Identification of host essential factors for recombinant AAV transduction of the polarized human airway epithelium.

IMPORTANCE: The essential steps of successful gene delivery by recombinant adeno-associated viruses (rAAVs) include vector internalization, intracellular trafficking, nuclear import, uncoating, double-stranded (ds)DNA conversion, and transgene expression. rAAV2.5T has a chimeric capsid of AAV2 VP1u and AAV5 VP2 and VP3 with the mutation A581T. Our investigation revealed that KIAA0319L, the multiple AAV serotype receptor, is not essential for vector internalization but remains critical for efficient vector transduction to human airway epithelia. Additionally, we identified that a novel gene WDR63, whose cellular function is not well understood, plays an important role in vector transduction of human airway epithelia but not vector internalization and nuclear entry. Our study also discovered the substantial transduction potential of rAAV2.5T in basal stem cells of human airway epithelia, underscoring its utility in gene editing of human airways. Thus, the knowledge derived from this study holds promise for the advancement of gene therapy in the treatment of pulmonary genetic diseases.

Inhibition of SARS-CoV-2 infection in human airway epithelium with a xeno-nucleic acid aptamer.

BACKGROUND: SARS-CoV-2, the agent responsible for the COVID-19 pandemic, enters cells through viral spike glycoprotein binding to the cellular receptor, angiotensin-converting enzyme 2 (ACE2). Given the lack of effective antivirals targeting SARS-CoV-2, we previously utilized systematic evolution of ligands by exponential enrichment (SELEX) and selected fluoro-arabino nucleic acid (FANA) aptamer R8-9 that was able to block the interaction between the viral receptor-binding domain and ACE2. METHODS: Here, we further assessed FANA-R8-9 as an entry inhibitor in contexts that recapitulate infection in vivo. RESULTS: We demonstrate that FANA-R8-9 inhibits spike-bearing pseudovirus particle uptake in cell lines. Then, using an in-vitro model of human airway epithelium (HAE) and SARS-CoV-2 virus, we show that FANA-R8-9 significantly reduces viral infection when added either at the time of inoculation, or several hours later. These results were specific to the R8-9 sequence, not the xeno-nucleic acid utilized to make the aptamer. Importantly, we also show that FANA-R8-9 is stable in HAE culture secretions and has no overt cytotoxic effects. CONCLUSIONS: Together, these results suggest that FANA-R8-9 effectively prevents infection by specific SARS-CoV-2 variants and indicate that aptamer technology could be utilized to target other clinically-relevant viruses in the respiratory mucosa.

Inhibition of SARS-CoV-2 Infection in Human Airway Epithelium with a Xeno-Nucleic Acid Aptamer.

Background: SARS-CoV-2, the agent responsible for the COVID-19 pandemic, enters cells through viral spike glycoprotein binding to the cellular receptor, angiotensin-converting enzyme 2 (ACE2). Given the lack of effective antivirals targeting SARS-CoV-2, we previously utilized systematic evolution of ligands by exponential enrichment (SELEX) and selected fluoro-arabino nucleic acid (FANA) aptamer R8-9 that was able to block the interaction between the viral receptor-binding domain and ACE2. Methods: Here, we further assessed FANA-R8-9 as an entry inhibitor in contexts that recapitulate infection in vivo. Results: We demonstrate that FANA-R8-9 inhibits spike-bearing pseudovirus particle uptake in cell lines. Then, using an in-vitro model of human airway epithelium (HAE) and SARS-CoV-2 virus, we show that FANA-R8-9 significantly reduces viral infection when added either at the time of inoculation, or several hours later. These results were specific to the R8-9 sequence, not the xeno-nucleic acid utilized to make the aptamer. Importantly, we also show that FANA-R8-9 is stable in HAE culture secretions and has no overt cytotoxic effects. Conclusions: Together, these results suggest that FANA-R8-9 effectively prevents infection by specific SARS-CoV-2 variants and indicate that aptamer technology could be utilized to target other clinically-relevant viruses in the respiratory mucosa.

Inhibition of DNA-dependent protein kinase catalytic subunit boosts rAAV transduction of polarized human airway epithelium.

