Can ACE2 expression explain SARS-CoV-2 infection of the respiratory epithelia in COVID-19?

Infection with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) leads to coronavirus disease 2019 (COVID-19), which poses an unprecedented worldwide health crisis, and has been declared a pandemic by the World Health Organization (WHO) on March 11, 2020. The angiotensin converting enzyme 2 (ACE2) has been suggested to be the key protein used by SARS-CoV-2 for host cell entry. In their recent work, Lindskog and colleagues (Hikmet et al, 2020) report that ACE2 is expressed at very low protein levels-if at all-in respiratory epithelial cells. Severe COVID-19, however, is characterized by acute respiratory distress syndrome and extensive damage to the alveoli in the lung parenchyma. Then, what is the role of the airway epithelium in the early stages of COVID-19, and which cells need to be studied to characterize the biological mechanisms responsible for the progression to severe disease after initial infection by the novel coronavirus?

Angiotensin-converting enzyme 2 in severe acute respiratory syndrome coronavirus and SARS-CoV-2: A double-edged sword?

Human angiotensin-converting enzyme 2 (ACE2) facilitates cellular entry of severe acute respiratory syndrome coronavirus (SARS-CoV) and SARS-CoV-2 as their common receptor. During infection, ACE2-expressing tissues become direct targets, resulting in serious pathological changes and progressive multiple organ failure or even death in severe cases. However, as an essential component of renin-angiotensin system (RAS), ACE2 confers protective effects in physiological circumstance, including maintaining cardiovascular homeostasis, fluid, and electrolyte balance. The absence of protective role of ACE2 leads to dysregulated RAS and thus acute changes under multiple pathological scenarios including SARS. This potentially shared mechanism may also be the molecular explanation for pathogenesis driven by SARS-CoV-2. We reasonably speculate several potential directions of clinical management including host-directed therapies aiming to restore dysregulated RAS caused by ACE2 deficiency. Enriched knowledge of ACE2 learned from SARS and COVID-19 outbreaks can provide, despite their inherent tragedy, informative clues for emerging pandemic preparedness.

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.

Novel severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) became pandemic by the end of March 2020. In contrast to the 2002-2003 SARS-CoV outbreak, which had a higher pathogenicity and lead to higher mortality rates, SARSCoV-2 infection appears to be much more contagious. Moreover, many SARS-CoV-2 infected patients are reported to develop low-titer neutralizing antibody and usually suffer prolonged illness, suggesting a more effective SARS-CoV-2 immune surveillance evasion than SARS-CoV. This paper summarizes the current state of art about the differences and similarities between the pathogenesis of the two coronaviruses, focusing on receptor binding domain, host cell entry and protease activation. Such differences may provide insight into possible intervention strategies to fight the pandemic.

Glycopeptide Antibiotics Potently Inhibit Cathepsin L in the Late Endosome/Lysosome and Block the Entry of Ebola Virus, Middle East Respiratory Syndrome Coronavirus (MERS-CoV), and Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV).

Ebola virus infection can cause severe hemorrhagic fever with a high mortality in humans. The outbreaks of Ebola viruses in 2014 represented the most serious Ebola epidemics in history and greatly threatened public health worldwide. The development of additional effective anti-Ebola therapeutic agents is therefore quite urgent. In this study, via high throughput screening of Food and Drug Administration-approved drugs, we identified that teicoplanin, a glycopeptide antibiotic, potently prevents the entry of Ebola envelope pseudotyped viruses into the cytoplasm. Furthermore, teicoplanin also has an inhibitory effect on transcription- and replication-competent virus-like particles, with an IC50 as low as 330 nm Comparative analysis further demonstrated that teicoplanin is able to block the entry of Middle East respiratory syndrome (MERS) and severe acute respiratory syndrome (SARS) envelope pseudotyped viruses as well. Teicoplanin derivatives such as dalbavancin, oritavancin, and telavancin can also inhibit the entry of Ebola, MERS, and SARS viruses. Mechanistic studies showed that teicoplanin blocks Ebola virus entry by specifically inhibiting the activity of cathepsin L, opening a novel avenue for the development of additional glycopeptides as potential inhibitors of cathepsin L-dependent viruses. Notably, given that teicoplanin has routinely been used in the clinic with low toxicity, our work provides a promising prospect for the prophylaxis and treatment of Ebola, MERS, and SARS virus infection.

