Virus
population
bottlenecks
during
within-host
progression
and
host-to-host
transmission
Serafı´n
Gutie´rrez
1,2,
Yannis
Michalakis
2and
Ste´phane
Blanc
1Despiterapidlygrowingtoimmensesizes,viruspopulations sufferrepeatedseverebottlenecks,bothwithinhostsandwhen transmittedfromhosttohost.Thepotentialeffectof bottleneckshasbeentheoreticallyandexperimentally documented,butformalestimationsoftheiractualsizesin naturalsituationsarescarce.Bottlenecksduringcolonizationof organsandduringtransmissionareinfluencedbythose occurringatthecellularlevel.Thestudyofthemultiplicityof cellularinfection(MOI)thusappearscentral,andthistraitmay bedifferentiallyregulatedbydifferentvirusspecies.Thevalues ofMOIandtheirputativeregulationdeserveimportantfuture efforts,inordertodisentanglethecomplexinteractions betweenthecontrolofgenecopynumbersandthepopulations dynamics/geneticsofviruses.
Addresses
1UMRBGPI,INRA-CIRAD-SupAgro,TA-A54K,CampusInternationalde Baillarguet,34398MontpellierCedex05,France
2Unite´ MixtedeRechercheGEMI2724,CNRS-IRD,AvenueAgropolis, B.P.64501,34394MontpellierCedex05,France
Correspondingauthor:Blanc,Ste´phane(blanc@supagro.inra.fr)
CurrentOpinioninVirology2012,2:546–555
ThisreviewcomesfromathemedissueonVirusevolution EditedbyRaulAndinoandMarcoVignuzzi
ForacompleteoverviewseetheIssueandtheEditorial
Availableonline22ndAugust2012 1879-6257#2012ElsevierB.V.
http://dx.doi.org/10.1016/j.coviro.2012.08.001
Introduction
Short generation times and high mutation rates allow adaptation of viruses to selective pressures at a speed matchedbynoother organisms.The remarkable popu-lation dynamics makes viruses excellent experimental models for evolution studies, where evolution at work canbemonitoredinreal-timeonalabbench.Inthereal world,fastandefficientadaptationallowstheseparasites to overcome natural or man-made barriers such as host defenses,hostresistance,ordrugtreatments,by generat-ingnewemergingvariantswithalteredbiological proper-ties.Consequently,viruseshavesuccessfullyinvadedall livingorganismsinallecosystems,withvirus–host inter-actionranging frompathogenicto mutualistic[1]. Both fundamental and health/sanitary concerns have fuelleda wealth of research onvirus evolution.Amost importantquestionthatisoftenaddressedistherelative
actionoftwoofthemainforcesdrivingevolution: deter-ministicnaturalselectionandrandomgeneticdrift.When natural selection is the predominant force shaping the evolutionaviruspopulation,adaptationcanbefast.On thecontrary,adaptationisgenerallysloweddownwhen genetic drift dominates because the resulting random variation in allele frequencies can distort the direction thatwouldbedrivenbyselection. Thereare important practicalimplications fromevaluatingtheselection/drift balance in a particular virus–host interaction. For example,facingman-madebarrierssuchasantiviraldrugs or virus-resistant crop varieties, viruses must adapt or disappear. Whether and howfast these barriers will be overcomedependstoalargeextentontheintensitywith whichselectionacts,as opposedtogeneticdrift. Estimatingtherelativeintensityofselectionanddriftina given viral population is not a trivial task. It requires determiningthesizeoftheviralpopulationreplicatingin theanalyzedsystem.Thepopulationsizeishighly infor-mativeherebecauseselectioncanactmore intenselyin largepopulations,whereastherandomeffectsofgenetic drift are more severe in small populations. It must be stressedthattheimportantfiguretobemeasuredhereis notnecessarilythewholepopulation,but only the frac-tion that multiplies. Since this parameter is obviously rather elusive, the effective size of the population is generally used instead. The effective population size canbedefined asthesize ofan idealpopulationwhere stochasticvariations in allele frequencies would bethe sameasthoseobservedinthepopulationunderstudy(for acomprehensive review on this parameter and its esti-mation see[2]) Although notexactly equivalent to the numberofreplicatingindividualsinallcircumstancesthe effectivepopulationsizeisavaluablequantitybecause(i) it captures the stochasticity involved in the observed changesinallelefrequencies,and(ii)itisan experimen-tallyaccessible parameter.
