**4. Empirical virucidal efficacy data for microbicides, including surface and hand hygiene agents, against the WHO Priority List viruses**

In this section, we review the literature with regard to inactivation of WHO Priority List viruses by microbicides intended for decontamination of surfaces, for hand hygiene, for decontamination of liquids, and for test sample disinfection. Our discussion is limited to chemical microbicides, and specifically to the efficacy of these microbicides against the viruses mentioned in the WHO Priority List. The stated purpose of this review was to identify knowledge gaps for virucidal efficacy against the WHO Priority List viruses. As such, information pertaining to surrogate viruses from other families, or even unlisted viruses from the same families, is considered out of scope for this chapter. In addition, physical inactivation approaches (e.g., heating, ultraviolet radiation, and gamma irradiation), are not in scope for this chapter. A review of physical inactivation approaches for SARS-CoV-2 and other coronaviruses can be found in this book [16].

Inactivation studies evaluate pathogens dried onto a surface or within a suspension, but also may investigate efficacy for inactivating pathogens suspended in the air. Studies evaluating decontamination of surfaces involve the application of viruses, in the absence or presence of a soil load, onto carriers representing different prototypic environmental surfaces of interest (e.g., glass, stainless steel, plastic, etc.). Following drying of the applied virus onto the carrier for a set time period, a small quantity of the microbicide is added and left on for the specified contact (dwell) time. Residual virus is collected using an appropriate medium, and the titer post-treatment is compared to the initial untreated virus titer, with log10 reduction results accounting for any cytotoxicity of the test microbicide or neutralizing reagents used on the host cells used in the respective viral assays.

For suspension inactivation studies, depending on the test methodology chosen, virus is added to a liquid matrix, again in the absence or presence of a soil load. The microbicide is added at the evaluated test concentration and the solution is incubated at the appropriate temperature for the planned contact times. Again, the virus titers post-treatment are compared to the titer applied, with log10 reduction results accounting for any cytotoxicity of the test microbicide or neutralizing reagents used on the host cells used in the respective viral assays. Hand hygiene agents may be

tested using suspension methodologies, or using specialized methods designed to recover virus directly from the skin. The hand hygiene agents are tested *in vitro*, or *in vivo* mimicking simulated-use (using *ex vivo* model), or under actual useconditions in human volunteers. Efficacies of virucidal products intended for administration orally or nasally, and other types of therapeutic virucides, are not addressed in this review.

Because of the differences in testing methodologies used for evaluation of surface disinfection vs. decontamination of liquids or test samples, extrapolations of efficacy from one application to another should be made with caution. Differences in virucidal efficacy testing of microbicides (hand and surface hygiene agents) in liquid vs. on surfaces (inanimate or animate) have been identified, but these differences are typically relative, and may depend on the challenge virus and the microbicide being tested [17].

The virucidal efficacy literature for microbicides against Lassa virus is summarized in **Table 2**, and that for the bunyaviruses (Crimean-Congo hemorrhagic fever virus and Rift Valley fever virus) is summarized in **Table 3**. Information on virucidal efficacy for the coronaviruses (SARS-CoV-2, SARS-CoV, and MERS-CoV) is presented in **Table 4**, and virucidal efficacy for filoviruses (Ebola virus and Marburg virus) is shown in **Table 5**. **Table 6** presents virucidal efficacy data for the flavivirus (Zika virus), and the limited information on virucidal efficacy of microbicides against paramyxoviruses (Nipah virus and other henipaviruses) is summarized in **Table 7**.

Not all of the virucidal efficacy information from the reviewed articles is shown in **Tables 2**–**7**. Wherever possible, the virucidal efficacy data shown are from conditions leading to the highest log10 reduction level, or complete-inactivation of the challenge virus to the limit of detection of the infectivity assays used. No data from studies using exclusively nucleic acid assays have been included, as the nucleic


*b Inactivation matrix was virus stock (virus in culture medium), unless otherwise indicated.*

#### **Table 2.**

*Efficacy of microbicides for inactivating the arenavirus Lassa virus.*


*b Inactivation matrix was virus stock (virus in culture medium), unless otherwise indicated.*

#### **Table 3.**

*Efficacy of microbicides for inactivating the bunyaviruses Rift Valley fever virus and Crimean-Congo hemorrhagic fever virus.*

acid endpoints are not useful for measuring infectious virus unless integrated cell culture-qPCR based assays [58] are used. The individual reports in papers referenced in this chapter should be consulted for complete information, including concentration/response information, time/inactivation kinetics information, and microbicidal product names (which have not been included here).

