WO2007020478A1

WO2007020478A1 - Virucidal solutions

Info

Publication number: WO2007020478A1
Application number: PCT/GB2006/050246
Authority: WO
Inventors: Nick Meakin; Robert Kenyon
Original assignee: Forum Bioscience Holdings Limited
Priority date: 2005-08-19
Filing date: 2006-08-18
Publication date: 2007-02-22
Also published as: GB0517044D0; GB2429152A

Abstract

The present invention relates to improved virucidal agents, and particularly to virucidal solutions, for killing harmful viruses such those causing avian viral diseases.

Description

Virucidal Solutions

The present invention relates to improved virucidal agents, and particularly to virucidal solutions, for killing harmful viruses such as avian viruses. Whilst virucidal agents are known, they are generally harmful substances and contact with them should be avoided. Furthermore, conventional virucidal agents are harmful to the environment and their use should therefore be kept to a minimum. The present invention relates to virucidal agents which are safe and which do not harm the environment or animals with which they may come into contact. Therefore, the virucidal agents of the invention may be used freely to prevent viral infection and to prevent the spread of viruses.

In particular, the present invention is concerned with killing and reducing the spread of avian and other viral diseases, such as Avian Influenza (commonly referred to as "bird 'flu"), Infective Bronchitis, Gumboro Disease, Chick Anaemia Merek's Disease and Newcastle Disease, Foot and Mouth Disease, Swine Vesicular Disease and Tuberculosis.

Viruses are non-living, infectious or pathogenic agents, containing nucleic acids (i.e., DNA or RNA), which are usually contained in a protein coat. Many different forms of virus exist. They are classified by shape, the nature of their nucleic acids (i.e., whether they have single or double-stranded RNA or DNA), the type of organisms that they infect and the way in which they infect them. Unlike bacteria or fungi, viruses cannot be controlled by the use of biopesticides or the like. Vaccines may be produced to increase protection against mammalian viral disease.

In the past, common disinfectant materials have been used as virucidal agents. For example, disinfectants containing hypochlorites, such as household bleach, can be effective against viruses. The virucidal activity usually comes from the agents' ability to bond with and destroy the outer surfaces (protein coatings) of viruses.

Influenza is a common virus and there are three types of influenza virus, namely Influenza A, B and C. Influenza A viruses are found in many different animals, including ducks, chickens, pigs, whales, horses, and seals. Influenza B viruses circulate widely only among humans. Influenza A or B viruses cause epidemics of disease almost every winter.

Wild birds are the primary natural reservoir for all subtypes of Influenza A viruses and are thought to be the source of Influenza A viruses in all other animals. Most influenza viruses cause asymptomatic or mild infection in birds. However, the range of symptoms in birds varies greatly depending on the strain of virus. Infection with certain avian influenza A viruses (for example, some strains of H5 and H7 viruses) can cause widespread disease and death among some species of wild and especially domestic birds such as chickens and turkeys.

While it is relatively rare for humans to get influenza infections directly from animals, sporadic human infections and outbreaks caused by certain avian Influenza A viruses have occurred. Because avian influenza viruses do not commonly infect humans, there is little or no immune protection against them in the human population. Therefore, when such cross-species infection does occur, the virus can spread easily from person to person, causing an influenza pandemic. It is thought that such outbreaks may be triggered by humans living in close proximity to birds, such as chickens, and pigs. This is commonly thought to be how the "Spanish Flu" pandemic of 1918-1920 started.

An outbreak of "bird 'flu" is currently affecting bird populations in countries throughout Asia and a number of human cases also have been reported. Key to containing the outbreak is the culling of sick and exposed birds. Patients are being treated and isolated, and investigations are underway to determine the source of infection. In addition to culling, and the possible development of a vaccine against this strain of bird 'flu, the spread of the outbreak could be further contained using the virucidal solutions of the present invention.

Merek's Disease is another avian viral disease. It is caused by a herpes virus and it occurs mainly in chickens under 16 weeks of age. At present there is no known treatment for Merek's Disease and its spread is controlled by vaccination of chicks when they are one day old. Infective Bronchitis is another avian viral disease and its symptoms include respiratory distress, sneezing and rattling. In laying birds, it is common to observe a drop in egg production, as well as misshaped eggs with low quality. Some strains of the virus may cause severe kidney damage. Once again, at present there is no known treatment for Infective Bronchitis, and its spread is controlled by vaccination.

A further avian viral disease is Chick or Chicken Anaemia. This condition is caused by the Chicken Anaemia Agent (CAA), a primary immune system pathogen which causes multiple anatomic and clinical immunopathologic lesions. Although birds of all ages are susceptible to the virus, embryos and chicks less than 14 days old are prime targets. CAA is extremely hardy and resistant to temperature extremes and most disinfectants, although commercial bleach solutions are reported to kill the virus.

Gumboro Disease (also known as Infectious Bursal Disease (IBD) or Infectious Bursitis) is an avian viral disease caused by a birnavirus. The serotype 1 virus occurs throughout the world in chickens, whilst the serotype 2 antibody (without disease) is present in turkeys. Different strains of the virus exhibit different virulence. The Gumboro Disease virus is mainly spread by faeces and litter. People, clothing, vehicles and equipment soiled with contaminated faeces or Utter are known to facilitate the spread of the virus. Litter beetles may also be a cause local spread and persistence. Effective cleansing and disinfection is required to remove infection and to reduce the risk of the spread of the virus. Vaccination of susceptible stock also provides protection from the disease.

Newcastle Disease is a highly contagious and fatal viral disease affecting most species of birds. The Newcastle Disease virus (NDV) is so virulent that many birds die without showing any clinical signs and the death rate can be as high as almost 100% in unvaccinated poultry flocks. Indeed, NDV can infect and cause death even in vaccinated poultry. NDV is primarily spread through direct contact with the bodily discharges of infected birds.

Foot and Mouth Disease (FMD) is a highly infectious viral disease that may even be transmitted through dust particles in the air and can prove fatal in pigs, cattle sheep and goats. Although there are vaccines, vaccination is generally ruled out for commercial reasons. Swine Vesicular Disease (SVD) is also a contagious viral disease. Clinically, SVD cannot be distinguished from FMD. The spread of the viruses responsible for FMD & SVD is usually prevented or reduced by disinfection, in conjunction with the slaughter of infected animals and those kept on premises where infection is found.

Tuberculosis (TB) is a Uf e- threatening infection that primarily affects the lungs and experts at the World Health Organization (WHO) believe that approximately 2 billion people are currently infected with TB. Despite advances in treatment, TB is a global pandemic, fuelled by the spread of HIV/AIDS, poverty, a lack of health services and the emergence of drug-resistant strains of Mycobacterium tuberculosis, the bacterium that causes the disease. Bacille Calmette Guerin (BCG) was developed in the 1930s and it remains the only vaccination available against TB today. BCG is made of a live, weakened strain of Mycobacterium bovis, (which is related to Mycobacterium tuberculosis). It is effective in reducing the likelihood and severity of TB in infants and young children, but it is mainly only used in areas of the world where TB is highly prevalent.

In light of the foregoing, it is clear that there are a number of viral diseases which have serious implications and for which vaccination is not possible or not an attractive or realistic option. The spread of the viruses is currently difficult to control and in some cases the only way to kill the virus is to use strong virucidal agents such as commercial bleach, in combination with treating the subjects with vaccines prior to infection and/or with antibiotics after infection. The strong virucidal agents are generally hazardous liquids and their use near animals such as birds and humans is undesirable. The agents tend to be highly toxic and emit harmful fumes. Therefore, the use of these conventional agents must be kept to a minimum and must be carefully monitored as it poses many risks, including, for example, the contamination of drinking water. Herein, "subjects" are animals including, for example, birds and mammals such as humans.

Birds, and in particular poultry, are widely bred for human consumption and this involves keeping large numbers of animals in close proximity to one another and a significant amount of transportation of the animals. It is therefore desirable to be able to disinfect the birds and their housing, transportation vehicles and anyone who comes into contact with them. This is currently not possible or not practical using conventional virucidal agents that are capable of destroying avian viruses.

It is therefore clear that an alternative means for killing viruses and controlling their spread is required. Such means needs to be safe for the subjects it seeks to protect, whilst being effective against the often resilient viruses.

In accordance with a first aspect of the present invention, a solution comprising electrochemically activated water is used to kill or destroy viruses and to control the spread of viral diseases. The viral diseases include avian viral diseases such as Avian Influenza, Infective Bronchitis, Gumboro Disease, Chick Anaemia Merek's Disease and Newcastle Disease. The viral diseases may also be Foot and Mouth Disease, Swine Vesicular Disease or Tuberculosis.

Electrochemically activated water (EAW), which is also sometimes referred to as "hydroactivated water" or "electrolysed water", is water which has undergone electrochemical activation (ECA), wherein the water and the natural salts therein, or salts added thereto, is exposed to a substantial electrical potential difference.

