View of Role of ACE2 in Infection by Corona Viruses

(1)

Role of ACE2 in Infection by Corona Viruses

Rahul Dev Ambedkar, Amar P. Garg*, Payal Mago** , Megha Gahlawat***

School of Biological Engineering and Life Sciences, Shobhit Institute of Engineering &

Technology, Deemed to-be-University, NH-58, Modipuram, Meerut-250110, India

** Principal Shaheed Rajguru College of Applied Sciences for Women, Vasundhara Enclave, Delhi-110096

*** School of Biological Engineering and Life Sciences, Shobhit Institute of Engineering &

Technology, Deemed to-be-University, NH-58, Modipuram, Meerut-250110, India

“^*Corresponding author: [email protected] / [email protected]”

Introduction

It is the beginning of 21st century when the species of SARS-CoV start spreading the deadly pneumonia into in humans (Drosten et al., 2003, Ksiazek et al., 2003), Middle- East respiratory syndrome corona virus (Zaki et al., 2012) (MERS-CoV), and SARS- CoV-2 (Huang et al., 2020, Zhu et al., 2020).

It was in China, Guangdong where SARS-CoV came to the fore in 2002, spreading via air, infecting 8,098 people and claiming 774 lives in five continents MERS-CoV followed shortly after in 2012 in Arabian Peninsula, expanding to 27 countries infecting 2494 individuals and causing 858 death To this date, it is a weighty health perturbation in the Middle-Eeast. Erstwhile unexplored corona virus, SARS-CoV-2 was ascertained in China, Wuhan (2019) and later sequenced in January 2020 (Zhou et al., 2020, Zhu et al., 2020). Right now SARS-CoV-2 is linked with an underway outbreak of an aberrant pneumonia (Covid-2019) which has affected 123,902,242 people including 2,727,837 deaths as of 9:07 am CET, 25 March 2021 according to WHO, which also proclaimed it as a public health emergency of international concern.

Although dromedary camels acted as a reservoir host unfurling the infection to humans but MERS-CoV was propounded to have emanated from bats. (Haagmans et.

al., 2014, Memish et al., 2013). SARS-CoV and SARS-CoV-2 are conveniently related to each other and originated in bats (Ge et al., 2013, Hu et al., 2017, Li et al., 2005b, Yang et al., 2015a, Zhou et al., 2020). Palm civets and racoon dogs on the other hand have been acknowledged as intermediary hosts for zoonotic conveyance of SARS-CoV between bats and humans (Guan et al., 2003, Kan et al., 2005, Wang et al., 2005), although recognition of multiple SARS-related corona viruses in bats put

(2)

forward the possibility of continuation of zoonotic transmissions (Anthony et al., 2017, Ge et al., 2013, Hu et al., 2017, Li et al., 2005b, Menachery et al., 2015, Menachery et al., 2016, Yang et al., 2015a, Zhou et al., 2020).

Four unhackneyed low morbific corona viruses are aboriginal in humans: HCoV- OC43, HCoV-HKU1, HCoV-NL63, and HCoV-229E besides highly infective SARS- CoV, MERS-CoV, and SARS-CoV-2, all members of β-corona virus genus.

Transmissibility

Reproductive numbers of SARS-CoV-2, SARS-CoV and MERS-CoV are 2–3.58, 1.7–1.9 and < 1, respectively (Wu P, Hao X, EHY L, Wong JY, KSM L, Wu JT, Cowling BJ, Leung GM, 2020) show the level of transmissibility, which increased for SARS-CoV-2 to 5.7 later due to upswing of cases ubiquitously around the world (Sanche S, Lin YT, Xu C, Romero-Severson E, Hengartner N, Ke R., 2020).

Transference by proximate contact between people via air droplets or contaminated veneers of devices (Olsen SJ, Chang HL, Cheung TY, Tang AF, Fisk TL, Ooi SP, Kuo HW, Jiang DD, Chen KT, Lando J, et al., 2003;Otter JA, Donskey C, Yezli S, Douthwaite S, Goldenberg SD, Weber DJ., 2016).

Spate of SARS-CoV in Hong Kong suggested transmission through airborne (Yu IT, Qiu H, Tse LA, Wong TW., 2014). Another outbreak proposed fecal contamination &

feco-oral transmission (Peiris JS, Chu CM, Cheng VC, Chan KS, Hung IF, Poon LL, Law KI, Tang BS, Hon TY, Chan CS, et al., 2003). Person to person transmission is the main event for MERS-CoV but conceptually MERS-CoV can be spreading through stool, vomitus, urine, serum and cerebrospinal fluid of infected individuals (Memish ZA, Perlman S, Van Kerkhove MD, Zumla A., 2020). In uttermost cases, a few rare pieces of evidence show maternal-fetal transmission of SARS-CoV-2 (Egloff C, Vauloup-Fellous C, Picone O, Mandelbrot L, Roques P., 2020). Recently enough, SARS-CoV-2 was found in gastrointestinal tissues alongside sputum, urine, blood/serum, ocular surface, saliva and aerosol as well (Wang W, Xu Y, Gao R, Lu R, Han K, Wu G, Tan W.,2020; Pan Y, Zhang D, Yang P, Poon LLM, Wang Q., 2020;

Xiao F, Tang M, Zheng X, Liu Y, Li X, Shan H.,2020; Lu CW, Liu XF, Jia ZF.,2020;To KK, Tsang OT, Leung WS, Tam AR, Wu TC, Lung DC, Yip CC, Cai JP, Chan JM, Chik TS, et al.,2020; Zhang R, Li Y, Zhang AL, Wang Y, Molina MJ.,2020). Of course we should be very careful while handling the samples in order to avoid any mishappening.

(3)

Structure & Genome

Showing a particular structure of a fringe like a spike onto the surface under electron microscope, the corona virus name was derived as this fringe take after solar corona (Masters, 2006). With an average diameter of about 80-120 nm, these viral organisms are curvilinear, having spikes projecting out to 17-20 nm with a swollen base of nigh 10 nm (Masters, 2006).

Figure 1: Diagrammatic view of corona virus (Source: Molecular biology of corona viruses: current knowledge I. Made Artika,a,b,∗ Aghnianditya Kresno Dewantari,c and Ageng Wiyatnoc)

There are about 4 major constructional proteins which are coded by Corona virus genome: the nucleocapsid (N) protein, the spike (S) protein, the membrane (M) protein & the envelope (E) protein, every single one is crucial to structure &

reproductive cycle of the virus in discussion. In other cases of a few corona viruses, not all the proteins are required to make a complete vicious viral structure. This stipulates how a few constructional proteins are inessential or code for indemnifiable functions (Schoeman and Fielding, 2019). As suggested by the figure shown, 3-4 main proteins are contained by viral envelope out of which important ones are S and

(4)

M. Hemagglutinin esterase (HE) is a third substantial envelope protein found in a few corona viruses. Nevertheless , E protein makes up a significant part of the viral envelope despite being minor (de Haan et al., 1999). Talking about post translational changes endured by Viral structural proteins help in playing title roles in regulating folding, stability, enzymatic activity, subcellular localization and interaction of the viral protein with other proteins (Fung and Liu, 2018). These changes go by change in protein anatomy by making disulfide bonds and cleaving with the help of proteases or increasing the chemical stockpile of amino acids by ushering in new functional groups via phosphorylation, glycosylation and lipidation (such as palmitoylation and myristoylation).

