scholarly journals High-throughput human primary cell-based airway model for evaluating influenza, coronavirus, or other respiratory viruses in vitro

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
A. L. Gard ◽  
R. J. Luu ◽  
C. R. Miller ◽  
R. Maloney ◽  
B. P. Cain ◽  
...  
Keyword(s):  
Influenza A ◽  
Rapid Assessment ◽  
Antiviral Agents ◽  
Antiviral Agent ◽  
Respiratory Viruses ◽  
Airway Epithelial Cells ◽  
Host Cells ◽  
Preclinical Model ◽  
Airway Epithelial

AbstractInfluenza and other respiratory viruses present a significant threat to public health, national security, and the world economy, and can lead to the emergence of global pandemics such as from COVID-19. A barrier to the development of effective therapeutics is the absence of a robust and predictive preclinical model, with most studies relying on a combination of in vitro screening with immortalized cell lines and low-throughput animal models. Here, we integrate human primary airway epithelial cells into a custom-engineered 96-device platform (PREDICT96-ALI) in which tissues are cultured in an array of microchannel-based culture chambers at an air–liquid interface, in a configuration compatible with high resolution in-situ imaging and real-time sensing. We apply this platform to influenza A virus and coronavirus infections, evaluating viral infection kinetics and antiviral agent dosing across multiple strains and donor populations of human primary cells. Human coronaviruses HCoV-NL63 and SARS-CoV-2 enter host cells via ACE2 and utilize the protease TMPRSS2 for spike protein priming, and we confirm their expression, demonstrate infection across a range of multiplicities of infection, and evaluate the efficacy of camostat mesylate, a known inhibitor of HCoV-NL63 infection. This new capability can be used to address a major gap in the rapid assessment of therapeutic efficacy of small molecules and antiviral agents against influenza and other respiratory viruses including coronaviruses.

scholarly journals Eosinophil Responses at the Airway Epithelial Barrier during the Early Phase of Influenza a Virus Infection in C57BL/6 Mice

Cells ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 509 ◽  
Author(s):  
Meenakshi Tiwary ◽  
Robert J. Rooney ◽  
Swantje Liedmann ◽  
Kim S. LeMessurier ◽  
Amali E. Samarasinghe
Keyword(s):  
Epithelial Cells ◽  
Influenza A Virus ◽  
Early Phase ◽  
Influenza A ◽  
Airway Epithelial Cells ◽  
Epithelial Barrier ◽  
Virus Infectivity ◽  
Airway Epithelial ◽  
In Vivo Models

Eosinophils, previously considered terminally differentiated effector cells, have multifaceted functions in tissues. We previously found that allergic mice with eosinophil-rich inflammation were protected from severe influenza and discovered specialized antiviral effector functions for eosinophils including promoting cellular immunity during influenza. In this study, we hypothesized that eosinophil responses during the early phase of influenza contribute to host protection. Using in vitro and in vivo models, we found that eosinophils were rapidly and dynamically regulated upon influenza A virus (IAV) exposure to gain migratory capabilities to traffic to lymphoid organs after pulmonary infection. Eosinophils were capable of neutralizing virus upon contact and combinations of eosinophil granule proteins reduced virus infectivity through hemagglutinin inactivation. Bi-directional crosstalk between IAV-exposed epithelial cells and eosinophils occurred after IAV infection and cross-regulation promoted barrier responses to improve antiviral defenses in airway epithelial cells. Direct interactions between eosinophils and airway epithelial cells after IAV infection prevented virus-induced cytopathology in airway epithelial cells in vitro, and eosinophil recipient IAV-infected mice also maintained normal airway epithelial cell morphology. Our data suggest that eosinophils are important in the early phase of IAV infection providing immediate protection to the epithelial barrier until adaptive immune responses are deployed during influenza.

scholarly journals Amino Acid 226 in the Hemagglutinin of H9N2 Influenza Viruses Determines Cell Tropism and Replication in Human Airway Epithelial Cells

2007 ◽  
Vol 81 (10) ◽  
pp. 5181-5191 ◽  
Author(s):  
Hongquan Wan ◽  
Daniel R. Perez
Keyword(s):  
Amino Acid ◽  
Influenza A ◽  
Airway Epithelial Cells ◽  
Influenza Viruses ◽  
Cell Tropism ◽  
Airway Epithelial ◽  
Receptor Binding Site ◽  
Human Virus ◽  
Human Airway

