**4. Inactivation by UVC irradiation**

As mentioned above, photons of light from various regions of the electromagnetic spectrum (i.e., visible, UVA, UVB, and UVC) have been used for inactivation of viruses. Available scientific literature indicates that light in the UVC range has the greatest efficacy for inactivating viruses, through a purely physical mechanism of action that does not depend on chemical radiation-sensitizing compounds. While visible light (405 nm) in the absence of photosensitizing agents has been shown to have efficacy for inactivating SARS-CoV-2, this activity is relatively weak, compared to that of UVC. For instance, a fluence of 288 mJ/cm2 was required to cause a 2.58 log10 inactivation [18], equating to about 0.0090 log10/(mJ/cm2 ), an order of magnitude greater than the UVC fluence required (see below). The reason for the unique efficacy of UVC light in the absence of sensitizing agents is thought to be the correspondence of the UVC light wavelength, typically 254 nm light from mercury vapor lamps, with the absorbance peaks of the target nucleic acids (265 nm) [9, 19].

Only the efficacy of UVC light is discussed in the tables below. Unlike gamma irradiation, which can penetrate solids, UVC irradiation is a line-of-sight approach, which depends on exposure of target organisms to the radiation. The impacts to the target organism depend on the absorbed dose. As with gamma irradiation, the dose of UVC light applied can be expressed in a single fluence term that takes into account both dose rate and time. A variety of units have been used in the literature, which can lead to confusion when attempting to compare results between labs. We use the units mJ/cm2 in this chapter, since most of the virus inactivation results to be found in the literature have been expressed in these units. Conversion of other fluence units, such as J/m2 to mJ/cm2 is straightforward, while exposures expressed in units of mW/cm2 must be multiplied by the exposure time (in seconds) to convert to mJ/cm2 .

The mechanism of inactivation of viruses by UVC radiation is thought to involve interaction of the energetic photons with nucleic acids comprising the viral genome. Pyrimidine nucleotides (uracil, thymine, cytosine) are especially susceptible to the formation of covalent dimers following exposure to UVC. A more thorough discussion of mechanisms and pyrimidine dimer formation, and relevance for predicting efficacy for viruses of different genomic structure, is beyond the scope of this chapter. Readers are referred to excellent source papers [5, 20, 21].

There is some literature on coronavirus inactivation in liquid matrices by UVC radiation, and rather scanty information on irradiation of these viruses on solid surfaces or in aerosols. A summary of the evaluation of UVC efficacy for inactivating SARS-CoV-2 and other coronaviruses in liquid matrices is displayed in **Table 2**. No attempt to cherry-pick the efficacy data has been made in assembling this table, although it will be readily apparent on review of this table that discrepant results in terms of *D* value and log10 inactivation per mJ/cm<sup>2</sup> have been reported. For an informed analysis of possible factors underlying these discrepant values, relating primarily to optical density of the liquid matrices and dosimetry difficulties, the reader is referred to Boegel et al. [19].

Neglecting the clearly discrepant values in this table, certain of which unfortunately have caused some confusion on the sensitivity of coronaviruses to UVC radiation [33], a consensus *D* value in the range of 0.5–2 mJ/cm<sup>2</sup> may be inferred. This *D* range corresponds to a consensus efficacy of 0.5–2 log10/mJ/cm<sup>2</sup> (**Table 2**). To put these *D* values into perspective, the most UVC-resistant viruses (adenoviruses and polyomaviruses), have UVC *D* values >50 mJ/cm<sup>2</sup> [6].

A summary of the evaluation of the inactivation of coronaviruses by UVC radiation on solid surfaces and in aerosols is provided in **Table 3**. As mentioned above, there are fewer reports for this topic within the literature. On a theoretical basis, UVC radiation accessibility to viruses dried on surfaces or present in aerosols should be optimal, therefore such considerations as impact of stirring or impact of matrix absorption of the radiation should not confound the efficacy results to the extent that these do in liquid matrix studies. Although the dataset in **Table 3** is limited, the agreement between observed *D* values between reports and between coronaviruses is fairly close, perhaps in keeping with the lessened impact of confounding factors mentioned above. The *D* values shown in **Table 3** also are in good agreement with the consensus *D* values (0.5–2 mJ/cm2 ) from the liquid matrix studies.


*a HCoV, human coronavirus; MHV, mouse hepatitis virus; PBS, phosphate buffered saline. SARS-CoV, severe acute respiratory syndrome coronavirus; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2. b The reported inactivation kinetics were not first-order.*

#### **Table 2.**

*Efficacy of Ultraviolet C (UVC) irradiation for inactivating coronaviruses in liquid matrices.*


*Physical Inactivation of SARS-CoV-2 and Other Coronaviruses: A Review DOI: http://dx.doi.org/10.5772/intechopen.103161*

*a HCoV, human coronavirus; IBV, infectious bronchitis virus; MHV, mouse hepatitis virus; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2*

#### **Table 3.**

*Efficacy of Ultraviolet C (UVC) irradiation for inactivating coronaviruses on surfaces or in aerosols.*

For general reviews of UVC inactivation of coronaviruses in various matrices, the reader may also consult Boegel et al. [19], Hadi et al. [20], Chiappa et al. [39], and Helßling et al. [40]. Pendyala et al. [21] used efficacy modeling based on pyrimidine dinucleotide content to predict UVC efficacy for inactivating various alpha-, beta-, and gamma-coronaviruses. The conclusion of the modeling was that coronaviruses, as a family, are highly susceptible to UVC, and the *D* values obtained in the modeling for the various coronaviruses ranged from 18.0 to 28.1 J/m<sup>2</sup> (1.8–2.8 mJ/cm<sup>2</sup> ), aligning well with the consensus *D* values from **Tables 2** and **3**.

