SARS-CoV-2 Coronavirus Spike Protein-Induced Apoptosis, Inflammatory, and Oxidative Stress Responses in THP-1-Like-Macrophages: Potential Role of Angiotensin-Converting Enzyme Inhibitor (Perindopril).

A purified spike (S) glycoprotein of severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) coronavirus was used to study its effects on THP-1 macrophages, peripheral blood mononuclear cells (PBMCs), and HUVEC cells. The S protein mediates the entry of SARS-CoV-2 into cells through binding to the angiotensin-converting enzyme 2 (ACE2) receptors. We measured the viability, intracellular cytokine release, oxidative stress, proinflammatory markers, and THP-1-like macrophage polarization. We observed an increase in apoptosis, ROS generation, MCP-1, and intracellular calcium expression in the THP-1 macrophages. Stimulation with the S protein polarizes the THP-1 macrophages towards proinflammatory futures with an increase in the TNFα and MHC-II M1-like phenotype markers. Treating the cells with an ACE inhibitor, perindopril, at 100 µM reduced apoptosis, ROS, and MHC-II expression induced by S protein. We analyzed the sensitivity of the HUVEC cells after the exposure to a conditioned media (CM) of THP-1 macrophages stimulated with the S protein. The CM induced endothelial cell apoptosis and MCP-1 expression. Treatment with perindopril reduced these effects. However, the direct stimulation of the HUVEC cells with the S protein, slightly increased HIF1α and MCP-1 expression, which was significantly increased by the ACE inhibitor treatment. The S protein stimulation induced ROS generation and changed the mitogenic responses of the PBMCs through the upregulation of TNFα and interleukin (IL)-17 cytokine expression. These effects were reduced by the perindopril (100 µM) treatment. Proteomic analysis of the S protein stimulated THP-1 macrophages with or without perindopril (100 µM) exposed more than 400 differentially regulated proteins. Our results provide a mechanistic analysis suggesting that the blood and vascular components could be activated directly through S protein systemically present in the circulation and that the activation of the local renin angiotensin system may be partially involved in this process. Graphical: Suggested pathways that might be involved at least in part in S protein inducing activation of inflammatory markers (red narrow) and angiotensin-converting enzyme inhibitor (ACEi) modulation of this process (green narrow).

Structure-based drug designing and immunoinformatics approach for SARS-CoV-2.

The prevalence of respiratory illness caused by the novel SARS-CoV-2 virus associated with multiple organ failures is spreading rapidly because of its contagious human-to-human transmission and inadequate globalhealth care systems. Pharmaceutical repurposing, an effective drug development technique using existing drugs, could shorten development time and reduce costs compared to those of de novo drug discovery. We carried out virtual screening of antiviral compounds targeting the spike glycoprotein (S), main protease (Mpro), and the SARS-CoV-2 receptor binding domain (RBD)-angiotensin-converting enzyme 2 (ACE2) complex of SARS-CoV-2. PC786, an antiviral polymerase inhibitor, showed enhanced binding affinity to all the targets. Furthermore, the postfusion conformation of the trimeric S protein RBD with ACE2 revealed conformational changes associated with PC786 drug binding. Exploiting immunoinformatics to identify T cell and B cell epitopes could guide future experimental studies with a higher probability of discovering appropriate vaccine candidates with fewer experiments and higher reliability.

Identifying Primate ACE2 Variants That Confer Resistance to SARS-CoV-2.

