First molecular analysis of rabies virus in Qatar and clinical cases imported into Qatar, a case report

Case

Report

First

molecular

analysis

of

rabies

virus

in

Qatar

and

clinical

cases

imported

into

Qatar,

a

case

report

Bas

B.

Oude

Munnink

a,1,

*

,

Elmoubashar

Abu

Baker

Abd

Farag

b,1

,

Corine

GeurtsvanKessel

a

,

Claudia

Schapendonk

a

,

Anne

van

der

Linden

a

,

Robert

Kohl

a

,

Georgina

Arron

a

,

Hisham

Ziglam

c

,

Wael

Goravey

Mhjoop

Goravey

c

,

Peter

V.

Coyle

c

,

Imad

Ibrahim

c

,

Khaled

A.

Mohran

d,e

,

Muneera

Mohammed

Saleh

Alrajhi

d

,

Md

Mazharul

Islam

d

,

Randa

Abdeen

d

,

A.

Aziz

Mahmoud

A.H.

Al-Zeyara

d

,

Nidal

Mahmoud

Younis

d

,

Hamad

Eid

Al-Romaihi

b

,

Mohammad

Hamad

J.

Al

Thani

b

,

Richard

Molenkamp

a

,

Reina

S.

Sikkema

a

_,

_Marion

_Koopmans

a

a

ErasmusMC,DepartmentofViroscience,WHOcollaboratingcentreforarbovirusandviralhemorrhagicfeverReferenceandResearch, Rotterdam,theNetherlands

b

MinistryofPublicofHealth,Doha,Qatar

c_{HamadMedicalCorporation,Doha,Qatar}

d_{DepartmentofAnimalResources,MinistryofMunicipalsandEnvironment,Doha,Qatar} e

AnimalHealthResearchInstitute,BiotechnologyDepartmentsERC,Dokki,Egypt

ARTICLE INFO Articlehistory:

Received19November2019

Receivedinrevisedform23April2020 Accepted25April2020 Keywords: Nanoporesequencing metagenomicsequencing rabiesvirus ABSTRACT

Identifyingtheoriginoftherabiesvirus(RABV)infectionmayhavesignificantimplicationsforcontrol measures.Here,weidentifiedthesourceofaRABVinfectionoftwoNepalesemigrantsinQatarby comparingtheirRABVgenomeswithRABVgenomesisolatedfromthebrainsofaRABVinfectedcamel andfoxfromQatar.

©2020TheAuthors.PublishedbyElsevierLtdonbehalfofInternationalSocietyforInfectiousDiseases. ThisisanopenaccessarticleundertheCCBY-NC-NDlicense(

http://creativecommons.org/licenses/by-nc-nd/4.0/).

1.Introduction

Rabies virus (RABV) is a single-stranded RNA virus with a genomiclengthofaround12kbandispartoftheRhabdoviridae family (Amarasinghe et al., 2017). Human infection may occur whenexposedtoinfectedanimals,mainlycarnivoresbutalsobats, andisalmostalwaysfatal,rankingrabiesamongthemostlethal diseases (Liu et al., 2019). RABV infection can be prevented throughvaccinationofhumansandwildanddomesticcarnivores, which hassuccessfully eliminated RABV from Western Europe (Muller and Freuling, 2018). However, RABV continues to be enzooticin largeparts oftheworld,suchasinAsiaand Africa, where it causes an estimated 35,172 cases and 21,476 human deaths yearly (WHO). Dogs are the main reservoir for human rabies(Hampsonetal.,2015).

RABV is commonly subdivided into six phylogeneticclades, namelytheAfrica2,Africa3,Arctic-related,Asian,Cosmopolitan, andIndiansubcontinentclades.InNepal,boththeArctic-relatedas wellastheIndiansubcontinentRABVcladearepresent(Pantetal., 2013),whiletheCosmopolitanRABVcladecurrentlycirculatesin theArabicPeninsula(Hortonetal.,2015;Troupinetal.,2016).

Recently, two RABV infections were diagnosed in Qatar in Nepalesemigrantworkers.Inthisstudy,wesoughttounravelthe source of these RABV infections. Therefore, whole genome sequences were generated from brain tissues of two human patientsaswellasfrombraintissuesofarabidcamelandarabid foxfromQatartodeterminetheirpotentialgeneticrelationship. 2.Thestudy

OnJuly18th_{,2018,thefirstpatient,a 33-year-oldNepaliwas}

admittedtotheHamadGeneralHospital.Thepatienthadarrived in Qatar onemonth earlierand reported ananimal biteby an unknownanimalthreemonthsagoinNepal.OnJuly27th_,aRABV

infectionwascon_firmedusingtwodifferentreal-timePCRassays

* Correspondingauthor.

