Propagation and titration of Alkhumra hemorrhagic fever virus in the brains of newborn Wistar rats 夽

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ContentslistsavailableatScienceDirect

Journal of Virological Methods

jou rn al h om ep ag e :w w w . e l s e v i e r . c o m / l o c a t e / j v i r o m e t

Propagation and titration of Alkhumra hemorrhagic fever virus in the brains of newborn Wistar rats ^夽

Tariq A. Madani

^a,∗

, Moujahed Kao

^b

, El-Tayeb M.E. Abuelzein

^b,c

, Esam I. Azhar

^b,d

, Hussein M.S. Al-Bar

^e

, Huda Abu-Araki

^f

, Rana Y. Bokhary

^g

, Thomas G. Ksiazek

^h

aDepartmentofMedicine,FacultyofMedicine,KingAbdulazizUniversity,Jeddah,SaudiArabia

bSpecialInfectiousAgentsUnit,KingFahdMedicalResearchCenter,KingAbdulazizUniversity,Jeddah,SaudiArabia

cTheScientiﬁcChairofSheikhMohammadHusseinAlamoudiforViralHemorrhagicFever,KingAbdulazizUniversity,Jeddah,SaudiArabia

dDepartmentofMedicalLaboratoryTechnology,FacultyofAppliedMedicalSciences,KingAbdulazizUniversity,Jeddah,SaudiArabia

eDepartmentofFamilyandCommunityMedicine,FacultyofMedicine,KingAbdulazizUniversity,Jeddah,SaudiArabia

fLaboratoryAnimalsUnit,KingFahdMedicalResearchCenter,KingAbdulazizUniversity,Jeddah,SaudiArabia

gDepartmentofPathology,FacultyofMedicine,KingAbdulAzizUniversity,Jeddah,SaudiArabia

hGalvestonNationalLaboratory,DepartmentsofPathologyandMicrobiologyandImmunology,UniversityofTexasBranch,Galveston,TX,USA

Articlehistory:

Received24March2013 Receivedinrevisedform 15November2013 Accepted11December2013 Availableonline18January2014

Keywords:

Alkhumrahemorrhagicfevervirus Propagation

Wistarrats

Medianratlethaldose

a b s t r a c t

Alkhumrahemorrhagicfevervirus(AHFV)isanovelflavivirusidentifiedfirstinSaudiArabia.Inthisstudy, successfulpropagationofAHFVinthebrainsofnewbornWistarratsisdescribedandthemedianratlethal dose(RLD50)isdetermined.AHFV-RNA-positivehumanseradiluted1:10wereinjectedintracerebrally into16,≤24holdrats.Post-inoculation,theratswereobserveddailyfor30days.Brainsofmoribund ratsweretestedforAHFV-RNAusingRT-PCRandculturedinLLC-MK2cells.Thetiteroftheisolated viruswasdeterminedandexpressedinmediantissuecultureinfectiousdose(TCID50).Todetermine theRLD50,AHFVbrainsuspensionwas10-folddilutedseriallyandeachdilutionwasinoculatedinthe cerebralhemispheresof10ratsforatotalof90rats.Threedayspost-inoculation,theratsdeveloped tremor,irritability,convulsion,opisthotonus,andspasticparesisstartinginthehindlimbsandascending toinvolvethewholebody.Allinfectedratsdiedwithin3–7dayswithhistopathologicallyconfirmed meningoencephalitis.AHFV-RNAwasdetectedinthebrainsofallinfectedratsandthevirustiterwas 10^9.4RLD50/ml.ThevirustiterinLLC-MK2was10^8.2TCID50/ml.Inconclusion,AHFVwaspropagated successfullytohightitersinthebrainsofnewbornWistarrats.

1. Introduction

Alkhumrahemorrhagicfevervirus(AHFV)isanewhemorrhagic feverflavivirusidentifiedfirstinSaudiArabia.Itisamemberofthe tick-borneencephalitisgroupinthegenusFlavivirusofthefam- ilyFlaviviridae.Itwasfirstisolatedin1995from6patientsliving

Abbreviations:AHFV,Alkhumrahemorrhagicfevervirus;CPE,cytopathiceffect;

EMEM,Eaglesminimumessentialmedium;FCS,fetalcalfserum;FITC,ﬂuorescein- isothiocyanate; ICTV,InternationalCommittee onTaxonomyof Viruses; IFAT, indirectﬂuorescentantibodytest;LLC-MK2,rhesusmonkeykidneycellline;PBS, phosphatebuffersaline;RT-PCR,reversetranscriptase-polymerasechainreaction;

RLD50,medianratlethaldose;TCID50,mediantissuecultureinfectiousdose.

