Adjusting Extracellular pH to Prevent Entry of SARS-CoV-2 into Human Cells

The frequent outbreaks of life-threatening RNA viruses, including the ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), pose tremendous challenges to humanity. The author proposes that creating a more alkaline extracellular environment that is unsuitable for the fusion between the envelope of SARS-CoV-2 and the host cell membrane is a promising method to prevent the entry of coronaviruses into human cells. The alkaline environment could be achieved by exposing the general public to water-clustered negative air ions (NAIs), both indoors and outdoors, to induce a gradual increase in the pH of the human body. Previous studies have demonstrated that there are no harmful effects of high-concentration NAIs on human health.

D r a f t 1

Adjusting Extracellular pH to Prevent Entry of SARS-CoV-2 into Human Cells
Bin Wang * Department of Chemistry, Marshall University, Huntington, WV, USA 25755 D r a f t Text RNA viruses use ribonucleic acids (RNAs) as their genetic material, and their genomes all encode an essential protein, RNA-dependent RNA polymerase (replicase) (de Farias et al., 2017). RNA viruses have remarkably high mutation rates, up to five orders of magnitude greater than those of DNA viruses (Duffy et al., 2008;Carrasco-Hernandez et al., 2017;Dolja and Koonin, 2018;Duffy, 2018;Wolf et al., 2018), due to the lack of proofreading mechanisms for most RNA viral replicases. Mutations are the building blocks of viral evolution, contributing to their fitness and virulence, while also rendering antiviral drugs and vaccines ineffective over time.
The outbreaks of highly pathogenic and contagious RNA viruses such as severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), influenza A virus subtype H1N1, and the current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), have posed tremendous challenges to humanity during the first 20 years of the 21 st century. To fight the pandemic of coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2, worldwide approaches have included regional lockdown, social distancing, mask wearing, and frequent disinfection and cleaning, along with the rapid development of antiviral drugs, antibody-based therapies, and vaccines (Polack et al., 2020;Sanders et al., 2020;Siddiqi and Mehra, 2020;Tai et al., 2020;Wang, 2020;Baden et al., 2021).
Because the goal of vaccination-induced herd immunity takes time to achieve, and SARS-CoV-2 mutations have already emerged Phan, 2020;Wu et al., 2021;Xie et al., 2021), the ultimate way to control the virus would be to interfere with its ability to cause infection.

D r a f t
In the author's opinion, one possible solution for overcoming infection by SARS-CoV-2 is to adjust the pH of the human extracellular environment so that coronaviruses can be blocked at the entry level. The spike glycoprotein on the surface of coronaviruses is responsible for viral binding to the entry receptors of host cells, and for the subsequent fusion between the viral envelope and the host cell membrane (Yang et al., 2004;Hoffmann et al., 2020a). Previous studies have demonstrated that the optimal extracellular pH for virus-cell fusion varies within the family Coronaviridae (Sturman et al., 1990;Gallagher et al., 1991;Zelus et al., 2003;Yang et al., 2004;Chu et al., 2006).
Researchers have discovered that SARS-CoV-2 enters host cells by pH-dependent endocytosis (Hoffmann et al., 2020b;Khan et al., 2020). Since an acidic pH environment is favored for fusion and penetration of viruses into acidified endocytic vesicles (Gallagher et al., 1991), raising the extracellular pH is a potential method for blocking SARS-CoV-2 invasions.
One approach for inducing an increase in pH involves exposing humans to an environment containing a high level of negative air ions (NAIs). Such an environment could be composed of several negatively charged ions, including O 2  , O  , O 3  , CO 3  , HCO 3  , NO 3  , NO 2  , and OH  , which can accept a proton to serve as Bronsted-Lowry bases. Among them, superoxide ions (O 2  ) are the most common naturally-generated negative ions. Iwama (Iwama, 2004) reported a decrease in blood lactate level (from 1.3  0.3 to 1.0  0.2 mmol/L) and an increase in blood pH level (from 7.388  0.025 to 7.417  0.036) in nine healthy adult volunteers exposed to NAIs (created by water shearing, similar to the Lenard effect) for one hour in an experimental chamber. Unlike the artificial corona discharge method that converts air molecules into negative ions with relatively short half-lives (of several seconds) and generates ozone as a by-product, the water shearing method produces only superoxide ions, which combine with water molecules to D r a f t 5 form negatively charged clusters (i.e., O 2  (H 2 O) n ). The substantially longer half-life of water shearing-generated NAIs (~60 seconds) provides negatively charged ions that last long enough to enter the circulatory system via the alveoli of the lungs, and thus have an effect on extracellular pH.
In addition to elevating the pH, NAIs can inhibit the growth of various viruses, bacteria, and fungi; remove particulate matter from polluted air; relieve symptoms of allergies; boost the human cardiovascular, respiratory, and immune systems; and benefit mental health (Byczkowski and Gessner, 1988;Mitchell and King, 1994;Seo et al., 2001;Fan et al., 2002;Fan et al., 2007;Fletcher et al., 2007;Sirota et al., 2008;Escombe et al., 2009;Yu, 2012;Hagbom et al., 2015;Erqou et al., 2018;Jiang et al., 2018). To the author's knowledge, NAIs do not cause harmful effects in humans at the concentrations in the cited articles, although certain artificial NAIgenerating methods, such as the corona discharge of air, do produce ozone as a by-product.
Little is known about the effects of an alkaline extracellular environment on various types of cells and signaling pathways. However, an excessive shift in the pH of bodily fluids would presumably cause abnormal conditions or disease. An elevation of extracellular pH may increase the intracellular pH of various cells, and thus alter their biological functions; these changes may not always be beneficial. In addition, RNA is unstable under higher pH conditions (Barshevskaia et al., 1987;AbouHaidar and Ivanov, 1999) because the 2'-OH group of the ribose becomes deprotonated and attacks the adjacent phosphorus in the phosphodiester bond of the sugar-phosphate backbone of the RNA. This breaks the phosphodiester bond, resulting in cleavage of the RNA backbone. Since the pH increase induced by NAIs is temporary, gradual, and mild, it would be unlikely to have severe adverse effects on the stability of RNAs, the immune function, or the general health of humans. To date, there have been no studies that D r a f t 6 identify the change in pH within the human body after a long-term and high-dose exposure to water-clustered NAIs. Future investigations are needed to fill this knowledge gap.
In summary, creating a more alkaline extracellular environment that is unsuitable for the fusion between the envelope of SARS-CoV-2 and the host cell membrane is a promising method for defeating COVID-19. The alkaline environment could be achieved by exposing the general public to a high level of water-clustered NAIs, both indoors and outdoors, to induce a gradual increase in the pH within the human body. Since most of the previous studies have focused on investigating the effects of extracellular acidification on human physiology and health, future research should be directed toward exploring the advantages and drawbacks of an alkaline extracellular environment, along with identifying the safe upper pH limits for the use of alkaline drugs and therapies.

Acknowledgments
This work is supported by the National Science Foundation under Award No. OIA-1458952. Any opinions, findings, and conclusions expressed in this material are those of the author and do not necessarily reflect the views of the National Science Foundation.

Conflicts of Interest Statement
The author declares that she has no competing financial interests.