Viruses in Research Flashcards
What are useful properties in viruses that we can exploit?
- natural gene delivery vehicle
- many have a cytopathic (killing) effect
- they can trigger an appropriate immune response
- we can remove components that make the virus harmful by using ‘attenuated’ viruses as vaccines and using non-human viruses as vectors/ vaccines.
What does attenuated mean?
This generally refers to human viruses that have lost properties to replicate efficiently in human cells.
Therefore, they wont cause disease.
An example is the MMR vaccine and polio vaccine.
How is virus gene expression helpful?
Viruses have very powerful gene expression and some viruses can express proteins that switch off cellular gene expression.
Elements of viruses are used to express many cellular proteins as they are powerful promoters/ enhancers - this is a routine process in many labs.
What is the process of viral gene expression modification i na lab?
A promoter region from a virion is cloned into a DNA plasmid vector (gene expression vectors) Modern vectors will have multiple restriction enzyme sites for ease of cloning. They will have a polyadenylation sequence (to add a poly A tail) and they may have a splice site.
What is pCDNA3.1?
It is a small plasma that replicates a bacteria. It has multiple cloning types that allows great flexibility.
A couple of the cloning sites are picked, a gene of interest is put between a promoter and polyadenylation signal in order to construct a gene that can go into a eukaryotic cell.
- Ampicillin and pUC ori - replication and selection in bacterial cells.
- Neomycin and SV40 ori - replication and selection in human cells.
What is transfection?
This is the process of getting DNA into a cell.
How do we use viruses as vectors?
Instead of using a plasmid, we would put the gene of interest in a virus-like particle (VLP) which is essentially a particle that looks and acts like a virus but isnt one.
This allows a more efficient delivery than chemical/ liposomal transfection as it uses the viruses own entry mechanism and can infect hard to transfect lines such as neurone delivered lines.
What is an adenoviral vector?
It is a virus with a small genome and has a high level of gene expression. It is easy to work with and purify and can infect many cell types. It is used as a vector in delivering the Covid vaccines.
However, it has a small genome which means only small amounts of DNA can be used. it is immunogenic, this isnt helpful when repeating a therapy as the body will have made antibodies to combat the vector, therefore not allowing the therapy to work. It has transient expression (it will express but turn off after a while).
What is the replication of adenoviruses?
We dont want the virus to replicate and form and get out of the cell, we only want it to deliver bits of DNA. So, we remove the E1 section of the genome which has replication factors in, thus ceasing replication of the adenovirus.
What are lentiviral vectors?
HIV is an example of a lentiviral (retroviral) virus. HIV can be used as a vector. We remove ALL of the HIV genes so it cant replicate and cause disease. We also remove the HIV surface proteins as these are what target and lyse T cells, causing AIDS.
Lentiviral vectors are stable unlike adenoviruses which means they can produce gene expression over a longer period of time which is excellent for gene therapy. They can also infect non-dividing cells.
Why are viruses used as vaccines?
Edward Jenner - infected 8yo James Phipps with material from cowpox lesion, then when challenged with smallpox, we was resistant to the infection.
This works as infecting with the virus produces the correct B and T cells to fight the virus when it actually infects the body. vaccines mimic initial exposure to a pathogen, by getting booster vaccines, it further enhances speed and memory of it and therefore your body can fight the infection quicker with each ‘booster’ or exposure.
What are the types of vaccine?
Live = attenuated e.g. MMR. It is heterologous which means it uses a closely related and less virulent virus.
Killed whole virus = via chemical or radiation, these can be used as vaccines e.g. polio.
Subunit vaccines = uses components purified from a whole virus e.g. influenza.
Gene delivery = gene therapy. The expression of a gene that encodes immunogenic targets from a pathogen i.e. coronavirus spike protein.
What are subunit vaccines?
The principle is to vaccinate with just the immunogenic viral proteins, rather than the whole virus.
An example is the influenza vaccine that consist of HA and NA proteins purified from virus stocks. Or, recombinant protein expressed from cloved viral genes e.g. HBV.
What is gene therapy?
It is the replacement of a defective gene function by introducing a ‘normal’ gene. There are 2 approaches:
Ex vivo - cells are removed from the body, modify them, and then return them.
In vivo - the replacement gene is introduced directly into the body via a gene vector or virus.
What is oncolytic vs oncogenic?
Oncolytic = viruses destroying tumour cells
Oncogenic = viruses promoting development of tumour cells.
