key: cord-0958754-9a3ax1zm
authors: Head, Richard J.; Lumbers, Eugenie R.; Jarrott, Bevyn; Tretter, Felix; Smith, Gary; Pringle, Kirsty G.; Islam, Saiful; Martin, Jennifer H.
title: Systems analysis shows that thermodynamic physiological and pharmacological fundamentals drive COVID‐19 and response to treatment
date: 2022-02-01
journal: Pharmacol Res Perspect
DOI: 10.1002/prp2.922
sha: 428c46c72133f342222679290f84e700c5666a99
doc_id: 958754
cord_uid: 9a3ax1zm

Why a systems analysis view of this pandemic? The current pandemic has inflicted almost unimaginable grief, sorrow, loss, and terror at a global scale. One of the great ironies with the COVID‐19 pandemic, particularly early on, is counter intuitive. The speed at which specialized basic and clinical sciences described the details of the damage to humans in COVID‐19 disease has been impressive. Equally, the development of vaccines in an amazingly short time interval has been extraordinary. However, what has been less well understood has been the fundamental elements that underpin the progression of COVID‐19 in an individual and in populations. We have used systems analysis approaches with human physiology and pharmacology to explore the fundamental underpinnings of COVID‐19 disease. Pharmacology powerfully captures the thermodynamic characteristics of molecular binding with an exogenous entity such as a virus and its consequences on the living processes well described by human physiology. Thus, we have documented the passage of SARS‐CoV‐2 from infection of a single cell to species jump, to tropism, variant emergence and widespread population infection. During the course of this review, the recurrent observation was the efficiency and simplicity of one critical function of this virus. The lethality of SARS‐CoV‐2 is due primarily to its ability to possess and use a variable surface for binding to a specific human target with high affinity. This binding liberates Gibbs free energy (GFE) such that it satisfies the criteria for thermodynamic spontaneity. Its binding is the prelude to human host cellular entry and replication by the appropriation of host cell constituent molecules that have been produced with a prior energy investment by the host cell. It is also a binding that permits viral tropism to lead to high levels of distribution across populations with newly formed virions. This thermodynamic spontaneity is repeated endlessly as infection of a single host cell spreads to bystander cells, to tissues, to humans in close proximity and then to global populations. The principal antagonism of this process comes from SARS‐CoV‐2 itself, with its relentless changing of its viral surface configuration, associated with the inevitable emergence of variants better configured to resist immune sequestration and importantly with a greater affinity for the host target and higher infectivity. The great value of this physiological and pharmacological perspective is that it reveals the fundamental thermodynamic underpinnings of SARS‐CoV‐2 infection.

This pandemic is an example of a complex systems analysis of the devasting intersection of human biological complexity with severe acute respiratory syndrome (SARS)-CoV-2 lethal simplicity. To better understand this intersection, there is a need to view this viral infection not from the perspective of the diverse disciplines underpinning medicine in isolation 1 but rather from the standpoint of an evolving series of physical and biological transformations in a fashion similar to that described by Trancossi et al. 2 Experimentally these transformations will depend on an understanding of the contribution of not from single cells but from appropriate multiple cell types and tissues in an integrated fashion. The importance of the underlying developmental biology has been highlighted recently by Chen et al. 3 

Homo sapiens is a successful multicellular natural complex of systems.

The success in complex systems is the essential design patterns that function to compete, survive, reproduce, and evolve over multiple generations toward fitness and growth. 4 A major design pattern in human development has been the devolution and automation of thousands of systemic processes to leave the brain free from overburdening decision-making, to leave it uncluttered. For this purpose, the common design pattern uses mechanisms in automation based on a binary balance/counter-balance process. The cornerstone of human physiology is this design that regulates and moderates minute to minute changes in an autonomous manner for complex systems that interact with their environments through sensors and actuators. 4 Although this essential design pattern for autonomous function in the human is efficient, it is vulnerable to dysregulation by pathogens that convert sophisticated binary regulation into a destructive nonbinary state. This is the essence of the patho- Prior to the current pandemic, complex systems approaches had been described for infectious disease surveillance and response in which the importance of systems modelling was stressed in predicting temporal spatial patterns as well as identifying underlying interactions as a key to decision-making. 5 It is these underlying interactions involving physicochemical fundamental principles that underpin the pathophysiology of infection with SARS-CoV-2. Events that provide the platform for SARS-CoV-2 to drive COVID-19 are in a physical/ chemical interplay between the complex and the simple ( Figure 1 ).

In viewing the current pandemic, one is drawn to the prevailing lethality of the simple. SARS-CoV-2 is constrained with few fundamental processes to drive its success in this pandemic. Systems thinking seeks to define what it is that is commonly used and repeated as the virus successfully progresses from a vector to a human, from cell to cell, organ to organ, and human to human. The lethality of SARS-CoV-2 relates, with simplicity, to a small but changeable viral surface architecture that triggers entry into the host cell, evasion of host defenses and subsequent replication. Specifically, it is the high-affinity interaction between the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein and the host cell angiotensin-converting enzyme 2 (ACE2) protein. It is the repeated SARS-CoV-2 occupancy of human ACE2 (hACE2) molecular sites together with the power of tropism, a biological effect causing a directed response, in this case the laws of thermodynamics and receptor affinity, that drives to humans in close proximity and then to global populations. The principal antagonism of this process comes from SARS-CoV-2 itself, with its relentless changing of its viral surface configuration, associated with the inevitable emergence of variants better configured to resist immune sequestration and importantly with a greater affinity for the host target and higher infectivity. The great value of this physiological and pharmacological perspective is that it reveals the fundamental thermodynamic underpinnings of SARS-CoV-2 infection.

ACE2, affinity, complex systems, COVID-19, dissociation constant, Gibbs free energy, innate immunity, kinetics, law of mass action, MERS, pandemic, pharmacology, pharmacology, physiology, physiology, renin angiotensin system, SARS-CoV-1, SARS-CoV-2, thermodynamics, tropism, variants, virus COVID-19 disease (Figure 1) . A frequent process in biology involving molecular recognition in which biological macro molecules form specific ligand interactions, comprehensively described as by Du et al. 6 and detailed in a similar fashion by Sepahvandi et al. 7 for SARS-CoV-2 in COVID-19 disease.

This manuscript will focus on both the vulnerability of the complex and lethality of the simple to outline the key intersections of basic ways of energy, chemistry and pharmacology that enable successful viral entry and replication.

Contrary to common thinking, ACE2 is not only the entry point for SARS-CoV-2 into host cells but the key to understanding SARS-CoV-2 pathogenesis. 5 It is an entry event facilitated by the fundamentals of thermodynamics and its attending laws of mass action; an entry facilitated by the architectural change in the surface of SARS-CoV-2 that evades sequestration from the host's immune armamentarium, and which, on binding to the host, unleashes a shift in entropy and enthalpy to initiate a thermodynamically spontaneous event.

SARS-CoV-2 is an obligate and, as such, is incapable of reproduction on its own. It is devoid of metabolic activity and depends on appropriating host cellular processes for self-assembly and release into the extracellular medium. It has been established for decades that ligand-receptor interaction and stabilization by enthalpy and/ or entropy can be analyzed by thermodynamics. 10 It is, therefore, not unreasonable to view the entire process of replication and selfassembly of SARS-CoV-2 from the standpoint of the fundamental laws of physics and chemistry. This is consistent with the view that thermodynamic studies are increasingly important in understanding the biological environment. 7 In a comprehensive analysis, Du et al. have drawn attention to Gibbs free energy (GFE) being the thermodynamic potential for characterizing driving force and the degree of protein-ligand association, which is determined by the extent of the negative GFE. 6 Bruinsma et al. have drawn attention to the fact that viral capsid assembly adheres to the law of mass action in chemical thermodynamics. 11 For example, it is the thermodynamic potentials that describe how a virus adsorbs to its host receptor. 7 Of importance in biological systems is the estimate of GFE-usable energy that predicts the spontaneous change. The change in GFE depends on the prevailing energy of the products and reactants and when negative, indicates a spontaneous reaction that is thermodynamically possible ( Figure 2 ).

