(Section A: Virology) Lecture 02: Viral Infectious Cycle + Virology Methods Flashcards
Describe:
The Viral Infectious Cycle
- Get inside the cell
- Expose viral nucleic acids
- Seek host ribosomes
- Multiply in host and escape host cell
Define:
Virions
Fully infectious virus that attaches and enters cell
What aspects of a host cell do viruses require?
- Energy (to move around)
- Transport vesicles (to move correctly)
- Protein translation machinery
Define:
Susceptible cell
Functional receptor for virus
Define:
Permissive cell
Allows the virus to replicate
Define:
Resistant cell
Not a receptor for virus
What aspects must a cell have in order for it to be infectible by viruses?
- Susceptibility
- Permissibility
What are examples of hosts for viruses?
- Whole animal hosts (for infection, vaccine and drug studies)
- Fertilized chicken eggs (for flu vaccine etc.)
- Cell culture
What do normal cells appear under microscope?
Stretched out, bright cells that adhere to the plate
What does a virus do to cells under the microscope?
Black spots appear in the culture
* Means cells cannot adhere or stretch
* Means small nuclei or necrosis has occurred
Define and List:
CPEs
Cytopathic Effects:
1. Cell lysis (bursting cells)
2. Syncytia (bunching cells)
3. Transformation (growing in piles, forming foci)
When measuring infectivity of a virus, what is assumed?
The virus has gone through the Virus Infectious Cycle
What procedure is used to measure viral infectivity?
Plaque Assay
How is a plaque assay set up?
- Agar plate with bacteria
- “Bacteria lawn” that is permissive and susceptible
What happens when virus infects bacteria in plaque assays?
Phage plaques occur
* Dark patches
* Areas where bacteria has died due to virus
When virus is added to the cells, why is another layer of gel overlayed on the plate?
Prevents progeny from lysing cells from infecting more cells
How is the number of infectious particles counted?
- 1 ml of concentrated virus is dilute in 9 mL of buffer
- This is a 10 fold dilution; this is done 8 times (a dilution of 10^8)
- Perform plaque assay
- Count plaques (average)
- Find pfu/mL
What is pfu/mL?
Plaque Forming Units / mL
What is the particle to PFU ratio?
Number of virus particles / Number of infectious particles
True or False:
If the particle to PFU ratio is higher, the virus is more infectious
False, the lower the ratio the more infectious the virus
What is an alternative method to measuring infectivity of viruses? (instead of using plaque assays)
Transformation assay
* Similar concept
* Count the foci to get Foci Forming Units / mL
What are methods to identify the presence of viruses?
- Hemoglutination
- Viral Enzyme Activity
- Immunostaining
- Immunoblotting
- Sequencing
- Fluorescent Proteins
Describe:
Hemagglutination test
Certain viruses attach to RBCs
* This forms distinct lattice structures
* These structures coat the sides of a test tube
* Lack of the virus results in the RBCs forming a dot in the test tube
Describe:
Viral Enzyme Activity test
There certain viral-only enzymes such as reverse transcriptase (RT) in retroviruses
* Presence of these show that the virus is present
Describe:
Immunostaining test
Using antibodies to detect viral proteins
Describe:
Immunoblotting test
Similar to immunostaining
* The cells are crushed and go through the blotting process to identify viral proteins
Describe:
Sequencing test
Finding viral genome sequence in samples
* Useful in low viral abundance genes
Describe:
Fluorescent proteins test
Lighting up areas of cell where viral replication is occurring