#### **4.1 Lassa virus**

There have appeared in the literature only few reports of the empirical testing of microbicides for efficacy as virucides for the arenavirus (Lassa virus). The literature that has been identified has been summarized in **Table 2**. In addition, some descriptions of the utility of microbicides can be found in the secondary literature. For example [59], "LASV [Lassa virus] is susceptible to inactivation by most detergents and disinfectants. Sodium hypochlorite (0.5–1%), phenolic compounds, 3% acetic acid, lipid solvents and detergents (e.g., SDS), formaldehyde/paraformaldehyde, glutaraldehyde (2%), and beta-propiolactone disrupt virion integrity." The source provided for these claims was another secondary source [60]. No primary






*a BKC, benzalkonium chloride; DBAC, dimethyl benzyl ammonium chloride; DBAS, dimethyl benzyl ammonium saccharinate; DNB, di-N-decyl dimethyl ammonium bromide; DNC, di-N-decyl dimethyl ammonium chloride; H2O2, hydrogen peroxide; MERS-CoV, Middle East respiratory syndrome coronavirus; ND, not determined; PBS, phosphate buffered saline; SARS-CoV, severe acute respiratory syndrome coronavirus; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; WHO, World Health Organization. Entries in blue font indicate formulations with microbicidal active ingredients.*

#### **Table 4.**

*Efficacy of microbicides for inactivating the coronaviruses SARS-CoV-2, SARS-CoV, and MERS-CoV.*

literature source was provided for these claims, and it should be noted that important information such as contact times, temperatures, inactivation matrices, or methodologies was not provided in these secondary sources [59, 60].

No primary literature (peer-reviewed) for virucidal efficacy of Lassa virus by microbicides on surfaces or in suspensions, or for efficacy of hand hygiene agents was identified during this literatures search. Characterization of the efficacy of microbicides for these purposes is required to resolve this knowledge gap. The few reports found related to agents intended for rendering laboratory samples safe for use in diagnostic assays [18–21].

#### **4.2 Crimean-Congo hemorrhagic fever virus and Rift Valley fever virus**

There are few reports of the empirical testing of microbicides for efficacy as virucides for the bunyaviruses [Crimean-Congo hemorrhagic fever virus (CCHFV) and Rift Valley fever virus (RVFV)]. The literature that has been identified has been summarized in **Table 3**. In addition, some descriptions of disinfectant utility can be found in the secondary literature. For example, "CCHFV can be inactivated by many disinfectants including 1% hypochlorite, 70% alcohol, hydrogen peroxide, peracetic acid, iodophors, glutaraldehyde, and formalin" [61]. No primary literature source was provided for these claims, and it should be noted that important information such as contact times, temperatures, inactivation matrices, or methodologies was not provided in this brief description [61]. Similar information is provided in the review by Bartoli et al. [62]. In that review, which has an emphasis on laboratory safety, attribution to the primary literature for CCHFV is provided for



*a FBS, fetal bovine serum; H2O2, hydrogen peroxide; QAC, quaternary ammonium compound; WHO, World Health Organization. Entries in blue font indicate formulations with microbicidal active ingredients. b Inactivation matrix was virus stock (virus in culture medium), unless otherwise indicated.*

#### **Table 5.**

*Efficacy of microbicides for inactivating the filoviruses Ebola and Marburg viruses.*

one of the eight references supporting the disinfectant efficacy section. The remaining references are either secondary literature or are related to the filovirus Ebola virus, not to CCHFV. Thus, the same disinfectant efficacy data, for which


*a QAC, quaternary ammonium compound: n-alkyl dimethyl benzyl ammonium chloride, n-alkyl ethyl benzyl ammonium chloride; H2O2, hydrogen peroxide; WHO, World Health Organization. Entries in blue font indicate formulations with microbicidal active ingredients.*

#### **Table 6.**

*Efficacy of microbicides for inactivating the flavivirus Zika virus.*


**Table 7.**

*Efficacy of microbicides for inactivating the paramyxoviruses Nipah virus and other henipaviruses.*

primary supporting data do not appear to be available, have appeared in numerous secondary sources and review articles on RVFV or CCHFV.

No primary reports describing efficacy of microbicides as surface or hand hygiene agents, or for inactivating these viruses in suspensions were identified during the literature search (**Table 3**). This represents a significant knowledge gap with respect to IPAC for these viruses. The available inactivation efficacy data relate to vaccine virus inactivation [22, 23] and sample disinfection reagents/cell fixatives [19, 21, 24, 25] for RVFV or CCHFV. The few microbicides that have been evaluated are solvents or detergents with expected efficacy for inactivating an enveloped virus, such as a bunyavirus.

