How Do Virus–Mosquito Interactions Lead to Viral Emergence?

2018·2.1 MB·English

by  Claudia Rückert

Review
–
How Do Virus Mosquito Interactions Lead to

Viral Emergence?

Claudia Rückert1,* and Gregory D. Ebel1,*

Arboviruses such as West Nile, Zika, chikungunya, dengue, and yellow fever Highlights

viruseshavebecomehighlysignificantglobalpathogensthroughunexpected, ArbovirusessuchasWestNile,Zika,

explosive outbreaks. While the rapid progression and frequency of recent chikungunya,dengue,andyellowfever
viruseshaveb ecomeh ighl ysignifi cant
arbovirus outbreaks is associated with long-term changes in human behavior
global pathogens through unex-
(globaliza tion, urban iza tion, climat e ch ange), ther e are dir ec t mosqu ito–virus pected ,explosiveo utbreaks.

interactions which drive shifts in host range and alter virus transmission. This
Recent advances in next-generation
review summarizes how virus–mosquito interactions are critical for these
sequencinghaveallowedrapiddetec-
viruses tobecomeg lobal pathogensatmo lecular,phys iolog ical,ev oluti onary, tion and id entific ation of virus es, as
well astr acingvirusm ove mentus ing
and epidemiological scales. Integrated proactive approaches are required in
phylogeneticanalysis.
order to effectively manage the emergence of mosquito-borne arboviruses,

which appears likely to continue into the indefinite future. Evolutionaryandmodelingapproaches,

combined with experiments and new

surveillancemodalities,areattempting
Mosquito-Borne Viruses as the New Global Pathogens
topredictvirusemergence.

Viral pathogens are major causes of morbidity and mortality among humans and animals.

Efficienttransmissionofvirusesbetweensusceptiblehostsisrequiredinorderfortheseagents Vector competence and virus evolu-
tionstu dies–usings elect edmo squito
to persist in nature and ultimately cause disease. Several mechanisms for this exist, and include spe cies and neglec ted arbo viruses –
direct contact, aerosol and sexual transmission, among others. A subset of viruses, termed arehelp ingto identifyw hichvirusma y

arthropod-borne viruses (arboviruses) requires hematophagous arthropods, mainly mosqui- causethenextoutbreak.

toesandticks,fortransmissionbetweenvertebrates.Ingeneral,perpetuationofarboviruses
Integrated proactive approaches are
requires vertebrate viremia so that arthropods acquire infectious virus along with nutrient-
requiredinordertoeffectivelymanage
containingbloodduringfeeding.Transmissionofvirustoanewhostbyanarthropodinfectedin the eme rg ence o f mosqui to-borne

thismannerrequiresthatthisarthropodbeacompetentvector.Inpublichealthentomology, arboviruses, which appears likely to
the term‘vec torcomp eten ce’ referstoth ein he rentability ofapart icu lararth ropod totransmita continue into the in definite fu ture.

particularvirus.Incompetentvectors,virusisacquiredduringfeeding,undergoesreplicationin

guttissue,disseminatestosecondarysitesofreplication,includingthesalivaryglandsandis

ultimatelyreleasedintothearthropod’ssalivarysecretions,whereitmaybeinoculatedintothe

skinandcutaneousvasculatureofthehostduringsubsequentfeeding(Figure1).Arboviruses,

therefore,arethosevirusesthathaveevolvedanintimateassociationwithbothavertebrateand

arthropod hostinorderto perpetuateinnature.

Mosquitoes are the most important vectors of arboviruses [1], although many are main-
1DepartmentofMicrobiology,
tainedbyticks[2],phlebotomines[3],andotherarthropods[4].Theglobalhealthburdenof
ImmunologyandPathology,College
mosquito-borne viruses is immense. It is commonly estimated that between 50 and 100 ofVeterinary Med icineandB iomedical
million cases of infection b y dengue vi rus (DENV) se rotypes 1– 4 oc cur per y ear . Re cent Sc iences,Co loradoSt ateU niversity,
FortCollin s,CO805 23,U SA
estimatesplacedtheburdenofDENVat1.14milliondisability-adjustedlife-years in2013

[5].MostoftheindividualswhoareatriskofDENVinfectionareatriskofotherarboviruses,

including yellow fever virus (YFV), chikungunya virus (CHIKV), and Zika virus (ZIKV) which *Correspondence:

sharethesamemosquitovector,Aedesaegypti[6].Additionalarbovirusesthatburdenthe [email protected]
(C.Rückert)and
healthofindividualslivingin,ortravelingto,thetropicsincludeJapaneseencephalitisvirus
[email protected]
(JEV), Venezuelan equine encephalitis virus (VEEV), Mayaro virus (MAYV), o’nyong nyong (G.D.Ebel).

310 TrendsinParasitology,April2018,Vol.34,No.4 https://doi.org/10.1016/j.pt.2017.12.004

©2017ElsevierLtd.Allrightsreserved.
virus(ONNV),andmanyothers.Temperateregionsalsoexperienceseasonalepidemicsof Glossary

arboviraldiseasecausedbyWestNilevirus(WNV),LaCrossevirus(LACV),easternequine
Antigenemia:describeslevelsof

encephalitis virus (EEEV), Jamestown Canyon virus (JCV) and related viruses. Although antigenintheblood.Intheparticular

quantitative estimates of the collective burden of mosquito-borne arboviruses on human caseofincreasedNS1antigenemia

mentionedhere,itmeansthatthe
health worldwide do not currently exist, it is clear that their burden is enormous, and
morerecentisolatesofZIKVresultin
increasing [5,7,8]. increa sedNS 1levels in theb loodo f

infectedindividuals.

