Firstsynthesizenewviruses thenregulatetheirrelease? Thecaseofthewild rabbit
E.ANGULO*andB.COOKE†
*EstaciónBiológicadeDoñana,Apdo. 1056,E-41080Sevilla,Spain andInstitutodeInvestigaciónenRecursosCinegéticos,Apdo.535, E-13080CiudadReal,Spain,†CSIROSustainableEcosystems,G.P.O.Box284,Canberra,ACT2601,Australia
Abstract
Europeanwild rabbitsoriginatedinsouthwesternEuropebuthavebeen introducedinto manyothercountries world-wide,becomingserious pests inmanyinstances. Asacon- sequenceofrabbitsbeing regardedsodifferently, appliedresearchfortheirmanagement oftenhasopposinggoals,namelytheirconservationortheircontrol.Furthermore,modern genetechnologyhasledtotheconceptofusing geneticallymodifiedmyxomaviruses for rabbitmanagement,againwithquitecontraryaimsinmind.Inthispaperweexplain the possibleecologicalandeconomicconsequencesofusingthesegeneticallymodifiedviruses inappropriatelyandwe consider whether national andinternationalregulationsare sufficienttopreventimproperuse.Ifinternationalregulationsareinadequate,molecular biologistsand ecologistsmust consider the consequencesof their research and advice beyondtheirowncountrytoavoidunwantedimpacts.
Keywords:conservation,GMO,myxomatosis,Oryctolaguscuniculus,rabbit haemorrhagicdisease, wildrabbit
Introduction
Organismsmodifiedusinggenetechnologyarecommonly referredtoasgenetically modifiedorganisms(GMOs). Theyarenowwidelyused inindustry,agriculture,health care,andbiologicalcontrol,usuallyinvolvingreleasesinto theenvironment.
Nevertheless, developmentsinthisareahave often progressedfaster than the legislationthat providesfor their safeuse and Tiedjeetal.(1989)have recommended thatresearchshouldbecarriedoutwithinaframeworkof science-based regulationthat encouragesinnovation withoutcompromisingenvironmentalvalues. AsGMOs may bespreadbeyondpolitical boundaries,itisessential toachieve internationalco-ordinationindevelopingsuch regulations.
Here wepresent thecaseofgeneticallymodifiedrabbit viruses,onedevelopedtohelpconserverabbits, theother developedfortheir control, inwhich thelackofeffective
Correspondence: Elena Angulo. Postal address: Estación
Biológica de Doñana,Apdo. 1056,E-41080Sevilla, Spain. Fax:
+34954621125;E-mail:
international co-ordinationandcontrol couldcompromise themanagementofrabbit populationsincountriesother than those inwhich theviruseswere developed(Angulo
2001).
Currentdistributionandecologicalproblems concerningwild rabbits
TheEuropean wild rabbit, Oryctolaguscuniculus, originated insouthwesternEurope ontheIberian peninsula(Fig.1).Itisaprolific species and has always supportedadiversepredatorcommunity.InSpainitisthe staple prey of two endangeredpredators,the imperial eagle(Aquilaadalberti)andtheIberianlynx(Lynxpardinus) (Delibes &Hiraldo1981).Humanshave alsotaken advantageofrabbit abundance:over onemillion hunters generate an estimated US$1.2×109 annually in Spain (Villafuerteetal.1998).However, inthelast50years wild rabbit populationshave undergoneasharp decline causedmainly bytheappearance oftwoviraldiseases, myxomatosisand rabbit haemorrhagicdisease (RHD) (Queneyetal.2000).Huntersandconservationistsalikeare concerned.
Fig.1Current distributionof Europeanwild rabbits. Arrows indicatesmallareaswhererabbits havebeenintroduced.Natural populationsmarkedingreyandintroduced populationsinblack.
Besides spreadingnaturallyinto other Europeancoun- tries,rabbits havebeendistributedworld-widebymanfor food and hunting(Fig.1). In many areas rabbits have becomearealpest,multiplying‘likerabbits’inanoptimal environment andwiththelackofeffectivepredators (Holland1999).InAustralia,rabbits cause erosion, land degradationand loss ofnative plants(Fenner Fantini
1999) and rabbit control and agricultural losses cost US$310 million annually(Robinsonetal.1997). InBritain, cropdamageisestimatedatoverUS$170millionannually (R.C.Trout,personalcommunication).