Theeffectivesizeofpopulationsislogicallysensitiveto fluctuations in the census (total) size, especially to dramatic reductions sometimes experienced by popu-lations, the so-called bottlenecks. A population bottle-neck can be defined as a transient reduction of the number of viral genomes within a population and has twomajoreffects.Ontheonehand,itinducesagenetic bottleneckthroughitsstochasticeffectonthenumberof genotypespassed down to the next round of infection. Thisstochasticeffectisinverselyproportionaltothesize ofthebottleneck.Ontheotherhand, itdeterminesthe Open access under CC BY-NC-ND license.
viral ‘gene copy number’ at the onset of infection, an utterly importantaspectinthebiologyofany organism, thatistoooftenoverlookedinvirology(furtherdiscussed later).
Viruspopulationsareperceivedasbeingextremelylarge, and this is usually the case for the census population within ahost. For example,the size of thevirus popu-lation in atobacco leaf infected byTobaccomosaic virus (TMV)hasbeenestimatedtoapproximately1012genome units[3].Similarfigureshavebeenreportedintheplasma of patientsinfected byHuman Immuno-deficiency virus-1 (HIV-1)duringviremiapeaks[4,5].However,virus popu-lations can endure very severe bottlenecks during the infection cycle. To start with, viruses must migrate repeatedly from onehost to another,aperilous journey during which only a fistful usually make it from the trillionsinthedonorhost.Onceinsidetherecipienthost, thesituationdoesnotimprove.Alargenumberoflimiting factors–likehostdefenses,intrinsicdecayratesofvirions [6],limitedavailabilityofsusceptiblecellsorofreceptors attheirsurface[7]–reducethenumberofindividualviral genomes actually contributing to the expansion of the population.Interestingly,virusesthemselvesrestricttheir effectivepopulationsizethroughwhatcanbeconsidered aterritorialbehavior.Onceacellbecomesinfected,the ‘resident’ virususually launchesmolecular mechanisms
precluding any new infection from incoming closely relatedgenomes.Thisprocessisknownassuperinfection exclusionandseemslargelyspreadamongviruses infect-ing animals, plants and bacteria [8–12]. Superinfection exclusion thus reduces theeffective size of viral popu-lations by limiting the number of genomes that can actuallyenterandreplicateinindividualsusceptiblecells. Populationsizecanthusgreatly dropatvariousstepsof the viral cycle, and the impact of such bottlenecks on virus evolutionhas beenaddressedin manytheoretical studies, plus several experimentaldemonstrations ofan associateddecreaseoftheviralfitness(reviewedin[13]). Quantitative dataonbottlenecksizes fromnatural situ-ations,however,remainsurprisinglyscarce(Table1).In this review, we present an overview of the information available. Despite being largely fragmentary, a blurred picture startsto emerge where,as it couldbeexpected from thevirusdiversity,drasticallydifferent population dynamics seem to be associated to diverging virus life styles.
Getting
in:
Are
severe
bottlenecks
the
rule
during
host-to-host
transmissions?
Thefirstquantitativeestimateofapopulationbottleneck during transmission was obtained for an insect-borne plant virus, the Potato virus Y (PVY), transmitted in a Table1
Comparisonofthequantitativeestimatesofbottlenecksavailableintheliterature
Typeofbottlenecka Virus Host N Ref. Vector/transmissionmode Bottleneckduringtransmission pop PVY Pepper 0.5–3.2 [14] Myzuspersicae
pop CMV Tomato 1.2–2 [15] Aphisgossypii
pop TMV Tobacco 1.3–3.3 [17] Leafcontact
gen HIV-1 Human 1 [19] Severalb
gen HCV Human 1–2 [20] N.S.c
Typeofbottleneck Virus Host N Ref. Organ/tissue
Bottlenecksduringorgancolonization pop WSMV Wheat 3–5 [47] Tiller
pop TMV Tobacco 3.1–5.6 [46] Leaf
pop CaMV Turnip 298–484 [39] Leaf
pop PVY Pepper 1–4 [45] Leaf
pop TEV Tobacco 1.2–47.9 [44] Leaf
pop TEV Pepper 1.1–5.4 [44] Leaf
pop CaMV Turnip 8.8–131 [43] Leaf
gen HIV-1 Human 103–105 [34] Blood
gen HCV Human 2 [20] Blood
Virus Host N Ref.