The geographic distribution of many arboviruses has expanded in recent decades [6,9], E1andE2glycoproteins:structural

resu lting in infec tion of naïv e p opula tions and pr ovidi ng opportu nit ies for new host –virus proteins incorporated into the
envelopeofthealphaviruses,such
relationshipstodevelop.Forexample,afterincursionsintoEuropeinthe1990s,WNV(genus asCHIKV .E 1a ndE2areimp ortant

Flavivirus)wasintroducedinto the Americasin1999andrapidly spreadfromasmallfocus forattachment,entry,andfusion
nearNew York Citythroug hou tthe NewWo rld .Simi larly, CHIKV (genus Alpha v irus)s pread eve nts of the vi rus.
Enzootictransmissioncycle:
fromanAfricanfocusintoAsiaduringthemid-2000sandwasintroducedintotheCaribbean
regio n in 2013 [10,11 ]. C HIKV is now en demic in th e Am eric as and has cau sed over one cdyecslceriobfeas tphaet hnoagtuernalb teratwnsemeniswsioildn
million inf ection s, many of whic h result in debilit ati ng a rthralgia [1 0]. Z IKV (genus Flaviv irus) anima ls. T heterm‘e nzootic’i s
equivalen tto thet erm‘endem ic’as
hasalsoemergedinrecentyears[12].FollowinganexpansionfromanAfricanfocusintothe
usedforhumandiseases.
Pacificislands,theviruswasintroducedintoSouthAmericaandhasspreadthroughoutmost
Epizootictransmissioncycle:
ofther angeof itsA e.ae gypt ivector[8]. ZIKV hasc ausedno tabl edis easeam ongdevelo ping referstod iseaseoutbreak samong

fetuses and unexpected neurological disease among adults [13]. WNV, CHIKV, and ZIKV, animalpopulations.Theterm
‘epizoo tic’isequiva lent totheterm
along with DENV and YFV, underscore the emergence of mosquito-borne viruses as truly ‘epidemic’ as usedforh um an
global pathogens. The combination of increased travel and trade has resulted in frequent
diseases.

exchangeofpathogensandvectorsacrosscontinents,suchthatthenotionof‘geographic Vectorcompetence:describesthe

diseases’ is increasingly irrelevant. Coupled with the rapid growth of tropical megacities, abilityofaparticulararthropodto
transm it a specificp athogen.
these exchanges continue to result in explosive epidemics of pathogens transmitted by
Vectorialcapacity:describesthe
mosquitovectorsthat requirethehuman footprint on theenvironment in order to survive – basicrepr oductivera teofavec tor-

vectors such as Ae. aegypti and Culex quinquefasciatus. The ongoing emergence of mos- bornepathogenbyaparticular
quito-bo rnev irus esis occurri ngo nasca le(geographic,eco nom ic,andh uman)thatis w ithout vector species/p op ula tion.

precedent in human history [7,8,14].

How,then,domosquito–virusrelationshipsleadtotheemergenceoftheseglobalpathogens

(Figure 2, Key Figure)? This review examines the ways that mosquitoes influence the emer-

genceofmosquito-bornevirusesinordertoprovideperspectivesonthehistoryandfutureof

thisphenomenon.Thefirstsectionexaminestwocentralconceptsinpublichealthentomology

–vectorcompetenceandvectorialcapacity–whichoutlinebasicmosquito–virusinteractions

andarekeyinunderstandinghowmosquitoesimpactvirusemergence.Theseconceptsare

alsorequiredforreaderstodevelopabasicunderstandingofthebiologyandepidemiologyof

arboviruses.Thesecondsectiondealswithnewknowledgeoftheevolutionaryrelationships

between viruses and their arthropod hosts. This section illuminates the complexities of

arthropod–host interactions and how these can influence virus population biology and phe-

notype,sometimesleadingtoemergence.Thethirdsectionprovidesahistoricalperspectiveon

howmosquitoesinfluencearbovirusemergencebyexaminingthecasesofWNV,CHIKV,and

ZIKV. Finally, we provide perspectives on the future emergence of mosquito-borne viruses,

highlighting emerging mosquito-borne viruses that have yet to capture the attention of the

generalpopulation.

Understanding Vector Competence and Vectorial Capacity

Arbovirusemergenceisdrivenbythehematophagousbehavioroftheirarthropodvectors.This

unique mode of transmission has important consequences for the ecology of arboviruses.

There aretwo conceptsthat arecentral toour understandingofarbovirus transmission and

epidemiology:vector competence(seeGlossary)andvectorial capacity.

TrendsinParasitology,April2018,Vol.34,No.4 311
Basal
lamina
Midgut
lumen
Salivary
d
o glands
o
Bl va
ai
S
Midgut
epithelium
Figure1.DifferentTissueBarriersDetermineVectorCompetenceinMosquitoes.Anarbovirusistakenupbythemosquitoduringaninfectiousbloodmeal.