The data presented suggest that UVC irradiation is a very effective physical approach for inactivating coronaviruses, such as SARS-CoV-2. It is not surprising, therefore, that UVC has been proposed for a variety of applications, including indoor air sanitization [36, 41–44], inactivation of coronaviruses in water [33] or other solutions, inactivation of biological samples for downstream use in assays [22], and surface hygiene [34, 45], including sanitization of personal protective equipment [35, 46, 47].

### **5. Thermal (heat) inactivation**

As is the case for gamma irradiation, heat can be highly penetrating, depending upon the inactivation matrix. For instance, heat transfer within liquids is typically efficient, so heat inactivation is a commonly employed method for inactivating adventitious agents (including viruses) in solutions. Heat inactivation is also commonly utilized for decontaminating non-porous surfaces. For some time, there has existed a dogma that heat inactivation of viruses is more effective when applied to solutions (liquid or wet inactivation) than to surfaces (carrier or dry inactivation). Exceptions to this have been noted recently [48, 49], and it is more correct to state that relative efficacy for wet vs. dry heating may depend upon the specific virus being inactivated.

The mechanisms underlying inactivation of viruses by heat are thought to be the same for both enveloped and non-enveloped viruses. The treatment is thought to result in leaky protein capsids, which allow penetration of the capsid by nucleases and loss of capsid contents to the environment. In either case, nucleases would be

expected to rapidly degrade the genomic material and render the viruses noninfectious [50]. If this mechanism is correct, heat inactivation efficacy should be similar for enveloped and non-enveloped viruses. Indeed, examination of wet heat inactivation data across virus families confirms this conclusion [6]. While certain viruses (e.g., animal parvoviruses and polyomaviruses) exhibit unusually high heat resistance, in general non-enveloped viruses do not appear to be significantly more resistant to heat than enveloped viruses [6].

The literature on heat inactivation of coronaviruses, including SARS-CoV-2, is extensive. The reports generally contain information on efficacy of one or more temperatures evaluated for one or more time periods. These studies [15, 29, 30, 51–65] generally do not report *D* values, only log10 reduction in titer obtained from heating at a given temperature for set time periods (e.g., 56°C for 30 min). Examples of this sort of heat inactivation data are given in **Table 4**. Note that in **Table 4**, data for temperatures greater than 45°C are displayed. Results at lower temperatures are associated with a great deal of variability. For readers interested in coronavirus stability at the lower temperatures (ambient to 45°C), the following review papers may be consulted [65–70]. The data in **Table 4** indicate that inactivation of coronaviruses at temperatures between 48 and 54°C may be incomplete at exposure times up to 60 min. Temperatures ≥56°C are generally quite effective at exposure times of 10 min or greater, while temperatures ≥80°C are very effective within 1 or 2 min of exposure. Similar efficacies of heat inactivation for various members of the *Coronaviridae* are observed.

Relatively few reports of heat inactivation on carriers (dry heat) have been published for coronaviruses (**Table 5**). These studies [35, 71–73] have been concerned primarily with decontamination of personal protective equipment (gowns, N95 respirators) for reuse, although Fischer et al. [35] and Biryukov et al. [72] also evaluated inactivation of SARS-CoV-2 on stainless steel carriers. Estimates of *D* values for heat inactivation on surfaces (**Table 5**) range from 7 min at 60°C (PEDV) to 11–35 min at 55–70°C (SARS-CoV-2).

The most useful heat inactivation results are expressed in terms of *D* values measured at three or more temperatures. The latter datasets enable the plotting of *D* vs. temperature curves, which, in turn, enable comparison of the efficacy of the heat inactivation results obtained in different laboratories, as well as estimation of *D* at non-measured temperatures. It should be noted that, while the kinetics of inactivation of viruses by heat at a given temperature are expected to be first-order with respect to time, the relationship between *D* and temperature is more complex [74]. In the past, the latter relationship has been plotted on semi-log scales (log10 *D* vs. time), resulting in linear plots from which *Z* values (°C per log10 change in *D*) could be calculated. These *Z* values could then be used to estimate *D* at non-measured temperatures. More recently, it has been discovered that the plot of *D* vs. temperature can be fit accurately with the power function. Examples of such plots for coronavirus heat inactivation are shown in **Figures 1** and **2**. The resulting line equation coefficients (**Table 6**) then may be used, in a more intuitive and straightforward manner, to estimate *D* at non-measured temperatures [74].

