**9. General order of susceptibility of non-enveloped viruses to chemical inactivation**

In the simplified hierarchy of susceptibility of pathogens to microbicides concept, small non-enveloped viruses are considered less susceptible than large nonenveloped viruses, and both groups of non-enveloped viruses are believed to be less


*Variability and Relative Order of Susceptibility of Non-Enveloped Viruses to Chemical… DOI: http://dx.doi.org/10.5772/intechopen.102727*

*a See Table 1 for abbreviations used for viruses. <sup>b</sup>*

*BSA, bovine serum albumin; medium, culture medium; RT, room temperature.*

*Entries in purple font indicate results from original or diluted formulations with microbicidal active ingredients.*

#### **Table 8.**

*Efficacy of aldehydes against non-enveloped viruses.*

susceptible than enveloped viruses. The hierarchy concept also assumes that the ranking applies to all types of microbicidal actives. Additionally, the hierarchy concept can generally lead to common notions that viruses that share similar virological properties (e.g., same family or genus of virus) may be expected to display similar degrees of susceptibility and that the smaller a virus is, the less susceptible it will be to microbicides in general.

These generalizations are correct, to a degree. For example, most enveloped viruses are indeed more susceptible than non-enveloped viruses to chemical inactivation. It should be noted though that exceptions to the hierarchy concept do exist, e.g., especially in the case of viral susceptibility to acids and alkalines [22], and exceptions are not uncommon for certain other chemistries. The hierarchy concept was never applied specifically to physical inactivation approaches, nor should it be. The evidence for heat inactivation, UV inactivation, and gamma irradiation indicates differing rankings of susceptibility to these modalities. Envelope status and

particle size do not, in each case, relate to susceptibility for inactivation by these physical approaches [22, 78–80].

The validity of the hierarchy concept *among non-enveloped viruses* is much more blurred. Firstly, the order of susceptibility among non-enveloped viruses, if any generalization may be made, is dependent upon the type of chemistry, and there is no universal order that holds true for all types of chemistries. Secondly, large nonenveloped viruses (adenoviruses, reovirus, rotavirus, etc.) are *not* always more susceptible than small non-enveloped viruses (parvoviruses, picornaviruses, caliciviruses, etc.). Thirdly, viruses within the same group (e.g., same family or genus) can exhibit profound and unequivocal differences in susceptibility. Finally, the rankings between viruses can be flipped (reversed), or nonexistent, depending on the type of microbicide. This implies that caution should be taken when interpreting the hierarchy concept for making predictions of efficacy for the nonenveloped viruses.

The accuracy and usefulness of a hierarchy concept can be improved if the model is broken into separate chemistries for non-enveloped viruses, since many viruses do exhibit a reliable and consistent trend of susceptibility for a specific type of chemical. **Table 9** and **Figure 2** provide a summary of the relative order of susceptibility for selected non-enveloped viruses under specific types of chemistry.



*Variability and Relative Order of Susceptibility of Non-Enveloped Viruses to Chemical… DOI: http://dx.doi.org/10.5772/intechopen.102727*

*Abbreviations used: H2O2, hydrogen peroxide; NaOCl, sodium hypochlorite; PAA, peracetic acid; QAC, quaternary ammonium compound.*

#### **Table 9.**

*Relative order of susceptibility of non-enveloped viruses to chemical inactivation.*


#### **Figure 2.**

*Relative order of susceptibility of non-enveloped viruses per microbicidal chemistry. Note: various types of adenoviruses exhibit different degrees of susceptibility to ethanol and quaternary ammonium compounds.*

## **10. Discussion**

The Spaulding concept of the hierarchy of susceptibility of pathogens to microbicidal inactivation, along with its modifications, has been widely influential. Multiple industries as well as regulatory agencies have adopted or referenced this concept to various degrees [9, 10, 81, 82]. The concept does provide a good tool for understanding the innate differences and trending of susceptibility among various types of pathogens. For the most part, the hierarchy is insightful and valuable. It is particularly helpful when a pathogen is newly emerged, and limited or no knowledge is yet available regarding its level of susceptibility to microbicides [83, 84]. In fact, the United States Environmental Protection Agency (U.S. EPA) and Centers for Disease Control and Prevention (U.S. CDC) use the hierarchy concept as the basis of the Emerging Viral Pathogen Guidance for Antimicrobial Pesticides and public hygiene [10, 82, 85, 86] specifically to deal with just such a possibility.

It should be cautioned, however, that the hierarchy concept is largely oversimplified and by no means perfect [87]. For viruses, although enveloped viruses are usually more susceptible than non-enveloped viruses, certain enveloped viruses such as bovine viral diarrhea virus can be less susceptible than some nonenveloped viruses (e.g., feline calicivirus) under certain chemistries (e.g., low pH and high pH).

