2.2. Classification and functions

Coronaviruses are mainly classified into 4 classes – α, β, γ, δ subtypes. β coronaviruses are further classified into SARS and SARS-CoV, Middle Eastern Respiratory Syndrome virus (MERS and MERS CoV) and COVID-19 causative agent SARS CoV-2. These SARS CoV-2 viruses contain many structural proteins of which the glucosylated spike (S) protein induces the immune response system of the host. S protein binds to a protein receptor known as the Angiotensin Converting Enzyme 2 (ACE 2) situated on the surface-membrane of the host cells mainly in the pulmonary tissues. This binding has a higher affinity when compared to the binding of SARS CoV and hence has higher transmissibility and contagiousness. The process f invasion also requires spike (S) protein priming, which is mainly carried out by host cell which produces serine protease TMPRSS211. The viral-genome has many other non-structural proteins like RNA — papain like protease (PLpro), coronavirus main protease (3CLpro) and dependent RNA polymerase (RdRp). Once the virus invades, it gains entry into the host cell as a single-stranded, positive sense-RNA. After gaining entry into the cytosol, the virus starts replicating inside the cell by using replicase (^{Fehr and Perlman, 2015}). This is followed by translation of viral polyproteins by using host cell protein translator machinery (Fig. 2). The product of translation is then sliced into effector-proteins by viral proteinases 3CLpro and PLpro. PLpro then deubiquitinises particular host cell proteins like INF factor 3 and NF-κ β and causes host immune-suppression. RdRp synthesize full-length negative stranded RNA template to make more viral genome, thus helping in viral replication (^{Li, 2016, Cynthia et al., 2020, Fehr and Perlman, 2015}).

These viruses are also known for homologous and non-homologous recombination (^{Fehr and Perlman, 2015}). Assembly and packaging inside the cell results in the formation of new viruses, ready to be released out and infect other cells. The viruses get released in the form of vesicles and get transported to the neighboring cells, but the S protein does not get incorporated in to the virus particle, making it going undetected by the virus-specific antibodies and helps in the creation of multinucleated giant cells (^{Fehr and Perlman, 2015}).

2.3. Disease pathology

Coronaviruses mainly attack the immune system by stimulating a cytokine storm (hypercytokinemia), which leads to plasma leakage, vascular permeability and disseminated intravascular coagulation. This cytokine storm results in high plasma levels of IL-1β, IL-1ra, IL-7, IL-8, IL-9, IL-10, basic FGF, G-CSF, GM-CSF, IFN-γ, IP-10, MCP-1, MIP-1α, MIP-1β, PDGF, TNF α, VEGF which injure the lung tissue. Hence the uncontrolled release of cytokines results in a ‘cytokine storm’ resembling Systemic Inflammatory Response Syndrome coupled with additional viral and bacterial infections because of dysregulated immunity, finally resulting in multiorgan failure and death (^{Catanzaro et al., 2020}).

ACE2 is mainly expressed in alveolar epithelial type II cells to facilitate replication of coronaviruses. They are also found in extra-pulmonary sites like heart, kidney, endothelium, and intestine (intestinal epithelial cells as a co-receptor for nutrient uptake) (^{Coccia, 2020b}). The binding of viral spike protein to host cells by ACE2 receptor triggers the detection of viral RNAs as Pathogen Associated Molecular Patterns (PAMPs) through pattern recognition receptors. These receptors belong to a family of Toll like receptors. This recognition happens through endosomal RNA receptors like TLR3 and TLR7/8 and cytosolic RNA sensor, retinoic acid-inducible gene (RIG-1)/MDA5. These TLRs activate signaling pathways in human CD 14+ monocytes and CD4+ T cells which include (a) IFN regulatory factor-3 which stimulate cytokines and chemokines, (b) Nuclear factor κ β and (c) JAK (Janus Kinase) STAT (Signal Transducer and Activator of Transcription). Nuclear factor κ β is normally inhibited by inhibitory proteins like Iκ βs. SARS CoV2 causes degradation of Iκ β α, thus activating NF κ β. NF κ β further causes activation of pro-inflammatory cytokines, chemokines, stress-response proteins and anti-apoptotic proteins. NF κ β is also activated by IL-6 and TNFα (^{Catanzaro et al., 2020}).

