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142 Cards in this Set
- Front
- Back
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What is the “Gold Standard Problem”?
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To be able to determine whether or not an animal is truly infected or uninfected.
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How might sensitivity of a test be affected?
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1. Different strains (local) of an organism which may differ from the test target.
2. The magnitude of infection: quantity of organism may not be enough |
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What 2 things cause predictive values to vary?
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1. Sensitivity and specificity of a test
2. Prevalence of disease |
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What happens to the predictive value of a positive test as prevalence decreases?
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It decreases unless specificity is 100%
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Why is there more opportunity for false positives to occur in a location where HW is low?
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Because the test gets stochastically overwhelmed.
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What is optimization of a diagnostic test?
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Balancing the specificity and sensitivity to reflect the intended use of a test.
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What is the 95% confidence limit?
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A range within which one can be 95% certain is the true value of that test parameter.
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What 2 considerations should be made when comparing test results?
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1. Should be run more or less simultaneously
2. Should be done using the same test specimens from the same animals |
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What test can be used to calculate the degree of certainty that two tests give different results?
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McNemar’s Paired Chi Square test (P-value)
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What are the 2 groups of diagnostic tests?
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1. Nucleic Acid (DNA) Sequence Analysis
2. Antigen/Antibody Reaction Analysis |
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What are the steps involved with DNA Sequence Analysis?
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1. Identify appropriate fragment
2. Clone the segment 3. Probe production |
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What can you put the fragment of DNA into for cloning?
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A bacterial plasmid or viral phage.
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What is hybridization?
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Binding of 2 complementary stands of DNA: both probe and target DNA
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What is a probe?
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A segment of DNA complimentary to target DNA that is labeled with a substance that will allow for identification once bound to target.
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What are some advantages and disadvantages of southern blotting?
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Adv: sensitive, highly specific
Disadv: does not tell the specific cell or tissue where the infection/neoplasia etc. is |
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What does in situ mean?
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In its normal or natural place.
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What is PCR?
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In vitro amplification of a specific region of target DNA that lies between flanking areas of a known sequence.
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Describe the steps involved with PCR
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1. Target DNA identified
2. Primers made 3. Target DNA is denatured 4. Primers anneal 5. Primer extension using DNA polymerase 6. process repeated |
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What are some PCR uses in veterinary practice?
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1. confirm a positive serology
2. diagnose latent or subclinical infections 3. identification of infections agents that cannot be cultured 4. organisms too low in number to see in microscopy 5. evaluate effectiveness of antimicrobial therapy |
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Why is PCR so specific?
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Because primers can be designed with desired specificity.
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What is the biggest disadvantage to PCR?
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False positives common due to incredible sensitivity and ease of contamination.
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What does a negative result mean in PCR?
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Target DNA was not identified in the sample that was submitted. May be due to the sensitivity of the test or the sample that was submitted or it may be a true negative.
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What is genetic clonality a hallmark sign of?
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Malignancy
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What are some different immunophenotypes of genetic clonality?
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B-cell tumors (CD21, CD79), T-Cell tumors (CD3, CD4, CD8)
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What is needed to be clonality?
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Amplicons all must be identical. A reactive population would have multiple types of cells all working together to get the infection or whatever under control.
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How would a clonal sample appear on a gel?
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As a single band of protein.
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What is chronic myelogenous leukemia and why is it difficult to diagnose?
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It is a hematopoietic tumor that results in excessive proliferation of neoplastic neutrophils. Problem is they all look normal so you have to rule out a reactive population to some infectious or inflammatory process.
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What predisposes humans (and potentially dogs) to CML?
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The Philadelphia chromosome. The 3’ end of chromosome 22 is translocated onto the 5’ end of chromosome 9.
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What is serology?
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The study of antigen-antibody reactions in vitro.
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How is diagnostic serology useful?
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It recognizes antibodies in the patient serum that are specifically produced against some antigens.
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Why are whole organisms the easiest method of serological assay development?
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Because there are various proteins (multiple antigens) available for antibody binding on a whole organism. It increases sensitivity,
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What can be a problem with using a whole antigen?
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Some antigens will be shared with other organisms and can result in false positives.
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What are the 3 serological assay formats?
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IFA: Immuno-Fluorescent
ELISA: Enzyme-linked Immunosorbant Assay Western Immunoblot |
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What are some advantages to western immunoblot?
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There are specific band patterns seen in different situations (ie. vaccinated vs. infected) so test specificity is enhanced.
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What are some disadvantages to western immunoblot?
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Time consuming and expensive, need equipment, need to know specific antigen.
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What are the characteristics of the ideal test antigen?
