Lecture 2

Question 1

What is a virion?

Answer 1

A virion is a vehicle used to transport the genome into another host

Question 2

What host machinery does the virus require?

Answer 2

It requires the host’s ribosomes, the host’s transport vesicles, and the host’s energy.

Question 3

In the viral life cycle, what is “absorption”?

Answer 3

Absorption is attachment of the virus to the host cell.

Question 4

What is the eclipse in a viral life cycle?

Answer 4

An eclipse is the time when infectivity disappears. It occurs when the virus is uncoating itself.

Question 5

What is the latent period of the viral infectious cycle?

Answer 5

Occurs with the replication of the genome, when protein synthesis is occurring.

Question 6

What is maturation in the viral life cycle?

Answer 6

Maturation is the assembly of the genome and proteins into the virion.

Question 7

How long does the viral replication cycle take?

Answer 7

Approximately 8-40 hours. Viruses make their bits and pieces (using the host) and then assemble into the infectious virion.

Question 8

What two characteristics does a host require to be infected by a virus?

Answer 8

It must be susceptible and permissive.

Question 9

What does it mean for a cell to be susceptible to a virus?

Answer 9

It means that the host has a functional receptor for the virus to attach to.

Question 10

What is a susceptible host cell?

Answer 10

A cell that has a functional extracellular receptor for the virus.

Question 11

What is a permissive cell?

Answer 11

A cell that has the organelles required for the virus to assemble into a virion.

Question 12

What is an advantage and a disadvantage of an animal host?

Answer 12

Advantage: The best type of host for a virus. Dramatically advanced our understanding of viral knowledge.

Disadvantage: Very costly laboratory model.

Question 13

What are some advantages of using a fertilized chicken egg as a viral host? What are they typically used for?

Answer 13

Fertilized chicken eggs are composed of multiple cell types; the virus can be injected into the yolk sack, the amnion, the embryo, etc. Less costly than animal models.

Perfect host for Influenza A. Chicken eggs are used to grow the influenza vaccine. Each individual egg hosts one single vaccine.

Question 14

How may cells be used as a host for a virus?

Answer 14

Cells can be used by adhering them to a slide, forming a monolayer. Continuous cell lines are formed by injecting the virus into a tumourous cell that is immortal and replicating uncontrollably.

Question 15

What are cytopathic effects (CPEs)?

Answer 15

The different changes that a virus induces in a cell.

Question 16

What does this photo demonstrate?

Answer 16

Demonstrates cytopathic effects. After the cell dies it no longer has the ability to adhere to the slide on the monolayer. Therefore, as time progresses, the virus kills the cells.

Question 17

What percentage of cancers are caused by human viruses?

Answer 17

20%

Question 18

What are three examples of CPEs?

Answer 18

1. Lysis
2. Syncytia
3. Transformation

Question 19

What is syncytia as a CPE?

Answer 19

When the plasma membranes of cells fuse together, producing multinucleated cells.

Question 20

What is transformation as a CPE?

Answer 20

When cells are no longer flat, but divide uncontrollably to become piles of cells.

Question 21

What is the next step that is to occur if a CPE does not occur?

Answer 21

Even if cells fail to show infection, they still need to be proven to be uninfected. Next, measure the infectivity.

Question 22

What is the general idea of a plaque assay in measuring the infectivity of a virus?

Answer 22

Adding a virus to cells, and overlaying with a substance (agar). When infected cells release their progeny, the spread is halted by the gel (the agar). The place where the infected cells released their progeny is the plaque.

Question 23

How is a plaque assay conducted?

Answer 23

There are 8 test tubes, each with 9 mL of buffer in them. Add 1 mL of concentrated virus to the first test tube (dilution = 10^1). Extract 1 mL of this and add to the next test tube (10^2). Continue until reaching the 8th test tube (10^8). Remove one mL of 10^8 diluted virus and add to a susceptible and permissive host. Cover with agar and allow for plaques to form. Conduct this in 4 petrie dishes, and count and average the number of plaques formed. Multiply this number by the dilution factor 10^8 to calculate the number of plaque forming units/mL.

Question 24

What is the unit of measurement in a plaque assay?

Answer 24

PFU/mL

PFU = plaque forming units

Question 25

What is a plaque assay used to measure?

Answer 25

The infectivity of a virus.

Question 26

How many clones does a plaque typically represent in a plaque assay?

Answer 26

**One** viral clone. This is due to the high degree of dilution. It indicated that only one viral clone may be required to initiate infection.

