Motivation

Viruses have caused many epidemics throughout human history. The novel coronavirus [10] is just the latest example. A new viral outbreak can be unpredictable, and development of specific defense tools and countermeasures against the new virus remains time-consuming even in today's era of modern medical science and technology. In the lack of effective and specific medication or vaccination, it would be desirable to have a nonspecific protocol or substance to render the virus inactive, a substance/protocol, which could be applied whenever a new viral outbreak occurs. This is especially important in cases when the emerging new virus is as infectious as SARS-CoV-2 [4].

Aim and structure of the present communication

In this editorial, we propose to consider the possibility of developing and implementing antiviral protocols by applying high purity aqueous chlorine dioxide (ClO₂) solutions. The aim of this proposal is to initiate research that could lead to the introduction of practical and effective antiviral protocols. To this end, we first discuss some important properties of the ClO₂ molecule, which make it an advantageous antiviral agent, then some earlier results of ClO₂ gas application against viruses will be reviewed. Finally, we hypothesize on methods to control the spread of viral infections using aqueous ClO₂ solutions.

Inactivating viruses with ClO₂ in aqueous phase

To our present knowledge, an aqueous solution of ClO₂ is able to inactivate all types of viruses. Disinfectants (in water phase) are compared by their CT values, which is the concentration (measured in mg/L) multiplied by the contact time (measured in minute). In CT tables, ClO₂ is indicated for viruses in general, without mentioning any exemptions. For example, according to [6], a CT value of 8.4 mg × min/L is needed to achieve a four-orders-of-magnitude (“4 log” or “99.99%”) inactivation of viruses in an aqueous medium at 25 °C.

Chemical mechanism of virus inactivation: reaction of ClO₂ with amino acid residues

In 1986, Noss et al. [19] proved that the inactivation of bacterial virus f2 by ClO₂ was due to its reactions with the viral capsid proteins, and almost no inactivation of the infectious viral RNA occurred [8] when that was treated with ClO₂ separately. They found [19], however, that three discrete chemical moieties in the viral protein, namely the cysteine, tyrosine, and tryptophan amino acid residues were able to react with ClO₂ rapidly. In 1987, Tan et al. [28] tested the reactivity of ClO₂ on 21 free amino acids. ClO₂ reacted only with six amino acids dissolved in 0.1 M sodium phosphate buffer, pH 6.0. The reaction with cysteine, tryptophan, and tyrosine was too rapid to be followed by their technique. Three further amino acids (histidine, hydroxyproline, and proline) reacted with ClO₂ much more slowly, at a measurable rate.

The reactivity of the three fast-reacting amino acids (cysteine [12], tyrosine [17], and tryptophan [27]) was studied in Margerum's laboratory between 2005 and 2008. They found that cysteine had the highest reactivity among these amino acids. From their experimental data they calculated second order-rate constants (at pH 7.0, 25 °C and 1 M ionic strength) and obtained the following sequence: cysteine 6.9 × 10⁶ M⁻¹ s⁻¹ >> tyrosine 1.3 × 10⁵ M⁻¹ s⁻¹ > tryptophan 3.4 × 10⁴ M⁻¹ s⁻¹ >> guanosine 5′-monophosphate 4.5 × 10² M⁻¹ s⁻¹. (They studied guanosine 5′-monophosphate [18] as a model compound for guanine in nucleic acids. Data presented here are taken from Table 3 of ref. [18]).

In 2007, Ogata [22] found that the antimicrobial activity of ClO₂ is based on denaturation of certain proteins, which is primarily due to the oxidative modification of the tryptophan and tyrosine residues of the two model proteins (bovine serum albumin and glucose-6-phosphate dehydrogenase) used in his experiments. In 2012, it was again Ogata who showed [23] that the inactivation of influenza virus by ClO₂ was caused by oxidation of a tryptophan residue (W153) in hemagglutinin (a spike protein of the virus), thereby abolishing its receptor-binding ability.

