Open access peer-reviewed chapter

Variability and Relative Order of Susceptibility of Non-Enveloped Viruses to Chemical Inactivation

Written By

Sifang Steve Zhou

Submitted: December 6th, 2021 Reviewed: January 17th, 2022 Published: March 22nd, 2022

DOI: 10.5772/intechopen.102727

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Viruses exhibit a marked variation in their susceptibilities to chemical and physical inactivation. Identifying a trend within these variations, if possible, could be valuable in the establishment of an effective and efficient infection control or risk mitigation strategy. It has been observed that non-enveloped viruses are generally less susceptible than enveloped viruses and that smaller sized viruses seem less susceptible than larger viruses. A theory of a “hierarchy” of pathogen susceptibility has been proposed and widely referenced. This concept provides a useful general guide for predicting the susceptibility of a newly emerged pathogen. It also serves as a theoretical basis for implementing a limited scale viral inactivation study that is to be extrapolated onto many other viruses. The hierarchy concept should be interpreted with caution since the actual viral inactivation efficacy may, in some cases, be different from the general prediction. The actual efficacy is dependent on the type of chemistry and application conditions. The order of susceptibility is not always fixed; and viruses within the same family or even the same genus may exhibit drastic differences. This chapter reviews viral inactivation data for several commonly used chemistries against non-enveloped viruses, highlighting the cases wherein the order of susceptibility varied or even flipped. Possible underlying mechanisms are also discussed.


  • enveloped viruses
  • non-enveloped viruses
  • hierarchy of susceptibility
  • disinfection
  • viral inactivation
  • virucidal efficacy

1. Introduction

Bacteria, fungi (yeasts and molds), mycobacteria, prions, protozoa, and viruses are common pathogens infecting humans and animals. They typically exist within the host or in the environment. It has been observed that these microorganisms exhibit a notable difference in the natural survivability in the environment, as well as susceptibility to chemical and physical inactivation. For example, under ambient and dried conditions, human coronaviruses seem to lose their infectivity in a matter of several hours to several days [1], whereas endospores and prions may remain infectious for years to decades or even indefinitely [2, 3].

As more and more data have become available regarding the survivability and susceptibility of pathogens to microbicides, it has been observed that the pathogens seem to demonstrate an order of susceptibility to chemical and physical inactivation. E. H. Spaulding first proposed a classification system for the sterilization and disinfection of medical instruments based on the infection risk in 1939 [4]. On the basis of this classification, the concept of a hierarchy of pathogen susceptibility was proposed, in which microorganisms are placed into several groups and ranked from least susceptible to most susceptible. In this hierarchy concept, bacterial spores were ranked the least susceptible, followed by mycobacteria, non-enveloped viruses, fungi, vegetative bacteria, and enveloped viruses. The susceptibility hierarchy was also believed to be related to the biochemical and biophysical characteristics of a pathogen [5, 6].

This hierarchy concept has been slightly modified and expanded over the years. For example, prions were added and considered less susceptible to inactivation by microbicides than bacterial spores; small non-enveloped viruses were considered less susceptible than large non-enveloped viruses; and the order between mycobacteria and small non-enveloped viruses was sometimes reversed (Figure 1) [7, 8, 9, 10]. Additionally, it has been suggested that the hierarchy concept may be applied either “vertically” (i.e., ranking of susceptibility betweenclasses of pathogens) and/or “horizontally” (i.e., ranking of susceptibility withina class of pathogens) [11].

Figure 1.

Proposed hierarchy of susceptibility of pathogens to microbicides. Note: slightly different versions of the hierarchy concept have been proposed in the literature. Mycobacteria have been placed above small non-enveloped viruses, and molds have been placed above large non-enveloped viruses in certain versions. In some versions, the small and large non-enveloped viruses are combined; and yeasts and molds may be combined.

The hierarchy concept has been quite useful for enabling scientists to better understand the innate difference among various types of pathogens. In the case of newly emerged pathogens, especially, the hierarchy concept has helped stakeholders design and implement a disinfection strategy swiftly with a reasonable level of confidence. The concept also helps the contaminant control for food, pharmaceutical, and biopharmaceutical products, as it is impractical to test every possible contaminating pathogen, and a robust infectivity assay system may be lacking for certain pathogens (e.g., hepatitis E virus).

Despite its usefulness, the hierarchy concept should be interpreted with caution, as it may oversimply the differences and trending of pathogen susceptibilities. Further examination and refinement of the concept may be necessary; and several important questions should be answered. For example, how often do exceptions to the hierarchy occur and what are the underlying reasons? Could a trending be specific to a given type of chemistry? Is the hierarchy the same between susceptibility to both chemical and physical inactivation? Why do pathogens in the same group, or even the same family or genus, sometimes exhibit striking differences in susceptibility? Is there a way to identify and separate reliable/consistent trending versus blurred/variable trending? A deeper look at the efficacy data for various types of microbicidal actives, especially for non-enveloped viruses, may help stakeholders understand the scope, reliability, and limitation of the hierarchy concept so that it can be best utilized.

