gradient in human Model B SE B b P-value Adj R2 AICc PUUV cases among districts. However, the interaction term between both predictors was positively correlated with the Methodological number of human PUUV cases (F = 22.11, adj. Step 1 3,36 2 = < Intercept À0.47 2.37 0.00 0.86 0.56 50.47 R 0.62, P 0.001), demonstrating that after beech mast Abundance 0.06 0.14 0.25 0.69 more human PUUV infections occurred in districts with RNA prevalence 0.16 0.12 1.00 1.30 higher cover of woodland than in districts with little beech Antibody À0.07 0.09 À0.42 À0.75 and oak forest. prevalence Step 2 Intercept À1.37 1.89 0.00 0.52 0.62 33.30 Discussion Abundance 0.08 0.13 0.32 0.57 RNA prevalence 0.10 0.08 0.59 0.33 We determined the presence of PUUV in bank voles from Step 3 (final) four H districts with a high number of recorded human Intercept À0.30 0.78 0.00 0.72 0.68 24.72 cases and further four L districts with no or a low number RNA prevalence 0.14 0.04 0.86 0.03* of recorded human cases during outbreak year 2012 and Environmental the following year. The serological and RT-PCR investiga- Step 1 tions in bank voles trapped during the outbreak year 2012 Intercept 2.65 1.96 0.00 0.18 0.62 confirmed the presence of PUUV at all four H districts, but Perc. Woodland À0.94 0.74 À0.16 0.21 Mast [1] À5.80 3.13 À1.92 0.07 also for three of four L districts. In line with previous inves- Perc. Woodland: 3.08 1.19 2.70 0.01* tigations (Bernshtein et al., 1999; Olsson et al., 2002; Augot Mast [1] et al., 2008), adult individuals showed a significantly higher RNA and seroprevalence. The average antibody and RNA B, regression coefficient; SE B, standard error B; b, standardized coeffi- prevalence was about 12.1% higher in H districts than in L cient; P-value, significance level; Adj. R2, adjusted r-squared; AICc, corrected Akaike’s information criterion. districts. *Significance level P < 0.05. The RNA prevalence in bank voles drove human PUUV infections in South-West Germany at the spatial scale of In 2013, a total of 161 bank voles were trapped at the districts. This was most likely governed by the interaction eight sites (Table 2). Trap success ranged from 2.0 ind./ of beech mast and the forest coverage per district because 100TN in L2 to 15.3 ind./100TN in the city district L3 more human PUUV infections occurred in districts with (Table 3). At seven sites the number of voles was lower higher cover of woodland. Such a relation seems plausible compared to the year 2012, except at site L3 (Table 2). One as bank vole abundance in temperate Germany is highly yellow-necked mouse and 48 shrews were collected as dependent on beech mast (Hansson et al., 2000; Clement bycatch at seven of the eight sites. et al., 2010). The importance of woodland is clearly linked There were no PUUV-positive bank voles in seven of to local human infection risks, as Reil et al. (2015) could eight districts in 2013 (Table 3). Only at site L1, PUUV- not detect any significant influence of woodland on human seropositive and RT-PCR-positive animals were detected incidences at the scale of federal states. The finding indi- with prevalences of 4.6% and 11.4%, respectively (Table 3). cates that variation in human PUUV infection at small spa- There was no statistically significant difference in the tial district scale can be explained by variation of PUUV PUUV infection rate between L and H districts (P = 1 for presence in host rodents and related environmental factors ELISA and P = 0.1798 for RT-PCR). such as beech mast and presence of forest. This can help to The average prevalence in 2012 and 2013 differed signifi- improve forecast systems that help to raise awareness cantly between bank voles from trapping sites L1–L4 and regarding human PUUV infections. H1–H4 (P < 0.001, odds ratio 2.14 (1.39–3.28) for IgG Another explanation for small-scale variation in the fre- ELISA and P < 0.001, odds ratio 3.69 (2.14–6.35) for RT- quency of human PUUV cases is a reduced awareness of PCR). medical personnel where human cases are rare. This Linear regression modelling revealed that the PUUV assumption is supported by reports of participants of focus RNA prevalence in bank voles was closely correlated with groups, which were conducted in PUUV endemic areas of the number of human PUUV infections (F1,4 = 11.5, adj. Germany (Dressel, 2014). In these groups, formerly severely R2 = 0.68, P < 0.001; Table 4). The trapping index of bank affected NE patients described the problem of being one of voles per district as well as the prevalence of PUUV-specific the first cases in a newly emerging area of the hantavirus

6 © 2016 Blackwell Verlag GmbH S. Drewes et al. Heterogeneous Distribution of Puumala virus disease, where it took several days before the medical staff In conclusion, this investigation indicates that the was able to diagnose the disease correctly. In addition, it heterogeneous geographical distribution of recorded takes 50–100 cases of an emerging vector-borne infection human PUUV cases in Baden-Wuerttemberg might be before it is recognized by the local general practitioners and caused by reduced presence or even absence of PUUV in eventually receives sufficient attention from the public local bank vole populations possibly mediated by the inter- health system (Schmidt et al., 2013). action of beech mast intensity and the extent of forested In the district L3, PUUV was not detected in bank voles area. It remains imperative to establish long-term rodent during the study period. An investigation of 40 bank voles and PUUV monitoring investigations (Jacob et al., 2014) collected during December 2009 at a site in the neighbour- that provide a solid database to test such hypotheses and ing district Breisgau-Hochschwarzwald, approx. 12 km may shed light on the stability of PUUV presence/absence, away from the trapping site L3, also failed to detect any the molecular evolution of PUUV strains and their fitness PUUV RNA- and antibody-positive bank voles (our consequences for bank voles as well as their virulence to unpublished data). The two recorded human PUUV infec- humans. Awareness of local physicians should be systemati- tions in this district may originate from patients who have cally raised by dedicated information campaigns released contracted the disease outside the district, as human cases by local public health authorities. This is especially impor- are recorded by place of residence (Robert Koch-Institut, tant for areas close to endemic regions where human cases 2015). have been rare. The findings can help to develop and vali- The promotion of bank vole populations by beech mast date early warning modules for hantavirus outbreaks and is one of the reasons for increased numbers of human associated human infection risk. PUUV infections in temperate Europe (Tersago et al., 2009; Clement et al., 2010; Reil et al., 2016). Indeed, the Acknowledgements high bank vole abundance in 2012 might be explained by the beech mast reported in 2011 for all eight districts of The support of Hans-Werner Maternowski, Brigitte Baden-Wuerttemberg. Additional factors might be Pehlke, Ewa Paliocha, Thomas Kuss, Michael Stauss, Katja involved, such as habitat or climatic factors enhancing or Wallmeyer and Jochen Blank in small mammal trapping impairing virus stability outside the reservoir host (Kallio and the excellent technical assistance of Dorte€ Kaufmann et al., 2006) and/or host and human behaviour. In addi- in serological and RT-PCR investigations are kindly tion, strong interactions between landscape features, acknowledged. We are very grateful to Dorte€ Kaufmann, immune gene expression and co-infections of bank voles Kathrin Baumann, Samuel Bernstein, Stefan Fischer, Nas- with the nematode Heligmosomum mixtum have been iden- tasja Kratzmann, Mathias Schlegel, Sabrina Schmidt, Julia tified previously (Guivier et al., 2014). Additional patho- Schneider, Hanan Sheikh Ali, Kerstin Tauscher and Chris- gens have been detected in bank voles from Germany, such tin Trapp for their help in dissection of the rodents. as herpesviruses (Ehlers et al., 2007), paramyxoviruses Beech mast data of the Staatsklenge Nagold, ForstBW, (Drexler et al., 2012), hepacivirus (Drexler et al., 2013), were kindly provided by Thomas Ebinger. The risk per- polyomavirus (Nainys et al., 2015), cowpox virus (Kin- ception study in Germany was assisted by Steffen Schule.€ nunen et al., 2011), tick-borne encephalitis virus (Achazi The investigations were funded in part by the German et al., 2011) and Leptospira spp. (Mayer-Scholl et al., Federal Ministry of Education and Research (BMBF) 2014), that might impact the fitness or susceptibility of the through the German Research Platform for Zoonoses bank vole for PUUV infection. (projects ‘Monitoring sylvatischer Zoonosen’, FKZ In a previous study, a high proportion of individuals car- 01KI1101, to CWW, and ‘Netzwerk “Nagetier-ubertragene€ rying maternal antibodies were found to constrain trans- Pathogene”’, FKZ 01KI1018 and 01KI1303, to RGU) and mission during cycle peak years of bank voles in Finland the Deutsche Forschungsgemeinschaft (SPP 1596 ‘Ecology (Voutilainen et al., 2016). In our study, the results of the and Species Barriers in Emerging Viral Diseases’, UL 405/ serological and RT-PCR investigations indicated the pres- 1-1 to RGU). This study was partially funded by EU grant ence of maternal antibodies only for two juvenile voles. FP7-261504 EDENext and is catalogued by the EDENext However, the average prevalence of PUUV infections in Steering Committee as EDENext461 (http://www.edenext.eu). bank voles detected by serology alone was mostly slightly The contents of this publication are the sole responsibility higher than prevalence detected by RT-PCR, despite the of the authors and do not necessarily reflect the views of fact that we corrected for maternal antibodies. This discrep- the European Commission. Collection of samples in ancy might be therefore explained by a lower viral load at district Breisgau-Hochschwarzwald were done in the the time of investigation, a lower sensitivity of the RT-PCR frame of the UFOPLAN project 3709 41 401 granted to assay or virus clearance. Jens Jacob.

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Pilaski, J., H. Feldmann, S. Morzunov, P. E. Rollin, S. L. Ruo, Tersago, K., R. Verhagen, A. Servais, P. Heyman, G. Ducoffre, B. Lauer, C. J. Peters, and S. T. Nichol, 1994: Genetic and H. Leirs, 2009: Hantavirus disease (nephropathia epidem- identification of a new Puumala virus strain causing severe ica) in Belgium: effects of tree seed production and climate. hemorrhagic fever with renal syndrome in Germany. J. Infect. Epidemiol. Infect. 137, 250–256. Dis. 170, 1456–1462. Ulrich, R. G., H. Meisel, M. Schutt,€ J. Schmidt, A. Kunz, R Core Team, 2015: R: A Language and Environment for Statis- B. Klempa, M. Niedrig, G. Pauli, D. H. Kruger,€ and J. Koch, tical Computing. Available at: https://www.R-project.org/. 2004: Prevalence of hantavirus infections in Germany. Bun- Razzauti, M., A. Plyusnina, H. Henttonen, and A. Plyusnin, desgesundheitsblatt Gesundheitsforschung Gesundheitsschutz 47, 2013: Microevolution of Puumala hantavirus during a com- 661–670. plete population cycle of its host, the bank vole (Myodes glare- Voutilainen, L., E. R. Kallio, J. Niemimaa, O. Vapalahti, and olus). PLoS One 8, e64447. H. Henttonen, 2016: Temporal dynamics of Puumala Reil, D., C. Imholt, J. A. Eccard, and J. Jacob, 2015: Beech fructi- hantavirus infection in cyclic populations of bank voles. Sci. fication and bank vole population dynamics – combined anal- Rep. 6, 21323. yses of promoters of human Puumala virus infections in Weber de Melo, V., H. Sheikh Ali, J. Freise, D. Kuhnert,€ S. Ess- Germany. PLoS One 10, e0134124. bauer, M. Mertens, K. M. Wanka, S. Drewes, R. G. Ulrich, Reil,D.,C.Imholt,S.Drewes,R.G.Ulrich,J.A.Eccard,and and G. Heckel, 2015: Spatiotemporal dynamics of Puumala J. Jacob, 2016: Environmental conditions in favour of a han- hantavirus associated with its rodent host, Myodes glareolus. tavirus outbreak in 2015 in Germany? Zoonoses Public Health Evol. Appl. 8, 545–559. 63, 83–88. Zeimes, C. B., S. Quoilin, H. Henttonen, O. Lyytik€ainen, Robert Koch-Institut, 2015: Infektionsepidemiologisches Jahr- O. Vapalahti, J. M. Reynes, C. Reusken, A. N. Swart, K. Vai- buch meldepflichtiger Krankheiten fur€ 2014, pp. 23–24. nio, M. Hjertqvist, and S. O. Vanwambeke, 2015: Landscape Robert Koch-Institut, Berlin. and regional environmental analysis of the spatial distribution Schilling, S., P. Emmerich, B. Klempa, B. Auste, E. Schnaith, H. of hantavirus human cases in Europe. Front. Public Health 3, Schmitz, D. H. Kruger,€ S. Gunther,€ and H. Meisel, 2007: Han- 54. tavirus disease outbreak in Germany: limitations of routine Zoller,€ L., M. Faulde, H. Meisel, B. Ruh, P. Kimmig, U. Schel- serological diagnostics and clustering of virus sequences of ling, M. Zeier, P. Kulzer, C. Becker, M. Roggendorf, E. K. F. human and rodent origin. J. Clin. Microbiol. 45, 3008–3014. Bautz, D. H. Kruger,€ and G. Darai, 1995: Seroprevalence of Schlegel, M., H. S. Ali, N. Stieger, M. H. Groschup, R. Wolf, and hantavirus antibodies in Germany as determined by a new R. G. Ulrich, 2012: Molecular identification of small mammal recombinant enzyme immunoassay. Eur. J. Clin. Microbiol. species using novel cytochrome B gene-derived degenerated Infect. Dis. 14, 305–313. primers. Biochem. Genet. 50, 440–447. Schmidt, K., K. M. Dressel, M. Niedrig, M. Mertens, S. A. Supporting Information Schule,€ and M. H. Groschup, 2013: Public health and vectorborne diseases – a new concept for risk governance. Zoonoses Additional Supporting Information may be found in the Public Health 60, 528–538. online version of this article: Schmidt, S., M. Saxenhofer, S. Drewes, M. Schlegel, K. M. Table S1. Abundance indices and results of serological Wanka, R. Frank, S. Klimpel, F. von Blanckenhagen, D. Maaz, and RT-PCR investigations of bank voles trapped at the C. Herden, J. Freise, R. Wolf, M. Stubbe, P. Borkenhagen, eight districts during 2012 and 2013 presented per trapping H. Ansorge, J. A. Eccard, J. Lang, E. Jourdain, J. Jacob, P. season and age/maturity groups according to Bajer et al. Marianneau, G. Heckel, and R. G. Ulrich, 2016: High genetic (2002). structuring of Tula hantavirus. Arch. Virol. 161, 1135–1149.