ABSTRACT Influenza A viruses of the H9N2 subtype are endemic in poultry in many Eurasian countries and have occasionally caused clinical respiratory diseases in humans. While some avian H9N2 viruses have glutamine (Q) at amino acid position 226 of the hemagglutinin (HA) receptor-binding site, an increasing number of isolates have leucine (L) at this position, which has been associated with the establishment of stable lineages of the H2 and H3 subtypes of viruses in humans. Little is known about the importance of this molecular trait in the infection of H9N2 viruses in humans. We show here that during the course of a single cycle of infection in human airway epithelial (HAE) cells cultured in vitro, the L-226-containing H9N2 viruses displayed human virus-like cell tropisms (preferentially infecting nonciliated cells) different from the tropisms showed by Q-226-containing H9N2 isolates (which infect both ciliated and nonciliated cells at ratios of 1:1 to 3:2) or other waterfowl viruses (which preferentially infect ciliated cells). During multiple cycles of replication in HAE cultures, L-226-containing H9N2 isolates grew consistently more efficiently and reached approximately 100-fold-higher peak titers than those containing Q-226, although peak titers were significantly lower than those induced by human H3N2 viruses. Our results suggest that the variation in residue 226 in the HA affects both cell tropism and replication of H9N2 viruses in HAE cells and may have implications for the abilities of these viruses to infect humans.

Nitrogen dioxide exposure: effects on airway and blood cells

2002 ◽  
Vol 282 (1) ◽  
pp. L155-L165 ◽  
Author(s):  
Mark W. Frampton ◽  
Joseph Boscia ◽  
Norbert J. Roberts ◽  
Mitra Azadniv ◽  
Alfonso Torres ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Nitrogen Dioxide ◽  
Airway Inflammation ◽  
Blood Cells ◽  
Respiratory Viruses ◽  
Airway Epithelial Cells ◽  
Airway Epithelial ◽  
Air Exposure ◽  
Syncytial Virus

This study examined the effects of nitrogen dioxide (NO2) exposure on airway inflammation, blood cells, and antiviral respiratory defense. Twenty-one healthy volunteers were exposed on separate occasions to air and 0.6 and 1.5 ppm NO2for 3 h with intermittent moderate exercise. Phlebotomy and bronchoscopy were performed 3.5 h after each exposure, and recovered cells were challenged with respiratory viruses in vitro. Blood studies revealed a 4.1% NO2dose-related decrease in hematocrit ( P = 0.003). Circulating total lymphocytes ( P = 0.024) and T lymphocytes ( P = 0.049) decreased with NO2exposure. Exposure to NO2increased the blood lymphocyte CD4+-to-CD8+ratio from 1.74 ± 0.11 to 1.85 ± 0.12 in males but decreased it from 1.88 ± 0.19 to 1.78 ± 0.19 in females ( P < 0.001 for gender difference). Polymorphonuclear leukocytes in bronchial lavage increased with NO2exposure ( P = 0.003). Bronchial epithelial cells obtained after exposure to 1.5 ppm NO2released 40% more lactate dehydrogenase after challenge with respiratory syncytial virus than with air exposure ( P = 0.024). In healthy subjects, exposures to NO2at levels found indoors cause mild airway inflammation, effects on blood cells, and increased susceptibility of airway epithelial cells to injury from respiratory viruses.

scholarly journals Multiple Classes of Antiviral Agents ExhibitIn VitroActivity against Human Rhinovirus Type C

2013 ◽  
Vol 58 (3) ◽  
pp. 1546-1555 ◽  
Author(s):  
Chris Mello ◽  
Esmeralda Aguayo ◽  
Madeleine Rodriguez ◽  
Gary Lee ◽  
Robert Jordan ◽  
...  
Keyword(s):  
Kinase Inhibitor ◽  
Antiviral Agents ◽  
Airway Epithelial Cells ◽  
Human Rhinovirus ◽  
Dependent Manner ◽  
Airway Epithelial ◽  
Content Type ◽  
Type C ◽  
Rna Replicon

