Ultrasound treatment inhibits SARS-CoV-2 in vitro infectivity (preprint)

Background: COVID-19 (coronavirus disease 2019) is a disease caused by infection with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), affecting millions of people worldwide, with a high rate of deaths. The present study aims to evaluate ultrasound (US) as a physical method for virus inactivation. Materials and methods: The UV-transductor was exposed to the SARS-CoV-2 viral solution for 30 minutes. Vero-E6 cells were infected with medium exposure or not with the US, using 3-12, 5-10, or 6-18MHz as frequencies applied. We performed confocal microscopy to determine virus infection and replicative process. Moreover, we detected the virus particles with a titration assay. Results: We observed an effective infection of SARS-CoV-2 Wuhan, Delta, and Gamma strains in comparison with mock, an uninfected experimental group. The US treatment was able to inhibit the Wuhan strain in all applied frequencies. Interestingly, 3-12 and 6-18MHz did not inhibit SARS-CoV-2 delta and gamma variants infection, on the other hand, 5-10MHz was able to abrogate infection and replication in all experimental conditions. Conclusions: These results show that SARS-CoV-2 is susceptible to US exposure at a specific frequency 5-10MHz and could be a novel tool for reducing the incidence of SARS-CoV-2 infection. Keywords: Ultrasound, SARS-CoV-2, virucidal effect, COVID-19

Targeting Neutrophils Extracellular Traps (NETs) reduces multiple organ injury in a COVID-19 mouse model (preprint)

COVID-19 is characterized by severe acute lung injury, which is associated with neutrophils infiltration and release of neutrophil extracellular traps (NETs). COVID-19 treatment options are scarce. Previous work has shown an increase in NETs release in the lung and plasma of COVID-19 patients suggesting that drugs that prevent NETs formation or release could be potential therapeutic approaches for COVID-19 treatment. Here, we report the efficacy of NET-degrading DNase I treatment in a murine model of COVID-19. DNase I decreased detectable levels of NETs, improved clinical disease, and reduced lung, heart, and kidney injuries in SARS-CoV-2-infected K18-hACE2 mice. Furthermore, our findings indicate a potential deleterious role for NETs lung tissue in vivo and lung epithelial (A549) cells in vitro, which might explain part of the pathophysiology of severe COVID-19. This deleterious effect was diminished by the treatment with DNase I. Together, our results support the role of NETs in COVID-19 immunopathology and highlight NETs disruption pharmacological approaches as a potential strategy to ameliorate COVID-19 clinical outcomes.

Efferocytosis of SARS-CoV-2-infected dying cells impairs macrophage anti-inflammatory programming and continual clearance of apoptotic cells (preprint)

COVID-19 is a disease of dysfunctional immune responses, but the mechanisms triggering immunopathogenesis are not established. The functional plasticity of macrophages allows this cell type to promote pathogen elimination and inflammation or suppress inflammation and promote tissue remodeling and injury repair. During an infection, the clearance of dead and dying cells, a process named efferocytosis, can modulate the interplay between these contrasting functions. Here, we show that engulfment of SARS-CoV2-infected apoptotic cells (AC) exacerbates inflammatory cytokine production, inhibits the expression of efferocytic receptors, and impairs continual efferocytosis by macrophages. We also provide evidence that monocytes from severe COVID-19 patients express reduced levels of efferocytic receptors and fail to uptake AC. Our findings reveal that dysfunctional efferocytosis of SARS-CoV-2-infected cell corpses suppress macrophage anti-inflammation and efficient tissue repair programs and provide mechanistic insights for the pathogenesis of the hyperinflammation and extensive tissue damage associated with COVID-19.

