Recently, SARS-CoV-2 was discovered in China and spread rapidly worldwide with greater virulence than the SARS-CoV outbreak in 2003 (Huang et al., 2020). Coronavirus infection can cause gastroenteritis, hepatitis, respiratory tract infections resembling the common cold, lower respiratory tract infections such as bronchitis and pneumonia, SARS, systemic diseases, and even death in humans (Owusu et al., 2014).

Since the first report of SARS-CoV-2 infection in December 2019 from the World Health Organization (WHO) Country Office in China, the number of COVID-19 cases has been growing steadily at an alarming pace (WHO, 2020b). According to the WHO, the cases identified outside of China in the early global spread occurred because infected travelers traveled abroad. Countries such as Australia, Canada, Cambodia, France, Finland, Germany, India, Italy, Japan, Nepal, Malaysia, the Philippines, the Republic of Korea, Singapore, Sri Lanka, Thailand, the United States of America (USA), United Arab Emirates, and Vietnam were the first to report Chinese traveler-associated COVID-19 cases (World-Health-Organization, 2020b). Some months after the initial report in China, the first SARS-CoV-2 infection notifications in South America emerged; Brazil, Peru, and Ecuador were the countries with the most significant number of cases (Dong et al., 2020). Currently, the USA and Brazil are the epicenters of the COVID-19 pandemic (World-Health-Organization, 2020a).

Data from China reveal that SARS-CoV-2 is highly virulent compared to SARS (Li Q. et al., 2020). The incubation period for SARS-CoV-2 is 4.5–5.8 days (Lauer et al., 2020). The infection then evolves to a pre-symptomatic stage (1–3 days) through symptomatic infection (2–4 weeks) to a prolonged post-symptomatic or recovery stage (2–8 weeks) (Subbarao and Mahanty, 2020). The median age of hospitalized patients with COVID-19 was 63 years (Bordallo et al., 2020). The SARS-CoV-2 infection affects people who are 2–72 years of age, with a high prevalence in men (Backer et al., 2020). People with chronic cardiovascular and pulmonary disease, immunodeficiency, hypertension (Ejaz et al., 2020; Flaherty et al., 2020), and diabetes, along with elderly patients (Wang et al., 2020) are more prone to infection and death by SARS-CoV-2 than healthy people. In Brazil, chronic heart disease is the deadliest comorbidity in COVID-19 patients (Pachiega et al., 2020).

The fatality rate tends to vary, ranging from 2.8 to 11% (Wang et al., 2020) with a median of 5% (Li L. Q. et al., 2020). There have been more than 15 million cases across the world and more than 625,000 people have died (World-Health-Organization, 2020c). However, sub-notification and delays to case confirmations can quickly change the statistics (Rocha-Filho, 2020).

The recovery time for COVID-19 is approximately 14 days (Noor et al., 2020; Wang et al., 2020). Asymptomatic SARS-CoV-2-infected people and patients in incubation or those who have recovered from COVID-19 may shed infectious virus particles (Hoehl et al., 2020) for up to 3 or 4 weeks (Lan et al., 2020), representing a considerable challenge for disease prevention and control.

SARS-CoV-2 has a broad spectrum of infection from asymptomatic patients to patients who develop mild, moderate, or severe forms of ARDS (Vardhana and Wolchok, 2020). Most patients have mild symptomatology without needing hospital treatment, with the appearance of fever, a dry cough, and tiredness. Less common symptoms are headache, aches and pains, nasal congestion, diarrhea, sore throat, loss of taste, or loss of smell (Struyf et al., 2020).

SARS-CoV-2 is transmitted predominantly via respiratory droplets and direct contact (Lai et al., 2020). SARS-CoV enters the host cell through interaction between the viral spike protein and the angiotensin-converting enzyme 2 (ACE2) of the host cell, triggering the virus's fusion with the cellular membrane and consequently releasing the viral genome into the cytoplasm (Belouzard et al., 2009). In the cytoplasm, replicase gene translation ensues from the virion genomic RNA, which encodes two large open reading frames: rep1a and rep1b. These express the polyproteins pp1a and pp1ab, posteriorly translated in nsps (Brierley et al., 1989).