JAK-STAT Pathway: Membrane bound IL-6 receptor and soluble form of IL-6 receptor which interacts with gp130 promotes the activation of JAK-STAT signaling. The signaling pathway is further enhanced by reactivation of IL-6. IL-6 produced by macrophages, endothelial and smooth muscle cells, promotes the secretion of cytokines like MCP-1, which induce atherogenesis further increasing the expression of cell adhesion molecules and stimulating the proliferation and migration of vascular smooth muscle cells. Inflamed cells further increase expression of ATII receptor, and its binding to ATII, which further activates IL-6 in a reciprocal manner, finally activating the JAK-STAT pathway (^{Catanzaro et al., 2020}).

The ACE2 receptor which helps in priming of viral cells is down regulated by the binding of viral spike protein, which results in overproduction of ATII, finally causing vascular inflammation and lung injury as mentioned before. ATII/ATII receptor axis also activates NF-κ β and ADAM17, and ADAM17 inactivates ACE2, hence amplifying ATII. ADAM17 is also known to process membrane form of IL-6 receptor α, which forms soluble form SIL-6Rα via SIL-6Rα-IL-6 complex which is also mediated by gp130. This reaction further activates STAT3 which finally activate NF-κ β in a variety of negative non-immune cells. Hence the signaling pathways interact with each other, to induce their co-activation. Further IL-6 provides a feedback loop and helps in amplifying many myeloid and lymphoid cells. ADAM17 in presence of ATII,trans-activates EGFR which stimulates the mature form of heparin to bind to EGF-like growth factor in vascular smooth cells which causes vascular remodeling (^{Catanzaro et al., 2020}).

3.2. Medical uses of ozone

Ozonized water is used as a spray or a compress; and used in dental medicine, geriatric developments, circulatory disorders, viral diseases, macular degeneration, rheumatism/arthritis, infected-wounds, AIDS, SARS and cancer. It also decreases the blood cholesterol, has antioxidant responses, used in treatment of hypoxic, it modifies the oxygenation in resting muscle and used in complementary treatment of ischemic syndromes (^{Elvis and Ekta, 2011}). It has been shown that a single subcutaneous injection of ozone in mouse with spared nerve injury of sciatic nerve reduces neuropathic pain (^{Elvis and Ekta, 2011}).

(a) Peripheral obstructive arterial diseases: Ozone induces glycolysis to increase ATP and 2, 3-DPG. This causes a shift in the sigmoidal O₂ – binding curve of hemoglobin to right in turn resulting in release of O₂ in ischemic tissues. This results in conversion of H₂O₂ to H₂O in RBC in presence of GSH. This reaction is mainly responsible for healing ischemic tissues and has been shown that even stage IV of the disease, where there is necrosis of toes and unbearable pain can be delayed by ozonated AHT in 50%. Ozone also stimulates HO-1, HSP70 and Nrf2 which induce antioxidants, nuclear transcription factor and hypoxia inducible factor – 1α (HIF – 1α). This induces VEGF, EPO and glycolytic enzymes to promote cell proliferation and survival. VEGF induces neo-angiogenesis to increase the blood flow which further increases EPO to enhance oxygen delivery. Moreover the glycolytic enzymes promote metabolism of glucose to increase the biosynthesis of NO and CO which also increase the blood flow and oxygen delivery to hypoxic tissues (^{Sagai and Bocci, 2011}).

(b) Age-related macular degeneration: The condition is mainly caused by death of photoreceptors in fovea centralis and of pigmented retinal epithelium to cause chronic hypoxia. Ozone (AHT) helps by increasing the delivery of oxygen to retina (^{Sagai and Bocci, 2011}).

(c) Diabetes mellitus: Ozone is found to reduce HbA₁C, CRP, nonenzymatic glycosylation, aldose reductase activity, advanced glycated end products and antioxidant–prooxidant​ balance (^{Sagai and Bocci, 2011}).

(d) Cancer: In a study by Sweet et al. it was shown that when an experimental attempt was done to insufflate an ozone and oxygen mixture into the peritoneum, it led to higher rate of survival of treated rabbits (^{Sagai and Bocci, 2011}).

