9 - Virus isolation and culture

Question 1

Direct virus detection

Answer 1

Measurement of virus particle
- Electron microscopy
- Cell culture
- Molecular methods
- Serological methods

Question 2

Indirect detection

Answer 2

Measurement of virus specific host immune response after infection

Question 3

Electron microscopy

Answer 3

Does not require organism-specific reagents (unlike serological or biochemical identification)

Question 4

Virus isolation

Answer 4

• Viruses can be isolated from infected tissues by propagation in animal or human cells, eggs or lab animals
• Cannot replicate outside of living cells

Question 5

Cell culture medium

Answer 5

An isotonic solution of salts, glucose, vitamins, and amino acids buffered to physiologic pH (7.2-7.4

Question 6

Three main kinds of cell cultures

Answer 6

1. Primary cell cultures
2. Diploid cell line
3. Continuous cell lines

Question 7

Primary cell cultures

Answer 7

• Prepared from human or animal tissues and have a life span of 5 -10 cell divisions.
• Derived from monkey kidney, human foreskin, embryos

Question 8

Diploid cell line

Answer 8

Consists of single cell types and can divide up to 100 times, retaining diploid chromosome number. E.g. WI-38 from human embryonic lung

Question 9

Continuous cell lines

Answer 9

• Consist of a single cell type that can be propagated indefinitely.
• Derived from a tumour or by treating cultured cells with a mutagen or transforming virus.
• Less differentiated than the cell of origin, and have abnormal chromosome numbers.
• E.g. Vero cells derived from African green monkey kidney epithelium

Question 10

Cytopathic effects

Answer 10

Virus induced cell changes

Question 11

Types of CPE

Answer 11

• Inclusion bodies
• Morphological alterations

Question 12

Examples of morphological alterations of CPE

Answer 12

• Adherent cells round up and detach from the supporting surface
• Cells fuse together to form multinucleated giant cells or syncytia
• Apoptosis

Question 13

Steps of PCR

Answer 13

• Denaturation of target DNA (98ºC)
• Annealing of primers (55ºC)
• Extension of primers with Taq polymerase (72ºC)

Question 14

RT PCR

Answer 14

Reverse Transcriptase PCR. Template RNA is first converted to DNA

Question 15

Immunofluorescence

Answer 15

An antibody specific for the antigen of interest is used to detect the presence of the antigen. Can be direct or indirect

Question 16

Direct IFA

Answer 16

the antibody is labeled with a fluorophore and attaches directly to viral antigen

Question 17

Indirect IFA

Answer 17

A second antibody, which recognises the Fc region of the first antibody (which is specific for the antigen of interest), is labeled with a flourophore

Question 18

Plaque assay

Answer 18

Areas of killed cells in a tissue culture vessel. Only viruses that cause visible damage to cells can be assayed this way

Question 19

Process of plaque assay

Answer 19

• 10 fold dilutions of virus stock are prepared and inoculated onto susceptible cell monolayers
• Cells are covered with gel that restricts spread of new viruses to neighbouring cells

Question 20

Detection of viral antigen by haemagglutination (HA)

Answer 20

• Some viruses can bind to sialic acid on RBC and agglutinate the RBC
• Non agglutinated RBC settle in the bottom of the test well as well as a button, agglutinated cells form a smooth lattice