SARS-CoV-2 infects humans through the binding of viral S-protein (spike protein) to human angiotensin I converting enzyme 2 (ACE2). The structure of the ACE2-S-protein complex has been deciphered and we focused on the 27 ACE2 residues that bind to S-protein. From human sequence databases, we identified nine ACE2 variants at ACE2-S-protein binding sites. We used both experimental assays and protein structure analysis to evaluate the effect of each variant on the binding affinity of ACE2 to S-protein. We found one variant causing complete binding disruption, two and three variants, respectively, strongly and mildly reducing the binding affinity, and two variants strongly enhancing the binding affinity. We then collected the ACE2 gene sequences from 57 nonhuman primates. Among the 6 apes and 20 Old World monkeys (OWMs) studied, we found no new variants. In contrast, all 11 New World monkeys (NWMs) studied share four variants each causing a strong reduction in binding affinity, the Philippine tarsier also possesses three such variants, and 18 of the 19 prosimian species studied share one variant causing a strong reduction in binding affinity. Moreover, one OWM and three prosimian variants increased binding affinity by >50%. Based on these findings, we proposed that the common ancestor of primates was strongly resistant to and that of NWMs was completely resistant to SARS-CoV-2 and so is the Philippine tarsier, whereas apes and OWMs, like most humans, are susceptible. This study increases our understanding of the differences in susceptibility to SARS-CoV-2 infection among primates.

Role of neutrophil chemoattractant CXCL5 in SARS-CoV-2 infection-induced lung inflammatory innate immune response in an in vivo hACE2 transfection mouse model.

Understanding the pathogenesis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and clarifying antiviral immunity in hosts are critical aspects for the development of vaccines and antivirals. Mice are frequently used to generate animal models of infectious diseases due to their convenience and ability to undergo genetic manipulation. However, normal adult mice are not susceptible to SARS-CoV-2. Here, we developed a viral receptor (human angiotensin-converting enzyme 2, hACE2) pulmonary transfection mouse model to establish SARS-CoV-2 infection rapidly in the mouse lung. Based on the model, the virus successfully infected the mouse lung 2 days after transfection. Viral RNA/protein, innate immune cell infiltration, inflammatory cytokine expression, and pathological changes in the infected lungs were observed after infection. Further studies indicated that neutrophils were the first and most abundant leukocytes to infiltrate the infected lungs after viral infection. In addition, using infected CXCL5-knockout mice, chemokine CXCL5 was responsible for neutrophil recruitment. CXCL5 knockout decreased lung inflammation without diminishing viral clearance, suggesting a potential target for controlling pneumonia.

Monoclonal antibodies for the S2 subunit of spike of SARS-CoV-1 cross-react with the newly-emerged SARS-CoV-2.

BackgroundA novel coronavirus, SARS-CoV-2, which emerged at the end of 2019 and causes COVID-19, has resulted in worldwide human infections. While genetically distinct, SARS-CoV-1, the aetiological agent responsible for an outbreak of severe acute respiratory syndrome (SARS) in 2002-2003, utilises the same host cell receptor as SARS-CoV-2 for entry: angiotensin-converting enzyme 2 (ACE2). Parts of the SARS-CoV-1 spike glycoprotein (S protein), which interacts with ACE2, appear conserved in SARS-CoV-2.AimThe cross-reactivity with SARS-CoV-2 of monoclonal antibodies (mAbs) previously generated against the S protein of SARS-CoV-1 was assessed.MethodsThe SARS-CoV-2 S protein sequence was aligned to those of SARS-CoV-1, Middle East respiratory syndrome (MERS) and common-cold coronaviruses. Abilities of mAbs generated against SARS-CoV-1 S protein to bind SARS-CoV-2 or its S protein were tested with SARS-CoV-2 infected cells as well as cells expressing either the full length protein or a fragment of its S2 subunit. Quantitative ELISA was also performed to compare binding of mAbs to recombinant S protein.ResultsAn immunogenic domain in the S2 subunit of SARS-CoV-1 S protein is highly conserved in SARS-CoV-2 but not in MERS and human common-cold coronaviruses. Four murine mAbs raised against this immunogenic fragment could recognise SARS-CoV-2 S protein expressed in mammalian cell lines. In particular, mAb 1A9 was demonstrated to detect S protein in SARS-CoV-2-infected cells and is suitable for use in a sandwich ELISA format.ConclusionThe cross-reactive mAbs may serve as useful tools for SARS-CoV-2 research and for the development of diagnostic assays for COVID-19.

Novel coronavirus SARS-CoV-2 (Covid-19) dynamics inside the human body.

