Polyomaviruses BK and JC DNA infection in peripheral blood cells from blood donors

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

Polyomaviruses

BK

and

JC

DNA

infection

in

peripheral

blood

cells

from

blood

donors

Malihe

Fathi

Haghighi

_,

_Noorossadat

_Seyyedi

_,

_Ali

_Farhadi

_,

_Farahnaz

_Zare

_,

Leila

Kasraian

_,

_Gholam

_Reza

_Refiei

_Dehbidi

_,

_Reza

_Ranjbaran

_,

Abbas

Behzad-Behbahani

a_{ShirazUniversityofMedicalSciences,SchoolofParamedicalSciences,DiagnosticLaboratorySciencesandTechnologyResearchCenter,}

Shiraz,Iran

b_{ShirazIslamicAzadUniversity,DepartmentofMicrobiology,Shiraz,Iran}

c_{HighInstituteforResearchandEducationinTransfusionMedicine,BloodTransfusionResearchCenter,Shiraz,Iran}

a

r

t

i

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l

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n

f

o

Received4September2018 Accepted16January2019 Availableonline23February2019

Keywords:

Blooddonors Leukocytes Mononuclear Polyomavirus

Real-timepolymerasechain reaction

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Objectives:Toinvestigatetheprevalenceofhumanpolyomavirus(BKandJCviruses) infec-tioninperipheralbloodmononuclearcellsofhealthyblooddonors.

Methods:Thestudyincluded250healthyblooddonors.Five-milliliterbloodwasdrawninto sterileEDTAtubesandPBMCswereisolatedfromwholeblood.TheisolatedPBMCswere countedandstoredat−70◦_{Cforfutureinvestigation.DNAwasextractedandsubjected}

tosimple,sensitiveandspecificsemi-nestedPCRaswellasQPCRusingbothgeneraland specificprimersfordifferentassays.

Results:Of250bloodsamples,66(26.4%)werepositiveforBKVDNA(146–34,514copies/106

cells).JCDNAwasfoundin45(18%)bloodsamples(65–21,250copies/106_{cells).Co-infection}

withtheseviruseswerefoundin11(4.4%)outof250bloodsamples.

Discussion:Ourstudyprovidesimportantdataonpolyomavirusinfectioninperipheralblood mononuclearleukocytesinimmunocompetentindividuals.Thesedataindicatesignificant differencesbetweentheprevalenceofBKVandJCVinfectioninhealthyblooddonors.The prevalenceofBKandJCvirusinfectionishigherintheagerange30–39yearscomparedto otherageranges.

©2019SociedadeBrasileiradeInfectologia.PublishedbyElsevierEspa ˜na,S.L.U.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/ licenses/by-nc-nd/4.0/).

∗ _{Correspondingauthor.}

E-mailaddresses:behzadba@sums.ac.ir,behzadba@gmail.com(A.Behzad-Behbahani).

https://doi.org/10.1016/j.bjid.2019.01.005

1413-8670/©2019SociedadeBrasileiradeInfectologia.PublishedbyElsevierEspa ˜na,S.L.U.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

ThehumanpolyomavirusBKVandJCVareprevalentglobally inpopulated areasbut arenoteasilytransmitted. Serocon-versiontoBKVinfectionoccursearlyinlife(5–7years)while to JCV occurs later. Human polyomavirus viruses, small, non-enveloped DNA, infect immunocompetent individuals withoutspecificsignsorsymptoms.Theyareableto estab-lishsubclinicalandpersistentinfectionsandmayreactivate fromlatencyincaseofimmunosuppression.1–4_{Viralinfection} ispersistentandusuallyestablishedinthekidneywithhigh levelsofvirusbeingexcretedintheurine.

