Phosphatidylserine vesicles enable efficient en bloc transmission of enteroviruses.

A central paradigm within virology is that each viral particle largely behaves as an independent infectious unit. Here, we demonstrate that clusters of enteroviral particles are packaged within phosphatidylserine (PS) lipid-enriched vesicles that are non-lytically released from cells and provide greater infection efficiency than free single viral particles. We show that vesicular PS lipids are co-factors to the relevant enterovirus receptors in mediating subsequent infectivity and transmission, in particular to primary human macrophages. We demonstrate that clustered packaging of viral particles within vesicles enables multiple viral RNA genomes to be collectively transferred into single cells. This study reveals a novel mode of viral transmission, where enteroviral genomes are transmitted from cell-to-cell en bloc in membrane-bound PS vesicles instead of as single independent genomes. This has implications for facilitating genetic cooperativity among viral quasispecies as well as enhancing viral replication.

Human rhinovirus 16 induces an ICAM-1-PKR-ATF2 axis to modulate macrophage functions.

Human rhinovirus (HRV) infections are the leading cause of disease exacerbations in individuals with chronic pulmonary diseases, primarily due to impaired macrophage functions, resulting in defective bacterial elimination. We previously demonstrated that HRV16 impairs macrophages' functions in an ARL5b-dependent manner. In permissive cells, ARL5b acted as an HRV16 restriction factor and was repressed. Here, we delve into the dual regulation of ARL5b by HRV16 in both cell types. We analyzed the effect of HRV16 on primary human macrophages using neutralizing antibodies, specific inhibitors, siRNA, and chromatin immune precipitation. Our study reveals that, while the virus does not replicate in macrophages, it induces interferon and pro-inflammatory responses. We identify the ICAM-1-PKR-ATF2 signaling axis as crucial for ARL5b induction in macrophages, whereas only ICAM-1 plays a role in ARL5b repression in permissive cells. Furthermore, HRV16 triggers epigenetic reprogramming in both cell types at the ARL5b promoter. In macrophages, epigenetic changes are ATF2 dependent. In conclusion, our findings highlight previously unknown signaling pathways activated by HRV16 in macrophages. Targeting these pathways could offer novel strategies to improve outcomes for individuals with respiratory conditions. IMPORTANCE: Human rhinovirus (HRV) infections are the leading cause of disease exacerbations in individuals with chronic pulmonary conditions and are frequently associated with bacterial superinfections due to defective bacterial elimination by macrophages. We previously identified ARL5b-induction by HRV16 to be responsible for the impairment of bacteria elimination. In contrast, in permissive cells, ARL5b is repressed and acts as a restriction factor for HRV16. Here, we investigated the dual regulation of ARL5b by HRV16 in these cells. Our study reveals that the ICAM-1-PKR-ATF2 signaling axis is crucial for ARL5b induction in macrophages. In permissive cells, only ICAM-1 plays a role in ARL5b repression. Moreover, HRV16 triggered epigenetic reprogramming in macrophages. ARL5b promoter was repressed in an ATF2-dependent manner. Collectively, our findings reveal previously unknown signaling pathways activated by HRV16 in macrophages. Targeting these pathways provides novel strategies to target ARL5b expression specifically in macrophages and improve outcomes for individuals with respiratory pathologies.

Developing a mouse model of human coronavirus NL63 infection: comparison with rhinovirus-A1B and effects of prior rhinovirus infection.

