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What is ISO18184, and what is it for? Suppose you’ve got a textile product and you’d like to know if it is antiviral. Then you need ISO18184. The ISO18184 test is a standardized experiment for testing the virucidal properties of textile products. ISOs (International Organization for Standardization) are standards internationally agreed upon by experts; they can be thought of as a formula describing the best way of doing something. So consider the ISO18184 as an expertly developed test that answers a very specific question: does my textile product have antiviral activity?

What happens during an ISO18184 test?
During the ISO18184 experiment, we add virus (you can choose among our virus library) onto the test textile (the textile you’re interested in) and reference textile (not expected to be antiviral, usually the test textile but without the active ingredient). The virus is then left in contact with the textiles, typically for 2 hours at room temperature. After the incubation period, any surviving virus is recovered using liquid media. The amount of surviving virus is then quantified, making it possible for us to say whether the test fabric is antiviral (relative to the reference fabric).

ISO18184 includes multiple controls
In addition to the main test, ISO18184 includes many controls and test criteria that make ISO18184 more reliable and reproducible. Below we describe each of these controls and how the antiviral activity of a tested fabric is calculated.

How is the ISO18184 assay performed?
When testing a textile for antiviral activity, we typically use 20 × 20 mm textile squares, stacking these so that we have0.4 g of fabric. The pieces are then added to a tube, where the test will be performed. All tests are done in triplicate, so we’ll set up three tubes with the test textile and three tubes with inert reference textile. We’ll also prepare tubes with samples for conducting the ISO18184 controls. A small volume of virus (200 µl, which is one-fifth of a milliliter) is added to each tube and should be absorbed into the textile. After the virus and textile have had 2 hours to interact in the tube, the surviving virus is recovered in liquid media. To then measure how well the virus survived, an indirect quantification method is used, where the damage caused by the infection to mammalian host cells is observed. 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 a sufficient period (days) to allow infection to progress. At the end of this period, the cells are fixed and stained with crystal violet, which allows us to discriminate between 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 is used to calculate the final R-value for the tested textile.

How is the antiviral activity of a textile calculated?
ISO18184 tests the reduction in a virus on the textile of interest relative to a control sample. This reduction is known as the Mv-Value and is the difference between the amount of virus recovered from the reference immediately after the inoculation (Back Titration) and from the test sample, both expressed as base 10 logarithms. To claim a mild antiviral activity, a sample must achieve an Mv ≥ 1. To claim a good antiviral activity, a sample must achieve an Mv ≥ 2. To claim an excellent antiviral activity, a sample must achieve an Mv ≥ 3. It’s important to note that for ISO18184 to be valid, the difference between the virus recovered from the reference immediately after the inoculation (Back Titration) and from the reference after the contact time must be less than 1 log. This is because the antiviral activity is always compared with the Back Titration, which can’t differ much from the reference sample values.

The cytotoxicity control
As discussed, to measure infectious viruses, the ISO18184 test relies on cultured host cells; the more infected host cells we identify, the more virus was recovered from the test. That happens because 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 textile, wait 5 minutes, recover the media, and add it to the host cells. The virus has been omitted, so if the host cells die, we know 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 important way to assess if the test is valid is to check if the test textile interferes with the host cells’ sensitivity to the virus. For example, if the test textile releases something into the culture media that makes the cells resistant to the virus, meaning the virus wouldn’t be able to infect the cells, and the cells would survive. This would give a false impression that the textile is antiviral. In the opposite scenario, the textile could make the host cells more sensitive to the virus. The infectivity control avoids these scenarios. In this control, we add the liquid media to the test and the reference textiles, wait 5 minutes, recover the media, add a virus to the recovered media, and wait 30 minutes. As a term of comparison, a media control is also added, where the media hasn’t been in contact with the textile at all. After the incubation period, the host cells are infected with the media plus virus mixture. In this control, the virus has never been in contact with the test textile, so we expect to have an infectious virus that will kill the host cells. If many fewer or many more host cells are killed than what is measured in the media control sample, the test can be invalidated. This control shows that 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)
There is no incubation period for this control, which means the virus is recovered from the textile reference right after its addition. The recovered virus collected will be used to calculate the initial virus’ dose in the experiment. If the recovery shows a low number of virus particles, this could indicate a problem with the virus stock and its concentration. This control also indicates the maximum amount of virus that can be recovered from the experimental procedure, providing a useful benchmark to test whether there was any reduction in infectivity in the reference control.

Our in-house controls
The controls described above are part of the ISO18184 criteria for a valid test. If these controls fail, the test can’t be trusted, and we go back to the drawing board. Here at Virology Research Services, we also include our in-house positive and negative controls as part of each experiment. We know these textiles well; we know exactly how they should behave in the ISO18184 test, and this lets us monitor how our ISO18184 is running. Our in-house positive control is a reliably antiviral textile. The negative control is an inert fabric with no antiviral properties, so we expect little or no virus inactivation with this sample. This can be important, particularly when our client’s reference material has antiviral activity(this can happen, especially when it comes to dry-washed or dyed fabrics, that’s why it is so important to have an inert reference material).

Can you ever 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 understanding where things might have gone wrong. So if your assay is valid and your Mv-Value is higher than 1, you can be confident that your samples have been thoroughly tested and proud of the results!

Blog by Thais Fuscaldi Reboucas
Edited by Reckon Better Scientific Editing