Data on human HEVin Brazil are limited as well.. This countryhasbeenclassifiedasmoderatelyendemicforHEV, with seroprevalence from 1% to 4% in blood donors or thegeneralpopulation,13%inind
Trang 1h tt p : / / w w w b j m i c r o b i o l c o m b r /
Veterinary Microbiology
Ana Maria Passos-Castilho∗, Celso Francisco Hernandes Granato
Federal University of Sao Paulo, Department of Medicine, Division of Infectious Diseases, São Paulo, SP, Brazil
a r t i c l e i n f o
Article history:
Received29April2016
Accepted19October2016
Availableonlinexxx
AssociateEditor:JoãoAraujoJr
Keywords:
Brazil
Genotype3
HepatitisEvirus
Pigs
Zoonosis
a b s t r a c t
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
∗ Corresponding author.
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/)
Trang 2iso-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.4Moreover,
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.18SpecificallyinParanáState,the
predomi-nantEuropeanancestry19,20leadstohigherporkconsumption
than inotherparts ofthecountry.21,22 Theimpactofsuch
habitsandthepotentialtransmissionofswineHEVtohumans
inthisregionremainunknown
Inthepresentstudy,weinvestigatedthefrequencyofHEV
infectioninpigsfromasmall-scalefarmintheruralareaof
ParanáState,SouthBrazil
Materials and methods
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%9witha95%confidenceinterval(CI).Additionally, samp-lingwasperformedattheagesof4,7,10,13and16weeks, according to the age distribution ofall the pigs raisedfor slaughter
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,23with
aprimerandprobesettargetingahighlyconserved70ntlong sequence withinoverlapping parts of ORF2 and ORF324 as wellasasetspecificfora113ntsequenceofORF2.25A pre-viouslycharacterizedplasmidclonefromaBrazilianhuman HEVstrain(KF152884)17wasconstructedwiththeTOPO®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 curvesweregeneratedusing10−1–1010copiesofplasmidRNA 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)
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
Trang 3Table 1 – Molecular detection of HEV-RNA in pigs from the rural area of Paraná State, South Brazil, using real-time and conventional RT-PCR.
Pigno Age(weeks) Real-timeRT-PCR ConventionalRT-PCR
ORF2/3(70nt) ORF2(113nt) ORF1(287nt) ORF2(348nt)
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
Statistical analysis
Inc., Chicago, IL, USA) Descriptive statistics consisted of
Table 2 – HEV-RNA detection frequency in pigs from the rural area of Paraná State, South Brazil, by age group.
Agegroup(weeks) HEV-RNApositive HEV-RNAnegative p
a Significantatp<0.05withPearson’sChi-squaretest
Trang 4Fig 1 – Phylogenetic tree reconstructed by the neighbor-joining method with common 242-nt ORF1 sequences from 29 isolates, including 8 porcine isolates from Brazil, 1 human isolate from Brazil, and the 2 swine isolates described in this study, Brazil79.1sw and Brazil82sw (highlighted in red) The GenBank accession number in parentheses, the name of the country of origin, the species from which it was isolated, and the genotype/subtype of the isolate identify each viral strain Bootstrap values of >50 are indicated for the major nodes as a percentage of the data obtained from 1000 replicates (bar, 0.02 substitutions per site) Major branches indicate genotypes Avian HEV is the outgroup.
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
Trang 5Fig 2 – Phylogenetic tree reconstructed by the neighbor-joining method with common 304-nt ORF2 sequences from 49 isolates, including 13 porcine isolates from Brazil, 4 human isolates from Brazil, and the 3 swine isolates described in this study, Brazil79sw, Brazil49sw and Brazil147sw (highlighted in red) The GenBank accession number in parentheses, the name of the country of origin, the species from which it was isolated, and the genotype/subtype of the isolate identify each viral strain Bootstrap values of >50 are indicated for the major nodes as a percentage of the data obtained from 1000 replicates (bar, 0.02 substitutions per site) Major branches indicate genotypes Avian HEV is the outgroup.
Trang 6HEV-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
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.7Thehigher
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.5Theseresultsarealsoinagreementwithastudy performedinCentralBrazilinswineaged20–30weeks,with 81%ofanti-HEVIgGpositivity.29
Theingestionofraworundercookedporkhasbeen asso-ciatedwithHEVinfection,30,31 andaprobablezoonoticHEV transmissionhasbeenreportedinBrazil.15Additionally,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
inrenaltransplantrecipientsinBrazil16and83–97%homology withswineHEVsequencesfromBrazil.5,9Amongallcompared 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
Theauthorsdeclarenoconflictsofinterest
Trang 7Acknowledgements
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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