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The SARS-CoV-2 infections are still imposing a great public health challenge despite the recent developments in vaccines and therapy. Searching for diagnostic and prognostic methods that are fast, low-cost and accurate is essential for disease control and patient recovery. The MALDI-TOF mass spectrometry technique is rapid, low cost and accurate when compared to other MS methods, thus its use is already reported in the literature for various applications, including microorganism identification, diagnosis and prognosis of diseases. Here we developed a prognostic method for COVID-19 using the proteomic profile of saliva samples submitted to MALDI-TOF and machine learning algorithms to train models for COVID-19 severity assessment. We achieved an accuracy of 88.5%, specificity of 85% and sensitivity of 91.5% for classification between mild/moderate and severe conditions. Then, we tested the model performance in an independent dataset, we achieved an accuracy, sensitivity and specificity of 67.18, 52.17 and 75.60% respectively. Saliva is already reported to have high inter-sample variation; however, our results demonstrates that this approach has the potential to be a prognostic method for COVID-19. Additionally, the technology used is already available in several clinics, facilitating the implementation of the method. Further investigation using a bigger dataset is necessary to consolidate the technique.

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SARS-CoV-2 papain-like protease (PLpro) covers multiple functions. Beside the cysteine-protease activity, PLpro has the additional and vital function of removing ubiquitin and ISG15 (Interferon-stimulated gene 15) from host-cell proteins to aid coronaviruses in evading the hosts innate immune responses. We established a high-throughput X-ray screening to identify inhibitors by elucidating the native PLpro structure refined to 1.42 [A] and performing co-crystallization utilizing a diverse library of selected natural compounds. We identified three phenolic compounds as potential inhibitors. Crystal structures of PLpro inhibitor complexes, obtained to resolutions between 1.7-1.9 [A], show that all three compounds bind at the ISG15/Ub-S2 allosteric binding site, preventing the essential ISG15-PLpro molecular interactions. All compounds demonstrate clear inhibition in a deISGylation assay, two exhibit distinct antiviral activity and one inhibited a cytopathic effect in a non-cytotoxic concentration range. These results highlight the druggability of the rarely explored ISG15/Ub-S2 PLpro allosteric binding site to identify new and effective antiviral compounds. Importantly, in the context of increasing PLpro mutations in the evolving new variants of SARS-CoV-2, the natural compounds we identified may also reinstate the antiviral immune response processes of the host that are down-regulated in COVID-19 infections.

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In 2019, the world witnessed the onset of an unprecedented pandemic. In September 2021, the infection by SARS-CoV-2 had already been responsible for the death of more than 4 million people worldwide. Recently, we and other groups discovered that SARS-CoV-2 infection induces ER-stress and activation of unfolded protein response (UPR) pathway. The degradation of misfolded/unfolded proteins is an essential element of proteostasis and occurs mainly in lysosomes or proteasomes. The N-terminal arginylation of proteins is characterized as an inducer of ubiquitination and proteasomal degradation by the N-end rule pathway. Here we present, for the first time, data on the role of arginylation during SARS-CoV-2 infection. We studied the modulation of protein arginylation in Vero CCL-81 and Calu-3 cells infected after 2h, 6h, 12h, 24h, and 48h. A reanalysis of in vivo and in vitro public omics data combined with immunoblotting was performed to measure the levels of ATE1 and arginylated proteins. This regulation is seen specifically during infections by coronaviruses. We demonstrate that during SARS-CoV-2 infection there is an increase in the expression of the ATE1 enzyme associated with regulated levels of specific arginylated proteins. On the other hand, infected macrophages showed no ATE1 regulation. An important finding revealed that modulation of the N-end rule pathway differs between different types of infected cells. We also confirmed the potential of tannic acid to reduce viral load, and furthermore, to modulate ATE1 levels during infection. In addition, the arginylation inhibitor merbromin (MER) is also capable of both reducing viral load and reducing ATE1 levels. Taken together, these data show the importance of arginylation during the progression of SARS-CoV-2 infection and open the door for future studies that may unravel the role of ATE1 and its inhibitors in pathogen infection.