If one places an anode (+) and a cathode (-) in pure water and applies a direct current, electrolysis of the water will occur at the poles leading to a breakdown of the water into its constituent elements, producing gaseous hydrogen and oxygen.

However, if sodium chloride is added to the water to form a solution, the dominant electrolysis end product is hypochlorite or hypochlorous acid (HOCl), a chlorine- based reagent which may be used to kill microorganisms.

The electrochemical activation process is improved by interposing an ion-permeable membrane between the positive and negative electrodes, forming an anode chamber and a cathode chamber. Preferably, the aqueous sodium chloride solution is fed into both the anode chamber and the cathode chamber and the sodium chloride, which is in its ionised form in solution (Na⁺ and Cl^") is exposed to the controlled electrical potential difference between the cathode and the anode. This potential difference causes the Na⁺ ions to migrate to the cathode and the Cl^" ions to migrate to the anode. The membrane which separates the anode chamber and the cathode chamber allows ions to pass unimpeded, whilst the un-ionised water and any organic molecules in the water are unable to pass through the membrane.

The presence of an ion-permeable non-ceramic membrane in the electrolysis apparatus allows the necessary ions to be concentrated in the anode and cathode chambers, which results in the formation of metastable ions with high biocidal activity and very low chlorine levels. Although a similar process takes place in conventional electrochemical activation processes, the presence of anion-permeable membrane prevents the complex reactive species formed at the cathode and anode from reacting with one another and being neutralised. The specific choice of a non- ceramic membrane further refines the chemical processes.

As the electrical potential is applied, high concentrations of Cl^" and OH^" build up on the anode side of the membrane and Na⁺ and H⁺ build up on the cathode side of the membrane. The unstable chemical state results in complex reactions which produce a metastable solution containing a wide variety of very reactive ions and molecules.

It is the formation of these complex chemical species which leads to the formation of solutions described as electrochemically activated water or hydroactivated water. Some of the reactive constituents which may be formed during the electrochemical activation of the sodium chloride solution include hypochlorite (HClO), hydrogen peroxide (H₂O₂), ozone (O₃), chlorine (Cl₂) and chloric acid (HClO₃). Most of these compounds are formed in the anode chamber. They are acidic, giving the anolyte a pH of between 2.4 and 4 and oxidising activity. In the cathode chamber, the reactive species are basic and are reducing agents. The catholyte will have a pH of between 10 and 12.

The anolyte and catholyte produced by the electrochemical activation of an aqueous sodium chloride solution also exhibit opposing potentials, the anolyte having a redox potential of approximately +105OmV, while the catholyte has a redox potential of approximately -85OmV. This is compared to the redox potential of the starting material of approximately +300 to 40OmV. In the apparatus with an ion-permeable membrane, two distinct solutions are formed, the catholyte and the anolyte, and these solutions can be separately extracted or they can be mixed.

The anolyte solution exhibits mild oxidative power and can destroy microorganisms, and therefore has useful sterilizing and disinfectant properties. The catholyte has properties which make it useful as a detergent and as a surfactant. Its reducing power also mean that the catholyte is effective in precipitating metal ions out of water and it can be used to soften hard water.

Whilst the oxidative and reductive properties of the anolytes and catholytes of the electrochemically activated water have previously been recognised, it has now unexpectedly been found that the anolyte of electrochemically activated water may be used to kill or destroy viruses, including resilient viruses such as avian viruses. What is more, the use of such anolyte solutions is safe. Whilst the mildly oxidative anolyte solutions are capable of rapidly breaking down the protein coating of a virus, they are benign in terms of fumes, corrosion and their effect on the skin of humans and other animals.

Therefore, in one embodiment of the present invention, a solution comprising the anolyte of electrochemically activated water is used to destroy or kill viruses and to control the spread of viral diseases. The anolyte is preferably produced by electrochemical activation of an aqueous salt solution and it may contain agents such as hypochlorite and hydrogen peroxide which have virucidal activity.

In another embodiment of the invention, the solution comprises a combination of the anolyte and the catholyte of electrochemically activated water. In a particular embodiment, the catholyte is added to the anolyte in order to adjust the pH of the solution to a desired value. Solutions with a pH of between 6 and 8 may be prepared and used.

What is more, it has been surprisingly found that the ability of the anolyte solutions of the present invention to kill or destroy viruses is not only provided when the solutions have been produced with high residual hypochlorite content of greater than 50ppm. Rather, it has now been recognised that electrochemically activated water with a much lower chlorine content also exhibits the disinfectant properties, despite containing only little, if any, hypochlorite when generated using non-ceramic membranes.

The present invention is described in greater detail below, with reference to the following figures.

Figure 1 is a graph indicating the ORP (oxidation-reduction potential) of electrochemically activated water in mV versus the electrical current used in its production for an ECA 2000 electrochemically activated water generator (available from Forum Bioscience Holding, Redhill, Surrey, UK).

Figure 2 is a graph indicating the increase in chlorine in ppm with current for an ECA 2000 machine.

Figure 3 is a graph showing the variation of pH with current for an ECA 2000 machine.

Figures 4 and 5 show the dissolved oxygen concentration in samples of electrochemically activated water produced using an ECA 2000 machine over time.

Figure 6 shows a block diagram of an ECA 2000 machine.

Figure 7 is a graph showing the results of the electrochemically activated water cytotoxicity for a host cell line.

It is generally believed that the disinfectant activity of electrochemically activated water is a function of the hypochlorite concentration of the water. As the concentration of hypochlorite increases, so does the ORP and the disinfectant activity of the electrochemically activated water. As a result, it is generally considered that the higher the hypochlorite content of the electrochemically activated water, the greater its disinfectant activity. Indeed, it is generally thought that the electrochemically activated water must include at least 50 to 200ppm hypochlorite in order for it to exhibit acceptable disinfectant activity. Where electrochemically activated water has been used in the past as a disinfectant, the hypochlorite concentration has been greater than 50ppm and is usually much greater, sometimes as high as 650ppm.

Therefore, it was previously believed that electrochemically activated water having a chlorine content of less than 8ppm would not exhibit any disinfectant activity and would not be effective against micro-organisms and certainly not effective against resilient viruses.

However, as can be seen from Table 1 below, the ORP of solutions produced on an ECA 2000 machine starts to plateau at 3-4 amps and from 6-8 amps onwards the figure remains almost constant whereas the chlorine content climbs rapidly. If the ORP is a measure of the disinfectant activity then the graphs of Figures 1, 2 and 3 and Table 1 indicate that electrochemically activated water solutions, produced on an ECA 2000 machine, should be effective disinfectants at total chlorine levels of less than 35ppm.

Table 1

This hypothesis has now been demonstrated. That is, when the electrochemically activated water produced using the above rationale to minimise the chlorine content is diluted with mains water to a chlorine content of less than 8ppm it still exhibits disinfectant activity.

The disinfectant activity of this water is not a result of the hypochlorite in the water, but rather it appears to be due to the presence of activated chlorine and oxygen species. This activity is thought to be "masked" in conventional electrochemically activated water by the levels of and the activity of the hypochlorite. It has now been found that the activated chlorine and oxygen species produce a redox potential high enough for the water to have disinfectant activity (greater than +90OmV), whilst having a minimal chlorine content.

It is thought that the active chlorine and oxygen species in the electrochemically activated water are highly reactive, reacting with many organic compounds including olefins, dienes, sulphides, aromatics, hetero-aromatics, terpenes, steroids, fatty acids, flavones, tetracyclines, vitamins, amino acids, proteins, nucleic acids, blood and bile pigments.

The activity of this minimal chlorine electrochemically activated water against bacteria, viruses and fungi species has previously been demonstrated by the inventors. It has been found that electrochemically activated water with a chlorine content of less than 8ppm can be used to kill or destroy viruses, including hardy or resilient viruses such as avian viruses.

It may be desirable to have electrochemically activated water with a low chlorine content for a number of reasons. The high chlorine content of conventional electrochemically activated water means that although the water is not harmful when it comes into contact with the animal subjects, such as birds, it should not be ingested. Where the chlorine content of the electrochemically activated water is between 8 and 120ppm, a bird drinking the water will increase its water intake, as this electrochemically activated water will make it more thirsty. This increased water intake leads to wet litter which is undesirable as it is more likely to stick to the birds and cause burning. What is more, the wet litter is more likely to provide a good breeding ground for microorganisms, which promotes the spread of disease. Where the chlorine content exceeds 120ppm, it will kill birds if ingested. Thus, in another embodiment of the present invention, the solution has a low chlorine content, preferably a chlorine content of less than 8ppm. In some embodiments, the chlorine content may be up to 10, 8, 6, 5 or 4ppm, and may be at least 0.1, 0.2, 0.3, 0.4 or 0.5ppm.

