SARS and Ozone Therapy: Theoretical Considerations

by G¨¦rard V. Sunnen, M.D.

May 2003

Abstract

SARS (Severe Acute Respiratory Syndrome) is a global disease of significant lethality with an expanding incidence and prevalence base. Of massive public health importance, SARS presents supremely challenging problems in light of its pathogenic capacity and mutational potential. Ozone, because of its special biological properties, has theoretical and practical attributes to make it a viable candidate as a SARS virus inactivator through a variety of physicochemical and immunological mechanisms.

The Family of Coronaviruses

The SARS virus belongs to the viral family Coronaviridae . which includes two genera, coronavirus and togovirus, each showing similar replication mechanisms and genomic organization but distinct genome lengths and viral architecture. First identified in the 60's, this family identifies itself by large, enveloped, positive-stranded RNA virions. Their appearance is characteristically distinct, with envelopes endowed with host cell membrane-tropic petal shaped spikes (peplomers). The large, amply spaced peplomers on the virion surface suggests a coronal (crown-like) appearance.

Prior to SARS, Coronaviridae were responsible for relatively mild cold-like syndromes in humans corresponding to their predilection for the ciliary epithelium of the trachea, nasal mucosa, and alveolar cells of the lungs. At times they were only rarely implicated in serious respiratory illnesses in frail older adults (Falsey 2002). SARS represents a quantum leap in Coronaviridae infectivity by way of its significant lethality. Widely seen in nature, coronaviruses infect a spectrum of animal hosts and are responsible for avian infectious bronchitis, murine hepatitis, and porcine gastroenteritis, among others. Of possible significance to humans is that animal coronaviruses are able to penetrate into the central nervous system.

SARS: Virion architecture and molecular biology

The SARS virion differs from other members of the Coronaviridae family in its genomic composition. The other viral structures, however, are similar, including virion architecture, and the fundamental composition of structural and non-structural proteins.

The software for viral replication is the nucleic acid core, a single strand long chain RNA nucleotide. The core is surrounded by the nucleic acid coat or capsid. The capsid is rigid and determines the shape of the virus; it is made of repeating units called capsomeres. The SARS viral nucleocapsid is tubular with a helical symmetry.

The nucleocapsid is surrounded by an envelope which forms the outer layer of the virion and maintains intimate contact with host bodily fluids. As such, it is sensitive to the composition and alterations in its milieu, such as temperature, pH, and ionic balance. The viral envelope is formed at the time of budding, an intricate process in which the nucleocapsid exits the host cell. In order to do this, it fuses with the host cell membrane, appropriating its components to form its own envelope. It is known that the lipid composition of viral membranes reflects the lipid composition through which the particles exit. Viral envelopes are composed of lipid bilayers associated with a union of carbohydrates and proteins, glycoproteins, and lipids and phosphates, phospholipids. Up to 60% of the lipid component of the envelope is composed of phospholipid and the remainder is mostly cholesterol. This lipid-carbohydrate envelope is closely articulated with the peplomers which determine attachment and penetration into host cells.

The genome composition and sequence of the SARS virus has recently been identified (Marra 2003; Rota 2003). Marra et al. described a viral genome configuration of 29,727 nucleotides in length, within which exists a gene order similar to other coronaviruses. However, because the genetic composition of SARS does not closely resemble any of the three known classes of coronaviruses, they propose a new and fourth class of coronaviruses, the SARS-CoV. Postulated, is a hypothesis that an animal virus recently mutated to successfully infect humans, or that the SARS virus mutated from a common human coronavirus.

Rota et al. reported a nucleotide sequence of 29,727 in SARS-CoV, with 11 open reading frames. Phylogenetic analyses and sequence comparisons showed that the SARS virus is not closely related to any of the previously characterized coronaviruses.

Virion structural proteins are essential elements in determining the morphological and functional dimensions of the SARS virus. Coronavirus structural proteins include the N nucleocapsid phosphoprotein which binds to viral RNA; the membrane glycoprotein M which forms the shell of the internal viral core and is responsible for triggering virus assembly ; the protein E associated with the virion envelope; the spike glycoprotein S which binds to specific cellular receptors and elicits cell-mediated immunity; and the Hemagglutinin-esterase glycoprotein HE forming small spikes on the coronavirus envelope (Knipe 2001).

