病毒感染的检查方法与防治原则e课件.ppt

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1、Laboratory Diagnosis,1,编辑版ppt,Laboratory Diagnosis 1编辑版pp,Category of Sample,Blood, Urine, Stool, nasal washing, nasal swab , throat swab, saliva , sputum, rectal swab, vesicle fluid( scraping or swab), tissue ,brain biopsy, cerebrospinal fluid, et al.,2,编辑版ppt,Category of SampleBlood, Urine,Laborat

2、ory Diagnosis,Microscopy IdentificationVirus isolation and identificationDetection of viral proteins( antigens and enzymes)Detection of viral genetic material Serologic procedures,3,编辑版ppt,Laboratory Diagnosis Microscop,Microscopy Identification,Light microscopyFluorescent microscopyElectron microsc

3、opy,4,编辑版ppt,Microscopy IdentificationLight,Light microscopy,Characteristic CPE Inclusion Bodies,5,编辑版ppt,Light microscopyCharacteristic,Cell deathCell roundingDegenerationAggregationLoss of attachments to substrateCharacteristic histological changes:inclusion bodies in the nucleus or cytoplasm, mar

4、gination of chromatinSyncytia: multinucleated giant cells caused by virus-induced cell-cell fusion,6,编辑版ppt,Cell death6编辑版ppt,Fluorescent microscopy,Fluorescent-antibody staining,7,编辑版ppt,Fluorescent microscopyFluoresc,Electron microscopy,Direct detection : Human rotavirus; HAV; HBV; Smallpox virus;

5、 Herpes virus.Immune Electron microscopy: Human rotavirus; HAV;,8,编辑版ppt,Electron microscopyDirect dete,Laboratory Diagnosis,Microscopy IdentificationVirus isolation and identificationDetection of viral proteins( antigens and enzymes)Detection of viral genetic material Serologic procedures,9,编辑版ppt,

6、Laboratory Diagnosis Microscop,Viral isolation and Identification,Viral Growth and Cell culture Viral Detection Viral Identification Interpretation of culture results,10,编辑版ppt,Viral isolation and Identifica,Systems for the Propagation of Viruses,PeopleAnimals: cows, chickens, mice,rats, suckling mi

7、ce Embryonated eggs Organ and tissue cultureOrgan culturePrimary tissue cultureCell lines: diploidTumor or （immortalized ）cell line,11,编辑版ppt,Systems for the Propagation of,Viral detection,CPEHemadsorptionInterfereMetabolize of cell,12,编辑版ppt,Viral detectionCPE12编辑版ppt,TCID50(Tissue culture infectiv

8、e dose),TCID50 is defined as that dilution of virus which will cause CPE in 50% of a given batch of cell cultureTCID50= log10 of highest dilution giving 100%CPE +1/2 (total number of test units showing CPE)/ (number of test units per dilution),13,编辑版ppt,TCID50(Tissue culture infecti,Viral identifica

9、tion,Complement fixation： Hemagglutination inhibitionNeutralizationImmunofluorescence ( direct or indirect)Latex agglutination In situ EIA ELISARIA(radioimmuno,14,编辑版ppt,Viral identificationComplement,Laboratory Diagnosis,Microscopy IdentificationVirus isolation and identificationDetection of viral

10、proteins( antigens and enzymes)Detection of viral genetic material Serologic procedures,15,编辑版ppt,Laboratory Diagnosis Microscop,Detection of viral proteins( antigens and enzymes),Antigen detection ( ELISA, RIA, Western blot)Hemagglutination and hemadsorptionEnzyme activities( reverse transcriptase)

11、Protein patterns( electrophoresis ),16,编辑版ppt,Detection of viral proteins(,Laboratory Diagnosis,Microscopy IdentificationVirus isolation and identificationDetection of viral proteins( antigens and enzymes)Detection of viral genetic material Serologic procedures,17,编辑版ppt,Laboratory Diagnosis Microsc

12、op,Detection of viral genetic material,PCR ( Polymerase chain reaction)RT-PCR (Reverse transcriptase polymerase chain reaction)Southern（DNA), Northern(RNA), and dot blots DNA genome hybridization in situ(cytochemistry)Electrophoretic mobilities of RNA for segmented RNA viruses( Electrophoresis) Rest