Multiplicityofcellularinfection phi6 Pseudomonas 2–3 Seerefin[38]
HIV-1 Spleencells 3d Seerefin[38]
HIV-1 Bloodcells 1d [42]
TMV Nicotianabenthamiana 1–6 [63]
SBWMV Chenopodiumquinoad 5–6 [64]
CaMV Turnip 2–13 [38]
aPop:populationbottleneck,numberofviralgenome;gen:geneticbottleneck,numberofgenotypes. b Transmissionmodesincludesexual,mother–childandsyringeexchange.
cN.S.:notspecified.
non-circulativewaybyaphids[14].Inthistransmission mode, the virus does not replicate within the insect vector.Itsimplyreversiblyinteractswithputative recep-torsinthemouthparts(stylets),whereitcanberetained infectious for only a few minutes [3]. Moury and col-leaguesusedasimpleandelegantmethodtodetermine thesizeofthepopulationsamplethatisactuallytakenup andtransmittedbyaphids.First,theyfedaphidsonvirus suspensionscontainingamixtureofvirusparticlesfrom twodifferentvariants,oneinfectiousinagivenvarietyof pepper and the other one not. Aphids fed on virus suspensions with different relative concentrations of thetwovariantshaddifferenttransmissionsuccessrates onthesepepperplants: thehighertheconcentration of the non-infectious variant, the lower the success rate. From these data, a stochastic model was then used to estimate that each individual aphid could efficiently transmitan averageof 0.5–3.2viralgenomes.In a com-parable approach, similar figures were calculated for Cucumber mosaic virus (CMV), another plant virus also transmittedin a non-circulativeway byaphids [15].An importantdifferenceofthelatterstudy,isthataphidsdid not acquire the virusfrom homogeneous artificial mix-tures, but from leaves co-infected by the two CMV variants. Because CMV variants have been shown to spatiallysegregatein co-infectedplants,alargefraction ofleafcellsaregenerallyinfectedbyasinglevariant[16]. AphidsusuallyacquireCMVafterfeedingfromoneora few cells. They may thus only access to the variant infecting the sampled cells, indeed generating a very strong geneticbottleneck reminiscent of what happens innaturalsituations,althoughitmayormaynotexactly correspondtotheactualnumberofgenomestransmitted. Populationbottlenecksduringcontacttransmissionhave alsobeenquantitativelyanalyzedinTMV,withasimilar approach [17]. Surprisingly, though the transmission mechanismistotallydifferent, thenumberofviral gen-omesinitiatinginfectionintherecipienthostplantshas beencalculatedto alsoliebetween1and4.
Inanimalviruses, thegeneticbottleneck duringHIV-1 transmissionhasbeen quantitativelyexaminedusingan approachinitiallydevelopedbyKeeleetal.[18].Inthis approach, a model of random viral evolution is imple-mented with the phylogeny data obtained from deep-sequencing the virus population early in infection. All followingstudiesconsistentlysuggestthatasingle HIV genotypeisusuallyattheoriginoftheviruspopulation withinthe patient(reviewed in [19]). Such an extreme geneticbottleneckhasbeenevidencedin70–90%ofthe casesofmothertochildtransmission,in60–90%ofboth heterosexualandhomosexualtransmissions,andevenin 40–70% of transmissions through syringe exchanges. A similar approach has also been used to estimate the founder population in four individuals infected with HepatitisCvirus(HCV),anotherblood-bornevirus,with
very similar results of 1 or 2 founder genotypes per infectedhost[20].