夽Thisisanopen-accessarticledistributedunderthetermsoftheCreativeCom- monsAttribution-NonCommercial-NoDerivativeWorksLicense,whichpermits non-commercialuse,distribution,andreproductioninanymedium,providedthe originalauthorandsourcearecredited.

∗ Correspondingauthorat:DepartmentofMedicine,KingAbdulazizUniversity, POBox80215,Jeddah21589,SaudiArabia.Tel.:+96626408348;

fax:+96626408344;mobile:+966557774666.

E-mailaddresses:[email protected],[email protected](T.A.Madani).

in Alkhumra districtin Jeddah, themain sea-port in thewest- ern borderof SaudiArabia (Qattanet al.,1996).In 2001–2003, Madanire-identiﬁedthediseaseanddescribed20conﬁrmedcases intheholycityofMakkah,75kmfromAlkhumradistrictinJeddah, andproposedthename‘Alkhumra’begiventothevirusafterthe geographiclocationfromwhichitwasisolatedoriginally(2005).

Unfortunately,Alkhumrahemorrhagicfeverviruswasmisnamed as‘Alkhurma’virusinmanyscientificpublicationsduetoatypo- graphicalerrorwheretheletters‘m’and‘r’weretranspositioned (Madani,2005;Madanietal.,2011,2012a,b;Liebert,2012).The InternationalCommitteeonTaxonomyofViruses(ICTV)hascor- rectedthismistakerecentlyandapprovedthename‘Alkhumra’as thecorrectnameofthevirus(Pletnevetal.,2011).From2003to 2007,8confirmedcasesofAHFVinfectionswerereportedsporadi- callyfromNajrannearthesouthernborderofSaudiArabia(Madani etal.,2011).Subsequently,asignificantoutbreakofAHFVinfection occurredinNajranin2008–2009with70confirmedcasesreported (Madanietal.,2011).Recently,twotravelersreturningtoItalyfrom southernEgyptwereconfirmedtobeinfectedwithAHFV(Carletti etal.,2010).

http://dx.doi.org/10.1016/j.jviromet.2013.12.004

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Epidemiological data suggest that AHFV is transmitted to humansfromlivestockanimals(sheep,goats,orcamels)bydirect contactwiththeseanimalsorthemosquitobites(Madani,2005;

Madani et al., 2011). Despite the close phylogenetic similarity betweenAHFVandKyasanurForestdiseasevirus,epidemiological datadidnotsuggestthattickswereplayinganimportantroleinthe transmissionofAHFVfromanimalstohumans,although,theirrole asareservoirofthevirusinitsecologicnicheandasvectorstrans- mittingthevirusamonganimalsisconceivablypossible(Madani, 2005;Madanietal.,2011).Manyaspectsofthisnewvirusarestill unknownincludingthenaturalreservoirofthevirus,themodeof transmissionfromthenaturalreservoirtolivestockanimalsand humans,theroleofticksandmosquitoesinthetransmissionand maintenanceofthevirus,thepathophysiologyofhumaninfection, andthetypeofimmunitythatdevelopspost-exposure.Allofthese aspectshavebeenpoorlyresearchedtodate.Thelackofsuitable experimentalanimalshasfurtherhinderedresearchinthisﬁeld.

Developmentofanimalmodelsisanimportantsteptostudythe pathophysiologyandotherepidemiologicalfeaturesofthisnewly describedvirus.Thepresentstudydescribessuccessfulpropaga- tionofAHFVinthebrainsofnewbornWistarrats,describesthe histopathologicalchangesthatoccurinthebrainandotherorgans, anddeterminesthemedianratlethaldose(RLD₅₀).

2. Materialsandmethods

2.1. Studypatients

FromMarch18thtoApril4th,2009,whenanAHFVoutbreakwas recognizedinitiallyinNajran,bloodsampleswerecollectedfrom sevenpatientswithsuspectedAHFVhemorrhagicfever.Detailsof thisoutbreakwerepublishedrecentlybytheauthors(Madanietal., 2011).

2.2. Bloodsamples

Whole blood was collected separately in EDTA and plain vacutainers(BD Vacutainers^®,Becton, Dickinson and Company, Plymouth,UK)fromeachofthesevenpatients.EDTAspecimens werespuninarefrigeratedcentrifugeat277×gfor10minandthe plasmawascollectedandstoredin0.5mlaliquotsat−86^◦C.The bloodsamplesintheplaincontainerswerelefttoclotandtheserum wasseparatedbylow-speedcooledcentrifugationandstoredat

−20^◦C.Theplasmaandserumspecimensweretransportedsub- sequentlyondry icein IATA-compliant containersfromNajran totheSpecialInfectiousAgentsUnit,abiosafetylevel3virology laboratory,atKingFahdMedicalResearchCentre,KingAbdulaziz University,Jeddah,SaudiArabia.