TMPRSS2 and ADAM17 cleave ACE2 differentially and only proteolysis by TMPRSS2 augments entry driven by the severe acute respiratory syndrome coronavirus spike protein.

The type II transmembrane serine proteases TMPRSS2 and HAT can cleave and activate the spike protein (S) of the severe acute respiratory syndrome coronavirus (SARS-CoV) for membrane fusion. In addition, these proteases cleave the viral receptor, the carboxypeptidase angiotensin-converting enzyme 2 (ACE2), and it was proposed that ACE2 cleavage augments viral infectivity. However, no mechanistic insights into this process were obtained and the relevance of ACE2 cleavage for SARS-CoV S protein (SARS-S) activation has not been determined. Here, we show that arginine and lysine residues within ACE2 amino acids 697 to 716 are essential for cleavage by TMPRSS2 and HAT and that ACE2 processing is required for augmentation of SARS-S-driven entry by these proteases. In contrast, ACE2 cleavage was dispensable for activation of the viral S protein. Expression of TMPRSS2 increased cellular uptake of soluble SARS-S, suggesting that protease-dependent augmentation of viral entry might be due to increased uptake of virions into target cells. Finally, TMPRSS2 was found to compete with the metalloprotease ADAM17 for ACE2 processing, but only cleavage by TMPRSS2 resulted in augmented SARS-S-driven entry. Collectively, our results in conjunction with those of previous studies indicate that TMPRSS2 and potentially related proteases promote SARS-CoV entry by two separate mechanisms: ACE2 cleavage, which might promote viral uptake, and SARS-S cleavage, which activates the S protein for membrane fusion. These observations have interesting implications for the development of novel therapeutics. In addition, they should spur efforts to determine whether receptor cleavage promotes entry of other coronaviruses, which use peptidases as entry receptors.

Tanshinones as selective and slow-binding inhibitors for SARS-CoV cysteine proteases.

In the search for anti-SARS-CoV, tanshinones derived from Salvia miltiorrhiza were found to be specific and selective inhibitors for the SARS-CoV 3CL(pro) and PL(pro), viral cysteine proteases. A literature search for studies involving the seven isolated tanshinone hits showed that at present, none have been identified as coronaviral protease inhibitors. We have identified that all of the isolated tanshinones are good inhibitors of both cysteine proteases. However, their activity was slightly affected by subtle changes in structure and targeting enzymes. All isolated compounds (1-7) act as time dependent inhibitors of PL(pro), but no improved inhibition was observed following preincubation with the 3CL(pro). In a detail kinetic mechanism study, all of the tanshinones except rosmariquinone (7) were identified as noncompetitive enzyme isomerization inhibitors. However, rosmariquinone (7) showed a different kinetic mechanism through mixed-type simple reversible slow-binding inhibition. Furthermore, tanshinone I (5) exhibited the most potent nanomolar level inhibitory activity toward deubiquitinating (IC(50)=0.7 µM). Additionally, the inhibition is selective because these compounds do not exert significant inhibitory effects against other proteases including chymotrysin, papain, and HIV protease. These findings provide potential inhibitors for SARS-CoV viral infection and replication.

A crucial role of angiotensin converting enzyme 2 (ACE2) in SARS coronavirus-induced lung injury.

During several months of 2003, a newly identified illness termed severe acute respiratory syndrome (SARS) spread rapidly through the world. A new coronavirus (SARS-CoV) was identified as the SARS pathogen, which triggered severe pneumonia and acute, often lethal, lung failure. Moreover, among infected individuals influenza such as the Spanish flu and the emergence of new respiratory disease viruses have caused high lethality resulting from acute lung failure. In cell lines, angiotensin-converting enzyme 2 (ACE2) has been identified as a potential SARS-CoV receptor. The high lethality of SARS-CoV infections, its enormous economic and social impact, fears of renewed outbreaks as well as the potential misuse of such viruses as biologic weapons make it paramount to understand the pathogenesis of SARS-CoV. Here we provide the first genetic proof that ACE2 is a crucial SARS-CoV receptor in vivo. SARS-CoV infections and the Spike protein of the SARS-CoV reduce ACE2 expression. Notably, injection of SARS-CoV Spike into mice worsens acute lung failure in vivo that can be attenuated by blocking the renin-angiotensin pathway. These results provide a molecular explanation why SARS-CoV infections cause severe and often lethal lung failure and suggest a rational therapy for SARS and possibly other respiratory disease viruses.

