Virus quantification is counting or calculating the number of virus particles (virions) in a sample to determine the virus concentration. It is used in both research and development (R&D) in academic and commercial laboratories as well as in production situations where the quantity of virus at various steps is an important variable that must be monitored. For example, the production of virus-based vaccines, recombinant proteins using viral vectors, and viral antigens all require virus quantification to continually monitor and/or modify the process in order to optimize product quality and production yields and to respond to ever changing demands and applications. Other examples of specific instances where viruses need to be quantified include clone screening, multiplicity of infection (MOI) optimization, and adaptation of methods to cell culture.
There are many ways to categorize virus quantification methods. Here, the methods are grouped according to what is being measured and in what biological context. For example, cell-based assays typically measure infectious units (active virus). Other methods may measure the concentration of viral proteins, DNA, RNA, or molecular particles, but do not necessarily measure infectivity. Each method has its own advantages and disadvantages, which often determine which method is used for specific applications.
Cell-based assays
Plaque assay
Plaque-based assays are a commonly used method to determine virus concentration in terms of infectious dose. Plaque assays determine the number of plaque forming units (PFU) in a virus sample, which is one measure of virus quantity. This assay is based on a microbiological method conducted in petri dishes or multi-well cell culture plates. Specifically, a confluent monolayer of host cells is infected by applying a sample containing the virus at varying dilutions and then covered with a semi-solid medium, such as agar or carboxymethyl cellulose, to prevent the virus infection from spreading indiscriminately, as would occur in a liquid medium. A viral plaque is formed after a virus infects a cell within the fixed cell monolayer. The virus-infected cell will lyse and spread the infection to adjacent cells, where the infection-to-lysis cycle is repeated. This will create an area of infected, lysed cells (viral plaque) surrounded by uninfected, intact cells. The plaque can be seen with an optical microscope or visually using cell staining techniques (e.g., staining with a crystal violet solution to visualize intact vs. lysed cells). Plaque formation can take 3–14 days, depending on the virus being analyzed. Plaques are generally counted manually, and the plaque count, in combination with the dilution factor of the infection solution (the sample initially applied to the cells), is used to calculate the number of plaque forming units per sample unit volume (PFU/mL). The PFU/mL number represents the concentration of infectious virus particles within the sample and is based on the assumption that each plaque formed is representative of an initial infection by one infectious virus particle.
Focus forming assay (FFA)
The focus forming assay (FFA) is a variation of the plaque assay, but instead of depending on cell lysis in order to detect plaque formation, the FFA employs immunostaining techniques using fluorescently labeled antibodies specific for a viral antigen to detect infected host cells and infectious virus particles before an actual plaque is formed. The FFA is particularly useful for quantifying classes of viruses that do not lyse the cell membranes, as these viruses would not be amenable to the plaque assay. Like the plaque assay, host cell monolayers are infected with various dilutions of the virus sample and allowed to incubate for a relatively brief incubation period (e.g., 24–72 hours) under a semisolid overlay medium that restricts the spread of infectious virus, creating localized clusters (foci) of infected cells. Plates are subsequently probed with fluorescently labeled antibodies against a viral antigen, and fluorescence microscopy is used to count and quantify the number of foci. The FFA method typically yields results in less time than plaque assays or fifty-percent-tissue-culture-infective-dose (TCID50) assays (see below), but it can be more expensive in terms of required reagents and equipment. Assay completion time is also dependent on the size of area that the user is counting. A larger area will require more time but can provide a more accurate representation of the sample. Results of the FFA are expressed as focus forming units per milliliter, or FFU/mL.
TCID50 endpoint dilution assay The TCID50 (50% tissue culture infectious dose) assay is the measure of infectious virus titer. This endpoint dilution assay quantifies the amount of virus required to infect 50% of infected hosts or to produce a cytopathic effect in 50% of inoculated tissue culture samples. This assay may be more common in clinical research applications where the lethal dose of the virus must be determined or if the virus does not form plaques. When used in the context of tissue culture, host cells are plated and serial dilutions of the virus are added. After incubation, the percentage of cell death (i.e. infected cells) is manually observed and recorded for each virus dilution, and results are used to mathematically calculate a TCID50 result. Due to distinct differences in assay methods and principles, TCID50 and pfu/mL or other infectivity assay results are not equivalent. This method can take up to a week due to cell infectivity time.
