Mechanisms of Pathogenesis

Question 1

What are animal models used for in viral research?

Answer 1

Used to measure and observe disease progression - can study host responses to infection

Question 2

What are some common animal models?

Answer 2

• Human receptors expressed in mice
• Viral gene or genome expressed in mice
• Immune cells depleted in mice
• Immune mediators deleted in mice
• Immune mediators stimulated in mice
• Clonal T cell receptor in mice

Question 3

What is the problem with using mice to model viral disease?

Answer 3

There are some differences between mice and humans - conclusions from studies using mice may not hold true in humans

Question 4

What is viral virulence?

Answer 4

The ability of a virus to cause disease in the host

Question 5

What quantifiable factors are used to define viral virulence?

Answer 5

• Time to death
• Time for disease sign to appear
• Measurement of fever or weight loss
• Measurement of lesions or circulating human cells

Question 6

What factors can cause study bias in viral virulence studies?

Answer 6

• Dose
• Route of infection
• Species
• Age
• Gender
• Host suceptibility

Question 7

Can we compare viral virulence of different viruses?

Answer 7

• Virulence of different viruses are hard to compare as they have different measurable outcomes
• For similar viruses, the assays must be the same

Question 8

What is the goal of viral virulence studies?

Answer 8

To identify host and viral genes that determine virulence

Question 9

How are virulence genes identified?

Answer 9

By mutations or deletions - can see whether it causes a reduction or absence of disease

Question 10

What are the functions of viral virulence genes?

Answer 10

• Viral replication (in target tissues)
• Invasiveness
• Tropism
• Alter host defence mechanisms
• Increase viral spread in the host
• Intrinsic cell killing effects

Question 11

What are restriction factors?

Answer 11

Factors that impair or reduce replication and pathogenesis

Question 12

How is genome wide screening used to identify restriction factors?

Answer 12

• Need a pool of long survival particles - viruses that can be used to infect eukaryotic cells that are expressing CRISPR library of guide RNA that have the ability to knock out every gene in the human genome
• Need reporter cells
• Need reporter virus - e.g. in Influenza one of the genes has been replaced with GFP so it’s fluorescent
• Transduce cells with lentivirus to generate a library of cells with individual gene knock outs
• Infect cells with fluorescent virus
• Cells that are supporting the infection will glow green - replicating the fluorescent virus and the more infected the cell is the more it glows green
• Use flow cytometry to separate the population of cells - sort specifically the top 10% of cells that are highly fluorescent
• Extracting RNA from the highly infected cells and sequence it - can identify the guide RNA that led to a high infection - allows identification which gene silencing increases replication
• Controls - is the adapted virus as efficient at replicating as wild type virus - does the adapted virus have the same virulence as the wild type virus
• Next step is to perform functional studies to see how the restriction factor works

Question 13

How do SERINC3 and SERINC5 restrict HIV?

Answer 13

• In wild type infections the virulence factor Nef counteracts the interaction of the SERINC proteins
• When Nef is expressed, it basically removes the SERINC proteins from the membrane - allows the virus to exit the cell and infect another cell
• When Nef isn’t expressed, SERINC can insert itself into the membrane of HIV virions - blocks the pores formed with the target cell - prevents delivery of capsid into new cells