High frequency of hepatitis E virus infection in swine from South Brazil and close similarity to human HEV isolates

h tt p : / / w w w . b j m i c r o b i o l . c o m . b r /

Veterinary

Microbiology

High

frequency

of

hepatitis

E

virus

infection

in

swine

from

South

Brazil

and

close

similarity

to

human

HEV

isolates

Ana

Maria

Passos-Castilho

,

Celso

Francisco

Hernandes

Granato

UniversidadeFederaldeSãoPaulo,DepartamentodeMedicina,DivisãodeDoenc¸asInfecciosas,SãoPaulo,SP,Brazil

a

r

t

i

c

l

e

i

n

f

o

Received29April2016 Accepted19October2016 Availableonline3January2017 AssociateEditor:JoãoAraujoJr.

Keywords: Brazil Genotype3 HepatitisEvirus Pigs Zoonosis

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HepatitisEvirusisresponsibleforacuteandchronicliverinfectionsworldwide.Swine hep-atitisEvirushasbeenisolatedinBrazil,andaprobablezoonotictransmissionhasbeen described,althoughdataarestillscarce.Theaimofthisstudywastoinvestigatethe fre-quencyofhepatitisEvirusinfectioninpigsfromasmall-scalefarmintheruralareaof ParanáState,SouthBrazil.Fecalsampleswerecollectedfrom170pigsandscreenedfor hepatitisEvirusRNAusingaduplexreal-timeRT-PCRtargetingahighlyconserved70nt longsequencewithinoverlappingpartsofORF2andORF3aswellasa113ntsequenceof ORF2.Positivesampleswithhighviralloadsweresubjectedtodirectsequencingand phy-logeneticanalysis.hepatitisEvirusRNAwasdetectedin34(20.0%)ofthe170pigsfollowing positiveresultsinatleastonesetofscreeningreal-timeRT-PCRprimersandprobes.The swinehepatitisEvirusstrainsclusteredwiththegenotypehepatitisEvirus-3breference sequencesinthephylogeneticanalysisandshowedclosesimilaritytohumanhepatitisE virusisolatespreviouslyreportedinBrazil.

©2017SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/

licenses/by-nc-nd/4.0/).

Introduction

Inendemic areas,hepatitisEvirus (HEV) causes large epi-demicsand sporadic casesin humans,including genotype HEV-1inAsiaandAfrica,genotypeHEV-2inMexicoandAfrica, andgenotypeHEV-4inAsia.Innon-endemicareas,isolated casesofgenotypeHEV-3occurinEurope,Japanandthe Amer-icas.GenotypesHEV-3andHEV-4aredescribedaszoonotic,as theyinfectnumerousmammalianspecies,including domes-ticpigs,andcanbetransmittedthroughtheingestionofraw orundercookedmeatfrominfectedanimals.1

∗ _{Correspondingauthor.}

E-mail:anampassos@gmail.com(A.M.Passos-Castilho).

HEVshowsnotableheterogeneitywithseveralgroupsand genotypes.Arecentconsensushasclassifiedthisvirusinone familyofHepeviridae,dividedin2generanamely Orthohep-evirusandPiscihepevirus.Orthohepevirusisfurtherdivided into 4speciesfromAtoD.OrthohepevirusAisthespecies infectinghumansandswineandotheranimals,suchasboar, deer,mongoose,rabbitandcamel.Theseincludetwo geno-types isolated from humans alone (HEV-1 and HEV-2), two genotypes reported in both humans and different animal speciesand associatedwiththe zoonoticcases(HEV-3 and HEV-4),twoisolatesfromwildboarinJapan(genotypeHEV-5 andHEV-6)andasingleisolatefromdromedarycamelinDubai

http://dx.doi.org/10.1016/j.bjm.2016.10.022

1517-8382/©2017SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

(genotypeHEV-7).RabbitHEVandcloselyrelatedhuman iso-late havebeen placedas adistant member inHEV-3. The moose virus appearsto cluster closely to genotype HEV-3, whileHEVisolatesfrom minktoferret virus (HEVC2)and HEVisolatesfromfoxtoratvirus(HEVC1).Theseviruseshave notbeenplacedinanyspecificgenotypeandneedcomplete genomic sequences fora definite taxonomic classification. OrthohepevirusBincludesall3avianHEVstrains, Orthohep-evirusConespeciesfromratandanotheronefromferretand OrthohepevirusDincludesbatHEV.Piscihepevirusincludes2 troutHEVstrainswithinasinglespecies.2,3