A knowledge-based cybernetic framework model representing the dynamics of SARS-CoV-2 inside the human body has been studied analytically and in silico to explore the pathophysiologic regulations. The following modeling methodology was developed as a platform to introduce a predictive tool supporting a therapeutic approach to Covid-19 disease. A time-dependent nonlinear system of ordinary differential equations model was constructed involving type-I cells, type-II cells, SARS-CoV-2 virus, inflammatory mediators, interleukins along with host pulmonary gas exchange rate, thermostat control, and mean pressure difference. This formalism introduced about 17 unknown parameters. Estimating these unknown parameters requires a mathematical association with the in vivo sparse data and the dynamic sensitivities of the model. The cybernetic model can simulate a dynamic response to the reduced pulmonary alveolar gas exchange rate, thermostat control, and mean pressure difference under a very critical condition based on equilibrium (steady state) values of the inflammatory mediators and system parameters. In silico analysis of the current cybernetical approach with system dynamical modeling can provide an intellectual framework to help experimentalists identify more active therapeutic approaches.

Blocking of the High-Affinity Interaction-Synapse Between SARS-CoV-2 Spike and Human ACE2 Proteins Likely Requires Multiple High-Affinity Antibodies: An Immune Perspective.

The pandemic of Coronavirus Disease 2019 (COVID-19) caused by SARS-CoV-2 has induced global eagerness to develop vaccines and therapeutics for treating COVID-19, including neutralizing antibodies. To develop effective therapeutic antibodies against SARS-CoV-2, it is critical to understand the interaction between viral and host's proteins. The human ACE2 (hACE2) protein is the crucial target for the SARS-CoV's Spike protein that allows the virus to adhere to host epithelial cells. X-ray crystal structures and biophysical properties of protein-protein interactions reveal a large interaction surface with high binding-affinity between SARS-CoV-2 and hACE2 (18 interactions), at least 15-fold stronger than between SARS-CoV-1 and hACE2 (eight interactions). This suggests that antibodies against CoV-1 infection might not be very efficient against CoV-2. Furthermore, interspecies comparisons indicate that ACE2 proteins of man and cat are far closer than dog, ferret, mouse, and rat with significant differences in binding-affinity between Spike and ACE2 proteins. This strengthens the notion of productive SARS-CoV-2 transmission between felines and humans and that classical animal models are not optimally suited for evaluating therapeutic antibodies. The large interaction surface with strong affinity between SARS-CoV-2 and hACE2 (dG-12.4) poses a huge challenge to develop reliable antibody therapy that truly blocks SARS-CoV-2 adherence and infection. We gauge that single antibodies against single epitopes might not sufficiently interfere with the strong interaction-synapse between Spike and hACE2 proteins. Instead, appropriate combinations of high-affinity neutralizing antibodies against different epitopes might be needed, preferably of IgA-class for optimal and prolonged activity at epithelial layers of respiratory and intestine tracts.

Strategies for Targeting SARS CoV-2: Small Molecule Inhibitors-The Current Status.

Severe Acute Respiratory Syndrome-Corona Virus-2 (SARS-CoV-2) induced Coronavirus Disease - 19 (COVID-19) cases have been increasing at an alarming rate (7.4 million positive cases as on June 11 2020), causing high mortality (4,17,956 deaths as on June 11 2020) and economic loss (a 3.2% shrink in global economy in 2020) across 212 countries globally. The clinical manifestations of this disease are pneumonia, lung injury, inflammation, and severe acute respiratory syndrome (SARS). Currently, there is no vaccine or effective pharmacological agents available for the prevention/treatment of SARS-CoV2 infections. Moreover, development of a suitable vaccine is a challenging task due to antibody-dependent enhancement (ADE) and Th-2 immunopathology, which aggravates infection with SARS-CoV-2. Furthermore, the emerging SARS-CoV-2 strain exhibits several distinct genomic and structural patterns compared to other coronavirus strains, making the development of a suitable vaccine even more difficult. Therefore, the identification of novel small molecule inhibitors (NSMIs) that can interfere with viral entry or viral propagation is of special interest and is vital in managing already infected cases. SARS-CoV-2 infection is mediated by the binding of viral Spike proteins (S-protein) to human cells through a 2-step process, which involves Angiotensin Converting Enzyme-2 (ACE2) and Transmembrane Serine Protease (TMPRSS)-2. Therefore, the development of novel inhibitors of ACE2/TMPRSS2 is likely to be beneficial in combating SARS-CoV-2 infections. However, the usage of ACE-2 inhibitors to block the SARS-CoV-2 viral entry requires additional studies as there are conflicting findings and severe health complications reported for these inhibitors in patients. Hence, the current interest is shifted toward the development of NSMIs, which includes natural antiviral phytochemicals and Nrf-2 activators to manage a SARS-CoV-2 infection. It is imperative to investigate the efficacy of existing antiviral phytochemicals and Nrf-2 activators to mitigate the SARS-CoV-2-mediated oxidative stress. Therefore, in this review, we have reviewed structural features of SARS-CoV-2 with special emphasis on key molecular targets and their known modulators that can be considered for the development of NSMIs.