Tothebestofourknowledge,thesearetheonlyformal quantitative estimates of population/genetic bottle-necks during viral transmission, currently available in the literature. One could conclude indeed to a very limited exploration of this question among the described viral diversity. Nevertheless, though they do not represent actual quantifications, additional stu-dies have detected drops in genetic diversity upon transmissionof severalviral species,indiverse genera and families (e.g. [21–23]). Despite this consistent trend, narrow bottlenecks might not always be the inevitable outcome of virus transmission. Aaskov et al. have shown that defective genotypes of Dengue virusaretransmittedamonghumansandmosquitoesin nature [24]. Two phenomena acting together can explainthisobservation:complementationofdefective genotypesbyfunctionalones,andrelaxedtransmission bottlenecksallowing thepresence ofbothtypesinthe recipient hosts. Likewise, a recent study analyzing changesinpopulationdiversityoftheEquineinfluenza virusbetweendonorandrecipienthorsesalsosuggests an absence of a severe transmission-associated bottle-neck[25].Finally,ithasbeenlogicallypostulatedthat when thenumber of transmissionevents perrecipient hostislarge,alargeviralpopulationcanbetransmitted whatever the bottleneck associated to single events. Thoughithasneverbeendirectlyinvestigated,thishas particularly been suggested for host plants visited by large numbers ofinsect vectors [26].
Inany case, there isa clearneed for more quantitative dataonbottlenecksduringnaturalviraltransmission.Asit stands,thecurrentliteratureonthesubjectisnot diver-sified enough to allow any inference of general trends which could reliably relate a specific mode of natural transmissiontosevereorto relaxedtransmission bottle-necks.
Beyondtheaveragesizeofabottleneck,thedistribution ofthesesizesamongtransmissioneventsshouldalsobe documentedfurther.Itmustbestressedthatevenasmall numberof multiplyinfectedhostscouldmattermostin virusevolution.Multiplyinfected hosts arethemelting pot where genetic exchange can take place between genotypes,aphenomenonthatcanradicallychangevirus properties (e.g. host range expansion) with important implications,evenif rare,in ecologyand epidemiology. Moreover, the detailed analysis of distribution of the bottleneck sizes during transmission could also reveal unknown aspects of the transmission mechanisms. For example, a bimodal distribution of the transmission-associated bottleneck sizes might reveal two different transmission routes for a given virus, as suggested by infectionof cellswithHIV [27].
Finally,webelieveitisof primeimportanceto keepin mindthat,althoughapproachesdescribedaboveon HIV-1,HCVandCMVarereliablyquantifyinggenetic bottle-necks during transmission [15,18,20], they might be partly blind to the actual number of transmitted viral genomes,asopposedtotransmittedgenotypes.Thisisan important point which deserves to be specifically addressed in the future because, as further discussed below, this number matters much more than usually considered.Itsetsthe(viral)genecopynumberatinitial phasesofthehostinfection.Boththeoreticaland exper-imental investigations have revealed the importanceof thenumberofcopyofageneinsideacell[28,29,30,31]. Eveninverysimplegenenetworkswithdiverseputative reciprocalregulations,slightchangesingenecopy num-berscandramaticallymodifytheirrelativeexpressionand sothephenotypicoutcomeofthenetwork.This phenom-enonappliestoviralgenesandcanchangethefateofthe infected cell, and thus of the viral cycle [28–31] (see below theSection onMOIfor furtherdiscussion).
How
host
colonization
shapes
population
structure
and
evolution
As briefly mentioned above, the genetic bottleneck associated to transmission may largely depend on the structure of the virus population in the donor host. If thispopulationisgeneticallyheterogeneous,transmission bottlenecks could be determined partly bythe genetic diversity available in the host compartment where the transmitted population is sampled.The extentof com-partmentalization ofviralpopulationsisa questionthat requiresdetailedanalysisatvariousstepsofhost coloni-zation. These analyses inform on dynamics of disease progression,onwithin-hostevolutionprocesses,and ulti-mately onthe historyand composition of the subpopu-lationavailablefor transmission.
Highlystructuredviralpopulationswithinindividual hosts
Afterprimaryinfectionofahost,virusesreplicatetolarge numbers,numerousmutants appearand,inmanycases, recombination shuffles mutations and generates new genotypes. This diversity ismost oftenloaded into the vascular system and transported away before entering new cells and replicatingfurther.Each celltype and/or organcanbeconsideredasadifferenthostcompartment that viruses infect with varyingsuccess, and where the developingviralsubpopulationsaremoreorlessisolated and may diverge from the initial source population [22,23,32–34] (Figure 1). Empirical evidence of popu-lationcompartmentalizationindifferentvirusmodelshas transformed theprevious panmicticview of within-host populations intoametapopulation view[35,36]. Never-theless, recent reports in both animals and plants have mitigatedthesituationandshownthatextremely differ-entdegreesofcompartmentalizationmayexistin differ-entvirusspecies[37–39].
