Heparin Inhibits SARS-CoV-2 Replication in Human Nasal Epithelial Cells

SARS-CoV-2 is the causative agent of the COVID-19 pandemic. Vaccination, supported by social and public health measures, has proven efficacious for reducing disease severity and virus spread. However, the emergence of highly transmissible viral variants that escape prior immunity highlights the need for additional mitigation approaches. Heparin binds the SARS-CoV-2 spike protein and can inhibit virus entry and replication in susceptible human cell lines and bronchial epithelial cells. Primary infection predominantly occurs via the nasal epithelium, but the nasal cell biology of SARS-CoV-2 is not well studied. We hypothesized that prophylactic intranasal administration of heparin may provide strain-agnostic protection for household contacts or those in high-risk settings against SARS-CoV-2 infection. Therefore, we investigated the ability of heparin to inhibit SARS-CoV-2 infection and replication in differentiated human nasal epithelial cells and showed that prolonged exposure to heparin inhibits virus infection. Furthermore, we establish a method for PCR detection of SARS-CoV-2 viral genomes in heparin-treated samples that can be adapted for the detection of viruses in clinical studies.     

SARS-CoV-2 Virion Infectivity and Cytokine Production in Primary Human Airway Epithelial Cells

The emergence of new SARS-CoV-2 variants and the replacement of preceding isolates have been observed through B.1.1.7, B.1.351, B.1.617.2, and B.1.1.529 lineages (corresponding to alpha, beta, delta, and omicron variants of concern (VoC), respectively). However, there is still a lack of biological evidence to which extent those VoC differ from the ancestral lineages. By exploiting human airway epithelial cell (HAEC) cultures, which closely resemble the human airway architecture and physiology, we report distinctive SARS-CoV-2 tropism in different respiratory tissues. In general, SARS-CoV-2 VoC predominantly infect and replicate in HAEC better than the progenitor USA-WA1 isolate or the BavPat1 isolate, which contains the D614G mutation, even though there is little to no difference between variants regarding their infectivity (i.e., virion-per-vRNA copy ratio). We also observe differential tissue-specific innate immunity activation between the upper and lower respiratory tissues in the presence of the virus. Our study provides better comprehension of the behavior of the different VoC in this physiologically relevant ex vivo model.     

SARS-CoV-2 Spike Expression at the Surface of Infected Primary Human Airway Epithelial Cells

Different serological assays were rapidly generated to study humoral responses against the SARS-CoV-2 Spike glycoprotein. Due to the intrinsic difficulty of working with SARS-CoV-2 authentic virus, most serological assays use recombinant forms of the Spike glycoprotein or its receptor binding domain (RBD). Cell-based assays expressing different forms of the Spike, as well as pseudoviral assays, are also widely used. To evaluate whether these assays recapitulate findings generated when the Spike is expressed in its physiological context (at the surface of the infected primary cells), we developed an intracellular staining against the SARS-CoV-2 nucleocapsid (N) to distinguish infected from uninfected cells. Human airway epithelial cells (pAECs) were infected with authentic SARS-CoV-2 D614G or Alpha variants. We observed robust cell-surface expression of the SARS-CoV-2 Spike at the surface of the infected pAECs using the conformational-independent anti-S2 CV3-25 antibody. The infected cells were also readily recognized by plasma from convalescent and vaccinated individuals and correlated with several serological assays. This suggests that the antigenicity of the Spike present at the surface of the infected primary cells is maintained in serological assays involving expression of the native full-length Spike.     

pEGFP-C3/SUMF2重组真核表达载体的构建及在人支气管上皮细胞中的表达

目的构建与增强型绿色荧光蛋白（enhanced green fluorescent protein,EGFP）融合的人硫酸酯酶修饰因子2（SUMF2）真核表达载体。方法用RT-PCR技术从人支气管上皮细胞（human bronchial epithelial cells,HBEC）获得SUMF2的cDNA模板,PCR方法扩增人SUMF2基因。用EcoRⅠ和BamHⅠ双酶切扩增人SUMF2基因及质粒pEGFP-C3;将2种酶切产物按常规方法连接、转化大肠杆菌Top10;挑取菌落培养,提取质粒,酶切鉴定,测序;将测序正确的重组载体用脂质体法转染HBEC,荧光倒置显微镜观察。提取转染细胞的总蛋白进行Western blot检测。结果扩增出1条约857bp的片段,与预期的SUMF2大小相符。酶切结果显示重组质粒pEGFP-C3/SUMF2被切成2条片段,其中1条为pEGFP-C3载体,另1条为目的片段。经测序鉴定,序列与GenBank（NM₀15411.2）中的序列高度同源。荧光倒置显微镜观察显示,人SUMF2基因主要在内质网表达。Western blot结果显示在相对分子质量为37×103处有一蛋白条带,与预期大小相符。结论成功构建重组表达载体pEGFP-C3/SUMF2,为进一步研究SUMF2对IL-13的作用奠定了实验基础。     

