scholarly journals Inactivation of Material from SARS-CoV-2-Infected Primary Airway Epithelial Cell Cultures

2021 ◽  
Vol 4 (1) ◽  
pp. 7
Author(s):  
Kaitlyn A. Barrow ◽  
Lucille M. Rich ◽  
Elizabeth R. Vanderwall ◽  
Stephen R. Reeves ◽  
Jennifer A. Rathe ◽  
...  
Keyword(s):  
Viral Entry ◽  
Uv Light ◽  
Sodium Dodecyl ◽  
Airway Epithelial Cells ◽  
Protein Isolate ◽  
Airway Epithelial ◽  
Innate Immune Responses ◽  
Triton X100 ◽  
Control And Prevention ◽  
Level 3

Given that the airway epithelium is the initial site of infection, study of primary human airway epithelial cells (AEC) infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) will be crucial to improved understanding of viral entry factors and innate immune responses to the virus. Centers for Disease Control and Prevention (CDC) guidance recommends work with live SARS-CoV-2 in cell culture be conducted in a Biosafety Level 3 (BSL-3) laboratory. To facilitate downstream assays of materials from experiments there is a need for validated protocols for SARS-CoV-2 inactivation to facilitate safe transfer of material out of a BSL-3 laboratory. We propagated stocks of SARS-CoV-2, then evaluated the effectiveness of heat (65 °C) or ultraviolet (UV) light inactivation. We infected differentiated human primary AECs with SARS-CoV-2, then tested protocols designed to inactivate SARS-CoV-2 in supernatant, protein isolate, RNA, and cells fixed for immunohistochemistry by exposing Vero E6 cells to materials isolated/treated using these protocols. Heating to 65 °C for 10 min or exposing to UV light fully inactivated SARS-CoV-2. Furthermore, we found in SARS-CoV-2-infected primary AEC cultures that treatment of supernatant with UV light, isolation of RNA with Trizol®, isolation of protein using a protocol including sodium dodecyl sulfate (SDS) 0.1% and Triton X100 1%, and fixation of AECs using 10% formalin and Triton X100 1%, each fully inactivated SARS-CoV-2.

The novel antiviral properties of brassicasterol against human adenovirus

2021 ◽  
Author(s):  
Peifeng Yu ◽  
Dan Lou ◽  
Lifeng Qi ◽  
Zewei Chen
Keyword(s):  
Dna Replication ◽  
Epithelial Cells ◽  
Viral Entry ◽  
Human Adenovirus ◽  
Airway Epithelial Cells ◽  
Dependent Manner ◽  
The Novel ◽  
Airway Epithelial ◽  
Infected Cells ◽  
Dose Dependent

Aim: To investigate whether brassicasterol has inhibitory effects against adenovirus (AdV). Materials and methods: The antiviral effects of brassicasterol against AdV 3 and 7 were tested in human airway epithelial cells. Brassicasterol cytotoxicity was assessed by WST-1 assay. AdV DNA was quantified by qPCR. Results: Brassicasterol inhibited AdV 3 and 7 infection of airway epithelial cells in a dose-dependent manner. Similarly, brassicasterol also inhibited AdV 3 and 7 production in infected cells. No apparent cytotoxicity of brassicasterol was detected. Further study showed that brassicasterol inhibited AdV DNA replication, but had no impact on viral entry of cells and viral genome import to nucleus. Conclusion: Brassicasterol exerts anti-AdV effects probably through the inhibition of viral DNA replication.

scholarly journals Mitoxantrone modulates a glycosaminoglycan-spike complex to inhibit SARS-CoV-2 infection

2021 ◽  
Author(s):  
Qi Zhang ◽  
Peter Radvak ◽  
Juhyung Lee ◽  
Yue Xu ◽  
Vivian Cao-Dao ◽  
...  
Keyword(s):  
Viral Entry ◽  
Alternative Therapy ◽  
Single Agent ◽  
Airway Epithelial Cells ◽  
Airway Epithelial ◽  
Dna Topoisomerase ◽  
Nucleosome Dynamics ◽  
Protein Receptors ◽  
Attractive Therapeutic Target ◽  
A Cell

Spike-mediated entry of SARS-CoV-2 into human airway epithelial cells is an attractive therapeutic target for COVID-19. In addition to protein receptors, the SARS-CoV-2 spike (S) protein also interacts with heparan sulfate, a negatively charged glycosaminoglycan (GAG) attached to certain membrane proteins on the cell surface. This interaction facilitates the engagement of spike with a downstream receptor to promote viral entry. Here, we show that Mitoxantrone, an FDA-approved topoisomerase inhibitor, targets a spike-GAG complex to compromise the fusogenic function of spike in viral entry. As a single agent, Mitoxantrone inhibits the infection of an authentic SARS-CoV-2 strain in a cell-based model and in human lung EpiAirway 3D tissues. Gene expression profiling supports the plasma membrane as a major target of Mitoxantrone but also underscores an undesired activity targeting nucleosome dynamics. We propose that Mitoxantrone analogs bearing similar GAG-binding activities but with reduced affinity for DNA topoisomerase may offer an alternative therapy to overcome breakthrough infections in the post-vaccine era.

