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Category of Sample
nasal swab , throat swab, saliva , sputum, rectal swab, vesicle fluid( scraping or swab), tissue ,brain biopsy, cerebrospinal fluid, et al.
Laboratory Diagnosis
Microscopy Identification Virus isolation and identification Detection of viral proteins( antigens and
enzymes)] Detection of viral genetic material Serologic procedures
Microscopy Identification
Light microscopy
Characteristic CPE Inclusion Bodies
Cell death
Cell rounding Degeneration Aggregation Loss of attachments to substrate
Fluorescent microscopy
Fluorescent-antibody staining
Electron microscopy
Laboratory Diagnosis
Microscopy Identification Virus isolation and identification Detection of viral proteins( antigens and
enzymes)] Detection of viral genetic material Serologic procedures
Viral detection
CPE Hemadsorption Interfere Metabolize of cell
+1/2 (total number of test units showing CPE)/ (number of test units per dilution)
Viral identification
Complement fixation Hemagglutination inhibition Neutralization Immunofluorescence ( direct or indirect) Latex agglutination In situ EIA ELISA RIA(radioimmuno
Laboratory Diagnosis
Microscopy Identification Virus isolation and identification Detection of viral proteins( antigens and
enzymes) Detection of viral genetic material Serologic procedures
Laboratory Diagnosis
Microscopy Identification Virus isolation and identification Detection of viral proteins( antigens and
enzymes)] Detection of viral genetic material Serologic procedures
chain reaction) SouthernDNA), Northern(RNA), and dot blots DNA genome hybridization in situ(cytochemistry) Electrophoretic mobilities of RNA for segmented RNA viruses( Electrophoresis) Restriction endonuclease cleavage patterns
Laboratory Diagnosis
Microscopy Identification Virus isolation and identification Detection of viral proteins( antigens and
enzymes)] Detection of viral genetic material Serologic procedures
Serologic procedures
If the antibody titer in the convalesentphase 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
Serologic procedures
Complement fixation Hemagglutination inhibition Neutralization Immunofluorescence ( direct or indirect) Latex agglutination In situ EIA ELISA RIA
Prevention
Active immunization
Vaccines
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)
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
LIVE VACCINES
Attributes live
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
Live recombinant
Vector 1. bovine vaccine 2. BCG
Quick immunity in majority of vaccinees In case of polio and adeno vaccines, easy administration
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 backmutated Poor "take" in tropics Problem in immunodeficiency disease (may spread to these patients)
Killed vaccines
The organism is propagated in bulk, in
vitro, and inactivated with either betapropiolactone 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.
Killed vaccines
Inactivated organism: rabies virus;
epidmic type B encephalitis virus. Subunit Vaccines: Influenza virus( HA and NA) Recombinant proteins: HBV
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
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
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
PB2 PB1 PA HA NA NP M NS
PB2 PB1 PA HA NA NP M NS
PB2 PB1 PA HA NA NP M NS
New Methods
Deletion mutants
Suppression unlikely (but caution in HIV)
Problems
Oncogenicity in some cases (adeno, retro)
New Methods
Recombinant DNA
Single gene (subunit) Hepatitis B vaccine raised in yeast
Express plasmid S-antigen mRNA cDNA
Better presentation
CTL response Vaccinia Attenuated Polio Being developed for anti-HIV vaccine
New Methods
Chemically synthesized peptide
malaria
poorly immunogenic
New methods
Anti-idiotype vaccine
Virus epitope
antibody
Antiidiotype antibody
antibody
Anti-idiotype antibody
cont 2 Use anti-idiotype antibody as injectable vaccine
Anti-idiotype antibody
Use as vaccine Binds and neutralizes virus
Anti-anti-idiotype antibody
Anti-anti-idiotype antibody Antibody to antiidiotype antibody Anti-anti-idiotype antibody
New Methods
New Jennerian Vaccines
Live vaccines derived from animal strains of similar viruses Naturally attenuated for humans
Ineffective in others
Due to differences in circulating viral
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
New Methods
Second Generation Jennerian Vaccines
Rotavirus 11 segments of double strand RNA Two encode: VP4 (hemagglutinin) Elicit neutralizing VP7 (glycoprotein) antibodies Co-infect tissue culture cells reassortment
Vaccines
1796 Jenner: wild type animaladapted virus
1800s Pasteur: Attenuated virus 1996 DNA vaccines
DNA vaccines
DNA vaccines are at present experimental ,
but hold promise for future therapy since they evoke both humoral and cellmediated immunity, without the dangers associated with live virus vaccines
DNA Vaccines
Gene for antigen
plasmid
Muscle cell
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
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
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-
DNA Vaccines
DNA vaccines produce a situation that reproduces a virally-infected cell
Gives:
Adjuvants
Certain substances, when administered
simultaneously with a specific antigen, will enhance the immune response to that antigen.