Thevirusinfectsthemidgutepitheliumandreplicatesbeforeitpassesthebasallaminaintothehemolymphanddisseminatesthroughoutthemosquito'sbody.Inorder

tobetransmittedtothenexthost,thevirushastoinfectthesalivaryglandsfromwhereitcanbereleasedintothesalivaandtransmittedtothenexthost.Viruspopulation

geneticdiversityisreducedstochasticallyasvirusespassthroughanatomicalbarrierstotransmission,suchasmidgutinfectionandescapebarriers,andsalivarygland

infectionandescapebarriers.Potentialchangesinviruspopulationsthathavepassedthroughsuchbottlenecksaredepictedasachangeincolor(increasinglydark

blue).Throughthisgeneticdrift,aswellaspositiveselectioninthemosquito,newgenotypesmayemerge.

Asdescribedabove,vectorcompetencedefinestheabilityofaparticulararthropodtotransmit

a given virus. Vector competence has been studied extensively in mosquitoes, and it is

determinedbybothgeneticandnongenetic(e.g.,environmental)factors.Itmayvarydepend-

ingonmosquitospecies,localmosquitopopulations,andevenindividualmosquitoes.Impor-

tantly, vector competence is a quantitative rather than a qualitative quantity: rarely do all

mosquitoesofagivenspeciesorpopulationtransmitagivenvirus.Thus,vectorcompetenceis

usuallyexpressedasaproportion(e.g.,34%ofCulextarsalismosquitoestransmitWNVafter

10daysincubation).Vectorcompetenceofaspecificmosquitopopulationisalsodependent

on the virus, and even different isolates of the same virus species may result in changes in

vectorcompetenceofamosquitopopulation[15,16].Generally,therearefourmajorbarriers

thatthevirusmustcrosswithinthemosquitoinordertobetransmitted(reviewedin[17]).First,

when a mosquito ingests an infectious bloodmeal, the virus must successfully infect and

replicateinthemidgutepithelialcells(Figure1).Themidgutisthefirsttissueinwhichvirus–

mosquitocellinteractionsoccurthatcanshapetheoutcomeofinfection.Mosquitoesinwhich

theviruscannotestablishaninfectionhaveamidgutinfectionbarrier(MIB).Thiscanoccurdue

togeneticfactors,suchaslackingexpressionofreceptorsonthecellsurface,ornongenetic

determinantssuchasmicrobiomedensityandcomposition[18–20].However,onceavirushas

establishedmidgutinfectionitmustcrossthebasallaminasurroundingthemidgutepitheliumin

ordertodisseminatethroughouttherestofthemosquito(Figure1).Whenvirusreplicationis

limitedtothemidgutanddisseminationdoesnotoccur,mosquitoesaresaidtohaveastrong

midgutescapebarrier(MEB).Thebasallaminaofthemidgutpresentsaphysicalbarrierforthe

virus,andthethicknessofthebasallaminahaspreviouslybeenlinkedtodecreaseddissemi-

nation of DENV-1 in different Aedes albopictus populations [21]. Furthermore, it has been

shownthatsmaller,nutritionallydeprivedAedestriseriatusmosquitoeshaveincreasedLACV

312 TrendsinParasitology,April2018,Vol.34,No.4
KeyFigure

Summary of Factors Influencing Arbovirus Emergence

Virus
Replica(cid:2)on
diversifica(cid:2)on
21nt
RNAi
Cellular virus–mosquito
Vector competence Host/vector switching Emergence as global pathogen
interac(cid:2)ons
Figure2.Cellularandmolecularinteractions,suchasRNAi,drivevirusdiversificationinthemosquito.Differencesinmosquitovectorcompetence,andbottlenecks

thatthevirusencountersduringdisseminationwithinamosquito,canresultinfurtherdivergenceoftheviruspopulationanddrivevirusevolutionandemergence.Dueto

urbanizationanddeforestation,humansandlivestockarefrequentlyincloseproximitytomosquitovectorsandtovertebratehostsmaintainingvirusesinsylvatic/

enzootictransmissioncycles.HumansettlementsmayalsobringalonganthropophagicmosquitoessuchasAedesaegyptiandAedesalbopictus,whichmay

encounterviremicreservoirhosts,suchasprimates.Interactionsbetweennewvectormosquitoesandvertebratehostscandrivearbovirusevolutionandemergence.

Duetointensetravel,andubiquitousdistributionofAedesspp.mosquitoes,suchspillovereventsmayeasilyleadtotheoutbreakofanewglobalpathogen.

dissemination rates compared to larger mosquitoes reared on a normal or rich larval diet