As highlighted recently by Popovic and Minceva, viral multiplication is a chemical process and through nonequilibrium thermodynamics permits GFE of growth comparative analysis. 12 In view of the effectiveness of SARS-CoV-2 in binding to hACE2 and subsequent entry into cells, viral replication and release, the entire process can be viewed in thermodynamic terms as an exergonic reaction. This follows from the view that thermodynamic potentials describe how a virus adsorbs to a host receptor 7 and that in coronaviruses it is the free energy released upon subsequent refolding of the fusion protein F I G U R E 1 Repeated SARS-CoV-2 self-assembly at scale drives the vulnerability of the complex and lethality of the simple. Illustration of the difference in scale and hence complexity between a human cell and a single SARS-CoV-2 virion. It shows that populations provide the platform to facilitate the repeated self-assembly at scale of SARS-CoV-2. The number of cells estimated to be present in a human is of the order of 37 trillion. 8 The diameter of a single SARS-CoV-2 is approximately 100 nm 9 or about a 1000 times smaller than the diameter of an average human cell (100 µm) to its most stable conformation that facilitates the close apposition of viral and cellular membranes and the actual membrane merger. 13 More recently, Popovic and Minceva in describing the three corona viruses SARS-CoV-1, Middle East Respiratory Syndrome (MERS), and SARS-CoV-2, highlighted that empirical formulas permitted calculation of the thermodynamic properties of the viruses for formation and growth and that GFE permits an estimate of the spontaneity of new virion formation. 12 In other words, infection by SARS-CoV-2 may be able to be explained as a nonequilibrium event characterized by an overall negative change in GFE and is as such spontaneous ( Figure 2 ).

As highlighted by Trancossi et al., a virus has more negative GFE than its host and this is a critical element in infection. 2 The discussion below explores the possibility that the SARS-CoV-2 surface architecture, its RBD together with its interaction with the host target protein (ACE2) drives the spontaneity for adhesion, entry, replication, and efflux in a thermodynamically acceptable path that powers COVID-19 disease (Figure 2 ).

It is convenient from a thermodynamic perspective to view the processes of SARS-CoV-2 binding to its receptor and the process of subsequent replication as linked discrete processes. In that regard, Popovic and Minceva demonstrated that the GFE of growth of the nucleocapsids of SARS, MERS, and SARS-CoV-2 was more negative than that of the host tissue. 12 The greater the negativity of the GFE of growth, the greater the spontaneous virus multiplication using host cell synthetic processes is. The new viral replication, assembly and release is also, in thermodynamic terms, biased in favor of the virus as an obligate parasite. It is noteworthy that a virus has no metabolic systems to generate energy and, in the absence of a host exhibits no energy flux and therefore there is no GFE change. However, when a virus binds to a host's surface proteins then the attending shift in enthalpy and entropy is associated with a negative GFE response. Popovic and Minceva hypothesized that the GFE for the virus is always more negative than the GFE of the host suggesting that the synthesis of viral components is favored thermodynamically 12 ( Figure 2 ).

The viral appropriation of the host cellular machinery is also made possible because of the prior energy costs borne by the infected cell in the provision of the cellular accoutrements for viral assembly and release. It can be concluded that this aspect of the cellular infection process obeys nonequilibrium dynamics. It is nonreversible and importantly, based on GFE considerations, is spontaneous in nature. What is fundamental is that this thermodynamically spontaneous multiplication of SARS-CoV-2 is only initiated after the virus binds to the host ACE2 protein, which facilitates cellular entry.

An enormous amount of information regarding the function and structure of the spike protein has been accumulated and published as this pandemic has progressed, and there are excellent descriptive F I G U R E 2 SARS-CoV-2 repeated self-assembly using thermodynamic spontaneity. The repeated self-assembly of SARS-CoV-2 is driven by Gibbs free energy (GFE), and it is this useable energy that predicts spontaneous change. Shown is the relationship between the dissociation constant (K d ) and the change in GFE of dissociation. 7 The negative GFE for binding of SARS-CoV-2 to ACE2 is illustrated. The GFE of the growth of the nucleocapsids for SARS-CoV-2 is more negative than that of the host tissue. 12 Illustrated is the view that populations provide the platform to facilitate the repeated self-assembly at scale of SARS-CoV-2. (ΔGd-GFE of dissociation, ΔG-Change in GFE, ΔH-change in enthalpy, ΔS-change in entropy, Kd-dissociation constant, R-ideal gas constant, T-absolute temperature, cθ = 1 mol/L) summary texts accompanied with informative illustrations. By way of summary, the key to infection of humans by SARS-CoV-2 is the role of the spike glycoprotein that binds to the ACE2 on the surface of targeted cells. The spike protein is comprised of two key subunitsone (S1) that houses the RBD and the other (S2) that orchestrates the binding of the virus to the host lipid membrane. Of fundamental importance is the binding affinity of the RBD for ACE2, which is related to Van der Waals interactions the electrostatic properties and the polar and nonpolar amino acid interactions. These are well described in Sepahvandi et al. 7 As highlighted by Sepahvandi et al., it is commonly thought that kinetics and thermodynamics influence the virus interaction with host receptors in as much as the thermodynamic potentials describe the adsorption of the virus onto the host receptor. 7 The forces that drive these molecular associations require measurement of changes of key thermodynamic parameters, including free energy of binding (ΔG), enthalpy (ΔH), and entropy (ΔS) of binding. 14 Of critical importance is the virus-host receptor interaction viewed initially as a reversible process the strength of which is defined as the bind- Details on the binding of SARS-CoV-2 to ACE2 are well documented and importantly can be interpreted in thermodynamic terms. As mentioned earlier, the thermodynamic interactions commence with the levels of energy of the reactants. During the binding of the S protein of SARS-CoV-2 to ACE2, the change in adsorption enthalpy reflects the sum of bond energy changes which determines the change in GFE. 7 When the GFE is negative, the thermodynamic considerations indicate this will be a spontaneous reaction. Of significance is the observation that the estimated binding energies for the spike protein structures of SARS-CoV-2 and SARS-CoV are both negative and the lowest binding energy value is associated with SARS-CoV-2. 15 Additionally, several mutations in the receptor-binding motif for SARS-CoV-2 lead to this change in binding energy when SARS-CoV-2 is compared with SARS-CoV. 15 In a detailed study, Koley et al. conducted sequence and structure-based analysis with SARS-CoV-2 spike RBD complex and hACE2 and reported a negative binding energy value. 16 Moreover, they compared the GFE of binding across a range of mammalian complexes using the human as a reference standard, demonstrating lower affinity between SARS-CoV-2 spike protein affinity and ACE2 from other mammalian species. 16 In a similar fashion, Piplani et al. also examined the binding free energies of SARS-CoV-2 spike protein across a range of species and demonstrated they were negative, supporting the view that the binding of SARS-CoV-2 spike protein to hACE2 was higher than any other species examined. 17 It also suggested that for ACE2 species within an upper affinity range, a correlation exists between binding free energies and infectivity, a fact critical in understanding the effect of mutations and contemplating pharmacological strategies to halt the infection.

