病毒感染的檢查方法與防治原則e

1、1,Laboratory Diagnosis,,2,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、.,3,Laboratory Diagnosis,Microscopy IdentificationVirus isolation and identificationDetection of viral proteins( antigens and enzymes)]Detection of viral genetic material Serologic procedures,4,Microscopy Identificat

3、ion,Light microscopyFluorescent microscopyElectron microscopy,5,Light microscopy,Characteristic CPE Inclusion Bodies,6,,Cell deathCell roundingDegenerationAggregationLoss of attachments to substrateCharac

4、teristic histological changes:inclusion bodies in the nucleus or cytoplasm, margination of chromatinSyncytia: multinucleated giant cells caused by virus-induced cell-cell fusion,7,Fluorescent microscopy,Fluorescent-anti

5、body staining,8,Electron microscopy,Direct detection : Human rotavirus; HAV; HBV; Smallpox virus; Herpes virus.Immune Electron microscopy: Human rotavirus; HAV;,9,Laboratory Diagnosis,Microscopy IdentificationVirus is

6、olation and identificationDetection of viral proteins( antigens and enzymes)]Detection of viral genetic material Serologic procedures,10,Viral isolation and Identification,Viral Growth and Cell culture Viral Detect

7、ion Viral Identification Interpretation of culture results,11,Systems for the Propagation of Viruses,PeopleAnimals: cows, chickens, mice,rats, suckling mice Embryonated eggs Organ and tissue cultureOrgan cultur

8、ePrimary tissue cultureCell lines: diploidTumor or （immortalized ）cell line,12,Viral detection,CPEHemadsorptionInterfereMetabolize of cell,13,TCID50(Tissue culture infective dose),TCID50 is defined as that

9、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),14,Vir

10、al identification,Complement fixation： Hemagglutination inhibitionNeutralizationImmunofluorescence ( direct or indirect)Latex agglutination In situ EIA ELISARIA(radioimmuno,15,Laboratory Diagnosis,Microscopy Ident

11、ificationVirus isolation and identificationDetection of viral proteins( antigens and enzymes)Detection of viral genetic material Serologic procedures,16,Detection of viral proteins( antigens and enzymes),Antigen det

12、ection ( ELISA, RIA, Western blot)Hemagglutination and hemadsorptionEnzyme activities( reverse transcriptase)Protein patterns( electrophoresis ),17,Laboratory Diagnosis,Microscopy IdentificationVirus isolation and id

13、entificationDetection of viral proteins( antigens and enzymes)]Detection of viral genetic material Serologic procedures,18,Detection of viral genetic material,PCR ( Polymerase chain reaction)RT-PCR (Reverse transcri

14、ptase 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 end

15、onuclease cleavage patterns,19,Laboratory Diagnosis,Microscopy IdentificationVirus isolation and identificationDetection of viral proteins( antigens and enzymes)]Detection of viral genetic material Serologic procedur

16、es,20,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

17、diseases, the presence of IgM antibody is used to diagnose current infectionOther nonspecific serologic tests are available,21,Serologic procedures,Complement fixation： Hemagglutination inhibitionNeutralizationImmuno

18、fluorescence ( direct or indirect)Latex agglutination In situ EIA ELISARIA,22,Viruses Diagnosed by Serology,Epstein-Barr virusRubella virusHepatitis A, B, C, D, and E virusesHIVHuman T-cell Leukemia virusArbovi

19、ruses ( Encephalitis viruses),23,Prevention,Successes of the Past Possibilities for the Future,24,Active immunization,Vaccines,25,Overview of Active immunization,Active immunization - administration of antigen resulting

20、 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 – Vaccinatio

21、n(vaccines),26,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 disease Stable ret

22、ain immunogenicity, despite adverse storage conditions prior to administrationIn-expensive,27,LIVE VACCINES,Live attenuated organismHeterologous vaccinesLive recombinant vaccines Attributes – live vaccines,28,L

23、ive attenuated organism,Organisms whose virulence has been artificially reduced by in vitro Culture under adverse conditions, such as reduced temperature.,29,Heterologous vaccines,Closely related organism of lesser virul

24、ence, 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 viru

25、s--- Variola virus,30,Live recombinant,Vector bovine vaccineBCG,31,Both cell mediated immunity and antibody responseActivates all phases of immune system. Can get humoral IgG and local IgARaises immune response to al

26、l protective antigens. Inactivation may alter antigenicity. More durable immunity; more cross-reactive Immunity is long livedSingle dose,Advantages of Attenuated Vaccines 2-1,32,Advantages of Attenuated Vaccines 2-2,L

27、ow 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 community,33,Disadvantages of L

28、ive 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%) S

29、pread vaccine not standardized--may be back-mutated Poor "take" in tropics Problem in immunodeficiency disease (may spread to these patients),34,Killed vaccines,The organism is propagated in bulk, in vitro, a

30、nd 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 in

31、duce immunity. In addition, they are usually expensive to prepare.,35,Killed vaccines,Inactivated organism: rabies virus; epidmic type B encephalitis virus.Subunit Vaccines: Influenza virus( HA and NA)Recombinant prote

32、ins: HBV,36,Advantages of inactivated vaccines,Gives 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 a

33、dverse storage conditions,Sometimes better in tropics,37,Disadvantages of inactivated vaccines,Many vaccinees do not raise immunitypoor, only antibody, no cell immediated immune responseresponse is short-lived and mult

34、iple 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,38,New

35、 Methods,Selection 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 tempera

36、ture-sensitive virus. Grows in upper respiratory tract but not lower ‘flu (new vaccine) respiratory syncytial virus,39,New Methods,Passage progressively at cold temperaturesTS mutant in internal proteinsCan be re-ass