Human coronavirus (HCoV)-NL63 causes respiratory tract infections in humans and uses angiotensin-converting enzyme 2 (ACE2) as a receptor. We sought to establish a mouse model of HCoV-NL63 and determine whether prior rhinovirus (RV)-A1B infection affected HCoV-NL63 replication. HCoV-NL63 was propagated in LLC-MK2 cells expressing human ACE2. RV-A1B was grown in HeLa-H1 cells. C57BL6/J or transgenic mice expressing human ACE2 were infected intranasally with sham LLC-MK2 cell supernatant or 1 × 105 tissue culture infectious dose (TCID50) units HCoV-NL63. Wild-type mice were infected with 1 × 106 plaque-forming units (PFU) RV-A1B. Lungs were assessed for vRNA, bronchoalveolar lavage (BAL) cells, histology, HCoV-NL63 nonstructural protein 3 (nsp3), and host gene expression by next-generation sequencing and qPCR. To evaluate sequential infections, mice were infected with RV-A1B followed by HCoV-NL63 infection 4 days later. We report that hACE2 mice infected with HCoV-NL63 showed evidence of replicative infection with increased levels of vRNA, BAL neutrophils and lymphocytes, peribronchial and perivascular infiltrates, and expression of nsp3. Viral replication peaked 3 days after infection and inflammation persisted 6 days after infection. HCoV-NL63-infected hACE2 mice showed increased mRNA expression of IFNs, IFN-stimulated proteins, and proinflammatory cytokines. Infection with RV-A1B 4 days before HCoV-NL63 significantly decreased both HCoV-NL63 vRNA levels and airway inflammation. Mice infected with RV-A1B prior to HCoV-NL63 showed increased expression of antiviral proteins compared with sham-treated mice. In conclusion, we established a mouse model of HCoV-NL63 replicative infection characterized by relatively persistent viral replication and inflammation. Prior infection with RV-A1B reduced HCoV-NL63 replication and airway inflammation, indicative of viral interference.NEW & NOTEWORTHY We describe a mouse model of human coronavirus (HCoV) infection. Infection of transgenic mice expressing human angiotensin-converting enzyme 2 (ACE2) with HCoV-NL63 produced a replicative infection with peribronchial inflammation and nonstructural protein 3 expression. Mice infected with RV-A1B 4 days before HCoV-NL63 showed decreased HCoV-NL63 replication and airway inflammation and increased expression of antiviral proteins compared with sham-treated mice. This research may shed light on human coronavirus infections, viral interference, and viral-induced asthma exacerbations.

Lactobacillus rhamnosus dampens cytokine and chemokine secretion from primary human nasal epithelial cells infected with rhinovirus.

AIMS: To investigate the effect of Lactobacillus rhamnosus on viral replication and cellular response to human rhinovirus (HRV) infection, including the secretion of antiviral and inflammatory mediators from well-differentiated nasal epithelial cells (WD-NECs). METHODS AND RESULTS: The WD-NECs from healthy adult donors (N = 6) were cultured in vitro, exposed to different strains of L. rhamnosus (D3189, D3160, or LB21), and infected with HRV (RV-A16) after 24 h. Survival and adherence capacity of L. rhamnosus in a NEC environment were confirmed using CFSE-labelled isolates, immunofluorescent staining, and confocal microscopy. Shed virus and viral replication were quantified using TCID50 assays and RT-qPCR, respectively. Cytotoxicity was measured by lactate dehydrogenase (LDH) activity. Pro-inflammatory mediators were measured by multiplex immunoassay, and interferon (IFN)-λ1/3 was measured using a standard ELISA kit. Lactobacillus rhamnosus was able to adhere to and colonize WD-NECs prior to the RV-A16 infection. Lactobacillus rhamnosus did not affect shed RV-A16, viral replication, RV-A16-induced IFN-λ1/3 production, or LDH release. Pre-exposure to L. rhamnosus, particularly D3189, reduced the secretion of RV-A16-induced pro-inflammatory mediators by WD-NECs. CONCLUSIONS: These findings demonstrate that L. rhamnosus differentially modulates RV-A16-induced innate inflammatory immune responses in primary NECs from healthy adults.

ICAM-1 induced rearrangements of capsid and genome prime rhinovirus 14 for activation and uncoating.

Most rhinoviruses, which are the leading cause of the common cold, utilize intercellular adhesion molecule-1 (ICAM-1) as a receptor to infect cells. To release their genomes, rhinoviruses convert to activated particles that contain pores in the capsid, lack minor capsid protein VP4, and have an altered genome organization. The binding of rhinoviruses to ICAM-1 promotes virus activation; however, the molecular details of the process remain unknown. Here, we present the structures of virion of rhinovirus 14 and its complex with ICAM-1 determined to resolutions of 2.6 and 2.4 Å, respectively. The cryo-electron microscopy reconstruction of rhinovirus 14 virions contains the resolved density of octanucleotide segments from the RNA genome that interact with VP2 subunits. We show that the binding of ICAM-1 to rhinovirus 14 is required to prime the virus for activation and genome release at acidic pH. Formation of the rhinovirus 14-ICAM-1 complex induces conformational changes to the rhinovirus 14 capsid, including translocation of the C termini of VP4 subunits, which become poised for release through pores that open in the capsids of activated particles. VP4 subunits with altered conformation block the RNA-VP2 interactions and expose patches of positively charged residues. The conformational changes to the capsid induce the redistribution of the virus genome by altering the capsid-RNA interactions. The restructuring of the rhinovirus 14 capsid and genome prepares the virions for conversion to activated particles. The high-resolution structure of rhinovirus 14 in complex with ICAM-1 explains how the binding of uncoating receptors enables enterovirus genome release.

