病毒感染的检查方法与防治原则e

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. 3 Laboratory Diagnosis Microscopy Identification Virus isolation and identification Detection of viral proteins( antigens and enzymes) Detection of viral genetic material Serologic procedures 4 Microscopy Identification Light microscopy Fluorescent microscopy Electron microscopy 5 Light microscopy Characteristic CPE Inclusion Bodies 6 Cell death Cell rounding Degeneration Aggregation Loss 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 fusion 7 Fluorescent microscopy Fluorescent-antibody staining 8 Electron microscopy Direct detection : Human rotavirus; HAV; HBV; Smallpox virus; Herpes virus. Immune Electron microscopy: Human rotavirus; HAV; 9 Laboratory Diagnosis Microscopy Identification Virus isolation and identification Detection of viral proteins( antigens and enzymes) Detection of viral genetic material Serologic procedures 10 Viral isolation and Identification Viral Growth and Cell culture Viral Detection Viral Identification Interpretation of culture results 11 Systems for the Propagation of Viruses People Animals: cows, chickens, mice,rats, suckling mice Embryonated eggs Organ and tissue culture Organ culture Primary tissue culture Cell lines: diploid Tumor or （immortalized ）cell line 12 Viral detection CPE Hemadsorption Interfere Metabolize of cell 13 TCID50 (Tissue culture infective dose) TCID50 is defined as that dilution of virus which will cause CPE in 50% of a given batch of cell culture TCID50= log10 of highest dilution giving 100%CPE +1/2 (total number of test units showing CPE)/ (number of test units per dilution) 14 Viral identification Complement fixation： Hemagglutination inhibition Neutralization Immunofluorescence ( direct or indirect) Latex agglutination In situ EIA ELISA RIA(radioimmuno 15 Laboratory Diagnosis Microscopy Identification Virus isolation and identification Detection of viral proteins( antigens and enzymes) Detection of viral genetic material Serologic procedures 16 Detection of viral proteins ( antigens and enzymes) Antigen detection ( ELISA, RIA, Western blot) Hemagglutination and hemadsorption Enzyme activities( reverse transcriptase) Protein patterns( electrophoresis ) 17 Laboratory Diagnosis Microscopy Identification Virus isolation and identification Detection 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 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 patterns 19 Laboratory Diagnosis Microscopy Identification Virus isolation and identification Detection of viral proteins( antigens and enzymes) Detection of viral genetic material Serologic procedures 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 diseases, the presence of IgM antibody is used to diagnose current infection Other nonspecific serologic tests are available 21 Serologic procedures Complement fixation： Hemagglutination inhibition Neutralization Immunofluorescence ( direct or indirect) Latex agglutination In situ EIA ELISA RIA 22 Viruses Diagnosed by Serology Epstein-Barr virus Rubella virus Hepatitis A, B, C, D, and E viruses HIV Human T-cell Leukemia virus Arboviruses ( 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 in production of a specific immune response with immunologic memory. Response may be cellular or humoral or both. v Natural immunity - to diseases you have caught and successfully fought v Artificial immunity Vaccination(vaccines) 26 Attributes of a good vaccine Ability to elicit the appropriate immune response for the particular pathogen Long term protection ideally life-long Safety vaccine itself should not cause disease Stable retain immunogenicity, despite adverse storage conditions prior to administration In-expensive 27 LIVE VACCINES Live attenuated organism Heterologous vaccines Live recombinant vaccines Attributes live vaccines 28 Live 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 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 virus 30 Live recombinant Vector 1. bovine vaccine 2. BCG 31 Both cell mediated immunity and antibody response Activates all phases of immune system. Can get humoral IgG and local IgA Raises immune response to all protective antigens. Inactivation may alter antigenicity. More durable immunity; more cross-reactive Immunity is long lived Single dose Advantages of Attenuated Vaccines 2-1 32 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 community 33 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 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, 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. 35 Killed vaccines Inactivated organism: rabies virus; epidmic type B encephalitis virus. Subunit Vaccines: Influenza virus( HA and NA) Recombinant proteins: 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 adverse storage conditions,Sometimes better in tropics 37 Disadvantages 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 immunity (important) Inactivated, therefore can not replicate in the host and cause disease Failure in inactivation and immunization with virulent virus Expense: Expensive to prepare 38 New Methods Selection of attenuated virus strain Varicella Hepatitis A Use monoclonal antibodies to select for virus with altered surface receptor Rabies Reo Use 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 39 New Methods Passage progressively at cold temperatures TS mutant in internal proteins Can be re-assorted to so that coat is the strain that is this years flu strain 40 PB2 PB1 PA HA NA NP M NS PB2 PB1 PA HA NA NP M NS PB2 PB1 PA HA NA NP M NS Attenuated Donor Master Strain New Virulent Antigenic Variant Strain X Attenuated Vaccine Strain: Coat of Virulent strain with Virulence Characteristics of Attenuated Strain 41 