[22,23],whichmayalsocorrelatewiththedevelopmentofathickbasallamina.However,ithas

recentlybeenshownthattheuptakeofabloodmealaltersexpressionofspecificenzymesinthe

mosquitomidgut,includingcollagenases,whichresultsintransientdegradationandincreased

permissibilityofthebasallamina,allowingCHIKVtodisseminate[24,25].Viruseswithslower

replicationrates,suchasDENV,maynotbenefitasmuchfromearlytransientdegradationof

thebasallaminafollowingabloodmeal.Anotherpossiblemidgutescaperouteforarboviruses

maybeviatrachealorneuronalcells.Dongandcolleagues[24]recentlyshowedthatCHIKV

caninfecttrachealcellsconnectedtoAe.aegyptimidguts.Whenthevirushasdisseminated

fromthemidgutitreplicatesinothermosquitotissues,includingthefatbody,hemocytes,nerve

tissue,andmuscletissue(dependingonthevirus)ultimatelyreachingthesalivaryglands,the

next crucialanatomicalbarriertoinfection,the‘salivarygland infectionbarrier’(SGIB).Upon

salivaryglandinfection,thevirusreplicatesandisdepositedintheapicalcavitiesofacinarcells

inordertobeexpectoratedwithsaliva(Figure1).However,notallmosquitoeswillbeableto

expectorate virus (for reasons yet unknown) and thus have a ‘salivary gland escape barrier’

(SGEB).
Other important mechanisms to limit virus replication throughout the mosquito body are

mosquito antiviral immune responses (reviewed in [26,27]). The most specific and potent

mosquitoantiviraldefenseisRNAinterference(RNAi).Duringinfectionofamosquitocell,viral

dsRNAintermediatesarerecognizedbytheendonucleaseDicer2andcleavedinto21ntvirus-

derivedsmallRNAs.ThesesmallRNAsareintegratedintotheRNA-inducedsilencingcomplex

(RISC)andcantargetviralRNAfordegradation.Thissequence-specificresponsecanbevery

efficient at controlling viral replication, and many viruses have evolved mechanisms to

TrendsinParasitology,April2018,Vol.34,No.4 313
antagonize or evade the RNAi response [28]. All mosquito antiviral responses may pose

selectivepressuresonthevirus,butRNAiisuniqueinitssequencespecificity,whichposes

adirectevolutionarypressureontheviralgenome.Mosquitobarriersandantiviralresponses

togethercontribute tothe overallphenotypeof vector competence.

Vectorialcapacityisasecondcriticalconceptthatdescribesthebasicreproductiverateofa

vector-bornepathogenbyaparticularvectorspecies/population(Figure3),andhighlightsthe

powerofmosquitoesasdriversofvirusemergence.Factorswhichinfluencevectorialcapacity

arevectordensitywithrespecttohost(m),thedailyprobabilityofthehostbeingfedupon(a),

vectorcompetence(VC),theprobabilityofdailysurvival(P)andtheextrinsicincubationperiod

(n).Thefactorsthatinfluencevectorialcapacity,andleadtoarbovirusemergence,havebeen

reviewedanddiscussedextensively[29,30].Briefly,themostinfluentialvariablesintheformula

aretheprobabilityofdailymosquitosurvivalandtheextrinsicincubationperiod(EIP).TheEIP

referstotheamountoftimethatittakesforavirustoinfectthemidgutanddisseminatetothe

saliva(i.e.,thetimebetweenuptakeofvirusandabilitytotransmit).Iftheprobabilityofmosquito

dailysurvivalislowandtheEIPlong,thelikelihoodoftransmissionislow.Extensivemosquito-

controlprogramsthatshortenthelifespancanthusefficientlyreducetransmission.However,

theEIPisaffectedbyenvironmentalconditions,suchastemperature[31–33],aswellasgenetic

factors[34–36]influencingvectorcompetenceofthemosquitopopulation.Virusesmayadapt

to fasterdisseminationinsusceptiblemosquitospeciesand thusshorten theEIP[37].

Anotherimportantvariableistheprobabilityofaparticularhostbeingfedupon,orthedegreeof

hostfocus.Thisvariable,‘a’inthevectorialcapacityequation,isaproportion(e.g.,80%,or0.8

ofmosquitoXbloodmealsaretakenfromhostY)andisraisedtothesecondpowertoreflect

theneedforsusceptiblehoststobebittentwiceinorderforperpetuationtooccur.Different

mosquito species vary in their blood-feeding behavior and host preference. The mosquito

speciesAe.aegypti,forexample,hasadaptedtolifearoundhumans,inparticularurbanareas

ofthetropicsandsubtropics,andmosquitoesofthisspeciesfeedalmostexclusivelyonhuman

m =vector density in rela(cid:2)on to host a =probability of host being fed upon
affected by climate change affected by differences in VC =vector competence
and globalisa(cid:2)on human behavior/living condi(cid:2)ons affected by climate, microbiome,
virus adapta(cid:2)on, mosquito gene(cid:2)cs
ma2 × VC × Pn
Vectorial capacity n =extrinsic incuba(cid:2)on period
P affected by climate, virus evolu(cid:2)on
-log
e
P = Probability of daily survival
affected by climate, mosquito control
Figure3.VectorialCapacity.Thevectorialcapacityformuladescribesthetotalnumberoffutureinfectiousbitesarisingfrommosquitoesbitinganindividual
infectio ush ostonasin gleday.Itc onsis tsoffive factors:ve ctorden sitywithre spe cttoh ost(m), the dailyp robabilityo fthe hostbe ingfed upon(a),ve ctorco mp etenceof

themosquitopopulation(VC),theprobabilityofdailysurvival(P)andtheextrinsicincubationperiod(n).Noneofthesefactorsareconstants,butvariableswhichdepend
on bothenvir onmentalin fluen ces aswellas s pecifi cvirus– mo squi toin teraction sasindica ted.