Coinfection with respiratory syncytial virus and rhinovirus increases IFN-λ1 and CXCL10 expression in human primary bronchial epithelial cells.

Acute respiratory tract infection (ARTI) is common in all age groups, especially in children and the elderly. About 85% of children who present with bronchiolitis are infected with respiratory syncytial virus (RSV); however, nearly one-third are coinfected with another respiratory virus, such as human rhinovirus (HRV). Therefore, it is necessary to explore the immune response to coinfection to better understand the molecular and cellular pathways involving virus-virus interactions that might be modulated by innate immunity and additional host cell response mechanisms. This study aims to investigate the host innate immune response against RSV-HRV coinfection compared with monoinfection. Human primary bronchial/tracheal epithelial cells (HPECs) were infected with RSV, HRV, or coinfected with both viruses, and the infected cells were collected at 48 and 72 hours. Gene expression profiles of IL-6, CCL5, TNF-α, IFN-ß, IFN-λ1, CXCL10, IL-10, IL-13, IRF3, and IRF7 were investigated using real-time quantitative PCR, which revealed that RSV-infected cells exhibited increased expression of IL-10, whereas HRV infection increased the expression of CXCL10, IL-10, and CCL5. IFN-λ1 and CXCL10 expression was significantly different between the coinfection and monoinfection groups. In conclusion, our study revealed that two important cytokines, IFN-λ1 and CXCL10, exhibited increased expression during coinfection.

Rhinovirus infection of the airway epithelium enhances mast cell immune responses via epithelial-derived interferons.

BACKGROUND: Mast cells (MCs) within the airway epithelium in asthma are closely related to airway dysfunction, but cross talk between airway epithelial cells (AECs) and MCs in asthma remains incompletely understood. Human rhinovirus (RV) infections are key triggers for asthma progression, and AECs from individuals with asthma may have dysregulated antiviral responses. OBJECTIVE: We utilized primary AECs in an ex vivo coculture model system to examine cross talk between AECs and MCs after epithelial rhinovirus infection. METHODS: Primary AECs were obtained from 11 children with asthma and 10 healthy children, differentiated at air-liquid interface, and cultured in the presence of laboratory of allergic diseases 2 (LAD2) MCs. AECs were infected with rhinovirus serogroup A 16 (RV16) for 48 hours. RNA isolated from both AECs and MCs underwent RNA sequencing. Direct effects of epithelial-derived interferons on LAD2 MCs were examined by real-time quantitative PCR. RESULTS: MCs increased expression of proinflammatory and antiviral genes in AECs. AECs demonstrated a robust antiviral response after RV16 infection that resulted in significant changes in MC gene expression, including upregulation of genes involved in antiviral responses, leukocyte activation, and type 2 inflammation. Subsequent ex vivo modeling demonstrated that IFN-ß induces MC type 2 gene expression. The effects of AEC donor phenotype were small relative to the effects of viral infection and the presence of MCs. CONCLUSIONS: There is significant cross talk between AECs and MCs, which are present in the epithelium in asthma. Epithelial-derived interferons not only play a role in viral suppression but also further alter MC immune responses including specific type 2 genes.

IL-1 receptor antagonist attenuates proinflammatory responses to rhinovirus in airway epithelium.

BACKGROUND: Rhinoviruses (RVs) are the most common trigger for asthma exacerbations, and there are currently no targeted therapies for viral-induced asthma exacerbations. RV infection causes neutrophilic inflammation, which is often resistant to effects of glucocorticoids. IL-1 receptor antagonist (IL-1RA) treatment reduces neutrophilic inflammation in humans challenged with inhaled endotoxin and thus may have therapeutic potential for RV-induced asthma exacerbations. OBJECTIVE: We sought to test the hypothesis that IL-1RA treatment of airway epithelium reduces RV-mediated proinflammatory cytokine production, which is important for neutrophil recruitment. METHODS: Human bronchial epithelial cells from deceased donors without prior pulmonary disease were cultured at air-liquid interface and treated with IL-13 to approximate an asthmatic inflammatory milieu. Human bronchial epithelial cells were infected with human RV-16 with or without IL-1RA treatment. RESULTS: RV infection promoted the release of IL-1α and the neutrophil-attractant cytokines IL-6, IL-8, and CXCL10. Proinflammatory cytokine secretion was significantly reduced by IL-1RA treatment without significant change in IFN-ß release or RV titer. In addition, IL-1RA reduced MUC5B expression after RV infection without impacting MUC5AC. CONCLUSIONS: These data suggest that IL-1RA treatment significantly reduced proinflammatory cytokines while preserving the antiviral response. These results provide evidence for further investigation of IL-1RA as a novel targeted therapy against neutrophil-attractant cytokine release in RV-induced airway inflammatory responses.

