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VIRUSAS POPULATIONS

Composition,Complexity,Quasispecies, Dynamics,andBiologicalImplications

SECONDEDITION

EstebanDomingo

CentrodeBiologíaMolecular “SeveroOchoa” (CSIC-UAM) Cantoblanco,Madrid,Spain

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Foreword

Virusesareminuteorganismsthat,although mostlyinvisible,ruletheworld.Scientistsmay arguewhethertheseobligatoryintracellular organismsbelonginthetreeoflifeorwhether theywerethe firstreplicatingentitiesonearth, butallagreethattheyhavedecisivelyinfluenced evolution.Thevastmajorityoftheplanet’s1031 virusesliveintheoceans,whereastheterrestrial virusesmakeuponlyaverysmallfraction.They were firstdiscovered,however,becausetheir “ghosts” wereexposedthroughdiseasesin plantsandmammals(researcherswereunable to “ see ” theseghostsuntilelectronmicroscopy revealedtheminthelate1930s).Followingthe discoveryoftobaccomosaicvirusandfoot-andmouthdiseasevirusin1898,abewildering numberofviruseshavebeenidentifiedmanyof whichcauseterriblehumandiseases,triggering immensefearandadmiration.Howcanthese minutecreatureslivesoefficientlyand,during pandemics,howcantheyspreadsorapidly acrossanentireglobe?

Inprinciple,allviruseshavethesame,simple architecture:arelativelysmallpieceofnucleic acid(theDNAorRNAgenome),containingall informationforproliferation,thatissurrounded byaproteinaceousshellforprotectionandcell attachment,oftenincombinationwithamembranouslayer.Yetthedifferencesinsizesand shapesofvirionsfornumerousdifferentvirus familiesareastounding.Evenmoremindbogglingisthewealthofgeneticinformation

storedintheirsmallgenomes,whichdirects verydifferentstrategiesofviralproliferation leadingtocelltransformation,tocelldeath,or thedeathoftheentirehost.Throughouthisprofessionallife,EstebanDomingohasstudiedthe highlycomplexissuesofviralgenetics,which qualifieshimsecondtononetosummarizethe entire field.

Domingobeginshisnarrativewithashort courseingeneralvirology,worthyofstudyfor anyoneinvolvedinteachingorcarryingout researchinvirology.Thisisfollowedbyanintroductiontotheoverwhelming,complexsecretsof geneticinformationhiddenineventhesmallest viralgenomes.Domingo’stextisnotdogmatic. Superblywritten,itoffersfactsandhypotheses andencouragesthereaderstoarrivethemselves atthe “truth,” howevershort-livedthattruth maybe.Thereaderispushedtoaskquestions likewhatexactlyisavirusandwheredoesit comefrom?Howcanweunderstandinfection andviraldiseases?Howcanwepreventtheir terror?

Beyondexperimentalbreakthroughsthat haveinformedourgrowingunderstandingof themolecularnatureofvirusesandtheirreplicationincellsandtissuesoftheirrespectivehosts, themorerecentdecadeshavebroughtforthnew conceptsinhowweperceiveviruses.Ashocking surpriseinvirusresearchwasthelandmark discoverybyHansEggersandIgorTammand byEstebanDomingoandCharlesWeissmann

ofthehighmutationrateinviralreplication, particularlyofRNAviruses.Therealizationof error-pronereplicationofvirusesheraldeda paradigmshiftinunderstandingmolecular evolution.Itchangedthelandscapeofallstudies regardingviralreplicationandpathogenesis. Ratherthan fixedentitiesoperatingwithasingle, uniquenucleicacidsequence,individualspecies andserotypesofviruseshavecometobeviewed asgeneticallyheterogeneous,complexpopulationscomprisedofaconsensussequencegenotype,calledbyManfredEigenandJohn Holland “quasispecies.” Asaresultoftheerrorpronereplicationandsubsequentselectionof mutationsthatconfervirus fitnessinresponse tocell-specifichostfactors,immunemodulation, orenvironmentalfactors,viruseshaveevolved toexistasquasispeciespopulations.Withthese observationsasabackdrop,EstebanDomingo offersacomprehensive,up-to-datetreatiseon themutablenatureofvirusesandhowthis propertyaffectsvirus hostinteractions,viral fitnessandadaptation,viralpathogenesis,and theever-changingprospectsforantiviraltherapies,includingtheintriguingpossibilityofmakinguseofwhatiscalledanerrorcatastrophe.

Thenarrativeontheerror-pronereplicationof virusesisamplifiedbybringingDarwinianprinciplestobearonthegenerationofvariantgenomes(and,asaresult,viralquasispecies)and howtheseviruspopulationsbehaveintermsof randomdrift,competition,andadaptation/ selectionfor fitnessindifferentenvironments. Thislattertopicisaddressedindepthviaadiscussionoftheinteractionsofviruspopulations withtheirhostsandhowbiologicalparameters suchasreceptorusage,codonusage/codon pairfrequencies,andthehostimmuneresponse canprovideselectivepressureandresultant coststoviral fitness.Inasubsequentsectionof thisvolume,viral fitnessisaddressedhead-on, withadiscussionofhowgenomicsequencesof

virusesimpactthe fitnesslandscapeduringviral replicationandhowsubpopulationsofvirus quasispeciesmayimpactthemolecularmemory ofanevolvingquasispecies.

Inthesecondhalfofthisvolume,Domingo describesdifferentexperimentalsystemsand approachesusedtoanalyzechangesinvirus populationcompositionresultingfromperturbationsandselectiveforcesappliedduringinfectionandreplication.Forexample,persistent viralinfectionsincellcultureallowthestudy ofexperimentalevolution,whileplaque-toplaquetransfersofvirusesexperimentallyrecapitulateinfectivitybottlenecksthatproduce low-fitnessviruseswhichmayhaveextreme phenotypes.Inaddition,theseexperimentalsystemsprovidetractableplatformstotestmoregeneralprinciplesofgenetics,forexample,Muller’ s ratchetortheRedQueenhypothesis,duringviral replication.Thisbookthenmovestodescribethe studyofvirustransmissionandevolutionin natureandthemanyparametersthatimpact evolutionrates.Emergenceandreemergenceof pathogensisdiscussedinthecontextofthe complexityofbehaviorofviralpopulationsintertwinedwithnonlineareventsderivedfromenvironmental,sociologic,andecologicalfactors.

The finalsectionsofthisbookaredevotedtoa discussionofsomeofthemoretangibleimplicationsofviralquasispecies/sequenceevolutionin consideringitsimpactondiseasepreventionand strategiesforvaccinedesignandantiviral therapeutics.Thislattertopicisanalyzedat boththetheoreticalandpragmaticlevelsas partofwhatDomingocalls “virusasmovingtargets,” duetothedynamicnatureofviralgenome sequencesandtheproteinstheyencode.Domingothenconcludesthisilluminatingmonograph bybringingthediscussionofquasispeciesand populationdynamicsfullcircletoafocuson nonviralsystems(cells,cancer,andotherinfectiousagents)thatleavethereaderwithanumber

of “big-picture” conceptsrelatedtogenetic variation,randomversusselectedreplication events,andinformationtheory.Itistheuniversal natureoftheseconceptsthatmakesthisvolume essentialreadingforvirologists,evolutionary biologists,populationgeneticists,andanyothers

whowishtobeexposedtoanintenselevelof scholarshiponafascinatingtopic. Fiatlux! BertSemler Irvine,California EckardWimmer StonyBrook,NewYork

Prefaceforthesecondedition

Asecondeditionofasciencebookisan indicationthatitdealswithatimelysubject thatattractstheinterestofthescienti ficcommunity.Virtuallyeachofthetopics(rangingfrom originoflife,roleofvirusesinbiologicalevolution,quasispeciesdynamics,toitsimplications forviraldiseaseemergenceandcontrol,orhow cancercelladaptationresemblestherapidly evolvingRNAviruses)maintaintheirinterest. Despitepromiseoferadicationofseveralviral diseases,theproblemsofselectionofescape mutants,andtheinaccessibilityoftreatmentsin manyareasoftheworldpersist.Furthermore, recognitionofdifferencesamongindividual componentsofbiologicalcollectivities(bethem virusesorcells)isbecomingincreasingly apparent. “Complexity” isthekeyterm,and theonethatprovidesamysterydosetotheunderstandingofpopulationsaswholes.Thisis whatthesecondeditionof VirusasPopulations isabout.