2.3. DetectionofAHFV-RNAbyrealtimereverse transcriptase-polymerasechainreaction(RT-PCR) 2.3.1. RNApreparation

Human sera, plasma, virus suspension from LLC-MK2 cell culture, passage 2, harvest, uninoculated LLC-MK2 cell culture suspension,and10%ratbrainsuspensionswereeachclearedby centrifugationinarefrigeratedtabletopcentrifugeat12298×gfor 10mintoobtaincell-freesamples.ViralRNAwasextractedfrom 140␮lofthecell-freeﬂuidusingtheQIAmpviralRNAkit(Qiagen, Hilden,Germany)withoutmodiﬁcation.RNAwaselutedin50␮l aliquots.

2.3.2. Primersandprobedesign

Apairofprimers(AHFVS1:5–GTGAGTGGCGCTTTGTTTGTA andAHFVR:5-CCCCCTTTCCTTTAAGGACG)andthecorrespond- ing5 –nucleasedetectionprobe(TBVTM:6FAM-ACAGCTTAG

GAGAACAAGAGCTGGGGAXT—PH)weredesignedwithPrimer Express software (Applied Biosystems, Weiterstadt, Germany), andsynthesizedbyTibMolbiol,Berlin,GermanybasedonAHFV sequencepublishedbyCharreletal.(2005).The5-nucleaseprobe waslabeledwith6-carboxyﬂuoresceinatthe5endandwith6- carboxy-N,N,N,N-tetramethylrhodamineatthe3end.The3end ofeachprobewasphosphorylatedtopreventelongationduring PCRasdescribedpreviously(Hollandetal.,1991;Livaketal.,1995).

2.3.3. RealtimeRT-PCRconditions

Theone-steprealtimeRT-PCRsystemcombiningsuperscript reversetranscriptasewithplatinum Taq-polymerase (LifeTech- nologies,Karlsruhe,Germany)wasusedin5-nucleaseassay.The reaction mix contained10␮l ofmaster mix provided withthe kit(including thebasiclevel of MgSO₄),40ngof bovineserum albumin (Sigma, Munich, Germany) per ␮l, and 2␮l of RNA.

The20-␮lassay oftheLightCyclerreaction capillaryfor AHFV RT-PCRwith5-nucleaseprobedetection involvedreversetran- scriptionat50^◦Cfor30min,initialdenaturationat95^◦Cfor15min, and 45 cyclesat 95^◦C for 1s and then at 57^◦C for 30s. Fluo- rescencewasread atthecombinedannealing-extensionstepat 57^◦C.

2.4. AHFVinoculationintonewbornWistarrats

Experimentalinfectionoftheratswasperformedinabiosafety level-3facility.AHFV-RNA-positivehumanserumfromtheﬁrst patient(patientno.1,Table1)wasdiluted1/10withphosphate buffersaline(PBS), pH7.4, and then 20␮lof this dilution was injectedintracerebrallyintoeachof16conventionalin-housebred newborn(≤24hold)Wistarrats.A controlgroupof4 newborn WistarratsweremockinjectedintracerebrallywithAHFV-RNA- negativehumanserum.The16experimentallyinfectednewborn rats,dividedintotwogroupsof8infectedrats,1controlrat,and theirtwo non-inoculated mothers werehoused separately and weighedandobserveddailyfor30daystodetectanysignsofillness ordeath.Thetworemainingcontrolratswerehousedseparately withtheirnon-inoculatedmother.Moribundratswereeuthanized andtheirbrainswereharvested,homogenized,suspendedin10%

Hanks’balancedsaltsolutionwith10%fetalcalfserum(FCS),and thencentrifugedat1107×gfor15min.Thesupernatantwastested for AHFV-RNA using RT-PCR and cultured in LLC-MK2 cells as describedbelow.Additionally,brains,livers,spleens,andkidneys frominfectedandcontrolratswereexaminedhistopathologically asdescribedbelow.