Rhinovirus C15 Attenuates Relaxation and cAMP Production in Human Airways and Smooth Muscle.

Rhinoviruses (RVs) evoke as many as 85% of acute asthma exacerbations in children and 50% in adults and can induce airway hyperresponsiveness and decrease efficacy of current therapeutics to provide symptom relief. Using human precision-cut lung slices (hPCLSs), primary human air-liquid interface-differentiated airway epithelial cells (HAECs), and human airway smooth muscle (HASM) as preclinical experimental models, we demonstrated that RV-C15 attenuates agonist-induced bronchodilation. Specifically, airway relaxation to formoterol and cholera toxin, but not forskolin (Fsk), was attenuated following hPCLS exposure to RV-C15. In isolated HASM cells, exposure to conditioned media from RV-exposed HAECs decreased cellular relaxation in response to isoproterenol and prostaglandin E2, but not Fsk. Additionally, cAMP generation elicited by formoterol and isoproterenol, but not Fsk, was attenuated following HASM exposure to RV-C15-conditioned HAEC media. HASM exposure to RV-C15-conditioned HAEC media modulated expression of components of relaxation pathways, specifically GNAI1 and GRK2. Strikingly, similar to exposure to intact RV-C15, hPCLS exposed to UV-inactivated RV-C15 showed markedly attenuated airway relaxation in response to formoterol, suggesting that the mechanism(s) of RV-C15-mediated loss of bronchodilation is independent of virus replication pathways. Further studies are warranted to identify soluble factor(s) regulating the epithelial-driven smooth muscle loss of ß2-adrenergic receptor function.

Molecular Determinants of Human Rhinovirus Infection, Assembly, and Conformational Stability at Capsid Protein Interfaces.

Human rhinovirus (HRV), one of the most frequent human pathogens, is the major causative agent of common colds. HRVs also cause or exacerbate severe respiratory diseases, such as asthma or chronic obstructive pulmonary disease. Despite the biomedical and socioeconomic importance of this virus, no anti-HRV vaccines or drugs are available yet. Protein-protein interfaces in virus capsids have increasingly been recognized as promising virus-specific targets for the development of antiviral drugs. However, the specific structural elements and residues responsible for the biological functions of these extended capsid regions are largely unknown. In this study, we performed a thorough mutational analysis to determine which particular residues along the capsid interpentamer interfaces are relevant to HRV infection as well as the stage(s) in the viral cycle in which they are involved. The effect on the virion infectivity of the individual mutation to alanine of 32 interfacial residues that, together, removed most of the interpentamer interactions was analyzed. Then, a representative sample that included many of those 32 single mutants were tested for capsid and virion assembly as well as virion conformational stability. The results indicate that most of the interfacial residues, and the interactions they establish, are biologically relevant, largely because of their important roles in virion assembly and/or stability. The HRV interpentamer interface is revealed as an atypical protein-protein interface, in which infectivity-determining residues are distributed at a high density along the entire interface. Implications for a better understanding of the relationship between the molecular structure and function of HRV and the development of novel capsid interface-binding anti-HRV agents are discussed. IMPORTANCE The rising concern about the serious medical and socioeconomic consequences of respiratory infections by HRV has elicited a renewed interest in the development of anti-HRV drugs. The conversion into effective drugs of compounds identified via screening, as well as antiviral drug design, rely on the acquisition of fundamental knowledge about the targeted viral elements and their roles during specific steps of the infectious cycle. The results of this study provide a detailed view on structure-function relationships in a viral capsid protein-protein interface, a promising specific target for antiviral intervention. The high density and scattering of the interfacial residues found to be involved in HRV assembly and/or stability support the possibility that any compound designed to bind any particular site at the interface will inhibit infection by interfering with virion morphogenesis or stabilization of the functional virion conformation.

Chemical Evolution of Rhinovirus Identifies Capsid-Destabilizing Mutations Driving Low-pH-Independent Genome Uncoating.