314 TrendsinParasitology,April2018,Vol.34,No.4
hostsandfrequentlyfeedindoors[38].Thisbehaviormakesthemextremelyefficientvectorsfor

viruses such as DENV, CHIKV, and ZIKV, which replicate to high titers in human hosts. In

contrast,Ae.albopictus,whichcanalsoserveasavectorforDENV,CHIKV,andZIKV,ismore

likelytofeedoutside,andwhileitisstillanthropophilic,itismoreofanopportunisticfeeder[39].

Othermosquitoes,suchasseveralspecieswithinthegenusCulex,mayfeedonahumanhost,

but tend to prefer birds [40]. This makes them efficientvectors for WNV and other zoonotic

arboviruses.WNVinfectionofhumanorhorsehostsisincidentalandresultsina‘deadend’for

thevirusduetoinefficientreplicationandconsequentlowviremia.Thus,thecompetenceofa

givenmosquitovectorforanarbovirusmaybeirrelevantifthatmosquitofeedsonlyinfrequently

on susceptiblevertebratehosts.

Virus adaptation to new mosquito vectors clearly shapes the patterns and dynamics of

arbovirusemergence.Generally,viruseswithanAe.aegypti-drivenurbanlifecycleemerged

fromenzooticprogenitorsthatcirculatedbetweennonhumanprimatesandsylvaticmosquito

speciessuchasAedesafricanusorAedesfurcifer[8,41].AdaptationtotransmissionbyAe.

aegyptimosquitoesprovidedaccesstoanewandabundantvertebratehost,humans.Thishas

resultedinexplosiveoutbreaksasseenbeforeforZIKV,whichoriginatedinasylvaticcyclein

Africa. Similarly, there are likely numerous viruses in current sylvatic viruses that may cause

diseaseoutbreaksinthefuture,someofwhichwealreadyknow,manyofwhichwemaybe

unawareof.

Mosquitoes as Sources of Virus Genetic Diversity

Arbovirustransmissionrequiresactivevirusreplicationbothinthearthropodvectorandthe

vertebrate host. Arboviruses are thus subject to selectivepressures from two evolutionarily

distant hosts. In vertebrates, where purifying selection is strong [42,43], these pressures

includeinnateandadaptiveimmuneresponses.Mosquitoeslackaclassicadaptiveimmune

system and do not produce interferon. However, Jak/STAT signaling and other signaling

pathways, such as Toll, have been identified as antiviral responses against DENV [44,45],

WNV [46], Semliki Forest virus [47], and ONNV [18]. RNAi is the main antiviral defense in

mosquitoes[27],whichhasdirectconsequencesforvirusintrahostevolutioninmosquitoes

(which can significantly impact virus emergence). Virus-derived small RNAs inhibit virus

replicationandtranslationbydirectlybindingtocomplementaryviralRNA[27].Thegenera-

tionandperpetuationofnovelsequencesofviralRNA(i.e.,containingmutations)maythusbe

beneficial forthe viruswithinthemosquitobecausesmallRNAswillnotmatch withperfect

complementarity. Experimental evolution studies have confirmed the hypothesis that RNAi

targetingbythearthropodvectorleadstodiversificationofarbovirusgenomes[48–50].Rapid

evolutionandgenerationofacomplexviruspopulationcouldbeparticularlybeneficialforthe

virusinordertoreducecomplementarybinding,andbecausesmallRNAshavebeenshown

tospreadfromcelltocellandmaythusprepareneighboringcellsforimminentvirusinfection

[51]. However, these rapidly generated complex viral populations encounter bottlenecks

withinthemosquitowhencrossingtheMEB,theSGIB,andduringtransmissionofsalivaitself

[52,53]. These bottlenecks vary in their size between different mosquito species and most

likelyvirus–vectorcombinations.InCx.quinquefasciatus,forexample,WNVdiversificationis

high, purifying selection is relatively weak, and bottlenecks are significant, resulting in high

divergenceofWNVpopulationsinthesalivacomparedtotheviruscontained intheblood-

meal [52]. Different mosquito species may thus alter the virus population that is ultimately

transmitted.Additionally,ithasbeenshownthatCulexmosquitoesmaytransmituniqueWNV

populations during each feeding episode [54], which is a further example of howmosquito

infection promotes virus diversification. However, it is not known if the same phenomenon

occurs with other viruses and mosquito species.

TrendsinParasitology,April2018,Vol.34,No.4 315
Rapid intrahostevolution ofamosquito-borne virusinthe mosquitomay result inthe emer-

gence of a new variant with a replication advantage in the vertebrate host or a new tissue

tropism resultingin increased disease severity. For instance, VEEV emerges frequently from

enzootictoepizootictransmissioncycles,withhorsesservingasamplifyinghostscausing

largeoutbreaksinbothhumansandhorses.Theseepizooticstrainsappeartohaveonlyafew

aminoacidchangesintheenvelopeglycoproteinE2incommoncomparedtotheenzootic

strains[55].TherearemanyVEEVstrainscurrentlybeingtransmittedinenzooticcycles,posing

a constant threatinSouthandCentralAmerica [56].