This chapter reviews the inactivation efficacy data from the literature against non-enveloped viruses for several commonly used types of chemistries, either in formulated or unformulated form, in an effort to generate a separate relative order of susceptibility among these non-enveloped viruses for each type of chemistry and to differentiate consistent versus variable trending. Physical inactivation approaches are not covered in this chapter, although a significant degree of variation also exists for physical treatments. It is not clear that the physical inactivation approaches, in general, are governed by the same hierarchy to susceptibility as is observed for chemical inactivation approaches [12].


2. Common families of mammalian non-enveloped viruses

Currently, there are a total of 21 families of viruses (including enveloped and non-enveloped) identified for humans [13], which represent only a small part of the entire paradigm of viruses in nature, whose host ranges extend from vertebrates to plants to bacteria. The most common families of non-enveloped viruses for humans and animals include Adenoviridae, Astroviridae, Caliciviridae, Circoviridae, Hepeviridae, Papillomaviridae, Parvoviridae, Picornaviridae, Polyomaviridae,and Reoviridae.The genome structure, size of viral particle, and some representative viruses for each viral family are presented in Table 1.

FamilyExample virusAbbreviationGenusGenomeSize (nm)
AdenoviridaeAdenovirus type 2AdV-2Mastadenovirusds DNA70–90
Adenovirus type 5AdV-5Mastadenovirusds DNA70–90
Adenovirus type 8AdV-8Mastadenovirusds DNA70–90
AstroviridaeHuman astrovirusHAstVMamastrovirusss RNA28–35
CaliciviridaeFeline calicivirusFCVVesivirusss RNA28–40
Human norovirusHuNoVNorovirusss RNA28–40
Murine norovirusMNVNorovirusss RNA28–40
Tulane virusTuVRecovirusss RNA28–40
CircoviridaePorcine circovirusPCVCircovirusss DNA∼17
HepeviridaeHepatitis E virusHEVOrthohepevirusss DNA32–34
PapillomaviridaeHuman papillomavirusHPVPapillomavirusds DNA50–60
ParvoviridaeBovine parvovirusBPVBocaparvovirusss DNA20–28
Canine parvovirusCPVProtoparvovirusss DNA20–25
Human parvovirus B19B19VErythroparvovirusss DNA23–26
Minute virus of miceMVM (MMV)Protoparvovirusss DNA20–25
Porcine parvovirusPPVProtoparvovirusss DNA20–25
PicornaviridaeBovine enterovirusBEVEnterovirusss RNA30–32
CoxsackievirusCoxEnterovirusss RNA30–32
Echovirus 11Echo11Enterovirusss RNA30–32
Encephalomyocarditis virusEMCVCardiovirusss RNA30–32
Enterovirus 71EV-71Enterovirusss RNA30–32
Enterovirus D68EV-D68Enterovirusss RNA30–32
Foot and mouth disease virusFMDVAphthovirusss RNA30–32
Hepatitis A virusHAVHepatovirusss RNA30–32
Poliovirus type 1PV1Enterovirusss RNA30–32
RhinovirusRVEnterovirusss RNA30–32
Seneca Valley virusSVVSenecavirusss RNA30–32
PolyomaviridaeBovine polyomavirusBPyVPolyomavirusds DNA40–50
Simian virus 40SV40Betapolyomavirusds DNA40–50
ReoviridaeBluetongue virusBTVOrbivirusds RNA60–80
Reovirus type 3REO-3Orthoreovirusds RNA60–80
RotavirusRotaRotavirusds RNA60–80

Table 1.

Common families of human and animal non-enveloped viruses.



Among these, the Adenoviridae and Reoviridaefamilies of viruses are generally considered large, non-enveloped viruses. Other non-enveloped viruses are generally considered small, non-enveloped viruses, although it should be noted that the particle sizes of Papillomavirusesand Polyomavirusesare notably larger than those for the rest of the small non-enveloped virus group (Table 1).

It is worth noting that viruses are typically classified taxonomically on the basis of virion properties (size, shape, envelope, physical, and chemical properties, etc.), genome organization, replication mechanism, antigenic properties, and biological properties [13, 14, 15]. The final classification is a combined consideration of these properties. However, the stability and susceptibility to inactivation of a virus may not relate to all of these properties and, as such, may not always align with the taxonomic classification system. For example, the susceptibility of a virus to surfactants may primarily be related to the envelope of the virion and not related to the genome structure or mode of replication.

