资源描述
Laboratory Diagnosis 1编辑版ppt Laboratory Diagnosis 1编辑版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编辑版pptCategory of SampleBlood,UrineLaboratory Diagnosis Microscopy IdentificationVirus isolation and identificationDetection of viral proteins(antigens and enzymes)Detection of viral genetic material Serologic procedures3编辑版pptLaboratory Diagnosis MicroscopMicroscopy IdentificationLight microscopyFluorescent microscopyElectron microscopy 4编辑版pptMicroscopy IdentificationLightLight microscopyCharacteristic CPE Inclusion Bodies 5编辑版pptLight microscopyCharacteristic Cell deathCell roundingDegenerationAggregationLoss of attachments to substrateCharacteristic histological changes:inclusion bodies in the nucleus or cytoplasm,margination of chromatinSyncytia:multinucleated giant cells caused by virus-induced cell-cell fusion6编辑版ppt Cell death6编辑版pptFluorescent microscopyFluorescent-antibody staining7编辑版pptFluorescent microscopyFluorescElectron microscopyDirect detection:Human rotavirus;HAV;HBV;Smallpox virus;Herpes virus.Immune Electron microscopy:Human rotavirus;HAV;8编辑版pptElectron microscopyDirect deteLaboratory Diagnosis Microscopy IdentificationVirus isolation and identificationDetection of viral proteins(antigens and enzymes)Detection of viral genetic material Serologic procedures9编辑版pptLaboratory Diagnosis MicroscopViral isolation and Identification Viral Growth and Cell culture Viral Detection Viral Identification Interpretation of culture results 10编辑版pptViral isolation and IdentificaSystems for the Propagation of VirusesPeopleAnimals:cows,chickens,mice,rats,suckling mice Embryonated eggs Organ and tissue cultureOrgan culturePrimary tissue cultureCell lines:diploidTumor or （immortalized）cell line11编辑版pptSystems for the Propagation ofViral detectionCPEHemadsorptionInterfereMetabolize of cell12编辑版pptViral detectionCPE12编辑版pptTCID50(Tissue culture infective 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pptTCID50(Tissue culture infectiViral identificationComplement fixation：Hemagglutination inhibitionNeutralizationImmunofluorescence(direct or indirect)Latex agglutination In situ EIA ELISARIA(radioimmuno14编辑版pptViral identificationComplementLaboratory Diagnosis Microscopy IdentificationVirus isolation and identificationDetection of viral proteins(antigens and enzymes)Detection of viral genetic material Serologic procedures15编辑版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编辑版pptDetection of viral proteins(Laboratory Diagnosis Microscopy IdentificationVirus isolation and identificationDetection of viral proteins(antigens and enzymes)Detection of viral genetic material Serologic procedures17编辑版pptLaboratory Diagnosis MicroscopDetection of viral genetic materialPCR(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)Restriction endonuclease cleavage patterns18编辑版pptDetection of viral genetic matLaboratory Diagnosis Microscopy IdentificationVirus isolation and identificationDetection of viral proteins(antigens and enzymes)Detection of viral genetic material Serologic procedures19编辑版pptLaboratory Diagnosis MicroscopSerologic proceduresIf 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 infectionOther nonspecific serologic tests are available20编辑版pptSerologic proceduresIf the antSerologic proceduresComplement fixation：Hemagglutination inhibitionNeutralizationImmunofluorescence(direct or indirect)Latex agglutination In situ EIA ELISARIA21编辑版pptSerologic proceduresComplementViruses Diagnosed by SerologyEpstein-Barr virusRubella virusHepatitis A,B,C,D,and E virusesHIVHuman T-cell Leukemia virusArboviruses(Encephalitis viruses)22编辑版pptViruses Diagnosed by SerologyEPreventionPrevention Successes of the Past Possibilities for the Future23编辑版pptPrevention23编辑版pptActive immunizationVaccines24编辑版pptActive immunizationVaccines24编Overview of Active immunizationActive 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 caught and successfully fought Artificial immunity Vaccination(vaccines)25编辑版pptOverview of Active immunizatioAttributes 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编辑版pptAttributes of a good vaccineAbLIVE VACCINESLive attenuated organismHeterologous vaccinesLive recombinant vaccines Attributes live vaccines27编辑版pptLIVE VACCINESLive attenuated oLive attenuated organismOrganisms whose virulence has been artificially reduced by in vitro Culture under adverse conditions,such as reduced temperature.28编辑版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 antigens of the virulent organism.Vaccinia virus/cowpox virus-Variola virus29编辑版pptHeterologous vaccinesClosely rLive recombinantVector 1.bovine vaccine2.BCG30编辑版pptLive