**3. Virucidal efficacy evaluation of Ag<sup>+</sup> formulations against SARS-CoV-2 and FeCV**

#### **3.1 Materials and methods**

1.Challenge viruses

• Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2): Isolate USA-WA1/2020, BEI Resources, NR-1586.

*Silver Ion (Ag+ ) Formulations with Virucidal Efficacy against Severe Acute… DOI: http://dx.doi.org/10.5772/intechopen.100268*

> • Feline calicivirus (FeCV): ATCC VR-782 (used as a surrogate for human norovirus).

2.Host (detector) cells


3.Test formulations

	- Each stock challenge virus had a titer of ≥6 log10 infectious units/mL, in a culture medium containing 5% fetal bovine serum as the organic soil challenge.
	- The test product was prepared for the use-dilution and an equal volume of the dilution medium (minimum essential medium containing 2% newborn calf serum) was prepared to serve a virus control.
	- The prepared viral inoculum was added to the test formulation and the virus recovery control at a ratio of virus (one part) + test formulation or dilution medium (nine parts).
	- Upon completion of the contact time, the test and recovery suspensions were neutralized by dilution into a chemical neutralizer (minimum essential medium +10% newborn calf serum +0.5% lecithin +1 mM EDTA).
	- For the cytotoxicity control, an aliquot of the use-dilution of the test formulation was mixed with the dilution medium (in lieu of the virus) and then neutralized in an identical manner to the test suspension.
	- For the neutralization control, an aliquot of the use-dilution of the test formulation was mixed with the dilution medium, neutralized, and then spiked with a low level of virus.
	- The neutralized test suspension, virus recovery control, cytotoxicity control, and neutralization control suspensions were serially diluted in the dilution medium. Each diluted solution was plated in quadruplicate to host cell monolayers in a 24-well plate. Maintenance medium was then added to each well, and the host cells with the inoculated virus were allowed to incubate for 4–9 days at 37°C with 5% CO2.

(1)

The virus load was calculated according to Eq. (1):

Virus Load Log10 TCID50 <sup>¼</sup> Virus Titer Log10 TCID50*=*mL <sup>þ</sup> Log10 <sup>½</sup>Volume mL ð Þ �Volume correction ð*e:g:*, neutralizationÞ�

The Log10 Inactivation was calculated according to Eq. (2):

Log10 Inactivation <sup>¼</sup> Virus Recovery Control Log10 TCID50 –Test Log10 TCID50 (2)

5.Virucidal test acceptance criteria [4]



#### **Table 1.**

*Virucidal efficacy of Ag+ formulations against SARS-CoV-2 and FeCV evaluated in suspension inactivation studies.*

*Silver Ion (Ag+ ) Formulations with Virucidal Efficacy against Severe Acute… DOI: http://dx.doi.org/10.5772/intechopen.100268*

### **3.2 Results of inactivation studies**

Virucidal activity studies were conducted according to a standardized quantitative suspension testing method ASTM E1052–20 [4]. Evaluation of virucidal activity against the enveloped coronavirus, SARS-CoV-2, demonstrated that 5 ppm Ag+ formulated without ethanol caused minimal (<1 log10) inactivation within 1-min contact time, but 3.1 log10 inactivations after 360 minutes (6 h) (**Table 1**). On the other hand, 5 ppm Ag+ formulated with a low (26% w/w) concentration of alcohol caused ≥3.72 log10 inactivations of SARS-CoV-2 within 1-min contact time (**Table 1**).

The 5 ppm Ag+ formulated with 26% w/w ethanol also demonstrated efficacy (≥ 4.3 log10 inactivations) against the non-enveloped calicivirus, FeCV, within 30-min contact time, but only minimal (<1 log10) inactivation of FeCV within 1 min contact time (**Table 1**).

