01-Course-Virology病毒學virus巴斯德研究所

1、IPS,CAS-20061VIROLOGY COURSE#1 General virology, History, Taxonomy,Techniques in virologyIPS,CAS-20062 Welcome to my world!IPS,CAS-20063 Virus : a noncellular genetic element that infects a cell for its own replication, it has an extracellular state. A virus particle containing nucleic acid surround

2、ed by protein and other macromolecular components is called virion. Viruses have a heavy dependence on host-cell structural and metabolic components. Viruses can confer important new properties on their host cell. Viruses may be non productive, latent, oncogenic, chronic or lytic.DefinitionIPS,CAS-2

3、0064I. GENERAL VIROLOGY1840s:Agents not visible under microscope1880-1900:Louis Pasteur: Absence of spontaneous generationAdolf Mayer and Dimitry Ivanovsky: Tobacco mosaic- Transmission of agent by the juice of sick plants to healthy plants- Not retained by porous glass filters (Charles Chamberland)

4、- But Robert Kochs postulate not validMartinus Beijerinck: Concept of filterable agent called virus (poison) not replicating outside host but amplified in host (contagious living liquid).TMV, FMDV, YFVConcept of virology. HistoryIPS,CAS-20065Kochs postulate:- Agent regularly found in the lesion of t

5、he disease- organism must be isolated in pure culture- inoculation of such culture to host should initiate the disease- the organism should again be isolated from the lesions of the hostIPS,CAS-200661900-1910: New conseptsViruses can be transmitted by mosquitoes 1901Viruses can cause cancer (Leukemi

6、a, Sarcoma) 1908-1911Viruses show lesions inside cells (Negri corpus 1903)1930-1955:1. Viruses contain proteins that can be: precipitated, concentrated, Inactivated (resistance to physical and chemical agents), neutralized (production of antibodies in animals),electrophoresed2. Viruses contain phosp

7、horus3. Viruses contain ribonucleic acids or deoxyribonucleic acids1917:Virus titration plaque assayIPS,CAS-200674. Electron microscopy (1937)5. Viruses have different structures 6.The use of animals for virus cultivation (1931)Mouse (leukemia lymphoid virus)Embryonated eggs (pox virus)7. The use of

8、 cell culture for virus cultivation (1948)Poliovirus Vaccine development in animals (rabies, YFV) embryonated chicken eggs (YFV, smallpox, influenza)cell culture (poliovirus) IPS,CAS-200681960now:Cell biology: eukaryotic transcription machinery and gene regulation (transcription factors, polymerase

9、promoters, RNA splicing, mRNA polyadenylation, capping and methylation, RNA transport from the nucleus to cytoplasm, regulation of translation and IRES, protein processing, IFN-regulated signals)1970now:Molecular biology and recombinant DNA(Reverse transcriptase, Restriction enzyme mapping chart, La

10、mbda phage for DNA library, protein expression vectors, site integration in host genome, gene therapy ) Role and benefit of virology in cell and molecular biology IPS,CAS-20069DNA Viruses:RNA Viruses:AdenovirusesCoronavirusesIridovirusesFilovirusesHerpesvirusesOrthomyxovirusesPapovavirusesParamyxovi

11、rusesParvovirusesPicornavirusesPoxvirusesRetrovirusesViral HepatitisRhabdovirusesArboviruses Arenaviruses, Bunyaviruses, Flaviviruses, Togaviruses Diarrhoea Viruses Astroviruses, Caliciviruses, Reoviruses (inc. Rotaviruses) Taxonomic order (Order, Family, Subfamily, Genus, Species, strain, variant)G

12、eneral host range (bacteria, plant, animal; host range, pathogenesis, transmission, habitat)Disease orderII. VIRUS TAXONOMYIPS,CAS-200610http:/ (DNA virus in Chinese)http:/ classificationICTV: -virus, -virinae, -viridae, -viralesCapitalized and italicized: ex: HRSVIPS,CAS-200611Most families have di