Adeno-associated virus 2.5T (AAV2.5T) was selected from the directed evolution of AAV capsid library in human airway epithelia. This study found that recombinant AAV2.5T (rAAV2.5T) transduction of well-differentiated primary human airway epithelia induced a DNA damage response (DDR) characterized by the phosphorylation of replication protein A32 (RPA32), histone variant H2AX (H2A histone family member X), and all three phosphatidylinositol 3-kinase-related kinases: ataxia telangiectasia mutated kinase, ataxia telangiectasia and Rad3-related kinase (ATR), and DNA-dependent protein kinase catalytic subunit (DNA-PKcs). While suppressing the expression of ATR by a specific pharmacological inhibitor or targeted gene silencing inhibited rAAV2.5T transduction, DNA-PKcs inhibition or targeted gene silencing significantly increased rAAV2.5T transgene expression. Notably, DNA-PKcs inhibitors worked as a "booster" to further increase rAAV2.5T transgene expression after treatment with doxorubicin and did not compromise epithelial integrity. Thus, our study provides evidence that DDR is associated with rAAV transduction in well-differentiated human airway epithelia, and DNA-PKcs inhibition has the potential to boost rAAV transduction. These findings highlight that the application of DDR inhibition-associated pharmacological interventions has the potential to increase rAAV transduction and thus to reduce the required vector dose.

SARS-CoV-2 infection of polarized human airway epithelium induces necroptosis that causes airway epithelial barrier dysfunction.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause the ongoing pandemic of coronavirus disease 2019 (COVID19). One key feature associated with COVID-19 is excessive pro-inflammatory cytokine production that leads to severe acute respiratory distress syndrome. Although the cytokine storm induces inflammatory cell death in the host, which type of programmed cell death mechanism that occurs in various organs and cells remains elusive. Using an in vitro culture model of polarized human airway epithelium (HAE), we observed that necroptosis, but not apoptosis or pyroptosis, plays an essential role in the damage of the epithelial barrier of polarized HAE infected with SARS-CoV-2. Pharmacological inhibitors of necroptosis, necrostatin-2 and necrosulfonamide, efficiently prevented cell death and epithelial barrier dysfunction caused by SARS-CoV-2 infection. Moreover, the silencing of genes that are involved in necroptosis, RIPK1, RIPK3, and MLKL, ameliorated airway epithelial damage of the polarized HAE infected with SARS-CoV-2. This study, for the first time, confirms that SARS-CoV-2 infection triggers necroptosis that disrupts the barrier function of human airway epithelia in vitro.

Severe pneumonia and pathogenic damage in human airway epithelium caused by Coxsackievirus B4.

Coxsackievirus B4 (CVB4) has one of the highest proportions of fatal outcomes of other enterovirus serotypes. However, the pathogenesis of severe respiratory disease caused by CVB4 infection remains unclear. In this study, 3 of 42 (7.2%, GZ-R6, GZ-R7 and GZ-R8) patients with severe pneumonia tested positive for CVB4 infection in southern China. Three full-length genomes of pneumonia-derived CVB4 were sequenced and annotated for the first time, showing their high nucleotide similarity and clustering within genotype V. To analyze the pathogenic damage caused by CVB4 in the lungs, a well-differentiated human airway epithelium (HAE) was established and infected with the pneumonia-derived CVB4 isolate GZ-R6. The outcome was compared with that of a severe hand-foot-mouth disease (HFMD)-derived CVB4 strain GZ-HFM01. Compared with HFMD-derived CVB4, pneumonia-derived CVB4 caused more intense and rapid disruption of HAE polarity, leading to tight-junction barrier disruption, loss of cilia, and airway epithelial cell hypertrophy. More pneumonia-derived CVB4 were released from the basolateral side of the HAE than HFMD-derived CVB4. Of the 18 cytokines tested, only IL-6 and IL-1b secretion significantly increased on bilateral sides of HAE during the early stage of pneumonia-derived CVB4 infection, while multiple cytokine secretions significantly increased in HFMD-derived CVB4-infected HAE. HFMD-derived CVB4 exhibited stronger neurovirulence in the human neuroblastoma cells SH-SY5Y than pneumonia-derived CVB4, which is consistent with the clinical manifestations of patients infected with these two viruses. This study has increased the depth of our knowledge of severe pneumonia infection caused by CVB4 and will benefit its prevention and treatment.