Ultrapotent human antibodies protect against SARS-CoV-2 challenge via multiple mechanisms.

Efficient therapeutic options are needed to control the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that has caused more than 922,000 fatalities as of 13 September 2020. We report the isolation and characterization of two ultrapotent SARS-CoV-2 human neutralizing antibodies (S2E12 and S2M11) that protect hamsters against SARS-CoV-2 challenge. Cryo-electron microscopy structures show that S2E12 and S2M11 competitively block angiotensin-converting enzyme 2 (ACE2) attachment and that S2M11 also locks the spike in a closed conformation by recognition of a quaternary epitope spanning two adjacent receptor-binding domains. Antibody cocktails that include S2M11, S2E12, or the previously identified S309 antibody broadly neutralize a panel of circulating SARS-CoV-2 isolates and activate effector functions. Our results pave the way to implement antibody cocktails for prophylaxis or therapy, circumventing or limiting the emergence of viral escape mutants.

Receptor-binding domain-specific human neutralizing monoclonal antibodies against SARS-CoV and SARS-CoV-2.

The outbreaks of severe acute respiratory syndrome (SARS) and Coronavirus Disease 2019 (COVID-19) caused by SARS-CoV and SARS-CoV-2, respectively, have posed severe threats to global public health and the economy. Treatment and prevention of these viral diseases call for the research and development of human neutralizing monoclonal antibodies (NMAbs). Scientists have screened neutralizing antibodies using the virus receptor-binding domain (RBD) as an antigen, indicating that RBD contains multiple conformational neutralizing epitopes, which are the main structural domains for inducing neutralizing antibodies and T-cell immune responses. This review summarizes the structure and function of RBD and RBD-specific NMAbs against SARS-CoV and SARS-CoV-2 currently under development.

Progress in Studies on Structural and Remedial Aspects of Newly Born Coronavirus, SARS-CoV-2.

The article highlights an up-to-date progress in studies on structural and the remedial aspects of novel coronavirus 2019-nCoV, renamed as SARS-CoV-2, leading to the disease COVID-19, a pandemic. In general, all CoVs including SARS-CoV-2 are spherical positive single-stranded RNA viruses containing spike (S) protein, envelope (E) protein, nucleocapsid (N) protein, and membrane (M) protein, where S protein has a Receptor-binding Domain (RBD) that mediates the binding to host cell receptor, Angiotensin Converting Enzyme 2 (ACE2). The article details the repurposing of some drugs to be tried for COVID-19 and presents the status of vaccine development so far. Besides drugs and vaccines, the role of Convalescent Plasma (CP) therapy to treat COVID-19 is also discussed.

Antibody-like proteins that capture and neutralize SARS-CoV-2.