New rabbitmanagementprogrammes: Australian vs.SpanishGMviruses
Given the two distinctlines of researchfor the management of wild rabbits, conservation and pest control, itisintriguingthat, foreach, asolutionisbeing soughtthroughthegenetic manipulationofthemyxoma virus (MV)originally derivedfrom cottontailrabbits (Sylvilagusspp.)intheAmericas.
Intryingtodealwith diseasesinwild rabbits insouth- west Europehuntersand conservationistshave increas- ingly turnedto moleculartechnology.Immunization of rabbits againstmyxomatosishaslongbeenpossibleusing cellculture-attenuatedMVstrains.However,duringthe lastfewyears,researchershaveexploredwaysof develop- ing recombinant vaccines that express the RHD virus (RHDV)capsidprotein.These includetheuseofbaculo- virus (Laurentetal.1994),poxivirus(Fischer etal.1997), plant viruses such as potyvirus(Fernández-Fernández etal.2001),orplants(Castañón etal.1999).Most import- antly, Bertagnolietal.(1996)produceda recombinant vaccinebased onanattenuatedMVthatexpressedRHDV capsid proteintoprotect simultaneouslyagainst both diseases.Mostofthesystemslistedrelyondirectinoculation ofindividualrabbits, and consequentlyarenotsuitedfor
large-scalewild rabbit vaccination. However,Spanish scientistshaverecentlydevelopedanalternativeGMvirus, based on an attenuatedbut transmissiblefield strain of MV,genetically modifiedtoprovideprotection against RHD aswell. Itiscapableofhorizontaltransmissionby contactbetweenrabbits;thus,onlyafewrabbitsneedtobe initially vaccinatedtoachieveimmunizationofthegreater population(Bárcena etal.2000). The Spanish National CommitteeofBiosafety authorizedthe experimentaltest releaseofthisrecombinantonaMediterraneanisland, Isla delAire,toassess itspotencyand safety. Infected rabbits producedantibodiesagainstboth viruses,and horizontal transmissiontoabout 50%ofuninoculatedrabbits inthe field was observedduringthe short trial period(Torres etal.2001).Scientists are hopefulofwidespreadrelease soon.
Thesame conceptofnaturalspreadofvirus toaffect rabbitpopulationsonawidescaleisalsobeingconsidered tocontrolrabbits.OneinitiativeofthePestAnimalControl CooperativeResearch Centre (PAC-CRC) inAustraliais theuseofGMMVtoreduce rabbit fertility through transmissible(virally vectored)immunocontraception. Thisconcept wasproposedattheConferenceonFertility ControlinWildlife held inMelbournein1990.Theidea wastodeveloprecombinantviralvectorsthatcantransmit immunogens toinduceaspecificimmuneresponseinthe target animal againstreproductiveproteins.Specificand contagiousviruses,inthiscaseMV,could disseminatethe contraceptiveagent into the population(Tyndale-Biscoe
1991).TherecombinantMVproduces therabbit zona pellucidaglycoproteinB and initial experimentshave induced temporaryinfertilityin 25% of female rabbits (Kerretal.1999).
ImpactofGMrabbitviruses:theworld-wide spreadofMVandRHD
WhilebothGMviruses could bevaluable inmanaging rabbits inthe countrieswherethey arebeing developed, theproblemisthat they may cause anentirelyunwanted effectinanothercountry,and thehistoryofrabbit viruses shows clearlythattheyarewellsuitedtoglobalspread.
MyxomatosiswasfirstrecordedinMontevideo,in1896 (Fig.2a)whenitwasspreadfromthenative South Amer- icancottontailrabbit, SylvilagusbrasiliensistoEuropean rabbits. Soonafter itsdiscovery,MVwas suggestedasa possibletoolforthecontrol ofrabbits inAustralia.During the1920s−1940s,therewasgreatdebateovertheuseofMV tocontrol rabbits, butitwasneverthelesslegally released inAustraliain1950(Ratcliffe etal.1952).Thesuccess of myxomatosis in Australia led a French landowner to release thevirus illegally in1952, andsubsequently myxomatosisspreadnaturallythroughtherestofEurope (Muñoz1960;Sellers 1987). Myxomatosiswas illegally
Fig.2(a)Origin and expansionofmyxomavirus. (b)Origin and expansionofrabbit haemorrhagicdisease.(Virus spreadin rabbitries outside the wild rabbit distribution has not been shown.)
used byArgentineanlandownerstocontrol thespread of wildrabbitsandwasillegallydistributedinBritain(Fenner
Fantini 1999).