#### **4. Silver ion (Ag<sup>+</sup> ) mechanisms of microbicidal activity**

The broad antimicrobial activity of silver nanoparticles (AgNP) includes efficacy against over 650 microorganisms, including bacteria, fungi, and viruses. This activity is primarily due to the leaching of Ag<sup>+</sup> ions from the outer surface of the AgNP [9]. Xiu et al. [10] demonstrated that anaerobic conditions (i.e., in the absence of oxygen) prevent the Ag0 oxidation and Ag+ leaching from AgNP that is favored in acidic environments [Eqs. (3) and (4)].

$$2\text{ Ag}^{0} + \text{O}\_{2} \rightarrow 2\text{ Ag}\_{2}\text{O} \tag{3}$$

$$2\text{ Ag}\_2\text{O} + 4\text{ }H^+ \to 4\text{ Ag}^+ + 2\text{ }H\_2\text{O} \tag{4}$$

Under anaerobic conditions, AgNP has no detectable effects on E. coli at concentrations 7665 times higher than the minimum lethal concentration of Ag<sup>+</sup> (0.025 mg/L) under the same exposure conditions. In addition, these authors found that the minimum lethal concentration for AgNP under anaerobic conditions was thousands of times higher than the minimum lethal concentration observed under aerobic conditions [10]. This discovery led the researchers to conclude that the antibacterial activity could be controlled by modulating the Ag<sup>+</sup> release (leaching) kinetics through modifications to the AgNP size, shape, and surface characteristics, including the presence of a coating [10].

There are four known antimicrobial actions of AgNP [11, 12]: 1) adhesion to the microbial cell membrane; 2) penetration of AgNP into the cell, causing disruption of biomolecules and intracellular damage; 3) induction of cellular toxicity mediated by reactive oxygen species (ROS), resulting in oxidative stress to the cell; and 4) disruption of signal transduction pathways of the cells.

When microbes are exposed to AgNP, the nanoparticles tend to stick or adhere to the cell wall or membrane due to the electrostatic attraction between the positive charge of Ag<sup>+</sup> generated during oxidation of AgNP and the negatively charged cell membrane of microorganisms (**Figure 1**). AgNP also displays a strong affinity for the sulfur-containing proteins in the microbial cell wall. The attachment of AgNP to the cell membrane causes irreversible morphological changes in the membrane structure. This can also cause a loss in the integrity of the lipid bilayer and changes in the permeability of the cell membrane. Alterations in such structures can cause increased permeability of the cell membrane, which, in turn, impacts the ability of the cell to regulate essential activities. For instance, the binding of AgNP and subsequent leaching of Ag+ can alter transport and release of potassium ion (K<sup>+</sup> ),

#### **Figure 1.**

*Schematic depiction of interaction of extracellular Ag+ with the bacterial cell membrane and subsequent entry of Ag+ into the cell. Ag+ and AgNP enter the bacterial cell membrane by porin proteins. Within the bacterial cell membrane, Ag<sup>+</sup> causes the formation of several highly oxidizing species, such as hydroxyl and superoxide freeradicals and hydrogen peroxide (H2O2), which quickly oxidizes DNA and RNA, and denature proteins. This results in cell membrane rupture and pore formation. Leakage of intracellular contents and cell lysis results [3].*

thus affecting the transport activity of cells. An increase in cell membrane permeability may also cause loss or leakage of cellular contents such as cytoplasmic proteins, ions, and cellular energy reservoirs (adenosine triphosphate; ATP).

Following the adhesion of AgNP to the microbial membrane, the nanoparticles can penetrate the cell and impact important biomolecules and cellular activities. AgNP is able to enter Gram-negative bacteria, such as E. coli*,* through water-filled channels in the membrane called porins*.* After penetration of AgNP into the cells, these nanoparticles will start to bind with cellular structures and biomolecules, such as proteins, lipids, and DNA, thus damaging the internal structure of the bacteria. Any leached Ag+ binds to negatively charged proteins, altering the proteins structurally and eventually resulting in denaturing of the proteins.