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1. Ab production seen early during disease
2. Ab level remains high 3. Ab levels decline when treated or cleared of infection 4. Antigen is specific for the organism desired 5. Vaccinated animals have no Ab to test Ag |
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What are recombinant proteins?
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Proteins that can be used as antigens to stimulate an immune response without actual infection and do not require the whole organism.
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What gene is essential in tumor angiogenesis?
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The c-myc gene. Over-expression increases angiogenesis.
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What occurs in cancer cells that is abnormal?
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1. Loss of contact inhibition
2. Increased growth factor secretion 3. Increased oncogene expression 4. Decreased tumor suppressor gene |
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What are the origins of the following tumors:
Carcinomas Sarcomas Leukemias/Lymphomas Gliomas/Retinoblastomas |
Carcinomas: epithelial
Sarcomas: muscle or connective tissue Leukemias/Lymphomas: hemopoietic cells Gliomas/Retinoblastomas: nervous system |
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What is “loss of heterozygosity”?
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Loss of an entire chromosome in the cancerous cells. They lose one allele so become homozygous.
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What are the 2 major class of alteration seen in genomic instability?
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1. Over-expression of proto-oncogene or conversion of proto-oncogenes to oncogenes
2. Inactivation or down regulation of tumor suppressor genes |
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Oncogene:
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A gene responsible for transformation of a cell to a cancerous phenotype
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Proto-oncogene:
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normal cellular gene that may become oncogene
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Give an example of how translocation can induce cancer.
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Burkitt’s Lymphoma: Translocation between chromosome 8 and any of 3 containing Ig genes. Immunoglobulin promoter then drives c-myc proto-oncogene.
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What gene in particular is classically associated with loss of tumor suppression?
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The Rb-gene. If Homozygous for loss of the gene, sufficient for oncogenic transformation.
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What is another example of a tumor suppressor gene? What does it do?
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The ING4 gene that regulates angiogenesis
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What are some different ways that tumor viruses can act?
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1. Bind tumor suppressor gene products and inactive them
2. Can cause activation of genes that induce DNA replication and/or cell cycling |
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What is an example of an epigenetic origin of cancer?
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Methylation (hyper usually malignant)
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What could potentially be used as a marker for tumorigenic potential in breast cancer?
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CD44 - no tumors vs. + had tumors. Also CD24 - had tumors. SO CD44 -/CD24 + much less likely to develop tumors.
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What else could potentially be used for prediction of tumorigenic potential?
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Transcriptional patterns: among tumors of similar oncogenic potential are very similar
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What affect do some tumors have on immune response?
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Can induce T-cell tolerance so immunization for cancer would be better suited before tumors exist.
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Hapten:
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chemical structure capable of eliciting a response but not being recognized on its own (too small)
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Epitope:
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chemical structure capable of eliciting an immune cell response and of being specifically recognized by molecules of immune recognition
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Paratope:
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the region within the Ag-binding site of an Ig molecule responsible for recognition of epitope structure
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Idiotope:
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Epitopes present in the unique variable sequences of an Ig molecule
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What is the difference between a linear epitope and a conformational one?
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Linear is only primary (and some secondary) structure while a conformational epitope is derived of amino acids from different regions within the molecule and all together make up one big epitope.
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How does the immune system get around the incredible number of epitopes that exist?
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1. Usually doesn’t have to anyway: Shouldn’t respond to self so that gets rid of a few, many potential epitopes aren’t biochemically feasible and antibodies aren’t 100% specific
2. Takes advantage of combinatorial genetics and mutations 3. Uses promiscuous binding molecules of broad specificity |
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What are combinatorial genetics?
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A mechanism by which multiple unique “parts” are assembled in many different potential combinations to make the whole.
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What are the 2 forms of diversity in immune recognition?
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1. Ig’s and T-Cell receptors-VERY specific
2. MHC Class I and II molecules-promiscuous |
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What are the 3 primary mechanisms of Ig diversity? Which species use which?
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1. Somatic recombination: mice, humans
2. Gene conversion: chicken, rabbit, cattle and pigs 3. Somatic Hypermutation: sheep |
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Which occurs first, H or L chain rearrangements?
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H chain
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What is somatic hypermutation?
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Random mutations that occur to increase affinity
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What 2 loci are found in T-Cell receptors?
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alpha and beta: beta similar to heavy chain
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What are some common structural elements seen within the Ig superfamily?
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-Ig domain
-TcR -MHC |
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What are some different mechanisms by which organisms can induce inefficient immune surveillance?