Coming to N protein,although it is responsible for establishing nucleoprotein by binding viral genome, it might not be needed for envelope formation but in a few corona viruses, it forms a fleeting expression of N protein helps in packaging and stabilizing and hence production of virus like particles by tempering of host cellular response to viral infection such as regulating the host cell cycle, affecting cell stress response, influencing the immune system, etc (Schoeman and Fielding, 2019). Even so, contemporary research shows the ability of N proteins being able to semble oligomers without binding to genomic RNA somehow. Thus, hypotheses about constitutive N protein oligomerization allowing the optimal loading of the genomic viral RNA into a ribonucleoprotein complex through the presentation of multiple viral RNA binding motifs came into existence (Cong et al., 2017)”. Into the bargain, Virus is able to fold its genome just like eukaryotic cells as it has a smaller structure & less space for uncondensed ribonucleoproteins as compared to other RNA viruses (Gui et al., 2017).

Concluding, SARS-CoV goes about for 3 functions: first, bringing the viral genome into the host cell crossing the plasma membrane; second, it sets out newly constructed genome orientation; third, guarding the genome unification during its passage between cells (Neuman and Buchmeier, 2016).

Spike Protein

A transmembrane spike (S) glycoprotein makes homotrimers obtruding out of the viral surface and facilitates the ingressing of the corona virus into the host cell (Tortorici and Veesler, 2019). Two in service proteins which bind to the host cell receptor (S1 subunit) & cause the fusion of viral and cellular membranes (S2 subunit) are possessed by S protein .S1 & S2 subunits continue to be in perfusion conformation non - covalently after being cleaved for multiple CoVs (Belouzard et

(5)

al., 2009, Bosch et al., 2003, Burkard et al., 2014, Kirchdoerfer et al., 2016, Millet and Whittaker, 2014, Millet and Whittaker, 2015, Park et al., 2016, Walls et al., 2016a).

S2 subunit contains the fusion machinery which is preserved by S1 subunit containing receptor binding domain(s) (Gui et al., 2017, Kirchdoerfer et al., 2016, Pallesen et al., 2017, Song et al., 2018, Walls et al., 2016a, Walls et al., 2017b, Yuan et al., 2017).

Additionally S is severed by host proteases at upstreamed S2 site situated next to fusion peptide (Madu et al., 2009, Millet and Whittaker, 2015). This chasm has been thought to actuate the protein through irrevocable configuration changes for membrane fusion (Belouzard et al., 2009, Heald-Sargent and Gallagher, 2012, Millet and Whittaker, 2014, Millet and Whittaker, 2015, Park et al., 2016, Walls et al., 2017b). Hence, as stated, infection and entry of corona virus into animal cells is a stepwise process which occurs only due to proper action of receptor binding and processing of S protein.

Furthermore, corona viruses use well defined domains accordingly within the S1 subunit to apprehend the entry receptores. There are several instances such as endemic human corona viruses OC43 and HKU1 fasten with the host cell surface by their S domain A (SA) to 5-N-acetyl-9-O-acetyl-sialosides present on glycoproteins (Hulswit et al., 2019, Tortorici et al., 2019, Vlasak et al., 1988). On the other hand, using domain A, MERS-CoV S recognizes non acetylated sialoside attachment receptors (Li et al., 2017, Park et al., 2019) that encourage domain B latching to entry receptor, dipeptidyl-peptidase 4 (Lu et al., 2013, Raj et al., 2013) whereas SARS-CoV use angiotensin-converting enzyme 2 (ACE2) and SB interrelation to infect the cells (Ge et al., 2013, Kirchdoerfer et al., 2018, Li et al., 2005a, Li et al., 2003, Song et al., 2018, Yang et al., 2015a).

Receptors

Moreover, ACE2 is a presiding receptor for SARS-CoV (Li, Wenhui, et al., 2003) along side DC-SIGN (CD209) and L-SIGN (CD209L) being employed as co- receptors for SARS-CoV (Gu J, Korteweg C.,2007). Furthermore, cellular receptor for MERS-CoV is dipeptidyl peptidase 4 (DPP4, also termed CD26), (Lu G, Hu Y, Wang Q, Qi J, Gao F, Li Y, Zhang Y, Zhang W, Yuan Y, Bao J, et al.,2013).

However ACE2 has natural higher accord for SARS-CoV-2 than to SARS-CoV (Wrapp D, Wang N, Corbett KS, Goldsmith JA, Hsieh CL, Abiona O, Graham BS, McLellan JS.,2020) but Christian et al. (Sigrist CJ, Bridge A, Le Mercier P.,2020) proposed SARS-CoV-2 could be substituting integrins as cell receptors which is deficient when it comes to legit experimental evidence. Although aiding corroboration

(6)

was put forward that CD147-SP could be an additional ingressing pathway for SARS-CoV-2 (Wang K, Chen W, Zhou Y-S, Lian J-Q, Zhang Z, Du P, Gong L, Zhang Y, Cui H-Y, Geng J-J, et al.,2020).

SARS-CoV, MERS-CoV and SARS-CoV-2 use serine protease TMPRSS2 &

endosomal cysteine proteases cathepsin B/L for spike protein priming, that is important for paving their way in host cells (Hoffmann M, Kleine-Weber H, Schroeder S, Krüger N, Herrler T, Erichsen S, Schiergens TS, Herrler G, Wu NH, Nitsche A, et al., 2020; Hoffmann M, Kleine-Weber H, Pöhlmann S.,2020).

Besides ACE2 has extensive assortment from respiratory tract, gastrointestinal tract, heart, kidney and olfactory neuroepithelium (Imai Y, Kuba K, Ohto-Nakanishi T, Penninger JM.,2010; Fodoulian L, Tuberosa J, Rossier D, Landis BN, Carleton A, Rodriguez I.,2020), aside from these DPP4 affects liver, thymus, prostate and bone marrow (Memish ZA, Perlman S, Van Kerkhove MD, Zumla A, 2020), repercussions in wide ranging cellular & tissue movement of SARS-CoV, MERS-CoV, and SARS- CoV-2 (Ding Y, He L, Zhang Q, Huang Z, Che X, Hou J, Wang H, Shen H, Qiu L, Li Z, et al., 2004; Widagdo W, Raj VS, Schipper D, Kolijn K, van Leenders G, Bosch BJ, Bensaid A, Segales J, Baumgartner W, Osterhaus A, et al.,2016; Wang W, Xu Y, Gao R, Lu R, Han K, Wu G, Tan W,2020). Summarising, these hCoVs are more than accoutered for producing an outspread range of symptoms.

