**2. Overview of physical viral inactivation approaches**

The most commonly employed physical approaches for inactivating viruses are thermal (heat) inactivation (applied either to viruses in solutions or dried on surfaces); irradiation (applied to viruses in solutions, in solids, or dried on surfaces); and high pressure (most often employed for disinfection of food items). The irradiation approaches include gamma irradiation, X-irradiation, electron beam irradiation, and 254 nm ultraviolet light (UVC) irradiation. Irradiation with ultraviolet light in the A range and with visible light typically requires the addition of a photoactive chemical and, therefore, these are not truly physical approaches, but rather mixed physical/chemical approaches. The latter will not be dealt with in this chapter. Electron beam irradiation and high-pressure treatment are most commonly used for food preservation and the efficacy data to be found in the literature necessarily involve viruses of food concern (e.g., caliciviruses, astroviruses, reoviruses, picornaviruses, and adenoviruses) [1, 2]. Coronaviruses are not considered viruses of food concern [3] and, therefore, there are little or no data for inactivation of coronaviruses by electron beam irradiation and high-pressure treatment. As a result, there will be little discussion of these approaches in this chapter.

Physical inactivation approaches display efficacy for a broad range of viruses, including both lipid-enveloped and non-enveloped viruses. The factors determining virucidal efficacy for one virus type over another differ among the physical approaches. For instance, particle size appears to be the major determinant for inactivation efficacy of gamma, X-ray, and electron beam irradiation [4], while genomic structure (single vs. double strand, circularity, and relative content of pyrimidine dinucleotides) appears to be more important for determining UVC inactivation efficacy [5]. Thermal inactivation appears to be effective for both lipidenveloped and non-enveloped viruses, and particle size does not appear to correlate with efficacy [6]. Having said this, the most highly resistant of viruses to heat inactivation are the non-enveloped parvoviruses, circoviruses, and polyomaviruses [6]. The orthogonality of mechanism of inactivation displayed by these physical approaches is convenient. If one approach is not practical for a given virus family, another approach may be applied. A good example is the parvovirus family of small non-enveloped viruses. These typically are highly resistant to thermal inactivation

and to gamma, X-ray, and electron beam irradiation but are quite susceptible to UVC irradiation [7].

Physical inactivation approaches also differ with respect to the types of sample matrices that may be treated. Thermal inactivation has the broadest range of matrix types, including liquids and surfaces. Of course, temperatures high enough to inactivate viruses may have adverse impacts on the sample matrices being irradiated. Gamma radiation has high penetrability, and can be used for liquids and solids, though the matrix to be irradiated must be brought in close contact with a gamma source, and such sources are available only at specialized irradiation facilities. In order to minimize potential side effects of gamma irradiation (free radicals, heat) and to maintain the integrity of the sample matrix (such as bovine serum), the typical gamma irradiation process requires keeping the sample to be irradiated at very cold temperature (typically, such samples are kept on dry ice during irradiation) [8]. Electron beam radiation has low penetrability, so is typically used for thin items such as food items [1]. Due to its low penetrability, ultraviolet light irradiation is effective only if the radiation reaches all portions of the matrix being irradiated [9]. It is a line-of-sight approach. It is used for inactivating viruses on non-porous surfaces and liquids which have low UVC-absorbance characteristics [9].

An advantage of physical inactivation approaches is the first-order behavior typically displayed for inactivation of viruses (see **Box 1**). This enables one to make informed predictions of inactivation efficacy at temperatures, times, fluences that have not specifically been tested empirically.

First-order viral inactivation by physical approaches. One commonality among the physical inactivation approaches is that, as a generality, the log10 reduction in virus titer observed following treatment is first-order (linear) with respect to time in the case of heat inactivation, or with applied dose (fluence) in the case of irradiation. Of course, there are exceptions, which are sometimes attributed to mixed virus populations with differential susceptibility to the inactivation approach. It is likely that the biphasic or non-linear behavior attributed to such mixed populations is due to experimental artifact, including the inclusion of data points which approach the limit of detection of the titration assays used, or simply the fact that most of the available virus has already been inactivated. The typical first-order behavior of the physical inactivation approaches enables the calculation of decimal reduction values (*D*) for a given virus, corresponding to the thermal treatment time or irradiation fluence associated with a 1-log10 reduction in virus titer. Knowing such a *D* value allows one to adjust the thermal contact time or the irradiation fluence such that a desired log10 reduction value may be achieved for a given virus. For instance, in the tables to follow in this chapter, thermal inactivation *D* values are plotted against temperature to allow estimation of log reduction at any given time and temperature. Similarly, gamma irradiation and UVC irradiation efficacy are expressed in terms of log10 reduction per kGy (gamma irradiation) or log10 reduction per mJ/cm<sup>2</sup> fluence (UVC). This enables one to estimate the effectiveness of the irradiation approach for inactivating a given virus under conditions not tested empirically.

#### **Box 1.**

*Left panel: calculation of a D value for heat inactivation of a parvovirus at 60 °C (from [10]); right panel, first-order behavior for two data sets ( and ◊) and one data set displaying non-linear behavior (*⧍) *for inactivation of a parvovirus by gamma irradiation (from [4]).*
