Applied Veterinary Virology: The isolation and identification of viruses using cell cultures
Applied Veterinary Virology:
The isolation and identification of viruses using cell
cultures
Authors: Prof Estelle Venter
Licensed under a Creative Commons Attribution license.
VIRAL QUANTIFICATION
Infectivity assays for viruses
Two general types of assays are based on the ability of the virus to infect cells. The first depends on
quantification of individual, localized infection in a layer of cells. The plaque assay is an example of
this. The second method depends on the detection of virus infection in cell cultures or animals,
inoculated with serial dilutions of the virus, is better known as the end-point dilution method.
Dose response curve and significance of ID50/TCID50
Figure 4: Dose response curve
Examine this graph
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Applied Veterinary Virology: The isolation and identification of viruses using cell cultures
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of the 50% area of response a very small alteration in the host is reflected by an exaggerated
difference in the percentage of the host affected
•
at high extremes of the curve, a much greater variation in the dose is required to affect the
•
number of hosts
thus the point of maximum sensitivity of the assay system can be established at the 50% level
Thus, the unit of measurement become a 50% dose and may be expressed in various ways, e.g.
•
LD5O – (lethal dose 50),
•
PD5O (paralytic dose 50)
•
TCID5O (tissue culture infected dose 50)
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It is also called the endpoint method - find the endpoint dilution at which 50% of the host cells
or organisms are damaged
This is only a statistical unit
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terms of the conditions, e.g. volume of inoculum, route of infection, host system must be
defined.
Example of the calculation of the 50% end point by the method of Reed & Muench (1938).
Table 3: Calculation of the TCID 50 of a virus suspension
DEATH / SURVIVAL
Virus dilution
Lab record
D
S
(P)
(N)
P
P+N
D/S
Cumulative
% ±
10-1
5/5
5
0
22
0
100
10-2
5/5
5
0
17
0
100
10-3
5/5
5
0
12
0
100
10-4
4/5
4
1
7
1
88
10-5
2/5
2
3
3
4
43
10-6
1/5
1
4
1
8
11
10-7
0/5
0
5
0
13
0
50 %
Proportionate distance =
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% 𝑚𝑜𝑟𝑡𝑎𝑙𝑖𝑡𝑦 > 50−50
% 𝑚𝑜𝑟𝑡𝑎𝑙𝑖𝑡𝑦 > 50− % 𝑚𝑜𝑟𝑡𝑎𝑙𝑖𝑡𝑦 < 50
Applied Veterinary Virology: The isolation and identification of viruses using cell cultures
=
=
=
88−50
88−42
38
45
0,8
(i.e. proportional distance between the 10-4 and 10-5 dilution at which 50 % of
the host system is affected)
By formula: LD50= log of lower dilution + (prof. distance x log. of dilution factor)= 4 + (0,8 x 1)
By same calculation the original suspension before dilution must contain 63100 LD 50 (Anti log of 8 =
0,6310) per unit volume.
 A dilution of 10-4,8 contains 1 LD50.
Plaque assay
In the plaque assay method, the number of plaques produced is proportional to the concentration of
the virus inoculated, that is, the dose-response curve is linear. This proportionally indicates that one
virus particle is responsible for the formation of each plaque. It also indicates that one virus particle is
sufficient to infect a cell.
The endpoint method is still used for certain viruses that do not cause sufficient CPE’s recognized as
plaques but whose presence can be detected in cultured cells, or for viruses that do not replicate in
tissue culture but do cause disease in embryos or adult animals. The virus is serially diluted, and a
constant volume of each dilution is inoculated into a number of similar test units (such as mice, chick
embryos or cell cultures). At each dilution the proportion of infected test units (infectivity ratio) is
scored e.g.
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death or disease of an animal or embryo,
•
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degeneration of a tissue culture, or
recognition of progeny virus in vitro
The lower dilutions of the virus infect most the test units, and the highest dilutions infect none. The
end-point is the last dilution at which virus can be detected in an inoculated culture or animal and that
dilution contains at least one infective unit in the volume inoculated. The transition is not sharp,
however, and only by combining the data from several dilutions is it possible to calculate the precise
endpoint at which 50% of the test units are infected. At this dilution each sample contains on the
average one ID50, i.e., one infectious dose for 50% of the test units.
