病毒感染的檢查方法與防治原則e課件

1、 Laboratory Diagnosis 1編輯版ppt Laboratory Diagnosis 1編輯版ppCategory of SampleBlood, 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編輯版pptCategory of SampleBlood, UrineLaboratory Di

2、agnosis Microscopy IdentificationVirus isolation and identificationDetection of viral proteins( antigens and enzymes)Detection of viral genetic material Serologic procedures3編輯版pptLaboratory Diagnosis MicroscopMicroscopy IdentificationLight microscopyFluorescent microscopyElectron microscopy 4編輯版ppt

3、Microscopy IdentificationLightLight microscopyCharacteristic CPE Inclusion Bodies 5編輯版pptLight microscopyCharacteristic Cell deathCell roundingDegenerationAggregationLoss of attachments to substrateCharacteristic histological changes:inclusion bodies in the nucleus or cytoplasm, margination of chrom

4、atinSyncytia: multinucleated giant cells caused by virus-induced cell-cell fusion6編輯版ppt Cell death6編輯版pptFluorescent microscopyFluorescent-antibody staining7編輯版pptFluorescent microscopyFluorescElectron microscopyDirect detection : Human rotavirus; HAV; HBV; Smallpox virus; Herpes virus.Immune Elect

5、ron microscopy: Human rotavirus; HAV; 8編輯版pptElectron microscopyDirect deteLaboratory Diagnosis Microscopy IdentificationVirus isolation and identificationDetection of viral proteins( antigens and enzymes)Detection of viral genetic material Serologic procedures9編輯版pptLaboratory Diagnosis MicroscopVi

6、ral isolation and Identification Viral Growth and Cell culture Viral Detection Viral Identification Interpretation of culture results 10編輯版pptViral isolation and IdentificaSystems for the Propagation of VirusesPeopleAnimals: cows, chickens, mice,rats, suckling mice Embryonated eggs Organ and tissue

7、cultureOrgan culturePrimary tissue cultureCell lines: diploidTumor or （immortalized ）cell line11編輯版pptSystems for the Propagation ofViral detectionCPEHemadsorptionInterfereMetabolize of cell12編輯版pptViral detectionCPE12編輯版pptTCID50(Tissue culture infective dose)TCID50 is defined as that dilution of v

8、irus 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編輯版pptTCID50(Tissue culture infectiViral identificationComplement fixation： Hemagglutination inhibitio

9、nNeutralizationImmunofluorescence ( direct or indirect)Latex agglutination In situ EIA ELISARIA(radioimmuno14編輯版pptViral identificationComplementLaboratory Diagnosis Microscopy IdentificationVirus isolation and identificationDetection of viral proteins( antigens and enzymes)Detection of viral geneti

10、c material Serologic procedures15編輯版pptLaboratory Diagnosis MicroscopDetection of viral proteins( antigens and enzymes)Antigen detection ( ELISA, RIA, Western blot)Hemagglutination and hemadsorptionEnzyme activities( reverse transcriptase)Protein patterns( electrophoresis )16編輯版pptDetection of viral

11、 proteins( Laboratory Diagnosis Microscopy IdentificationVirus isolation and identificationDetection of viral proteins( antigens and enzymes)Detection of viral genetic material Serologic procedures17編輯版pptLaboratory Diagnosis MicroscopDetection of viral genetic materialPCR ( Polymerase chain reactio

12、n)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) Restriction endonuclease cleavage patterns18編輯版pptDetection of viral gene

13、tic matLaboratory Diagnosis Microscopy IdentificationVirus isolation and identificationDetection of viral proteins( antigens and enzymes)Detection of viral genetic material Serologic procedures19編輯版pptLaboratory Diagnosis MicroscopSerologic proceduresIf the antibody titer in the convalesent-phase se

14、rum 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 infectionOther nonspecific serologic tests are available20編輯版pptSerologic proceduresIf the

15、 antSerologic proceduresComplement fixation： Hemagglutination inhibitionNeutralizationImmunofluorescence ( direct or indirect)Latex agglutination In situ EIA ELISARIA21編輯版pptSerologic proceduresComplementViruses Diagnosed by SerologyEpstein-Barr virusRubella virusHepatitis A, B, C, D, and E virusesH