Non-Polio Enterovirus C Replicate in Both Airway and Intestine Organotypic Cultures.

Non-polio enteroviruses (EV) belonging to species C, which are highly prevalent in Africa, mainly among children, are poorly characterized, and their pathogenesis is mostly unknown as they are difficult to culture. In this study, human airway and intestinal organotypic models were used to investigate tissue and cellular tropism of three EV-C genotypes, EV-C99, CVA-13, and CVA-20. Clinical isolates were obtained within the two passages of culture on Caco2 cells, and all three viruses were replicated in both the human airway and intestinal organotypic cultures. We did not observe differences in viral replication between fetal and adult tissue that could potentially explain the preferential infection of infants by EV-C genotypes. Infection of the airway and the intestinal cultures indicates that they both can serve as entry sites for non-polio EV-C. Ciliated airway cells and enterocytes are the target of infection for all three viruses, as well as enteroendocrine cells for EV-C99.

A novel type of serum­free medium for culturing human airway epithelium cells.

The present study aimed to evaluate the ability of a novel serum-free medium (SFM) to culture human airway epithelium cells (hAECs). hAECs were cultured in the novel SFM as the experimental group in the PneumaCult-Ex medium and Dulbecco's modified eagle medium (DMEM) and fetal bovine serum (FBS) as the control groups. Cell morphology, proliferative capacity, differentiation capacity and expression levels of basal cell markers were assessed accordingly in both culture systems. Optical microscope photos of hAECs were collected for cell morphology assessment. Cell Counting Kit-8 assay was conducted to assess the proliferation ability, and an air-liquid interface (ALI) assay was conducted to assess the differentiation capacity. Markers for proliferating basal and differentiated cells were relatively identified by immunohistochemical and immunofluorescent analysis. The results show that whether grown in the novel SFM or Ex medium, hAECs exhibited similar morphology at every passage, whereas cells could hardly form colonies in the DMEM + FBS group. Cells typically exhibited cobblestone shape, while a proportion of them in the novel SFM at late passage exhibited a larger shape. White vesicles appeared in the cytoplasm of some control cells at the later stage of culture. Basal cell markers (P63+KRT5+KI67+CC10-) for proliferating ability were found in the hAECs cultured by the novel SFM and Ex medium. hAECs at passage 3 cultured in the novel SFM and Ex medium both had the capacity to differentiate into ciliated cells (acetylated tubulin+), goblet cells (MUC5AC+) and club cells (CC10+) in the ALI culture assay. In conclusion, the novel SFM was capable of culturing hAECs. The hAECs cultured by the novel SFM could proliferate and differentiate in vitro. The novel SFM does not change the morphological characteristics or biomarkers of hAECs. The novel SFM has the potential for the amplification of hAECs for scientific research and clinical application.

Human Airway Basal Cells Undergo Reversible Squamous Differentiation and Reshape Innate Immunity.

Histological and lineage immunofluorescence examination revealed that healthy conducting airways of humans and animals harbor sporadic poorly differentiated epithelial patches mostly in the dorsal noncartilage regions that remarkably manifest squamous differentiation. In vitro analysis demonstrated that this squamous phenotype is not due to intrinsic functional change in underlying airway basal cells. Rather, it is a reversible physiological response to persistent Wnt signaling stimulation during de novo differentiation. Squamous epithelial cells have elevated gene signatures of glucose uptake and cellular glycolysis. Inhibition of glycolysis or a decrease in glucose availability suppresses Wnt-induced squamous epithelial differentiation. Compared with pseudostratified airway epithelial cells, a cascade of mucosal protective functions is impaired in squamous epithelial cells, featuring increased epithelial permeability, spontaneous epithelial unjamming, and enhanced inflammatory responses. Our study raises the possibility that the squamous differentiation naturally occurring in healthy airways identified herein may represent "vulnerable spots" within the airway mucosa that are sensitive to damage and inflammation when confronted by infection or injury. Squamous metaplasia and hyperplasia are hallmarks of many airway diseases, thereby expanding these areas of vulnerability with potential pathological consequences. Thus, investigation of physiological and reversible squamous differentiation from healthy airway basal cells may provide critical knowledge to understand pathogenic squamous remodeling, which is often nonreversible, progressive, and hyperinflammatory.

