The Three Subtypes of Tick-Borne Encephalitis Virus Induce Encephalitis in a Natural Host, the Bank Vole (Myodes glareolus)

The Th ee Sub ypes o Tick-Bo ne Encephali is Vi us Induce Encephali is in a Na u al Hos , he Bank Vole ( Myodes gla eolus ) Elina Ton e i 1,2,3 *, Anja Kipa 4,5,6 , Liina Vou ilainen 1,7 , Si kka Vene 3 , An i Vahe i 1,8 , Olli Vapalah i 1,6,8 , A ˚ke Lundk is 2,3,9 1Depa men o Vi ology, Haa man Ins i u e, Uni e si y o Helsinki, Helsinki, Finland, 2Depa men o Mic obiology, Tumo and Cell Biology, Ka olinska Ins i u e , S ockholm, Sweden, 3Swedish Ins i u e o In ec ious Disease Con ol, Solna, Sweden, 4Finnish Cen e o Labo a o y Animal Pa hology, Facul y o Ve e ina y Medicine, Uni e si y o Helsinki, Helsinki, Finland, 5Ve e ina y Pa hology, School o Ve e ina y Science and Depa men o In ec ion Biology, Ins i u e o In ec ion and Global Heal h, Uni e si y o Li e pool, Li e pool, Uni ed Kingdom, 6Depa men o Ve e ina y Biosciences, Facul y o Ve e ina y Medicine, Helsinki, Finland, 7Finnish Fo es Resea ch Ins i u e, Van aa, Finland, 8Depa men o Vi ology and Immunology, Helsinki Uni e si y Hospi al Labo a o y (HUSLAB), Helsinki, Finland, 9Depa men o Medical Biochemis y and Mic obiology, Uppsala Uni e si y, Uppsala, Sweden Abs ac Tick-bo ne encephali is i us (TBEV) in ec s bank oles (Myodes gla eolus) in na u e, bu he ele ance o oden s o TBEV ansmission and main enance is unclea . We in ec ed colonized bank oles subcu aneously o s udy and compa e he in ec ion kine ics, acu e in ec ion, and po en ial i al pe sis ence o he h ee known TBEV sub ypes: Eu opean (TBEV-Eu ), Sibe ian (TBEV-Sib) and Fa Eas e n (TBEV-FE). All s ains ep esen ing he h ee sub ypes we e in ec i e and highly neu o opic. They induced (meningo)encephali is in some o he animals, howe e mos o he cases did no p esen wi h appa en clinical symp oms. TBEV-RNA was clea ed signi ican ly slowe om he b ain as compa ed o o he o gans s udied. Suppo ing ou ea lie indings in na u al oden popula ions, TBEV-RNA could be de ec ed in he b ain o up o 168 days pos in ec ion, bu we could no demons a e in ec i i y by cell cul u e isola ion. Th oughou all ime poin s pos in ec ion, RNA o he TBEV-FE was de ec ed signi ican ly mo e o en han RNA o he o he wo s ains in all o gans s udied. TBEV-FE also induced p olonged i emia, indica ing dis inc i e kine ics in oden s in compa ison o he o he wo sub ypes. This s udy shows ha bank oles can de elop a neu oin asi e TBEV in ec ion wi h pe sis ence o i al RNA in b ain, and moun an an i-TBEV IgG esponse. The indings also p o ide u he e idence ha bank oles can se e as sen inels o TBEV endemici y. Ci a ion: Ton e i E, Kipa A, Vou ilainen L, Vene S, Vahe i A, e al. (2013) The Th ee Sub ypes o Tick-Bo ne Encephali is Vi us Induce Encephali is in a Na u al Hos , he Bank Vole (Myodes gla eolus). PLoS ONE 8(12): e81214. doi:10.1371/jou nal.pone.0081214 Edi o : Che yl A. S odda , Uni e si y o Cali o nia, San F ancisco, Uni ed S a es o Ame ica Recei ed July 25, 2013; Accep ed Oc obe 9, 2013; Published Decembe 13, 2013 Copy igh : ß2013 Ton e i e al. This is an open-access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion License, which pe mi s un es ic ed use, dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal au ho and sou ce a e c edi ed. Funding: The wo k was suppo ed by he Niemi Founda ion (no URL a ailable); he Helsinki Biomedical G adua e School (h p://www.hbgs.helsinki. i/HBGS_2/ Home.h ml); Biocen e Finland (www.biocen e . i); he Academy o Finland (www.aka. i) (122827 and 251836) and he Swedish Ins i u e o Communicable Diseases (www.smi skyddsins i u e .se). All unde s ha e con ibu ed o und he labou cos s and Swedish Ins i u e o Communicable Diseases and Academy o Finland did also und he expe imen s. The unde s had no ole in s udy design, da a collec ion and analysis, decision o publish, o p epa a ion o he manusc ip . Compe ing In e es s: The au ho s ha e decla ed ha no compe ing in e es s exis . * E-mail: elina. in ala@helsinki. i In oduc ion Tick-bo ne encephali is i us (TBEV) is a zoono ic la i i us ha occu s on he Eu asian con inen and causes ick-bo ne encephali is (TBE) in humans [1]. TBEV is conside ed he medically mos impo an a h opod ec o ansmi ed i us (a bo i us) in Eu ope [2]. Th ee TBEV sub ypes ha e been iden i ied: Eu opean (TBEV-Eu ), Sibe ian (TBEV-Sib) and Fa Eas e n (TBEV-FE) [3]. TBEV-Sib and TBEV-FE o m wo sepa a e lineages, which sha e a common ances o . These wo sub ypes ha e adia ed conside ably ea lie han he TBEV-Eu s ains ha a e ci cula ing oday [4,5]. The cou se and se e i y o human disease appea s o di e be ween he h ee sub ypes. While o TBEV-FE, a mo ali y o 30% has been epo ed, TBEV-Eu and TBEV-Sib in ec ions a e a al in only 1–2% [6] and 6–8% [7] o he cases espec i ely. Howe e , TBEV-Sib has been associa ed wi h an ele a ed isk o ecu en in ec ions in humans [7,8]. Phylogene ic analysis allows iden i ica ion o Bal ic and Sibe ian sublineages o TBEV-Sib. These a e po en ially also disce nable based on hei pa hogenici y o humans [5,8]. TBEV is main ained in na u e by Ixodes