The solutions used in the present invention preferably have a redox potential of at least +90OmV, and preferably of at least +100OmV or +105OmV. Preferably, the solutions used in the present invention include active chlorine and oxygen species and the concentration of the active oxygen is preferably between 11 and 20mg/l.

In a preferred embodiment, the level of chlorine dioxide (ClO₂) present in the solutions is less than lOppm, preferably less than 5ppm, and most preferably less than lppm. For example, the level of chlorine dioxide is no more than 9, 8, 7, 6, 5, 4, 3, 2 or lppm.

In a further preferred embodiment, the level of hypochlorite in the solutions is less than lOppm, preferably less than 5ppm, most preferably less than lppm. For example, the level of hypochlorite is no more than 9, 8, 7, 6, 5, 4, 3, 2 or lppm.

In a yet further embodiment, the virucidal solutions according to the present invention contain no ozone or substantially no ozone (where substantially no ozone means that the ozone is present in an amount so small that it has no discernible effect on virucidal activity). Alternatively or additionally, the virucidal solutions according to the present invention contain no radicals or substantially no radicals (where substantially no radicals means that they are present in an amount so small that they have no discernible effect on virucidal activity). It is important to note that both ozone and radicals are harmful to living organisms and so their inclusion in the solutions according to the present invention is undesirable, especially where the solutions are to come into contact with the skin of animal subjects, or where they are to be ingested by the subjects.

In another embodiment of the invention, the solutions have a pH which is adjusted to a desired value or range. The anolyte of the electrochemically activated water may have a pH of below 4. However, in many applications of the solutions, the pH is preferably higher than this. For example, in certain preferred embodiments, the pH of the solution is between 3 and 8, or between 4 and 7. It is also desirable for the pH to be kept at a constant pH, with variations of 0.5 of a unit or less, and preferably of 0.1 of a unit or less. Compositions which have a stable pH value of 3- 8 or 4-7 are particularly useful in the present invention, as they are advantageous for use in physiological systems, as proposed herein. Solutions having a pH of 2.85-3.5 are also particularly useful.

It is important to note that the solutions according to the present invention do not depend upon their acidic pH for their virucidal activity. In the prior art, solutions are disclosed which have a very acidic pH, for example 2.15 or less. These solutions rely upon their acidity to have a virucidal effect. Indeed, very large volumes of these known solutions are required in order to achieve the desired virucidal effect and this is indicative of an acid kill. What is more, these known solutions also have a low chlorine content, often less than 7.15ppm, and this chlorine content would appear to be too low to be responsible for the alleged virucidal effect.

The compositions disclosed herein have the advantage that their pH may be adjusted without significant loss of disinfectant and biocidal activity. In one embodiment, the pH of the anolyte solution, optionally with a minimal chlorine content, is adjusted by adding an amount of the catholyte of the electrochemically activated water sufficient to produce the desire pH value. This combination of the anolyte and the catholyte of electrochemically activated water retains the disinfectant and biocidal activity of the anolyte. The pH of the solutions according to the present invention may also be adjusted by the inclusion of buffers.

The electrochemically activated water used in the present invention can be produced in a number of ways, including those well known in the art.

Conventional electrolysis processes operate at voltages and/or power densities at which HClO^" and/or ClO₂ are produced. Indeed, it is these species in conventional electrochemically activated water which is traditionally relied upon for the biocidal activity.

As discussed above, electrochemically activated water with a minimal chlorine content is preferably used in the present invention. The chlorine content of the electrochemically activated water is affected by the amount of sodium chloride in the water prior to the electrochemical activation process, the current used to electrolyse the solution (the size of the current and the length of time the water is exposed to it) and the rate of flow of the salt solution through the different chambers of the electrolytic cell.

Compositions for use in the present invention may be produced by running the electrolysis process at a voltage and/or power density which is higher than that at which O₂ and Cl₂ are produced, but which is lower than that at which HClO^" and ClO₂ are produced. Preferably, the voltage adjacent to the electrode face is between 1.35 and 1.63V, more preferably between 1.4 and 1.5V and most preferably between 1.42 and 1.47V. It is important to note that these values do not refer to the voltage across the whole system. The chemical reactions are taking place at the face of the electrodes and so it is the voltage here that is significant.

A person skilled in the technical field of the present invention would have no difficulty adjusting the parameters of the electrolysis process in order to achieve the voltage and/or power density at the electrodes required to produce the compositions of the present invention, once he is aware of the required voltage and/or power density at the electrodes relevant to the membrane in use.

The ratio of the anolytexatholyte flow can be from 5:95 to 95:5 with the optimum flow ratio being be 90:10, whilst the current should be between 1 and 20 amps with the preferred range being 6 to 12 amps.

The variation of the chlorine concentration in ppm with water flow at constant current is shown in Table 2.

Table 2

In addition, the application of a low current during the electrochemical activation process also results in a solution with a reduced chlorine content. This is likely to be due to the fact that the lower magnetic field has a lesser ability to attract the negative chloride ions during the brief period that any specific chloride ion is within the electromagnetic field. As a result there is a far greater probability that the electrolysis will involve the water molecules will which will lie alongside the electrodes and produce oxygen based species than chlorine based species.

That the electrochemically activated water produced by the ECA 2000 series machines has an exceptionally high concentration of oxygen is shown in Figures 4 and 5. For comparison, the normal saturated concentration for oxygen in water is approximately 8.3mg/litre. In Figures 4 and 5, "Hydroactive" is the electrochemically activated water anolyte stream from an ECA 2000 machine and "Cell Waste Stream" is the electrochemically activated water catholyte stream from an ECA 2000 machine.

Regardless of the method used to produce the electrochemically activated water, the water has the beneficial properties utilised by the present invention.

Where the chlorine content of the electrochemically activated water is to be limited by using a low sodium chloride concentration in the water fed into the apparatus carrying out the electrochemical activation, the water fed into the apparatus and electrochemically activated preferably has a sodium chloride concentration of between 1000 and 5000ρpm, and preferably between 2000 and 3000ppm chloride ion concentration.

The conventional electrochemical activation processes apply a current of approximately 10-20 amps to the sodium chloride solution. In order to reduce the chlorine content of the electrochemically activated water, it is recommended that the current be reduced to between 1 and 10 amps with the preferred range being between 5 and 9 amps. The ECA 2000 machine may be preferably used to generate the electrochemically activated water. This system significantly reduces the formation of undesirable trihalomethane and chloroamine by-products.

The main principle of the ECA 2000 machines is an electrochemical synthesis of gaseous mixture of oxidants from a dilute solution of sodium chloride under pressure in diaphragm modular electrochemical elements, each of which is a separate electrochemical rector

As shown in Figure 6, sodium chloride solution is delivered into the electrochemical reactor. The process results in a partial division of sodium chloride solution into activated chlorine and oxygen based oxidants in the anode chamber, and hydrogen and sodium hydroxide formed in the cathode chamber. The oxidants produced in the anode chamber together with the remaining un-electrolysed saline solution are collected (preferably in a darkened acid resistant air tight chemical drum). The electrochemically activated water is then delivered by the injection pump into the part of water to be processed resulting in a dilute solution of oxidants in the final treated water. Hydrogen is generated in the cathode chambers of electrochemical elements and is vented to air through the catholyte discharge tube.

Previous equipment used a ceramic membrane to separate the solutions resulting from close contact with the electrodes. The applicant has developed the "flat" core which consists of two half cells working at +15 to 0 volts and 0 to -15V, working back to back where the water to be processed flows across the electrodes and the electrode pairs (and thus the solutions) are separated by an ion exchange membrane. It is this core which can preferably be used to produce the electrochemically activated water which is described herein. It is non-ceramic.

The apparatus preferably has a feedback mechanism to maintain a constant current, which is dependent upon the ionic strength of the water, which is in turn dependent upon the salt content and the nature of the water used.

Provided that the storage conditions are suitable, the electrochemically activated water produced according to the present invention can be stored for periods of weeks or months. In a preferred embodiment, the electrochemically activated water can be stored for more than 72 hours.

As discussed above, the disinfectant activity of the minimal chlorine electrochemically activated water is due to active chlorine and oxygen species in the water. It is clear that if gas is allowed to escape from the water during its storage or use, the activity will be diminished or lost. Therefore, it is necessary to ensure that the water is stored in a closed system which will keep the loss of gas from the water to a minimum. Any stirring or other agitation of the water should also be avoided, as this will encourage gas to escape from the water.

When stored in sealed bottles with minimal head space, the dissolved active oxygen species will remain in solution above the saturated oxygen in water concentration of approx 8.3mgs/litre for a considerable period of time, thus maintaining the activity of the product (see Figures 4 and 5).

The electrochemically activated water solutions of the present invention are ecologically friendly and present no problems for the environment. This is shown by the concentrations of chlorite (ClO₂), chlorate (ClO₃) and perchlorate (ClO₄) measured in neat, freshly prepared solution, recorded in Table 3.

