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1.
Int J Mol Sci ; 23(14)2022 Jul 12.
Article in English | MEDLINE | ID: covidwho-1964003

ABSTRACT

The mosquito-borne disease caused by the Rocio virus is a neglected threat, and new immune inputs for serological testing are urgently required for diagnosis in low-resource settings and epidemiological surveillance. We used in silico approaches to identify a specific antigenic peptide (p_ROCV2) in the NS1 protein of the Rocio virus that was theoretically predicted to be stable and exposed on its surface, where it demonstrated key properties allowing it to interact with antibodies. These findings related to the molecular dynamics of this peptide provide important insights for advancing diagnostic platforms and investigating therapeutic alternatives.


Subject(s)
Flavivirus , Molecular Dynamics Simulation , Animals , Immunologic Tests , Molecular Docking Simulation , Peptides , Viral Nonstructural Proteins/chemistry
2.
Photodiagnosis Photodyn Ther ; 39: 103015, 2022 Jul 14.
Article in English | MEDLINE | ID: covidwho-1937066

ABSTRACT

Ultraviolet (UV) light can inactivate SARS-CoV-2. However, the practicality of UV light is limited by the carcinogenic potential of mercury vapor-based UV lamps. Recent advances in the development of krypton chlorine (KrCl) excimer lamps hold promise, as these emit a shorter peak wavelength (222 nm), which is highly absorbed by the skin's stratum corneum and can filter out higher wavelengths. In this sense, UV 222 nm irradiation for the inactivation of virus particles in the air and surfaces is a potentially safer option as a germicidal technology. However, these same physical properties make it harder to reach microbes present in complex solutions, such as saliva, a critical source of SARS-CoV-2 transmission. We provide the first evaluation for using a commercial filtered KrCl excimer light source to inactivate SARS-CoV-2 in saliva spread on a surface. A conventional germicidal lamp (UV 254 nm) was also evaluated under the same condition. Using plaque-forming units (PFU) and Median Tissue Culture Infectious Dose (TCID50) per milliliter we found that 99.99% viral clearance (LD99.99) was obtained with 106.3 mJ/cm2 of UV 222 nm for virus in DMEM and 2417 mJ/cm2 for virus in saliva. Additionally, our results showed that the UV 254 nm had a greater capacity to inactivate the virus in both vehicles. Effective (after discounting light absorption) LD99.99 of UV 222 nm on the virus in saliva was ∼30 times higher than the value obtained with virus in saline solution (PBS), we speculated that saliva might be protecting the virus from surface irradiation in ways other than just by intensity attenuation of UV 222 nm. Due to differences between UV 222/254 nm capacities to interact and be absorbed by molecules in complex solutions, a higher dose of 222 nm will be necessary to reduce viral load in surfaces with contaminated saliva.

3.
Virulence ; 13(1): 1031-1048, 2022 12.
Article in English | MEDLINE | ID: covidwho-1900978

ABSTRACT

The ongoing COVID-19 pandemic caused a significant loss of human lives and a worldwide decline in quality of life. Treatment of COVID-19 patients is challenging, and specific treatments to reduce COVID-19 aggravation and mortality are still necessary. Here, we describe the discovery of a novel class of epiandrosterone steroidal compounds with cationic amphiphilic properties that present antiviral activity against SARS-CoV-2 in the low micromolar range. Compounds were identified in screening campaigns using a cytopathic effect-based assay in Vero CCL81 cells, followed by hit compound validation and characterization. Compounds LNB167 and LNB169 were selected due to their ability to reduce the levels of infectious viral progeny and viral RNA levels in Vero CCL81, HEK293, and HuH7.5 cell lines. Mechanistic studies in Vero CCL81 cells indicated that LNB167 and LNB169 inhibited the initial phase of viral replication through mechanisms involving modulation of membrane lipids and cholesterol in host cells. Selection of viral variants resistant to steroidal compound treatment revealed single mutations on transmembrane, lipid membrane-interacting Spike and Envelope proteins. Finally, in vivo testing using the hACE2 transgenic mouse model indicated that SARS-CoV-2 infection could not be ameliorated by LNB167 treatment. We conclude that anti-SARS-CoV-2 activities of steroidal compounds LNB167 and LNB169 are likely host-targeted, consistent with the properties of cationic amphiphilic compounds that modulate host cell lipid biology. Although effective in vitro, protective effects were cell-type specific and did not translate to protection in vivo, indicating that subversion of lipid membrane physiology is an important, yet complex mechanism involved in SARS-CoV-2 replication and pathogenesis.