recombinantVector 30编辑版ppBoth 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 dose Advantages of Attenuated Vaccines 2-131编辑版pptBoth cell mediated immunity anAdvantages 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 community32编辑版pptAdvantages of Attenuated VacciDisadvantages 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 vaccine not standardized-may be back-mutated Poor take in tropics Problem in immunodeficiency disease(may spread to these patients)33编辑版pptDisadvantages 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 needed to induce immunity.In addition,they are usually expensive to prepare.34编辑版pptKilled vaccinesThe organism isKilled vaccinesInactivated organism:rabies virus;epidmic type B encephalitis virus.Subunit Vaccines:Influenza virus(HA and NA)Recombinant proteins:HBV35编辑版pptKilled vaccinesInactivated 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编辑版pptAdvantages of inactivated vaccDisadvantages 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 inactivation and immunization with virulent virusExpense:Expensive to prepare37编辑版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 degrees.Selects for temperature-sensitive virus.Grows in upper respiratory tract but not lower flu(new vaccine)respiratory syncytial virus 38编辑版pptNew MethodsSelection of attenuNew MethodsPassage progressively at cold temperaturesTS mutant in internal proteinsCan be re-assorted to so that coat is the strain that is this years flu strain39编辑版pptNew MethodsPassage progressivePB2PB1PAHANANPMNSPB2PB1PAHANANPMNSPB2PB1PAHANANPMNSAttenuated Donor Master StrainNew Virulent Antigenic Variant StrainXAttenuated Vaccine Strain:Coat of Virulent strain with Virulence Characteristics of Attenuated Strain40编辑版pptPB2PB1PAHANANPMNSPB2PB1PAHANANNew Methods Deletion mutants Suppression unlikely(but caution in HIV)Viable but growth restrictionsProblems Oncogenicity in some cases(adeno,retro)41编辑版pptNew Methods Deletion mutants41New Methods Recombinant DNASingle gene(subunit)S-antigen mRNAcDNAExpress plasmidS-antigen mRNA proteinHepatitis B vaccineraised in yeast42编辑版pptNew Methods Recombinant DNAS-aSingle gene(subunit)-problems Surface glycoprotein poorly soluble-deletion?Poorly immunogenic Post-translational modifications Poor CTL response43编辑版pptSingle gene(subunit)-probleSingle gene(subunit)in expression vectorVaccinate with live virusCanary Pox Infects human cells but does not replicate Better presentation CTL responseVacciniaAttenuated PolioBeing developed for anti-HIV vaccine44编辑版pptSingle gene(subunit)in expreNew MethodsChemically synthesized peptide malariapoorly immunogenic45编辑版pptNew MethodsChemically synthesiantibodyNew methodsAnti-idiotype vaccineepitopeAntibody with epitope binding siteVirus46编辑版pptantibodyNew methodsAnti-idiotyantibodyAnti-idiotype vaccine contMake antibody against antibody idiotypeAnti-idiotypeantibodyAnti-idiotype antibody mimics the epitope47编辑版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编辑版pptAnti-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编辑版pptNew MethodsNew“Jennerian VaccNew MethodsNew Jennerian VaccinesBovine parainfluenza Type 3Bovine virus is:Infectious to humans Immunogenic(61%of children get good response)Poorly transmissablePhenotypicaly stable50编辑版pptNew MethodsNew Jennerian VacciNew MethodsSecond Generation Jennerian VaccinesRotavirus11 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编辑版pptNew MethodsSecond Generation JVaccines 1796 Jenner:wild type animal-adapted virus 1800s Pasteur:Attenuated virus 1996 DNA vaccinesThe third vaccine revolution52编辑版pptVaccines 1796 Jenner:wild tDNA vaccinesDNA vaccines are at present experimental,but hold promise for future therapy since they evoke both humoral and cell-mediated immunity,without the dangers associated with live virus vaccines53编辑版pptDNA vaccinesDNA vaccines are aDNA VaccinesplasmidMuscle cellGene for antigenMuscle cell expresses protein-antibody madeCTL response54编辑版pptDNA VaccinesplasmidMuscle cellDNA 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 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 and using it as an immunogen.55编辑版pptDNA Vaccines Plasmids 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 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编辑版pptDNA 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 the host may lead to specific non-responsiveness to that antigen)57编辑版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 be against internal proteinIn mice a nucleoprotein DNA vaccine is effective against a range of viruses with different hemagglutinins58编辑版pptDNA VaccinesDNA vaccines