Mymaintaskforthesecondeditionhasbeen toupdateobservations,emphasizesomepoints, correctmistakes,andaddliteraturereferences. Eachchaptershouldbeunderstandableinits own,althoughIhaveaimedatconnectingconceptsthroughreferencestootherchapters.The neweditionisfaithfultotheconvictionthatthe maininterestofthisbookliesintheconcepts, asthesupportingexperimentsandmodels changemonthaftermonth.Iwasverypleased

thatElsevierwaspositiveabouttheupdating of VirusasPopulations;bookpreparationwas facilitatedbythegenerousandefficientinvolvementofPatGonzalez,KattieWashington,and LindaVersteeg-Buschman.

TheAcknowledgmentsectionofthe first editionremainsentirelyvalid.Somescientists whocontributedtothefoundationsofthetopics coveredinthisbookarenolongerwithus. JohnJ.Holland(1929 2013)andChristofK. Biebricher(1941 2009)passedawaybeforethe firsteditionwaswritten.Unfortunately,the greatManfredEigen(1927 2019)hastobe added.Oneofhislast(probablytheverylast) piecesofscientificwritingwashisForeword forvolume293of CurrentTopicsinMicrobiology andImmunology thatIeditedwithPeterSchuster. Inthatvolume,aswellasinpages317to498 ofvolume47of EuropeanBiophysicsJournal (dedicatedtohimontheoccasionofhis90th birthday),thewidthofEigen’scontributionsto molecularevolutionbecomesevident.Without Eigen,Holland,andBiebricher,thisbook wouldnotexist.Equallysadwasthepassing awayofJoséAntonioMelero(1949 2018),an outstandingvirologistandcolleaguefromInstitutodeSaludCarlosIIIwhomaintainedavivid interestinscienceandvirology,includingthe subjectsofthisbook.

VirusasPopulations couldnothavebeenwrittenwithouttheinvolvementandgeneroushelp

ofCeliaPerales(nowwithherowngroup),Ana IsabeldeAvila,andIsabelGallego,withthe participationMaríaEugeniaSoriaofCelia’ s team.Mydeepestappreciationgoestothem andtothestudentswhohavebeenorare involvedinourresearch.

Itismyhopethatthisbookcontinuesbeing ausefulintroductiontostudentsofmany

scienti fi careas,aswellastoprofessionals whoseexperiencetellsthemthatitistimetounderstandbiologicalentities(mainly,butnotonly, viruses)atthepopulationlevel.

EstebanDomingo Cantoblanco,Madrid,September2019

Acknowledgments

Thisbookisalongstory,withalottobe acknowledged.Ibeginwiththemostimmediate andthengobackintime,withsomedeviations fromlinearity.Thepresentcoreteaminmy laboratoryatCentrodeBiologíaMolecular “SeveroOchoa” (CBMSO)inMadrid,composed ofCeliaPerales,AnaIsabeldeAvila,andIsabel Gallego,havedoneanimmensejobthathas permittedthetimelycompletionofthisbook (photographyattheendoftheAcknowledgments).Inadditiontoherscientificleadership, Celiahasuniqueskillstoconvertscienti fic conceptsintoimages.Hersarealltheoriginal figuresinthisbook.AnaandIsabelhave diligentlycomplementedmycomputerage inabilitiesbykeepingtrackofend-noteand professionaltyping,alwaysalerttomistakes andinconsistenciesinmydrafts.Celia,Ana, andIsabelhaveworkedonthisbookwhile continuingwithexperimentsandactingasa survivalbrigadeinverydifficulttimesfor Spanishscienceduetodrasticbudgetrestrictions.Iamdeeplythankfultothethreeofthem.

IamalsoindebtedtoEckardWimmerand BertSemlerfortakingtimetoreadthisbook andtowriteaForeword.Ideeplyappreciate theirhelp,notonlynowbutalsoatdifferent stagesofmyactivityasapicornavirologist.We sharedfriendshipwithJohnHolland,adecisive nameinthescientificcontentsofthisbook.

ManythanksgoalsotoElsevierstafffor theirsupportandinvolvement.Inparticular,to ElizabethGibsonintheearlystagesofbook

proposal,JillLeonardforhercontinuousencouragement,HalimaWilliamsforpushingmethe rightdose(neverbeyondacatastrophe threshold),andJuliaHaynesforanintelligent andpositiveattitudeduringthelaststagesof bookproduction.IamindebtedtoElsevierfor publishingthisbookandalsoforlong-time supportreflectedinthepublicationofthetwo editionsof “OriginandEvolutionofViruses” and myinvolvementintheeditorialboardofVirus ResearchinitiallywithBrianMahy,andrecently withLuisEnjuanes,AlinaHelsloot,and distinguishedcolleagues.

Ourlaboratoryhashadtheprivilegeto engageinmultiplecollaborationsthathave broadenedthescopeofourresearchwhilemaintainingthefocusonimplicationsofvirus complexity.Webelongtoanactivenetworkof expertsinliverdisease(Spanishacronym CIBERehd),thankstotheinterestofJordi Gomez,JaumeBosch,JuanIgnacioEsteban, andJosepQuerinourworkonviralquasispecies andmodelstudieswithhepatitisCvirus(HCV). Ourcurrentcooperationinvolvesliverdisease expertsofdifferentinstitutions,particularly JosepQuer,JosepGregori,JavierGarciaSamaniego,AuroraSanchez-Pacheco,Antonio Madejon,ManuelLeal,AntonioMas,Pablo Gastaminza,XavierForns,andtheirteams. OurconnectionwithHCVgoesbacktotheearly 1990swhenJordiGomezandJuanIgnacio EstebanfromHospitalValld’Hebronin Barcelonacametoourlaboratorytodiscussthe

resultsthatbecamethe firstevidenceofHCV quasispeciesininfectedpatients(thenowclassic Martelletal.,1992,J.Virol.66,3225 3229).The cellculturesystemforHCVwasimplemented inMadridwiththehelpofCharlesRiceand theeffortsofCeliaPeralesandJulieSheldon. Theseongoingstudiesareasourceofknowledge andinspirationforus.

BeforeHCV,therewere35yearsofresearch onfoot-and-mouthdiseasevirus(FMDV).About 100students,postdocs,andvisitorscontributed totheresearchwhosemajoraimwastoexplore ifquasispeciesdynamicsappliedtoanimal viruses.Icannotmentionallpeopleinvolved, butmostofthemarecoauthorsofpublications quotedinthebook.JuanOrtín,thenowfamous influenzavirus(IV)expert,andIorganizedand sharedour firstindependentlaboratory(“down atthecorner” ofC Vblock)atCBMSO, animportantinstitutionforSpanishbiomedical sciencescreatedinthe1970sundertheauspices ofEladioVinuela,MargaritaSalas,David Vazquez,CarlosAsensio,FedericoMayor Zaragoza,SeveroOchoa,andothers.Eladio suggestedtoJuanandItoworkonIVand FMDVbecauseoftheunknownsunderlying antigenicvariationofthesevirusesandthe problemofproducingeffectivevaccines.And sowedid.JuanonIVandmeonFMDV. MercedesDavilajoinedaslaboratoryassistant andremainedintheFMDVlaboratoryfor 31years(!),onlytoleaveasaresultofan appointmenttomanageacentralfacilityat CBMSO.The firstgraduatestudentswere FranciscoSobrino(Pachi)andJuanCarlosdela Torre.TheyinitiatedtheFMDVquasispecies workatthepre-PCRage.MauricioG.Mateu wasdeterminanttoexpandthescopeofour researchbecauseatthesuggestionandwith helpofLuisEnjuanes,weextendedthestudies ofgeneticvariationtoantigenicvariationby

producingandusingmonoclonalantibodies againstFMDV.Inadditiontoourown antibodies,wereceivedsomefromPanaftosa, Brasil,andEmilianaBrocchifromBrescia, Italy.Collaborationswereestablishedwith PanaftosaandCentrodeVirologíaAnimaland INTA-CastelarfromArgentina.Wealsobegan productiveprojectswithErnestGiraltandDavid Andreuonpeptideantigens,withIgnasiFita, NuriaVerdagueronthestructureofantigen antibodycomplexes,andDavidStuartonthe structureoftheFMDVserotypeusedinour research.ThecollaborationwithNuriaVerdaguerandherteamcontinuestodateonviral polymerases,particularlywiththeworkof CristinaFerrer-Orta,inalineofworkinitiated byArmandoAriasatthebiochemicallevel.

