Practical Virology

Question 1

What is MOI (Multipicity of infection)?

Answer 1

the ratio of infective virus to susceptible host cells

Question 2

How do you calculate MOI?

Answer 2

total number of bacteriophages in staring culture
/
Total number of bacterial cells in starting culture

Question 3

3 ways of studing quantity of viruses?

Answer 3

Plaque Assay
Hemagluttination
Quantal dose response (LD50)

Question 4

2 ways of studing structure of viruses?

Answer 4

Electron microscopy / X-ray crystallography

Density gradient centrifugation

Question 5

2 ways of studying replication of viruses?

Answer 5

Viral proteins (serological methods)
Viral nucleic acid e.g. qRCR, sequencing

Question 6

3 ways of studying function of viruses?

Answer 6

Tissue culture / animal studies / rational drug design

Question 7

What are the steps of a plaque assay?

Answer 7

Make serial dilutions of the virus
Plate dilutions onto susceptible cells.
After attachment, overlay cells with semi solid media to restrict diffusion of virus particles
Restricted cell-to-cell spread of virus results in localized destruction of cell monolayers visible as “plaques”

Question 8

How do you calculate the amount of virus on the plate?

Answer 8

PFU/ml = no. of PFU on plate/ (volume of sample plated in ml x diltution)

Question 9

What is LD50?

Answer 9

Dose at which 50% infected organisms die

Question 10

What are examples of some seminal experiments performed using bacteriophages?

Answer 10

Ellis & Delbrück, 1939: One step growth curve

Luria & Delbrück, 1943: Fluctuation test

Hershey & Chase, 1952: Nature of genetic material

Fiers et al., 1976: First genome sequence (ssRNA phage MS2)

Question 11

What happened in the 1952 Hershey and Chase experiment on the Nature of genetic material?

Answer 11

Phage T2 propogated into E.Coli
E.Coli labelled with one of two radioisotopes
35S (into sulphur-containing amino acids in proteins)
32P (into nucleic acids)

Allowed to attach to cells, then mix was homogenised briefly

35S particles stayed in the supernatant but 32P particles entered the cells

So they concluded that the DNA genome had entered the cells and initiated the infection

Question 12

What happened in the Luria & Delbrück, 1943: Fluctuation test?

Answer 12

Observed resistant bacteria in phage experiments

Hypothesised that mutations would be highly variable if if
spontaneous and lack varirability if induced by media

Demonstrated mutations arise spontaneously

Question 13

What happened in the Ellis & Delbrück, 1939: One step growth curve?

Answer 13

First experiment to show virus replication in stages

Phages added to rapidly growing bacteria

Culture then diluted after a few minutes

Samples taken at intervals and plated on agar plates to analyse bacterial cells and onto bacteria lawns for phage analysis

Question 14

Regarding the one step growth curve what is the latent period?

Answer 14

The time between the start of the infection and the appearance of the first new extracellular virus particles

Latent period = eclipse period + intracellular accumulation period

Question 15

What happens during the eclipse period

Answer 15

no infectious particles detectable

Question 16

How long is the latent period for most bacteriophages?

Answer 16

~20-25mins

Question 17

What happens during intracellular accumulation?

Answer 17

progeny viruses assembled but not released

Question 18

What happens during the rise period of the one step growth curve?

Answer 18

Release of mature virions from the infected cell

Question 19

What is the burst size?

Answer 19

number of virus particles produced per infected cell

Question 20

How does low MOI affect the one step multiplication curve?

Answer 20

First and second burst

Second burst is curved on graph

Question 21

What is the Poisson distribution?

Answer 21

distribution of bacteriophages among host bacterial cells

Question 22

Poisson distribution

P(n) = (m^n x e^-m) / n!

Answer 22

P(n) = fraction of cells infected by the n virus particles
m = MOI
e = 2.71828182845904
n! = n factorial

Use P(n>0) = 1 - P(0)