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1、PART IIIThe VirusesChapter 8TheViruses:IntroductionandGeneralCharacteristicsViruseshavehadenormousimpactonhumansandotherorganisms,yetverylittlewasknownabouttheirnatureuntilfairlyrecently.Abriefhistoryoftheirdiscoveryandrecognitionasuniquelydifferentinfectiousagentscanhelpclarifytheirnature.8.1 Early
2、 Development of VirologyThedevelopmentin1884oftheporcelainbacterialfilterbyCharlesChamberland,oneofPasteurscollaboratorsandinventoroftheautoclave,madepossiblethediscoveryofwhatarenowcalledviruses.TobaccomosaicdiseasewasthefirsttobestudiedwithChamberlandsfilter.In1892,DimitriIvanowskipublishedstudies
3、showingthatleafextractsfrominfectedplantswouldinducetobaccomosaicdiseaseevenafterfiltrationtoremovebacteria.In1898,MartinusW.Beijerinckpublishedtheresultsofextensivestudiesontobaccomosaicdisease.Becausethefilteredsapofdiseasedplantswasstillinfectious,heproposedthatthediseasewascausedbyanentitydiffer
4、entfrombacteria,afilterablevirus.Heobservedthattheviruswouldmultiplyonlyinlivingplantcells,butcouldsurviveforlongperiodsinadriedstate.In1900,FriedrichLoefflerandPaulFroschinGermanyfoundthatthefoot-and-mouthdiseaseofcattlewasalsocausedbyafilterablevirusratherthanbyatoxin.Itwassoondiscoveredthatbacter
5、iathemselvesalsocouldbeattackedbyviruses.Thefirstpublishedobservationsuggestingthatthismightbethecasewasmadein1915byFrederickW.Twort.Twortisolatedbacterialvirusesthatcouldattackanddestroymicrococciandintestinalbacilli.In1917,DHerelleisolatedbacterialvirusesfrompatientswithdysentery,probablycausedbyS
6、higella dysenteriae. Hedemonstratedthatthesevirusescouldreproduceonlyinlivebacteria;thereforehenamedthembacteriophagesbecausetheycouldeatholesinbacterial“lawns.”In1935,WendellM.Stanleycrystallizedthetobaccomosaicvirus(TMV)andfoundittobelargelyorcompletelyprotein.AshorttimelaterFrederickC.BawdenandNo
7、rmanW.PiriemanagedtoseparatetheTMVvirusparticlesintoproteinandnucleicacid.In1940，KauschewatchedtheTMVvirusparticlesinelectronicmicroscope.8.2 General Properties of VirusesVirusesdifferfromlivingcellsinatleastthreeways:(1)theirsimple,acellularorganization;(2)thepresenceofeitherDNAorRNA,butnotboth.(3)
8、theirinabilitytoreproduceindependentofcellsandcarryoutcelldivisionasprocaryotesandeucaryotesdo.Althoughbacteriasuchaschlamydiaandrickettsiaareobligatelyintracellularparasiteslikeviruses,theydonotmeetthefirsttwocriteria.Virion,theextracellularphase,possesfewifanyenzymesandcannotreproduceindependently
9、oflivingcells.Intheintracellularphase,virusesexistprimarilyasreplicatingnucleicacidsthatinducehostmetabolismtosynthesizevirioncomponents;eventuallycompletevirusparticlesorvirionsarereleased.extracellularandintracellularViruses can exist in two phases Hosts Three main classes - animal viruses, bacter
10、ial viruses (bacteriophages), and plant viruses.Theparticularhostrangeofavirusisdeterminedbythevirussrequirementsforitsspecificattachmenttothehostcellandtheavailabilitywithinthepotentialhostofcellularfactorsrequiredforviralmultiplication.8.3 The Cultivation of VirusesBecausetheyareunabletoreproducei
11、ndependentoflivingcells,virusescannotbeculturedinthesamewayasbacteriaandeucaryoticmicroorganisms.1.TheCultivation of Animal VirusesFormanyyearsresearchershavecultivatedanimalvirusesbyinoculatingsuitablehostanimalsorembryonatedeggsfertilizedchickeneggsincubatedabout6to8daysafterlaying.2.TheCultivatio
