1、 Laboratory Diagnosis 1编辑版pptCategory 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pptLaboratory Diagnosis Microscopy Identification Virus isolation and identification Dete
2、ction of viral proteins(antigens and enzymes)Detection of viral genetic material Serologic procedures3编辑版pptMicroscopy Identification Light microscopy Fluorescent microscopy Electron microscopy 4编辑版pptLight microscopy Characteristic CPE Inclusion Bodies 5编辑版ppt Cell deathCell roundingDegenerationAgg
3、regationLoss of attachments to substrate Characteristic histological changes:inclusion bodies in the nucleus or cytoplasm,margination of chromatin Syncytia:multinucleated giant cells caused by virus-induced cell-cell fusion6编辑版pptFluorescent microscopy Fluorescent-antibody staining7编辑版pptElectron mi
4、croscopy Direct detection:Human rotavirus;HAV;HBV;Smallpox virus;Herpes virus.Immune Electron microscopy:Human rotavirus;HAV;8编辑版pptLaboratory Diagnosis Microscopy Identification Virus isolation and identification Detection of viral proteins(antigens and enzymes)Detection of viral genetic material S
5、erologic procedures9编辑版pptViral isolation and Identification Viral Growth and Cell culture Viral Detection Viral Identification Interpretation of culture results 10编辑版pptSystems for the Propagation of Viruses People Animals:cows,chickens,mice,rats,suckling mice Embryonated eggs Organ and tissue cult
6、ureOrgan culturePrimary tissue cultureCell lines:diploidTumor or （immortalized）cell line11编辑版pptViral detection CPE Hemadsorption Interfere Metabolize of cell12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 c
7、ulture TCID50=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pptViral identification Complement fixation：Hemagglutination inhibition Neutralization Immunofluorescence(direct or indirect)Latex agglutination In situ EIA ELI
8、SA RIA(radioimmuno14编辑版pptLaboratory Diagnosis Microscopy Identification Virus isolation and identification Detection of viral proteins(antigens and enzymes)Detection of viral genetic material Serologic procedures15编辑版pptDetection of viral proteins(antigens and enzymes)Antigen detection(ELISA,RIA,We
9、stern blot)Hemagglutination and hemadsorption Enzyme activities(reverse transcriptase)Protein patterns(electrophoresis)16编辑版pptLaboratory Diagnosis Microscopy Identification Virus isolation and identification Detection of viral proteins(antigens and enzymes)Detection of viral genetic material Serolo
10、gic procedures17编辑版pptDetection 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(Electrop
11、horesis)Restriction endonuclease cleavage patterns18编辑版pptLaboratory Diagnosis Microscopy Identification Virus isolation and identification Detection of viral proteins(antigens and enzymes)Detection of viral genetic material Serologic procedures19编辑版pptSerologic procedures If the antibody titer in t
12、he 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 infection Other nonspecific serologic tests are available20编辑版pptSero
13、logic procedures Complement fixation：Hemagglutination inhibition Neutralization Immunofluorescence(direct or indirect)Latex agglutination In situ EIA ELISA RIA21编辑版pptViruses Diagnosed by Serology Epstein-Barr virus Rubella virus Hepatitis A,B,C,D,and E viruses HIV Human T-cell Leukemia virus Arbovi
14、ruses(Encephalitis viruses)22编辑版pptPrevention Successes of the Past Possibilities for the Future23编辑版pptActive immunizationVaccines24编辑版pptOverview of Active immunizationActive immunization-administration of antigen resulting in production of a specific immune response with immunologic memory.Respon
15、se 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pptAttributes of a good vaccine Ability to elicit the appropriate immune response for the particular pathogen Long term protection ideally life-lon
16、g Safety vaccine itself should not cause disease Stable retain immunogenicity,despite adverse storage conditions prior to administration In-expensive26编辑版pptLIVE VACCINES Live attenuated organism Heterologous vaccines Live recombinant vaccines Attributes live vaccines27编辑版pptLive attenuated organism
17、 Organisms whose virulence has been artificially reduced by in vitro Culture under adverse conditions,such as reduced temperature.28编辑版pptHeterologous vaccines Closely related organism of lesser virulence,which shares many antigens with the virulent organism.The vaccine strain replication in the hos
18、t and induces an immune response that cross reacts with antigens of the virulent organism.Vaccinia virus/cowpox virus-Variola virus29编辑版pptLive recombinantVector 1.bovine vaccine2.BCG30编辑版pptBoth cell mediated immunity and antibody responseActivates all phases of immune system.Can get humoral IgG an
19、d 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pptAdvantages of Attenuated Vaccines 2-2 Low cost Quick immunity in majority of vacci
20、nees 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pptDisadvantages of Live Attenuated Vaccine Mutation;reversion to virulence(often frequent)Spread to contacts of vaccinee who have not consented to b
21、e 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pptKilled vaccines The organism is propagated in bulk,in vitro,and inact
22、ivated 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 vaccines Ina
23、ctivated organism:rabies virus;epidmic type B encephalitis virus.Subunit Vaccines:Influenza virus(HA and NA)Recombinant proteins:HBV35编辑版pptAdvantages of inactivated vaccines Gives sufficient humoral immunity if boosters given No mutation or reversion Can be used with immuno-deficient patients These