(e) Neurodegenerative diseases: Nrf2 and HIF – 1α induced by ozone through activation of HO-1 play an important role in neuroprotection and suppression of neuro-inflammation in neurodegenerative diseases (^{Sagai and Bocci, 2011}).

(f) Skin and mucosal infections: Ozone is used in diabetic foot, ischemic ulcers, necrosis, decubitus, abscesses, anal fissures, fistulae, aphthous stomatitis, osteomyelitis, stomatitis, vulvovaginitis and onychomycosis (^{Sagai and Bocci, 2011}).

(g) Backache and orthopedic diseases: when 30–35 μg/ml of ozone is directly administered into the center of intersomatic space corresponding to disc herniation, it dissolves in the water of nucleus pulposus and reacts with macromolecular glycoproteins composed of carbohydrates and polypeptide chains (proteoglycans, collagen II and IV). This generates OH− which reabsorbs hydrolytic products and free water, thus causing shrinkage of nucleus and disappearance of herniated material (^{Sagai and Bocci, 2011}).

3.3. Mechanism of ozone in killing the viruses

Ozone disrupts viral proteins, lipoproteins, lipids, glycolipids, or glycoproteins, making them susceptible to oxidizing effect and participates in redox reactions. It also damages the viral envelope glycoproteins and peplomers, which are essential for the attachment of virus to host receptors. Coronaviruses contain cysteine residues in their spike and envelope proteins (^{Rowen and Robins, 2020}). These cysteine residues are made of sulfhydryl groups called thiol groups (R-SH). These thiol groups in a reduced state in the virus play a pivotal role in the viral entry and viral membrane fusion with the host cell membrane as described in the viral structure. Hence, when ozone reacts with these thiol groups/sulfhydryl groups, it oxidizes them into to sulphonic acid residues (R-SO₃-H) as shown in Fig. 3 (^{Fernández-Cuadros et al., 2020}). This process can be enhanced at higher temperatures. Ozone by altering these cysteine residues decreases the growth of the virus by 2 log lower than the wild type virus (^{Rowen and Robins, 2020}). In addition, coronaviruses have a cysteine dependent papain (a cysteine dependent protein enzyme) which is oxidized and degraded to sulfonate/sulphonic acid.

The spike proteins also have tryptophan which can be degraded by ozone (^{Rowen and Robins, 2020, Fernández-Cuadros et al., 2020}). Ozone incorporation into the serum of the human body produces lipid and protein peroxides which have antiviral effects. Ozone also alters viral envelope and viral genome significantly enough resulting in nonpathogenic dysfunctional viruses. These attenuated viruses help in evoking a unique immunological response to various strains of a particular virus creating a host-specific auto vaccine (^{de Wit et al., 2016, Bocci, 1992}). Besides the antibodies produced in the body to respond to any virus itself, it produces many highly active forms of ozone. Therefore, the addition of ozone essentially enhances the antigen–antibody response against viral infections in the host (^{Di Paolo et al., 2004}).

3.4. Mechanism of therapeutic actions of ozone

In blood, ozone combines and gets dissolved in the plasma propagating the decomposition of the gas into a variety of reactive oxygen species like superoxide anion (O2∙¯), hydrogen peroxide (H2O₂) and hydroxyl radical (OH∙) ions (^{Di Paolo et al., 2004, Elvis and Ekta, 2011}). Moreover, the peroxidation of plasma lipids results in formation of Lipid Oxidation Products (LOPS) (^{Di Paolo et al., 2004}). The above two mechanisms mainly help in enhancing the defense mechanisms (^{Di Paolo et al., 2004, Azuma et al., 2014}). Ozone activates cytoplasmic transcription factors by secondary messengers like (a) hypoxia-inducible factor 1-α (HIF-1α), (b) Nuclear factor kappa beta (NF- κ β) and (c) Nuclear factor erythroid – 2 – related factor 2 (Nrf2) (^{Hernández et al., 2020}).