SARS: Viral replication

The viral replication cycle follows the pattern seen in mammalian viruses and may be divided into several stages (Cann 1997; Evans 1997; Knipe 2001). The coronavirus attaches to the membrane of the host cells by binding the S and HE proteins of its peplomers to receptor glycoproteins or glycans.

Once cell entry is achieved, the virion sheds its envelope to commence its replication in the host cell cytoplasm. It binds to cellular ribosomes and released viral polymerase begins the RNA replication cycle. Newly formed nucleocapsids continue their assembly with the acquisition of new envelopes by means of budding through membranes of the cell's endoplamic reticulum.

Virions are then released into the general blood and lymphatic circulation, ready to infect new cells, other organ systems, and new hosts.

SARS: Clinical findings

Recently, the clinical manifestations of SARS have been comprehensively described (Peiris 2003). In this study of 50 hospitalized patients, fever, chills, myalgia, and dry cough were the most frequent presenting complaints. Also reported, were rhinorrhea, sore throat, and gastrointestinal symptoms.

Radiological examination showed evidence of pulmonary consolidation approximately 5 days after the onset of symptoms. Laboratory examination showed leucopenia and lymphopenia, despite the presence of fever; also anemia, thrombocytopenia, liver enzyme elevations (alanine aminotransferase), and skeletal and heart muscle enzyme elevation (creatinine phosphokinase). All these features point to severe systemic inflammatory insults.

The incubation of SARS is 2 to 10 days, and in some patients perhaps longer. Viral transmission is achieved by the respiratory route where it may infect the new host through aerosol and droplet contact with mucosal surfaces of the mouth, nose, throat, and probably the conjunctiva. SARS virions have been found in feces and the importance of this route of tranmission is being evaluated, as it is known that several animal coronaviruses use this propagation venue. Moreover, since it is appreciated that SARS particles remain viable on fomites for 48 hours or longer, any eradication effort must address the infectivity of objects in the environment.

The syndrome progresses to severe disease with respiratory distress and oxygen desaturation requiring ventilatory support in over a third of patients, approximately 8 days after symptom onset. Mortality has been noted to vary according to transmission clusters, ranging from 3 to 20%. This suggests that the etiology of SARS depends upon a heterogenous population of viral quasispecies with variable degrees of virulence.

SARS: Genetic creativity

As is the case in the majority of RNA viruses, coronaviruses mutate at a high rate (Steinhauer 1986). Within any one afflicted individual, coronaviruses particles do not show a homogeneous population. Instead, they function as a pool of genetically variant strains known as quasispecies. This is due to the high error frequency of RNA polymerases, the presence of deletion mutants, the high frequency of RNA recombination and point mutations, and the occurrence of defective-interfering RNA (DI RNA). The net result of these diverse and complex mechanisms is the continuous spawning of novel virions and divergent quasispecies. Some of the genetic creations will find themselves at an advantage in negotiating new host antibody responses and pharmacological antiviral countermeasures; and they will propagate accordingly, thus expanding their ecological terrain. Other genetic creations will be too lethal to their hosts, work against their own survival, and will prove to be non-adaptive. If we can speak of a viral psychology, an efficient survival balance aims somewhere between defeat by host defenses on one hand, and viral suicide through aggressive lethality on the other.

Ozone: Physical and physiological properties

The oxygen atom exists in nature in several forms: (1) as a free atomic particle (O), it is highly reactive and unstable; (2) Oxygen (O2), its most common and stable form, is colorless as a gas and pale blue as a liquid; (3) Ozone (O3), has a molecular weight of 48, a density one and a half times that of oxygen and contains a large excess of energy in its molecule (O3 ( 3/2 O2 + 143 KJ/mole). It has a bond angle of 127 ( 3(, which resonates among several forms, is distinctly blue as a gas and dark blue as a solid; (4) O4 is a very unstable, rare, nonmagnetic pale blue gas which readily breaks down into two molecules of oxygen.

Ozone (O3), a naturally occurring configuration of three oxygen atoms, has a half life of about one hour at room temperature, reverting to oxygen. A powerful oxidant, ozone has unique biological properties. Since medicinal ozone is administered by interfacing it with blood, basic research on ozone's biological dynamics have centered upon its effects on blood cellular elements (erythrocytes, leucocytes, and platelets), and to its serum components (proteins, lipids, lipoproteins, glycolipids, carbohydrates, electrolytes).