RESEARCH LETTERS

This case highlights 2 issues: the unknown epidemiol- Puumala Virus in Bank Voles, ogy of CHIKV in Africa and the difficulty of diagnosing one arboviral infection during an outbreak of another ar- Lithuania boviral infection. Further research is necessary to elucidate the true extent of CHIKV in African countries and to un- Petra Straková, Sandra Jagdmann, derstand the public health implications of co-infection and Linas Balčiauskas, Laima Balčiauskienė, co-distribution of multiple arboviruses. Stephan Drewes, Rainer G. Ulrich

This work was supported by a grant from the National Center for Author affiliations: Academy of Sciences, Brno, Czech Republic Global Health and Medicine (27-6001). (P. Straková); Masaryk University, Brno (P. Straková); Friedrich-Loeffler-Institut, Greifswald-Insel Riems, Germany Dr. Takaya is a medical doctor at the National Center for Global (P. Straková, S. Jagdmann, S. Drewes, R.G. Ulrich); Nature Health and Medicine, Disease Control and Prevention Center. Research Centre, Vilnius, Lithuania (L. Balčiauskas, Her main research interest is tropical infectious diseases. L. Balčiauskienė)

DOI: http://dx.doi.org/10.3201/eid2301.161400 References 1. Filipe AF, Pinto MR. Arbovirus studies in Luanda, Angola. 2. Little is known about the presence of human pathogenic Virological and serological studies during an outbreak of dengue- like disease caused by the chikungunya virus. Bull World Health Puumala virus (PUUV) in Lithuania. We detected this virus Organ. 1973;49:37–40. in bank voles (Myodes glareolus) in a region of this country 2. World Health Organization. Situation report: yellow fever in which previously PUUV-seropositive humans were identi- outbreak in Angola W30, 29 July 2016 [cited 2016 Aug 18]. fied. Our results are consistent with heterogeneous distribu- http://www.afro.who.int/en/yellow-fever/sitreps/item/8866-situation- tions of PUUV in other countries in Europe. report-yellow-fever-outbreak-in-angola-29-july-2016.html 3. World Health Organization. Rift Valley fever in China [cited 2016 Aug 18]. http://www.who.int/csr/don/02-august-2016-rift-valley- uumala virus (PUUV) (family Bunyaviridae) is an fever-china/en/ 4. Ross RW. The Newala epidemic. III. The virus: isolation, Penveloped hantavirus that contains a single-stranded pathogenic properties and relationship to the epidemic. J trisegmented RNA genome of negative polarity (1). PUUV Hyg (Lond). 1956;54:177–91. http://dx.doi.org/10.1017/ harbored by the bank vole (Myodes glareolus) is the most S0022172400044442 prevalent human pathogenic hantavirus in Europe (2). A 5. Moyen N, Thiberville SD, Pastorino B, Nougairede A, Thirion L, Mombouli JV, et al. First reported chikungunya fever outbreak in high population density of bank voles can lead to disease the republic of Congo, 2011. PLoS One. 2014;9:e115938. clusters and possible outbreaks of nephropathia epidemica, http://dx.doi.org/10.1371/journal.pone.0115938 a mild-to-moderate form of hantavirus disease (3). 6. Ochieng C, Ahenda P, Vittor AY, Nyoka R, Gikunju S, In contrast to the Fennoscandian Peninsula and parts Wachira C, et al. Seroprevalence of infections with dengue, Rift Valley fever and chikungunya viruses in Kenya, 2007. PLoS One. of central Europe (4,5), little is known about the epidemi- 2015;10:e0132645. http://dx.doi.org/10.1371/journal.pone.0132645 ology of PUUV in Poland and the Baltic States. Recent 7. Gudo ES, Pinto G, Vene S, Mandlaze A, Muianga AF, Cliff J, investigations confirmed the presence of PUUV in certain et al. Serological evidence of chikungunya virus among acute parts of Poland (5,6). A molecular study of bank voles in febrile patients in southern Mozambique. PLoS Negl Trop Dis. 2015;9:e0004146. http://dx.doi.org/10.1371/journal.pntd.0004146 Latvia identified 2 PUUV lineages (Russian and Latvian) 8. Centers for Disease Control and Prevention. Geographic (7). In Estonia, serologic and molecular screening provided distribution. Where has chikungunya virus been found? [cited 2016 evidence of the Russian PUUV lineage (8). For Lithuania, Aug 18]. https://www.cdc.gov/chikungunya/geo/index.html a previous serosurvey indicated the presence of PUUV- 9. Furuya-Kanamori L, Liang S, Milinovich G, Soares Magalhaes RJ, Clements AC, Hu W, et al. Co-distribution and co-infection of specific antibodies in humans from 3 counties (online chikungunya and dengue viruses. BMC Infect Dis. 2016;16:84. Technical Appendix Figure 1, http://wwwnc.cdc.gov/EID/ http://dx.doi.org/10.1186/s12879-016-1417-2 article/23/1/16-1400-Techapp1.pdf). However, molecular 10. Parreira R, Centeno-Lima S, Lopes A, Portugal-Calisto D, evidence of PUUV in humans or in voles is lacking (9). Constantino A, Nina J. Dengue virus serotype 4 and chikungunya virus coinfection in a traveller returning from Luanda, We report a molecular survey of rodent populations in Angola, January 2014. Euro Surveill. 2014;19:20730. Lithuania at 5 trapping sites, including 2 sites in counties http://dx.doi.org/10.2807/1560-7917.ES2014.19.10.20730 where PUUV-specific antibodies were previously detected in humans (online Technical Appendix Figure 1). A total Address for correspondence: Satoshi Kutsuna or Saho Takaya, Disease of 134 bank voles, 72 striped field mice (Apodemus agrar- Control and Prevention Center, National Center for Global Health and ius), and 59 yellow-necked field mice A.( flavicollis) were Medicine, 1-21-1, Toyama, Shinjuku, Tokyo 162-8655, Japan; email: captured during 2015. Three trapping sites (Juodkrantė, [email protected] or [email protected] Elektrėnai, and Lukštas) were located in forests at or near

158 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 23, No. 1, January 2017 RESEARCH LETTERS a cormorant colony, and 2 trapping sites (Žalgiriai and Rusnė) were located in a wet forest and flooded meadows. All applicable institutional and national guidelines for the care and use of animals were followed. For PUUV detection, we extracted RNA from bank vole lung tissue samples by using the Qiazol Protocol (QIAGEN, Hilden, Germany) and conducting screening by using a small segment RNA–specific reverse transcription PCR (RT-PCR) and primers Pu342F and Pu1102R (6). We detected PCR products for 5 (LT15/164, LT15/165, LT15/166, LT15/174, and LT15/201) of 45 bank voles from the Lukštas trapping site. All 9 striped field mice and 2 yellow-necked field mice from Lukštas showed negative results for the PUUV RT-PCR. We amplified the complete nucleocapsid protein– Figure. Phylogenetic tree based on complete nucleocapsid gene encoding region for 3 of the 5 samples positive by sequences of Puumala virus (PUUV) strains from Lithuania (LT), RT-PCR with 3 primer pairs: PuNCRS (5′-TAGTAG- Latvia (Jelgava1), and other PUUV clades. Tula virus (TULV) TAGACTCCTTGAA-3′)/Pu255R (5′-TGGACACAG- was used as the outgroup. The tree was generated by Bayesian CATCTGCCA-3′), Pu40F (5′-CTGGAATGAGTGACTTA- and maximum-likelihood analysis using MrBayes 3.2.6 (http:// AC-3′)/Pu393R (5′-TATGGTAATGTCCTTGATGT-3′), and mrbayes.sourceforge.net/download.php) and MEGA6 software (http://www.megasoftware.net/). The optimal substitution Pu1027F (5′-ATGGCAGAGTTAGGTGCA-3′)/Pu1779R model was calculated by using jModelTest 2.1.4 (https://code. (5′-TCAGCATGTTGAGGTAGT-3′). RT-PCR products google.com/p/jmodeltest2). The Bayesian tree was based on were directly sequenced by using the BigDye Terminator transition model 2 with invariant sites and gamma distribution Version 1.1 Cycle Sequencing Kit (Applied Biosystems, and 4 million generations. For maximum-likelihood analysis, the Darmstadt, Germany). We deposited the sequences of the Kimura 2-parameter model and 1,000 bootstrap replicates were used. Posterior probabilities are indicated before slashes, and 5 samples in GenBank under accession nos. KX757839, bootstrap values are indicated after slashes. Scale bar indicates KY757840, KX 757841, KX751706, and KX751707 (Fig- nucleotide substitutions per site. ALAD, Alpe-Adrian lineage; CE, ure; online Technical Appendix Figure 2). Central European lineage; DAN, Danish lineage; FIN, Finnish The 3 nucleocapsid protein–encoding nucleotide se- lineage; HOKV, Hokkaido virus; LAT, Latvian lineage; N-SCA, quences showed identities of 98.2%–99.8%, and the 3 de- North-Scandinavian lineage; RUS, Russian linage; S-SCA, South- Scandinavian lineage. duced nucleocapsid protein amino acid sequences showed identities of 99.8%–100% (online Technical Appendix Table). We found the highest similarity of the 3 nucleotide under accession nos. KX769843 (LT15/164), KX769844 and corresponding amino acid sequences for the PUUV (LT15/165), KX769845 (LT15/166), KX769846 strain from Latvia (Jelgava1/Mg149/2008; JN657228): nu- (LT15/174), and KX769847 (LT15/201), and compared cleotide sequence 89.8%–90.4% and amino acid sequence them with cytochrome b prototype sequences of evolution- 99.8%–100% (online Technical Appendix Table). ary lineages. Consistent with results for northern Poland We generated phylogenetic trees by using MrBayes (6), we identified 2 bank vole lineages at Lukštas, and the 3.2.6 software (http://mrbayes.sourceforge.net/download. PUUV sequences were detected in 4 bank voles of the Car- php) and MEGA6 software (http://www.megasoftware. pathian phylogroup and in 1 vole of the Eastern lineage. net/) for complete (1,302 nt; Figure) and partial (465 nt; In conclusion, we detected PUUV in bank voles at 1 online Technical Appendix Figure 2) nucleocapsid pro- site (Lukštas) in Lithuania (prevalence of 11.1%). This site tein–encoding sequences. Phylogenetic analysis confirmed is located in a region where PUUV-seropositive persons results of pairwise nucleotide sequence divergence analy- were identified 9( ) and near the border with Latvia (online sis, which indicated clustering of PUUV sequences from Technical Appendix Figure 1). The absence of PUUV in Lithuania with sequences from northern Poland (online bank voles at 4 other sites might have been caused by the Technical Appendix Figure 2) and the Jelgava 1 strain from small number of voles tested. However, our results are con- Latvia (Figure). These sequences of the Latvian clade are sistent with heterogeneous distributions of PUUV in other well separated from the Russian and all other European countries (10). PUUV clades. Detection of this novel PUUV strain by using a spe- To evaluate a potential association of PUUV with evo- cific RT-PCR confirms the reliability of this assay for -mo lutionary lineages of the bank vole, we determined vole lecular diagnostic and epidemiologic studies of this virus in cytochrome b gene sequences, deposited them in GenBank Lithuania. Future large-scale monitoring studies are needed

Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 23, No. 1, January 2017 159 RESEARCH LETTERS to evaluate the geographic distribution and temporal fluc- Loiasis in US Traveler tuation of PUUV in bank vole populations in Lithuania. Returning from Bioko Island, Acknowledgment Equatorial Guinea, 2016 We thank Nicole Reimer for generating Technical Appendix Figure 1. David H. Priest, Thomas B. Nutman P.S. was supported by a stipend from the Erasmus Programme. Author affiliations: Novant Health, Winston-Salem, North Carolina, USA (D.H. Priest); National Institute of Allergy and Infectious Ms. Straková is a doctoral student at Masaryk University, Brno, Diseases, National Institutes of Health, Bethesda, Maryland, USA Czech Republic. Her research interests are zoonotic viruses, (T.B. Nutman) vectorborne diseases, and molecular diagnostics. DOI: http://dx.doi.org/10.3201/eid2301.161427

References 1. Plyusnin A, Beaty BJ, Elliot RM, Goldbach R, Kormelink R, The filarial parasite Loa loa overlaps geographically with Lundkvist A, et al. Family Bunyaviridae. In: King AM, Adams MJ, Onchocera volvulus and Wuchereria bancrofti filariae in Carstens EB, Lefkowitz EJ, editors. Virus taxonomy: ninth report central Africa. Accurate information regarding this overlap of the international committee on taxonomy of viruses. San Diego: is critical to elimination programs targeting O. volvulus and Elsevier Academic Press; 2012. p. 725–41. W. bancrofti. We describe a case of loiasis in a traveler 2. Heyman P, Ceianu CS, Christova I, Tordo N, Beersma M, returning from Bioko Island, Equatorial Guinea, a location João Alves M, et al. A five-year perspective on the situation of haemorrhagic fever with renal syndrome and status of the heretofore unknown for L. loa transmission. hantavirus reservoirs in Europe, 2005-2010. Euro Surveill. 2011;16:19961. 3. Clement J, Maes P, van Ypersele de Strihou C, van der Groen G, oiasis (African eye worm disease) is caused by infec- Barrios JM, Verstraeten WW, et al. Beechnuts and outbreaks of Ltion with Loa loa, a parasitic vector-borne filarial worm nephropathia epidemica (NE): of mast, mice and men. Nephrol endemic to 10 countries in central and western Africa, in- Dial Transplant. 2010;25:1740–6. http://dx.doi.org/10.1093/ndt/ cluding Equatorial Guinea (1). The worm, spread by the gfq122 4. Klempa B, Radosa L, Krüger DH. The broad spectrum of bite of Chrysops dimidiata and C. silacea flies, is of public hantaviruses and their hosts in central Europe. Acta Virol. health concern because of its geographic overlap with On- 2013;57:130–7. http://dx.doi.org/10.4149/av_2013_02_130 chocerca volvulus and Wuchereria bancrofti worms, which 5. Michalski A, Niemcewicz M, Bielawska-Drózd A, Nowakowska cause onchocerciasis and lymphatic filariasis, respectively A, Gaweł J, Pitucha G, et al. Surveillance of hantaviruses in Poland: a study of animal reservoirs and human hantavirus disease (2). Mass drug administration programs for onchocercia- in Subcarpathia. Vector Borne Zoonotic Dis. 2014;14:514–22. sis and lymphatic filariasis often include ivermectin, which http://dx.doi.org/10.1089/vbz.2013.1468 can cause serious and occasionally fatal adverse neurologic 6. Ali HS, Drewes S, Sadowska ET, Mikowska M, Groschup MH, reactions in persons with high levels of circulating L. loa Heckel G, et al. First molecular evidence for Puumala hantavirus in Poland. Viruses. 2014;6:340–53. http://dx.doi.org/10.3390/ microfilariae (3). To avoid such reactions, an accurate pic- v6010340 ture of the geographic distribution of L. loa infection is 7. Razzauti M, Plyusnina A, Niemimaa J, Henttonen H, Plyusnin A. needed. Given the importance of epidemiologic data in the Co-circulation of two Puumala hantavirus lineages in Latvia: a management of filarial infections, we report a case of loia- Russian lineage described previously and a novel Latvian lineage. J Med Virol. 2012;84:314–8. http://dx.doi.org/10.1002/jmv.22263 sis in a US woman who had traveled to Equatorial Guinea. 8. Golovljova I, Sjölander KB, Lindegren G, Vene S, Vasilenko V, In May 2016, a 25-year-old woman sought care in Plyusnin A, et al. Hantaviruses in Estonia. J Med Virol. Winston-Salem, North Carolina, USA, for fatigue, swelling 2002;68:589–98. http://dx.doi.org/10.1002/jmv.10231 of her left ankle, right knee pain, and intensely pruritic skin 9. Sandmann S, Meisel H, Razanskiene A, Wolbert A, Pohl B, Krüger DH, et al. Detection of human hantavirus infections in lesions on her lower extremities. She had lived on Bioko Lithuania. Infection. 2005;33:66–72. http://dx.doi.org/10.1007/ Island, Equatorial Guinea, during October 2015–March s15010-005-4058-8 2016 while studying local wildlife. On Bioko Island, she 10. Drewes S, Turni H, Rosenfeld UM, Obiegala A, Strakova P, frequented local water sources to bathe and wash clothes Imholt C, et al. Reservoir-driven heterogeneous distribution of recorded human Puumala virus cases in South-West Germany. and consistently took atovaquone/proguanil for malaria Zoonoses and Public Health. In press 2016. prophylaxis. She did not spend time on Equatorial Guinea’s mainland or travel to other nations in central or western Address for correspondence: Rainer G. Ulrich, Friedrich-Loeffler- Africa. Her flight from the United States to Bioko Island Institut, Federal Research Institute for Animal Health, Institute for Novel connected in Ethiopia; she did not leave the airport. and Emerging Infectious Diseases, Südufer 10, 17493 Greifswald-Insel Symptoms developed soon after her return to North Riems, Germany, email: [email protected] Carolina in late March 2016. Laboratory evaluations

160 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 23, No. 1, January 2017

Infection, Genetics and Evolution 48 (2017) 127–130

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Infection, Genetics and Evolution

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Short communication Novel hantavirus identiﬁed in European bat species Nyctalus noctula

Petra Straková a,b,c,d, Lucie Dufkova a,JanaŠirmarová a,Jiří Salát a,Tomáš Bartonička b,BorisKlempae,f, Florian Pfaff g,DirkHöperg, Bernd Hoffmann g,RainerG.Ulrichd,DanielRůžek a,h,⁎ a Department of Virology, Veterinary Research Institute, Brno, Czech Republic b Faculty of Science, Masaryk University, Brno, Czech Republic c Institute of Vertebrate Biology, Czech Academy of Sciences, Brno, Czech Republic d Institute for Novel and Emerging Infectious Diseases, Friedrich-Loefﬂer-Institut, Greifswald-Insel Riems, Germany e Biomedical Research Center, Slovak Academy of Sciences, Bratislava, Slovakia f Charité Medical School, Berlin, Germany g Institute of Diagnostic Virology, Friedrich-Loefﬂer-Institut, Greifswald-Insel Riems, Germany h Institute of Parasitology, Biology Centre of the Czech Academy of Sciences, České Budějovice, Czech Republic article info abstract

Article history: Hantaviruses are emerging RNA viruses that cause human diseases predominantly in Asia, Europe, and the Received 27 September 2016 Americas. Besides rodents, insectivores and bats serve as hantavirus reservoirs. We report the detection and ge- Received in revised form 21 December 2016 nome characterization of a novel bat-borne hantavirus isolated from insectivorous common noctule bat. The Accepted 22 December 2016 newfound virus was tentatively named as Brno virus. Available online 23 December 2016 © 2016 Elsevier B.V. All rights reserved.

Keywords: Hantavirus Bat Phylogenetic analysis Emerging virus Bat-borne virus

1. Introduction report the detection of a novel hantavirus, tentatively named Brno virus (BRNV), in the European insectivorous bat species Nyctalus noctula Hantaviruses (genus Hantavirus, family Bunyaviridae) are responsi- collected in the Czech Republic, Central Europe. For genetic characteri- ble for life-threatening human diseases: hantavirus cardiopulmonary zation, the three genome segments were sequenced by high-through- syndrome (HCPS) in the Americas and hemorrhagic fever with renal put sequencing (HTS). syndrome (HFRS) in Asia and Europe (Krüger et al., 2011). Rodents are natural reservoirs of hantaviruses; however, recent studies have 2. The study demonstrated that insectivores and bats also represent hosts for divergent hantaviruses (Xu et al., 2015; Zhang, 2014; Witkowski et al., 2016). A total of 53 bats were collected during the years 2008–2013 in the Bats are considered the natural reservoir of a large variety of zoonotic vi- South Moravia region in the Czech Republic. The sample collection ruses causing serious human diseases, such as lyssaviruses, contained bats that died accidentally or were found dead in the field. henipaviruses, severe acute respiratory syndrome coronavirus, and Bats represented 14 different species from two families: Eptesicus Ebola virus (Li et al., 2010). Genetically divergent bat-borne hantavi- nilssonii (n = 1), E. serotinus (n = 1), Hypsugo savii (n = 4), Myotis ruses have been identified in Africa – Mouyassue virus (MOYV) in bechsteinii (n = 1), M. daubentonii (n = 3), M. emarginatus (n = 1), Cote d'Ivoire (Sumibcay et al., 2012), Magboi virus (MGBV) in Sierra M. mystacinus (n = 1), M. myotis (n = 1), Nyctalus noctula (n = 12), Leone (Weiss et al., 2012), and Makokou virus (MAKV) in Gabon Pipistrellus pipistrellus (n = 15), Plecotus auritus (n = 2), Pl. austriacus (Witkowski et al., 2016)andinAsia– Xuan Son virus (XSV) in Vietnam (n = 2), Vespertilio murinus (n = 6) of the family Vespertilionidae and (Arai et al., 2013), Huangpi virus (HUPV), Longquan virus (LQUV), Rhinolophus hipposideros (n = 3) of the family Rhinolophidae. Laibin virus (LBV) in China (Xu et al., 2015; Guo et al., 2013), and Total RNA was extracted from the lungs, kidneys and livers of all an- Quezon virus (QZNV) in the Philippines (Arai et al., 2016). Here we imals using QIAamp viral RNA Mini Kit (QIAGEN) or QIAZOL/TRIZOL method and screened for the presence of hantavirus RNA by a broad- spectrum RT-PCR targeting the large (L) genome segment of hantavi- ⁎ Corresponding author at: Veterinary Research Institute, Hudcova 70, CZ-62100 Brno, fi Czech Republic. ruses (Klempa et al., 2006). Hantavirus RNA was identi ed in two E-mail address: [email protected] (D. Růžek). noctule bats (N. noctula – the species was determined morphologically

