ICL 1.17: Methods in Virology

Question 1

what are the 3 ways in which viruses can be cultured?

Answer 1

1. animal hosts
2. eggs
3. cell culture

**but not all viruses can be cultured in labs!!! this makes it really hard to study some of them

Question 2

what is the purpose of culturing a virus?

Answer 2

1. to isolate and identify viruses in clinical samples
2. for research
3. to prepare viruses for vaccines

Question 3

what are the different methods/tests used to detect and analyze viruses?

Answer 3

1. plaque assay
2. ELISA
3. western blot
4. RT-PCR/PCR
5. sequence analyses

Question 4

what does CPE stand for?

Answer 4

cytopathic effect

Question 5

what is CPE?

Answer 5

morphological changes in a cell caused by virus infection

ex. cell rounding, swelling, fusion, multi-nucleated giant cells, inclusion bodies, etc.

not all viruses cause CPE!!

CPE can be diagnostic but not always

Question 6

what are inclusion bodies?

Answer 6

a DNA virus replicates in the nucleus of a host cell so when you look at an infected cell, there will be an inclusion in the nucleus where the virus is replicating!

on the other hand, viruses that replicate in the cytoplasm can cause cytoplasmic inclusions

Question 7

what is syncytia?

Answer 7

multi-nucleated giant cells (it’s a CPE of some viruses)

it’s when a cell has not fully divided so you have multiple nuclei and one really large body

it’s caused by the expression of certain viral proteins on the surface of the cell leading to their fusion

Question 8

what are the two hallmarks of CPE?

Answer 8

1. syncytia

2. loss of cell shape

Question 9

which virus families are characterized by syncytia formation under physiological conditions?

Answer 9

paramyxoviruses and herpesviruses

other families can also cause syncytia formation, but for these two cytopathic effect is used for diagnosis

Question 10

how does a plaque assay work?

Answer 10

1. a single PFU infects a susceptible cell and produces viral progeny
2. the agar prevents the progeny from diffusing but neighboring cells can be infected and release a second batch of viral progeny
3. the process continues during a third round of replication
4. the infected cells detech from the flask leaving an empty space; a plaque!

the identification of plaques can improve using a dye to stain remaining cells so you can see the hole where the plaque once was

slide 9

Question 11

what is a PFU?

Answer 11

PFU = plaque forming unit

one virus can infect a single cell, spread and kill surrounding cells to cause a plaque (a clear area of dead cells) surrounded by live cells

Question 12

what’s the formula for amount of virus in the plaque assay?

Answer 12

titer (PFU/mL) = plaque #/ (dilution * volume)

ex. 30 plaques/.2 mL of sample = 150

Question 13

how do neutralizing antibodies work to increase specificity of plaque assays?

Answer 13

in our own body, we have antibodies that bind to the surface of a virus and prevent it from binding to any host cells; aka they neutralize the virus

so in the lab, if we add the patient’s serum with neutralizing antibodies in it to a panel of different rhinoviruses, we can see which ones react and this would be diagnostic for a specific virus!

with a plaque assay, it’s hard to say that the plaque in the dish was formed due to a particular virus; we need it to be more specific

so if we incubate the patient’s serum with cells that are infected with a certain virus, we can see if plaques form!

if plaques form this means that there’s aren’t antibodies against that virus but if no plaques form, it means that the patient can full neutralize the test virus!

so neutralizing antibodies can add specificity to plaque assays!

Question 14

what are neutralizing antibodies?

Answer 14

in our own body, we have antibodies that bind to the surface of a virus and prevent it from binding to any host cells; aka they neutralize the virus

Question 15

what does ELISA stand for?

Answer 15

enzyme immunosorbent assay

Question 16

what are steps in an ELISA for HIV?

Answer 16

1. HIV antigen is coated onto the walls of a microtiter plate
2. the patient’s serum is placed in the wells; if it contains HIV antibdodies these will bind with the antigens
3. the serum is then washed from the wells
4. antihuman antibody bound to an enzyme is now added

this “indication conjugate” attaches to the HIV antibodies already bound to the HIV antigen

5. the wells are then washed to remove any unbound conjugate
6. finally, a substrate is placed in the wells

enzyme attached to the antibody/antigen complex catalyzes a color-producing reaction, indicating the presence of HIV antibody in the patient’s serum

samples are considered positive when the color has twice as much intensity as the negative control

Question 17

what is ELISA used for?

Answer 17

ELISA is commonly used as a quantitative method to determine the amount of diverse proteins, including viral antigens, cytokines and hormones

the concentration of an unknown can be calculated by running in parallel known amounts of the protein in question

the intensity of the color is directly correlated to the amount of protein present

Question 18

what could cause the wells of an ELISA to remain clear and not turn any color when testing for a certain virus?

Answer 18

1. the disease is not due to a viral infection
2. the disease is not influenza
3. the disease is due to an influenza virus that has an HA that is not 1,2, 3, or 5, and an NA that is not 1, 2, or 3
4. the child has not yet developed antibodies against influenza

Question 19

what are western blots used for?