Berberine and Obatoclax Inhibit SARS-Cov-2 Replication in Primary Human Nasal Epithelial Cells In Vitro

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged as a new human pathogen in late 2019 and it has infected over 100 million people in less than a year. There is a clear need for effective antiviral drugs to complement current preventive measures, including vaccines. In this study, we demonstrate that berberine and obatoclax, two broad-spectrum antiviral compounds, are effective against multiple isolates of SARS-CoV-2. Berberine, a plant-derived alkaloid, inhibited SARS-CoV-2 at low micromolar concentrations and obatoclax, which was originally developed as an anti-apoptotic protein antagonist, was effective at sub-micromolar concentrations. Time-of-addition studies indicated that berberine acts on the late stage of the viral life cycle. In agreement, berberine mildly affected viral RNA synthesis, but it strongly reduced infectious viral titers, leading to an increase in the particle-to-pfu ratio. In contrast, obatoclax acted at the early stage of the infection, which is in line with its activity to neutralize the acidic environment in endosomes. We assessed infection of primary human nasal epithelial cells that were cultured on an air-liquid interface and found that SARS-CoV-2 infection induced and repressed expression of specific sets of cytokines and chemokines. Moreover, both obatoclax and berberine inhibited SARS-CoV-2 replication in these primary target cells. We propose berberine and obatoclax as potential antiviral drugs against SARS-CoV-2 that could be considered for further efficacy testing.     

Comparison of SARS-CoV-2 Evolution in Paediatric Primary Airway Epithelial Cell Cultures Compared with Vero-Derived Cell Lines

SARS-CoV-2 can efficiently infect both children and adults, albeit with morbidity and mortality positively associated with increasing host age and presence of co-morbidities. SARS-CoV-2 continues to adapt to the human population, resulting in several variants of concern (VOC) with novel properties, such as Alpha and Delta. However, factors driving SARS-CoV-2 fitness and evolution in paediatric cohorts remain poorly explored. Here, we provide evidence that both viral and host factors co-operate to shape SARS-CoV-2 genotypic and phenotypic change in primary airway cell cultures derived from children. Through viral whole-genome sequencing, we explored changes in genetic diversity over time of two pre-VOC clinical isolates of SARS-CoV-2 during passage in paediatric well-differentiated primary nasal epithelial cell (WD-PNEC) cultures and in parallel, in unmodified Vero-derived cell lines. We identified a consistent, rich genetic diversity arising in vitro, variants of which could rapidly rise to near fixation within two passages. Within isolates, SARS-CoV-2 evolution was dependent on host cells, with paediatric WD-PNECs showing a reduced diversity compared to Vero (E6) cells. However, mutations were not shared between strains. Furthermore, comparison of both Vero-grown isolates on WD-PNECs disclosed marked growth attenuation mapping to the loss of the polybasic cleavage site (PBCS) in Spike, while the strain with mutations in Nsp12 (T293I), Spike (P812R) and a truncation of Orf7a remained viable in WD-PNECs. Altogether, our work demonstrates that pre-VOC SARS-CoV-2 efficiently infects paediatric respiratory epithelial cells, and its evolution is restrained compared to Vero (E6) cells, similar to the case of adult cells. We highlight the significant genetic plasticity of SARS-CoV-2 while uncovering an influential role for collaboration between viral and host cell factors in shaping viral evolution and ultimately fitness in human respiratory epithelium.     