2.5. DeterminationoftheRLD50

AHFVisolatedinthebrainsofnewbornWistarratsinoculated withserum fromthe ﬁrst patient (patientno. 1, Table 1) was selectedastheprototypeofthisoutbreakanditwaspassaged2sub- sequenttimesinthebrainsofnewbornrats.A10%brainsuspension ofthisthirdpassagewaspreparedandusedasthevirusstockupon whichfurthertitrationswereperformed.Thesuspensionwas10- folddilutedseriallywithPBS,pH7.4.Aliquotsof20␮Lfromeach dilutionwereeachinoculatedintheleftcerebralhemispheresof 10newbornWistarrats.Atotalof90rats,10ratsforeachdilution, werethusinoculated.Allinoculatedratswereobserveddailyfor anyclinicalmanifestationsordeathforatotalof30days.Thebrains ofanydeadratswereharvested,homogenizedin10%Hanks’bal- ancedsaltsolutionwith10%FCS,andthencentrifugedat1107×g for15min.ThesupernatantwastestedforAHFV-RNAusingRT-PCR.

TheRLD50titerwascalculatedaccordingtothemethodofReedand Muench(1938).

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Table1

Alkhumrahemorrhagicfevervirus(AHFV)RNAdetectioninpatients’serumandplasma,andinthebrainsofnewbornWistarratsbyRT-PCRandAHFVcultureinLLC-MK2 inoculatedwithbrainsuspensionfrommoribundinfectedrats.

Patients DirectdetectionofAHFV-RNAbyRT-PCR DetectionofAHFVRNAinthebrainsof newbornWistarratsbyRT-PCR

RecoveryofAHFVfromratbrain suspensionculturedinLLC-MK2cells

Plasma Serum

1 + + + +

2 + − + +

3 − + + +

4 + + + +

5 + + + +

6 − − − −

7 − − − −

+,positive;−,negative.

2.6. AdaptationoftheAHFVtogrowintheLLC-MK2cells

Theprototypevirusintheformofnewbornratbrainsuspension, passage1,wasinoculatedontothecontinuousmonkeykidneycell line(LLC-MK2)monolayersasfollows:thecellmonolayerswere growninCorningscrew-captissue cellculturetubesat37^◦Cin EaglesMinimumEssentialMedium(EMEM)supplemented with 10%FCSandantibiotics(100U/mlpenicillinand100␮g/mlstrep- tomycin).Whenthemonolayers were70%conﬂuent, eachtube hadthegrowthmediumremovedandwasinoculatedwith0.1ml oftheundilutednewbornratbrainsuspensionandincubatedat 37^◦Cfor1htoadsorb.Thiswasfollowedbytheadditionof1mlof EMEMmaintenancemediumcontaining2%FCSandincubationat 37^◦Cwithdailyobservationfordiscerniblecytopathiceffect(CPE).

WhentheCPEaffected90%orgreaterofthemonolayer,thetubes wereplacedat−86^◦C.Thiswasfollowedbythawing,vortexingthe contents,spinningfor10minat429×g.Thesupernatantﬂuidwas collectedandusedundilutedtoinoculatenewmonolayersofthe LLC-MK2cellsasdescribedabove.

2.7. VirustitrationintheLLC-MKcellculture

Themicrosystemfortitrationoftheisolatedprototypevirus yield,passage2,fromtheLLC-MK2cellculturewasemployeduti- lizing96-welltissueculturemicroplates.Thevirussuspensiontobe titratedwasdilutedinEMEMsupplementedwith2%FCS.Toeach wellofthemicrotiterplate,50␮lofEMEM,supplemented with 2%FCS,wereadded.Ten-folddilutionseriesofthevirussuspen- sionwasmadeinsterilevials.Foreachvirusdilution,5replicates wereused.Startingfromthehighesttothelowestdilution,50␮l ofthevirusdilutionswereaddedtotherelevantwells.Thiswas followedbytheadditionof50␮lofcellsuspensioncontaining10⁶ cellspermlinEMEMsupplementedwith2%FCStoeachwellat aconcentrationof10⁶perml.Theplateswerecoveredandincu- batedat37^◦CinaCO₂incubator.Theplateswereexamineddaily fordiscernibleCPEandtheﬁnalreadingwasmadeafter7days.The virustiterwascalculatedaccordingtoReedandMuench(1938).

Controlwellscontaininguninoculatedcellculturewereincluded inthetest.

2.8. Histopathologicalexamination

Thebrain,liver, spleen,and kidneyfromcontrol andeutha- nizedmoribundinfectedratswerefixedin10%neutral-buffered formalinforatleast24h.Tissuesweredehydratedingradedalco- hols,clearedwithxylene,andinfiltratedandembeddedinparaffin.

Tissuesembeddedinparafﬁnwerecutat4␮mandmountedon glassslides.Sectionswerestainedwithhematoxylinandeosin(H

&E)stainandexaminedfor microscopicchanges undera light microscope.

