Genotypes of Epstein–Barr virus (EBV1/EBV2) in individuals with infectious mononucleosis in the metropolitan area of Belém, Brazil, between 2005 and 2016

brazjinfectdis2020;24(4):322–329

w w w . e l s e v ie r . c o m / l o c a t e / b j i d

The

Brazilian

Journal

of

INFECTIOUS

DISEASES

Original

article

Genotypes

of

Epstein–Barr

virus

(EBV1/EBV2)

in

individuals

with

infectious

mononucleosis

in

the

metropolitan

area

of

Belém,

Brazil,

between

2005

and

2016

Talita

Antonia

Furtado

Monteiro

_,

_Iran

_Barros

_Costa

_,

_Igor

_Brasil

_Costa

_,

Thais

Letícia

dos

Santos

Corrêa

_,

_Beatriz

_Monteiro

_Rodrigues

_Coelho

_,

Amanda

Emanuelle

Santos

Silva

_,

_Francisco

_Lúzio

_de

_Paula

_Ramos

_,

Arnaldo

Jorge

Martins

Filho

_,

_José

_Luiz

_Furtado

_Monteiro

_,

Jones

Anderson

Monteiro

Siqueira

_,

_Yvone

_Benchimol

_Gabbay

_,

Rita

Catarina

Medeiros

Sousa

a_{MinistériodaSaúde,SecretariadeVigilânciaemSaúde,InstitutoEvandroChagas,Ananindeua,PA,Brazil} b_{InstitutoEvandroChagas,LaboratóriodeRotavírus,Ananindeua,PA,Brazil}

c_{UniversidadeEstadualdoPará,ResidênciaMédica(UEPA),Belém,PA,Brazil} d_{UniversidadeFederaldoPará,NúcleodeMedicinaTropical,Belém,PA,Brazil}

e_{InstitutoEvandroChagas,Pós-graduac¸ãoemVirologia,(PPGV/IEC),Ananindeua,PA,Brazil}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received10March2020 Accepted6June2020 Availableonline30June2020

Keywords: EBNA3Cgene EBV1 EBV2 EBV1+EBV2 Infectiousmononucleosis Northernregion Brazil

a

b

s

t

r

a

c

t

TwotypesofEpsteinBarrvirus(EBV1/EBV2)havebeenshowntoinfecthumans.Although theirgenomesaresimilar,theregionscontainingtheEBNAgenesdiffer.Thisstudyaimedto characterizetheEBVgenotypesofinfectiousmononucleosis(IM)casesinthemetropolitan regionofBelém,Brazil, from2005to2016. Atotalof8295suspectedcaseswith symp-toms/signsofIMwereinvestigatedbyinfectiousdiseasephysiciansatEvandroChagas Institute,HealthCareService,fromJanuary2005toDecember2016.Outofthetotal,1645 (19.8%)sampleshadpositiveresultsforEBVbyenzymeimmunoassayand251(15.3%)were submittedtopolymerasechainreaction(PCR)technique,usingtheEBNA3Cregion,inorder todeterminethetypeofEBV.Biochemicaltestinginvolvingaspartateaminotransferase, ala-nineaminotransferaseandgamma-glutamyltransferasewerealsoperformed.EBVtypewas identifiedbyPCRin30.3%(76/251)ofindividuals;ofthose,71.1%(54/76)wereclassifiedas EBV1,17.1%(13/76)asEBV2,and11.8%(9/76)asEBV1+EBV2.Themainsymptoms/signs observed with EBV1 infection were cervical lymphadenopathy (64.8%, 35/54), fever

Abbreviations:EBV,Epstein–Barrvirus;LCLs,lymphoblasticcelllines;AST,aspartateaminotransferase;ALT,alanineaminotransferase; GGT,gamma-glutamyltransferase;IM,infectiousmononucleosis.

∗ _{Correspondingauthor.}

E-mailaddress:talitamonteiro@iec.gov.br(T.A.Monteiro). https://doi.org/10.1016/j.bjid.2020.06.004

