The Lazarus virus

infected cellThere 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.

Are all virus particles infectious?

particle to pfu ratioChris Upton, a contributor to the virology toolbox, has raised an important point about multiplicity of infection:

Perhaps this is a place to bring up particle to pfu ratio? The above is great for when talking about phage, for example, when the ratio approaches 1. But with something like polio when it can be very high (>1000 ??), then it’s not that all cells don’t receive “a particle” at MOI=1 – but that they don’t get an “infectious dose”. Not sure how to say it better – enough to initiate an infection.

So why does polio require 1000 virions to make an infectious dose? I don’t buy the idea that most of the particles are not “viable”.

If we take the titer of a virus preparation (in plaque forming-units per milliliter) and divide it by into the number of virus particles in the sample, we obtain a number known as the particle-to-PFU ratio. It is a measure of the fraction of virus particles in a given sample that can complete an infectious cycle. For many bacteriophages, the particle-to-PFU ratio approaches 1, which is the lowest value that can be obtained. A value of 1 means that every virus particle in the sample is able to form a plaque.

For animal viruses, the particle-to-pfu ratio is often much higher, from 1 to 10,000 (the image shows values for different animal viruses – click to enlarge). These high values complicate the study of animal viruses. When the particle-to-pfu ratio is high, one can never be certain that properties measured in infected cells are those of the infectious or the non-infectious viral particles.

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 or that have been damaged during growth or purification. Another explanation is that although all viruses in a preparation are in fact capable of initiating infection, not all of them succeed because of the complexity of the infectious cycle. Failure at any one step in the cycle prevents completion.

A high particle-to-pfu ratio does not indicate that most particles are defective, but that they failed to complete the infection.