Caliciviruses in hospitalized children, São Luís, Maranhão, 1997-1999: detection of norovirus GII.12

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

Genetics

and

Molecular

Microbiology

Caliciviruses

in

hospitalized

children,

São

Luís,

Maranhão,

1997–1999:

detection

of

norovirus

GII.12

Thayara

Morais

Portal

_,

_Jones

_Anderson

_Monteiro

_Siqueira

_,

Larissa

Cristina

Prado

das

Neves

Costa

_,

_Ian

_Carlos

_Gomes

_de

_Lima

_,

Maria

Silvia

Sousa

de

Lucena

_,

_Renato

_da

_Silva

_Bandeira

_,

Alexandre

da

Costa

Linhares

_,

_Claudia

_Regina

_Nunes

_Eloi

_da

_Luz

_,

Yvone

Benchimol

Gabbay

_,

_Hugo

_Reis

_Resque

a_{ProgramadePós-Graduac¸ãoemBiologiaParasitárianaAmazônia,UniversidadedoEstadodoPará,Belém,Pará,Brazil} b_{ProgramadePós-Graduac¸ãoemVirologia,InstitutoEvandroChagas/SVS/MS,Ananindeua,Pará,Brazil}

c_{FaculdadeIntegradaBrasilAmazônia,Belém,Pará,Brazil}

d_{Sec¸ãodeVirologia,InstitutoEvandroChagas/SVS/MS,Ananindeua,Pará,Brazil} e_{UniversidadeFederaldoMaranhão,SãoLuís,Maranhão,Brazil}

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Articlehistory:

Received17April2015 Accepted4January2016 Availableonline22April2016 AssociateEditor:MaurícioLacerda Nogueira Keywords: Norovirus Sapovirus Gastroenteritis Children

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Gastroenteritisisoneofthemostcommondiseasesduringchildhood,withnorovirus(NoV) andsapovirus(SaV)beingtwoofitsmaincauses.Thisstudyreportsforthefirsttimethe incidenceofthesevirusesinhospitalizedchildrenwithandwithoutgastroenteritisinSão Luís,Maranhão.Atotalof136fecalsamplesweretestedbyenzymeimmunoassays(EIA)for thedetectionofNoVandbyreversetranscription-polymerasechainreaction(RT-PCR)for detectionofbothNoVandSaV.Positivesamplesforbothagentsweresubjectedto sequenc-ing.TheoverallfrequencyofNoVasdetectedbyEIAandRT-PCRwas17.6%(24/136)and 32.6%(15/46),respectivelyindiarrheicpatientsand10.0%(9/90)innon-diarrheicpatients (p<0.01).Ofthediarrheicpatients,17%hadfever,vomitingandanorexia,and13% devel-opedfever,vomitingandabdominalpain.Ofthe24NoV-positivesamples,50%(12/24)were sequencedandclassifiedasgenotypesGII.3(n=1),GII.4(6),GII.5(1), GII.7(2),GII.12 (1) andGII.16(1).SaVfrequencywas9.8%(11/112),with22.6%(7/31)indiarrheicpatientsand 4.9%(4/81)innondiarrheic(p=0.04)ones.Indiarrheiccases,27.3%hadfever,vomitingand anorexia,whereas18.2%hadfever,anorexiaandabdominalpain.OneSaV-positivesample wassequencedandclassifiedasGII.1.TheseresultsshowahighgeneticdiversityofNoV andhigherprevalenceofNoVcomparedtoSaV.OurdatahighlighttheimportanceofNoV andSaVasenteropathogensinSãoLuís,Maranhão.

