Exam 2 - Lecture 4

Question 1

What is meant by virus cultivation?

Answer 1

Taking virions from a sample and “growing” more of them

Question 2

What limitations do we face “growing” viruses as opposed to bacteria?

Answer 2

Cultivating viruses requires suitable host cells or a whole organism, and they can’t be grown on standard nutrient agar plates or in nutrient broth

Question 3

Growing viruses in animals
- What are some examples of lab animals used?
- What are transgenic animals and why are they especially useful?

Answer 3

Common lab animals include mice, guinea pigs, rabbits, hamsters, and primates

Transgenic animals are genetically altered to make them more suitable for cultivating viruses when the original host is not able to be used

Question 4

Growing viruses in animals
- What are benefits of using animals?
- What are drawbacks of using animals?

Answer 4

Benefits : can see virus in its native environment and study the immune response
Drawbacks : expense, difficulty in purifying, and animal rights concerns

Question 5

Growing viruses in plants
- What are difficulties when using plants?

Answer 5

The cuticle and cell walls of plant cells makes infection difficult to achieve
- usually requires damage to the plant cells
- difficult to mimic in cells grown in Petri dishes

Question 6

Growing viruses in plants
- How do we usually inoculate plants?

Answer 6

By scratching virions into the plant and creating damage so the virus can penetrate better

Question 7

Know the procedure for growing viruses in embryonic eggs (and parts of the egg) well

Answer 7

1. Disinfect the outer shell
2. Drill small hole into the shell and inject the virus into the chorioallantoic membrane, allantoic cavity, amniotic sac, and yolk sac
3. Seal hole with wax
4. Look for signs of infection
5. Harvest virions from egg

Question 8

Growing viruses in embryonated eggs
- What are benefits of using eggs?
- What are drawbacks?

Answer 8

Benefits : cheap and unlimited supply, the inside of the egg is naturally sterile and very nutrient rich
Drawbacks: can’t be used for pathogenesis studies, need to purify virions from tissue

Question 9

Growing lytic bacteriophages in cultured bacterial cells
- How does the purification process work if done in broth?

Answer 9

Add phage to bacteria growing in broth culture, and the phage will infect, kill, and release virions so that it can be purified

Question 10

Growing lytic bacteriophages in cultured bacterial cells
- What are plaques and how do they form?

Answer 10

A plaque is an area of dead host cells that the phage has killed

Question 11

Growing lytic eukaryotic viruses
- What is cell culture, where are cells grown during cell culture, and what are benefits of cell culture?

Answer 11

Cell culture is the process of growing cells outside of the animal
- Cells are grown in special dishes/ flasks with buffered growth media
- Benefits : cheap, limitless supply, and total control over conditions

Question 12

Growing lytic eukaryotic viruses
- What are primary vs continuous cell lines (what are differences between them)?

Answer 12

Primary cell lines consist of cells that have been isolate from a plant or animal, and they’ll only last a few generations. Continuous cell lines use tumor cells that can divide indefinitely

Question 13

Growing lytic eukaryotic viruses
- How do we grow plant cells in culture?

Answer 13

You first have to strip away the cell wall

Question 14

What is meant by the concept of viral purification?

Answer 14

Isolating virions from their host cells, other types of virions, and any other contaminants for further identification and study

Question 15

How can we use plaque formation as a way of purification?

Answer 15

Plaque will form in the cell monolayer and can then be easily transferred

Question 16

How is filtration used to purify virions?

Centrifugation – what is it, what is the difference between differential and density?

Answer 16

Filtration : virions and contaminants are passed through a membrane filter, the virions pass through and leave behind the contaminants

Centrifugation: a sample is spun so that its different components will separate
- differential – mixture is only spun
- density – a partially pure virion sample is added to a density gradient

Question 17

Know the different forms of light microscopy

Answer 17

bright field, fluorescence (regular), and confocal

Question 18

How is electron microscopy different from light microscopy?

Answer 18

Electron microscopy can be used to observe fine details of individual virions while light microscopy cannot get that specific

Question 19

What is the differences between transmission, scanning, and cryo EM?