12、n of Bacterial VirusesBacterialvirusesorbacteriophages (phages forshort)arecultivatedineitherbrothoragarculturesofyoung,activelygrowingbacterialcells.Somanyhostcellsaredestroyedthatturbidbacterialculturesmayclearrapidlybecauseofcelllysis.Agarculturesarepreparedbymixingthebacteriophagesamplewithcool,
13、liquidagarandasuitablebacterialculture.Themixtureisquicklypouredintoapetridishcontainingabottomlayerofsterileagar.Afterhardening,bacteriainthelayeroftopagargrowandreproduce,formingacontinuous,opaquelayeror“lawn.”Whereveravirioncomestorestinthetopagar,thevirusinfectsanadjacentcellandreproduces.Eventu
14、ally,bacteriallysisgeneratesaplaqueorclearinginthelawn.3.TheCultivation of Plant VirusesPlantvirusesarecultivatedinavarietyofways.Planttissuecultures,culturesofseparatedcells,orculturesofprotoplastsmaybeused.Virusesalsocanbegrowninwholeplants.Leavesaremechanicallyinoculatedwhenrubbedwithamixtureofvi
15、rusesandanabrasivesuchascarborundum.Whenthecellwallsarebrokenbytheabrasive,thevirusesdirectlycontacttheplasmamembraneandinfecttheexposedhostcells.8.4 Virus Purification and AssaysVirologistsmustbeabletopurifyvirusesandaccuratelydeterminetheirconcentrationsinordertostudyvirusstructure,reproduction,an
16、dotheraspectsoftheirbiology.Thesemethodsaresoimportantthatthegrowthofvirologyasamoderndisciplinedependedontheirdevelopment.1.Virus PurificationPurificationmakesuseofseveralvirusproperties.Virionsareverylargerelativetoproteins,areoftenmorestablethannormalcellcomponents,andhavesurfaceproteins.Becauseo
17、fthesecharacteristics,manytechniquesusefulfortheisolationofproteinsandorganellescanbeemployedinvirusisolation.Fourofthemostwidelyusedapproachesare:(1)differentialanddensitygradientcentrifugation,(2)precipitationofviruses,(3)denaturationofcontaminants,(4)enzymaticdigestionofcellconstituents.(1)differ
18、entialanddensitygradientcentrifugation(2)precipitationofvirusesViruses,likemanyproteins,canbepurifiedthroughprecipitationwithconcentratedammoniumsulfate.Initially,sufficientammoniumsulfateisaddedtoraiseitsconcentrationtoaleveljustbelowthatwhichwillprecipitatethevirus.Afteranyprecipitatedcontaminants
19、areremoved,moreammoniumsulfateisaddedandtheprecipitatedvirusesarecollectedbycentrifugation.Virusessensitivetoammoniumsulfateoftenarepurifiedbyprecipitationwithpolyethyleneglycol.(3)denaturationofcontaminantsVirusesfrequentlyarelesseasilydenaturedthanmanynormalcellconstituents.Contaminantsmaybedenatu
20、redandprecipitatedwithheatorachangeinpHtopurifyviruses.Becausesomevirusesalsotoleratetreatmentwithorganicsolventslikebutanolandchloroform,solventtreatmentcanbeusedtobothdenatureproteincontaminantsandextractanylipidsinthepreparation.Thesolventisthoroughlymixedwiththeviruspreparation,thenallowedtostan
21、dandseparateintoorganicandaqueouslayers.Theunalteredvirusremainssuspendedintheaqueousphasewhilelipidsdissolveintheorganicphase.Substancesdenaturedbyorganicsolventscollectattheinterfacebetweentheaqueousandorganicphases.(4)enzymaticdigestionofcellconstituentsCellularproteinsandnucleicacidscanberemoved
22、frommanyviruspreparationsthroughenzymaticdegradationbecausevirusesusuallyaremoreresistanttoattackbynucleasesandproteasesthanarefreenucleicacidsandproteins.Forexample,ribonucleaseandtrypsinoftendegradecellularribonucleicacidsandproteinswhileleavingvirionsunaltered.2.Virus AssaysThequantityofvirusesin
