Lecture 29: Virology IV

Question 1

What was the earlier way of growing viruses?

Answer 1

Most viruses were grown in laboratory animals, in embryonated eggs (fertilized eggs that started development). Different viruses were inserted into different parts of the egg.

Question 2

Why were viruses able to be grown in embryonated eggs?

Answer 2

Because the interior is a sterile (aseptic) and nutrient-rich environment

Question 3

Aside from embryonated eggs, what is the other way of culturing viruses?

Answer 3

Growing them in tissue culture cells

Question 4

When did culturing viruses in cells become possible?

Answer 4

In 1949.

Question 5

Who first grew viruses in cells? What kind of virus?

Answer 5

Enders, Weller, and Robbins. They cultured poliovirus.

Question 6

What are the 3 steps involved in virus cultivation in tissue culture cells?

Answer 6

1. A tissue is treated with enzymes to separate the cells.
2. Cells are suspended in a culture medium.
3. Normal cells or primary cells grow in a monolayer across the container OR continuous cells grow but not in a monolayer.

Question 7

What are the two types of cell cultures for viruses? Name an advantage and disadvantage of each.

Answer 7

Primary cells (non cancerous): cells don’t last for very long but physiologically relevant
Continuous cell lines (cancerous): cells grow infinitely but are less physiologically relevant

Question 8

Give an example of a continuous cell line commonly used for viral growth.

Answer 8

HeLa cells.

Question 9

What are cytopathic effects?

Answer 9

They are the morphological changes observed in cells infected with a virus.

Question 10

Give two examples of cytopathic effects.

Answer 10

Cell lysis and cell fusion (syncytia).

Question 11

Viruses that kill cells by lysis are known as […]

Answer 11

Lytic viruses

Question 12

What is syncytia?

Answer 12

It is a type of cytopathic effect where the virions produced in an infected cell will cause the fusion of cells.

Question 13

What is the central notion behind the plaque assay idea?

Answer 13

That a single virus is sufficient to produce a plaque from cell lysis.

Question 14

Explain how plaque assays work.

Answer 14

A serial dilution is done using a virus stock, and a small amount is applied to a few areas on a plate. Knowing the dilution factor and assuming that each plaque was produced by one viral particle, you can calculate how many viruses were in the biological sample.

Question 15

The quantification of plaque assays is reported as […]

Answer 15

Plaque-forming units (PFUs)

Question 16

The plaque assay method serves to quantify […]. Explain why.

Answer 16

The number of infectious particles. This is not the same as the true number of viral particles, as some might not be infectious and thus not cause a plaque.

Question 17

What is a plaque?

Answer 17

It is an area of cell lysis.

Question 18

When using the plaque assay method, how do you ensure that the virions only infect adjacent cells to the infected cell?

Answer 18

You cover the cells with a thin layer of agarose to reduce diffusion of the virions.

Question 19

What is MOI?

Answer 19

It is a concept in the plaque assay method that refers to the average number of infectious particles added per cell.

Question 20

What is the relevance of MOI to the plaque assay method?

Answer 20

It reflects how when cells are mixed with virus, although the average might reflect the goal of one virus per cell, but in reality, some cells will be uninfected while others might receive several particles. This is important given that the method is based on one virion per plaque.

Question 21

The distribution of virus per cell is best described using the […] distribution

Answer 21

Poisson

Question 22

What % of cells infected can you expect from an MOI of:
a) 0.25
b) 0.5
c) 1.0
d) 2.0
e) 3.0
f) >4.0

Answer 22

a) 20%
b) 40%
c) 60%
d) 85%
e) 95%
f) Near 100% (aymptotic)

Question 23

What is the purpose of plaque purification?

Answer 23

It is a method used to produce a pure virus stock using plaque assays. It is a way of reducing the probability of accidentally including more than one virus in the stock.

Question 24

Explain how plaque purification works.

Answer 24

The liquid in one of the plaques, which hypothetically should be caused by one virus but might not be, is diluted and is used to infect fresh cells. This process is done 3 times.

Question 25

If you are dealing with a non-lytic virus, what method can be used as an alternative to the plaque assay?

Answer 25

Focus forming assay (FFA)

Question 26

How does FFA work?

Answer 26

Instead of relying on cell lysis, it uses immunostaining techniques using antibodies specific for a viral antigen to detect infected host cells.

Question 27

What is a one-step growth curve?

Answer 27

It is when you put pure viral stock on the cells and watch how long it takes to start reproducing and lysing cells.

Question 28

In the one-step growth curve, how will the PFU change intracellularly over time?

Answer 28

It will initially be flat, then start climbing linearly after a few hours. It will eventually plateau.

Question 29

In the one-step growth curve, how will the PFU change extracellularly over time?

Answer 29

It will start climbing linearly a few hours after it starts climbing intracellularly.

Question 30

What is the eclipse period in the one-step growth curve? Explain what is happening.

Answer 30

It is the phase during which intracellular PFU hasn’t started climbing. It is the phase between virus entry into the cell and the appearance of virus progeny inside the cell.

Question 31

What is the latent period of the one-step growth curve?

Answer 31

It is the phase from the start time to when the extracellular PFU starts climbing. It is the period between infection and cell lysis/release of infectious virions.

Question 32

What is the yield of a virus?

Answer 32

It is the point in time at which the amount of virus produced is fixed, as all cells have been infected and lysed.