©2016SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/

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

∗ _{Correspondingauthor.}

E-mail:hugoresque@iec.pa.gov.br(H.R.Resque).

http://dx.doi.org/10.1016/j.bjm.2016.04.008

1517-8382/©2016SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

Acutegastroenteritis(AG)isconsideredtobethesecond lead-ingcauseofdeathduetoinfection1_{mainlyinchildrenunder} fiveyearsold,leadingtonearly760,000deathseachyear,with 75%ofepisodescausedbyviruses.2–4

Amongthemaincausesofviralgastroenteritis,two mem-bersoftheCaliciviridaefamilyareepidemiologicallyrelevant: norovirus(NoV)andsapovirus(SaV).TherelevanceofNoVas amajorcauseofAGoutbreakshasalreadybeenestablished,5 aswellasitsroleinepisodesofdiarrheathatrequire hospital-izationorvisitstooutpatientclinics.6_{SaVarepredominantly} related to sporadic episodes and outbreaks of diarrhea in indoorsenvironments,suchasdaycarecenters,andarealso associatedwithAGatdifferentages.7

NoVandSaVarenon-envelopedviruseswithdiametersof 27–40nm,andbotharecomposedofasingle-strandedRNA genome.TheNoVisclassifiedintosixgenogroups,with sev-eralgenotypesinthe GI,GIIandGIVgenogroupsknown to causeAGinhumans.8,9_{GII.4iscurrentlyregardedasthemost} frequentgenotype,beingrelatedtoapproximately70%of out-breaks,andtheglobalepidemicsthathaveoccurredsince1995 havebeencausedbysevenGII.4variants.9–11_{TheSaVgenusis} dividedintofivegenogroups,fourofwhichcaninfecthumans (GI-II,GIVandGV),withGIdescribedasthemostcommon.8,12 Thetransmissionofbothvirusesoccursprimarilybythe fecal-oralroutethroughdirectinterpersonalcontact,ingestionof contaminatedfoodandwaterandpossiblybyaerosolized par-ticlesfromvomiting.13_{ItisbelievedthatforNoV,freshfoods} suchasfruitsarethosemostlikelytobecontaminated,and thisprobablyoccursduringirrigation.6

Casesofasymptomaticinfection vary from 12% to30%, whichmayrepresentatransmissionfacilitator.14,15_Theperiod ofincubationofthesepathogensrangesfrom24to48h,with symptomspersistingfrom12to72h.Theclinical manifesta-tionsvaryfromseveretomoderate,mainlyinNoVcases,with diarrhea,vomiting,nausea,andfeverthatsometimesleadto dehydration,particularlyinchildrenandthe elderly,which mayleadtofataloutcomes.16

Researchconductedinhospitals,clinicsandcommunities inseverallocationsinBrazilshowedNoVoccurrenceranging from8.6%to39.7%.15,17–20 _{However,studiesinvolvingthese} virusesarescarceinthenorthandnortheastregions.

Inthisstudy,wesoughttodeterminetheprevalenceofand partiallycharacterizeNoVandSaVinhospitalizedchildren withorwithout diarrheainthecityofSãoLuís, Maranhão, Brazil,duringtheperiodfrom1997to1999.

Materials

and

methods

Locationofstudyandpatients

Atotalof250stoolsampleswereobtainedfromchildrenless thanthreeyearsoldwhowerehospitalizedwithdiarrheaor othersymptomsattheMaternoInfantilUnitatthe Univer-sitaryHospital(HUMI)oftheFederalUniversityofMaranhão (UFMA)fromJune1997toJune1999.Previously,these sam-plesweretestedforrotavirus(RV)21 _{andastrovirus(AstV),}22

andonlythesampleswithnegativeresultsforbothviruses were usedinthe presentstudy.Allsampleswere storedat −20◦_{C,and136specimens(46diarrheicand90without}

diar-rhea)wereselectedforthisresearch.Thisstudywasapproved bytheEvandroChagasInstituteEthicalCommitteeunderthe number284.852.