23、asamplecanbedeterminedeitherbycountingparticlenumbersorbymeasurementoftheinfectiousunitconcentration.Althoughmostnormalvirionsareprobablypotentiallyinfective,manywillnotinfecthostcellsbecausetheydonotcontactthepropersurfacesite.Thusthetotalparticlecountmaybefrom2to1milliontimestheinfectiousunitnumbe
24、rdependingonthenatureofthevirionandtheexperimentalconditions.Despitethis,bothapproachesareofvalue.Virusparticlescanbecounteddirectlywiththeelectronmicroscope.Inoneprocedurethevirussampleismixedwithaknownconcentrationofsmalllatexbeadsandsprayedonacoatedspecimengrid.Thebeadsandvirionsarecounted;thevir
25、usconcentrationiscalculatedfromthesecountsandfromthebeadconcentration.Thistechniqueoftenworkswellwithconcentratedpreparationsofvirusesofknownmorphology.Virusescanbeconcentratedbycentrifugationbeforecountingifthepreparationistoodilute.However,ifthebeadsandvirusesarenotevenlydistributed(assometimeshap
26、pens),thefinalcountwillbeinaccurate.Themostpopularindirectmethodofcountingvirusparticlesisthehemagglutination assay. Manyvirusescanbindtothesurfaceofredbloodcells.Iftheratioofvirusestocellsislargeenough,virusparticleswilljointheredbloodcellstogether,forminganetworkthatsettlesoutofsuspensionoraggluti
27、nates.Thisassayisanaccurate,rapidmethodfordeterminingtherelativequantityofvirusessuchastheinfluenzavirus.Avarietyofassaysanalyzevirusnumbersintermsofinfectivity,andmanyofthesearebasedonthesametechniquesusedforviruscultivation.Forexample,intheplaque assay severaldilutionsofbacterialoranimalvirusesare
28、platedoutwithappropriatehostcells.Whenthenumberofvirusesplatedoutaremuchfewerthanthenumberofhostcellsavailableforinfectionandwhenthevirusesaredistributedevenly,eachplaqueinalayerofbacterialoranimalcellsisassumedtohavearisenfromthereproductionofasinglevirusparticle.Thereforeacountoftheplaquesproduced
29、ataparticulardilutionwillgivethenumberofinfectiousvirionsorplaque-forming units (PFU), andtheconcentrationofinfectiousunitsintheoriginalsamplecanbeeasilycalculated.Supposethat0.10mlofa106dilutionoftheviruspreparationyields75plaques.Theoriginalconcentrationofplaque-formingunitsisPFU/ml=(75PFU/0.10ml)
30、(106)=7.5108.Virusesproducingdifferentplaquemorphologytypesonthesameplatemaybecountedseparately.AlthoughthenumberofPFUdoesnotequalthenumberofvirusparticles,theirratiosareproportional:apreparationwithtwiceasmanyviruseswillhavetwicetheplaque-formingunits.8.5 The Structure of VirusesVirusmorphologyhasb
31、eenintenselystudiedoverthepastdecadesbecauseoftheimportanceofvirusesandtherealizationthatvirusstructurewassimpleenoughtobeunderstood.Progresshascomefromtheuseofseveraldifferenttechniques:electronmicroscopy,X-raydiffraction,biochemicalanalysis,andimmunology.Althoughourknowledgeisincompleteduetothelar
32、genumberofdifferentviruses,thegeneralnatureofvirusstructureisbecomingclear.1.Virion SizeVirusparticles(virions)varywidelyinsizeandshape.Virusesaresmallerthancells,ranginginsizefrom0.02to0.3m.Acommonunitofmeasureforvirusesisthenanometer.Smallpoxvirus,oneofthelargestviruses,isabout200nmindiameter(abit
33、smallerthanthesizeofthesmallestbacteria);poliovirus,oneofthesmallest,isonly28nmindiameter(aboutthesizeofaribosome).Virusesvaryconsiderablyinsize.Althoughmostarequiteabitsmallerthanbacteria,someofthelargerviruses(suchasthesmallpoxvirus)areaboutthesamesizeassomeverysmallbacteria(suchasthemycoplasmas,r