Answer 19

Scanning - gives 3D view of virion surface
Transmission - gives a 2D slice through a virion
cryoEM uses freezing to prepare samples

Question 20

What is X-ray crystallography?

Answer 20

Virion crystals zapped w/ X rays

Question 21

How does atomic force microscopy work?

Answer 21

A small probe is moved over the virions, creating a contour map of the surface structure – very high resolution

Question 22

Counting virions
- What are benefits and drawbacks of using EM?

Answer 22

Benefits: can take a sample of a virus and observe it directly, which is useful for viruses that can’t be cultivated as well as those that don’t kill the host cells (any virus that you can’t use a plaque assay for

Drawbacks: not overly accurate due to unknown exact volumes, and doesn’t distinguish between infectious vs. noninfectious

Question 23

Know how a plaque assay is done and know how to calculate titer

Answer 23

Dilute virus sample and infect the monolayer of cells, and count the plaques produced

Titer = (# pfu x inverse of the dilution factor)/ vol plated) = ____ pfu/volume unit

Question 24

What is different about the concept of “titer” in plaque assays versus indirect measures like quantal assays?

Answer 24

Plaque assays actually give you the number of virions per amount of a given sample. Indirect assays give titers that are a dilution of a sample

Question 25

How is a quantal assay measured (e.g. what is meant by an LD50)? What is the relationship between titer and dilution used and why?

Answer 25

LD50 (lethal dose) is the measure of how much of a given virus would be needed to kill or infect 50% of hosts The higher the dilution, the higher the titer. The more you can dilute it and still see the LD50, the more virus that is present in that sample

Question 26

What is a hemagglutination assay, how is it done, /what does the titer represent/?

Answer 26

A hemagglutination assay is done to estimate the number of virions that are present from a virus that causes red blood cells to clump How: dilute virus sample and add to RBCs -- the more virions present, the greater clumping ability

Question 27

Detecting virions - What are antibodies and why are they used? What are antigens?

Answer 27

Antibodies: protein complexes that are designed to attach to anything foreign and alert the immune system - useful for detecting specific antigens Antigens: the foreign bodies that antibodies attach to

Question 28

For ELISA, know how the assays are done, what the data looks like, and how to interpret the data. Understand the concept of false positive results.

Answer 28

Uses plastic wells that provide a location for attached antigens and antibodies to interact -- you will have either the antibody or the antigen, and use it to find the other Two antibodies are added, one with an enzyme that changes color when it is bound to the antigen - The wells that produce the most significant color change have more antigen in the sample The results show that the host may have been exposed to the virus at some point, but they may not currently be infected

Question 29

For SDS-PAGE/Western blot, know how the assays are done, what the data looks like, and how to interpret the data. Understand the concept of false positive results.

Answer 29

1) take a sample and separate the proteins on a gel and then send a current through it so that the proteins separate out based on size 2) transfer proteins from the gel to a paper and add 2 antibodies, one with an enzyme that will change color when it is bound to the 1st antibody - enzyme turns color on that spot False positive: colored band is not the right size

Question 30

What is immunofluorescence (how does it work) and why would one use it?

Answer 30

An antibody specific for a viral protein is tagged with a fluorescent dye. The antibody binds to the target antigen and makes it visible because of the fluorescence This process can be useful to detect viral proteins in their native location within cells or tissues

Question 31

What is PCR, how is it done, and what occurs as a result? Where does the specificity come in with PCR?

Answer 31

PCR (polymerase chain reaction) is a method of targeting a specific sequence of DNA and making many copies of it Can amplify 1 copy into several billion in a couple of hours -- extremely sensitive, allows you to detect a very small amount of virus Uses sequence-specific primers that will only target the virus that they're designed to bind to

Question 32

What does the data look like with PCR?

Answer 32

After running the PCR, the sample is placed on an agarose gel. If the viral genome is present, it will be amplified

Question 33

What is FISH, how is it done, and why would someone use it?

Answer 33

FISH = fluorescence in situ hybridization - like immunofluorescence, but uses a DNA probe instead of an antibody Can be used for detecting infection and associated location information

Question 34

What are some other methods used for analyzing viruses?

Answer 34

DNA microassays, mutagenesis and siRNA, and DNA sequencing