Fecalsuspensionsandenzymeimmunoassay(EIA)

Fecal suspensions were prepared at a dilution of 10% (weight/volume) and were tested for the presence of NoV usingacommercialenzymeimmunoassay(EIA)(3rd gener-ation RIDASCREEN® _{NorovirusEIA,R-Biopharm,Darmstadt,}

Germany)accordingtothemanufacturer’srecommendations.

ViralRNAextractionandreversetranscription(RT)

Avolumeof300␮loffecalsuspensionwasusedfornucleic acidextractionbythesilicamethod23_{withafinalvolumeof} 45␮lthatwasstoredat−20◦_{Cuntiluse.Avolumeof6␮lof}

RNAwasusedtoobtainthecomplementaryDNA(cDNA)by reversetranscription(RT)usingarandomprimer.

Moleculardetection

Thespecimenswerescreenedbypolymerasechainreaction (PCR)withtheprimersMon431/Mon433andMon432/Mon434, whicharespecificfortheNoVRNApolymerasesequencesof genogroupsIandII,respectively.24_{Samplesthattested} neg-ativewiththispoolofprimerswerealsotestedwithprimers P289/P290forthedetectionofSaV.25

AllNoV-positivesamplesweresubjectedtotwootherPCR analyses:thefirstoneamplifiedpartoftheviralcapsidregion ofNoV (Cap A, B2,B1 and CapC,D3, D1)26 _{and theother} amplifiedtheB(polymerase)andC(capsid)junctionregions (primersG2SKRandMon/432).27

PurificationandsequencingofRT-PCRcalicivirus amplicons

Thesamplessubjectedtonucleotidesequencingwerepurified withtheQIAquick®_{PCRPurification(Qiagen)andQIAquick}®

GelExtraction(Qiagen)commercialkitsaccordingtothe man-ufacturer’s instructions and sequenced using the Big Dye Terminator kit (v.3.1)(Applied Biosystems) inanautomatic sequencer. Theanalysisand editingofthe chromatograms were performed by the BioEdit Sequence Alignment Editor (v.7.0)programandcomparedtoothersequencesstoredin GenBankwiththeBLASTprogram.Phylogeneticgroupingwas constructedusingmaximumlikelihoodanalysis.The evolu-tionarymodelselectedforthepolymerasetreewasTPM3+G4 andforthecapsidtreewastheTIM2e+G4.Forbothtrees, test-ing was performedwith 1000bootstrapreplicates, and the cut-offvaluewas70%.

Statisticalanalysis

Statistical analysis was performed using BioEstat 5.0 software.28_{Theoddsratiotestwasusedtocorrelateinfection} withageranges.Thechi-squaretestandFisher’sexacttest

Table1–Detectionofnorovirusandsapovirusinfecalsamplescollectedfromhospitalizedchildrenwithandwithout diarrheafromSãoLuís,MaranhãofromJune1997toJune1999.

Samples NoV SaV

POS(%) NEG(%) POS(%) NEG(%)

Diarrheal 15(32.6) 31(67.4) 7(22.6) 24(77.4)

Non-diarrheal 9(10) 81(90) 4(4.9) 77(95.1)

Total 24(17.6) 112(82.4) 11(9.8) 101(90.2)

Table2–Distributionofnorovirusgenotypesdetectedinfecalsamplescollectedfromhospitalizedchildrenwithand withoutdiarrheainSãoLuís,MaranhãofromJune1997toJune1999.

Samples Nucleotidesequencing Total

OnlyregionB–RdRp OnlyregionD–viralcapsid Posbothregions Junctionregion

Diarrheal GII.P4(2) GII.P7 GII.12 GII.3 GII.16 GII.4 GII.5 8 Non-diarrheal GII.P4 GII.P7 GII.4 GII.4 4 Total 5 1 4 2 12

were used to determine whether the results for the two groups(NoV and SaV) were statisticallysignificant. Simple regressionanalysiswasusedtorelateeachsymptomtoNoV andSaVinfections.