The interaction between a virus and host cell involves an interaction of thermodynamic evolution with time. 2 It follows that for SARS-CoV-2 infection, the adsorption of the spike protein to the ACE2 receptor is the initiating event necessary for subsequent cellular replication of the virus. The synchrony between binding and subsequent replication drives the host cell infection. The two critical elements in that process are both characterized by negative GFE and as such are thermodynamically spontaneous. It has been proposed that there is a greater spontaneous virus multiplication rate which produces an enhanced reservoir of virus and permits greater population transmission. 12 It is the thermodynamics that is likely to determine much of the binding of the SARS-CoV-2 to the host. However, it is the negative GFE that determines the stability of the protein-ligand complex and, as such, the binding affinity of a ligand to a receptor. 6 It is this binding energy that is converted to the dissociation constant, measured by the binding of the spike protein for ACE2. 7 As highlighted by Du et al., the ratio of the kinetic parameters (k on and k off) determines the thermodynamic properties including the stability of the complex and the binding affinity between the protein and ligand. 6 In this way, the thermodynamic nature (GFE) of the adsorption of SARS-CoV-2 to the receptor domain of ACE2 is integrated with the rate kinetics embodied in the law of mass action. In doing so, they draw attention to the complexity of the SARS-CoV-2 spike protein binding that is related to the size and shape of the protein interaction. Regardless, the binding of the spike proteinbinding domain for ACE2 has been well documented, 18, 19 and the K D values for high affinity binding to ACE2 protein were determined to be in the order of 3.0 nM 20 to 4.6 nM. 19 The ability to quantify the binding of the SARS-CoV-2 spike protein to ACE2, the triggering event for infectivity, is the most powerful measure in COVID-19 disease for three reasons:

1. Outcome prediction. The extent of the initial binding of SARS-CoV-2 spike protein to ACE2 establishes the pattern by which subsequent linked processes quantitatively follow. To that extent, it is not unreasonable to assume that the kinetics of the initial binding of SARS-CoV-2 to ACE2 obey the laws of mass action and, as such, the kinetics of that initial binding step will predict the cellular and/or tissue potency of this virus. Support for this view comes from a comparison between the binding of SARS-CoV-2 and the less infectious SARS-CoV as it is commonly seen that the affinity of the SARS-CoV-2 RBD is greater than that for the SARS-CoV RBD. This is precisely the same phenomenon of a predictive role of initial ligand binding already demonstrated in the cancer field, where initial in vitro biochemical binding affinities predict the concentration range in which kinase inhibitors will be active in intact cells. 21 In addition, as discussed by Walls et al., the rate of viral replication in distinct species, transmissibility, and disease severity correlate with the binding affinity of SARS-CoV-2 for hACE2. 22 Moreover, the binding affinity of the spike protein for ACE2 has been suggested to be a major determinant of SARS-CoV-2 replication and disease severity. 23 It is fortuitous that the measurement of affinity by way of the measurement Kd has underpinned drug development for decades and that experience in predicting potency and comparative potencies is transferrable to characterizing the binding of SARS-CoV-2 and its variants to the ACE2 receptor. 

It is this combination of cellular and host tropism, together with the thermodynamics and kinetics of the binding of SARS-CoV-2 to ACE2 that underpins the development of COVID-19. Previous experience with the influenza A virus illustrates how viruses must change their tropism to preferentially target a new species. 27 An appreciation of these determinates requires an understanding of the nature of SARS-CoV-2 tropism in the human, the molecular basis that defines that viral tropism and the degree to which these structural features provide the platform for thermodynamic spontaneity.

The efficiency of SARS-CoV-2 in recognizing binding to a host receptor determines the preference of this virus for a given species, tissue or cell type. 28 The lethality and targeting of hosts by SARS-CoV-2 is driven by simplicity, namely the ability to manipulate and change a very small portion of its viral surface such that it can infect a susceptible host. Rawat et al. demonstrated, in a comparison between three coronaviruses that bind to ACE2, that the interface surface area of the spike protein for the ACE2 complex was smaller for HCoV-NL63 than that of SARS-CoV and SARS-CoV-2, concluding that the mild HCoV-NL63 has less binding affinity than the two other strains. 29 As indicated by Zhang et al. the binding affinities for ACE2 are clinically relevant, in the nanomolar region from 5 to 95 nM. 30 Additionally, the strong interaction between ACE2 and the SARS-CoV-2 spike protein is a characteristic of this virus that is reflected in its high transmissibility rate, infectivity, and global spread. 30, 31 As accentuated by Zhao et al. viral spike protein recognition is the key determinant of the host range. 32 In a similar fashion, Liu et al.

provided data indicating a broad host tropism for SARS-CoV-2. 33 Within humans a significant cellular tropism with SARS-CoV-2 exists. Importantly, the hACE2 protein is expressed on many different cell types and thus, ACE2 is present in many organs including blood vessels, the lung, heart, kidney, testis, placenta, gastrointestinal tract, and brain, all of which are, therefore, potential targets for SARS-CoV-2 infection. 30 This extensive target disposition provides the opportunity for very broad-based human cellular tropism.

The molecular basis for understanding SARS-CoV-2 tropism is fundamental in predicting what cells in the human are prone to infection and this information informs the pathophysiology of COVID-19 disease. It is also highly likely that SARS-CoV-2 tropism largely determines the nature of the progression of COVID-19 in the human. This follows from the observation that SARS-CoV-2 infection is associated with early nasopharyngeal viral shedding raising the possibility of a tropism to the throat with this virus. 34 Much has been written on the structural biological characteristics of the interaction of the SARS-CoV-2 spike glycoprotein with hACE2 and the focus here is briefly on the molecular features of this interaction that underpin SARS-CoV-2 tropism.

SARS-CoV-2 appears well suited to binding to hACE2 in that five amino acid changes on the SARS-CoV-2 spike glycoprotein are associated with the natural selection for critical binding sites (L455, F486, Q493, S494, N501) and responsible for the high tropism with hACE2. 28 However, it is becoming apparent that the precise nature of the interaction of the amino acid residues on the SARS-CoV-2 spike protein depends on its configuration. There is accumulating evidence suggesting that the RBD of SARS-CoV-2 can oscillate between an "up" or a "down" configuration. 30, 35 In the "down" position the RBD is associated with ineffective receptor binding, immune evasion 35 and the interaction with ACE2 inhibited with stearic inhibition. 30 In the "up" position greater than 16 amino acids in the RBD interact with hACE2. 30 The change in RBD conformation between the open and closed states in SARS-CoV-2 occurs with exposure of the binding interface to ACE2, which causes the rotational motion of the whole RBD. Additionally, whereas SARS-CoV and SARS-CoV-2 both have flexible regions in their RBD, SARS-CoV-2 also has flexible regions within the binding interface, which favor or disfavor binding. 29 It is important to recognize that SARS-CoV-2 infection is dependent not only on the virus binding to ACE2 but the assistance of the pro-protein convertase furin in the polybasic cleavage at the junction of S1 and S2. This allows the subsequent cleavage of the S2 site by TMPRSS2, which exposes the internal fusion motif peptide that is required for membrane fusion. 30 This pre-activation of SARS-CoV-2, unlike that of SARS-CoV reduces its dependence on target cell proteases for entry. 5 Although the efficiency of furin in SARS-CoV-2 S protein cleavage may be a distinguishing feature of SARS-CoV-2 in being more aggressive than other coronaviruses, 30 the TMPRSS2 proteins have high homology across hosts and therefore would not appear to be involved in host selectivity. 36 However, an important throat tropism seen with SARS-CoV-2 and not SARS-CoV may be due to the presence of the polybasic furin cleavage site at the S1/S2 junction present in the SARS-CoV-2 virus. 34 It follows that when viewing SARS-CoV-2 infection within the human, the affinity and high suitability for ACE2 binding on cells and tissues together with the furin mediated efficiency are the fundamental determinants of tropism. The co-location of TMPRSS2 is a necessary attending requirement for this tropism to be functional.

The amino acids involved in these protein interactions determine the thermodynamic potentials. 7

The association of the virus ligand with the ACE2 receptor is determined by the extent of the negative GFE value and this in turn determines the stability of the complex or the binding affinity of the ligand.