http://dx.doi.org/10.1016/j.meegid.2016.12.025 1567-1348/© 2016 Elsevier B.V. All rights reserved. 128 P. Straková et al. / Infection, Genetics and Evolution 48 (2017) 127–130 and confirmed by HTS-based results of three host genes) collected in the 6528 nt, respectively, encoding nucleocapsid protein (N), glycoprotein city of Brno. The obtained sequences (369 base pairs, bp) were designat- precursor (GPC) and viral RNA-dependent RNA polymerase (L) proteins ed as Brno 7/2012/CZE (amplified from liver tissue) and Brno 11/2013/ of 423, 1136 and 2145 amino acids in length, respectively (GenBank ac- CZE (identical sequences were obtained from liver and kidney samples) cession numbers: KX845678, KX845679, KX845680). Sequence com- and deposited in GenBank (accession numbers KR920359 and parison revealed that the three genome segments and the encoded KR920360, respectively). proteins of BRNV showed 54.7–78.3% nucleotide and 44.5–81.7% Attempts to isolate the virus were done in Vero cells and in suckling amino acid sequence identity with other bat-borne hantaviruses while mice, but were negative. sequence identity with hantaviruses from rodents, shrews and moles In order to determine the whole genome sequence of BRNV, ranged between 50.1 and 64.8%at the nucleotide and between 38.9 IonTorrent HTS was conducted. The sample Brno 7/2012/CZE was select- and 64.1% at amino acid level (Table 1). ed as most suitable for sequencing based on high viral loads in a novel The observed amino acid sequence differences clearly exceed one of BRNV-specific real-time RT-qPCR (qScript XLT 1-step RT-PCR Kit (Quan- the current species demarcation criteria of the current International ta/VWR)). Briefly, RNA was extracted from the liver tissue (QIAamp Committee on Taxonomy of Viruses (ICTV) for the genus Hantavirus, RNeasy mini Kit, Qiagen) and its concentration was measured with a 7% difference in amino acid sequences of the N and GPC proteins. The Nanodrop 1000 photometer (RNA concentrations in the two samples current ICTV criteria also include serological differences in virus neutral- were of 1545.91 ng/μl and 896.98 ng/μl). A purification and concentration ization, preventing virus classification without a cell culture isolate. step with RNA clean beads (Beckman Coulter) followed. Synthesis of However, a new proposal for species demarcation criteria (assigned cDNA was performed with a cDNA Synthesis kit (Roche) and resulting code of the proposal: 2016.023a,bM; available for download at https:// cDNA was fragmented by M220 Focused-ultrasonicator (Corvaris). This talk.ictvonline.org), submitted by the ICTV Bunyaviridae Study Group, fragmented cDNA with an average length of about 500 bp was used for li- is currently under consideration which allows the classification solely brary preparation (Gene Read Library Prep Kit, Qiagen). The library was based on genetic data. It is based on a concatenated multiple sequence purified and size selected using AMPure XP Beads (Beckman Coulter). Op- alignment of complete amino acid sequences of the N and GPC proteins timal size distribution and quality of the resulting library were verified on which is used to calculate PED (pairwise evolutionary distances) values a Bioanalyzer in combination with a DNA High Sensitivity Chip (Agilent). using WAG amino acid substitution matrix. A species of the genus Han- The library was quantified with the Ion Library Taqman Quantitation Kit tavirus is defined by a PED value greater than 0.1. According to this cri- (ThermoFisher). Sequencing was performed on an Ion Personal Genome terion, BRNV clearly represents a new hantavirus species because the Machine (ThermoFisher) according to manufacturer's guidelines. lowest PED value, observed for the most closely related LQUV, is 0.5. A metagenomics analysis of the complete dataset was conducted The obtained BRNV sequences were subjected to phylogenetic anal- using RIEMS software (Scheuch et al., 2015). Reads classified as related yses within the Maximum Likelihood framework using MEGA7 (Kumar to the family Bunyaviridae were extracted and de-novo assembled into et al., 2016). Unfortunately, only partial L segment sequences are avail- contigs using 454 Sequencing System Software (version 3.0). Resulting able for the majority of bat-borne hantaviruses. Therefore, we first contigs were subsequently analyzed using the BioEdit software 7.2.5 based our analysis on a short L segment dataset (352 nt) which contains (Hall, 1999)andGeneious9.0.5(Kearse et al., 2012). The lengths of all currently recognized bat-borne hantaviruses (Fig. 1). BRNV clustered the sequences of S, M and L gene segments were 1441, 3575 and within the clade containing all bat-borne hantaviruses and shared the

Table 1 Nucleotide and amino acid sequence identities (%) of three genome segments and corresponding encoded nucleocapsid protein (N), glycoprotein precursor (GPC) and RNA-dependent RNA polymerase (RdRp) between Brno virus (BRNV) and other representative bat-, insectivore- and rodent-borne hantavirusesa.

Host Virus strain Country S segment/N M segment/GPC L segment/RdRp

1272 nt 424 aa 3411 nt 1137 aa 6435 nt 2145 aa

Bats Longquan virus China 65.9 65 66.3 62.5 78.3b 80.5b Laibin virus China 58.7 56.3 55.4 45.6 66 66.7 Huangpi virus China 65.6b 64b ––71.7b 81.7b Xuan Son virus Vietnam 58.5 54.3 61.2b 54.4b 70b 75.1b Mouyassue virus Côte d'Ivoire ––––73.4b 78.9b Magboi virus Sierra Leone ––––74.2b 75.7b Makokou virus Gabon ––––66.8b 67.6b Quezon virus Philippines 59.2 55.5 54.7 44.5 65.4 66.6 Shrews Uluguru virus Tanzania 50.9 40.9 54.8b 42.9b 64.5 62.7 Altai virus Russia 56.9b 52.6b 54.8b 48.9b 63.3 62.3 Cao Bang virus Vietnam 57.1 51.7 51.9 40.4 63.7 62.1 Seewis virus Switzerland 57.8 49.4 54.2b 46.9b 60.7b 60.3b Thottapalayam virus India 54 46.5 50.1 38.9 62.8 62.2 Moles Nova virus Belgium 57.5 51.7 54.6 44.2 64.5 63.3 Asama virus Japan 55.6 51 52.2b 40.3b 64.8b 64.1b Rodents Puumala virus Finland 58.3 51.9 51 40.1 63.2 60.3 Sin Nombre virus USA 58.8 53 52.5 41.6 63.4 61.3 Seoul virus Korea 55.4 48.8 51.6 39.1 62.2 60.7 Hantaan virus Korea 56.6 50.1 51 40 62.2 60.6 Dobrava-Belgrade virus Greece 55.7 49.8 50.4 40.1 62.5 61.1 Tula virus Czech Republic 57.2 52.9 51.9 40.9 63.1 61.3

– no sequence available. a Viral sequences used to generate sequence identities: Bat-borne hantaviruses: Longquan virus (JX465415, JX465397, JX465381), Laibin virus (KM102247, KM102248, KM102249), Huangpi virus (JX473273, JX465369), Xuan Son virus (KF704710, KJ000539, KF704715), Mouyassue virus (JQ287716), Magboi virus (JN037851), Makokou virus (KT316176), Quezon virus (KU950713, KU950714, KU950715); Shrew-borne hantaviruses: Uluguru virus (JX193695, JX193696, JX193697), Altai virus (KM361048, KM361053, KM361061), Cao Bang virus (EF543524, EF543526, EF543525), Thottapalayam virus (NC_010704, NC_010708, NC_010707), Seewis virus (EF636024, EF636025, EF636026); Mole-borne hantaviruses: Nova virus (KT004445, KT004446, KT004447), Asama virus (EU929072, EU929075, EU929078); Rodent-borne hantaviruses: Puumala virus (NC_005224, NC_005223, NC_005225), Sin Nombre virus (NC_005216, NC_005215, NC_005217), Seoul virus (NC_005236, NC_005237, NC_005238), Hantaan virus (NC_005218, NC_005219, NC_005222), Dobrava-Belgrade virus (NC_005233, NC_005234, NC_005235), Tula virus (NC_005227, NC_005228, NC_005226). b Comparison based on shorter sequences. P. Straková et al. / Infection, Genetics and Evolution 48 (2017) 127–130 129

Fig. 1. Maximum-Likelihood phylogenetic tree showing the phylogenetic position of Brno virus (BRNV; marked by black arrow and bold face) constructed on the basis of partial L segment nucleotide sequences (352 nt). Evolutionary analysis was conducted in MEGA7 (15). The evolutionary history was inferred by using the Maximum-Likelihood method based on the General Time Reversible (GTR) model with using a discrete Gamma distribution (+G) with 5 rate categories and by assuming that a certain fraction of sites are evolutionarily invariable (+I) which was estimated to be the Best-Fit substitution model according to Bayesian Information Criterion. The scale bars indicate an evolutionary distance in substitutions per position in the sequence. Bootstrap values ≥70%, calculated from 500 replicates, are shown at the tree branches. The insert shows the phylogenetic group containing all bat-borne hantaviruses (marked by bat pictogram) in greater detail. Rodent-borne hantaviruses associated with members of the families Muridae or Cricetidae are indicated by grey-shaded background. The list of the accession numbers used in the analysis is available from the authors upon request. Abbreviations: ALTV, Altai virus; ANDV, Andes virus; ARRV, Ash River virus; ARTV, Artybash virus; ASAV, Asama virus; ASIV, Asikkala virus; AZGV, Azagny virus; BAYV, Bayou virus; BCCV, Black Creek Canal virus; BOGV, Boginia virus; BOWV, Bowé virus; BRNV, Brno virus; CBNV, Cao Bang virus; CDV, Cano Delgadito virus; CHOV, Choclo virus; DOBV, Dobrava-Belgrade virus; HOKV, Hokkaido virus; HUPV, Huanqpi virus; HTNV, Hantaan virus; JEJV, Jeju virus; JMSV, Jemez Springs virus; KILV, Kilimanjaro virus; KKMV, Kenkeme virus; LAIV, Laibin virus; LHEV, Lianghe virus; LNV, Laguna Negra virus; LQUV, Longquan virus; MAKV, Makokou virus; MAPV, Maporal virus; MGBV, Magboi virus; MJNV, Imjin virus; MONV, Montano virus; MOUV, Mouyassué virus; MUJV, Muju virus; NVAV, Nova virus; OXBV, Oxbow virus; PHV, Prospect Hill virus; PUUV, Puumala virus; QDLV, Qiandao Lake virus; QZNV, Quezon virus; RIOMV, Rio Mamore virus; RKPV; Rockport virus; RPLV, Camp Ripley virus; SANGV, Sangassou virus; SEOV, Seoul virus; SERV, Serang virus; SNV, Sin Nombre virus; SWSV, Seewis virus; TANGV, Tanganya virus; TIGV, Tigray virus; THAIV, Thailand virus; TPMV, Thottapalayam virus; TULV, Tula virus; ULUV, Uluguru virus; VLAV, Vladivostok virus; XSV, Xuan Son virus. In cases of HTNV, PUUV, SWSV, and XSV, several sequences of the same virus were marked by grey curve to allow unambiguous designation of the taxa. most recent common ancestor with LQUV found in insectivorous acid sequences for all three segments (phylogenetic trees based on Rhinolophus sp. bats in China. This pattern has been consequently ob- partial sequences of S and M segments are shown in Supplementary served in all other phylogenetic analyses including complete amino Figs. 1 and 2). 130 P. Straková et al. / Infection, Genetics and Evolution 48 (2017) 127–130