Influenza A and Respiratory Syncytial Virus Trigger a Cellular Response That Blocks Severe Acute Respiratory Syndrome Virus 2 Infection in the Respiratory Tract.

BACKGROUND: Multiple viruses cocirculate and contribute to the burden of respiratory disease. Virus-virus interactions can decrease susceptibility to infection and this interference can have an epidemiological impact. As humans are normally exposed to a community of cocirculating respiratory viruses, experimental coinfection studies are necessary to understand the disease mechanisms of multipathogen systems. We aimed to characterize interactions within the respiratory tract between severe acute respiratory syndrome virus 2 (SARS-CoV-2) and 2 major respiratory viruses: influenza A virus (IAV), and respiratory syncytial virus (RSV). METHODS: We performed single infections and coinfections with SARS-CoV-2 combined with IAV or RSV in cultures of human bronchial epithelial cells. We combined microscopy with quantification of viral replication in the presence or absence of an innate immune inhibitor to determine changes in virus-induced pathology, virus spread, and virus replication. RESULTS: SARS-CoV-2 replication is inhibited by both IAV and RSV. This inhibition is dependent on a functional antiviral response and the level of inhibition is proportional to the timing of secondary viral infection. CONCLUSIONS: Infections with other respiratory viruses might provide transient resistance to SARS-CoV-2. It would therefore be expected that the incidence of coronavirus disease 2019 (COVID-19) may decrease during periods of high circulation of IAV and RSV.

BPIFA1 is a secreted biomarker of differentiating human airway epithelium.

In vitro culture and differentiation of human-derived airway basal cells under air-liquid interface (ALI) into a pseudostratified mucociliated mucosal barrier has proven to be a powerful preclinical tool to study pathophysiology of respiratory epithelium. As such, identifying differentiation stage-specific biomarkers can help investigators better characterize, standardize, and validate populations of regenerating epithelial cells prior to experimentation. Here, we applied longitudinal transcriptomic analysis and observed that the pattern and the magnitude of OMG, KRT14, STC1, BPIFA1, PLA2G7, TXNIP, S100A7 expression create a unique biosignature that robustly indicates the stage of epithelial cell differentiation. We then validated our findings by quantitative hemi-nested real-time PCR from in vitro cultures sourced from multiple donors. In addition, we demonstrated that at protein-level secretion of BPIFA1 accurately reflects the gene expression profile, with very low quantities present at the time of ALI induction but escalating levels were detectable as the epithelial cells terminally differentiated. Moreover, we observed that increase in BPIFA1 secretion closely correlates with emergence of secretory cells and an anti-inflammatory phenotype as airway epithelial cells undergo mucociliary differentiation under air-liquid interface in vitro.

The interplay between the airway epithelium and tissue macrophages during the SARS-CoV-2 infection.

The first line of antiviral immune response in the lungs is secured by the innate immunity. Several cell types take part in this process, but airway macrophages (AMs) are among the most relevant ones. The AMs can phagocyte infected cells and activate the immune response through antigen presentation and cytokine release. However, the precise role of macrophages in the course of SARS-CoV-2 infection is still largely unknown. In this study, we aimed to evaluate the role of AMs during the SARS-CoV-2 infection using a co-culture of fully differentiated primary human airway epithelium (HAE) and human monocyte-derived macrophages (hMDMs). Our results confirmed abortive SARS-CoV-2 infection in hMDMs, and their inability to transfer the virus to epithelial cells. However, we demonstrated a striking delay in viral replication in the HAEs when hMDMs were added apically after the epithelial infection, but not when added before the inoculation or on the basolateral side of the culture. Moreover, SARS-CoV-2 inhibition by hMDMs seems to be driven by cell-to-cell contact and not by cytokine production. Together, our results show, for the first time, that the recruitment of macrophages may play an important role during the SARS-CoV-2 infection, limiting the virus replication and its spread.

Comprehensive single cell analysis of pandemic influenza A virus infection in the human airways uncovers cell-type specific host transcriptional signatures relevant for disease progression and pathogenesis.

