CMB1003/L16 Viruses as Tools

Question 1

Give 2 useful properties of viruses that we can exploit.

Answer 1

Natural gene-delivery vehicle
Trigger appropriate immune response
Many have cytopathic effect

Question 2

What does the term ‘attenuated’ mean?

Answer 2

Human viruses that have lost the properties to replicate efficiently in human cells

Question 3

Give 2 powerful promoters/enhancers.

Answer 3

Human cytomegalovirus (HCMV)
Simian virus 40 (SV40)
Human immunodeficiency virus LTRs

Question 4

How can viruses be used for gene expression?

Answer 4

A promoter region from a virus cloned into a DNA plasmid vector
Generate cDNA of protein wish to express from mRNA using PCR

Question 5

How are viruses used to express genes? (4)

Answer 5

Isolate ORF of interest
Clone into expression vector
Isolate/purify plasmid DNA
Transfect target cells & detect gene expression

Question 6

What is a VLP and why are they used as vectors?

Answer 6

A virus-like particle
Allow more efficient deliver than chemical/liposomal transfection

Question 7

Give 2 advantages to adenoviral vectors.

Answer 7

Small genome, easy to work with & purify
Will infect many cell types, high level of gene expression

Question 8

Give 2 disadvantages to adenoviral vectors.

Answer 8

Limited to ORFs <8kb
Immunogenic, means single shot
Transient expression

Question 9

Describe adenovirus replication in the lab. (2)

Answer 9

Most of late phase ORF are removed
Replace viral protein ORFs with ORF/protein of interest

Question 10

What is modified in adenoviral vectors before use? (3)

Answer 10

Missing E1 gene region (replication)
Missing all viral proteins
Require a ‘helper’ or ‘packaging’ cell-line

Question 11

Give 2 advantages to lentiviral vectors.

Answer 11

Small genome, easy to work with
Will infect many cell types, high level of gene expression
Infect/express in non-dividing cells
Stable gene expression over time, gene integrates into genome of target cell

Question 12

Give 2 disadvantages of lentiviral vectors.

Answer 12

Limited to ORFs <10kb
Safety concerned if transgene inserts near a proto-oncogene
Constitutive expression

Question 13

Give 3 different types of vaccine.

Answer 13

Live
Killed whole virus
Subunit vaccines
Gene delivery

Question 14

Describe the synthesis of attenuated vaccines. (4)

Answer 14

Pathogenic virus isolated and cultured on host cells
Virus incubated on cells from another host
Virus spontaneously mutates and grows on (monkey) cells
Virus can be used as a vaccine as it cannot grow on human cells

Question 15

Give 3 advantages to attenuated vaccines.

Answer 15

Mimic wildtype infection
Strong humoral & cellular responses
Induces innate immunity
Fewer doses required for complete immunity

Question 16

Give 3 disadvantages to attenuated vaccines.

Answer 16

Reversion can occur to wildtype
Cannot be used in immunosuppressed individuals
Required careful handling/storage (low temps)

Question 17

Define gene therapy.

Answer 17

Replacement of a defective gene function by introducing a ‘normal’ gene

Question 18

What is an oncolytic virus?

Answer 18

Virus which destroys tumour cells

Question 19

What is an oncogenic virus?

Answer 19

Virus which promotes development of tumour cells

Question 20

What can occur once a tumour cell lyses and releases oncolytic viruses (oncolytic virotherapy)? (3)

Answer 20

Destruction of tumour microenvironment
Infect more tumour cells
Systemic anti-tumour immune response

Question 21

Give 3 barriers to oncolytic therapy.

Answer 21

Overcoming vector neutralisation
Enriching T cell response
Increasing speed
Enhancing tumour uptake

Question 22

Give 3 advantages to phage therapy.

Answer 22

Specific to a bacteria spp.
Can evolve and adapt to resistance
Self-amplifying until all bacteria are dead

Question 23

Give 3 disadvantages to phage therapy.

Answer 23

Lysis of Gram -ve bacteria, release of endotoxins (sepsis)
Theoretical potential to transfer DNA between bacteria (Ab resistance)
May need cocktail of phage
Individualised

Question 24

What are lysins/endolysins?

Answer 24

Enzymes produced by some bacteriophages to cleave host cell wall during final stage of lytic cycle

Question 25

What is phage therapy used for? (2)

Answer 25

Treat burn infections
Treatment of biofilms