by Gérard V.
Sunnen, M.D.
BACK
TO HOME
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
§
Ackey D, Walton TE. Liquid-phase study of ozone inactivation
of Venezuelan Equine Encephalomyelitis virus. Appl Environ Microbiol 1985; 50:
882-886
§
Amato G, Sacchetta A, Borrelli E, et al. Ruolo
dell'ozonoterapia mediante grande autoemotrasfusione nel trattamiento delle
epatiti croniche post-epatiti virale (II parte), In: Proceedings: I Congresso
IMOS, Italia, Siena, 2-4 Nov 2000, p 11
§
Bocci V. Oxygen-Ozone Therapy: A Critical Evaluation. Kluwer
Academic Publishers, Dordrecht, 2002
§
Bocci V. Biological and clinical effects of ozone. Br J
Biomed Sci 01 Jan 1999; 56(4): 270-279
§
Bocci V, Luzzi E, Corradeschi F, Paulesu, et al. Studies on the
biological effects of ozone: 5. Evaluation of immunological parameters and
tolerability in normal volunteers receiving ambulatory autohaemotherapy.
Biotherapy 1994; 7:83-90
§
Bocci V. Ozonation of blood for the therapy of viral diseases
and immunodeficiencies. A hypothesis. Medical Hypotheses 1992 Sept; 39(1):30-34
§
Bocci V. Autohemotherapy after treatment of blood with ozone:
A reappraisal. The Journal of International Medical Research 1994; 22: 131-144
§
Bolton DC, Zee YC, Osebold JW. The biological effects of
ozone on representative members of five groups of animal viruses. Environmental
Research 1982; 27:476-48
§
Buckley RD, Hackney JD, Clarck K, Posin C. Ozone and human
blood. Archives of Environmental Health 1975; 30:40-43
§
Cann AJ. Principles of Molecular Virology, Second Edition.
Academic Press, New York, 1997
§
Cardile V, et al. Effects of ozone on some biological
activities of cells in vitro. Cell Biology and Toxicology 1995 Feb; 11(1):11-21
§
Clarke LM, Bromberg K. Human Respiratory Viruses. In Armstrong.
Infectious Diseases, First Ed. Mosby, Philadelphia, 2000
§
Carpendale MT, Freeberg JK. Ozone inactivates HIV at
noncytotoxic concentrations. Antiviral Research 1991; 16:281-292
§
Chun TW, Fauci AS. Latent reservoirs of HIV; obstacles to the
eradication of virus. Proc Natl Acad Sci U.S.A. 1999; 96:10958-10961
§
Cross CE, Reznick AZ, Packer L, et al. Oxidative damage to
human plasma proteins by ozone. Free Rad Res Commun 1992; 15:347-352
§
Dailey JF. Blood. Medical Consulting Group, Arlington MA,
1998
§
de Haan CA. Assembly of the coronavirus envelope: homotypic
interactions between the M proteins. J Virol 01 Jun 2000; 74(11): 4967-78
§
Diadori A, Nuti A, Ferrari G et al. Ozone therapy: a new
perspective in ophthalmology. Vision Res 1996; 36 (suppl.):418
§
Di Paolo N. Extracorporeal blood oxygenation and ozonation
(EBOO) in man. Preliminary report. Int J Artif Organs 01 Feb 2000; 23(2):
131-141
§
Evans AS, Kaslow RA (Eds). Viral Infections in Humans:
Epidemiology and Control, Fourth Edition, Plenum, New York, 1997
§
Falsey AR. Rhinovirus and coronavirus infection-associated
hospitalizations among older adults. J Infec Dis 1 May 2002; 185(9):1338-41
§
Fukagawa NK. Aging: is oxidative stress a marker or is it
causal? Proc Soc Exp Biol Med 1999; 222:293-298
§
Goheen SC, O'Rourke L, Larkin EC. Ozone and the peroxidation
of polyunsaturated fatty acids in vivo. Environ Res 1986: 40: 47-57
§
Gumulka J, Smith L. Ozonation of cholesterol. J Am Chem Soc
1983; 105(7): 1972-1979
§
Hernandez F, Menendez S, Wong R. Decrease of blood
cholesterol and stimulation of antioxidative response in cardiopathy patients
treated with endovenous ozone therapy. Free Rad Biol Med 1995; 19:115-119
§
Hurst CJ. Viral Ecology. Academic Press, New York, 2000
§
Knipe DM, Howley PM. Fundamental Virology, Fourth Edition.