34、ickettsias,andchlamydias).Virusesrangefrom20to300nmindiameter2.Genome in virionViralgenomes.ThegenomesofvirusescanbecomposedofeitherDNAorRNA,andsomeusebothastheirgenomicmaterialatdifferentstagesintheirlifecycle.However,onlyonetypeofnucleicacidisfoundinthevirionofanyparticulartypeofvirus.Thiscanbesin
35、gle-stranded(ss),double-stranded(ds),orinthecaseofthehepadnaviruses,partiallydouble-stranded.A virus can have either DNA or RNA but never both !Virusesareexceptionallyflexiblewithrespecttothenatureoftheirgeneticmaterial.Theyemployallfourpossiblenucleicacidtypes:single-strandedDNA,double-strandedDNA,
36、single-strandedRNA,anddouble-strandedRNA.Allfourtypesarefoundinanimalviruses.Plantvirusesmostoftenhavesingle-strandedRNAgenomes.Althoughphagesmayhavesingle-strandedDNAorsingle-strandedRNA,bacterialvirusesusuallycontaindouble-strandedDNA.3.Structure of viruses Most viruses are too small to be seen un
37、der light microscope. All viruses consists of an RNA or DNA core genome surrounded by a protein coat capsid. The combined viral genome and capsid is called the nucleocapsid.Thenucleicacidofavirusissurroundedbyaproteincoatcalledthecapsid.Eachcapsidiscomposedofproteinsubunitscalledcapsomeres.Insomevir
38、uses,thecapsidiscoveredbyanenvelope,whichusuallyconsistsofsomecombinationoflipids,proteins,andcarbohydrates.Dependingonthevirus,envelopesmayormaynotbecoveredbyspikes, whicharecarbohydrateproteincomplexesthatprojectfromthesurfaceoftheenvelope.Virusesmaybeclassifiedintoseveralmorphologicaltypesontheba
39、sisoftheircapsidarchitectureasrevealedbyelectronmicroscopyandatechniquecalledx-raycrystallography.GENERAL MORPHOLOGYA, Some viruses, such as tobacco mosaicvirus, have a helical symmetry with thecapsidsurroundinganRNAgenome.B, Manyvirusesthatinfectbacteria,suchastheT-evenbacteriophage,haveacomplexcap
40、sidwithDNAcontainedwithintheheadstructure.C,Someanimalviruses,suchasadenovirus,haveisometricsymmetryandaDNAgenome.D, others, such as coronavirus, havecomplex capsids and an envelope withprotruding proteins surrounding an RNAgenome.Helical viruses resemble long rods that may be rigid or flexible. Sur
41、rounding the nucleic acid, their capsid is a hollow cylinder with a helical structure. An example of a helical virus that is a rigid rod is the tobacco mosaic virus (1)Helical virusesThe capsid of most polyhedral viruses is in the shape of a regular polyhedron with 20 triangular faces and 12 corners
42、. The capsomeres of each face form an equilateral triangle. An example of a polyhedral virus in the shape of an icosahedron is the adenovirus. Another icosahedral virus is the poliovirus. (2)Polyhedral virusesThecapsidofsomevirusesiscoveredbyanenvelope.Envelopedvirusesareroughlysphericalbuthighlyvar
43、iableinshapebecausetheenvelopeisnotrigid.Whenhelicalorpolyhedralvirusesareenclosedbyenvelopes,theyarecalledenvelopedhelicalandenvelopedpolyhedralviruses. Anexampleofanenvelopedhelicalvirusistheinfluenzavirus.(3)Enveloped virusesSomeviruses,particularlybacterialviruses,haveverycomplicatedstructuresan
44、darecalledcomplexviruses.Examplesofcomplexvirusesarepoxviruses,whichdonotcontainclearlyidentifiablecapsidsbuthaveseveralcoatsaroundthenucleicacid.Certainbacteriophageshavecapsidstowhichadditionalstructuresareattached. (4) Complex virusesComplexbacterialviruseswithbothheadsandtailsaresaidtohavebinal
45、symmetry becausetheypossessacombinationoficosahedral(thehead)andhelical(thetail)symmetry.4.Viral EnvelopesManyanimalviruses,someplantviruses,andatleastonebacterialvirusareboundedbyanoutermembranouslayercalledanenvelope.Animalvirusenvelopesusuallyarisefromhostcellnuclearorplasmamembranes;theirlipidsa
