Contaminated surfaces and materials can be an important route for virus spread. During the COVID-19 pandemic, we’ve become accustomed to regularly wiping down any regularly touched surface.

There is also growing demand for next-generation antiviral surfaces able to rapidly inactivate any contaminating virus particles. In response, material developers have taken two principal approaches: using naturally antiviral materials (e.g., copper and various copper derivatives) or incorporating antiviral additives into their products.

ISO 21702
ISO 21702 is a method designed to test the virucidal properties of plastic and other non-porous surfaces. This involves loading a pre-determined concentration of virus onto the test surfaces and a reference surface, which are then left in a humidified chamber at room temperature for up to 24 hours. Any surviving virus is then recovered by washing the samples with liquid media. The amount of infectious virus recovered from these samples is then quantified, informing on whether the tested surface is antiviral by comparison with the reference control. That all sounds straightforward, but let’s dig a little deeper into the experimental process. Beyond the basic antiviral test, there are also many control experiments and pass/fail criteria built into the ISO21702 test. These controls have been carefully designed to ensure the test results are reliable, reproducible, and meaningful. Below we describe each of these controls, and then how the antiviral activity of a tested surface is calculated.

How the assay is performed
When testing a surface for antiviral activity, we typically use 5 × 5 cm squares and always test in triplicate: so, three square pieces of the test surface and three squares of the inert control surface (plus others for the controls described below). A small volume of virus (200 µl, which is one-fifth of a millilitre) is added to each piece of test surface and control surface. After the agreed upon contact time between the virus and test surfaces (which can be anything up to 24 h), the surviving virus is recovered in liquid media.

Next, we measure how well the virus survived on the test and control surfaces. This is not done directly, but by observing the damage caused by infection to mammalian host cells. The surviving virus is loaded onto cells cultured in 96-well plates: four columns for each of the three replicates, with each column consisting of a 10-fold dilution series.

The virus is then left with the cultured cells for sufficient time (days) to allow infection to progress. At the end of this period, the cells are fixed and stained with crystal violet, a stain that allows us to discriminate those wells containing cells infected with the virus (white well) or not infected (violet well). Each well is scored as either infected or not infected, and this data used to calculate the final R value for the tested surface.

How the antiviral activity of a surface is calculated
ISO21702 tests the reduction in virus on the surface of interest relative to a control sample. This reduction is known as the R-Value, and is the difference between the amount of virus recovered from the reference and from the test sample, both expressed as base 10 logarithms. To claim antiviral activity, a sample must achieve an R-value of 1 or more. R = 1 is equivalent to a 90% reduction of infectious virus in the sample-of-interest relative to the control sample, R = 2 a 99% reduction, and R = 3 a reduction of 99.9%, and so on.

It’s important that the R-value is a relative measurement (relative to an inert control) because viruses tend to degrade and become inactive on any surface over time. So, the R value tells us the ‘extra’ virus inactivation due to the sample-of-interest. Other reasons for reporting a relative rather than an absolute reduction in virus is because the lab conditions might differ from day to day, and there might be batch variability in the virus stock that is used. By reporting a relative value (the R-value), we control for such variations and test only the contribution of the treatment to the inactivation of the virus.

The cytotoxicity control
As discussed, the ISO21702 test relies on cultured host cells to measure infectious virus. At its simplest, we measure the amount of infectious virus recovered from the test surfaces by adding the recovered virus on the host cells. The virus kills these host cells in a characteristic way (called cytopathic effect or CPE); so the more infectious virus recovered, the more host cells will be killed. For this to work, we must be certain it’s the virus (and only the virus) that’s killing the host cells. Suppose a test sample leaches a toxic substance into the test media that subsequently kills the host cells. Without an appropriate control, we might falsely conclude the test material is not antiviral: “the host cells died, so there must have been lots of surviving virus”.

This is where the cytotoxicity control comes in. In this control we add the liquid media to the test surface, wait 5 min, recover the media, and add it to host cells. The virus has been omitted, so if the host cells die, we know that there must be toxicity coming from the test material. A result like this can invalidate the whole test. We should also note that this test only assesses the cytotoxicity toward cultured cells growing in media under specific lab conditions, and is designed to support the conclusions of the antiviral test. It is not an attempt to assess the toxicity of the test material in a real-world setting.

The infectivity control
Another way the ISO21702 test might be compromised is if the test material interferes with the host cells’ sensitivity to the virus. Suppose the test material released something into the culture media that made the cells resistant to the virus. The cells would live even if the virus itself was unaffected, and the researchers might falsely conclude that the material is antiviral: “the host cells live, so the virus must have been killed”. It would also be a problem if the material somehow made the host cells especially sensitive to the virus. The infectivity control is there to guard against these scenarios.

In this control we add the liquid media to the test and the reference surfaces, wait 5 min, recover the media, then add virus to the recovered media and wait 30 minutes. We also add the virus to the same volume of media that has not been in touch with either the test or the reference material, as a term of comparison. We then combine the media plus virus with the host cells. Note that here the virus has never been in contact with the test material, so we expect to have infectious virus that will kill our host cells. If many fewer or many more host cells are killed than what is measured in the media only sample, the test can be invalidated. Put simply, we cannot test whether the material inactivates the virus if something has leached into the culture media that then affects the normal host-virus interaction.

The Back Titration (or virus recovery control at time zero)
Here the virus is recovered from the test material immediately after adding to the test surface (time zero). This recovered virus is then used to calculate the initial dose of virus in the experiment. If there is too little virus, this could indicate a problem with the virus stock. It also informs on the maximum amount of virus that can be recovered from the experimental procedure, providing a useful benchmark to identify whether there was any reduction in infectivity in the reference control.

VRS Controls
All these controls are important to ensure that the results of the antiviral test are correctly interpreted, and that the conditions for a valid assay are all checked. In addition to the ISO21702 controls just described, at VRS we include one positive and one negative control, which are treated as the test and reference materials. The positive control is represented by a validated material with robust and reproducible antiviral properties. The negative control is inert plastic, with no antiviral properties. The negative control confirms that, in inert conditions, only marginal (if any) virus inactivation is observed. This can be important, particularly when the reference material is itself antiviral (this can happen…). The positive control ensures that the assay has been performed correctly, and the conditions to reveal the virucidal properties of a material are met. In addition, it allows us to compare the performance of our assay over time, and across different operators.

You can never have enough controls…
As in any well performed scientific test, the number of controls easily exceeds the number of test conditions! Controls are critical for understanding and interpreting the test, and also to understand where things can go wrong. So if your assay is valid and your R is higher than 1, you can be confident that your samples have been thoroughly tested, and proud of the results!