The effects of ozonation on whole blood are extraordinarily complex and are far from adequately elucidated. If the biochemical configuration of serum - with its proteins, including enzymes, immunoglobulins, clotting factors; its hormones, vitamins, lipoproteins and cholesterol; its carbohydrates including glucose, and electrolytes, among others (Dailey 1998) - can be compared to an orchestra, ozone administration can be likened to the introduction of a novel and powerful musical instrument, affecting the interactions of all the other instruments.

Even though an in-depth analysis of ozone's multifaceted effects upon the panoply of blood constituents is beyond the intent and scope of this article (The reader is referred to Bocci, 2002; Sunnen, 1988), the following points of research interest are advanced:

Erythrocytes have been extensively studied in relation to ozone administration. Many studies which have used erythrocyte suspension in physiologic saline (Kourie 1998; Fukunaga 1999) have found hemolysis at relatively low ozone dosages (10 to 30 ug/ml). When ozone is administered in whole blood, however, the dynamics of ozone interaction are such that hemolysis begins to be observed at significantly higher doses, implying a buffering action of blood constituents. Moreover, the functionality of erythrocyte enzymes are maintained, suggesting a protective role of antioxidant systems (Cross 1992). There is some evidence that ozone administration may stimulate erythrocyte formation and release (Hernandez 1999).

Leucocytes, intimately connected to immune function, show good resistance to ozone because they possess enzymes which protect them from oxidative confrontation. These enzymes include superoxide dismutase, glutathione, and catalase. A promising area of research centers on cytokine and interferon stimulation in ozone administration and its implication for enhancing immune function (Paulesu 1991; Bocci 1994; Larini 2001). A classical adage of ozone therapy is that lower ozone dosages are stimulating to immune action while higher dosages become inhibitory (Viebahn 1999). Further research will need to clarify the parameters of this phenomenon, as well as the effects of ozone infusion upon different types of leucocytes in relation to the disease process being treated.

Ozone: Antipathogenic properties

Recently, there has been renewed interest in the potential of ozone for viral inactivation in vivo. It has long been established that ozone neutralizes bacteria, viruses, fungi, and parasites in aqueous media. This has prompted the creation of water purification processing plants in numerous major municipalities worldwide. Ozone's unique physicochemical and biological properties, and environmentally-friendly aspects, have since been applied to a panoply of industrial uses such as the packaging of pharmaceuticals, the fumigation of homes and buildings (sick building syndrome), the treatment of indoor air in operating rooms and nursing homes, and the disinfection of large scale air conditioning systems in hospitals (Rice 2002).

Ozone's remarkable capacities for pan-antipathogenic action has been applied to the treatment of poorly healing wounds and burns (Sunnen 1999). A partial list of organisms susceptible to ozone inactivation in these clinical situations includes aerobic and anaerobic bacteria, Bacteroids, Campylobacter, Clostridium, Corynebacteria, Escherichia, Klebsiella, Legionella, Mycobacteria, Propriobacteria, Pseudomonas, Salmonella, Shigella, Staphylococcus, Streptococcus, and Yersinia. Susceptible viruses include Adenoviridae, Filiviridae , Hepnaviridae, Herpesviridae, Orthomyxoviridae, Picornaviridae, Reoviridae, and Retroviridae. Ozone-sensitive fungi include Actinomycoses, Aspergillus, Candida, Cryptococcus, Epidermophyton, Histoplasma, Microsporum, and Trichophyton.

Some viruses are more susceptible to ozone's action than others. It has been found that lipid-enveloped viruses are the most sensitive. This makes intuitive sense, since enveloped viruses are designed to blend into the dynamically constant milieu of their mammalian hosts. This group includes, hepatitis B and C, herpes 1 and 2, Cytomegalus (Epstein-Barr), HIV 1 and 2, Influenza A and B, West Nile virus, Togaviridae, Eastern and Western equine encephalitis, rabies, and Filiviridae (Ebola, Marburg), among others.

The envelopes of viruses provide for intricate cell attachment, penetration, and cell exit strategies. Peplomers, finely tuned to adjust to changing receptors on a variety of host cells, constantly elaborate slightly new glycoprotein configuration under the direction of portions of the viral genome, thus adapting to host cell defenses. Envelopes are fragile. They can be disrupted by ozone and its by-products.

