(Section A: Virology) Lecture 02: Viral Infectious Cycle + Virology Methods

Question 1

Describe:

The Viral Infectious Cycle

Answer 1

1. Get inside the cell
2. Expose viral nucleic acids
3. Seek host ribosomes
4. Multiply in host and escape host cell

Question 2

Define:

Virions

Answer 2

Fully infectious virus that attaches and enters cell

Question 3

What aspects of a host cell do viruses require?

Answer 3

1. Energy (to move around)
2. Transport vesicles (to move correctly)
3. Protein translation machinery

Question 4

Define:

Susceptible cell

Answer 4

Functional receptor for virus

Question 5

Define:

Permissive cell

Answer 5

Allows the virus to replicate

Question 6

Define:

Resistant cell

Answer 6

Not a receptor for virus

Question 7

What aspects must a cell have in order for it to be infectible by viruses?

Answer 7

1. Susceptibility
2. Permissibility

Question 8

What are examples of hosts for viruses?

Answer 8

• Whole animal hosts (for infection, vaccine and drug studies)
• Fertilized chicken eggs (for flu vaccine etc.)
• Cell culture

Question 9

What do normal cells appear under microscope?

Answer 9

Stretched out, bright cells that adhere to the plate

Question 10

What does a virus do to cells under the microscope?

Answer 10

Black spots appear in the culture
* Means cells cannot adhere or stretch
* Means small nuclei or necrosis has occurred

Question 11

Define and List:

CPEs

Answer 11

Cytopathic Effects:
1. Cell lysis (bursting cells)
2. Syncytia (bunching cells)
3. Transformation (growing in piles, forming foci)

Question 12

When measuring infectivity of a virus, what is assumed?

Answer 12

The virus has gone through the Virus Infectious Cycle

Question 13

What procedure is used to measure viral infectivity?

Answer 13

Plaque Assay

Question 14

How is a plaque assay set up?

Answer 14

• Agar plate with bacteria
• “Bacteria lawn” that is permissive and susceptible

Question 15

What happens when virus infects bacteria in plaque assays?

Answer 15

Phage plaques occur
* Dark patches
* Areas where bacteria has died due to virus

Question 16

When virus is added to the cells, why is another layer of gel overlayed on the plate?

Answer 16

Prevents progeny from lysing cells from infecting more cells

Question 17

How is the number of infectious particles counted?

Answer 17

1. 1 ml of concentrated virus is dilute in 9 mL of buffer
2. This is a 10 fold dilution; this is done 8 times (a dilution of 10^8)
3. Perform plaque assay
4. Count plaques (average)
5. Find pfu/mL

Question 18

What is pfu/mL?

Answer 18

Plaque Forming Units / mL

Question 19

What is the particle to PFU ratio?

Answer 19

Number of virus particles / Number of infectious particles

Question 21

True or False:

If the particle to PFU ratio is higher, the virus is more infectious

Answer 21

False, the lower the ratio the more infectious the virus

Question 22

What is an alternative method to measuring infectivity of viruses? (instead of using plaque assays)

Answer 22

Transformation assay
* Similar concept
* Count the foci to get Foci Forming Units / mL

Question 23

What are methods to identify the presence of viruses?

Answer 23

1. Hemoglutination
2. Viral Enzyme Activity
3. Immunostaining
4. Immunoblotting
5. Sequencing
6. Fluorescent Proteins

Question 24

Describe:

Hemagglutination test

Answer 24

Certain viruses attach to RBCs
* This forms distinct lattice structures
* These structures coat the sides of a test tube
* Lack of the virus results in the RBCs forming a dot in the test tube

Question 25

Describe:

Viral Enzyme Activity test

Answer 25

There certain viral-only enzymes such as reverse transcriptase (RT) in retroviruses
* Presence of these show that the virus is present

Question 26

Describe:

Immunostaining test

Answer 26

Using antibodies to detect viral proteins

Question 27

Describe:

Immunoblotting test

Answer 27

Similar to immunostaining
* The cells are crushed and go through the blotting process to identify viral proteins

Question 28

Describe:

Sequencing test

Answer 28

Finding viral genome sequence in samples
* Useful in low viral abundance genes

Question 29

Describe:

Fluorescent proteins test

Answer 29

Lighting up areas of cell where viral replication is occurring