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1.
Front Cell Infect Microbiol ; 12: 849017, 2022.
Article in English | MEDLINE | ID: covidwho-1952255

ABSTRACT

SARS-CoV-2 is an emerging virus from the Coronaviridae family and is responsible for the ongoing COVID-19 pandemic. In this work, we explored the previously reported SARS-CoV-2 structural membrane protein (M) interaction with human Proliferating Cell Nuclear Antigen (PCNA). The M protein is responsible for maintaining virion shape, and PCNA is a marker of DNA damage which is essential for DNA replication and repair. We validated the M-PCNA interaction through immunoprecipitation, immunofluorescence co-localization, and PLA (Proximity Ligation Assay). In cells infected with SARS-CoV-2 or transfected with M protein, using immunofluorescence and cell fractioning, we documented a reallocation of PCNA from the nucleus to the cytoplasm and the increase of PCNA and γH2AX (another DNA damage marker) expression. We also observed an increase in PCNA and γH2AX expression in the lung of a COVID-19 patient by immunohistochemistry. In addition, the inhibition of PCNA translocation by PCNA I1 and Verdinexor led to a reduction of plaque formation in an in vitro assay. We, therefore, propose that the transport of PCNA to the cytoplasm and its association with M could be a virus strategy to manipulate cell functions and may be considered a target for COVID-19 therapy.


Subject(s)
COVID-19 , Coronavirus M Proteins , Proliferating Cell Nuclear Antigen , COVID-19/drug therapy , Coronavirus M Proteins/metabolism , Humans , Proliferating Cell Nuclear Antigen/metabolism , SARS-CoV-2
2.
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
3.
Viruses ; 13(11)2021 10 22.
Article in English | MEDLINE | ID: covidwho-1481018

ABSTRACT

A SARS-CoV-2 B.1.1.7 variant of concern (VOC) has been associated with increased transmissibility, hospitalization, and mortality. This study aimed to explore the factors associated with B.1.1.7 VOC infection in the context of vaccination. On March 2021, we detected SARS-CoV-2 RNA in nasopharyngeal samples from 14 of 22 individuals vaccinated with a single-dose of ChAdOx1 (outbreak A, n = 26), and 22 of 42 of individuals with two doses of the CoronaVac vaccine (outbreak B, n = 52) for breakthrough infection rates for ChAdOx1 of 63.6% and 52.4% for CoronaVac. The outbreaks were caused by two independent clusters of the B.1.1.7 VOC. The serum of PCR-positive symptomatic SARS-CoV-2-infected individuals had ~1.8-3.4-fold more neutralizing capacity against B.1.1.7 compared to the serum of asymptomatic individuals. These data based on exploratory analysis suggest that the B.1.1.7 variant can infect individuals partially immunized with a single dose of an adenovirus-vectored vaccine or fully immunized with two doses of an inactivated vaccine, although the vaccines were able to reduce the risk of severe disease and death caused by this VOC, even in the elderly.


Subject(s)
COVID-19 Vaccines , COVID-19/immunology , COVID-19/virology , SARS-CoV-2/classification , SARS-CoV-2/genetics , Vaccination , Adenoviridae , Adult , Aged , Aged, 80 and over , Antibodies, Neutralizing/immunology , Brazil/epidemiology , COVID-19/prevention & control , COVID-19 Serological Testing , Cohort Studies , Disease Outbreaks/statistics & numerical data , Female , Genetic Vectors , Humans , Immunoglobulin G/blood , Male , Middle Aged , RNA, Viral , Vaccines, Inactivated , Whole Genome Sequencing , Young Adult
4.
J Photochem Photobiol ; 8: 100072, 2021 Dec.
Article in English | MEDLINE | ID: covidwho-1446893

ABSTRACT

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is mainly transmitted by airborne droplets generated by infected individuals. Since this and many other pathogens are able to remain viable on inert surfaces for extended periods of time, contaminated surfaces play an important role in SARS-CoV-2 fomite transmission. Cosmetic products are destined to be applied on infection-sensitive sites, such as the lips and eyelids. Therefore, special biosafety precautions should be incorporated into the routine procedures of beauty parlors and shops. Indeed, innovative cosmetics companies are currently searching for disinfection protocols that ensure the customers' safety in makeup testing. Here, we propose an ultraviolet germicidal irradiation (UVGI) strategy that can be used to reduce the odds of COVID-19 fomite transmission by makeup testers. It is well-known that UVGI effectively inactivates pathogens on flat surfaces and clear fluids. However, ultraviolet-C (UVC) radiation at 254 nm penetrates poorly in turbid and porous materials, such as makeup and lipstick formulations. Thus, we investigated the virucidal effect of UVGI against SARS-CoV-2 deposited on such substrates and compared their performance to that of flat polystyrene surfaces, used as controls. Concentrated infectious SARS-CoV-2 inoculum (106 PFU/mL) deposited on lipstick and makeup powder was completely inactivated (>5log10 reduction) following UVC exposures at 1,260 mJ/cm2, while flat plastic surfaces required 10 times less exposure (126 mJ/cm2) to reach the same microbicidal performance. We conclude that UVGI comprises an effective disinfection strategy to promote biosafety for cosmetics testers. However, appropriate UVC dosimetry must be implemented to overcome inefficiencies caused by the optical properties of turbid materials in lipsticks and makeup powders.