Lipid enveloped viruses in aqueous media are readily inactivated by ozone via the oxidation of their envelope lipoproteins and glycoproteins (Akey 1985; Shinriki 1988; Vaughn 1990; Wells 1991; Carpendale 1991). In whole blood, however, ozone's virucidal actions are buffered by the spectrum of its components and ozone becomes less effective. This situation is further complicated in the case of retroviruses which ensconce themselves within host DNA (Chun 1999), and in Herpesviridae, where virions have the capacity to persist indefinitely in their host through the formation of an episome in the nuclei of the cells that harbor them (White 1994).

Several studies have reported the safety and the benefits of ozone administration in vivo. Wells et al. (1991) showed that ozone-treated HIV-spiked Factor VIII maintained its biological capacity; and that, concomitantly, there was an 11 log reduction in detectable virions. The improvement of liver enzymes in hepatitis C patients after several months of ozone therapy was described (Viebahn 1999; Amato 2000). An 80% hepatitis C viral load reduction in 82 patients using AHT was reported by Luongo et al., 2000.

It is remarkable, however, that to date, no adequate double blinded study has addressed ozone therapy in viral conditions such as hepatitis B and C, HIV, or herpes.

Ozone: Clinical methodology

Ozone may be utilized for the therapy of a spectrum of clinical conditions (Viebahn 1999). Routes of administration are varied and include external, and internal (blood interfacing) methods. In the technique of ozone major autohemotherapy (AHT), an aliquot of blood (50 to 300 ml) is withdrawn from a virally-afflicted patient, anticoagulated, interfaced with an ozone/oxygen mixture, then re-infused. This process is repeated serially, in a manner consonant with the treatment protocol until viral load reduction and symptom abatement are observed.

Recently there has been interest in new methods of interfacing oxygen-ozone mixtures with whole blood, serum, and serum components (Sunnen and Robinson, 2001).

Another, more experimental and more intensive technique of ozone administration, is called the Extracorporeal Blood Circulation Versus O2-O3 (EBOO), which treats the entire blood volume using a hollow-fibre oxygenator-ozonizer (Di Paolo 2000).

Ozone: Possible mechanisms of anti-viral action

The average adult has 4 to 6 liters of blood, accounting for about 7% of body weight. How can any viral load reduction reported via AHT ozone therapy be explained in the face of a technique that treats relatively small percentages of blood volume, albeit serially?

The viral culling effects of ozone in infected blood may recruit a variety of mechanisms. Research is needed to ascribe relative importance to these, and possibly other mechanisms of ozone's anti-viral action:

  1. The denaturation of virions through direct contact with ozone. Ozone, via this mechanism, disrupts viral proteins, lipoproteins, lipids, glycolipids, or glycoproteins. The presence of numerous double bonds in these molecules makes them vulnerable to the oxidizing effects of ozone which readily donates its oxygen atom and accepts electrons in redox reactions. Unsaturated bonds are thus reconfigured, molecular architecture is disrupted, and breakage of the envelope ensues. Deprived of an envelope, virions cannot sustain nor replicate themselves.
  2. Ozone proper, and the peroxide compounds it creates, may alter structures on the viral envelope which are necessary for attachment to host cells. Peplomers, the viral glycoproteins protuberances which connect to host cell receptors are likely sites of ozone action. Even minimal alteration in peplomer integrity through glycoprotein peroxidation could impair attachment to host cellular membranes foiling viral attachment and penetration.
  3. Introduction of ozone into the serum portion of whole blood induces the formation of lipid and protein peroxides. While these peroxides are not toxic to the host in quantities produced by ozone therapy, they nevertheless possess oxidizing properties of their own which persist in the bloodstream for several hours. Peroxides created by ozone administration show long-term antiviral effects which may serve to further reduce viral load.
  4. The immunological effects of ozone have been documented (Bocci 1992; Paulesu 1991). Cytokines are proteins manufactured by several different types of cells which, in turn, regulate the functions of other cells. Mostly released by leucocytes, they are important in mobilizing immune reactivity. Ozone-induced release of cytokines may constitute an avenue for the reduction of circulating virions.
  5. Ozone action on viral particles in infected blood yield several possible outcomes. One outcome is the modification of virions so that they remain structurally grossly intact yet sufficiently dysfunctional as to be nonpathogenic. This attenuation of viral particle functionality through slight modifications of the viral envelope, and possibly the viral genome itself, not only modifies pathogenicity, but allows the host to diversify its immune response. The creation of dysfunctional viruses by ozone offers unique therapeutic possibilities. In view of the fact that so many mutational variants exist in any one afflicted individual, the creation of an antigenic spectrum of crippled virions could provide for a unique host-specific stimulation of the immune system, thus designing what may be called a host-specific autovaccine.
  6. A very exiting avenue of research suggests that the virucidal properties of antibodies is predicated upon their ability to catalyse highly active forms of oxygen including ozone (Marx 2002; Wentworth 2002). A key element in the viral-inactivating capacity of antibodies may thus reside in the formation of ozone integral to antigen-antibody reactions. Exogenously administered ozone may, in this model, amplify the efficacy of antigen-antibody dynamics.
SARS and Ozone: Special considerations