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1. fetal exposure-induction of immunotolerance
2. molecular mimicry 3. antigenic masking with host components 4. infection of immune cells 5. adhesion to antigen presenting cells |
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Antigenic drift:
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continued expression of genes undergoing continual low-level mutation
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Antigenic shift:
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mixing of genomes with sudden changes in phenotype
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Phase variation:
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off and on variation of presentation
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Antigenic variation:
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expression of different or altered genes of a multi-gene family
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What is a good example of an organism that practices fetal exposure?
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Bovine viral diarrhea
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What is molecular mimicry?
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Production by an infectious agent of a component that resembles and behaves similarly to a normal host component.
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What is a good example of molecular mimicry?
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Vaccinia virus: produces a protein that acts as if it were IL-1R
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What is antigen masking? What is an example?
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Some microbes can bind host components to “mask” their own surface. Babesia bigemia binds IgM to its surface.
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What is cytoadhesion?
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Some parasites adhere to host cells and prevent circulation and detection in the spleen.
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What is needed for cytoadhesion to occur?
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Specific receptor-ligand interactions between infected erythrocytes and endothelium.
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Why is antigenic drift important?
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Because some infectious agents can undergo continuous slow mutations and eventually will appear nothing like the original agent thus avoiding the immune system. Can result in drug-resistance. Example is HIV.
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Why is antigenic shift important? What disease in particular are we worried about?
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It is a sudden change of phenotype brought about by mixing of genomes from 2 microbes of differing genotypes. A potential problem with avian influenza meeting human influenza.
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What is “true/programmed” antigenic variation?
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A mechanism that specifically targets a multi-gene family for variation at rates 2-3 orders of magnitude more rapid than random mutation.
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What agents has “true/programmed” antigenic variation been observed in?
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Protozoa and bacteria. NOT viruses.
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What are some diseases caused by retroviruses?
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FeLV, BLV, Avian Leukosis Virus, HIV
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What are the 4 different viral genes?
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gag, prt, pol, env
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What are the 2 unique components of the viral genome?
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U5 and U3 region that are “unique” to the 5’ and 3’ region respectively.
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What else exists at the 5’ end? The 3’?
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5’: Repeat sequences with the U5 region and a cap
3’: U3 with repeat sequences and then a polyA tail |
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What is the LTR? What 3 sequences make up the LTR?
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Long terminal repeats that gets integrated into the host genome and is known as a provirus. Includes U3, U5 and R
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Where does reverse transcription occur?
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In the cytoplasm
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What happens during reverse transcription?
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The virus gets duplicated and the LTR segment ends up at both ends of the linear DNA.
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What happens to the double stranded DNA?
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It gets taken into the nucleus and integrated into the host genome.
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What are the functions of the LTR?
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1. contains viral promoter
2. contains transcriptional initiation and termination sites 3. required for integration into the host genome |
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Where is the promoter found?
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In the U3 region
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What signals termination?
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The poly A tail at the 5’ end
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What is the minimum number of proteins a viral transcript can make?
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8
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What proteins can be made from the following segments:
Gag: Prt: Pol: Env: |
Gag: Matrix (MA), Capsid (CA), Nucleocapsid (NC)
Prt: Protease Pol: Reverse transcriptase (RT), Integrase (IN) Env: Surface glycoprotein (Su), Transmembrane (Tm) |
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What is the protein that gets spliced out on its own?
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Envelope which then cleaves into Su and Tm
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What is integrase needed for?
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Integration into the host genome
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With GAG-PRT-POL translation, what is 80% of the product? 20%?
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80% is just GAG and the other 20% stays as the whole
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Why does it happen this way?
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There is a stop codon at the end of Gag and Pol but usually just stops at Gag
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What can protease do?
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Cut itself out and start cleaving other products
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What surrounds a retroviral particle when its not in a cell?
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A lipid bilayer from when it is exocytosed from the host.
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What other genes are present in complex retroviruses? What do they do?
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Tax and Tat: Activate gene expression directed by LTR, enhance viral replication
Rex and Rev: serves as switch from early to late gene expression |
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Describe the virus-host interaction in the following situations:
non-cytopathic cytopathic |
Non-cytopathic: cell permanently produces virus at low level, virus becomes dormant and then transforms cells and results in malignancy
Cytopathic: extensive virus replication, direct toxicity of viral products, immune-mediated killing of infected cell |
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What are the 3 mechanisms of oncogenicity?
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1. Transducing retroviruses
2. cis-activating 3. trans-activating |
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How do transducing retroviruses induce oncogenecity?
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They have a high efficiency of tumor formation and cell derived oncogene is carried in viral genome. Only a few days needed to cause tumors.