Twoinitialparametersdefinethegeneticsof the popu-lation that invades a given compartment: the genetic diversityavailableinthesourcepopulationandthesize of the bottleneck during colonization. The population colonizinganeworganortissueoftenderivesfromthatin the vascular system. Unfortunately, the viral genetic diversity flowingin sapor plasma isnoteasy to charac-terizebecauseitcanamplyvaryalongtime.Spectacular changesofviralgeneticsinthevasculaturearethe recur-ringselectivesweepsresultingfromtheattackofthehost immunesystemonavirusserotype,whichisthenrapidly replaced byanewone[40].The relationshipsbetween the viral dynamicswithin the vasculatureand the viral migration withinand in betweenthevarious host com-partments remains to be investigated, although some recentstudiesstarttotacklethequestion[41,42].This paucityofdataisparticularlydramaticinthecaseofplant viruses, probablybecause simple methodsfor sampling thevascularsystemhavelongbeenmissing[43]. Theextenttowhichtheviralpopulationpassesfromthe vasculaturetoagivencompartmentdependsonthe mech-anisms of entry. Two contrasted scenarioscan be envi-sioned(Figure2):(i)organsarehighlypermeabletovirus infectionandpopulationbottlenecksdependonlyonthe viralload(theviraldoseortiter)inthevasculature,or(ii) limitingbarriersleadto severebottleneckswhateverthe viral load in sap or serum. Deciding which of these scenariosmostoftentakesplacerequirescomparativedata estimating in parallel thepopulation bottlenecks during organ colonization and the viral load in the vasculature irrigatingthisorgan.Theonlytwoexamplesavailableare Figure1
Current Opinion in Virology
CompartmentalizationofTurnipmosaicvirus(TuMV)variantswithina systemicallyinfectedleaf.
EachTuMVvariantencodesandthusproducesadifferentfluorescent protein(mGFP5ingreenandmRFP1inred).Compartmentalizationis particularlyeasytovisualizehere,aswellasinmanyotherplantviruses, owingtothepossibilitytoexpressdifferentfluorescentproteinsviatwo otherwiseidenticalclones,co-inoculatedintothesamehostplant.Each clonecanbeobservedtoseparatelyinfectleafcells,yieldinga patchworkofinfectedregionswithasinglefluorescence.Bar=0.5cm.
dealing with plant virus models [43,44]. Both studies indicatethatbottlenecksreportedduringleafcolonization byvariousplantviruses[39,45–47]couldbeinfactlargely drivenbytheviralloadinthesap.Zwartandcolleagues have quantified the bottleneck sizes in populations of Tobacco etch virus (TEV) invading the first systemically infectedleafoftobaccohosts.Plantswereinitially inocu-latedwithdifferentviraldoses,inducingdifferentnumbers ofinfectionfociontheinoculatedleaves(doserangedfrom 1to50fociperplant).Thesenumberspositively corre-lated with the size of the bottlenecks during ulterior systemiccolonizationofleaves,suggestingthatthe bottle-neckswereinthiscasemainlydeterminedbytheviralload, though not directly demonstrated. With another virus species, the Cauliflower mosaic virus (CaMV), we have analyzedinparallelthebottlenecksattheentryofleaves successivelyappearingontheinfectedhostplantsandthe virustiterinthesapflowingintotheseleaves[43].Our results show that successive leaves are colonized by increasingpopulationsizes,rangingfromunitstohundreds of genomes, and that this colonizing population size directly depends on the virus load in the sap. In this situation,themostdrasticbottlenecksufferedbyviruses duringtheirlifecycle might bethetransmission bottle-necks.Indeed,thesetwostudiestogetherindicatethatthe limitingfactorforthegrowthoftheviralpopulationisthe
viralloadwithinthesap[43],whichisprobablyinfluenced bytheinitialinoculum[44].