ABSTRACTHuman rhinovirus type C (HRV-C) is a newly discovered enterovirus species frequently associated with exacerbation of asthma and other acute respiratory conditions. Until recently, HRV-C could not be propagatedin vitro, hampering in-depth characterization of the virus replication cycle and preventing efficient testing of antiviral agents. Herein we describe several subgenomic RNA replicon systems and a cell culture infectious model for HRV-C that can be used for antiviral screening. The replicon constructs consist of genome sequences from HRVc15, HRVc11, HRVc24, and HRVc25 strains, with the P1 capsid region replaced by aRenillaluciferase coding sequence. Following transfection of the replicon RNA into HeLa cells, the constructs produced time-dependent increases in luciferase signal that can be inhibited in a dose-dependent manner by known inhibitors of HRV replication, including the 3C protease inhibitor rupintrivir, the nucleoside analog inhibitor MK-0608, and the phosphatidylinositol 4-kinase IIIβ (PI4K-IIIβ) kinase inhibitor PIK93. Furthermore, with the exception of pleconaril and pirodavir, the other tested classes of HRV inhibitors blocked the replication of full-length HRVc15 and HRVc11 in human airway epithelial cells (HAEs) that were differentiated in the air-liquid interface, exhibiting antiviral activities similar to those observed with HRV-16. In summary, this study is the first comprehensive profiling of multiple classes of antivirals against HRV-C, and the set of newly developed quantitative HRV-C antiviral assays represent indispensable tools for the identification and evaluation of novel panserotype HRV inhibitors.

scholarly journals High-Throughput Human Primary Cell-Based Airway Model for Evaluating Influenza, Coronavirus, or other Respiratory Viruses in vitro

2020 ◽  
Author(s):  
A.L. Gard ◽  
R. Maloney ◽  
B.P. Cain ◽  
C.R. Miller ◽  
R.J. Luu ◽  
...  
Keyword(s):  
High Throughput ◽  
Influenza A ◽  
Respiratory Viruses ◽  
Therapeutic Agents ◽  
Liquid Interface ◽  
Primary Cell ◽  
Host Cells ◽  
Successful Infection ◽  
Air Liquid Interface

AbstractInfluenza and other respiratory viruses represent a significant threat to public health, national security, and the world economy, and can lead to the emergence of global pandemics such as the current COVID-19 crisis. One of the greatest barriers to the development of effective therapeutic agents to treat influenza, coronaviruses, and many other infections of the respiratory tract is the absence of a robust preclinical model. Preclinical studies currently rely on high-throughput, low-fidelity in vitro screening with cell lines and/or low-throughput animal models that often provide a poor correlation to human clinical responses. Here, we introduce a human primary airway epithelial cell-based model integrated into a high-throughput platform where tissues are cultured at an air-liquid interface (PREDICT96-ALI). We present results on the application of this platform to influenza and coronavirus infections, providing multiple readouts capable of evaluating viral infection kinetics and potentially the efficacy of therapeutic agents in an in vitro system. Several strains of influenza A virus are shown to successfully infect the human primary cell-based airway tissue cultured at an air-liquid interface (ALI), and as a proof-of-concept, the effect of the antiviral oseltamivir on one strain of Influenza A is evaluated. Human coronaviruses NL63 (HCoV-NL63) and SARS-CoV-2 enter host cells via ACE2 and utilize the protease TMPRSS2 for protein priming, and we confirm expression of both in our ALI model. We also demonstrate coronavirus infection in this system with HCoV-NL63, observing sufficient viral propagation over 96 hours post-infection to indicate successful infection of the primary cell-based model. This new capability has the potential to address a gap in the rapid assessment of therapeutic efficacy of various small molecules and antiviral agents against influenza and other respiratory viruses including coronaviruses.

scholarly journals Human metapneumovirus induces autophagy through JNK and MEK/ERK pathway

2019 ◽  
Author(s):  
Pan Zhang ◽  
Suhua Chen ◽  
Hui Yang ◽  
Bin Tan ◽  
Yao Zhao
Keyword(s):  
Airway Epithelial Cells ◽  
Human Metapneumovirus ◽  
Host Cells ◽  
Respiratory Pathogen ◽  
Erk Pathway ◽  
Airway Epithelial ◽  
Host Interaction ◽  
Hmpv Infection ◽  
Lc3 Puncta