1

Theseauthorscontributedequallytothismanuscript.

https://doi.org/10.1016/j.ijid.2020.04.070

1201-9712/©2020TheAuthors.PublishedbyElsevierLtdonbehalfofInternationalSocietyforInfectiousDiseases.ThisisanopenaccessarticleundertheCCBY-NC-ND license(http://creativecommons.org/licenses/by-nc-nd/4.0/).

InternationalJournalofInfectiousDiseases96(2020)323–326

ContentslistsavailableatScienceDirect

International

Journal

of

Infectious

Diseases

onsalivasamples(Wakeleyetal.,2005;Wadhwaetal.,2017).The patienthadnohistoryofRABVvaccinationanddiedonAugust6th, 2018. On February 24th_{, 2019, a second patient, a 25-year-old}

NepalifromtheNuwakotdistrict,visitedtheQatarRedCrescent worker's health center in Mesaimeer after which he was transferred to the Hamad General Hospital. According to his relatives,hehadbeenbittenbyaraccoon(Procyonlotor)inearly October2018inNepal.RABVinfectionwasconfirmedonthe18th_of

April using the RT-PCRs described above. The patient died on March19th_{, 2019.There wereno RABV neutralizing antibodies}

detectableinserumbyfluorescentantibodyvirusneutralizationin both patients. Post-mortem tissue samples were taken after obtainingconsentfromtherelatives.

InSeptember2018,theAnimalHealthDepartmentinQatarwas notified of a fox (Vulpes vulpes) attack at a camel (Camelus dromedarius) farm in the Alkharsah area. Approximately two weeksafterthefoxbite,acamelstartedtodevelopneurological symptomsanddied.Thefoxwascaptured,andbraintissuefrom boththefoxand thecameltestedpositivefor RABVbyRT-PCR (Hoffmannetal.,2010)attheAnimalHealthDepartmentinQatar

Figure1.PhylogeneticanalysisoftheN-geneoftherabiesvirusgenomessequencedinthisstudy.Greenindicatesthevaccinestrains,redindicatesthenewlysequenced samples,andblueindicatesthevirusesfromNepal.Amaximum-likelihoodtreewasconstructedundertheGTR+F+I+G4modelasthebest-predictedmodelusingthebest modelpredictionfunction,ultrafastbootstrapping,and1000replicates.Thetreeismidpointrooted,andthescalebarrepresentsthenumberofsubstitutionspersite. 324 B.B.OudeMunninketal./InternationalJournalofInfectiousDiseases96(2020)323–326

inSeptember2018andNovember2018,respectively.Eventhough theNepalihad nodirect animal contact inQatar, theseanimal sampleswereincludedasthereisnoRABVgenomicsequencedata fromQataravailable for comparison and sourcetracking.Brain material from both animals was collected, stored in Virus Transport Medium (VTM), and shipped to the Erasmus MC togetherwiththesamples fromboth humanpatientsfor virus isolationandsequencing.

Humanandanimalbrainsamplesweresuspendedin supple-mentedDMEM(Thermofisher).Frompatient1materialfromthe leftbrainwas cultured while frompatient2 materialfrom the hippocampus, the midbrainand the cerebellum werecultured. Cerebellum samples were taken from the animals. Mouse neuroblastoma astrocytes cells were seeded in supplemented DMEMina24-wellplate.Upon80%confluency,200

m

lofbrain suspensionwasinoculated onthecells,followed by15minutes centrifugation at 3500xg. The plate was centrifuged in a plate centrifugefor15minutesat3500xg,andtheculturemediumwas refreshed.Cultureswereplacedat36,5Cina5%CO2humidified incubatorandcheckedforcytopathiceffectdaily.OnceCPEwas visible,thesupernatantoftheculturewascollected,centrifuged for fiveminutes at 5000xg, and filtered using a 0.45

m

mfilter. NucleicAcidwasextracted,andcDNAwasmadeusingsuperscript IV (ThermoFisher)and random primers(ThermoFisher). dsDNA wasmadeusingKlenow(NEB)andusedasinputforamultiplexed metagenomic Nanopore sequencing using the SQK-PBK004 kit (Nanopore)onanR9flowcell.