The susceptibilities of non-enveloped viruses to chemicals have been found to be highly variable and somewhat hard to predict, since they do not always agree with the hierarchy concept. For example, according to the hierarchy concept as modified by Sattar [8], small non-enveloped viruses should be less susceptible than large non-enveloped viruses. Additionally, if there is a fixed hierarchy, all small non-enveloped viruses should either display similar levels of susceptibility or should demonstrate a definitive trend of relative susceptibility, regardless of the type of microbicide. Based on the literature, neither of these predictions appear to hold in every case. The relative order of susceptibility seems chemistry-dependent; and sometimes viruses within the same family or even genus have been found to exhibit unequivocal differences in their susceptibilities (reviewed in [16]). Any trending or hierarchy, therefore, must be reviewed in the context of the type of chemistry, and it should not be assumed that non-enveloped viruses within the same family or genus will always display similar susceptibilities to a given microbicide.


3. Overview of chemical viral inactivation approaches

Viral inactivation may be achieved by chemical and/or physical methods. The subset of chemicals commonly used for inactivation of non-enveloped viruses includes alcohols, oxidizers, halogen compounds, quaternary ammonium compounds, phenolics, aldehydes, acids, and alkalines [17, 18, 19]. These differ with respect to efficacy, stability, toxicity, material or surface compatibility, cost, and sensitivity to organic soil load. Soil load is a term used to signify an organic matrix used to challenge the inactivating efficacy of a microbicide. It is intended to mimic secretions or excretions in which the virus would be released from an infected person or animal. Some chemistries (e.g., sodium hypochlorite, phenolics, and aldehydes) are mostly used for environmental or medical device disinfection. Other chemistries (e.g., ethanol) are more commonly used for hand hygiene, while some others (e.g., quaternary ammonium compounds) may be used for both environmental disinfection and skin antisepsis (Table 2).

ClassChemicalTypical conc.UsageMechanism of viral inactivationSensitivity to soil load
AlcoholsEthanol50–95%Disinfection; AntisepsisProtein denaturation+
Isopropanol70–90%DisinfectionProtein denaturation+
OxidizersSodium hypochlorite0.01–0.5%DisinfectionProtein/genome damage++
Chlorine dioxide0.1–1 mg/LDisinfection; Water treatmentProtein/genome damage
Hydrogen peroxide0.1–10%Disinfection; AntisepsisLipid/protein/genome damage+
Hypochlorous acid0.002–0.1%Disinfection; Water treatmentProtein/genome damage++
Peracetic acid0.01–1%Disinfection; SterilizationProtein denaturation
Povidone-iodine0.02–8%Disinfection; AntisepsisProtein/genome damage++
Chlorohexidine0.02–0.2%AntisepsisProtein denaturation+
QACBKC, DDAC, etc.0.01–0.2%DisinfectionLipid/protein damage+
Low pHAcids≤ pH 4Sanitization; BiomanufacturingCapsid/protein damage
High pHNaOH, etc.≥ pH 10Disinfection; Tissue processingCapsid/genome damage
AldehydesGlutaraldehyde0.02–2%HLD; SterilizationCrosslinking/protein & genome damage
Formaldehyde0.1–5%Disinfection/PreservationAlkylating/protein & genome damage
OPA0.02–2%HLD; SterilizationCrosslinking/protein damage
PhenolicsPhenylphenol, etc.0.05–5%DisinfectionProtein damage

Table 2.

Common types of chemistries used for non-enveloped viral inactivation.

Abbreviations used: BKC, benzalkonium chloride; Conc, concentration; DDAC, didecyldimethylammonium chloride; HLD, high-level disinfection; NaOH, sodium hydroxide; OPA, ortho-phthaldehyde; QAC, quaternary ammonium compounds.

The virucidal efficacy of a product is not only determined by the type and concentration of the chemical, but is also heavily influenced by the formulation, pH, exposure (contact or dwell) time, organic soil load, temperature, and surface characteristics (as applicable), etc. [10, 20, 21, 22]. Given the differences between various testing methods, as well as the intrinsic variability of viral infectivity (titration) assays, a general conclusion on the efficacy of a particular type of active ingredient will be enhanced if the efficacy is derived from multiple sets of data and under various application conditions (such as the concentration of the microbicidal active(s), contact time, formulation matrix (as applicable), and organic soil load, etc.) Additionally, in order best to explore the relative ranking of susceptibility between viruses, or the lack thereof, efficacy data from side-by-side studies wherein the same test methodologies and conditions were used would be preferable. Care should be taken when comparing data from different studies, especially if the formulations, test methods, and test conditions were different.