Myxomatosisinitially reducedBritishwildrabbit popu- lations by99%(Flowerdewetal.1992).InAustraliaMV was alsohighly effective atfirstbutattenuatedintoless virulentstrains and rabbits developedgenetic resistance to the diseasesothat todaythere isadynamicbalance betweenvirulenceand hostresistanceinwhich myxoma- tosis kills between40and 60%ofinfectedsusceptible rabbits (KerrBest1998).Thisalsoexplainswhy rabbits were relativelyrare inBritain for about 25years (Lloyd
1981)andwhyMVcontinuestoregulatetheirpopulations today(Troutetal.1992).
MVhascaused majordeclines innative wild rabbit populationsofsouthwest Europe.InSpain,itresultedina reductionofhuntingactivity(Muñoz1960),andnegatively
affected endangeredpredators(Delibes &Hiraldo1981). MV also had negativeenvironmental(Flowerdewetal.
1992)andeconomicimpacts(Fenner Fantini 1999).
In1984,a newdisease,RHD[alsoknownasrabbitcalici- virusdisease(RCD)inAustralasia],appearedinrabbitries inChina (Fig.2b).In1987itappearedinItalyand broke out simultaneouslyinseveral other Europeancountries, transmittedlargely throughtrade indomesticrabbits. It quicklyexpandedintowildrabbitpopulations,evencross- ing the English Channelinto Britain by1992(Chasey Trout1995).In1995,beforeitwasfullyevaluatedasanew rabbit control agent inAustralia,RHD escapedfrom an experimentaltrial on a quarantinedisland and crossed
5kmofseatomainlandAustraliawhereitsoon became established (Kovaliski 1998). In1997 itwasillegally introducedinNewZealand(O’Keefeetal.1999).
InAustralia, theinitialeffectivenessofRHDwas variable,withthehighestlevelsinaridandsemiaridareas wheremortalityreached95%,leadingtothe collapse of rabbit commerce (Fenner Fantini 1999). Meanwhile, RHDhad sharply reducednative wild rabbit populations insouthwestEurope. ThefirstRHDepizooticscaused mortalityrates between70and 90%indomesticrabbits, and between50and 60%inwild rabbits (Villafuerteetal.
1994),althoughMarchandeauetal.(1998)detectedmortal- ityrates up to80%inwild rabbits. InSpain, fewpopula- tions have recoveredto prior levels, directlyaffecting huntingactivity and endangeredpredators(Fernández
1993;Villafuerteetal.1998;MartínezCalvo2001).
Asingle pathogenicRHDV serotypeseems toexist to date (Asgari etal.1999).But a nonpathogenicrabbit calicivirusrelatedtoRHDVhasbeendescribedindomestic rabbits (Capucci etal.1996).Besides, seropositiverabbits, apparentlycarryingantibodies raisedagainstarelated nonvirulentcalicivirusand protectedfrom severe RHD, have been foundinEurope(Trout etal.1997),Australia (Nageshaetal.2000) and New Zealand(O’Keefe etal.
1999).Mutationofanavirulentformofthecalicivirusisa possibleexplanationforthe origin ofRHD (Rodak etal.
1990).
Canvirusesbecontainedwithintargetpopulations ordistinctgeographicalareas?
Forboth theSpanishGMvirus, which vaccinatesrabbits againstmyxomatosisand RHD,and theAustralianvirus, aimedatreducingthe fertility ofrabbits, itisenvisaged that active virusesthat retain their capacity to spread wouldbemost useful. Thisisimportantbecauseitwould notbenecessary tovaccinate every rabbit. Anaturally spreadingvaccine could beintroducedinto some rabbits then spreadtoagreater part ofthepopulation. However, itispreciselythischaracteristicthat would make them so difficult tocontain.
Thereadyspreadofboth MVand RHDVraises many questionsaboutourabilitytocontainsuchviruses.Clearly landholdersinterestedinreducing rabbit problems deliberatelyspreadMVandRHDV.Thereisalsoarisk,as happenedwithRHDVinAustralia,ofunderestimatingthe role ofinsect vectors intransmittingthe virus over very longdistances.Seabirds have alsobeenimplicatedinthe spreadofboth MVand RHDV. Itisthereforequite con- ceivable that recombinantMV could be used in areas wheresuch risks were not fully considered.Indeed,the most recent trialswith aGMMVwere carried outonIsla delAire,only1kmoffshore fromoneof thelargerBalearic Islandsand wherethere isaseagull colony and regular huntingactivity.