The respiratory epithelium constitutes the first line of defense against invading respiratory pathogens, such as the 2009 pandemic strain of influenza A virus (IAV, H1N1pdm09), and plays a crucial role in the host antiviral response to infection. Despite its importance, however, it remains unknown how individual cell types within the respiratory epithelium respond to IAV infection or how the latter may influence IAV disease progression and pathogenesis. Here, we used single cell RNA sequencing (scRNA-seq) to dissect the host response to IAV infection in its natural target cells. scRNA-seq was performed on human airway epithelial cell (hAEC) cultures infected with either wild-type pandemic IAV (WT) or with a mutant version of IAV (NS1R38A) that induced a robust innate immune response. We then characterized both the host and viral transcriptomes of more than 19,000 single cells across the 5 major cell types populating the human respiratory epithelium. For all cell types, we observed a wide spectrum of viral burden among single infected cells and a disparate host response between infected and bystander populations. Interestingly, we also identified multiple key differences in the host response to IAV among individual cell types, including high levels of pro-inflammatory cytokines and chemokines in secretory and basal cells and an important role for luminal cells in sensing and restricting incoming virus. Multiple infected cell types were shown to upregulate interferons (IFN), with type III IFNs clearly dominating the antiviral response. Transcriptional changes in genes related to cell differentiation, cell migration, and tissue repair were also identified. Strikingly, we also detected a shift in viral host cell tropism from non-ciliated cells to ciliated cells at later stages of infection and observed major changes in the cellular composition. Microscopic analysis of both WT and NS1R38A virus-infected hAECs at various stages of IAV infection revealed that the transcriptional changes we observed at 18 hpi were likely driving the downstream histopathological alterations in the airway epithelium. To our knowledge, this is the first study to provide a comprehensive analysis of the cell type-specific host antiviral response to influenza virus infection in its natural target cells - namely, the human respiratory epithelium.

Microcystin-LR aerosol induces inflammatory responses in healthy human primary airway epithelium.

Harmful algal blooms plague bodies of freshwater globally. These blooms are often composed of outgrowths of cyanobacteria capable of producing the heptapeptide Microcystin-LR (MC-LR) which is a well-known hepatotoxin. Recently, MC-LR has been detected in aerosols generated from lake water. However, the risk for human health effects due to MC-LR inhalation exposure have not been extensively investigated. In this study, we exposed a fully differentiated 3D human airway epithelium derived from 14 healthy donors to MC-LR-containing aerosol once a day for 3 days. Concentrations of MC-LR ranged from 100 pM to 1 µM. Although there were little to no detrimental alterations in measures of the airway epithelial function (i.e. cell survival, tissue integrity, mucociliary clearance, or cilia beating frequency), a distinct shift in the transcriptional activity was found. Genes related to inflammation were found to be upregulated such as C-C motif chemokine 5 (CCL5; log2FC = 0.57, p = 0.03) and C-C chemokine receptor type 7 (CCR7; log2FC = 0.84, p = 0.03). Functionally, conditioned media from MC-LR exposed airway epithelium was also found to have significant chemo-attractive properties for primary human neutrophils. Additionally, increases were found in the concentration of secreted chemokine proteins in the conditioned media such as CCL1 (log2FC = 5.07, p = 0.0001) and CCL5 (log2FC = 1.02, p = 0.046). These results suggest that MC-LR exposure to the human airway epithelium is capable of inducing an inflammatory response that may potentiate acute or chronic disease.

Krüppel-Like Factor 5 Regulates CFTR Expression Through Repression by Maintaining Chromatin Architecture Coupled with Direct Enhancer Activation.

Single cell RNA-sequencing has accurately identified cell types within the human airway that express the Cystic Fibrosis Transmembrane Conductance regulator (CFTR) gene. Low abundance CFTR transcripts are seen in many secretory cells, while high levels are restricted to rare pulmonary ionocytes. Here we focus on the mechanisms coordinating basal CFTR expression in the secretory compartment. Cell-selective regulation of CFTR is achieved within its invariant topologically associating domain by the recruitment of cis-regulatory elements (CREs). CRE activity is coordinated by cell-type-selective transcription factors. One such factor, Krüppel-Like Factor 5 (KLF5), profoundly represses CFTR transcript and protein in primary human airway epithelial cells and airway cell lines. Here we reveal the mechanism of action of KLF5 upon the CFTR gene. We find that depletion or ablation of KLF5 from airway epithelial cells changes higher order chromatin structure at the CFTR locus. Critical looping interactions that are required for normal gene expression are altered, the H3K27ac active chromatin mark is redistributed, and CTCF occupancy is modified. However, mutation of a single KLF5 binding site within a pivotal airway cell CRE abolishes CFTR expression. Hence, KLF5 has both direct activating and indirect repressive effects, which together coordinate CFTR expression in the airway.