13、riction endonuclease cleavage patterns,18,编辑版ppt,Detection of viral genetic mat,Laboratory Diagnosis,Microscopy IdentificationVirus isolation and identificationDetection of viral proteins( antigens and enzymes)Detection of viral genetic material Serologic procedures,19,编辑版ppt,Laboratory Diagnosis Mi

14、croscop,Serologic procedures,If the antibody titer in the convalesent-phase serum sample is at least 4-fold higher than the titer in the acute-phase serum sample, the patient is considered to be infected.In certain viral diseases, the presence of IgM antibody is used to diagnose current infectionOth

15、er nonspecific serologic tests are available,20,编辑版ppt,Serologic proceduresIf the ant,Serologic procedures,Complement fixation： Hemagglutination inhibitionNeutralizationImmunofluorescence ( direct or indirect)Latex agglutination In situ EIA ELISARIA,21,编辑版ppt,Serologic proceduresComplement,Viruses D

16、iagnosed by Serology,Epstein-Barr virusRubella virusHepatitis A, B, C, D, and E virusesHIVHuman T-cell Leukemia virusArboviruses ( Encephalitis viruses),22,编辑版ppt,Viruses Diagnosed by SerologyE,Prevention,Successes of the Past Possibilities for the Future,23,编辑版ppt,Prevention23编辑版ppt,Active immuniza

17、tion,Vaccines,24,编辑版ppt,Active immunizationVaccines24编,Overview of Active immunization,Active immunization - administration of antigen resulting in production of a specific immune response with immunologic memory. Response may be cellular or humoral or both. Natural immunity - to diseases you have c

18、aught and successfully fought Artificial immunity Vaccination(vaccines),25,编辑版ppt,Overview of Active immunizatio,Attributes of a good vaccine,Ability to elicit the appropriate immune response for the particular pathogenLong term protection ideally life-longSafety vaccine itself should not cause dise

19、ase Stable retain immunogenicity, despite adverse storage conditions prior to administrationIn-expensive,26,编辑版ppt,Attributes of a good vaccineAb,LIVE VACCINES,Live attenuated organismHeterologous vaccinesLive recombinant vaccines Attributes live vaccines,27,编辑版ppt,LIVE VACCINESLive attenuated o,Liv

20、e attenuated organism,Organisms whose virulence has been artificially reduced by in vitro Culture under adverse conditions, such as reduced temperature.,28,编辑版ppt,Live attenuated organismOrgani,Heterologous vaccines,Closely related organism of lesser virulence, which shares many antigens with the vi

21、rulent organism. The vaccine strain replication in the host and induces an immune response that cross reacts with antigens of the virulent organism.Vaccinia virus /cowpox virus- Variola virus,29,编辑版ppt,Heterologous vaccinesClosely r,Live recombinant,Vector bovine vaccineBCG,30,编辑版ppt,Live recombinan

22、tVector 30编辑版pp,Both cell mediated immunity and antibody responseActivates all phases of immune system. Can get humoral IgG and local IgARaises immune response to all protective antigens. Inactivation may alter antigenicity. More durable immunity; more cross-reactive Immunity is long livedSingle dos

23、e,Advantages of Attenuated Vaccines 2-1,31,编辑版ppt,Both cell mediated immunity an,Advantages of Attenuated Vaccines 2-2,Low cost Quick immunity in majority of vaccinees In case of polio and adeno vaccines, easy administration Easy transport in field Can lead to elimination of wild type virus from the

24、 community,32,编辑版ppt,Advantages of Attenuated Vacci,Disadvantages of Live Attenuated Vaccine,Mutation; reversion to virulence (often frequent)Spread to contacts of vaccinee who have not consented to be vaccinated (could also be an advantage in communities where vaccination is not 100%) Spread vaccin

25、e not standardized-may be back-mutated Poor take in tropics Problem in immunodeficiency disease (may spread to these patients),33,编辑版ppt,Disadvantages of Live Attenuat,Killed vaccines,The organism is propagated in bulk, in vitro, and inactivated with either beta-propiolactone or formaldehyde. These

