Virus quantification

Virus quantification involves counting the number of viruses in a specific volume to determine the virus concentration. It is utilized in both research and development (R&D) in commercial and academic laboratories as well as production situations where the quantity of virus at various steps is an important variable. For example, the production of viral vaccines, recombinant proteins using viral vectors and viral antigens all require virus quantification to continually adapt and monitor the process in order to optimize production yields and respond to ever changing demands and applications. Examples of specific instances where known viruses need to be quantified include clone screening, multiplicity of infection (MOI) optimization and adaptation of methods to cell culture. This page discusses various techniques currently used to quantify viruses in liquid samples. These methods are separated into two categories, traditional vs. modern methods. Traditional methods are industry-standard methods that have been used for decades but are generally slow and labor-intensive. Modern methods are relatively new commercially available products and kits that greatly reduce quantification time. This is not meant to be an exhaustive review of all potential methods, but rather a representative cross-section of traditional methods and new, commercially available methods. While other published methods may exist for virus quantification, non-commercial methods are not discussed here. Virus quantification involves counting the number of viruses in a specific volume to determine the virus concentration. It is utilized in both research and development (R&D) in commercial and academic laboratories as well as production situations where the quantity of virus at various steps is an important variable. For example, the production of viral vaccines, recombinant proteins using viral vectors and viral antigens all require virus quantification to continually adapt and monitor the process in order to optimize production yields and respond to ever changing demands and applications. Examples of specific instances where known viruses need to be quantified include clone screening, multiplicity of infection (MOI) optimization and adaptation of methods to cell culture. This page discusses various techniques currently used to quantify viruses in liquid samples. These methods are separated into two categories, traditional vs. modern methods. Traditional methods are industry-standard methods that have been used for decades but are generally slow and labor-intensive. Modern methods are relatively new commercially available products and kits that greatly reduce quantification time. This is not meant to be an exhaustive review of all potential methods, but rather a representative cross-section of traditional methods and new, commercially available methods. While other published methods may exist for virus quantification, non-commercial methods are not discussed here. Plaque-based assays are the standard method used to determine virus concentration in terms of infectious dose. Viral 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 plates. Specifically, a confluent monolayer of host cells is infected with the virus at varying dilutions and covered with a semi-solid medium, such as agar or carboxymethyl cellulose, to prevent the virus infection from spreading indiscriminately. A viral plaque is formed when 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. The infected cell area will create a plaque (an area of infection surrounded by uninfected cells) which can be seen visually or with an optical microscope. Plaque formation can take 3–14 days, depending on the virus being analyzed. Plaques are generally counted manually and the results, in combination with the dilution factor used to prepare the plate, are used to calculate the number of plaque forming units per sample unit volume (pfu/mL). The pfu/mL result represents the number of infective particles within the sample and is based on the assumption that each plaque formed is representative of one infective virus particle. The focus forming assay (FFA) is a variation of the plaque assay, but instead of relying 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 or TCID50 assays, 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. 50% Tissueculture Infective Dose (TCID50) is the measure of infectious virus titer. This endpoint dilution assay quantifies the amount of virus required to kill 50% of infected hosts or to produce a cytopathic effect in 50% of inoculated tissue culture cells. This assay may be more common in clinical research applications where the lethal dose of 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: 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: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.