46、ndcarbohydratesarenormalhostconstituents.Incontrast,envelopeproteinsarecodedforbyvirusgenesandmayevenprojectfromtheenvelopesurfaceasspikes orpeplomers.Thesespikesmaybeinvolvedinvirusattachmenttothehostcellsurface.Sincetheydifferamongviruses,theyalsocanbeusedtoidentifysomeviruses.Becausetheenvelopeis
47、aflexible,membranousstructure,envelopedvirusesfrequentlyhaveasomewhatvariableshapeandarecalledpleomorphic.8.6 Principles of Virus TaxonomyTheclassificationofvirusesisinamuchlesssatisfactorystatethanthatofeitherbacteriaoreucaryoticmicroorganisms.Inpart,thisisduetoalackofknowledgeoftheiroriginandevolu
48、tionaryhistory.Usuallyvirusesareseparatedintoseverallargegroupsbasedontheirhostpreferences:animalviruses,plantviruses,bacterialviruses,bacteriophages,andsoforth.Inthepastvirologistsworkingwiththesegroupswereunabletoagreeonauniformsystemofclassificationandnomenclature.Beginningwithits1971report,theIn
49、ternationalCommitteeforTaxonomyofViruseshasdevelopedauniformclassificationsystemandnowdividesvirusesintothreeorders,56families,9subfamilies,233genera,and1,550virusspecies.Thecommitteeplacesgreatestweightonafewpropertiestodefinefamilies:nucleicacidtype,nucleicacidstrandedness,presenceorabsenceofanenv
50、elope,andthehost.Virusesaredividedintodifferenttaxonomicgroupsbasedoncharacteristicsthatarerelatedtothetypeofhostused,virionstructureandcomposition,modeofreproduction,andthenatureofanydiseasescaused.1.Natureofthehost-animal,plant,bacterial,insect,fungal2.Nucleicacidcharacteristics-DNAorRNA,singleord
51、oublestranded,molecularweight3.Capsidsymmetry-icosahedral,helical,binal4.Presenceofanenvelopeandethersensitivity5.Diameterofthevirionornucleocapsid6.Numberofcapsomersinicosahedralviruses7.Immunologicproperties8.IntracellularlocationofviralreplicationSome important characteristics for viral classific
52、ation(1)nucleic acid type(2) nucleic acid strandedness(3) presence or absence of an envelope Recently,theInternationalCommitteeforTaxonomyofViruseshasdevelopedauniformclassificationsystemanddividedvirusesinto56families.Thecommitteeplacesgreatestweightonthreeproperties:Schematic representations of th
53、e main types of bacterial virusesTailed bacteriophage:Genome:DNA,double-stranded.Virion:complexshape,binarysymmetry,variablenumberofcapsomers.ThetailsofthephagearelongandcontractileingroupA,longandnoncontractileingroupB,andveryshortingroupC.Example:T-evencoliphages.Cubic bacteriophage:Group1Genome:D
54、NA,single-stranded.Virion:icosahedral,cubicsymmetry,12capsomers.Example:X174Group2Genome:DNA,double-stranded.Virion:cubicsymmetry,enveloped.Example:PM-2.Group3Genome:RNA,single-stranded.Virion:icosahedral,cubicsymmetry,32capsomers.Group4Genome:RNA,double-stranded.Virion:cubicsymmetry,enveloped.Examp
55、le:06.Filamentous bacteriophage:Genome:DNA,single-stranded.Virion:rod-shaped,helicalsymmetry.Example:fd.Plant Viruses : Tobaccomosaicvirus(TMV)asanexample (1) penetration by the virus of a susceptible plant cell-generally through abrasions or insect bites, (2) tincoating of the viral nucleic acid wi
56、thin the plant cell,(3) assumption by the viral genome of control of the synthetic activities of the host cell, (4) expression of the viral genome so that viral nucleic acid and capsid components are synthesized, (5) assembly of the viral particles within the host cell, and, (6) release of the compl
