A D V A N C E D M A T E R I A L S & P R O C E S S E S | J U L Y / A U G U S T 2 0 2 1 2 9 of viral structures must involve the specific components of the substrate itself. From the standpoint of antiviral materials development, this effect should be investigated further. VIRUSES ON MATERIALS Viruses can live on inanimate materials even though there are no virus-receptor interactions as there are in biological systems. The virus generally maintains its activity for a couple of days—during which time it can still infect human beings. The duration of infectious capabilities depends on the properties of the specific material involved. As described above, there must be some reaction or interaction between the virus and the material. To find the mechanism of the interaction or potential reaction, an evaluation system must be developed so that new and advanced antiviral materials can be created. MEASURING VIRUS ACTIVITY Various methods are available to evaluate the behavior of viruses on material surfaces. For example, transmission electron microscopes (TEMs) are often used to visualize viruses. There are also detection methods such as the antigen-antibody reaction (AAR) (called immunochromatography) and enzyme-linked immunosorbent assay (ELISA). However, these techniques are not suitable for evaluating the anti- viral characteristics of materials. This is because those evaluation methods usually depend on expensive and high-performance apparatus requiring experienced operators, and quantitative evaluation is difficult. Fortunately, a few other evaluation methods have been developed to determine the antiviral characteristics of materials. Because viruses are particles on the nanoscale, it is difficult to count their numbers. Such a process requires a high-resolution microscope such as a TEM. Evaluation of viral infectivity is usually achieved by measuring the cellular degeneration of the virus. When viruses are active, they retain the ability to attack cells, and this is the phenomenon used for evaluation. This type of evaluation is called the tissue culture infectious dose 50 assay, or TCID50 assay[4,5]. In this method, a series of diluted viral solutions are prepared and inoculated into the cellular mediums (Fig. 3). When half of the prepared cellular mediums for each series of diluted viral solutions is destroyed, the concentration of the viral solution is defined as infectivity. For example, in Fig. 3, suppose the amount of inoculation is x ml and for the 1% diluted series, half of the inoculatedcellswouldbe destroyed. In this case, we could calcu- late the infectivity of the virus as follows: Viral infectivity = 100 (dilution factor) / x (1) Or, Viral infectivity = log (100/x) (2) An alternative technique is a plaque assay[6,7]. This method exploits the phenomenon of viruses forming visible destroyed areas, which appear as white circular shapes on the surface of cellular cultures. The area is called plaque. The number of plaque shapes is countable and the logarithmic number (plaque-forming unit or PFU) is defined as infectivity. Figure 4 shows an example of plaque formations. Consider an example where the plaque number of a target specimen would be PT, while that for a non-worked specimen, or the initial plaque number as a control, would be P0. Then the viral infectivity could be defined by the following equation: Viral infectivity of materials = -(log PT – log P0) (3) It is important to understand the infectivity of materials, and another type of evaluation involves the film covering method (Fig. 5). In a previous article in this magazine, one of the authors highlighted the use of this technique to evaluate the antibacterial effect of different materials[8]. In this method, a designated amount of viral solution (containing a Fig. 3 — TCID50 assay. Fig. 4 — Plaque formation.
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