Abstract Background: Human metapneumovirus (hMPV) is a ubiquitous respiratory pathogen, especially in infants and young children. Virus-host interaction affects viral replication and host immune responses. Autophagy plays an important role in virus-host interaction. Airway epithelial cells serve as the first line in host to against respiratory virus infection. However, it is still unknown whether autophagy is activated in human metapneumovirus (hMPV) infected host cells. Methods: In this study, we demonstrated the occurrence of autophagy through some autophagic features including the conversion of GFP-RFP-LC3 plasmid, RFP-LC3 puncta and expression of autophagic-related gene. The pathways involved in autophagy were detected by Western Blot and verified by specific pathway inhibitors. The relationship between the replication of hMPV and autophagy was tested by Rapamycin, 3-MA, siRNA-LC3 and pathway inhibitors. We also used BALB/c mice to verified the autophagy and pathways in hMPV infection. Results: We found that the GFP-RFP-LC3 plasmid, RFP-LC3 puncta and expression of ATG5, ATG7, Becllin1, LC3 were increased in hMPV group in vitro . JNK and MEK/ERK signaling pathway are activated in hMPV induced-autophagy and we confirmed the two pathways with specific inhibitors SP600125 and PD98059. Furthermore, we found that rapamycin suppressed hMPV infection significantly, but reversed when treated with autophagy inhibitor 3-MA, siRNA-LC3 and pathway inhibitors. hMPV infected mice also induce autophagy through JNK and MEK/ERK pathways. Conclusion: Taken together, our results show strong evidence that autophagy involved in hMPV infection through JNK, MEK/ERK pathway which plays an antiviral role in the process of hMPV infection.

scholarly journals E-cigarette vapour enhances pneumococcal adherence to airway epithelial cells

2018 ◽  
Vol 51 (2) ◽  
pp. 1701592 ◽  
Author(s):  
Lisa Miyashita ◽  
Reetika Suri ◽  
Emma Dearing ◽  
Ian Mudway ◽  
Rosamund E Dove ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Epithelial Cells ◽  
Pneumococcal Infection ◽  
Platelet Activating Factor ◽  
Airway Epithelial Cells ◽  
Host Cells ◽  
Airway Epithelial ◽  
Oxidative Potential ◽  
Airway Cells

E-cigarette vapour contains free radicals with the potential to induce oxidative stress. Since oxidative stress in airway cells increases platelet-activating factor receptor (PAFR) expression, and PAFR is co-opted by pneumococci to adhere to host cells, we hypothesised that E-cigarette vapour increases pneumococcal adhesion to airway cells.Nasal epithelial PAFR was assessed in non-vaping controls, and in adults before and after 5 min of vaping. We determined the effect of vapour on oxidative stress-induced, PAFR-dependent pneumococcal adhesion to airway epithelial cells in vitro, and on pneumococcal colonisation in the mouse nasopharynx. Elemental analysis of vapour was done by mass spectrometry, and oxidative potential of vapour assessed by antioxidant depletion in vitro.There was no difference in baseline nasal epithelial PAFR expression between vapers (n=11) and controls (n=6). Vaping increased nasal PAFR expression. Nicotine-containing and nicotine-free E-cigarette vapour increased pneumococcal adhesion to airway cells in vitro. Vapour-stimulated adhesion in vitro was attenuated by the PAFR blocker CV3988. Nicotine-containing E-cigarette vapour increased mouse nasal PAFR expression, and nasopharyngeal pneumococcal colonisation. Vapour contained redox-active metals, had considerable oxidative activity, and adhesion was attenuated by the antioxidant N-acetyl cysteine.This study suggests that E-cigarette vapour has the potential to increase susceptibility to pneumococcal infection.

scholarly journals Antiviral activity of lambda-carrageenan against influenza viruses in mice and severe acute respiratory syndrome coronavirus 2 in vitro

2020 ◽  
Author(s):  
Yejin Jang ◽  
Heegwon Shin ◽  
Myoung Kyu Lee ◽  
Oh Seung Kwon ◽  
Jin Soo Shin ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Antiviral Activity ◽  
Influenza A ◽  
Antiviral Agent ◽  
Influenza Viruses ◽  
Host Cells ◽  
Progeny Virus ◽  
Dependent Manner ◽  
Viral Attachment

ABSTRACTInfluenza virus and coronavirus, belonging to enveloped RNA viruses, are major causes of human respiratory diseases. The aim of this study was to investigate the broad spectrum antiviral activity of a naturally existing sulfated polysaccharide, lambda-carrageenan (λ-CGN), purified from marine red algae. Cell culture-based assays revealed that the macromolecule efficiently inhibited both influenza A and B viruses, as well as currently circulating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), with EC50 values ranging from 0.3–1.4 μg/ml. No toxicity to host cells was observed at concentrations up to 300 μg/ml. Plaque titration and western blot analysis verified that λ-CGN reduced expression of viral proteins in cell lysates and suppressed progeny virus production in culture supernatants in a dose-dependent manner. This polyanionic compound exerts antiviral activity by targeting viral attachment to cell surface receptors and preventing entry. Moreover, intranasal administration to mice during influenza A viral challenge not only alleviated infection-mediated reductions in body weight but also protected 60% of mice from virus-induced mortality. Thus, λ-CGN could be a promising antiviral agent for preventing infection by several respiratory viruses.