Neuraminidase inhibitors rewire neutrophil function in murine sepsis and COVID-19 patient cells (preprint)

Neutrophils overstimulation plays a crucial role in tissue damage during severe infections. Neuraminidase-mediated cleavage of surface sialic acid has been demonstrated to regulate leukocyte responses. Here, we report that antiviral neuraminidase inhibitors constrain host neuraminidase activity, surface sialic acid release, ROS production, and NETs released by microbial-activated human neutrophils. In vivo, treatment with Oseltamivir results in infection control and host survival in murine models of sepsis. Moreover, Oseltamivir or Zanamivir treatment of whole blood cells from severe COVID-19 patients reduces host NEU-mediated shedding of surface sialic acid and neutrophil overactivation. These findings suggest that neuraminidase inhibitors are host-directed interventions to dampen neutrophil dysfunction in severe infections.

Nanodroplet-Benzalkonium Chloride Formulation Demonstrates In Vitro and Ex Vivo Broad-Spectrum Antiviral Activity Against SARS-CoV-2 and Other Enveloped Viruses (preprint)

The Covid-19 pandemic has highlighted the importance of aerosolized droplets inhaled into the nose in the transmission of respiratory viral disease. Inactivating pathogenic viruses at the nasal portal of entry may reduce viral loads, thereby reducing transmission and contagion. We have developed an oil-in-water nanoemulsion (nanodroplet) formulation containing the potent antiseptic 0.13% Benzalkonium Chloride (NE-BZK) which demonstrates safe and broad anti-viral activity. While The Centers for Disease Control and Prevention (CDC) have reported that BZK may have less reliable activity than ethyl alcohol against certain viruses, including coronaviruses, we have demonstrated that NE-BZK exhibits broad-spectrum, long-lasting antiviral activity with >99.99% in vitro killing of enveloped viruses including SARS-CoV-2, human coronavirus, RSV and influenza B. Furthermore, in vitro studies demonstrated that NE-BZK continues to kill >99.99% of human coronavirus even when diluted 20-fold, while 0.13% aqueous BZK solution (AQ-BZK) did not. Ex vivo studies of NE-BZK on human cadaver skin demonstrated persistent >99.99% killing of human coronavirus for at least 8 hours after application. AQ-BZK failed to demonstrate durable antimicrobial activity on skin over time. The repeated application of NE-BZK, twice daily for 2 weeks on to rabbit nostrils indicated safety with no irritation. These findings demonstrate that formulating BZK on the surface of proprietary nanodroplets offers a safe and effective antiviral as a significant addition to strategies to combat the spread of respiratory viral infectious diseases.

Bifurcations and mutation hot-spots in the SARS-CoV-2 spike protein (preprint)

The spike protein is a most promising target for the development of vaccines and therapeutic drugs against the SARS-CoV-2 infection. But the apparently high rate of mutations makes the development of antiviral inhibitors a challenge. Here a methodology is presented to try and predict mutation hot-spot sites, where a small local change in spike protein's structure can lead to a large scale conformational effect, and change the protein's biological function. The methodology starts with a systematic physics based investigation of the spike protein's C backbone in terms of its local topology. This topological investigation is then combined with a statistical examination of the pertinent backbone fragments; the statistical analysis builds on a comparison with high resolution Protein Data Bank (PDB) structures. Putative mutation hot-spot sites are identified as proximal sites to bifurcation points that can change the local topology of the C backbone in an essential manner. The likely outcome of a mutation, if it indeed occurs, is predicted by a comparison with residues in best-matching PDB fragments together with general stereochemical considerations. The detailed methodology is developed using the already observed D614G mutation as an example. This is a mutation that could have been correctly predicted by the present approach. Several additional examples of potential hot-spot residues are identified and analyzed in detail, some of them are found to be even better candidates for a mutation hot-spot than D614G.

Identifying and prioritizing potential human-infecting viruses from their genome sequences (preprint)

Rapid assessment of which animal viruses may be capable of infecting humans is currently intractable, but would allow their prioritization for further investigation and pandemic preparedness. We developed machine learning algorithms that identify candidate zoonoses using evolutionary signals of host range encoded in viral genomes. This reduces lists of hundreds of viruses with uncertain human infectivity to tractable numbers for prioritized research, generalizes to virus families excluded from model training, can distinguish high risk viruses within families that contain a minority of zoonotic species, and could have identified the exceptional risk of SARS-CoV-2 prior to its emergence. Genome-based risk assessment allows identification of high-risk viruses immediately upon discovery, increasing both the feasibility and likelihood of downstream virological and ecological characterization and allowing for evidence-driven virus surveillance.