The firstnucleotidesequencesofourFMDV referenceviruswereobtainedbyNieves VillanuevaandEncarnacionMartínez-Salas.In thelate1980s,CristinaEscarmís(mywifeand thepersonwhointroducednucleotide sequencinginSpainandinourlaboratory) joinedourgrouptoexpandoursequencing know-howandtoworkonthemolecularbasis ofMuller’sratchet.Usingroutinelynucleotide sequencesratherthanT1-fingerprints(atransitionfacilitatedbyJohnSkehel,withasummer visittoMillHill)wasasexcitingachangeas weexperiencenowadaysseeingdeep sequencingdata.AdditionalhelptotheFMDV researchwasobtainedfromJoanPlana,Eduardo L.Palma,MaríaTeresaFranze-Fernandez,Elisa C.Carrillo,AlexDonaldson,JoaquínDopazo, AndrésMoya,PedroLowenstein,andMaría ElisaPiccone,amongothers.

Inthe1980s,thereweretwoeventsthatledtothe connectionofourlaboratorywiththoseofJohn HollandandManfredEigen.OnewasthepublicationbyJohnHollandandcolleaguesin1982ofthe Science (1982)articleon “Rapidevolutionof

RNAgenomes ” Thepaperwasbroughttomeby mycolleaguefromInstitutodeSaludCarlosIII JoséAntonioMelero.WhatIthoughtwasthe forgottenQb workofZurichsuddenlyrevived ina Science articlethatproposedanumberof biologicalimplicationsofhighmutationrates andrapidRNAevolution.Notwithout hesitation,someyearslater,Iwrotealetterto John.Ashewasextremelyreceptive,wetalked onthephoneandmetforthe firsttimeatthe InternationalCongressofVirologyinEdmonton, Canada,in1987.JuanCarlosdelaTorrewentto workwithJohnasapostdoctoralstudent,and thenIspentasabbaticalstayin1988 89, followedbyseveralothervisitstoUCSanDiego. OurfriendshipandsharedviewofhowRNA virusesworklasteduntilhisdeathin2013.John andhisteam(whichatthetimeofmyvisits includedDavidSteinhauer,DavidClarke, ElizabethDuarte,JuanCarlosdelaTorre,Isabel Novella,ScottWeaver,andseveralstudents, andvisitsofSantiagoElenaandJosepQuer) werepioneersinlinkingbasicconceptsof populationgeneticswithviralevolution.Some oftheexperimentsmarkedthebeginning of fitnessassaysandlethalmutagenesis,so importantincurrentvirology.Johnwasagreat supporttoourworkinMadridinthosetimes ofincredulityofhighmutationratesandquasispecies.Imisshistimelyandencouraging commentsenormously.ThevisitstoSanDiego openedalsolinkswiththeScrippsResearch InstituteatLaJolla,whenJuanCarlosdelaTorre joinedMichaelB.A.Oldstonedepartment.Each visittoScrippsisanimportantscientificstimulus, andthefriendshipandsupportofJuanCarlos andMichaelremainunforgettable.

ThecontactwithManfredEigenbeganina coincidentalmanner.TheColombianphysicist AntonioM.RodriguezVargasinvitedseveral EuropeanandUSscientiststoparticipateinthe firstLatinAmericanSchoolofBiophysicsheld

inBogotain1984.Invitedspeakersincluded themembersofSolSpiegelman’steam,Fred KramerandDonaldMills,andChristof BiebricherfromGottingen.Thiswasthe first timeImetChristof,andourfriendshiplasted untilhisdeathin2009.Heintroducedmeto ManfredEigenandIparticipatedinseveral Max-PlanckWinterSeminarsthatManfred organizesatKlosters,Switzerland.Iwillnever forgetthediscussionswithChristofonthe theory experimentinterphaseofquasispecies, atfreezingtemperaturescombinedwithahot soupatamountainrestauranthut.Oneofthe years,JohnDrake,thepioneerofmutationrates, joinedusinextremelylivelydiscussions.At Klosters,ImetPeterSchusterwithwhomI havekeptcontactsince.Thediscussionswith Peteronquasispeciesanderrorthresholdhave beenextremelyhelpfulandclarifying.The WinterSeminarswereakeyinconvincingme thatourMadridresearchwasontherighttrack, andthatIcouldsurvivegivingatalkinfrontof NobelPrizeawardeeswhichatthetimeI perceivedasanachievement.

Thetransdisciplinary flavoroftheseminarsat KlostersrevivedyearslaterinMadridwhenin the1990sthephysicistJuanPerezMercader,at thesuggestionofFedericoMoran,invitedme tojoinintheorganizationofanewcentertermed CentrodeAstrobiología(CAB),inTorrejonde Ardoz,nearMadrid.Excitingdiscussionswith manyscientistsconformedanewinterest inthenascentscienceofAstrobiologywith participationofnotedscientistssuchasAndrés Moya,RicardSolé,FedericoMoran,Ricardo Amils,DavidHochberg,AlvaroGiménez,Luis Vazquez,RicardoGarcía-Pelayo,Ramon Capote,andFranciscoAnguita,amongothers. ThescienceatCABopenedseveralcollaborationswithSusannaManrubia,EsterLazaro, andCarlosBrionesthatcontinuetoday,with amonthlyseminarwithFranciscoMontero,

CecilioLopez-Galíndez,andothercolleaguesas participants.

Theviewonviralpopulationsthatthisbook conveyshaditsorigininworkwithbacteriophageQb inthelaboratoryofCharlesWeissmanninZ € urich.I firmlybelievethatdespite thegreatrecognitionthatCharleshashadasa scientistduetomanyachievementsindifferent fields,theearlyQb workthatcontributedthe firstsite-directedmutagenesisprotocols,the birthofreversegenetics,andthe firstevidence ofhighmutationratesandquasispecies dynamicsisstillunderappreciated.Perhaps,as thenotedmolecularbiologistandvirologist RichardJacksononceputit,theworkcame20 yearstooearly.Fundamentalgroundworkon theearlygeneticsofbacteriophageQb was

performedinZurichbyMartinBilleter,Hans Weber,EricBandle,DonnaSabo,Tadatsugu Taniguchi,andRichardFlavell.Yearslater, whenWeissmannreadHolland’s1982paperin Science,hetoldmethatanew fieldofresearch hadbegun.

Icomebacktothepresent.Thereasonto namesomanypeopleinpreviousparagraphs isnottopublicizeabiosketchthatwillinterest onlymyfamily(andminimally).Thereasonis thateachofthepersons,institutions,events, andencountersmentionedhashadsomeinfluenceinthecontentsofthisbook.Theideaof writingabookoccurredtomeyearsback whenIproducedachapterforFieldsVirology thatIhadtoreducebyafactorof fiveto fitthe requiredlength.Otherincentivescamefrom

whatwouldbeusuallyconsideredinconsequentialepisodes.Forexample,about10yearsago, beforeatalkinFrance,oneofthehostscientists toldmethathewaseagertohearmy “ new ” ideasaboutvirusevolution,whenIhadbeen workingonquasispeciesforalmost30years! Inaddition,atameetingonantiviralagents, somebodytoldmethathewasunabletoselect aresistantviralmutant,andhefoundagood ideamysuggestionofincreasingtheviral populationsizeintheserialpassages. Theseandafewothereventsreinforcedinme thethoughtthatperhapsabookcouldbeof someuse.Thisbookisintendedtobeanintroductiontofundamentalconceptsrelatedtothe rolethatviralpopulationnumbersplayin severalfeaturesofvirusbiology.Relatedaspects ofagivenconceptareexplainedindifferent chapters.Thisiswhymanyboxesandcrossreferencesamongchaptersareincluded.Ingenetics language,Ihaveaimedatcomplementation amongchapters.Thespeci ficexamples(some ofwhichattainaconsiderabledegreeofdetail andareunderstandablybiasedtowardmy expertise)arejustanexcusetoillustrategeneral points.Hopefully,thereaderwillbeableto applythemtospecificcases.