Coronavirus-specific antibody production in middle-aged mice requires phospholipase A2G2D.

Worse outcomes occur in aged compared with young populations after infections with respiratory viruses, including pathogenic coronaviruses (SARS-CoV, MERS-CoV, and SARS-CoV-2), and are associated with a suboptimal lung milieu ("inflammaging"). We previously showed that a single inducible phospholipase, PLA2G2D, is associated with a proresolving/antiinflammatory response in the lungs, and increases with age. Survival was increased in naive Pla2g2d-/- mice infected with SARS-CoV resulting from augmented respiratory dendritic cell (rDC) activation and enhanced priming of virus-specific T cells. Here, in contrast, we show that intranasal immunization provided no additional protection in middle-aged Pla2g2d-/- mice infected with any of the 3 pathogenic human coronaviruses because virtually no virus-specific antibodies or follicular helper CD4+ T (Tfh) cells were produced. Using MERS-CoV-infected mice, we found that these effects did not result from T or B cell intrinsic factors. Rather, they resulted from enhanced, and ultimately, pathogenic rDC activation, as manifested most prominently by enhanced IL-1ß expression. Wild-type rDC transfer to Pla2g2d-/- mice in conjunction with partial IL-1ß blockade reversed this defect and resulted in increased virus-specific antibody and Tfh responses. Together, these results indicate that PLA2G2D has an unexpected role in the lungs, serving as an important modulator of rDC activation, with protective and pathogenic effects in respiratory coronavirus infections and immunization, respectively.

The Janus kinase 1/2 inhibitor ruxolitinib in COVID-19 with severe systemic hyperinflammation.

A subgroup of patients with severe COVID-19 suffers from progression to acute respiratory distress syndrome and multiorgan failure. These patients present with progressive hyperinflammation governed by proinflammatory cytokines. An interdisciplinary COVID-19 work flow was established to detect patients with imminent or full blown hyperinflammation. Using a newly developed COVID-19 Inflammation Score (CIS), patients were prospectively stratified for targeted inhibition of cytokine signalling by the Janus Kinase 1/2 inhibitor ruxolitinib (Rux). Patients were treated with efficacy/toxicity guided step up dosing up to 14 days. Retrospective analysis of CIS reduction and clinical outcome was performed. Out of 105 patients treated between March 30th and April 15th, 2020, 14 patients with a CIS ≥ 10 out of 16 points received Rux over a median of 9 days with a median cumulative dose of 135 mg. A total of 12/14 patients achieved significant reduction of CIS by ≥25% on day 7 with sustained clinical improvement in 11/14 patients without short term red flag warnings of Rux-induced toxicity. Rux treatment for COVID-19 in patients with hyperinflammation is shown to be safe with signals of efficacy in this pilot case series for CRS-intervention to prevent or overcome multiorgan failure. A multicenter phase-II clinical trial has been initiated (NCT04338958).

Ectodomain shedding of angiotensin converting enzyme 2 in human airway epithelia.

Angiotensin-converting enzyme 2 (ACE2) is a terminal carboxypeptidase and the receptor for the SARS and NL63 coronaviruses (CoV). Loss of ACE2 function is implicated in severe acute respiratory syndrome (SARS) pathogenesis, but little is known about ACE2 biogenesis and activity in the airways. We report that ACE2 is shed from human airway epithelia, a site of SARS-CoV infection. The regulation of ACE2 release was investigated in polarized human airway epithelia. Constitutive generation of soluble ACE2 was inhibited by DPC 333, implicating a disintegrin and metalloprotease 17 (ADAM17). Phorbol ester, ionomycin, endotoxin, and IL-1beta and TNFalpha acutely induced ACE2 release, further supporting that ADAM17 and ADAM10 regulate ACE2 cleavage. Soluble ACE2 was enzymatically active and partially inhibited virus entry into target cells. We determined that the ACE2 cleavage site resides between amino acid 716 and the putative transmembrane domain starting at amino acid 741. To reveal structural determinants underlying ACE2 release, several mutant and chimeric ACE2 proteins were engineered. Neither the juxtamembrane stalk region, transmembrane domain, nor the cytosolic domain was needed for constitutive ACE2 release. Interestingly, a point mutation in the ACE2 ectodomain, L584A, markedly attenuated shedding. The resultant ACE2-L584A mutant trafficked to the cell membrane and facilitated SARS-CoV entry into target cells, suggesting that the ACE2 ectodomain regulates its release and that residue L584 might be part of a putative sheddase "recognition motif." Thus ACE2 must be cell associated to serve as a CoV receptor and soluble ACE2 might play a role in modifying inflammatory processes at the airway mucosal surface.