InBrazil,swineHEVwasfirstisolatedfrompigfecal sam-plesfromtheSãoPauloState,SoutheasternBrazil.4_Moreover,

genotypeHEV-3wasfoundinpigsandeffluentsfromapig slaughterhouseinRiodeJaneiroState,SoutheasternBrazil,5,6

and inpigs from the Eastern BrazilianAmazon7 _{and from}

Southern Brazil.8,9 _{Nonetheless, hepatitis E has only been}

studiedasapotentialzoonoticdiseaseinLatinAmerica in thelasttenyears,anddataonthissubjectarestillscarce.

Data on human HEVin Brazil are limited as well. This countryhasbeenclassifiedasmoderatelyendemicforHEV, with seroprevalence from 1% to 4% in blood donors or thegeneralpopulation,13%inindividualsfrom an agricul-tural settlement in the Amazon Basin, and 15% in renal transplant recipients.10–13 _{Amore recent study observed a}

seroprevalence of anti-HEV IgG antibodies of 10% among blooddonorsinthemetropolitanareaofItajaiValley, South-ernBrazil,aregionofpredominantGermanancestrywhere culturalhabits resultinahigh pork consumption.14

Geno-type HEV-3 infection has been described in the country, both amongimmunocompetent and immunocompromised individuals.15–17

Brazilisthefourthbiggestporkexporterintheworld,with animportantincreaseinrecentyears.TheStateofParanáhas oneofthelargestswine productionsinthecountry,and in 2014,thisregionwassolelyresponsiblefor20%ofthenational pigslaughter.Inadditiontoexport,porkconsumptionhasalso increasedinBrazil.18_{SpecificallyinParanáState,the}

predomi-nantEuropeanancestry19,20_{leadstohigherporkconsumption}

than inotherparts ofthecountry.21,22 _{Theimpactofsuch}

habitsandthepotentialtransmissionofswineHEVtohumans inthisregionremainunknown.

Inthepresentstudy,weinvestigatedthefrequencyofHEV infectioninpigsfromasmall-scalefarmintheruralareaof ParanáState,SouthBrazil.

Materials

and

methods

Fecalsamples

ThestudyprotocolwasapprovedbytheInstitutional Commit-teeforEthicsintheUseofResearchAnimals(CEUA-UNIFESP 2014/1004300914).

InSeptember2014,fecalsampleswerecollectedfrom170 pigsfromasmall-scalefarminthemunicipalityofItapejara d’Oeste,inaruralareaofParanáState,SouthernBrazil.This farmisoneofthemanysmallfarmsintheregion,witha cur-rentpopulationofapproximately70sows,5boarsand580pigs thataresoldtoslaughterhouseswhenraisedtoamedianage

of22weeks.Thesamplesizeof170animalswascalculated toallowdeterminingaHEVRNAprevalenceofapproximately 15%9_{witha95%confidenceinterval(CI).Additionally,}

samp-lingwasperformedattheagesof4,7,10,13and16weeks, according to the age distribution ofall the pigs raisedfor slaughter.

RNAextraction,nestedRT-PCRandquantitativeRT-PCR HEV RNA was extracted from the fecal samples using the QIAampviralRNAminikit(QIAGEN,Hilden,Germany).Briefly, fecal samples were first suspended in a 50% solution in ultrapure nuclease-free distilled water, then centrifuged at 20,000×gfor15min,andthesupernatantwasusedtoextract viralRNAaccordingtothemanufacturer’sinstructions. Quan-titative RT-PCR was performed according to a previously describedmodifiedone-stepduplexreal-timeprotocol,23_with

aprimerandprobesettargetingahighlyconserved70ntlong sequence withinoverlapping parts of ORF2 and ORF324 _as

wellasasetspecificfora113ntsequenceofORF2.25_A

pre-viouslycharacterizedplasmidclonefromaBrazilianhuman HEVstrain(KF152884)17_{wasconstructedwiththeTOPO}®_TA

