Aedes albopictus Mosquito: The Main Vector of the 2007ChikungunyaOutbreakinGabonFre de ricPage` s1*,ChristopheN.Peyrefitte2,Me dardToungMve3,FannyJarjaval1,SylvainBrisse4,IsabelleIteman4,PatrickGravier2,5,Dieudonne Nkoghe3,MarcGrandadam21Institut de me decine tropicale du Service de santedes arme es, Unitedentomologie medicale, URMITE UMR 6236, Marseille, France, 2Institut de me decine tropicale duService de santedes arme es, Unitede virologie tropicale, Marseille, France, 3Ministe` re de la Santepublique, Libreville, Gabon, 4Institut Pasteur, plate-forme genomique,Paris, France, 5Serviceme dical du6eBIMA, Libreville, GabonAbstractThe primary vector at the origin of the 2007 outbreak in Libreville, Gabon is identified as Aedes albopictus, trapped aroundthenearbyFrenchmilitarycamp. TheChikungunyaviruswasisolatedfrommosquitoesandfoundtobeidentical totheA226V circulating human strain. This is the first field study showing the role of the recently arrived species Aedes albopictusin Chikungunya virus transmission in Central Africa, and it demonstrates this species role in modifying the epidemiologicalpresentationofChikungunyainGabon.Citation: Page` sF, PeyrefitteCN, MveMT, Jarjaval F, BrisseS, etal. (2009)AedesalbopictusMosquito: TheMainVectorofthe2007ChikungunyaOutbreakinGabon. PLoSONE4(3): e4691. doi:10.1371/journal.pone.0004691Editor:DeeA. Carter, UniversityofSydney, AustraliaReceivedNovember6, 2008; AcceptedFebruary2, 2009; PublishedMarch4, 2009Copyright: 2009Pages et al. This is anopen-access articledistributedunder theterms of theCreativeCommons AttributionLicense, whichpermitsunrestricteduse, distribution, andreproductioninanymedium, providedtheoriginal authorandsourcearecredited.Funding:Thisworkissupportedbythefrenchsurgeongeneral. Thefundershadnoroleinstudydesign, datacollectionandanalysis, decisiontopublish, orpreparationofthemanuscript.CompetingInterests:Theauthorshavedeclaredthatnocompetinginterestsexist.*E-mail: frederic_pages@yahoo.comIntroductionFirst isolated in Tanzania in 1952, Chikungunya virus(CHIKV), an Alphavirus member of the Togaviridae, is now aworld-wide public health problem[1,2]. ACHIKVoutbreakbegan in Kenya in 2004, spread to populated islands in the IndianOceanandlater jumpedtoIndia, andEurope [36]. Thoughpreviously considered to be a vector with poor efficiency, Ae.albopictuswasidentifiedasthemajorvectorinLaReunionIslandandEurope [2,7]. Sequence analysisof the virusgenome revealedthat these recent outbreaks were caused by a new variantcharacterizedbya mutation in the E1 envelope glycoproteingene(A226V)[4].ThismutationhasfavoredimprovedtransmissibilityofthevirusbythemosquitoAe. albopictus[8].In Africa, while recent serological surveys suggest a highprevalence of Togaviridae, Flaviviridae and Bunyaviridae [9] andthere is recent evidence of CHIKVcirculation in RepublicDemocratic of Congo [10] and Cameroon [11], detailedinformationabout CHIKVcirculationremains imprecise. TheChikungunyavirus was classicallyisolatedfromseveral sylvaticAedes sp. includingAedes africanus, Aedes furcifer, Aedes luteocephalus,Aedes neoafricanus, Aedes taylori [12,13]. Rural outbreaks werefound to be heavily dependent upon the densities of these sylvaticmosquitos, which increase during periods of heavy rainfall.Duringthelate1970s, serological surveys conductedinGabonrevealedaCHIKVexposureseroprevalenceof 8.5%inurbanLibreville (the capital of Gabon), rising from20%to 44%inrural areas[14]. Inrecent times, theAsiantigermosquitoAedesalbopictushassucceededincolonizingsomepartsofsub-SaharanAfrica.IncentralAfrica,thisspreadingvectorwasfirstcollectedin2000 inCameroon, in2003 inEquatorial Guinea, andin2006inGabon[15].Duringthelast6years,theurbancyclehastended