In this context it is interesting to remark that the spike protein of the new coronavirus SARS_CoV-2 contains 54 tyrosine, 12 tryptophan, and 40 cysteine residues [29]. If we assume that in an aqueous solution all of these residues are able to react with ClO₂ just like the free amino acids, then the inactivation of the viruses can be extremely rapid even in a very dilute (e.g., in a 0.1 mg/L) ClO₂ solution.

ClO₂ is a water soluble gas

Although chlorinedioxide in itself is a gas, it is highly soluble in water. When both air and water are present, ClO₂ is distributed between the two phases in an equilibrium ratio determined by the temperature. This distribution coefficient of ClO₂ was determined by Ishi [11] in 1958. The distribution coefficient, γ = [ClO₂]_G/[ClO₂]_L gives the ratio of the concentrations expressed in the same units in the gas and the aqueous phases (e.g., g/L) and changes as a function of the temperature. For example, at 20 °C γ = 0.0316, indicating that in equilibrium 1 cm³ aqueous phase contains (0.0316)⁻¹ = 31.6 times more ClO₂ molecules than 1 cm³ gas phase.

Inactivating viruses with ClO₂ in gas phase

The virus-inactivating reactions (the reactions of ClO₂ with the three amino acids) take place in an aqueous medium; consequently, ClO₂ can inactivate microbes in their wet state only. Therefore, ClO₂ gas that is moisturized can be an ideal agent against viruses both in their wet and dry states. Viruses that are carried by water droplets could be easily inactivated even by ClO₂ gas owing to the high solubility of ClO₂ in water [11]. A dry ClO₂ gas would be inappropriate as the water content of the aqueous droplet could evaporate, and in the absence of aqueous medium the reactions of ClO₂ slow down extremely. Indeed, Morino et al. [16] reported that when applying a low concentration of ClO₂ in the gas phase against FCV in dry state, atmospheric moisture – at least a 75–85% relative humidity – is indispensable to inactivate viruses. The advantage of using a moisturized ClO₂ gas is that its water content is also able to wet viruses in dry environment. Most viruses are found on hard surfaces indoor, but a small fraction of viruses are “airborn”, attached to dust particles, which can also carry a single microbe or an aggregate of microbes. Therefore, it is a prerequisite of an effective disinfection that all microbes in all parts of the room should be wet and should be in contact with ClO₂. If enough aqueous ClO₂ solution is sprayed into the room, the droplets will saturate the atmosphere with water vapor everywhere, moreover, the atmosphere will also contain gaseous ClO₂ everywhere. The great advantage of this method is that H₂O and ClO₂ molecules of the gas phase can reach the microbes in every small corner of the room. Finely dispersed water droplets containing dissolved ClO₂ can create an advantageous environment to maintain such conditions for a longer time.

This method using high ClO₂ concentration allows fast disinfection of rooms when people are not present, e.g., intensive care units, buildings used as quarantine, or public transport vehicles. However, the application of ClO₂ gas is limited when people are present, as it is harmful for humans and animals above certain concentrations. The US Occupational Safety and Health Administration (OSHA) limits the concentration of ClO₂ gas allowed in workplace air to 0.1 ppm (V/V) time-weighted average (TWA) for an 8-h exposure, and to a temporarily higher 0.3 ppm Short-Term Exposure Limit (STEL) only for a 15-min period [30].

Previous research on preventing viral infections with gaseous ClO₂

Ogata [21] realized first that ClO₂ is able to inactivate viruses even under the 0.1 ppm (OSHA TWA) limit that is in concentrations which are not harmful to humans. In 2008, Ogata and Shibata [25] demonstrated that infection of mice with influenza A virus applied in an aerosol can be prevented by ClO₂ gas present at 0.03 ppm concentration in the air, which is only 30% of the permissible TWA exposure level for humans at a workplace. They concluded that “ClO₂ gas could, therefore, be useful as a preventive tool against influenza in places of human activity without necessitating evacuation.” They have even made attempts to decrease the incidence of flue infections among schoolchildren by applying low concentrations of ClO₂ gas in a classroom [24].