icks; TBEV-Eu mainly in I. icinus and TBEV-Sib and TBEV-FE in I. pe sulca us. Ticks ac as ec o s and also cons i u e he main ese oi o TBEV [1,9]. Small oden s, in Eu ope mos impo an ly he bank ole (Myodes gla eolus) and he yellow-necked mouse (Apodemus la icollis) a e conside ed as b idge hos s o TBEV be ween he di e en li e s ages o icks, in a p ocess called non- i emic ansmission [2,10–14]. Ticks can also acqui e he i us when eeding on a i emic oden hos o ia e ical ansmission, al hough ick co- eeding on non- i emic o e en immune oden hos s is conside ed he mos ele an ou e o in ec ion o he TBEV ecology, a leas o TBEV-Eu [2,11]. Co- eeding is dependen on local mic oclima ic condi ions and, consequen ly, he geog aphical dis ibu ion o TBEV is ocal [1]. TBEV-RNA has been shown o pe sis in oden s in bo h TBEV-Eu and TBEV-Sib oci [13,15–19]. Roden s ha e also PLOS ONE | www.plosone.o g 1 Decembe 2013 | Volume 8 | Issue 12 | e81214 been ound o se e as excellen sen inels o TBEV oci [15,20– 22]. Fu he mo e, e ical ansmission o TBEV-Sib has been demons a ed in he no he n ed-backed ole (Myodes u ilus) [23]. S ill, he ele ance o i al pe sis ence in oden s o TBEV ansmission and main enance emains unclea . We p e iously de ec ed TBEV-RNA in o gans o ee- anging Myodes gla eolus and Mic o us ag es is ou side he ick- eeding season, du ing wo subsequen win e s, bo h in TBEV-Eu and TBEV-Sib oci in Finland [19]. In he p esen s udy, we ha e u he assessed he kine ics o TBEV in ec ion in a na u al oden hos , he bank ole (Myodes gla eolus). Expe imen al in ec ion wi h all h ee sub ypes was unde aken o allow cha ac e iza ion o he cou se o in ec ion and pe sis ence o TBEV including he compa ison o he sub ypes in a con olled app oach. Ma e ials and Me hods Vi uses The ollowing s ains o he h ee known TBEV sub ypes (TBEV-Eu , TBEV-Sib and TBEV-FE) we e used in he p esen s udy: TBEV-Sib, Kokkola-8 [24]; TBEV-Eu , Isosaa i-5 and TBEV-FE, Bu ya ia-169 [25]. All h ee s ains o igina e om icks ha we e collec ed by lagging by us. The i us s ains ha e i s been isola ed and hen passaged once in suckling NMRI-mouse b ains a he Haa man Ins i u e, Uni e si y o Helsinki, using he same p o ocols and acili ies. Suckling NMRI-mouse b ains we e homogenized in Dulbecco’s PBS+0.2% bo ine se um albumin and u he dilu ed in Hank’s Bu e ed Sal Solu ion (HBSS; Li e Technologies). Vi us i e s we e de e mined by apid luo escen ocus inhibi ion es (RFFIT) as p e iously desc ibed [26]. The h ee sub ypes we e used a equal i e s p ede e mined by RFFIT. A i us copy numbe op imal o in ec ion was de e mined in a pilo expe imen , in which bank oles we e inocula ed wi h a en- old se ies ollowing he same p o ocol as in he inal expe imen s. Expe imen al in ec ion o bank oles (Myodes gla eolus) Eigh y- wo colonized, inb ed, pa hogen- ee, young and sexu- ally ma u e M. gla eolus oles we e included in he s udy. All animal handling was in compliance wi h he guidelines o The Swedish Ins i u e o Communicable Disease Con ol, Solna, Sweden, and he expe imen al s udies we e app o ed by he au ho i y o animal s udy e hics in S ockholm (#N419/10). All e o s we e made o minimize su e ing and animals we e eu hanized immedia ely i any symp oms we e seen. Two in ec ion expe imen s we e unde aken, a i s examining he acu e phase o in ec ion and a second o assess pe sis en in ec ion (Table 1). In bo h s udies, oles ecei ed 100 mL i us solu ion, con aining 100 luo escen ocus- o ming uni s ( u), as a subcu aneous injec ion in he neck. In ec ed animals we e housed in isola ed cages unde BSL-3 condi ions wi h wa e and ood p o ided ad libi um. Two unin ec ed animals se ed as con ols in each s udy. The s udies we e unde aken on g oups o 3–4 animals in ec ed wi h one o he h ee i us sub ypes. Fo acu e phase in ec ion, animals we e eu hanized a 4, 8, 14 and 25 days pos -in ec ion (dpi), espec i ely (Table 1). Howe e , wo TBEV-FE in ec ed indi iduals housed oge he we e eu hanized a 12 dpi due o se e e acu e disease. Fo he pe sis ence s udy, oles we e eu hanized a 53, 109, 133 and 168 dpi (Table 1), excep o one TBEV-Eu in ec ed animal scheduled o eu hanasia a 133 dpi ha died a 110 dpi. F om all animals, ca diac blood was collec ed du ing eu hana- sia. In addi ion, oles om he pe sis ence s udy we e bled om he e o-o bi al sinus a 18, 53 and 84 dpi. The blood was collec ed in Mic o aine H ubes (BD) and spun o gain he se um ac ion ha was subsequen ly s o ed a 280uC. Voles we e nec opsied immedia ely a e dea h and b ain, spleen, lung, kidneys and u e us ( emale animals om he pe sis ence s udy) we e collec ed. B ains we e cu in hal longi udinally. One hal o he b ain, each hal o spleen, lung and u e us as well as one kidney we e s o ed a 280uC o RNA ex ac ion. The o he hal o he issues as well as one kidney was ixed in 4% pa a o maldehyde (PFA; pH 7.4) o his opa hological examina ion. Du ing he pe sis ence s udy, u ine and ecal samples we e collec ed om 31 dpi un il he e mina ion o he expe imen whene e animals we e handled o cage cleaning o blood sampling. Samples we e s o ed a 280uC. All wo k wi h ac i e i