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.

TWiV 129: We’ve got mail

rich unwindsHosts: Vincent Racaniello, Alan Dove, Dickson Despommier, and Rich Condit

Vincent, Alan, Dickson and Rich answer listener questions about XMRV, yellow fever vaccine, virus-like particles, West Nile virus, amyotrophic lateral sclerosis and human endogenous retroviruses, multiplicity of infection, and how to make a poxvirus.

Click the arrow above to play, or right-click to download TWiV #129 (67 MB .mp3, 93 minutes).

Subscribe to TWiV (free) in iTunes , at the Zune Marketplace, by the RSS feed, by email, or listen on your mobile device with the Microbeworld app.

Links for this episode:

Weekly Science Picks

Rich – Polyxeni Potter and EID covers
Dickson – American Museum of Natural History
Alan –
Moon Trees (EurekAlert! article)
Vincent – Infection Landscapes

Listener Picks of the Week

Didier  – The Vaccines (MySpace)
/Sven-Urban –
The Science of Discworld by Terry Pratchett
GarrenOmega Tau podcast

Send your virology questions and comments (email or mp3 file) to twiv@microbe.tv, or call them in to 908-312-0760. You can also post articles that you would like us to discuss at microbeworld.org and tag them with twiv.

TWiV 118: The virus always rings twice

Hosts: Vincent Racaniello, Alan Dove, and Rich Condit

On episode #118 of the podcast This Week in Virology, Vincent, Alan, and Rich answer listener questions about vaccinia virus, fungal viruses, synthetic viruses, influenza vaccine, HeLa cells, multiplicity of infection, and much more.

Click the arrow above to play, or right-click to download TWiV #118 (68 MB .mp3, 94  minutes).

Subscribe to TWiV (free) in iTunes , at the Zune Marketplace, by the RSS feed, or by email, or listen on your mobile device with the Microbeworld app.

Links for this episode:

Weekly Science Picks

Alan – What you need to know about infectious disease
Rich – Bad Project (YouTube)
Vincent – Federal research center will help develop medicines

Send your virology questions and comments (email or mp3 file) to twiv@microbe.tv. You can also post articles that you would like us to discuss at microbeworld.org and tag them with twiv.

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.

Multiplicity of infection

Multiplicity of infection (MOI) is a frequently used term in virology which refers to the number of virions that are added per cell during infection. If one million virions are added to one million cells, the MOI is one. If ten million virions are added, the MOI is ten. Add 100,000 virions, and the MOI is 0.1. The concept is straightforward.

But here is the tricky part. If you infect cells at a MOI of one, does that mean that each cell in the cutlure receives one virion?

The answer is no.

Here is another way to look at this problem: imagine a room containing 100 buckets. If you threw 100 tennis balls into that room – all at the same time – would each bucket get one ball? Most likely not.

How many tennis balls end up in each bucket, or the number of virions that each cell receives at different MOI, is described by the Poisson distribution:

P(k) = e-mmk/k!

In this equation, P(k) is the fraction of cells infected by k virus particles, and m is the MOI. The equation can be simplified to calculate the fraction of uninfected cells (k=0), cells with a single infection (k=1), and cells with multiple infection (k>1):

P(0) = e-m

P(1) = me-m

P(>1) = 1-e-m(m+1)*

*this value is obtained by subtracting from unity (the sum of all probabilities for any value of k) the probabilities P(0) and P(1)

Here are some examples of how these equations can be used. If we have a million cells in a culture dish and infect them at a MOI of 10, how many cells receive 0, 1, and more than one virion? The fraction of uninfected cells – those which receive 0 particles – is

P(0) = e-10

= 4.5 x 10-5

In a culture of one million cells this is 45 uninfected cells. That’s why an MOI of 10 is used in many virology experiments – it assures that essentially every cell is infected.

At the same MOI of 10, the number of cells that receive 1 particle is calculated by

P(1) = 10e-10

= 10 x 4.5 x 10-5

= 4.5 x 10-4

In a culture of one million cells, 450 cells receive 1 particle.

How many cells receive more than one particle is calculated by

P(>1) = 1-e-10(10+1)

=0.995

In a culture of one million cells, 999,500 cells receive more than one particle.

Using the same formulas, we can determine the fraction of cells receiving 0, 1, and more than one virus particle if we infect one million cells at a MOI of 1:

P(0) = e-1 = 0.37 = 37% of cells are uninfected

P(1) = 1 x e-1 = 37% of cells receive one virion

P(>1) = 1 – e-1(1+1) = 26% of cells are multiply infected

An assumption inherent in these calculations is that all cells in a culture are identical in their ability to be infected. In a clonal cell culture (such as HeLa cells) the deviations in size and surface properties are small enough to be negligible. However, in a multicellular animal there are substantial differences in cell types that affect susceptibility to infection. Under these conditions, it is experimentally difficult to determine how many virions infect different cells.

High MOI is used when the experiment requires that every cell in the culture is infected. By contrast, low MOI is used when multiple cycles of infection are required. However, it is not possible to calculate the MOI unless the virus titer can be determined – for example by plaque assay or any other method of quantifying infectivity.