Methods in Virology

Techniques• Detection of a meaningful immune response to the virus (antibody

or cell-mediated) by immunologic assay(s) • Identification of the agent by staining of specimens or sections of

tissue (light and electron microscopy)• Isolation and identification of the agent (cell culture or fertile eggs)• Detection of viral nucleic acid (probes or amplification)

Infected Cells (Immunohistochemistry)

Cell Culture• Cell culture is still the most common method for the

propagation of viruses.

• Tissues are dissociated into a single-cell suspension by mechanical disruption, followed by treatment with a proteolytic enzyme.

• Cells are cultured in a plastic flask in Minimum Essential Media (MEM). As the cells divide, they cover the plastic surface.

• Epithelial and fibroblastic cells attach to the surface of the plastic and form a monolayer.

Developing a cell line

Tissue cells to be cultured Culture vessel with appropriate growth media

Cell adhere to vessel and grow to form a monolayer

Lift cells into solution with enzyme

Seed cells into new culture vessels

Virus Dilution

1 1/10 1/100 1/1000 1/10000 1/100000

10-1 10-2 10-3 10-4 10-5

0.1 ml 0.1 ml 0.1 ml 0.1 ml 0.1 ml

101

0.9 ml 0.9 ml 0.9 ml 0.9 ml 0.9 mlStock

Cytopathic Effect

• Some viruses kill the cells in which they replicate, and infected cells may eventually detach from the cell culture plate.

• As more cells are infected, the changes become visible and are called cytopathic effects.

Examples of Cytopathic Effects of Viral Infection

• Nuclear shrinking (pyknosis)• Proliferation of nuclear membrane• Vacuoles in cytoplasm• Syncytia (cell fusion)

• Margination and breaking of chromosomes• Rounding up and detachment of cultured cells• Inclusion bodies

Quantification of CPE

• Tissue Culture Infective Dose 50 (TCID50): a measure of virulence of virus

• Why Quantify?– Virulence– Immunity– Strain

Infectivity Assays

Multi-well Plates

1 2 3 4 5 6

10-2

10-3

10-4

10-1 10-5

10-6

10-7

Control

B

C

D

A

TCID50 Procedure

• Count wells exhibiting CPE • Ideally you would know all the dilution

factors to get infection rates of zero to 100 percent

0

100

Decreasing Dilution

CPE

Calculation of TCID50

• In any biological quantification, the most desirable endpoint is one representing a situation in which half of the inoculated animals or cells show the reaction (death in the case of animals and in CPE case of cells) and the other half do not.

• Reed-Muench Method of computing a 50% endpoint of a virus titration

• Calculates the proportionate distance between dilutions which infect above and below 50% of the wells

TCID50

10-4

1 2 3 4 5 6

10-1 10-5

10-6

10-7

Control

B

C

D

A

10-2

10-3

Dilution Infected % Infected10-1 3/3 10010-2 3/3 10010-3 3/3 10010-4 2/3 6610-5 1/3 3310-6 0/3 010-7 0/3 0

Log PD = 66-50 x (Log10)66-33

Log PD = 0.48Log Dilution above 50 % Infection 10-4.48

Plaque Forming Units

• Areas where infected cells are being lysed by virus are seen as plaques, or areas of clearing in the cell monolayer.

• When stained with Crystal Violet these areas are easily identified as areas without stain

Plaque forming Units• A single virus infective dose can cause an area

of cell destruction• Movement of virus within cell is restricted by an

agar overlay• This causes areas of localized destruction• Plaques are enumerated under a microscope to

determine the plaque forming units per ml (PFU/ml)

• This allows comparison of different viruses in the same unit

Calculating viral titer based on the plaque assay method

• The viral titer is a quantitative measurement of the biological activity of a virus and is expressed as plaque forming units (pfu) per ml.

• To calculate the viral titer, count the number of well isolated plaques.

• Then use the following formula to determine the titer (pfu/ml) of your viral stock.

Average # Plaques = PFU/ml D x V

D = Dilution factorV = Volume of diluted virus added to the well

Wells observed 7 days after inoculation with of 0.1ml viral solution

PFU’s/ml = 42 plaques observed plaques (10-7 dilution factor)(0.1ml virus added)PFU’s/ml = 4.2 x109

Calculation of PFU/mL• Plaques are

enumerated• Plaque Counts are

averaged over wells• The average is then

divided by the dilution times the volume

(43+40+38)/3(10-4 x 0.1)

= 3,730,000 pfu/ml

43 4 1 040 3 0 038 6 2 0Plaques formed per well

Detection of Viral Nucleic Acid