2.9. Indirectﬂuorescentantibodytest(IFAT)

TheIFATwasusedtoconfirmidentityoftheprototypeAHFV isolate,passage2, in theLLC-MK2 cells which wereinoculated withbrainsuspensionfrommoribundnewbornrats.Forty-eight hours after inoculation, the cell culture monolayers were har- vested. The cells werepelleted by centrifugation for 10minat 429×gand depositedonTefloncoated8-wellslides.Theslides wereairdriedinsideabiosafetycabinetandfixedinchilledace- tone/methanol(1:1)for20min.Thewellswereoverlaidwith20␮l (1:200dilutioninPBSpH7.4)ofhyperimmunemouseasciticfluid containingpolyclonalantibodiesagainstAHFVpreparedusingthe sameprocedurepreviouslydescribed(Brandtetal.,1967).Slides wereincubatedinamoistchamberat37^◦Cfor60minbeforethey werewashedthreetimesinPBSpH7.4.Theboundantibodywas detectedwithfluorescein-isothiocyanate (FITC)-conjugated goat anti-mouseIgG(Sigma,St.Louis,USA)with0.2%Evansblue(Sigma, St.Louis,USA).Theslideswerewashed,mountedwithFluoprep (BioMerieux,MarcyL’Etoile,France)andexaminedusingaLeitz fluorescencemicroscopewithappropriateexcitationandbarrier filtersforFITC.

2.10. Ethicalapproval

KingAbdulazizUniversity’spolicyonthecareanduseoflabo- ratoryanimalswasfollowed.Ethicalapprovalwasobtainedfrom theResearchEthicsCommitteeattheFacultyofMedicine,King AbdulazizUniversity,Jeddah,SaudiArabia.

3. Results

3.1. AHFV-RNAdetectioninpatients’seraandplasmabyRT-PCR Fiveof thesevenpatientshaddetectableAHFVRNAintheir plasma or serum (Table1). AHFV RT-PCRwas positive both in plasmaandserumin3patients(patient1,4,5),inplasmaalonein onepatient(patient2),andinserumaloneinonepatient(patient 3).

3.2. AHFVinoculationintonewbornWistarrats

Threedays postinoculation,theratsinoculated withAHFV- infectedspecimensdevelopedneurologicalmanifestationscharac- terizedbytremor,irritability,convulsion,opisthotonus,andspastic paresis startingin thehind limbsandascending toinvolve the wholebody (video1).AllAHFV-inoculatedratsdiedwithin3–7 dayspostinoculation;8ratsdied3dayspost-inoculation,5rats died 6 days post-inoculation, and the remaining 3 rats died 7 dayspost-inoculation.Viral-RNAwasdetectedinthebrainsofall AHFV-inoculatedrats(Table1).AllAHFV-inoculatednewbornrats atepoorlyandtheyweredehydrated,emaciated,andsmallerin

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Fig.1.FiveAlkhumrahemorrhagicfevervirus(AHFV)-intracerebrallyinoculated(left-handside)andonecontrol(right-handside)newbornWistarratsﬁvedayspost- inoculationshowingfailuretothriveandemaciationoftheAHFV-inoculatedrats.

sizewhencomparedtothecontrolrats(Fig.1).Pre-inoculation, theweightoftheratsrangedfrom8.6to10.4g,withameanof 9.5±0.6g.Sevendayspost-inoculation,theweightofthesurviv- ingAHFV-inoculatedratsrangedfrom6.0to9.2gwithameanof 7.5±1.2g,whereasthatofthecontrolratsrangedfrom9.1to11.0g, withmeanof10.0±0.7g.

The three non-inoculated mothers, the two control (mock- inoculated)newbornratsthatwerehousedinthesamecagewith theratsinoculatedwithAHFV-positivespecimens,andtheother twocontrolnewbornratsandtheirmotherthatwerehousedsep- aratelyremainedhealthyfortheentire30daysofobservationand wereeuthanizedattheendoftheobservationperiod.Noviral- RNAwasdetectedintheblood,brains,hearts,lungs,livers,spleens, kidneys,testes,orovariesprocuredfromtheeuthanizedcontrol rats.

Table2shows theincubationperiod,durationofillness, and mortalityamongthe90newbornWistarratsthatwereinoculated

intracerebrallywith10-foldseriallydilutedbrainsuspension(10%) ofAHFV.Thevirustiterwas10^9.4RLD₅₀/ml.