us o po en ially i us-con aining issues was pe o med in BSL-3 acili ies. ELISA To con i m TBEV in ec ion and moni o he p oduc ion o TBEV-speci ic IgG, he comme cial IMMUNOZYMHFSME (TBE) IgG All Species ki (P ogen Bio echnik GmbH) was used acco ding o he manu ac u e ’s ins uc ions. RNA ex ac ion and eal ime e e se ansc ip ase PCR o TBEV RNA ex ac ion was pe o med using he T iPu e isola ion eagen (Roche Diagnos ics Co p.) acco ding o he manu ac u - e ’s ins uc ions. F om all animals excep o hose examined a 168 dpi, issue samples we e ini ially mixed wi h 1 mL T iPu e Isola ion Reagen and homogenized using a Tissuelyze (Qiagen). F om oles eu hanized a 168 dpi, issue samples we e homoge- nized in 500 mL Dulbecco’s PBS+0.2% bo ine se um albumin. Subsequen ly, 300 mL o he homogena e was added o 1 mL T iPu e Isola ion Reagen and he RNA ex ac ion comple ed acco ding o he manu ac u e ’s ins uc ions. The emainde o he homogena e was s o ed a 280uC. Feces samples we e homogenized in 500 ml 0.89% NaCl by adding a glass bead and o exing. The homogena e was cen i uged a 40006g o 30 min a 4uC, using an Eppendo cen i uge 5417C (Eppendo ). RNA was ex ac ed om 140 mLo he eces homogena e supe na an and om u ine samples, using he QIAamp Vi al RNA Mini Ki (Qiagen) acco ding o he manu ac u e ’s ins uc ions. The se um samples unde wen one eeze- haw cycle be o e inal eezing. RNA was ex ac ed using he QIAamp Vi al RNA Mini Ki (Qiagen) acco ding o he manu ac u e ’s ins uc ions. Immedia ely a e RNA ex ac ion, eal- ime RT-PCR o TBEV was pe o med as p e iously desc ibed [27], bu wi h 150 nmol/L o wa d p ime , 500 nmol/L e e se p ime , 400 nmol/L p obe and 25 ml eac ion olume. PCR he mal cycling was pe o med using he ABIP ism 7900HT Fas Sys em (Li e Technologies). RNA concen a ion was de e mined using a NanoD op spec opho ome e (The mo Scien i ic). His opa hological and immunohis ological examina ion Tissue specimens we e ixed in PFA o 72–96 h, hen immed and ou inely pa a in wax embedded. Sec ions (3–5 mm) we e p epa ed and s ained wi h hema oxylin-eosin (HE) o his ological e alua ion o used o immunohis ological s aining. Immunohis ology o he demons a ion o TBEV an igen was pe o med on he b ain o all animals, using a abbi polyclonal an ibody gene a ed agains he Hochos e wi z TBEV isola e (kindly dona ed by P o . F anz X. Heinz, Uni e si y o Vienna, In ec ion o Myodes gla eolus wi h TBEV PLOS ONE | www.plosone.o g 2 Decembe 2013 | Volume 8 | Issue 12 | e81214 Aus ia [28,29] and he ho se adish pe oxidase me hod (EnVi- sion TM ; Dako). Fo malin- ixed and pa a in wax-embedded Ve o E6 cell pelle s in ec ed wi h each i us s ain and ha es ed a 8 dpi se ed as posi i e con ols o he immunohis ological examina ion. The an ibody ecognized all h ee TBEV s ains and esul ed in a g anula cy oplasmic as well as a pe iphe al eac ion in in ec ed cells. Consecu i e sec ions incuba ed wi h an un ela ed abbi an ibody agains Toxoplasma gondii se ed as nega i e con ols. Selec ed b ains o oles sac i iced a 8 and 12 dpi ha exhibi ed an in lamma o y in il a e we e also s ained o leukocy e ma ke s (CD3 o T cells, CD79a o B cells and lysozyme o mac ophages and neu ophils), o cha ac e ize he in il a ing leukocy e popu- la ion, and we e s ained o clea ed caspase-3 o demons a e apop o ic cell dea h, ollowing p e iously published p o ocols [30,31]. Cell cul u e i us isola ion Ve o E6 cells we e incuba ed wi h homogenized (b ain) issue specimens om oles eu hanized a 168 dpi ha had been es ed posi i e by RT-PCR. Cells we e passaged up o 9 imes, e e y 4–7 days. A each passaging, a p opo ion o cells was ixed on a mic oscope slide wi h ace one o 7 min, ollowed by an immuno luo escence assay (IFA) o de ec in ec ed cells, incuba ing slides wi h an i-TBEV abbi polyclonal an ibodies ( aised agains TicoVac Junio accine, Bax e ) and polyclonal swine an i- abbi FITC conjuga e (Dako) o 30 min a 37uC. Real- ime RT-PCR o TBEV was pe o med as desc ibed abo e, on RNA ex ac ed om cell supe na an s using he QIAamp Vi al RNA Mini Ki (Qiagen) acco ding o he manu ac u e ’s ins uc ions. S a is ical analysis The pe sis ence o he h ee TBEV sub ypes in di e en o gans o in ec ed bank oles was s udied using GLMM (gene alized linea mixed modeling) wi h binomial e o dis ibu ions and a logi link unc ion. In he ull model, he h ee TBEV sub ypes (TBEV-Eu , TBEV-Sib and TBEV-FE), ime as dpi, issue ype (b ain, lung, spleen, kidney, se um), and all hei wo- and h ee- way in e ac ions we e used as ixed ac o s. To accoun o epea ed measu emen s om he same oles, a andom in e cep was allowed o each animal. The ull model was educed by emo ing ixed e ec s sequen ially i hei inclusion did no dec ease AICc (sample numbe adjus ed in o ma ion c i e ion) by mo e han wo uni s [32]. All models we e i ed using he Laplace app oxima ion me hod (lme unc ion o lme4 package [33] in he R so wa e [34]. Resul s Acu e phase in ec ion s udy The i s , acu e phase s udy a ge ed TBEV-in ec ed bank oles be ween 4 and 25 dpi (Table 1). Th ee s ains o igina ing om icks and ep esen ing he h ee sub ypes o TBEV we e used. Each g oup o 13 