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123 Cards in this Set
- Front
- Back
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Contagionist Theory
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Disease caused Inanimate objects or organisms
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Zymotic Theory
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Disease is caused by substances outside the body
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Miasmatic Theory
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"Tainted" air and water, by Hyppocrates
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van Leeuwenhoek
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Father of microscopy, one lens, convex, was able to see single cell organisms
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Agostino Bassi
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discovered that a disease killing silk worms was caused by a fungus, which encouraged people to look for more pathological agents. Possible father of germ theory
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Spontaneus Generation
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Non-living objects can give rise to living organisms.Francesco Redi preformed the first major experiment to challenge SG
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Louis Pasteur
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1860s finally disproved spontaneous generation by using air traps to prevent contamination. Also developed rabies vaccine.
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Joseph Lister
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Used carbolic acid spray to sterilize air during surgery. Reduced number of fatalities due to infections. Antiseptic Surgery
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Variolation
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Using smallpox pus to inoculate other people. The practice of injecting dried exudate from recovering smallpox patients into an immunologically naive individual in order to generate protective immunity.
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Edward Jenner
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developed smallpox vaccine from cowpox pus.
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John Buist
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Scottish pathologist. In 1886 drew fluid from postulates of vaccinia and variola. First man to see a virus.
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Robert Koch
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1884 and 1890s created postulates for infectious agent for a disease, causal relationship
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Germ Theory
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Disease is caused by microorganisms.
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Koch's Postulates
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1. The agent must be present in every case of the disease.
2. The agnet must be isolated from the host & grown in vitro. 3. The disease must be reproduced when a pure culture of the agent is inoculated into a healthy susceptible host. 4. The same agent must be recovered once again from the experimentally infected host. |
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Filterable agents
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disease causing agents that passed through a filter so fine that not even bacteria would fit. Porcelain filters
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Dmitri Ivanovski
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Beginning of virology, identified organisms that could pass through filters causing tobacco mosaic virus
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Martinus Willem Beijerinck
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tobacco mosaic virus- independently replicated Ivanovski’s work. Named the biological entity “virus”
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Friedrich Loeffler and Frosch
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Worked with Koch, identified organism causing Diphtheria.and Foot and mouth disease. Filterable agents in animals.
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Lansteiner & Popper
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Discovered polio, 1st human filterable agent
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Walter Reed
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yellow fever in mosquitoes
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Twort & d'Herelle
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Co-discoverers in 1915 of bacteriophages
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W. M. Stanley
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Nobel prize laureate crystallized Tobacco mosaic virus
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Hershey & Chase
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made important discoveries on the replication of DNA during their studies on a bacteriophage
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Meselson & Stahl
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Showed that DNA replicates semi-conservatively. How DNA replicates, recombines and is repaired in cells. Meselson-Stahl experiment.
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Describe the development of the germ theory and explain its importance.
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-First spontaneous generation.
-Agostino Bassi silkworm disease postulates germ theory. -Leewenhoek, miscroscopy. -Redi, Snow, Pasteur No knowledge of how disease spread before. Changed the way we combat infection. |
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How has the development of microscopy impacted the field of virology?
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-Leewenhoek, miscroscopy.
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List Kochʼs postulates and explain the difficulties that researchers encountered trying to fulfill these requirements when working with viruses
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1. The agent must be present in every case of the disease.
2. The agnet must be isolated from the host & grown in vitro. 3. The disease must be reproduced when a pure culture of the agent is inoculated into a healthy susceptible host. 4. The same agent must be recovered once again from the experimentally infected host. Not Appropriat for viruses because they need a cell to grow |
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Capsid
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Protein coat around nucleic acid genome. Made up of many identical copies of 1 or a few proteins.
Function: protects fragile nucleic acid genome from physical, chemical or enzymatic damage |
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Enveloped
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provides protection from dessication and enzymatic damage.
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Naked
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Have capsid proteins exposed to external environment
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Glycoproteins
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Transmembrane proteins with sugar residue. Mediate immunity, cellular recognition, cell signaling, and cell adhesion.
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Helical Symmetry
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Simplest way to arrange multiple, identical protein subunits. Irregular shaped proteins arranged around the circumference of a circle.
P = mu x p |
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Icosahedral symmetry
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Icosahedron: a solid shape consisting of 20 triangular faces arranged around the surface of a sphere. Most efficient symmetrical arrangement.