24、 vaccines tend to be able to withstand more adverse storage conditions,Sometimes better in tropics36编辑版pptDisadvantages of inactivated vaccines Many vaccinees do not raise immunity poor,only antibody,no cell immediated immune response response is short-lived and multiple doses are needed No local im
25、munity(important)Inactivated,therefore can not replicate in the host and cause disease Failure in inactivation and immunization with virulent virus Expense:Expensive to prepare37编辑版pptNew MethodsSelection of attenuated virus strain Varicella Hepatitis AUse monoclonal antibodies to select for virus w
26、ith 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 MethodsPassage progressively at cold temperaturesTS mutant in internal protei
27、nsCan be re-assorted to so that coat is the strain that is this years flu strain39编辑版pptPB2PB1PAHANANPMNSPB2PB1PAHANANPMNSPB2PB1PAHANANPMNSAttenuated Donor Master StrainNew Virulent Antigenic Variant StrainXAttenuated Vaccine Strain:Coat of Virulent strain with Virulence Characteristics of Attenuate
28、d Strain40编辑版pptNew 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 Recombinant DNASingle gene(subunit)S-antigen mRNAcDNAExpress plasmidS-antigen mRNA proteinHepatitis B vaccineraised i
29、n yeast42编辑版pptSingle gene(subunit)-problems Surface glycoprotein poorly soluble-deletion?Poorly immunogenic Post-translational modifications Poor CTL response43编辑版pptSingle gene(subunit)in expression vectorVaccinate with live virusCanary Pox Infects human cells but does not replicate Better present
30、ation CTL responseVacciniaAttenuated PolioBeing developed for anti-HIV vaccine44编辑版pptNew MethodsChemically synthesized peptide malariapoorly immunogenic45编辑版pptantibodyNew methodsAnti-idiotype vaccineepitopeAntibody with epitope binding siteVirus46编辑版pptantibodyAnti-idiotype vaccine contMake antibo
31、dy against antibody idiotypeAnti-idiotypeantibodyAnti-idiotype antibody mimics the epitope47编辑版pptAnti-anti-idiotypeantibodyAnti-idiotype antibody cont 2Use anti-idiotype antibody as injectable vaccineAntibody to anti-idiotype antibodyBinds and neutralizes virusAnti-idiotypeantibodyAnti-anti-idiotyp
32、eantibodyAnti-anti-idiotypeantibodyUse as vaccine48编辑版pptNew 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 vir
33、al serotypes49编辑版pptNew 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 MethodsSecond Generation Jennerian VaccinesRotavirus11 segments of double strand RNATw
34、o 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pptVaccines 1796 Jenner:wild type animal-adapted virus 1800s P
35、asteur:Attenuated virus 1996 DNA vaccinesThe third vaccine revolution52编辑版pptDNA vaccines DNA 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 Vaccinesp
36、lasmidMuscle cellGene for antigenMuscle cell expresses protein-antibody madeCTL response54编辑版pptDNA 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 l
37、aboratory.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 i
38、s 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 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
39、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 dise
40、ases caused by certain obligate intracellular pathogens(e.g.Mycobacterium tuberculosis)56编辑版pptDNA 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 immunolo
41、gic 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 VaccinesDNA vaccines produce a situation that reproduces a virally-infected cellGives:Broad based immune response Long lasting CTL response Advantage of new DNA vacci
42、ne 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pptAdjuvants Certain substances,when administered simultaneously with a specific antigen,will enhance the immune response to that antig
43、en.59编辑版pptAdjuvants in common use Aluminium salts Liposomes and immunostimulating complexes Complet Freunds adjuvant is an emulsion of mycobacteria,oil and water Incomplete Freunds adjuvant Muramyl di-peptide Cytokines60编辑版pptPossible action modes of adjuvant By trapping antigen in the tissues,thus
44、 allowing maximal exposure to dendritic cells and specific T and B lymphocytes By 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pptSmallpox62编辑版pptSmallpox Variolation1%v.25%mortalityLife-long im
45、munity No drift or shift63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pptSmallpox No animal reservoir Lifelong immunity Subclinical cases rare I
46、nfectivity does not precede overt symptoms One Variola serotype Effective vaccine Major commitment by governments65编辑版ppt polio Killed 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pptSmal
47、l 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
48、immunity Cannot wipe out wt virusPolio Vaccine67编辑版pptReported cases per 100000 population1001010.10.0010.0119501960197019801990Inactivated(Salk)vaccineOral vaccineCases per 100,000 population United States68编辑版ppt1000010001001010Reported cases195019551960196519701975Killed(Salk)vaccineTotal casesSw
49、eden and Finland69编辑版pptReciprocal virus antibody titer51212832821Serum IgGSerum IgGSerum IgMSerum IgMNasal and duodenal IgANasal IgASerum IgASerum IgADuodenal IgADaysVaccinationVaccination48489696Killed(Salk)VaccineLive(Sabin)Vaccine70编辑版pptSabin Polio VaccineAttenuation by passage in foreign hostM
50、ore 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pptSalk Polio Vaccine Formaldehyde-fixed No rev