Rhinoviruses (RVs) cause recurrent infections of the nasal and pulmonary tracts, life-threatening conditions in chronic respiratory illness patients, predisposition of children to asthmatic exacerbation, and large economic cost. RVs are difficult to treat. They rapidly evolve resistance and are genetically diverse. Here, we provide insight into RV drug resistance mechanisms against chemical compounds neutralizing low pH in endolysosomes. Serial passaging of RV-A16 in the presence of the vacuolar proton ATPase inhibitor bafilomycin A1 (BafA1) or the endolysosomotropic agent ammonium chloride (NH4Cl) promoted the emergence of resistant virus populations. We found two reproducible point mutations in viral proteins 1 and 3 (VP1 and VP3), A2526G (serine 66 to asparagine [S66N]), and G2274U (cysteine 220 to phenylalanine [C220F]), respectively. Both mutations conferred cross-resistance to BafA1, NH4Cl, and the protonophore niclosamide, as identified by massive parallel sequencing and reverse genetics, but not the double mutation, which we could not rescue. Both VP1-S66 and VP3-C220 locate at the interprotomeric face, and their mutations increase the sensitivity of virions to low pH, elevated temperature, and soluble intercellular adhesion molecule 1 receptor. These results indicate that the ability of RV to uncoat at low endosomal pH confers virion resistance to extracellular stress. The data endorse endosomal acidification inhibitors as a viable strategy against RVs, especially if inhibitors are directly applied to the airways. IMPORTANCE Rhinoviruses (RVs) are the predominant agents causing the common cold. Anti-RV drugs and vaccines are not available, largely due to rapid evolutionary adaptation of RVs giving rise to resistant mutants and an immense diversity of antigens in more than 160 different RV types. In this study, we obtained insight into the cell biology of RVs by harnessing the ability of RVs to evolve resistance against host-targeting small chemical compounds neutralizing endosomal pH, an important cue for uncoating of normal RVs. We show that RVs grown in cells treated with inhibitors of endolysosomal acidification evolved capsid mutations yielding reduced virion stability against elevated temperature, low pH, and incubation with recombinant soluble receptor fragments. This fitness cost makes it unlikely that RV mutants adapted to neutral pH become prevalent in nature. The data support the concept of host-directed drug development against respiratory viruses in general, notably at low risk of gain-of-function mutations.

Rhinovirus induces airway remodeling: what are the physiological consequences?

BACKGROUND: Rhinovirus infections commonly evoke asthma exacerbations in children and adults. Recurrent asthma exacerbations are associated with injury-repair responses in the airways that collectively contribute to airway remodeling. The physiological consequences of airway remodeling can manifest as irreversible airway obstruction and diminished responsiveness to bronchodilators. Structural cells of the airway, including epithelial cells, smooth muscle, fibroblasts, myofibroblasts, and adjacent lung vascular endothelial cells represent an understudied and emerging source of cellular and extracellular soluble mediators and matrix components that contribute to airway remodeling in a rhinovirus-evoked inflammatory environment. MAIN BODY: While mechanistic pathways associated with rhinovirus-induced airway remodeling are still not fully characterized, infected airway epithelial cells robustly produce type 2 cytokines and chemokines, as well as pro-angiogenic and fibroblast activating factors that act in a paracrine manner on neighboring airway cells to stimulate remodeling responses. Morphological transformation of structural cells in response to rhinovirus promotes remodeling phenotypes including induction of mucus hypersecretion, epithelial-to-mesenchymal transition, and fibroblast-to-myofibroblast transdifferentiation. Rhinovirus exposure elicits airway hyperresponsiveness contributing to irreversible airway obstruction. This obstruction can occur as a consequence of sub-epithelial thickening mediated by smooth muscle migration and myofibroblast activity, or through independent mechanisms mediated by modulation of the ß2 agonist receptor activation and its responsiveness to bronchodilators. Differential cellular responses emerge in response to rhinovirus infection that predispose asthmatic individuals to persistent signatures of airway remodeling, including exaggerated type 2 inflammation, enhanced extracellular matrix deposition, and robust production of pro-angiogenic mediators. CONCLUSIONS: Few therapies address symptoms of rhinovirus-induced airway remodeling, though understanding the contribution of structural cells to these processes may elucidate future translational targets to alleviate symptoms of rhinovirus-induced exacerbations.