4. Inactivation of non-enveloped viruses by alcohols

Alcohols, primarily ethanol and isopropanol, are widely used for hand hygiene and environmental disinfection, and their efficacies against bacteria and viruses have been extensively studied [23, 24, 25]. Ethanol at a concentration of 70–90% and isopropanol at 70% have been broadly shown to be effective against enveloped viruses; however, their efficacies against non-enveloped viruses are much more variable.

The trending of the degree of susceptibility of non-enveloped viruses to ethanol and isopropanol is generally clearer and more consistent than it is for many other types of chemistries, thanks to the large amount of data in the literature. The relative ranking of susceptibility of non-enveloped viruses seems to differ between ethanol and isopropanol; and the ranking does not appear to align well with the classical virological taxonomy.

For ethanol, parvoviruses and the polyomavirus simian virus 40 have low susceptibility, while rotavirus (a reovirus) is susceptible (Table 3). Viruses in the Picornaviridaefamily display clear differences in their susceptibilities to ethanol; and even viruses within the same genus display marked differences. For example, hepatitis A virus and human enterovirus 71 are much less susceptible than rhinovirus; and poliovirus, foot-and-mouth disease virus, and coxsackie virus seem to exhibit intermediate levels of susceptibility compared with the aforementioned viruses. The viral family Caliciviridaealso has shown drastic differences among family members in the susceptibility to ethanol. Murine norovirus is quite susceptible to ethanol, whereas feline calicivirus, human norovirus, and Tulane virus are significantly more difficult to inactivate with ethanol. The Adenoviridaeis another non-enveloped virus family that has shown intrafamily differences, wherein adenovirus 5 is rather susceptible but adenovirus 2 and adenovirus 8 are much less susceptible. The relative order of susceptibility between murine norovirus (a small, non-enveloped virus) and adenovirus types 2 and 8 (two large, non-enveloped viruses) clearly conflicts with the simplified hierarchy concept (Figure 1).

VirusaMethodSoil/MatrixbLog10 Reduction afterReferences
30 s1 min5 min10 min
70% Ethanol
PPVStainless steelErythrocytes + BSA0.30.6[26]
MVMStainless steelErythrocytes + BSA0.30.7[26]
HEV71Suspension testMedium< 1[27]
HAVSuspension testMedium0.4[28]
HAVSuspension test20% fecal0.4[28]
HuNoVSuspension test20% stool<0.5[29]
TuVSuspension testMedium<0.5[30]
PV1Suspension test20% fecal0.3[28]
PV1Suspension testMedium0.4[31]
PV1Stainless steelErythrocytes + BSA2.11.8[26]
PV1Suspension testMedium4[28]
FCVSuspension testMedium0.52.6[32]
FCVSuspension testMedium1.72.2[30]
AdV-8Suspension testMedium1.9[33]
AdV-5Stainless steelErythrocytes + BSA2.4>4.1[26]
AdV-5Stainless steelMedium∼5[34]
MNVSuspension testMedium> 3.6[32]
MNVSuspension testMedium5[30]
RotavirusSuspension testMedium> 3.1[28]
75% Ethanol
80% Ethanol
CPVStainless steelMedium0.1[36]
SV40Suspension testMedium<1[37]
FCVSuspension testMedium1.3[38]
FMDVSuspension testMedium2.3[39]
PV1Suspension testMedium4.2[40]
PV1Suspension testMedium4.2[41]
70% Isopropanol
TuVSuspension testMedium<0.5[30]
FCVSuspension testMedium<0.5[30]
FCVSuspension testMedium0.10.2[32]
HEV71Suspension testMedium<1[27]
PV1Suspension testmedium<1[37]
AdV-5Stainless steelMedium∼1[34]
AdV-8Suspension testMedium2.0[33]
MNVSuspension testMedium2.6>2.6[32]
MNVSuspension testMedium1.83.1[30]
SV40Suspension testMedium>4[37]
RotavirusSuspension testMedium> 4[42]

Table 3.

Efficacy of alcohols against non-enveloped viruses.

See Table 1 for abbreviations used for viruses.

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

Entries in purple font indicate results from undiluted or diluted formulations with the indicated microbicidal active ingredients.

Interestingly, the above order of susceptibility does not appear to hold the same for isopropanol (Table 3). For example, the polyomavirus simian virus 40 is much more susceptible to isopropanol than many other non-enveloped viruses; and poliovirus appears to display a lower susceptibility, similar to that of hepatitis A virus and human enterovirus 71. Murine norovirus is still more susceptible than feline calicivirus to isopropanol, but not as susceptible as simian virus 40 or rotavirus. The apparent difference between adenovirus 5 and adenovirus 8 that has been observed for ethanol has not been observed for isopropanol.


