Lecture 2- One Step Growth Experiment, Virion Structure

Question 1

What is a one-step growth experiment?

Answer 1

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.

Question 2

How do you ensure every cell is infected in a one step growth experiment?

Answer 2

The ratio of infecting virus particles to cells is called the multiplicity of infection and is abbreviated m.o.i.
moi= total number of pfu’s (viral particles)/total number of cells= average pfu/cell
Question is, how high does the moi have to be to get every cell infected?

Question 3

How to use the poisson with moi?

Answer 3

Relationship between moi and fraction of cells infected
Poison: relationship of moi and fraction of cells infected
Calculate number of cells receiving each virus particle number
Fraction of cells receiving r virus particles = Pr (r)
To calculate Pr (r) need to know the average number of pfu’s per cell and this value is abbreviated “s” (s=moi) (s = average number of plaque forming units per cell)
Pr (r) = ((s^r)(e^-s))/(r!)
Pr (0) and Pr (1) are the same, will decrease after that
double check this: result is percent of cells left uninfected

Question 4

What is the design of a one-step growth experiment?

Answer 4

At each of a series of time points, remove a sample from the infected culture, split the sample in two and make two different determinations.

1) Extracellular virus. Centrifuge; cells at bottom and viruses will be elsewhere. Look at supernatant.
2) Disrupt cells (get both) and look at intracellular and extracellular virus

Question 5

Graphing results of one-step growth experiment

Answer 5

Pfu/cell vs time
Pfu/cell starts high (moi of 10), centrifuge and line goes down b/c no ex virus, goes down as viruses infect host cells. From beginning until lysis is called latent period. Within latent period is eclipse phase, which has early phase and late phase. Early phase is before genome replication starts and late phase is after. Eventually goes up as viral particles get released. “Intracellular and extracellular virus” line starts to go up first, then “extracellular virus only” line. Both plateau at same place (lysate- which is what you do a plaque assay on)
Note: early stages: int and extracellular virus disappear at same time. Once inside host, virus loses identity.

Question 6

Latent period

Answer 6

Bacteriophages 15-60 minutes

Animal viruses 3-72 hours

Question 7

Burst size

Answer 7

Virus yield (pfu)/number of infected cells
(10's to millions)

Question 8

Infectivity ratio

Answer 8

number of infectious particles/total number of particles (phage: 0.1 to 1.0; animal viruses: 10^-2 - 10^-6)

Question 9

Naked capsid viruses- different structures

Answer 9

Helical symmetry- cylindrical shape
(ex: tobacco mosaic virus TMV))
Cubic symmetry- spherical shape
(ex: polio and adenovirus)

Question 10

Enveloped viruses- different structures

Answer 10

Helical symmetry
(ex: VSV)
Cubic symmetry
(ex: herpes)

Question 11

TMV structure

Answer 11

helical/cylindrical

not all same size, but same diameter

Question 12

Poliovirus structure

Answer 12

same size and shape

knobs/bumps

Question 13

Bacteriophage phiX174 structure

Answer 13

12 bumps

Question 14

Adenovirus structure

Answer 14

12 spikes

Question 15

Influenza A structure

Answer 15

enveloped, squishy envelope, not all same size

Question 16

Bacteriophage T4 structure

Answer 16

phage structure (head, fibers, etc.)

Question 17

Archaeal structure

Answer 17

Odd shapes, lots of variety

Question 18

Genome diversity

Answer 18

Single-stranded DNA (ssDNA), can be linear or circular
Double stranded DNA (dsDNA), can be linear or circular
Single-stranded RNA (ssRNA), linear or linear segments
Double stranded RNA (dsRNA), linear segments