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The nucleocapsid (N) and the receptor binding domain (RBD) of the Spike (S) proteins elicit robust antibody and T cell responses either in vaccinated or COVID-19 convalescent individuals. We generated a chimeric protein that comprises the sequences of RBD from S and N antigens (SpiN). SpiN was highly immunogenic and elicited a strong IFN{gamma} response from T cells and high levels of antibodies to the inactivated virus, but no neutralizing antibodies. Importantly, hamsters and the human Angiotensin Convertase Enzyme-2-transgenic mice immunized with SpiN were highly resistant to challenge with the wild type SARS-CoV-2, as indicated by viral load, clinical outcome, lung inflammation and lethality. Thus, the N protein should be considered to induce T-cell-based immunity to improve SARS-CoV-2 vaccines, and eventually to circumvent the immune scape by variants.

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Genetic variants of SARS-CoV-2 have been emerging and circulating in many places across the world. Rapid detection of these variants is essential since their dissemination can impact transmission rates, diagnostic procedures, disease severity, response to vaccines or patient management. Sanger sequencing has been used as the preferred approach for variant detection among circulating human immunodeficiency and measles virus genotypes. Using primers to amplify a fragment of the SARS-CoV-2 genome encoding part of the Spike protein, we showed that Sanger sequencing allowed us to rapidly detect the introduction and spread of three distinct SARS-CoV-2 variants in two major Brazilian cities. In both cities, after the predominance of variants closely related to the virus first identified in China, the emergence of the P.2 variant was quickly followed by the identification of the P1 variant, which became dominant in less than one month after it was first detected.

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INTRODUCTIONCOVID-19 convalescent plasma (CCP) transfusion has emerged in the past months as an alternative approach to treat pneumonia cases of SARS-CoV-2. Current evidence regarding characteristics of the plasma product, the titer of neutralizing antibodies (nAbs) in the transfused units, time to onset of intervention, and impact of nAbs produced by the patient are limited and heterogeneous. MATERIAL AND METHODSWe describe the preliminary results of 104 patients with severe pneumonia due to SARS-CoV-2 transfused with CCP at three medical centers in Brazil. All enrolled patients were transfused with doses between 200 mL through 600mL of ABO compatible CCP on days 0-2 after enrolment. Clinical parameters were monitored and nAbs titration was performed using the cytopathic effect-based virus neutralization test with SARS-CoV-2 (GenBank MT126808.1). RESULTSForty-one patients achieved clinical improvement on day 14, and multivariable logistic regression showed that nAbs T (from CCP units transfused) (p= 0.001), nAbs P0 (on day of enrolment) (p=0.009) and use of other supportive therapies (p<0.001) were associated with higher odds for this clinical improvement. Considering ICU length of stay (LOS) and length of mechanical ventilation, in our analysis, nAbs P0 were associated with a significant reduction in ICU LOS (p=0.018) and duration of mechanical ventilation (p<0.001). Administration of CCP after 10 days of symptom onset was associated with increases in ICU length of stay (p<0.001). DISCUSSION/CONCLUSIONDespite the study limitations, our data have shown an association between patients previously acquired nAbs and clinical outcomes. The potential value of timely administration of CCP transfusion before day 10 of disease onset was demonstrated and nAbsP0, but not nAbsT, were associated with ICU LOS, and duration of mechanical ventilation on the improvement of clinical outcomes was also demonstrated. In conclusion, we consider these data are useful parameters to guide future CPP transfusion strategies to COVID-19.

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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.

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PurposeSARS-CoV-2 infection poses a global public health problem. There is a critical need for improvements in the noninvasive prognosis of COVID-19. We hypothesized that matrix-assisted laser desorption ionization mass spectrometry (MALDI-TOF MS) analysis combined with bottom-up proteomic analysis of plasma proteins might identify features to predict high and low risk cases of COVID-19. Patients and MethodsWe used MALDI-TOF MS to analyze plasma small proteins and peptides isolated using C18 micro-columns from a cohort containing a total of 117 cases of high (hospitalized) and low risk (outpatients) cases split into training (n = 88) and validation sets (n= 29). The plasma protein/peptide fingerprint obtained was used to train the algorithm before validation using a blinded test cohort. ResultsSeveral sample preparation, MS and data analysis parameters were optimized to achieve an overall accuracy of 85%, sensitivity of 90%, and specificity of 81% in the training set. In the blinded test set, this signature reached an overall accuracy of 93.1%, sensitivity of 87.5%, and specificity of 100%. From this signature, we identified two distinct regions in the MALDI-TOF profile belonging to the same proteoforms. A combination of 1D SDS-PAGE and quantitative bottom-up proteomic analysis allowed the identification of intact and truncated forms of serum amyloid A-1 and A-2 proteins. Conclusions: We found a plasma proteomic profile that discriminates against patients with high and low risk COVID-19. Proteomic analysis of C18-fractionated plasma may have a role in the noninvasive prognosis of COVID-19. Further validation will consolidate its clinical utility. Key messageO_ST_ABSWhat is the key question?C_ST_ABSDo individuals infected with SARS-CoV-2 harboring different degree of disease severity have a plasma protein profile that differentiate them and predict the COVID-19 outcome? What is the bottom line?In a series of 117 patients with COVID-19 divided in hospitalized (60) and outpatients (57), differential expression of serum amyloid A-1 (SAA1) and A-2 (SAA2) predict their outcome. Why read on?The high mortality rate in SARS-CoV-2 infected individuals requires accurate markers for predicting COVID-19 severity. Plasma levels of SAA1 and SAA2 indicate higher risk of hospitalization and can be used to improve COVID-19 monitoring and therapy.

