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1.
International Journal of Molecular Sciences ; 23(16):9488, 2022.
Article in English | MDPI | ID: covidwho-1997648

ABSTRACT

The aims of our study are to: (i) investigate the ability of nicotine to modulate the expression level of inflammatory cytokines in A549 cells infected with SARS-CoV-2;(ii) elucidate the ultrastructural features caused by the combination nicotine+SARS-CoV-2;and (iii) demonstrate the mechanism of action. In this study, A549 cells pretreated with nicotine were either exposed to LPS or poly(I:C), or infected with SARS-CoV-2. Treated and untreated cells were analyzed for cytokine production, cytotoxicity, and ultrastructural modifications. Vero E6 cells were used as a positive reference. Cells pretreated with nicotine showed a decrease of IL6 and TNFαin A549 cells induced by LPS or poly(I:C). In contrast, cells exposed to SARS-CoV-2 showed a high increase of IL6, IL8, IL10 and TNFα, high cytopathic effects that were dose- and time-dependent, and profound ultrastructural modifications. These modifications were characterized by membrane ruptures and fragmentation, the swelling of cytosol and mitochondria, the release of cytoplasmic content in extracellular spaces (including osmiophilic granules), the fragmentation of endoplasmic reticulum, and chromatin disorganization. Nicotine increased SARS-CoV-2 cytopathic effects, elevating the levels of inflammatory cytokines, and inducing severe cellular damage, with features resembling pyroptosis and necroptosis. The protective role of nicotine in COVID-19 is definitively ruled out.

3.
Viruses ; 14(5)2022 05 17.
Article in English | MEDLINE | ID: covidwho-1869812

ABSTRACT

Lipids play a crucial role in the entry and egress of viruses, regardless of whether they are naked or enveloped. Recent evidence shows that lipid involvement in viral infection goes much further. During replication, many viruses rearrange internal lipid membranes to create niches where they replicate and assemble. Because of the close connection between lipids and inflammation, the derangement of lipid metabolism also results in the production of inflammatory stimuli. Due to its pivotal function in the viral life cycle, lipid metabolism has become an area of intense research to understand how viruses seize lipids and to design antiviral drugs targeting lipid pathways. Palmitoylethanolamide (PEA) is a lipid-derived peroxisome proliferator-activated receptor-α (PPAR-α) agonist that also counteracts SARS-CoV-2 entry and its replication. Our work highlights for the first time the antiviral potency of PEA against SARS-CoV-2, exerting its activity by two different mechanisms. First, its binding to the SARS-CoV-2 S protein causes a drop in viral infection of ~70%. We show that this activity is specific for SARS-CoV-2, as it does not prevent infection by VSV or HSV-2, other enveloped viruses that use different glycoproteins and entry receptors to mediate their entry. Second, we show that in infected Huh-7 cells, treatment with PEA dismantles lipid droplets, preventing the usage of these vesicular bodies by SARS-CoV-2 as a source of energy and protection against innate cellular defenses. This is not surprising since PEA activates PPAR-α, a transcription factor that, once activated, generates a cascade of events that leads to the disruption of fatty acid droplets, thereby bringing about lipid droplet degradation through ß-oxidation. In conclusion, the present work demonstrates a novel mechanism of action for PEA as a direct and indirect antiviral agent against SARS-CoV-2. This evidence reinforces the notion that treatment with this compound might significantly impact the course of COVID-19. Indeed, considering that the protective effects of PEA in COVID-19 are the current objectives of two clinical trials (NCT04619706 and NCT04568876) and given the relative lack of toxicity of PEA in humans, further preclinical and clinical tests will be needed to fully consider PEA as a promising adjuvant therapy in the current COVID-19 pandemic or against emerging RNA viruses that share the same route of replication as coronaviruses.


Subject(s)
COVID-19 , SARS-CoV-2 , Amides , Antiviral Agents/pharmacology , Antiviral Agents/therapeutic use , COVID-19/drug therapy , Ethanolamines , Humans , Palmitic Acids/pharmacology , Pandemics , Peas , Peroxisome Proliferator-Activated Receptors , Spike Glycoprotein, Coronavirus
5.
Virol J ; 19(1): 79, 2022 05 13.
Article in English | MEDLINE | ID: covidwho-1846849

