Lecture 20: Cultivation of Viruses

Question 1

Why do we cultivate viruses?

Answer 1

To isolate and identify viruses in clinical samples.

To study their structures and features

For vaccine production (although this is now possible without growing viruses)

Question 2

What are the 2 culture systems for cultivating viruses/

Answer 2

In vivo (animal, plant, bacteria, chicken eggs)

In vitro (cell cultures that have been isolated and grown from hosts)

Question 3

What animals are usually used for in vivo culturing?

Answer 3

Mice, as well as monkeys, rabbits, and hampsters

Question 4

After hosts, such as mice, are inoculated with viruses, what happens?

Answer 4

The animals are observed for symptoms of disease and death.

The virus can also then be isolated by sacrificing the animals and purifying the viruses from their tissue.

Question 5

Describe how viruses are cultivated in eggs

Answer 5

• Fertilized eggs that are 8-11 days old are used
• The virus is inoculated into any of the compartments of the egg, this is dependant on the virus
• Viral growth is indicated by the death of the embryo, by embryo cell damage, changes in fluids or by the formation of typical pocks or lesions on membranes.
• Egg cultivation is most commonly used for influenza virus vaccine production, due to the excellent yield of the virus when grown this way

Question 6

Describe how viruses are cultured in cell cultures (in vitro)

Answer 6

• Primary cell culture, where a culture of normal cells is obtained from animals organs or tissues. (To prepare, tissues are minced, using enzymes to break up the bonds between the cells, in order to obtain isolated cells).
• These cells will then be added to petri dishes and incubated with broth media to allow them to grow
• Finally , a mono layer will be formed
• These cells have a short life span of 2-3 days, as they’er primary cells
• If this time passes, they have to be subcultured in another petri dish and growth media should be added for them to be grown again

Question 7

Why can’t primary cells be subcultured indefinitely?

Answer 7

Because primary cells have a limited number of divisions

Question 8

Which cells can undergo continuous cell culture?

Answer 8

cancer cells.

Question 9

Is it true that cancer cells can be passaged/subcultured indefinitely?

Answer 9

Yes

Question 10

Why can’t cancer cells be used for vaccine production?

Answer 10

Because they have an altered and irregular number of chromosomes

Question 11

What are HeLA cells?

Answer 11

the first continuous tissue-culture cell line. They were used to establish tissue culture as an important technology for research in cell biology, virology, and medicine.

Question 12

Are HeLA cells still alive?

Answer 12

Yes, and they are actively being used for commercial and research purposes. Including research discoveries related to polio, cancer, and AIDS, among other diseases.

Question 13

What are CytoPatheic Effect (CPE)?

Answer 13

Any observable, distinct change in shape, appearance, morphological features in infected cells due to growth and replication of viruses. (CPE) produced by different types of viruses are characteristic and help in the initial identification of virus isolates.

Question 14

Can CPE confirm a diagnosis?

Answer 14

No, other tests will need to be carried out as well

Question 15

What is an example of a CytoPatheic Effect (CPE)?

Answer 15

Cytopathic Effect-Syncytia

Question 16

How can viruses be quantified?

Answer 16

Using Non-infective Assays, which are physical measurements of viruses.

Using infective assays, which measure the number of infectious viral particles

Question 17

what is cytopathic effect syncytia?

Answer 17

Where multinucleated giant cells formed when infected cells fuse with neighboring cells.

Question 18

What are some examples of non-infective assays?

Answer 18

Electron microscope

ELISA

Immunofluorescence

Haemagglutinin assay

Question 19

What is the quantification of viruses?

Answer 19

Determining their number

Question 20

Why are EMs rarely used for the quantification of viruses?

Answer 20

• Preparing samples for the electron microscope is difficult
• An expert is needed to identify the viruses viewed
• Very expensive

Question 21

ELISA

Answer 21

• ELISA is used to view antigens and antibodies (their binding)
• The rapid test for COVID is based on a modified version of ELISA

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Question 22

Haemagglutinin assay

Answer 22

• An assay used to determine the number of viruses, based on the ability of some viruses to agglutinate red blood cells.
• Some viruses are able to cross link the red blood cells.
• This is done by incubating the viruses with red blood cells in tubes with rounded bottoms, to allow the red blood cells to precipitate.
• The agglutination will result in the formation of a red layer
• Prepare serial dilutions of the virus and then incubate each of the dilutions with a fixed amount of red blood cells. Look for agglutination
• Choose the highest dilution that causes complete agglutination

Question 23

What is the disadvantage of non-infective assays?

Answer 23

Can’t be used to determine the number of infectious particles

Question 24

Plaque assay

Question 25

What were plaque assays used for in the 1930s?

Answer 25

to study the multiplication of bacteriophages

Question 26

What did Renato Dulbecco do in 1952?

Answer 26

He developed a plaque assay for animal viruses

Answer 27

Seed host cells into plates (6- or 12-well) and incubate until they form a confluent monolayer (typically 80–100% coverage).

Perform 10-fold serial dilutions of the viral sample in sterile medium to achieve a range of viral concentrations. (Example: Add 100 µL of the viral sample to 900 µL of medium for a 10⁻¹ dilution, then repeat for subsequent dilutions.)

Remove the growth medium from the cell monolayer.

Add 100–200 µL of each viral dilution to individual wells.

Gently rock the plate to ensure even distribution.

Incubate the plate for 30 minutes to 1 hour at 37°C to allow virus adsorption to cells.

After adsorption, remove the viral inoculum and add an overlay medium (e.g., growth medium mixed with 0.5–1% agar or carboxymethylcellulose).

Incubate the plates at the appropriate temperature and conditions for the virus and host cells (e.g., 37°C with 5% CO₂).

Once plaques have formed, fix and stain the cells with a dye such as crystal violet or neutral red to visualize the plaques:

Count the number of plaques in wells with distinct, countable plaques (usually 10–100 plaques per well). Each plaque corresponds to one infectious virion in the initial inoculum.

Calculate the plaque-forming units per mL (PFU/mL) using the formula: PFU/mL=
Volumeofinoculum(mL)
Numberofplaques×Dilutionfactor
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Question 28

1 plaque = 1 PFU (plaque forming unit)