Whilevirusadaptationtoreplicationinmosquitoesmayreducetheabilityofthevirustoreplicate

to high titers in the mammalian host, such adaptations can also affect pathogenesis in

undesirableways.ThiscanbeseeninEEEV,forexample,whichdoesnotreachhighviremia

inhumans,butcancauseseveretofatalencephalitis.Ithasrecentlybeenshownthatthevirus

cannotreplicateinmyeloidcellsofmiceduetoamiRNAtargetsiteinthe3ʹUTRformiRNA142-

3p,amyeloidcell-specificmiRNA[57].However,whilemutatingthistargetsequenceledtoan

increase in viremia and replication in peripheral tissues in mice, it resulted in a decrease in

replicationinmosquitocellsandininfectivityofmosquitoes,suggestingthatthissequenceis

importantforreplicationinmosquitoes.Thisisoneexampleofhowadaptationtothevector

may interfere withreplicationina mammalian host.

Conversely,insect-specificvirusesmayhaveatsomepointdevelopedmechanismstoinhibit

mosquitoantiviraldefenses, whichmayalsohelp thevirusreplicateefficientlyinmammalian

cells. One such mechanism is the generation of subgenomic flaviviral RNA (sfRNA) during

replication.Flaviviruseshavecomplexsecondarystructuresinthe3ʹUTR,whichstallthe3ʹ!5ʹ

exonucleaseXRN1,resultinginthegenerationofsfRNAs.ThesesfRNAsinhibitmRNAdegra-

dationbysequesteringXRN1,bothinmosquitoandmammaliancells.sfRNAsalsosuppress

interferonsignalinginvertebratecellsandareimportantforcytopathicityaswellaspathoge-

nicityinmice[58].ProductionofsfRNAsisalsoimportantforreplicationanddisseminationof

flavivirusesinmosquitoes[59,60],whichmaybeduetobothrepressionofXRN1andtheirother

functionassuppressorsofthemosquitoRNAimachinery[61,62].However,ithasrecentlybeen

shownthatDENVsfRNApatternsdifferwhenvirusispassagedinmosquitocellscomparedto

mammaliancells[63],suggestingthatwhilesfRNAgenerationisimportantinbothcelltypesitis

notoptimizedforoneovertheother.Overall,sfRNAsmayhaveevolvedinmosquitoes(similar
yet lesscomp lex 30UT Rst ruct urese xistinin sect-spe cific flaviv iruses),b u thaveanim portant

role invertebratecells thatisrelated to,but distinctfrom,itsroleinmosquitoes.

Virus–Mosquito Interactions Have Previously Shaped Virus Emergence

Inrecentdecades,WNV,CHIKV,andZIKVhaveemergedasglobalpathogensinexplosive

outbreaksthathavecausedsignificanthumanmorbidityandmortality.Thegeneralphenom-

enon of formerly geographically restricted arboviruses emerging on a global scale has

become common enough, and it is perhaps best considered a new status quo. Several

factorsthathaveledtothisphenomenonhavebeenextensivelydiscussed,includinginthis

review.Theseincludeglobalincreasesintravelandtrade,theriseoftropicalmegacities,and

thedeclineofpublichealthprogramstomanagevectormosquitoes[9,64,65].Further,aswe

have seen above, arboviruses have the capacity for explosive outbreaks due to their

coadaptation to mosquitoes, which carry these agents between individuals with direction

(i.e.,asavector).However,ineachcase(WNV,CHIKV,andZIKV)thevirusadaptedtolocal

conditionsduringglobalspread,whichmaximizedtransmissionpotential(andhealthburden).

Thesechangeshighlightthewaysthattheassociationofviruseswithmosquitoescanleadto

their emergence.

316 TrendsinParasitology,April2018,Vol.34,No.4
WNVwasintroducedintotheAmericasin1999,mostlikelyfromtheMiddleEast[66].Molecular

epidemiologic studies confirmed that the virus remained fairly homogeneous until approxi-

mately2001,whenanewvirusgenotypeemergedincentralUSA.Duringthenext2years,this

genotype (somewhat erroneously called the ‘WN02’ lineage because it was first recognized

during 2002) outcompeted the introduced ‘NY99’ genotype to the extent that the NY99

genotypeseemstohavebecomeextinct.Themostnotabledifferencebetweenthisgenotype

andtheintroducedgenotypewasamutationthatresultedinaconservativevalinetoalanine

change on the exposed surface of the WNV envelope glycoprotein [67,68]. This mutation

conferredfastertransmissionbyCulexpipiensmosquitoes,importantvectorsofWNVinNorth

America[37].ThereducedEIPoftheWN02genotypeenhancesvectorialcapacity(seeabove)

becausemortalityhaslesstimetoactonWN02-infectedmosquitoesbeforetheycantransmit

virustoanewhost.ThisenhancedvectorialcapacityoftheWN02genotypecoincidedwitha

massiveWNVoutbreakinNorthAmericain2002.Thus,specificvirus–mosquitointeractions

between WNV and Cx. pipiens led to epidemiological changes (via vectorial capacity) that

contributedto a significant and,at thetime, unprecedentedarbovirus outbreakintheUSA.