Some authors [66, 75] have taken the interesting and informative approach of combining the heat inactivation data from multiple individual reports to create summary plots of *D* vs. temperature. An example for heat inactivation of coronaviruses in liquids and on surfaces has been reported by Guillier et al. [66]. The portion of the dataset within the temperature range 40°C–70°C has been reproduced as **Figure 1** below. As can be appreciated from this figure, there is


#### *Physical Inactivation of SARS-CoV-2 and Other Coronaviruses: A Review DOI: http://dx.doi.org/10.5772/intechopen.103161*

*a SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; SARS-CoV, severe acute respiratory syndrome coronavirus; MERS-CoV, Middle East respiratory syndrome coronavirus; PEDV, porcine epidemic diarrhea virus; CaCoV, canine coronavirus; MHV, mouse hepatitis virus; FIPV (Wt), feline infectious peritonitis coronavirus (wildtype).*

#### **Table 4.**

*Efficacy of heat inactivation for inactivating coronaviruses in liquid matrices.*


#### **Table 5.**

*Efficacy of heat inactivation for inactivating coronaviruses on surfaces.*

#### **Figure 1.**

*Relationship between decimal reduction value (*D*; time required for 1 log10 inactivation) and temperature for heating studies involving various coronaviruses. Data are from reference [66].*

#### **Figure 2.**

*Relationship between decimal reduction value (*D*; time required for 1 log10 inactivation) and temperature for heating studies involving SARS-CoV-2. Data are from reference [75] (□), [76] (*◆*), and [77]* ■*).*

considerable variability in response at the lower temperatures, while greater concurrence is seen at temperatures of 50°C and above. The ability of the power function (*<sup>D</sup>* <sup>=</sup> *<sup>a</sup>* temperature*<sup>b</sup>* ; where *D* is the decimal reduction value and *a* and *b* are calculated coefficients) to fit the combined coronavirus dataset is similar to

*Physical Inactivation of SARS-CoV-2 and Other Coronaviruses: A Review DOI: http://dx.doi.org/10.5772/intechopen.103161*


*a Abbreviations used:* a *and* b*, coefficients for power function line equation* D *=* a *Temperature;* D*, decimal reduction value; CaCoV, canine coronavirus; PEDV, porcine epidemic diarrhea virus; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; TGEV, transmissible gastroenteritis virus.*

#### **Table 6.**

*Power function coefficients for* D *vs. temperature curves for thermal inactivation of coronaviruses.*

the ability of this function to fit data for multiple temperatures for SARS-CoV-2 generated within a given laboratory (**Figure 2**).

In cases where a laboratory has generated *D* vs. temperature data for three or more temperatures, these data may be plotted as shown in **Figure 2**. This figure compiles line fit data from two empirical liquid inactivation studies for SARS-CoV-2 [76, 77]. The third line on this plot is the line fit obtained from modeling of SARS-CoV-2 inactivation at various temperature by Yap et al. [75]. The modeling by Yap and coworkers was performed on the basis of heat inactivation data generated by various labs, using as challenge viruses a variety of coronaviruses (SARS-CoV, SARS-CoV-2, MERS-CoV, MHV, PEDV, and TGEV) [75]. The agreement between the line fits for these three datasets in striking. It is apparent from the plots in **Figure 2** that it takes hours to achieve 1 log10 inactivation of SARS-CoV-2 at temperatures ≤40°C, while inactivation at temperatures greater than 50°C requires only min.

In **Table 6**, the power function line fit coefficients for heat inactivation studies evaluating various coronaviruses are displayed. The estimation of *D* at 56°C is shown as a means of demonstrating the utility of the power function line fitting approach for enabling comparison of datasets generated at different laboratories. Note that at 56°C, *D* values for the various coronaviruses range from 3 to 39 min, with the 39 min required for TGEV considered to be atypical.

Taken together, the data in **Figures 1** and **2** and **Tables 4**–**6** support the expectation that similar heat sensitivities are to be expected for various members of the *Coronaviridae* family. To put the *D* values shown in **Table 6** into perspective, more heat susceptible virus families include the *Rhabdoviridae* (*D*56 °C ranging from 0.2 to 1.9 min) and *Retroviridae* (*D*56 °C of 1.4 min), while less susceptible viruses include animal members of the *Parvoviridae* (*D*56 °C > 10 hours) [6]. The heat susceptibilities displayed by the *Coronaviridae* are fairly typical of enveloped and non-enveloped viruses in general, except as noted above.

The literature that has been reviewed above indicate that heat inactivation is typically utilized for inactivation of coronaviruses in solutions, but this physical approach has also been used for decontamination of these viruses on surfaces, such as stainless steel and N95 respirator material. In addition, hot (≥63°C), humid (95% relative humidity) air exposure for 1 hour has been described for decontaminating enveloped RNA virus (bacteriophage Phi6 used as a surrogate for SARS-CoV-2) dried on surfaces within aircraft [81].
