Measurement of viruses by end-point dilution assay

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 this example of an end-point dilution assay, 10 monolayer cell cultures

were infected with each virus dilution. After an incubation period, plates

that displayed cytopathic effects were scored with a +. At high dilutions,

none of the cell cultures are infected because no particles are present. At

low dilutions, every cell culture is infected. Half of the cell cultures showed

cytopathic effects at the 10-5 dilution. This is the end point: the dilution of

virus at which 50% of the cell cultures are infected. This number can be

calculated from the data and expressed as 50% infectious dose (ID50) per

milliliter. The virus stock in this example contains 105 ID50 per ml.

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.

End-point dilution methods can also be used to determine the virulence of

a virus in animals. The same approach is used: serial dilutions of viruses are

made and inoculated into multiple test animals. Infection of the animal can

be determined by death or clinical symptoms such as fever, weight loss, or

paralysis. The results are expressed as 50% lethal dose (LD50) per ml or 50%

paralytic dose (PD50) per ml when lethality or paralysis are used as end

points.

The following example illustrates the use of end point dilution to measure

the lethality of poliovirus in mice. Eight mice were inoculated per virus

dilution, and the end point was death. The statistical method of Reed and

Muench was used to determine the 50% end point. In this method, the

results are pooled, and the mortality at each dilution is calculated. The 50%

end point, which falls between the fifth and sixth dilutions, is calculated to

be 10-6.5. Therefore the virus sample contains 106.5 LD50 units.

Reed, L.J., & Muench, H. (1938). A simple method of estimating fifty

percent endpoints. Am. J. Hygiene, 27, 493-497

Detecting viruses: the plaque assay

One of the most important procedures in virology is measuring the virus

titer – the concentration of viruses in a sample. A widely used approach for

determining the quantity of infectious virus is the plaque assay. This

technique was first developed to calculate the titers of bacteriophage

stocks. Renato Dulbecco modified this procedure in 1952 for use in animal

virology, and it has since been used for reliable determination of the titers

of many different viruses.

To perform a plaque assay, 10-fold dilutions of a virus

stock are prepared, and 0.1 ml aliquots are inoculated onto susceptible cell

monolayers. After an incubation period, to allow virus to attach to cells, the

monolayers are covered with a nutrient medium containing a substance,

usually agar, that causes the formation of a gel. When the plates are

incubated, the original infected cells release viral progeny. The spread of

the new viruses is restricted to neighboring cells by the gel. Consequently,

each infectious particle produces a circular zone of infected cells called a

plaque. Eventually the plaque becomes large enough to be visible to the

naked eye. Dyes that stain living cells are often used to enhance the

contrast between the living cells and the plaques. Only viruses that cause

visible damage of cells can be assayed in this way. An example of plaques

formed by poliovirus on a monolayer of HeLa cells is shown at left. In this

image, the cells have been stained with crystal violet, and the plaques are

readily visible where the cells have been destroyed by viral infection.

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.

Dulbecco, R., & Vogt, M. (1953). Some problems of animal virology as

studied by the plaque technique. Cold Spring Harbor Symp. Quant. Biol., 18,

273-279

The Tissue Culture Infectious Dose50 (TCID50) Assay:

Plaque assays for viruses have largely replaced endpoint dilution techniques such as the TCID50 assay, which are statistical ways of measuring virus populations. However, endpoint techniques are still used in certain circumstances, e.g. for viruses which do not grow in culture, when 'Lethal Dose50' (LD50) or 'Infectious Dose50' (ID50) values must be calculated. They are also used for viruses which are not cytopathic or do not produce plaques, (e.g. HIV). There are several statistical methods for analysing such data (e.g. Spearman-Karber formula or Probit analysis).

Using the method of Reed & Muench (below), determine the TCID50 value (TCID50/50µl) in the experiment shown below:

VIRUS ASSAY

Virus infectivity

In order to ‘reproduce’, viruses need to be capable of replicating in a susceptible host cell. This replicative cycle isaccompanied by a number of biochemical and morphological changes within the cell which usually results is the death of the cell. The accompanying morphological changes (e.g. cell rounding or fusion) are referred to as the cytopathic effect (CPE). A particular type of CPE is often a characteristic of specific virus growth and can be used when attemption to identify an unknown virus. The appearance and detection of CPE regularly forums the basis of infectivity assays, designed to determine the number of infectious units of virus per unit volume, and is the infectivity titer (e.g. plaque forming units (pfus) per milliliter). An infectious unit is thought of as being the smallest amount of virus that will produce a detectable biological effect in the assay (e.g. tissue culture infective dose 50 (TCID50))or focal, detection of a focus of infection (e.g. a plaque assay).

Virus dilution

Virus titers are determined by making accurate serial dilutions of virus suspensions. Such dilutions are usually done using factors of two, five or 10. For routine use, 10-fold dilutions are usually carried out. It is important to use a new sterile pipette for the transfer of volumes between each dilution and to mix thoroughly the dilution before further transfer. Once diluted, ivrus should be assayed as soon as possible as most viruses rapidly lose infectivity at room temperature.

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 =

For example, if there is a mean number of 100 plaques form monolayers infected with 0.1 ml of a 10-6 dilution then the calculation is:

TCID50

The TCID50 is defined as that dilution of virus required to infect 50% of a given batch of inoculated cell cultures. The assay relies on the presence and

detection of CPE. Host cells are grown in cenfouent healthy monolayers, usually in tubes, to which aliquots of virus dilutions are made. it is usual to use either five or 10 repetitions per dilution. During incubation the virus replicates and releases progeny virus particles into the supernatant which in turn infect other healthy cells in the monolayer. The CPE is allowed to develop over a perild of days, at which time the cell monolayers are observed microscopically. Tubes (the ‘test units’) are scored for the presence or absence of CPE. In this quantal assay the data are used to calculate the TCID50 i.e. the dilution of virus which will give CPE in 50% of the cells inoculated. Table 1 shows some typical data.

Table 1. Data used to calculate TCID50

Log10 of virus dilution lnfected test units (e.g. infected tubes)

-6 -7 -8

-9

By using the data in Table 1 the following calculation can be made:

TCID50 = log 10 of highest dilution giving 100% CPE +1/2－

total number of test units showing CPE

number of test units per dilution

= -6 + 1/2－9/5 = -7.3 TCID50

or 10-7.3 TCID50 unit vol.-1

The titer is therefore 107.3 TCID50 per unit vol.-1

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.