Emerging SARS-CoV-2 Genotypes Show Different Replication Patterns in Human Pulmonary and Intestinal Epithelial Cells

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) quickly spread worldwide following its emergence in Wuhan, China, and hit pandemic levels. Its tremendous incidence favoured the emergence of viral variants. The current genome diversity of SARS-CoV-2 has a clear impact on epidemiology and clinical practice, especially regarding transmission rates and the effectiveness of vaccines. In this study, we evaluated the replication of different SARS-CoV-2 isolates representing different virus genotypes which have been isolated throughout the pandemic. We used three distinct cell lines, including Vero E6 cells originating from monkeys; Caco-2 cells, an intestinal epithelium cell line originating from humans; and Calu-3 cells, a pulmonary epithelium cell line also originating from humans. We used RT-qPCR to replicate different SARS-CoV-2 genotypes by quantifying the virus released in the culture supernatant of infected cells. We found that the different viral isolates replicate similarly in Caco-2 cells, but show very different replicative capacities in Calu-3 cells. This was especially highlighted for the lineages B.1.1.7, B.1.351 and P.1, which are considered to be variants of concern. These results underscore the importance of the evaluation and characterisation of each SARS-CoV-2 isolate in order to establish the replication patterns before performing tests, and of the consideration of the ideal SARS-CoV-2 genotype–cell type pair for each assay.     

Chloroquine and Sulfadoxine Derivatives Inhibit ZIKV Replication in Cervical Cells

Despite the severe morbidity caused by Zika fever, its specific treatment is still a challenge for public health. Several research groups have investigated the drug repurposing of chloroquine. However, the highly toxic side effect induced by chloroquine paves the way for the improvement of this drug for use in Zika fever clinics. Our aim is to evaluate the anti-Zika virus (ZIKV) effect of hybrid compounds derived from chloroquine and sulfadoxine antimalarial drugs. The antiviral activity of hybrid compounds (C-Sd1 to C-Sd7) was assessed in an in-vitro model of human cervical and Vero cell lines infected with a Brazilian (BR) ZIKV strain. First, we evaluated the cytotoxic effect on cultures treated with up to 200 µM of C-Sds and observed CC₅₀ values that ranged from 112.0 ± 1.8 to >200 µM in cervical cells and 43.2 ± 0.4 to 143.0 ± 1.3 µM in Vero cells. Then, the cultures were ZIKV-infected and treated with up to 25 µM of C-Sds for 48 h. The treatment of cervical cells with C-Sds at 12 µM induced a reduction of 79.8% ± 4.2% to 90.7% ± 1.5% of ZIKV–envelope glycoprotein expression in infected cells as compared to 36.8% ± 2.9% of infection in vehicle control. The viral load was also investigated and revealed a reduction of 2- to 3-logs of ZIKV genome copies/mL in culture supernatants compared to 6.7 ± 0.7 × 10⁸ copies/mL in vehicle control. The dose–response curve by plaque-forming reduction (PFR) in cervical cells revealed a potent dose-dependent activity of C-Sds in inhibiting ZIKV replication, with PFR above 50% and 90% at 6 and 12 µM, respectively, while 25 µM inhibited 100% of viral progeny. The treatment of Vero cells at 12 µM led to 100% PFR, confirming the C-Sds activity in another cell type. Regarding effective concentration in cervical cells, the EC₅₀ values ranged from 3.2 ± 0.1 to 5.0 ± 0.2 µM, and the EC₉₀ values ranged from 7.2 ± 0.1 to 11.6 ± 0.1 µM, with selectivity index above 40 for most C-Sds, showing a good therapeutic window. Here, our aim is to investigate the anti-ZIKV activity of new hybrid compounds that show highly potent efficacy as inhibitors of ZIKV in-vitro infection. However, further studies will be needed to investigate whether these new chemical structures can lead to the improvement of chloroquine antiviral activity.     

Ferristatin II Efficiently Inhibits SARS-CoV-2 Replication in Vero Cells

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to have a significant impact on global public health. Multiple mechanisms for SARS-CoV-2 cell entry have been described; however, the role of transferrin receptor 1 (TfR1) in SARS-CoV-2 infection has received little attention. We used ferristatin II to induce the degradation of TfR1 on the surface of Vero cells and to study the consequences of such treatment on the viability of the cells and the replication of SARS-CoV-2. We demonstrated that ferristatin II is non-toxic for Vero cells in concentrations up to 400 µM. According to confocal microscopy data, the distribution of the labeled transferrin and receptor-binding domain (RBD) of Spike protein is significantly affected by the 18h pretreatment with 100 µM ferristatin II in culture medium. The uptake of RBD protein is nearly fully inhibited by ferristatin II treatment, although this protein remains bound on the cell surface. The findings were well confirmed by the significant inhibition of the SARS-CoV-2 infection of Vero cells by ferristatin II with IC₅₀ values of 27 µM (for Wuhan D614G virus) and 40 µM (for Delta virus). A significant reduction in the infectious titer of the Omicron SARS-CoV-2 variant was noted at a ferristatin II concentration as low as 6.25 µM. We hypothesize that ferristatin II blocks the TfR1-mediated SARS-CoV-2 host cell entry; however, further studies are needed to elucidate the full mechanisms of this virus inhibition, including the effect of ferristatin II on other SARS-CoV-2 receptors, such as ACE2, Neuropilin-1 and CD147. The inhibition of viral entry by targeting the receptor on the host cells, rather than the viral mutation-prone protein, is a promising COVID-19 therapeutic strategy.     