Beforeending,Ihaveadditionalnamesto thankfortheirsupportondifferentoccasions. Attheriskofforgettingsomenames,theyare SimonWain-Hobson,NoelTordo,JeanLouis Virelizier,MarcoVignuzzi,CarlaSaleh,and othercolleaguesatPasteurInstituteinParis, RafaelNajera,MiguelAngelMartínez,Enrique Tabarés,AlbertBosch,RosaPinto,RaulAndino, CraigCameron,KarlaKirkegaard,OlenKew, ErnstPeterhans,EtienneThiry,Paul-PierrePastoret,FranciscoRodriguez-Frias,XavierForns, LuisMenéndez-Arias,VincentSoriano,Noemi Sevilla,StevenTracy,DavidRowlands,Louis M.Mansky,RobertoCattaneo,StefanG.Sarafianos,KamalendraSingh,UlrichDesselberger, AlexanderPlyusnin,MargaritaSalas,Jesus Avila,AntonioGarcíaBellido,CarlosLopez Otín,andPedroGarcíaBarreno.Inaddition, mythanksgotoboardmembersoftheSpanish SocietyofVirologyandoftheRoyalAcademy ofSciencesofSpain.Lastbutnotleast,colleaguesandstaffatCBMSO,andfunding agencies,whohavemadeourworkpossible. Mydeepestappreciationgoestoall.

EstebanDomingo Cantoblanco,Madrid,Summer2015 ForIker,Laia,Héctor,Jorge

Introductiontovirusoriginsandtheirrole inbiologicalevolution

Abbreviations

AIDS acquiredimmunedeficiencysyndrome

APOBEC apolipoproteinBmRNAediting complex

CCMV cowpeachloroticmottlevirus

dsRNA double-strandedRNA

E.coli Escherichiacoli

eHBV endogenoushepatitisBviruses

HBV hepatitisBvirus

HCV hepatitisCvirus

HDV hepatitisdeltavirus

HIV-1 humanimmunodeficiencyvirustype1

ICTV InternationalCommitteeonTaxonomyof Viruses

Kbp thousandbasepairs

mRNA messengerRNA

PMWS postweaningmultisystemicwasting syndrome

RT reversetranscriptase

RdRp RNA-dependentRNApolymerase

ssRNA single-strandedRNA

T7 bacteriophageT7

tRNA transferRNA

UV ultraviolet

1.1Considerationsonbiological diversity

Toapproachthebehaviorofvirusesactingas populations,wemust firstexaminethediversity

ofthepresent-daybiosphereandthephysical andbiologicalcontextinwhichprimitiveviral formsmighthavearisen.Evolutionpervadesnature.Thankstonewtheoriesandtotheavailabilityofpowerfulinstruments,newexperimental procedures,andincreasingcomputingpower whichtogetherconstitutetheveryrootsofscienti ficprogress weknowthatthephysicaland biologicalworldsareconstantlyevolving. Severalclassesofenergyhavegraduallyshaped matterandlivingentities,basicallyasthe outcomeofrandomeventsandDarwiniannaturalselectioninitsbroadestsense.TheidentificationofDNAasthegeneticmaterialandthe adventofgenomicsinthesecondhalfofthe twentiethcenturyunveiledanastonishing degreeofdiversitywithinthelivingworldthat derivesmainlyfromcombinationsoffourclasses ofnucleotides.Biodiversity,atermcoinedbyO. Wilsonin1984andemphasizedbyT.Lovejoy andothers,isafeatureofalllivingbeings,be differentiatedmulticellularorganisms,singlecellorganisms,orsubcellulargeneticelements, amongthemtheviruses.Next-generation sequencingmethodsdevelopedatthebeginning ofthe21stcenturyallowthousandsofsequences fromthesamebiologicalsample(amicrobial communityinasoiloroceansample,atumor, oraninfectedhost)tobedetermined.Theseprocedureshavedocumentedthepresenceof

1.Introductiontovirusoriginsandtheirroleinbiologicalevolution

myriadsofvariantsina “singlebiologicalentity” orin “communitiesofbiologicalentities.” Differencesextendtoindividualsthatbelongtothe samebiologicalgroup,beit Homosapiens, Drosophilamelanogaster, Escherichiacoli,orhuman immunodeficiencyvirustype1(HIV-1).No exceptionshavebeendescribed.Diversityis extensiveandnotrestrictedtothegenotypic level.Italsoaffectsphenotypictraits.

Duringdecades,inthe firsthalfofthetwentiethcentury,populationgeneticshadasoneof itstenetsthatgeneticvariationduetomutation hadforthemostpartbeenoriginatedinaremote past.Itwasgenerallythoughtthatthepresentdaydiversitywasessentiallybroughtaboutby thereassortmentofchromosomesduringsexual reproduction.Thisviewwasweakenedbythe discoveryofextensivegeneticpolymorphisms, firstin Drosophila andhumans,throughsecondaryanalysesofelectrophoreticmobilityofenzymes,detectedbyinsituactivityassaysto yieldzymogramsthatweredisplayedaselectromorphs.Theseearlystudiesonallozymeswere soonextendedtootherorganisms.Assuming thatnoproteinmodificationshadspeci fically occurredinsomeindividuals,theresultssuggestedthepresenceofseveraldifferent(allelic) formsofagivengeneamongindividualsof thesamespecies,beithumans,insects,or bacteria.IntheabsenceofinformationonDNA nucleotidesequences,the firstestimatesofheterogeneityfromthenumbersofelectromorphs werecollatedwiththeproteinsequenceinformationavailable.Anexcellentreviewofthesedevelopments(Selander,1976)endedwiththe followingpremonitorysentenceontheroleof molecularbiologyinunveilingevolutionarily relevantinformation: “Consideringthemagnitudeofthiseffect,wemaynotbeoverfanciful tothinkthatfuturehistorianswillseemolecular biologymoreasthesalvationforthan,asit first seemed,thenemesisofevolutionarybiology.”

Theconceptualbreakwasconfirmedand accentuatedwhenmolecularcloningand

nucleotidesequencingtechniquesproduced genomicnucleotidesequencesfrommultipleindividualsofthesamebiologicalspecies.Variety hasshakenourclassificationschemes,openinga debateonhowtodefineanddelimitbiological “species” inthemicrobialworld.Fromamedical perspective,ithasopenedthewayto “personalized” medicine,sodifferentaretheindividual contextsinwhichdiseaseprocesses(infectious orother)unfold.Diversityisageneralfeatureof thebiologicalworld,withmultipleimplications forinteractionsintheenvironment,andalsofor humanhealthanddisease(Bernstein,2014).

1.2Somequestionsofcurrentvirology andthescopeofthisbook

Viruses(fromtheLatin “virus,” poison)are noexceptionregardingdiversity.Thenumber ofdifferentvirusesandtheirdissimilarityin shapeandbehaviorisastounding.Currentestimatesindicatethatthetotalnumberofvirusparticlesinourbiospherereaches1032,exceedingby oneorderofmagnitudethetotalnumberofcells. Virusesarefoundinsurfaceanddeep-seaand lakewaters,belowtheEarthsurface,inany typeofsoil,indeserts,andinmostenvironments designatedasextremeregardingionicconditions(i.e.,hypersaline)andtemperature(thermophilic)(Breitbartetal.,2004;Villarreal,2005; Lopez-Buenoetal.,2009; Box1.1).Theviruses thathavebeenstudiedareprobablyaminimal andbiasedrepresentationofthosethatexist, withatleasthundredthousandmammalianvirusesawaitingdiscovery(Anthonyetal.,2013; EpsteinandAnthony,2017).Thisisbecause high-throughputscreeningprocedureshave onlyrecentlybecomeavailable,andalsobecause preventionofdiseasehasprovidedthemain incentivetostudyviruses.Disease-associatedvirusesarethosemostdescribedinthescientific literature.

BOX1.1

SOMENUMBERSCONCERNINGVIRUSESINTHEEARTH BIOSPHERE

• Totalnumberofviralparticles: w 1032.Thisis 10timesmorethancells,andtheyare equivalentto2 108 tonsofcarbon.

• Virusparticlesin1cm3 ofseawater: w 108.

• Virusparticlesin1m3 ofair: w 2 106 to 40 106

• Rateofviralinfectionsintheoceans: w 1 1023/s.

Currentvirologyposessomegeneraland fascinatingquestions,whicharenoteasily approachableexperimentally.Herearesome:

• Whatistheoriginofviruses?

• Didtheyoriginatebeforeorafteracellular worldwasinplace?