1413-8670/©2020SociedadeBrasileiradeInfectologia.PublishedbyElsevierEspa ˜na,S.L.U.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

brazj infect dis.2020;24(4):322–329

323

(63%,34/54),headache(20.4%,11/54),arthralgia(20.4%,11/54),andexanthema(18.5%,10/54). EBV2infectionwasdetectedinallbuttwoagegroups,withanaverageageof24years.The mostcommonsigns/symptomsofEBV2werefever(76.9%,10/13),averagedurationof18 days,andlymphadenopathy(69.2%,9/13).Incontrast,EBV1+EBV2coinfectionsweremore frequentinthoseagedfiveyearsorless(20.0%,2/10).ThesymptomsofEBV1+EBV2 coin-fectionincludedfever(66.7%,6/9),andcervicallymphadenopathyandheadache(33.3%,3/9) each.ThemeanvaluesofhepaticenzymesaccordingtotypeofEBVwassignificantly differ-ent(p<0.05)inthoseEBV1infectedover14yearsofage.Thus,thispioneeringstudy,using molecularmethods,identifiedtheEBVgenotypesin30.3%ofthesamples,withcirculation ofEBV1,EBV2,andEBV1+EBV2co-infectionincasesofinfectiousmononucleosisinthe northernregionofBrazil.

©2020SociedadeBrasileiradeInfectologia.PublishedbyElsevierEspa ˜na,S.L.U.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/ licenses/by-nc-nd/4.0/).

Background

Epstein Barr virus (EBV)belongs tothe order Herpesvirales,

family Herpesviridae, subfamily Gammaherpesvirinae, genus

Lymphocryptovirus and species Human gammaherpesvirus 4.1 EBVwasthefirstoncogenicvirusshowntoinfecthumans.2,3 Thehexagonalnucleocapsidviralparticles areformedwith linear, double-stranded, enveloped DNA, with a diameter rangingfrom180to200nm.4

Althoughsymptomaticinfectionswiththesevirusescan occurbenignly,EBVhasbeenimplicatedinthegenesisofa varietyoflymphoproliferativedisorders and severe epithe-lial neoplasms, such as African Burkitt’s lymphoma5 _and nasopharyngealcarcinoma6_.

InBrazil,severalstudieshavedocumentedhighlevelsof antibodiesto EBVinthe studiedpopulations. Investigation conducted by Monteiro et al.7 _{found that at least 70% of} serumsamplesanalyzedinthecityofBelém,stateofPará, containedIgGantibodiestoEBV,withratesinoutpatient clin-icsranging from 53.8% to95.6% and at communitiesfrom 81.1% to 100%. Positivity rates were high evenin younger agegroupswith10.6%(25/234)ofchildrenandadolescentsin northernBrazilshowingactiveandrecentinfection(infectious mononucleosis).8

DatafromYoungandMurray9 _{demonstratedthatEBVis} presentinapproximately90%ofindividualsandiscontrolled bytheimmunesystem,mainlybycellularimmunity,which maymakethepersonmoresusceptibletovirusproliferation andmaytriggerlymphoproliferativedisorders.10

ThephenotypicdifferencebetweenEBV1andEBV2ismore evidentduringimmortalizationofBcellsinlymphoblastoid celllines(LCLs)byEBV1comparedtoEBV2.Thisfactreinforces thebiologicalandfunctionaldifferencebetweenthetwoviral types,whereBcellimmortalizationinvitrowasshowntobe moreeffectivebyEBV1.11 _{AccordingtoMandelletal.}12 _the differentiationofgenotypescanclarifythedifferentimmune responsesduringviralpersistence.

EpidemiologicalstudiesonEBV1andEBV2types character-izingclinical,demographic(sex,ageandorigin)andmolecular findingsinthemetropolitanregionofBelém,arestilllacking. Duetotheircharacteristicsintermsofviralpersistence,EBVs may inducechronicinfections and reactivations inhuman

populations with genetic competence, possibly leading to oncogenicoutcomes.

TheaimofthisstudywastodeterminethetypesofEBV (EBV1/EBV2)inclinicalcasesofIMinthemetropolitanareaof Belémbetween2005and2016.

Material

and

methods

Patients

This was a retrospective study involving serum samples collected from January 2005 to December 2016, from 8295 clinicallysuspectedindividuals ofhavingIM.Patientswere screened byinfectious diseases physiciansfrom the Evan-dro Chagas Institute, Ministry ofHealth ofBrazil (IEC/MS), ReferenceCenterfortheDiagnosisofInfectiousDiseasesin NorthernBraziltoidentifythosewithserological confirma-tion.

Laboratoryanalysis

Serumsampleswerecollected andtestedforEBVusingan enzymeimmuneassayaswellasforbiochemicalanalyses. Peripheralbloodmononuclearcells(PBMCs)wereseparatedby Ficoll-Hipaque densitygradientcentrifugation(Lymphoprep NycomedPharmaAS,Norway)foruseinfuturepolymerase chainreaction(PCR)tests.