indi iduals was inocula ed subcu aneously wi h one o he s ains using 100 u o i us. In ec ed animals we e housed in isola ed cages unde BSL-3 condi ions. All oles inocula ed wi h TBEV-Eu o TBEV-FE p oduced speci ic IgG an ibodies, i.e. se ocon e ed o TBEV and/o we e shown o be sys emically TBEV in ec ed based on he p esence o i al RNA in hei o gans. Howe e , only 8/13 bank oles inocula ed wi h TBEV-Sib we e ound posi i e o TBEV by ei he o he wo me hods (Table S1). A 4 dpi, all animals excep o one TBEV-FE in ec ed ole, we e i emic, as shown by he p esence o i al RNA in he se um. They all es ed posi i e o i al RNA in he b ain. O he o gans we e also posi i e o i al RNA, i.e. he spleen in all animals, he lungs in all bu one TBEV-Sib in ec ed animal, and he kidneys in all TBEV-FE, wo TBEV-Eu and one TBEV-Sib in ec ed ole. The his ological assessmen o he b ain om TBEV-Eu and -FE in ec ed animals did no de ec any pa hological changes, no did immunohis ology de ec i al an igen exp ession. One TBEV-Sib in ec ed bank ole exhibi ed a ocal mac ophage-domina ed in lamma o y in il a ion in he on al co ex, bu i al an igen was no de ec ed by immunohis ology. The animals did no exhibi TBEV se um an ibodies (Figu e 1). Howe e , spleens gene ally exhibi ed ela i ely la ge ollicles wi h de eloping ge minal cen e s ha con ained nume ous apop o ic cells. A 8 dpi, he EIA iden i ied TBEV-IgG in all TBEV-Eu and - FE in ec ed animals, bu only in one o he h ee -Sib in ec ed bank oles, and a bo de line le el (Figu e 1, Table S1). Howe e , in none o he animals was he e his ological e idence o seconda y ollicle o ma ion in he spleen. All TBEV-FE in ec ed animals we e ound o be i emic, based on he de ec ion o TBEV RNA in he se um. In con as , only one o he h ee TBEV-Eu in ec ed Table 1. Timescales o he expe imen al in ec ion s udies. Pe sis ence s udy Days pos in ec ion 18 53 83 109 133 168 Time, weeks pos in ec ion 0 3 6 8 10 12 14 16 18 20 22 24 Numbe o animals in o al 39 39 39 30 30 30 30 21 21 21 12 12 Animals eu hanized/s ain 0 0 0 30003 03 04 Animalseu hanizedin o al 00090009 09 012 Blood sampling x x x x x x U ine and exc emen s we e collec ed a e e y men ioned ime poin h oughou he pe sis ence s udy. Acu e phase in ec ion s udy Days pos in ec ion 0 4 8 14 25 Numbe o animals eu hanized/s ain 0 3 3 4 3 Numbe o animals eu hanized in o al 0 9 9 12 9 doi:10.1371/jou nal.pone.0081214. 001 In ec ion o Myodes gla eolus wi h TBEV PLOS ONE | www.plosone.o g 3 Decembe 2013 | Volume 8 | Issue 12 | e81214 animals and no TBEV-Sib in ec ed animal exhibi ed i al RNA in he se um. Apa om he b ain, spleen, lung and kidney we e ound posi i e o i al RNA e en i i emia was no de ec ed, which would sugges in ec ion o pa enchymal cells in hese o gans. One o he TBEV-Sib in ec ed indi iduals was nega i e in all es s (Table S1). All TBEV-Eu and -FE in ec ed oles, bu only he IgG-posi i e -Sib in ec ed ole exhibi ed i al RNA in he b ain; hese animals all showed a sligh o mode a e non-suppu a i e o ocally mixed (meningo)encephali is (Table S1) ha was gene ally a ec ing he ce eb al co ex, in mos cases also he hippocampus and in one case he ce ebellum. The in lamma o y p ocess was ep esen ed by he p esence o leukocy es wi hin smalle eins, and hei olling and a achmen o ac i a ed endo helial cells, emig a ion and pe i ascula accumula ion wi h sp eading in o he adjacen pa enchyma (Figu e 2A, B). In some cases, ma ked leukocy e apop osis was obse ed (Figu e 2B, C). Mic oglial nodules and di use mic oglial ac i a ion we e also seen. In il a ing leukocy es we e mainly mac ophages (lysozyme-posi i e), wi h a a iable p opo ion o neu ophils (Figu e 2B, D, E). In he TBEV-Eu and -FE in ec ed oles, mac ophages we e also seen in he pa enchy- ma, occasionally su ounding indi idual neu ons (sa elli osis; Fig. 2E), and he e was occasional e idence o neu onal degene a ion. Lymphocy es we e less nume ous among in il a ing leukocy es and we e mainly T cells, whe eas B cells we e e y a e and only seen in pe i ascula and lep omeningeal in il a es. Vi al an igen exp ession was obse ed in sca e ed neu ons in he co ex (mainly in e nal py amidal cell laye ; Figu e 2F), he hippocampus (py amidal cells; Figu e 2G) and he den a e gy us (g anule cells). The an ibody le els we e inc easing om 8 dpi un il he end poin o he s udy (Figu e 1). Two TBEV-FE in ec ed oles ha we e housed oge he in one cage and scheduled o eu hanasia a 14 dpi de eloped acu e gene alized symp oms and we e eu hanized a 12 dpi. Bo h oles had de eloped an ibodies agains TBEV and exhibi ed i al RNA in b ain, spleen, lung, and kidney, and one animal was also i emic (Table S1). The pa hological indings in hese oles we e es ic ed o e y mild non-suppu a i e encephali is, mainly ep esen ed by sca e ed mic oglial nodules in co ex and ce ebellum, wi h e y a e sa elli osis (Figu e 3A). Vi al an igen exp ession was mo e widesp ead and was obse ed no only in neu ons in he co ex, bu also in indi idual Pu kinje cells in he ce ebellum (Figu e 3B), and in ne e ib es in he ol ac o y bulb in one animals. S aining o clea ed caspase-3 iden i