The Spike Glycoprotein of SARS-CoV-2 Binds to β1 Integrins Expressed on the Surface of Lung Epithelial Cells

The spike glycoprotein attached to the envelope of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) binds to and exploits angiotensin-converting enzyme 2 (ACE2) as an entry receptor to infect pulmonary epithelial cells. A subset of integrins that recognize the arginyl–glycyl–aspartic acid (RGD) sequence in the cognate ligands has been predicted in silico to bind the spike glycoprotein and, thereby, to be exploited for viral infection. Here, we show experimental evidence that the β1 integrins predominantly expressed on human pulmonary epithelial cell lines and primary mouse alveolar epithelial cells bind to this spike protein. The cellular β1 integrins support adhesive interactions with the spike protein independently of ACE2, suggesting the possibility that the β1 integrins may function as an alternative receptor for SARS-CoV-2, which could be targeted for the prevention of viral infections.     

Molecular Genetics and Cytotoxic Responses to Titanium Diboride and Zinc Borate Nanoparticles on Cultured Human Primary Alveolar Epithelial Cells

Titanium diboride (TiB₂) and zinc borate (Zn₃BO₆) have been utilized in wide spectrum industrial areas because of their favorable properties such as a high melting point, good wear resistance, high hardness and thermal conductivity. On the other hand, the biomedical potentials of TiB₂ and Zn₃BO₆ are still unknown because there is no comprehensive analysis that uncovers their biocompatibility features. Thus, the toxicogenomic properties of TiB₂ and Zn₃BO₆ nanoparticles (NPs) were investigated on human primary alveolar epithelial cell cultures (HPAEpiC) by using different cell viability assays and microarray analyses. Protein-Protein Interaction Networks Functional Enrichment Analysis (STRING) was used to associate differentially expressed gene probes. According to the results, up to 10 mg/L concentration of TiB₂ and Zn₃BO₆ NPs application did not stimulate a cytotoxic effect on the HPAEpiC cell cultures. Microarray analysis revealed that TiB₂ NPs exposure enhances cellular adhesion molecules, proteases and carrier protein expression. Furthermore, Zn₃BO₆ NPs caused differential gene expressions in the cell cycle, cell division and extracellular matrix regulators. Finally, STRING analyses put forth that inflammation, cell regeneration and tissue repair-related gene interactions were affected by TiB₂ NPs application. Zn₃BO₆ NPs exposure significantly altered inflammation, lipid metabolism and infection response activator-related gene interactions. These investigations illustrated that TiB₂ and Zn₃BO₆ NPs exposure may affect different aspects of cellular machineries such as immunogenic responses, tissue regeneration and cell survival. Thus, these types of cellular mechanisms should be taken into account before the use of the related NPs in further biomedical applications.     

Repurposing Probenecid to Inhibit SARS-CoV-2, Influenza Virus,and Respiratory Syncytial Virus (RSV) Replication

Viral replication and transmissibility are the principal causes of endemic and pandemic disease threats. There remains a need for broad-spectrum antiviral agents. The most common respiratory viruses are endemic agents such as coronaviruses, respiratory syncytial viruses, and influenza viruses. Although vaccines are available for SARS-CoV-2 and some influenza viruses, there is a paucity of effective antiviral drugs, while for RSV there is no vaccine available, and therapeutic treatments are very limited. We have previously shown that probenecid is safe and effective in limiting influenza A virus replication and SARS-CoV-2 replication, along with strong evidence showing inhibition of RSV replication in vitro and in vivo. This review article will describe the antiviral activity profile of probenecid against these three viruses.     