Effect of SARS-CoV-2 Infection on Renin/Angiotensin System

It’s of utmost crucial understanding that ACE2 receptor is the point of entry of virus in the body, so studying it will give us the outline of the pathophysiological changes because of the SARS-CoV-2. Getting a hold of molecular downstream effects of Angiotensin can lead to the mapping of “multiple respiratory distress”, “myocardial injury”, “renal failure”, and “increased mortality” due to SARS-CoV-2 (Zhou et al., 2020; Zhu et al., 2020).

ACE Genes

ACE & ACE2 have 42% resemblance of amino acids as maintained by the study of sequences depicting ACE2 materialize because of gene duplication (Donoghue et al., 2000). Being present on chromosome Xp22, ACE2 possess 18 exons & 20 introns (Turner et al., 2002), also, type 1 membrane glycoprotein made up of 805 amino acids is coded by ACE2 itself (Marian, 2013). Domains on N-terminal & C-terminal which are functional carry out signaling task for peptide present on side of HEXXH zinc binding metalloprotease motif (catalytic domain) (Li et al., 2003; Cerdà-Costa and

(7)

Xavier Gomis-Rüth, 2014). While manifesting almost all tissues, ACE2 is mostly found in epithelial cells of alveoli along endothelial cells of blood capillaries, additionally, it is present in blood vessel rich organs like kidneys & lungs (Hamming et al., 2004; Tikellis and Thomas, 2012; Roca-Ho et al., 2017). Although variegated, ACE and ACE2 are related to the diseases such as diabetes , hypertension & stroke (Crackower et al., 2002; Ramachandran et al., 2008; Jang and Kim, 2012; Fehr and Perlman, 2015). Apart from this, both receptors have different functions despite being kindred in constitution, as in ACE2 is responsible for tackling with perilous consequences of ACE/RAS pathway through ACE2/Angiotensin (1-7)/MAS axis and ACE looks out for RAS system (Renin Angiotensin Aldosterone system). In the development of metabolic disorders, RAS plays an important role (de Kloet et al., 2010). ACE & renin bring out angiotensin II where Prorenin is the dronish herald of renin. After Prorenin is converted to renin by special proteins due to actuation of afferent arteriole’s Juxtaglomerular apparatus, it is released into blood breaking angiotensinogen in angiotensin (Ang) I, which is torpid physically & acts as a progenitor for Ang II due to catalytic activity of ACE. Ang II connects to arterioles, adrenal cortex, kidney, and the brain’s type II & type I (AT) receptors (Figure 1A).

While being detected in the upper respiratory system & lungs’ epithelium, ACE is majorly found in kidneys & lungs (Wakahara et al., 2007). There’s a complete body water, sodium hike along with soaring vascular tone due to RAS activation.

Endothelial injury (Watanabe et al., 2005), vasoconstriction (Gustafsson and Holstein- Rathlou, 1999), endovascular thrombosis (Tay and Lip, 2008) & increment in volume of blood are the results of Ang II self indulgence to AT receptors. Multiple signaling reactions are initiated at cellular level by Angiotensin II which encompass JNK/MAPK & PKC, serine/threonine kinase, ERK (Malhotra et al., 2001). Several different researchers claimed that IL-6 is prompted by Ang II & maybe “serine tyrosine kinases”,G protein coupled receptor activation”, ERK/JNK MAPK activation or interaction with mineralocorticoid receptors starting off TNF- α (Funakoshi et al., 1999; Han et al., 1999; Ruiz-Ortega et al., 2002; Luther et al., 2006). Activator of NADPH oxidase is Ang II as it is a catalyst of reactive oxygen species production (ROS) (Garrido and Griendling, 2009). Furthermore, macrophage & neutrophil fluidity to target cells is commenced by Ang II leading to vascular injuries (Kato et al., 1996; Nabah et al., 2004). Studies of Ang II as an efficient multipotent agent to increase endothelial injury, oxidative injury, cytokines etc have been done for it to not be impede with target tissues, although trials at a clinical level for swotting the

(8)

effect MABs against IL-6 receptor are going on (ClinicalTrials.gov Identifier:

NCT04317092).

A few definite number of tissues such as myocardium, lungs, brain, kidney and arterioles have proteases which are insensitive to ACE inhibitors which leads to the shortlisting of a few inhibitors in order to decrease Ang II levels for a less amount of time (Mento and Wilkes, 1987).

Besides the proteases mentioned are “neutral endopeptidase”, “tonin”, “tryptensin”,

“cathepsin G”, “kallikrein”, and “chymase: (Figure1A), these can cleave Ang I to Ang II (Kramkowski et al., 2006; Lorenz, 2010; Becari et al., 2011; Uehara et al., 2013).

Model animals had ACE2 articulation administered by Ang II receptor blocker (ARB) inhibitors and ACE (Ishiyama et al., 2004).

Safeguarding component of ARB & ACE is because of increment in number of ACE2 giving poor prognosis with higher viral load due to more receptors for entry of virus (Chu et al., 2004) connecting it with substitute pathways might play a part in structuring ANG II (Diaz, 2020).

Meanwhile, there have been clinical trials to study the upshots of ACE/ARB inhibitors related to SARS-CoV-2 (ClinicalTrials.gov Identifier: NCT04330300). If by chance there’s a different route to the making of Ang II, it is implausible that ACE/ARB inhibitors take part in clinical trail of SARS-CoV-2 infection. Ang II was minced into 1-7 a heptapeptide & Ang I into Ang 1-9 a nonapeptide by ACE2 (Santos et al., 2003; Marian, 2013). Multiple uses such as antiproliferative, vasodilatory, and protective by prompting MAS receptor (a G protein coupled receptor) are illustrated by heptapeptides & nonapeptides discussed previously (Donoghue et al., 2000;

Santos et al., 2003). Neutralization of the RAS system mechanism balancing ACE/Ang II/AT1 receptor axis’s derogatory outcomes is done by the axis of ACE2 / Ang / MAS1 (Santos et al., 2003). MAS1 or ACE2 receptor scarce mice depicted several cardiac & metabolic anomalies (Yamamoto et al., 2006; Tikellis and Thomas, 2012). Thus ACE2 is major in warding off the deleterious effects of Ang II (Fraga- Silva et al., 2010; Tikellis and Thomas, 2012).

Necropsy of people died due to SARS-CoV-2 infection showed severely impaired alveoli with capillary clogging alongside clotting in vessels (Menter et al., 2020). In association with MAS, ACE 2 produces Ang 1-7 which sets off multiple advantageous actions such as inhibition of cell growth, vasodilation,anti-thrombotic, anti arrhythmogenic and antifibrotic effects (le Tran and Forster, 1997; Schindler et al., 2007; Li et al., 2008) and as stated by other researchers, it also shows guardian effect on brain and protecting against ischemic stroke (Jiang et al., 2013).