5. Inactivation of non-enveloped viruses by oxidizers

An oxidizer or oxidizing agent is a chemical that has the ability to oxidize other molecules, i.e., to accept their electrons. Common oxidizing agents used for disinfection, sterilization, or antisepsis include hydrogen peroxide, peracetic acid, ozone, and halogen-containing compounds such as sodium hypochlorite (bleach), hypochlorous acid, povidone-iodine, chlorohexidine, and chlorine dioxide, etc. These compounds can react with and alter the proteins and nucleic acids of non-enveloped viruses and render them noninfectious. Oxidizers comprise a large group of chemicals, and the relative order of susceptibility of non-enveloped viruses to oxidizers seems to vary by specific type of active ingredient (Table 4).

VirusaMethodSoil/MatrixbLog10 Reduction afterReferences
≤ 1 min2 min5 min10 min
Sodium hypochlorite, 0.05%
FCVSuspension testMedium3[29]
FCVSuspension test20% stool0.5[29]
MNVSuspension testMedium3[29]
MNVSuspension test20% stool0.0[29]
Sodium hypochlorite, 0.1%
CPVStainless steel90% plasma< 1[43]
MNVStainless steel10% stool< 1[44]
MNVStainless steelmedium1.4[30]
TuVStainless steelmedium1.2[30]
CPVStainless steel5% serum5[43]
FCVStainless steelmedium5.3[30]
FCVStainless steel10% stool∼2[44]
HAVStainless steel5% serum5[43]
HAVStainless steel90% plasma<15[43]
HAVSuspension testPBS/20% fecal4[28]
PV1Suspension testPBS/20% fecal4[28]
RV14Stainless steelMucin2.5[45]
Sodium hypochlorite, 0.25%
PPVStainless steelErythrocytes + BSA0.61.0[26]
MVMStainless steelErythrocytes + BSA3.04.4[26]
PV1Stainless steelErythrocytes + BSA2.84.5[26]
AdV-5Stainless steelErythrocytes + BSA4[26]
Sodium hypochlorite, ∼0.3%
Cox A16GlassMedium> 3[46]
EV71GlassMedium> 3[46]
Sodium hypochlorite, 0.5%
MNVStainless steel10% stool< 1∼3.2[44]
MVMStainless steelMedium1.22.2[47]
MVMSuspension testMedium2.5> 4[47]
FCVStainless steel10% stool3.2> 5[44]
Hydrogen peroxide, ∼0.05%
HAVStainless steelMedium∼3.8[47]
MVMStainless steelMedium>4.6[47]
Hydrogen peroxide, ∼0.1%
FCVSuspension testMedium>3[48]
Hydrogen peroxide, 1%
RotavirusStainless steelNon-purified virus1[49]
RotavirusStainless steelPurified virus>3[49]
MNVStainless steelMedium1.12.0[50]
Hydrogen peroxide, 3%
PV1Suspension testMedium<0.5<0.5[31]
Hydrogen peroxide, 7.5%
PPVStainless steelErythrocytes + BSA0.5[26]
MVMStainless steelErythrocytes + BSA1.5[26]
PV1Stainless steelErythrocytes + BSA3.9[26]
AdV-5Stainless steelErythrocytes + BSA2.3[26]
Peracetic acid, 100 ppm
Peracetic acid, 500 ppm
MNVSuspension testMedium∼3[52]
Peracetic acid, 640 ppm
HAVSuspension testMedium∼3[53]
PVSuspension testMedium>3[53]
Peracetic acid, 1000 ppm
CPVStainless steelBSA1.6[34]
MVMStainless steelBSA2.3-2.9[34]
PPVStainless steelBSA3.8-5.5[34]
AdV-5Stainless steelBSA4.9-5.8[34]

Table 4.

Efficacy of oxidizers against non-enveloped viruses.

See Table 1 for abbreviations used for viruses.

BSA, bovine serum albumin; PBS, phosphate buffered saline; medium, culture medium; RT, room temperature.

Viral-inoculated lettuce was washed with PAA solution for a defined period of time.

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

Parvoviruses are generally among the least susceptible viruses to various types of oxidizers, including sodium hypochlorite, hydrogen peroxide, and peracetic acid. However, for sodium hypochlorite, minute virus of mice appears to be more susceptible than porcine parvovirus and canine parvovirus. All picornaviruses appear to exhibit a similar degree of susceptibility to sodium hypochlorite; but within the family of Caliciviridae, feline calicivirus appears to be more susceptible than murine norovirus. Both adenovirus and rotavirus are susceptible to sodium hypochlorite.