to play an increasing role in CHIKVtransmission inCentral Africa[11], bringingintoquestiontheparticipationofurbanAedes sp. suchas Aedes aegypti or the recentlydiscoveredAedesalbopictusasaprimaryvector.InApril 2007, anoutbreakofDengue-likesymptomsoccurredinLibrevillewhereAedesalbopictuswasfirstdetectedinDecember2006. The majority of patients were presumptively treated formalariawithnosymptomimprovement. Whenmalariadiagnosistests (bloodthicksmear, slideormalariaCoreHrapidtest) wereperformed, theresults werenegativeandCHIKVwasidentifiedfrompatientbloodsamplesasthemaincausativeagent[16].TheGabon Ministry of Public Health commissioned a crisis committeeto manage the outbreak; patients were identified as suspectedCHIK cases if they presented with a sudden fever over 39uC whichlastedformorethan48hinspiteof anti-malarial treatment, inadditiontoaheadacheandseverejointandmusclepain,withorwithout a rash. From May 2007 throughSeptember 2007, 13,802suspectedcaseswererecordedinhealthcentersand829patientswerehospitalized, thoughfortunatelynodeaths occurred. FromApril 2007 through May 2007, 5,000 additional cases wereretrospectively included by the Gabon health authorities afterfurtheranalysis(Figure1).ThetransmissiondecreaseobservedinJune was presumably due to the end of the rains andcorresponding decrease in vector densityboth Aedes albopictusadult abundanceandovipositionratearecloselycorrelatedwithrainfall [17]. During the peak of the May outbreak, CHIKVtransmission was also recorded in Bitam, the capital of theWoleumNtemprovince(northGabon). SinceAugust 2007, nosuspectedcaseshavebeenrecordedinLibrevilledespitethestartof the rainy seasoninSeptember of that year. However, newCHIKcases wereconfirmedinNovember 2007inLastourville,OgoueLoloprovince(south-eastGabon).PLoSONE | www.plosone.org 1 March2009| Volume4| Issue 3 | e4691In Libreville, Aedes albopictus and Aedes aegypti are potentialvectorsofCHIKandDENviruses. Toassesstherespectiverolesof Aedes albopictus andAedes aegypti inDengue andchikungunyatransmission in Libreville, we proceeded in two steps. Anexperimental study was conducted in the laboratory, usingexperimental infections todetermine the ability of Ae. albopictusand Ae. aegypti collected in Libreville to transmit CHIKandDenguevirus[18]. Ae. albopictusshowedahighersusceptibilitytoCHIKvirus thanAe. aegypti. Thus, Ae. albopictus appears tobeabetter vector for CHIKvirus than Ae. aegypti. Moreover, asdemonstrated by the rates of disseminated infection obtained,GaboneseAe. albopictuswaslesssusceptibletoDENvirus, leadingustoconsiderAe. albopictusasasecondarydenguevector. Duringthesameperiod,afieldstudywasconductedtoresearchDENV-andCHIKV-infectedmosquitoes. The objective of the presentstudy is to identify the primary vector at the origin of the Librevilleoutbreak.MethodsBg-sentinel odor traps (Biogents AGRegensburg, Germany)were used in the French military camp of Libreville (lat: 0.44 long:9.43) and its neighboring areas (Figure 2). Traps were set upduringtheentiredayfor15days.Catchbagswerecollectedeachmorning (7 am) and evening (8 pm). Mosquito identifications werecarried out using one of several keys [19,20,21]. The averagenumbers of Aedes sp. caught inside andoutside the campwerecomparedusing Students t-test. Eachdays collections of Aedesmosquitoesweresortedbytrap, speciesandsex. Mosquitopoolswere mixed with sterile PBS, crushed and 0.22 mmfiltered(disposable0.22 mmfilter,DutscherDominique,67172Brumath,France). For samples of fewer than 5 mosquitoes, a PBS volume of200 ml wasusedforcrushing; forlargersamples, thevolumewasincreasedtopreventthemixturebecomingtooviscous. GenomicCHIKVdetectionwas performedfor eachpool