3.3. ViruspropagationandtitrationinLLC-MK2cells

TheCPEproducedbythevirusintheLLC-MK2cellsstartedascell roundingfollowedbycellaggregation,syncytiaformationandcell destruction.Thetimespanfrominoculationtocompletedestruc- tionofthecellmonolayerwas7days.ThevirustiteroftheLLC-MK2 was10^8.2TCID50/ml.Viral-RNAwasdetectedintheinoculatedLLC- MK2cellculturebutnotintheuninoculatedcontrols.

3.4. Histopathologicalexamination

Histopathologicalexaminationofsectionstakenfromnewborn controlandAHFV-inoculatedratsshowedinﬂammatorychanges inthebrainoftheinfectedratswithperivascularandparenchymal

Fig.2.Comparisonbetweencontrolratsandinfectedrats’brains:(a)normalcerebralparenchymaofacontrolrat(hematoxylinandeosinstain,10×);(bandc)infected rats’brainsshowinginfiltrationoftheparenchymabymononuclearinflammatorycellswithperivascularcuffing(hematoxylinandeosinstain,20×);and(d)infectedrats’

brainsshowingmeningealinﬂammatoryinﬁltrate(hematoxylinandeosinstain,4×).

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Fig.3.Sectionfromtheliver(a,hematoxylinandeosinstain,20×)andthespleen (b,hematoxylinandeosinstain,4×)ofacontrolratshowing extramedullary hematopoiesis.

mononuclearcellinfiltratesinthecerebralhemispheres,aswellas moderateleptomeningealmononuclearcellinfiltratecompatible withmeningoencephalitis(Fig.2a–d).Theinfiltrationwaspatchy withnonecrosisseen.Noviralinclusionbodieswereseenwithin thebraincells.Thecerebellarparenchymawasunaffected.

Theliversandspleensfrombothcontrolandinfectednewborn ratsshowedmarkedextramedullary hematopoiesis expectedin thisage(Fig.3aandb).However,theliversofinfectedratsshowed moderatepatchyportalmixedinflammatoryinfiltratewithnoevi- denceofinterfacehepatitis,parenchymalinfiltrate,viralinclusions, ornecrosis(Fig.4).Thespleensandkidneysofinfectedratsshowed noinflammatorychanges,viralinclusions,orotherchangescom- paredtothoseofthecontrolrats(Figs.4band5a).

Fig.4. Infectedrats’livershowingmixedinﬂammatoryinﬁltratewithinoneofthe portaltractswithnointerfacehepatitis(hematoxylinandeosinstain,20×).

3.5. IFAT

Fig.6shows theIFAT resultsbeforeand afterinoculationof theLLCMK-2cellculturewithinfectedratbrainsuspension.The inoculatedcellsclearlyexpressAHFVantigensinthecytoplasm.

4. Discussion

Adearthofinformationisthusfaravailableregardingisolation ofAHFVindifferentlaboratoryanimalspeciesorcellculture.Inthe presentstudy,thesuitabilityofnewbornWistarratswasexamined forpropagationofAHFV.TheresultsdemonstratedthatAHFVvirus waspropagatedsuccessfullyinthebrainsofnewbornWistarrats resultinginneurologicalsignsbeginning3daysafterinoculation with100%mortality3–7dayspostinoculation.Thevirusfromthe inoculatedratbrainswaspropagatedsuccessfullyinmonolayersof theLLC-MK2cellline,identiﬁedbytheIFAT,andtheviralRNAwas detectedbytheRT-PCR.

Histopathologicalexaminationofthebrainsoftheinoculated ratsconfirmedmeningoencephalitis.Theinflammatoryinfiltration waspatchywithnonecrosisseen.Thepathologicalchangesseen inthebrainweresimilartothosedescribedinthebrainsofPowas- sanvirus-infectedmice,althoughinthelattersomeareasofthe cerebrumshowednecrosisintheformofacuteneuronalinjury, edema,andkaryorrhexis(Holbrooketal.,2005).TheOmskhem- orrhagicfevervirus(OHFV)-infectedmiceweredescribedtohave patchymeningoencephalitiswithsomeevidenceofperivascular cuffingbut noevidence of necrotizinglesions (Holbrook et al., 2005).AlthoughthelesionsdescribedinOHFV-infectedmicewere notdifferentinnaturefromthosein AHFV-infectedWistar rats

Table2

Incubationperiod,durationofillness,andmortalityamong90newbornWistarratsinoculatedintracerebrallywith20␮lofserialdilutionsofAlkhumrahemorrhagicfever virus(AHFV).