In spite of these promising early results we are not aware of any wider-scale application of this method in the last decade. There are two problems which could hinder the widespread adoption of this method:

1. With the technique applied by the above-cited authors it is not an easy task to achieve and maintain a very low ClO₂ concentration in a large space and for a long time, which is a prerequisite of achieving a satisfactory level of virus inactivation.

2. It is not understood why low ClO₂ levels are not harmful to humans or animals and still effective against viruses.

Size selective effect of ClO₂

Although cysteine, tyrosine, and tryptophan residues can also be found in human tissues, ClO₂ is much less toxic for humans or animals than for microbes (bacteria, fungi, and viruses). Noszticzius et al. [20] found that the main reason for this selectivity between humans and microbes is based not on their different biochemistries but on their different sizes. Based on experiments and calculations using a reaction-diffusion model Noszticzius et al. [20] found that the killing time of a living organism is proportional to the square of its characteristic size (e.g., its diameter), thus small ones will be killed extremely fast. Their calculations indicated that a bacterium 1 μm in diameter would be killed in a 300 mg/L ClO₂ solution within 3 ms, and even in a much more dilute, 0.25 mg/L ClO₂ solution it would be eliminated in only 3.6 s. During this time, ClO₂ reaches all parts of the cell and kills it by destroying its cysteine-, tyrosine-, and tryptophan-containing proteins, which are essential for life processes.

The protective role of glutathione against ClO₂ oxidation in a living cell

According to Ison et al. [12] glutathione reacts with ClO₂ at a rate, which is even higher than the rate of the very fast ClO₂ – cysteine reaction. When ClO₂ contacts a living cell containing glutathione, at first the ClO₂ concentration remains very low even at the point of entry into the cell due to this rapid reaction. As a small molecule, glutathione can also diffuse rapidly to the point of entry from other parts of the cell consuming most of the ClO₂ there, and preventing it from reaching the cysteine, tyrosine, and tryptophan residues of the proteins in the bulk of the cytoplasm. Consequently, the initial low ClO₂ concentration cannot make too much harm. However, continuous ClO₂ entry can finally exhaust the cell's glutathione (and other antioxidant) capacity even if the cell produces such antioxidants continuously. At this point, ClO₂ can enter into the previously protected zones of the cell and react with the reactive amino acid residues, causing denaturation of the affected proteins and ultimately cell death.

The effect of glutathione and other small antioxidant molecules present in living cells was not taken into account in the theoretical calculations of Noszticzius et al. [20]. Their experiments were done on a non-living and washed animal membrane, where membrane-fixed reactive proteins were present, but glutathione and other small molecules were absent. A living cell, however, continuously produces these antioxidants, thus their role cannot be neglected. Indeed, looking at the experimentally measured disinfection dynamics of a 0.25 mg/L ClO₂ solution against Escherichia coli bacteria [2] we can see a disinfection rate, which is surprisingly fast but still about one order of magnitude slower than the theoretical estimate. It is reasonable to assume that the delaying effect of these small reducing molecules is responsible for that deviation.

Protection of human tissues against the oxidative effect of ClO₂

Human cells also contain glutathione in mM concentrations, as well as other antioxidants like vitamin C and E, which work together with glutathione to reduce ClO₂ [7]. As a human cell is much larger than a bacterium, consequently its glutathione reserve and glutathione production potential are also greater, so even an isolated human cell can survive much longer in a ClO₂ environment than a planktonic bacterium. Considering that human cells are not isolated but form tissues, their glutathione stock may be many orders of magnitude greater than that of a planktonic bacterium. Additionally, in multicellular organisms circulation transports antioxidants continuously to the cells of the tissue affected by a ClO₂ attack, helping them to survive. This strengthens the size-selectivity effect, and explains the surprising observation [15] that ClO₂ solutions that are able to kill planktonic bacteria in a fraction of a second may be consumed, because they are safe for humans to drink in a small amount (e.g., drinking 1 L of 24 mg/L ClO₂ solution in two portions on a single day caused no observable effects in humans [15]).