Immunoenzymaticassay(EIA)

EBV screening in samples was carried out using the

RIDASCREEN® enzymeimmunoassaykit(R-Biopharm, Darm-stadt,Germany),whichdetectsIgGandIgMantibodiestoviral capsidantigen(VCA),accordingtothemanufacturer’s guide-lines.

Nucleicacidextraction

PBMCDNA was extractedusing the QIAampDNA MiniKit (Qiagen,Germantown,MD)accordingtothe manufacturer’s protocol.

324

Table1–DetectionoftheEBNA3CgenebyPCRin251patientswithinfectiousmononucleosisandanti-IgMpositivefor EBV,accordingtoage,sexandtype.Samplescollectedfrom2005to2016.

Age range

PCR/ELISA Pos(%)

SEX EBVtypes

Male/PosPCR(%) Female/PosPCR(%) EBV1PosPCR(%) EBV2/PosPCR(%) EBV1+2/PosPCR(%) ≤5 10/37(27.0) 4/18(22.2) 6/19(31.5) 8/10(80.0) 0/10(0.0) 2/10(20.0) >5–10 15/54(27.8) 9/29(31.0) 6/25(24.0) 10/15(66.6) 3/15(20.0) 2/15(13.3) >10–15 8/28(28.5) 6/17(35.2) 2/11(18.2) 2/8(25.0) 5/8(62.5) 1/8(12.5) >15–20 10/27(37.0) 4/13(30.7) 6/14(42.8) 8/10(80.0) 1/10(10.0) 1/10(10.0) >20–30 11/47(23.4) 3/13(23.7) 8/34(23.5) 11/11(100.0) 0/11(0.0) 0/11(0.0) >30 22/58(37.9) 8/21(38.1) 14/37(37.8) 15/22(68.1) 4/22(18.2) 3/22(13.6) Total 76/251(30.3) 34/111(30.6) 42/140(30.0) 54/76(71.1) 13/76(17.1) 9/76(11.8)

IdentificationoftheEBVEBNA3Cgene

TheprimerEBNA-3CwasusedtoidentifybothtypesofEBV,13 becausetheprimaryflankingregioncandifferentiatethem basedontheresultingproduct:EBV1with153basepairs(bp) andEBV2with246bp.

Five microliters of the eluted DNA was used for

PCR amplification with a primer concentration of 0.05␮M (EBNA3C1, forward: 5-GCCAGAGGTAAGTGGACTTT-3 and EBNA3C2,reverse:5-TGGAGAGGTCAGGTTACTTA-3).PCRwas performedin0.5mLmicrocentrifugetubesinafinalvolume of25␮Lofmixturecontaining0.125␮L(5U/␮L)ofPlatinum Taq DNA Polymerase (InvitrogenTM_{, Brazil), 1.5mM MgCl}

2

(InvitrogenTM_{, Brazil), 0.2mM dNTPs (Invitrogen}TM_{, Brazil),}

5␮Lof10Xbuffer(InvitrogenTM_{, Brazil)and2␮L(20␮M/␮L)}

ofeachtheabovementionedprimers.

The PCR cycle conditions (PTC 100/Peltier Effect Cycle, ThermostableController)wereasfollows:denaturationofthe DNAat94◦Cfor1min;40cyclesofdenaturationat94◦Cfor 30s, annealing at 58◦C for30s, and extensionat72◦C for 1min;andafinalextensionstepat72◦Cfor7min.ForallPCR analyses,waterwasusedasnegativecontrol,andB958and P3HR1celllineswereusedaspositivecontrolsforEBV1and EBV2,respectively.Theamplifiedproductsweresubjectedto 2%agarosegelelectrophoresisusingethidiumbromideand visualizedbyUVillumination.

Biochemicaldosagesanalyses

Biochemicaltestssuchasaspartateaminotransferase(AST; reference:4–40U/L),alanineaminotransferase(ALT;reference: 2–41U/L),andgamma-glutamyltransferase(GGT;reference: 5–55U/L)wereperformedonanautomatedclinical biochem-istryanalyzer(COBASINTEGRAclinicalPLUS400/ROCHE).

Statisticalanalysis

ResultswereorganizedandstoredinadatabaseinMicrosoft OfficeAccess2016,StatisticalPackageforSocialScience—SPSS 17.0.Ap-valuelessthan0.05wasconsideredsignificant.14

Ethicalconsiderations

This study was approved by the research ethics com-mittees of the Evandro Chagas Institute (CAAE number 65332717.2.0000.0019,withlegalopinionnumber2098453,of June4,2017).