JCV reactivation in immunocompromised patients may causeademyelinatingdiseaseofthecentralnervoussystem (CNS), named progressive multifocal leukoencephalopathy (PML),whereasBKVreactivationmaycausehemorrhagic cys-titisandpolyomavirusassociatednephropathy(PVAN)inrenal transplant patients.5,6 _{Since no specific antiviral drugs are} availabletotreatpolyomavirusdisease,treatment depends onimprovingimmunefunctionstoregaincontroloverviral replication.7

ThemainsanctuaryofBKVandJCVpersistenceinhealthy subjects is the kidney. However, identification ofBKV and JCV DNA in peripheral blood mononuclear cells (PBMC) likelyindicates recentinfectionbyorre-activationofthese viruses.8,9 _{ThefirstevidenceofaninteractionbetweenBKV} and hematopoieticcells wasgivenbystudies onthe stim-ulatoryeffectofBKVinfection onlymphocyteculturesand on BKVreceptors on the surfaceof peripheralblood cells. Thesedatasuggestedtheparticipationoflymphoid cellsin the pathogenesis of BKV infection. Nevertheless, the data onthe prevalenceofBKVand JCVDNA inperipheral lym-phocytesishighlydiscordant and contradictory.BloodBKV positivityvaries from 0 to90%.10,11 _{Moreover, detection of} BKVsequences in lymphocyteshas disputable significance for the pathogenesis, as it is not yet clear whether these cells are persistently or transiently infected and had inte-grated or episomal copies ofviral DNA.It isreported that 2–3% of the population have JCV in their PBMC, particu-larlyinB-lymphocytes.However,thelikelihoodofsecondary transmission is unknown. Overall, immunocompromised patients,pregnantwomen,diabetics,andtheelderlyappear to be at higher risk of disease and transmission of poly-omaviruses.

Polyomavirus infection and viral replication have been reportedinplasmaandurinesamplesofhealthy, immuno-competent blood donors. However, transmission by blood transfusionhasnotbeendocumented.12 _{Sinceanumberof} immunocompromisedtransplantrecipientsneedblood trans-fusionduringorgantransplantationprocedure,thetransfused bloodmaybecontaminatedwithanactiveformofthevirus. WeinvestigatedtheprevalenceofpolyomavirusBKVandJCV infectioninhealthy blood donors.Moreover,the DNAlevel atthetimeofblooddonationwasdetermined byreal-time polymerasechainreaction.

Methods

Studypopulationandthesamples

A total of 250 healthy blood donors randomly selected at theblood donationcenter,Shiraz,Iran,were includedfrom SeptemberthroughDecember2017,andcategorizedintofour agegroups(20–29,n=44,30–39,n=85,40–49,n=67,and50–59 years,n=54).Therewereonly11females (age19–57years). IncludeddonorswerenegativeserologiesforhepatitisBvirus (HBV), hepatitisC virus (HCV),HIV,and Treponema pallidum

infections.Participantsgavewritteninformedconsentandthe studywasapprovedbythelocalethicscommittee.

From each participant, 5mLofblood wascollected into sterile EDTA tubes at the time of regular blood donation. Peripheral blood mononuclear cells (PBMCs) were isolated usingFicoll-PaqueTM _{(GEHealthcare,Uppsala,Sweden)}

gra-dientcentrifugation(density1.077g/mL)andstoredat−70◦_C

forfutureinvestigation.

Semi-nestedPCR

DNAextractionandPCRamplification

pBKV(34-2) plasmid(ATCC® 45025)andpJCV (1–4)plasmid (ATCC® 45027TM_{)wasused asthereferencesourceofDNA}

for the PCR assays. Viral DNA from PBMCs was extracted usingcommerciallyavailableQIAmpDNABloodMinikit (Qia-gen,Hilden,Germany).Fortheamplificationoftargetgenes, PCRwasrunintwoseparatesteps,asdescribedpreviously.14 The outer primers were used toamplify parts of the con-servedearlyregionsforbothvirusgenomes.PCRamplification wascarriedoutwith0.5–0.8␮gofextractedDNAfrom1×106

PBMCs,1×PCRbuffer,1.5mMMgCl2,200␮MofdNTPs,0.5␮M

ofeachouterprimer(PEP1andPEP2),and2.5UofTaqDNA polymerase,inafinalvolumeof50␮L.Theouterprimerpair 5-AAGTCTTTAGGGTCTTCTAC-3and5-GTGCCAACCTAT GGAACAGA-3wasusedtoamplifya176-bpfragment com-montobothviruses.Forspecificdetection,theinnerprimer paircombinedthecommonprimer5-AAGTCTTTAGGGTCT TCTAC-3witheithertheBKV-specificprimer5-GAGTCCTGG TGGAGTTCC-3 toobtain aBKVfragmentof149bp,orthe JCVspecificprimer5-GAATCCTGGTGGAATACA-3 yield-ingaJCVfragmentof146bp.Thermalcyclerconditionswere asfollows:afirstdenaturingcycleat94◦Cfor5min,followed by40cyclesofamplificationdefinedbydenaturationat94◦C for30s,55◦Cannealingtemperaturefor45s,andextensionat 72◦Cfor1min.Afinalextensioncycleof72◦Cfor5minwas included.