Using homology modelling based on atomic details, Sakkiah et al. determined the interactions between the trimeric spike protein and ACE2 and demonstrated that the spike protein binds tightly with ACE2 with an estimated binding free energy of −60.54 kcal/mol. 37 It follows that the properties associated with SARS-CoV-2 tropism are associated with a negative GFE value for the binding of this virus to its ACE2 receptor.

humans is insightful regarding the free energy of binding of this virus to the ACE2 target. Although at least seven human coronaviruses have been identified, three (HCoV-NL63, SARS-CoV, and SARS-CoV-2) display binding to ACE2. It is noteworthy that the spike protein binding region of HCoV-NL63 has a low sequence identity with SARS-CoV and SARS-CoV-2. 29 Of importance, SARS-CoV and SARS-CoV-2 displayed a high sequence identity of about 73% using multiple sequence alignment analysis. 29 The difference in binding affinity between SARS-CoV and SARS-CoV-2 offers important insights into the thermodynamic spontaneity and SARS-CoV-2 tropism. For example, He et al. have reported that the binding free energy of the SARS-CoV-2 RBD-ACE2 interaction is −50.43 kcal/mol, which is lower than that of the SARS-CoV RBD-ACE2 interaction (−36.75 kcal/mol), consistent with the higher binding affinity of SARS-CoV-2 for ACE2. 38 In addition, the binding free energy contributions indicate that this higher binding affinity is due to the solvation energy contribution.

Shang et al. indicated that the SARS-CoV-2 RBD has a higher binding affinity to hACE2 than the SARS-CoV RBD and these differences are related to structural features that change the type of bonds between the RBD and ACE2. 5, 7 As described earlier, the RBD of SARS-CoV-2 can oscillate between an "up" or a "down" configuration and the less exposed entire SARS-CoV-2 spike has comparable or lower binding than SARS-CoV due to this lower exposure. It follows that the significant free binding energy of SARS-CoV-2 is related to these conformational dynamics. Overcoming the energy barrier associated with this conformational change would be expected to facilitate the binding of SARS-CoV-2 to ACE2 and thereby subsequent entry into the host cell. 7

In summary, although knowledge of prevailing viral and host tropism is extremely important, it becomes very powerful when combined with the measures of receptor kinetics. It is this combination of cellular and host tropism, thermodynamics and kinetics that drives the temporal and spatial characteristics of COVID-19.

As discussed, SARS-CoV-2 cellular and host tropism, thermodynamics and kinetics are the fundamentals that underpin COVID-19 disease. However, alone they are insufficient to drive the passage of SARS-CoV-2 from the host reservoir to the human.

In viewing the efficient passage of a virus from one species to another, the following three considerations deserve attention.

There must exist a shared highly conserved viral binding target Focus has been drawn to bat species by virtue of their ability to accommodate many viruses including zoonotic coronaviruses and to harbor more zoonotic pathogens than any other known mammalian species. 39 Bats can transmit viruses within-host with minimal pathology 40 and can display ACE2 receptor viral binding. 41 Recent emerging viral disease outbreaks including Hendra, Nipah, Marburg, Ebola, SARS, and MERS have been linked to batborne viruses. 39 Although it is believed that SARS-CoV-2 may infect bats, direct evidence has been lacking and the molecular basis is still not fully understood. 42 It is important to consider that there are ap- Moreover, there is at least a 79.6% shared genome sequence identity between the two viruses as well as ACE2. 43 As highlighted by Irving et al., SARS-CoV-2 is thought to have ancestral origins in bats. 39 Finally, understanding the relationships between hosts and viruses in bats provides the potential to gain important insights into the mechanisms used to avoid the pathology from virulent pathogens.

The passage of SARS-CoV-2 from one species to another is the genesis for the spatial development of COVID-19 disease in humans.

Woolhouse et al. showed that a potential determinant of whether a virus will transit from one species to another is its ability to use a common receptor which is conserved across both species. 44 The ability of the virus to recognize the host binding site dictates the preference of the virus for both species and tissues. 28 The high rates of mutation and recombination lead to variability in RNA viruses consistent with a more frequent jump between species than with other pathogens.

As mentioned earlier, the initiating event with SARS-CoV-2 viral infection involves the binding of the SARS-CoV-2 spike protein with ACE2. In 2010 Yu et al. highlighted that ACE2 proteins is highly conserved across mammalian species and a group of key amino acid residues are associated with the susceptibility of ACE2 to SARS-CoV infection. 45 Consistent with the predeterminants outlined by residues of ACE2 and viral binding propensity. 46 An extremely broad host range for SARS-CoV-2 may be a consequence of the conservation of ACE2 in mammals. 46 Damas et al. noted that only mammals were defined by the medium to very high categories and that vertebrate classes other than mammals are not likely to be hosts for the virus. 46 Furthermore, in exploring the evolution of ACE2 variation in vertebrates, it was concluded that the major ACE2 codons are significantly conserved, possibly reflecting the critical function of the renin-angiotensin system. 46 Moreover, they observed that 10 residues in the ACE2 binding domain are exceptionally conserved in the Chiroptera (bat) family.

Based on an analysis of 70 ACE2 placental orthologues and using 30 critical ACE2 binding sites, Fam et al. concluded that there existed a high diversity of ACE2 between mammalian species. 28 The broad host range in mammals is also dramatically illustrated in the bat. In contrast to a single human species, there are approximately 1400 species of bat, with ACE2 receptor viral usage, that is species dependent. 47 Fam et al.'s comprehensive study of binding and infection assays examined 46 ACE2 orthologues from phylogenetically diverse bat species found that even closely related bat species showed different ACE2 proteins with some failing to support infection by either SARS-CoV or SARS-CoV-2.

Human variation in ACE2 in the population is rare and intolerant to loss of function mutations. 46 This is in stark contrast to other mammals that display a broad host range of ACE2 binding propensity for SARS-CoV-2 as discussed above. Importantly the studies of 

The inflammatory response plays a key role in pathogen-driven infective disease. Inflammation can serve as a protective response on the one hand but if dysregulated can act to enhance pathophysiology.

Thus, host regulation of the physiological consequences of viral infection is an important determinant in viral infection and spread. The view that the bat is believed to host more zoonotic pathogens than any other known mammalian species may be a consequence, in part, of the bats' ability to regulate host infection to prevent excessive immune-driven pathology and a failure to display the clinical signs of inflammatory-based disease when infected by viruses. 39 Intriguingly, bats may limit viral load with the antiviral cytokine interferon-alpha (IFNα) which predictably in mammals would normally be associated with inflammation; however, in bat adaption has apparently curtailed this inflammatory response. In a key study using viral dynamics, Brook et al. explored the transmission rates of IFN-mediated immunity in bat cell lines that displayed either a constitutive or induced IFN response. 40 They demonstrated that cells were protected from mortality with the antiviral state induced by the IFN pathway and suggested that the enhanced IFN capabilities achieve a more rapid within-host transmission rate without pathology. Similarly, mammalian cells will induce the secretion of type I IFN proteins (IFNα and IFNβ), which affect the expression of interferon-stimulated genes (ISGs) in bystander cells, promoting an antiviral response and a predicted harmful immune inflammation. 40 By way of summary, the bat may limit its viral load by way of antiviral cytokines and concurrently regulate the predicted inflammatory response through adaption. In contrast, lower respiratory serious COVID-19 disease is associated with severe cytokine-driven inflammation.

Since a lower respiratory cytokine storm is a hallmark of serious COVID-19 disease, it is not surprising that focus has centered upon the NLRP3 inflammasome response with SARS-CoV-2 infection. 18 There is general agreement that in serious COVID-19 disease, dysregulation of the angiotensin (Ang) II/AT 1 R (Ang II type 1 receptor) and Ang-(1-7) axis precipitates a hyper-inflammatory state.

There are possible links between the angiotensin-based dysfunction and the NLRP3 inflammasome response in SARS-CoV-2. The role of SARS-CoV-2 in interacting with ACE2 and escalating the host response to infection into a dysregulated uncontrolled inflammatory response has been highlighted recently. 53 In pulmonary tissue, Ang II binds to the AT 1 R and activates the NLRP3 inflammasome through intermediates. 54 Hyperactivation of showing that bat ACE2s were less efficient overall than the hACE2 in relation to the susceptibility of SARS-CoV entry. 58 This lower efficiency of bat ACE2 (bACE2) compared with hACE2 may also occur with SARS-related CoVs (SARSr-CoVs). As highlighted by Guo et al., the bat R. sinicus carries SARSr-CoVs, and they demonstrated that the SARSr-CoV spike proteins had a higher binding affinity to hACE2 than to bACE2. 60 They provided additional evidence to suggest that SARSr-CoV co-evolved with the host R. sinicus for a long time. 60 It also suggests the possibility of infection into humans is, therefore, possible.

