Virus cultivation in the virology laboratory is fundamental to virological research, serving key roles in diagnosis, basic studies, and vaccine or drug development. It enables the isolation and identification of disease-causing viruses from animal samples by observing cytopathic effects (CPE) and confirming results with molecular or immunological methods such as RT-PCR.
In basic research, virus culture enables investigation of viral infection, replication, and host cell responses, providing insights into disease mechanisms and preventive strategies. It is also essential for producing large quantities of virus for use in attenuation or inactivation during vaccine production, such as for rabies, influenza, and African horse sickness.
Moreover, in vitro virus culture provides a safe and efficient system for evaluating antiviral compounds, prototype vaccines, and neutralizing antibodies, supporting ongoing efforts in disease control and surveillance.
Before using propagated viruses for various applications, an essential step is to accurately quantify the virus. Several quantification methods are available, and the choice depends on the intended purpose of the virus. Generally, these methods can be classified into two main categories:
1. Infectious Virus Quantification Methods — These techniques measure only infectious (viable) viruses capable of replicating and causing infection in host cells. Common examples include the Plaque Assay, Tissue Culture Infectious Dose 50% (TCID50) Assay, Focus Forming Assay (FFA), and Endpoint Dilution (Quantitative Infection) Assay.
2. Total Virus Quantification Methods — These approaches determine the total number of viral particles or their components, such as viral proteins or nucleic acids, without distinguishing between infectious and inactivated viruses. Representative methods include the Hemagglutination (HA) Assay, Enzyme-Linked Immunosorbent Assay (ELISA), Quantitative Polymerase Chain Reaction (qPCR or RT-qPCR), Transmission Electron Microscopy (TEM), and Flow Cytometry.
Among these methods, infectious virus quantification is particularly important when the goal is to measure the number of viruses capable of causing infection. Within this group, the Plaque Assay is widely regarded as the gold standard, providing accurate and reproducible measurements of infectious viral particles. Its reliability and ability to quantify viable viruses make it an essential tool in virology research.
Plaque Assay
Virus quantification by the plaque assay is a highly reliable method and is considered the gold standard for determining the concentration of viable viruses, as it allows accurate identification and quantification of infectious viral particles. The principle of the plaque assay is to measure the number of viruses capable of causing infection and cell death.
In this method, a sample of the virus is first inoculated onto a prepared monolayer of cultured cells. After the virus infects the cells, an overlay medium or substrate is applied to restrict viral spread. This setup enables progeny viruses originating from each initially infected particle to replicate locally, forming clear zones known as plaques.
Each plaque represents an area where cells have been destroyed by viral infection and can be observed as a visible hole or a transparent region on the cell monolayer. The shape of plaques may be circular or irregular with indistinct edges, depending on the virus type and the host cells used. Finally, the number of plaques can be counted to determine the viral concentration, with each plaque corresponding to a single infectious viral particle, defined as one plaque-forming unit (PFU). Examples of plaques produced by different viruses in various cell types are shown in Figure 1.
Figure 1 Plaque formation of different viruses in various cell lines, a) Porcine epidemic diarrhea virus (PEDV) in Vero cells, b) Duck Tembusu virus (DTMUV) in Vero cells, and c) Canine coronavirus (CCoV) in A72 cells.
The general steps of the plaque assay procedure, following virus isolation and propagation, are as follows:
Plaque counting is typically conducted on dilutions that yield an appropriate number of plaques, usually within the range of 10–50 or 10–100 per well, depending on the virus and plaque size. Viral samples should be diluted across a sufficiently broad range to ensure an optimal concentration of infectious viral particles. If the viral concentration is too high, plaques may overlap, while very low concentrations can result in considerable variability in plaque counts, as shown in Figure 2.
Figure 2 Plaque formation of Duck Tembusu virus (DTMUV) in Vero cells at different viral dilutions (10-4 – 10-6).
The number of plaques formed depends on the viral concentration. Plaque counting should be performed using dilutions that yield an appropriate number of plaques. The plaques suitable for counting should be well separated, clearly visible, and free from overlap. To minimize plaque overlap during incubation, plates can be gently rocked every 30 minutes throughout the incubation period. Examples of clearly defined plaques suitable for counting are shown in Figure 3.
Figure 3 Plaque formation of each virus, showing clear and well-separated plaques without overlap.
In addition to plaque numbers, plaque size is also an important factor influencing counting accuracy, as excessively large plaques can overlap and cause counting errors. Plaque size is influenced by the incubation period, which varies among different viruses, and even for the same virus, variations in incubation duration can lead to plaques of different sizes under identical cell types and experimental conditions. Therefore, the incubation period should be selected appropriately. For example, in a plaque assay of Porcine epidemic diarrhea virus (PEDV) on Vero cells, incubation for 3, 4, and 6 days produced plaques of different sizes, as shown in Figure 4.
Figure 4 Plaque morphology of Porcine epidemic diarrhea virus (PEDV) in Vero cells at 3, 4, and 6 days of incubation
The number of plaques counted is used to determine the viral concentration, expressed as plaque-forming units (PFU). This calculation considers the number of plaques, the volume of virus added, and the corresponding dilution factor. For example, if 1 ml of virus at a 10-5 dilution is added per well and 35 plaques are observed, the viral concentration is 3.5 × 106 PFU/ml. The viral titer can then be calculated using the following formula:
The plaque assay is a method used to quantify live viruses capable of infecting cultured cells (infectious virus), making it a gold-standard technique for measuring infectious viral particles. When performed under consistent conditions, it yields reproducible results. The assay can also be used to characterize viruses, as plaque size, shape, and clarity differ among viral types. Furthermore, the plaque assay is relatively low-cost and requires only basic cell culture equipment, supporting its widespread use in general virology laboratories.
Although the plaque assay is considered the gold standard for quantifying infectious viruses, it has several limitations. The procedure is time-consuming, typically taking 3–7 days depending on the virus, and is applicable only to viruses that induce cytopathic effects (CPE) in the cultured cells used. This requires highly susceptible cell lines and strict aseptic techniques. Skilled personnel are also needed to ensure proper execution, including preparing uniform cell monolayers, performing accurate serial dilutions of the virus, and carefully applying the overlay medium, as even minor errors can lead to significant variations in plaque counts.
In conclusion, the plaque assay is a widely used method for quantifying infectious viruses, with both advantages and limitations. It provides reliable and reproducible measurements, but its time-consuming nature and requirement for viruses that induce cytopathic effects should be considered. The decision to use this assay should be guided by the study’s purpose, as well as the availability of suitable equipment, laboratory facilities, and skilled personnel. In laboratories equipped for cell culture, the plaque assay can be performed efficiently, making it a practical and valuable tool for virus quantification and virology research.
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Jarupha Taowan
Scientist (Senior Professional Level), The Monitoring and Surveillance Center for Zoonotic Diseases in Wildlife and Exotic Animals (MoZWE)