SARS-CoV-2 Is More Efficient than HCoV-NL63 in Infecting a Small Subpopulation of ACE2+ Human Respiratory Epithelial Cells.

Human coronavirus (HCoV)-NL63 is an important contributor to upper and lower respiratory tract infections, mainly in children, while severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of COVID-19, can cause lower respiratory tract infections, and more severe, respiratory and systemic disease, which leads to fatal consequences in many cases. Using microscopy, immunohistochemistry (IHC), virus-binding assay, reverse transcriptase qPCR (RT-qPCR) assay, and flow cytometry, we compared the characteristics of the susceptibility, replication dynamics, and morphogenesis of HCoV-NL63 and SARS-CoV-2 in monolayer cultures of primary human respiratory epithelial cells (HRECs). Less than 10% HRECs expressed ACE2, and SARS-CoV-2 seemed much more efficient than HCoV-NL63 at infecting the very small proportion of HRECs expressing the ACE2 receptors. Furthermore, SARS-CoV-2 replicated more efficiently than HCoV-NL63 in HREC, which correlates with the cumulative evidence of the differences in their transmissibility.

Human Air-Liquid-Interface Organotypic Airway Cultures Express Significantly More ACE2 Receptor Protein and Are More Susceptible to HCoV-NL63 Infection than Monolayer Cultures of Primary Respiratory Epithelial Cells.

Human coronavirus NL63 (HCoV-NL63) is commonly associated with mild respiratory tract infections in infants, being that the respiratory epithelial cells are the main target for infection and initial replication of this virus. Standard immortalized cells are highly permissive to HCoV-NL63, and they are routinely used for isolation and propagation of the virus from clinical specimens. However, these cell lines are not the natural cell target of the virus and lack sufficient complexity to mimic the natural infection process in vivo. This study comparatively evaluated the differences on the susceptibility to HCoV-NL63 infection and virus replication efficiency of submerged monolayer cultures of LLC-MK2 and primary human respiratory epithelial cells (HRECs) and organotypic airway cultures of respiratory cells (ALI-HRECs). Productive viral infection and growth kinetics were assessed by morphologic examination of cytopathic effects, immunofluorescence, reverse transcription quantitative real-time PCR, and flow cytometry. Results from this study showed higher susceptibility to HCoV-NL63 infection and replication in LLC-MK2 cells followed by ALI-HRECs, with very low susceptibility and no significant virus replication in HRECs. This susceptibility was associated with the expression levels of angiontensin-converting enzyme 2 (ACE2) receptor protein in LLC-MK2, ALI-HRECs, and HRECs, respectively. Remarkably, organotypic ALI-HREC cultures expressed significantly more ACE2 receptor protein and were more susceptible to HCoV-NL63 infection than monolayer cultures of HREC. The ACE2 receptor is, therefore, a critical factor for susceptibility to HCoV-NL63 infection and replication, as is the type of culture used during infection studies. IMPORTANCE HCoV-NL63 is widespread globally, accounting for a significant number of respiratory infections in children and adults. HCoV-NL63 gains entrance into respiratory epithelial cells via the ACE2 receptor, the same cell receptor used by severe acute respiratory syndrome coronavirus (SARS-CoV) and SARS-CoV-2. Thus, HCoV-NL63 has been suggested as safe surrogate for studying disease mechanisms and therapeutic interventions against SARS-like CoVs, while working under BSL-2 conditions. The present study not only showed the critical role of ACE2 for effective HCoV-NL63 infection and replication, but also shed light on the need of more refined and complex in vitro organotypic models that recapitulate the proxy of air-liquid respiratory epithelia cell composition, structure, and functionality. These cultures have broaden virological studies toward improving our understanding of how coronaviruses cause disease and transmission not just within humans but also in animal populations.

Immunobiotic Lactobacilli Improve Resistance of Respiratory Epithelial Cells to SARS-CoV-2 Infection.

Previously, we reported that immunomodulatory lactobacilli, nasally administered, beneficially regulated the lung antiviral innate immune response induced by Toll-like receptor 3 (TLR3) activation and improved protection against the respiratory pathogens, influenza virus and respiratory syncytial virus in mice. Here, we assessed the immunomodulatory effects of viable and non-viable Lactiplantibacillus plantarum strains in human respiratory epithelial cells (Calu-3 cells) and the capacity of these immunobiotic lactobacilli to reduce their susceptibility to the acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Immunobiotic L. plantarum MPL16 and CRL1506 differentially modulated IFN-ß, IL-6, CXCL8, CCL5 and CXCL10 production and IFNAR2, DDX58, Mx1 and OAS1 expression in Calu-3 cells stimulated with the TLR3 agonist poly(I:C). Furthermore, the MPL16 and CRL1506 strains increased the resistance of Calu-3 cells to the challenge with SARS-CoV-2. L. plantarum MPL16 induced these beneficial effects more efficiently than the CRL1506 strain. Of note, neither non-viable MPL16 and CRL1506 strains nor the non-immunomodulatory strains L. plantarum CRL1905 and MPL18 could modify the resistance of Calu-3 cells to SARS-CoV-2 infection or the immune response to poly(I:C) challenge. To date, the potential beneficial effects of immunomodulatory probiotics on SARS-CoV-2 infection and COVID-19 outcome have been extrapolated from studies carried out in the context of other viral pathogens. To the best of our knowledge, this is the first demonstration of the ability of immunomodulatory lactobacilli to positively influence the replication of the new coronavirus. Further mechanistic studies and in vivo experiments in animal models of SARS-CoV-2 infection are necessary to identify specific strains of beneficial immunobiotic lactobacilli like L. plantarum MPL16 or CRL1506 for the prevention or treatment of the COVID-19.