Other issues also need to be fully understood.These includethepotentialforinteractionbetweenGMviruses and field strainsofMV(Tyndale-Biscoe1994)including genetic exchangebetweenGMvirusesand wild viruses which may have differentvirulenceorgreaterability to compete.Itisalso necessarytounderstandand counter anypotentialimpact ofGMvirusesonSylvilagusspp.,the originalhostsofMV.
Althoughsuchquestions arebeingconsidered with the ideaofdevelopingsafeguardsintheGMviruses,theidea ofusing actively spreadingvirusesremainsproblematic. Aswehave seen, MVand RHDVaredifficult tocontain withindistinct geographical areas. Itisessential toask whetheritwouldbepossibletopreventthepotentialfor spread ofGMrabbit virusesintoinappropriateregions throughcurrentlyavailable mechanismssuch asinter- national controlsandregulations.
Whatregulationscoverresearchandreleaseof
GMOsatnationallevels?
Duringthe1970smany countrieslaunchedbiotechnology policiesandmanagementplans.Mostdistinguishbetween contained GMOwork and deliberate releases intothe environmentwithseparatelegislation.Anationalauthority generally regulatesapprovalforrelease followingrisk assessmentthat mayinclude scientificand ethical con- siderationsaswellaspublic consultation.
Forexample,in1990,theEuropeanUnion allowedfor releases of GMOs throughDirective 90/219/EEC(EEC
1990).Within that frameworkanationalauthoritycould evaluaterisks. Thisdirectiveresolvedthe problemsona nationallevelbutcreatedaproblemontheEuropeanlevel, asother MemberStatescould notdiscussthedecision.In April2001anewDirective 2001/18/ECwasadopted(EC
2001),wherebythereleaseofaGMOinanycountryneeds theagreementoftheEuropean Commissionandtherestof the memberstates. The final date for MemberStates to comply with this Directive isOctober 2002(althoughit hasnotyetbeenadoptedinSpain).Untilthisdate, GMO
applications(i.e.recombinantvaccineMV)maybesubject totheDirective 90/220/EEC.
InNew Zealand,the HazardousSubstancesand New Organisms Act covers the importation, development, field-testingand theintentionalrelease ofGMOsintothe environment( viruses, anassessmentwouldobviouslybemadeinterms oftheir capacity tocausedisease.But,itisnotclearwhetherinter- nationalrisksorconsequencesareconsideredbythislegis- lation.
Genetechnologywassubjecttovoluntaryassessmentin Australiafrom1975until June2001.Responsibilitieswere held bydifferentcommittees,buttheir recommendations werenotenforced.In1997Australiabeganpreparingnew legislationtotightenassessment.Called theGeneTechno- logyAct2000,itcommencedoperationinJune2001(Radke
2001).Forthe release ofGMOinto the environment,the Gene Technology Regulatormay consultinternational experts.TheGeneTechnology Regulatorcanimpose conditionstolimitthespreadorpersistenceof theGMOin theenvironment.However, therelease maybeapproved, claimingisolationdistancesorphysicalbarrierstoother continents.Currently,researchonmodifiedMVdone by thePestAnimal Control CRCand Australian National Universityislicensedas adealingnotinvolvingintentional releaseintotheenvironment.
International agreements onresearchandrelease ofGMOs
Internationalorganizationssuch asthe Organizationfor Economic Cooperationand Development(OECD),World Trade Organization(WTO), the World Organizationfor Animal Health(OIE),the World HealthOrganization, ortheConvention onBiologicalDiversity,trytounify nationalregulations.However,internationalorganiza- tionsonlydevelop recommendationsand guidelines,and thesemayormaynotnecessarilybeadoptedbyindividual countries.
TheOECDseekstoensure safety,developeffective regulatoryoversightand facilitate trade inbiotechnology productsbetweenthe29membercountries.TheOECDhas organizedinternationalmeetings onGMOs,mainlyon modifiedfood and crops. Similarly, the WTOhas devel- oped the Agreementonthe ApplicationofSanitaryand Phytosanitary Measures (SPS) to protect animals and plantsfrompestsanddiseasesandGMOswerediscussed duringSPSCommitteemeetingsinNovember2001.The OIEinformscountriesoftheoccurrenceofanimaldiseases, harmonizes regulations fortrade inanimals oranimal products, and developsrecommendationstoprevent diseasespread.
TheConventionon Biological Diversity adoptedan agreementknownastheCartagenaProtocolofBiosafetyin
January2000,toprotectbiological diversityfrompotential risks posedby GMOs. Itestablishedaprocedurespeci- fically focusingon cross-bordermovementofGMOs in which risks areassessedbynationalauthoritiesbut final decisionsregardingthe importationorrelease ofGMOs must becommunicatedtotheConvention.ByNovember
2001onlyeightcountrieshadratifiedtheCartagenaProtocol, butneithercountries oftheEuropean Community nor AustraliaandNewZealandweresignatories.