• Whatselectiveforceshavemaintained multiplevirusesasparasitesofunicellular andmulticellularorganisms?

• Dovirusesexistessentiallyassel fish parasites,orhavetheyplayedconstructive rolesinthebiosphere?

• Whyhaveafewviralformsnotoutcompeted mostotherforms?Orhavethey?

• Havevirusesbeenmaintainedasmodulators ofthepopulationnumbersoftheirhost species?

• Doesvirusvariationplayaroleinthe unfoldingofviraldiseaseprocesses?

• Isitquasispeciesdynamicsthatcharacterize RNAandmanyDNAviruses,aremnantof anadaptivestrategythatpresidedalllife formsintheremotepast?

• AstringwiththevirusesonEarthwouldbe about w2 108 light-yearslong (w1.9 1024 m).Thisisthedistancefrom EarthofthegalaxyclustersCentaurus,Hydra, andVirgo.

Basedon: Suttle(2007),Whonetal.(2012),andKooninand Dolja(2013)

• Isthebehaviorofpresent-dayvirusesatthe populationlevelonlyaninheritanceoftheir originsorapresent-daynecessity?

Thisbookdealswithsomeoftheseissues, mainlythosethatareamenabletoexperimental testing.Topicscoveredincludemolecularmechanismsofgeneticvariation,withemphasison highmutationrates,Darwinianprinciplesacting onviruses,quasispeciesdynamicsanditsimplications,consequencesforvirus-hostinteractions, fitnessasarelevantparameter,experimental modelsystemsincellculture,ex-vivoandin vivo,long-termvirusevolution,thecurrentsituationofantiviralstrategiestoconfrontquasispeciesswarms,andconceptualextensionsof quasispeciestononviralsystems.Thesesubjects haveasacommonthreadthatDarwiniannaturalselectionhasanimmediateimprinton them,observableinthetimescaleofdaysor evenhours.Thecapacityforrapidevolutiondisplayedbyvirusesrepresentsanunprecedented andoftenunderappreciateddevelopmentin biology:thedirectobservationofDarwinian principlesatplaywithinshorttimes.

1.Introductiontovirusoriginsandtheirroleinbiologicalevolution

Evolutionisdefinedasachangeinthegenetic compositionofapopulationovertime.Inthis bookevolutionwillbeusedinitsbroadersense tomeananychangeinthegeneticcomposition ofavirusovertime,irrespectiveofthetime frameinvolved,andthetransienceofthechange. Wetreatasevolutionboththegenomevariation thatpoliovirushasundergonefromthemiddle ofthe20thcenturytopresentdaysandthe changesthatthesamevirusundergoeswithin aninfectedindividual.Itisremarkablethat onlyafewdecadesagovirusevolution(orfor thatmattermicrobialevolutioningeneral)was notconsideredasignificantfactorinviralpathogenesis.Evolutionwaslargelyoverlookedinthe planningofstrategiesformicrobialdiseasecontrol.Alucidhistoricalaccountofthedifferent perceptionsofvirusevolution,includingearly evidenceofphenotypicvariationofviruses, withemphasisontheimpactofthecomplexity ofRNAviruspopulations,waswrittenbyJ.J. Holland(2006).Despitehavingbeenlargely ignoredbyvirologists,thepresentbookwas partlystimulatedbytheconvictionthatthe conceptofcomplexityispertinenttotheunderstandingofvirusesatthepopulationlevel,havingdirectconnectionswithviraldiseaseand diseasecontrol.

1.3Thestaggeringubiquityand

diversityofviruses:limited

morphotypes

Despitepleomorphismincellsandviruses (presenceornotofenvelopes,andvirusesbeing sphericalorevendisplayingalemon-likeshape, orbeingelongated),thesizeofviralparticlesand theirhostcellstendstobecommensuratewith theamountofgeneticmaterialthattheycontain andtransmittoprogeny(Fig.1.1).The2017 reportfromInternationalCommitteeonTaxonomyofViruses(ICTV)dividesvirusesinto10

Orders,andeachofthemissubdividedinto severalfamilies,subfamilies,genera,species, andisolates(https://talk.ictvonline.org/),and eachisolateincludesamultitudeofvariants. Thetaskofclassifyingvirusesmeetswith considerablehurdlesandrequiresperiodicrevisionsbytheICTV,anorganizationwhoserole hasbeenessentialtoprovideconceptualorder inthevastviralworld.Oneofitsobjectivesis theassignmentofnewlydiscoveredvirusesto theadequategroup.Aremarkablenumberof isolatesremainunclassified,anechoofthe

FIGURE1.1 Representativeaveragediametervaluesand genomecomplexityofvirusesandsomecelltypes.Diameters areexpressedinmicrons(m),lengthofDNAinbasepairs (bp),andofRNAinnucleotides(nt).Viralgenomescanbe linear,circular,diploid,segmented,orbipartite(multipartite ingeneral;genomesegmentsencapsidatedinseparateparticles);inthelattercaseatleasttwoparticles,eachwithone kindofgenomicsegment,mustinfectthesamecellforprogenyproduction.Thebottomboxesdescribefourgroupsof virusesaccordingtothetypeofnucleicacidthatactsasreplicativeintermediate.

naturaldiversityofviruses,evenamongthe limitedsubsetthathasbeenisolatedand characterized.

Virusescanbedividedintotwobroadgroups: thosethathaveRNAasgeneticmaterial,termed the RNAviruses,andthosethathaveDNAas geneticmaterialtermedthe DNAviruses.They canhavelinear,circular,orsegmentedgenomes ofsingle-strandedordouble-strandednucleic acid(Fig.1.1).Allevidencesuggeststhatthe polynucleotidechain(orchains)thatconstitute theviralgenomehasalltheinformationto generateinfectiousprogenyinacell,asevidenced bytheproductionofinfectiouspoliovirusfrom syntheticDNAcopiesassembledtorepresent thegenomicnucleotidesequence(Celloetal., 2002).

Withregardtotheconceptsofgenomestabilityversusvariationaddressedinthisbook,itis helpfultodividevirusesintofourgroups, dependingonwhetheritisDNAorRNAthe typeofgeneticmaterial,whichactsasareplicativeintermediateintheinfectedcell(bottom grayshadedboxesin Fig.1.1).Thenucleicacids writteninthefourschemesarethoseinvolvedin the flowofgeneticinformation(indicatedby arrows),notingeneexpressionsinceallofthem usemessengerRNA(mRNA)forvirus-specific proteinsynthesis.Mistakesintheformofmisincorporationofnucleotidesduringthereplication stepsindicatedbyarrowsaretransmittedtoprogenygenomes.RNAsproducedbytranscriptionto servesolelyasmRNAsareessentialforgene expressionandvirusmultiplication,butmisincorporationsinsuchtranscriptsarenottransmittedtoprogeny.Itcouldbeconsideredthat somemRNAmoleculeswhensynthesizedfrom thecorrespondingRNAorDNAtemplatemay acquiremutationsandthatthesemutatedmolecules(e.g.,anmRNAencodingaviralpolymerase),whentranslated,mayproducea polymerasewithlowercopying fidelitythatwill evokeadditionalmutations;wewillignorethis

possibilitysinceasinglemRNAmoleculeshould havearatherlimitedcontributiontotheoverall geneticvariationofareplicatingviruspopulation withthousandsofpolymeraseandtemplatemoleculesinthereplicationcomplexes(orreplication factories).

Group1(withareplicativeschemeabbreviatedasRNA/RNA)includesRNAviruses whosegenomicreplicationcycleinvolvesonly RNA.Theyaresometimescalled riboviruses.Examplesaretheinfluenzaviruses,hepatitisAand Cviruses,poliovirus,coronaviruses,Ebolavirus, foot-and-mouthdiseasevirus,ortobaccomosaic virus,amongmanyotherimportanthuman,animal,andplantpathogens.Theirreplicationis catalyzedbyanRNA-dependentRNApolymerase(RdRp)encodedintheviralgenome,often organizedasareplicationcomplexwithviral andhostproteinsincellularmembrane structures.