Dolosigranulum pigrum Modulates Immunity against SARS-CoV-2 in Respiratory Epithelial Cells.

In a previous work, we demonstrated that nasally administered Dolosigranulum pigrum 040417 beneficially modulated the respiratory innate immune response triggered by the activation of Toll-like receptor 3 (TLR3) and improved protection against Respiratory Syncytial Virus (RSV) in mice. In this work, we aimed to evaluate the immunomodulatory effects of D. pigrum 040417 in human respiratory epithelial cells and the potential ability of this immunobiotic bacterium to increase the protection against Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). The respiratory commensal bacterium D. pigrum 040417 differentially modulated the production of IFN-ß, IL-6, CXCL8, CCL5 and CXCL10 in the culture supernatants of Calu-3 cells stimulated with poly(I:C) or challenged with SARS-CoV-2. The differential cytokine profile induced by the 040417 strain was associated with a significant reduction in viral replication and cellular damage after coronavirus infection. Of note, D. pigrum 030918 was not able to modify the resistance of Calu-3 cells to SARS-CoV-2 infection, indicating a strain-specific immunomodulatory effect for respiratory commensal bacteria. The findings of this work improve our understanding of the immunological mechanisms involved in the modulation of respiratory immunity induced by respiratory commensal bacteria, by demonstrating their specific effect on respiratory epithelial cells. In addition, the results suggest that particular strains such as D. pigrum 040417 could be used as a promising alternative for combating SARS-CoV-2 and reducing the severity of COVID-19.

Dual-Fluorescence Labeling Pseudovirus for Real-Time Imaging of Single SARS-CoV-2 Entry in Respiratory Epithelial Cells.

The pseudovirus strategy makes studies of highly pathogenic viruses feasible without the restriction of high-level biosafety facility, thus greatly contributing to virology and is used in the research studies of SARS-CoV-2. Here, we generated a dual-color pseudo-SARS-CoV-2 virus using a human immunodeficiency virus-1 pseudovirus production system and the SARS-CoV-2 spike (S) glycoprotein, of which the membrane was labeled with a lipophilic dye (DiO) and the genomic RNA-related viral protein R (Vpr) of the viral core was fused with mCherry. With this dual-color labeling strategy, not only the movement of the whole virus but also the fate of the labeled components can be traced. The pseudovirions were applied to track the viral entry at a single-particle level in four types of the human respiratory cells: nasal epithelial cells (HNEpC), pulmonary alveolar epithelial cells (HPAEpiC), bronchial epithelial cells (BEP-2D), and oral epithelial cells (HOEC). Pseudo-SARS-CoV-2 entered into the host cell and released the viral core into the cytoplasm, which clearly indicates that the host entry mainly occurred through endocytosis. The infection efficiency was found to be correlated with the expression of the known receptor of SARS-CoV-2, angiotensin-converting 2 (ACE2) on the host cell surface. We believe that the dual-color fluorescently labeled pseudovirus system created in this study can be applied as a useful tool for many purposes in SARS-CoV-2/COVID-19.

PDE4 inhibition as a therapeutic strategy for improvement of pulmonary dysfunctions in Covid-19 and cigarette smoking.

Angiotensin-converting enzyme 2 (ACE2) is the binding-site and entry-point for SARS-CoV-2 in human and highly expressed in the lung. Cigarette smoking (CS) is the leading cause of pulmonary and cardiovascular diseases. Chronic CS leads to upregulation of bronchial ACE2 inducing a high vulnerability in COVID-19 smoker patients. Interestingly, CS-induced dysregulation of pulmonary renin-angiotensin system (RAS) in part contributing into the potential pathogenesis COVID-19 pneumonia and acute respiratory distress syndrome (ARDS). Since, CS-mediated ACE2 activations is not the main pathway for increasing the risk of COVID-19, it appeared that AngII/AT1R might induce an inflammatory-burst in COVID-19 response by up-regulating cyclic nucleotide phosphodiesterase type 4 (PDE4), which hydrolyses specifically the second intracellular messenger 3', 5'-cyclic AMP (cAMP). It must be pointed out that CS might induce PDE4 up-regulation similarly to the COVID-19 inflammation, and therefore could potentiate COVID-19 inflammation opening the potential therapeutic effects of PDE4 inhibitor in both COVID-19-inflammation and CS.