Lippincott Williams & Wilkins, Philadelphia, 2001
§
Konrad H. Ozone therapy for viral diseases. In: Proceedings
10th Ozone World Congress 19-21 Mar 1991, Monaco. Zurich: International Ozone
Association 1991:75-83
§
Ksiasek TG, Erdman D, Goldsmith CS, et al. A novel
coronavirus associated with severe acute respiratory syndrome. N Engl J Med
2003; 348:1947-1958
§
Kourie JI. Interaction of reactive oxygen species with ion
transport mechanisms. Am J Physiol 1998; 275:1-24
§
Lai MM, Holmes KV. Coronaviridae: The Viruses and Their
Replication. In: Knipe DM, Howley PM. Fundamental Virology. Lippincott
Williams&Wilkins, Philadelphia, 2001
§
Larini A, Aldinucci C, Bocci V. Ozone as a modulator of the
immune system, In : Proceedings of the 15th Ozone World Congress, London, UK,
11th-15th September 2001 (International Ozone Association 2001, Speedprint
Macmedia Ltd, Ealing, London, UK
§
Luongo C, Sammartino A, Lauritano et al. Trattamento e
monitoraggio dei potenziali redox nelle membrane cellulari nello studio delle
infezioni da HCV. In: Proceedings: I Congresso IMOS, Italia, Siena, 2-4 Nov
2000, p 42
§
Maeda J. Membrane topology of coronavirus E protein. Virology
15 Mar 2001; 281(2): 163-169
§
Marra MA, Jones SJ, Astell CR. The genome sequence of the
SARS-Associated coronavirus. Science. Sciencexpress. 1 May 2003
www.sciencexpress.org
§
Max J. Antibodies kill by producing ozone. Science 15 Nov
2002; 298: 1319
§
Monto AS. Coronaviruses. In Evans AS, Kaslow RA. Viral
Infections in Humans, Fourth edition, Plenum, New York, 1997
§
Olwin JH, Ratajczak HV, House RV. Successful treatment of
herpetic infections by autohemotherapy. J Altern Complement Med 1997; 3:
155-158
§
O'Neil CA, van der Vliet A, Hu ML, et al. Oxidation of
biologic molecules by ozone: the effect of Ph. J Lab Clin Med 1993; 122:
495-505
§
Paulesu L, Luzzi L, Bocci V. Studies on the biological
effects of ozone: Induction of tumor necrosis factor (TNF-alpha) on human
leucocytes. Lymphokine Cytokine Research 1991; 5:409-412
§
Peiris JS, Lai ST, Poon LL, et al. Coronavirus as a possible
cause of severe acute respiratory syndrome. Lancet. Ap 19, 2003; 361: 1319-1325
§
Razumovskii SD, Zaikov GE. Ozone and its reaction with
organic compounds. Elsevier, New York, 1984
§
Rice RG. Century 21 - Pregnant with ozone. Ozone Science and
Engineering 2002; 24: 1-15
§
Romero A, Menendez C, Gomez M, et al. Ozone therapy in the
advanced stages of arteriosclerosis obliterans. Angiologia 1993; 45: 146-148
§
Roy D, Wong PK, Engelbrecht RS, Chian ES. Mechanism of
enteroviral inactivation by ozone. Applied Environmental Microbiology 1981; 41:
728-733
§
Shinriki N, Suzuki T, Takama K, et al. Susceptibilities of
plasma antioxidants and erythrocyte constituents to low levels of ozone.
Haematologia 1998; 29; 229-239
§
Siddell SG. The Coronaviruses. Plenum Press, New York, 1995
§
Steiberg D. Low density lipoprotein oxidation and its
pathobiological significance. J Biol Chem 1997; 272: 20963-66
§
Steinhauer DA, Holland GG. Direct method for quantation of
extreme error frequencies at selected single base sites in viral RNA. J Virol
1986; 57:219-228
§
Sunnen GV. Ozone in Medicine. Journal of Advancement in
Medicine. 1988 Fall; 1(3): 159-174
§
Sunnen G. Possible mechanisms of viral inactivation by ozone.
Townsend Letter for Doctors. Ap 1994: 336
§
Sunnen G. Apparatus for the application of ozone/oxygen for
the treatment of external pathogenic conditions. Patent pend. PCTUS99-17286,
Jul 1999
§
Sunnen G, Robinson J. Method and apparatus for ozone
decontamination of biological fluids. Patent pend. 10-002943, Jul 2001
§
Sunnen G., http://www.triroc.com/sunnen
§
Thanomsub B. Effects of ozone treatment on cell growth and ultrastructural
changes in bacteria. J Gen Appl Microbiol 01 Aug 2002; 48(4): 193-199
§
Valentine GS, Foote CS, Greenberg A, Liebman JF (Eds). Active
Oxygen in Biochemistry. Blackie Academic and Professional, London, 1995
§
Vaughn JM, Chen Y, Linburg K, Morales D. Inactivation of
human and simian rotaviruses by ozone. Applied Environmental Microbiology 1987;
48: 2218-2221
§
Vaughn JM, Chen YS, Novotny JF. Effects of ozone treatment on
the infectivity of hepatitis A virus. Can J Microbiol 1990; 36: 557-560
§
Viebahn R. The Use of Ozone in Medicine. Odrei Publishers,
Iffezheim, 1999
§
Wells KH, Latino J, Gavalchin J, Poiesz BJ. Inactivation of
human immunodeficiency virus Type 1 by ozone in vitro. Blood 1991 Oct;
78(7):1882-1890
§
Wentworth P, McDunn JE, Wentworth AD, et al., Evidence for
antibody-catalysed ozone formation in bacterial killing and inflammation.
Science 13 Dec 2002; 298: 2195-2199
§
White DO, Fenner FJ. Medical Virology, Fourth edition.
Academic Press, New York, 1994
§
Yamamoto M, et al. The effect of ozone on treatment of 4
patients suffering from hepatitis C. Bulletin of Japan Research Association for
the Medical Use of Ozone. 1996; 3: 1-2
§
Yu BP. Cellular defenses against damage from reactive oxygen
species. Physiological Reviews 1994 Jan; 74(1):139-162
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