3. Conclusions Arai, S., Taniguchi, S., Aoki, K., Yoshikawa, Y., Kyuwa, S., Tanaka-Taya, K., et al., 2016. Mo- lecular phylogeny of a genetically divergent hantavirus harbored by the Geoffroy's rousette (Rousettus amplexicaudatus), a frugivorous bat species in the Philippines. In- In the present study a novel bat-borne hantavirus has been identi- fect. Genet. Evol. 45:26–32. http://dx.doi.org/10.1016/j.meegid.2016.08.008. fied and its complete sequence of all coding genomic regions of has Guo, W.P., Lin, X.D., Wang, W., Tian, J.H., Cong, M.L., Zhang, H.L., et al., 2013. Phylogeny and origins of hantaviruses harbored by bats, insectivores, and rodents. PLoS Pathog. been determined. The successful determination of the genome segment 9, e1003159. http://dx.doi.org/10.1371/journal.ppat.1003159. sequences of BRNV provide reference data for improving detection Hall, T.A., 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis methods and determining the genome sequences of further European program for Windows 95/98/NT. Nucleic Acids Symp. Ser. 41, 95–98. bat-borne hantaviruses. Kearse, M., Moir, R., Wilson, A., Stones-Havas, S., Cheung, M., Sturrock, S., et al., 2012. Geneious Basic: an integrated and extendable desktop software platform for the or- Its phylogenetic relatedness to other bat-borne hantaviruses, high ganization and analysis of sequence data. Bioinformatics 25:1647–1649. http://dx. genetic distance to other known hantaviruses, and its independent de- doi.org/10.1093/bioinformatics/bts199. tection in two distinct animals of the same species and in two organs Klempa, B., Fichet-Calvet, E., Lecompte, E., Auste, B., Aniskin, V., Meisel, H., et al., 2006. Hantavirus in African wood mouse, Guinea. Emerg. Infect. Dis. 12:838–840. http:// led us to conclude that BRNV is an indigenous bat-borne hantavirus as- dx.doi.org/10.3201/eid1205.051487. sociated with common noctule bat (N. noctula). This study provided a Krüger, D.H., Schönrich, G., Klempa, B., 2011. Human pathogenic hantaviruses and pre- missing molecular proof that bat-borne hantaviruses occur in Europe vention of infection. Hum. Vaccin. 7:685–693. http://dx.doi.org/10.4161/hv.7.6. 15197. too, and need to be considered as putative public health threat. Kumar, S., Stecher, G., Tamura, K., 2016. MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol. Biol. Evol. 33:1870–1874. http://dx.doi.org/. 10. Acknowledgments 1093/molbev/msw054. Li, L., Victoria, J.G., Wang, C., Jones, M., Fellers, G.M., Kunz, T.H., et al., 2010. Bat guano virome: predominance of dietary viruses from insects and plants plus novel mamma- This study was funded by grant no. LO1218 of the MEYS of the Czech lian viruses. J. Virol. 84:6955–6965. http://dx.doi.org/10.1128/JVI.00501-10. Republic under the NPU I program (to D.R.), Ministry of Agriculture of Scheuch, M., Höper, D., Beer, M., 2015. RIEMS: a software pipeline for sensitive and com- prehensive taxonomic classification of reads from metagenomics datasets. BMC the Czech Republic (RO0516 to D.R.), by the contract-research-project Bioinf. 16:69. http://dx.doi.org/10.1186/s12859-015-0503-6. for the Bundeswehr Medical Service FV E/U2AD/CF512/DF557 META- Sumibcay, L., Kadjo, B., Gu, S.H., Kang, H.J., Lim, B.K., Cook, J.A., et al., 2012. Divergent lin- InfRisk (to R.G.U.) and partially funded by the Slovak Scientific Grant eage of a novel hantavirus in the banana pipistrelle (Neoromicia nanus) in Cote Agency VEGA, grant number 2/0174/15 (to B.K.). The continuous sup- d'Ivoire. Virol. J. 9:34. http://dx.doi.org/10.1186/1743-422X-9-34. Weiss, S., Witkowski, P.T., Auste, B., Nowak, K., Weber, N., Fahr, J., et al., 2012. Hantavirus port of Martin Beer is kindly acknowledged. Petra Straková acknowl- in bat, Sierra Leone. Emerg. Infect. Dis. 18:159–161. http://dx.doi.org/10.3201/ edges support by Erasmus + stipendium. eid1801.111026. Witkowski, P.T., Drexler, J.F., Kallies, R., Ličková, M., Bokorová, S., Mananga, G.D., et al., 2016. Phylogenetic analysis of a newfound bat-borne hantavirus supports a Appendix A. Supplementary data laurasiatherian host association for ancestral mammalian hantaviruses. Infect. Genet. Evol. 41:113–119. http://dx.doi.org/10.1016/j.meegid.2016.03.036. Supplementary data to this article can be found online at http://dx. Xu, L., Wu, J., He, B., Qin, S., Xia, L., Qin, M., et al., 2015. Novel hantavirus identified in black-bearded tomb bats, China. Infect. Genet. Evol. 31:158–160. http://dx.doi.org/ doi.org/10.1016/j.meegid.2016.12.025. 10.1016/j.meegid.2015.01.018. Zhang, Y.Z., 2014. Discovery of hantaviruses in bats and insectivores and the evolution of References the genus Hantavirus. Virus Res. 187:15–21. http://dx.doi.org/10.1016/j.virusres. 2013.12.035. Arai, S., Nguyen, S.T., Boldgiv, B., Fukui, D., Araki, K., Dang, C.N., et al., 2013. Novel bat- borne hantavirus, Vietnam. Emerg. Infect. Dis. 19:1159–1161. http://dx.doi.org/10. 3201/eid1907.121549.

Research in Veterinary Science 102 (2015) 159–161

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Research in Veterinary Science

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Short Communication The common coot as sentinel species for the presence of West Nile and Usutu ﬂaviviruses in Central Europe

Petra Straková a,b, Silvie Šikutová a, Petra Jedličková a, Jiljí Sitko c, Ivo Rudolf a,b,⁎,ZdenekHubáleka,b a Institute of Vertebrate Biology v.v.i., Academy of Sciences, Brno, Czech Republic b Department of Experimental Biology, Faculty of Science, Masaryk University, Brno, Czech Republic c Comenius Museum, Ornithological Station, Přerov, Czech Republic article info abstract

Article history: We examined 146 common coots (Fulica atra)onfishponds in central Moravia, Czech Republic, for antibodies to Received 23 March 2015 West Nile (WNV) and Usutu (USUV) flaviviruses. Eighteen birds reacted in the plaque-reduction neutralization Received in revised form 20 July 2015 test against WNV; these WNV seropositive samples were then titrated in parallel against USUV and tick-borne Accepted 2 August 2015 encephalitis virus (TBEV) to exclude flavivirus cross-reactivity. Two birds (1.4% overall) had the highest titers against WNV while 9 birds (6.2% overall) were seropositive for USUV, and in 7 birds the infecting flavivirus Keywords: could not be differentiated with certainty. Our results indicate that both WNV and USUV infections occur in West Nile virus ‘ ’ fi Usutu virus common coots; these birds might serve as a sentinel species indicating the presence of these viruses at shpond Common coot and wetland habitats in Central Europe. Fulica atra © 2015 Elsevier Ltd. All rights reserved. Surveillance Mosquito-borne viruses Culex spp.

West Nile virus (WNV) and Usutu virus (USUV) belong to the genus In a previous study, we found that among 391 wild birds in Moravia Flavivirus (family Flaviviridae)(Hubálek, 2008). Both viruses circulate in (Czechland, i.e. territory of the Czech Republic), 13 had specific antibod- nature between birds and bird-feeding mosquitoes. Migratory birds ies to WNV — including several common coots (Fulica atra), and one may be infected with WNV or USUV in their African wintering grounds coot had specific antibodies also against USUV (Hubálek et al., 2008a). and could carry the virus during spring migrations northward We decided to assess prevalence of antibodies against WNV and USUV to European sites (Hannoun et al., 1972; Watson et al., 1972; Calistri in this particular bird species in Moravia by examining a greater number et al., 2010). of individuals. Usutu virus is an African virus but in 2001 it emerged surprisingly in The birds were legally shot by fishermen and gamekeepers (they Austria, causing fatal outbreaks in blackbirds (Turdus merula)andsome received a permit from Přerov and Kojetín municipalities) at fishponds other avian species (Weissenböck et al., 2002). In the following years, it in Záhlinice (49°17′ N, 17°29′ E) near Přerov in central Moravia, Czech spread to Hungary, Italy, Switzerland, Germany, Spain and Czechland Republic, during September to October 2011. The serum samples were (Bakonyi et al., 2007; Calzolari et al., 2010, 2012; Manarolla et al., maintained at −20 °C. 2010; Steinmetz et al., 2011; Jost et al., 2011; Becker et al., 2012; All serum samples were inactivated at 56 °C for 30 min and diluted Vazquez et al., 2011; Hubálek et al., 2014). WNV and USUV can circulate 1:5 in Leibowitz L-15 medium. In a plaque-reduction neutralization together in certain ecosystems (Calzolari et al., 2010). In contrast to microtest (PRNT: Hubálek et al., 2008a), the diluted serum samples WNV, USUV has rarely caused human disease — only in immunocom- (30 µl) were mixed in the microtiter plate wells with test dose of promised persons (Vazquez et al., 2011). However, neutralizing virus (30 µl, containing about 20 to 40 PFU) and incubated at 37 °C for antibodies against Usutu virus were documented recently in sera of 3 60 min. Three viruses were used for neutralization tests – WNV Eg- patients with neuroinvasive disease (one patient presented with men- 101, TBEV Hypr, and USUV 939 – all prepared as infected suckling ingitis and two with meningoencephalitis) in Croatia (Vilibic-Cavlek mouse brain suspension in L-15 medium with 2% of fetal calf serum. et al., 2014). During an initial screening (all sera diluted 1:5 and 1:10, i.e. final dilutions were 1:10 and 1:20), only WNV was used. Vero E6 cells grown at 37 °C for 3 days in L-15 medium with 10% fetal calf serum and antibi- otics were added to each well and incubated at 37 °C for 4 h. After incu- ⁎ Corresponding author at: Institute of Vertebrate Biology, v.v.i., Academy of Sciences of μ the Czech Republic, Kvetna 8, CZ-603 65 Brno, Czech Republic. bation, 120 l of carboxymethylcellulose overlay was poured into each E-mail address: [email protected] (I. Rudolf). well, and after 3 to 5 days at 37 °C, the cells were stained with 0.1%

http://dx.doi.org/10.1016/j.rvsc.2015.08.002 0034-5288/© 2015 Elsevier Ltd. All rights reserved. 160 P. Straková et al. / Research in Veterinary Science 102 (2015) 159–161 solution of naphthalene black. The controls were titrations of test doses dead bird (mostly blackbirds) cases appeared in 2011 (Jost et al., of the Eg-101 strain of WNV, immune mouse WNV reference serum, 2011; Becker et al., 2012). In the same year, several blackbirds killed control negative bird serum and the cells without viruses (to reveal po- by USUV were reported in Czechland (Hubálek et al., 2014). Recent tential cytotoxic effect of individual avian sera). A 90% reduction of evidence of USUV RNA in Culex modestus in South Moravian fishponds plaque-forming units (PFU) was used in this study as a measure of neu- indicates possible establishment of this virus in that country (Rudolf tralization (PRNT90), and reciprocal serum titers 20 or higher were con- et al., in preparation). It is interesting that USUV strains from sidered positive. All WNV positive sera were then titrated in parallel Germany, Switzerland, Austria, Hungary, Italy and Czechland are nearly with two other flaviviruses present in Czechland, i.e. USUV and TBEV, identical in nucleotide sequence. Serological surveys sporadically to exclude serological cross reactions. detected antibodies to USUV in wild and game birds in additional Serum samples from 146 common coots (F. atra)werefirst examined European countries — Great Britain (Buckley et al., 2003, 2006), Spain for the presence of WNV antibodies. During this initial screening, 18 (Llorente et al., 2013) and Poland (Hubálek et al., 2008b). coots were positive for WNV. However, when these sera were titrated Reports on mosquito-borne viruses in the target bird of this study – against all three viruses in parallel, 9 tested birds were found to have spe- the common coot – are sporadic. In India, Mishra et al. (2012) did a cific antibodies against USUV, two birds had specific antibodies against serosurvey of 1058 wild birds for WNV: 26 samples (2.5%) were positive WNV, while the prevalence of antibodies in 7 birds could not be differen- (including common coots). In southern Spain, a total of 1213 birds tiated by PRNT with certainty (Table 1). Local circulation of WNV in belonging to 72 species were examined during preliminary screening Czechland was first proved indirectly in 1985 and then in 1990 by for antibodies against WNV and 43 common coots reached positive hemagglutination-inhibition test in free-living wetland birds and senti- WNV titres ranging from 1:20 to 1:640 (Figuerola et al., 2008). On the nel ducks on South Moravian fishponds (Hubálek et al., 1989; Juřicová basis of this finding they focused on coots in Doñana NP, Spain, and de- and Halouzka, 1993; Juřicová et al., 1993). After a big flood in Moravia tected WNV seroconversion in nine birds during the 2004–2005 season in 1997, a higher prevalence rate of arboviruses in local mosquitoes (Figuerola et al., 2007). They also did parallel neutralization against was observed, and WNV-3 (Rabensburg) was isolated in that area re- USUV but all titers of 47 serum samples from the coots were higher to peatedly (Hubálek et al., 2000; Bakonyi et al., 2005). Moreover, WNV-2 WNV than to USUV. According to an experimental study, American was detected in south Moravia recently (Rudolf et al., 2014). In another coots (Fulica americana) have very low competence to WNV (but only study, 13 WNV specifically seroreactive birds were found, including 5 one bird was tested) and therefore another transmission mechanism common coots (Hubálek et al., 2008a). These common coots came should be taken into account, such as fecal–oral transmission of WNV from fishponds at Zahlinice near Přerov. Interestingly, WNV antibodies (Komar et al., 2003). A very interesting finding is that of Alkhovskij were detected in coots also in other countries — Spain (Figuerola et al., et al. (2003) who detected RNA of WNV in 15% of coots examined in 2007), southern Russia (Lvov et al., 2008), Iran (Fereidouni et al., 2011) the Volga delta which might indicate significant role of common coots and India (Mishra et al., 2012). in circulation and spread of WNV in that region. There are not enough data on the prevalence of USUV and antibodies Detection of antibodies in migratory birds such as the common coot against it because USUV is relatively new to Europe. Weissenböck et al. need not mean that the bird was infected at the place of sampling. For (2013) did a retrospective analysis of archived bird tissue samples and instance, the coots occurring in central Moravia during autumn found USUV to be present in northern Italy as early as 1996. In migration (this study) breed in Czechland, but also in Poland and Baltic Austria, USUV is endemic since its first occurrence in 2001 (Chvala countries, while the coots breeding in Czechland usually migrate south- et al., 2007; Meister et al., 2008). Bakonyi et al. (2007) tested dead west to Austria, Switzerland, Italy, France and Spain (Cepák et al., 2008), birds in Hungary between years 2003 and 2006: they found one positive where USUV might occur. This fact must be taken into account at inter- blackbird in 2005 and six positive blackbirds in 2006. Llorente et al. pretation of findings. Herein we examined serum samples obtained (2013) tested in parallel antibodies against WNV, Bagaza virus and from 146 common coots in central Moravia for the specific WNV and