ABSTRACT

BACKGROUND: Torquetenovirus (TTV), a widespread anellovirus recognized as the main component of the healthy human virome, displays viremia that is highly susceptible to variations in immune competence. TTV possesses microRNA (miRNA)-coding sequences that might be involved in viral immune evasion. Among TTV-encoded miRNAs, miRNA t1a, t3b, and tth8 have been found in biological fluids. Here, the presence of TTV DNA and TTV miRNAs in the plasma of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected subjects was investigated to monitor the possible association with coronavirus disease 2019 (COVID-19) severity. METHODS: Detection of TTV DNA and miRNA t1a, t3b, and tth8 was investigated in plasma samples of 56 SARS-CoV-2-infected subjects with a spectrum of different COVID-19 outcomes. TTV DNA and TTV miRNAs were assessed with a universal single step real-time TaqMan PCR assay and miRNA quantitative RT-PCR miRNA assay, respectively. RESULTS: The TTV DNA prevalence was 59%, whereas at least one TTV miRNA was found in 94% of the patients tested. miRNA tth8 was detected in 91% of subjects, followed by miRNAs t3b (64%) and miRNAt1a (30%). Remarkably, although TTV DNA was unrelated to COVID-19 severity, miRNA tth8 was significantly associated with the degree of disease (adjusted incidence rate ratio (IRR) 2.04, 95% CI 1.14-3.63, for the subjects in the high severity group compared to those in the low severity group). CONCLUSIONS: Our findings encourage further investigation to understand the potential role of TTV miRNAs in the different outcomes of COVID-19 at early and late stages.


Subject(s)
COVID-19 , MicroRNAs , Torque teno virus , DNA, Viral/genetics , Humans , MicroRNAs/genetics , SARS-CoV-2/genetics , Torque teno virus/genetics
6.
Comput Struct Biotechnol J ; 19: 6140-6156, 2021.
Article in English | MEDLINE | ID: covidwho-1734314

ABSTRACT

We exploited a multi-scale microscopy imaging toolbox to address some major issues related to SARS-CoV-2 interactions with host cells. Our approach harnesses both conventional and super-resolution fluorescence microscopy and easily matches the spatial scale of single-virus/cell checkpoints. After its validation through the characterization of infected cells and virus morphology, we leveraged this toolbox to reveal subtle issues related to the entry phase of SARS-CoV-2 variants in Vero E6 cells. Our results show that in Vero E6 cells the B.1.1.7 strain (aka Alpha Variant of Concern) is associated with much faster kinetics of endocytic uptake compared to its ancestor B.1.177. Given the cell-entry scenario dominated by the endosomal "late pathway", the faster internalization of B.1.1.7 could be directly related to the N501Y mutation in the S protein, which is known to strengthen the binding of Spike receptor binding domain with ACE2. Remarkably, we also directly observed the central role of clathrin as a mediator of endocytosis in the late pathway of entry. In keeping with the clathrin-mediated endocytosis, we highlighted the non-raft membrane localization of ACE2. Overall, we believe that our fluorescence microscopy-based approach represents a fertile strategy to investigate the molecular features of SARS-CoV-2 interactions with cells.

7.
The New Microbiologica ; 44(4):205, 2021.
Article in English | ProQuest Central | ID: covidwho-1696320

ABSTRACT

The SARS-CoV-2 pandemic is ongoing worldwide, causing prolonged pressure on molecular diagnostics. Viral antigen (Ag) assays have several advantages, ranging from lower cost to shorter turnaround time to detection. Given the rare occurrence of low-load viremia, antigen assays for SARSCoV-2 have focused on nasopharyngeal swab and saliva as biological matrices, but their effectiveness must be validated. We assayed here the performances of the novel quantitative Liaison® SARSCoV-2 Ag assay on 119 nasopharyngeal swabs and obtained results were compared with Hologic Panther and Abbott m2000 RT-qPCR. The Ag assay demonstrated a good correlation with viral load, shorter turnaround time, and favorable economics. The best performance was obtained in the acute phase of disease.