As with SARS-CoV, the SARS-CoV-2 virus enters cells by binding its spike to ACE2 (Hoffmann et al., 2020). After this first contact, SARS-CoV-2 uses an enzyme, the cellular serine protease TMPRSS2, to prime the spike protein (Hoffmann et al., 2020). The spike protein of SARS-CoV-2 contains a receptor-binding domain (RBD) that specifically recognizes ACE2 as its receptor. The SARS-CoV-2 RBD contains a core and a receptor-binding motif that mediates tight binding with ACE2 (Shang et al., 2020). The C-terminal domain (CTD) of SARS-CoV-2 also binds to ACE2. Wang et al. (2020) demonstrated that key residue substitutions in the SARS-CoV-2 CTD slightly strengthen the interaction with ACE2 and leads to a higher affinity for receptor binding than with the SARS RBD (Wang et al., 2020). This interaction between the S protein and its receptor is responsible for the species specificity and tissue tropism of the virus (Li et al., 2005). The cleavage of the viral protein by TMPRSS2 is crucial for the fusion between the virus and cellular membrane, starting the viral infection process (Hoffmann et al., 2020). Some cells show accentuated vulnerability to infection, such as type II alveolar cells, myocardial cells, esophagus epithelial cells, proximal tubule cells of the kidney and ileum, and urothelial bladder cells (Zou et al., 2020).

The SARS-CoV-2 S-protein sequence contains 12 additional nucleotides upstream of the single Arg cleavage site 1, which is associated with a canonical furin-like cleavage site (Coutard et al., 2020). This furin-like cleavage site may support virus egress by being cleaved for S-protein priming and may provide an advantage in transmission when compared to other lineages of beta-coronaviruses (Coutard et al., 2020). Coronavirus replication occurs entirely in the cytoplasm (Subbarao and Mahanty, 2020). After entry and uncoating, the virus genome is translated into the pp1a and pp1ab replicase polyproteins to start RNA synthesis in the replicase–transcriptase complex (de Wilde et al., 2018). Viral RNA synthesis produces genomic and subgenomic RNAs, and the viral structural proteins are inserted into the endoplasmic reticulum, where mature virions are formed (Fehr and Perlman, 2015).

Higher expression of ACE2 may prolong the virus multiplication cycle, enhance virus replication, and mediate penetration of the virus into the host cell (Tian et al., 2020). SARS-CoV and SARS-CoV-2 bind to ACE2, leading to downregulation (Kumar et al., 2020). The virus appears to enter cells via the membrane receptor, which is functionally removed from the external site of the membrane, consequently increasing the bioavailability of angiotensin 2 (Verdecchia et al., 2020). Angiotensin 2 is associated with other pulmonary diseases such as ARDS by triggering significant inflammatory lesions in the respiratory tree-like inflammatory infiltrate, edema, and alveolar wall thickening, which contribute to heightened viral pathogenesis (Imai et al., 2005; Kuba et al., 2005).

In the lungs, over-activation of angiotensin II receptor type 1 (Ang2-AT1R) leads to severe lung injury and lung failure due to pneumonia (Banu et al., 2020). Activation of the Ang2-AT1R pathway activates NFκB, increasing the expression of proinflammatory cytokines (Albini et al., 2020). It also activates the JAK/STAT pathway and ADAM17, leading to downstream production and release of IL-6 (Catanzaro et al., 2020) and inactivating ACE2 (Eguchi et al., 2018), respectively. Additionally, the Ang2-AT1R pathway mediates the conversion of a soluble form of IL-6 (Murakami et al., 2019) and is vasoconstrictive and pro-fibrotic (Albini et al., 2020; Banu et al., 2020), playing an essential role in the pathogenesis of COVID-19. The vasoconstrictive effect caused by the increase in angiotensin 2 and subsequent dysfunction of the renin-angiotensin-aldosterone system could be the reason some patients have shallow blood oxygen levels but are not breathless (Subbarao and Mahanty, 2020). One of the suggested mechanisms behind this is that oxygen uptake is obstructed because of congested and constricted blood vessels in the lungs, but not due to accumulation of edema fluid in the alveoli (Subbarao and Mahanty, 2020). The most severe symptom observed in COVID-19 patients is interstitial pulmonary edema, which is common in 90% of cases (Lu et al., 2020). Some studies have shown that this clinical manifestation is supported by ACE2 downregulation, which increases alveolar capillary permeability and leads to interstitial and alveolar edema (Imai et al., 2005).

ACE2 expression is mainly associated with innate and acquired immune responses: regulation of B cell-mediated immunity, secretion of the cytokines IL-1, IL-10, TNFα, IL- 6, and IL-8, and activation of neutrophils, NK cells, and T cells (Costela-Ruiz et al., 2020). ACE-2 downregulation induces macrophage activation syndrome (MAS) (Banu et al., 2020), which is characterized by uncontrolled activation and proliferation of T lymphocytes and macrophages, leading to a cytokine storm and multiple organ failure (Bracaglia et al., 2017).