Table 3 - Concentrations of chlorine species found in neat electrochemically activated water as produced in ECA 2000 machine

In one embodiment of the present invention, the solutions comprising electrochemically activated water are used as one might use a conventional disinfectant. The solutions may be sprayed on any surface which might be contaminated. Thus, the walls, floor and ceiling of animal housing may be sprayed, as well as other surfaces, such as feeding troughs and the like. A fine mist of the solution is sufficient for the virucidal effect. Also, vehicles used to transport animals may also be sprayed, as well as any other buildings, vehicles, etc. with which the animals, excrement and the like could come into contact. The solutions may be used liberally, due to their non-hazardous nature. This is also of benefit to the people working with the animals or in the vicinity of the animals. The electrochemically activated water is not harmful if it comes into contact with their clothes or skin and it does not produce harmful or unpleasant fumes. The solutions may even be used to wash the workers' clothes, shoes or even hands, without causing harm.

Where they are used to clean surfaces as described above, the solutions according to the present invention may have a chlorine content that is greater than about 8ppm. The electrochemically activated water may be produced using known or conventional processes and apparatus. Alternatively, the solutions may have a relatively low chlorine content, that is no higher than about 8ppm, produced using the methods and apparatus discussed above.

In a further embodiment of the invention, virucidal solutions with a low chlorine content may be used in further useful ways. Animals, including birds, can be washed or directly sprayed with the solutions. As the solutions contain little or no hypochlorite or hydrogen peroxide, such direct application is safe. In another embodiment of the invention, the animals' food is treated with the low-chlorine solutions; for example, it may be sprayed with the solutions before being fed to the animals. This results in the low-chlorine solutions being ingested. Finally, in yet another embodiment, an effective dose of the solutions may be added to the animals' normal drinking water. At effective concentrations, the low-chlorine solutions may be safely ingested and this type of use is especially useful for birds.

In yet another embodiment of the present invention, the electrochemically activated water is used in conjunction with one or more antibiotic agent or drug. As indicated in the experimental results discussed below, the electrochemically activated water does not interfere with the activity of antibiotics such as lincospectin or amoxycillin and so it is envisaged that the water may be used in conjunction with such antibiotics. This could involve simultaneous, sequential or separate administration of the electrochemically activated water and one or more antibiotic agent or drug. In one embodiment, the antibiotic may be added to the solution comprising electrochemically activated water. The amount of antibiotic used in conjunction with the electrochemically activated water may represent the conventional dose of said antibiotic. Alternatively, the dose may be less than the dose required when the antibiotic is administered on its own, that is, without the electrochemically activated water of the present invention.

According to a second aspect of the present invention, methods of killing or destroying viruses are provided, the methods comprising exposing viruses to the solutions comprising electrochemically activated water, including solutions according to the first aspect of the invention and as described herein.

According to a third aspect of the present invention, solutions comprising electrochemically activated water, including solutions according to the first aspect of the invention and as described herein, are provided for use in the treatment or prevention of viral infections. Preferably, the solutions are to be administered to a subject prior to, during or after exposure to a virus. The subjects are animals such as birds and mammals, including humans.

According to a fourth aspect of the present invention, there is provided the use of solutions comprising electrochemically activated water, including solutions according to the first aspect of the invention and as described herein, in the manufacture of a medicament for treating or preventing viral infection.

Details of the studies conducted into the efficacy of the solutions comprising electrochemically activated water against viruses and its interaction with known antibiotics are set out below.

Experiments - Influenza A Virus

Experimental Procedure The study was performed to evaluate the virucidal efficacy of electrochemically activated water against Influenza A virus. Influenza A virus subtype (ATCC-VR- 1520) was chosen as a cell culture adapted prototype strain representative of Avian Influenza A virus subtype HlNl for virucidal efficacy testing purposes. The electrochemically activated water (also referred to in the discussion of this study as the test substance) was generated using an ECA 2000 machine (available from Forum Bioscience Holding, Redhill, Surrey, UK). The machine was set 9 amps (± 0.1 amp) and was fed with an aqueous salt solution comprising 2400 to 3000ppm NaCl.

The study was conducted using standard test procedures for determination of the efficacy of antimicrobial agents against viruses in suspension (American Standard Test Method, ASTM E 1052-96). The test procedure incorporated three elements; evaluation of cytotoxicity of the test substance for the host cell line, evaluation of test substance interference in the assay system, and quantitative suspension tests in the presence and absence of organic soil. Influen2a A virus was assayed in cell culture and quantified by calculation of 50% Tissue Culture Infectious Dose (TCID₅₀). Virucidal efficacy of the test substance was assessed by calculation of log₁₀ reduction in cytopathic effect by subtraction of TCID₅₀ values obtained from treated samples and positive (untreated) controls.

Influenza A virus VR-1520 (sourced from LGC Promochem, Queens Road, Teddington, Middlesex, TWl 1 OLY, England) was received in the form of a lyophihsed preparation. Influenza A virus cell culture fluid lysate was prepared by passage of ATCC-VR-1520 twice in Madin Darby Canine Kidney (MDCK) cells at 33°C ± 2°C. Cell culture medium (virus growth medium) was harvested when cytopathic effect (CPE) was maximal (approximately 48 hours post infection), aliquoted and stored at -75°C.

The electrochemically activated water was produced on the day of analysis using a machine as described above. Following priming of the machine for several minutes to achieve illumination of the green 'work' LED display. The electrochemically activated water was produced for approximately 30 minutes and run to waste before collection of approximately lOOmls of the water into a sterile container.

Five lots of the electrochemically activated water were prepared for use in this study. Each lot was used on the same day as manufacture. Prior to testing, the test substance was stored at room temperature. Analysis of free chlorine content of the test substance was carried out using the Palintest®. Free chlorine with diethyl-p-phenylene diamine (DPD) in buffered solution to produce a pink colouration. The intensity of the colour is proportional to the free chlorine concentration and was measured using a comparator disc (CD/011/05). Three dilutions of test substance were prepared using distilled water, namely 1/10, 1/50 and 1/100.

lOmls of each dilution was added to a clean square test tube and one DPD tablet added. The tablet was broken up to dissolve using a plastic probe. The cell was placed into a comparator and the colour of the solution matched with the disc. Free chlorine concentration (mg/1), corresponding to the colour match was recorded for each dilution.

Cytotoxicity of the test substance for the detector cell line (MDCK cells), was assessed in triplicate samples. An initial 1/5 dilution of the test substance was prepared in cell culture dilution medium made up as follows. To 50OmIs of Modified Eagle Medium (MEM, Sigma M-5650), 5OmIs of Foetal Bovine Serum (FBS, Sigma F-2442), 5mls of antibiotic/antimycotic solution (10OX, Sigma A- 5955), 5mls of L-Glutamine (20OmM, Sigma G-7513) and 5mls of non-essential amino acids (NEAA, Sigma M-7145) were added using aseptic procedures and swirled to mix.

Further two fold dilutions of the test substance were prepared in cell culture medium from 1/10 to 1/160. lOOμl of each dilution was inoculated into 10 replicate wells of 96-well cell culture plate containing a confluent monolayer of MDCK cells. lOOμl of cell culture dilution medium was inoculated into appropriate wells (up to 8 wells/plate) as negative controls. Plates were placed in a moist container and incubated at 33°C ± 2°C. Plates were examined microscopically for signs of cytotoxicity after 48 hours incubation.

The potential for disinfectant to interfere with the ability of the virus to infect the detector cell line used to assay the virus was assessed. Medium from 3 x 96-well cell culture plates containing confluent monolayers of MDCK cells was discarded. Cell monolayers were then washed by addition of lOOμl of PBS to each well and discarded. 1/10 and 1/00 (v/v) dilutions of the electrochemically activated water were prepared in virus growth medium and lOOμl of each dilution was added to (3 x 12) replicate wells (per dilution) of a 96-well cell culture plate containing confluent monolayers of MDCK cells. lOOμl of untreated virus growth medium was added to 3 x 12 replicate wells as untreated controls.

Cells treated with the electrochemically activated water and untreated control cells were challenged with Influenza A virus suspension (10^~6 dilutions). lOOμl of each virus dilution was added to 10 replicate wells containing untreated and treated (with the electrochemically activated water) virus growth medium. Plates were placed in a moist container and incubated at 33 + 2°C for 7 days. Following incubation, cells were examined microscopically for signs of virus induced CPE. Individual wells were scored as positive or negative for virus CPE.

A quantitative suspension test for the assessment of virucidal efficacy of the electrochemically activated water against Influenza A virus was set up in 2 phases. In the first phase, a range of dilutions of electrochemically activated water was tested for virucidal efficacy in the absence of organic load. In the second phase of the study dilutions showing virucidal efficacy in phase 1 were tested for efficacy in the presence and absence of organic load (no added protein, 0.3% Foetal Bovine Serum and 1% Foetal Bovine Serum). Test samples and controls (excluding virus) were prepared as shown in Tables 4 and 5.