Lessons from SARS: control of acute lung failure by the SARS receptor ACE2.

Angiotensin-converting enzyme 2 (ACE2), a second angiotensin-converting enzyme (ACE), regulates the renin-angiotensin system by counterbalancing ACE activity. Accumulating evidence in recent years has demonstrated a physiological and pathological role of ACE2 in the cardiovascular systems. Recently, it has been shown that severe acute respiratory syndrome (SARS) coronavirus, the cause of SARS, utilizes ACE2 as an essential receptor for cell fusion and in vivo infections in mice. Intriguingly, ACE2 acts as a protective factor in various experimental models of acute lung failure and, therefore, acts not only as a key determinant for SARS virus entry into cells but also contributes to SARS pathogenesis. Here we review the role of ACE2 in disease pathogenesis, including lung diseases and cardiovascular diseases.

Angiotensin-converting enzyme 2 in lung diseases.

The renin-angiotensin system (RAS) plays a key role in maintaining blood pressure homeostasis, as well as fluid and salt balance. Angiotensin II, a key effector peptide of the system, causes vasoconstriction and exerts multiple biological functions. Angiotensin-converting enzyme (ACE) plays a central role in generating angiotensin II from angiotensin I, and capillary blood vessels in the lung are one of the major sites of ACE expression and angiotensin II production in the human body. The RAS has been implicated in the pathogenesis of pulmonary hypertension and pulmonary fibrosis, both commonly seen in chronic lung diseases such as chronic obstructive lung disease. Recent studies indicate that the RAS also plays a critical role in acute lung diseases, especially acute respiratory distress syndrome (ARDS). ACE2, a close homologue of ACE, functions as a negative regulator of the angiotensin system and was identified as a key receptor for SARS (severe acute respiratory syndrome) coronavirus infections. In the lung, ACE2 protects against acute lung injury in several animal models of ARDS. Thus, the RAS appears to play a critical role in the pathogenesis of acute lung injury. Indeed, increasing ACE2 activity might be a novel approach for the treatment of acute lung failure in several diseases.

COVID-19. La historia se repite y seguimos tropezando con la misma piedra / COVID-19. History repeats itself and we keep stumbling on the same stone

La pandemia por COVID-19 ha provocado un desajuste en todos los sistemas de salud. La mayoría de los países habían olvidado cómo comportarse ante una epidemia de estas características sin disponer de los recursos adecuados. Es preciso realizar un balance de todo lo sucedido, instruir a la población y generar un nuevo conocimiento que nos permita afrontar nuevas epidemias

The COVID-19 pandemic has caused a mismatch in all health systems. Most countries had forgotten how to behave in the face of such an epidemic without adequate resources. We need to take stock of everything that has happened, instruct the population and generate a new knowledge that allows us to face new epidemics

Use of simple laboratory features to distinguish the early stage of severe acute respiratory syndrome from dengue fever.

BACKGROUND: The diagnosis of severe acute respiratory syndrome (SARS) is difficult early in the illness, because its presentation resembles that of other nonspecific viral fevers, such as dengue. Dengue fever is endemic in many of the countries in which the large SARS outbreaks occurred in early 2003. Misdiagnosis may have serious public health consequences. We aimed to determine simple laboratory features to differentiate SARS from dengue. METHODS: We compared the laboratory features of 55 adult patients with SARS at presentation (who were all admitted before radiological changes had occurred) and 147 patients with dengue. Features independently predictive of dengue were modeled by multivariate logistic regression to create a diagnostic tool with 100% specificity for dengue. RESULTS: Multivariate analysis identified 3 laboratory features that together are highly predictive of a diagnosis of dengue and able to rule out the possibility of SARS: platelet count of <140 x 10(9) platelets/L, white blood cell count of <5x10(9) cells/L, and aspartate aminotransferase level of >34 IU/L. A combination of these parameters has a sensitivity of 75% and a specificity of 100%. CONCLUSIONS: Simple laboratory data may be helpful for the diagnosis of disease in adults admitted because of fever in areas in which dengue is endemic when the diagnosis of SARS needs to be excluded. Application of this information may help to optimize the use of isolation rooms for patients presenting with nonspecific fever.