Subject(s)
COVID-19 , SARS-CoV-2 , Animals , Antiviral Agents/pharmacology , COVID-19/drug therapy , Chlorocebus aethiops , HEK293 Cells , Humans , Lipids , Mice , Pandemics , Quality of Life , Vero Cells , Virus Replication
4.
PLoS Comput Biol ; 18(5): e1010121, 2022 05.
Article in English | MEDLINE | ID: covidwho-1846916

ABSTRACT

The nucleocapsid (N) protein of the SARS-CoV-2 virus, the causal agent of COVID-19, is a multifunction phosphoprotein that plays critical roles in the virus life cycle, including transcription and packaging of the viral RNA. To play such diverse roles, the N protein has two globular RNA-binding modules, the N- (NTD) and C-terminal (CTD) domains, which are connected by an intrinsically disordered region. Despite the wealth of structural data available for the isolated NTD and CTD, how these domains are arranged in the full-length protein and how the oligomerization of N influences its RNA-binding activity remains largely unclear. Herein, using experimental data from electron microscopy and biochemical/biophysical techniques combined with molecular modeling and molecular dynamics simulations, we show that, in the absence of RNA, the N protein formed structurally dynamic dimers, with the NTD and CTD arranged in extended conformations. However, in the presence of RNA, the N protein assumed a more compact conformation where the NTD and CTD are packed together. We also provided an octameric model for the full-length N bound to RNA that is consistent with electron microscopy images of the N protein in the presence of RNA. Together, our results shed new light on the dynamics and higher-order oligomeric structure of this versatile protein.


Subject(s)
Coronavirus Nucleocapsid Proteins , SARS-CoV-2 , COVID-19 , Coronavirus Nucleocapsid Proteins/chemistry , Coronavirus Nucleocapsid Proteins/metabolism , Humans , Microscopy, Electron , Molecular Dynamics Simulation , Nucleocapsid Proteins/chemistry , Nucleocapsid Proteins/metabolism , Phosphoproteins/metabolism , Protein Binding , RNA, Viral/genetics , SARS-CoV-2/chemistry , SARS-CoV-2/genetics , SARS-CoV-2/metabolism
5.
Nat Commun ; 12(1): 6844, 2021 11 25.
Article in English | MEDLINE | ID: covidwho-1537310

ABSTRACT

COVID-19 manifests as a milder disease in children than adults, but the underlying mechanisms are not fully characterized. Here we assess the difference in cellular or humoral immune responses of pediatric and adult COVID-19 patients to see if these factors contribute to the severity dichotomy. Children's non-specific immune profile is dominated by naive lymphocytes and HLA-DRhighCX3CR1low dendritic cells; meanwhile, children show strong specific antibody and T cell responses for viral structural proteins, with their T cell responses differing from adults by having weaker CD8+TNF+ T cells responses to S peptide pool but stronger responses to N and M peptide pools. Finally, viral mRNA is more abundant in pediatric patients. Our data thus support a scenario in which SARS-CoV-2 infected children contribute to transmission yet are less susceptible to COVID-19 symptoms due to strong and differential responses to the virus.


Subject(s)
Antibodies, Viral/immunology , COVID-19/immunology , Immunity, Humoral , RNA, Viral , SARS-CoV-2/genetics , Vaccines, Synthetic/immunology , Adolescent , Adult , Aged , Antibodies, Viral/blood , Brazil , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes , COVID-19/prevention & control , COVID-19 Vaccines , Child , Child, Preschool , Cytokines/blood , Female , Humans , Immunity, Innate , Male , Middle Aged , RNA, Messenger , Spike Glycoprotein, Coronavirus/immunology , T-Lymphocytes , Viral Structural Proteins/immunology , Young Adult
6.
JCI Insight ; 7(1)2022 01 11.
Article in English | MEDLINE | ID: covidwho-1523122

ABSTRACT

Neutrophils are recognized as important circulating effector cells in the pathophysiology of severe coronavirus disease 2019 (COVID-19). However, their role within the inflamed lungs is incompletely understood. Here, we collected bronchoalveolar lavage (BAL) fluids and parallel blood samples of critically ill COVID-19 patients requiring invasive mechanical ventilation and compared BAL fluid parameters with those of mechanically ventilated patients with influenza, as a non-COVID-19 viral pneumonia cohort. Compared with those of patients with influenza, BAL fluids of patients with COVID-19 contained increased numbers of hyperactivated degranulating neutrophils and elevated concentrations of the cytokines IL-1ß, IL-1RA, IL-17A, TNF-α, and G-CSF; the chemokines CCL7, CXCL1, CXCL8, CXCL11, and CXCL12α; and the protease inhibitors elafin, secretory leukocyte protease inhibitor, and tissue inhibitor of metalloproteinases 1. In contrast, α-1 antitrypsin levels and net proteolytic activity were comparable in COVID-19 and influenza BAL fluids. During antibiotic treatment for bacterial coinfections, increased BAL fluid levels of several activating and chemotactic factors for monocytes, lymphocytes, and NK cells were detected in patients with COVID-19 whereas concentrations tended to decrease in patients with influenza, highlighting the persistent immunological response to coinfections in COVID-19. Finally, the high proteolytic activity in COVID-19 lungs suggests considering protease inhibitors as a treatment option.