Group2(RNA/DNA/RNA)comprisesthe retroviruses[suchasHIV-1,theacquiredimmune deficiencysyndrome(AIDS)virus,andseveral tumorviruses]thatretrotranscribetheirRNA intoDNA.Retrotranscriptioniscatalyzedby reversetranscriptase(RT),anRNA-dependent DNApolymeraseencodedintheretroviral genome.Itreversesthe firststepinthenormal flowofexpressionofgeneticinformationfrom DNAtoRNAtoprotein,onceknownasthe dogma ofmolecularbiology.Thisenzymewas instrumentalintheunderstandingofcancer, andforgeneticengineeringandtheoriginof modernbiotechnology.Asahistoricalaccount oftheimpactofH.Temin’swork(codiscoverer ofRTwithD.Baltimore),thereaderisreferred to Cooperetal.(1995).RetrovirusesincludeaprovirusstageinwhichtheviralDNAisintegrated intohostDNA.Whensilentlyinstalledincellular DNA,theviralgenomebehavesmostlyasa cellulargene.

Group3(DNA/DNA)containsmostDNA viruses,suchasherpesviruses,poxviruses, 1.3Thestaggeringubiquityanddiversityofviruses:limitedmorphotypes

iridoviruses,andpapillomaviruses,andthe extremelylargevirusesofamebae(i.e.,Mimivirus,Megavirus,orPandoravirus,generically termed giantviruses)(LaScolaetal.,2008;Colson etal.,2017).Theirreplicationiscatalyzedbya DNA-dependentDNApolymeraseeither encodedintheviralgenomeorinthecellular DNA.CellularDNApolymerasesareinvolved inthereplicationofDNAvirusesthatdonot encodetheirownDNApolymerase.

Finally,Group4(DNA/RNA/DNA)includesviruseswhichdespitehavingDNAas geneticmaterial,produceanRNAasareplicativeintermediate,themostsignificantexamples beingthehumanandanimalhepatitisBviruses (HBVs)andthecauli flowermosaicvirusof plants,termed hepadnaviruses. Mostviruses,fromthemorecomplexDNA viruses[i.e.,1200Kbp(1000basepairs)forthe amebaMimivirus,752Kbpforsometailedbacteriophages,andupto370Kbpforpoxviruses,iridoviruses,andherpesviruses],thevirophages thatareparasitesofthegiantDNAviruses,the simplestDNAviruses(thecircularsinglestranded1760residueDNAofporcinecircovirus),RNAbacteriophages(4220nucleotidesof ssRNAforbacteriophageQb),orsubviralelements(viroids,virusoids,satellites,andhelperdependentdefectivereplicons)showremarkable geneticdiversity.However,RNAvirusesthat replicateentirelyviaRNAtemplates(Group1 in Fig.1.1);retroviruses(Group2);andthehepadnaviruses(Group4)displaysalientgenetic plasticity,mainlyinthewayofahighrateof introductionofpointmutations(Chapter2). Theirmutabilitymaybeaninheritanceofuniversal fl exibilitythatprobablycharacterized primitiveRNAorRNA-likemolecules,thought tohavepopulatedanancestralRNAworldatan earlystageoflifeonEarth(Section1.4.2 ).Thus, thepresenceofRNAatanyplaceinthereplicativeschemes(Group1,2,and4in Fig.1.1)implieserror-pronereplicationandthepotential

ofveryrapidevolution. “Potential” mustbe underlinedbecausehigherrorratesdonot necessarilyresultinrapidlong-termevolution innature(Chapter7).

Theextentofgeneticvariationanditsbiologicalconsequenceshavebeenlessinvestigatedfor DNAvirusesthanforRNAviruses.TheavailabledatasuggestthatDNAvirusesarecloser toRNAvirusesthansuspectedonlyafewyears ago,regardingtheircapacityofvariationand adaptation.Thisisparticularlytrueofthe single-strandedDNAvirusesofanimalsand plants.Evolutionarytheorypredictsthathighfidelitypolymerasemachineryisnecessaryto maintaintheinformationalstabilityofcomplex genomes(thosethatcarryalargeamountofgeneticinformation).Thisnecessityaccomplished byproofreading-repairandpostreplicativerepairactivitiesthatassistthereplicativeDNA polymerasesandtheircellularandviralDNA progeny.

Ourcurrentcapacitytosamplemanythousandsviralgenomesinshorttimes(atrend thatiscontinuouslyexpanding)isrevealingan astonishingnumberofslightlydifferentviral genomeswithinasingleinfectedhost,andeven withinanorganorwithinindividualcellsofan organ!Intrahostdiversityofvirusescanbethe resultnotonlyofdiversificationwithinthehost butalsoofcoinfectionwithdifferentviruses(or variantsofonevirus),oraninfectionthattriggers reactivationofarelatedorunrelatedvirusfroma latentreservoir,orcombinedeffectsofthese mechanisms.Inturn,interhost(long-term)virus diversificationcanresultfromselectionacting onvariantsgeneratedbymutation,recombinationorreassortment,andrandomsampling events(independentofselection)withinhosts andinhost-to-hosttransmission,ortheircombinedeffects(Chapters2and3).

Adifferentpictureofdiversityisobtainedby comparingthemorphologicalcharacteristicsof viralparticles(termed virions).Thehundredsof

thousandsofbacterialandarchaealvirusesthat havebeenrecognizedcanbeassignedtoasfew as20morphotypes.Thecapsidsofnonenveloped(naked)virusesdisplayhelicaloricosahedralsymmetrythatdeterminesthearchitecture ofthevirion.Variationinsizeandsurfaceproteindistributionscanbeattainedfromlimited proteinfoldsandthesamesymmetryprinciples (Mateu,2013, Fig.1.2).Divergentprimaryamino acidsequencesinproteinscanfoldinclosely relatedstructures.The “structuralspace”

availabletovirusesparticlesismuchmore restrictedthanthe “ sequencespace ” available toviralgenomes(Abresciaetal.,2012).Sequence spaceanditsmappingintoaphenotypicspace arekeyconceptsfortheunderstandingofevolutionarymechanisms(Chapter3).

Three-dimensionalstructuresofentirevirions ortheirconstituentproteinscanprovideanoverviewofphylogeneticlineagesandevolutionary stepsincasesinwhichtheinformationcannot beattainedbyviralgenomics(Ravanttietal.,

FIGURE1.2 Examplesofsphericalbacterial,fungal,plant,andanimalvirusparticlesreconstructedfromcryo-electronmicroscopyimages.L-Av, Saccharomycescerevisae virus;PcV, PenicillumChrysogenum virus;RHDV,rabbithemorrhagicdisease virus;HRV-2humanrhinovirustype2;CCMV,cowpeachloroticmottlevirus;HPV-16,humanpapillomavirustype16;T7, headofbacteriophageT7;SINV,Sindbisvirus;IBVD,Infectiousbursaldiseasevirus;RV,rotavirus. Picturemodifiedfromone kindlysuppliedbyJ.R.Caston,Carrascosa,J.L.,2013.Thebasicarchitectureofviruses.In:Mateu,M.G.(Ed.),StructureandPhysics ofViruses.Springer,Dordrecht,Heidelberg,NewYork,London,pp.53 75withpermission. 1.3Thestaggeringubiquityanddiversityofviruses:limitedmorphotypes

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BROILING.

Broiling is the best possible mode of cooking and of preserving the flavour of several kinds of fish, amongst which we may specify mackerel and whitings; it is also incomparably superior to frying for steaks and cutlets, especially of beef and mutton; and it is far better adapted also, to the preparation of food for invalids; but it should be carefully done, for if the heat be too fierce, the outside of the meat will be scorched and hardened so as to render it uneatable; and if, on the contrary, it be too gentle, the gravy will be drawn out, and yet the flesh will remain so entirely without firmness, as to be unpleasant eating. A brisk fire, perfectly free from smoke, a very clean gridiron, tender meat, a dish and plates as hot as they can be, and great despatch in sending it to table when done, all are essential to the serving of a good broil. The gridiron should be heated, and rubbed with mutton suet before the meat is laid on, and it should be placed slopingly over the fire, that the fat may run off to the back of the grate, instead of falling on the live coals and smoking the meat; if this precaution should not prevent its making an occasional blaze, lift the gridiron quickly beyond the reach of the smoke, and hold it away until the fire is clear again. Steaks and chops should be turned often, that the juices may be kept in, and that they may be equally done in every part. If, for this purpose, it should be necessary for want of steak-tongs to use a fork, it should be passed through the outer skin or fat of the steak, but never stuck into the lean, as by that means much of the gravy will escape. Most eaters prefer broiled beef or mutton, rather underdressed; but lamb or pork cutlets should always be thoroughly cooked. When a fowl or any other bird is cut asunder before it is broiled, the inside should first be laid to the fire: this should be done with kidneys also. Fish is less dry and of better flavour, as well as less liable to be smoked, if it