We concludethat, while there are some regulations focusedontheresearchandreleaseofGMOsthereappear to be few agreements that specifically address safe research,handlingandreleaseofGMOsataninternational level.Moreover,regulationsarevery general,orfocuson safety issues regardingGMfood trade and importation, andtheireffectivenessisweak,asshownbythediscovery ofGMcrops growinginareas wherepermissionhad not beengranted(Dalton 2001;Jayaraman2001).Regulations fornontradableGMOsorGMvirusesseemtobeevenless wellconsidered.
Conclusion
Appliedresearchforthe managementofwild rabbits in differentparts oftheworldhasopposing goals.Thismay leadtothecreationandreleaseofantagonisticGMviruses, one aimed at conservation, the other aimed at rabbit control. The use ofvirally vectoredimmunocontracep- tiontocontrol pests iscurrentlybeing investigatedfor anumber ofdifferentspecies (Tyndale-Biscoe1991), includingpossums (Sutherlandetal.1996),foxes(Holland
1999),cats(Courchamp Cornell 2000)and rodents (Ylönen 2001).Bearing in mindthe facility with which virusesspreadorcanbeintentionally spreadand the difficultyofvirus control inthe field (Fenner Fantini
1999)merelegislationisnotenough.Toavoid unexpected effectsoftheintroductionofnewGMOsforwildlife man- agement,itisessentialtogetinternational agreementand co-ordinationinthedevelopmentanduseofsuchstrategies. Itisessentialthat researchonrabbit control and con- servationtrytoachieve realisticmanagementgoalswhere risks areminimized(Angulo2001).Certainly,Australian and Spanishscientistsfollow theguidelinesestablished intheir respectivecountries(Robinsonetal.1997;Bárcena etal.2000),butagreatereffortshouldbemadetopromote the internationalcommunicationbetweenscientistsand national and international authorities (Tyndale-Biscoe
1994).Evaluatingthepossibleimpact ofrelease ofGMOs intotheenvironmentrequiresexpertiseinmany scientific disciplines.Between them, molecularbiologists,veterin- arians and ecologistsmust considerthe consequencesof theirdecisions,beyondtheirowncountry.
Thispapertakesastepinthatdirectionbypointingout some potentialimpactsofGMOs being developedfor
managingwildrabbitpopulations.Paststudieshavefocused ongeneralecologicalandevolutionaryaspects(Tiedjeetal.
1989)orparticularlegalandethicalissues(Tyndale-Biscoe
1994),but none has providedathoroughassessmentof therisks. Wemake nospecific recommendationsabout a course ofaction that canbetaken other than tolistsome questionsthatmightberaisedininternationalscientificor regulatory meetings.These includeasking: (i) whether accidentalorillegalspreadcould bepreventedbyexisting internationalcontrolsorconventionsthat regulatecross- borderGMOmovements; (ii)what international scientific structuresshouldbeestablishedtoenabletherationaldevel- opmentofGMOsforwildlifemanagement;(iii)howcaninter- nationalregulationsonGMO releases bedesignedtobe acceptabletoandimplementedwithinindividualcountries? Inessence, there isaneed forscientific and regulatory structures that guide the development and release of GMOs by (i)evaluatingtheir potentialto escape and establishabroad;(ii)assessingwhetheror not risks are internationally acceptableat scientific, economicand environmental levels;and (iii)developing specificregula-
tionoftheiruse.
Inthemeantime,ecologists,veterinariansandmolecular biologistsmust keep aninternationalperspectiveontheir work anddevise measurestoreducetheriskofunwanted ecological and economicimpacts,ofthe kind illustrated hereforvirusesbeingdesignedtomanagewildrabbits.
Acknowledgements
Fundswere providedbyJunta deAndalucía-CSIC(ref.2413/99/ M00)and theSpanishMinistryofScienceand Technology.Xim Cerdá encouragedustowrite this paper,and with Marc Artois andRogerTroutprovideduseful commentsandinsights.Thanks goalsotoAnaAngulo, CarlosCalvete, IvánGómez-Mestre, Christian Gortázar, JavierJuste,SacramentoMoreno, Antonio Tugores, Ramón Soriguer and RafaelVillafuerte.Wearegrateful toTonyPeacock,RobertWayneandtwounknownrefereeswhose commentsgreatlyimprovedthemanuscript.
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