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37 Cards in this Set
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- Back
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Sources of specimen
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1. Respiratory tract
2. GI tract 3. Skin 4. CNS 5. Blood 6. saliva, urine, biopsy |
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Diagnosis of Viral Infections
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1. Infectivity(need live virus in the specimen)
2. Visualization-electron microscopy 3. Detection of viral components-proteins/genomes 4. Evaluation of patient's immune response -serology |
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Infectivity methods of ID
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CPE: virus induced morph changes in cells(rounding, shrinkage, syncytia) via light microscopy
Hemadsoption: viral protein hemagglutinin on infected cells binds RBCs |
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CPE
Positives/downfalls used in clinical setting |
+ characteristic of each virus, sensitive
- takes too long to develop not every virus can grow in culture or produce CPE need technical expertise need additional test not used in clinical setting |
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CPE: Morphology characterisitics:
Syncytia, protein, examples? Plaques? Can also be used for what? Hemadsorption? proteins? Cells infected by what? |
Syncytia: multinuclear cell, fusion via protein paramyxoviruses
EX. Measles Plaques: focal areas of cell destruction, each plaque is from one virus-->can be used to quantify viral particles Cells, infected by influenza, mups, parainfluenza, express hemagglutinin, will stick to RBCs. |
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Visualization uses what? is based on the fact that?
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Electron microscopy
each virus has a unique shape |
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Visualization positives/downfalls
used in clinical setting? where/when is it useful? |
+ useful to ID unknown, each virus is unique in shape
- Need skilled staff Costly/complicated no, great for research to ID unknown viruses |
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Detection of viral components, what are you detecting?
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Viral Proteins: capsid, glycoprotein, nucleoprotein
Nucleic Acids: RNA/DNA |
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Ways to detect viral proteins? examples of these test?
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Antibodies: immunoassay, ELISA, immunofluorescence, western blot
Protein activity: enzymatic fxn, hemagglutination(binding to RBCs) |
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Immunoassay of viral antibodies methods?
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Captured antibody immoblized to solid support: binds viral protein
Dye/enzyme linked detection antibody binds viral protein as well |
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Protein detection by ELISA
method? example? |
Precoat with capture antibody
Add Viral proteins which will be captured by antibody wash Add enzyme-linked antibody which will bind viral proteins wash Add substrates: Give signal after reaction anti-influenza nuceloprotein Ab viral protein another anti-influenza nucleoprotein AB substrates added, reaction produces color change |
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Rapid Detection using ELISA-like method
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Lower end: mobile detection antibody, dye labeled or enzyme linked
Middle: capture antibody immoblized Sample added to lower end mixed with detection. complex flows along strip, if match, trapped by immobilized capture antibody. if trapped, dye/enzyme will form strip |
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Western Blot
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Antibodies used to attach to HIV infected cells. Line show up?
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Immunfluorescence assay
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Antibody tagged with certain color/indicator, mixed with viral antigen, wash, if color = ID
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Detection of Protein Activity
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1. Enzymatic Activity
2. Hemagluttination |
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Enzymatic Activity methods:
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1. Reverse transcriptase(retrovirus) RNA-->DNA
2. Neuraminidase(Influenza)- cleaves sialic acid |
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Hemagglutination theory?
For which viruses? |
hemagglutinin allows infected cells to bind RBCs. Cells stuck to each other form a lattice. This will show up as all red on a agar plate. If no lattice, you will just see RBCs sink towards the bottom of the plate forming a red dot.
Usually use dilutions, Titer will = the most dilution which still shows lattice Mumps, Influenza, Parainfluenza |
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Detection of Viral Nucleic Acids: classical
Theory? methods? |
Use labeled complement DNA/RNA sequence to detect viral genomes
classical(no amp) -in situ hybridization |
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Detection of Viral Nucleic Acids: Amplification:
Benefits/downfalls |
PCR
+ sensitive - false positive, contamination |
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Virus Specific Antibody:
theory? |
Use body's own immune response via antibodies to detect if person has had former infection. Often used as a blood screen
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Virus Specific Antibody
theory: Methods: |
Use viral proteins to determine if person has antibodies
Western Blot Hemagglutination inhibition assay(IFA): some antibodies block hemagglutinin specifically ELISA IFA(immunofluorescence assay) Neutralization: some antibodies inhibit viral infection |
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Indirect Detection: ELISA
steps example |
Precoated with viral proteins
Add specimen containing viral specific antigen wash Add enzyme-linked antibody which binds to virus specific antiboddy antibody wash Substrate Ex. HIV Capsid, Blood, anti-antibody, substrate |
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Indirect Detection: Neutralization Theory:
what does neutralization mean? |
Virus specific antibody blocks virus to infect cells, inhbiting CPE/plaque/haemagluttination
Neutralization: loss of infectivty via antibody |
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Indirect Detection: Hemagglutination Inhibition
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virus specific antibody blocks virus from binding to RBCs
titer level = highest dilution in which no lattice is found |
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During Disease Acute Phase what kind of experiments should be used?
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choosing anti-viral therapy and decide prevention
PCR of viral nucleic acids ELISA of viral proteins |
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During chronic infection/after resolution
what kind of experiments should be used? |
blood screening/epiedmiology studies
serological studies of virus-specific antibodies |
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Virochip theory?
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dots = nucleic acid of known virus, a match will show color change.
1. extract rna/dna samples 2. lable with fluorecent dye 3. apply chip 4. find match |
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SARS outbreak
what did they think it was originally? Kind of tests they used? what was it? |
influenza
isolation cell culutre ECM Virochip/PCR Immunofluorescence Coronoavirus- SARS-CoV |
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Anti-Viral Therapy
Target? Issues? Common targets? |
1. any step of viral rep which is essential for virus but not for cells
2. virus uses host cells machinery and enzymes, too much overlap 3. virus encoded enzymes, structures of virus |
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Targets: Neutralizing antibodies
theory? ex? |
natural host response
binds to viral envelope proteins and blocks binding to cells. Pooled human immuneglobulin ex. Hep A, B, Rabies |
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Uncoating:
Pleconaril for? fxn? Amantadine for? fxn? |
-picornavriuses(polio), blocks release of viral genomes
influenza A, Rimantadine blocks release of viral vesicles wit nucleic acids into cells |
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Why most anti-viral target nucleic acid synthesis?
common targets? most drugs of this type are? |
viruses encode own enzymes for this step
DNA poly Reverse Transcriptase RNA poly nucleoside analogues. Bind DNA/RNA to block translation/transcription |
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Inhibit nucleic acid synthesis drugs:
DNA POLY |
Acyclovir
Ganciclovir |
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Inhibit nucleic acid synthesis drugs:
Reverse Transcriptase |
AZT
3TC(lamivudine) |
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Inhibit nucleic acid synthesis drugs:
RNA Polymerase |
Ribavirin
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Inhibits Protein Synthesis:
fxn? by? used to treat? why ideal to use? |
inhibit translation
1. degrade viral mRNA, stop protein syn, enhance host immune HBV and HCV infections Can create interferons with long 1/2 lives which then are very stable in the body |
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Inhibits Assembly and Release of Virus?
example? |
1. Assembly:Protease inhibitor
HIV, prevents maturity of HIV 2. Neuraminidase inhibitor Influenza H1N1, release Tamiflu prevents infeciton of new cells |