26、vaccines are not infectious and are therefore relatively safe. However, they are usually of lower immunogenicity and multiple doses may be needed to induce immunity. In addition, they are usually expensive to prepare.,34,编辑版ppt,Killed vaccinesThe organism is,Killed vaccines,Inactivated organism: rab

27、ies virus; epidmic type B encephalitis virus.Subunit Vaccines: Influenza virus( HA and NA)Recombinant proteins: HBV,35,编辑版ppt,Killed vaccinesInactivated org,Advantages of inactivated vaccines,Gives sufficient humoral immunity if boosters given No mutation or reversion Can be used with immuno-deficie

28、nt patients These vaccines tend to be able to withstand more adverse storage conditions,Sometimes better in tropics,36,编辑版ppt,Advantages of inactivated vacc,Disadvantages of inactivated vaccines,Many vaccinees do not raise immunitypoor, only antibody, no cell immediated immune responseresponse is sh

29、ort-lived and multiple doses are neededNo local immunity (important)Inactivated, therefore can not replicate in the host and cause diseaseFailure in inactivation and immunization with virulent virusExpense: Expensive to prepare,37,编辑版ppt,Disadvantages of inactivated v,New Methods,Selection of attenu

30、ated virus strain Varicella Hepatitis AUse monoclonal antibodies to select for virus with altered surface receptor Rabies ReoUse mutagen and grow virus at 32 degrees. Selects for temperature-sensitive virus. Grows in upper respiratory tract but not lower flu (new vaccine) respiratory syncytial virus

31、,38,编辑版ppt,New MethodsSelection of attenu,New Methods,Passage progressively at cold temperaturesTS mutant in internal proteinsCan be re-assorted to so that coat is the strain that is this years flu strain,39,编辑版ppt,New MethodsPassage progressive,40,编辑版ppt,PB2PB1PAHANANPMNSPB2PB1PAHANAN,New Methods,D

32、eletion mutants Suppression unlikely (but caution in HIV) Viable but growth restrictionsProblems Oncogenicity in some cases (adeno, retro),41,编辑版ppt,New Methods Deletion mutants41,New Methods,42,编辑版ppt,New Methods Recombinant DNAS-a,Single gene (subunit) - problems,Surface glycoprotein poorly solubl

33、e - deletion? Poorly immunogenic Post-translational modifications Poor CTL response,43,编辑版ppt,Single gene (subunit) - proble,Single gene (subunit) in expression vector,Vaccinate with live virusCanary Pox Infects human cells but does not replicate Better presentation CTL responseVacciniaAttenuated Po

34、lioBeing developed for anti-HIV vaccine,44,编辑版ppt,Single gene (subunit) in expre,New Methods,Chemically synthesized peptide malariapoorly immunogenic,45,编辑版ppt,New MethodsChemically synthesi,New methods,Anti-idiotype vaccine,epitope,Antibody with epitope binding site,Virus,46,编辑版ppt,antibodyNew meth

35、odsAnti-idioty,Anti-idiotype vaccine cont,Make antibody against antibody idiotype,Anti-idiotype antibody mimics the epitope,47,编辑版ppt,antibodyAnti-idiotype vaccine,Anti-idiotype antibody cont 2,Use anti-idiotype antibody as injectable vaccine,Antibody to anti-idiotype antibody,Binds and neutralizes

36、virus,Use as vaccine,48,编辑版ppt,Anti-anti-idiotypeantibodyAnt,New Methods,New “Jennerian Vaccines” Live vaccines derived from animal strains of similar viruses Naturally attenuated for humansRotavirus: Monkey Rota80% effective in some human populationsIneffective in othersDue to differences in circul

37、ating viral serotypes,49,编辑版ppt,New MethodsNew “Jennerian Vacc,New Methods,New Jennerian VaccinesBovine parainfluenza Type 3Bovine virus is: Infectious to humans Immunogenic (61% of children get good response) Poorly transmissablePhenotypicaly stable,50,编辑版ppt,New MethodsNew Jennerian Vacci,New Meth