(9)

Vaccine & Drug Development

Being surface exposed, S glycoprotein is counterpoised by antibodies(Abs) which is now th+e main focus of drug and vaccine development. S trimers are equipped with N-linked glycans which are crucial for folding (Rossen et al., 1998) and providing convenience to host proteases alongside neutralizing antibodies (Walls et al., 2016b, Walls et al., 2019, Xiong et al., 2018, Yang et al., 2015b). Earlier cogent human- neutralizing Abs were distinguished from scarce memory B cells of SARS-CoV (Traggiai et al., 2004) or MERS-CoV (Corti et al., 2015) patients to communicate molecular level understanding of competitive inhibition of SB attachment to the host receptor (Walls et al., 2019). However, ACE2 has a natural accord to SARS-CoV-2 than to SARS-CoV (Wrapp, Daniel, et al., 2020).

Manipulation of ACE2/Ang(1-7) and Protease Activity as Novel Therapeutic Targets

In view of the significant SARS-CoV-2 related periling factors for hospitalization and deaths among patients with metabolic diseases, including obesity, cardiovascular diseases, arterial hypertension, and diabetes that could depict overall activation of the RAS system, tweaking of RAS activation through the ACE2/(Ang1-7)/MAS pathway should be reviewed for treatment of this disease. Moreover, the clinical observation and published clinical data paint a unique clinical presentation of SARS-CoV-2 patients: many patients with relatively preserved hemodynamics and lack of lactic acidosis. Albeit they have respiratory distress, are in a hypercoagulable state “(Liu et al., 2020; Menter et al., 2020)”, show progressivekidney failure “(Cheng et al., 2020)”, have stroke-like characteristics and myocardial injury “(Zhou et al., 2020)”.

Clinical experimental studies pertain that in most cases the respiratory distress happen many days (in general about 14 days) after the infection, indicating that this may not be a direct effect of the initial viral infection albeit the hosts reaction to the forfeiture of function of ACE2 and misregulation of Ang II/ACE2 pathways as well prompting of host proteases. Our main hypothesis is that the binding of the corona virus spike protein to ACE2 results in shedding of ACE2 receptors vis various proteases, which in result in leads to the loss of preserving function of the ACE2/MAS axis in the lungs and other organs (Figure 1B). Furthermore to the loss of protective function of ACE2/MAS, prompting of classical pathway (ACE/RAS/Ang II) and different pathways through tissue specific proteases, including cathepsins, chymase-like proteases, leads to an excessive production of Ang II at the tissue level. These actions may further tilt the balance of protective Ang (1-7)/MAS and ACE2 function to the

(10)

harmful effects of increased Ang II contributing to lung epithelial and endovascular injury. Thus, installment of the downstream pathway of ACE2, by triggering the ACE2/Ang1-7/MAS axis could prove a meaningful strategy in averting lung and cardiovascular damage associated with SARS-CoV-2 infections. As less ACE2/MAS activity augments the Ang II/AT1R activity and its deleterious outcomes on increased pulmonary vascular endothelial/epithelial injury and lung pathology. Baring the activity of proteases important for splintering of viral spike proteins: for example hindrance of enzymatic activity of ADAM17 and TMPRSS2 could serve as other novel therapeutic targets. This could potently block viral interaction with the receptor and its entry into the cells. Recognising specific proteases and advancement of inhibitors targeting proteases important for cleavage of spike proteins could prove to be viable. In addition,using the protective effect of Ang1-7 or its analogs, such as AVE0991 AVE0991 “(Pinheiro et al., 2004)” against harmful effect of increased Ang II is feasible and could be effective for the symptomatic treatment of these patients.

Figure 1. Dysregulation of Ang II and Ang (1-7) by loss of protective function of ACE2 receptor. (A) under physiological condition there is a balance in ACE and ACE2 receptor activity. ACE regulates the Renin Angiotensin Aldosterone system (RAS) and cleaves Ang I to produce Ang II. Ang II is a potent vasoconstrictor and detrimental for endothelial and epithelial function through activating AT1 and AT2 receptors. The counterbalance of the RAS/Ang II output is regulated by ACE2 and

(11)

Mas/G protein coupled receptor activity. ACE2 cleaves Ang I and Ang II into Ang-1- 9 and Ang1-7, respectively, thereby it activates MAS/G protein coupled receptor that protect cell death. (B) SARS-CoV-2 binds to ACE2 to gain entry to epithelial cells of the lungs. Cleavage of spike proteins by a protease such as trypsin/cathepsin G and or ADAM17 on ectodomain and TMPRSS2 of endodomain sites facilitate viral entry into the cells. This process leads to shedding of host ACE2 receptors and loss of its protective function. Loss of function of ACE2 activity prevents production of Ang 1-9 and Ang1-7. Lack of Ang1-7 diminishes the activity of MAS/G receptor, leading to the loss of its protective functions including vasodilatation, cell protection both at the epithelial and endothelial sites. Loss of ACE2 function leads to an imbalance and unchecked effects of Ang II and upregulation of RAS/Ang II pathway. Upregulation of Ang II leads to vasoconstriction, thrombophilia, microthrombosis, alveolar epithelial injury and respiratory failure. Therefore, inhibiting the proteolytic function of trypsin/cathepsin and ADAM17 or TMPRSS2 and or direct activation of MAS/G receptor by enhancing Ang-(1-7) can overcome the loss of function ACE2 and are viable targets to prevent tissue damage to the host.

Source “(http://www.frontiersin.org/articles/10.3389/fcimb.2020.00317full)”

Another suggestion from the studies is that if mutations occur at the site of attachment of SARS-CoV-2 with ACE2 receptor, the vaccine will be doubtful.

So all the sites of attachment of SARS-CoV-2 in all the available strains should be studied.

References

1. ⦁ Rota, P. A., Oberste, M. S., Monroe, S. S., Nix, W. A., Campagnoli, R., Icenogle, J. P., ... & Bellini, W. J. (2003). Characterization of a novel corona virus associated with severe acute respiratory syndrome. science, 300(5624), 1394-1399.

2. ⦁ Zaki, A. M., Van Boheemen, S., Bestebroer, T. M., Osterhaus, A. D., &

Fouchier, R. A. (2012). Isolation of a novel corona virus from a man with pneumonia in Saudi Arabia. New England Journal of Medicine, 367(19), 1814- 1820.

3. ⦁ Wu, A., Peng, Y., Huang, B., Ding, X., Wang, X., Niu, P., ... & Jiang, T.

(2020). Genome composition and divergence of the novel corona virus (2019- nCoV) originating in China. Cell host & microbe, 27(3), 325-328.

(12)

4. ⦁ Pan, Y., Guan, H., Zhou, S., Wang, Y., Li, Q., Zhu, T., ... & Xia, L.

(2020). Initial CT findings and temporal changes in patients with the novel corona virus pneumonia (2019-nCoV): a study of 63 patients in Wuhan, China.

European radiology, 30(6), 3306-3309.