Cloning®Kit(Invitrogen,Carlsbad,CA,USA)andthedescribed primers. PlasmidDNA waspurifiedusingtheQIAprep Spin MiniprepKit(Qiagen,Hilden,Germany) andthenlinearized andquantifiedwiththeNanodropND-1000instrument (Wil-mington, DE, USA), followed by transcription to RNA with T7RNApolymerase(Promega,Madison,WI,USA).Standard curvesweregeneratedusing10−1–1010_{copiesofplasmidRNA.}

HEVviralloadswere determinedbasedonstandard curves and were reported as the log10 number of copies of HEV RNApermLoffecalsuspension.Thedetection limitofthe real-timeRT-PCRwasthreecopiesofviralRNAperreaction (2.40log10copiespermLoffecalsuspension),whilethe

quan-tificationlimitwassetat10copiesofviralRNAperreaction (3.00log10 copiespermLoffecalsuspension).Allscreening

reactionswereruninduplicatewithpropercontrols,whereas positive results were confirmed in separate confirmatory reactions.

ThequalitativenestedRT-PCRone-stepreactionwas con-ducted with primers to amplify partial regions of ORF1 and ORF2 of 287nt and 348 nt,respectively, after second-roundPCR.26,27 _{Allprecautionsandproceduressuggestedto}

avoidthepossibilityofcross-contaminationwereemployed. Amplified products were visualized in a 1.5% agarose gel stained with SYBR® Safe (Life Technologies, Austin, TX, USA).

Sequencingandphylogeneticanalysis

Final fragments obtained from the nested RT-PCRanalysis (ORF1287ntandORF2348nt)werepurifiedusingthe ExoSAP-ITPCRClean-upKit(GEHealthcare,ChalfontSt.Giles,UK),and sequencedusingtheBigDye®Terminatorv3.1Cycle Sequenc-ingKitandtheautomatedABI3100DNASequencer(Applied Biosystems,FosterCity,CA,USA),accordingtothe manufac-turer’sinstructions.SequencesfromhumanandswineHEV werecollectedfrompublicdatabases,andphylogenetictrees were constructed using the neighbor-joining method with

Table1–MoleculardetectionofHEV-RNAinpigsfromtheruralareaofParanáState,SouthBrazil,usingreal-timeand

conventionalRT-PCR.

Pigno. Age(weeks) Real-timeRT-PCR ConventionalRT-PCR ORF2/3(70nt) ORF2(113nt) ORF1(287nt) ORF2(348nt)

31 7 + − − − 42 7 + − − − 49 7 − 3.20log10 − + 51 7 − + − − 59 7 − + − − 63 7 + − − − 69 10 − + − − 71 10 + − − − 74 10 + − − − 77 10 + − − − 78 10 + − − − 79 10 − 6.48log10 + + 80 10 − + − − 82 10 5.98log10 5.54log10 + − 84 10 + − − − 85 10 − + − − 86 10 + − − − 91 10 + − − − 96 10 + − − − 100 13 − + − − 108 13 − + − − 115 13 − + − − 118 13 + + − − 119 13 + + − − 123 13 + + − − 126 13 + − − − 130 13 − + − − 132 13 + + − − 133 13 + − − − 134 13 − + − − 136 13 − + − − 138 13 + − − − 146 13 + + − − 147 13 + 3.37log10 − + n/N(%) 21/170(12.4%) 20/170(11.8%) 2/170(1.2%) 3/170(1.8%) Forpositivesamples,viralloadisexpressedasthelog10numberofcopiesofHEV-RNApermLoffecalsuspensionifhigherthanthequantification limitofthereal-timeRT-PCR(>3.00log10copies).Positivesampleswithviralloadbetween2.40and3.00log10copiesareexpressedasthesymbol

+.

theKimura2-parametermodelofnucleotidesubstitutionin MEGAv.5.0(TheBiodesignInstitute,USA).Statisticswas per-formedbybootstrapanalysiswith1000pseudoreplicates.The sequencesreportedinthisstudyareavailableintheGenBank

databaseundertheaccessionnumbersKP966825–KP966829.