8.
EuropePMC;
Preprint in English | EuropePMC | ID: ppcovidwho-327541

ABSTRACT

Several compounds have been tested against SARS-CoV-2;at present, COVID-19 treatments decrease the deleterious inflammatory response and acute lung injury. However, the best therapeutic response would be expected by combining anti-inflammatory properties, while concomitantly blocking viral replication. These combined effects should drastically reduce both infection rate and severe complications induced by novel SARS-CoV-2 variants. Therefore, we explored the antiviral potency of a class of anti-inflammatory compounds that inhibit the N-Acylethanolamine acid amidase (NAAA). This enzyme catalyzes the hydrolysis of palmitoylethanolamide (PEA), a bioactive lipid that mediates anti-inflammatory and analgesic activity through the activation of peroxisome proliferator receptor-α (PPAR-α). Similarly, this pathway is likely to be a significant target to impede viral replication since PPAR-α activation leads to dismantling of lipid droplets, where viral replication of Flaviviruses and Coronaviruses occurs. Here, we show that either genetic or pharmacological inhibition of the NAAA enzyme leads to five-fold reduction in the replication of both SARS-CoV-2 and ZIKV in various cell lines. Once NAAA enzyme is blocked, both ZIKV and SARS CoV-2 replication decrease, which parallels a sudden five-fold decrease in virion release. These effects induced by NAAA inhibition occurs concomitantly with stimulation of autophagy during infection. Remarkably, parallel antiviral and anti-inflammatory effects of NAAA antagonism were confirmed in ex-vivo experiments, within SARS-CoV-2 infected human PBMC cells, in which both viral genomes and TNF-α production drop by ~60%. It is known that macrophages contribute to viral spread, excessive inflammation and macrophage activation syndrome that NAAA inhibitors might prevent, reducing the macrophage-induced acute respiratory distress syndrome and subsequent death of COVID-19 patients.

10.
Transfus Med ; 31(5): 371-376, 2021 Oct.
Article in English | MEDLINE | ID: covidwho-1356901

ABSTRACT

BACKGROUND: Pathogen reduction technologies (PRT) based on nucleic-acid damaging chemicals and/or irradiation are increasingly being used to increase safety of blood components against emerging pathogens, such as convalescent plasma in the ongoing COVID-19 pandemic. Current methods for PRT validation are limited by the resources available to the blood component manufacturer, and quality control rely over pathogen spiking and hence invariably require sacrifice of the tested blood units: quantitative real-time PCR is the current pathogen detection method but, due to the high likelihood of detecting nonviable fragments, requires downstream pathogen culture. We propose here a new molecular validation of PRT based on the highly prevalent human symbiont torquetenovirus (TTV) and rolling circle amplification (RCA). MATERIALS AND METHODS: Serial apheresis plasma donations were tested for TTV before and after inactivation with Intercept® PRT using real-time quantitative PCR (conventional validation), RCA followed by real-time PCR (our validation), and reverse PCR (for cross-validation). RESULTS: While only 20% of inactivated units showed significant decrease in TTV viral load using real-time qPCR, all donations tested with RCA followed by real-time PCR showed TTV reductions. As further validation, 2 units were additionally tested with reverse PCR, which confirmed absence of entire circular genomes. DISCUSSION: We have described and validated a conservative and easy-to-setup protocol for molecular validation of PRT based on RCA and real-time PCR for TTV.


Subject(s)
DNA, Viral , Real-Time Polymerase Chain Reaction , Torque teno virus , Virus Inactivation , COVID-19/blood , COVID-19/genetics , DNA, Viral/blood , DNA, Viral/genetics , Humans , SARS-CoV-2/genetics , SARS-CoV-2/metabolism , Torque teno virus/genetics , Torque teno virus/metabolism
11.
J Clin Virol Plus ; 1(3): 100035, 2021 Sep.
Article in English | MEDLINE | ID: covidwho-1347690

ABSTRACT

Background: Several ABO blood groups have been associated with the likelihood of infection, severity, and/or outcome of COVID-19 in hospitalized cohorts, raising the hypothesis that anti-A isoagglutinins in non-A-group recipients could act as neutralizing antibodies against SARS-CoV-2. Materials and methods: We run live virus neutralization tests using sera from 58 SARS-CoV-2 seronegative blood donors (27 O-group and 31 A-group) negatives for SARS-CoV-2 IgG to investigate what degree of neutralizing activity could be detected in their sera and eventual correlation with anti-A isoagglutinin titers. Results: We could not find clinically relevant neutralizing activity in any blood group, regardless of anti-isoagglutinin titer. Discussion: Our findings suggest that mechanisms other than neutralization explain the differences in outcomes from COVID19 seen in different ABO blood groups.

12.
Emerg Microbes Infect ; 10(1): 1254-1256, 2021 Dec.
Article in English | MEDLINE | ID: covidwho-1263637

ABSTRACT

We report 11 cases of SARS-CoV-2 infection in healthcare workers (HCW) naïve for COVID-19 and seropositive after the second dose of the BNT162b2 mRNA vaccine. Based on voluntary-based surveillance, they tested positive for different strains of SARS-CoV-2, as Spike gene sequencing showed. Five of them reported mild symptoms. Given the risk for SARS-CoV-2 introduction from asymptomatic vaccinees, this case series suggests the need to continue nasopharyngeal screening programmes.