16、IVHuman T-cell Leukemia virusArboviruses ( Encephalitis viruses)22編輯版pptViruses Diagnosed by SerologyEPrevention Successes of the Past Possibilities for the Future23編輯版pptPrevention23編輯版pptActive immunizationVaccines24編輯版pptActive immunizationVaccines24編Overview of Active immunizationActive immuniza

17、tion - 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 caught and successfully fought Artificial immunity Vaccination(vaccines)25編輯版pptOverview of Active immu

18、nizatioAttributes of a good vaccineAbility to elicit the appropriate immune response for the particular pathogenLong term protection ideally life-longSafety vaccine itself should not cause disease Stable retain immunogenicity, despite adverse storage conditions prior to administrationIn-expensive26編

19、輯版pptAttributes of a good vaccineAbLIVE VACCINESLive attenuated organismHeterologous vaccinesLive recombinant vaccines Attributes live vaccines27編輯版pptLIVE VACCINESLive attenuated oLive attenuated organismOrganisms whose virulence has been artificially reduced by in vitro Culture under adverse condi

20、tions, such as reduced temperature. 28編輯版pptLive attenuated organismOrganiHeterologous vaccinesClosely related organism of lesser virulence, which shares many antigens with the virulent organism. The vaccine strain replication in the host and induces an immune response that cross reacts with antigen

21、s of the virulent organism.Vaccinia virus /cowpox virus- Variola virus29編輯版pptHeterologous vaccinesClosely rLive recombinantVector bovine vaccineBCG30編輯版pptLive recombinantVector 30編輯版ppBoth cell mediated immunity and antibody responseActivates all phases of immune system. Can get humoral IgG and lo

22、cal IgARaises immune response to all protective antigens. Inactivation may alter antigenicity. More durable immunity; more cross-reactive Immunity is long livedSingle dose Advantages of Attenuated Vaccines 2-131編輯版pptBoth cell mediated immunity anAdvantages of Attenuated Vaccines 2-2 Low cost Quick

23、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 community32編輯版pptAdvantages of Attenuated VacciDisadvantages of Live Attenuated Vaccine Mutation; reversion to virulence (often frequ

24、ent)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 vaccine not standardized-may be back-mutated Poor take in tropics Problem in immunodeficiency disease (may spread to these patients)33編輯版pptDisa

25、dvantages of Live AttenuatKilled vaccinesThe organism is propagated in bulk, in vitro, and inactivated with either beta-propiolactone or formaldehyde. These vaccines are not infectious and are therefore relatively safe. However, they are usually of lower immunogenicity and multiple doses may be need

26、ed to induce immunity. In addition, they are usually expensive to prepare.34編輯版pptKilled vaccinesThe organism isKilled vaccinesInactivated organism: rabies virus; epidmic type B encephalitis virus.Subunit Vaccines: Influenza virus( HA and NA)Recombinant proteins: HBV35編輯版pptKilled vaccinesInactivate

27、d orgAdvantages of inactivated vaccinesGives sufficient humoral immunity if boosters given No mutation or reversion Can be used with immuno-deficient patients These vaccines tend to be able to withstand more adverse storage conditions,Sometimes better in tropics36編輯版pptAdvantages of inactivated vacc

28、Disadvantages of inactivated vaccinesMany vaccinees do not raise immunitypoor, only antibody, no cell immediated immune responseresponse is short-lived and multiple doses are neededNo local immunity (important)Inactivated, therefore can not replicate in the host and cause diseaseFailure in inactivat

29、ion and immunization with virulent virusExpense: Expensive to prepare37編輯版pptDisadvantages of inactivated vNew MethodsSelection of attenuated virus strain Varicella Hepatitis AUse monoclonal antibodies to select for virus with altered surface receptor Rabies ReoUse mutagen and grow virus at 32 degre

30、es. Selects for temperature-sensitive virus. Grows in upper respiratory tract but not lower flu (new vaccine) respiratory syncytial virus 38編輯版pptNew MethodsSelection of attenuNew MethodsPassage progressively at cold temperaturesTS mutant in internal proteinsCan be re-assorted to so that coat is the

31、 strain that is this years flu strain39編輯版pptNew MethodsPassage progressivePB2PB1PAHANANPMNSPB2PB1PAHANANPMNSPB2PB1PAHANANPMNSAttenuated Donor Master StrainNew Virulent Antigenic Variant StrainXAttenuated Vaccine Strain: Coat of Virulent strain with Virulence Characteristics of Attenuated Strain40編輯