Statisticalanalysis

All data were analyzed using SPSS version 11.0 (SPSS

Inc., Chicago, IL, USA). Descriptive statistics consisted of

the characterization of the studied population and the

Table2–HEV-RNAdetectionfrequencyinpigsfromtheruralareaofParanáState,SouthBrazil,byagegroup.

Agegroup(weeks) HEV-RNApositive HEV-RNAnegative p

n(%) n(%) 4 0(0.0) 30(100.0) 0.001a 7 6(9.1) 30(90.9) 10 13(40.6) 19(59.4) 13 15(20.8) 34(79.2) 16 0(0.0) 23(100.0) Total 34(20.0) 136(80.0) 170(100)

Fig.1–Phylogenetictreereconstructedbytheneighbor-joiningmethodwithcommon242-ntORF1sequencesfrom29 isolates,including8porcineisolatesfromBrazil,1humanisolatefromBrazil,andthe2swineisolatesdescribedinthis

study,Brazil79.1swandBrazil82sw(highlightedinred).TheGenBankaccessionnumberinparentheses,thenameofthe

countryoforigin,thespeciesfromwhichitwasisolated,andthegenotype/subtypeoftheisolateidentifyeachviralstrain. Bootstrapvaluesof>50areindicatedforthemajornodesasapercentageofthedataobtainedfrom1000replicates(bar,0.02 substitutionspersite).Majorbranchesindicategenotypes.AvianHEVistheoutgroup.

frequency of HEV-RNA detection with the respective per-centages and 95% CI. The bivariate analysis to compare categorical values consisted of Pearson’s Chi-square test. Non-conditionallogisticregressionwasusedtoidentify asso-ciations between dependent and independentvariables by

the means of odds ratio (OR). For this analysis, the ages of 4 and 7 weeks were grouped to avoid empty cells, as well as the ages of 13 and 16 weeks. The statistical significance levelwas p<0.05.All reported valuesare two-tailed.

Fig.2–Phylogenetictreereconstructedbytheneighbor-joiningmethodwithcommon304-ntORF2sequencesfrom49 isolates,including13porcineisolatesfromBrazil,4humanisolatesfromBrazil,andthe3swineisolatesdescribedinthis

study,Brazil79sw,Brazil49swandBrazil147sw(highlightedinred).TheGenBankaccessionnumberinparentheses,the

nameofthecountryoforigin,thespeciesfromwhichitwasisolated,andthegenotype/subtypeoftheisolateidentifyeach viralstrain.Bootstrapvaluesof>50areindicatedforthemajornodesasapercentageofthedataobtainedfrom1000 replicates(bar,0.02substitutionspersite).Majorbranchesindicategenotypes.AvianHEVistheoutgroup.

Results

HEV-RNAdetectionandsequencing

HEV-RNAwasdetectedin34(20.0%)of170pigsfollowinga positiveresultinatleastonesetofscreeningreal-time RT-PCRprimersandprobes.Seven(20.6%)ofthesesampleswere positivewithbothsetsofprimersandprobes.The70ntORF2/3 setamplified21(12.4%)of170samples,whilethe113ntORF2 setamplified20samples(11.8%).Amongthe34positive sam-ples,only4(11.8%)presentedviralloadshigherthan3.00log10

copiespermLoffecalsuspension(Table1).

Table2showsthedetectionofHEV-RNAbyagegroup.Inthe

agegroupfrom4to7weeks,only9.1%ofpigshaddetectable HEV-RNA,whereas40.6%haddetectableHEV-RNAat10weeks ofage.Theseresultsshowanapproximate7-foldhigherrisk ofundergoingaHEVinfectionattheageof10weeksthanat theagesof4or7weeks(OR=6.84,95%CI2.29–20.48).

Duetothe lowviralloadofthemajorityofthepositive samplesand the difference between the detectionlimit of thescreeningreal-timeRT-PCRandtheconventionalnested RT-PCR,only4(11.8%;4/34)samplescouldbeamplifiedwith conventionalnestedRT-PCRandsubjectedtodirect sequenc-ing.Amongthesesamples,twowereamplifiedwiththesetof primersresultinginafinalproductof287nt,andthreewere amplifiedwiththe384ntset.Sample079wasamplifiedusing bothsetsofprimersontheconventionalnestedRT-PCR.