Human Airway Epithelium Responses to Invasive Fungal Infections: A Critical Partner in Innate Immunity.

The lung epithelial lining serves as the primary barrier to inhaled environmental toxins, allergens, and invading pathogens. Pulmonary fungal infections are devastating and carry high mortality rates, particularly in those with compromised immune systems. While opportunistic fungi infect primarily immunocompromised individuals, endemic fungi cause disease in immune competent and compromised individuals. Unfortunately, in the case of inhaled fungal pathogens, the airway epithelial host response is vastly understudied. Furthering our lack of understanding, very few studies utilize primary human models displaying pseudostratified layers of various epithelial cell types at air-liquid interface. In this review, we focus on the diversity of the human airway epithelium and discuss the advantages and disadvantages of oncological cell lines, immortalized epithelial cells, and primary epithelial cell models. Additionally, the responses by human respiratory epithelial cells to invading fungal pathogens will be explored. Future investigations leveraging current human in vitro model systems will enable identification of the critical pathways that will inform the development of novel vaccines and therapeutics for pulmonary fungal infections.

Hydroxychloroquine and azithromycin used alone or combined are not effective against SARS-CoV-2 ex vivo and in a hamster model.

Drug repositioning has been used extensively since the beginning of the COVID-19 pandemic in an attempt to identify antiviral molecules for use in human therapeutics. Hydroxychloroquine and azithromycin have shown inhibitory activity against SARS-CoV-2 replication in different cell lines. Based on such in vitro data and despite the weakness of preclinical assessment, many clinical trials were set up using these molecules. In the present study, we show that hydroxychloroquine and azithromycin alone or combined does not block SARS-CoV-2 replication in human bronchial airway epithelia. When tested in a Syrian hamster model, hydroxychloroquine and azithromycin administrated alone or combined displayed no significant effect on viral replication, clinical course of the disease and lung impairments, even at high doses. Hydroxychloroquine quantification in lung tissues confirmed strong exposure to the drug, above in vitro inhibitory concentrations. Overall, this study does not support the use of hydroxychloroquine and azithromycin as antiviral drugs for the treatment of SARS-CoV-2 infections.

Long-Term Modeling of SARS-CoV-2 Infection of In Vitro Cultured Polarized Human Airway Epithelium.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replicates throughout human airways. The polarized human airway epithelium (HAE) cultured at an airway-liquid interface (HAE-ALI) is an in vitro model mimicking the in vivo human mucociliary airway epithelium and supports the replication of SARS-CoV-2. Prior studies characterized only short-period SARS-CoV-2 infection in HAE. In this study, continuously monitoring the SARS-CoV-2 infection in HAE-ALI cultures for a long period of up to 51 days revealed that SARS-CoV-2 infection was long lasting with recurrent replication peaks appearing between an interval of approximately 7 to 10 days, which was consistent in all the tested HAE-ALI cultures derived from 4 lung bronchi of independent donors. We also identified that SARS-CoV-2 does not infect HAE from the basolateral side, and the dominant SARS-CoV-2 permissive epithelial cells are ciliated cells and goblet cells, whereas virus replication in basal cells and club cells was not detected. Notably, virus infection immediately damaged the HAE, which is demonstrated by dispersed zonula occludens-1 (ZO-1) expression without clear tight junctions and partial loss of cilia. Importantly, we identified that SARS-CoV-2 productive infection of HAE requires a high viral load of >2.5 × 105 virions per cm2 of epithelium. Thus, our studies highlight the importance of a high viral load and that epithelial renewal initiates and maintains a recurrent infection of HAE with SARS-CoV-2.IMPORTANCE The pandemic of coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has led to >35 million confirmed cases and >1 million fatalities worldwide. SARS-CoV-2 mainly replicates in human airway epithelia in COVID-19 patients. In this study, we used in vitro cultures of polarized human bronchial airway epithelium to model SARS-CoV-2 replication for a period of 21 to 51 days. We discovered that in vitro airway epithelial cultures endure a long-lasting SARS-CoV-2 propagation with recurrent peaks of progeny virus release at an interval of approximately 7 to 10 days. Our study also revealed that SARS-CoV-2 infection causes airway epithelia damage with disruption of tight junction function and loss of cilia. Importantly, SARS-CoV-2 exhibits a polarity of infection in airway epithelium only from the apical membrane; it infects ciliated and goblet cells but not basal and club cells. Furthermore, the productive infection of SARS-CoV-2 requires a high viral load of over 2.5 × 105 virions per cm2 of epithelium. Our study highlights that the proliferation of airway basal cells and regeneration of airway epithelium may contribute to the recurrent infections.