5. ⦁ Memish, Z. A., Cotten, M., Meyer, B., Watson, S. J., Alsahafi, A. J., Al Rabeeah, A. A., ... & Drosten, C. (2014). Human infection with MERS corona virus after exposure to infected camels, Saudi Arabia, 2013. Emerging infectious diseases, 20(6), 1012.

6. ⦁ Gao, Y., Yan, L., Huang, Y., Liu, F., Zhao, Y., Cao, L., ... & Rao, Z.

(2020). Structure of the RNA-dependent RNA polymerase from COVID-19 virus. Science, 368(6492), 779-782.

7. ⦁ Wang, M., Yan, M., Xu, H., Liang, W., Kan, B., Zheng, B., ... & Xu, J.

(2005). SARS-CoV infection in a restaurant from palm civet. Emerging infectious diseases, 11(12), 1860.

8. ⦁ Walls, A. C., Park, Y. J., Tortorici, M. A., Wall, A., McGuire, A. T., &

Veesler, D. (2020). Structure, function, and antigenicity of the SARS-CoV-2 spike glycoprotein. Cell, 181(2), 281-292.

9. ⦁ Wu, P., Hao, X., Lau, E. H., Wong, J. Y., Leung, K. S., Wu, J. T., ... &

Leung, G. M. (2020). Real-time tentative assessment of the epidemiological characteristics of novel corona virus infections in Wuhan, China, as at 22 January 2020. Eurosurveillance, 25(3), 2000044.

10. ⦁ Sanche S, Lin YT, Xu C, Romero-Severson E, Hengartner N, Ke R.

High contagiousness and rapid spread of severe acute respiratory syndrome corona virus 2. Emerg Infect Dis. 2020;26:1470–7.

11. ⦁ Olsen SJ, Chang HL, Cheung TY, Tang AF, Fisk TL, Ooi SP, Kuo HW, Jiang DD, Chen KT, Lando J, et al. Transmission of the severe acute respiratory syndrome on aircraft. N Engl J Med. 2003;349:2416–22.

12. ⦁ Otter JA, Donskey C, Yezli S, Douthwaite S, Goldenberg SD, Weber DJ. Transmission of SARS and MERS corona viruses and influenza virus in healthcare settings: the possible role of dry surface contamination. J Hosp Infect. 2016;92:235–50.

13. ⦁ Yu IT, Qiu H, Tse LA, Wong TW. Severe acute respiratory syndrome beyond Amoy gardens: completing the incomplete legacy. Clin Infect Dis.

2014;58:683–6.

14. ⦁ Peiris JS, Chu CM, Cheng VC, Chan KS, Hung IF, Poon LL, Law KI, Tang BS, Hon TY, Chan CS, et al. Clinical progression and viral load in a

(13)

community outbreak of corona virus-associated SARS pneumonia: a prospective study. Lancet. 2003;361:1767–72.

15. ⦁ Memish ZA, Perlman S, Van Kerkhove MD, Zumla A. Middle East respiratory syndrome. Lancet. 2020;395:1063–77.

16. ⦁ Wang W, Xu Y, Gao R, Lu R, Han K, Wu G, Tan W. Detection of SARS-CoV-2 in different types of clinical specimens. Jama. 2020;323:1843–4.

17. ⦁ Pan Y, Zhang D, Yang P, Poon LLM, Wang Q. Viral load of SARS- CoV-2 in clinical samples. Lancet Infect Dis. 2020;20:411–2.

18. ⦁ Xiao F, Tang M, Zheng X, Liu Y, Li X, Shan H. Evidence for Gastrointestinal Infection of SARS-CoV-2. Gastroenterology. 2020;158:1831–

3.

19. ⦁ Lu CW, Liu XF, Jia ZF. 2019-nCoV transmission through the ocular surface must not be ignored. Lancet. 2020;395:e39.

20. ⦁ To KK, Tsang OT, Leung WS, Tam AR, Wu TC, Lung DC, Yip CC, Cai JP, Chan JM, Chik TS, et al. Temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by SARS-CoV-2: an observational cohort study. Lancet Infect Dis.

2020;20:565–74.

21. ⦁ Zhang R, Li Y, Zhang AL, Wang Y, Molina MJ. Identifying airborne transmission as the dominant route for the spread of COVID-19. Proc Natl Acad Sci U S A. 2020;117:14857–63.

22. ⦁ Masters, P. S. (2006). The molecular biology of corona viruses.

Advances in virus research, 66, 193-292.

23. ⦁ Schoeman D., Fielding B.C. Corona virus envelope protein: current knowledge. Virol. J. 2019;16:1–22.

24. ⦁ de Haan C.A.M., Smeets M., Vernooij F., Vennema H., Rottier P.J.M.

Mapping of the corona virus membrane protein domains involved in interaction with the spike protein. J. Virol. 1999;73:7441–7452.

25. ⦁ Fung T.S., Liu D.X. Post-translational modifications of corona virus proteins: roles and function. Future Virol. 2018;13:405–443.

26. ⦁ Schoeman D., Fielding B.C. Corona virus envelope protein: current knowledge. Virol. J. 2019;16:1–22

27. ⦁ Cong Y., Kriegenburg F., de Haan C.A.M., Reggiori F. Corona virus nucleocapsid proteins assemble constitutively in high molecular oligomers. Sci.

Rep. 2017;7:1–10.

(14)

28. ⦁ Gui M., Liu X., Guo D., Zhang Z., Yin C.-C., Chen Y., Xiang Y.

Electron microscopy studies of the corona virus ribonucleoprotein complex.

Protein Cell. 2017;8:219–224.

29. ⦁ Neuman B.W., Buchmeier M.J. Supramolecular architecture of the corona virus particle. Adv. Virus Res. 2016;96:1–27.

30. ⦁ Tortorici, M. A., & Veesler, D. (2019). Structural insights into corona virus entry. In Advances in virus research (Vol. 105, pp. 93-116). Academic Press.

31. ⦁ Belouzard, S., Chu, V. C., & Whittaker, G. R. (2009). Activation of the SARS corona virus spike protein via sequential proteolytic cleavage at two distinct sites. Proceedings of the National Academy of Sciences, 106(14), 5871-5876.

32. ⦁ Walls, A. C., Park, Y. J., Tortorici, M. A., Wall, A., McGuire, A. T., &

Veesler, D. (2020). Structure, function, and antigenicity of the SARS-CoV-2 spike glycoprotein. Cell, 181(2), 281-292.

33. ⦁ Millet, J. K., & Whittaker, G. R. (2018). Physiological and molecular triggers for SARS-CoV membrane fusion and entry into host cells. Virology, 517, 3-8.

35. ⦁ Park, A., & Iwasaki, A. (2020). Type I and type III interferons–

induction, signaling, evasion, and application to combat COVID-19. Cell host

& microbe.

37. ⦁ Raj, V. S., Mou, H., Smits, S. L., Dekkers, D. H., Müller, M. A., Dijkman, R., ... & Haagmans, B. L. (2013). Dipeptidyl peptidase 4 is a functional receptor for the emerging human corona virus-EMC. Nature, 495(7440), 251-254.

