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
Seed cells into new
culture vessels
Lift cells into solution
with enzyme
Culture vessel with
appropriate growth media
Cell adhere to vessel and
grow to form a monolayer
Virus Dilution
0.1 ml
1
0.1 ml
0.1 ml
0.1 ml
0.1 ml
1/10
1/100
1/1000
1/10000 1/100000
Stock
0.9 ml
0.9 ml
0.9 ml
0.9 ml
0.9 ml
101
10-1
10-2
10-3
10-4
10-5
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
A
10-1
10-5
B
10-2
10-6
C
10-3
10-7
D
10-4
Control
6
TCID50 Procedure
• Count wells exhibiting CPE
• Ideally you would know all the dilution
factors to get infection rates of zero to
100 percent
100
CPE
0
Decreasing Dilution
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
1
TCID50
Dilution
10-1
10-2
10-3
10-4
10-5
10-6
10-7
Infected
3/3
3/3
3/3
2/3
1/3
0/3
0/3
2
3
4
5
A
10-1
10-5
B
10-2
10-6
C
10-3
10-7
D
10-4
6
Control
% Infected
100
100
Log PD =
66-50 x (Log10)
100
66-33
66
Log PD =
0.48
33
Log Dilution above 50 %
0
Infection 10-4.48
0
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
DxV
D = Dilution factor
V = 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
0
40
3
0
0
38
6
2
0
Plaques formed per well
Detection of Viral Nucleic Acid