One of the leading causes of death from COVID-19 is a cytokine storm or cytokine release syndrome, which is also associated with ARDS (Wang et al., 2020). Severe cases tend to demonstrate lymphocytopenia and a higher leukocyte count (Vabret et al., 2020). Recognition of SARS-CoV-2 activates downstream transduction pathways such as NFκB, JAK-STAT, and IRF3, leading to a large release of inflammatory cytokines, such as IL-6, IL-1β, IL-2, IL-8, IL-17, CCL3, and TNFα; such cytokines are increased in severely ill patients (Catanzaro et al., 2020). The expression of proinflammatory genes, such as chemokines, are elevated in COVID-19 patients compared to in community-acquired pneumonia patients and healthy controls, causing chemokine-dominant hypercytokinemia (Zhou et al., 2020).

During infection of host cells by viruses, IFN-stimulated genes (ISGs) and type I IFNs are expressed to induce an antiviral state (Subbarao and Mahanty, 2020). IFN-stimulated genes (ISGs) are increased in COVID-19 patients; however, compared to other viruses, SARS-CoV infections induced fewer ISGs. In addition, SARS-CoV-2 may have developed mechanisms to delay the IFN response by inhibiting innate immune signaling (Zhou et al., 2020).

Inflammatory cytokine IL-6 levels are significantly elevated in critically ill patients' serum; this is related to the need for mechanical ventilation and mortality (Zhou et al., 2020). This cytokine's consistent elevation might function as a predictive biomarker for disease severity (Gao et al., 2020). High IL-6 levels amplify the innate immune system response by inducing the release of more cytokines, heightening the influx of neutrophils (Hunter and Jones, 2015), activating downstream JAK/STAT3 and MAPK pathways, and subsequently activating several genes involved in inflammation and immunity (Garbers et al., 2015; Jordan et al., 2017).

Beyond immune cells, IL-6 causes endothelial cell activation and dysfunction by inducing the expression of chemoattractant proteins and adhesion molecules that recruit immune cells into the sub-intimal space (Qu et al., 2014). IL-6 also induces excessive vascular endothelial growth factor (VEGF) production, leading to enhanced angiogenesis (Tanaka et al., 2014). The effects induced by IL-6 are associated with cardiovascular diseases; several studies have already reported IL-6 alterations in such diseases and atherosclerosis (Qu et al., 2014; Wainstein et al., 2017; Zhang et al., 2018).

There is a high prevalence of cardiovascular diseases among fatal cases of COVID-19 (Clerkin et al., 2020). The most common manifestations are acute myocardial injury and arrhythmias (Bansal, 2020). Myocardial injury is mainly manifested as an increase in high-sensitivity cardiac troponin I levels (>28 pg/ml) (Huang et al., 2020). The increase in some inflammatory biomarkers (IL-6, ferritin, lactate dehydrogenase, and D-dimer) concomitantly raises the possibility of this being associated with the cytokine storm. Angiotensin II activates JAK/STAT (Schieffer et al., 2000) and the NFκB pathway in COVID-19 patients (Kranzhofer et al., 1999), increasing IL-6 production. Another mechanism is the myocardial dysfunction from the direct effect of SARS-CoV-2 on the heart, mediated by ACE2 (Clerkin et al., 2020).

Another striking characteristic of SARS-CoV-2 infection is the high incidence of interstitial pulmonary edema and pulmonary alveolar edema (Figure 2) (Tian et al., 2020), that is, leakage of fluid from pulmonary capillaries into the interstitium and alveoli. Lu et al. (2020) demonstrated that 90% of COVID-19 patients showed interstitial pulmonary edema in the lungs. Some pulmonary edema mechanisms may include ACE2 downregulation with increased alveolar-capillary permeability, leading to interstitial and alveolar edema (Imai et al., 2005).

The enzyme NKA plays a pivotal role in edema clearance in the lungs (Goncalves-de-Albuquerque et al., 2015). Active transport of Na⁺ by basolateral NKA drives the transepithelial fluid from the airspaces to the capillaries, improving edema clearance (Gonçalves-de-Albuquerque et al., 2016). The decreased expression of NKA results in the accumulation of pulmonary fluid (Zhang et al., 2018). Activation of NKA is also linked to lung inflammation, as reported by our group (Goncalves-de-Albuquerque et al., 2012; Goncalves-de-Albuquerque et al., 2013; Goncalves-de-Albuquerque et al., 2014). The SARS-CoV infection has already been shown to hamper pulmonary edema clearance by NKA due to the flaking of the bronchiolar barrier, resulting in the displacement of the enzyme (Nieto-Torres et al., 2015).