DilutionofAHFV(10%brainsuspension) No.ofintracerebrally inoculatedrats

Incubationperiodin days(mean±SD, range)

Durationofillnessindays (mean±SD,range)

Mortality(%)

10⁻¹ 10 3.7±0.48(3–4) 3.0±0.82(2–4) 10(100)

10⁻² 10 4.6±0.97(4–7) 2.7±0.82(2–4) 10(100)

10⁻³ 10 7.0±4.24(3–16) 3.9±2.02(2–8) 10(100)

10⁻⁴ 10 5.6±0.52(5–6) 5.2±1.03(3–6) 10(100)

10⁻⁵ 10 5.7±0.48(5–6) 4.9±1.91(1–7) 10(100)

10⁻⁶ 10 5.8±0.42(5–6) 5.1±0.99(4–7) 10(100)

10⁻⁷ 10 7.5±2.80(6–15) 3.4±0.84(3–5) 9(90)

10⁻⁸ 10 13.6±5.38(6–18) 3.7±1.57(2–6) 3(30)

10⁻⁹ 10 0 0 0

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Fig.5.Infectedrats’spleen(a,hematoxylinandeosinstain,20×)andkidney(b, hematoxylinandeosinstain,4×)showingnohistopathologicalabnormalities.

andPowassanvirus-infectedmice,thelocalizationwasdifferent, withastrikinginvolvementofthecerebelluminOHFVinfection (Holbrooketal.,2005).

Histopathologyofthelivers showedmoderateinﬂammatory response around the portal tracts without interface hepatitis, whereasthatofthespleensandkidneyswascompletelynormal.

SpleensofthePowassanvirus-infectedmiceshowedprominent activationofthecellsinthewhitepulpwithformationofill-deﬁned germinalcenterscontainingtangiblebodymacrophages.Thered pulpcontainedlargemacrophage-likecells(Holbrooketal.,2005).

Thespleen’swhitepulpoftheOHFV-infectedmiceappearedtobe lessorganizedandlessactivatedthanthatofthePowassanvirus- infectedmiceandtheredpulpwascongestedwithmildincreasein numberofhematopoieticcells(Holbrooketal.,2005).Theliversof AHFV-infectedratsshowedpatchymixedinflammatoryinfiltrate intheportaltracts.ThisissimilartothechangesseeninPowassan virus-infectedmice(Holbrooketal.,2005).Incontrast,theliversof theOHFV-infectedmiceappearedtohavemoresevereinflamma- tion,withevidenceofmononuclear-cellinfiltrationandprominent Kupffercells(Holbrooketal.,2005).SimilartoAHFV-infectedrats, thekidneyswereessentiallynormalinPowassanvirus-andOHFV- infectedmice(Holbrooketal.,2005).

RecoveryofAHFVfromthebrainsofnewbornWistarratsinoc- ulated with serum of infected patients was superior to direct detectionAHFV-RNA fromtheserumorplasma.Ratinoculation waspositiveinallfivepatientswithconfirmedinfection,whereas onlyfourofthefivepatientswereconfirmedwithdirectAHFV-RNA detectionfromeitherserumorplasma.Previousreportsalsoindi- catedthatratswereutilizedsuccessfullyinpropagationofanother

Fig.6. Indirectﬂuorescentantibodytest(IFAT)resultsbefore(a)andafter(b) inoculationofLLCMK-2cellculturewithAlkhumrahemorrhagicfevervirus(AHFV)- infectedratbrainsuspension.TheinoculatedcellsclearlyexpressAHFVantigensin thecytoplasm.

ﬂavivirus,theJapaneseencephalitisvirus(Duffy,1953;Ogataetal., 1991;Kimura-Kurodaetal.,1993).

TheexactreservoirsofAHFVremainunknown(Madani,2005;

Madanietal.,2011).Currentepidemiologicaldatasuggestclear associationofhumaninfectionwithlivestockanimalsparticularly sheep,goat,andcamelsdespitetheabsenceofanymanifestations ofillnessinsuchanimals(Madanietal.,2011).Directcontactwith theseanimalsorhandlingoftheirfreshraw meatisa strongly suspectedprimarymodeoftransmission.Additionally,mosquitoes seemalsotobeimportantvectorsinthetransmissionofthevirus fromanimalstohumans(Madani,2005;Madanietal.,2011).The recentreportofsuccessfulpropagationofAHFVinmosquitocells lendsfurthersupporttothespeculatedmosquito-bornemodeof transmission(Madanietal.,2012a,b).Becauseoftheclosephyloge- neticsimilaritybetweenAHFVandKyasanurForestdiseasevirus, ticksarebelievedtoplayanimportantrolein thetransmission cycleofAHFV(Charreletal.,2001).Thisisfurthersupportedby thePCR-baseddetectionofaviruscloselyrelatedtoAHFVfrom anOrnithodorossp.tickinJeddah,andOrnithodorossavignyiand HyalommadromedariiticksinNajran,SaudiArabia(Charreletal., 2007; Mahdietal., 2011; Doddet al.,2011).However,clinico- epidemiological studiesindicatethat ticksdo not seem toplay animportantroleintransmissionfromanimalstohumanseven though,theroleofticksasreservoirsofthevirusinitsecologicniche andasvectorstransmittingthevirusbetweenanimalsandperhaps alsofromanimalstohumansisconceivablypossible(Madani,2005;