Zebrafish studies on the vaccine candidate to COVID-19, the Spike protein: Production of antibody and adverse reaction (preprint)

Establishing new experimental animal models to assess the safety and immune response to the antigen used in the development of COVID-19 vaccine is an imperative issue. Based on the advantages of using zebrafish as a model in research, herein we suggest doing this to test the safety of the putative vaccine candidates and to study immune response against the virus. We produced a recombinant N-terminal fraction of the Spike SARS-CoV-2 protein and injected it into adult female zebrafish. The specimens generated humoral immunity and passed the antibodies to the eggs. However, they presented adverse reactions and inflammatory responses similar to severe cases of human COVID-19. The analysis of the structure and function of zebrafish and human Angiotensin-converting enzyme 2, the main human receptor for virus infection, presented remarkable sequence similarities. Moreover, bioinformatic analysis predicted protein-protein interaction of the Spike SARS-CoV-2 fragment and the Toll-like receptor pathway. It might help in the choice of future therapeutic pharmaceutical drugs to be studied. Based on the in vivo and in silico results presented here, we propose the zebrafish as a model for translational research into the safety of the vaccine and the immune response of the vertebrate organism to the SARS-CoV-2 virus.

Isolation of cross-reactive monoclonal antibodies against divergent human coronaviruses that delineate a conserved and vulnerable site on the spike protein (preprint)

The coronavirus spike glycoprotein, located on the virion surface, is the key mediator of cell entry. As such, it is an attractive target for the development of protective antibodies and vaccines. Here we describe two human monoclonal antibodies, 1.6C7 and 28D9, that display a remarkable cross-reactivity against distinct species from three Betacoronavirus subgenera, capable of binding the spike proteins of SARS-CoV and SARS-CoV-2, MERS-CoV and the endemic human coronavirus HCoV-OC43. Both antibodies, derived from immunized transgenic mice carrying a human immunoglobulin repertoire, blocked MERS-CoV infection in cells, whereas 28D9 also showed weak cross-neutralizing potential against HCoV-OC43, SARS-CoV and SARS-CoV-2 in a neutralization-sensitive virus pseudotyping system, but not against authentic virus. Both cross-reactive monoclonal antibodies were found to target the stem helix in the spike protein S2 fusion subunit which, in the prefusion conformation of trimeric spike, forms a surface exposed membrane-proximal helical bundle, that is antibody-accessible. We demonstrate that administration of these antibodies in mice protects from a lethal MERS-CoV challenge in both prophylactic and/or therapeutic models. Collectively, these antibodies delineate a conserved, immunogenic and vulnerabe site on the spike protein which spurs the development of broad-range diagnostic, preventive and therapeutic measures against coronaviruses.

SARS-CoV-2 infects brain astrocytes of COVID-19 patients and impairs neuronal viability (preprint)

COVID-19 patients may exhibit neuropsychiatric and/or neurological symptoms. We found that anxiety and cognitive impairment are manifested by 28-56% of SARS-CoV-2-infected individuals with mild or no respiratory symptoms and are associated with altered cerebral cortical thickness. Using an independent cohort, we found histopathological signs of brain damage in 19% of individuals who died of COVID-19. All of the affected brain tissues exhibited foci of SARS-CoV-2 infection, particularly in astrocytes. Infection of neural stem cell-derived astrocytes changed energy metabolism, altered key proteins and metabolites used to fuel neurons and for biogenesis of neurotransmitters, and elicited a secretory phenotype that reduces neuronal viability. Our data support the model where SARS-CoV-2 reaches the brain, infects astrocytes and triggers neuropathological changes that contribute to the structural and functional alterations in the brain of COVID-19 patients.

