Detection/Quantification of Viruses

Question 1

Viruses are Propagated to

Answer 1

Study them
Diagnose diseases
Create vaccines

Question 2

Viruses can be propagated in

Answer 2

Cell culture
In animals - lab animals or final host
In embryonated eggs

Question 3

Primary Culture

Answer 3

The tissue which can support viral growth is excised from a live animal, digested with enzymes to separate cells and cultured artificially with cell growth media. (Short lived)

Question 4

Transformation

Answer 4

The process by which primary cells acquire an almost indefinite life or cancerous. Achieved using chemicals or exposure to radiation.

Question 5

Continuous Cell Lines

Answer 5

Transformed cells that have an indefinite life. They can be cultured several times in the lab.

Question 6

Monolayer

Answer 6

A continuous cell sheet in a cell culture flask

Question 7

Suspension Cells

Answer 7

Do not attach to a surface but can be grown in a flask. BHK21

Question 8

Cell Lines

Answer 8

Used to culture viruses as they are infected and support viral replication. Therefore, they can also be used to isolate viruses from clinical specimens collected from patients.

Question 9

Virus Culture

Answer 9

Permissive cells are grown to a monolayer or suspension, the virus is incubated with the cells so that it infects them, virus can be harvested after 1-3 days by lysing the cells.

Question 10

Cytopathic Effect (CPE)

Answer 10

On infecting cells some viruses cause bunching, rounding off or syncytia (fused cells)

Question 11

Column Chromatography

Answer 11

Separation of components of a mixture using a matrix in which each component has a different migration pattern.

Question 12

Size Exclusion

Answer 12

The separation is based on size

Question 13

Affinity

Answer 13

The separation is based on affinity or binding of a ligand to a receptor (virus specific antibodies are bound to the column to bind viruses)

Question 14

Centrifugation

Answer 14

Separation by centrifugal force, based on mass and size. Heavier particles will move more quickly in a centrifugal field than lighter particles. Max 15-20,000 RPM/ min

Question 15

Ultracentrifugation

Answer 15

Viruses are extremely small. They require very high speed [G force ]and vacuum for separation. Ultracentrifuges can spin at 80,000-100,000 RPM/ min.

Question 16

TCID50

Answer 16

Tissue culture infective dose 50. The dose of the virus required to kill 50% of the cells, animals, or egg embryos is calculated.

Question 17

ID50

Answer 17

Animal infectious dose 50. The dose of the virus required to infect 50% of the experimental animals.

Question 18

LD50

Answer 18

Lethal dose 50. The dose of the virus required to kill 50% of the experimental animals

Question 19

EID50

Answer 19

Egg infectious dose 50. The does of the virus required to infect 50% of the experimental egg embryos.

Question 20

PFU

Answer 20

Plaque forming units

Question 21

Test Antigen

Answer 21

The virus or a viral protein from the sample to be identified.

Question 22

Primary Antibody

Answer 22

An antibody which is specific to the birus

Question 23

Secondary Antibody

Answer 23

Antibody raised against the IgG of the species in which the primary antibody was made. It is conjugated to an enzyme [HRPO] which can produce a color reaction and help to detect the specific interaction between the antigen [virus] and the primary antibody

Question 24

Immuno-fluorescence Assay

Answer 24

Virus is cultured on a glass slide containing a monolayer of cells

The detection system is similar to the ELISA, except that the secondary antibody is conjugated to a florescent tag instead of HRPO

Requires a florescent microscope for visualization.

Question 25

PCR - Polymerase Chain Reaction

Answer 25

Targets the specific and exponential amplification of target DNA/RNA sequences.

Question 26

PCR Method

Answer 26

The DNA or RNA or the suspected viral sample is extracted. If the virus has an RNA genome, reverse-transcription is required before the PCR

DNA/cDNA is added to “specific” primers that can only amplify the viral sequence, along with ingredients required to the PCR; DNA polymerase and nucleotides and amplified in a PCR themocycler

The thermocycling conditions include a) heating to separate the DNA strands b) Annealing – to attach the primers c) Extension – to complete amplification. Repeated about 30 times. DNA is visualized on a gel.

Question 27

Quantitative PCR

Answer 27

Used for viral quantification (Real time PCR or qPCR)

Question 28

In-situ Hybridization (ISH)

Answer 28

Viruses in infected tissue can be detected using a labelled DNA or RNA probe which specifically attaches to the nucleic acid of the virus during hybridization.