viral infectious cycle & virology methods

Question 1

describe the general viral infection cycle

Answer 1

attachment
- virus binds to receptors on the surface of a host cell
- interaction is often highly specific

entry
- virus or its genetic enters the host cell
- can be done thru direct fusion with the cell membrane or endocytosis (cell engulfs virus in a vesicle)

uncoating
- virus’s outer shell (capsid) is removed to reveal genetic material into the cytoplasm/nucleus

replication/transcription
- viral genome hijacks host cell machinery to replicate and transcribe the mRNA
- uses host cell’s ribosomes to synthesize viral proteins

assembly
- new viral particles are assembled from the viral genome and viral proteins

budding/release
- new viruses are released
- can occur thru lysis (burst) or budding (virus exits the cell gradually, taking part of the cell membrane with it)

— repeat

Question 2

describe what is meant by a susceptible and permissive cell… what is a resistant cell?

Answer 2

a cell that takes up a virus and allows the virus to replicate is susceptible and permissive

susceptible cell: functional receptor for virus

permissive cell: allows the virus to replicate

resistant cell: no receptor

Question 3

what host is generally used in labs when studying the infectious cycle?

Answer 3

in a lab setting, animal hosts can be costly, thus, we use fertilized chicken eggs which are composed of multiple cell types – they’re still used to replicate influenza virus to make the flu vaccine

we can also replicate vaccines in cell culture (bacteria)

Question 4

what are cytopathic effects? give examples

Answer 4

cytopathic effects are the different changes that a virus induces inside a cell

examples include:

cell lysis (burst)

1. syncytia
   - fusion of adjacent plasma membranes, resulting in a multinucleated array of cells
2. transformation
   - cells are no longer flat, but divide uncontrollably to become piles of round cells (cells are on top of each other)
3. cells no longer adhering to surface

(MAKE SURE YOU CAN LOOK AT IMAGES AND TELL WHICH IS OCCURING!)

Question 5

describe how one can measure infectivity via plaque assay

Answer 5

first used for bacteriophages

plaques
- an area where bacteria have been infected with viruses

you can count the number of plaques to establish the plaque forming units/ml (pfu/ml)

overlaying cells with agar, a gel-like substance prevents infected cells from releasing progeny (budding), effectively halting the spread and build up of plaque

in plaque assays where crystal violet will stain cells that are alive, white is infected

Question 6

what are the steps of measuring infectivity via plaque assays

Answer 6

1 ml of concentrated virus is diluted in 9ml of buffer - for optimal measurements
(this is considered a 10-fold dilution)

the step is repeated 8 times
hence, dilution of the final tube is 10^8

Question 7

how to calculate the degree of infectivity when you are using plaque assays

Answer 7

number of plaques x dilution

Question 8

describe measuring infectivity via particle to PFU

Answer 8

number of virus particles / number of infectious particles

a smaller ratio = more infective

larger ratio = less infective!!

Question 9

describe measuring infectivity via transformation assay

Answer 9

certain viruses do not form plaques but instead form foci

foci: localized concentrations of proteins or other cellular components that form within the cell nucleus

e.g. occurs in Rous Sarcoma Virus (RSV)

you can count foci and get foci forming units / ml

Question 10

describe the physical measurement of viruses via hemagglutination – why is this not a measure of infectivity

Answer 10

certain viruses contain proteins (hemagglutinin) that bind to the surface of the red blood cells

if a sample contains viruses, they will bind RBC and form a distinct crystal lattice structure that coats the side of the tube

lack of virus results in RBCs forming a “dot”

note that red blood cells can’t be infected as they lack the cell machinery – but hemagglutination is due to binding - hence, a physical measurement and not on of infectivity

Question 11

describe the physical measurement via viral enzyme activity

Answer 11

for example, could use the activity of reverse transcriptase (converts RNA to cDNA) in retroviruses (such as HIV)

in this situation, we will measure the amount of cDNA and not gDNA

Question 12

describe physical measurement via immunoblotting

Answer 12

separating proteins by size via electrophoresis

blot proteins onto a membrane

add specific antibodies to detect viral proteins

Question 13

describe physical measurement via fluorescent proteins

Answer 13

lights up viral sequences, indicating whether viral proteins are expressed or not

Question 14

what are examples of methods to measure infectivity?

Answer 14

plaque assays

particle-to-PFU

transformation assay

Question 15

what are examples of physical measurements for viruses?

Answer 15

hemagglutination assay

viral enzyme activity

immunostaining of proteins

immunoblotting

genomic sequencing

fluorescent proteins