Results

The NoV frequency was 17.6% (24/136), with 75% (18/24) detectedonlybyPCRand25%(6/24)detectedbybothPCRand EIE.ForSaV,thefrequencywas9.8%(11/112).Ofthetwogroups studied(i.e.,withandwithoutdiarrhea),thefirstdisplayeda higherincidenceofinfection forbothviruses:NoV(32.6%–

p<0.012)andSaV(22.6%–p<0.04)(Table1).

Ofthe24NoV-positivesamples,50%(12/24)wereusedto performnucleotidesequencing,with66.7%(8/12)from diar-rheicpatientsand33.3%(4/12)fromthenon-diarrheicgroup. Inthediarrheicgroup,threesampleswerecharacterized asgenotypeGII.4andfiveasgenotypesGII.3,GII.5,GII.7,GII.12 andGII.16(oneeach).Inthenon-diarrheicgroup,twosamples werecharacterizedasGII.4andtwoasGII.7.

Ofthe12samplescharacterized,fourweresequencedwith primerstargetingtworegions:regionB(RdRp)andregionD (viralcapsid),withthegenotypesconfirmedinbothregionsas GII.3andGII.16andtwoasGII.4.Twosamplesweresequenced usingthe junctionregion and wereclassified asgenotypes GII.4andGII.5.Fiveothersampleswerecharacterizedbythe

RdRpregionB,withthreeclassifiedasGII.P4andtwoasGII.P7. OnesamplewascharacterizedasGII.12byregionDoftheviral capsid(Table2andFigs.1and2).

NopositivesampleswerecharacterizedasNoVgenogroup I.OnediarrheicsamplewassequencedforSaVandclassified asGII.1.

ThehighestNoVfrequencyaccordingtoagegroupswas foundbetween6and12monthsofage(OD=1.7%,p<0.2)and forSaVfrequency,between 18monthstotwoyears ofage

(Table3).

Inadditiontodiarrhea,theotherfourclinicalsymptoms (fever,vomiting,anorexiaandabdominalpain)inthe symp-tomaticpatientswereanalyzed.Feverwastheonlysymptom that showed a statistically significant association (p<0.04). OfthepositivecasesforNoV,28.6%hadfever,vomitingand anorexia,and21.6%hadfever,vomitingandabdominalpain. OfthecasespositiveforSaV,42.8%wereassociatedwithfever, vomitingand anorexia,and 28.6%withfever,anorexia and abdominalpain.TheanalysisofthesymptomsofNoV-and SaV-positivecasesisshowninTable4.

Discussion

Currently,studiesrelatedtothecirculationofthesepathogens innortheasternBrazilarescarce,andthisisthefirststudyto

Table3–Frequencybyageofpositivecasesfornorovirusandsapovirusinhospitalizedchildrenwithandwithoutacute gastroenteritis,June1997toJune1999inSãoLuís,Maranhão.

Age Diarrheicpatients Non-diarrheicpatients Total

NoV+/total(%) SaV+/total(%) NoV+/total(%) SaV+/total(%)

0<6 1/5(20) 1/5(20) 1/12(8.3) 2/12(16.6) 5/17(29.4) 6<12 11/23(47.9) 1/23(4.5) 5/47(10.7) 0/47(0) 17/70(24.3) 12<18 3/13(23.1) 2/13(15.5) 0/18(0) 2/18(11.1) 7/31(22.6) 18<24 0/5(0) 3/5(60) 1/7(14.2) 0/7(0) 4/12(33.3) Nospecified – – 2/6(33.3) 0/6(0) 2/6(33.3) Total 15/46(32.6) 7/46(15.2) 9/90(10) 4/90(4.4) 35/136(25.7)

Fig.1–Phylogenetictreebasedonthepolymerasesequenceofnorovirus.Thestudysamplesaremarkedinbold,andthe

treewasconstructedusingmaximumlikelihoodanalysis.Theevolutionarymodelselectedforthepolymerasetreewas

TPM3+G4.Thetestwasperformedwith1000bootstrapreplicates,andthecut-offvaluewas70%.