38、ods,51,编辑版ppt,New MethodsSecond Generation J,Vaccines,1796 Jenner: wild type animal-adapted virus 1800s Pasteur: Attenuated virus 1996 DNA vaccinesThe third vaccine revolution,52,编辑版ppt,Vaccines 1796 Jenner: wild t,DNA vaccines,DNA vaccines are at present experimental , but hold promise for future t

39、herapy since they evoke both humoral and cell-mediated immunity, without the dangers associated with live virus vaccines,53,编辑版ppt,DNA vaccinesDNA vaccines are a,DNA Vaccines,plasmid,Muscle cell,Gene for antigen,Muscle cell expresses protein - antibody madeCTL response,54,编辑版ppt,DNA VaccinesplasmidM

40、uscle cell,DNA Vaccines,Plasmids are easily manufactured in large amounts DNA is very stable DNA resists temperature extremes so storage and transport are straight forward DNA sequence can be changed easily in the laboratory. This means that we can respond to changes in the infectious agent By using

41、 the plasmid in the vaccinee to code for antigen synthesis, the antigenic protein(s) that are produced are processed (post-translationally modified) in the same way as the proteins of the virus against which protection is to be produced. This makes a far better antigen than purifying that protein an

42、d using it as an immunogen.,55,编辑版ppt,DNA Vaccines Plasmids are easi,DNA Vaccines,Mixtures of plasmids could be used that encode many protein fragments from a virus/viruses so that a broad spectrum vaccine could be produced The plasmid does not replicate and encodes only the proteins of interest No

43、protein component so there will be no immune response against the vector itself Because of the way the antigen is presented, there is a CTL response that may be directed against any antigen in the pathogen. A CTL response also offers protection against diseases caused by certain obligate intracellul

44、ar pathogens (e.g. Mycobacterium tuberculosis),56,编辑版ppt,DNA Vaccines Mixtures of plasm,DNA Vaccines,Possible Problems Potential integration of plasmid into host genome leading to insertional mutagenesis Induction of autoimmune responses (e.g. pathogenic anti-DNA antibodies) Induction of immunologic

45、 tolerance (e.g. where the expression of the antigen in the host may lead to specific non-responsiveness to that antigen),57,编辑版ppt,DNA VaccinesPossible Problems5,DNA Vaccines,DNA vaccines produce a situation that reproduces a virally-infected cellGives: Broad based immune response Long lasting CTL

46、response Advantage of new DNA vaccine for flu:CTL response can be against internal proteinIn mice a nucleoprotein DNA vaccine is effective against a range of viruses with different hemagglutinins,58,编辑版ppt,DNA VaccinesDNA vaccines produ,Adjuvants,Certain substances, when administered simultaneously

47、with a specific antigen, will enhance the immune response to that antigen.,59,编辑版ppt,AdjuvantsCertain substances, w,Adjuvants in common use,Aluminium saltsLiposomes and immunostimulating complexesComplet Freunds adjuvant is an emulsion of mycobacteria, oil and waterIncomplete Freunds adjuvantMuramyl

48、 di-peptideCytokines,60,编辑版ppt,Adjuvants in common useAlumini,Possible action modes of adjuvant,By trapping antigen in the tissues, thus allowing maximal exposure to dendritic cells and specific T and B lymphocytesBy activating antigen-presenting cells to secrete cytokines that enhance the recruitme

49、nt of antigen-specific T and B cells to the site of inoculation,61,编辑版ppt,Possible action modes of adju,Smallpox,62,编辑版ppt,Smallpox62编辑版ppt,Smallpox,Variolation1% v. 25% mortalityLife-long immunity No drift or shift,63,编辑版ppt,Smallpox Variolation63编辑版ppt,Smallpox,Vaccination Jenner 1796 : Cowpox/Swi

50、nepox 1800s Compulsory childhood vaccination 1930s Last natural UK case 1940s last natural US case 1958 WHO program October 1977: Last case (Somalia),64,编辑版ppt,SmallpoxVaccination64编辑版ppt,Smallpox,No animal reservoir Lifelong immunity Subclinical cases rare Infectivity does not precede overt symptom