To combat severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) and any unknown emerging pathogens in the future, the development of a rapid and effective method to generate high-affinity antibodies or antibody-like proteins is of critical importance. We here report high-speed in vitro selection of multiple high-affinity antibody-like proteins against various targets including the SARS-CoV-2 spike protein. The sequences of monobodies against the SARS-CoV-2 spike protein were successfully procured within only 4 days. Furthermore, the obtained monobody efficiently captured SARS-CoV-2 particles from the nasal swab samples of patients and exhibited a high neutralizing activity against SARS-CoV-2 infection (half-maximal inhibitory concentration, 0.5 nanomolar). High-speed in vitro selection of antibody-like proteins is a promising method for rapid development of a detection method for, and of a neutralizing protein against, a virus responsible for an ongoing, and possibly a future, pandemic.

Competitive SARS-CoV-2 Serology Reveals Most Antibodies Targeting the Spike Receptor-Binding Domain Compete for ACE2 Binding.

As severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to spread around the world, there is an urgent need for new assay formats to characterize the humoral response to infection. Here, we present an efficient, competitive serological assay that can simultaneously determine an individual's seroreactivity against the SARS-CoV-2 Spike protein and determine the proportion of anti-Spike antibodies that block interaction with the human angiotensin-converting enzyme 2 (ACE2) required for viral entry. In this approach based on the use of enzyme-linked immunosorbent assays (ELISA), we present natively folded viral Spike protein receptor-binding domain (RBD)-containing antigens via avidin-biotin interactions. Sera are then competed with soluble ACE2-Fc, or with a higher-affinity variant thereof, to determine the proportion of ACE2 blocking anti-RBD antibodies. Assessment of sera from 144 SARS-CoV-2 patients ultimately revealed that a remarkably consistent and high proportion of antibodies in the anti-RBD pool targeted the epitope responsible for ACE2 engagement (83% ± 11%; 50% to 107% signal inhibition in our largest cohort), further underscoring the importance of tailoring vaccines to promote the development of such antibodies.IMPORTANCE With the emergence and continued spread of the SARS-CoV-2 virus, and of the associated disease, coronavirus disease 2019 (COVID-19), there is an urgent need for improved understanding of how the body mounts an immune response to the virus. Here, we developed a competitive SARS-CoV-2 serological assay that can simultaneously determine whether an individual has developed antibodies against the SARS-CoV-2 Spike protein receptor-binding domain (RBD) and measure the proportion of these antibodies that block interaction with the human angiotensin-converting enzyme 2 (ACE2) required for viral entry. Using this assay and 144 SARS-CoV-2 patient serum samples, we found that a majority of anti-RBD antibodies compete for ACE2 binding. These results not only highlight the need to design vaccines to generate such blocking antibodies but also demonstrate the utility of this assay to rapidly screen patient sera for potentially neutralizing antibodies.

Neurological injuries in COVID-19 patients: direct viral invasion or a bystander injury after infection of epithelial/endothelial cells.

A subset of patients with coronavirus 2 disease (COVID-19) experience neurological complications. These complications include loss of sense of taste and smell, stroke, delirium, and neuromuscular signs and symptoms. The etiological agent of COVID-19 is SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2), an RNA virus with a glycoprotein-studded viral envelope that uses ACE2 (angiotensin-converting enzyme 2) as a functional receptor for infecting the host cells. Thus, the interaction of the envelope spike proteins with ACE2 on host cells determines the tropism and virulence of SARS-CoV-2. Loss of sense of taste and smell is an initial symptom of COVID-19 because the virus enters the nasal and oral cavities first and the epithelial cells are the receptors for these senses. Stroke in COVID-19 patients is likely a consequence of coagulopathy and injury to cerebral vascular endothelial cells that cause thrombo-embolism and stroke. Delirium and encephalopathy in acute and post COVID-19 patients are likely multifactorial and secondary to hypoxia, metabolic abnormalities, and immunological abnormalities. Thus far, there is no clear evidence that coronaviruses cause inflammatory neuromuscular diseases via direct invasion of peripheral nerves or muscles or via molecular mimicry. It appears that most of neurologic complications in COVID-19 patients are indirect and as a result of a bystander injury to neurons.