ied apop o ic cells in he mic oglial nodules, bu no apop o ic neu ons. A 14 dpi, all ou TBEV-Eu , he emaining wo -FE, bu only one o he ou -Sib in ec ed animals we e posi i e o TBEV-IgG an ibodies in EIA (Figu e 1). Bo h TBEV-FE, one -Eu - and one - Sib (IgG nega i e) in ec ed ole we e i emic a his s age (Table S1). All non- i emic TBEV-Eu and -FE in ec ed oles and one TBEV-Sib in ec ed animal exhibi ed i al RNA in one o all o he es ed o gans, i.e. spleen, lung and/o kidney (Table S1). Also, ega dless o he EIA esul s, when examined, he spleen o mos animals exhibi ed well dema ca ed ollicles wi h e idence o ge minal cen e o ma ion. Bo h TBEV-FE-in ec ed oles ha - bo ed i al RNA in he b ain, bu wi hou associa ed pa hological changes; i al an igen was only ound in one animal, in one weakly posi i e Pu kinje cell. The b ains o h ee animals inocula ed wi h TBEV-Sib we e nega i e o i al RNA and an igen and wi hou any pa hological changes, bu he ou h animal exhibi ed a sligh non-suppu a i e encephali is and an igen exp ession oge he wi h posi i e PCR esul o i al RNA (Table S1) in occasional neu ons in co ex, den a e gy us, and ce ebellum, also in associa ion wi h sa elli osis. All TBEV-Eu in ec ed oles exhibi ed i al RNA in he b ain and wo showed a mild non-suppu a i e encephali is, a ec ing he co ex and hippocampus, and o a e y low ex en he ce ebellum and b ain s em. Vi al an igen was de ec ed in Pu kinje cells in he ce ebellum (Figu e 3B, D) and, Figu e 1. An ibody esponse in TBEV-in ec ed bank oles. An ibody esponse (Vienna uni s o EIA, mean 695% con idence in e al) in TBEV- in ec ed bank oles du ing he sho - e m (closed symbols) and he long- e m expe imen (open symbols). S ains ha di e s a is ically signi ican ly wi hin a day pos in ec ion (P,0.05 in pai wise Tukey con as s) a e ma ked wi h a and b. The g ey ba indica es he bo de line a ea o EIA posi i i y. No ice he di e en scales on he sepa a ed pa s o he x axis. EUR = Eu opean, FE = Fa -Eas e n, SIB=Sibe ian TBEV sub ype. doi:10.1371/jou nal.pone.0081214.g001 In ec ion o Myodes gla eolus wi h TBEV PLOS ONE | www.plosone.o g 4 Decembe 2013 | Volume 8 | Issue 12 | e81214 Figu e 2. B ain o bank oles a 8 dpi. A–D. Co ex wi h mode a e non-suppu a i e o mixed in lamma ion, mainly cen ed a ound eins. A. Vein packed wi h leukocy es ha also emig a e om he essel, o m a pe i ascula cu and a e p esen in he adjacen pa enchyma (a ows). HE s ain; Ba = 20 mm. B. Vein wi h ma ked pe i ascula and pa enchymal accumula ion o leukocy es, nume ous o which exhibi degene a i e changes consis en wi h apop osis (a ows). HE s ain; Ba = 10 mm. C. S aining o clea ed caspase-3 con i ms ha leukocy es in bo h pe i ascula cu s and adjacen pa enchyma die ia apop osis (a ows). Pe oxidase an i-pe oxidase me hod, Papanicolaou’s hema oxylin coun e s ain; Ba = 10 mm. D. S aining o lysozyme iden i ies he as majo i y o in il a ing leukocy es in he pe i ascula in il a es as mac ophages and neu ophils. Pe oxidase an i-pe oxidase me hod, Papanicolaou’s hema oxylin coun e s ain; Ba = 10 mm. E. Mac ophages (lysozyme posi i e) a e also ound in pa enchymal in il a es and su ounding neu ons (a ows) in sa elli osis. Pe oxidase an i-pe oxidase me hod, Papanicolaou’s hema oxylin coun e s ain; Ba = 10 mm. F, G. Exp ession o i al an igen. F. Co ex wi h sca e ed posi i e neu ons, one o which is su ounded by mic oglia/mac ophages (sa elli osis). G. Hippocampus. Vi al an igen in py amidal cells and hei p ocesses (a ows). Ho se adish pe oxidase me hod, Papanicolaou’s hema oxylin coun e s ain; Ba s = 10 mm. doi:10.1371/jou nal.pone.0081214.g002 In ec ion o Myodes gla eolus wi h TBEV PLOS ONE | www.plosone.o g 5 Decembe 2013 | Volume 8 | Issue 12 | e81214 al hough gene ally only in low numbe s, in co ex and hippocam- pus, mainly in associa ion wi h sa elli osis and/o glial nodules (Figu e 3A–D) and wi h mild ocal as ogliosis (Figu e 3E). The hi d PCR-posi i e TBEV-Eu in ec ed ole did no exhibi any pa hological changes in he b ain, bu i al an igen exp ession was ound in wo neu ons in he inne py amidal laye o he co ex. A 25 dpi, all TBEV-Eu and -FE in ec ed, and one o h ee TBEV-Sib in ec ed animals, showed TBEV-speci ic an ibodies in he EIA. Two o he h ee TBEV-Sib in ec ed animals we e nega i e in all es s. All IgG-posi i e oles exhibi ed i al RNA in b ain and lung, some also in spleen and kidney, bu i emia was only de ec ed in he TBEV-FE in ec ed animals (Figu e 1, Table S1). The spleens o all animals, ega dless o he EIA esul s, exhibi ed ollicles wi h small ge minal cen e s. Vi al an igen exp ession was no de ec ed in he b ain o any ole despi e o en ela i ely high i al RNA i e s. Howe e , wo TBEV-FE and wo -Eu in ec ed oles exhibi ed in lamma o y changes in he b ain, which in he -Eu in ec ed animals ep esen ed a sligh and mild non-suppu a i e (meningo)encephali is espec i ely, wi h some pe i ascula lymphocy e cu ing in hippocampus and ce ebellum as well as sca e ed small mic oglial nodules, whe eas in he TBEV-FE in ec ed animals, i was es ic ed o sca e ed mic oglial nodules. Pe sis ence s udy A sepa a e s udy ocused