Phylogeneticanalysis

Thetwosamplesfromthe287ntORF1partialregionshared 99%homologywitheachotherandclusteredwiththe geno-typeHEV-3breferencesequencesinthephylogeneticanalysis (Fig.1).Thethreesamplesfromthe384ntORF2partialregion alsogroupedwiththegenotypeHEV-3breferencesequences inthephylogeneticanalysis.Nucleotideidentityamongthese threesamplesrangedfrom98%to99%(Fig.2).

Discussion

ThepresentdatashowahigherfrequencyofHEVinfection (20.0%) inpigs than previously reported inBrazil. A study performedduring2009inthesameregionwith170fecal sam-plesfrom14pigfarmsfoundHEV-RNAin15.3%ofsamples.9

Anotherstudy thatinvestigatedserum,bileand fecal sam-plesfrom151pigsfromtheeasternBrazilianAmazon,North Brazil,detectedHEV-RNAin9.9%oftheanimals.7_Thehigher

infectionrateobservedinourstudycouldbearesultofthe sanitaryconditionsofthesmall-scalepigfarmsintherural areaofParanáState,wherethecontactofpigsofdifferentages mayoccur.However,itcouldalsobeduetothedifferencein methodology,sincepreviousstudiesemployedconventional RT-PCRtechniquesasopposedtothemoresensitivereal-time RT-PCRused inthe present study.Additionally, the duplex RT-PCRtechniqueemployedinthisstudydemonstratedthe importanceofusingmorethanonesetofprimersandprobes forhigherdetectionduringHEVscreening,asonly20.6%of

thepositivesampleshaddetectableHEV-RNAwithbothsets ofprimersandprobes.

PreviousstudiesreportedthatHEVinfection inpigswas morefrequentfrom12to16weeksofageandthatatslaughter age(20–24 weeks),theanimalshadalreadydeveloped anti-HEVantibodies.28 _{Inthe presentstudy,HEV-RNAwasmore}

frequentinpigsaged10weeks(40.6%),althoughthefrequency inpigsaged13 wasstillhigh(20.8%).Nonetheless,noneof the samplesfromthe pigsaged4or16 weekstested posi-tive.Theseobservationsconferwithastudyperformedamong swineherdsinRiodeJaneiro,SoutheastBrazil,showingthat newbornpigsbecamesusceptibletoHEVbetweenweeks7and 9,anageinwhichtheserumlevelsofthematernalantibodies declined.5_{Theseresultsarealsoinagreementwithastudy}

performedinCentralBrazilinswineaged20–30weeks,with 81%ofanti-HEVIgGpositivity.29

Theingestionofraworundercookedporkhasbeen asso-ciatedwithHEVinfection,30,31 _{andaprobablezoonoticHEV}

transmissionhasbeenreportedinBrazil.15_{Additionally,there}

is a known riskof HEVtransmission to people who come in contactwithfeces from infected pigs,which have been reported asanimportantsourceofinfectionfor slaughter-houseworkersandbutchersandhavebeenassociatedwith infection in non-endemic regions.32,33 _{HEV has also been}

describedinsewagesamplesfromaslaughterhousein South-easternBrazil.6

The ORF1 HEV isolates foundin this study shared 88% homologywithahumanHEVsequencefromBrazil15and86% to96%homologywithswineHEVsequencesfromBrazil.5,8,9

TheORF2HEVisolatesshared86–93%homologywithhuman HEVsequencespreviouslycharacterizedbyourresearchgroup inrenaltransplantrecipientsinBrazil16_{and83–97%homology}

withswineHEVsequencesfromBrazil.5,9_{Amongallcompared}

HEV sequences, the highest homology (98–99%) was with human sequences recently isolated in Southeastern Brazil fromapediatriclivertransplantrecipientwithchronicHEV infection.17

Although morestudies are neededto elucidatethe real impactofswineHEVinfectionandzoonotictransmissionin Brazil,takentogether,theseresultsreinforcethehypothesis that domesticpigsmay beanimportantsourceforhuman hepatitisEvirusinfectioninthissetting.