Table4–Analysisoftheclinicalsymptomspresentedby childrenhospitalizedwithacutegastroenteritispositive fornorovirusandsapovirusinSãoLuís,Maranhão,from June1997toJune1999.

Symptoms Positivecases

NoV(%) SaV(%) Vomit 1(7.1) – Vomit/anorexia 1(7.1) – Vomit/anorexia/abdominalpain 1(7.1) – Vomit/fever/anorexia 4(28.6) 3(42.8) Vomit/fever/abdominalpain 3(21.6) – Vomit/fever/anorexia/abdominalpain 2(14.2) 1(14.3) Fever/anorexia 1(7.1) 1(14.3) Fever/anorexia/abdominalpain 1(7.1) 2(28.6) Total 14 7

Inthenon-diarrheiccases,ninesampleswerepositiveforNoVand fourtoSaV.

investigatetheprevalenceofthesevirusesinSãoLuís,state ofMaranhão.

In the present study, the prevalence of NoV infection (17.6%)in casesof hospitalized childrenwith and without gastrointestinal infection washigher than that detectedin childrenlessthanfiveyearsoldinthestateofAcre(north)

(12.7%),29_{similartotheresultsfoundinanAfrican} descend-antcommunityinPará(north)(19.7%)30_{andlowerthanthat} detectedinNiterói(southeast) (30.3%),15 _wheremost infec-tionsbyNoVwererecordedinchildrenlessthantwoyearsof age.ForSaV,theincidencewashigher(11%)thanthatdetected intheUnitedStates(5%)31_{andinItaly(3%).}32

Ofthetotalsamplesanalyzed,thehighestprevalencewas observedamongdiarrheicpatients,whichwassignificantfor bothNoV(32.6%–p<0.012)andSaV(22.6%–p<0.04).These findingscorroboratetheresultsobtainedforNoVinastudy conductedinSãoPaulo(southeast)(36.2%)17_{andalsoinBelém} (north)(35.4%),33 _{bothreportingincreasedfrequencyofthis} pathogen insymptomaticpatients. WithregardtoSaV, the prevalence detected inthe present study forsymptomatic patientswaslessthanthatobservedinIndia(39%).34 Detec-tion of asymptomatic casesemphasizes thateven without clinicalsigns,viraldispersionmaybetakingplace,whichis anothersourceoftransmissionofthispathogen.

Inthepresentstudy,NoVwasdeterminedbythreedifferent setsofprimers,onespecificfortheRNApolymerase(regionB, ORF1),anotherspecificforthecapsid(regionD,ORF2)andthe thirdspecificforthejunctionregion(regionB,ORF1andregion C,ORF2).Nucleotidesequencesallowedclassificationofthe sixsamplesasgenotypeGII.4.Accordingtotheliterature, cur-rently,strainsbelongingtothisgenogroupareshowinghigher

Fig.2–Phylogenetictreebasedonthecapsidregionofnorovirus.Thestudysamplesaremarkedinbold,andthetreewas

constructedusingmaximumlikelihoodanalysis.TheevolutionarymodelselectedforthepolymerasetreewasTIM2e+G4,

andthetestwasperformedwith1000bootstrapreplicates,andthecut-offvaluewas70%.

frequenciesincasesofacutegastroenteritisaroundtheworld, accountingforupto80%ofoutbreaksworldwideandleading totheemergenceofnewvariantseverytwoorthreeyears.35–37 TwosamplesweregroupedasgenotypeGII.12andGII.16. GII.12hasbeenassociatedwithoutbreaksintheyears2009 and2010,with16%ofgastroenteritisintheUnitedStates38 causedbyNoVandwasfirstdescribedinBrazilin2009, simul-taneouslywithGII.16.20 _{Ourstudydemonstratesthat these} twostrainswerealreadycirculatinginSãoLuís,Maranhãoin theyears1997and1999,respectively.