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Complex symmetry
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More complex type of virus like a Pox virus with outer envelope, tubules, palisade layer, core envelope
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Amplitude & Pitch
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A helical virus is defined by amplitude (diameter) and pitch (distance covered by each complete turn of helix).
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2-3-5 symmetry
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axes of symmetry of capsid
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Triangulation number
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Icosohedral capsids are built using three identical subunits to form each triangular face.
T = 1, 60 subunits T = 3, 180 subunits |
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Budding
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process that allow a virus to exit from the infected cell without its total destruction.
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Matrix proteins
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Internal virion proteins.
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Viroids
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small circular RNA molecules with a rod-like secondary structure, no capsid nor enveloped. Depend on host plant cell for replication.
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Virusoids
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satellite, viriod-like molecules, somewhat larger than viriods. Depend on the presence of virus replication for multiplication. Packaged into virus capsids as passengers.
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Prions
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infectious agents generally believed to consist of a single type of protein molecule with no nucleic acid component.
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Describe how viruses differ from most living organism.
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Submicroscopic, obligate intracellular parasites.
Assembled from preformed components. Virions do not undergo division. Need a living host. Lack genetic apparatus. Energy parasites. |
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What is the function of the capsid?
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Protects fragile nucleic acid genome from physical, chemical, or enzymatic damage.
Recognition and first interaction with the host cell. |
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Explain the differences between naked and enveloped viruses.
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Some viruses have a more complex lipid bi-layer called an envelope, derived from the cell that the virus replicates from. naked viruses do not have an envelope
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Explain helical and icosahedral symmetry
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2 symmetries for viruses.
Helix shaped and amplitude and pitch. Solid shape with 20 triangular faces |
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Explain the Baltimore system of virus replication.
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1971 David Baltimore proposed scheme for classification of viruses, based on the way that viruses produce mRNA during infection.
Positive sense mRNA, genome just like mRNA. Negative sense mRNA, complementary as mRNA 7 groups and subviral agents |
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Attachment
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1st Steps: binding of a virus attachment protein (or antireceptor) on the surface of the virus particle to a cellular receptor molecule.
Target receptors are proteins or carbohydrate residues. |
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Assembly
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occurs from subunits often sequestered in the cell
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Eclipse
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Period when can't detect infectious virus. Time between disappearance of infecting virus and appearance of new intracellular viruses.
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Penetration
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Enveloped viruses
(A) Some enveloped viruses fuse directly with the plasma membrane. Thus, the internal components of the virion are immediately delivered to the cytoplasm of the cell (B) Viruses that require an acid pH are taken up by invagination of the membrane into endosomes. Acidification occurs, and virion membrane fuses with the endosome membrane. The internal components of the virus are then delivered into the cell Non-enveloped viruses may cross the plasma membrane directly or may be taken up into endosomes. They then cross (or destroy) the endosomal membrane. |
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Glycoproteins
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transmembrane proteins.
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Latent period
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time between disappearance of infecting virus and appearance of virus in surrounding medium.
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uncoating
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Capdis is completely or partially removed to expose the genome.
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antireceptor
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virus attachment protein on the surface of the virus particle.
Attaches to a cellular receptor molecule during attachment |
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inclusion bodies
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subunits often sequestered in the cell
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List and explain the steps of virus replication
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APURA
Attachment Penetration Uncoating Replication Assembly and Release |
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Describe a 1-step multiplication curve
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Lab study for virus replication. Amount of viruses vs. time. Can show eclipse period and latent period.
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What does phenotypic mixing demonstrate?
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Replication with mixed progeny virions. Only phenotipycally mixed, no cross-over.
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Explain the process of transfection.
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Infection of cell by nucleic acid alone.
Efficiency of infection is lower. Host range is wider. Infectious nucleic acid can be extracted from heat inactivated virus. |
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Organ culture
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slices of organs, maintain tissue architecture.
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Primary cell line
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isolated from embryonic tissue or newborn animal tissue. cell line that preserves number of chromosomes. Useful because they are nearly identical to the tissues from which they derive, but they required a lot of food and have a limited lifespan.
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Continuous cell line
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isolated for a long time, lose resemblance of host, aneuploid, inmortal. a single cell type that can be serially propagated. Cell lines of finite life are diploid, survive about 30 cycles. Tumor cells can be immortal. Tissue is minced and homogenized.
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Transformation
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tumor viruses transform cells rather than killing them. Lose inhibition, form microtumors.morphological and kinetic alteration. DNA viruses transform cells so that normal growth is altered. Form clumps of proliferating cells.