SARS-CoV-2 Assays To Detect Functional Antibody Responses That Block ACE2 Recognition in Vaccinated Animals and Infected Patients.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global pandemic of COVID-19, resulting in cases of mild to severe respiratory distress and significant mortality. The global outbreak of this novel coronavirus has now infected >20 million people worldwide, with >5 million cases in the United States (11 August 2020). The development of diagnostic and research tools to determine infection and vaccine efficacy is critically needed. We have developed multiple serologic assays using newly designed SARS-CoV-2 reagents for detecting the presence of receptor-binding antibodies in sera. The first assay is surface plasmon resonance (SPR) based and can quantitate both antibody binding to the SARS-CoV-2 spike protein and blocking to the Angiotensin-converting enzyme 2 (ACE2) receptor in a single experiment. The second assay is enzyme-linked immunosorbent assay (ELISA) based and can measure competition and blocking of the ACE2 receptor to the SARS-CoV-2 spike protein with antispike antibodies. The assay is highly versatile, and we demonstrate the broad utility of the assay by measuring antibody functionality of sera from small animals and nonhuman primates immunized with an experimental SARS-CoV-2 vaccine. In addition, we employ the assay to measure receptor blocking of sera from SARS-CoV-2-infected patients. The assay is shown to correlate with pseudovirus neutralization titers. This type of rapid, surrogate neutralization diagnostic can be employed widely to help study SARS-CoV-2 infection and assess the efficacy of vaccines.

Prospect of SARS-CoV-2 spike protein: Potential role in vaccine and therapeutic development.

The recent outbreak of the betacoronavirus SARS-CoV-2 has become a significant concern to public health care worldwide. As of August 19, 2020, more than 22,140,472 people are infected, and over 781,135 people have died due to this deadly virus. In the USA alone, over 5,482,602 people are currently infected, and more than 171,823 people have died. SARS-CoV-2 has shown a higher infectivity rate and a more extended incubation period as compared to previous coronaviruses. SARS-CoV-2 binds much more strongly than SARS-CoV to the same host receptor, angiotensin-converting enzyme 2 (ACE2). Previously, several methods to develop a vaccine against SARS-CoV or MERS-CoV have been tried with limited success. Since SARS-CoV-2 uses the spike (S) protein for entry to the host cell, it is one of the most preferred targets for making vaccines or therapeutics against SARS-CoV-2. In this review, we have summarised the characteristics of the S protein, as well as the different approaches being used for the development of vaccines and/or therapeutics based on the S protein.

Animal and translational models of SARS-CoV-2 infection and COVID-19.

COVID-19 is causing a major once-in-a-century global pandemic. The scientific and clinical community is in a race to define and develop effective preventions and treatments. The major features of disease are described but clinical trials have been hampered by competing interests, small scale, lack of defined patient cohorts and defined readouts. What is needed now is head-to-head comparison of existing drugs, testing of safety including in the background of predisposing chronic diseases, and the development of new and targeted preventions and treatments. This is most efficiently achieved using representative animal models of primary infection including in the background of chronic disease with validation of findings in primary human cells and tissues. We explore and discuss the diverse animal, cell and tissue models that are being used and developed and collectively recapitulate many critical aspects of disease manifestation in humans to develop and test new preventions and treatments.

COVID-19 Vaccine: A comprehensive status report.

The current COVID-19 pandemic has urged the scientific community internationally to find answers in terms of therapeutics and vaccines to control SARS-CoV-2. Published investigations mostly on SARS-CoV and to some extent on MERS has taught lessons on vaccination strategies to this novel coronavirus. This is attributed to the fact that SARS-CoV-2 uses the same receptor as SARS-CoV on the host cell i.e. human Angiotensin Converting Enzyme 2 (hACE2) and is approximately 79% similar genetically to SARS-CoV. Though the efforts on COVID-19 vaccines started very early, initially in China, as soon as the outbreak of novel coronavirus erupted and then world-over as the disease was declared a pandemic by WHO. But we will not be having an effective COVID-19 vaccine before September, 2020 as per very optimistic estimates. This is because a successful COVID-19 vaccine will require a cautious validation of efficacy and adverse reactivity as the target vaccinee population include high-risk individuals over the age of 60, particularly those with chronic co-morbid conditions, frontline healthcare workers and those involved in essentials industries. Various platforms for vaccine development are available namely: virus vectored vaccines, protein subunit vaccines, genetic vaccines, and monoclonal antibodies for passive immunization which are under evaluations for SARS-CoV-2, with each having discrete benefits and hindrances. The COVID-19 pandemic which probably is the most devastating one in the last 100 years after Spanish flu mandates the speedy evaluation of the multiple approaches for competence to elicit protective immunity and safety to curtail unwanted immune-potentiation which plays an important role in the pathogenesis of this virus. This review is aimed at providing an overview of the efforts dedicated to an effective vaccine for this novel coronavirus which has crippled the world in terms of economy, human health and life.