Previous experience with the single-stranded RNA influenza A virus with its high mutation rate provides valuable insights into interspecies viral transmission. For example, the role of a change in viral tropism in interspecies transmission that occurs with a shift in receptor binding specificity of the influenza A virus is mutation determined. 27 It has been the molecular insights into the haemagglutinin (HA) viral glycoproteins that bind to sialylated host cell receptors (and mediates membrane fusion) that have provided insights into the ability of several of the influenza A virus subtypes to jump from avian to human hosts. 27 It is evident that the same broad principles relating to a shift in receptor binding characteristics and viral tropism with interspecies transmission may be applicable to SARS-CoV-2 infection.

A key study used the structural analysis of the interfaces of the SARS-CoV RBD and host receptors to determine the principles that govern host adaptions and cross-species infections and, importantly, the ability of SARS-CoV to engage ACE2. 61 

The coronaviruses SARS-CoV-1 and MERS are thought to have used palm civets (Paguma larvata) and dromedary camels (Camelus dromedarius), respectively, as intermediary hosts 63 and are thought to have played a key role in transmission to humans. 64 The possibility of a similar bridge host from natural reservoir host to humans is open to speculation; however, with regard to the importance of a cross-species affinity gradient it deserves focus. It has been suggested that horseshoe bats (R. affinis) seem to be natural reservoir hosts. 65 The studies highlighted above point to the existence of a very significant variation in susceptibility to SARS-CoV and SARS-CoV-2 by way of binding to the ACE2 receptor. The available evidence thus points to the possibility of a higher affinity of SARS-CoV-2 binding to hACE2 than the equivalent binding in a natural reservoir, suggesting that the interspecies transmission for SARS-CoV-2 adheres to a pattern similar to that described for the influenza A virus, namely a change in tropism occurring with a shift in receptor-binding specificity. It is this potential gradient in affinity of binding that may provide the thermodynamic spontaneity for the passage of SARS-CoV-2 from the natural reservoir to the human in COVID-19 disease (Figure 3 ).

This discussion highlights the importance of the thermodynamic changes that occur with SARS-CoV-2 spike protein binding to hACE2 as an initiating or triggering event in infectivity associated with COVID-19. However, as well as thermodynamics, there may be other complexities coming to light that explain the pivotal role of the RBD-ACE2 interaction, including, at least in bats, the fact that SARS-CoV may use an alternative receptor to ACE2. 67 

The relentless drive underpinning the infection of a single cell, the passage from reservoir vectors to a new host, the success of viral variants and the infectious spread globally in a pandemic is a thermodynamics-based complex system. The clinical properties of COVID-19 display a time-based evolution with three following timebased phases. (i) The initial 1 to 2 days of infection with an asymptomatic state, (ii) followed by an upper airway and then a lower airway response, and (iii) progression for those with serious illness to acute respiratory distress syndrome (ARDS) and multiorgan failure. 68 The evolution in a complex system has been described previously as time-based thermodynamic evolution. 2 F I G U R E 3 Gibbs free energy of binding and K d (affinity constant) for viral binding of SARS-CoV-2 to ACE2 as potential faciliatory mediators of species jump. Illustration of a potential gradient underpinning the interspecies transmission for SARS-CoV-2 based on favorable thermodynamic spontaneity due to a higher affinity of the virus for hACE2. As indicated in the text, this pattern is not dissimilar to that described for influenza A, with a change in tropism and a shift in receptor binding specificity F I G U R E 4 A schematic representation of a mucosal gradient for the passage of SARS-CoV-2 from the upper respiratory tract mucosal airway intersection to the epithelial cell-bound ACE2 target. The polybasic cleavage site at the junction of S1 and S2 present in SARS-CoV-2 likely provides for efficient cell entry and removal from the mucosal layer, thus ensuring that the concentration of the virus will be minimal compared with that which may be present at the mucosal airway surface. The figure also highlights the importance of Gibbs free energy in both the diffusion of SARS-CoV-2 across the mucosal layers and the subsequent interaction with its ACE2 receptor

The major mechanism of SARS-CoV-2 transmission in humans is by way of infected respiratory droplets and/or aerosols with nasopharyngeal viral shedding very early in COVID-19 disease. Santos et al. drew attention to the observation that nasal swabs from patients with COVID-19 display higher viral loads than do throat swabs, inferring a potential role of the nasal epithelium as an entry point for infection and transmission. 69 

One of the remarkable distinguishing features between SARS-CoV and SARS-CoV-2 is the degree to which they infect the upper respiratory airways in the human. SARS-CoV-2 is more efficient in its 74 One hypothesis to explain this extension of tropism is the presence of a polybasic furin cleavage site at the S1-S2 junction within SARS-CoV-2 but not SARS-CoV, with the potential that it may lead to a gain-of-fusion. 34 Earlier, we highlighted that the SARS-CoV-2 RBD can exist in an "up" or "down" configuration. The balance between having high infectivity as well as limiting the immune accessibility of the SARS-CoV-2 RBD is achieved by using host protease activation. 75 

The first interaction between an individual and airborne SARS-CoV-2 is at the mucosal surface in the upper respiratory tract. This These are localized adjacent to the tethered mucins within the PCL with a gradient in infectivity from the upper to lower respiratory tract. However, the extent to which SARS-CoV-2 can infect the multiciliated airway epithelial cells is determined by the effectiveness of an additional diffusion gradient, namely, a diffusional gradient spanning the mucosal barrier from the air interface to the underlying ACE2 enriched multiciliate airway epithelial cells.

The following processes will influence that diffusion:

The binding affinity of SARS-CoV-2 for ACE2 is viewed as one of the contributors to COVID-19. As mentioned earlier, the binding free energy for the SARS-CoV-2 RBD-ACE2 interaction is approximately 24 kcal/mol more negative than that for SARS-CoV, a virus that displays minimal interaction with the upper respiratory tract. 38 Moreover, the polybasic cleavage site at the junction of S1 and S2 present in SARS-CoV-2 and not SARS-CoV, provides for efficient cell entry mediated by furin and TMPRSS2. In kinetic terms, one can anticipate that in the region of the nasal epithelium, the high affinity of SARS-CoV-2 for ACE2 and the efficiency of the extracellular removal of the virus is a powerful combination that in kinetic terms (high affinity and product removal) facilitates the passage of this virus across the mucosal layers. This combination is expressed as SARS-CoV-2 tropism for the upper respiratory tract.

As indicated earlier the diameter of SARS-CoV-2 is approximately 100 nm and the thickness of the mucus layer that covers the epithelial cells in the airways is in the range of 7 to 70 µm 83 suggesting that SARS-CoV-2 has to traverse a barrier some 70 to 700 times its diameter to be within the region of the epithelial cell tethered ACE2 receptor.

If the passage of the virus is impeded in the mucosal layer and the diffusion of virus particles is not sufficient to achieve an equilibrium concentration across the mucus layers including the PCL adjacent to the epithelial bound ACE2 target, then a gradient will be established that is analogous to the well-described pharmacological agonist concentration gradients (Figure 4) . Of significance is that the nonequilibrium state implies that there is free energy available to do work and that this viral mucosal migration can be described by the

It should be noted that the passage of a virus particle across the mucosal layers is not totally assured despite this high affinity and efficiency. This follows from the fact that mucins may serve as binding sites for pathogens and may, through steric hindrance, also modulate the binding of a virus to its epithelial cell bound receptor. 84 In this context, understanding the nature of glycosylation of the virus and the mucins is fundamental. Likewise, the S2 subunit N-linked glycosylation is also conserved. 85 In contrast to bacteria where the glycans are encoded by the bacterial genome, the glycosylation of SARS-CoV-2 is the product of a previous mammalian host's cellular glycosylation processes. As At the epithelial surface, glycans may play an additional role in the docking of SARS-CoV-2 with ACE2. The ACE2 target has been reported to have seven N-glycosylation sites and several Oglycosylation sites and importantly, the glycan at N322 interacts tightly with the bound spike protein. 86 Collectively, these suggest a role for glycosylation in the binding of SARS-CoV-2 to its receptor.