32、版pptPB2PB1PAHANANPMNSPB2PB1PAHANANNew Methods Deletion mutants Suppression unlikely (but caution in HIV) Viable but growth restrictionsProblems Oncogenicity in some cases (adeno, retro)41編輯版pptNew Methods Deletion mutants41New Methods Recombinant DNASingle gene (subunit)S-antigen mRNAcDNAExpress pla

33、smidS-antigen mRNA proteinHepatitis B vaccineraised in yeast42編輯版pptNew Methods Recombinant DNAS-aSingle gene (subunit) - problems Surface glycoprotein poorly soluble - deletion? Poorly immunogenic Post-translational modifications Poor CTL response43編輯版pptSingle gene (subunit) - probleSingle gene (s

34、ubunit) in expression vectorVaccinate with live virusCanary Pox Infects human cells but does not replicate Better presentation CTL responseVacciniaAttenuated PolioBeing developed for anti-HIV vaccine44編輯版pptSingle gene (subunit) in expreNew MethodsChemically synthesized peptide malariapoorly immunog

35、enic45編輯版pptNew MethodsChemically synthesiantibodyNew methodsAnti-idiotype vaccineepitopeAntibody with epitope binding siteVirus46編輯版pptantibodyNew methodsAnti-idiotyantibodyAnti-idiotype vaccine contMake antibody against antibody idiotypeAnti-idiotypeantibodyAnti-idiotype antibody mimics the epitop

36、e47編輯版pptantibodyAnti-idiotype vaccine Anti-anti-idiotypeantibodyAnti-idiotype antibody cont 2Use anti-idiotype antibody as injectable vaccineAntibody to anti-idiotype antibodyBinds and neutralizes virusAnti-idiotypeantibodyAnti-anti-idiotypeantibodyAnti-anti-idiotypeantibodyUse as vaccine48編輯版pptAn

37、ti-anti-idiotypeantibodyAntNew MethodsNew “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 circulating viral serotypes49編輯版pptNew Me

38、thodsNew “Jennerian VaccNew MethodsNew Jennerian VaccinesBovine parainfluenza Type 3Bovine virus is: Infectious to humans Immunogenic (61% of children get good response) Poorly transmissablePhenotypicaly stable50編輯版pptNew MethodsNew Jennerian VacciNew MethodsSecond Generation Jennerian VaccinesRotav

39、irus11 segments of double strand RNATwo encode: VP4 (hemagglutinin) VP7 (glycoprotein)Co-infect tissue culture cells reassortment10 segments from monkey rotavirus 1 segment outer capsid protein of each of four major rotavirus strainsEfficacy 80%Elicit neutralizing antibodies51編輯版pptNew MethodsSecond

40、 Generation JVaccines 1796 Jenner: wild type animal-adapted virus 1800s Pasteur: Attenuated virus 1996 DNA vaccinesThe third vaccine revolution52編輯版pptVaccines 1796 Jenner: wild tDNA vaccinesDNA vaccines are at present experimental , but hold promise for future therapy since they evoke both humoral

41、and cell-mediated immunity, without the dangers associated with live virus vaccines53編輯版pptDNA vaccinesDNA vaccines are aDNA VaccinesplasmidMuscle cellGene for antigenMuscle cell expresses protein - antibody madeCTL response54編輯版pptDNA VaccinesplasmidMuscle cellDNA Vaccines Plasmids are easily manuf

42、actured 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 the plasmid in the vaccinee to code for antigen

43、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 and using it as an immunogen.55編輯版pptDNA Vaccines P

44、lasmids are easiDNA 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 protein component so there will be no immune response

45、 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 intracellular pathogens (e.g. Mycobacterium tuberculosis)56編輯版pp

46、tDNA Vaccines Mixtures of plasmDNA VaccinesPossible 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 tolerance (e.g. where the expression of the antigen in th

47、e host may lead to specific non-responsiveness to that antigen)57編輯版pptDNA VaccinesPossible Problems5DNA VaccinesDNA vaccines produce a situation that reproduces a virally-infected cellGives: Broad based immune response Long lasting CTL response Advantage of new DNA vaccine for flu:CTL response can