scholarly journals TMPRSS2 structure-phylogeny repositions Avoralstat for SARS-CoV-2 prophylaxis in mice

2021 ◽  
Author(s):  
Young Joo Sun ◽  
Gabriel Velez ◽  
Dylan Parsons ◽  
Kun Li ◽  
Miguel Ortiz ◽  
...  
Keyword(s):  
Viral Entry ◽  
Serine Proteases ◽  
Tertiary Structure ◽  
Airway Epithelial Cells ◽  
Airway Epithelial ◽  
Cell Infection ◽  
Effective Inhibition ◽  
Transmembrane Serine Protease

Drugs targeting host proteins can act prophylactically to reduce viral burden early in disease and limit morbidity, even with antivirals and vaccination. Transmembrane serine protease 2 (TMPRSS2) is a human protease required for SARS-CoV-2 viral entry and may represent such a target. We hypothesized drugs selected from proteins related by their tertiary structure, rather than their primary structure, were likely to interact with TMPRSS2. We created a structure-based phylogenetic computational tool 3DPhyloFold to systematically identify structurally similar serine proteases with known therapeutic inhibitors and demonstrated effective inhibition of SARS-CoV-2 infection in vitro and in vivo. Several candidate compounds, Avoralstat, PCI-27483, Antipain, and Soybean-Trypsin-Inhibitor, inhibited TMPRSS2 in biochemical and cell infection assays. Avoralstat, a clinically tested Kallikrein-related B1 inhibitor, inhibited SARS-CoV-2 entry and replication in human airway epithelial cells. In an in vivo proof of principle, Avoralstat significantly reduced lung tissue titers and mitigated weight-loss when administered prophylactically to SARS-CoV-2 susceptible mice indicating its potential to be repositioned for COVID-19 prophylaxis in humans.

scholarly journals Klebsiella pneumoniae Increases the Levels of Toll-Like Receptors 2 and 4 in Human Airway Epithelial Cells

2008 ◽  
Vol 77 (2) ◽  
pp. 714-724 ◽  
Author(s):  
Verónica Regueiro ◽  
David Moranta ◽  
Miguel A. Campos ◽  
Javier Margareto ◽  
Junkal Garmendia ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Klebsiella Pneumoniae ◽  
Sodium Dodecyl ◽  
Airway Epithelial Cells ◽  
Lymphoid Cells ◽  
Airway Epithelial ◽  
Tlr4 Expression ◽  
Toll Like Receptors ◽  
Human Airway ◽  
Human Airway Epithelial Cells

ABSTRACT Airway epithelial cells act as the first barrier against pathogens. These cells recognize conserved structural motifs expressed by microbial pathogens via Toll-like receptors (TLRs) expressed on the surface. In contrast to the level of expression in lymphoid cells, the level of expression of TLR2 and TLR4 in airway epithelial cells is low under physiological conditions. Here we explored whether Klebsiella pneumoniae upregulates the expression of TLRs in human airway epithelial cells. We found that the expression of TLR2 and TLR4 by A549 cells and human primary airway cells was upregulated upon infection with K. pneumoniae. The increased expression of TLRs resulted in enhancement of the cellular response upon stimulation with Pam3CSK4 and lipopolysaccharide, which are TLR2 and TLR4 agonists, respectively. Klebsiella-dependent upregulation of TLR expression occurred via a positive IκBα-dependent NF-κΒ pathway and via negative p38 and p44/42 mitogen-activated protein kinase-dependent pathways. We showed that Klebsiella-induced TLR2 and TLR4 upregulation was dependent on TLR activation. An isogenic capsule polysaccharide (CPS) mutant did not increase TLR2 and TLR4 expression. Purified CPS upregulated TLR2 and TLR4 expression, and polymyxin B did not abrogate CPS-induced TLR upregulation. Although no proteins were detected in the CPS preparation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and colloidal gold staining, we could not rule out the possibility that traces of protein in our CPS preparation could have been responsible, at least in part, for the TLR upregulation.

scholarly journals Viral and host factors determine innate immune responses in airway epithelial cells from children with wheeze and atopy

