There is an excellent question in the comments to “Are all virus particles infectious?“: if the particle-to-PFU ratio for a virus stock is 10,000:1, and I infect 1,000,000 cells with 10,000 particles, how many plaques would I expect to observe? Answering this question provides insight into the particle-to-PFU ratio of viruses.
If we take 10,000 particles of our virus stock and infect 1,000,000 cells, we are adding just one infectious particle. Therefore a correct answer to the question is one plaque. But would you be wrong if you answered 100 plaques? That would depend on how you justified your answer.
To understand why 100 plaques could be correct, we need to do some math, and calculate the number of virus particles that each cell receives. If we add 10,000 particles to 1,000,000 cells, the MOI is 0.01. At that MOI, 0.01% of the cells will receive more than one virus particle. In a culture of 1 million cells, 100 cells will receive at least two virus particles and could, in theory, become productively infected. Let’s explore why.
The linear nature of the dose-response curve indicates that a single virion is capable of initiating an infection. However, the high particle-to-pfu ratio of many viruses shows that not all virions are successful. A high particle-to-pfu ratio is sometimes caused by the presence of noninfectious particles with genomes that harbor lethal mutations.
To simplify this problem, let’s assume that among the 10,000 noninfectious particles in our sample, half of them have a mutation in gene A and half have a mutation in gene B. This scenario is illustrated in the figure, which shows a cell infected with two viruses (only the viral genomes are shown). Both mutations are lethal – cells infected with either viral mutant do not produce new virus particles. However, when a cell is infected with both virus mutant A and virus mutant B, complementation of the defects might occur. The virus with mutant gene A produces a fully functional gene B product; and the virus with mutant gene B produces a fully functional gene A product. The result is that the infected cell contains functional versions of proteins A and B, and viral replication can occur. It’s also possible that the two viral genomes might undergo recombination, producing a new genome that does not contain any lethal mutations. Either mechanism could explain why we might expect to observe up to 100 plaques in this experiment.
The reality is that the 10,000 noninfectious virus particles in our stock likely have mutations in many genes, not just two. Therefore the probability that complementation or recombination can correct the defects is remote. This is the reason why we are likely to observe just one plaque in our experiment.
The plaque assay is an essential tool for determining virus titers. The concept is simple: virus infection is restricted to neighboring cells by a semisolid overlay. By counting the number of plaques, the virus titer can be calculated in PFU per ml. A key question is: how many viruses are needed to form a single plaque?
For most animal viruses, one infectious particle is sufficient to initiate infection. This conclusion can be reached by studying the relationship between the number of infectious virus particles and the plaque count. A linear relationship means that one infectious particle can form a plaque. In this case the virus is said to infect cells with one-hit kinetics. This concept is illustrated below. In this figure, the number of plaques produced by a virus with one-hit kinetics or two-hit kinetics is plotted versus the relative concentration of the virus.
There are some examples of viruses with two-hit kinetics: in other words, two different types of viral particles must infect a cell to initiate the infectious cycle. Examples include the genomes of some (+) strand RNA viruses of plants, which consists of two RNA molecules that are packaged in different particles. The dose-response curve of such viruses is parabolic rather than linear.
When a single virus particle can form a plaque, the viral progeny within the plaque are clones. Virus stocks prepared from a single plaque are called plaque purified virus stocks. To prepare such virus stocks, the tip of a small pipette is inserted into the agar overlay above the plaque. The plug of agar is removed and placed in buffer. The viruses within the agar plug move into the buffer, which can then be used to infect cultured cells. To ensure purity, this process is usually repeated at least one more time. Plaque purification is used extensively in virology to establish clonal virus stocks. The ability to prepare clonal virus stocks was an essential development that permitted genetic analysis of viruses.