Conditionally reprogrammed asthmatic bronchial epithelial cells express lower FOXJ1 at terminal differentiation and lower IFNs following RV-A1 infection.

Primary bronchial epithelial cells (pBECs) obtained from donors have limited proliferation capacity. Recently, conditional reprogramming (CR) technique has overcome this and has provided the potential for extended passaging and subsequent differentiation of cells at air-liquid interface (ALI). However, there has been no donor-specific comparison of cell morphology, baseline gene expression, barrier function, and antiviral responses compared with their "parent" pBECs, especially cells obtained from donors with asthma. We, therefore, collected and differentiated pBECs at ALI from mild donors with asthma (n = 6) for the parent group. The same cells were conditionally reprogrammed and later differentiated at ALI. Barrier function was measured during the differentiation phase. Morphology and baseline gene expression were compared at terminal differentiation. Viral replication kinetics and antiviral responses were assessed following rhinovirus (RV) infection over 96 h. Barrier function during the differentiation phase and cell structural morphology at terminal differentiation appear similar in both parent and CR groups, however, there were elongated cell structures superficial to basal cells and significantly lower FOXJ1 expression in CR group. IFN gene expression was also significantly lower in CR group compared with parent asthma group following RV infection. The CR technique is a beneficial tool to proliferate pBECs over extended passages. Considering lower FOXJ1 expression, viral replication kinetics and antiviral responses, a cautious approach should be taken while choosing CR cells for experiments. In addition, as lab-to-lab cell culture techniques vary, the most appropriate technique must be utilized to best match individual cell functions and morphologies to address specific research questions and experimental reproducibility across the labs.

Transcriptomics of rhinovirus persistence reveals sustained expression of RIG-I and interferon-stimulated genes in nasal epithelial cells in vitro.

BACKGROUND: Human rhinoviruses (HRVs) are frequently associated with asthma exacerbations, and have been found in the airways of asthmatic patients. While HRV-induced acute infection is well-documented, it is less clear whether the nasal epithelium sustains prolonged HRV infections along with the associated activation of host immune responses. OBJECTIVE: To investigate sustainably regulated host responses of human nasal epithelial cells (hNECs) during HRV persistence. METHODS: Using a time-course study, HRV16 persistence and viral replication dynamics were established using an in vitro infection model of hNECs. RNA sequencing was performed on hNECs in the early and late stages of infection at 3 and 14 days post-infection (dpi), respectively. The functional enrichment of differentially expressed genes (DEGs) was evaluated using gene ontology (GO) and Ingenuity pathway analysis. RESULTS: HRV RNA and protein expression persisted throughout prolonged infections, even after decreased production of infectious virus progeny. GO analysis of unique DEGs indicated altered regulation of pathways related to ciliary function and airway remodeling at 3 dpi and serine-type endopeptidase activity at 14 dpi. The functional enrichment of shared DEGs between the two time-points was related to interferon (IFN) and cytoplasmic pattern recognition receptor (PRR) signaling pathways. Validation of the sustained regulation of candidate genes confirmed the persistent expression of RIG-I and revealed its close co-regulation with interferon-stimulated genes (ISGs) during HRV persistence. CONCLUSIONS: The persistence of HRV RNA does not necessarily indicate an active infection during prolonged infection. The sustained expression of RIG-I and ISGs in response to viral RNA persistence highlights the importance of assessing how immune-activating host factors can change during active HRV infection and the immune regulation that persists thereafter.

Human Rhinovirus Infection Blocks Severe Acute Respiratory Syndrome Coronavirus 2 Replication Within the Respiratory Epithelium: Implications for COVID-19 Epidemiology.

Virus-virus interactions influence the epidemiology of respiratory infections. However, the impact of viruses causing upper respiratory infections on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication and transmission is currently unknown. Human rhinoviruses cause the common cold and are the most prevalent respiratory viruses of humans. Interactions between rhinoviruses and cocirculating respiratory viruses have been shown to shape virus epidemiology at the individual host and population level. Here, we examined the replication kinetics of SARS-CoV-2 in the human respiratory epithelium in the presence or absence of rhinovirus. We show that human rhinovirus triggers an interferon response that blocks SARS-CoV-2 replication. Mathematical simulations show that this virus-virus interaction is likely to have a population-wide effect as an increasing prevalence of rhinovirus will reduce the number of new coronavirus disease 2019 cases.

Rhinovirus-induced CCL17 and CCL22 in Asthma Exacerbations and Differential Regulation by STAT6.