complexes Complet Freunds adjuvant is an emulsion of mycobacteria, oil and water Incomplete Freunds adjuvant Muramyl di-peptide Cytokines
Smallpox
Smallpox
Variolation
No drift or shift
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
Smallpox
No animal reservoir
Lifelong immunity Subclinical cases rare Infectivity does not precede overt symptoms One Variola serotype Effective vaccine Major commitment by governments
polio
Killed virus vaccine(Salk, 1954) Live attenuated oral polio vaccine( Sabin,
1957) The inactivated Salk vaccines is recommended for children who are immunosuppressed.
Polio Vaccine
Small RNA virus viable Some driftbut not too far as non-
paralytic polio
No gut immunity
100
10
0. 1
0.01 0.00 1 195
0
1960
1970
1980
1990
10000
Reported cases
1000
100
1 0 1 0 195 0
1955
1960
196 5
197 0
1975
512
Serum IgG
Serum IgG
128
32
Serum IgM
Serum IgM
Serum IgA
Duodenal IgA
48 9 6
Days Vaccinatio
Formaldehyde-fixed No reversion
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
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
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
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
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)
Hepatitis A
Formalin-inactivated , cell culturedderived virus,
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.
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 .
Influenza
Varicella-Zoster virus
Not licensed vaccines
Passive Immunisation
Modes of immunization
Passive immunization - administration of
antibody-containing serum to provide immediate, but temporary protection. Doesn't activate a lasting specific immune response.
Natural
Provides immunity for diphtheria, tetanus,
streptococcus, rubeola (red measles), rubella (German measles), mumps, polio, and others.
Artificial
Often used as antitoxins for things such as
black widow spider and snake bites, botulism, and tetanus. Important for some infectious diseases such as rabies, since it provides immediate protection rather than waiting the 7-10 days for a protective response to develop from active immunization.
Immunoglobulin
NormalImmune globulin Hyper-immune globulin
NormalImmune globulin
Low titres of antibody to a wide range of human viruses Hepatitis A virus infection Parvovirus infection Enterovirus infections (in neonates) HIV-infected babies
viruses Zoster immune globulin: prevention of varicella in immunocompromised children and neonates Human rabies immunoglobulin: post-exposure prophylaxis in an individual who has been bitten by a rabid animal Hepatitis B immune globulin:non-immune individal who has been exposed to HBV RSV immune globulin: treatment of respiratory syncitial virus infections in the very young
Antiviral Therapy
Antiviral Therapy
Influenza: Amantadine Herpes simplex virus: Acyclovir Varicella-Zoster virus: Acyclovir Cytomegalovirus : Gancyclovir, Foscarnet Respiratory syncitial virus: Ribavirin
Nucleotide analogues
Nucleotide analogues competes with
normal nucleotide for incorporation into viral DNA or RNA.
Interferon
Direct antiviral effect ( prevents the
infection of new cells) by a) degradation of viral mRNA, and b) inhibition of protein synthesis Enhancement of the specofic immuneresponse by increasing the expression of MHC class I molecules on the surface of infected cells, the interferons increase the opportunity for specifif cytotoxic T cells to recognise and kill infected cells
Chinese Herbs