Prognostic factors for severe acute respiratory syndrome: a clinical analysis of 165 cases.

This study analyzes single factors that affect the prognosis of severe acute respiratory syndrome (SARS) and establishes a prognosis model by multivariate analysis. We retrospectively analyzed the clinical features of SARS in 165 clinically confirmed severe cases. Both age and existence of other diseases before SARS were significantly correlated with prognosis (r=0.506 and r=0.457, respectively; P<.001). During the acute phase of SARS, lactate dehydrogenase level, degree of hypoxemia, respiratory rate, alpha -hydroxybutyric dehydrogenase level, creatine kinase isoenzyme-MB, platelet count, and number of involved lobes noted on chest radiographs, and so on, correlated markedly with the prognosis (r=0.257-0.788; P<.05). The multivariate prognosis regression model was associated with degree of hypoxemia and platelet count. The model was defined by the formula Py=1=es/(1+es), where S is [2.490 x degree of hypoxemia]-[0.050 x number of platelets], and it had a high sensitivity (91.67%), specificity (98.33%), and accuracy (96.42%). The model could be used to effectively judge the state of illness and the prognosis.

Influenza and SARS-coronavirus activating proteases TMPRSS2 and HAT are expressed at multiple sites in human respiratory and gastrointestinal tracts.

The type II transmembrane serine proteases TMPRSS2 and HAT activate influenza viruses and the SARS-coronavirus (TMPRSS2) in cell culture and may play an important role in viral spread and pathogenesis in the infected host. However, it is at present largely unclear to what extent these proteases are expressed in viral target cells in human tissues. Here, we show that both HAT and TMPRSS2 are coexpressed with 2,6-linked sialic acids, the major receptor determinant of human influenza viruses, throughout the human respiratory tract. Similarly, coexpression of ACE2, the SARS-coronavirus receptor, and TMPRSS2 was frequently found in the upper and lower aerodigestive tract, with the exception of the vocal folds, epiglottis and trachea. Finally, activation of influenza virus was conserved between human, avian and porcine TMPRSS2, suggesting that this protease might activate influenza virus in reservoir-, intermediate- and human hosts. In sum, our results show that TMPRSS2 and HAT are expressed by important influenza and SARS-coronavirus target cells and could thus support viral spread in the human host.

Computational approaches for the discovery of cysteine protease inhibitors against malaria and SARS.

Cysteine proteases are implicated in a variety of human physiological processes and also form an essential component of the life cycle of a number of pathogenic protozoa and viruses. The present review highlights the drug design approaches utilized to understand the mechanism of inhibition and discovery of inhibitors against protozoal cysteine protease, falcipain (a cysteine protease of P. falciparum which causes malaria), and viral cysteine protease, SARS-CoV M(pro) (a cysteine protease of severe acute respiratory syndrome corona virus). The article describes rational approaches for the design of inhibitors and focuses on a variety of structure as well as ligand-based modeling strategies adopted for the discovery of the inhibitors. Also, the key features of ligand recognition against these targets are accentuated. Although no apparent similarities exist between viral and protozoal cysteine proteases discussed here, the goal is to provide examples of rational drug design approaches adopted to design inhibitors against these proteases. The current review would be of interest to scientists engaged in the development of drug design strategies to target the cysteine proteases present in mammals and other lower order organisms.

[Study on interaction between SARS-CoV N and MAP19].

AIM: Severe acute respiratory syndrome coronavirus (SARS-CoV) is the etiological agent of SARS, an emerging disease characterized by atypical pneumonia. Using a yeast two-hybrid screen with the nucleocapsid (N)protein of SARS-CoV as a bait, the N protein was found to interact with MAP19, a non-enzymatic protein of MASP(mannan-associated serine protease). The interaction between SARS-CoV N and MAP19 would be further tested in cells in this article. METHODS: The interaction between SARS-CoV N and MAP19 was demonstrated by immuno- coprecipitation, and the amount of MAP19 influenced by SARS-CoV N was investgated by Western blot. RESULTS: The interaction between SARS-CoV N and MAP19 was already demonstrated by immuno-coprecipitation. SARS-CoV N greatly increased the amount of MAP19. CONCLUSION: SARS-CoV N can bind with MAP19 in cells. Our study may be conductive to further research into the molecular mechanism of action between SARS-CoV N and MAP19.