38. ⦁ Ge, X. Y., Li, J. L., Yang, X. L., Chmura, A. A., Zhu, G., Epstein, J. H., ... & Shi, Z. L. (2013). Isolation and characterization of a bat SARS-like corona virus that uses the ACE2 receptor. Nature, 503(7477), 535-538.

39. ⦁ Kirchdoerfer, R. N., Wang, N., Pallesen, J., Wrapp, D., Turner, H. L., Cottrell, C. A., ... & Ward, A. B. (2018). Stabilized corona virus spikes are

(15)

resistant to conformational changes induced by receptor recognition or proteolysis. Scientific reports, 8(1), 1-11.

40. ⦁ Li, F., Li, W., Farzan, M., & Harrison, S. C. (2005). Structure of SARS corona virus spike receptor-binding domain complexed with receptor. Science, 309(5742), 1864-1868.

41. ⦁ Li, W., Moore, M. J., Vasilieva, N., Sui, J., Wong, S. K., Berne, M. A., ... & Farzan, M. (2003). Angiotensin-converting enzyme 2 is a functional receptor for the SARS corona virus. Nature, 426(6965), 450-454.

42. ⦁ Song, W., Gui, M., Wang, X., & Xiang, Y. (2018). Cryo-EM structure of the SARS corona virus spike glycoprotein in complex with its host cell receptor ACE2. PLoS pathogens, 14(8), e1007236.

43. ⦁ Yang, X. L., Hu, B., Wang, B., Wang, M. N., Zhang, Q., Zhang, W., ...

& Shi, Z. L. (2016). Isolation and characterization of a novel bat corona virus closely related to the direct progenitor of severe acute respiratory syndrome corona virus. Journal of virology, 90(6), 3253-3256.

44. ⦁ Gu, J., & Korteweg, C. (2007). Pathology and pathogenesis of severe acute respiratory syndrome. The American journal of pathology, 170(4), 1136- 1147.

45. ⦁ Lu G, Hu Y, Wang Q, Qi J, Gao F, Li Y, Zhang Y, Zhang W, Yuan Y, Bao J, et al. Molecular basis of binding between novel human corona virus MERS-CoV and its receptor CD26. Nature. 2013;500:227–31.

46. ⦁ Wrapp D, Wang N, Corbett KS, Goldsmith JA, Hsieh CL, Abiona O, Graham BS, McLellan JS. Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation. Science. 2020;367:1260–3.

47. ⦁ Sigrist CJ, Bridge A, Le Mercier P. A potential role for integrins in host cell entry by SARS-CoV-2. Antivir Res. 2020;177:104759.

48. ⦁ Wang K, Chen W, Zhou Y-S, Lian J-Q, Zhang Z, Du P, Gong L, Zhang Y, Cui H-Y, Geng J-J, et al. SARS-CoV-2 invades host cells via a novel route:

CD147-spike protein. bioRxiv. 2020; 2020.2003.2014.988345.

49. ⦁ Hoffmann M, Kleine-Weber H, Schroeder S, Krüger N, Herrler T, Erichsen S, Schiergens TS, Herrler G, Wu NH, Nitsche A, et al. SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor. Cell. 2020;181:271–80.

50. ⦁ Hoffmann M, Kleine-Weber H, Pöhlmann S. A Multibasic Cleavage Site in the Spike Protein of SARS-CoV-2 Is Essential for Infection of Human Lung Cells. Mol Cell. 2020;78:779–84.

(16)

51. ⦁ Imai Y, Kuba K, Ohto-Nakanishi T, Penninger JM. Angiotensin- converting enzyme 2 (ACE2) in disease pathogenesis. Circ J. 2010;74:405–10.

52. ⦁ Fodoulian L, Tuberosa J, Rossier D, Landis BN, Carleton A, Rodriguez I. SARS-CoV-2 receptor and entry genes are expressed by sustentacular cells in the human olfactory neuroepithelium. bioRxiv. 2020.

53. ⦁ Memish ZA, Perlman S, Van Kerkhove MD, Zumla A. Middle East respiratory syndrome. Lancet. 2020;395:1063–77.

54. ⦁ Ding Y, He L, Zhang Q, Huang Z, Che X, Hou J, Wang H, Shen H, Qiu L, Li Z, et al. Organ distribution of severe acute respiratory syndrome (SARS) associated corona virus (SARS-CoV) in SARS patients: implications for pathogenesis and virus transmission pathways. J Pathol. 2004;203:622–30.

55. ⦁ Zhou, F., Yu, T., Du, R., Fan, G., Liu, Y., Liu, Z., ... & Cao, B. (2020).

Clinical course and risk factors for mortality of adult inpatients with COVID- 19 in Wuhan, China: a retrospective cohort study. The lancet, 395(10229), 1054-1062.

56. ⦁ Donoghue, M., Hsieh, F., Baronas, E., Godbout, K., Gosselin, M., Stagliano, N., ... & Acton, S. (2000). A novel angiotensin-converting enzyme–

related carboxypeptidase (ACE2) converts angiotensin I to angiotensin 1-9.

Circulation research, 87(5), e1-e9.

57. ⦁ Turner, A. J., Tipnis, S. R., Guy, J. L., Rice, G. I., & Hooper, N. M.

(2002). ACEH/ACE2 is a novel mammalian metallocarboxypeptidase and a homologue of angiotensin-converting enzyme insensitive to ACE inhibitors.

Canadian journal of physiology and pharmacology, 80(4), 346-353.

58. ⦁ Marian, A. J. (2013). The discovery of the ACE2 gene. Circulation research, 112(10), 1307-1309.

59. ⦁ Li, W., Moore, M. J., Vasilieva, N., Sui, J., Wong, S. K., Berne, M. A., ... & Farzan, M. (2003). Angiotensin-converting enzyme 2 is a functional receptor for the SARS corona virus. Nature, 426(6965), 450-454.

60. ⦁ Cerdà‐Costa, N., & Xavier Gomis‐Rüth, F. (2014). Architecture and function of metallopeptidase catalytic domains. Protein Science, 23(2), 123- 144.

61. ⦁ Hamming, I., Timens, W., Bulthuis, M. L. C., Lely, A. T., Navis, G. V.,

& van Goor, H. (2004). Tissue distribution of ACE2 protein, the functional receptor for SARS corona virus. A first step in understanding SARS pathogenesis. The Journal of Pathology: A Journal of the Pathological Society of Great Britain and Ireland, 203(2), 631-637.

(17)

62. ⦁ Tikellis, C., & Thomas, M. C. (2012). Angiotensin-converting enzyme 2 (ACE2) is a key modulator of the renin angiotensin system in health and disease. International journal of peptides, 2012.

63. ⦁ Roca-Ho, H., Riera, M., Palau, V., Pascual, J., & Soler, M. J. (2017).