The trending for hydrogen peroxide seems more complex than that for sodium hypochlorite. For example, there seems a higher level of variability within the Picornaviridaefamily. Rhinovirus is quite susceptible to hydrogen peroxide, whereas hepatitis A virus is much less susceptible. Poliovirus seems to be more susceptible than hepatitis A virus but less susceptible than rhinovirus. Similar to the case for sodium hypochlorite, feline calicivirus seems more susceptible than murine norovirus to hydrogen peroxide. Interestingly, adenovirus and rotavirus, two larger non-enveloped viruses, seem to be less susceptible than rhinovirus, a smaller virus, to inactivation by hydrogen peroxide. This is another case where the size of viral particle alone does not appear to dictate the level of susceptibility to a microbicide.

For peracetic acid, hepatitis A virus also seems less susceptible than poliovirus. Both feline calicivirus and murine norovirus are susceptible to peracetic acid and so is adenovirus.


6. Inactivation of non-enveloped viruses by quaternary ammonium compounds

Quaternary ammonium compounds (QAC) are widely used as active ingredients for disinfectants. Among the advantages of QAC are good stability, dual function of disinfection and cleaning, surface activity, low toxicity, and lack of odor, etc. The potential limitation in the microbicidal efficacy and possible effect in promoting antimicrobial resistance of QAC have also been discussed in the literature [54, 55].

Quaternary ammonium compounds are generally efficacious on most vegetative bacteria and enveloped viruses. Their efficacies against non-enveloped viruses, however, are generally much weaker. Nevertheless, several non-enveloped viruses, such as rotavirus, rhinovirus, and coxsackievirus A11, have been shown to be susceptible to QAC. The susceptibility levels among the Adenoviridaefamily of viruses seem to vary, with adenovirus 8 displaying less susceptibility than adenovirus 5. Both feline calicivirus and murine norovirus display low susceptibility to QAC (Table 5). The relative order of susceptibility of non-enveloped viruses to QAC does not seem to align well with the relative size of the virions; and the efficacy of QAC is often dependent on the product formulation.

VirusaMethodSoil/matrixbLog10 reduction afterReferences
30 s1 min10 min60 min
QAC 0.05%
PPVStainless steelErythrocytes + BSA0.4[26]
MVMStainless steelErythrocytes + BSA0.5[26]
PV1Stainless steelErythrocytes + BSA0.5[26]
AdV-5Stainless steelErythrocytes + BSA1.8[26]
QAC 0.1%
AdV-8Suspension testMedium1.0-1.8[57]
AdV-5Suspension testMedium3.7-5.3[57]
TuVSuspension testMedium<0.5[30]
QAC 0.2%
PV1Suspension testBSA/yeast extract0.0[58]
AdV-25Suspension testBSA/yeast extract0.3[58]
Cox A11Suspension testBSA/yeast extract>5.1[58]
QAC 0.9%
FCVSuspension testMedium<0.5[29]
MNVSuspension testMedium<0.5[29]
Mixed QACs
FCVSuspension testMedium0.52.6[44]

Table 5.

Efficacy of QAC against non-enveloped viruses.

See Table 1 for abbreviations used for viruses.

BSA, bovine serum albumin; medium, culture medium; QAC, quaternary ammonium compound.

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


7. Inactivation of non-enveloped viruses by low pH and high pH

Acids and alkalines, either used alone or in combination with other active ingredients in formulated products, can be an effective means for viral inactivation. Acids may be used for disinfection, sanitization, textile or face mask pretreatment, or viral clearance during biopharmaceutical manufacturing. Alkalines may also be used for disinfection, sanitization, and viral clearance during biopharmaceutical manufacturing and can be effective against even the least susceptible of pathogens, the prions [58].

It has been widely reported that a low-pH treatment (typically at pH 4 and below) can effectively inactivate most enveloped viruses, although some enveloped viruses, such as bovine viral diarrhea virus, still exhibit a relatively low susceptibility to this treatment pH [22]. The range of susceptibilities of non-enveloped viruses to low pH seems quite scattered and often goes against the “conventional wisdom” that non-enveloped viruses are not susceptible to acidic pH (Table 6). For instance, in the family of Parvoviridae, human parvovirus B19 has been found to be markedly susceptible to low pH (completely inactivated after 1–2 h treatment at pH 4), whereas animal parvoviruses, such as minute virus of mice, are not inactivated at all under the same conditions. Interestingly, another human parvovirus (type 4) appears to be less susceptible than B19, but more susceptible than minute virus of mice.