Table 4 - Quantitative suspension test: Preparation of samples and controls (Study Phase 1)

Table 5 - Quantitative suspension test: Preparation of samples and controls (Study Phase 2)

Foetal Bovine serum (30% solution) Test substance diluted in 1/9 in de-ionised water to produce a final dilution of /10 in the test samples Virus suspension was added to appropriate samples and vortex mixed. 200μl aliquots of test sample and positive and negative controls were taken at specified timed intervals following vortex mixing and immediately diluted in 1800μl of virus growth medium (ICT¹ dilution). Further tenfold dilutions of test samples and positive controls were prepared from 10^"2 to 10^"8 in virus growth medium (10^~2 and 10^"3 dilutions of negative control samples were prepared in virus growth medium). Medium from 96-well cell culture plates containing confluent monolayers of MDCK cells was discarded and cell monolayers washed with PBS (1 OOμl/well) . lOOμl of virus growth medium was added to each well.

Dilutions of test samples, positive and negative controls were added to appropriate wells of a 96-well cell culture plate containing confluent monolayers of MDCK cells (10 replicate wells per dilution). lOOμl of virus growth medium was added to wells designated for cell controls. Plates were placed in a moist container and incubated at 33°C. After 7 days incubation, plates were examined for virus induced CPE and individual wells scored positive or negative for CPE.

Virus inactivation as a result of the action of the disinfectant was quantified by subtraction of the virus titres obtained from test samples and the positive controls. Standard errors and 95% confidence interval were calculated for each titration. The virus titre was calculated as follows. For virus titration, a tenfold dilution series was prepared and each dilution inoculated into 8 replicate wells containing detector cells. At the end of the culture period, each well is scored as positive or negative and each set of dilution is evaluated for the proportion of positive and negative cultures. The titre is expressed at 50% Tissue Culture Infectious Dose (TCID₅₀). The TCID₅₀ is a standard measure of virus titre and is defined as the virus dose capable of infecting one half of the inoculated cultures. The TCID₅₀ value is calculated using the method of Karber:

-log₁₀ TCID₅₀ = log₁₀ starting dilution- (Sp-0.5) log₁₀ dilution factor.

Thus for the data presented in Table 7 below, if 8 replicate wells were inoculated with lOOμl volumes;

-1Og₁₀ TCID₅₀ = 4 - (1.75 - 0.5).! = -4 - 1.25 = -5.25 Le. TCID₅₀ occurs at a dilution of 10 ^{5 25}

Table 6 - Example end point dilution assay data

τ P> - — proportion of wells showing positive for cytopathic effect.

The standard error in this type of assay depends on the number of replicate wells used per dilution of the sample i.e. the more replicates used, the more accurate the determination of virus titre.

The standard error of the estimate of virus titre (S TCID)₅₀ is calculated as follows:

S lOg₁₀ TCID₅₀ = ΔV ∑p(l-p)/n-l

Where Δ = the constant interval between log dilutions p = the proportion of infected wells n = the number of wells tested

Thus for the data in Table 7:

S lOg₁₀ TCID₅₀ = W 0.343/7 = 0.221 and the standard error at 95% probability level is calculated as: 1.96 x 0.221 = 0.434 logs

Therefore, the titre of the sample from the data shown in Table 4 (per lOOμl) can be expressed at 5.25+ 0.434 logs, giving a minimum titre of 4.82 and a maximum titre of 5.68.

To express virus titre per ml, values are adjusted to 6.25 ± 0.434 logs, giving a minimum titre of 5.82 and a maximum titre of 6.68.

Results and Discussion Results of available chlorine analysis on the test substance using the Palintest are shown in Table 7. Table 7 - Available chlorine results (Palintest)

Test substance Date / Available Chlorine (mg/L) dilution

14.05.2003 15.05.2003 09.09 2003 10. 09 .2003 17. 09 .2003

1 /10 v/v 3.0 3.0 3. 5 3 5 3 0

1/50 v/v 0.5 1.0 1 5 1 5 >1 o, <1.5

1/100 v/v <0.5 >0.5, <1.0 1 0 1 0 >0 .5, <1.0

Variation in the available chlorine levels in the electrochemically activated water was noted between days and between dilutions. Levels of available chlorine (mg/L) varied from 30-35mg/L for the 1/10 dilution from 25 to 75mg/L for the 1/50 dilution and from <50-100 mg/L for the 1 /100 dilution. The Palintest is a broad indicator of available chorine and had previously been noted that variations in result were obtained from different dilutions. The subjective nature of the Palintest may contribute to the slight observed variation in levels of available chlorine level between test substance generated on different days.

Results of the electrochemically activated water cytotoxicity for the host cell line are shown in Table 8.

Table 8 - Cytotoxicity Test

Electrochemically activated water was not cytotoxic for the host cell line over the dilution range tested (1/5 to 1/160). Cytotoxicity was therefore not a factor in quantification of virucidal activity of the test substance.

Results of the effect of electrochemically activated water on the ability of the virus to infect the host cells are shown in Table 9.

Table 9 - Virus interference test

Host cells (MDCK) treated with low dilutions (10^'1 and 10^~2) of test substance were able to support the growth of test virus at high dilution (10 ⁵ and 10^"6). The test substance did not compromise the ability of the host cell line to support the growth of virus was therefore not a factor in quantification of virucidal activity.

Virucidal efficacy of electrochemically activated water was quantified by suspension test. Phase 1 of this study assessed a range of dilutions of electrochemically activated water (undiluted, 1/10, 1/100 and 1/250) for virucidal activity against Influenza A virus in the absence of organic load (see Tables 10 and 11).

Table 10 - Virucidal efficacy against Influenza A virus: Virus recovery

Table 11 - Virucidal efficacy against Influenza A virus: Reduction in virus infectivity

The electrochemically activated water (undiluted and diluted 1/10) showed significant virucidal activity achieving >51og₁₀ reduction in virus infectivity after contact times of 1 minute and 30 minutes respectively. Based on these results, electrochemically activated water undiluted and diluted 1/10 (v/v) were selected for testing in Phase 2 of the study in the absence and presence of organic load. Electrochemically activated water diluted 1/100 showed marginal virucidal activity (1.7 log₁₀ reduction in virus infectivity after a contact time of 30 minutes). Electrochemically activated water diluted 1/250 (v/v) showed insignificant virucidal (<0.51og₁₀ after 30 minutes).

Phase 2 of this study assessed electrochemically activated water (undiluted and 1/10) in the absence and presence of organic load (0.3% FBS and 1% FBS). The electrochemically activated water (undiluted) showed efficient virucidal activity reducing virus titre by greater than 5 log₁₀ after all contact times tested (1, 5, 10 and 30 minutes) in the absence and presence of organic load (see Tables 13 and 14).

Table 12 - Electrochemically activated water (undiluted): Virucidal efficiency against Influenza A virus: Virus recovery

Table 13 - Electrochemically activated water (ECA) (undiluted): Virucidal efficiency against Influenza A virus: Reduction in virus infectivity

Sample Reduction in Infectivity (-log_in TCID,₀)7Contact time

1 min 5 tnins 10 mins 30 mins

ECA water, undiluted (no protein) > 5 37 > 5.33 ≥ 5.4 > 5 .1

ECA water, undiluted (0.3% FBS) > 5 37 > 5.33 > 5.4 > 5 .1

ECA water, undiluted (1% FBS) > 5 37 > 5.33 ≥ 5.4 > 5 .1

¹ Mean of triplicate samples

The electrochemically activated water (diluted 1/100) showed virucidal efficacy dependent on time and the absence and presence of organic load. Greater than 51og₁₀ reduction in virus titre was achieved after contact times of 5 minutes in the absence of organic load. The presence of organic load delayed virucidal effect with >51og₁₀ reduction in virus infectivity achieved after contact times of 10 and 30 minutes in the presence of 0.3% FBS and 1% FBS respectively (see Tables 15 and 16).

Table 14 - Electrochemically activated water (1/10 v/v): Virucidal efficiency against Influenza A virus: Virus recovery

Table 15 - Electro chemically activated water (ECA) (1:10 v/v): Virucidal efficiency against Influenza A virus: Reduction in virus infectivity

¹ Mean of triplicate samples

Statistical data gathered from the above discussed studies are set out in Tables 16, 17 and 18.

Table 16 - Electro chemically activated water (ECA): Virucidal efficacy against Influenza A virus: Virus recovery (Study Phase 1)

Table 17 - Electrochemically activated water (undiluted): Virucidal efficacy against Influenza A virus: Virus recovery (Study Phase 2)

Table 18 - Electrochemically activated water (1/10 dilution): Virucidal efficacy against Influenza A virus: Virus recovery (Study Phase 2)

Conclusions

The tested electrochemically activated water was not cytotoxic to the host cell line at dilution tested. Furthermore, it did not interfere with the ability of the host cell line to support the growth of the test virus.