That physical barriers sometimes impose bottlenecks, andthus favorcompartmentalization of the virus popu-lation,evenwhenthe viraldoseavailable in thesap or bloodisnotlimitingmakeslittledoubt.Forexample,still inplant viruses,Li andRoossinck [23] observeda con-tinuouslossof geneticmarkersin apopulation ofCMV colonizing successive leaves of tobacco host plants, suggestingthepersistenceofstrongbottlenecksallalong theinfection.Similarly,inanimalviruses,Kussetal.[48] monitored the genetics of an artificial population of poliovirusduringthecolonizationofimmuno-suppressed miceexpressingapoliovirushumanreceptor.Theyalso observed a constant decrease in the genetic diversity during virus progression, thus postulating that physical barriers imposed bottlenecks at several stages of this process.
Overall,becausehintsinfavorofoneortheotherscenario canbefound bothin animals[37,42]andplant viruses [39], it would not be surprising that a whole range of differentsituationsexistsinnature.Complexinteractions betweentheviralrushforhostinvasion,theonsetofhost defenses, the availability of susceptible cells and the Figure2
Increasing viral load
(a) (b) V ascular system V ascular system Organ Organ
Current Opinion in Virology
Twooppositescenariosduringviruscolonizationoforgansfromthevasculature.
(A)Thesizeofthepopulationthatinvadestheorgandependsontheconcentrationofvirusinfectiousunits(colouredcircles)inthevascularsystem.In thiscase,nolimitingbarriersexistandtheviruscanfreelymovefromonecompartmenttotheother.
(B)Thesizeofthepopulationinvadingtheorganisconstantlylow,whatevertheviralloadinthevasculature,owingtobarriersimposedbythehost (e.g.limitingnumberofsusceptiblecells,limitingnumberofreceptors,etc.),orbythevirusitself(e.g.mechanismsinhibitingsurperinfection).
inhibition of superinfection might haveoutcomes alter-natingbetweenstrongandrelaxedviruspopulation bot-tlenecks at various stages of the infection. It is also possible that ‘going alone’ or ‘going together’ might representadaptiveviralstrategies.Moreexplicitly,virus specieswithmanygeneproductsacting(or complement-ing)intrans,and/orwithahighrecombinationrate,may gain benefits from relaxed bottlenecks whereas other speciespoorlycomplementingorrecombiningmayonly get thecosts of competition. The question of whether such adaptivestrategiesexist ishighlychallenging, and will require increased efforts in characterizing the dynamics of within-host colonization for a wide and diversepanelof virusspecies.
Gettingout:thetransmissiblepopulation
After infectionof a compartment, the colonizing popu-lationcanfurtherevolvedifferentially,owingto compart-ment specific conditions, to isolation, or conversely throughmigrationofadditionalvirusgenomes(reviewed in [34]for HIV-1). Compartmentsof particular interest are those fromwhere thetransmission to thenext host will actually occur. The population contained in such compartmentsishereaftercalledthe‘transmissible popu-lation’. It is obvious that these compartments differ among virus–host models, but whether they docontain subpopulations specifically adapted to transmission is only anascent question, despite numerous occurrences ofsuchaphenomenoninothernon-viralparasites[49,50]. Theadventofhigh-throughputsequencingtechnologies has facilitated analysis of viralpopulation genetics, and these tools are currently used to decipher the genetic structure of transmissible-populations for different viruses: HIV-1 in the genital tract and in the blood [41,42,51], HCV in the blood [20], rhinoviruses [52] orequineinfluenzavirus[25]innasalswabs, Foot-and-mouthdiseasevirusinhooves[53]orWestNilevirusin salivaryglandsofmosquitoes [37](note thatthereisno comparable example published from plant viruses thus far). Unfortunately, for obvious practical reasons, most studiescitedabovehavefocusedonalimitednumberof infectedindividuals.Theconsequenceisthat,foragiven virus, no common feature among transmissible popu-lations in different individual hosts could be detected, althoughthetransmissiblepopulationsprovedtobe dis-tinct fromthose inothercompartments.