48、be against internal proteinIn mice a nucleoprotein DNA vaccine is effective against a range of viruses with different hemagglutinins58編輯版pptDNA VaccinesDNA vaccines produAdjuvantsCertain substances, when administered simultaneously with a specific antigen, will enhance the immune response to that an

49、tigen. 59編輯版pptAdjuvantsCertain substances, wAdjuvants in common useAluminium saltsLiposomes and immunostimulating complexesComplet Freunds adjuvant is an emulsion of mycobacteria, oil and waterIncomplete Freunds adjuvantMuramyl di-peptideCytokines60編輯版pptAdjuvants in common useAluminiPossible actio

50、n 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 recruitment of antigen-specific T and B cells to the site of inoculation61編輯版pptPossi

51、ble action modes of adjuSmallpox62編輯版pptSmallpox62編輯版pptSmallpox Variolation1% v. 25% mortalityLife-long immunity No drift or shift63編輯版pptSmallpox Variolation63編輯版pptSmallpoxVaccination Jenner 1796 : Cowpox/Swinepox 1800s Compulsory childhood vaccination 1930s Last natural UK case 1940s last natura

52、l US case 1958 WHO program October 1977: Last case (Somalia)64編輯版pptSmallpoxVaccination64編輯版pptSmallpox No animal reservoir Lifelong immunity Subclinical cases rare Infectivity does not precede overt symptoms One Variola serotype Effective vaccine Major commitment by governments65編輯版pptSmallpox No a

53、nimal reservoir65 polioKilled virus vaccine(Salk, 1954)Live attenuated oral polio vaccine( Sabin, 1957)The inactivated Salk vaccines is recommended for children who are immunosuppressed. 66編輯版ppt polioKilled virus vaccine(SalSmall RNA virus Some driftbut not too far as non-viableSabin attenuated vac

54、cine 10 cases vaccine-associated disease per year 50% vaccinees feces 50% contacts Vaccine-associated cases: revertants 1 in 4,000,000 vaccine infections paralytic polio 1 in 100 of wt infectionsScandinavia: Salk dead vaccine No gut immunity Cannot wipe out wt virusPolio Vaccine67編輯版pptSmall RNA vir

55、us SomeReported cases per 100000 population1001010.10.0010.0119501960197019801990Inactivated (Salk) vaccineOral vaccineCases per 100,000 population United States68編輯版pptReported cases per 100000 popu1000010001001010Reported cases195019551960196519701975Killed (Salk) vaccineTotal casesSweden and Finl

56、and69編輯版ppt1000010001001010Reported casesReciprocal virus antibody titer51212832821Serum IgGSerum IgGSerum IgMSerum IgMNasal and duodenal IgANasal IgASerum IgASerum IgADuodenal IgADaysVaccinationVaccination48489696Killed (Salk) VaccineLive (Sabin) Vaccine70編輯版pptReciprocal virus antibody titeSabin P

57、olio VaccineAttenuation by passage in foreign hostMore suited to foreign environment and less suited to original hostGrows less well in original hostPolio: Monkey kidney cells Grows in epithelial cells Does not grow in nerves No paralysis Local gut immunity (IgA)Pasteur rabies vaccine also attenuate

58、d71編輯版pptSabin Polio VaccineAttenuationSalk Polio Vaccine Formaldehyde-fixed No reversion72編輯版pptSalk Polio Vaccine FormaldehydPolio VaccineWhy use the Sabin vaccine?: Local immunity: Vaccine virus just like natural infection Stopping replication in G.I. Tract stops viral replication TOTALLY Dead Sa

59、lk vaccine virus has no effect on gut replication No problem with selective inactivation Greater cross reaction as vaccine virus also has antigenic drift Life-long immunity73編輯版pptPolio VaccineWhy use the Sabin MeaslesLive attenuated virus grown in chick embryo fibroblasts, first introduced in the 1

60、960s.Etiology: Measles virus Incubation: 8 to 12 days Clinical Manifestations: cough, coryza, conjunctivitis , erythematous maculopapular rash fever ,Koplik Spots ,complictions include Encephalitis, Pneumonia, and SSPE Treatment: Supportive 74編輯版ppt MeaslesLive attenuated virus MumpsLive attenuated