In contrast, CHIKV provides evidence of how an arbovirus can acquire the capacity for

efficienttransmissionbyanewvectormosquitospecieswithdisastrousconsequences.The

currentglobalCHIKVepidemicbeganwhenthevirusemergedincoastalKenyaaround2004.

Fromthere,itspreadthroughouttheIndianOceanregion.Duringthisprocessofemergence,

thevirusacquiredamutationtothecodingsequenceoftheenvelopeglycoproteinE1that

resultedinthesubstitutionofavalineforanalanineatposition226(A226V)oftheprotein[69].

Thismutation rendered the virus more transmissible by the highly abundant Ae. albopictus

mosquito[70](i.e.,increased thecompetence ofAe. albopictus).Since thecompetenceof

any given vector (again, see above) clearly impacts the reproductive rate of the virus, the

acquisition of the A226V mutation conferred an advantage and spread rapidly [70]. Initial

adaptationtoAe.albopictuslaterallowedformutationsinCHIKVE2todevelop,whichfurther

enhanced infection of the Ae. albopictus midgut [71]. Interestingly, the CHIKV that was

introducedintotheCaribbeanin2013lackedtheA226Vmutation,andthismutationhasnot

yet been detected in CHIKV from the Americas.

Mostrecently,ZIKVhasemergedasaglobalpathogeninamannersimilartothatofWNVand

CHIKV before it. The consequences of ZIKV infection were not fully understood prior to this

emergence, and are now notorious: microcephaly in developing fetuses and Guillain–Barré

syndromeinsomeinfectedadults[13].Further,itisnowclearthatZIKVmaybetransmitted

sexually[72,73].SeveralmutationshavebeendescribedintheliteraturethatimpacttheZIKV

phenotypeinpotentiallysignificantways.Amutationthatchangesanalanineatposition188of

theZIKVNS1proteintoavalinehasbeenassociatedwithenhancedinfectivitytoAe.aegypti

mosquitoesviaincreasedNS1antigenemia[74].Similarly,theserinetoasparaginemutation

ataminoacid139(S139N)oftheprMprotein,thatappearstohavearisenaround2013,has

beenshowntocontributetopathogenesisindevelopingfetuses[75].Itisnotyetclearwhat

specificinteractionscouldhaveproducedeitherofthesechangesandhowtheymayimpact

other importantZIKVphenotypes. Thispromisesto beafruitful areafor futureresearch.

Future Arbovirus Threats

Therecenthistoryofmosquito-bornevirusemergence,coupledwithwhatweknowaboutthe

molecular and ecological interactions that facilitate their transmission, indicate that new

explosiveoutbreaksofarbovirusdiseasearelikelytooccurinthefuture.Severalarboviruses

(Table 1) currently circulate at low levels in geographically limited settings that result in few

humancases.Whilealloftheseviruseshavethe(theoretical)capacitytospreadrapidlyand

TrendsinParasitology,April2018,Vol.34,No.4 317
Table1.SelectedArboviruseswithCurrentlyLimitedGeographicDistributionandDiseaseIncidence

Genus Virus Suspectedmosquitovector Geographicdistribution Disease

Alphavirus Mayarovirus(MAYV) Haemagogusspp. SouthandCentralAmerica Febrileillness,arthralgia,myalgia

Unavirus(UNAV) Psorophoraspp. SouthAmerica Febrileillness,arthralgia,myalgia

Pixunavirus(PIXV) Unclear SouthAmerica Febrileillness,encephalitis

RioNegrovirus(RNV) Unclear SouthAmerica Febrileillness,myalgia

Tonatevirus(TONV) Unknown FrenchGuiana Febrileillness,encephalitis

Evergladesvirus(EVEV) Culexcedecei Florida Fever,headache,myalgia

Mucambovirus(MUCV) Culexspp. SouthAmerica Febrileillness,arthralgia,myalgia

Trocaravirus(TROCV) Aedesserratus SouthAmerica Unknown

O’nyongnyongvirus(ONNV) Anophelesspp. Sub-SaharanAfrica Febrileillness,arthralgia,myalgia

Flavivirus Spondwenivirus(SPONV) Aedescircumluteolus Sub-SaharanAfrica Febrileillness,arthralgia,myalgia

Denguevirustype5(DENV-5) Aedesspp. SouthEastAsia Highfever,arthralgia,myalgia

Rociovirus(ROCV) Aedesspp.,Psorophoraspp. SouthAmerica Febrileillness,encephalitis

Orthobunyavirus Oropouchevirus(OROV) MainlyCulicoidesbitingmidgesa SouthAmerica Highfever,arthralgia,myalgia

Phlebovirus RiftValleyfevervirus(RVFV) >40speciesofmosquitoes Sub-SaharanAfrica Febrileillness,arthralgia,myalgia

aOropouchevirusispossiblyalsotransmittedbyAedesspp.andCoquillettidiaspp.mosquitoes.

causeoutbreaks,someofthemseemmorelikelythanotherstocauselarge-scaleepidemicsin

the near future.