In addition to the above, there is also production of hyper soluble antioxidants (^{Di Paolo et al., 2004}) and ozonides (^{Rowen and Robins, 2020}). The hydrogen peroxide produced by oxidizing effect of ozone mainly results in shifting the hemoglobin dissociation curve in the RBCs to the right and releasing oxygen, inducing glycolysis which increase ATP, thus reducing hypoxia (^{Rowen and Robins, 2020, Hernández et al., 2020}). They also cause production of interleukins (IL-6), interferon gamma (antiviral action) (^{Rowen and Robins, 2020}), TNF, TGF, nitrogen oxide in the leukocytes, release of growth factors in platelets, and propagates the antioxidant effect in all cells (^{Di Paolo et al., 2004, Bocci, 1992}). Ozone also stimulates Kreb’s cycle enhancing the oxidation carboxylation of pyruvate to produce ATP, reduces the amount of NADH, thus promoting the oxidation of cytochrome C, production of a vasodilator and prostacycline, activation of glutathione peroxide, catalase and superoxide dismutase (^{Rowen and Robins, 2020, Elvis and Ekta, 2011}).

Ozone also activates the macrophages and neutrophils, accelerates the manifestation of major histocompatibility complex (MHC) Class I and II on diseased cells, lymphocytes and monocytes, and is also responsible for the improvement of cyto-toxicity either antibody-mediated or MHC-inhibited, or via natural killer (NK) cells enhancing the immune responses against many micro-organisms including viruses (^{Bocci, 1992}). Ozone by activating H₂O₂ in neutrophils and lymphocytes causes activation of tyrosine kinase which phosphorylates NF-κ β, thus activating cytokines (^{Hernández et al., 2020}). Effect of ozone on lung tissue can be varied. Ozone can increase specific and mean airway resistance and respiratory rate, and also reduce the maximal trans-pulmonary pressure and tidal volume (^{Elvis and Ekta, 2011}). Ozone when given at appropriate dosage levels, mainly adds on to the preexisting oxidative stress in the body thus reaching the calculated dosage necessary to combat the infections. It is thus said to have a precise therapeutic window (^{Di Paolo et al., 2004}). Therefore, very low doses are not effective as they are masked by the existing antioxidants and very high doses can be toxic mainly to the pulmonary system. In terms of atherosclerosis, chronic viral infections, neurodegenerative diseases, tumor growth and aging; ozone administration can actually balance and maintain the antioxidant levels in the body, while the endogenous ROS go down during chronic oxidative stresses. Furthermore, it can also slow down the process of depletion of endogenous ROS and hence a long term calculated therapy with ozone can restore their levels in the body in the above mentioned conditions (^{Di Paolo et al., 2004}). If the period of stress continues, ozone therapy can enhance the production of existing heat-shock proteins (HSP), glucose-regulated proteins (GRP) and oxidative shock proteins (OSP) (^{Di Paolo et al., 2004}). O2–O3 acts on nitric oxide and iNOS signaling pathways. NO inhibits palmitoylation of S (spike) protein of SARS CoV2, hence prevents the binding of S protein to ACE2 receptor primarily in lung tissue (^{Franzini et al., 2020}).

Ozone therapy (OT) utilizes around 1 to 5% ozone in 95 to 99% trioxygen as a gas (around 10 to 70 mcg ozone/cc gas). This blend is called “medical ozone” (^{Rowen and Robins, 2020}). It is suggested that ozone in the form of autohemotherapy can combat infections (^{de Wit et al., 2016, Di Paolo et al., 2004}), where the minor ozone autohemotherapy and major ozone autohemotherapy involves the use of 5 to 10 mL and 200 to 250 mL of blood respectively as described by Wolff in 1974. The process is relatively straightforward: first, blood will be collected in a glass having sodium citrate of heparin, kept in vicinity of O2∕O3 mixture at concentrations varying from 15 to 80 μg/mL for around 5 to 10 min and then reinfused into the intend patients. This is typically carried out two times a week for about 7 to 8 weeks. Besides being used in various vascular and cardiovascular diseases, it is found to be effective in chronic viral infections like Herpes Zoster, Hepatitis, Herpes I and II, Acinetobacterbaumanni, C.difficile, MRSA (^{Elvis and Ekta, 2011}) and also in fungal and bacterial infections (^{Di Paolo et al., 2004, Rowen and Robins, 2020}). It is also seen that activation of immunity occurs at 30–55 μg/cc (^{Elvis and Ekta, 2011}).