Madanietal.,2011,2012a,b).

(7)

Theroleofrodents,shrews,andotheranimalsinthetransmis- sioncycleofAHFVisyettobeelucidated.Noanimalexperiments todeﬁnetherolesofsuchanimalshavebeenreported.Adaptation toananimalmodelisanessentialsteptostudythisvirusindepth.

Wistarratsareanoutbredstrainofalbinoratsbelongingtothe speciesRattusnorvegicus(Krinke,2000).Thisstrainwasdeveloped attheWistarInstituteattheUniversityofPennsylvania, USAin 1906foruseinbiologicalandmedicalresearch.Itistheﬁrstrat straindevelopedtoserveasamodellaboratoryanimalatatime whenlaboratoriesprimarilyusedMusmusculus,thecommonhouse mouse(Krinke,2000).Inthisstudywewereabletopropagatethe virusinthebrainsofnewbornWistarratswhichmaythusbeuti- lizedintheprimaryisolationofAHFVfromclinicalspecimensand befurtherexaminedinpathogenesisstudiesofAHFV.

In conclusion, this study is the ﬁrst report to demonstrate that AHFV is capable of replicating in vivo in the brains of newborn Wistar rats causing neurological manifestations fol- lowed by death. The resultant virus titer of brain suspension of the infected rats was 10^9.4 RLD₅₀/ml. Newborn Wistar rat may thus be utilized for the primary isolation of AHFV from clinicalspecimens andpropagationof hightiteredvirusstocks.

Wistarratsmayalsobeusefulforfurtherexperimentalstudiesof AHFV.

Conﬂictsofinterest

Theauthorsdeclarethattheyhavenoconﬂictsofinterests.

Financialsupport

This study is one of the research products of the Scientiﬁc Chair of Mohammad Hussein Alamoudi for Viral Hemorrhagic Fevers,KingAbdulazizUniversity,Jeddah,SaudiArabia.Thespon- sor,SheikhMohammadHusseinAlamoudi,hadnoinvolvementin thestudydesign,inthecollection,analysisandinterpretationof data,inthewritingofthemanuscript,orinthedecisiontosubmit themanuscriptforpublication.

Authors’contributions

T.A.M. conceived and designed the study, carried out clinical assessment of the patients and collection of the samples, andwrotethemanuscript;M.K.andE.M.E.A.performedtheani- mal experiment and viral isolation and assisted in writing the manuscript; E.M.E.A., assisted in the conception and design of thestudy, analysisand interpretation ofdata, collection ofthe samples,andcriticalrevisionofthemanuscript;E.I.A.performed theReal TimeRT-PCRandassisted in collectionofthesamples andinterpretationofdata;H.M.S.A.assistedininterpretationof data and critical revision of themanuscript; H.A. provided the Wistar rats used in theexperiment and assisted in interpreta- tionofdata;R.Y.B.performedthehistopathologicalexamination and interpreted the histopathological changes. T.G.K. provided hyperimmunemouseasciticfluidcontainingpolyclonalantibodies againstAHFVfortheindirectfluorescentantibodytestandcritically revisedthemanuscript;allauthorsread andapprovedthefinal manuscript.

Acknowledgments

TheauthorsthankSheikhMohammadHusseinAlamoudifor sponsoringtheScientiﬁcChair forAlkhumraHemorrhagicFever VirusResearchatKingAbdulazizUniversity,Jeddah,SaudiArabia.

WethankthetechnologistAhmedM.Hassanforhisexcellenttech- nicalassistanceintheanimalexperimentsandviralisolationand

forcapturingimagesandﬁlmingtherats.Wethankthetechnol- ogistsBadrMasriandNooraA.Al-Kaiediforperformingthereal timeRT-PCR.

AppendixA. Supplementarydata

Supplementarydataassociatedwiththisarticlecanbefound, in the online version, at http://dx.doi.org/10.1016/j.jviromet.

2013.12.004.

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