A Conjuror.

be wrapped in a thickly buttered sheet of writing paper before it is placed on the gridiron. For the more delicate-skinned kinds, the bars should be rubbed with chalk instead of suet when the paper is omitted. Cutlets, or meats of any other form, when egged and crumbed for broiling, should afterwards be dipped into clarified butter or sprinkled with it plentifully, as the egg-yolk and bread will otherwise form too dry a crust upon it. French cooks season their cutlets both with salt and pepper, and brush a little oil or butter over them to keep them moist; but unless this be done, no seasoning of salt should be given them until they are just ready to be dished: the French method is a very good one. Steaks or cutlets may be quickly cooked with a sheet or two of lighted paper only, in the apparatus shown in the preceding page, and called a conjuror. Lift off the cover and lay in the meat properly seasoned, with a small slice of butter under it, and insert the lighted paper in the aperture shown in the plate; in from eight to ten minutes the meat will be done, and found to be remarkably tender, and very palatable: it must be turned and moved occasionally during the process. This is an especially convenient mode of cooking for persons whose hours of dining are rendered uncertain by the nature of their avocations. For medical men engaged in extensive country practice it has often proved so; and we would especially recommend it to the notice of emigrants, to whom it would often prove invaluable. The part in which the meat is placed is of block tin, and fits closely into the stand, which is of sheet iron. The conjuror from which our design was drawn, was purchased in a country town in Essex, and was exceedingly well made, and very cheap. We find on inquiry that the maker has quitted the place, or we would insert his address.

FRYING.

This is an operation, which, though apparently very simple, requires to be more carefully and skilfully conducted than it commonly is. Its success depends principally on allowing the fat to attain the exact degree of heat which shall give firmness, without too quick browning or scorching, before anything is laid into the pan; for, if this be neglected, the article fried will be saturated with fat, and remain pale and flaccid. When the requisite degree of colour is acquired before the cooking is complete, the pan should be placed high above the fire, that it may be continued slowly to the proper point. Steaks and cutlets should be seasoned with salt and pepper and dredged on both sides lightly with flour before they are laid into the pan, in which they should be often moved and turned that they may be equally done, and that they may not stick nor burn to it. From ten to fifteen minutes will fry them. They should be evenly sliced, about the same thickness as for broiling, and neatly trimmed and divided in the first instance. Lift them into a hot dish when done; pour the fat from the pan, and throw in a small slice of butter; stir to this a large teaspoonful of flour, brown it gently, and pour in by degrees a quarter of a pint of hot broth or water; shake the pan well round, add pepper, salt, and a little good catsup, or any other store sauce which may be preferred to it, and pour the gravy over the steaks: this is the most common mode of saucing and serving them.

Minute directions for fish, vegetables, omlets, and different preparations of batter, are given in their proper places; but we must again observe, that a very small frying pan (scarcely larger than a dinner-plate) is necessary for many of these; and, indeed, the large and thick one suited to meat and fish, and used commonly for them is altogether unfit for nicer purposes.

The sauté-pan, shown in the preceding page, is much used by French cooks instead of a frying-pan; it is more particularly

convenient for tossing quickly over the fire small collops, or aught else which requires but little cooking.

All fried dishes, which are not sauced, should be served extremely dry upon a neatly-folded damask cloth: they are best drained upon a sieve reversed placed before the fire.

A wire basket of this form is convenient for frying parsley and other herbs. It must be placed in a pan well filled with fat, and lifted out quickly when the herbs are done: they may likewise be crisped in it over a clear fire, without any fat.

Wire Lining of Fryingpan.

Wire Basket for Frying.

The frying-pans fitted with wire linings that lift in and out of them, which have lately come much into use in good kitchens, are so excellently adapted to save trouble, and so convenient for preparing delicately light patties, croquettes, rissoles, and other dishes of a similar nature, that no cook who is expected to serve them in the best manner should be without one. They should all be arranged upon this wire lining, and plunged together into the boiling fat; and well drained on it when they are lifted out.

Modern Sauté Pan.

BAKING, OR OVEN COOKERY

Nottingham Jar.

The improved construction of the ovens connected with all modern cooking stoves, gives great facility at the present day for home baking, even in very small establishments; and without this convenience it is impossible for justice to be done to the person who conducts the cookery; as many and great disadvantages attend the sending to a public oven; and it is very discouraging to a servant who has prepared her dishes with nicety and skill, to have them injured by the negligence of other persons. One of the best modes of cooking with which we are acquainted is by means of a jar, resembling in form that shown above, well pasted down, and covered with a fold of thick paper, and then placed in a gentle oven. Rice is most excellent when thus slowly baked with a certain proportion of liquid, either by itself, or mingled with meat, fish, or fruit; but we must reserve for another volume particulars of this little system of slow oven-cookery, in which for some years past we have had numberless experiments made with almost uniform success: it is especially suited to invalids, from preserving the entire amount of nourishment contained in the articles of food dressed by it; and it is to their use that we hope to appropriate it.

American Oven.[73]

73 We have scarcely done justice in the former editions of this work to these very useful little ovens, which we have found, after long trial, better adapted to some purposes than brick or iron ones, because preparations which require it, (those of Indian corn, for example) can be heated in them more gradually; and when once the management of them is understood, they will answer admirably for delicate sweet puddings, and for cakes, with the advantage of requiring but a very moderate fire

The oven may be used with advantage for many purposes for which it is not commonly put into requisition. Calves’ feet, covered with a proper proportion of water, may be reduced to a strong jelly if left in it for some hours; the half-head, boned and rolled, will be found excellent eating, if laid, with the bones, into a deep pan and baked quite tender in sufficient broth or water, to keep it covered in every part until done; good soup also may be made in the same way, the usual ingredients being at once added to the meat, with the exception of the vegetables, which will not become tender if put into cold liquid, and should therefore be thrown in after it begins to simmer. Baking is likewise one of the best modes of dressing various kinds of fish: pike and red mullet amongst others. Salmon cut into thick slices, freed from the skin, well seasoned with spice, mixed with salt (and with minced herbs, at pleasure), then arranged evenly in a dish, and covered thickly with crumbs of bread, moistened with clarified butter, as directed in Chapter II., for baked soles, and placed in the oven for about half an hour, will be found very rich and highly flavoured. Part of the middle of the salmon left entire, well cleaned, and thoroughly dried, then seasoned, and securely wrapped in two or three folds of thickly buttered paper, will also prove excellent eating, if gently baked. (This may likewise be roasted in a Dutch oven, either folded in the paper, or left without it, and basted with butter.) Hams, when freshly cured, and not over salted, if neatly trimmed, and covered with a coarse paste, are both more juicy, and of finer flavour baked than boiled. Savoury or pickled beef too, put into a deep pan with a little gravy, and plenty of butter or chopped suet on the top, to prevent the outside from becoming dry; then covered with paste, or with several folds of thick paper, and set into a moderate oven for four or five hours, or even longer, if it be of large weight, is an excellent dish. A goose, a leg of pork, and a sucking pig, if properly attended to while in the oven, are said to be nearly, or quite as good as if roasted; but baking is both an unpalatable and an unprofitable mode of cooking joints of meat in general, though its great convenience to many persons who have but few other facilities for obtaining the luxury of a hot dinner renders it a very common one. It is usual to raise meat from the dish in which it is sent to the oven by placing it, properly skewered, on a stand, so as to allow

potatoes or a batter pudding to be baked under it. A few button onions, freed from the outer skin, or three or four large ones, cut in halves, are sometimes put beneath a shoulder of mutton. Two sheets of paper spread separately with a thick layer of butter, clarified marrow, or any other fat, and fastened securely over the outside of a joint, will prevent its being too much dried by the fierce heat of the oven. A few spoonsful of water or gravy should be poured into the dish with potatoes, and a little salt sprinkled over them. A celebrated French cook recommends braising in the oven; that is to say, after the meat has been arranged in the usual manner, and just brought to boil over the fire, that the braising pan, closely stopped, should be put into a moderate oven, for the same length of time as would be required to stew the meat perfectly tender.

BRAISING.