Ozone is also said to have anti-hypoxic effects wherein, (i) it improves the transportation of oxygen and in cellular metabolism like glycolysis, Krebs cycle, (ii) it induces negative charge in RBCs increasing the blood cell elasticity and (iii) it improves the metabolism of inflamed tissues by increasing their oxygenation and reduces local inflammatory processes (^{Gupta and Mansi, 2012}). It also increases the synthesis of ribosomes, and mitochondria in the cells thus enhancing aerobic respiration and enhances the protein anabolism. Ozone is also known for the increased production of NO which helps in vasodilation of arterioles and veins and also in angiogenesis (^{Gupta and Mansi, 2012}).

4.2. Ozone therapy as a novel treatment for “conventionally untreatable” viral illnesses

^{Rowen and Robins (2020)} have proposed the use of ozone on treating the coronavirus in the context that the coronaviruses are abundance in cysteine, which must not be damaged for effective viral properties. However, the sulfhydryl functional groups are susceptible to oxidation and the O3 therapy can make use of this vulnerability to deactivate the virus. They have highlighted that ozone therapy could assist the human system in deactivating the thiols in viruses in tissues and in blood by creating more oxidized environment. Notably, the spike protein of coronavirus is also abundance in tryptophan (^{Broer et al., 2006}), which is vulnerable to oxidation  (^{Virender and Nigel, 2010}) next to cysteine.

Further, in this report they have mentioned that how ozone was used to treat patients infected with Ebola epidemic (dated back in October 2014), where they employed the Directly Administered Oxygen/Ozone gas Intravenously (DIV) using ozone of concentrations of 30–55 mcg of ozone/cc in a quantity of 20 cc and administered it to 5 patients on board over few min and found that all the 5 patients survived the attack of the virus. They mentioned that the treatment required an ozone generator, which could be run on car battery, medical-grade compressed O2, a syringe (and butterfly-type needle for the DIV technique). However, this method was found to cause few side effects including transient chest tightness, cough and irritation of veins. Nevertheless this method carried negligible cost and produced low waste materials in comparison to the autohaemotherapy and could be effectively employed during epidemics.

4.3. A cohort study on ozone therapy for SARS-CoV-2 pneumonia patients

^{Hernandez et al. (2020)} have conducted cohort study on 18 COVID-19 confirmed patients with severe pneumonia. The mean age of the patients was 68 years with SD of 15 years, and 72.2% (n = 13) of the patients were male. Out of 18 patients, the ozonated autohemotherapy was given to 9 patients (50%). In the study, they observed that in an unadjusted comparison, the ozonated autohemotherapy required significantly shorter time to achieve the clinical improvement with higher proportion of patients, who achieved clinical improvements in 14 days. Similarly, in risk adjusted studies, it required shorter mean time to achieve the clinical improvement (Fig. 4).

Notably, the following were highlighted as limitations of this study; small sample size, wide adjusted estimates of 95% Cls, possibility of unmeasured residuals, requires external validity. On the other hand, the key strengths of the study were considered as its observations on the real world COVID-19 patients and the use of regression modeling for the primary clinical outcome and risk-adjustments​ analyses, where it is claimed to provide new data denoting to the effective contribution of ozonated-autohaemotherapy towards treating severe COVID-19 pneumonia.

4.4. Cytoprotective properties of ozone therapy in treating the COVID-19

The report by ^{Martínez-Sánchez et al. (2020)} highlights that the action mechanism of ozone involves the modulation of O2∕O3 as the Keap1/Nrf2/ARE pathways and the reduction of IL-6 and IL-1. In such process, as the homeostasis of free radicals and antioxidant balance are associated with the O2∕O3 modulation (typically known as the ozone therapy), it can induce the cytoprotective effect which can be effectively exploited to treat the clinical conditions caused by SARS-CoV/COVID-19 (Fig. 5(a)–(b)). It has also been highlighted that the underlying principles and action mechanisms of cytoprotective activity of ozone therapy have been demonstrated clinically with other viruses and therefore future clinical studies are highly needed to validate and establish the potential use of ozone therapy in treating COVID-19.

