Lecture 1- Virus Definition, Virus-host Interactions, and Plaque Assay

Question 1

Virus

Answer 1

A genetic element that replicates intracellularly and programs the synthesis of particles that transmit its genome from one cell to another

Question 2

Bacteriophage

Answer 2

Bacterial virus, also called phage. Virus that infects bacterial cells

Question 3

Chemical constituents of cells and viruses

Answer 3

Cells: mostly ribosomal RNA, not much DNA, lots of protein
Viruses: 1-50% dna/rna, 50-90% everything else protein, barely anything else

Question 4

Expanded Definition of a Virus

Answer 4

Small particle (20-1000 times smaller than cells). Particle called a virion
Genetic element that moves from cell to cell
Genes enclosed in some kind of shell (minimally protein)
No energy metabolism or ribosomes
Intracellular parasite (infects host cell)

Question 5

Cellular Multiplication

Answer 5

Exponential/logarithmic growth. This can be rapid, viruses more rapid

Question 6

Virus Multiplication

Answer 6

In the time it would take for host cell to divide, 100’s to 1000000’s viral particles have been created

Question 7

Virus-Host Relationships

Answer 7

1) Viruses generally can only infect particular types of cells
2) The types of cells a virus can infect called host range
3) Circumstances of infection can determine outcome (environmental influences, cell physiology, etc.)

Question 8

What are the two possible outcomes after uncoating (capsid breaks down)?

Answer 8

First steps: 
1) adsorption
2) penetration
3) uncoating
Next: either productive response or nonproductive response

Question 9

What is the productive response? (next few steps and potential outcome for the host cell)

Answer 9

4) component production
5) assembly (encapsidation) (maturation)
6) Release. Two possible outcomes for release
a) Cell death (most of the time). Occurs with lytic infection, cytocidal virus.
b) Cell survival (some of the time). Chronic or persistent infection (filamentous bacteriophages and retroviruses).
In productive response, more viruses are produced

Question 10

What is the non productive response?

Answer 10

No viruses are produced. Goes into a latent state. This occurs with temperate viruses (lysogen by phages, latent infection or transformation by animal viruses). From latent state, can experience induction.
Induction brings back to beginning of productive response.

Question 11

Plaque Assay

Answer 11

A plaque assay is used to determine the number of infectious visions in a virus stock. One infectious particle = one plaque.

Question 12

What is a plaque?

Answer 12

A clearing in the middle of an agar plate, caused by 1 infected bacterial cell.

Question 13

What does the number of plaques mean?

Answer 13

Number of plaques is the original number of infectious viruses. This is why you must dilute stock solution of viruses before mixing with host cells (serial dilution).

Question 14

Why aren’t all cells infected on the plate?

Answer 14

Limited diffusion by agar (limited movement). Viruses are limited to zone where they are placed. Also, each plaque came from 1 virus particle infecting one cell.

Question 15

What is the overall plaque assay protocol?

Answer 15

1. Serially dilute virus stock
2. Mixx diluted virus with cell suspension
3. Incubate mixture to allow adsorption
4. Imbed the mixture in agar on a petri plate
5. Incubate to allow plaques to form
6. Count plaques and calculate titer
7. Express titer as plaque-forming units per ml (pfu/ml)

Question 16

What is the overall plaque assay protocol?

Answer 16

1. Serially dilute virus stock
2. Mixx diluted virus with cell suspension
3. Incubate mixture to allow adsorption
4. Imbed the mixture in agar on a petri plate
5. Incubate to allow plaques to form
6. Count plaques and calculate titer
7. Express titer as plaque-forming units per ml (pfu/ml)

Question 17

What is a one-step growth experiment?

Answer 17

1. Single cycle of infection- host cells and virus, single cycle of infection
2. Infect every cell in the population- don’t want a mixed population, must infect all cells.
3. Achieve synchrony for infected cell population- at each moment during infection, want what’s happening in each cell to be the same.