Inasecond-roundPCR,1␮Lofthe1:10-dilutedfirstround PCRproductwasamplifiedundertheabovesamecondition withthespecificinnerprimerpairs.Thereactionwas visu-alized on 2% agarose gel and stained with GelRedTM _and

visualizedunderUVlight.

ToinvestigateefficientDNAextractionandPCR amplifica-tion,human␤-actingenewasalsoamplifiedalongwiththe

samplesusingprimers,whichamplifieda177-bpfragment. Onlypositivesampleswereinvestigatedfurtherfor amplifica-tionofthetargetDNAsequences.

SYBRgreen-basedreal-timePCRassay

ASYBRgreen-basedreal-timeqPCRassay,targetingthehighly conserved regions of BKV and JCV genomes, was devel-opedfordetectionand quantificationofBKVandJCVDNA extracted from PBMCs. Using SYBR green PCRmaster mix fromTakara(Shiga,Japan),theoptimizationofthePCR reac-tionwas performedbydeterminingthe optimalamount of primers,aswellasthecyclingcondition.Quantitative real-time PCR was performed in a final volume of25␮Lusing QiagenRotor-GeneQ5-Plex(QIAGEN,Hilden,Germany).The outerforwardprimer5-AAGTCTTTAGGGTCTTCTAC-3of semi-nestedPCR(0.2␮M/reaction)witheithertheBKV-specific primer5-GAGTCCTGGTGGAGTTCC-3 ortheJCVspecific primer 5-GAA TCC TGG TGG AATACA-3 (0.2␮M/reaction) wasusedforamplificationinreal-timePCRassay.TheDNA fromclinicalsampleswasextractedasdescribedabove (Sec-tion2.2.1)andtested(2␮L/reaction)induplicatewiththeQPCR assay.

Thermalcyclingwasinitiatedwithincubationat95◦Cfor 10min,followedby40cyclesof95◦Cfor30s,55◦Cfor30s, and72◦Cfor30s,attheendofwhichfluorescencewasread. Anon-templatecontrolwasincludedtomonitorpossible con-tamination.

Calibrationcurve

Forthecalibrationcurve,thethresholdcycle(Ct)valueswere plottedagainstthelogarithmoften-foldserialdilutionsofa plasmidcontainingtheBKV(6×106_to6×100_{copies/reaction)}

andpJCV(1–4)plasmid(9×16_to9×100_{copies/reaction).}

Dataanalysis

ThestatisticalpackageSPSSVersion17.0forWindows(SPSS, Chicago,IL,USA)was useddata analysis.Theassociations withthestudiedcategoricalvariablesandBKVandJCV infec-tionwereassessedusingX2_{testorFisher’sexacttest,when}

indicated.

Results

Limitofdetection

Thelimitdetectionofthesemi-nestedPCRassayforBKVand JCVDNAwas sixandnine copiesofplasmaDNA/reaction, respectively.Serial10-folddilutionsofpBKVandpJCVwere runinQPCR.Standardcurvesdisplayedalinearrelationship forqPCRassay,withefficienciesof99%forbothBKVandJCV targets.Undertheseconditions,thelimitsofdetectionwere 1–60copies/PCRreactionforbothBKVandJCVDNA.No cross-reactionswerefoundwhenBKVprimerswereusedagainst JCVDNAandviceversa.

PrevalenceofBKVandJCVDNAinPBMCs

Semi-nestedPCRwascarriedoutcarefullytoavoidpossible cross-contamination ofBKVand JCVDNA.All thesamples showedapositiveresultinthe␤-actinPCR,indicatingabsence ofinhibitorsandadequateamountofDNA.Noneofthe nega-tivecontrolswasamplified.Resultsfromsemi-nestedPCRfor detectionofBKVandJCVDNAinallsamplesarepresentedin

Table1.Overall,polyomavirusesBKandJCDNAwerefoundin 111/250PBMCs(44.4%)ofhealthyblooddonors.BKVandJCV DNAwerefoundin66/250(26.4%)andin45/250samples(18%), respectively.DistributionofBKVandJCVinfectioninvarious agerangesisshowninTable1.