Electrochemically activated water (undiluted) showed efficient virucidal activity reducing virus titre by greater than 51og₁₀ after all contact times tested (1, 5, 10 and 30 minutes) in the absence and presence of organic load.

Electrochemically activated water (diluted 1/10) showed virucidal efficacy dependant on time and the absence and presence of organic load. Greater than 51og_]0 reduction in virus titre was achieved after contact times of 5 minutes in the absence of organic load. The presence of organic load delayed virucidal effect, with >51og₁₀ reduction in virus infectivity achieved after contact times of 10 minutes and 30 minutes in the presence of 0.3% FBS and 1% FBS respectively.

Experiments - Infections Bronchitis Virus

Experimental Procedure

This study was performed to evaluate the virucidal efficacy of the electrochemically activated water against Infectious Bronchitis Virus (IBV). The Infectious Bronchitis Virus used in the study was the Beaudette strain ATCC-VR-22. The host cell line used was Vero cells (African green monkey kidney cells), ECACC number 84113001.

The test procedure incorporated three elements, namely the evaluation of the cytotoxicity of the electrochemically activated water (also referred to herein as the test substance) for the host cell line, evaluation of the test substance interference with the assay system, and a quantitative suspension test for the determination of virucidal activity of the test substance against IBV. The IBV was assayed in cell culture (Veto cells) and quantified by calculation of 50% Tissue Culture Infectious Dose (TCID₅₀). Virucidal efficacy of the test substance was assessed by calculation of log₁₀ reduction in TCID₅₀ of the virus following treatment with the test substance and quantified by subtraction of TCID₅₀ values obtained from treated samples and positive (untreated) controls.

The IBV (sources from LGC Promochem, Queens Road, Teddington, Middlesex, TWl 1 OLY, England) was received as a lyophilised preparation. The virus was adapted to cell culture by repeated passage (7 passages) in Vero cells. After each passage, virus suspension (cell culture medium) was harvested when cytopathic effect was maximal (approximately 72 hours post infection) and stored at -75°C. Virus suspension harvested from the 7^th passage in Vero cells was concentrated by polyethylene glycol precipitation. The precipitate was centrifuged and the pellet resuspended in phosphate buffered saline and stored at -75°C.

The electrochemically activated water was produced on the day of analysis using a machine as described above. Following priming of the machine for several minutes to achieve illumination of the green 'work' LED display, the electrochemically activated water was produced for approximately 10 minutes and run to waste before collection of approximately 200ml of the water into a sterile container. The water was used on the same day as manufacture. Prior to testing, the water was stored at room temperature.

Analysis of free chlorine content of the test substance was carried out using the Palintest® at two dilutions, namely 1/10 and 1/50. The free chlorine concentration (mg/1) was measured, as discussed above.

Cytotoxicity of the test substance for the host cell line (Vero cells) was tested. An initial 1/5 dilution of the test substance was prepared in cell culture dilution medium made up as follows. To 500ml of Dulbecco's Modified Eagle Medium (DMEM, Sigma M-5671), 10ml of Foetal Bovine Serum (FBS, Sigma F-7524), 5ml of antibiotic/antimycotic solution (10OX, Sigma A-5955) and 5ml of L-Glutamine (20OmM, Sigma G-7513) were added using aseptic procedures and swirled to mix. Further four-fold dilutions of the test substance were prepared in dilution medium. lOOμl of each dilution was inoculated into 10 replicate wells of 96-well cell culture plate containing a confluent monolayer of Vero cells. lOOμl of dilution medium was inoculated into appropriate wells (up to 8 wells/plate) as negative controls. Plates were placed in a moist container and incubated at 37 ± 2°C.

Cell monolayers were examined microscopically for signs of cytotoxicity after 7 days incubation. Cell viability was tested using Methylthiatetra2olium (MTT) assay. MTT solution was added to monolayers and incubated at 37 + 2°C for 2 hours. The coloured product (formazan) was eluted using Dimethylsulphoxide (DMSO) and absorbance read at 540nm. Dilution showing 50% end point of cytotoxic effect was calculated by non-linear regression using binary data.

The potential for the test substance to interfere with the ability of the host cell line to support the growth of the virus was tested in an interference test. Medium from two 96-well cell culture plates containing confluent monolayers of Vero cells was discarded. Cell monolayers were then washed by addition of lOOμl of PBS to each well and discarded. Dilutions of the test substance were prepared in dilution medium and lOOμl of each dilution was added to 11 replicate wells (per dilution) of a 96-well cell culture plate containing confluent monolayers of Vero cells. In a second (control) plate, lOOμl of dilution medium was added to each well.

Cells treated with the test substance and untreated control cells were challenged with IBV suspension. lOOμl of each virus dilution was added to 10 replicate wells containing treated cells and untreated control cells. The plates were placed in a moist container and incubated at 37 ± 2°C for 7 days. Following incubation, cells were examined microscopically for signs of virus induced cytopathic effect (CPE). Individual wells were scored as positive or negative for virus CPE.

A quantitative suspension test was used to determine virucidal efficacy of the test substance against IBV. Test samples and controls were prepared as shown in Table 19. Table 19 - Quantitative suspension test: Preparation of samples and controls

Dilutions of the product were prepared to produce a final dilution as specified, accounting for the volume of the virus suspensions in the test procedure.

Virus suspension was added to appropriate samples and vortex mixed. 500μl aliquots of test sample and positive and negative controls were taken at specified timed intervals following vortex mixing and immediately diluted in 1500μl of dilution medium, producing an initial 1/5 dilution. Further four-fold dilutions of test samples and positive controls were prepared from 1/2 to 1/327680 in dilution medium. For the negative controls, 1/20 and 1/80 dilutions were prepared in dilution medium. Dilutions of test samples, positive and negative controls were added to appropriate wells of a 96-well cell culture plate containing confluent monolayers of Vero cells (10 replicate wells per dilution). lOOμl of dilution medium was added to wells designated for cell controls. The plates were placed in a moist container and incubated at 37 + 2°C. After 7 days incubation, plates were examined for virus induced CPE and individual wells scored positive or negative for CPE.

TCID₅₀ values were calculated using the method of Karber. Virus inactivation as a result of the action of the disinfectant was quantified by subtraction of the virus titres obtained from test samples and the positive controls. Standard errors and 95% confidence interval were calculated for each titration. These calculations are all explained above.

Results and Discussion

Results of available chlorine analysis on the test substance using the Palintest® are shown in Table 20. Table 20 - Available chlorine results (Palintest)

Levels of available chlorine varied from 30mg/l for the 1/10 dilution to 75mg/l for the 1 /50 dilution.

Results of the electrochemically activated water cytotoxicity for the host cell line are shown in Figure 7 and in Table 21 below.

Table 21 - Cytotoxicity Test

Data taken from Figure 7, 50% end point of cytotoxic effect

Although the undiluted test substance showed cytotoxicity fro the host cell line, no cytotoxicity was observed when the solution was diluted from 1/5 to 1/20480. In quantitative suspension tests, the lowest dilution of the test substance inoculated onto host cells for virus recovery studies was 1/20 (demonstrated as non-cytotoxic). Cytotoxicity of the test substance for the host cell line was therefore not a factor in quantification of virucidal activity of the test substance.

The results of the interference test conducted to show the potential of the test substance to affect the ability of the host cell line to support the growth of the virus are shown in Table 22.

Table 22 - Virus interference test

TCID₅₀/ml

Untreated cells Treated cells

5.42 5.42

No difference was observed in virus recovered from cells treated with dilutions of the test substance (from 1/20 to 1/327680) in comparison to the untreated controls. TCID₅₀ values were the same from the two titrations, indicating that the test substance did not compromise the ability of the host cell line to support the growth of virus.

Virucidal activity of the test substance, diluted (1/10, final dilution) and undiluted, against IBV using a quantitative suspension test is shown in Tables 23 and 24 below.

Table 24 - Virucidal efficiency against IBV: Reduction in virus titre (Log_]0 CCID₅₀), mean of triplicate samples

Undiluted test substance showed efficient virucidal activity against IBV with no virus recovered after all contact times tested (1, 5, 10 and 30 minutes). Log₁₀ reduction in virus titre of ≥2.97 log₁₀ was achieved after a contact time of 1 minute and ≥3 log₁₀ after a contact time of 30 minutes, equivalent to ≥99.9% virus inactivation. Test substance diluted 1/10 was less efficient with some virus recovery from the lower dilutions. However, after a contact time of 30 minutes, >3 log₁₀ reduction in virus titire was observed, equivalent to ≥99.9% virus inactivation.

When tested undiluted, virus inactivation was demonstrated to assay baseline levels (no virus recovered). The relatively low titre of the IBV challenge virus may have prevented demonstration of greater log reduction in virus titre.