6.
Lancet Microbe ; 2(10): e527-e535, 2021 10.
Article in English | MEDLINE | ID: covidwho-1307293

ABSTRACT

BACKGROUND: Mutations accrued by SARS-CoV-2 lineage P.1-first detected in Brazil in early January, 2021-include amino acid changes in the receptor-binding domain of the viral spike protein that also are reported in other variants of concern, including B.1.1.7 and B.1.351. We aimed to investigate whether isolates of wild-type P.1 lineage SARS-CoV-2 can escape from neutralising antibodies generated by a polyclonal immune response. METHODS: We did an immunological study to assess the neutralising effects of antibodies on lineage P.1 and lineage B isolates of SARS-CoV-2, using plasma samples from patients previously infected with or vaccinated against SARS-CoV-2. Two specimens (P.1/28 and P.1/30) containing SARS-CoV-2 lineage P.1 (as confirmed by viral genome sequencing) were obtained from nasopharyngeal and bronchoalveolar lavage samples collected from patients in Manaus, Brazil, and compared against an isolate of SARS-CoV-2 lineage B (SARS.CoV2/SP02.2020) recovered from a patient in Brazil in February, 2020. Isolates were incubated with plasma samples from 21 blood donors who had previously had COVID-19 and from a total of 53 recipients of the chemically inactivated SARS-CoV-2 vaccine CoronaVac: 18 individuals after receipt of a single dose and an additional 20 individuals (38 in total) after receipt of two doses (collected 17-38 days after the most recent dose); and 15 individuals who received two doses during the phase 3 trial of the vaccine (collected 134-230 days after the second dose). Antibody neutralisation of P.1/28, P.1/30, and B isolates by plasma samples were compared in terms of median virus neutralisation titre (VNT50, defined as the reciprocal value of the sample dilution that showed 50% protection against cytopathic effects). FINDINGS: In terms of VNT50, plasma from individuals previously infected with SARS-CoV-2 had an 8·6 times lower neutralising capacity against the P.1 isolates (median VNT50 30 [IQR <20-45] for P.1/28 and 30 [<20-40] for P.1/30) than against the lineage B isolate (260 [160-400]), with a binominal model showing significant reductions in lineage P.1 isolates compared with the lineage B isolate (p≤0·0001). Efficient neutralisation of P.1 isolates was not seen with plasma samples collected from individuals vaccinated with a first dose of CoronaVac 20-23 days earlier (VNT50s below the limit of detection [<20] for most plasma samples), a second dose 17-38 days earlier (median VNT50 24 [IQR <20-25] for P.1/28 and 28 [<20-25] for P.1/30), or a second dose 134-260 days earlier (all VNT50s below limit of detection). Median VNT50s against the lineage B isolate were 20 (IQR 20-30) after a first dose of CoronaVac 20-23 days earlier, 75 (<20-263) after a second dose 17-38 days earlier, and 20 (<20-30) after a second dose 134-260 days earlier. In plasma collected 17-38 days after a second dose of CoronaVac, neutralising capacity against both P.1 isolates was significantly decreased (p=0·0051 for P.1/28 and p=0·0336 for P.1/30) compared with that against the lineage B isolate. All data were corroborated by results obtained through plaque reduction neutralisation tests. INTERPRETATION: SARS-CoV-2 lineage P.1 might escape neutralisation by antibodies generated in response to polyclonal stimulation against previously circulating variants of SARS-CoV-2. Continuous genomic surveillance of SARS-CoV-2 combined with antibody neutralisation assays could help to guide national immunisation programmes. FUNDING: São Paulo Research Foundation, Brazilian Ministry of Science, Technology and Innovation and Funding Authority for Studies, Medical Research Council, National Council for Scientific and Technological Development, National Institutes of Health. TRANSLATION: For the Portuguese translation of the abstract see Supplementary Materials section.


Subject(s)
COVID-19 , SARS-CoV-2 , Antibodies, Neutralizing , Antibodies, Viral , Brazil/epidemiology , COVID-19/prevention & control , COVID-19 Vaccines , Humans , SARS-CoV-2/genetics , United States , Vaccination
8.
Gut Microbes ; 13(1): 1-9, 2021.
Article in English | MEDLINE | ID: covidwho-1069185

ABSTRACT

Microbiota-derived molecules called short-chain fatty acids (SCFAs) play a key role in the maintenance of the intestinal barrier and regulation of immune response during infectious conditions. Recent reports indicate that SARS-CoV-2 infection changes microbiota and SCFAs production. However, the relevance of this effect is unknown. In this study, we used human intestinal biopsies and intestinal epithelial cells to investigate the impact of SCFAs in the infection by SARS-CoV-2. SCFAs did not change the entry or replication of SARS-CoV-2 in intestinal cells. These metabolites had no effect on intestinal cells' permeability and presented only minor effects on the production of anti-viral and inflammatory mediators. Together our findings indicate that the changes in microbiota composition of patients with COVID-19 and, particularly, of SCFAs do not interfere with the SARS-CoV-2 infection in the intestine.


Subject(s)
COVID-19/virology , Fatty Acids, Volatile/metabolism , Gastrointestinal Microbiome , Intestinal Mucosa/virology , Adult , Aged , Caco-2 Cells , Colon/virology , Epithelial Cells/virology , Female , Humans , In Vitro Techniques , Male , Middle Aged , SARS-CoV-2/pathogenicity , SARS-CoV-2/physiology , Viral Load , Virus Internalization , Young Adult
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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