Sequential infection with influenza A virus followed by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) leads to more severe disease and encephalitis in a mouse model of COVID-19. (preprint)

COVID-19 is a spectrum of clinical symptoms in humans caused by infection with SARS-CoV-2, a recently emerged coronavirus that has rapidly caused a pandemic. Coalescence of a second wave of this virus with seasonal respiratory viruses, particularly influenza virus is a possible global health concern. To investigate this, transgenic mice expressing the human ACE2 receptor driven by the epithelial cell cytokeratin-18 gene promoter (K18-hACE2) were first infected with IAV followed by SARS-CoV-2. The host response and effect on virus biology was compared to K18-hACE2 mice infected with IAV or SARS-CoV-2 only. Infection of mice with each individual virus resulted in a disease phenotype compared to control mice. Although, SARS-CoV-2 RNA synthesis appeared significantly reduced in the sequentially infected mice, these mice had a more rapid weight loss, more severe lung damage and a prolongation of the innate response compared to singly infected or control mice. The sequential infection also exacerbated the extrapulmonary manifestations associated with SARS-CoV-2. This included a more severe encephalitis. Taken together, the data suggest that the concept of "twinfection" is deleterious and mitigation steps should be instituted as part of a comprehensive public health response to the COVID-19 pandemic.

A 3D Structural Interactome to Explore the Impact of Evolutionary Divergence, Population Variation, and Small-molecule Drugs on SARS-CoV-2-Human Protein-Protein Interactions (preprint)

The recent COVID-19 pandemic has sparked a global public health crisis. Vital to the development of informed treatments for this disease is a comprehensive understanding of the molecular interactions involved in disease pathology. One lens through which we can better understand this pathology is through the network of protein-protein interactions between its viral agent, SARS-CoV-2, and its human host. For instance, increased infectivity of SARS-CoV-2 compared to SARS-CoV can be explained by rapid evolution along the interface between the Spike protein and its human receptor (ACE2) leading to increased binding affinity. Sequence divergences that modulate other protein-protein interactions may further explain differences in transmission and virulence in this novel coronavirus. To facilitate these comparisons, we combined homology-based structural modeling with the ECLAIR pipeline for interface prediction at residue resolution, and molecular docking with PyRosetta. This enabled us to compile a novel 3D structural interactome meta-analysis for the published interactome network between SARS-CoV-2 and human. This resource includes docked structures for all interactions with protein structures, enrichment analysis of variation along interfaces, predicted {Delta}{Delta}G between SARS-CoV and SARS-CoV-2 variants for each interaction, predicted impact of natural human population variation on binding affinity, and a further prioritized set of drug repurposing candidates predicted to overlap with protein interfaces. All predictions are available online for easy access and are continually updated when new interactions are published. NOTE: Some sections of this pre-print have been redacted to comply with current bioRxiv policy restricting the dissemination of purely in silico results predicting potential therapies for SARS-CoV-2 that have not undergone thorough peer-review. The results section titled 'Prioritization of Candidate Inhibitors of SARS-CoV-2-Human Interactions Through Binding Site Comparison,' Figure 4, Supplemental Table 9, and all links to our web resource have been removed. Blank headers left in place to preserve structure and item numbering. Our full manuscript will be published in an appropriate journal following peer-review.

Molecular characterization of SARS-CoV-2 from Bangladesh: Implications in genetic diversity, possible origin of the virus, and functional significance of the mutations (preprint)