In Vitro Combinations of Baloxavir Acid and Other Inhibitors against Seasonal Influenza A Viruses.

Two antiviral classes, the neuraminidase inhibitors (NAIs) and polymerase inhibitors (baloxavir marboxil and favipiravir) can be used to prevent and treat influenza infections during seasonal epidemics and pandemics. However, prolonged treatment may lead to the emergence of drug resistance. Therapeutic combinations constitute an alternative to prevent resistance and reduce antiviral doses. Therefore, we evaluated in vitro combinations of baloxavir acid (BXA) and other approved drugs against influenza A(H1N1)pdm09 and A(H3N2) subtypes. The determination of an effective concentration inhibiting virus cytopathic effects by 50% (EC50) for each drug and combination indexes (CIs) were based on cell viability. CompuSyn software was used to determine synergism, additivity or antagonism between drugs. Combinations of BXA and NAIs or favipiravir had synergistic effects on cell viability against the two influenza A subtypes. Those effects were confirmed using a physiological and predictive ex vivo reconstructed human airway epithelium model. On the other hand, the combination of BXA and ribavirin showed mixed results. Overall, BXA stands as a good candidate for combination with several existing drugs, notably oseltamivir and favipiravir, to improve in vitro antiviral activity. These results should be considered for further animal and clinical evaluations.

Human Bocavirus 1 Infection of Well-Differentiated Human Airway Epithelium.

Human bocavirus 1 (HBoV1) is a small DNA virus that belongs to the Bocaparvovirus genus of the Parvoviridae family. HBoV1 is a common respiratory pathogen that causes mild to life-threatening acute respiratory tract infections in children and immunocompromised individuals, infecting both the upper and lower respiratory tracts. HBoV1 infection causes death of airway epithelial cells, resulting in airway injury and inflammation. In vitro, HBoV1 only infects well-differentiated (polarized) human airway epithelium cultured at an air-liquid interface (HAE-ALI), but not any dividing human cells. A full-length HBoV1 genome of 5543 nucleotides has been cloned from DNA extracted from a human nasopharyngeal swab into a plasmid called HBoV1 infectious clone pIHBoV1. Transfection of pIHBoV1 replicates efficiently in human embryonic kidney 293 (HEK293) cells and produces virions that are highly infectious. This article describes protocols for production of HBoV1 in HEK293 cells, generation of HAE-ALI cultures, and infection with HBoV1 in HAE-ALI. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Human bocavirus 1 production in HEK293 cells Support Protocol 1: HEK293 cell culture and transfection Support Protocol 2: Quantification of human bocavirus 1 using real-time quantitative PCR Basic Protocol 2: Differentiation of human airway cells at an air-liquid interface Support Protocol 3: Expansion of human airway epithelial cell line CuFi-8 Support Protocol 4: Expansion of human airway basal cells Support Protocol 5: Coating of plastic dishes and permeable membranes of inserts Support Protocol 6: Transepithelial electrical resistance measurement Basic Protocol 3: Human bocavirus 1 infection in human airway epithelium cultured at an air-liquid interface Support Protocol 7: Isolation of infected human airway epithelium cells from inserts Basic Protocol 4: Transduction of airway basal cells with lentiviral vector.