All the above-cited reports suggest that transmissible populations containarelativelyelevatedgenetic diver-sity,indicatingthat dramaticgeneticbottlenecks often observed after transmission are not owing to homo-geneous transmissible populations. One would expect then thatthemostfrequentgenotypesinthe transmis-siblepopulationwouldbemostoftentransmitted.One study [41], however,seems to uncover an intriguing process. In an analysis comparing theHIV-1 transmis-siblepopulations inthegenital tractofdonorsand the
actually transmitted populations in recipients, it was clearly demonstrated that the frequent genotypes are nottheonestransmitted.Thisratherunexpectedresult suggests two potential scenarios: (i) either numerous genotypes are transferred to the recipient and only specific ones (present at low frequencies) are able to initiate infection, or (ii) specific genotypes (though present at low frequencies) are the only ones trans-ferred.Interestingly,aseriesofpapershaveshownthat founder HIV-1 virus genotypes have distinct features like fewer glycosylated sites in the envelope proteins [54,55,56,57]. These observations point to the exist-ence of viral ‘morphs’ specifically adapted to trans-mission in HIV-1 populations. Transmission morphs have been described for baculoviruses, a viral family infectinginsects.Baculoviruses havealife cycle invol-ving two types ofvirions with specific envelopes, one dedicated to within-host colonization and the other essential for transmission. Thegenerality ofthe exist-ence of viral transmission morphs, whether they only appear or localize in specific host compartments, and whethertheirspecificityhasbothaphenotypicand/ora genetic determinism, represent particularly appealing future prospects.
Themultiplicityofcellularinfectionplaysaseminalrole inbottlenecks
We have thus far reviewed bottlenecks during trans-missionandduringorgancolonization.However,perhaps themostimportantlevelinviralpopulationdynamicsis that of individualcells [34].The populationbottleneck duringcellinfection,thatisthenumberofviralgenomes entering andreplicatingwithinacell, isherecalled the multiplicity of cellular infection (MOI). The impact of MOI on population bottlenecks at higher organization levelscanbestraightforwardormoresubtle.When colo-nizing new organs, the bottleneck results from the additionoftheMOIintheinitiallyinfectedcells.During transmission, genetic bottlenecks depend on both the MOI in donorcell compartments and that in recipient susceptible cells. Through its ruling of the number of genomes enteringcells,thiskeyparameteralsoimpacts ontheinteractionsbetweenvirusgenotypes[58–61],and on their phenotype when sensitive to the gene copy number[29,31,62].
Forviruses,thecellisthemainarenawheregenomesofa given population can meet and interact. These inter-actionsincluderecombination,competitionand comple-mentation, all major phenomena in the evolution and epidemiology of viruses, which can take place or not depending on MOIvalues. For example, for a MOIof one,recombinationwillbeprecluded,complementation ofdefectiveinterferingparticleslargelyalleviated,while competitionforcellularresourceswillberelaxed.These are commonly discussed implications of MOI in recent availablepublications [38,42,63,64].
TheMOIalsodefinesthegenecopynumberand,though thisparametercangreatlyinfluencethevirusbiology,itis most often overlooked in the MOI-related literature [30]. A spectacular phenotype sensitive to gene copy numbervariation(CNV)hasbeendescribedforlysogenic bacterial viruses [29,31,62]. If the MOI is one (gene copynumberisone),thephagemultipliesand killsthe host.IftheMOIisgreaterthanone,thephagestendto integrate into the host genome leaving the cell alive. Thus,achange in theMOIcan changethe fateof the infectedcellanddefinewhether transmissionisvertical or horizontal. Such dramaticalteration of viralbehavior resultingfrompotentiallysmallchangesintheMOI(or, moregenerally,ingenecopynumbers),havebeen theor-eticallystudiedandpredictedtobeverycommon[30]. The important point here is that phenotype switches triggered by CNV do occur even if the co-infecting genomes arestrictly identical.Hence, thetotalnumber ofgenomespassingthroughabottleneck(thepopulation bottleneck)istheonethatiscriticalhere,notthenumber of distinct genotypes (the genetic bottleneck). CNV-regulatedphenotypeshavebeen showninorganisms as different as phages [29,31,62], bacteria [65,66] and mammals[67,68],sotheymighthavebeenlargely under-estimatedinthebiologyofotherviruses.Thestudyofthe MOIduring viruslifecycleshasevenbroader perspect-iveswhenviewedunderthis newlight.