Two methods commonly used to calculate TCID50 (can also be used to calculate other types of 50% endpoint such EC50, IC50, and LD50) are: Spearman–Kärber Reed–Muench method
The theoretical relationship between TCID50 and PFU is approximately 0.69 PFU = 1 TCID50 based on the Poisson distribution, a probability distribution which describes how many random events (virus particles) occurring at a known average rate (virus titer) are likely to occur in a fixed space (the amount of virus medium in a well). However, it must be emphasized that in practice, this relationship may not hold even for the same virus + cell combination, as the two types of assay are set up differently and virus infectivity is very sensitive to various factors such as cell age, overlay media, etc. But the following reference defines the relationship differently:
From ATCC: "Assuming that the same cell system is used, that the virus forms plaques on those cells, and that no procedures are added which would inhibit plaque formation, 1 mL of virus stock would be expected to have about half of the number of plaque forming units (PFUs) as TCID50. This is only an estimate but is based on the rationale that the limiting dilution which would infect 50% of the cell layers challenged would often be expected to initially produce a single plaque in the cell layers which become infected. In some instances, two or more plaques might by chance form, and thus the actual number of PFUs should be determined experimentally.
"Mathematically, the expected PFUs would be somewhat greater than one-half the TCID50, since the negative tubes in the TCID50 represent zero plaque forming units and the positive tubes each represent one or more plaque forming units. A more precise estimate is obtained by applying the Poisson distribution. Where is the proportion of negative tubes and m is the mean number of infectious units per volume (PFU/ml), . For any titer expressed as a TCID50, . Thus and which is ~ 0.7.
"Therefore, one could multiply the TCID50 titer (per ml) by 0.7 to predict the mean number of PFU/ml. When actually applying such calculations, remember the calculated mean will only be valid if the changes in protocol required to visualize plaques do not alter the expression of infectious virus as compared with expression under conditions employed for TCID50.
"Thus as a working estimate, one can assume material with a TCID50 of 1 × 105 TCID50/mL will produce 0.7 × 105 PFUs/mL."
Protein and antibody-based assays
There are several variations of protein- and antibody-based virus quantification assays. In general, these methods quantify either the amount of all protein or the amount of a specific virus protein in the sample rather than the number of infected cells or virus particles. Quantification commonly relies on colorimetric or fluorescence detection. Some assay variations quantify proteins directly in a sample, while other variations require host cell infection and incubation to allow virus growth prior to quantification. The variation used depends primarily on the amount of protein (i.e. viral protein) in the initial sample and the sensitivity of the assay itself. If incubation and virus growth are required, cell and/or virus lysis/digestion are often conducted prior to analysis. Most protein-based methods are relatively fast and sensitive but require quality standards for accurate calibration, and quantify protein, not actual virus particle concentrations. Below are specific examples of widely used protein-based assays.
Hemagglutination assay
The hemagglutination assay (HA) is a common non-fluorescence protein quantification assay specific for influenza. It relies on the fact that hemagglutinin, a surface protein of influenza viruses, agglutinates red blood cells (i.e. causes red blood cells to clump together). In this assay, dilutions of an influenza sample are incubated with a 1% erythrocyte solution for one hour and the virus dilution at which agglutination first occurs is visually determined. The assay produces a result of hemagglutination units (HAU), with typical PFU to HAU ratios in the 106 range. This assay takes ~1–2 hours to complete.
The hemagglutination inhibition assay is a common variation of the HA assay used to measure flu-specific antibody levels in blood serum. In this variation, serum antibodies to the influenza virus will interfere with the virus attachment to red blood cells. Therefore, hemagglutination is inhibited when antibodies are present at a sufficient concentration.
Bicinchoninic acid (BCA) assay
The bicinchoninic acid assay (BCA; a.k.a. Smith assay) is based on a simple colorimetric measurement and is a commonly used protein quantification assay.
BCA is similar to the Lowry or Bradford protein assays. The BCA assay reagent was first developed and made commercially by Pierce Chemical Company (now owned by Thermo Fisher Scientific) which held the patent until 2006.
In the BCA assay, a protein's peptide bonds quantitatively reduce Cu2+ to Cu1+, which produces a light blue color. BCA chelates Cu1+ at a 2:1 ratio resulting in a more intensely colored species that absorbs at562 nm. Absorbance of a sample at 562 nm is used to determine the bulk protein concentration in the sample. Assay results are compared with known standard curves after analysis with a spectrophotometer or plate reader. Total assay time is 30 minutes to one hour. While this assay is ubiquitous and fast, it lacks specificity to viral proteins since it counts all protein in the sample. Thus the virus preparation to be quantified must be highly purified, and free from host, serum, carrier or other proteins.
Enzyme-linked immunosorbent assay (ELISA)
Enzyme-linked immunosorbent assay (ELISA) is an antibody-based assay that utilizes an antigen-specific antibody chemically linked to an enzyme (or bound to a second antibody linked to an enzyme) to detect the presence of an unknown amount of the antigen (e.g., viral protein) in a sample. The antibody-antigen binding event is detected and/or quantified through the enzyme's ability to convert a substrate reagent to produce a detectable signal that can then be used to calculate the concentration of the target antigen in the sample. Horseradish peroxidase (HRP) is a common enzyme utilized in ELISA schemes due to its ability to amplify signal and increase assay sensitivity.