In a try to understand the pathogenesis, evolution, and epidemiology of the SARS-CoV-2 virus, scientists from all over the world are tracking its genomic changes in real-time. Genomic studies can be helpful in understanding the disease dynamics. We have downloaded 324 complete and near-complete SARS-CoV-2 genomes submitted in the GISAID database from Bangladesh which were isolated between 30 March to 7 September 2020. We then compared these genomes with the Wuhan reference sequence and found 4160 mutation events including 2253 missense single nucleotide variations, 38 deletions, and 10 insertions. The C>T nucleotide change was most prevalent possibly due to selective mutation pressure to reduce CpG sites to evade CpG targeted host immune response. The most frequent mutation that occurred in 98% of the isolates was 3037C>T which is a synonymous change that almost always accompanied 3 other mutations that include 241C>T, 14408C>T (P323L in RdRp), and 23403A>G (D614G in spike protein). The P323L was reported to increase mutation rate and D614G is associated with increased viral replication and currently the most prevalent variant circulating all over the world. We identified multiple missense mutations in B-cell and T-cell predicted epitope regions and/or PCR target regions (including R203K and G204R that occurred in 86% of the isolates) that may impact immunogenicity and/or RT-PCR based diagnosis. Our analysis revealed 5 large deletion events in ORF7a and ORF8 gene products that may be associated with less severity of the disease and increased viral clearance. Our phylogeny analysis identified most of the isolates belonged to the Nextstrain clade 20B (86%) and GISAID clade GR (88%). Most of our isolates shared common ancestors either directly with European countries or jointly with middle eastern countries as well as Australia and India. Interestingly, the 19B clade (GISAID S clade) was unique to Chittagong which was originally prevalent in China. This reveals possible multiple introductions of the virus in Bangladesh via different routes. Hence more genome sequencing and analysis with related clinical data are needed to interpret the functional significance and better predict the disease dynamics that may be helpful for policymakers to control the COVID-19 pandemic in Bangladesh.

Structural basis for bivalent binding and inhibition of SARS-CoV-2 infection by human potent neutralizing antibodies (preprint)

Neutralizing monoclonal antibodies (nAbs) to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) represent promising candidates for clinical intervention against coronavirus virus diseases 2019 (COVID-19). We isolated a large number of nAbs from SARS-CoV-2 infected individuals capable of disrupting proper interaction between the receptor binding domain (RBD) of the viral spike (S) protein and the receptor angiotensin converting enzyme 2 (ACE2). In order to understand the mechanism of these nAbs on neutralizing SARS-CoV-2 virus infections, we have performed cryo-EM analysis and here report cryo-EM structures of the ten most potent nAbs in their native full-length IgG or Fab forms bound to the trimeric S protein of SARS-CoV-2. The bivalent binding of the full-length IgG is found to associate with more RBD in the "up" conformation than the monovalent binding of Fab, perhaps contributing to the enhanced neutralizing activity of IgG and triggering more shedding of the S1 subunit from the S protein. Comparison of large number of nAbs identified common and unique structural features associated with their potent neutralizing activities. This work provides structural basis for further understanding the mechanism of nAbs, especially through revealing the bivalent binding and their correlation with more potent neutralization and the shedding of S1 subunit.

Degradation of SARS-CoV-2 receptor ACE2 by tobacco carcinogen-induced Skp2 in lung epithelial cells (preprint)

An unexpected observation among the COVID-19 pandemic is that smokers constituted only 1.4-18.5% of hospitalized adults, calling for an urgent investigation to determine the role of smoking in SARS-CoV-2 infection. Here, we show that cigarette smoke extract (CSE) and carcinogen benzo(a)pyrene (BaP) increase ACE2 mRNA but trigger ACE2 protein catabolism. BaP induces an aryl hydrocarbon receptor (AhR)-dependent upregulation of the ubiquitin E3 ligase Skp2 for ACE2 ubiquitination. ACE2 in lung tissues of non-smokers is higher than in smokers, consistent with the findings that tobacco carcinogens downregulate ACE2 in mice. Tobacco carcinogens inhibit SARS-CoV-2 Spike protein pseudovirions infection of the cells. Given that tobacco smoke accounts for 8 million deaths including 2.1 million cancer deaths annually and Skp2 is an oncoprotein, tobacco use should not be recommended and cessation plan should be prepared for smokers in COVID-19 pandemic.