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The disease caused by SARS-CoV2, covid-19, rapidly spreads worldwide, causing the greatest threat to global public health in the last 100 years. This scenario has become catastrophic as there are no approved vaccines to prevent the disease, and the main measures to contain the virus transmission are confinement and social distancing. One priority strategy is based on drug repurposing by pursuing antiviral chemotherapy that can control transmission and prevent complications associated with covid-19. With this aim, we performed a high content screening assay for the discovery of anti-SARS-CoV-2 compounds. From the 65 screened compounds, we have found four drugs capable to selectively inhibit SARS-CoV-2 in vitro infection: brequinar, abiraterone acetate, neomycin, and the extract of Hedera helix. Brequinar and abiraterone acetate had higher inhibition potency against SARS-CoV-2 than neomycin and Hedera helix extract, respectively. Drugs with reported antiviral activity and in clinical trials for covid-19, chloroquine, ivermectin, and nitazoxanide, were also included in the screening, and the last two were found to be non-selective. We used a data mining approach to build drug-host molecules-biological function-disease networks to show in a holistic way how each compound is interconnected with host node molecules and virus infection, replication, inflammatory response, and cell apoptosis. In summary, the present manuscript identified four drugs with active inhibition effect on SARS-CoV-2 in vitro infection, and by network analysis, we provided new insights and starting points for the clinical evaluation and repurposing process to treat SARS-CoV-2 infection. Summary sentenceDiscovery of drug repurposing candidates, inhibitors of SARS-CoV-2 infection in vitro, using a phenotypic screening strategy and network analysis.

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Pathogens (bacteria, fungus and virus) are becoming a potential threat to the health of human beings and environment worldwide. They widely exist in the environment, with characteristics of variety, spreading quickly and easily causing adverse reactions. In this work, an Ag-based material is used to be incorporated and functionalized in polycotton fabrics using pad-dry-cure method. This composite proved to be effective for inhibiting the SARS-CoV-2 virus, decreasing the number of replicates in 99.99% after an incubation period of 2 minutes. In addition, it caused 99.99% inhibition of the pathogens S. aureus, E. coli and C. albicans, preventing cross-infections and does not cause allergies or photoirritation processes, demonstrating the safety of its use.

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SARS-CoV-2 is the cause of the ongoing COVID-19 pandemic. Here, we investigated the interaction of this new coronavirus with Vero cells using high resolution scanning electron microscopy. Surface morphology, the interior of infected cells and the distribution of viral particles in both environments were observed 2 and 48 hours after infection. We showed areas of viral processing, details of vacuole contents, and viral interactions with the cell surface. Intercellular connections were also approached, and viral particles were adhered to these extensions suggesting direct cell-to-cell transmission of SARS-CoV-2. HighlightsWe used high resolution scanning electron microscopy to investigate Vero cells infected with SARS-CoV-2 at 2 and 48 hours post-infection. The central conclusions of this work include: O_LIInfected cells display polarization of their cytosol forming a restricted viroplasm-like zone dedicated to virus production and morphogenesis. C_LIO_LIThis is the first demonstration of SARS-CoV-2 attachment by scanning electron microscopy. C_LIO_LIThis is the first scanning electron microscopy images of the interior of SARS-CoV-2 infected cells and exploration of their vacuole contents. C_LIO_LIPerspective-viewing of bordering vesicles in close association with vacuoles. C_LIO_LIObservation of membrane ruffles and structures suggestive of exocytosis on the surface of infected cells. C_LIO_LIThe first demonstration of viral surfing in cell-to-cell communication on SARS-CoV-2 infection. C_LI

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