scholarly journals Bromodomain and Extraterminal Protein Inhibitor, Apabetalone (RVX-208), Reduces ACE2 Expression and Attenuates SARS-Cov-2 Infection In Vitro

Biomedicines ◽  
2021 ◽  
Vol 9 (4) ◽  
pp. 437
Author(s):  
Dean Gilham ◽  
Audrey L. Smith ◽  
Li Fu ◽  
Dalia Y. Moore ◽  
Abenaya Muralidharan ◽  
...  
Keyword(s):  
Antiviral Agents ◽  
Host Cells ◽  
Protein Inhibitor ◽  
Angiotensin Converting Enzyme 2 ◽  
Dipeptidyl Peptidase 4 ◽  
Surface Receptors ◽  
Bet Inhibitor ◽  
Epigenetic Machinery ◽  
Unexplored Area

Effective therapeutics are urgently needed to counter infection and improve outcomes for patients suffering from COVID-19 and to combat this pandemic. Manipulation of epigenetic machinery to influence viral infectivity of host cells is a relatively unexplored area. The bromodomain and extraterminal (BET) family of epigenetic readers have been reported to modulate SARS-CoV-2 infection. Herein, we demonstrate apabetalone, the most clinical advanced BET inhibitor, downregulates expression of cell surface receptors involved in SARS-CoV-2 entry, including angiotensin-converting enzyme 2 (ACE2) and dipeptidyl-peptidase 4 (DPP4 or CD26) in SARS-CoV-2 permissive cells. Moreover, we show that apabetalone inhibits SARS-CoV-2 infection in vitro to levels comparable to those of antiviral agents. Taken together, our study supports further evaluation of apabetalone to treat COVID-19, either alone or in combination with emerging therapeutics.

scholarly journals Differential Expression of Serum Exosome microRNAs and Cytokines in Influenza A and B Patients Collected in the 2016 and 2017 Influenza Seasons

Pathogens ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 149
Author(s):  
Sreekumar Othumpangat ◽  
William G. Lindsley ◽  
Donald H. Beezhold ◽  
Michael L. Kashon ◽  
Carmen N. Burrell ◽  
...  
Keyword(s):  
Healthy Volunteers ◽  
Influenza A ◽  
Influenza Infection ◽  
Cell Types ◽  
Airway Epithelial Cells ◽  
Differentially Expressed ◽  
Influenza B ◽  
Airway Epithelial ◽  
Monocyte Chemoattractant Protein 1 ◽  
Novel Approach

MicroRNAs (miRNAs) have remarkable stability and are key regulators of mRNA transcripts for several essential proteins required for the survival of cells and replication of the virus. Exosomes are thought to play an essential role in intercellular communications by transporting proteins and miRNAs, making them ideal in the search for biomarkers. Evidence suggests that miRNAs are involved in the regulation of influenza virus replication in many cell types. During the 2016 and 2017 influenza season, we collected blood samples from 54 patients infected with influenza and from 30 healthy volunteers to identify the potential role of circulating serum miRNAs and cytokines in influenza infection. Data comparing the exosomal miRNAs in patients with influenza B to healthy volunteers showed 76 miRNAs that were differentially expressed (p < 0.05). In contrast, 26 miRNAs were differentially expressed between patients with influenza A (p < 0.05) and the controls. Of these miRNAs, 11 were commonly expressed in both the influenza A and B patients. Interferon (IFN)-inducing protein 10 (IP-10), which is involved in IFN synthesis during influenza infection, showed the highest level of expression in both influenza A and B patients. Influenza A patients showed increased expression of IFNα, GM-CSF, interleukin (IL)-13, IL-17A, IL-1β, IL-6 and TNFα, while influenza B induced increased levels of EGF, G-CSF, IL-1α, MIP-1α, and TNF-β. In addition, hsa-miR-326, hsa-miR-15b-5p, hsa-miR-885, hsa-miR-122-5p, hsa-miR-133a-3p, and hsa-miR-150-5p showed high correlations to IL-6, IL-15, IL-17A, IL-1β, and monocyte chemoattractant protein-1 (MCP-1) with both strains of influenza. Next-generation sequencing studies of H1N1-infected human lung small airway epithelial cells also showed similar pattern of expression of miR-375-5p, miR-143-3p, 199a-3p, and miR-199a-5p compared to influenza A patients. In summary, this study provides insights into the miRNA profiling in both influenza A and B virus in circulation and a novel approach to identify the early infections through a combination of cytokines and miRNA expression.

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