The accuracy and applicability of the hierarchy concept are more complex and limited among non-enveloped viruses. The trending is highly dependent on the type of chemistry; and the size of the virion is not always a primary determinant of viral susceptibility among non-enveloped viruses. If a clearer and more consistent trending can be identified among non-enveloped viruses, albeit only specific to a given type of chemistry, the knowledge should be useful.

To generalize an order of susceptibility, for a specific chemistry, data from sideby-side studies wherein viruses are evaluated concurrently by the same test method and under the same conditions should, ideally, be used. When results from different studies are used, caution should be taken to exclude conditional or case-specific differences that result from the test methodology and/or condition. For instance, a surface (carrier) test may give different log10 reduction results than a suspension test of the same microbicide or formulation under certain situations [88]. For example, the data of Kindermann et al. [47] and Tyler et al. [31] indicate that sodium hypochlorite causes a higher log10 reduction value (LRV) when tested in a suspension test than in a surface test. On the other hand, glutaraldehyde has been found to cause similar log reduction in either methodology, while hydrogen peroxide causes higher LRV in the surface test, which is thought to be likely related to the consumption of hydrogen peroxide by the protein in the virus-suspending solution [31].

The organic soil load in which the challenge virus is suspended prior to inoculation can also impact the viral inactivation outcome, especially for oxidizers, alcohols, and QAC. It would be inaccurate or even misleading if a result from a light organic load (e.g., 5% animal serum or phosphate-buffered saline) were to be directly compared with a test that used a heavier organic load (e.g., 90% blood or 20% fecal suspension). Tung *et al.* [29] reported that 500 ppm sodium hypochlorite inactivated MNV and FCV by 3-log10 in the absence of fecal suspension but only 0–0.5 log10 for these viruses in the presence of 20% fecal suspension.

Other testing conditions may also affect the reduction results. For instance, a higher contact temperature may work in the favor of the virucide under investigation, which may result in a higher log reduction. Nemoto et al. [56] reported that a 0.125% glutaraldehyde solution completely inactivated rotavirus after 10 min under ambient temperature, but not when evaluated on ice. The pH and other

### *Variability and Relative Order of Susceptibility of Non-Enveloped Viruses to Chemical… DOI: http://dx.doi.org/10.5772/intechopen.102727*

components in the product formulation could also affect the viral reduction outcome, presumably by activating the chemical and/or by a synergistic or additive effect between the pH and the active chemical [22, 39, 89]. The efficacy of formulated versus non-formulated microbicides may differ even within the same type and concentration of active(s). For example, formulated QAC and ethanol products have been reported to exhibit strong activities against certain non-enveloped viruses albeit the efficacy may be weaker for non-formulated solutions [45, 54, 90, 91]. Therefore, the formulation of the microbicidal active must be considered. The viral stock (i.e., inoculum) preparation method and the challenge viral titer may also affect the reported viral reduction efficacy. For example, purified virus may be more susceptible than crude virus preparations [49]; viral clumps can make the virus less susceptible [92]; and a higher viral challenge titer could make the chemical harder to achieve an expected log10 reduction. Sometimes, viruses propagated in different host cell types may behave differently. It would therefore be ideal if all studies could use a standardized viral preparation and infectivity assay protocol. This is, of course, practically challenging. Last, but not least, the method for preparing the microbicide and the verification of the active concentration might also differ from lab to lab, thus potentially influencing the efficacy results obtained.

Despite these practically hard-to-avoid differences in test methodology and conditions, some generalizations on the pattern of susceptibility among non-enveloped viruses can still be made with confidence. For instance, it is quite apparent that the *Picornaviridae* family of viruses do not always exhibit a similar level of susceptibility to each other [16, 93]; and even the genus is not a good predictor for susceptibility to microbicides within this family. This reflects the ability of certain members of this family to infect the gastrointestinal tract (i.e., enteroviruses), while others infect primarily the respiratory system. The variation of susceptibility within this viral family is particularly striking for ethanol, hydrogen peroxide, QAC, and low pH.

The family *Caliciviridae* is another example of the existence of unequivocal intrafamily differences in susceptibility to microbicides [16]. For feline calicivirus and murine norovirus (the two most commonly used surrogate viruses for human norovirus), not only can their levels of susceptibility be very different, but the relative order of susceptibility between these two family members can be entirely reversed. For instance, murine norovirus is susceptible to ethanol but not very susceptible to low pH, whereas feline calicivirus is not very susceptible to ethanol but quite susceptible to low pH. For some other types of chemicals, such as peracetic acid and QAC, notable differences in susceptibility to these two viruses are not observed. Given the importance of human norovirus to public health and the lack of a convenient and robust tissue culture model for the virus, a more detailed research and discussion are needed with respect to the choice of feline calicivirus and murine norovirus as the best surrogate for evaluating inactivation products against human norovirus. This topic has been discussed extensively [94–96].