Results

Serologicalanalysis

Out of8295serumsamplescollected andtestedforEBVby EIA, 19.8% (1645/8295) were positive for IgMantibodies.Of these samples,a subgroupof251 (15.3%)was usedin this researchandtestedforthedetectionofEBVbyPCR.IgG anti-body screeningwas alsocarried outin162(64.5%)ofthese positivesamplesandallofthemhadnegativeresults.

Moleculardetection

ThetypeofEBVwasidentifiedbyPCRin30.3%(76/251)ofthe IgMpositivesampleswiththeEBNA-3Cprimer.Theagegroups 15–20years and >30 yearshad slightly higherpositivityby PCR(37.0%,p=0.557)(Table1).PCRpositivityrateswere sim-ilaramongmales(30.6%,34/111)andfemales(30.0%,42/140). Moreover,themajorityofPCRpositivecasesweremaleaged >30(38.1%)years;however,amongfemalesthepositivityrate washigherintheagegroup15–20(42.8%)years.

ClassificationofthepositivesamplesbyEBVtypeshowed that71.1%(54/76)wereEBV1,17.1%(13/76)EBV2,and11.8% (9/76) EBV1+EBV2 coinfection; 76.3% (58/76) PCR positive patients camefrom thecities ofBelém,22.4% (17/76)from Ananindeua,and 1.3%(1/76) fromMarituba. Multiple signs andsymptomswereobservedinthesecases(Table2), includ-ingfeverin65.8%(50/76),cervicallymphadenomegalyin61.8% (47/76),exanthemain13.1%(10/76),arthralgiain17.1%(14/76), andheadachein21.0%(16/76).

Regarding the number of days with fever, 22% (11/50) reportedfeverforuptofivedays,16%(8/50)for10days,20% (10/50)for15days,16%(8/50)for20days,and16%(8/50)for morethan20days,while10%(5/50)hadnosuchinformation intheirrecord.

EBV1wasdetectedinallagegroups.Ontheotherhand, EBV2 was verifiedat alower rate or absent, exceptin the agegroup10–15years,whenitpresentedamuchhigherrate (62.5%)than EBV1(25.0%).Withtheexception ofthegroup 20–30 years,all other age groups had atleast one caseof coinfection(Table1).EBV1accountedfor79.1%(34/43)of indi-viduals over15yearsold,withanaverageageof22.6years; 61.1%(33/54)ofwomenwereinfectedbyEBV1,whereas38.9% (21/54)weredetectedinmen.Additionally,75.9%(41/54)EBV1 infected individuals came from Belém, 22.2% (12/54) from Ananindeua,and1.9%(1/54)Marituba.

b r a z j i n f e c t d i s . 2 0 2 0; 2 4(4) :322–329

325

Table2–Mainsymptomsobservedin76EBV-infectedcases,byageandtype,whoprovidedsamplescollectedfrom2005to2016.

Age Signsandsymptoms/EBVsubtypes

Fever(65.8%—50/76) Cervicallymphadeno*(61.8%—47/76) Exanthema(13.1%—10/76) Arthralgia(17.1%—14/76) Headache(21.0%—16/76) EBV1(%) EBV2(%) EBV1+2(%) EBV1(%) EBV2(%) EBV1+2(%) EBV1(%) EBV2(%) EBV1+2(%) EBV1(%) EBV2(%) EBV1+2(%) EBV1(%) EBV2(%) EBV1+2(%)

≤5 4/7 – 1/2 6/7 – 1/2 0/7 – 0/2 0/7 – 0/2 0/7 – 0/2 57.1 50.0 85.7 50.0 – – – – – – 6–10 9/10 3/3 1/2 7/10 3/3 2/2 0/10 0/30 0/2 2/10 0/3 0/2 2/10 0/3 0/2 90.0 100 50.0 70.0 100 100 – – – 20.0 – – 20.0 – – 11–15 1/2 3/5 1/1 2/2 4/5 0/1 1/2 0/5 0/1 1/2 0/5 1/1 0/2 0/5 1/1 50.0 60.0 100 100 80.0 – 50.0 – – 50.0 – 100 – – 100 16–20 5/8 1/1 1/1 5/8 1/1 0/1 2/8 0/1 0/1 2/8 0/1 1/1 2/8 1/1 1/1 62.8 100 100 62.8 100 – 25.0 – – 25.0 – 100 25.0 100 100 ≥20 15/27 3/4 2/3 15/27 1/4 0/3 7/27 0/4 0/3 6/27 1/4 0/3 7/27 1/4 1/3 60.0 75.0 66.6 60.0 25.0 – 28.0 – – 22.2 25.0 – 28.0 25.0 33.3 Total 34/54 10/13 6/9 35/54 9/13 3/9 10/54 0/13 0/9 11/54 1/13 2/9 11/54 2/13 3/9 63.0 76.9 66.7 64.8 69.2 33.3 18.5 0.0 0.0 20.4 7.7 22.2 20.4 15.4 33.3

326

Table3–Hepaticenzymes(AST,ALTandGGT)elevationsaccordingtoageandEBVtypes,in76PCRpositivesamplesusingtheEBNA3Cgene,collectedfrom2005to 2016.