57、ete viral particles from the host plant cell. Animal Viruses:Viroids and Prions:Viroids are small, circular, single-stranded RNA molecules that are the smallest known pathogens.The extracellular form of the viroid is naked RNA-there is no capsid of any kind.Prions have a distinct extracellular form,
58、 but the extracellular form seems to be entirely protein. It apparently does not contain any nucleic acid, or if it does, the molecule is not long enough to encode the single kind of protein of which the prion is composed .人的库鲁病(kuru)、克雅氏病(CreutzfeldtJakobdisease,CJD)等羊搔痒症(scrapie)牛海绵状脑病(spongiforme
59、ncephalopathy)引起人与动物的致死性中枢神经系统疾病Prusiner (1982)提出羊搔痒病因子是一种蛋白质侵染颗粒（proteinaceous infectious particle），并将之称做Prion或Virino。 -朊病毒1997年，StanleyB.Prusiner荣获诺贝尔奖Summery1.Chamberlandsinventionofaporcelainfilterthatcouldremovebacteriafromvirussamplesenabledmicrobiologiststoshowthatvirusesweredifferentfrombact
60、eria.2.Inthelate1930sStanley,Bawden,andPiriecrystallizedthetobaccomosaicvirusanddemonstratedthatitwascomposedonlyofproteinandnucleicacid.3.AvirioniscomposedofeitherDNAorRNAenclosedinacoatofprotein(andsometimesothersubstancesaswell).Itcannotreproduceindependentlyoflivingcells.4.Virusesarecultivatedus
61、ingtissuecultures,embryonatedeggs,bacterialcultures,andotherlivinghosts.5.Sitesofanimalviralinfectionmaybecharacterizedbycytopathiceffectssuchaspocksandplaques.Phagesproduceplaquesinbacteriallawns.Plantvirusescancauselocalizednecroticlesionsinplanttissues.6.Virusescanbepurifiedbytechniquessuchasdiff
62、erentialandgradientcentrifugation,precipitation,anddenaturationordigestionofcontaminants.7.Virusparticlescanbecounteddirectlywiththetransmissionelectronmicroscopeorindirectlybythehemagglutinationassay.8.Infectivityassayscanbeusedtoestimatevirusnumbersintermsofplaque-formingunits.9.Allvirionshaveanuc
63、leocapsidcomposedofanucleicacid,eitherDNAorRNA,heldwithinaproteincapsidmadeofoneormoretypesofproteinsubunitscalledprotomers.10.Therearefourtypesofviralmorphology:nakedicosahedral,nakedhelical,envelopedicosahedralandhelical,andcomplex.11.Viralnucleicacidscanbeeithersinglestrandedordoublestranded,DNAo
64、rRNA.MostDNAviruseshavedouble-strandedDNAgenomesthatmaybelinearorclosedcircles.12.RNAvirusesusuallyhavessRNAthatmaybeeitherplus(positive)orminus(negative)whencomparedwithmRNA(positive).ManyRNAgenomesaresegmented.13.Virusescanhaveamembranousenvelopesurroundingtheirnucleocapsid.Theenvelopelipidsusuall
65、ycomefromthehostcell;incontrast,manyenvelopeproteinsareviralandmayprojectfromtheenvelopesurfaceasspikes.14.Althoughviruseslacktruemetabolism,somecontainafewenzymesnecessaryfortheirreproduction.15.Currentlyvirusesareclassifiedwithataxonomicsystemplacingprimaryemphasisonthehost,typeandstrandednessofvi
66、ralnucleicacids,andonthepresenceorabsenceofanenvelope.HOMEWORKI. MULTIPLE-CHOICE QUESTIONS1.Avirusisatinyinfectiousa.cellb.particlec.livingthingd.nucleicacid2.Thecapsidiscomposedofproteinsubunitscalleda.spikesb.virionsc.plaquesd.capsomers3.Theenvelopeofananimalvirusisderivedfromthe()ofitshostcell.a.
67、cellwallb.glycocalyxc.cellmembraned.receptors4.Thenucleicacidofavirusisa.DNAonlyb.RNAonlyc.bothDNAandRNAd.eitherDNAorRNAII.CONCEPT QUESTIONS1.Describe10unique characteristicsofviruses(canincludestructure,behavior,multiplication).2.Characterizethevirusesaccordingtosizerange.Whatdoesitmeantosaythattheyareultramicroscopic?Thattheyarefilterable?