Thorax ◽  
2014 ◽  
Vol 69 (10) ◽  
pp. 918-925 ◽  
Author(s):  
Kirsten M Spann ◽  
Engin Baturcam ◽  
Johanna Schagen ◽  
Carmen Jones ◽  
Claire P Straub ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Immune Responses ◽  
Airway Epithelial Cells ◽  
Innate Immune ◽  
Host Factors ◽  
Airway Epithelial ◽  
Innate Immune Responses

scholarly journals SARS-CoV-2 inhibition using a mucoadhesive, amphiphilic chitosan that may serve as an anti-viral nasal spray

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Krzysztof Pyrć ◽  
Aleksandra Milewska ◽  
Emilia Barreto Duran ◽  
Paweł Botwina ◽  
Agnieszka Dabrowska ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Viral Entry ◽  
Statistical Significance ◽  
Airway Epithelial Cells ◽  
Host Cells ◽  
In Vivo Studies ◽  
Repeat Dose ◽  
Airway Epithelial ◽  
Viral Inhibition

AbstractThere are currently no cures for coronavirus infections, making the prevention of infections the only course open at the present time. The COVID-19 pandemic has been difficult to prevent, as the infection is spread by respiratory droplets and thus effective, scalable and safe preventive interventions are urgently needed. We hypothesise that preventing viral entry into mammalian nasal epithelial cells may be one way to limit the spread of COVID-19. Here we show that N-palmitoyl-N-monomethyl-N,N-dimethyl-N,N,N-trimethyl-6-O-glycolchitosan (GCPQ), a positively charged polymer that has been through an extensive Good Laboratory Practice toxicology screen, is able to reduce the infectivity of SARS-COV-2 in A549ACE2+ and Vero E6 cells with a log removal value of − 3 to − 4 at a concentration of 10–100 μg/ mL (p < 0.05 compared to untreated controls) and to limit infectivity in human airway epithelial cells at a concentration of 500 μg/ mL (p < 0.05 compared to untreated controls). In vivo studies using transgenic mice expressing the ACE-2 receptor, dosed nasally with SARS-COV-2 (426,000 TCID50/mL) showed a trend for nasal GCPQ (20 mg/kg) to inhibit viral load in the respiratory tract and brain, although the study was not powered to detect statistical significance. GCPQ’s electrostatic binding to the virus, preventing viral entry into the host cells, is the most likely mechanism of viral inhibition. Radiolabelled GCPQ studies in mice show that at a dose of 10 mg/kg, GCPQ has a long residence time in mouse nares, with 13.1% of the injected dose identified from SPECT/CT in the nares, 24 h after nasal dosing. With a no observed adverse effect level of 18 mg/kg in rats, following a 28-day repeat dose study, clinical testing of this polymer, as a COVID-19 prophylactic is warranted.

scholarly journals SARS-CoV-2 inhibition in human airway epithelial cells using a mucoadhesive, amphiphilic chitosan that may serve as an anti-viral nasal spray

2020 ◽  
Author(s):  
Krzysztof Pyrć ◽  
Aleksandra Milewska ◽  
Emilia Barreto Duran ◽  
Paweł Botwina ◽  
Rui Lopes ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Viral Entry ◽  
Airway Epithelial Cells ◽  
Host Cells ◽  
Repeat Dose ◽  
Airway Epithelial ◽  
Viral Inhibition ◽  
Human Airway ◽  
Adverse Effect Level ◽  
Human Airway Epithelial Cells

AbstractThere are currently no cures for coronavirus infections, making the prevention of infections the only course open at the present time. The COVID-19 pandemic has been difficult to prevent, as the infection is spread by respiratory droplets and thus effective, scalable and safe preventive interventions are urgently needed. We hypothesise that preventing viral entry into mammalian nasal epithelial cells may be one way to limit the spread of COVID-19. Here we show that N-palmitoyl-N-monomethyl-N,N-dimethyl-N,N,N-trimethyl-6-O-glycolchitosan (GCPQ), a positively charged polymer that has been through an extensive Good Laboratory Practice toxicology screen, is able to reduce the infectivity of SARS-COV-2 in A549ACE2+ and Vero E6 cells with a log removal value of −3 to −4 at a concentration of 10 – 100 μg/ mL (p < 0.05 compared to untreated controls) and to limit infectivity in human airway epithelial cells at a concentration of 500 μg/ mL (p < 0.05 compared to untreated controls). GCPQ is currently being developed as a pharmaceutical excipient in nasal and ocular formulations. GCPQ’s electrostatic binding to the virus, preventing viral entry into the host cells, is the most likely mechanism of viral inhibition. Radiolabelled GCPQ studies in mice show that at a dose of 10 mg/ kg, GCPQ has a long residence time in mouse nares, with 13.1% of the injected dose identified from SPECT/CT in the nares, 24 hours after nasal dosing. With a no observed adverse effect level of 18 mg/ kg in rats, following a 28-day repeat dose study, clinical testing of this polymer, as a COVID-19 prophylactic is warranted.

Sign in / Sign up

Export Citation Format

Share Document