13、stinct virion morphology, genome structure, and replication strategyVIII. Subviral agents (Viroids, satellitesPrions) IX Unassigned virusIPS,CAS-200612Host range:AlgaeArchealBacteriaFungiInvertebratePlantAnimalProtozoaVertebratesSome viruses can replicate in different types of hosts: ex: Flaviviruse

14、s, Bunyaviridae, and AlphaviridiaeIPS,CAS-200613Virus StructureViruses consist of nucleic acids, capsid, and/or envelope.http:/:8002/project/Viruses/256,1,Chapter 6 VirusesIPS,CAS-200614Nature of the virionVirus Size: 20 nm to 300 nm (Ebola: 1000 nm long)Largest virus: Smallpox (天花病毒天花病毒) is 200 nmS

15、mallest virus: Polio virus (脊髄灰質(zhì)炎病毒脊髄灰質(zhì)炎病毒) is 28 nmVirus genome: Smaller than those of cells, the largest known viral genome: vaccinia (牛痘牛痘) is only 190 KB.Bacterial genomes: 1000-9000 KBIPS,CAS-200615Virus SymmetryHelical symmetry: Tobacco mosaic virus (TMV).Icosahedron: The most efficient arrang

16、ement for subunits in a closed shell.Enveloped viruses: envelope consists of lipid bilayer with glycoproteins embedded in it. Virus symmetry refers to its nucleocapside.Complex viruses: T4 bacteriophageIPS,CAS-200616Virus MorphologyIPS,CAS-200617 Icosahedric symetry of viruses by cryo-electron micro

17、scopyT=3 symetryIPS,CAS-200618III. VIRUSES AND DISEASESCancer:Epstein Barr: Burkitt lymphoma (1962-68) and mononucleosisHuman T-cell leukemia (1977-1980)Hepatocellular carcinoma-Hepatitis B (1967)PapillomavirusHepatitis C (1985): a different story from RNA virusAcute diseases:Ex: Influenza, dengueCh

18、ronic disease:Ex: Herpes, relapsing virusesSymptomatic diseases:Acute respiratory diseaseViral encephalitisHepatitisDiarrheaIPS,CAS-200619IV. VIRUS LIFE CYCLE1. Attachment (adsorption)2. Penetration3. Uncoating3. Early steps in replication4. Replication5. Synthesis of protein subunits6. Assembly and

19、 packaging7. ReleaseIPS,CAS-200620Virus Life CycleThe one-step growth curve of virus replicationIPS,CAS-200621Virus infectionIPS,CAS-200622Major determinants in viral tropismReceptor and co-receptorCell surface receptors binding directly to native virusCo-receptors bind as a result of primary bindin

20、g to native or altered form of virus proteinEntry Injection of genetic material Membrane disruptionPoresFusionEndocytosisUncoatingRNA replicationIPS,CAS-200623G-protein-coupled receptors (chemokine receptors for retroviruses)Proteins with multiple membrane-spanning domains (Transporter for retroviru

21、ses)Immunoglobin-related proteins (UK function or ICAM and CD4 for Picorna-, Adeno-, Corona-, Retro-, Herpes-, Flavi-viruses) Low-density lipoprotein receptor-related proteins (Retro- and Picorna- viruses)Integrins (Vitroectin and laminin binding for Adeno-, Picorna-viruses)TNF-receptors (Retro-, He

22、pers viruses)Small consensus repeat-containing proteins (Complement inhibition for Picorna-, Herpes-, Paramyxo-viruses)Miscellaneous (Metalloprotease:Coronavirus, a-dystroglycan:Arenaviruses, Laminin binding:Togavirus; Ephrin2 TKR:Nipah virus)Virus receptors (1)Enzymatic inactivation, Monoclonal ant

23、ibodies, soluble antigens competition, gene transfer, VOPBAIPS,CAS-200624Sialic acid-containing oligosaccharides (Orthomyxo-, Paramyxo-, Reo-, Papova-, Parvo-, Corona-viruses)Galactosyl ceramide (Retroviruses)Heparane sulfate (Herpes-, flavi-viruses)Carbohydrate destroying enzymes, Monoclonal antibo