USUV in partridges and pheasants in South Spain and recorded overall USUV antibodies by PNRT90. Two birds (1.4%) had specific antibodies prevalence 10% against USUV. Steinmetz et al. (2011) noticed a mass against WNV and nine birds (6.2%) had specific antibodies against mortality due to USUV in wild and captive songbirds and owls around USUV. the Zurich Zoo in Switzerland. In 2010, a strain of USUV was isolated In conclusion, common coots might serve as a ‘sentinel’ species indi- from mosquitoes Culex pipiens pipiens in Germany where the first cating the presence of WNV and USUV at fishpond and wetland habitats in Central Europe and serological examinations of this species could be a potentially useful tool for surveillance of mosquito-borne viruses in Table 1 Europe. Antibody reciprocal titers (PRNT90) of 18 bird sera tested against three flaviviruses (West Nile, tick-borne encephalitis, Usutu). Specific reactions for particular viruses are printed in bold. Conflicts of interest Bird no. WNV TBEV USUV 42 20 b20 80 The authors declare that they have no competing interests. 43 40 40 80 45 40 b20 40 46 40 20 80 Acknowledgments 47 40 20 40 50 40 80 80 56 40 40 80 This study was funded by the EU grant FP7-261504 EDENext and is 57 20 b20 80 cataloged as EDENext313. 60 20 b20 40 155 20 20 40 175 40 b20 40 References 176 40 40 80 178 40 b20 80 Alkhovskij, S.V., Lvov, D.N., Samokhvalov, E.I., Prilipov, A.G., Lvov, D.K., Aristova, V.A., 179 20 b20 20 Gromashevskiĭ, V.L., Dzharkenov, A.F., Kovtunov, A.I., Deriabin, P.G., Odolevskiĭ, E.I., 182 40 20 40 Ibragimov, R.M., 2003. Screening of birds in the Volga delta (Astrakhan region, 184 160 20 20 2001) for the West Nile virus by reverse transcription-polymerase chain reaction. – 186 20 b20 20 Vopr. Virusol. 48, 14 17 (in Russian). Bakonyi, T., Hubálek, Z., Rudolf, I., Nowotny, N., 2005. Novel flavivirus or new lineage of 187 80 20 20 West Nile virus, Central Europe. Emerg. Infect. Dis. 11, 225–231. P. Straková et al. / Research in Veterinary Science 102 (2015) 159–161 161

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For Peer ReviewVector-Borne Only/Not and Zoonotic Diseases for Distribution

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Serologic survey for West Nile virus in wild artiodactyls, central Europe

Journal: Vector-Borne and Zoonotic Diseases

Manuscript ID Draft

Manuscript Type: Original Research

Date Submitted by the Author: n/a

Complete List of Authors: Hubálek, Zdenek; Institute of Vertebrate Biology, Laboratory of Medical Zoology Juřicová, Zina; Institute of Vertebrate Biology, Academy of Sciences, Brno, Department of Medical Zoology; Strakova, Petra; Ustav Biologie Obratlovcu Akademie Ved Ceske Republiky vvi, Medical Zoology Blazejova, Hana; Ustav Biologie Obratlovcu Akademie Ved Ceske Republiky vvi, Medical Zoology Betášová, Lenka; Institute of Vertebrate Biology, v.v.i., Academy of Sciences, Medical Zoology Rudolf, Ivo; Institute of Vertebrate Biology, v.v.i., Academy of Sciences, Medical Zoology

Keyword: Arbovirus(es), Flaviviridae, Surveillance, West Nile, Zoonosis

Manuscript Keywords (Search wildlife, serosurvey, deer, mouflon, wild boar, mammals Terms):

Antibodies neutralizing West Nile virus (WNV) were tested in 1,023 wild Artiodactyla: 105 roe deer (Capreolus capreolus), 148 red deer (Cervus elaphus), 287 fallow deer (Dama dama), 71 mouflons (Ovis musimon), and 412 wild boars (Sus scrofa). The blood sera, sampled in SouthMoravian district of Břeclav (Czech Republic) in the years 19902008, were examined Abstract: by plaquereduction neutralization test. Specific antibodies to WNV were detected in 5.9% of wild ruminants (4.8% roe deer, 4.1% red deer, 6.3% fallow deer, 9.9% mouflons), and 4.1% of wild boars, with titres between 1:20 and 1:320. The results indicate that WNV has circulated in wild artiodactyls at a variable frequency during the years in the area.

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1 2 3 Serologic survey for West Nile virus in wild artiodactyls, central Europe 4 5 6 7 Zdenek Hubálek 1,2 , Zina Juřicová 1, Petra Straková 1,2 , Hana Blažejová 1, Lenka Betášová 1, Ivo 8 9 1,2 10 Rudolf * 11 12 13 14 1Institute of Vertebrate Biology, Academy of Sciences, v.v.i., Brno, Czech Republic 15 16 2Masaryk University, Faculty of Science, Department of Experimental Biology, Brno, Czech 17 18 Republic 19 20 21 22

23 24 25 26 27 *Corresponding author: 28 29 30 Medical Zoology Laboratory, Institute of Vertebrate Biology ASCR, Klášterní 2, 31 32 691 42 Valtice, Czech Republic 33 34 Fax: 420519352387 35 36 Phone: 420519352961 37 38 Email: [email protected] 39 40 41 42 43 Key words: West Nile virus; Flavivirus ; Moravia; serosurvey; wildlife; mammals; roe deer; 44 45 red deer; fallow deer; mouflon; wild boar 46 47 48 49 50 Running title: West Nile virus antibodies in wild artiodactyls 51

52 53 54 55 56 57 58 59 60 Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801 Vector-Borne and Zoonotic Diseases Page 2 of 13 For Peer Review Only/Not for Distribution

1 2 3 Abstract 4 5 6 7 Antibodies neutralizing West Nile virus (WNV) were tested in 1,023 wild Artiodactyla : 105 8 9 10 roe deer ( Capreolus capreolus ), 148 red deer ( Cervus elaphus ), 287 fallow deer ( Dama 11 12 dama ), 71 mouflons (Ovis musimon ), and 412 wild boars ( Sus scrofa ). The blood sera, 13 14 sampled in SouthMoravian district of Břeclav (Czech Republic) in the years 19902008, were 15 16 examined by plaquereduction neutralization test. Specific antibodies to WNV were detected 17 18 in 5.9% of wild ruminants (4.8% roe deer, 4.1% red deer, 6.3% fallow deer, 9.9% mouflons), 19 20 21 and 4.1% of wild boars, with titres between 1:20 and 1:320. The results indicate that WNV 22 23 has circulated in wild artiodactyls at a variable frequency during the years in the area. 24 25 26 27 28 29 30 31 32 33 34 Introduction 35 36 37 38 39 The mosquitoborne Flavivirus West Nile (WNV, family Flaviviridae ) is the etiologic 40 41 agent of West Nile fever or encephalitis in humans, certain other mammals (e.g., horse), and 42 43 birds (Kramer et al. 2007). WNV is transmitted to vertebrates through the bite of an infected 44 45 mosquito, usually of the genus Culex but sometimes also by other mosquito genera (Hubálek 46 47 48 and Halouzka 1999). WNV occurs in Africa, Eurasia, Australia and, since 1999, also in the 49 50 Americas. 51 52 In Czechland (Czech Republic), WNV was isolated from Culex pipiens mosquito in the 53 54 district of Břeclav (southern Moravia) after a big flood in 1997; specific antibodies against 55 56 57 this virus were detected in 2.1% of local human population in that year, and five cases of 58 59 60 Mary Ann Liebert, Inc., 140 Huguenot2 Street, New Rochelle, NY 10801 Page 3 of 13 Vector-Borne and Zoonotic Diseases For Peer Review Only/Not for Distribution

1 2 3 WNV fever were recorded – two confirmed and three probable (Hubálek et al. 1998, 2000). 4 5 More recently, four strains of WNV lineage 2 were isolated from Culex modestus mosquitoes 6 7 collected in reed beds on the SouthMoravian fishponds during August 2013 (Rudolf et al. 8 9 10 2014). 11 12 WNV was also isolated from Aedes rossicus mosquito during virological examination for 13 14 arboviruses between 2006 −2008 (Hubálek et al. 2010). This mosquito species feeds 15 16 preferably on mammals including man (Becker 2010) whereas a majority of competent 17 18 19 mosquito vectors of WNV are ornithophilic. This raises a question whether WNV might be 20 21 adapted to an alternative mosquitomammal cycle in the SouthMoravian natural focus. 22 23 The aim of the present study was to evaluate retrospectively the activity of WNV in 24 25 southern Moravia in wild mammals prior to the flood year 1997 and later, using serologic 26 27 survey of archived wildlife sera. 28 29 30 31 32 Materials and Methods 33 34 35 36 37 Study sites 38 39 Several hunting areas were surveyed, all situated in the district of Břeclav, South Moravian 40 41 42 region. "Soutok" is an extensive area of floodplain forest with meadows at the confluence of 43 44 the rivers Dyje (Thaya) and Morava (March: 48.6348.74 N, 16.9016.98 E) − a game reserve 45 46 with wild deer (roe, red and fallow) and wild boars. "Palava" (Pavlovske vrchy hills and 47 48 foothills) is a Protected Landcape Area (48.8048.87 N, 16.6516.74 E) that also includes a 49 50 game reserve with deer (roe, red and fallow), mouflons and wild boars. In addition, a number 51 52 53 of smaller, dispersed other hunting grounds were sampled in the district of Břeclav (marked as 54 55 "BVother"). Most of the sampling sites were situated in habitats with abundant mosquito 56 57 populations (Šebesta et al. 2010). 58 59 60 Mary Ann Liebert, Inc., 140 Huguenot3 Street, New Rochelle, NY 10801 Vector-Borne and Zoonotic Diseases Page 4 of 13 For Peer Review Only/Not for Distribution

1 2 3 4 5 Blood sampling 6 7 8 The wild animals were shot by hunters during hunting seasons, and blood samples were 9 10 collected from the heart or from the thoracic cavity. After clotting, the samples were 11 12 centrifuged in the laboratory and the sera stored at 20 ºC until use. 13 14 15 16 17 Plaque-reduction neutralization microtest (PRNT) 18 19 PRNT was originally proposed by Madrid and Porterfield (1969), and later adopted to a 20 21 microtechnique on 96well (flatbottomed) microplates for cell culture (Hubálek et al. 1979). 22 23 24 Briefly, tested sera were thermally inactivated at 56 ºC for 30 min, and diluted 1:10 in L15 25 26 medium for screening; 35 µl of the diluted serum was mixed in a microplate (Sarstedt) well 27 28 with 35µl test dose of the virus (containing 2030 PFU of WNV strain Eg101) in L15 29 30 medium, and incubated at 37 °C for 60 min; 60 µl of Vero E6 cell suspension (in L15 with 31 32 33 3% fetal calf serum FCS) was then added to each test well (20,000 to 30,000 cells), and 120 34 35 µl of carboxymethylcellulose sodium salt (1.5% CMC of medium viscosity) was overlayed 36 37 after an incubation at 37°C for 4 h in each well. The microplates were sealed in plastic bags 38 39 and incubated at 37 °C for 45 days, and the microcultures were then stained with 0.1% acidic 40 41 solution of naphthalene blue black. Sera were tested in duplicate, and controls included the 42 43 44 virus test dose and its titration, immune WNV reference serum; control negative serum; and 45 46 cells without virus. Cytotoxic sera were excluded from the analysis. Sera, revealing 80% or 47 48 greater reduction in the number of WNV plaques at the 1:20 dilution during the screening, 49 50 were titrated by twofold dilutions, and those dilutions corresponding to 80% reduction of 51 52 53 plaque counts were regarded as the serum titres (PRNT 80 ). Reciprocal titres ≥20 were 54 55 considered positive. In addition, those sera reacting with WNV were then also tested in PRNT 56 57 against two other flaviviruses occurring in Central Europe, i.e. tickborne encephalitis virus 58 59 60 Mary Ann Liebert, Inc., 140 Huguenot4 Street, New Rochelle, NY 10801 Page 5 of 13 Vector-Borne and Zoonotic Diseases For Peer Review Only/Not for Distribution