4.5. Ozone therapy-major autohemotherapy (MAH): A preliminary evaluation

Study 1 ^{Zheng et al. (2020)} have demonstrated their experience in treating two patients confirmed with COVID-19 by ozone therapy called major autohemotherapy (MAH). In this treatment, the MAH was given to two patients (two males of 53 and 66 years old) once on a daily basis consequently for 7 days. In each turn, 100 mL of venous blood was taken and blended with ozone gas at 1:1 ratio of O2∕O3 to blood volume, with the end-concentration of O2∕O3 being 20 μg/mL. Upon the MAH-treatment, their symptoms disappeared rapidly and started recovering. It was observed that as compared to the baseline chest-imaging, the subsequent serial CT-scans exhibited progressively absorbed bilateral lung lesions. Later, the patients were discharged after meeting the necessary various criteria. Further, no adverse effects associated with MAH-treatment were observed and reported. In addition to this, they also highlighted that these two results were compared with two other cases of similar age and illness without subjecting to MAH-treatment, where they realized that the patients without MAH required extended duration of viral disinfection and treatment. They concluded that the ozone therapy-MAH may be a promising modality in treating and limiting the COVID-19 infections and further clinical studies are needed to evaluate the parameters such as dosage, effectiveness and duration of treatment. Notably, this clinical study was later approved by the Clinical Research Ethics Committee of Renmin Hospital of Wuhan University (WDRY2020-K020).

Study 2: In a study conducted by ^{Franzini et al. (2020)} 78 positive elderly patients were considered. Among them many were also suffering from ARDS and interstitial pneumonia. O₂–O3 treatment was given to all these patients and their lab parameters were drawn and subjected to analysis. It was observed that there was a major reduction in inflammation burst (CRP ≤ 30%, IL-6 ≤ 25%), a reduction in D-dimer (≤35%) and an elevation in pulmonary function indicated by O2 saturation% ≥ 10%, and PaO₂/FiO₂ ≥ 6%. Ozone was delivered to these patients by withdrawing 200 ml of blood from patients, then they were collected in a CE certified SANO₃ bag, treated with 45 μg/ml of O₂–O3 mixture and then introduced into patient’s blood. This autohaemotherapy reported 0.7 adverse events for every 100,000 treatments. The treatment lasted for 30 min and was also found to be cost-effective. This treatment lasted from 5–5 cycles with 100-200 ml of O₂–O3 once a day for 5 consecutive days (dosage: 45 μg/ml). Reduction was observed in inflammatory parameters like pro-calcitonin, PCT and fasting glucose. The greatest variability was seen with respect to serum lactate (mean: −8.220% ± 39.91SD) and PaO2/FiO2 (mean: ＋13.812% ± 58.897SD). In the same study patients having aortic dissection were also studied, in whom an increase in PO₂ and SatO2 were observed with ozone concentrations of 40 μg/ml, 80 μg/ml and 160 μg/ml and hence concluded that ozone does not harm erythrocytes (^{Franzini et al., 2020}).

4.6. Intravenous ozonated saline: A novel therapy for COVID-19

Among many adjunctive ozone therapies for COVID-19, one of the recognized mechanisms is to improve the oxygenation of blood/tissue to avert the failures in multi-organ due to hypoxia. In this direction, ^{Thorp et al. (2020)} have examined the effect of ozonated-saline, which administered intravenously, on the oxygenation in human body. The obtained results showed that the intravenously administered ozonated-saline improved the oxygenation of blood/tissues and extended the ability of person to stay in a hypoxic-environment.

4.7. Ozone as a non-drug therapy

^{Wang et al. (2020a)} have proposed non-drug therapy based techniques using light, thermal and gas-phase sources to combat the coronaviruses. Their proposal includes the following three techniques; (i) A new ventilator based on thermal and micro ozone, (ii) A treatment cabin based on far-infrared (IR), thermal-oxygen, thermal-air and thermal-micro ozone and (iii) Cold plasma physical treatment based on the pulse plasma to produce micro ozone to be inhaled to kill the viruses. However, these treatment plans are still in infant state and they have to be optimized and tested before the human use. It has been highlighted that as compared with the drug-based therapies, these physical techniques are safer as they would not cause any side effects, user-friendly and cost-effective.