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Cytopathic Eeefct
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CPE, caused by viruses growing in cell culture, cells fuse together, no membrane
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Hemadsorption
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Ability of infected cells to bind to red cells. Some viruses have HA receptor, bind to sialic acid.
The ability of a virus membrane or a virus infected cell to stick to red blood cells caused by the action of several glycoproteins. |
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Interference
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results of smaller fragmentary genome being able to reproduce, move copies in a given time that the complete genome.
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Infectivity assay
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1. Qualitative: (PTIP)
-plaque -transformation -infectious center -pock assays Quantal: result is none or all, can use endpoint calculation method |
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Infectious center assay
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modification of plaque assay. used to determine the fraction of cells in a culture that are infected with a virus.
Place dilutions of infected cells on a monolayer of susceptible cells Number of plaques is an indicator of how many cells in the original sample were infected. |
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Plaque Assay
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Simple and precisely quantitative.
Looking for cytopathic effect. Based on agility of single virus particle to give rise to area of CPE on otherwise normal monolayer. Use a semisolid medium Report as Plaque Forming Unit/ml |
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Pock Assay
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Titration of virus on chorioallantoic membrane (CAM).
Viruses will form focal lesions. Mostly replaced by cell culture techniques. Still used in vaccine production. |
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Quantal Assay
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Used if virus can't be adapted to either a plaque or focus assay.
Virus serially diluted and multiple replicate samples of each dilution are inoculated into the appropriate assay system. |
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Physical assays
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Don't measure infectivity.
Include both hemagglutination and electron microscopy. |
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Hemagglutination assay
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Used for viruses with proteins that can bind to RBCs
Virion attaches to multiple RBCs and links them forming a lattice. Unadsorbed RBCs settle in bottom of well and form a bitton. 30 minutes, not sensitive. |
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Direct particle count
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mix virus with latex beads, and count concentration. *Cannot distinguish between infectious and non infectious virus.
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Serological assays
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Different assay methods:
-Neutralization tests -Immunodiffusion -Complement fixation test -Hemagglutination inhibition -Radioimmunoassay -Immnoflouresence -Enzyme-linked immunoabsorvent assay (ELISA) -Western Blots |
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Neutralization test
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Incubate serum dilutions with a constant dose of virus.
Assay for residual infectivity. Sensitive and specific. |
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Immunodifussion
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use Ouchterlony plates. Reacts diffuse through agar.
Line of precipitate where Antibody and antigen meet. Can test several antibodies or antigens at once. No quantification. Requires high concentration of reactants. |
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Complement fixation test
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mix patient serum with complement fixation antigen.
Add complement. Immune complex fixes complement. preventing lysis of red blood cells. |
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Hemagglutination inhibition
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dilutions of serum mixed with constant amount of virus.
Titer is the highest dilution of serum that inhibits hemagglutination. Quick, easy, cheap. |
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Immunofluorescence Assay
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antibodies are used to visualize viral proteins in infected cells or tissues.
-Direct: antibody is labeled with fluorescent dye. -Indirect: second antibody is labeled (more sensitive) |
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ELISA and EIA
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tests to detect antigens or antibodies by producing an enzyme triggered color change.
Rapid, high specificity and sensitivity. |
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Western blot
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analyze specific virus protein from a complex mixture of antigens.
Electrophoresis followed by transfer to membrane. Probe with antibodies |
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Multiplicity of infection
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is the ratio of infectious agents (e.g. phage or virus) to infection targets (e.g. cell).
Calculated using Poisson distribution. |
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Radioimmunoassay
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compare binding of specific antibodies to a pure antigen, to antibody binding to a patient antigen sample.
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Ultracentrifugation
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-Concentration and purification of virus through centrifugal force
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Differential centrifugation
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-Centrifuge materials at different speeds
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Density gradient centrifugation
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Each particle moves to equilibrium zone
-Rate zonal: sucrose gradients -Isopycnic: caesium chloride gradients |
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Characterizing Virus genomes
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-PCR and RT-PCR
-Sequence analysis -Microarrays |
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PCR
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scientific technique to amplify a single or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence.
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RT-PCR
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Reverse transcriptase.
To detect RNA, RT is used to produce single strand complimentary DNA |
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Sequence Analysis
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the process of subjecting a DNA, RNA or peptide sequence to any of a wide range of analytical methods to understand its features, function, structure or evolution.