1 2 3 (TBEV) strain Hypr and Usutu virus (USUV) strain 939, in order to exclude cross reactions 4 5 with these antigenically related viruses. 6 7 Differences in proportions among positive sera were statistically evaluated by χ 2 test. 8 9 10 11 12 Results 13 14 15 16 17 In total, specific antibodies against WNV were detected in 53 out of the 1,023 wild 18 19 artiodactyls examined (5.2%). Table 1 shows specific WNV seropositivity: no significant 20 21 crossreactions with the two other flaviviruses (TBEV, USUV) in that titres to WNV were in 22 23 all cases higher than those to the other viruses. Specific antibodies to WNV were thus 24 25 detected in 5.9% of wild ruminants (4.8% of 105 roe deer Capreolus capreolus , 4.1% of 148 26 27 28 red deer Cervus elaphus , 6.3% of 287 fallow deer Dama dama , 9.9% of 71 mouflons Ovis 29 30 musimon ), and 4.1% of 412 wild boars Sus scrofa (nonruminant artiodactyl), with titres 31 32 between 1:20 and 1:320. Table 2 compares the WNVseropositivity including titre ranges and 33 34 mean titres in the five artiodactyl species. 35 36 37 Overall differences in proportions among the artiodactyl species did not attain significance 38 2 39 level (χ = 5.21; P = 0.266) but significantly higher frequency of antibodies was detected in 40 41 mouflons (9.9%) than in wild boars (4.1%; χ2 = 4.21; P = 0.040). All the other differences 42 43 among pairs of the five species were insignificant. 44 45 Significant difference (χ2 = 6.05; P = 0.014) in the frequency of WNV seropositive animals 46 47 48 was found between the study sites "Palava" (7.6% of 340 animals examined) and "BV other 49 50 hunting areas" (2.3% of 175 animals), but not between "Palava" and "Soutok" (4.5% of 508 51 52 animals; χ2 = 3.64; P = 0.056). 53 54 55 56 57 58 59 60 Mary Ann Liebert, Inc., 140 Huguenot5 Street, New Rochelle, NY 10801 Vector-Borne and Zoonotic Diseases Page 6 of 13 For Peer Review Only/Not for Distribution

1 2 3 There was considerable fluctuation in seropositivity rate among sampling periods (Table 4 5 3): overall value of χ 2 = 19.80 (P = 0.0005). The high prevalence of antibodies in wild animals 6 7 was found especially in the period 19941997, and then in 2008 (12.5% rate in that year). 8 9 10 11 12 Discussion 13 14 15 16 17 A few WNV serosurveys were published that included wild ruminants and boars in 18 19 Europe. Kozuch et al. (1976) detected WNV neutralizing antibodies in 6.2% of 65 roe deer, 20 21 3.0% of 101 red deer, 8.3% of 24 fallow deer, and 2.6% of 38 wild boars in western Slovakia 22 23 during the years 19691972. Juřicová and Hubálek (1999) found 1315% of 400 wild 24 25 ruminants and 150 boars with WNV hemagglutinationinhibiting (HI) antibodies in South 26 27 28 Moravia, Czechland. However, HI test with flaviviruses (as well as ELISA) is considered less 29 30 specific than neutralization test due to cross reactions among these viruses (Calisher et al. 31 32 1989, Niedrig et al. 2007). In the studied region, Halouzka et al. (2008) detected antibodies 33 34 neutralizing WNV in 6.5% of 93 wild boars, the figure much closer to the data in the present 35 36 37 study. In Spain, the proportion of WNVseropositive (in ELISA) individuals was 4.0% among 38 39 742 wild boars, while only 0.2% of 887 juvenile wild red deer reacted between 2003 and 2011 40 41 (Boadella et al. 2012). The authors suggest that wild boars are potentially useful as sentinel 42 43 animals for the WNV surveillance in southern Europe. Other Spanish serosurveys on WNV in 44 45 wild artiodactyls include those of GutiérrezGuzmán et al. (2012) and GarcíaBocanegra et al. 46 47 48 (2016), with WNV antibody prevalence rates similar to our study. In Serbia, Escribano 49 50 Romero et al. (2015) used ELISA combined with a confirmatory neutralization test, and found 51 52 specific WNV antibodies in 5.5% of 91 roe deer and in 10.4% of 318 wild boars. 53 54 It is worth mentioning that sampling of sera was not homogeneous in our survey due to 55 56 57 inability to collect regularly samples for such a long period, and the results might thus be 58 59 60 Mary Ann Liebert, Inc., 140 Huguenot6 Street, New Rochelle, NY 10801 Page 7 of 13 Vector-Borne and Zoonotic Diseases For Peer Review Only/Not for Distribution

1 2 3 biased to certain extent. It is therefore difficult to discuss or explain adequately the differences 4 5 in seroprevalence rates among individual species of artiodactyls or between hunting grounds. 6 7 In conclusion, data of the present retrospective study indicate fluctuating WNV activity in 8 9 10 southern Moravia between the years 1990 and 2008 and they suggest a moderate contact of 11 12 WNV with wild artiodactyls. 13 14 15 16 Acknowledgments 17 18 Technical assistance of L. Ševčíková and J. Peško is gratefully acknowledged. The 19 20 21 USUV strain 939 was generously supplied by Prof. N. Nowotny and Dr. T. Bakonyi from 22 23 Vienna Veterinary University. 24 25 26 27 Author Disclosure Statement 28 29 30 All authors declare that no competing financial interests exist. 31 32 33 34 REFERENCES 35 36 37 38 Becker N, Petrič D, Zgomba M, Boase C, et al. Mosquitoes and Their Control , 2nd ed. 39 40 41 HeidelbergDordrechtLondonNew York: Springer; 2010. 42 43 Boadella M, DiezDelgado I, GutierrezGuzman AV, Hoefle U, et al. Do wild ungulates allow 44 45 improved monitoring of flavivirus circulation in Spain? Vectorborne Zoonot Dis 46 47 2012;12:490495. 48 49 50 Calisher CH, Karabatsos N, Dalrymple JM, Shope RE, et al. Antigenic relationships between 51 52 flaviviruses as determined by crossneutralization tests with polyclonal antisera. J Gen 53 54 Virol 1989;70:37–43. 55 56 57 58 59 60 Mary Ann Liebert, Inc., 140 Huguenot7 Street, New Rochelle, NY 10801 Vector-Borne and Zoonotic Diseases Page 8 of 13 For Peer Review Only/Not for Distribution

1 2 3 EscribanoRomero E, Lupulović D, MerinoRamos T, Blázquey AH, et al. West Nile virus 4 5 serosurveillance in pigs, wild boars, and roe deer in Serbia. Vet Microbiol 2015;176:365 6 7 369. 8 9 10 GarcíaBocanegra I, Paniagua J, GutiérrezGuzmán AV, Lecollinet S, et al. Spatiotemporal 11 12 trends and risk factors affecting West Nile virus and related flavivirus exposure in 13 14 Spanish wild ruminants. BMC Vet Res. 2016;12:249258. 15 16 GutiérrezGuzmán AV, Vicente J, Sobrino R, PerezRamírez E, et al. Antibodies to West Nile 17 18 virus and related flaviviruses in wild boar, red foxes and other mesomammals from 19 20 21 Spain.Vet Microbiol. 2012;159:291297. 22 23 Halouzka J, Juřicová Z, Janková J, Hubálek Z. Serologic survey of the wild boars for 24 25 mosquitoborne viruses in South Moravia (Czech Republic). Vet Med 2008;53:266271. 26 27 Hubálek Z, Chanas AC, Johnson BK, Simpson DIH. Crossneutralization study of seven 28 29 30 California group ( Bunyaviridae ) strains in homoiothermous (PS) and poikilothermous 31 32 (XTC2) vertebrate cells. J Gen Virol 1979;42:357–362. 33 34 Hubálek Z, Halouzka J. West Nile fever a reemerging mosquitoborne viral disease in 35 36 Europe. Emerg Infect Dis 1999;5:643650. 37 38 Hubálek Z, Halouzka J, Juřicová Z. West Nile fever in Czechland. Emerg Infect Dis 1999;5: 39 40 41 594595. 42 43 Hubálek Z, Halouzka J, Juřicová Z, Šebesta O. First isolation of mosquitoborne West Nile 44 45 virus in the Czech Republic. Acta Virol 1998;42:119–120. 46 47 Hubálek Z, Rudolf I, Bakonyi T, Kazdová K, et al. Mosquito (Diptera: Culicidae ) 48 49 50 surveillance for arboviruses in an area endemic for West Nile (lineage Rabensburg) and 51 52 Ťahyňa viruses in Central Europe. J Med Entomol 2010;47:466472. 53 54 Juřicová Z, Hubálek Z. Serological surveys for arboviruses in the game animals of southern 55 56 Moravia (Czech Republic). Folia Zool 1999;48:185189. 57 58 59 60 Mary Ann Liebert, Inc., 140 Huguenot8 Street, New Rochelle, NY 10801 Page 9 of 13 Vector-Borne and Zoonotic Diseases For Peer Review Only/Not for Distribution

1 2 3 Kozuch O, Nosek J, Gresikova M, Ernek E. Surveillance of mosquitoborne natural focus in 4 5 Záhorská Lowland. In: Sixl W. (ed.): 2 International Arbeitskolloquium über die 6 7 Naturherde von Infektionskrankheiten in Zentraleuropa . Hygiene Institut der Universität: 8 9 10 Graz; 1976;115118. 11 12 Kramer LD, Li J, Shi PY. West Nile virus. Lancet Neurol 2007;6(2):171181. 13 14 Madrid AT, Porterfield JS. A simple microculture method for the study of group B 15 16 arboviruses. Bull WHO 1969;40:113–121. 17 18 Niedrig M, Sonnenberg K, Steinhagen K, Paweska JT. Comparison of ELISA and 19 20 21 immunoassays for measurement of IgG and IgM antibody to West Nile virus in human 22 23 sera against virus neutralisation. J Virol Meth 2007;139:103–105. 24 25 Rudolf I, Bakonyi T, Šebesta O, Peško J, et al. West Nile virus lineage 2 isolated from Culex 26 27 modestus mosquitoes in the Czech Republic, 2013: expansion of the European WNV 28 29 30 endemic area to the North? EuroSurveill 2014;19 (31):pii=20867. 31 32 Šebesta O., Halouzka J., Hubálek Z., Juřicová Z., et al. Mosquito (Diptera: Culicidae) fauna in 33 34 an area endemic for West Nile virus. J Vector Ecol 2010;35:156162. 35 36 37 38 39 40 41 Correspondence address: Dr. Ivo Rudolf, Institute of Vertebrate Biology Acad. Sci., 42 43 Klasterni 2, 69142 Valtice, Czech Republic. Email: [email protected] 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Mary Ann Liebert, Inc., 140 Huguenot9 Street, New Rochelle, NY 10801 Vector-Borne and Zoonotic Diseases Page 10 of 13 For Peer Review Only/Not for Distribution