The interplay of type-2 inflammation and antiviral immunity underpins asthma exacerbation pathogenesis. Virus infection induces type-2 inflammation-promoting chemokines CCL17 and CCL22 in asthma; however, mechanisms regulating induction are poorly understood. By using a human rhinovirus (RV) challenge model in human airway epithelial cells in vitro and mice in vivo, we assessed mechanisms regulating CCL17 and CCL22 expression. Subjects with mild to moderate asthma and healthy volunteers were experimentally infected with RV and airway CCL17 and CCL22 protein quantified. In vitro airway epithelial cell- and mouse-RV infection models were then used to define STAT6- and NF-κB-mediated regulation of CCL17 and CCL22 expression. Following RV infection, CCL17 and CCL22 expression was higher in asthma, which differentially correlated with clinical and immunological parameters. Air-liquid interface-differentiated primary epithelial cells from donors with asthma also expressed higher levels of RV-induced CCL22. RV infection boosted type-2 cytokine-induced STAT6 activation. In epithelial cells, type-2 cytokines and STAT6 activation had differential effects on chemokine expression, increasing CCL17 and suppressing CCL22, whereas NF-κB promoted expression of both chemokines. In mice, RV infection activated pulmonary STAT6, which was required for CCL17 but not CCL22 expression. STAT6-knockout mice infected with RV expressed increased levels of NF-κB-regulated chemokines, which was associated with rapid viral clearance. Therefore, RV-induced upregulation of CCL17 and CCL22 was mediated by NF-κB activation, whereas expression was differentially regulated by STAT6. Together, these findings suggest that therapeutic targeting of type-2 STAT6 activation alone will not block all inflammatory pathways during RV infection in asthma.

Utility of Three Nebulizers in Investigating the Infectivity of Airborne Viruses.

Laboratory-generated bioaerosols are widely used in aerobiology studies of viruses; however, few comparisons of alternative nebulizers exist. We compared aerosol production and virus survival for a Collison nebulizer, vibrating mesh nebulizer (VMN), and hydraulic spray atomizer (HSA). We also measured the dry size distribution of the aerosols produced and calculated the droplet sizes before evaporation and the dry size distribution from normal saline solution. Dry count median diameters of 0.11, 0.22, and 0.30 µm were found for normal saline from the Collison nebulizer, VMN, and HSA, respectively. The volume median diameters were 0.323, 1.70, and 1.30 µm, respectively. The effect of nebulization on the viability of two influenza A viruses (IAVs) (H1N1 and H3N2) and human rhinovirus 16 (HRV-16) was assessed by nebulization into an SKC BioSampler. The HSA had the least impact on surviving fractions (SFs) of H1N1 and H3N2 (89% ± 3% and 94% ± 2%, respectively), followed by the Collison nebulizer (83% ± 1% and 82% ± 2%, respectively). The VMN yielded SFs of 78% ± 2% and 76% ± 2%, respectively. Conversely, for HRV-16, the VMN produced higher SFs (87% ± 8%). Our findings indicate that there were no statistical differences between SFs of the viruses nebulized by these nebulizers. However, VMN produced higher aerosol concentrations within the airborne size range, making it more suitable where high aerosol mass production is required. IMPORTANCE Viral respiratory tract infections cause millions of lost days of work and physician visits globally, accounting for significant morbidity and mortality. Respiratory droplets and droplet nuclei from infected hosts are the potential carriers of such viruses within indoor environments. Laboratory-generated bioaerosols are applied in understanding the transmission and infection of viruses, modeling the physiological aspects of bioaerosol generation in a controlled environment. However, little comparative characterization exists for nebulizers used in infectious disease aerobiology, including Collison nebulizer, vibrating mesh nebulizer, and hydraulic spray atomizer. This study characterized the physical features of aerosols generated by laboratory nebulizers and their performance in producing aerosols at a size relevant to airborne transmission used in infectious disease aerobiology. We also determined the impact of nebulization mechanisms of these nebulizers on the viability of human respiratory viruses, including IAV H1N1, IAV H3N2, and HRV-16.

Rhinovirus and Bacteria Synergistically Induce IL-17C Release from Human Airway Epithelial Cells To Promote Neutrophil Recruitment.