on animals ha we e sac i iced be ween 53 and 168 dpi (Table 1) and was expec ed o p o ide in o ma ion on he pe sis ence o he i uses in hei oden hos s ou side he ick- eeding season. G oups o 13 bank oles we e inocula ed subcu aneously each wi h one s ain. All TBEV-Eu and -FE in ec ed bank oles se ocon e ed o TBEV, whe eas wo o he 13 TBEV-Sib in ec ed animals emained nega i e in he EIA. Bo h nega i e oles had appa en ly Figu e 3. B ain o bank oles a 12 and 14 dpi. A, B. Animal eu hanized a 12 dpi due o gene alized symp oms. A. Co ex wi h mic oglial nodule (a ow) and mic oglial ac i a ion. HE s ain; Ba = 10 mm. B. S aining o TBEV an igen. Ce ebellum wi h Pu kinje cell exp essing i al an igen in he cell body (a ows) and in cell p ocesses (a owheads). Inse : Vi al an igen exp ession in cell body and p ocesses (a owheads) o an indi idual neu on su ounded by mic oglial cells/mac ophages (sa elli osis). Ho se adish pe oxidase me hod, Papanicolaou’s hema oxylin coun e s ain; Ba s = 10 mm. C–E. Ce ebellum o animal scheduled sac i iced a 14 dpi. C. Sa elli osis a ound Pu kinje cells (a ows). HE s ain; Ba = 10 mm. D. Pu kinje cells exp essing i al an igen, su ounded by mic oglial cells/mac ophages (sa elli osis). Ho se adish pe oxidase me hod, Papanicolaou’s hema oxylin coun e s ain; Ba = 10 mm. E. Mild ocal as ogliosis in a ea wi h inc eased cellula i y (a ow: mic oglial nodule/in il a ing mac ophages), indica ed by he p esence o GFAP-posi i e s a -shaped eac i e as ocy es (a owheads) wi h nume ous long p ocesses. Pe oxidase an i-pe oxidase me hod, Papanicolaou’s hema oxylin coun e s ain; Ba = 10 mm. doi:10.1371/jou nal.pone.0081214.g003 In ec ion o Myodes gla eolus wi h TBEV PLOS ONE | www.plosone.o g 6 Decembe 2013 | Volume 8 | Issue 12 | e81214 no become in ec ed, as hey we e also nega i e o i al RNA in all es ed o gans (Figu e 1, Table S1). A 53 dpi none o he animals exhibi ed i al an igen o any pa hological changes in he b ain, al hough TBEV-RNA could be de ec ed by PCR. The o he o gans we e also unal e ed and only in wo oles (one TBEV-Sib and one -Eu in ec ed; bo h EIA- posi i e) did he spleen exhibi seconda y ollicles (Figu e 1, Table S1). F om 109 dpi onwa ds, TBEV-RNA was s ill de ec ed in he b ain (bu only in he b ain) o all animals (N = 9), ega dless o he TBEV sub ype used o in ec ion (Table S1). Voles sac i iced a day 109 dpi did no show any pa hological changes in he b ain o any o he o gan, and he e was no e idence o ge minal cen e o ma ion in splenic ollicles. Vi al an igen was no de ec ed in he b ain. One TBEV-Eu in ec ed animal scheduled o be eu hanized a 133 dpi, was ound dead a 110 dpi, wi hou p io clinical symp oms. The b ain was posi i e o TBEV RNA and he his ological examina ion e ealed a se e e mul i ocal neu ophil- domina ed and nec o izing en iculi is and co ical lep omenin- gi is wi h he p esence o i al an igen, bo h cell- ee and wi hin mac ophages and occasional neu ophils (Figu e 4). Adjacen o hese in il a es, a sligh pe i ascula mixed cellula pa enchymal in il a ion was seen. A simila , al hough less in ense in lamma ion was obse ed in ano he TBEV-EUR in ec ed ole, which had been sac i iced as scheduled, a 133 dpi. In his animal, which also ha bo ed i al RNA in he b ain, he lep omeningeal in il a es we e ound o e he co ex and b ain s em, su ounding a la ge essel ha exhibi ed ex ensi e ib inoid nec osis (Figu e 4A). Vi al an igen was seen wi hin mac ophages and occasional neu ophils (Figu e 4B). Also a 168 dpi, ocal lep omeningeal lymphocy e accumula ions we e ound a co ex and ce ebellum in one o he TBEV-Eu in ec ed animals (mild ocal lep omeningi is; Table S1). All o he oles sac i iced a 133 dpi and animals eu hanized a 168 dpi did no show any pa hological changes in he b ain o any o he o gan. Howe e , i al RNA was de ec ed in he b ain o wo o he h ee TBEV-FE in ec ed animals examined a 133 dpi and in one o he wo emaining -Eu in ec ed oles, whe eas he -Sib in ec ed oles we e PCR-nega i e and no i al an igen was de ec ed in he b ain by immunohis ology (Table S1). A 168 dpi, he TBEV-Eu in ec ed oles es ed nega i e o i al RNA and only one o he ou TBEV-Sib and –FE in ec ed oles each es ed posi i e. Also, RNA was de ec ed in he se um o he TBEV-Sib in ec ed ole s ill a 84 dpi (Table S1). Voles ha we e posi i e in bo h EIA and RT-PCR a 133 o 168 dpi exhibi ed highe IgG i e s han hose posi i e only in he EIA (Figu e 1). Ve o E6 cell cul u es inocula ed wi h PCR-posi i e b ain homogena es o animals sac i iced a 168 dpi es ed nega i e o TBEV bo h in IFA and RT-PCR un il 10 passages, con i ming ha cells we e no in ec ed. The u e i we e nega i e in he PCR a all ime poin s and did no show any his ological changes. All non-in ec ed con ol animals we e nega i e in all es s and did no exhibi any his ological changes in any examined issues. P esence o TBEV RNA in issues In he bes suppo ed GLMM (Table 2, Figu e 5), i ed o he da a om bo h in ec ion ials, TBEV RNA was ini ially (4 dpi) ound in he b ains mo e o en han in kidneys o se um. A his s age, lungs and spleens did no con ain TBEV RNA any less equen ly