Conclusions

The tested electrochemically activated water was not cytotoxic to the host cell line at the dilutions used for virus recovery studies (≥ 1/20). Furthermore, it did not interfere with the ability of the host cell line to support the growth of the test virus.

Electrochemically activated water (undiluted) showed efficient virucidal activity reducing virus titre by greater than 2.88- 3.0 log₁₀ after all contact times tested (1, 5, 10 and 30 minutes).

Experiments - Effect of Electrochemically Active Water on Amoxycillin Experimental Procedure

This study was carried out to determine the effect of electrochemically activated water on the minimum inhibitory concentrations (MIC) and the minimum microbiocidal concentrations (MMC) of amoxycillin, when tested against the bacterial organism Salmonella abony (also referred to herein as the test organism).

The experiments were carried out using Salmonella (enteritidis) abony NCTC 6017

(from the National Collection of Type Cultures, London, England). The bacterial inoculum was prepared from a fresh working culture grown on Tryptone Soya Agar (TSA - Oxoid CMl 31) prepared by suspending 4Og of TSA in 1 litre of distilled water and sterilising by autoclaving at 121°C for 15 minutes.

The culture was incubated at 37 ± 2°C for approximately 24 hours. Immediately before the test, surface growth was harvested and directly suspended into sterile 0.1% Peptone saline (prepared by dissolving l.Og/litre bacteriological peptone (Oxoid L37) and 8.9g/litre sodium chloride in 1 litre of distilled water and sterilising by autoclaving at 121°C for 15 minutes) and standardised to give 35-40% light transmission at 520 nm on a Jenway 6105 Spectrophotometer - an appropriate yield of 10⁸ colony family units (cfu) per ml. The suspension was diluted further using Mueller Hinton Broth (MHB) to give approximately 10⁵ cfu/ml. The MHB was prepared by suspending 21g of MHB (Oxoid CM405) in 1 litre of distilled water and sterilising it by autoclaving at 121⁰C for 15 minutes. The final suspension was then used immediately as the inoculum.

The electrochemically activated water was generated immediately before use and was diluted with sterile distilled water to give 3ppm free chlorine. The chlorine content was measured using a Palintest® kit. The amoxycillin was formulated in sterile distilled water at 100 mg/ml. It was also formulated in the electrochemically activated water (3ppm chlorine) at 100 mg/ml. Sixteen halving dilutions of each formulation were prepared, using sterile distilled water and electrochemically activated water (3ppm chlorine) respectively. Dilutions were prepared in glass containers and were stored protected from light.

The MIC test was conducted approximately 1 hour, 6 hours and 24 hours after formulation of the amoxycillin, in order to determine whether its activity was affected in a time-dependent manner by the electrochemically activated water.

For each formulation of amoxycillin, the test organism was added to a single row of wells on a microtitre plate to give a final viable inoculum concentration of approximately 10⁴ cfu/ml. The amoxycillin was then added. The final test concentrations were prepared by transferring 20μl of the amoxycillin dilutions to the appropriate wells on the test plate, each well containing 180μl of inoculated MHB. These additions represented a 10-fold dilution of the amoxycillin. The final concentrations of the amoxycillin in the wells were, therefore, 10000, 5000, 2500, 1250, 625, 312.5, 156.25, 78.13, 39.06, 19.53, 9.77, 4.88, 2.44, 1.22, 0.61, 0.31 and 0.15 μg/ml. The highest three concentrations were insoluble in MHB.

Negative control wells to demonstrate growth of the test organism in the absence of amoxycillin were established. Sterility controls for both media and amoxycillin were also established. The electrochemically activated water (3ppm chlorine) was also added to wells containing the test organism, in order to assess its effect on the growth of the organism. The lid and base of each test plate were sealed together using masking tape and the plates secured to an automatic plate shaker and shaken for 2 minutes. The masking tape was then removed and the plates incubated at 37 ± 2°C for approximately 24 hours.

AU wells showing no growth of the test organism and also some wells showing growth were subcultured using a sterile 1 μl inoculating loop onto TSA and incubated at 37 ± 2°C for approximately 24 hours.

After incubation, the wells of each microtitre plate were examined for the presence of microbial growth (turbidity) and the end point (MIC) for the amoxycillin recorded as the lowest concentration that completely inhibited growth of the test organism. The lowest concentration of the amoxycillin showing no growth on subculture onto agar media was recorded as the MMC.

Results & Conclusion

The results of the tests to determine the MIC and MMC values of amoxycillin in the absence and presence of electrochemically activated water (3ppm chlorine) against Salmonella abony 1, 6 and 24 hours after formulation are shown in Table 25.

Table 25 - MIC and MMC values of amoxycillin tested against Salmonella abony

Amoxycillin demonstrated good antimicrobial activity towards Salmonella abony, both in the absence and presence of electrochemically activated water. The MMC values were found to be the same as the MIC values, demonstrating that amoxycillin showed bactericidal rather than bacteriostatic activity.

The wells treated with the electrochemically activated water showed that it did not affect the growth of the test organism. The growth control wells demonstrated adequate growth of the test organism, whereas the sterility control wells demonstrated that both the amoxycillin and the growth medium were sterile.

Although the MIC value doubled when amoxycillin was formulated in electrochemically activated water compared with water, it was considered that this increase probably fell within the range of variation expected for a test system of this nature and may not have been due to the deactivation of amoxycillin by the electrochemically activated water.

It was concluded that the electrochemically activated water did not substantially affect the activity of amoxycillin, when challenged with Salmonella abony.

Experiments - Efficacy of Electr ochemically Active Water and Lincospectin

Experimental Procedure

This study was carried out to determine the minimum inhibitory concentrations (MIC) and the minimum microbiocidal concentrations (MMC) of lincospectin and electrochemically activated water, when tested against three bacterial organisms, namely Escherichia coli, Salmonella abony and Campylobacter jejuni. The effect of the electrochemically activated water on the activity of lincospectin was also determined by pre-incubating the lincospectin in the presence of electrochemically activated water before testing the two substances together.

The experiments were carried out using Salmonella (enteritidis) abony NCTC 6017 and Campylobacter jejuni NCTC 11351, both from the National Collection of Type

Cultures, London, England, and Escherichia coli NCIMB 8545 from the National Collection of Industrial and Marine Bacteria, Aberdeen, Scotland.

The bacterial inocula were prepared from fresh working cultures grown on Mueller Hinton Agar (MHA) for Escherichia coli and Salmonella abony, and on Campylobacter Agar containing 5% lysed horse blood (CA) for Campylobacter jejuni. The MHA was prepared by suspending 38g of MHA (Oxoid CM337) in 1 litre of distilled water, bringing the suspension to the boil to dissolve completely and then sterilising by autoclaving at 121 °C for 15 minutes. The CA was prepared by suspending 14.8g of Campylobacter Agar in 380ml of distilled water, bringing the suspension to the boil to dissolve completely and then sterilising by autoclaving at 121°C for 15 minutes. This was then cooled at 50°C or below and 20ml of Lysed Horse Blood (Oxoid - SR48) aseptically added.

AU cultures were incubated at 37 ± 2°C for approximately 24 hours. Campylobacter jejuni was grown under microaerophilic conditions (Oxoid Campylobacter Gas Generating Kit - CampyGen CN035A and an Oxoid Anaerobe Jar). Surface growth was harvested and directly suspended into sterile 0.1% Peptone saline (discussed above) and standardised to give 30-40% light transmission at 520nm on a Jenway 6105 Spectrophotometer - an approximate yield of 10⁸ cfu/ml. These suspensions were further diluted using Mueller Hinton Broth (MHB) as discussed above, to give approximately 10⁵ cfu/ml. These final suspensions were then used immediately as inocula.

A preliminary test was conducted in order to determine MIC values for both lincospectin and electrochemically activated water, prior to a main test with the two test substances added together.

Lincospectin was formulated in sterile distilled water to give a concentration of 8 mg/ml. Dilutions from this concentration were prepared on a 96-well microtitre plate in serial 1:1 v/v steps by transferring lOOμl test substance solution into the adjacent well on the microtitre plate containing lOOμl sterile distilled water. This gave a range of test substance concentrations from 8mg/ml to 3.9μg/ml, that were equivalent to ten times the final test concentrations.

The electrochemically activated water was generated immediately before use and was diluted with sterile distilled water to give 3ppm free chlorine. The chlorine content was measured using a Palintest® kit and was found to be 35-40mg/ml using a 1/10 dilution and 150mg/ml using a 1/100 dilution.

The electrochemically activated water was then diluted on a separate dilution plate using sterile distilled water to give a range of halving concentrations from 100% v/v to 0.05% v/v.