Viral titres obtained by the endpoint method are expressed in various equivalents of the ID 50: LD50
(lethal dose) if the criterion is death, PD50 (paralysis dose) if the criterion is paralysis, TC 50 or TCID50
(tissue culture dose) if the criterion is degeneration of a culture.
Statistical methods according to Reed & Muench, and Kaber.
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Applied Veterinary Virology: The isolation and identification of viruses using cell cultures
The interpolation to obtain the ID50 can be carried out in a variety of ways. The method of Reed
and Muench (1938), though not mathematically derived, yields results in fair agreement with
more rigorous methods. This method is used in conjunction with a series of progressive
dilutions of a virus. If the 50% endpoint lies between two dilutions, the dilution containing one
ID50 is obtained by interpolation between the two dilutions that straddle the 50% value of the
infectivity ratio. The interpolation assumes that in the proximity of the ID 50 the infectivity ratio
varies linearly with the log dilution. The interpolated value is given by:
% animals affected at dilution next above 50% - 50%
% animals affected at dilution next above 50% - % animals affected at dilution below 50%
This is multiplied by h, which is the log of the dilution factor employed at each step of the serial
dilution of the virus.
This method of calculation is only justified if one assumes that the fluctuations observed at
each dilution are purely due to variations in host sensitivity to the agent under test and not to
the chance presence or absence of discrete material particles.
Plaque assay technique and factors which influence infective particle counts
The plaque method is the fundamental assay method in virus research, and is of great value in
diagnosis for it combines simplicity with accuracy and high reproducibility. It was first used with
bacteriophages and these were assayed as follows: A phage-containing sample was mixed
with a drop of dense liquid culture of suitable bacteria and a few millilitres of melted soft agar at
44°C. The mixture was poured over the surface of a plate or Petri-dish containing a layer of
hard nutrient agar. The soft agar spreads in a thin layer and sets, and the bacteriophages
diffuse through it until each meets and infects a bacterium. After 20-30 min the bacterium
undergoes lysis, releasing several hundred progeny virions. These, in turn, infect neighbouring
bacteria, which again lyse and infect new virus. In the meantime, the uninfected bacteria grow
to form a dense, opaque lawn and after a day’s incubation the lysed areas stand out as
transparent plaques against the dense background. The soft agar permits diffusion of phage to
nearby cells, but prevents convection to other regions of the plate and secondary centres of
infection cannot form.
With animal viruses, a similar method is possible, the bacteria being replaced by a monolayer
of cells growing on a solid support, and the medium is replaced by a solution containing serial
dilutions of virus. Within an hour or so, most of the virions attach to cells. Soft nutrient agar or
some other gelling mixture is poured over the cell layer to minimize the spread of the virus by
dispersion, which would occur in liquid medium. A single virus particle infects a cell, and
progeny virus produced by that virus spreads to adjacent cells. This process repeats itself and
results in a localized area of infection and cell damage that can be visually identified and
counted (plaques) after a period of incubation.
Plaques are detected in a variety of ways:
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Applied Veterinary Virology: The isolation and identification of viruses using cell cultures
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The virus often kills the infected cells and produces the so-called CPE. The plaques are
the detected by staining the cell layer with a dye that stains only the live cells (e.g. neutral
red) or only the dead cells (trypan blue)
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With certain viruses, the cells in the plaques are not killed but acquire the ability to absorb
red blood cells. The plaques are revealed by haemadsorption (flooding the cell layer with a
suspension of red blood cells and then washing out those not attached to infected cells)
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The infected cells may fuse with neighbouring uninfected cells to form polykaryocytes
which are microscopically detectable (syncytial plaques)
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Often the cells of the plaque contain large amounts of viral antigens, which can be
detected by immunofluorescence
Each lesion is caused by a single virus particle, and the titre of the viral preparation can be
directly calculated from the number of plaques and the dilution of the sample. The amount of
virus quantitated by the plaque assay method is expressed as plaque-forming units (PFU).
A variation of the plaque assay that is used for tumour viruses that do not cause cell death is
the focus assay. A tumour virus causes cell proliferation and the formation of localized area of
transformed, proliferating cells called a focus. The units are expressed as focus-forming units
(FFU).
Some viruses cause localized lesions on the chorioallantoic membrane of the chick embryo,
which are called pocks. Each pock is formed by a single virion.
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