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What happens when there is a transduced oncogene?
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The oncogene is now under control of the viral promoter so could have continuous transcription of that protein.
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What are some tumors that are produced by transducing retroviruses?
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Feline, Avian and Murine sarcomas
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What do cis-activating retroviruses do?
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Take weeks to months to initiate tumor formation. Cellular oncogene is activated by a provirus.
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What are some cis-activated tumors?
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B cell lymphoma in birds, Erythroleukemia in cats
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Are Trans-activating viruses efficient at inducing tumors?
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Very poor, takes months to years. Adult T-cell leukemia
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Describe lentiviruses.
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Cytopathic, all infect macrophage and many CD+4 T cells, complex genome, all have Tat and Rev. High amounts of antigenic drift.
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What are the 4 accessory genes?
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1. Nef: Negative factor
2. Vif: Viral infectivity 3. Vpr: Viral protein R 4. Vpu: Viral protein U |
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What effect does the nef gene have on HIV/AIDS?
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Nef + results in AIDS while Nef - is asymptomatic.
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What are some functions of Nef?
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-reduce expression of MHC Class I and the CD4 and CD28 receptor
-Affects T-cell activation -enhances virion infectivity |
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What is the function of Vif?
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-Helps overcome cellular restriction to virus replication
-prevents packaging of host protein into virus particles because if it were in there, would be a target for enzyme cleavage |
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What are some functions of VPR?
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Promotes cell arrest and apoptosis.
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Effective antiviral therapy must be directed at what 2 types of viruses?
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1. free
2. cell-associated |
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What are the 2 potential places for genetic changes to occur?
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1. nuclei of fertilized embryo
2. stem cells (embryonic) |
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What are transgenic animals?
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Introduction of new genetic material into germ line of an animal
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How can transgenics be used in Pompe’s disease?
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Can synthesize an artificial intron with a casein promoter and the alpha-glycosidase gene and a poly A tail. Inject it into mice embryos and it will be expressed in the mammary glands.
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How can you make a chimera?
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Take 2 separate morulas and push them together into one 16 cell organism.
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What does the Agouti gene do?
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Controls the way pigment is laid down.
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Describe the process involved with using a SCID mouse and the ability to make it no longer a SCID mouse. Include genes.
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Rag2 -/- mouse is a SCID mouse. You can grow a culture of cells and do nuclear transfer into an enucleated oocyte. Activate and culture the -/- ES cells and recombine them with a wild type to make it Rag2 +/+. Differentiate into hematopoietic stem cells and do a bone marrow transfusion back into the SCID mouse.
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What is gene therapy?
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Ability to cure a disease or harmful condition by using a large molecule (DNA).
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What is the general principle of gene therapy?
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Put the virus DNA on either side of the gene you want (make your DNA look like a virus) and make it integrate into the genome but your gene is the only genetic coding.
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What is one of the favorite vectors to use for gene therapy? How does it work?
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Adeno Associated Virus (its a DNA virus). It doesn’t integrate into the cell but forms an episomal body.
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What disease has had great success using an AAV-mediated correction?
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Hemophilia in dogs.
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What gene in brain tumors is the target of the new viral anti-cancer therapy?
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Herpes Simplex thymidine kinase gene.
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What is the general idea behind using a virus to kill brain tumors?
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If you inject a virus that only grows in replicating cells and have Gangcyclovir in the virus, it will get inorporated by the brain tumor and all the cells will undergo apoptosis.
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What 2 receptors are needed by the HIV virus to be effective?
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CD4 and CCR-5
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True positive:
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The animal tested positive and is infected
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False negative:
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An infected animal tested negative
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True negative:
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An uninfected animal tested negative
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False positive:
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An uninfected animal tested positive.
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What is the sensitivity of a test?
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The proportion of positive test results from a group of infected animals.
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What is the equation?
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True positives
True (+) + False (-) |
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What is the specificity of a test?
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The proportion of a negative test result in a group of uninfected animals.
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What is the equation?
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True Negatives
True neg + False pos |
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What is the accuracy of a test? Equation?
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the proportion of correct test results from a group that includes both infected and uninfected animals.
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What is the predictive value of a positive test?
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The proportion of all positive test results coming from animals infected with the disease agent of interest.
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How is this different from test sensitivity?
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Because it is specific for a desired agent and tells you if the results are actually going to be an effective diagnostic tool for that agent.
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What is the equation?
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True Positives
True pos + false pos |
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What is the predictive value of a negative test?
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The proportion of all negative test results coming from animals not infected with the disease agent of interest.
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What is the equation?
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True Negatives
True neg + false neg |