#### **4.3 SARS-CoV-2, SARS-CoV, and MERS-CoV**

In the case of SARS-CoV-2, an extensive literature for virucidal efficacy of microbicides has been developed over the past year and a half. To a lesser extent, literature for original SARS-CoV and for MERS-CoV was identified. Data on the inactivation of these beta-coronaviruses by microbicides are summarized in **Table 4**. The information displayed in **Table 4** considers microbicides intended for disinfection of HITES [15, 26, 27], inactivation in liquid suspension [15, 28–37], and microbicides intended for hand hygiene [15, 27, 30, 31, 33, 35, 38–41, 63] and for laboratory sample decontamination [19, 34, 35, 37, 42, 43]. Additional reports on disinfection of laboratory samples which did not report results in terms of log10 reduction in titer include the following [64, 65]. The inactivation literature for SARS-CoV-2 and other coronaviruses has been reviewed extensively [66–75]. Readers interested in the virucidal efficacy of these microbicides for coronaviruses under different testing conditions, carrier types, contact times, temperatures, and the presence or absence of a challenge soil load are advised to examine these review papers, as well as the primary literature sources indicated in **Table 4**. It was not possible to display all useful information from these sources within one summary table, so **Table 4** should be used as a guide for pursuing additional detail for the listed microbicides and applications.

The types of microbicides that display virucidal efficacy for SARS-CoV-2, SARS-CoV, and MERS-CoV-2 are those expected to be lipid-disrupting agents (e.g., solvents, alcohols, detergents, phenolics, and quaternary ammonium compounds) and broad-spectrum microbicides (oxidizing agents, and organic and inorganic acids and

bases). Inactivation conditions leading to complete inactivation to the limit of detection of the infectivity assays have been described in **Table 4**, enabling researchers and healthcare workers to implement cleaning regimens with the greatest chances of limiting onward transmission of the virus through contaminated fomites, solutions, hands, and diagnostic samples. The primary knowledge gap identified during this literature review is around the efficacy of plain soap and water inactivation of the beta-coronaviruses. This gap has been discussed previously [76].

### **4.4 Ebola virus and Marburg virus**

Ebola virus and Marburg virus are members of the *Filoviridae* family. These are enveloped viruses which cause relatively lethal hemorrhagic fevers in humans. Most of the available literature on inactivation of Ebola virus variants by microbicides has been generated in carrier studies [44–49]. Very little data for inactivation of Ebola virus in suspension studies was identified during the literature search [50, 51]. Few reports of the efficacy of hand hygiene agents for inactivating Ebola virus were found [40, 51, 52], while efficacy of laboratory sample decontamination agents has been reported both for Ebola virus variants [18, 19, 53, 54] and Marburg virus strains [18, 19]. The data from these reports have been summarized in **Table 5**. Fortunately, a variety of Ebola variants have been used as challenge viruses, and at least two strains of Marburg virus have been evaluated. Where side-by-side comparisons of efficacy between variants has been evaluated [45], any differences in virucidal efficacy identified have been relative; that is, differences have been in degree of inactivation (i.e., log10 reduction in titer) only.

Knowledge gaps for Ebola virus inactivation include evaluation of the efficacy of plain soap and water hand washing. In the case of Marburg virus, little virucidalefficacy data of microbicides (surface and hand hygiene agents) have been generated. This knowledge gap is, therefore, relatively profound. The secondary literature [77] suggests that "Ebola viruses and Marburg viruses are both reported to be susceptible to sodium hypochlorite, glutaraldehyde, β-propiolactone, 3% acetic acid (pH 2.5), formaldehyde, and paraformaldehyde. Recommended dilutions of sodium hypochlorite may vary with the use. Calcium hypochlorite, peracetic acid, methyl alcohol, ether, sodium deoxycholate, and some other agents have also been tested against Ebola viruses, and found to be effective." The only source provided in support of the above was Mitchell and McCormick [18]. As is apparent, much of the current knowledge in such secondary sources [77, 78] pertains to inactivating agents for rendering laboratory samples safe for use. It should be noted that for most of the listed microbicides, important information such as microbicide concentration, contact time, temperature, inactivation matrix, or study methodology was not provided in these secondary sources.

#### **4.5 Zika virus**

Zika virus is a member of the *Flaviviridae* family of enveloped viruses, which includes such common pathogens as hepatitis C virus, West Nile virus, hog cholera virus, and bovine viral diarrhea virus. Data on the inactivation of Zika virus by microbicides have been summarized in **Table 6**. The information displayed in **Table 6** considers microbicides intended for surface disinfection [55, 56], inactivation in liquid suspension [56], and microbicides intended for hand hygiene [40], and for laboratory sample decontamination [57]. While the totality of the data is relatively minimal, a variety of lipid-disrupting agents have been evaluated and found effective. The oxidizing agents (chlorine, sodium hypochlorite, and hydrogen peroxide) also proved effective, as expected per the hierarchy of susceptibility to

microbicides (**Figure 1**). Note that the peracetic acid- and chlorine-containing microbicides displayed limited efficacy when the virus was dried on carriers within a blood matrix (**Table 6**). Since Zika virus is transmitted primarily through insect vectors and fomite (indirect) transmission plays a lesser role, the surface hygiene, suspension inactivation, and hand hygiene efficacy data are mainly relevant to IPAC under health-care and laboratory settings (i.e., handling clinical samples containing bodily fluids for analysis). Relevance for the publicat-large is perhaps lesser, compared with the other viruses discussed within this chapter.