Characterization of the SARS-CoV-2 Host Response in Primary Human Airway Epithelial Cells from Aged Individuals

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19), a global pandemic characterized by an exaggerated immune response and respiratory illness. Age (>60 years) is a significant risk factor for developing severe COVID-19. To better understand the host response of the aged airway epithelium to SARS-CoV-2 infection, we performed an in vitro study using primary human bronchial epithelial cells from donors >67 years of age differentiated on an air–liquid interface culture. We demonstrate that SARS-CoV-2 infection leads to early induction of a proinflammatory response and a delayed interferon response. In addition, we observed changes in the genes and pathways associated with cell death and senescence throughout infection. In summary, our study provides new and important insights into the temporal kinetics of the airway epithelial innate immune response to SARS-CoV-2 in older individuals.     

Influenza A(H1N1)pdm09 Virus but Not Respiratory Syncytial Virus Interferes with SARS-CoV-2 Replication during Sequential Infections in Human Nasal Epithelial Cells

The types of interactions between severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other respiratory viruses are not well-characterized due to the low number of co-infection cases described since the onset of the pandemic. We have evaluated the interactions between SARS-CoV-2 (D614G mutant) and influenza A(H1N1)pdm09 or respiratory syncytial virus (RSV) in the nasal human airway epithelium (HAE) infected simultaneously or sequentially (24 h apart) with virus combinations. The replication kinetics of each virus were determined by RT-qPCR at different post-infection times. Our results showed that during simultaneous infection, SARS-CoV-2 interferes with RSV-A2 but not with A(H1N1)pdm09 replication. The prior infection of nasal HAE with SARS-CoV-2 reduces the replication kinetics of both respiratory viruses. SARS-CoV-2 replication is decreased by a prior infection with A(H1N1)pdm09 but not with RSV-A2. The pretreatment of nasal HAE with BX795, a TANK-binding kinase 1 inhibitor, partially alleviates the reduced replication of SARS-CoV-2 or influenza A(H1N1)pdm09 during sequential infection with both virus combinations. Thus, a prior infection of nasal HAE with SARS-CoV-2 interferes with the replication kinetics of A(H1N1)pdm09 and RSV-A2, whereas only A(H1N1)pdm09 reduces the subsequent infection with SARS-CoV-2. The mechanism involved in the viral interference between SARS-CoV-2 and A(H1N1)pdm09 is mediated by the production of interferon.     

Nafamostat–Interferon-α Combination Suppresses SARS-CoV-2 Infection In Vitro and In Vivo by Cooperatively Targeting Host TMPRSS2

SARS-CoV-2 and its vaccine/immune-escaping variants continue to pose a serious threat to public health due to a paucity of effective, rapidly deployable, and widely available treatments. Here, we address these challenges by combining Pegasys (IFNα) and nafamostat to effectively suppress SARS-CoV-2 infection in cell culture and hamsters. Our results indicate that Serpin E1 is an important mediator of the antiviral activity of IFNα and that both Serpin E1 and nafamostat can target the same cellular factor TMPRSS2, which plays a critical role in viral replication. The low doses of the drugs in combination may have several clinical advantages, including fewer adverse events and improved patient outcome. Thus, our study may provide a proactive solution for the ongoing pandemic and potential future coronavirus outbreaks, which is still urgently required in many parts of the world.     

Primary SARS-CoV-2 Infections,Re-infections and Vaccine Effectiveness during the Omicron Transmission Period in Healthcare Workers of Trieste and Gorizia (Northeast Italy), 1 December 2021–31 May 2022