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Microarray
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2D array on a solid substrate (usually a glass slide or silicon thin-film cell) that assays large amounts of biological material using high-throughput screening methods.
DNA chip, large arrays for hybridization. |
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Pulse labeling
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short pulse of radiolabel.
Produces snapshot of proteins being synthesized. Visualize synthesis of host cell proteins, and viral proteins |
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Monoclonal antibody
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monospecific antibodies that are the same because they are made by identical immune cells that are all clones of a unique parent cell.
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polyclonal antibodies
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antibodies that are obtained from different B cell resources. They are a combination of immunoglobulin molecules secreted against a specific antigen, each identifying a different epitope.
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Explain the differences between quantal and quantitative assays.
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Quantitative assays look at number of plaque froming units (PFUs) that can be identified in a plaque assay. Some viruses can be diluted so much that there are no PFUs so Quantal assays are used. Quantal assays look are statistical measures no need to localize plaques. Number of subjects must be infected with increasing dilutions of virus and the scored for illness, death, or cytopathicity. ID 50 or LD50
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Describe how infectivity, physical and serological assays differ.
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Infectivity: quantal and quantitative
Physical assays do not measure infectivity, they use electron microscopy, and hemagglutination. |
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Why is it important to know what type of assay is used in a study?
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To obtain the best one for sensitivity and specificity
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You apply a virus stock solution containing 3 X10^6 particles to a flask containing 3X10^5 cells. What is the MOI for this infection?
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10
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Explain how dilution endpoint methods are calculated.
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In this case, one adds serially diluted aliquots of virus to cultured cells, which are typically plated into multiwell plates. One then allows the cells to grow for some fixed period of time, after which the wells are scored for the presence of viral replication.
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Compare differential and density gradient centrifugation
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different speeds
Differential won't yield total pure fractions so use gradient |
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What is the difference between PCR and RT-PCR?
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look back
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Explain why you would do a pulse-chase experiment
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examining a cellular process occurring over time by successively exposing the cells to a labeled compound (pulse) and then to the same compound in an unlabeled form (chase).
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How could you use a monoclonal antibody to analyze a specific viral protein.
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find
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SDS polyacrylaminade gel electrophores
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is a technique widely used in biochemistry, forensics, genetics and molecular biology to separate proteins according to their electrophoretic mobility (a function of length of polypeptide chain or molecular weight). SDS gel electrophoresis of samples that have identical charge per unit mass due to binding of SDS results in fractionation by size.
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Innate immunity
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Generalized response that senses certain proteins or molecules that are found on pathogens.
Only immune system for first few days of infection. Only system that can discern the nature of invader. Informs adaptive immune system when infection dangerous. |
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Adaptive immunity
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Generates efficient and selective immune responses.
Lymphocytes are key B cells and T cells Humoral and cell-mediated |
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Neutrophils
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60-70% of white blood cells, first responders,
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Monocytes
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5% of white blood cells, develop into macrophages, some migrate some reside in tissue
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Natural killer cells
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destroy virus-infected cells, cause cell to lyse
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Complement system
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serum proteins that carry out a cascade of events leading to lysis of infected cell
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Apoptosis
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Programmed cell death
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Inflammation
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localized to injured area, chemical signals start it. Phagocytosis is key element.
Fever by pyrogens |
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Interferon
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Cytokines that function in the defense against viruses.
Cytokines are soluble proteins that bind to receptor and trigger resistance to infection |
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Antiviral state
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a state induced by interferon in a susceptible cell. The cell is partially refractory to virus replication.
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Necrosis
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is the premature death of cells and living tissue. Necrosis is caused by factors external to the cell or tissue, such as infection, toxins, or trauma.
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Interferon stimulated genes
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antiviral activity, inhibit viral penetration, inhibits primary viral gene transmission, inhibits cell transformation by retroviruses
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RNAi
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RNA interference: process of mRNA degradation induced by dsRNA in a sequence specific manner.
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Immunological memory
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primary immune response will create memory, secondary response much greater than original
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IgM, IgG, IgA
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Antibodies
IgM: fast response, aglutination, indicates current infection IgG:activate complemet system and efficiently opsonize viruses for ingestion by macrophages and neutrophils, provide history of infection. IgA: produces by cells in mucous membranes |
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C3a, C3b
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Part of complement system, proteins
C3b: binds to surface of viruses, makes viruses attractive to macrophages. C3a: recruits macrophages and neutrophils |
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Opsinization
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is the process by which a pathogen is marked for ingestion and destruction by a phagocyte
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