The overall prevalence of BKV DNA was significantly higher than that of JCV DNA in PBMCs of healthy blood donors(p=0.03).AlthoughthefrequencyofbothBKVandJCV infectionswerehigherinagerange30–39,nostatistically sig-nificantdifferenceswerefoundrelatedtoagegroup(p>0.05). Amongthe11femalehealthyblooddonors,BKVandJCVDNA werefoundinPBMCsinsix(54.5%)andtwo(18%),respectively; withco-infectionintwoofthem.BKandJCvirusDNAwerenot detectedinbloodsamplesofthreefemaleblooddonors.On theotherhand,theincidenceofBKVandJCVinfectioninmale donorswere59/239(24.9%)and42/239(17.6%),respectively.

BKandJCDNAviralloadinsemi-nestedPCRpositive PBMCssamples

Inordertomeasureviralload,allsemi-nestedpositive sam-plesweretestedbyquantitativereal-timePCR.Amongthe66 PBMCsamplespositiveforBKVDNAwithsemi-nestedPCR, viralloadrangedfrom146×100_to34×103_copies/106_PBMC

bySYBRgreenreal-timePCR.ForJCV,viralloadrangedfrom 65×100_to21×103_copies/106_{PBMCs.MeanviralloadforBKV}

and JCVDNA/106 _{copiesofPBMNwere5773.56 and3652.92}

withthemodeof654and952respectively.

ResultsofQPCRforthesampleswithco-infectionofBKV andJCVareshowninTable2.

DescriptivestatisticsofBKandJCVdetectionintotal sam-plesareshowninTable3.

Discussion

ThepresentstudyshowsahighfrequencyofBKVandJCVDNA inPBMCofhealthyblooddonors.Peripheralleukocyteswere isolatedfrombloodsamplesofhealthyindividualsand cellu-larDNAwasextracted.TheprevalenceofBKVandJCVDNA inperipheralleukocytesfrom250healthyadultblooddonors wasevaluatedbysemi-nestedPCR.Thesensitivityand speci-ficityofthemethodusingspecificinner PCRprimersallow ustodetermineiftheseviruseswerepresentinthesample tested.

In addition to the kidney, several studies have demon-stratedthatperipheralbloodleukocytesarecommonsitesof latentBKVandJCVinfectioninhealthyindividuals.However, persistenceofBKVandJCVpolyomavirusesinlymphocytes ofhealthyhumansisstilldebated.TherateofPBMCinfection by BKV and JCV have been reported between 0 to 90% in

Table1–PrevalenceofBKVandJCVDNAinperipheralbloodleukocytesofhealthyblooddonors. SamplespositiveforpolyomavirusDNA(%)

Agerange(year) Total,n(%) BKV,n(%) JCV,n(%) Co-infection,n(%)

20–29 44(17.6%) 10/44(22.7) 7/44(15.9) 0

30–39 85(34%) 26/85(30.6) 21/85(24.7) 7(8.2)

40–49 67(26.8%) 16/67(23.9) 8/67(12) 3(4.5)

50–59 54(21.6%) 14/54(30) 9/54(16.7) 1(1.9)

Total 250 66/250(26.4) 45/250(18) 11/250(4.4%)

Table2–ViralloadofBKVandJCVco-infectedPBMCssamples.

DonorNo. Age(year) Real-timePCRresults

JCV/BKVDNA JCVDNA copies/106_cells BKVDNA copies/106_cells 1 55 (+) 21,250 11,876 2 44 (+) 4359 1429 3 39 (+) 3580 9954 4 40 (+) 1290 2345 5 36 (+) 2190 365 6 39 (+) 12,683 14,900 7 31 (+) 876 14,953 8 35 (+) 4912 34,514 9 31 (+) 384 12,003 10 36 (+) 1200 654 11 48 (+) 948 6212

Table3–FrequencyanddescriptivestatisticsofJCVandBKVinfectionamongpositivesamples.