Answer 19

they identify specific viral products based on the size of the protein(s) detected

they disregard false positives due to binding of serum antibodies to non-viral proteins which are identified as bands with incorrect molecular weight

Question 20

what are the steps in a western for HIV?

Answer 20

1. purified HIV antigen mixture is layered onto a gel slab and electrophoresed

the antigens migrate through the gel, with the higher molecular weight proteins forming bands near the top

3. the protein bands are then transferred onto nitrocellulose filter paper which is cut into strips and incubated with the patient’s serum

if the serum contains HIV antibodies, they will bind with their corresponding anitgen bands

3. after washing, the paper strip is incubated with an antihuman antibody bound to an enzyme – this conjugate attaches to the HIV antibodies
4. the strip is incubated with a substrate that turns color when acted upon by the enzyme conjugate

so colored bands will appear on the strip wherever there is an antigen-antibody complex

if all bands have developed color, presence is indicated of antibodies in the patient’s serum to all principal HIV antigens

Question 21

what would recommend for someone who has an indeterminate diagnosis from a western blot?

Answer 21

Assuming that you got a proper weakly-positive control and that you did identify indeterminates, RT-PCR of blood samples may be helpful, or a second visit some time later for another western blot analysis

sometimes the patients have not developed a good antibody response at the time of analysis – for instance, if the infection occurred recently or if the patient has underlying immune deficiencies

another possibility is that the patient may have been infected with a strain from a very different clade, so the immune response against the infecting virus may recognize poorly (or not at all) virus of the clade used for the test

Question 22

how do you use PCR to sequence viral target proteins?

Answer 22

they can tell us both what virus is infecting a patient and, whether the virus is sensitive or resistant to a drug

Question 23

how do you isolate RNA from a sample to do PCR on it?

Answer 23

1. extraction of irrelevant material with organic solvents like phenol
2. column purification

Question 24

how does RT-PCR work?

Answer 24

1. isolate RNA
2. once pure RNA is available, it is copied into a cDNA molecule by reverse transcription

this reaction is mediated by purified retroviral reverse transcriptases, which use RNA as a template to synthesize DNA

3. there is a single round of copying
4. cDNA is amplified with specific primer pairs
5. a sample is mixed with primer pairs, nucleotides and a thermal-resistant polymerase (Taq)

aliquots of the sample can be used to test multiple primer pairs belonging to different viral species

6. after the required number of amplification cycles are finished, an aliquot is loaded on an agarose gel
7. the DNA bands get seperated based on their molecular weight because light ones will move faster
8. the separated bands can be seen under UV light with ethidium bromide that binds to DNA

Question 25

what are the basic steps that happen during PCR amplification?

Answer 25

1. dsDNA is denatured
2. anealed
3. elongatured

repeat x100

Question 26

why are viruses harder to treat than bacterial infections?

Answer 26

viruses use the host-cell machinery to replicate

this property makes it very difficult to find drugs that specifically target viral replication without damaging the cell

thus, there are very few antiviral drugs compared to antibacterial drugs

Question 27

what do the results of a PCR mean?

Answer 27

a negative result and absence of infection by the corresponding viral species

if a band is present, but its molecular weight does not correspond to the expected molecular weight, the amplification is considered as due to non-specific priming and the test is also considered negative

if a band of the expected molecular weight is present, the test is positive

Question 28

what makes viruses resistant to drugs?

Answer 28

resistance to drugs is in most cases well characterized and due to specific amino acid changes in target viral proteins

so by determining the gene sequence that codes for these proteins we can see whether there are mutations causing relevant amino acid substitutions, and decide whether the virus is sensitive or not to a particular antiviral drug

this is why gene sequencing is so important!

Question 29

what is a sequencing reaction?

Answer 29

a sequencing reaction is essentially a PCR reaction in which the template is the amplified cDNA

in addition to ATP, CTP, GTP and TTP, there are color-labeled analogs of each nucleotide that prevent further growth of the DNA strand

this reaction results in a collection of molecules of increasing length, depending on where a terminator is introduced, and these molecules are separated by chromatography

the labels, with one color corresponding to each of the nucleotides, are read and produce the order of nucleotides of the DNA under investigation

once the genomic sequence is available there is software to produce the encoded amino acid sequence, which can be aligned with sequences of other viruses that are sensitive or resistant

the nucleotide positions that code for amino acids involved in resistance can be compared to controls

Question 30

what do the results of gene sequencing tell you?

Answer 30

gene sequencing gives you the order of the nucleotides in a virus –> AA sequence

but the presence of mutations does not necessarily indicate resistance

some mutations are the product of errors during replication and are irrelevant, or they are relevant to other biological properties of the virus, such as virulence or host range

synonymous mutations in relevant codons will not produce any amino acid change, and thus, will not cause resistance = silent mutation

only non-synonymous mutations in specific nucleotides indicate resistance to a drug