Subject(s)
Bacterial Infections , Bronchoalveolar Lavage Fluid , COVID-19 , Coinfection , Influenza, Human , Adult , Aged , Bacterial Infections/complications , Bacterial Infections/immunology , Bacterial Infections/metabolism , Bacterial Infections/pathology , Bronchoalveolar Lavage Fluid/chemistry , Bronchoalveolar Lavage Fluid/cytology , Bronchoalveolar Lavage Fluid/immunology , COVID-19/complications , COVID-19/diagnosis , COVID-19/immunology , COVID-19/pathology , Coinfection/immunology , Coinfection/metabolism , Coinfection/pathology , Cytokines/analysis , Female , Humans , Inflammation , Influenza, Human/complications , Influenza, Human/diagnosis , Influenza, Human/immunology , Influenza, Human/pathology , Lung/immunology , Lung/metabolism , Lung/pathology , Male , Middle Aged
7.
Clin Transl Immunology ; 10(4): e1271, 2021.
Article in English | MEDLINE | ID: covidwho-1525427

ABSTRACT

OBJECTIVES: Emerging evidence of dysregulation of the myeloid cell compartment urges investigations on neutrophil characteristics in coronavirus disease 2019 (COVID-19). We isolated neutrophils from the blood of COVID-19 patients receiving general ward care and from patients hospitalised at intensive care units (ICUs) to explore the kinetics of circulating neutrophils and factors important for neutrophil migration and activation. METHODS: Multicolour flow cytometry was exploited for the analysis of neutrophil differentiation and activation markers. Multiplex and ELISA technologies were used for the quantification of protease, protease inhibitor, chemokine and cytokine concentrations in plasma. Neutrophil polarisation responses were evaluated microscopically. Gelatinolytic and metalloproteinase activity in plasma was determined using a fluorogenic substrate. Co-culturing healthy donor neutrophils with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) allowed us to investigate viral replication in neutrophils. RESULTS: Upon ICU admission, patients displayed high plasma concentrations of granulocyte-colony-stimulating factor (G-CSF) and the chemokine CXCL8, accompanied by emergency myelopoiesis as illustrated by high levels of circulating CD10-, immature neutrophils with reduced CXCR2 and C5aR expression. Neutrophil elastase and non-metalloproteinase-derived gelatinolytic activity were increased in plasma from ICU patients. Significantly higher levels of circulating tissue inhibitor of metalloproteinase 1 (TIMP-1) in patients at ICU admission yielded decreased total MMP proteolytic activity in blood. COVID-19 neutrophils were hyper-responsive to CXCL8 and CXCL12 in shape change assays. Finally, SARS-CoV-2 failed to replicate inside human neutrophils. CONCLUSION: Our study provides detailed insights into the kinetics of neutrophil phenotype and function in severe COVID-19 patients, and supports the concept of an increased neutrophil activation state in the circulation.

9.
Cell Metab ; 32(3): 437-446.e5, 2020 09 01.
Article in English | MEDLINE | ID: covidwho-670096

ABSTRACT

COVID-19 can result in severe lung injury. It remained to be determined why diabetic individuals with uncontrolled glucose levels are more prone to develop the severe form of COVID-19. The molecular mechanism underlying SARS-CoV-2 infection and what determines the onset of the cytokine storm found in severe COVID-19 patients are unknown. Monocytes and macrophages are the most enriched immune cell types in the lungs of COVID-19 patients and appear to have a central role in the pathogenicity of the disease. These cells adapt their metabolism upon infection and become highly glycolytic, which facilitates SARS-CoV-2 replication. The infection triggers mitochondrial ROS production, which induces stabilization of hypoxia-inducible factor-1α (HIF-1α) and consequently promotes glycolysis. HIF-1α-induced changes in monocyte metabolism by SARS-CoV-2 infection directly inhibit T cell response and reduce epithelial cell survival. Targeting HIF-1ɑ may have great therapeutic potential for the development of novel drugs to treat COVID-19.


Subject(s)
Betacoronavirus/physiology , Blood Glucose/metabolism , Coronavirus Infections/complications , Diabetes Complications/complications , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Monocytes/metabolism , Pneumonia, Viral/complications , Adult , COVID-19 , Cell Line , Coronavirus Infections/metabolism , Diabetes Complications/metabolism , Diabetes Mellitus/metabolism , Female , Glycolysis , Humans , Inflammation/complications , Inflammation/metabolism , Male , Middle Aged , Monocytes/virology , Pandemics , Pneumonia, Viral/metabolism , Reactive Oxygen Species/metabolism , SARS-CoV-2 , Signal Transduction
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