Objective: To evaluate the incidence of primary and recurrent COVID-19 infections in healthcare workers (HCWs) routinely screened for SARS-CoV-2 by nasopharyngeal swabs during the Omicron wave. Design: Dynamic Cohort study of HCWs (N = 7723) of the University Health Agency Giuliano Isontina (ASUGI), covering health services of the provinces of Trieste and Gorizia (Northeast Italy). Cox proportional hazard model was employed to estimate the risk of primary as well as recurrent SARS-CoV-2 infection from 1 December 2021 through 31 May 2022, adjusting for a number of confounding factors. Results: By 1 December 2021, 46.8% HCWs of ASUGI had received the booster, 37.2% were immunized only with two doses of COVID-19 vaccines, 6.0% only with one dose and 10.0% were unvaccinated. During 1 March 2020–31 May 2022, 3571 primary against 406 SARS-CoV-2 recurrent infections were counted among HCWs of ASUGI, 59.7% (=2130/3571) versus 95.1% (=386/406) of which occurring from 1 December 2021 through 31 May 2022, respectively. All HCWs infected by SARS-CoV-2 during 1 December 2021 through 31 May 2022 presented mild flu-like disease. Compared to staff working in administrative services, the risk of primary as well as recurrent SARS-CoV-2 infection increased in HCWs with patient-facing clinical tasks (especially nurses and other categories of HCWs) and in all clinical wards but COVID-19 units and community health services. Regardless of the number of swab tests performed during the study period, primary infections were less likely in HCWs immunized with one dose of COVID-19 vaccine. By contrast, the risk of SARS-CoV-2 re-infection was significantly lower in HCWs immunized with three doses (aHR = 0.58; 95%CI: 0.41; 0.80). During the study period, vaccine effectiveness (VE = 1-aHR) of the booster dose declined to 42% against re-infections, vanishing against primary SARS-CoV-2 infections. Conclusions: Though generally mild, SARS-CoV-2 infections and re-infections surged during the Omicron transmission period. Compared to unvaccinated colleagues, the risk of primary SARS-CoV-2 infection was significantly lower in HCWs immunized just with one dose of COVID-19 vaccines. By Italian law, HCWs immunized only with one dose were either suspended or re-assigned to job tasks not entailing patient facing contact; hence, while sharing the same biological risk of unvaccinated colleagues, they arguably had a higher level of protection against COVID-19 infection. By contrast, SARS-CoV-2 re-infections were less likely in HCWs vaccinated with three doses, suggesting that hybrid humoral immunity by vaccination combined with natural infection provided a higher level of protection than vaccination only. In this stage of the pandemic, where SARS-CoV-2 is more infectious yet much less pathogenic, health protection measures in healthcare premises at higher biological risk seem the rational approach to control the transmission of the virus.     

SARS-CoV-2 infection of airway cells causes intense viral and cell shedding,two spreading mechanisms affected by IL-13