ThevaluesoftheviralMOIinnatureremainunknown, probablyowing to thetechnical challengeof their esti-mation.Currently,formalestimationshavebeenreported foraphage,aninsectvirus,threeplantvirusesandHIV-1 (seereferencesin[38]andTable1).Figuresaltogether rangebetween3and13genomespercell.Withsuchlittle information,itisimpossibletopredictwhatMOIvalues canbeinthediversityofthe‘virosphere’.Nevertheless, webelievethattheinherenttrade-offsassociatedtothe MOI implyits tight regulation. Thus, theMOI values determined might be considered adaptive traits. This hypothesis predicts that different virus species would have evolved ahigh or a low MOIstrategy depending on their biological properties, as proposed above for severeorrelaxedbottlenecks.SincetheMOIisdirectly controlledbyviralmechanisms ofsuperinfection exclu-sion(seeIntroduction), controllingtheMOIcouldbea mean for viruses to implement its above-mentioned strategy of‘going alone’ (low MOI)or ‘going together’ (highMOI).
Some data from plant viruses are consistent with this hypothesis. Numerous reports of spatial segregation suggest that low-MOI scenarios do exist (reviewed in [12]).Indeed,toourview,thespatialsegregationshown inFigure1canonlybeexplainedbyaMOIclosetoone when the virus exits the vasculature (sieve tubes) and enters the first leaf companion cells, inducing the observedindividualizationofthetwofluorescentclones.
The subsequent invasion of neighboring cells in the mesophyl creates leaf territories occupied by specific clones, the overlap between territories being probably preventedbyinhibitionofsuperinfection.Becausespatial segregation is observed all along infection of the host plant(ourownobservationonTuMV)aMOIclosetoone seemstobeconstantlymaintainedatleastinthe compa-nioncellsthroughwhichthevirusexitsthesap.Wehave observed a very different scenarioduring CaMV infec-tion. In plants co-inoculated with two distinct CaMV variants, the proportion of observed co-infected cells rapidly reached 100% in systemically infected leaves [38].Thisisclearlyprecludinganypossibilityofspatial segregationas observedwith TuMV(Figure1).Infact, we have demonstrated that CaMV colonizes cells in groups and that group size (i.e. the MOI) is variable anddependsontheviralloadinthevasculature[38,43]. FromthesesimpleobservationswithTuMVandCaMV infectingthesamehostspecies,wecansuggestthatsome virusescolonizecellsingroupswhereasothersgolonely. Ifonedarestorespectivelyassimilatethesetwoscenarios to contrasted social behaviors, then the rationale for adapting such a distinct way of life in the two species isatthispointelusive.Additionalexhaustivecomparisons betweenspecieswith differentgenomestructure, repli-cation,andgeneexpressionstrategieswillberequiredin ordertodetectanycorrelationbetweenaparticularviral featureandoneortheothertypeofpopulationdynamics. Inaddition,itisverylikelythatarangeofintermediate situations exists between the two opposite scenarios sketchedhere,andthiswillcertainlyaugmentthe diffi-culty in deciphering the parameters driving viruses towardsoneortheother.
Conclusions
Callingformorequantitativedataonbottlenecksizesin different virus models, we now possibly surmise that different viral species might cope differentially with bottlenecks. If our speculation is sound, some viral speciesdomaintainnarrowbottlenecks,atleastatsome specificstepsoftheircycle,whileothersappearto main-tainthemaswideaspossible.Theenigmaofthe mech-anisms, and the respective benefits and costs, that are hiddenbehindthesedifferentviralstrategiesrepresentsa verystimulatingdirectionforforthcomingresearchinthis field.
Wewouldliketoconcludebyhighlightingtheneed for increased consideration of the gene copy number vari-ationsinviruses,withregardstobottlenecksand particu-larly to MOI. As theoretically predicted [30], and demonstrated experimentally in phages [29,31,62], the biological properties of a virus can totally change depending on the initial number of genomes within a cell.Thisuncoversthepossibilityofcomplexregulations in the viral cycle through changing MOI in time or in
differenthosttissues.Ifso,CNVwould certainlybean importantunforeseenplayerinthetrade-offsinvolvedin MOI regulation,and more generally in viralpopulation dynamics/genetics.
Acknowledgements
SBandSGacknowledgesupportfromINRAdepartmentSPEandfromthe grantNo.2010-BLAN-170401fromtheFrenchANR.YMacknowledges supportformtheCNRSandIRD.
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