Question 27

What is the purpose of using crystal violet staining when conducting a plaque assay?

Answer 27

Crystal violet stains the **living host cells.** Therefore the plaques can be easier to see.

Question 28

What does the particle:PFU ratio measure?

Answer 28

The infectivity of a virus. It tells you whether or not all of the virions are infectious or not (ex. you may produce 40 virions but not all of them may be infectious).

Question 29

How is the particle:PFU ratio used? What does it mean if the number obtained is big? Small?

Answer 29

Ratio = number of viral particles/number of infectious particles The smaller the ratio, the more infectious the virus is.

Question 30

What is an example of a virus that forms foci?

Answer 30

The Rous Sarcoma Virus (RSV)

Question 31

What is a transformation assay? What are its units? What does it measure?

Answer 31

Used for viruses that do not form plaques, but form foci. Measured in foci forming units/mL (FFU/mL). Transformation assays are used to measure the infectivity of a virus.

Question 32

Between a plaque assay and a transformation assay, which is the most effective way to measure viral infectivity?

Answer 32

A plaque assay, by far. Much more accurate.

Question 33

What type of viruses always kills the host cell by burst/lysis? When does it occur?

Answer 33

Cytopathic viruses. Each host cell has a finite ability to produce virions, and eventually the cell is killed through its lysis.

Question 34

Can a non-cytopathic virus cause cell lysis?

Answer 34

No.

Question 35

If a virus is **not cytopathic**, can infectivity be measured?

Answer 35

No, infectivity cannot be measured, physical measures must be used instead.

Question 36

What is the difference between measuring physical viral measures and measuring viral infectivity?

Answer 36

Physical: measuring how well the virus can produce virions. Ex. a virus can produce 40 virions. Infectivity: measuring how infectious the virions produced by a virus are. Measures of infectivity include the entire viral infectious cycle. Ex. virus produces 40 virions, how many of these virions are infectious?

Question 37

What is a hemaglutination assay in terms of physical viral measurement? Why is this considered physical measurement and not a measure of infectivity?

Answer 37

A hemaglutination assay involved adding red blood cells to a buffer, along with a virus. If recognition occurs between the red blood cell surface and the virus, a lattice will form (positive). If recognition does not occur, the red blood cells will remain at the bottom in a drop of blood (negative). Keep in mind that with decreasing dilution of the virus, less lattice is formed. This measures physical infectivity, of the first step of the viral infectious cycle: recognition.

Question 38

How does measuring viral enzyme activity occur? How is to a form of physical measurement?

Answer 38

Ex. Measuring the activity of reverse trasncriptase in a retrovirus. When reverse transcriptase is used, add radioactive nucleotides. The host cell cannot convert mRNA to DNA, but retroviruses can. This can proove the presence of radioactive reverse transcriptase, or proove that it is functional.

Question 39

What is immunostaining? How is it a form of physical measurement?

Answer 39

Can be direct (one antibody binding to one viral protein) or indirect (an antibody binding to another antibody which binds to the viral protein). The antibody is tagged flourescently. The antibody is then incubated on the monolayer, and the protein of interest can be visualized if the antibody binds. Allows you to detect the **location** of specific proteins.

Question 40

What is immunoblotting? How is it a method of physical measurement?

Answer 40

Immunoblotting involves taking antibodies and the cell lysate (post-viral infection) and incubating them. It involves running them on a gel and separating by size. Used to detect the **amount** of the protein of interest.

Question 41

What is the difference between immunostaining and immunoblotting?

Answer 41

Immunostaining: measures the location of the viral protein of interest. Immunoblotting: measures the amount of the viral protein of interest.

Question 42

What is sequencing as a form of physical measurement? When is it typically used?

Answer 42

Typically used for low abundance viral genes. Exponential growth of the gene product, and the revolution of deep sequencing. Viral gene sequencing is used to detect the presence of a specific mRNA. If transcription is occurring, it is present. If not, absent.

Question 43

How is flourescent tagging used as a form of physical measurement?

Answer 43

Take the green fluorescent protein gene and insert it before the viral gene. Virus will transcribe it, and the protein will fluoresce. Can be used to determine which cells have been infected.

Question 44

What are the different measures of infectivity? What are the different physical measures?

Answer 44

Infectivity: plaque assay, transformation assay Physical: immunoblotting, immunostaining, gene sequencing, hemaglutination assay, viral enzyme activity, fluorescent proteins