Conclusions

Inconclusion,thisstudyconfirmsthecirculationofthe hep-atitisEvirusandshowsahighfrequencyofHEVinfectionin pigsofdifferentagesraisedforslaughterintheruralareaof ParanáState,SouthBrazil.Theclosesimilaritybetweenthe humanHEVstrainsandthosefoundhereinindicatesthat ade-quatesafetyactionsshouldbetakentopreventHEVinfection whenhandlingpigsand/orconsumingpork.

Conflicts

of

interest

Acknowledgements

This study was supported by the Fundac¸ão de Amparo à PesquisadoEstadodeSãoPaulo(GrantNos.2012/22925-3and 2013.03701-0).WewishtothankMr.Voitena,hisfamily,and VeterinaryDoctor Jessica Voitenaforall theircollaboration andtechnicalassistance.

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1.KamarN,DaltonHR,AbravanelF,IzopetJ.HepatitisEvirus

infection.ClinMicrobiolRev.2014;27:116–138.

2.SmithDB,SimmondsP,MembersoftheInternational

CommitteeontheTaxonomyofVirusesHepeviridaeStudy

G.,etal.Consensusproposalsforclassificationofthefamily

Hepeviridae.JGenVirol.2014;95:2223–2232.

3.KhurooMS,KhurooMS.HepatitisE:anemergingglobal

disease–fromdiscoverytowardscontrolandcure.JViral

Hepat.2016;23:68–79.

4.PaivaHH,TzanevaV,HaddadR,YokosawaJ.Molecular

characterizationofswinehepatitisEvirusfrom

SoutheasternBrazil.BrazJMicrobiol.2007;38:693–698.

5.dosSantosDR,VitralCL,dePaulaVS,etal.Serologicaland

molecularevidenceofhepatitisEvirusinswineinBrazil.Vet

J.2009;182:474–480.

6.dosSantosDR,dePaulaVS,deOliveiraJM,MarchevskyRS,

PintoMA.HepatitisEvirusinswineandeffluentsamples

fromslaughterhousesinBrazil.VetMicrobiol.

2011;149:236–241.

7.deSouzaAJ,Gomes-GouvêaMS,SoaresMC,etal.HEV

infectioninswinefromEasternBrazilianAmazon:evidence

ofco-infectionbydifferentsubtypes.CompImmunolMicrobiol

InfectDis.2012;35:477–485.

8.GardinaliNR,BarryAF,OtonelRA,AlfieriAF,AlfieriAA.

HepatitisEvirusinliverandbilesamplesfromslaughtered

pigsofBrazil.MemInstOswaldoCruz.2012;107:935–939.

9.GardinaliNR,BarryAF,daSilvaPF,deSouzaC,AlfieriAF,

AlfieriAA.Moleculardetectionandcharacterizationof

hepatitisEvirusinnaturallyinfectedpigsfromBrazilian

herds.ResVetSci.2012;93:1515–1519.

10.EchevarríaJM,GonzálezJE,Lewis-XimenezLL,etal.

HepatitisEvirusinfectioninLatinAmerica:areview.JMed

Virol.2013;85:1037–1045.

11.HeringT,PassosAM,PerezRM,etal.Pastandcurrent

hepatitisEvirusinfectioninrenaltransplantpatients.JMed

Virol.2014;86:948–953.

12.VitralCL,daSilva-NunesM,PintoMA,etal.HepatitisAand

Eseroprevalenceandassociatedriskfactors:a

community-basedcross-sectionalsurveyinruralAmazonia.

BMCInfectDis.2014;14:458.

13.Passos-CastilhoAM,deSenaA,ReinaldoMR,GranatoCF.

HepatitisEvirusinfectioninBrazil:resultsof

laboratory-basedsurveillancefrom1998to2013.RevSocBras

MedTrop.2015;48:468–470.

14.Passos-CastilhoAM,deSenaA,GeraldoA,SpadaC,Granato

CF.HighprevalenceofhepatitisEvirusantibodiesamong

blooddonorsinSouthernBrazil.JMedVirol.2016;88:361–364.

15.LopesDosSantosDR,Lewis-XimenezLL,daSilvaMF,de

SousaPS,GasparAM,PintoMA.Firstreportofahuman

autochthonoushepatitisEvirusinfectioninBrazil.JClin

Virol.2010;47:276–279.