One of these viruses is MAYV, which has been circulating in parts of South and Central

Americasinceatleast1954 when itwasfirstisolatedin Trinidad[76].Diseasesymptoms

aresimilartothoseofCHIKVinfection,includingfever,rash,myalgia,andarthralgia.MAYV

issuspectedofbeingtransmittedbetweennonhumanprimatesbyvectormosquitoesofthe

genusHaemagogus;however,Ae.aegyptiandAe.albopictusarecapableoftransmission

inanexperimentalsetting[77,78].Whilefewhumaninfectionshavebeenreportedoverall,a

relatively large outbreak of 77 cases occurred in 2010 in Venezuela [79], and MAYV

received some media attention due to a human case in Haiti in 2015 [80]. Historically,

MAYVinfections occurredpredominantlyinnorthernSouthAmerica,inregionsbordering

the Amazon basin [81]. Since there was no history of travel in the Haitian case, infection

probably occurred through local mosquito transmission. The patient was also coinfected

withDENV-1,possiblysuggestingthatinfectionoccurredthroughthebiteofanAe.aegypti

mosquito. Since there are no nonhuman primates on Hispaniola, this may indicate the

presenceofalocalhuman–mosquitotransmissioncycle.However,MAYVwas previously

isolatedfromamigratingbirdinColorado[82],suggestinganotherpotentialrouteofMAYV

introductionintoHaiti(andasourceofinfectionoflocalmosquitoes).Whethercontinuous

local transmission was established remains unknown. While reports of potential recent

recombinantMAYVstrains[83]areofsomeconcern,recombinationofalphavirusesislikely

a rare event. The only recombinant alphaviruses described are the three members of the

Westernequineencephalitisvirusserocomplex,whicharerecombinantsofastrainofEEEV

andaSindbis-likevirus[84].However,withamultitudeofalphavirusescirculatinginSouth

and Central America, and therecent additionof CHIKV, a smallchance of recombination

eventsremains.Thiscouldresultinrapidemergenceofnewviruseswithalteredtransmis-

sion phenotypes and pathogenesis. The risk of MAYV emergence has been discussed in

detail in several recent reviews [85–87].

318 TrendsinParasitology,April2018,Vol.34,No.4
Inaddition,alittle-knownflavivirusofsomeconcernisSpondwenivirus(SPONV).SPONVis Outstanding Questions

currently circulating solely in sub-Saharan Africa in sylvatic cycles involving zoophilic Aedes Can the emergence of mosquito-

mosquitoes. Peri-domestic mosquitoes, such as Ae. aegypti, Ae. albopictus, and Cx. quin- borne arboviruses be predicted and

pre-emptivelymanaged?
quefasciatus,donotappeartobecompetentvectors[88].However,aspreviouslydiscussed,

adaptationtoanurbantransmissioncyclemaybeachievedbyonlyoneorafewmutationsin
Which sylvatic/enzootic arboviruses
the virus genome that increase the infection potential for Ae. aegypti or Ae. albopictus can be maintained in a transmission

mosquitoes. Current research on SPONV is limited, but recent evidence shows its potential cycle exclusively between humans
tobesexuall ytransm ittedamo ng mice,alb eit inefficie ntly [89].Ou rcurrent lackof und erstand- andm osquitoes?

ing of SPONV pathogenesis and transmission warrants further investigation. The zoonotic
Whatvector-controlmechanismsand
potential of arbovirusescurrently circulatinginAfrica hasbeen reviewedelsewhere [90].
otherpreventativemeasurescanspe-
cifical ly help to p revent or co ntain

Concluding Remarks futureoutbreaks?

Overrecentdecades,arboviruseshavetrulyemergedasglobalpathogens.Virusespreviously
existi ngonly inlocaltra nsmissionc ycles inru raltropica lse ttingsa renowdist ributedw orldwide, What are the molecular mechanisms
thatunderpinvirushostshifts?
causing devastating disease outbreaks. This is largely due to the now global distribution of

mosquitospecieswithhighvectorpotential,suchasAe.aegyptiandAe.albopictus,aswellas
human tra vel and pop ulatio n dens ity in trop ical m eg aci ties. Viru s–m osq uito interac tio ns a nd aTpop rowahcahte sexctoennttr ibcuaten toevoplruetdioicntaivrey
mosqu ito ad apta tion to hum ans co nt ribute s ignificantly to arboviral em ergence. Fu ture efforts?

research should be aimed at increasing our understanding of neglected arboviruses, novel
Defining transmission and/or patho-
surveillance methods, and implementation of surveillance programs to recognize spillover
genesis-altering mutations to a viral
eventsandarbovirusoutbreaksearlyon(seeOutstandingQuestions).Surveillanceforarbo-
genome using experimental studies
viruses and also inva sive mosq uito s pec ies can help with implement ation of rapi d v ector- is, in so me ca ses, highly t ractable.

controlresponses.Aswithallarboviruses,vectorcontrolwillbecrucialforthepreventionand However, frequently, these studies
mayresul t inso-calle d‘gain -of-func-
containment of future arbovirus emergence. The development of simple inexpensive diagnostic tion’ varia nts . Are th e benefits of
tests for a large selection of pathogens may also help to identify and contain an outbreak.
understanding the plasticity of viral
More ove r, the m osquito– viru s interactio n ha s fa cilitat ed the rap id e mergenc e of several genotypesand phe notypesw ort hthe

arboviruses,apatternthatislikelytocontinue,requiringanintegratedapproachtooutbreak risk of generating gain-of-function

variants?
management.
Isoursocietywillingtoinvestindeter-
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