Numberofpositivessamples Range Minimum Maximum Mean Std.deviation

JCV 44 30,169.00 65.00 30,234.00 4418.3 6555.3

BKV 66 34,404.00 146.00 34,550.00 6673.1 9347.9

the various studies.10,13–17 _{In our investigation, the overall} detection of BKV and JCV DNA in PBMC of healthy blood donors was 24.6 and 18%, respectively, which is the first reportfrom ourgeographicalregion.Inonestudy inChina, using semi-nestedPCR assay with the same primers used inourstudy,BKVand JCVDNAwasreported in42.1%and 7.8%ofPBMCfrom healthyblood donors, respectively(16). SuchfindingsindicatethatBKVandJCVinfectioninvariesin differentgeographicareasoftheworld.

Furthermore,theresultsofthisstudy pointoutthatthe prevalenceofBKVDNAwashighlysignificantlyhigher com-paredtothatofJCVDNA(p=0.03).Becauseofthesmallsample offemaleblooddonors(11/250donors)theimpactofsexon theresultscouldnotbeascertained,BKvirusinfectionseemed tobemorecommonthanJCvirusinfection(3:1).Althoughthe prevalenceofBKVandJCVDNAinPBMCwashigherinthe agerange30–39years,therewasnostatisticallysignificant differenceamongagegroups.

BK polyomavirus is associated with nephropathy and ureteric stenosis in kidney transplant recipients as well in other types of transplant recipients.18,19 _{BKV causes} hemorrhagic cystitis in allogenic hematopoietic cell trans-plant recipients.20 _{However, less than 5% ofpolyomavirus} nephropathy among kidney transplant recipients may be

caused by JC, perhaps with more inflammation and focal fibrosis, and is typically associated with a milder clinical course.21

Generally, primary infection ofBKV and JCV in healthy individuals isfollowedbylifelong viralpersistence.Bothof thesevirusesareshedintheurineofhealthypeople.However, immunosuppressionmay reactivate latentvirus in healthy individualsandcausedisease.

Real-timequantitativePCRwasusedtoquantitateDNAof polyomavirusesBK(BKV)andJC(JCV)inPBMCsofhealthy indi-viduals.ThereisnostandardlevelofpolyomavirusDNAin healthyoreveninpatients.Manyvariablesincludingsample processingprotocoland sensitivityand specificityof detec-tionmethodsmayaffecttheultimateresultsofquantitative PCRassays.Environmentalfactorssuchasage,sex,personal hygiene, individual’simmune statuscaninterferewith the resultoftheassay.Basedonthefindings ofthis study,the viruspersistsinbloodmononuclearleukocytesinupto25% ofhealthyblooddonorswhoarelifelongcarriersofthevirus. Itcanbetransmittedbytransfusionofcellularblood com-ponents althoughthis may bedifficult todistinguishfrom reactivation of a previous infection. BKV infection,in par-ticular, can cause severe infection in renal transplant and immunocompromised individuals.Therefore, polyomavirus

diseasetransmittedthroughPBMCsduringbloodtransfusion shouldnotbeneglected.

Wehave shown that BKVand JCVcan infect PBMCsof healthy individuals and establish alatentinfection. In our experiment,theDNAlevelindifferentindividualsvariesfrom 146×100_to34×103_copies/106_{PBMCforBKVand65×10}0_to

21×103_copies/106_{PBMCforJCVDNA.Therefore,when}

trans-fusingbloodtoanimmunosuppressedpatienttransmissionof thehighlevelofinfectedcellswithpolyomavirusmayresult inseveredisease.

In conclusion, our study provides important data on polyomavirus infection in peripheral blood mononuclear leukocytes in immunocompetent individuals living in the southwestofIran.Thesedataindicatesignificantdifferences betweentheprevalenceofBKVandJCVinfectioninhealthy blooddonors.TheprevalenceofBKandJCvirusinfectionis higherintheagerange30–39yearscomparedtootherage ranges.DifferentlevelsofBKandJCvirusDNAwasfoundin PBMCsofhealthyblooddonors.However,viralDNA quanti-tationinplasmasamplesbyreal-timePCRhelpsidentifying ongoingviralreactivation.

Funding

Thisresearchdidnotreceiveanyspecificgrantfromfunding agenciesinthepublic,commercial,ornot-for-profitsectors.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

ThisresearchwassupportedbyShirazUniversityofMedical SciencesGrantNumber97-01-45-17407.

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