Another mechanism of action of AgNP is the production of ROS, which causes cellular oxidative stress in microbes. Reactive oxygen species is a general term for oxygenated compounds that are involved in various cellular biological events. These can include but are not limited to superoxide, hydrogen peroxide, and hydroxyl radicals. The antibacterial potential of AgNP is usually related to the ability of the nanoparticles to produce ROS and increase the oxidative stress in the cells. Production of intercellular ROS is thought to be the most important indicator of toxicity related to AgNP, as the ROS may induce lipid damage and leakage of cellular biomolecules, and may eventually lead to cell apoptosis [11, 12].

The virucidal efficacy of Ag+ and AgNP is mediated by the following types of interactions: 1) the Ag+ /AgNP bind to spike proteins of enveloped viruses, inhibiting the attachment of these viruses to host cell receptors (**Figure 2**); and 2) Ag+ /AgNP bind to the genomic DNA or RNA of both enveloped and non-enveloped viruses, inhibiting the replication or propagation of the virus inside the host cells.

For example, in the case of the human immunodeficiency virus (HIV-1; family *Retroviridae*, enveloped), the AgNP binds to the sulfur groups of gp120 protein spikes on the viral envelope, thereby preventing infectivity due to the fusion of the viral envelope with the host cell membrane [13]. Similarly, the attachment and entry of herpes simplex virus type 1 (HSV-1; family *Herpesviridae*, enveloped) into cells involve interaction between viral envelope glycoproteins and cell surface heparan sulfate (HS). Viral entry can be prevented by AgNP capped with mercaptoethane sulfonate

*Silver Ion (Ag+ ) Formulations with Virucidal Efficacy against Severe Acute… DOI: http://dx.doi.org/10.5772/intechopen.100268*

**Figure 2.** *Schematic depiction of interaction of extracellular Ag+ with enveloped viruses, such as SARS-CoV-2.*

targeting the virus and competing for its binding to cellular HS through their sulfonate end groups [14]. The antiviral mechanism of inorganic metals such as copper and silver against influenza A viruses appears to be mediated through the inactivation of hemagglutinin (HA) and neuraminidase (NA) cell surface proteins [15].

It was demonstrated in experimental RSV infection studies by Morris et al. [16] that AgNP caused a reduction in RSV. In the mouse model, the antiviral activity appeared to be mediated to a large extent by neutrophils, which were recruited in higher numbers to the airways and activated *via* a neutrophil-specific program of cytokines. This was reported as the first *in vivo* study demonstrating antiviral activity of AgNP during RSV infection.

## **5. Discussion**

Silver ion (Ag+ ) has been used since ancient times for various purposes [1]. Silver plays a certain role in mythology and has found various usages as a metaphor. In folklore, silver was commonly thought to have mystic powers. For example, a bullet cast from silver was supposed in such folklore to be the only weapon effective against a werewolf, witch, or other monsters. From this mythology, the idiom of the silver bullet resulted in figuratively referring to any simple solution with very high effectiveness or almost miraculous results, as in the widely discussed software engineering paper "No Silver Bullet" [17]. Other mythic powers attributed to silver have included detection of poisons and facilitation of passage into the mythical realm of the fairies.

In medicine, silver has been incorporated into wound dressings and used as an antibiotic coating in medical devices. Wound dressings containing silver sulfadiazine or silver nanoparticles have been used to treat external infections. Silver has also been used in urinary catheters for reducing catheter-related urinary tract infections and in endotracheal breathing tubes for reducing ventilator-associated pneumonia [18, 19]. Silver ion is bioactive and, at sufficient concentration, readily kills bacteria *in vitro*. Silver and silver nanoparticles are used as antimicrobial ingredients in a variety of

industrial, health care, and domestic applications. For example, infusing clothing with AgNP allows the items to remain odorless longer [20].