Structural impact on SARS-CoV-2 spike protein by D614G substitution (preprint)

Substitution for aspartic acid by glycine at position 614 in the spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the ongoing pandemic, appears to facilitate rapid viral spread. The G614 variant has now replaced the D614-carrying virus as the dominant circulating strain. We report here cryo-EM structures of a full-length S trimer carrying G614, which adopts three distinct prefusion conformations differing primarily by the position of one receptor-binding domain (RBD). A loop disordered in the D614 S trimer wedges between domains within a protomer in the G614 spike. This added interaction appears to prevent premature dissociation of the G614 trimer, effectively increasing the number of functional spikes and enhancing infectivity. The loop transition may also modulate structural rearrangements of S protein required for membrane fusion. These findings extend our understanding of viral entry and suggest an improved immunogen for vaccine development.

Beneficial effects of colchicine for moderate to severe COVID-19: an interim analysis of a randomized, double-blinded, placebo controlled clinical trial (preprint)

Introduction. Neutrophilia and high levels of proinflammatory cytokines and other mediators of inflammation are common finds in patients with severe acute respiratory syndrome due to COVID-19. By its action on leukocytes, we propose colchicine as an intervention worthy of being tested. Objective. To evaluate whether the addition of colchicine to standard treatment for COVID-19 results in better outcomes. Methods. We present the interim analysis of a single-center randomized, double-blinded, placebo controlled clinical trial of colchicine for the treatment of moderate to severe COVID-19, with 38 patients allocated 1:1 from April 11 to July 06, 2020. Colchicine regimen was 0.5 mg thrice daily for 5 days, then 0.5 mg twice daily for 5 days. The first dose was 1.0 mg whether body weight was [≥] 80 kg. Endpoints. The primary endpoints were the need for supplemental oxygen; time of hospitalization; need for admission and length of stay in intensive care units; and death rate and causes of mortality. As secondary endpoints, we assessed: serum C-reactive protein, serum Lactate dehydrogenase and relation neutrophil to lymphocyte of peripheral blood samples from day zero to day 7; the number, type, and severity of adverse events; frequency of interruption of the study protocol due to adverse events; and frequency of QT interval above 450 ms. Results. Thirty-five patients (18 for Placebo and 17 for Colchicine) completed the study. Both groups were comparable in terms of demographic, clinical and laboratory data at baseline. Median (and interquartile range) time of need for supplemental oxygen was 3.0 (1.5-6.5) days for the Colchicine group and 7.0 (3.0-8.5) days for Placebo group (p = 0.02). Median (IQR) time of hospitalization was 6.0 (4.0-8.5) days for the Colchicine group and 8.5 (5.5-11.0) days for Placebo group (p = 0.03). At day 2, 53% vs 83% of patients maintained the need for supplemental oxygen, while at day 7 the values were 6% vs 39%, in the Colchicine and Placebo groups, respectively (log rank; p = 0.01). Hospitalization was maintained for 53% vs 78% of patients at day 5 and 6% vs 17% at day 10, for the Colchicine and Placebo groups, respectively (log rank; p = 0.01). One patient per group needed admission to ICU. No recruited patient died. At day 4, patients of Colchicine group presented significant reduction of serum C-reactive protein compared to baseline (p < 0.001). The majority of adverse events were mild and did not lead to patient withdrawal. Diarrhea was more frequent in the Colchicine group (p = 0.17). Cardiac adverse events were absent. Discussion. The use of colchicine reduced the length of supplemental oxygen therapy and the length of hospitalization. Clinical improvement was in parallel with a reduction on serum levels of C-reactive protein. The drug was safe and well tolerated. Colchicine may be considered a beneficial and not expensive option for COVID-19 treatment. Clinical trials with larger numbers of patients should be conducted to further evaluate the efficacy and safety of colchicine as an adjunctive therapy for hospitalized patients with moderate to severe COVID-19.