produAdjuvantsCertain substances,when administered simultaneously with a specific antigen,will enhance the immune response to that antigen.59编辑版pptAdjuvantsCertain substances,wAdjuvants in common useAluminium saltsLiposomes and immunostimulating complexesComplet Freunds adjuvant is an emulsion of mycobacteria,oil and waterIncomplete Freunds adjuvantMuramyl di-peptideCytokines60编辑版pptAdjuvants in common useAluminiPossible 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 recruitment of antigen-specific T and B cells to the site of inoculation61编辑版pptPossible action modes of adjuSmallpox62编辑版pptSmallpox62编辑版pptSmallpox Variolation1%v.25%mortalityLife-long immunity No drift or shift63编辑版pptSmallpox Variolation63编辑版pptSmallpoxVaccination Jenner 1796:Cowpox/Swinepox 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pptSmallpoxVaccination64编辑版pptSmallpox No animal reservoir Lifelong immunity Subclinical cases rare Infectivity does not precede overt symptoms One Variola serotype Effective vaccine Major commitment by governments65编辑版pptSmallpox No animal 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编辑版ppt polioKilled virus vaccine(SalSmall RNA virus Some driftbut not too far as non-viableSabin attenuated vaccine 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编辑版pptSmall RNA virus SomeReported cases per 100000 population1001010.10.0010.0119501960197019801990Inactivated(Salk)vaccineOral vaccineCases per 100,000 population United States68编辑版pptReported cases per 100000 popu1000010001001010Reported cases195019551960196519701975Killed(Salk)vaccineTotal casesSweden and Finland69编辑版ppt1000010001001010Reported casesReciprocal virus antibody titer51212832821Serum IgGSerum IgGSerum IgMSerum IgMNasal and duodenal IgANasal IgASerum IgASerum IgADuodenal IgADaysVaccinationVaccination48489696Killed(Salk)VaccineLive(Sabin)Vaccine70编辑版pptReciprocal virus antibody titeSabin Polio 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 attenuated71编辑版pptSabin Polio VaccineAttenuationSalk Polio Vaccine Formaldehyde-fixed No reversion72编辑版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 Salk 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编辑版pptPolio VaccineWhy use the Sabin MeaslesLive attenuated virus grown in chick embryo fibroblasts,first introduced in the 1960s.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编辑版ppt MeaslesLive attenuated virus MumpsLive attenuated virus developed in the 1960sMMR vaccineEtiology:Mumps Virus Incubation:16 to 18 days Clinical Manifestations:swelling of the salivary glands complications include Meningitis,Orchitis,Encephalitis,and Deafness 75编辑版ppt MumpsLive attenuated virus de rubellaLive attenuated virusEtiology:Rubella Virus Incubation:14 to 21 days Clinical Manifestations:Congenital,cataracts patent ductus arteriosus,deafness mental retardation,Postnatal mild disease,erythematous maculopapular rash,postauricular lymphadenopathy transient polyarthralgias 76编辑版ppt rubellaLive attenuated virus Hepatitis BTwo vaccines are in current use:A serum derived vaccineA recombinant vaccineEtiology:Hepatitis B Virus Incubation:120 days(average)Clinical Manifestations:jaundice;anorexia nausea and vomiting;malaise complications include the development of a chronic carrier state with a high risk for Hepatocellular Carcinoma(liver cancer)77编辑版ppt Hepatitis BTwo vaccines are i Hepatitis AFormalin-inactivated,cell cultured-derived virus,78编辑版ppt Hepatitis AFormalin-inactivat Yellow feverThe 17D strain is a live attenuated vaccine developed in 1937.It is a highly effective vaccine which is administered to residents in the tropics and travellers to endemic areas.79编辑版ppt Yellow feverThe 17D strain is RabiesNo safe attenuated strain of rabies virus has yet been developed for human.Vaccines in current use include:a The neurotissue vaccine b human diploid cell culture-derived vaccine,which is much safer.There are two situation where vaccine is given:a Post-exposure prophylaxis,followinf the bite of a rabid animal,Hyperimmune rabies globulin may also administered.b Pro-exposure prophylaxis is used for protection of those occupation puts them at risk of infection with rabies.80编辑版ppt RabiesNo safe attenuated stra InfluenzaNew vaccines are produced every year81编辑版ppt InfluenzaNew vaccines are pro Varicella-Zoster virusNot licensed vaccines82编辑版ppt Varicella-Zoster virusNot li83编辑版ppt83编辑版pptPassive Immunisation84编辑版pptPassive Immunisation84编辑版pptModes of immunizationPassive immunization-administration of antibody-containing serum to provide immediate,but temporary protection.Doesnt activate a lasting specific immune respo
展开阅读全文