Silver ion (Ag<sup>+</sup> ) displays broad-spectrum antimicrobial action, with efficacy against various bacteria, fungi, and viruses. Due to their versatility, AgNP is currently used as microbicides in wound dressings, medical devices, deodorant sprays, and fabrics. Studies have demonstrated the virucidal efficacy of AgNP against human pathogenic viruses, including enveloped viruses such as respiratory syncytial virus (RSV), influenza virus, hepatitis B virus (HBV), and human immunodeficiency virus (HIV), as well as non-enveloped viruses such as human norovirus [2]. In addition, Ag<sup>+</sup> has been shown to possess virucidal efficacy against severe acute respiratory syndrome coronavirus (SARS-CoV) [21] and SARS-CoV-2 [22, 23]. AgNP formulations have been proposed for cleaning inanimate surfaces to efficiently control the ongoing COVID-19 pandemic [23]. The hypothesis was based on the proposed mechanism of action of AgNP, involving binding to the spike glycoprotein of the virus, thereby inhibiting the binding of the virus to the host cells. Dissemination of respiratory pathogens such as SARS-CoV-2 from infected to susceptible individuals is believed to occur directly, *via* respiratory droplets and droplet nuclei/aerosols, and indirectly, through contaminated high-touch environmental surfaces (HITES) [24]. SARS-CoV-2 has been reported to remain infectious on contaminated HITES for hours to days [25–27].

The Ag+ formulations discussed in this chapter are not considered silver zeolites or silver zirconium phosphate. These Ag<sup>+</sup> formulations are, therefore, not in scope for the European Union ban on the use of certain silver compounds in antimicrobial products [28].

Until the virucidal efficacy of microbicides is empirically demonstrated for SARS-CoV-2 specifically, the EPA has allowed agents to be used on the basis of their activity against other enveloped and non-enveloped viruses (**Box 1**). Virucidal efficacy of a selection of formulated microbicidal actives against SARS-CoV-2 has, to date, been assumed based on efficacy data obtained using other coronaviruses, or based on non-standardized methods of assessing viral inactivation (i.e., log10 reduction in infectious titer) in suspension without details of the testing method used, including use of appropriate controls. To date, only limited virucidal testing against SARS-CoV-2 has been demonstrated definitively through testing conducted per standardized surface and suspension methodologies [27].

On March 5, 2020, the US Environmental Protection Agency (EPA) announced the release of a new list [29] of EPA-registered disinfectant products that were considered as qualified for use against SARS-CoV-2, the coronavirus that causes the disease COVID-19. Products on EPA's "List N: Disinfectants for Use Against SARS-CoV-2" are registered disinfectants qualified for use against SARS-CoV-2 through EPA's Emerging Viral Pathogen Program (EVPP) [30]. Currently, there are 85 products listed that are qualified for use against SARS-CoV-2. Of note, EPA states that if the directions for use for viruses/virucidal activity of the listed products provide different contact times or dilutions, the longest contact time or most concentrated solution should be used. The EPA initially issued guidance for the EVPP in 2016; the program was intended to "expedite the process for registrants to provide useful information to the public" regarding products that should be effective against emerging viral pathogens.

According to the EVPP, in the event of an outbreak, companies with pre-approved products can make off-label claims (e.g., technical literature distributed exclusively to healthcare facilities, physicians, nurses, and public health officials; 1–800 consumer information services; company websites (non-label related); and social media) for use of these products against the outbreak virus. These emerging pathogen statements do not appear on marketed (final print) product labels. Products that meet EPA's criteria for use against SARS-CoV-2 can be searched using the EPA database [29]. This database allows users to search by criteria such as EPA registration number, the active ingredient, use site, surface types, contact time, and keywords. *Silver Ion (Ag+ ) Formulations with Virucidal Efficacy against Severe Acute… DOI: http://dx.doi.org/10.5772/intechopen.100268*