Sequences were demultiplexed and mapped to a randomly derivedNepaleseRABVgenomefromGenBank(KX148228)using minimap2(Li,2018).Theconsensussequenceswerecomparedto the non-redundant database using BLASTn, and the reference-based alignment was repeated with the closest reference sequence, using a 100x read coverage cut-off (Oude Munnink etal.,2019).Phylogeneticanalysiswasperformedonthe10closest BLASThitsforthedifferentviruses,andalsoallcompleteRABV genomes from the Arabian Peninsula, Nepal, and neighboring countries were included (GenBank, 27-04-2019). Phylogenetic analysiswasalsoperformedonallfull-lengthN-genesequences fromNepal,asthishasbeenstudiedpreviouslyinNepal(Pantetal., 2013).Sequences were aligned using MUSCLE,after which the alignment was manually inspected. Phylogenetic analysis was performedin IQ-TREE(Nguyen etal., 2015)undertheGTR+F+ I+G4modelas thebest-predictedmodel usingthebestmodel predictionfunction,ultrafastbootstrapping,and1000replicates. Phylogeneticanalysisbasedonthefull-lengthN-geneandon full-lengthsequencesrevealedthattheRABVfrompatient1clustered withsequences fromthe Indian subcontinent clades while the RABV sequence frompatient 2 clusteredwith theArctic-like3 clades (Figure 1 and Supplementary Figure 1). Both human patient viruses clustered most closely with viruses previously detectedinNepal.TheRABVsequencesfromthefoxandcamel cluster within the Cosmopolitan clades, with viruses from the ArabicPeninsula.

3.Conclusions

Here,wedescribethefirsttwocompleteanimalRABVsequences from Qatar and two RABV sequences from Nepalese patients diagnosedwith RABVin Qatar. Weshow that thehumanRABV strainsclusterwithRABVsequencespreviouslyidentifiedinNepal. In contrast, theanimal RABV sequences fromQatar are part of anothercladeandclusterwithsequencesfromtheArabicPeninsula, whichis inline with epidemiologicalinformationand exposure history.Therefore,weconcludethatthesehumanRABVinfections were acquiredin Nepalandnot in Qatar. However,given theobserved diversityofRABV,moreinformationaboutthediversityofRABV

circulating in Qatar is needed for morerobust conclusions. The identificationof twoimportedcasesof RABVdemonstrates that QatarisatriskofintroducingRABV,especiallyinmigrantworkers. ThelastreportofananimalRABVinfectioninQatardatesfrom 2009,accordingtotheWorldOrganisationofAnimalHealth(OIE

World Animal Health Information System, 2019). Our report demonstratesthatRABVisstillpresentinQatar.Thisshowsthe needforfurtherepidemiologicalresearchandcontrolmeasureson thehuman-animalinterface.IfRABVisdetectedinregionswith stray dogsand foxes,wildcarnivorevaccinationisanoptionto preventeconomicdamagetocamels.Also,inareaswhereRABVis identi_fied, human and animal health care workers should be informed and alerted to the presence of RABV and veterinary surveillanceshouldbeputinplace,aswellascarefulassessment andpossibletreatmentofhumansafteranimalbites.

Ethicalapproval

Ethicalapprovalforallactivitiesdescribedinthismanuscript wasobtainedbytheHealthResearchGovernanceDepartmentat theMinistryofPublicHealthinQatarundertheprotocolID MRC-04-19-387.

Conflictofinterest

Theauthorshavenoreportedconflictofinterest. Acknowledgments

This work hasreceived funding fromthe European Union's Horizon 2020 research and innovation program under grant agreementNo.643476(COMPARE).WewouldliketothankSaleh JarallaAlmarri,AbdullaMohdZNAL-Marri,TarekMAMElsherbini, Ahmed Alatafi Almutawali Aldasuki and Ibrahim Mahmoud IbrahimAliforthesamplecollections andTonyVincentChawla andJalaluddinBhuiyanforfacilitatingtheshipment.Wewouldlike tothanksAnnemiekvanderEijkforclinicalsupport.

AppendixA.Supplementarydata

Supplementarymaterialrelatedtothisarticlecanbefound,in theonlineversion,athttps://doi.org/10.1016/j.ijid.2020.04.070. References

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