Muco-obstructive lung diseases are typically associated with high risks of COVID-19 severity; however, allergic asthma showed reduced susceptibility. To investigate viral spread, primary human airway epithelial (HAE) cell cultures were infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and host–virus interactions were examined via electron microscopy, immunohistochemistry, RNA in situ hybridization, and gene expression analyses. In HAE cell cultures, angiotensin-converting enzyme 2 (ACE2) expression governed cell tropism and viral load and was up-regulated by infection. Electron microscopy identified intense viral egress from infected ciliated cells and severe cytopathogenesis, culminating in the shedding of ciliated cells packed with virions, providing a large viral reservoir for spread and transmission. Intracellular stores of MUC5AC, a major airway mucin involved in asthma, were rapidly depleted, likely to trap viruses. To mimic asthmatic airways, HAE cells were treated with interleukin-13 (IL-13), which reduced viral titers, viral messenger RNA, and cell shedding, and significantly diminished the number of infected cells. Although mucus hyperproduction played a shielding role, IL-13–treated cells maintained a degree of protection despite the removal of mucus. Using Gene Expression Omnibus databases, bulk RNA-sequencing analyses revealed that IL-13 up-regulated genes controlling glycoprotein synthesis, ion transport, and antiviral processes (albeit not the typical interferon-induced genes) and down-regulated genes involved in cilial function and ribosomal processing. More precisely, we showed that IL-13 reduced ACE2 expression, intracellular viral load, and cell-to-cell transmission while increasing the cilial keratan sulfate coating. In conclusion, intense viral and cell shedding caused by SARS-CoV-2 infection was attenuated by IL-13, which affected viral entry, replication, and spread.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus causing COVID-19, is the third coronavirus outbreak affecting the human population since the beginning of the 21st century and was preceded by SARS-CoV in 2002 and Middle East respiratory syndrome (MERS)–CoV in 2012 (1–3). More than 2 y after the initial outbreak, SARS-CoV-2 continues to be actively transmitted in the human population and has already killed >5 million people. The clinical symptoms of COVID-19 vary greatly between infected individuals, ranging from asymptomatic disease to severe respiratory illness and even death in nearly 2% of cases (4, 5). Despite similarities in transmission, infection, and clinical symptoms with other human coronaviruses, the elevated transmissibility and global burden posed by SARS-CoV-2 warrant further investigation into its tropism, pathogenesis, and spreading mechanism.SARS-CoV-2 utilizes the angiotensin-converting enzyme 2 (ACE2) receptor and a transmembrane serine protease (TMPRSS2) to enter host cells, and both proteins are coexpressed in respiratory epithelia (6, 7). ACE2 expression has been detected in ciliated and goblet cells, two important cell types involved in airway clearance (8, 9). Using human airway epithelial (HAE) cell models, we have shown that SARS-CoV-2 efficiently infected cells from the upper airways, suggesting that the nose and large airways are preferred sites for viral transmission and replication (10, 11). Furthermore, scanning electron microscopy (SEM) confirmed direct interactions between SARS-CoV-2 viruses and ciliated epithelial cells (12).In the lungs, ciliated cells coordinate the movement of secretions to clear inhaled particles, a process referred to as mucociliary transport (MCT). Thus, viral infections targeting this particular cell type can affect airway clearance. Tropism for ciliated cells has been reported for other respiratory viruses (e.g., respiratory syncytial virus, influenza, and other coronaviruses), which was associated with cell shedding (13–15). Another important player in viral infection is the glycocalyx coating the periciliary (PCL) region to provide a barrier while facilitating cilia beating (16, 17). In response to infection, goblet cells increase the secretion of mucins, the large polymeric glycoproteins responsible for the viscoelastic properties of mucus (18, 19). MUC5AC, a major gel-forming mucin expressed in the lungs, is secreted in small amounts in healthy individuals and is up-regulated during respiratory infections, suggesting a protective role against pathogens (20–23). In diseases such as chronic obstructive pulmonary disease (COPD) and asthma, MUC5AC is up-regulated, and has been associated with reduced MCT (24–26). COPD patients infected with SARS-CoV-2 are at higher risk of severe clinical outcomes, but, for asthmatic patients, the data are more contradictory (27–31). The variability within the asthmatic population may originate from pathophysiologic differences based on Th2-low (nonallergic) and Th2-high (allergic) inflammatory profiles, with allergic asthma being protective (32, 33). IL-13, a type 2 cytokine associated with allergic asthma, has been shown to increase MUC5AC secretion (34, 35) and down-regulate ACE2 expression (30, 36), and the effects of IL-13 on SARS-CoV-2 infection are only beginning to be explored (37).In this study, we infected HAE cell cultures with SARS-CoV-2 and examined the fate of infected airway cells using fluorescent labeling, combined with SEM and transmission electron microscopy (TEM). These approaches characterized the fate of infected cells by examining the mode of virion cellular release and the detachment of infected cells. In addition, we investigated whether IL-13 protects against viral spread. The effects of IL-13 on airway cultures were determined by measuring viral titers, viral gene replication, and epithelial cell damage. Mucus hyperproduction provided a physical barrier; however, after removing mucus either by cell washing or using MUC5AC knockout (KO) cells, lower viral loads were maintained in IL-13–treated cells. To investigate how IL-13 modified host antiviral responses, the effects of IL-13 administration on gene expression in HAE cultures were analyzed from three independent bulk RNA-sequencing studies available on the Gene Expression Omnibus (GEO) database. These analyses revealed that critical processes for viral entry, replication, and spread, pertinent to asthma and COVID-19 severity, were affected by IL-13 treatment.     

Enrichment of SARS-CoV-2 Entry Factors and Interacting Intracellular Genes in Tissue and Circulating Immune Cells

SARS-CoV-2 uses ACE2 and TMPRSS2 to gain entry into the cell. However, recent studies have shown that SARS-CoV-2 may use additional host factors that are required for the viral lifecycle. Here we used publicly available datasets, CoV-associated genes, and machine learning algorithms to explore the SARS-CoV-2 interaction landscape in different tissues. We found that in general a small fraction of cells express ACE2 in the different tissues, including nasal, bronchi, and lungs. We show that a small fraction of immune cells (including T cells, macrophages, dendritic cells) found in tissues also express ACE2. We show that healthy circulating immune cells do not express ACE2 and TMPRSS2. However, a small fraction of circulating immune cells (including dendritic cells, monocytes, T cells) in the PBMC of COVID-19 patients express ACE2 and TMPRSS2. Additionally, we found that a large spectrum of cells (in tissues and circulation) in both healthy and COVID-19-positive patients were significantly enriched for SARS-CoV-2 factors, such as those associated with RHOA and RAB GTPases, mRNA translation proteins, COPI- and COPII-mediated transport, and integrins. Thus, we propose that further research is needed to explore if SARS-CoV-2 can directly infect tissue and circulating immune cells to better understand the virus’ mechanism of action.