It is the SARS-CoV-2/ACE2 binding efficiency that determines SARS-CoV-2 transmissibility. 85 SARS-CoV-2 but not SARS-CoV-1 infection is directed toward the upper respiratory tract. Based on the similarity of glycan shielding for both viruses, it seems unlikely that this difference in infectivity is due to differences in the ability of both viruses to diffuse across the mucosal layers. It seems rather more likely to be due to the 10-to-20-fold higher target binding affinity for SARS-CoV-2 for the ACE2 receptor. 85 It is this higher binding affinity coupled with the efficient furin facilitated cellular fusion and the removal of the virus at the surface of the epithelium that drives the glycan shielded SARS-CoV-2 to cross the mucosal layers. In this way, with exposure, the concentration of the virus particles will be minimal at the epithelial mucosal layer interface and higher within the mucosal bilayer.

The mucosal viral gradient driven by viral tropism and receptor affinity is the first critical event in infection of humans with SARS-CoV-2. It is the opportunity to enhance the efficiency of this process that is the primary objective of mutant variants of SARS-CoV-2 ( Figure 4 ).

CoV-2 mutants and hACE2 to determine the effect of naturally occurring RBD mutations on receptor binding affinity and infectivity. 87 Of importance was the observation that the GFE (ΔG) of the V367F mutant was significantly lower (approximately 13 kJ/mol) than that of the original wild-type strain. Moreover, the affinity constant (K D ) of the wild-type RBD was reported to be about two orders of magnitude higher than the K D of the V367F mutant (14.7 and 0.11 nM, respectively) indicating an increased affinity of the mutant for hACE2. That is, compared with the K D (14.7 nM) of the prototype RBD, the K D of the V367F mutant was 0.11 nM, which is two orders of magnitude lower than for the prototype strain, indicating an increased affinity to hACE2. Subsequently, it has been established that mutations within the RBD focus on key sites (K417, L452, E484, N501) which enable the spike protein to avoid antibody neutralization and concurrently maintain or enhance binding to ACE2. 88 A comprehensive description of the nature and significance of spike protein mutations as they relate to SARS-CoV-2 has been described in detail by Winger and Caspari, with relatively small changes in RBD having profound effects on viral infectivity. 88 Evidence for increased infectious titers in nasal washes but not the lungs of hamsters infected with SARS-CoV-2 expressing spike D614G support the clinical data that these mutations enhanced viral loads in the upper respiratory tract of patients with COVID- 19. 89 There is growing evidence that SARS-CoV-2 variants bind ACE2 with Additionally, there is increasing evidence that the SARS-CoV-2 variants of concern have higher viral loads, longer viral shedding time, and shorter incubation periods. 93 It is the extent to which changes in GFE with variants favors the passage of SARS-CoV-2 and its variants across the mucosal gradient and the changing mutational-driven efficiency in binding to the ACE2 receptor that is the bedrock for the initial dynamics and seriousness of COVID-19 disease in humans ( Figure 5 ).

As highlighted above, COVID-19 disease is the activation of a complex system that can be described by a time-based thermodynamic evolution. 2 The initial site for entry of SARS-CoV-2 is the mucosal surface. This is followed by diffusion across the mucosal bilayers, attachment to the ACE2 receptor, and entry within the nasal epithelium. The immediate events that follow upper airway infection dictate whether this disease is either mild or asymptomatic or alternatively progresses to serious disease.

What is generally accepted is the initial high viral loads in the upper respiratory tract suggesting a high viral RNA shedding potential for transmission 94 and this pharyngeal shedding occurs at a time when symptoms are mild. 34 What is at first consideration surprising is the absence of an early and significant nasopharyngeal An alternative explanation for a dampened nasal response comes from kinetic studies in bat cells on the transmission rates of IFNmediated immunity, where it was suggested that it is possible to F I G U R E 5 Gibbs free energy of binding and Kd (affinity constant) for enhanced ACE2 binding and infectivity of SARS-CoV-2 variants. A schematic representation illustrating how the greater affinity of SARS-CoV-2 variants lowers the concentration of virus particles required to complete the passage across the mucosal barrier and bind with epithelial cell-bound ACE2 and achieve successful infectivity. The schematic also portrays the efficiency of variant (▲) spread throughout bystander cells driven by repeated thermodynamic spontaneity as a consequence of the availability of Gibbs free energy (ΔG) associated with viral target binding. Wild type SARS-CoV-2 is schematically represented by the circular virus achieve a more rapid host transmission rate without the pathology. 40 It is highly likely that this delayed inflammatory response prolongs viral replication 95 and would explain why asymptomatic patients with COVID-19 disease have significant viral loads and can infect others.

Consistent with this view are the findings of Mick et al. that the IL-1 and NLRP3 inflammasome pathways were nonresponsive to SARS-CoV-2 consistent with impaired neutrophil and macrophage recruitment, explaining high viral load prior to symptom onset. 104 This nasal airway regulatory response is also entirely consistent with earlier studies with rhinovirus that demonstrated regional differences in epithelial airway cells and in particular, a trade-off between viral defense and oxidative stress protection detailed by Mihaylova et al. 105 These authors demonstrated that nasal cells display a predominately IFN response, whereas bronchial cells exhibit a predominant oxidative stress response.

The potential activation of the NLRP3 inflammasome in response to SARS-CoV-2 infection has been discussed in detail and it is noteworthy that one of the pathways includes the loss of a capacity to hydrolyze Ang II by virtue of the SARS-CoV-2/ACE2 interaction and, consequently, the activation of the NLRP3 inflammasome. 18 It is likely that this NLRP3 mediated dampened response adheres to a broader response pattern for two reasons. Firstly, blunted activation of the IL-1 and NLRP3 pathways is associated with an asymptomatic course of infection with human influenza challenge. 104 Secondly, and of particular significance, is the demonstration that the NLRP3 inflammasome is dampened in bat primary immune cells compared with those of human or mouse and this dampened response does not affect viral load. 51 In addition to the nasopharyngeal tropism outlined earlier, SARS-CoV-2 has used suppression of the predominately IFN-driven upper airway response to enable greater viral titers with minimal inflammatory responses and greater asymptotic infectiveness in the human.

It has been suggested that the virus migrates down the respiratory tract with the triggering of a more robust innate immune response and about 80% of those infected will have mild disease restricted to the upper respiratory tract and conducting airways. 106 It could be argued that on viral shedding and luminal release of the virus that inhalation may lead to infection of alveolar cells by way of SARS-CoV-2 binding to ACE2. In a detailed analysis, Hou et al. drew attention to the importance of oral-lung aspiration as a major contributor to many lower airway infectious diseases and the combination of muco-ciliary clearance, accumulation of a bolus with viral titer in the oral cavity followed by aspiration to the lower lung. 82 After progression to the lower respiratory tract, the SARS-CoV-2-mediated targeting and impairment of ACE2 function is fundamental in the progression to serious disease in the lower respiratory tract for two reasons. Firstly, ACE2 is localized on the alveolar type II cells that are responsible for the production of surfactants, stabilization of the epithelial barrier, immune defense and regeneration following injury. 70 With significant impairment of these cells by SARS-CoV-2 using ACE2 as the cellular target, the stage is set for the progression to ARDS. This progression has been well covered in the literature and involves the hyper-inflammatory response associated with the "cytokine storm" associated with the initial exudative 

The principal mechanism of SARS-CoV-2 infection is via respiratory droplets that contact nasal, conjunctival or oral mucosa. 107 From the preceding discussions, it is apparent that SARS-CoV-2 has a higher affinity for ACE2, and the presence of the furin cleavage site imparts a high level of efficiency for entry of SARS-CoV-2 into cells containing the ACE2 receptor and predictably simultaneously reduce the con- Each of these binding events, as discussed earlier, is described by the GFE considerations that determine the stability, binding affinity of the SARS-CoV-2 and binding energy that is reflected in the thermodynamic spontaneity.