Characterization of ACE and ACE2 expression within different organs of the NOD mouse. International journal of molecular sciences, 18(3), 563.

64. ⦁ Crackower, M. A., Sarao, R., Oudit, G. Y., Yagil, C., Kozieradzki, I., Scanga, S. E., ... & Penninger, J. M. (2002). Angiotensin-converting enzyme 2 is an essential regulator of heart function. Nature, 417(6891), 822-828.

65. ⦁ Ramachandran, V., Ismail, P., Stanslas, J., Shamsudin, N., Moin, S., &

Mohd Jas, R. (2008). Association of insertion/deletion polymorphism of angiotensin-converting enzyme gene with essential hypertension and type 2 diabetes mellitus in Malaysian subjects. Journal of the Renin-Angiotensin- Aldosterone System, 9(4), 208-214.

66. ⦁ Jang, Y., & Kim, S. M. (2012). Influences of the G2350A polymorphism in the ACE Gene on cardiac structure and function of ball game players.

Journal of negative results in biomedicine, 11(1), 1-7.

67. ⦁ Fehr, A. R., & Perlman, S. (2015). Corona viruses: an overview of their replication and pathogenesis. Corona viruses, 1-23.

68. ⦁ de Kloet, A. D., Krause, E. G., & Woods, S. C. (2010). The renin angiotensin system and the metabolic syndrome. Physiology & behavior, 100(5), 525-534.

69. ⦁ Wakahara, S., Konoshita, T., Mizuno, S., Motomura, M., Aoyama, C., Makino, Y., ... & Miyamori, I. (2007). Synergistic expression of angiotensin- converting enzyme (ACE) and ACE2 in human renal tissue and confounding effects of hypertension on the ACE to ACE2 ratio. Endocrinology, 148(5), 2453-2457.

70. ⦁ Gustafsson, F., & Holstein-Rathlou, N. H. (1999). Angiotensin II modulates conducted vasoconstriction to norepinephrine and local electrical stimulation in rat mesenteric arterioles. Cardiovascular research, 44(1), 176- 184.

71. ⦁ Watanabe, T., Barker, T. A., & Berk, B. C. (2005). Angiotensin II and the endothelium: diverse signals and effects. Hypertension, 45(2), 163-169.

72. ⦁ Tay, K. H., & Lip, G. Y. (2008). What “Drives” the Link Between the Renin–Angiotensin–Aldosterone System and the Prothrombotic State in Hypertension?.

(18)

73. ⦁ Malhotra, A., Kang, B. P., Cheung, S., Opawumi, D., & Meggs, L. G.

(2001). Angiotensin II promotes glucose-induced activation of cardiac protein kinase C isozymes and phosphorylation of troponin I. Diabetes, 50(8), 1918- 1926.

74. ⦁ Funakoshi, Y., Ichiki, T., Ito, K., & Takeshita, A. (1999). Induction of interleukin-6 expression by angiotensin II in rat vascular smooth muscle cells.

Hypertension, 34(1), 118-125.

75. ⦁ Han, Y., Runge, M. S., & Brasier, A. R. (1999). Angiotensin II induces interleukin-6 transcription in vascular smooth muscle cells through pleiotropic activation of nuclear factor-κB transcription factors. Circulation research, 84(6), 695-703.

76. ⦁ Ruiz-Ortega, M., Ruperez, M., Lorenzo, O., Esteban, V., Blanco, J., Mezzano, S., & Egido, J. (2002). Angiotensin II regulates the synthesis of proinflammatory cytokines and chemokines in the kidney. Kidney International, 62, S12-S22.

77. ⦁ Luther, J. M., Gainer, J. V., Murphey, L. J., Yu, C., Vaughan, D. E., Morrow, J. D., & Brown, N. J. (2006). Angiotensin II induces interleukin-6 in humans through a mineralocorticoid receptor–dependent mechanism.

Hypertension, 48(6), 1050-1057.

78. ⦁ Garrido, A. M., & Griendling, K. K. (2009). NADPH oxidases and angiotensin II receptor signaling. Molecular and cellular endocrinology, 302(2), 148-158.

79. ⦁ Kato, H., Hou, J., Chobanian, A. V., & Brecher, P. (1996). Effects of angiotensin II infusion and inhibition of nitric oxide synthase on the rat aorta.

80. ⦁ Yamamoto, K., Ohishi, M., Katsuya, T., Ito, N., Ikushima, M., Kaibe, M., ... & Ogihara, T. (2006). Deletion of angiotensin-converting enzyme 2 accelerates pressure overload-induced cardiac dysfunction by increasing local angiotensin II. Hypertension, 47(4), 718-726.

81. ⦁ Kramkowski, K., Mogielnicki, A., & Buczko, W. (2006). The physiological significance of the alternative. J. Physiol. Pharmacol, 57, 529- 539.

82. ⦁ Lorenz, J. N. (2010). Chymase: the other ACE?.

83. ⦁ Becari, C., Oliveira, E. B., & Salgado, M. C. O. (2011). Alternative pathways for angiotensin II generation in the cardiovascular system. Brazilian Journal of Medical and Biological Research, 44(9), 914-919.

(19)

84. ⦁ Uehara, Y., Miura, S. I., Yahiro, E., & Saku, K. (2013). Non-ACE pathway-induced angiotensin II production. Current pharmaceutical design, 19(17), 3054-3059.

85. ⦁ Mento, P. F., & Wilkes, B. M. (1987). Plasma angiotensins and blood pressure during converting enzyme inhibition. Hypertension, 9(6_pt_2), III42.

86. ⦁ Ishiyama, Y., Gallagher, P. E., Averill, D. B., Tallant, E. A., Brosnihan, K. B., & Ferrario, C. M. (2004). Upregulation of angiotensin-converting enzyme 2 after myocardial infarction by blockade of angiotensin II receptors.

87. ⦁ Chu, C. M., Poon, L. L., Cheng, V. C., Chan, K. S., Hung, I. F., Wong, M. M., ... & Yuen, K. Y. (2004). Initial viral load and the outcomes of SARS.

Cmaj, 171(11), 1349-1352.

88. ⦁ Diaz, J. H. (2020). Hypothesis: angiotensin-converting enzyme inhibitors and angiotensin receptor blockers may increase the risk of severe COVID-19. Journal of travel medicine.

89. ⦁ Donoghue, M., Hsieh, F., Baronas, E., Godbout, K., Gosselin, M., Stagliano, N., ... & Acton, S. (2000). A novel angiotensin-converting enzyme–

related carboxypeptidase (ACE2) converts angiotensin I to angiotensin 1-9.

Circulation research, 87(5), e1-e9.

90. ⦁ Santos, R. A., e Silva, A. C. S., Maric, C., Silva, D. M., Machado, R. P., de Buhr, I., ... & Walther, T. (2003). Angiotensin-(1–7) is an endogenous ligand for the G protein-coupled receptor Mas. Proceedings of the National Academy of Sciences, 100(14), 8258-8263.

