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The plaque assay is a terrific method for determining virus titers, but it
doesn’t work for all viruses. Fortunately there are several alternative
methods available, including the end-point dilution assay.
The end-point dilution assay was used to measure virus titer before the
development of the plaque assay, and is still used for viruses that do not
form plaques. Serial dilutions of a virus stock are prepared and inoculated
onto replicate cell cultures, often in multi-well formats (e.g. 96 well plastic
plates). The number of cell cultures that are infected is then determined for
each virus dilution, usually by looking for cytopathic effect.
In real life, the 50% end point does not usually fall exactly on a dilution as
shown in the example. Therefore statistical procedures are used to
calculate the end point of the titration.
The titer of a virus stock can be calculated in plaque-forming units (PFU) per
milliliter. To determine the virus titer, the plaques are counted. To minimize
error, only plates containing between 10 and 100 plaques are counted,
depending on the size of the cell culture plate that is used. Statistical
principles dictate that when 100 plaques are counted, the sample titer will
vary by plus or minus 10%. Each dilution is plated in duplicate to enhance
accuracy. In the example shown below, there are 17 plaques on the plate
made from the 10-6 dilution. The titer of the virus stock is therefore 1.7 x
108 PFU/ml.
Next we’ll consider how the plaque assay can be used to prepare clonal
virus stocks, a step that is essential for studying viral genetics.
Using the method of Reed & Muench (below), determine the TCID 50 value
(TCID50/50µl) in the experiment shown below:
VIRUS ASSAY
Virus infectivity
Plaque assay
The plaque assay quantifies the number of infectious units in a given
suspension of virus. Plaques are localized discrete foci of infection denoted
by zones of cell lysis or cytopathic effect (CPE) within a monolayer of
otherwise healthy tissue culture cells. Each plaque originates from a single
infectious virion, thus allowing a very precise calculation of the virus titer.
The most common plaque assay is the monolayer assay. Here, a small
volume of virus diluent (0.1 ml) is added to a previously seeded confluent
tissue culture cell monolayer. Following adsorption of virus to the cells, an
overlay medium is added to prevent the formation of secondary plaques.
Followig incubation, the cell sheets are ‘fixed’ in formol saline and stained,
and the plaques counted. For statistical reasons, 20-100 plaques per
monolayer are ideal to count, although the actual number that can be
easily counted is often dependent on the size of the plaque and the size of
the vessel used for the assay. Thypical plaques are shown in Fig. 1, and the
assay procedure is summarized in Fig 2. The infectivity titer is expressed as
the number of plaque forming units per ml (pfu ml-1) and is obtained in the
following way:
pfu ml-1 =
TCID50
Particle counting
Not all virus particles are infectious. Indeed in many cases for every one
infectious particle up to 100 or more non-infectious particles may be
produced from an infected cell. The total number of particles can only be
determined by counting them with the aid of an electruon microscope. The
counting procedure relies on the use of reference particles which are
usually latex beads of uniform diameter. The principle is that if viruses can
be mixed with reference particles of known concentration (i.e. a number
per unit volume), a sipmle determination of the ratio of virus to reference
particles will yield the virus coumt. Latex and virus particles are
distinguished after negative staining with phosphotungstate. The ratio of
total particles to infectious particles is termed the particle/infectivity ratio,
which is important to know when, for example, monitoring virus
purification, or determingig the state or age of a virus suspension.
Hemagglutination
Many viruses have the ability to agglutinate RBCS, this being referred to as
hemagglutin- ation. In oredr for the reaction to occur, the virus should be in
sufficient concentration to form cross-bridegs between RBCS, causing their
agglutination. Non-agglutinated RBCS will form a pellet in a hemispherical
eell, whereas agglutinated RBCS form a lattice-work structure which coats
the sides of the well. This phenomenon forms the basis of an assay which
determines the number of hemagglutinating particles in a given suspension
of virus. It is not a measure of infectivity, but is one of the most commonly
used indirect methods for the determination of virus titer. The assay is
done by end-point titration.Serial two-fold dilutions of virus are mixed with
an equal volume of RBCS and the wells are observed for agglutination. The
end point of the titration is the last dilutin sho0wing complete
agglutination, which by definition is said to contain one HA unit. The HA
titer of a virus suspension is therefore defined as being the reciprocal of the
highest dilution which causes complete agglutination and is expressed as
the number of HA units per unit volume. An example upon which a
calculation of the HA titer can be made is shown in Fig. 3. The end point in
this figure is 1/512. If 0.2 ml virus dilution was added per well the HA titer
would be 512 HA units per 0.2 ml or 2560 HA units ml-1.