VirusaMethodSoil/MatrixbLog10 Reduction afterReferences
20 min30 min45 min1–2 hr
pH < 2
REO-3Suspension testMedium1–3[59]
PCVSuspension testMedium>3[60]
pH 2.0
MVMSuspension testMedium<1[61]
MNVSuspension testMedium<0.5[30]
TuVSuspension testMedium<0.5[30]
PARV4Suspension testMedium2–3[61]
B19VSuspension testMedium> 4[61]
FCVSuspension testMedium6.3[30]
FCVSuspension testMedium>5[62]
pH ∼ 2.6
PVSuspension testMedium<1[63]
PVSuspension testMedium<1[64]
HAVSuspension testMedium<1[64]
pH 3.0
MNVSuspension testMedium<0.5[30]
TuVSuspension testMedium<0.5[30]
Cox A9Suspension testMedium<1[65]
FCVSuspension testMedium∼3[30]
FCVSuspension testMedium∼4.7[62]
RVSuspension testMedium>3[65]
FMDVSuspension testMedium>3[65]
pH 4.0
MVMSuspension testMedium<1[66]
EV71Suspension testMedium<1[67]
EV-D68Suspension testMedium∼4–5<5[67]
B19VSuspension testMedium[66]

Table 6.

Efficacy of low pH against non-enveloped viruses.

See Table 1 for abbreviations used for viruses.

Medium, culture medium.

The Picornaviridaefamily also exhibits disparity with respect to susceptibility to low pH. For instance, hepatitis A virus, poliovirus, human enterovirus 71, and coxsackievirus A9 display low susceptibility (less than 1-log10 reduction at pH 3–4 for 1–2 h), whereas rhinovirus, foot-and-mouth disease virus, and enterovirus EV-D68 are highly susceptible (more than 4-log10 reduction or complete inactivation at pH 3–4 after 20–45 min). Note that human enterovirus 71, coxsackievirus A9, rhinovirus, and enterovirus EV-D68 are all members of the same genus (Enterovirus).

Feline calicivirus and murine norovirus in the family Caliciviridaerepresent another interesting and convincing example that not all viruses within the same family exhibit the same degree of susceptibility. As an example, feline calicivirus is susceptible to low pH, whereas murine norovirus is much less susceptible. Rotavirus and reovirus (family Reoviridae) also display low susceptibility to low pH. The low susceptibility of murine norovirus and rotavirus to low pH may not be a surprise, since these viruses naturally exist in the digestive track, which has an acidic environment. Feline calicivirus, on the other hand, acts more like a respiratory virus.

Viruses, both enveloped and non-enveloped, are generally susceptible to high pH. At an environment of pH 12 or above, most if not all non-enveloped viruses would be inactivated, with extent depending both on temperature and contact time. Reovirus, simian virus 40, hepatitis A virus, canine parvovirus, poliovirus, murine norovirus, and Tulane virus seem to be less susceptible than minute virus of mice, feline calicivirus, adenovirus, rotavirus, and foot-and-mouth disease virus. It may be worth noting that the order of susceptibility to high pH seems to be in discord with the hierarchy concept by the greatest degree: in this case, an enveloped virus, bovine viral diarrhea virus, seems to be less susceptible than most, if not all, non-enveloped viruses [22]; parvoviruses are not necessarily less susceptible than many other non-enveloped viruses; and the size of the viral particle does not seem to matter much with regard to the degree of susceptibility (Table 7).

VirusaMethodSoil/MatrixbLog10 Reduction afterReferences
≤ 1 min10 min30 min1 hr
pH 10
MNVSuspension testMedium∼2[30]
TuVSuspension testMedium∼2.2[30]
FCVSuspension testMedium>5.5[30]
pH 12–12.5
REO-3Suspension testMedium3[68]
Cox BSuspension testMedium5[69]
Echo 11Suspension testMedium6[68]
FMDVSuspension testMedium>3.5[39]
NaOH, 0.1 M (∼pH 13)
BVDVSuspension testMedium2.5[70]
HAVSuspension testMedium2.7[59]
SV40Suspension testMedium3.9[70]
HAVStainless steel5% serum3.0[43]
HAVStainless steel90% plasma3.6[43]
CPVStainless steel5% serum3.5[43]
CPVStainless steel90% plasma5.2[43]
MVMSuspension testMedium>4.7[71]
MVMSuspension testMedium>4[66]
CPVSuspension testMedium5.6[70]
PVSuspension testMedium5.9[70]
AdV-2Suspension testMedium>6.9[70]
AdV-5Suspension testMedium>6[72]
NaOH, 0.5 M (∼pH 13.7)
HAVsuspension testMedium2.4[59]
PVsuspension testMedium4.1[63]
NaOH, 0.75 M (∼pH 13.9)
Avian ReoSuspension testMedium4[73]
PVSuspension testMedium5.1[73]
Bovine RotaSuspension testMedium>6[73]

Table 7.

Efficacy of high pH against non-enveloped viruses.

See Table 1 for abbreviations used for viruses.

Medium, culture medium.