In-depth Analysis of Laboratory Parameters Reveals the Interplay Between Sex, Age and Systemic Inflammation in Individuals with COVID-19 (preprint)

Introduction: The progression and severity of the coronavirus disease 2019 (COVID-19), an infectious disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), varies significantly in the population. While the hallmarks of SARS-CoV-2 and severe COVID-19 within routine laboratory parameters are emerging, little is known about the impact of sex and age on these profiles. Methods: We performed multidimensional analysis of millions of records of laboratory parameters and diagnostic tests for 178,887 individuals, of which 33,266 tested positive for SARS-CoV-2. These included complete blood cell count, electrolytes, metabolites, arterial blood gases, enzymes, hormones, cancer biomarkers, and others. Results: COVID-19 induced more alterations in the laboratory parameters in males compared to females between 13 and 60 years old, in contrast to older individuals, where several parameters were altered by COVID-19 in both men and women. Biomarkers of inflammation, such as C-reactive protein (CRP) and ferritin, were increased especially in older men with COVID-19, whereas other markers such as abnormal liver function tests were common across several age groups, except for young women. Low peripheral blood basophils and eosinophils were also more common in the elderly with COVID-19. Both male and female COVID-19 patients admitted to the intensive care unit (ICU) displayed alterations in the coagulation system, and higher levels of neutrophils, CRP, lactate dehydrogenase (LDH), among others. Discussion: Our study uncovers the laboratory profile of a large cohort of COVID-19 patients that underly discrepancies influenced by aging and biological sex. These profiles directly link COVID-19 disease presentation to an intricate interplay between sex, age and the immune response.

Inflammasome activation in COVID-19 patients (preprint)

Severe cases of COVID-19 are characterized by a strong inflammatory process that may ultimately lead to organ failure and patient death. The NLRP3 inflammasome is a molecular platform that promotes inflammation via cleavage and activation of key inflammatory molecules including active caspase-1 (Casp1p20), IL-1{beta} and IL-18. Although the participation of the inflammasome in COVID-19 has been highly speculated, the inflammasome activation and participation in the outcome of the disease is unknown. Here we demonstrate that the NLRP3 inflammasome is activated in response to SARS-CoV-2 infection and it is active in COVID-19, influencing the clinical outcome of the disease. Studying moderate and severe COVID-19 patients, we found active NLRP3 inflammasome in PBMCs and tissues of post-mortem patients upon autopsy. Inflammasome-derived products such as Casp1p20 and IL-18 in the sera correlated with the markers of COVID-19 severity, including IL-6 and LDH. Moreover, higher levels of IL-18 and Casp1p20 are associated with disease severity and poor clinical outcome. Our results suggest that the inflammasome is key in the pathophysiology of the disease, indicating this platform as a marker of disease severity and a potential therapeutic target for COVID-19.

Infection of human lymphomononuclear cells by SARS-CoV-2 (preprint)

Although SARS-CoV-2 severe infection is associated with a hyperinflammatory state, lymphopenia is an immunological hallmark, and correlates with poor prognosis in COVID-19. However, it remains unknown if circulating human lymphocytes and monocytes are susceptible to SARS-CoV-2 infection. In this study, SARS-CoV-2 infection of human peripheral blood mononuclear cells (PBMCs) was investigated both in vitro and in vivo. We found that in vitro infection of whole PBMCs from healthy donors was productive of virus progeny. Results revealed that monocytes, as well as B and T lymphocytes, are susceptible to SARS-CoV-2 active infection and viral replication was indicated by detection of double-stranded RNA. Moreover, flow cytometry and immunofluorescence analysis revealed that SARS-CoV-2 was frequently detected in monocytes and B lymphocytes from COVID-19 patients, and less frequently in CD4+T lymphocytes. The rates of SARS-CoV-2-infected monocytes in PBMCs from COVID-19 patients increased over time from symptom onset. Additionally, SARS-CoV-2-positive monocytes and B and CD4+T lymphocytes were detected by immunohistochemistry in post mortem lung tissue. SARS-CoV-2 infection of blood circulating leukocytes in COVID-19 patients may have important implications for disease pathogenesis, immune dysfunction, and virus spread within the host.