Lecture 2 Flashcards
_____ assays are used to measure the number of infectious viral particles are present in a stock sample. It is done via serial _____, whereby each subsequent _____ has a concentration that is 10 times less (one order of magnitude) than the previous. The idea is to be able to visualize individual _____ once plated. The plating requires an overlay of agarose to prevent viral particles released from lysed cells from infecting neighboring cells. Keep in mind for one-hit kinetics the number of _____ is directly proportional to the concentration of virus inoculated (this is what allows calculation of viral particles in stock solution from number of ______.
Plaque
Dilution
Dilution
Plaques
Plaques
Plaques
Plaque assays are also important for ______ viruses of interest. The idea here is that one plaque is formed from one specific virus, so it can be purified in this way. This can be useful when trying to make ______ viruses.
Isolation
Recombinant
For viruses that do not exhibit CPE (cytopathic effects), ______-focus assay can be used. It involves permeabilizing cells and incubating them with ____ specific to the virus. Then, a second ____ is added, which is _____ labeled, and is specific to the first ____. You can determine number of titres in this way.
Fluorescent-Focus assay
Ab
Ab
Fluorescently
Ab
Another method for quantifying viruses is ______ center assay, which involves counting the number of infected _____ in your stock, as opposed to the number of viruses directly. This is useful for viral particles that are always associated with cells (i.e. Varicella zoster virus.)
Infectious Center assay
Cells
_____ assay is only used for viruses that can transform cells (i.e. Rous Sarcoma virus.) The idea here is the monolayer of normal cells in the culture will transform and exhibit non-normal qualities (i.e. loss of ______ inhibition.)
Transformation
Contact
Efficiency of plating = ____ / ____.
Paricle to PFU ration = ____ / ____
PFU / # of viral particles
of viral particles / PFU
Molecular _____ are used to detect viral genomes. They have a stem-and-loop structure that prevents the label from ______. When they hybridize to a target sequence, the ______ group is freed and will ______.
Beacons
Fluorescing
Fluorescent
Fluoresce
____ represents the percentage of uninfected cells. The lower the Multiplicity of Infection (moi or m) value, the higher the ____ value (more uninfected cells.)
P(0)
P(0)
For ____-step growth curves, you want as close to 100% of cells infected, so you’ll want a ____ moi value.
One-step growth curve
High
DNA ______ assays are used to detect viral gene expression and regulation and how that differs between wild type and mutant virus. The idea is that the WT genes are labeled with one color and the mutant with another. An overlay of the genes would show a mix of the colors for genes expressed in both and just the individual colors for genes expressed in one but not the other.
DNA Microarray assay
One of the benefits of the fact that ____ viruses are highly error prone is in vitro cultures of these mutated viruses allow for creation of vaccines because the mutations prevent the virus from causing disease while allowing it to elicit an immune response. A good example is the live attenuated _____ vaccine. However, these types of mutations can also make these viruses hard to treat, as some mutations make them resistant to treatments.
RNA
Poliovirus
_______ refers to the ability of multiple viruses to infect the same cell and compensate for what would otherwise be a deleterious mutation. That is, if virus A has a mutation in one gene, and virus B has a mutation in a different gene, they can assort their genomes in such a way that a combination of A and B would produce viable virus. If the mutation is in the same gene for both A and B, they cannot make up for one another.
Complementation
Bacterial Artificial Chromosome (BAC) is a method used to make ______ viruses. The idea is viral DNA is transfected into E. coli, mutations are introduced into the viral DNA within the E. coli, and then the viral DNA is purified from the bacteria.
Recombinant