37、orted to so that coat is the strain that is this years flu strain,40,41,New Methods,Deletion mutants Suppression unlikely (but caution in HIV) Viable but growth restrictionsProblems Oncogenicity in some cases (adeno,

38、 retro),42,New Methods,43,Single gene (subunit) - problems,Surface glycoprotein poorly soluble - deletion? Poorly immunogenic Post-translational modifications Poor CTL response,44,Single gene (subunit) in expression v

39、ector,Vaccinate with live virusCanary Pox Infects human cells but does not replicate Better presentation CTL responseVacciniaAttenuated PolioBeing developed for anti-HIV vaccine,45,New Methods,Chemically synthesiz

40、ed peptide malariapoorly immunogenic,46,New methods,Anti-idiotype vaccine,,epitope,,Antibody with epitope binding site,Virus,47,Anti-idiotype vaccine cont,Make antibody against antibody idiotype,Anti-idiotype antibody

41、mimics the epitope,,48,Anti-idiotype antibody cont 2,Use anti-idiotype antibody as injectable vaccine,Antibody to anti-idiotype antibody,Binds and neutralizes virus,,,Use as vaccine,49,New Methods,New “Jennerian Vaccine

42、s” 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

43、al serotypes,50,New Methods,New Jennerian VaccinesBovine parainfluenza Type 3Bovine virus is: Infectious to humans Immunogenic (61% of children get good response) Poorly transmissablePhenotypicaly stable,51,New Met

44、hods,52,Vaccines,1796 Jenner: wild type animal-adapted virus 1800’s Pasteur: Attenuated virus 1996 DNA vaccinesThe third vaccine revolution,53,DNA vaccines,DNA vaccines are at present experimental , but hold promi

45、se for future therapy since they evoke both humoral and cell-mediated immunity, without the dangers associated with live virus vaccines,54,DNA Vaccines,,,,,plasmid,,,Muscle cell,,,Gene for antigen,Muscle cell expresses p

46、rotein - antibody madeCTL response,55,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

47、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 p

48、rocessed (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.,

49、56,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

50、 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 pat

51、hogen. A CTL response also offers protection against diseases caused by certain obligate intracellular pathogens (e.g. Mycobacterium tuberculosis),57,DNA Vaccines,Possible Problems Potential integration of plasmid into

52、 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 lea

53、d to specific non-responsiveness to that antigen),58,DNA Vaccines,DNA vaccines produce a situation that reproduces a virally-infected cellGives: Broad based immune response Long lasting CTL response Advantage of new

54、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,59,Adjuvants,Certain substances, when administer

55、ed simultaneously with a specific antigen, will enhance the immune response to that antigen.,60,Adjuvants in common use,Aluminium saltsLiposomes and immunostimulating complexesComplet Freund’s adjuvant is an emulsion o

56、f mycobacteria, oil and waterIncomplete Freund’s adjuvantMuramyl di-peptideCytokines,61,Possible action modes of adjuvant,By trapping antigen in the tissues, thus allowing maximal exposure to dendritic cells and spec

57、ific 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 inoculation,62,Smallpox,63,Smallpox,Variolation1% v. 25

58、% mortalityLife-long immunity No drift or shift,64,Smallpox,Vaccination Jenner 1796 : Cowpox/Swinepox 1800’s Compulsory childhood vaccination 1930’s Last natural UK case 1940’s last natural US case 1958 WHO progra

59、m October 1977: Last case (Somalia),65,Smallpox,No animal reservoir Lifelong immunity Subclinical cases rare Infectivity does not precede overt symptoms One Variola serotype Effective vaccine Major commitme

60、nt by governments,66,polio,Killed virus vaccine(Salk, 1954)Live attenuated oral polio vaccine( Sabin, 1957)The inactivated Salk vaccines is recommended for children who are immunosuppressed.,67,Polio Vaccine,68,,,,,,,,

61、,Reported cases per 100000 population,,100,10,1,0.1,0.001,0.01,,1950,1960,1970,1980,1990,,,,Inactivated (Salk) vaccine,,Oral vaccine,Cases per 100,000 population United States,69,,,,,,10000,1000,100,10,1,0,,,Reported cas

62、es,1950,1955,1960,1965,1970,1975,,,,,,,,,,,,,,,,,,,,Killed (Salk) vaccine,Total casesSweden and Finland,70,71,Sabin Polio Vaccine,Attenuation by passage in foreign hostMore suited to foreign environment and less suited

63、 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 attenuated,72,Salk P

64、olio Vaccine,Formaldehyde-fixed No reversion,73,Polio Vaccine,Why use the Sabin vaccine?: Local immunity: Vaccine virus just like natural infection Stopping replication in G.I. Tract stops viral replication TOTALLY D

65、ead 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 immunity,74,Measles,Live attenuated virus grown

66、 in chick embryo fibroblasts, first introduced in the 1960’s.Etiology: Measles virus Incubation: 8 to 12 days Clinical Manifestations: cough, coryza, conjunctivitis , erythematous maculopapular rash fever ,Koplik Spo

67、ts ,complictions include Encephalitis, Pneumonia, and SSPE Treatment: Supportive,75,Mumps,Live attenuated virus developed in the 1960’sMMR vaccineEtiology: Mumps Virus Incubation: 16 to 18 days Clinical Manifestatio

68、ns: swelling of the salivary glands complications include Meningitis, Orchitis, Encephalitis, and Deafness,76,rubella,Live attenuated virusEtiology: Rubella Virus Incubation: 14 to 21 days Clinical Manifestations: C