Biochemical parameters (referencevalue)

Agegroup(mean±standarddeviations) Total/p-valor

2to5years 6to14years >14years

EBV1 EBV2 EBV1+2 EBV1 EBV2 EBV1+2 EBV1 EBV2 EBV1+2

+/Total(%) +/Total(%) +/Total(%) +/Total(%) +/Total(%) +/Total(%) +/Total(%) +/Total(%) +/Total(%)

Aspartateaminotransferase 1/7(14.2) – 0/2 0/11 1/7(14.2) 0/3 7/36(19.4) 0/6 0/4 9/76(11.8) AST(5–40U/L)1 _{(28.00±8.77) – (26.50±13.44) (25.55±11.11) (30.53±7.51) (27.66±2.27) (44.44±89.51) (24.94±7.16) (24.42±4.99) <0.05} Alanine aminotransferase 0/7 – 0/2 0/11 0/7 0/3 12/36(33.3) 0/6 0/4 12/76(15.8) ALT(2–41U/L)1 _{(17.29±7.16) – (16.50±3.54) (17.27±8.60) (20.57±2.88) (25.52±5.64) 48.61±93.87) (17.67±3.63) (24.17±5.17) <0.05} Gammaglutamyltransferase 0/7 – 0/2 1/11(9.1) 2/7(28,6) 0/3 15/36(41.7)b _{.1/6(16.6) 0/4 19/76(25.0)} GGTa(5–55U/L) (13.57±3.87)a _{– (28.86±14.33)}a _{(27.91±20.63)}a _{(41.71±17.74)}a _{(24.79±4.69)}a b_(12–43U/L)1 _{(77.28±137.66)}b _{(31.69±11.84)}b _{(11.00±7.07)}b _<0.05 Total 40/76(52.3)

brazj infect dis.2020;24(4):322–329

327

Table4–MainvariablesanalyzedofEBVtype1infected caseswhoprovidedthesamplescollectedfrom2005to 2016.

Variable Pvalue Oddsratio ICde95% Age>30y 0,7272 0,7750 0,19–3,24

Male 0,0137 0,1667 0,04–0,69

Female 0,0137 6,0003 1,44–24,95

Fever 0,2503 0,4219 0,10–1,84

Adenopathy 0,9524 0,9575 0,23–3,99

Regardingclinicalsymptomsorsignsobservedinpatients

infectedwithEBV1,the prevailing symptomswere cervical

lymphadenopathy in 64.8% (35/54), fever in 63.0% (34/54),

headacheandarthralgiain20.4%(11/54),andexanthemain 18.5%(10/54)(Table2)

EBV2infectionwasalsodetectedinallbuttwoagegroups groups,withameanageof24years.ThisEBVtypewasmore frequentamong males (76.9%, 10/13) than females (23.1%, 3/13)(Table1).

EBV1+EBV2co-infectedpatientspresentedwithfever, cer-vicallymphadenopathyandheadacheasthemostfrequent symptoms(66.7%;6/9)and33.3%(3/9),respectively(Table2). Coinfectionwasmorecommoninindividualsagedfiveyears orless(20.0%;2/10)(Table1).ThemeanageofEBV1+EBV2 coinfectedpatientswas 21years.Theremorecasesamong women(66.7%;6/9)thanamongmen(33.3%;3/9).

Regarding hepatic functiontests, overall 14.8% (8/54) of EBV1-infectedcaseshad elevatedASTlevels (normalrange 5−40U/L),whiletheagegroupof>14yearsabnormalvalues in19.4%(7/36)ofthecases.ForEBV2infection,7.7%(1/13)had elevatedASTcomparedto14.2%,(1/7)intheagegroupsixto 14years(Table3).

Concerning ALT, values above the reference range (2–41U/L) were significantly more common among EBV1-infectedcasesaged14yearsandabove(33.3%;12/36)thanin agegoups2–5and6–14years(Table3).