Different types of adenoviruses seem to exhibit varying degrees of susceptibility to ethanol and QAC. For example, adenovirus type 5 appears to be notably more susceptible to ethanol than are adenovirus types 2 and 8. In general, however, adenoviruses are more susceptible than many other non-enveloped viruses. Considering that adenovirus type 5 is listed as one of the allowable challenge viruses for a generic or "broad-spectrum" virucidal efficacy claim (i.e., a product that is effective for adenovirus type 5 may be considered effective against all viruses) [97, 98], this practice may not represent a challenge and lead to an insufficient safety margin, which is not supported by the published data.

Parvoviruses are among the smallest of non-enveloped viruses. The animal parvoviruses (e.g., minute virus of mice, porcine parvovirus, bovine parvovirus, canine parvovirus, etc.) are considered to exhibit very low susceptibility to chemical

inactivation [99] and are commonly used as a worst-case model for viral inactivation studies. This literature review generally supports this notion, although it should be noted that the animal parvoviruses do not appear to represent a worst-case challenge for high-pH inactivation, and porcine parvovirus seems less susceptible than minute virus of mice at times. Additionally, human parvovirus B19 seems especially susceptible to acid treatment [100].

It has been observed that the particle size of a virus is not an exclusive or even a primary determinant of susceptibility to microbicides for non-enveloped viruses, albeit this characteristic may play a role. There are numerous reports demonstrating that larger non-enveloped viruses, such as adenoviruses and reoviruses, are less susceptible than some of the smaller non-enveloped viruses for certain chemistries. Interestingly though, rotavirus, a large non-enveloped virus, indeed seems to be the most susceptible among non-enveloped viruses, except to low pH.

The mechanisms underlying the large variation in susceptibility among nonenveloped viruses and the chemistry dependency are not always clear, but they could presumably be related to the physicochemical properties of the virus as well as the mechanisms of action of the chemical inactivants. For alcohols, for instance, it has been proposed that the hydrophobicity or hydrophilicity of the viral particles is an important determinant of susceptibility [101]. Poliovirus, which is hydrophilic, is more susceptible to ethanol than it is to isopropyl alcohol. This is attributed to the fact that ethanol is more hydrophilic than isopropanol. In comparison, the hydrophobic simian virus 40 is susceptible to isopropanol but not to ethanol [101]. Enterovirus 71 (EV71) and enterovirus EV-D68 (EV-D68) are both enteroviruses in the family *Picornaviridae.* Despite both infecting the gastrointestinal tract, EV71 displays low susceptibility to low pH, while EV-D68 is acid-labile. This can be explained by the observed acid-induced uncoating for EV-D68 but not for EV71 [67].

A review of the relative order of susceptibility for non-enveloped viruses under each chemistry reveals that the order for some chemicals (e.g. aldehydes) seems to fit the traditional hierarchy concept well (e.g., parvoviruses are less susceptible than larger viruses); but the order for some other chemistries (e.g., low pH) does not seem to agree with the concept as well.

The variability in viral susceptibility to physical treatments is not covered in this chapter; however, a marked degree of variation also exists for physical treatments, both within non-enveloped viruses and between enveloped and non-enveloped viruses [12, 16, 21, 49]. A comparison of the order of susceptibility of viruses to chemical versus physical treatments and an exploration of the underlying mechanisms would be interesting and revealing.

### **11. Conclusions**

This chapter reviewed the literature on chemical inactivation of non-enveloped viruses, with an emphasis on the relative difference and trending of susceptibility among some relevant (from a public health perspective) non-enveloped viruses under each type of chemistry. The traditional concept of a hierarchy of susceptibility to microbicides provides a useful tool in understanding and predicting the susceptibility of a pathogen; however, the concept tends to be oversimplified. The order of susceptibility among non-enveloped viruses depends on the type of chemistry, and there is no universal order that holds true for all types of chemistries. Picornaviruses and caliciviruses exhibit a particularly high degree of intrafamily variation, and the order may even be reversed between viruses, depending on the chemistry. Additionally, larger non-enveloped viruses are not always more susceptible than some of the smaller non-enveloped viruses. It may be inappropriate to

*Variability and Relative Order of Susceptibility of Non-Enveloped Viruses to Chemical… DOI: http://dx.doi.org/10.5772/intechopen.102727*

consider adenovirus type 5 as a worst-case non-enveloped virus; and even the animal parvoviruses, universally considered among the least susceptible to chemical inactivation, do not actually represent the least susceptible virus type for certain chemistries.