16.PassosAM,HeringerTP,Medina-PestanaJO,FerrazML,

GranatoCF.Firstreportandmolecularcharacterizationof

hepatitisEvirusinfectioninrenaltransplantrecipientsin

Brazil.JMedVirol.2013;85:615–619.

17.Passos-CastilhoAM,PortaG,MiuraIK,etal.Chronic

hepatitisEvirusinfectioninapediatricfemaleliver

transplantrecipient.JClinMicrobiol.2014;52:4425–4427.

18.BrazilianInstituteofGeographyandStatistics.Livestock ProductionEstimates;2015.Availableat:

http://www.ibge.gov.br/home/estatistica/indicadores/

agropecuaria/producaoagropecuaria/default.shtmAccessed

14.05.15.

19.BrazilianInstituteofGeographyandStatistics(IBGE).Brasil:

500anosdepovoamento[Brazil:500YearsofSettlement].2nded.

RiodeJaneiro:IBGE;2007[inPortuguese].

20.LazierH.Análisehistóricadapossedeterranosudoeste

paranaense[HistoricalAnalysisofLandPossessioninthe SoutheasternParanáState].FanciscoBeltrão:GrafitGráficae

EditoraLtd.;1997[inPortuguese].

21.SchlindweinMM,KassoufAL.Análisedainfluênciade

algunsfatoressocioeconômicosedemográficosnoconsumo

domiciliardecarnesnoBrasil[AnalysisoftheInfluenceof

SomeSocio-EconomicandDemographicFactorsin

HouseholdConsumptionofMeatinBrazil].RevEconSociol

Rural.2006;44:549–572[inPortuguese].

22.VerbekeW,Perez-CuetoFJ,BarcellosMD,KrystallisA,

GrunertKG.Europeancitizenandconsumerattitudesand

preferencesregardingbeefandpork.MeatSci.

2010;84:284–292.

23.VasickovaP,KralikP,SlanaI,PavlikI.Optimisationofa

triplexrealtimeRT-PCRfordetectionofhepatitisEvirus

RNAandvalidationonbiologicalsamples.JVirolMethods.

2012;180:38–42.

24.JothikumarN,CromeansTL,RobertsonBH,MengXJ,HillVR.

Abroadlyreactiveone-stepreal-timeRT-PCRassayforrapid

andsensitivedetectionofhepatitisEvirus.JVirolMethods.

2006;131:65–71.

25.GyarmatiP,MohammedN,NorderH,BlombergJ,BelákS,

WidénF.UniversaldetectionofhepatitisEvirusbytwo

real-timePCRassays:TaqManandPrimer-ProbeEnergy

Transfer.JVirolMethods.2007;146:226–235.

26.WangY,LingR,ErkerJC,etal.Adivergentgenotypeof

hepatitisEvirusinChinesepatientswithacutehepatitis.J

GenVirol.1999;80:169–177.

27.HuangFF,HaqshenasG,GuenetteDK,etal.Detectionby

reversetranscription-PCRandgeneticcharacterizationof

fieldisolatesofswinehepatitisEvirusfrompigsindifferent

geographicregionsoftheUnitedStates.JClinMicrobiol.

2002;40:1326–1332.

28.NakaiI,KatoK,MiyazakiA,etal.Differentfecalshedding

patternsoftwocommonstrainsofhepatitisEvirusatthree

Japaneseswinefarms.AmJTropMedHyg.2006;75:1171–1177.

29.GuimaraesFR.HepatitisEvirusantibodiesinswineherdsof

MatoGrossostate,CentralBrazil.BrazJMicrobiol.

2005;36:223–226.

30.ColsonP,BorentainP,QueyriauxB,etal.Pigliversausageas

asourceofhepatitisEvirustransmissiontohumans.JInfect

Dis.2010;202:825–834.

31.Legrand-AbravanelF,KamarN,Sandres-SauneK,etal.

CharacteristicsofautochthonoushepatitisEvirusinfection

insolid-organtransplantrecipientsinFrance.JInfectDis.

2010;202:835–844.

32.HepatitisJaryC.Eandmeatcarcasses.BrJGenPract.

2005;55:557–558.

33.Perez-GraciaMT,MateosML,GalianaC,etal.Autochthonous

hepatitisEinfectioninaslaughterhouseworker.AmJTrop