Accordingly, it is not unreasonable to assume that the epidemiological characteristics of the COVID-19 pandemic must, in part or fully reflect the fundamental characteristics of SARS-CoV-2 target binding, assembly, and organization that occurs in a single cell, tissues or collectively in the human. Support for this view comes from several sources. Firstly, Ghanbari et al. assumed that the spread of F I G U R E 6 Schematic representation illustrating the progression of SARS-CoV-2-mediated COVID-19 disease from mild or asymptomatic infection to either full recovery or serious illness. Highlighted is viral self-assembly at scale and population spread is mediated by upper respiratory tract infection. The fundamental pathophysiology of serious COVID-19 disease is driven by lower respiratory tract infection and does not favor viral self-assembly at scale COVID-19 is a thermodynamic system focused on entropy (a measure of the disorder of a system) and used this to predict behavior and model COVID-19 propagation. 110 Secondly, Lucia et al., building on the thermodynamics of complex systems, also focused on entropy as the function to determine the evolution of the infectious disease and the time of spread. 111 They reasoned that the spread of infection can be examined as an open thermodynamic system and in doing so, they focused only on the Gibbs entropy shape. Moreover, they were able to demonstrate that the model has been confirmed for the COVID-19 pandemic.

What is apparent from these considerations is that the measure of entropy predicts outcomes at a population level -it is the thermodynamic interplay between entropy and enthalpy that governs the spontaneity of infection of a single cell with SARS-CoV-2.

There has been a significant focus on identifying therapeutic agents that would be of benefit in treating patients with COVID-19 disease.

Generally, this has often involved identifying on or off-target (repurposed) existing and approved drugs that may be useful in COVID- 19. This has been well described by Sultana et al., where they classified the approaches into those that inhibit key steps in the SARS-CoV-2 life cycle (viral replication, virion assembly/release) or those that counteract the effects of infection and the attending inflammation (anti-inflammatory and immunomodulating drugs). 112 This is an important area that is to be encouraged and one on which we have commented upon previously. 113, 114 The one cautionary area that we suggest needs further discussion relates to the initial dampened IFN response with the suppressed immune response in the nasal passage at the very commencement of infection. It could be argued that this initial dampened inflammatory response facilitates viral replication, bystander cell spread and viral shedding. It follows that further suppression of inflammation through therapeutics may not achieve the desired outcome at this initial stage of infection but may do so immediately after symptoms become apparent.

An advantage in exploring the thermodynamic spontaneity, mass action, and tropism of SARS-CoV-2 in COVID-19 disease is that it has the potential to highlight the areas of vulnerability of this virus and to identify areas of intervention. From the considerations discussed in this review, the passage of SARS-CoV-2, from its shedding from the epithelium in the nasopharyngeal tissues of an infected individual to its attachment to ACE2 on the nasal epithelial cells in a new host, represents an area of viral vulnerability with respect to infectivity. It is self-evident that social isolation and the wearing of masks interferes with the aerosol mediated passage of the virus from infected individual to new host. An additional important approach has been suggested by Hou et al. and involves therapeutic strategies that decrease viral titers in the nasal tissue early in the progression of the disease. 82 The key focus of this type of intervention is to prevent viral seeding of the lower respiratory tract with its attending serious disease. In a similar fashion, Bridges et al. have suggested that because the ciliated cells in the sino-nasal airway are an initial infection site, this is where treatments should be designed to block infection and limit viral propagation. 96 Additionally, Hou et al. have suggested strategies that involve nasal lavages, topical antivirals, or immune modulation. 82 Although it is beyond the scope of this review to detail the potential therapeutic approaches, the role of mucosal IgA deserves comment. IgA protects the epithelial cell barriers from pathogens and is active against rotavirus, poliovirus, influenza virus and SARS-CoV-2. 115 As pointed out by Russell et al., the first interactions that occur between SARS-CoV-2 and the immune system must occur at the respiratory mucosal surface, and they propose there is a significant role for mucosal immunity and for secretory as well as circulating IgA antibodies. 79 We hypothesize that the upper respiratory tract nasopharyngeal mucosal interface may represent a potential novel therapeutic and immunological target for preventing progression to serious disease in COVID-19.

Epidemiological characteristics of the COVID-19 pandemic reflect repeated SARS-CoV-2 target binding at enormous scale. Illustrates how entropy considerations predict outcomes not only at a cellular level but across infected populations reflecting the enormity of the repetition scale of thermodynamic spontaneity associated with the high affinity of SARS-CoV-2 for the ACE2 target in single cells, in multiple cells, in organs and tissues and across multiple cells in populations

The passage of SARS-CoV-2 within humans and across human populations displays the key characteristics of a complex system that underpins COVID-19 disease. This complex system is powerfully described by the thermodynamic considerations underpinning the physiological and pharmacological properties involved in SARS-CoV-2, from infection of a single cell to its global spread (Figure 7) .

It is the synchrony of the simplicity of the viral surface change together with the liberation of GFE causing thermodynamic spontaneity that fundamentally drives COVID-19 disease.

This understanding of thermodynamic spontaneity, mass action, and tropism provides the key platform to observe and understand the severity of disease that SARS-CoV-2 has on a human. This platform enables an 

We thank Professor Linfa (Lin-Fa) WANG, PhD FTSE FAAM, Duke-NUS Medical School, Singapore 169857 for helpful discussions on SARS-CoV-2.

The authors have no conflicts of interest to declare.

No funding was received for this article

The quest for system-theoretical medicine in the COVID-19 era

A critical review on heat and mass transfer modelling of viral infection and virion evolution: the case of SARS-COV2

Studying SARS-CoV-2 infectivity and therapeutic responses with complex organoids

Complex systems biology

Structural basis of receptor recognition by SARS-CoV-2

Insights into protein-ligand interactions: mechanisms, models, and methods

COVID-19: insights into virus-receptor interactions

An estimation of the number of cells in the human body

Science forum: SARS-CoV-2 (COVID-19) by the numbers

Thermodynamic aspects of drug-receptor interactions

Physics of viral dynamics

Thermodynamic insight into viral infections 2: empirical formulas, molecular compositions and thermodynamic properties of SARS, MERS and SARS-CoV-2 (COVID-19) viruses

The coronavirus spike protein is a class I virus fusion protein: structural and functional characterization of the fusion core complex

Thermodynamics of proteinligand interactions: history, presence, and future aspects

In silico investigation of critical binding pattern in SARS-CoV-2 spike protein with angiotensinconverting enzyme 2

Structural analysis of COVID-19 spike protein in recognizing the ACE2 receptor of different mammalian species and its susceptibility to viral infection

In silico comparison of SARS-CoV-2 spike protein-ACE2 binding affinities across species and implications for virus origin

The NLRP3 inflammasome and COVID-19: activation, pathogenesis and therapeutic strategies

Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptor

Engineered ACE2 receptor traps potently neutralize SARS-CoV-2

Features of selective kinase inhibitors

Structure, function, and antigenicity of the SARS-CoV-2 spike glycoprotein

SARS-CoV-2 entry factors are highly expressed in nasal epithelial cells together with innate immune genes

SARS-CoV-2 D614G spike mutation increases entry efficiency with enhanced ACE2-binding affinity

SARS-CoV-2 receptor binding mutations and antibody mediated immunity

SARS-CoV-2 B. 1.1. 7 and B. 1.351 spike variants bind human ACE2 with increased affinity

Enabling the'host jump': structural determinants of receptor-binding specificity in influenza A viruses

ACE2 diversity in placental mammals reveals the evolutionary strategy of SARS-CoV-2

Why are ACE2 binding coronavirus strains SARS-CoV/SARS-CoV-2 wild and NL63 mild? Proteins: Structure, Function

Molecular mechanism of interaction between SARS-CoV-2 and host cells and interventional therapy

Thermodynamics of the interaction between the spike protein of severe acute respiratory syndrome Coronavirus-2 and the receptor of human angiotensin-converting enzyme 2. Effects of possible ligands