usingTaqManRT-PCR [22], while Dengue virus genome detection wasconductedusingapreviouslydescribedmethod[23]. Toisolatethevirus,thesupernatantsofeachgenomicCHIKVpositivepoolwere inoculatedontoVeroE6andC6/36cell lines [22]. TheCHIKVgenomeswerepartiallysequenced(E1-30UTRjunction)andcomparedtohumanisolates collectedduringthis outbreak(GenBankaccessionnumbers EF613342, EF613343, EF613344);alignmentwasperformedwithClustalW1.7software.Figure 1. Rainfall in Libreville, Gabon from September 2006 toSeptember 2007 (grey bars) and monthly numbers of suspect-ed Chikungunya cases in Gabon, March 2006September 2007(blackcurve).doi:10.1371/journal.pone.0004691.g001Figure2. Locationofmosquitotrappingsitesbothinside[49] andoutside[13] themilitarycampofFrencharmedforcesinLibreville,Gabon.doi:10.1371/journal.pone.0004691.g002ChikungunyaFeverinGabonPLoSONE | www.plosone.org 2 March2009| Volume4| Issue 3 | e4691ResultsDuringthetwoweeks studiedinJuly2007, atotal of 10,605mosquitoes werecaught: Culex quinquefasciatus (5642females and3807males),Mansoniauniformis(78femalesand4males),Anophelesgambiae (7 females and 1 male), Anopheles sp. (8 females and 1 male),Aedes sp. (10females), 1046Aedes albopictus (801females and245males) and80Aedesaegypti (71femalesand9males). Theaveragenumber of Ae. albopictus specimens per trap caught inside andoutside the camp were 1.7 and 16.3, respectively (p,0.0001).There was no statistical difference between the average number ofAe. Aegypti specimens caught inside andoutside the camp. PoolsizesandcompositionsaregiveninTable1,withanaveragepoolsize of 3.81, and a range from 1 to 41. Neither the Aedes aegypti norAedes albopictus pools were positive for Dengue virus. The twoAedes albopictus female pools (N=21, N=12) were positive forCHIKVaccordingtogenome detectionbyTaqManRT-PCR,and Chikungunya virus was also isolated when their filteredsupernatantswereinoculatedontoVeroE6andC6/36celllines.The female Aedes albopictus minimal infection rate (calculated as theratioof positivepools tothe totalnumber of Aedes albopictusfemalemosquitoes tested) was approximately 2.5%. The CHIKVgenomes were partially sequenced(GenBank accessionnumberEU403052). Whencomparedtothehumanisolates frombloodsamples of Gabonese patients [16], the Aedes albopictus isolatedCHIKV displayed 98.7% to 99.1% identity at the nucleotide leveland 99.4% to 99.6% identity at the amino-acid level. Both humanandmosquitoisolates displayedtheA226VmutationwithintheE1gene[4].DiscussionSequence analysis of the virus genome revealed that therecent CHIKV outbreaks were caused by a new variantcharacterizedby a mutationinthe E1 envelope glycoproteingene (A226V) [4]. The A226Vmutationhas favoredabettertransmissibilityofthevirusbythemosquitoAe.albopictus[8].InGabon, wefoundthesamevirusvectorassociationasdetectedduring the 2006 Reunion Island Outbreak: A226VmutatedCHIKVandanAedes albopictusstrain. Interestingly, ananalysisof full-length viral sequences reveals three independent instancesof Ae. albopictus exposuretothevirus inIndia, CameroonandGabon, eachfollowedby the acquisitionof a single adaptivemutationproviding a selective advantage for transmissionbythis mosquito[24]. Therefore, theA226Vmutationis stronglybelievedtoplayaroleintheepidemiologicalsuccessofCHIKVinGabon.Inside the camp, the vector control programagainst Aedesmosquitoes is based on the destruction or elimination of unwantednatural and artificial water containers. The efficacy of this strategyis highlightedbythe loweraverage numberof Ae. albopictus caughtintraps insidethecamp. This studyconfirms that reducingthenumber of such containers in and around homes significantlydecreasesthesizeofAe. albopictuspopulations.Thelownumberof