GGT was withinthe normalrange (5–55U/L) inthe age group2–5 years.GGTwaselevatedin9.1%(1/11)and28.6% (2/7)inEBV1-andEBV2-infectedcases,respectively,intheage group6–14years.Inthoseover14yearsofage,GGTwasabove theupperlimitofnormalityforthatage-range(12–43U/L)in 41.7%(15/36)and16.6%(1/6),respectively,ofEBV1-and EBV2-infectedcases(Table3).

Multiplelogisticregression(MLR)showedthatamong76 positivecasesfortheEBNA3Cgene,femalesexwassix-fold more likely to be infected with EBV subtype 1 (p=0.0137; OR=6.003),afteradjustingforageandclinicaldata(feverand adenopathy)(Table4).

Discussion

Primary EBV infection is usually asymptomatic and may progresstoabenignlymphoproliferativediseaseknownasIM, especiallyinlatechildhoodorearlyadulthoodofdeveloping countries.4

Infectious mononucleosis ischaracterized bysignificant clinicalpolymorphismsinwhichfactorssuchasage,immune status and comorbidities are associated with the clinical

outcome of the disease, which can vary from

asymp-tomatic infection to more severe conditions. This disease can be manifested by acute complications, such as mul-tiple organ failure, disseminated intravascular coagulation, ulcer/perforation of the digestive tract, coronary artery aneurysm, lymphomas and lymphohistiocytes, and EBV-associatedhemophagocytosis.15,16

Mendoza et al.17 _{confirmed that the incubation period} of the EBV infection ranges from 4 to 6weeks. Prodromal symptoms includeasthenia, anorexia, headache and chills oftenfollowedbythesignsandsymptomsofmononucleosis such as fever (up to 39–40◦C) with concomitant pharyn-gotonsillitis and lymphadenopathy.18,19 Alsoin the present studywith76EBV-infectedpatients,themainclinicalfindings werefever(65.8%;50/76),cervicallymphadenomegaly(61.8%; 47/76),arthralgia(17.1%;14/76),andheadache(21.0%;16/76).

TherearetwodifferenttypesofEBV,20_{namely,type1and} type2whicharerelatedtovariationsintheEBNA2andEBNA3 genesequences.13,21

Ourfindings demonstrated thatEBV1was themost fre-quenttype(82.9%,63/76)detectedbyPCRusingtheEBNA3C geneinIMcasesreportedinthemetropolitanregionofBelém, Pará,Brazil,where71.1%ofcases(54/76)hadEBV1aloneand 11.8%(9/76)associatedwithEBV2.

Itis worthmentioning that the present study,obtained insymptomaticpatients,isthefirstreportonEBVtypesin thisregion.AninvestigationconductedinChineseindividuals usingthesametechniqueshowedslightlylowerratesofEBV1 (76.3%,45/59).22Inthiscontext,otherstudiescarriedoutby Dengetal.(2014)23_{inJapanesepatientsandbySmattietal.} (2017)24_{inQatarhavealsodescribedEBV1withratesof73.3%} (107/146)and72.5%(37/51),respectively.

Our analyses, using multiple logistic regression model (MLR) showed that womenwere six-fold morelikely tobe infectedbyEBV1,incontrasttothefindingsofCorreaetal.25 Thisdetailcanbeexplainedbythefactthatwomenstaylonger in the household, where asymptomatic transmission can occurthroughsaliva,directoralcontactorsalivaryresidues leftinfood,glasses,toys,etc.Anotherpassiveformof trans-missioncanbethroughsexualcontact.

EBV2 was detected in28.9% (22/76)of the positive EBV cases, with 17.1% monoinfection and 11.8% (9/76) in coin-fections with EBV1(Table 1). The rate ofEBV2 was higher thanthosereportedbyDengetal.,23_{andCorreaetal.,}25 _in Japan(18.5%, 27/146)and Argentina(14.6%,29/199), respec-tively.Notably,EBV2positivityrateintheagegroupcomprising individualsaged10–15yearsoldwasmuchhigher(62.5%,5/8) than EBV1positivityrate(25.0%,2/8).However,thesecases weredetectedindifferentmonthsoftheyearsinwhichtesting wasconducted.