han he b ain, bu as he in ec ion p oceeded, he occu ence o i al RNA d opped signi ican ly soone in hese o gans han in he b ain. O e all, TBEV RNA was signi ican ly mo e o en p esen in issues o animals in ec ed wi h TBEV-FE han in hose in ec ed wi h TBEV-Eu o -Sib. The la e wo sub ypes did no di e om each o he in his espec (Table 2, Figu e 5). Discussion TBEV has been shown o pe sis in wild oden s [13,15,16,19], bu he in ec ion kine ics and he di e ences be ween he h ee sub ypes o he i us ha e ye no been sys ema ically s udied. We in ec ed bank oles (M. gla eolus), which ha e been shown o ca y TBEV in na u e [19,20] wi h all h ee TBEV sub ypes o s udy p ospec i ely and compa a i ely he in ec ion pa ame e s in an acu e and p olonged se ing unde con olled condi ions. Animals we e in ec ed subcu aneously and, ega dless o he sub ype, he as majo i y de eloped i emia, which was ollowed Figu e 4. B ain o bank ole a 133 dpi. A. Ma ked ocal, p edominan ly mononuclea lep meningi is (a ows), su ounding a medium-sized a e y ha exhibi s ocal ex ensi e ib inoid nec osis o he wall (*) and ocal degene a i e changes o endo helial cells (a owheads). HE s ain; Ba = 20 mm. B. TBEV an igen is exp essed cell ee and by mac ophages (a ows) and occasionally also by neu ophils (inse : a owhead) in he in il a e. Ho se adish pe oxidase me hod, Papanicolaou’s hema oxylin coun e s ain; Ba = 20 mm (inse : 10 mm). doi:10.1371/jou nal.pone.0081214.g004 In ec ion o Myodes gla eolus wi h TBEV PLOS ONE | www.plosone.o g 7 Decembe 2013 | Volume 8 | Issue 12 | e81214 by in ec ion o he b ain 8 dpi a la es . Neu ons we e he only cells ound o be in ec ed, and he in ec ion appea ed o i s occu in he co ex and hippocampus and hen sp ead mainly o he ce ebellum, whe e posi i e Pu kinje cells we e ound on days 12 and 14 pi hus p o ing ha TBEV is clea ly neu o opic. Only e y a ely was he e e idence ha he i us cause neu onal dea h in his na u al hos . A la e s ages, i al an igen was no u he de ec ed in neu ons, al hough pe sis ence o he TBEV RNA in he b ain was con i med by RT-PCR, suppo ing ou ea lie indings [19]. In all examined species so a , and bo h in na u al and expe imen al in ec ion, TBEV is exclusi ely neu o opic. In bank oles, we iden i ied he i us in neu ons in co ex, ce ebellum and hippocampus/den a e gy us. While i al an igen has been ound in hese loca ions in o he species as well, he a ge neu onal popula ions a e mo e a ied in humans and dogs, and he e appea s o be a p e e ence o he b ain s em [28,29]. Howe e , subcu aneous in ec ion o labo a o y mice wi h TBEV yielded a simila neu onal in ec ion pa e n as in he bank oles [35]. The ype and dis ibu ion o changes obse ed in he b ain o in ec ed bank oles in his s udy is e y simila o ha obse ed in o he species a e na u al and expe imen al in ec ion. We obse ed a mac ophage and T cell media ed in lamma o y esponse, bu wi h neu ophil con ibu ion. The la e was also Figu e 5. The obse ed p opo ion and he p edic ed p obabili y o TBEV-RNA occu ing in issues o bank oles. Lines ep esen p edic ed alues, i.e., es ima es o ixed e ec s in Table 2. Ci cles indica e he obse ed p opo ions o TBEV RNA posi i e animals, hei size being p opo ional o numbe o animals. doi:10.1371/jou nal.pone.0081214.g005 In ec ion o Myodes gla eolus wi h TBEV PLOS ONE | www.plosone.o g 8 Decembe 2013 | Volume 8 | Issue 12 | e81214 obse ed in na u al canine cases and in mice [29,35], bu was no a signi ican ea u e in human pa ien s [36]. Ea ly neu ophil ec ui men is also obse ed in o he la i i al in ec ions o he b ain, such as Wes Nile i us (WNV) in ec ion, whe e i has been a ibu ed o ma ked ele a ion o neu ophil- ec ui ing chemo- kines by mac ophages [37]. WNV eplica es in neu ophils a high le els and is likely dissemina ed ia neu ophils [37]. A simila mechanism appea s possible o TBEV, since i has been shown o in ec neu ophils [38,39], al hough in he p esen s udy immu- nohis ology did no de ec i us in in il a ing neu ophils in he ea ly s age o in ec ion when he e was e idence o cell ec ui men in o he b ain pa enchyma. Like in humans, i al an igen was mainly obse ed in in ac and only a ely in dying, apop o ic neu ons which u he con i ms ha he i us is no likely o ha e a di ec neu opa hic e ec and ha di ec ac i a ion o he apop o ic cascade is no a p ominen mode o cell dea h in TBE [36]. This may be di e en in dogs and in labo a o y mice, whe e subs an ial neu onal dea h has been obse ed, in he la e a leas wi h a high inocula ion dose [29,35]. In e es ingly, we obse ed a ocal se e e neu ophil-domina ed lep omeningi is (and en iculi is) in wo animals la e a e in ec ion (110 and 133 dpi, espec i ely) wi h he p esence o i al an igen bo h cell- ee and in mac ophages, as well as e idence o immune complex asculi is in one animal. I is possible ha his is due o local con inemen and eplica ion o he i us, bu canno be eadily explained. Mos animals in he p esen s udy did no de elop any clinical symp oms and unde wen scheduled eu hanasia. This was mos likely a consequence o he ela i ely limi ed in lamma o y esponse ha was gene ally seen. The esul