91. ⦁ Yamamoto, K., Ohishi, M., Katsuya, T., Ito, N., Ikushima, M., Kaibe, M., ... & Ogihara, T. (2006). Deletion of angiotensin-converting enzyme 2 accelerates pressure overload-induced cardiac dysfunction by increasing local angiotensin II. Hypertension, 47(4), 718-726.

92. ⦁ Tikellis, C., & Thomas, M. C. (2012). Angiotensin-converting enzyme 2 (ACE2) is a key modulator of the renin angiotensin system in health and disease. International journal of peptides, 2012.

93. ⦁ Menter, T., Haslbauer, J. D., Nienhold, R., Savic, S., Hopfer, H., Deigendesch, N., ... & Tzankov, A. (2020). Postmortem examination of COVID‐19 patients reveals diffuse alveolar damage with severe capillary congestion and variegated findings in lungs and other organs suggesting vascular dysfunction. Histopathology, 77(2), 198-209.

(20)

94. ⦁ Oudit, G. Y., Kassiri, Z., Jiang, C., Liu, P. P., Poutanen, S. M., Penninger, J. M., & Butany, J. (2009). SARS‐corona virus modulation of myocardial ACE2 expression and inflammation in patients with SARS.

European journal of clinical investigation, 39(7), 618-625.

95. ⦁ le Tran, Y., & Forster, C. (1997). Angiotensin-(1-7) and the rat aorta:

modulation by the endothelium. Journal of cardiovascular pharmacology, 30(5), 676-682.

96. ⦁ Schindler, C., Bramlage, P., Kirch, W., & Ferrario, C. M. (2007). Role of the vasodilator peptide angiotensin-(1–7) in cardiovascular drug therapy.

Vascular health and risk management, 3(1), 125.

97. ⦁ Li, X., Molina-Molina, M., Abdul-Hafez, A., Uhal, V., Xaubet, A., &

Uhal, B. D. (2008). Angiotensin converting enzyme-2 is protective but downregulated in human and experimental lung fibrosis. American Journal of Physiology-Lung Cellular and Molecular Physiology, 295(1), L178-L185.

98. ⦁ Jiang, T., Gao, L., Lu, J., & Zhang, Y. D. (2013). ACE2-Ang-(1-7)-Mas axis in brain: a potential target for prevention and treatment of ischemic stroke.

Current neuropharmacology, 11(2), 209-217.

99. ⦁ Rossen, J. W. A., De Beer, R., Godeke, G. J., Raamsman, M. J. B., Horzinek, M. C., Vennema, H., & Rottier, P. J. M. (1998). The viral spike protein is not involved in the polarized sorting of corona viruses in epithelial cells. Journal of virology, 72(1), 497-503.

100. ⦁ Walls, A. C., Tortorici, M. A., Frenz, B., Snijder, J., Li, W., Rey, F. A., ... & Veesler, D. (2016). Glycan shield and epitope masking of a corona virus spike protein observed by cryo-electron microscopy. Nature structural &

molecular biology, 23(10), 899.

101. ⦁ Walls, A. C., Xiong, X., Park, Y. J., Tortorici, M. A., Snijder, J., Quispe, J., ... & Veesler, D. (2019). Unexpected receptor functional mimicry elucidates activation of corona virus fusion. Cell, 176(5), 1026-1039.

102. ⦁ Xiong, X., Tortorici, M. A., Snijder, J., Yoshioka, C., Walls, A. C., Li, W., ... & Veesler, D. (2018). Glycan shield and fusion activation of a deltacorona virus spike glycoprotein fine-tuned for enteric infections. Journal of virology, 92(4).

103. ⦁ Yang, X. L., Hu, B., Wang, B., Wang, M. N., Zhang, Q., Zhang, W., ...

& Shi, Z. L. (2016). Isolation and characterization of a novel bat corona virus closely related to the direct progenitor of severe acute respiratory syndrome corona virus. Journal of virology, 90(6), 3253-3256.

(21)

104. ⦁ Yang, Y., Liu, C., Du, L., Jiang, S., Shi, Z., Baric, R. S., & Li, F. (2015).

Two mutations were critical for bat-to-human transmission of Middle East respiratory syndrome corona virus. Journal of virology, 89(17), 9119-9123.

105. ⦁ Traggiai, E., Becker, S., Subbarao, K., Kolesnikova, L., Uematsu, Y., Gismondo, M. R., ... & Lanzavecchia, A. (2004). An efficient method to make human monoclonal antibodies from memory B cells: potent neutralization of SARS corona virus. Nature medicine, 10(8), 871-875.

106. ⦁ Corti, D., Zhao, J., Pedotti, M., Simonelli, L., Agnihothram, S., Fett, C., ... & Lanzavecchia, A. (2015). Prophylactic and postexposure efficacy of a potent human monoclonal antibody against MERS corona virus. Proceedings of the National Academy of Sciences, 112(33), 10473-10478.

107. ⦁ Walls, A. C., Xiong, X., Park, Y. J., Tortorici, M. A., Snijder, J., Quispe, J., ... & Veesler, D. (2019). Unexpected receptor functional mimicry elucidates activation of corona virus fusion. Cell, 176(5), 1026-1039.

108. ⦁ Wrapp, D., Wang, N., Corbett, K. S., Goldsmith, J. A., Hsieh, C. L., Abiona, O., ... & McLellan, J. S. (2020). Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation. Science, 367(6483), 1260-1263.

109. ⦁ Liu, X., Li, Z., Liu, S., Chen, Z., Zhao, Z., Huang, Y. Y., ... & Luo, H. B.

(2020). Therapeutic effects of dipyridamole on COVID-19 patients with coagulation dysfunction. MedRxiv.

110. ⦁ Menter, T., Haslbauer, J. D., Nienhold, R., Savic, S., Hopfer, H., Deigendesch, N., ... & Tzankov, A. (2020). Postmortem examination of COVID‐19 patients reveals diffuse alveolar damage with severe capillary congestion and variegated findings in lungs and other organs suggesting vascular dysfunction. Histopathology, 77(2), 198-209.

111. ⦁ Cheng, Y., Luo, R., Wang, K., Zhang, M., Wang, Z., Dong, L., ... & Xu, G. (2020). Kidney disease is associated with in-hospital death of patients with COVID-19. Kidney international, 97(5), 829-838.

112. ⦁ Zhou, F., Yu, T., Du, R., Fan, G., Liu, Y., Liu, Z., ... & Cao, B. (2020).

Clinical course and risk factors for mortality of adult inpatients with COVID- 19 in Wuhan, China: a retrospective cohort study. The lancet, 395(10229), 1054-1062.

113. ⦁ Pinheiro, S. V. B., Simoes e Silva, A. C., Sampaio, W. O., de Paula, R.

D., Mendes, E. P., Bontempo, E. D., ... & Santos, R. A. S. (2004). Nonpeptide AVE 0991 is an angiotensin-(1–7) receptor Mas agonist in the mouse kidney.