A pharmacological perspective of chloroquine in SARS-CoV-2 infection: An old drug for the fight against a new coronavirus?

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is having serious consequences on health and the economy worldwide. All evidence-based treatment strategies need to be considered to combat this new virus. Drugs need to be considered on scientific grounds of efficacy, safety and cost. Chloroquine (CQ) and hydroxychloroquine (HCQ) are old drugs used in the treatment of malaria. Moreover, their antiviral properties have been previously studied, including against coronaviruses, where evidence of efficacy has been found. In the current race against time triggered by the COVID-19 pandemic, the search for new antivirals is very important. However, consideration should be given to old drugs with known anti-coronavirus activity, such as CQ and HCQ. These could be integrated into current treatment strategies while novel treatments are awaited, also in light of the fact that they display an anticoagulant effect that facilitates the activity of low-molecular-weight heparin, aimed at preventing acute respiratory distress syndrome (ARDS)-associated thrombotic events. The safety of CQ and HCQ has been studied for over 50 years, however recently published data raise concerns for cardiac toxicity of CQ/HCQ in patients with COVID-19. This review also re-examines the real information provided by some of the published alarming reports, although concluding that cardiac toxicity should in any case be stringently monitored in patients receiving CQ/HCQ.

Functional prediction and comparative population analysis of variants in genes for proteases and innate immunity related to SARS-CoV-2 infection.

New coronavirus SARS-CoV-2 is capable to infect humans and cause a novel disease COVID-19. Aiming to understand a host genetic component of COVID-19, we focused on variants in genes encoding proteases and genes involved in innate immunity that could be important for susceptibility and resistance to SARS-CoV-2 infection. Analysis of sequence data of coding regions of FURIN, PLG, PRSS1, TMPRSS11a, MBL2 and OAS1 genes in 143 unrelated individuals from Serbian population identified 22 variants with potential functional effect. In silico analyses (PolyPhen-2, SIFT, MutPred2 and Swiss-Pdb Viewer) predicted that 10 variants could impact the structure and/or function of proteins. These protein-altering variants (p.Gly146Ser in FURIN; p.Arg261His and p.Ala494Val in PLG; p.Asn54Lys in PRSS1; p.Arg52Cys, p.Gly54Asp and p.Gly57Glu in MBL2; p.Arg47Gln, p.Ile99Val and p.Arg130His in OAS1) may have predictive value for inter-individual differences in the response to the SARS-CoV-2 infection. Next, we performed comparative population analysis for the same variants using extracted data from the 1000 Genomes project. Population genetic variability was assessed using delta MAF and Fst statistics. Our study pointed to 7 variants in PLG, TMPRSS11a, MBL2 and OAS1 genes with noticeable divergence in allelic frequencies between populations worldwide. Three of them, all in MBL2 gene, were predicted to be damaging, making them the most promising population-specific markers related to SARS-CoV-2 infection. Comparing allelic frequencies between Serbian and other populations, we found that the highest level of genetic divergence related to selected loci was observed with African, followed by East Asian, Central and South American and South Asian populations. When compared with European populations, the highest divergence was observed with Italian population. In conclusion, we identified 4 variants in genes encoding proteases (FURIN, PLG and PRSS1) and 6 in genes involved in the innate immunity (MBL2 and OAS1) that might be relevant for the host response to SARS-CoV-2 infection.