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


8. Inactivation of non-enveloped viruses by aldehydes

Aldehydes, such as glutaraldehyde, formaldehyde, and ortho-phthaldehyde, are widely used for sterilization, high-level disinfection for critical and semi-critical medical devices, biomanufacturing, and preservation. Their use for regular disinfection, sanitization, or antisepsis has been more limited, primarily due to human toxicity concerns. The efficacy of aldehydes, similar to the case for other types of actives, is concentration-dependent. There have been limited side-by-side comparison studies of the susceptibility of non-enveloped viruses to aldehydes; however, it may be concluded that animal parvoviruses seem to be less susceptible than other viruses, such as poliovirus, hepatitis A virus, feline calicivirus, adenovirus, reovirus, and rotavirus [74]. Within the parvoviruses, porcine parvovirus seems to be less susceptible to aldehydes than minute virus of mice (Table 8).

VirusaMethodSoil/MatrixbLog10 Reduction afterReferences
5 min10 min30 min60 min
Glutaraldehyde, 0.02%
HAVSuspension testMedium3.0[75]
Glutaraldehyde, 0.05%
MVMSuspension testMedium0.51.52.8[47]
MVMStainless steelMedium0.51.21.4[47]
REO-3Suspension testMedium3.3>5[47]
REO-3Stainless steelMedium3.35.3[47]
Glutaraldehyde, ∼0.1%
PPVStainless steelBSA1.7–2.8[34]
MVMStainless steelBSA2.5–3.3[34]
PV1Suspension testMedium>3[76]
FCVSuspension testMedium5[48]
AdV-5Stainless steelBSA4.9–6.3[34]
RotavirusSuspension testMedium>5[56]
Glutaraldehyde, 2%
PPVStainless steelErythrocytes + BSA3.6[26]
MVMStainless steelErythrocytes + BSA>4.4[26]
Formaldehyde, 2%
AdV-5Suspension testMedium>5.0[77]
Ortho-phthaldehyde, 0.55%
PPVStainless steelErythrocytes + BSA3.6[26]
MVMStainless steelErythrocytes + BSA>4.[26]

Table 8.

Efficacy of aldehydes against non-enveloped viruses.

See Table 1 for abbreviations used for viruses.

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.


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 non-enveloped viruses, and both groups of non-enveloped viruses are believed to be less 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, 79, 80].

The validity of the hierarchy concept among non-enveloped virusesis 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 non-enveloped viruses (adenoviruses, reovirus, rotavirus, etc.) are notalways 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 non-enveloped 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.

ChemicalLower susceptibilityMedium susceptibilityHigher susceptibility
EthanolAnimal parvovirusPoliovirusMurine norovirus
Simian virus 40Foot and mouth disease virusRhinovirus
Hepatitis A virusHuman norovirusAdenovirus 5
Enterovirus 71Feline calicivirusRotavirus
Adenovirus 2, 8
IsopropanolAnimal parvovirusAdenovirus 5, 8Simian virus 40
Hepatitis A virusMurine norovirusRotavirus
Enterovirus 71
Feline calicivirus
NaOClPorcine parvovirusMinute virus of miceFeline calicivirus
Hepatitis A virusHepatitis A virusAdenovirus
Enterovirus 71
Murine norovirus
H2O2Animal parvovirusPoliovirusRhinovirus
Hepatitis A virusMurine norovirusFeline calicivirus
PAAAnimal parvovirusPoliovirusFeline calicivirus
Hepatitis A virusMurine norovirus
QACAnimal parvovirusFeline calicivirusRotavirus
PoliovirusMurine norovirusRhinovirus
Adenovirus 8, 25Adenovirus 5Coxsackievirus A11
Low pHMinute virus of miceHuman parvovirus 4Feline calicivirus
Hepatitis A virusRhinovirus
PoliovirusFoot and mouth disease virus
Enterovirus 71Enterovirus EV-D68
Coxsackievirus A9Human parvovirus B19
Murine norovirus
High pHBovine viral diarrhea virusReovirus
(enveloped virus)
Murine minute virus
Simian virus 40Feline calicivirus
Hepatitis A virusAdenovirus
Canine parvovirusRotavirus
PoliovirusFoot and mouth disease virus
Murine norovirus
Tulane virus
AldehydesPorcine parvovirusMinute virus of micePoliovirus
Hepatitis A virus
Feline calicivirus

Table 9.

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

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

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 non-enveloped 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 side-by-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 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 Picornaviridaefamily 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 Caliciviridaeis 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, 95, 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 non-enveloped 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 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.


The author thanks Drs. Raymond Nims and M. Khalid Ijaz for the critical review of the manuscript and discussion.

Conflict of interest

The author declares no conflict of interest.


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Written By

Sifang Steve Zhou

Submitted: December 6th, 2021 Reviewed: January 17th, 2022 Published: March 22nd, 2022