Virus-bacteria coinfections are associated with more severe exacerbations and increased risk of hospital readmission in patients with chronic obstructive pulmonary disease (COPD). The airway epithelium responds to such infections by releasing proinflammatory and antimicrobial cytokines, including IL-17C. However, the regulation and role of IL-17C is not well understood. In this study, we examine the mechanisms regulating IL-17C production and its potential role in COPD exacerbations. Human bronchial epithelial cells (HBE) obtained from normal, nontransplanted lungs or from brushings of nonsmokers, healthy smokers, or COPD patients were exposed to bacteria and/or human rhinovirus (HRV). RNA and protein were collected for analysis, and signaling pathways were assessed with pharmacological agonists, inhibitors, or small interfering RNAs. HBE were also stimulated with IL-17C to assess function. HRV-bacterial coinfections synergistically induced IL-17C expression. This induction was dependent on HRV replication and required NF-κB-mediated signaling. Synergy was lost in the presence of an inhibitor of the p38 MAP kinase pathway. HBE exposed to IL-17C show increased gene expression of CXCL1, CXCL2, NFKBIZ, and TFRC, and release CXCL1 protein, a neutrophil chemoattractant. Knockdown of IL-17C significantly reduced induction of CXCL1 in response to HRV-bacterial coinfection as well as neutrophil chemotaxis. HBE from healthy smokers release less IL-17C than cells from nonsmokers, but cells from COPD patients release significantly more IL-17C compared with either nonsmokers or healthy smokers. These data suggest that IL-17C may contribute to microbial-induced COPD exacerbations by promoting neutrophil recruitment.

Rhinovirus induces an anabolic reprogramming in host cell metabolism essential for viral replication.

Rhinoviruses (RVs) are responsible for the majority of upper airway infections; despite their high prevalence and the resulting economic burden, effective treatment is lacking. We report here that RV induces metabolic alterations in host cells, which offer an efficient target for antiviral intervention. We show that RV-infected cells rapidly up-regulate glucose uptake in a PI3K-dependent manner. In parallel, infected cells enhance the expression of the PI3K-regulated glucose transporter GLUT1. In-depth metabolomic analysis of RV-infected cells revealed a critical role of glucose mobilization from extracellular and intracellular pools via glycogenolysis for viral replication. Infection resulted in a highly anabolic state, including enhanced nucleotide synthesis and lipogenesis. Consistently, we observed that glucose deprivation from medium and via glycolysis inhibition by 2-deoxyglucose (2-DG) potently impairs viral replication. Metabolomic analysis showed that 2-DG specifically reverts the RV-induced anabolic reprogramming. In addition, treatment with 2-DG inhibited RV infection and inflammation in a murine model. Thus, we demonstrate that the specific metabolic fingerprint of RV infection can be used to identify new targets for therapeutic intervention.

Human TH1 and TH2 cells targeting rhinovirus and allergen coordinately promote allergic asthma.

BACKGROUND: Allergic asthmatic subjects are uniquely susceptible to acute wheezing episodes provoked by rhinovirus. However, the underlying immune mechanisms and interaction between rhinovirus and allergy remain enigmatic, and current paradigms are controversial. OBJECTIVE: We sought to perform a comprehensive analysis of type 1 and type 2 innate and adaptive responses in allergic asthmatic subjects infected with rhinovirus. METHODS: Circulating virus-specific TH1 cells and allergen-specific TH2 cells were precisely monitored before and after rhinovirus challenge in allergic asthmatic subjects (total IgE, 133-4692 IU/mL; n = 28) and healthy nonallergic controls (n = 12) using peptide/MHCII tetramers. T cells were sampled for up to 11 weeks to capture steady-state and postinfection phases. T-cell responses were analyzed in parallel with 18 cytokines in the nose, upper and lower airway symptoms, and lung function. The influence of in vivo IgE blockade was also examined. RESULTS: In uninfected asthmatic subjects, higher numbers of circulating virus-specific PD-1+ TH1 cells, but not allergen-specific TH2 cells, were linked to worse lung function. Rhinovirus infection induced an amplified antiviral TH1 response in asthmatic subjects versus controls, with synchronized allergen-specific TH2 expansion, and production of type 1 and 2 cytokines in the nose. In contrast, TH2 responses were absent in infected asthmatic subjects who had normal lung function, and in those receiving anti-IgE. Across all subjects, early induction of a minimal set of nasal cytokines that discriminated high responders (G-CSF, IFN-γ, TNF-α) correlated with both egress of circulating virus-specific TH1 cells and worse symptoms. CONCLUSIONS: Rhinovirus induces robust TH1 responses in allergic asthmatic subjects that may promote disease, even after the infection resolves.