24、dies, Heparine competitionVirus receptors (2)IPS,CAS-200625Virus entryEndocytosis at the plasma membraneFusion at the cell membrane. Endocytosis and fusion at the membrane of the endosome (acidification) Tranportation into the cytoplasm, decapsidation Transportation to the nucleusFusion requires con

25、formational changes of the virus coat protein(s) One or two proteins (one or 2 receptors) acidic or neutral pH The use of microtubules (HIV, Herpes, Adeno) or cellular proteins like Karyopherin (Influenza) for transportation of the capsid to the nucleusIPS,CAS-200626Fusion of Influenza virusIPS,CAS-

26、200627Fusion of flavivirusIPS,CAS-200628DNA virus replicationmRNA is equal to RNA(+)DNA (+) is the template DNA for making mRNAIPS,CAS-200629+strand RNA virus replicationFlow of events during multiplication of RNA virusIPS,CAS-200630Replication strategies (1)dsRNA viruses (IFN induction)ssRNA viruse

27、s positive-sense and negative-sense (ratio, IR, RNP, encapsidation)Circular RNA vs linear RNA (CAP, polyA, Ambisens, CIS, palindromes, repeats)Segmented vs non segmented RNA viruses (conserved sequences, subgenomics, coordination of replication and packaging, shifts, )Cis-acting signals and secondar

28、y structures (subversion of cellular machinery,replication selectivity and efficiency for protein synthesis, transcription,packaging)Satellite RNA and defective viral RNAHost specificity and cell specificity IPS,CAS-200631ssRNA positive-sense viruses (used directly as mRNA, +/- subgenomes). Regulati

29、on processssRNA negative-sense (RdRp and NP associated with viral RNA, subgenomes, ambisens strategy, ortho and borna in the nucleus, splicing, Retroviruses (reverse transcriptase, cDNA integration, mRNA splicing, no shut off)DNA viruses (nucleus, maturation, early and late phases)Cell RNA/protein i

30、nhibition (shut off): block splicing and extranuclear transport, RNA or protein degradation, overhelming translation machinery) and anti-viral Strategies (siRNA, IFN, and apoptosis)Regulation of transcription? (Cis-acting elements, Vpg, sc-oligos, DI, editing) Replication strategies (2)IPS,CAS-20063

31、2Viral enzymes:Some viruses contain their own enzymes for their own purposes, such as: Polymerase (RdRp, reverse transcriptase): similarities & evolutionNeuraminidase:break down glycoprotein, helping liberation of the virusHelicaseATPaseMethyl- and Guaninyl-transferaseCAP_specific endonucleasePr

32、oteasePolyA polymeraseNucleaseIntegraseLysosome: bacteriophageReplication strategies (3)IPS,CAS-200633-strand RNA virus replicationCharacteristic: ambisens strategy(Arenavirus, Bunyavirus)IPS,CAS-200634Filovirus genome3-PyPuCU CUUCUAAUU Dpart 3-UAAUUC(U)5/6 PolyadnylationAC 0 1 2 3 4 5 6 7 8 9 10 11

33、 12 13 14 15 16 17 18 19IRVP35NPVP4053GPVP24VP30LLeaderOverlapOverlapIRStopIROverlapTrailerVP35NPVP405IRGPVP24VP30LOverlapIRIRTrailer3LeaderIREbola MarburgIRGenome editingIPS,CAS-200635Site de dpart GP VP30 VP40 VP35 VP24 L NPHairpin structures at the 5 of mRNA of filivirusesIPS,CAS-200636Sites of v

34、irus morphogenesisNucleusCytoplasmRERGolgiMembraneIncorporation of nucleic acid materialPost-assembly modifications and virus releaseFacilitation of release and prevention to reinfection (downregulation of receptors)IPS,CAS-200637IV VIRUS CULTURE Bacteriophage: bacterial Cultures Animal viruses: Tis

35、sue or cell culturesIPS,CAS-200638Quantification of virusesPlaque assay technique for quantification of bacterial viruses.IPS,CAS-200639Quantification of animal virusesIPS,CAS-200640Efficiency of Plating Plaque assay has low efficiency: With bacteriophages: 50% With animal virues: 0.1-1% Therefore, to quantify virus, it is accurate to express the concentration