SARS is produced by a novel coronavirus which has succeeded in finding breaches in the immunological defenses in our contemporary human population. It appears to have developed an aggressive balance between viral propagation and lethality.

The universal strategy in mastering infections, whether bacterial or viral, is the culling of pathogenic organisms to the point where they no longer represent an invasive and replicative threat; and, concomitantly, the elaboration of systems of immune defense capable of neutralizing subsequent viral attacks. This goal is achieved mainly through direct pathogen inactivation on one hand, and by the actuation of host immune competence on the other.

SARS, as an acute, rapidly progressing, pan-inflammatory infection which, predicated upon the quasispecies involved, may present distressful mortality outcomes. A salient clinical configuration of this disease rests upon its acute involvement of the respiratory system in its disruption of the harmonious homeostasis of blood gases. When pO2 and Pco2 are sufficiently compromised, central nervous system cortical changes in the level of consciousness occur which impair volition to breathe, along with depression of the respiratory chemoreceptors in the medulla.

Antiviral agents and inhibitors to inflammation (steroids) have, thus far, not been effective in significantly softening the virulence of SARS.

Because of its acuteness, SARS is likely to require proactive viral culling. With an estimated 10 billion SARS viral particles generated daily - a reproductive magnitude commonly observed in viremic episodes in enveloped viruses - it is suggested that ozone administration may likely need to be more intensive than in chronic infections, such as hepatitis B and C. Whereas the latter conditions have been addressed with AHT frequencies ranging from once daily to once weekly, SARS may require more accelerated attention, either with AHT or with EBOO.

SARS and sterilization of the environment

The recent findings that the SARS virus has the capacity to remain infectious on fomites for up to several days indicates that it is a hardier organism than most of its other lipid enveloped colleagues.

Predictably, disinfectants such as bleach, phenol, and formaldehyde have been found to be effective in deactivating the SARS virus; detergents, however, were less capable.

Caustic liquid agents have the disadvantage of faring poorly in decontaminating complex medical equipment and the complex hospital room milieu of SARS patients.

Ozone, in light of its pan-virucidal profile, offers the advantage of existing as a gas, with its attendant ability to disinfect poorly accessible spaces. Moreover, ozone has the distinct benefit of reverting to oxygen, while liquid-based disinfectants are likely to injure the surfaces to which they are applied, and to leave toxic residues. Ozone-mediated environmental decontamination, however, needs to respect stringent protocols to insure that the ambient ozone in the process of sterilizing the target environment has time to revert to its stable parent, oxygen, without inflicting toxicity to the personnel.

Summary and conclusions

SARS is an acute pan-inflammatory multi-system syndrome caused by a hitherto unknown coronavirus. This virion incorporates a novel RNA genome and a lipid bilayered glycoprotein envelope. The SARS virus, based upon what is known about Coronaviridae is likely to have a high rate of mutation allowing any one individual to harbor numerous quasispecies.

Ozone is a naturally occurring energy-rich molecule embodying unique physico-chemical and biological properties suggesting a possible role in the therapy of SARS, either as a monotherapy or, more realistically, as an adjunct to standard treatment regimens.

This paper outlines six possible mechanisms by which ozone may exert its antiviral actions. Due to the excess energy contained within the ozone molecule, it is theoretically likely that ozone, unlike organism-specific antiviral options available today, will show effectiveness across the entire genotype and subtype spectrum of SARS.

Ozone has unique disinfectant properties. As a gas, it has a penetration capacity that liquids do not possess. In view of the fact that SARS persists on fomites for up to several days, it is suggested that ozone technology be applied to the decontamination of SARS-contaminated medical environments.

In conclusion, it is suggested that this treatment modality, which has been demonstrated to be innocuous to humans and animals in contemporary treatment protocols, be granted research consideration for SARS. It may then be found therapeutically useful not only in SARS, but also in future epidemics caused by novel organisms which, unfortunately, are certain to emerge.

Bibliography