We also detected the genotypes GII.3 and GII.7. GII.3 strainsareusuallyassociatedwithsporadiccasesofinfection amongbreastfeedinginfants,especiallythoseyoungerthan sixmonths old,besidesbeing reported together with pan-demicstrainsofGII.4infoodborneoutbreaks.39_Allsamples showednegativeresultsforNoVGI.Moststudieshave demon-stratedhigherprevalenceofNoVGII,whichmayberelatedto theirbroadgeneticdiversity.40

TheGII.4genotypewaspredominant,inaccordancewith other studies that reported a higher prevalence of this

genotype;however,fiveothertypeswerealsoobserved dur-ingtheperiodof1997–1999,showinggeneticdiversityamong strainscirculatinginthispopulation.Thisfactcouldinfluence thefuturedevelopmentandimplementationofapossible vac-cineagainstNoV.

OneSaVsamplewassequencedandclassifiedasGII.1.This genotypewasalsodetectedinthestoolsampleofachildwith AG inastudy carriedout inSalvador,Brazil,in200241 _and describedinthemetropolitanregionofBelém,beingpresent intenofthefifteen(66.7%)samplescharacterized.42

TheagegroupmostaffectedbyNoVwaschildrenbetween sixmonthsandoneyearofage(47.9%).Thisprevalencewas higherthanthatfoundinPeru(9%)43_{andreinforcesthedata} foundinBurkinaFaso44_andinBelém,45_{where36%and45.1%,} respectively,ofchildrenatthisagewereinfectedwithNoV, whilemuchlowerfrequencieswerefoundinthosethatwere olderthantwoyears.SaVwasmoreprevalentamongchildren eighteenmonthstotwoyearsold.Theseresultsdifferfrom thosedescribedinJapan,whereSaVwasassociatedwith chil-dren36monthsofage(91%).46_{However,itisimportanttotake}

intoaccountthelimited numberofcasesanalyzedineach groupinthepresentstudy.

Consideringdiarrheaastheinclusion criteria,thisstudy examinedtheprevalenceofotherclinicalmanifestationsthat werepresentinthemajorityofpositivecases,suchas vomi-ting,anorexiaandfever.Thesesymptomsmayberelatedto theclinicalconditioncausedbyNoVandSaV,asdemonstrated byNordgrenetal.,44_{whofoundanassociationwithanorexia} (62%),fever(49%)andvomiting(49%)amongthepositivecases forNoV.Inadditiontothat,distress wasdescribedinsome positivecases.

Onelimitationofthepresentstudyisthatweonlytested samplesnegativeforRVandHAstV,eventhoughco-infection involving enteric viruses is relatively common in cases of acutegastroenteritis assuggestedby somereportson this matter.18,47,48_{InBrazil,Victoriaetal.}49_reportedin hospital-izedchildrenfromRiodeJaneiroarateof4%co-infectionby RVandNoV.

Althoughthisstudywasconductedwithsamplescollected in1997–1999,ourdatamaybeusefulasareferenceforpossible future molecular epidemiological studies (including evolu-tionaryanalyses)onNoVandSaVinfectionsinBrazil.

Conclusion

ThepresenceofNoVandSaVinhospitalizedchildreninSão Luís,Maranhão,reinforcesthatthisagegroupissusceptible toinfection bytheseagents,inlinewithgeneralreportsin theliterature.Thehighernumberofpositivecasesin symp-tomaticpatientswasexpected,andourresultsalsoemphasize thediversityofgenotypesfoundinthesamples.Theseresults demonstratethecirculationofNoVandSaVandindicatethat morestudiesarenecessary,consideringthatepidemiological informationaboutthesevirusesinSãoLuis,Maranhãoisstill limited.

Conflicts

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interest

Theauthorsdeclarenoconflictsofinterest.

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