Inthisinvestigation weshowedthat EBV2infection had alongerclinicalcoursethanEBV1,withameandurationof 17.6days(range:1–90days)offeverforEBV2comparedto14.7 days(range:1–30days)forEBV1.TheintheEBV2-infected indi-vidualsaged10–15yearhadahighrate(62.5%)offeverand lymphadenopathy.EBV-2hasfrequentlybeenassociatedwith specificclinicalconditionsinpatientswithweakenedimmune systems, suchasHIV-infectedpersons withneoplastic dis-eases.Invitro,severalstudieshaverecordedalowfrequency ofEBV2inBlymphocytesdemonstratingareduced or less efficientcapacityforreplicationincellcultures.13

328

ThepresenceofcoinfectionsbyEBV1+EBV2wasobserved in11.8%oftheIgMpositivecasesofIM.Ofthose,20.0%(2/10) werechildrenlessthanfiveyearsold(Table1).Thisfinding emphasizes that coinfections are not limited to immuno-compromisedindividuals.Thisassociationwasalsoverified byCorreaetal.,25 _{in10.5%ofhealthy individualswho} par-ticipated in a study conducted in Argentina. Coinfection may result from simultaneous transmission ofboth geno-typesor bycontactwith twopeople infected withdistinct strains.

Anothernotablefindingofthepresentinvestigationwas theelevationsofhepatic enzymes(AST,ALT andGGT) lev-els.Theproportionofindividualswiththesealterationsvaried from19.4%to41.7%(Table3)amongthosewithEBV1intheage groupover14years.Thesesubjectsshowedincreasedlevels oftheseenzymes,consistentwiththefindingsofHerbinger etal.26

Smallelevationsinhepaticenzymescanoccurinnormal individualswithoutinfection (lessthantwicethereference value),whileinindividualswithEBV-infectivemononucleosis, thesevaluescanincreasefiveto10timestheupperlimitand mayevenincreasetolevelsindicatingfulminatehepatitis.27

Inaddition,inthis studythemeansofhepaticenzymes were significantlyhigherinEBV1-infected subjectsaged14 years and above (p<0.05) compared to EBV2-infected or EBV1+EBV2coinfectedsubjects.Similardatahavealsobeen reportedbyZhangetal.,28_{comparingIMcasesandcontrols,as} theyobservedhighlevelsofALT,ASTandGGTonlyincases ofIM,indicatingthathepaticenzymeslevelsshouldalsobe observedasariskalertinIMcausedbyEBVinfection.

Conclusion

In this pioneering study, conducted from 2005 to 2016, the predominance of EBV1, the presenceof EBV2 and the co-circulation of both types (EBV1+EBV2) were observed. Additionally, the relevant clinical signs/symptoms of lym-phadenopathyandfeverandexpressiveenzymaticchanges aredescribed.ThisknowledgeregardingthemainEBV geno-typeswithlocalcirculationcansupporttheformulation of new clinical approaches, especially in cases of infectious mononucleosisthatcanevolvewithaseverecourse. Determi-nationofthetypesofEBVinthepresentstudyhasenabled,for thefirsttime,theabilitytodistinguishthemolecular epidemi-ologyandthecirculationoftheseviralagentsinindividuals fromnorthernBrazil

Funding

Theauthorsreceivednospecificfundingforthiswork.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

Wearegratefultothosewhocontributedtothedevelopment ofthisstudy,especiallyAntoniodeMoura,AlessandraAlves PolaroandDr.ManuelGomes.

r

e

f

e

r

e

n

c

e

s

1.InternationalCommitteeonTaxonomyofViruses(ICTV). https://talk.ictvonline.org/ictv-reports/ictvonlinereport/, 2017.

2.RickinsonAB,KieffED.Epstein-Barrvirusanditsreplication. In:FieldsBN,KnipeDM,HowleyPM,editors.Fieldsvirology. Philadelphia:Lippincott,Williams&Wilkins;2007.p.2603–54. 3.JavierRT,ButelJS.Thehistoryoftumorvirology.CancerRes.

2008;68:7693–706.

4.RickinsonAB,KieffED.Epstein-Barrvirus.In:KnipeDM, HowleyPM,editors.Fieldsvirology.Philadelphia:Lippincott, WilliamsandWilkins;2007.p.2657–701.

5.HassanR,WhiteLR,StefanoffCG,etal.Epstein-Barrvirus (EBV)detectionandtypingbyPCR:acontributionto diagnosticscreeningofEBV-positiveBurkitt’slymphoma. DiagnPathol.2006;1:1–7.

6.WolfH,HausenHZ,BeckerV.EBviralgenomesinepithelial nasopharyngealcarcinomacells.NatNewBiol.

1973;244:245–7.

7.MonteiroTAF,GomesMLC,NoronhaVLC,etal.Prevalenciade anticorposparaovírusdeEpsteinBarr(EBV)emBelém,Pará, Brasil.RevistaParaensedeMedicina.1998;12:8–12.