So far, there are some vaccines in the final test phase (Sharma et al., 2020). Among these, the sputnik V has already been widely used in Russia to vaccinate doctors and teachers (Logunov et al., 2020). Several drugs have also been suggested to treat patients infected with SARS-CoV-2 (Elfiky, 2020; Hossain et al., 2020), including chloroquine and its analogue, hydroxychloroquine. However, some studies have shown that these antimalarial drugs can cause adverse reactions, such as skin changes, neuromyopathy (Estes et al., 1987), cardiotoxicity (Mahon et al., 2004), dysfunction of lysosomal enzymes that leads to impairment of intracellular degradation processes and accumulation of glycogen and phospholipids (Homewood et al., 1972) and retinopathy (Marmor et al., 2011). Moreover, in vitro tests using chloroquine and hydroxychloroquine showed that these compounds did not decrease acute SARS-CoV-2 infection (Touret and De Lamballerie, 2020). In vivo tests have also shown that chloroquine and hydroxychloroquine are not effective in inhibiting SARS-CoV replication in a mouse model (Barnard et al., 2006). Other potential compounds include lopinavir–ritonavir; however, a study in hospitalized adult COVID-19 patients showed that treatment with these drugs did not significantly improve clinical improvement or decrease mortality and viral RNA detection (Cao et al., 2020). Remdesivir is effective against SARS, MERS, and SARS-CoV-2 in vitro; clinical studies show promising results, but they are still under investigation (Chatterjee, 2020).

Other prophylactic/therapeutic options should be explored, including human monoclonal antibodies, IFNs, and siRNAs, and low-molecular-weight SARS-CoV inhibitors targeted at any of the specific processes involved in the viral replication cycle (such as viral entry into the cells, proteolytic cleavage, RNA replication, and transcription) (De Clercq and Field, 2006). In this review, we have discussed the effect of CGs on respiratory viruses; these compounds inhibit viral entry and replication without inducing cell death. Additionally, they inhibit NKA, impairing Na⁺ and K⁺ movement (Buckalew, 2015). Intracellular transport of molecules and ions (Boulant et al., 2015), such as K⁺ (Hartley et al., 1993) and Na⁺ (Hoffmann et al., 2008) is essential for the viral replication process, making NKA a possible molecular target and CG an anti-SARS-CoV-2 drug candidate.

Some studies have shown the efficacy and safety of CGs. Digoxin use reduced recurrent hospitalizations to treat congestive heart failure (CHF) and decreased the severity of CHF (Digitalis Investigation Group, 1997). Digoxin decreased all-cause 30-day hospital admissions in older patients (mean age, 72 years) with chronic systolic CHF. Kaur et al. (2009) reported a significant reduction in cerebral infarct size and prevention of ischemia/reperfusion-induced cognitive and motor deficits after digoxin treatment. An in vitro study has also shown the inhibition of prostate cancer cell proliferation by digoxin, digitoxin, and ouabain without damaging normal cells (Yeh et al., 2001).

A low concentration of CG triggers Src signaling via NKA, resulting in the inhibition of SARS-CoV infection (Figure 1B) (Burkard et al., 2015). Src modulates PI3K-AKT signaling (Liu et al., 2007) and is involved in ouabain-induced Ca²⁺ oscillation by affecting the interaction between the IP3 receptor and α1 NKA (Fontana et al., 2013). The activation of NKA-dependent PI3K-PDK1 signaling by ouabain is an essential contribution to anti-transmissible gastroenteritis coronavirus activity (Yang et al., 2018). Protein kinase C also regulates NKA affinity by compounds (Ridge et al., 2003). The interaction of ouabain and NKA also activates a cellular signaling cascade involving Src, mitoKATP, and reactive oxygen species (ROS). In this pathway, ouabain protects outer mitochondrial membrane integrity, adenine nucleotide compartmentation, and energy transfer efficiency, boosting the myocardium’s resistance to oxygen deficiency (Pasdois et al., 2007). In addition, Src activation by CG can trigger MAPK extracellular signal-regulated kinase 1/2 signaling through the Raf-MEK cascade, causing effects independent of alterations to intracellular calcium (Wong et al., 2018). Nimmerjahn et al. (2004) demonstrated the importance of NFκB for influenza virus (A/FPV/Bratislava (H7N7) and A/WSN/33 (H1N1) strains) infection in human cells, reporting that cells with low NFκB activity are resistant to influenza virus infection and a normalization increase in NFκB activity suppresses viral infection. In a mouse model of SARS-CoV infection, blockage of NFκB activation prevented cytokine storms, resulting in increased survival (DeDiego et al., 2014). Digoxin blocks the host cytokine storm induced by influenza (A/Wuhan/H3N2/359/95 strain), inhibiting NFκB and nuclear factor of activated T-cells (NFAT). Inhibition of NFκB and NFAT blocks TNFα, GRO/KC, MCP1, MIP2, IL-1β, TGFβ, and IFNγ, decreasing the inflammatory process and consequently improving the infection outcome. Ouabain and digitoxin show antiviral activity, inhibiting mRNA and viral protein expression of SARS-CoV-2 (Cho et al., 2020). Based on these data, CGs can be considered anti-SARS-CoV-2 drug candidates.