The effect of ClO₂ on the lung

While human tissues are not very sensitive to ClO₂ in general, lungs should be considered differently. This is because the interalveolar septum separating the airspace of an alveolus from the blood stream of a capillary lumen is very thin. That diffusion barrier in the human lung is a mere 2 μm thick [1] in order to facilitate an efficient diffusional exchange of oxygen and carbon dioxide between the air and blood. The alveolus is covered by a thin layer of lining fluid called epithelial lung lining fluid (ELF) or hypophase. The ELF is only 0.2 μm thick in rat alveoli [1, 13]. It contains glutathione [3] and other antioxidants such as ascorbic and uric acids [5]. It is remarkable, that the ascorbic acid concentration is 2.5 times, and the glutathione concentration is more than 100 times higher in the ELF than in the plasma. The normal function of these non-enzymatic antioxidants in the ELF is to protect the epithelial cells from reactive oxygen species (ROS) like superoxide radicals or hydrogen peroxide, which are toxic products of the metabolism. They can also defend the lung against other toxic gases such as ozone (O₃), nitrogen dioxide (NO₂) or ClO₂. However, high amounts of ClO₂ can consume all reducing agents in the ELF, at which point ClO₂ starts to react with the epithelial cells causing a continuously growing damage to these cells. It is known that higher concentrations of ClO₂ gas can be lethal. However, it is reasonable to assume that the effect of ClO₂ on the lung depends not only on its concentration in the gas phase but also on the contact time. Thus, when considering the impact of ClO₂ on the lung, it would be logical to regard the CT (concentration) × (contact time) product in a similar way as in the case of the microbes.

Estimating the inactivation time of viruses

In the case of viruses, the inactivation mechanism differs from that of bacteria or other cells. It is feasible to assume that the inactivation time of a virus is probably much shorter than the inactivation time of a bacterium under the same conditions (ClO₂ concentration, temperature, etc.). The following arguments support this assumption:

1. Viruses are about one order of magnitude smaller than bacteria e.g., the diameter of SARS_CoV-2 is about 120 nm [9]. The killing time of a virus as introduced in [20] would be 1–2 orders of magnitude shorter than that of a bacterium, i.e., the diffusion-controlled reaction with ClO₂ would happen on a shorter time scale in the entire volume of the virus.

2. It is not necessary for ClO₂ gas to penetrate the virus in order to inactivate it. It is enough if ClO₂ reacts with one or some of the cysteine, tyrosine, and tryptophan amino acid residues of the spike, which are located on the surface of the virus. This means that the theoretical approach of ref. [20] overestimates the inactivation time of viruses. On the one hand, because diffusion is extremely fast over a 0.1 μm length scale, thus probably it is not limiting the rate of the reactions. On the other hand, ClO₂ can reach a large part of the reactive amino acid residues of the spike without permeating through the protein envelope of the virus.

3. Viruses do not contain protective small molecular thiols like glutathione or other small molecular protective metabolic products, because viruses have no metabolism. In this respect viruses should be much more vulnerable than bacteria to an attack by ClO₂.

These facts all suggest that once ClO₂ contacts the surface of a virus, its inactivation is quick. However, a virus ready to infect a cell is typically in aqueous phase, e.g., in a fluid droplet, or in the epithelial lining fluid covering the mucous membranes. The size of these aqueous phases is much larger than that of the virus. Therefore, in such cases, the rate-limiting step probably is the diffusion of ClO₂ in the water and the reaction with other substances. The time required to inactivate the virus itself would be short compared to the time needed to transport enough ClO₂ molecules to the virus.

Global prevention

Local prevention. Personal disinfection techniques against viral infections