8.MonteiroTAF,GomesMLC,GusmãoRHP,OliveiraCS,Freitas RB,NoronhaVLC.Mononucleoseinfecciosaemcrianc¸ase adolescentesdeBelém,Pará,Brasil:abordagemclínicae soroepidemiológica.RevistaParaensedeMedicina. 1998;12:34–44.

9.YoungLS,MurrayPG.Epstein–Barrvirusandoncogenesis: fromlatentgenestotumours.Oncogene.2003;22:5108–21. 10.FanH,GulleyML.Epstein-Barrviralloadmeasurementasa

markerofEBV-relateddisease.MolDiagn.2001;6:279–89. 11.FarrellPJ.Epstein-Barrvirusstrainvariation.CurrTop

MicrobiolImmunol.2015;390:45–69.

12.MandellGL,BennettJE,DolinR.PrinciplesandPracticeof InfectiousDisease.5thed.NewYork:ChurchillLivingstone; 2001.p.1364–77,v.2.

13.SampleJ,YoungL,MartinB,etal.Epstein-Barrvirustypes1 and2differintheirEBNA-3A,EBNA-3B,andEBNA-3Cgenes.J Virol.1990;64:4084–92.

14.SPSS—StatisticalPackagefortheSocialSciences. https://www.taylorfrancis.com/books/9780203180990,2017. 15.SonKH,ShinMY.ClinicalfeaturesofEpstein-Barr

virus-associatedinfectiousmononucleosisinhospitalized Koreanchildren.KoreanJPediatr.2011;54:409–13.

16.FujiwaraS,KimuraH,ImadomeK,etal.Currentresearchon chronicactiveEpstein-BarrvirusinfectioninJapan.Pediatr Int.2014;56:159–66,http://dx.doi.org/10.1111/ped.12314. 17.MendozaN,DiamantisM,AroraA,etal.Mucocutaneous

manifestationsofEpstein-Barrvirusinfection.AmJClin Dermatol.2008;9:295–305.

18.LuzuriagaK,SullivanJL.Infectiousmononucleosis.NEnglJ Med.2010;362:1993–2000.

19.TattevinP,LeTulzoY,MinjolleS,etal.Increasingincidenceof severeEpstein-Barrvirus-relatedinfectiousmononucleosis: surveillancestudy.JClinMicrobiol.2006;44:1873–4.

20.ChoY,GordadzeAV,LingPD,WangF.Evolutionoftwotypes ofrhesuslymphocryptovirussimilartotype1andtype2 Epstein-Barrvirus.JVirol.1999;73:9206–12.

brazj infect dis.2020;24(4):322–329

329

21.DambaughT,HennessyK,ChamnankitL,KieffE.U2regionof Epstein-BarrvirusDNAmayencodeEpstein-Barrnuclear antigen2.ProcNatlAcadSciUSA.1984;81:7632–6.

22.CuiY,WangY,LiuX,etal.GenotypicanalysisofEpstein-Barr virusisolatesassociatedwithnasopharyngealcarcinomain NorthernChina.Intervirology.2011;54:131–8.

23.DengZ,UeharaT,MaedaH,etal.Epstein-Barrvirusand humanpapillomavirusinfectionsandgenotypedistribution inheadandneckcancers.PLoSOne.2014;10:e0118439, http://dx.doi.org/10.1371/journal.pone.0118439.

24.SmattiMK,YassineHM,AbuOdehR,etal.Prevalenceand molecularprofilingofEpsteinBarrvirus(EBV)amonghealthy blooddonorsfromdifferentnationalitiesinQatar.PLoSOne. 2017;12:e0189033.

25.CorreaRM,FellnerMD,AlonioLV,DurandK,TeyssiéAR, PicconiMA.Epstein-barrvirus(EBV)inhealthycarriers:

distributionofgenotypesand30bpdeletioninlatent membraneprotein-1(LMP-1)oncogene.JMedVirol. 2004;73:583–8.

26.HerbingerKH,HanusI,FelbingerTW,etal.Elevatedvaluesof clinicallyrelevanttransferasesinducedbyimported

infectiousdiseases:acontrolledcross-sectionalstudyof 14,559diseasedgermantravelersreturningfromthetropics andsubtropics.AmJTropMedHyg.2016;95:481–7.

27.VouloumanouEK,RafailidisPI,FalagasME.Currentdiagnosis andmanagementofinfectiousmononucleosis.CurrOpin Hematol.2012;9:14–20.

28.ZhangL,ZhouP,MengZ,PangC,GongL,ZhangQ,JiaQ,Song K.Infectiousmononucleosisandhepaticfunction.ExpTher Med.2018;15:2901–9.