Glycans of SARS-CoV-2 spike protein in virus infection and antibody production

Human coronavirus-229E,-OC43,-NL63, and-HKU1 (Coronaviridae). Encyclopedia of Virol

Virological assessment of hospitalized patients with COVID-2019

Differences and similarities between SARS-CoV and SARS-CoV-2: spike receptor-binding domain recognition and host cell infection with support of cellular serine proteases

SARS-CoV-2 host tropism: an in silico analysis of the main cellular factors

Elucidating interactions between SARS-CoV-2 trimeric spike protein and ACE2 using homology modeling and molecular dynamics simulations

Molecular mechanism of evolution and human infection with SARS-CoV-2

Lessons from the host defences of bats, a unique viral reservoir

Accelerated viral dynamics in bat cell lines, with implications for zoonotic emergence

ACE2 receptor usage reveals variation in susceptibility to SARS-CoV and SARS-CoV-2 infection among bat species

Functional and genetic analysis of viral receptor ACE2 orthologs reveals a broad potential host range of SARS-CoV-2

Severe acute respiratory syndrome (SARS) related coronavirus in bats

Human viruses: discovery and emergence

Identification of key amino acid residues required for horseshoe bat angiotensin-I converting enzyme 2 to function as a receptor for severe acute respiratory syndrome coronavirus

Broad host range of SARS-CoV-2 predicted by comparative and structural analysis of ACE2 in vertebrates

Many bat species are not potential hosts of SARS-CoV and SARS-CoV-2: Evidence from ACE2 receptor usage

Isolation of SARS-CoV-2-related coronavirus from Malayan pangolins

Pathological changes in masked palm civets experimentally infected by severe acute respiratory syndrome (SARS) coronavirus

Inflammasomes and pyroptosis as therapeutic targets for COVID-19

Dampened NLRP3-mediated inflammation in bats and implications for a special viral reservoir host

Age-induced NLRP3 inflammasome over-activation increases lethality of SARS-CoV-2 pneumonia in elderly patients

The interacting physiology of COVID-19 and the renin-angiotensin-aldosterone system: key agents for treatment

Mir-21 mediates the inhibitory effect of Ang (1-7) on AngII-induced NLRP3 inflammasome activation by targeting Spry1 in lung fibroblasts

SARS-CoV-2 entry receptor ACE2 is expressed on very small CD45− precursors of hematopoietic and endothelial cells and in response to virus spike protein activates the Nlrp3 inflammasome

The angiotensin-(1-7)/Mas receptor axis protects from endothelial cell senescence via klotho and Nrf2 activation

Sex-related overactivation of NLRP3 inflammasome increases lethality of the male COVID-19 patients

Angiotensin-converting enzyme 2 (ACE2) proteins of different bat species confer variable susceptibility to SARS-CoV entry

Mutations derived from horseshoe bat ACE2 orthologs enhance ACE2-Fc neutralization of SARS-CoV-2

Evolutionary arms race between virus and host drives genetic diversity in bat SARS related coronavirus spike genes

Structural analysis of major species barriers between humans and palm civets for severe acute respiratory syndrome coronavirus infections

Cross-species recognition of SARS-CoV-2 to bat ACE2

SARS-CoV-2 jumping the species barrier: zoonotic lessons from SARS, MERS and recent advances to combat this pandemic virus

SARS-CoV-2 in animals: potential for unknown reservoir hosts and public health implications

minks and other animals-villains or victims of SARS-CoV-2?

The Rhinolophus affinis bat ACE2 and multiple animal orthologs are functional receptors for bat coronavirus RaTG13 and SARS-CoV-2

Pan-sarbecovirus neutralizing antibodies in BNT162b2-immunized SARS-CoV-1 survivors

Defensive properties of mucin glycoproteins during respiratory infections-relevance for SARS-CoV-2

In nasal mucosal secretions, distinct IFN and IgA responses are found in severe and mild SARS-CoV-2 infection

Alveolar epithelial cell type II as main target of SARS-CoV-2 virus and COVID-19 development via NF-Kb pathway deregulation: a physio-pathological theory

SARS-CoV-2 and the pathophysiology of coronavirus disease 2019 (COVID-19)

SARS-CoV-2, SARS-CoV, and MERS-CoV viral load dynamics, duration of viral shedding, and infectiousness: a systematic review and metaanalysis

Asymptomatic transmission, the achilles' heel of current strategies to control Covid-19

Mechanisms of infection by SARS-CoV-2, inflammation and potential links with the microbiome

Cell entry mechanisms of SARS-CoV-2

COVID-19, SARS and MERS: are they closely related?

Back to the future: lessons learned from the 1918 influenza pandemic

The central role of the nasal microenvironment in the transmission, modulation, and clinical progression of SARS-CoV-2 infection

Mucosal immunity in COVID-19: a neglected but critical aspect of SARS-CoV-2 infection

The interaction between respiratory pathogens and mucus

ACE2 localizes to the respiratory cilia and is not increased by ACE inhibitors or ARBs

SARS-CoV-2 reverse genetics reveals a variable infection gradient in the respiratory tract

Quantifying diffusion in mucosal systems by pulsed-gradient spin-echo NMR

Mucin signature as a potential tool to predict susceptibility to COVID-19

The role of the SARS-CoV-2 S-protein glycosylation in the Interaction of SARS-CoV-2/ACE2 and immunological responses

Dual nature of human ACE2 glycosylation in binding to SARS-CoV-2 spike

V367F mutation in SARS-CoV-2 spike RBD emerging during the early transmission phase enhances viral infectivity through increased human ACE2 receptor binding affinity

The spike of concern-the novel variants of SARS-CoV-2

Spike mutation D614G alters SARS-CoV-2 fitness

Threat Assessment Brief: Emergence of SARS-CoV-2 B.1.617 variants in India and situation in the EU/EEA

How Dangerous Is the Delta Variant

A heavily mutated SARS-CoV-2 variant exhibits stronger binding to ACE2 and potently escape approved COVID-19 therapeutic antibodies

SARS-CoV-2 variants with shortened incubation periods necessitate new definitions for nosocomial acquisition

Upper respiratory tract levels of severe acute respiratory syndrome coronavirus 2 RNA and duration of viral RNA shedding do not differ between patients with mild and severe/critical coronavirus disease 2019

Gene expression profiling reveals the shared and distinct transcriptional signatures in human lung epithelial cells infected with SARS-CoV-2, MERS-CoV, or SARS-CoV: potential implications in cardiovascular complications of COVID-19

Respiratory epithelial cell responses to SARS-CoV-2 in COVID-19

Immune determinants of COVID-19 disease presentation and severity

Imbalanced host response to SARS-CoV-2 drives development of COVID-19

Untuned antiviral immunity in COVID-19 revealed by temporal type I/III interferon patterns and flu comparison

Roles of type I and III interferons in COVID-19

Differential roles of interferons in innate responses to mucosal viral infections

Impaired local intrinsic immunity to SARS-CoV-2 infection in severe COVID-19. bioRxiv

The interferon landscape along the respiratory tract impacts the severity of COVID-19

Upper airway gene expression reveals suppressed immune responses to SARS-CoV-2 compared with other respiratory viruses

Regional differences in airway epithelial cells reveal tradeoff between defense against oxidative stress and defense against rhinovirus

Pathogenesis of COVID-19 from a cell biology perspective

Virology, transmission, and pathogenesis of SARS-CoV-2

One year of COVID-19 pandemic: case fatality ratio and infection fatality ratio. A Systematic Analysis of 219 Countries and Territories

The total number and mass of SARS-CoV-2 virions

Mathematical prediction of the spreading rate of COVID-19 using entropy-based thermodynamic model

Entropy-based pandemics forecasting

Challenges for drug repurposing in the COVID-19 pandemic era

A pharmacological framework for integrating treating the host, drug repurposing and the damage response framework in COVID-19

Buying time: drug repurposing to treat the host in COVID-19H

IgA antibodies and IgA deficiency in SARS-CoV-2 infection

Systems analysis shows that thermodynamic physiological and pharmacological fundamentals drive COVID-19 and response to treatment