The final test concentrations were prepared by transferring 20μl of each test substance dilution from the corresponding wells on the dilution plates to the appropriate wells on separate test plates. Each well already contained 160μl of MHB, to which 20μl of the test organism inoculum, prepared in MHB, had been added. One test organism was used per row on the plate at a final viable inoculum concentration of approximately 10⁴ cfu/ml. These additions represented a 10-fold dilution of the test substance concentrations such that the final test concentrations were in the range of 800-0.39μg/ml for lincospectin and 10-0.005% for the electrochemically activated water.

20μl of each test organism were inoculated into single wells containing only 160μl MHB, plus 20μl sterile distilled water, to act as positive growth controls. Wells containing 200μl MHB only were incubated as media sterility controls. Wells containing 180μl MHB plus 20μl of the test substance dilutions only were incubated as test substance sterility controls.

The lid and base of each test plate were sealed together using masking tape and the plates secured to an automatic plate shaker and shaken for 2 minutes. The plates were then incubated at 37 ± 2°C for approximately 24 hours.

Following visual assessment of the test plates for MIC concentrations, all wells showing no growth of test organisms were subcultured using a sterile lμl inoculating loop onto MHA (discussed above). Wells showing growth of the test organism were also included as a control to determine the time required to see visible growth. The plates were incubated as described above for growth of test organisms in order to determine MMC concentrations.

The main test was conducted using all three test organisms. Dilutions of lincospectin and electrochemically activated water in sterile water were prepared as previously described. In order to evaluate whether the electrochemically activated water affected the MIC of lincospectin, lincospectin was formulated at 8mg/ml in electrochemically activated water at concentrations of 100%, 10%, 1% and 0.1% v/v. These solutions were left for one hour at room temperature in order to allow time for any reaction between the lincospectin and the electrochemically activated water. After this time, the formulations of lincospectin were diluted with the same concentrations of electrochemically activated water that they had been dissolved in. This was performed on a dilution plate as previously described. Test plates were prepared as previously described. A separate test plate was prepared for each test organism. Rows therefore contained: lincospectin dissolved and diluted with electrochetnically activated water at 100%, 10%, 1% and 0.1% v/v; lincospectin dissolved and diluted with sterile distilled water; a growth control; and a media sterility control. Two separate plates were established in order to act as sterility controls for all test substance dilutions.

The plates were sealed, shaken and incubated as previously described, except the plate containing Campylobacter jejuni and its sterility control plate were incubated under microaerophilic conditions for approximately 72 hours at 37 ± 2°C.

MIC and MMC concentrations were assessed as previously described. For

Campylobacter jejuni the wells were subcultured onto CA plus 5% lysed horse blood plates and incubated under microaerophilic conditions for approximately 48 hours at 37 ± 2°C.

Results & Conclusion Results of the tests to determine the MIC and MMC values are shown in Table 26.

Table 26 - MIC and MMC values from preliminary test

It was not possible to determine the MMC using the range of concentrations tested as light growth was seen on plates subcultured from wells treated with 50 to 800μg/ml. This indicated that lincospectin was bacteriostatic rather than bacteriocidal at these concentrations.

The electrochemically activated water did not inhibit growth at any of the concentrations tested, despite the fact that it was expected, by its nature, to exhibit antimicrobial activity. One possible explanation is that the electrochemically activated water was deactivated by the organic load present in the broth. Therefore, this type of study may not be suitable for determining the antimicrobial activity for substances of this nature. The results of the main test are shown in Tables 27 and 28 below.

Table 27 - MIC values (μg/ml) for lincospectin treated with a range of concentrations of electrochemically activated water

It can be seen that the electrochemically activated water did not affect the activity. It was not possible to determine MMC values for Escherichia coli and Salmonella abony as light growth was seen on plates from all wells treated with the MIC and above. Therefore, a bacteriostatic rather than a bacteriocidal effect was seen at these concentrations. However, for Campylobacter jejuni the MMC values suggest a bacteriocidal effect.

It was concluded that the electrochemically activated water did not substantially affect the activity of lincospectin, when challenged with the three bacterial organisms tested. It was not possible to determine the MIC or MMC of the electrochemically activated water in this study.

The results of the discussed experiments show that the electrochemically activated water is capable of killing various viruses and these results can be interpreted as indicating that the water will also be effective in killing other types of avian virus, such as those mentioned specifically herein.

Claims

1. Use of a solution comprising electrochemically activated water to kill or destroy a virus.

2. A use as claimed in claim 1, wherein the solution comprises the anolyte solution of electrochemically activated water.

3. A use as claimed in claim 2, wherein the solution further comprises the catholyte solution of electrochemically activated water.

4. A use as claimed in any one of the preceding claims, wherein the virus is an avian virus.

5. A use as claimed in claim 4, wherein the virus is a virus causing Avian

Influenza, Infective Bronchitis, Gumboro Disease, Chick Anaemia, Merek's Disease or Newcastle Disease.

6. A use as claimed in any one of claims 1 to 3, wherein the virus is a virus causing Foot and Mouth Disease, Swine Vesicular Disease or Tuberculosis.

7. A use as claimed in any one of the preceding claims, wherein the solution has a redox potential of at least +90OmV.

8. A use as claimed in any one of the preceding claims, wherein the solution has a chlorine content of less than 8ppm.

9. A use as claimed in any one of the preceding claims, wherein the solution includes an active oxygen species in an amount between 13 and 20mg/l.

10. A use as claimed in any one of the preceding claims, wherein the solution further comprises one or more antibiotic agent or drug.

11. A use as claimed in claim 10, wherein the antibiotic agent is lincospectin or amoxycillin.

12. A method of killing or destroying a virus comprising exposing the virus to a solution comprising electrochemically activated water.

13. A method as claimed in claim 12, wherein the solution comprises the anolyte solution of electrochemically activated water.

14. A method as claimed in claim 13, wherein the solution further comprises the catholyte solution of electrochemically activated water.

15. A method as claimed in any one of claims 12 to 14, wherein the virus is an avian virus, such as a virus causing Avian Influenza, Infective Bronchitis, Gumboro Disease, Chick Anaemia, Merek's Disease or Newcastle Disease.

16. A method as claimed in any one of claims 12 to 14, wherein the virus is a virus causing Foot and Mouth Disease, Swine Vesicular Disease or Tuberculosis.

17. A method as claimed in any one of claims 12 to 16, wherein the solution has a redox potential of at least +90OmV.

18. A method as claimed in any one of claims 12 to 17, wherein the solution has a chlorine content of less than 8ppm.

19. A method as claimed in any one of claims 12 to 18, wherein the solution includes an active oxygen species in an amount between 13 and 20mg/l.

20. A method as claimed in any one of claims 12 to 19, wherein the method further comprises exposing the virus to one or more antibiotic agent or drug.

21. A method as claimed in claim 20, wherein the solution comprising the electrochemically activated water further comprises one or more antibiotic agent or drug.

22. A method as claimed in claim 20 or 21, wherein the antibiotic agent is lincospectin or amoxycillin.

23. A solution comprising electrochemically activated water, for use in the treatment or prevention of a viral infection, wherein the solution is administered to a subject prior to, during or after exposure to the virus.

24. A solution as claimed in claim 23, wherein the solution has a redox potential of at least +90OmV.

25. A solution as claimed in any one of claims 23 or 24, wherein the solution has a chlorine content of less than 8ppm.

26. A solution as claimed in any one of claims 23 to 25, wherein the solution includes an active oxygen species in an amount between 13 and 20mg/l.

27. A solution as claimed in any one of claims 23 to 26, wherein the solution is administered as part of the subject's food or drink.

28. A solution as claimed in any one of claims 23 to 27, for use in conjunction with one or more antibiotic agent or drug.

29. A solution as claimed in claim 28, wherein the solution further comprises one or more antibiotic agent or drug.

30. A solution as claimed in claim 28 or 29, wherein the antibiotic agent is lincospectin or amoxycillin.

31. Use of a solution comprising electrochemically activated water, in the manufacture of a medicament for the treatment or prevention of infection of a subject by a virus, wherein a therapeutically effective dose of the electrochemically activated water is administered to the subject.

32. A use as claimed in claim 31, wherein the solution has a redox potential of at least +90OmV.

33. A use as claimed in any one of claims 31 or 32, wherein the solution has a chlorine content of less than 8ppm.

34. A use as claimed in any one of claims 31 to 33, wherein the solution includes an active oxygen species in an amount between 13 and 20mg/l.

35. A use as claimed in any one of claims 31 to 34, wherein the solution is administered orally.

36. A use as claimed in any one of claims 31 to 35, wherein the solution is to be administered in conjunction with one or more antibiotic agent or drug.

37. A use as claimed in claim 36, wherein the solution and the one or more antibiotic agent or drug are administered simultaneously, sequentially or separately.

38. A use as claimed in any one of claims 36 or 37, wherein the solution comprising the electrochemically activated water further comprises one or more antibiotic agent or drug.

39. A use as claimed in any one of claims 36 to 38, wherein the antibiotic agent is lincospectin or amoxycillin.