Subject(s)
COVID-19 Vaccines/immunology , COVID-19/prevention & control , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/genetics , Vaccination , Adult , COVID-19/virology , Female , Health Personnel , Humans , Italy , Male , Middle Aged , Nasopharynx/virology , SARS-CoV-2/genetics
13.
ERJ Open Res ; 7(2)2021 Apr.
Article in English | MEDLINE | ID: covidwho-1183500

ABSTRACT

The coronavirus disease 2019 (COVID-19) pandemic has a variable degree of severity according to underlying comorbidities and life-style. Several research groups have reported an association between cigarette smoking and increased severity of COVID-19. The exact mechanism of action is largely unclear. We exposed low angiotensin-converting enzyme 2 (ACE2)-expressing human pulmonary adenocarcinoma A549 epithelial cells to nicotine and assessed ACE2 expression at different times. We further used the nicotine-exposed cells in a virus neutralisation assay. Nicotine exposure induces rapid and long-lasting increases in gene and protein expression of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) receptor ACE2, which in turn translates into increased competence for SARS-CoV-2 replication and cytopathic effect. These findings show that nicotine worsens SARS-CoV-2 pulmonary infection and have implications for public health policies.

14.
Life (Basel) ; 11(4)2021 Apr 01.
Article in English | MEDLINE | ID: covidwho-1159500

ABSTRACT

Antibody-dependent enhancement (ADE) of severe acute respiratory syndrome coronavirus-2 (SARS CoV-2) infection has been hypothesized. However, to date, there has been no in vitro or in vivo evidence supporting this. Cross-reactivity exists between SARS CoV-2 and other Coronaviridae for both cellular and humoral immunity. We show here that IgG against nucleocapsid protein of alphacoronavirus NL63 and 229E correlate with the World Health Organization's (WHO) clinical severity score ≥ 5 (incidence rate ratios was 1.87 and 1.80, respectively, and 1.94 for the combination). These laboratory findings suggest possible ADE of SARS CoV-2 infection by previous alphacoronavirus immunity.

15.
Emerg Infect Dis ; 27(4): 1249-1251, 2021 04.
Article in English | MEDLINE | ID: covidwho-1145546
16.
Emerg Microbes Infect ; 10(1): 710-712, 2021 Dec.
Article in English | MEDLINE | ID: covidwho-1142611

ABSTRACT

We report here an imported case of SARS-CoV-2 variant of concern B.1.1.351 (also known as 20H/501Y.V2 or "South African variant" or VOC 202012/02) in a 66-years old symptomatic male who returned from Malawi to Italy.


Subject(s)
COVID-19/virology , SARS-CoV-2/isolation & purification , Aged , Humans , Italy , Malawi , Male , Phylogeny , SARS-CoV-2/classification , SARS-CoV-2/genetics , SARS-CoV-2/physiology , Travel
17.
Front Oncol ; 10: 1428, 2020.
Article in English | MEDLINE | ID: covidwho-782025

ABSTRACT

SARS-CoV-2 is the viral agent responsible for the pandemic that in the first months of 2020 caused about 400,000 deaths. Among compounds proposed to fight the SARS-CoV-2-related disease (COVID-19), tyrosine kinase inhibitors (TKIs), already effective in Philadelphia-positive acute lymphoblastic leukemia (Ph+ ALL) and chronic myeloid leukemia (CML), have been proposed on the basis of their antiviral action already demonstrated against SARS-CoV-1. Very few cases of COVID-19 have been reported in Ph+ ALL and in CML Italian cohorts; authors suggested that this low rate of infections might depend on the use of TKIs, but the biological causes of this phenomenon remain unknown. In this study, the CML model was used to test if TKIs would sustain or not the viral replication and if they could damage patient immunity. Firstly, the infection and replication rate of torquetenovirus (TTV), whose load is inversely proportional to the host immunological control, have been measured in CML patients receiving nilotinib. A very low percentage of subjects were infected at baseline, and TTV did not replicate or at least showed a low replication rate during the follow-up, with a mean load comparable to the measured one in healthy subjects. Then, after gene expression profiling experiments, we found that several "antiviral" genes, such as CD28 and IFN gamma, were upregulated, while genes with "proviral" action, such as ARG-1, CEACAM1, and FUT4, were less expressed during treatment with imatinib, thus demonstrating that TKIs are not detrimental from the immunological point of view. To sum up, our data could offer some biological explanations to the low COVID-19 occurrence in Ph+ ALL and CML patients and sustain the use of TKIs in COVID-19, as already proposed by several international ongoing studies.

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