Aedes aegypti captureddidnot allowustodetermineif thisspecieshadparticipatedasasecondvectorforCHIKVtransmission during the outbreak. In Libreville, Ae.aegypti andAe. albopictusareoftensampledinthesamecontainer[15], but Ae. albopictus exploits awider range of breedingsitesthanAe. aegypti, whichisclassicallymoredependentonartificialbreeding sites than Ae. albopictus. During the dry season, thereductionofartificialbreedingsiteshasastrongerimpactonAe.aegypti populationsize. Ourfieldstudyis not abletoclarifytheroleof Ae. aegypti inCHIKVtransmissionduringtheoutbreaknor to identify Ae. aegypti as the DENvector in Libreville.Nevertheless, the results of experimental infectionsuggest thatAe. aegypti wasprobablyabetterDENvectorinLibreville[18].Ae. albopictus was amoreefficient experimental vectorof CHIKvirus than Ae. aegypti. formosus, and is assumed to be a betterCHIKvectorinthefield, notplayingasignificant roleinDENtransmissioninGabon.Thehighlevel of identitybetweenthehumanandmosquitovirus isolates fromLibreville indicates a strong relationshipbetweenthese CHIKVisolates, while the finding of CHIKVinfectedAedesalbopictusduringthemiddleofthedryseasonandthe highexperimental infectionrate highlight the role of thismosquito as the primary vector during the 2007 outbreak inGabon. This was the first field study showing the role of recentlyarrivedAedesalbopictusinthetransmissionofChikungunyavirusinCentral Africa.Aedesalbopictushascurrentlybeencollectedinfour of the Gabons nine provinces, indicating an ongoingcolonization of the country similar to the colonization ofCameroon[25]. Interestingly, epidemic CHIKVtransmissiononly occurred in the major population centers of theseprovinces, such as Libreville (Estuaire Province), Bitam(Wo-leumNtemProvince) andLastourville(OgoueLoloProvince),emphasizing the role of Aedes albopictus inthe modificationofChikungunyaepidemiologicalpresentationinGabonfromruralandendemic tourbanandendemo-epidemic. This trendwasobserved as early as 2000 in outbreaks in the DemocraticRepublic of the Congo [10]. As Ae. albopictus continues tospread,displacingAe.aegyptiinAfricancountrieslikeCameroon[25],theoccurrenceofthisvectorintheAfricanrainforestisofconcern, as yellowfever andother arboviruses areendemicincentralAfrica.InGabon, Ae.albopictusshouldnotbeanefficientDENvector [18], but we cannot forecast the results as itencounters West Nile virus, Sindbi virus, Orungo virus orSemliki forest virus[26,27].AuthorContributionsConceivedanddesignedtheexperiments: FP.Performedtheexperiments:FPMTMFJPGDNMG. Analyzedthedata: FPCPMG. Contributedreagents/materials/analysistools:CPFJSBIIMG. Wrotethepaper:FPCP.Table 1. Total number of Aedes albopictus and Aedes aegyptitrappedbylocationbothinside[49]andoutside[13]theFrenchmilitarycamp, Libreville, Gabon, July2007.trapnumberAedesaegypti Aedesalbopictusfemales males females males1 9 0 373a482 12 2 257b1493 17 4 38 304 5 0 6 25 4 0 7 06 7 0 9 17 5 2 25 18 6 1 48 109 6 0 38 4All traps 71 9 801 245aonepool(21mosquitoes)infectedbyCHIKV(July14th2007).bonepool (12mosquitoes)infectedbyCHIKV(July20th2007).doi:10.1371/journal.pone.0004691.t001ChikungunyaFeverinGabonPLoSONE | www.plosone.org 3 March2009| Volume4| Issue 3 | e4691References1. Ross RW(1956) The Newalaepidemic. III. The virus: isolation, pathogenicpropertiesandrelationshiptotheepidemic. JHyg(Lond) 54: 177191.2. ReiterP,FontenilleD,PaupyC(2006)Aedesalbopictusasanepidemicvectorofchikungunyavirus: anotheremergingproblem?LancetInfectDis6: 463464.3. Chretien JP, Anyamba A, Bedno SA, Breiman RF, Sang R, et al. (2007)Drought-associated chikungunya emergence along coastal East Africa. 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