New Methods Deletion mutants Suppression unlikely (but caution in HIV) Viable but growth restrictions Problems Oncogenicity in some cases (adeno, retro) 42 New Methods Recombinant DNA Single gene (subunit) S-antigen mRNA cDNA Express plasmid S-antigen mRNA protein Hepatitis B vaccine raised in yeast 43 Single gene (subunit) - problems Surface glycoprotein poorly soluble - deletion? Poorly immunogenic Post-translational modifications Poor CTL response 44 Single gene (subunit) in expression vector Vaccinate with live virus Canary Pox Infects human cells but does not replicate Better presentation CTL response Vaccinia Attenuated Polio Being developed for anti-HIV vaccine 45 New Methods Chemically synthesized peptide malaria poorly immunogenic 46 antibody New methods Anti-idiotype vaccine epitope Antibody with epitope binding site Virus 47 antibody Anti-idiotype vaccine cont Make antibody against antibody idiotype Anti- idiotype antibody Anti-idiotype antibody mimics the epitope 48 Anti-anti-idiotype antibody Anti-idiotype antibody cont 2 Use anti-idiotype antibody as injectable vaccine Antibody to anti-idiotype antibody Binds and neutralizes virus Anti-idiotype antibody Anti-anti-idiotype antibody Anti-anti-idiotype antibody Use as vaccine 49 New Methods New “Jennerian Vaccines” Live vaccines derived from animal strains of similar viruses Naturally attenuated for humans Rotavirus: Monkey Rota 80% effective in some human populations Ineffective in others Due to differences in circulating viral serotypes 50 New Methods New Jennerian Vaccines Bovine parainfluenza Type 3 Bovine virus is: Infectious to humans Immunogenic (61% of children get good response) Poorly transmissable Phenotypicaly stable 51 New Methods Second Generation Jennerian Vaccines Rotavirus 11 segments of double strand RNA Two encode: VP4 (hemagglutinin) VP7 (glycoprotein) Co-infect tissue culture cells reassortment 10 segments from monkey rotavirus 1 segment outer capsid protein of each of four major rotavirus strains Efficacy 80% Elicit neutralizing antibodies 52 Vaccines 1796 Jenner: wild type animal-adapted virus 1800s Pasteur: Attenuated virus 1996 DNA vaccines The third vaccine revolution 53 DNA 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 vaccines 54 DNA Vaccines plasmid Muscle cell Gene for antigen Muscle cell expresses protein - antibody made CTL 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 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. 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 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) 57 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 tolerance (e.g. where the expression of the antigen in the host may lead to specific non-responsiveness to that antigen) 58 DNA Vaccines DNA vaccines produce a situation that reproduces a virally- infected cell Gives: Broad based immune response Long lasting CTL response Advantage of new DNA vaccine for flu: CTL response can be against internal protein In mice a nucleoprotein DNA vaccine is effective against a range of viruses with different hemagglutinins 59 Adjuvants Certain substances, when administered simultaneously with a specific antigen, will enhance the immune response to that antigen. 60 Adjuvants 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 Cytokines 61 Possible action modes of adjuvant By trapping antigen in the tissues, thus 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 inoculation 62 Smallpox 63 Smallpox Variolation 1% v. 25% mortality Life-long immunity No drift or shift 64 Smallpox Vaccination 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) 65 Smallpox No animal reservoir Lifelong immunity Subclinical cases rare Infectivity does not precede overt symptoms One Variola serotype Effective vaccine Major commitment 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 Small RNA virus Some driftbut not too far as non-viable Sabin 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 infections Scandinavia: Salk dead vaccine No gut immunity Cannot wipe out wt virus Polio Vaccine 68 Reported cases per 100000 population 100 10 1 0.1 0.001 0.01 19501960 197019801990 Inactivated (Salk) vaccine Oral vaccine Cases per 100,000 population United States 69 10000 1000 100 10 1 0 Reported cases 195019551960196519701975 Killed (Salk) vaccine Total cases Sweden and Finland 70 Reciprocal virus antibody titer 512 128 32 8 2 1 Serum IgG Serum IgG Serum IgMSerum IgM Nasal and duodenal IgA Nasal IgA Serum IgA Serum IgA Duodenal IgA Days Vaccination Vaccination 48489696 Killed (Salk) Vaccine Live (Sabin) Vaccine 71 Sabin Polio Vaccine Attenuation by passage in foreign host More suited to foreign environment and less suited to original host Grows less well in original host Polio: 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 Polio 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 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 immunity 74 Measles Live 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 75 Mumps Live attenuated virus developed in the 1960s MMR vaccine Etiology: Mumps Virus Incubation: 16 to 18 days Clinical Manifestations: swelling of the salivary glands complications include Meningitis, Orchitis, Encephalitis, and Deafness 76 rubella Live attenuated virus Etiology: 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 77 Hepatitis B Two vaccines are in current use: A serum derived vaccine A recombinant vaccine Etiology: 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) 78 Hepatitis A Formalin-inactivated , cell cultured-derived virus, 79 Yellow fever The 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. 80 Rabies No 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. 81 In