1 2 3 Table 1. WNV-positive sera (PRNT reciprocal titres) of wild artiodactyls. 4 5 6 Age Study 7 No. Species yrs. Sex Date coll. site WNV TBEV USUV 8 9 10 24 C. capreolus 7 m 10.08.1990 BV-other 80 20 nt 11 27 C. capreolus 5 m 22.08.1990 BV-other 40 <20 <20 12 88 O. musimon ng m 29.09.1991 Palava 160 40 nt 13 161 O. musimon 2 m 07.12.1993 Palava <20 14 40 20 15 277 D. dama 1 ng 08.01.1994 Palava 80 20 nt 16 297 D. dama 2 ng 13.01.1994 Palava 20 <20 <20 17 299 D. dama 5 m 13.01.1994 Palava 20 <20 <20 18 300 O. musimon 1 ng 13.01.1994 Palava 160 <20 40 19 20 301 D. dama 5 m 13.01.1994 Palava 80 <20 <20 21 302 C. elaphus 7 f 15.01.1994 Palava 20 <20 <20 22 309 D. dama 1 ng 15.01.1994 Palava 40 <20 <20 23 319 D. dama 4 f 19.01.1994 Palava 20 <20 <20 24 329 D. dama 8 f 18.01.1994 Palava 320 40 nt 25 26 339 D. dama 4 f 03.02.1994 Palava 80 <20 20 27 350 D. dama 1 ng 22.01.1994 Palava 20 <20 <20 28 403 C. capreolus 3 f 01.12.1995 Palava 80 20 nt 29 503 S. scrofa 2 f 01.09.1996 Soutok 40 <20 <20 30 506 S. scrofa 2 m 28.08.1996 BV-other 80 <20 <20 31 32 511 C. elaphus ng m 15.09.1996 Soutok 160 40 <20 33 512 C. elaphus 3 m 20.09.1996 Soutok 40 <20 <20 34 515 C. elaphus ng m 14.09.1996 Soutok 80 <20 <20 35 528 O. musimon 3 m 19.10.1996 Palava 80 20 nt 36 542 D. dama 7 f 28.10.1996 Palava 80 <20 <20 37 38 577 D. dama 7 f 30.12.1996 Palava 20 <20 <20 39 582 D. dama 8 f 30.12.1996 Palava 40 <20 20 40 631 O. musimon 1 ng 31.10.1997 Palava 80 40 <20 41 638 C. capreolus 1 ng 07.11.1997 Palava 160 80 <20 42 642 D. dama 1 ng 07.11.1997 Palava <20 43 80 40 44 643 D. dama 8 m 14.11.1997 Palava 80 20 <20 45 654 S. scrofa 2 f 16.09.1997 BV-other 40 20 nt 46 681 D. dama 5 f 21.11.1997 Palava 80 <20 20 47 689 O. musimon 1 ng 27.11.1997 Palava 80 <20 <20 48 49 698 O. musimon 1 ng 05.12.1997 Palava 40 <20 <20 50 717 D. dama 6 f 18.12.1997 Palava 80 40 <20 51 795 S. scrofa ng m 05.12.2002 Soutok 20 <20 <20 52 1039 S. scrofa 1 ng 19.02.2007 Soutok 40 <20 <20 53 1084 S. scrofa 1 ng 05.11.2007 Soutok 40 <20 nt 54 55 1101 S. scrofa 1 ng 22.11.2007 Soutok 40 <20 nt 56 1104 S. scrofa 3 f 22.11.2007 Soutok 80 <20 nt 57 1166 S. scrofa 1 ng 12.12.2007 Soutok 160 40 20-40 58 59 60 Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801 Page 11 of 13 Vector-Borne and Zoonotic Diseases For Peer Review Only/Not for Distribution

1 2 3 1169 S. scrofa 1 ng 12.12.2007 Soutok 80 80 nt 4 1170 S. scrofa 1 ng 12.12.2007 Soutok 160 <20 <20 5 1193 S. scrofa 1 ng 10.01.2008 Soutok 40 <20 nt 6 7 1196 C. elaphus 2 m 12.01.2008 Soutok 160 <20 nt 8 1204 S. scrofa 2 m 18.01.2008 Soutok 80 <20 <20 9 1215 S. scrofa 1 ng 20.01.2008 Soutok 160 <20 20 10 1220 D. dama 4 f 20.01.2008 Soutok 40 <20 <20 11 1226 C. capreolus ng f 28.01.2008 Soutok <20 <20 12 40 13 1245 S. scrofa 1 ng 09.02.2008 Soutok 160 <20 20 14 1249 S. scrofa 1 ng 09.02.2008 Soutok 20-40 <20 20 15 1250 S. scrofa 1 ng 09.02.2008 Soutok 20 <20 <20 16 1269 D. dama 1 ng 19.02.2008 Soutok 80 20-40 <20 17

18 19 20 Legend: nt, not tested (not enough serum); ng, data not given by the hunters 21 22 23 24 Evaluation: 25 26 PRNT titer was defined as the reciprocal dilution of serum that produced an 80% decrease in 27 28 virus PFU count as compared with a virus-only titration control. The titres 20 or higher were 29 30 31 regarded positive, and printed in bold. 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801 Vector-Borne and Zoonotic Diseases Page 12 of 13 For Peer Review Only/Not for Distribution

1 2 3 Table 2. Presence of specific antibodies to WNV in wild artiodactyls. 4 5 6 7 Species: Roe deer Red deer Fallow deer Mouflon Wild boar 8 9 Capreolus Cervus Dama Ovis Sus scrofa 10 11 capreolus elaphus dama musimon 12 Total examined 105 148 287 71 412 13 14 15 No. (%) 5 (4.8% ) 6 (4.1% ) 18 (6.3% ) 7 (9.9% ) 17 (4.1% ) 16 seropositive 17 18 Reciprocal titre, 40-160 20-160 20-320 40-160 20-160 19 20 range 21 Geometric mean 70 57 54 80 63 22 23 titre 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801 Page 13 of 13 Vector-Borne and Zoonotic Diseases For Peer Review Only/Not for Distribution

1 2 3 4 5 Table 3. WNV seropositivity of wild artiodactyls according to sampling periods. 6 7 8 9 10 Years: 1990 −93 1994 −95 1996 −97 2002 −0 6 2007 −08 11 12 Total examined 150 117 260 226 270 13 14 No. (%) seropositive 5 (3.3%) 12 (10.3%) 18 (6.9%) 1 (0.4%) 17 (6.3%) 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Mary Ann Liebert, Inc., 140 Huguenot Street, New Rochelle, NY 10801

Další publikační aktivita uchazečky

1) Venclíková K., Mendel J., Betášová L., Blažejová H., Jedličková P., Straková P., Hubálek Z., Rudolf I. (2016): Neglected tick-borne pathogens in the Czech Republic, 2011-2014. Ticks Tick. Borne Dis. 7: 107-112. 2) Rudolf I., Bakonyi T., Šebesta O., Mendel J., Peško J., Betášová L., Blažejová H., Venclíková K., Straková P., Nowotny N., Hubálek Z. (2015): Co-circulation of Usutu virus and West Nile virus in a reed bed ecosystem. Parasit. Vectors. 8: 520-525.

3) Dufková L., Straková P., Širmanová J., Salát J., Moutelíková R., Chrudimský T., Bartonička T., Nowotny N., Růžek D. (2015): Detection of diverse novel bat Astrovirus sequences in the Czech Republic. Vector Borne Zoonotic Dis. 15: 518-521.

4) Rudolf I., Šebesta O., Straková P., Betášová L., Blažejová H., Venclíková K., Seidel B., Toth S., Hubálek Z., Schaffner F. (2015): Overwintering of Uranotaenia unguiculata adult females in central Europe: a possible way of persistence of the putative new lineage of West Nile virus? J. Am. Mosq. Control Assoc. 31: 364-365.

5) Kríž B., Hubálek Z., Marek M., Daniel M., Straková P., Betášová L. (2015): Results of the screening of tick-borne encephalitis virus antibodies in human sera from eight districts collected two decades apart. Vector Borne Zoonotic Dis. 15: 489-493.

6) Rudolf I., Bakonyi T., Šebesta O., Mendel J., Peško J., Betášová L., Blažejová H., Venclíková K., Straková P., Nowotny N., Hubálek Z. (2014): West Nile virus lineage 2 isolated from Culex modestus mosquitoes in the Czech Republic, 2013: expansion of the European WNV endemic area to the North? Euro. Surveill. 19: 2-5.

7) Straková P., Kříž B., Rudolf I., Hubálek Z. (2014): Seroprevalence study of hepatitis E virus infection in two districts of the Czech Republic. Epidemiol. Mikrobiol. Imunol. 63: 92-94.

8) Straková P., Rudolf I., Pavliš O., Hubálek Z. (2014): The use of immunoenzymatic method for detection of antibodies against zoonotic diseases in Czech soldiers returning from Afghanistan. Mil. Med. Sci. Lett. 83: 67-72.

Účast na konferencích, projektech a stážích

1) Straková P.. Hantaviruses – still surprising! In Czechoslovak virology conference, České Budějovice 2017 - prezentace 2) Straková P., Schmidt S., Saxenhofer M., Wen C., Balčiauskiené L., Balčiauskas L., Heroldová M., Beerli O., Marianneau P., Heckel G., Ulrich R.G.. Hantaviruses in Microtus voles. In 15th International Conference on Rodent Biology, Olomouc 2016 - poster 3) Straková P., Drewes S., Dafalla M., Schmidt S., Rosenfeld U.M., Schlegel M., Sheikh Ali H., Ulrich R.G.. Rodent- and shrew-borne hantaviruses in Germany. In X International Conference on HFRS, HPS and Hantaviruses, Fort Collins, Colorado 2016 - poster 4) Straková P., Schmidt S., Schlegel M., Drewes S., Ulrich R.G.. Detection of Dobrava- Belgrade hantavirus genotype Kurkino in Apodemus agrarius in Germany during 2009-2015. In 15th Medical Biodefence Conference, Munich 2016 – poster 5) Drewes S., Turni H., Rosenfeld U.M., Obiegala A., Straková P., Imholt C., Glatthaar E., Dressel K., Pfeffer M., Jacob J., Wagner-Wiening, Ulrich R.G.. Inhomogeneous distribution of Puumala virus in South-West Germany. In 15th Medical Biodefence Conference, Munich 2016 – poster 6) Daffala M., Straková P.. Monitoring of shrew borne Seewis hantavirus in Germany. In 15th Medical Biodefence Conference, Munich 2016 – poster 7) Straková P.. Hantavirus infection in rodents from Germany. In Graduate Meeting Evolutionary Biology and Ecology, Responses to Environmental Change, Greifswald 2016 - prezentace 8) Straková P., Dufková L., Širmanová J., Salát J., Moutelíková R., Chrudimský T., Bartonička T., Nowotny N., Růžek D.. Detection of diverse novel bat astrovirus sequences in the Czech Republic. In National Symposium on Zoonoses Research, Berlin 2015 - poster 9) Venclíková K., Rudolf I., Betášová L., Blažejová H., Straková P., Hubálek Z.. Neglected tick-borne pathogens in the Czech Republic – EDENext prevalence study. In EDENext Annual Meetings, Herkalion 2015 - poster 10) Venclíková K., Rudolf I., Betášová L., Blažejová H., Straková P., Hubálek Z.. Tick- borne pathogens in Ixodes ricinus in the Czech Republic. In EDENext Annual Meetings, Rovaniemi 2014 - poster 11) Venclíková K., Rudolf I., Betášová L., Blažejová H., Straková P., Hubálek Z.. The prevalence of “ Candidatus Neoehrlichia mikurensis“ in Ixodes ricinus ticks and sheep in the Czech Republic. ESOVE conference, Thessaloniki 2014 - poster 12) Straková P.. Využití imunoenzymatické metody pro průkaz protilátek k vybraným zoonózám. In 26. kongres československé společnosti mikrobiologické, Brno 2013 – prezentace

Pasivní účast Symposium on Species Barriers in Emerging Viral Diseases, Berlin 2016 Workshop „AKTimo – Alternative Kleinsäuger-Tierversuchsmodelle“, Insel Riems 2016 ECCMID - The 24th European Congress of Clinical Microbiology and Infectious Diseases, Barcelona 2014 10. ročník celostátní conference Medicína katastrof, Hradec Králové 2013 Český kongres o infekčních nemocech, Brno 2013

Účast na grantových projektech EDENext (Biology and Control of Vector-borne Infections in Europe) Doba řešení: 2011-2015 Pozice: v letech 2014-2015 člen výzkumného týmu GAČR standartní projekt (Pokročilé studie patogeneze západonilské horečky směrem k novým možnostem terapie) Doba řešení: 2016-2018 Pozice: člen výzkumného týmu

Stáže ERASMUS+ pracovní pobyt, Friedrich-Loeffler Institut (INNT), Insel Riems - Greifswald, Německo (11 měsíců) Fakultní nemocnice Brno, Jihlavská 20, Brno, týdenní praxe na oddělení Klinické mikrobiologie Ústřední kontrolní a zkušební ústav zemědělský, Hroznová 2, Brno, semestrální stáž v rámci předmětu Environmentální praxe

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Výuka během studia

Masarykova univerzita, Přírodovědecká fakulta, oddělení Mikrobiologie 1) Obecná mikrobiologie - laboratorní cvičení pro studenty oborů biochemie a experimentální biologie (kromě oboru mikrobiologie a molekulární biotechnologie), jarní a podzimní semestr 2) Mikrobiální zoonózy a sapronózy – laboratorní cvičení pro studenty oboru Mikrobiologie, cvičení probíhá ve Valticích, podzimní semestr 3) Letní škola pro gymnázia (2014) – třídenní cyklus přednášek a laboratorních cvičení pro studenty gymnázií 4) MjUNI (2017) – workshop v rámci výuky Dětské univerzity MjUNI