The virology pages are organized like this:
- What is a virus?
- Review of genomes, RNA, DNA and replication
- Viral genomes
- Structures of viruses
- Attachment & entry
- Viral replication
- Basic immunology
An assay for infectious virus particles
Many assays exist for determining the number of virus particles in a sample, or even fragments of virus particles. For example, in the SARS-CoV-2 epidemic of 2020-21, PCR (polymerase chain reaction) was a common test performed on patients and samples. But these tests, sensitive as they are, could give positive results even if the sample contained only remnant mRNAs or fragments of them. That's not a test for infectious virus, and that's ultimately what we'd like to know: How much infectious virus is in, say, tha nasal passages of an infected person?
The plaque assay is a handy and pretty simple way to measure (to assay) the number of infectious virus particles in a sample. The basic idea is that several serial dilutions of a virus-containing sample are made and applied to a sample of living host cells.
The virus samples are prepared in an agarose gel, which reduces their ability to diffuse throughout the sample, so any virus particle can only infect the closest cell, and any further infections of neighboring cells will likely come from new viruses leaked from that original infected cell.
Once several cells (hundreds or thousands because they're small) have been infected, a visible plaque (a hole in a confluent "lawn" of cells) is formed, and those holes can be counted, yielding the number of infectious particles applied to that sample. Knowing the dilution factor gives the number of infectious particles in the original virus sample.
On this page we'll go through the basics of how to design and perform a plaque assay to measure the quantity of infectious virus in a sample.
In the context of a plaque assay, a plaque is a region on a culture plate in which cells have died or become damaged, making them visually distinct from the intact cells around them.
Infectious particle or viral fragment?
Here's just a little more about the motivation for doing a plaque assay. consider the figure below, which shows the approximate course of infection by measuring "viral load." That is, we might use PCR or some antigen test (exposing a sample to antibodies known to bind to certain protein sequences of viral surface proteins) to measure the relative amount of virus in a sample.
The problem is that what we measure over the course of an infection can change. In the early stages of an infection, we often observer some "incubation phase," during which a few viruses are multiplying and getting the infection going, a disease phase during which many of the signs and symptoms of the disease might be manifested by the host, and a phase during which the adaptive immune system has got the upper hand in reducing the viral load.
During that last phase, in particular, it might be possible to be amplifying or detecting just fragments of viruses, leading to the impression that there are more infectious virions than there really are. That's why we do experiments like the plaque assay.
Preparing the assay
A plaque assay can be performed in a Petri dish (usually plastic) or a plastic, multi-welled plate, like a six-well plate. Petri dishes are often just called "plates." Here are the basic steps in preparing the plates or wells (plate shown)
- A petri dish or multi-well plate is partially filled with a sterile mixture of agar and nutrient medium. The medium is specific for the cell type that needs to be supported.
- A cell culture is applied to the medium+agar on the plate, in sufficient quantity that colonies will merge (become confluent) and cover the entire surface. In the beginning individual colonies might be visible, ...
- ... but eventually there will be a "lawn" of confluent cells covering the agar + medium mixture.
- Once the cells have grown, and before they exhaust the medium, a top agar containing a known dilution of the virus sample (and cooled sufficiently so as not to kill the live cells on the plate) is poured over the grown cells and allowed to gel. This is a crucial step in the plaque assay. It severley limits their diffusion across the plate, so that if a particle is infected, viruses formed from the infection are limited to infecting neighboring cells. That's how the plaque forms.
The dilutions
First: Why do we need to make dilutions? It's because we don't know the concentration of infectious virus particles in our sample. By making dilutions and performing plaque assays for each, we guarantee that we'll find at least one set of conditions that forms separate plaques that can be easily counted and that don't overlap. Generally, we shoot for more than 20 but less than 100, depending on the size of the plate used. If fewer than ten plaques are found, we worry about the statistical validity of the sample.
The serial dilutions are fairly simple to do. What's important is to keep track of the dilution factor, as this is a quantitative assay of infectious particles. We'll work out the math below. Equivalent plaque plates are prepared with equal volumes of the virus-solution dilutions, so that what's different between plates is just that dilution factor. Generally, serial dilutions are prepared using factors of two or ten.
Plaque formation
Explanation of how the plaques form
Imagine that two infectious viruses are deposited on the plate so that they can infect cells. These are marked by
The top agar restricts diffusion of viruses so the only way for more cells to be infected is from viruses replacating in and escaping from one cell – the center cell of a growing circle of degraded cells, a hole in the lawn of cells.
Over time, the holes will grow larger. Part of the art of the plaque assay is to analyze the plates while the plaques are big enough to count but small enough to have merged and become uncountable.
An example
Let's say that each of our serial dilutions contained a total volume of 1 mL, and that we made 10-fold dilutions. That's 100 μL of the original sample plus 900 μL of buffer solution in the first dilution, and so on down the line — 100 μL of the previous solution placed into 900 μL of buffer and mixed. Now for each plate, we transfer 100 μL of the corresponding dilution to the top-agar solution. That last part is an additional dilution because we only took 10% of each solution. That's easy to overlook.
Now let's say that we obtained a nice plate with 21 distinct plaques from our 10-6 dilution. That means:
- There were 21 infectious particles in the 100 μL of solution transferred to the plate, so there were 210 in the dilution sample.
- The dilution factor was 106, so that's $210 \times 10^6$ infectious particles in the original sample. In proper scientific notation that would be $2.1 \times 10^8$ infectious particles per milliliter.
The advantages of the plaque assay are that (1) it's quantitative, and (2) It counts infectious viruses. Recall that not all virus particles in any given sample are actually capable of infecting cells. It's extremely valuable to get a good estimate of the fraction that are, so long as we can also assay for the total number of particles, infectious or not.
Note: Sometimes infectious particles, in the context of a plaque assay, are called plaque-forming units (PFU).
One complication: multiplicity of infection
One small complication to this picture is that we tend to assume that it takes a single infectious virus particle to cause an infection in a host cell. That's not necessarily the case. If it takes, on average, three viruses to produce an infected cell, we say that the multiplicity of infection (MOI) is 3 in that situation.
We approach MOI from a statistical perspective, and we'll save that for another (as yet future) section. The possibility of an MOI greater than one is part of the motivation for referring to the number of infectious particles as the number of plaque-forming units.
Confluent
Confluent means flowing together or merging.
Two rivers flow together at their confluence.
When cells grow together into a continuous "lawn" on a culture plate, they are said to be confluent.
Serial dilutions
A dilution of a solution (containing one or more solutes in a solvent) is made by placing a small quantity of a more concentrated solution into a known amount of solvent (or equivalently, just adding more solvent).
Serial dilutions are made by diluting a solution, then diluting the dilution in the same way, so as to make a series weaker solutions. Here's a diagram of a two-fold serial dilution:
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