English Braising-pan.

Braising is but a more expensive mode of stewing meat. The following French recipe will explain the process. We would observe, however, that the layers of beef or veal, in which the joint to be braised is imbedded, can afterwards be converted into excellent soup, gravy, or glaze; and that there need, in consequence, be no waste nor any unreasonable degree of expense attending it; but it is a troublesome process, and quite as good a result may be obtained by simmering the meat in very strong gravy. Should the flavour of the bacon be considered an advantage, slices of it can be laid over the article braised, and secured to it with a fillet of tape.

“To braise the inside (or small fillet, as it is called in France) of a sirloin of beef: Raise the fillet clean from the joint; and with a sharp knife strip off all the skin, leaving the surface of the meat as smooth as possible; have ready some strips of unsmoked bacon, half as thick as your little finger, roll them in a mixture of thyme finely minced, spices in powder, and a little pepper and salt. Lard the fillet quite through with these, and tie it round with tape in any shape you choose. Line the bottom of a stewpan (or braising-pan) with slices of bacon; next put in a layer of beef or veal, four onions, two bayleaves, two carrots, and a bunch of sweet herbs, and place the fillet on them. Cover it with slices of bacon, put some trimmings of meat all round it, and pour on to it half a pint of good beef broth or gravy. Let it stew as gently as possible for two hours and a half; take it up, and keep it very hot; strain, and reduce the gravy by quick boiling until it is thick enough to glaze with; brush the meat over with it; put the rest in the dish with the fillet, after the tape has been removed from it, and send it directly to table.”

Equal parts of Madeira and gravy are sometimes used to moisten the meat.

No attempt should be made to braise a joint in any vessel that is not very nearly of its own size.

74 The line which passes round this stewpan just above the handle, is a mistake of the designer, and conveys an erroneous idea of the form of the cover, and it ought to have been omitted.

Copper Stewpan.[74]

A round of buttered paper is generally put over the more delicate kinds of braised meat, to prevent their being browned by the fire, which in France is sometimes put round the lid of the braising-pan, in a groove made on purpose to contain it. The embers of a wood fire mixed with the hot ashes, are best adapted to sustain the regular but gentle degree of heat required for this mode of cooking.

Braising pans are of various forms. They are often shaped like a ham-kettle, and sometimes like the design at the commencement of this section; but a stewpan of modern form, or any other vessel which will admit of embers being placed upon the lid, will answer for the purpose as well. Common cooks sometimes stew meat in a mixture of butter and water, and call it braising.

LARDING.

Larding Pins.

Cut into slices, of the same length and thickness, some bacon of the finest quality; trim away the outsides, place the slices evenly upon each other, and with a sharp knife divide them obliquely into small strips of equal size. For pheasants, partridges, hares, fowls, and fricandeaux, the bacon should be about the eighth of an inch square, and two inches in length; but for meat which is to be larded quite through, instead of on the outside merely, the bits of bacon (properly called lardoons) must be at least the third of an inch square.

In general, the breasts only of birds are larded, the backs and thighs of hares, and the whole of the upper surface of a fricandeau: these should be thickly covered with small lardoons, placed at regular intervals, and in lines which intersect each other, so as to form rather minute diamonds.

The following directions for larding a pheasant will serve equally for poultry, or for other kinds of game:—

Secure one end of the bacon in a slight larding-needle, and on the point of this take up sufficient of the flesh of the bird to hold the lardoon firmly; draw the needle through it, and part of the bacon, of which the two ends should be left of equal length. Proceed thus, until the breast of the pheasant is entirely garnished with lardoons, when it ought to resemble in appearance a cake thickly stuck with strips of almonds.

The larger strips of bacon, after being rolled in a high seasoning of minced herbs and spices, are used to lard the inside of meat, and they should be proportioned to its thickness, as they must be passed quite through it. For example: a four-inch slice from a rump of beef will require lardoons of very nearly that length, which must be drawn through with a large larding-pin, and left in it, with the ends just out of sight on either side.

In France, truffles, anchovies, slices of tongue, and of fat, all trimmed into proper shape, are occasionally used for larding. The bacon employed there for the purpose is cured without any saltpetre (as this would redden the white meats), and it is never smoked: the receipt for it will be found in Chapter XIII.

A turkey is sometimes larded with alternate lardoons of fat bacon and of bullock’s tongue, which has been pickled but not dried: we apprehend that the lean of a half-boiled ham, of good colour, would answer the purpose quite as well, or better.

Larding the surface of meat, poultry, or game, gives it a good appearance, but it is a more positive improvement to meat of a dry nature to interlard the inside with large lardoons of well-seasoned, delicate, striped English bacon.

BONING.

Very minute directions being given in other parts of our volume for this, we confine ourselves here to the following rules:—in disengaging the flesh from it, work the knife always close to the bone, and take every care not to pierce the outer skin.

TO BLANCH MEAT OR VEGETABLES.

This is merely to throw either into a pan of boiling water for a few minutes, which gives firmness to the first, and is necessary for some modes of preparing vegetables.

The breast only of a bird is sometimes held in the water while it boils, to render it firm for larding. To preserve the whiteness of meat, and the bright green of vegetables, they are lifted from the water after they have boiled a few minutes, and are thrown immediately into spring water, and left till cold.

5 to 10 minutes.

GLAZING

This process we have explained at the article Glaze, Chapter IV. The surface of the meat should be covered evenly, with two or three separate layers of the glaze, which, if properly made, soon becomes firm. A ham should be well dried in the oven before it is laid on. Cutlets of all kinds may be glazed before they are sent to table, with very good effect. The figure above represents a glazepot and brush, used for heating and applying the preparation: a jar placed in a pan of boiling water may be substituted for the first, when it is not at hand.

TOASTING.

A very cheap apparatus, by which chops can be dressed before a clear fire, is shown by the first of these figures; and the second is peculiarly convenient when bread or muffins are required to be toasted expeditiously and in large quantities, without much time and attention being bestowed upon them.

TO BROWN THE SURFACE OF A DISH WITHOUT BAKING OR PLACING IT AT THE FIRE.

This is done with a salamander, as it is called, formed like the engraving below; it is heated in the fire, and held over the dish sufficiently near to give it colour. It is very much used in a superior order of cookery. A kitchen shovel is sometimes substituted for it on an emergency.

CHAPTER X. Beef.

1. Sirloin.

2. Rump.

3. Edge-bone.

4. Buttock, or Round.

5. Mouse Buttock.

6. Veiny Piece.

7. Thick Flank.

8. Thin Flank.

9. Leg.

10. Fore Rib. (Five Ribs.)

11. Middle Rib. (Four Ribs.)

12. Chuck Rib. (Three Ribs.)

13. Shoulder, or Leg of Mutton Piece.

14. Brisket.

15. Clod.

16. Neck.

17. Shin.

18. Cheek.

TO CHOOSE BEEF.

Beef is in reality in season through the entire year, but it is best during the winter months, when it will hang a sufficient time to become tender before it is dressed. Meat of a more delicate nature is better adapted for the table in summer. The Christmas beef of England is too much celebrated to require any mention here

If young and freshly killed, the lean of ox-beef will be smoothly grained, and of a fine, healthy, carnation-red, the fat rather white than yellow, and the suet white and firm. Heifer-beef is more closely grained, and rather less bright of colour, the bones are considerably smaller, and the fat of a purer white.

Of bull-beef we only speak to warn our readers that it is of all meat the coarsest and the most rank in flavour. It may be known by its dark hue, its close tough fibre, and the scanty proportion, bad appearance, and strong odour of its fat.

In choice and well-fed beef, the lean will be found intergrained with fat: very lean meat is generally of an inferior quality.

The ribs, the sirloin, and the rump, are the proper joints for roasting. The round, or buttock, the edgebone, the second round, or mouse-buttock, the shin, the brisket, the shoulder or leg of mutton piece, and the clod, may be boiled or stewed. The neck is generally used for soup or gravy; and the thin flank for collaring. The best steaks are cut from the middle of the rump; the next best from the veiny piece, or from the chuck-rib. The inside of the sirloin, commonly used for the purpose in France, makes by far the most delicate steaks; but though exceedingly tender, they are considered by some English epicures to be wanting in flavour

The finest part of the sirloin is the chump-end, which contains the larger portion of the fillet; of the ribs, the middle ones are those generally preferred by experienced housekeepers.

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