s o ou p e ious s udy on wild-caugh oden s suppo s he inding ha he in ec ion is mainly asymp oma ic also du ing na u al in ec ion o bank oles, as we could de ec i al RNA in animals caugh se e al mon hs a e he ick eeding season. We would no expec animals ha had de eloped a signi ican disease o o he wise educed i ness o s ay ali e un il o o e he win e [19]. So a , s udies on he oden ese oi s ha e no assessed whe he TBEV in ec ion ( h ough he induc ion o encephali is) can ha e an e ec on he hos oden popula ion ia inc eased mo ali y. The bu den o TBEV in ec ion on hos oden popula ion dynamics needs o be conside ed in a con ex o cos o pa asi e de ences. Also, in he p esen s udy, animals we e ed ad libi um, whe eas in na u e, limi ed nu i ion may ha e an e ec on immune unc ions [40–42]. A sepa a e s udy add essing he e ec o TBEV in a oden popula ion should be conduc ed in p e iously epo ed geog aph- ically es ic ed TBEV oci. Roden s ha e been shown o se e as excellen sen inels o TBEV oci du ing ick- eeding season [15,20–22]. In ou ea lie s udy, TBEV an ibodies we e seldom de ec ed in wild oden s apped in win e , 4–9 mon hs a e he p obable in ec ion pe iod [19]. In he p esen s udy we could de ec high le els o an ibodies in animals in ec ed wi h all sub ypes un il 168 dpi, oge he wi h mo phological e idence o a sys emic immune esponse o he i us, ep esen ed by seconda y ollicle o ma ion in he spleen. These indings may indica e a di e ence be ween na u al and expe imen al in ec ion. Chunikhin and Ku eko [12] epo ed i emia las ing 2–3 days in expe imen ally in ec ed M. gla eolus, while Knap e al. [21] sugges ed ha he du a ion o i emia migh ha e been unde es ima ed in olde s udies. Howe e , Chunikhin and Ku eko [12] desc ibed, ha s ains isola ed om Bu ya ia, ep esen ing he TBEV-FE sub ype, gene ally p oduced highe LD50 alues on day 2–3 dpi han s ains isola ed in TBEV-Sib (Bal ic) and TBEV-Eu egions. In ou s udy, p olonged i emia was gene ally de ec ed in TBEV-FE–in ec ed animals and in one indi idual TBEV-Sib in ec ed ole ha was s ill TBEV-RNA- posi i e on 168 dpi. Acco ding o ou p esen s udy, TBEV-FE- RNA was mo e o en p esen in issues independen o he ime poin . TBEV-FE is endemic in he a ea anging om he Lake Baikal egion o No he n Japan and he eas e n pa o No he n China. Some indi idual oci ha e been ound also in he Bal ics and Wes e n Sibe ia [3,43,44]. The geog aphical dis ibu ion o M. gla eolus co e s only he endemic a eas o TBEV-Eu and TBEV-Sib, while o he Myodes species, M. u ilus and M. u ocanus inhabi he a ea endemic o TBEV-FE [45]. None heless, because o he close ela edeness be ween Myodes species, he di e ences seen in TBEV-FE in ec ion kine ics compa ed o he o he wo sub ypes seen in ou s udy we e likely no due o di e en Myodes hos species inocula ed in his expe imen al in ec ion. We ha e no been able o in ec suckling mice o Ve o E6 cells wi h TBEV-RNA posi i e b ain homogena es o pe sis en ly in ec ed bank oles. This could be due o a possible loss o i al in ec i i y in associa ion wi h i s pe sis ence in he b ain. Simila esul s we e ob ained in hesus monkeys a e subcu aneous TBEV in ec ion [46,47]. So a , he de e minan s o pe sis ence and i s po en ial ele ance o he ansmission o TBEV emain unclea . Pe sis ed TBEV could only be isola ed om na u ally in ec ed Myodes u ilus a e ea men wi h an immunosupp essan [48]. I could hus be specula ed ha a igge , such as immunosupp es- sion by ho mones o s ess, is needed o push he i us om pe sis ence o i emia he eby becoming accessible o icks. This hypo hesis should be es ed in na u al hos oden s. I is also possible ha he ou e o in ec ion ( ick bi e s. injec ion) is o majo ele ance o he cou se o in ec ion as ick sali a is known o con ain se e al immunomodula ing compounds [49] In o de o in es iga e whe he icks can become in ec ed di ec ly by eeding on pe sis en ly in ec ed oles, we unde ook a pilo expe imen as a pa o he p esen s udy. We se colonized TBEV- ee icks (Ixodes Table 2. The bes suppo ed GLMM analyzing he p obabili y o a issue sample o a TBEV-inocula ed bank ole o con ain TBEV RNA. Explana o y a iable Fac o le el Es ima e (SE) a z P alue In e cep b 4.86 (0.97) 5.0 , 0.001 Sub ype Eu opean 2 1.16 (0.43) 2 2.7 0.007 Sibe ian 2 1.72 (0.49) 2 3.5 , 0.001 Days pos in ec ion 2 0.03 (0.01) 2 4.0 , 0.001 Tissue Lung 20.58 (1.19) 20.5 0.627 Spleen 20.23 (1.26) 20.2 0.854 Kidney 2 2.34 (1.10) 2 2.1 0.033 Se um 2 2.85 (1.02) 2 2.8 0.005 Days pos in ec ion:Tissue Lung 2 0.05 (0.02) 2 2.1 0.033 Spleen 2 0.08 (0.03) 2 2.6 0.009 Kidney 20.08 (0.14) 21.9 0.052 Se um 20.04 (0.02) 21.9 0.059 Va iance a ibu able o a andom e ec o animal iden i y (71 g oups) was ,0.01 wi h s anda d de ia ion ,0.01. a Es ima es a e gi en on logi scale and s anda d e o s o es ima es a e in pa en heses. Signi ican coe icien s a e in bold. b In e cep is calcula ed o b ain issue o a bank ole inocula ed wi h Fa - eas e n TBEV sub ype ou days pos in ec ion. doi:10.1371/jou nal.pone.0081214. 002 In ec ion o Myodes gla eolus wi h TBEV PLOS ONE | www.plosone.o g 9 Decembe 2013 | Volume 8 | Issue 12 | e81214