Respiratory Syncytial Virus Pathogenesis Research Articles

Landscape of respiratory syncytial virus.

Respiratory syncytial virus (RSV) is an enveloped, negative-sense, single-stranded RNA virus of the Orthopneumovirusgenus of the Pneumoviridae family in the order Mononegavirales. RSV can cause acute upper and lower respiratory tract infections, sometimes with extrapulmonary complications. The disease burden of RSV infection is enormous, mainly affecting infants and older adults aged 75 years or above. Currently, treatment options for RSV are largely supportive. Prevention strategies remain a critical focus, with efforts centered on vaccine development and the use of prophylactic monoclonal antibodies. To date, three RSV vaccines have been approved for active immunization among individuals aged 60 and above. For children who are not eligible for these vaccines, passive immunization is recommended. A newly approved prophylactic monoclonal antibody, Nirsevimab, which offers enhanced neutralizing activity and an extended half-life, provides exceptional protection for high-risk infants and young children. This review provides a comprehensive and detailed exploration of RSV's virology, immunology, pathogenesis, epidemiology, clinical manifestations, treatment options, and prevention strategies.

Characterization of a Panel of Monoclonal Antibodies Targeting the F-Protein of the Respiratory Syncytial Virus (RSV) for the Typing of Contemporary Circulating Strains.

Respiratory syncytial virus (RSV) is the most common cause of upper and lower respiratory tract infections in infants and young children. Virus-specific monoclonal antibodies (mAbs) can be used for diagnosis, prophylaxis, and research of RSV pathogenesis. A panel of 16 anti-RSV mAbs was obtained from mice immunized by RSV strain Long. Half of them had virus-neutralizing activity. According to Western blot all of these mAbs effectively bound native oligomeric (homodimeric and homotrimeric) forms of the RSV fusion (F) protein. Only five of the mAbs interacted with the monomeric form, and only one of these possessed neutralizing activity. None of these mAbs, nor the commercial humanized neutralizing mAb palivizumab, reacted with the denaturated F protein. Thus, interaction of all these mAbs with F protein had clear conformational dependence. Competitive ELISA and neutralization assays allowed the identification of nine antigenic target sites for the interaction of mAb with the F protein. Five partially overlapping sites may represent a complex spatial structure of one antigenic determinant, including one neutralizing and four non-neutralizing epitopes. Four sites (three neutralizing and one non-neutralizing) were found to be distinct. As a result of virus cultivation RSV-A, strain Long, in the presence of a large amount of one of the neutralizing mAbs, an escape mutant with a substitution, N240S, in the F protein, was obtained. Thus, it was shown for the first time that position 240 is critical for the protective effect of an anti-RSV antibody. To assess the ability of these mAbs to interact with modern RSV strains circulating in St. Petersburg (Russia) between 2014 and 2022, 73 RSV-A and 22 RSV-B isolates were analyzed. Six mAbs were directed to conserved epitopes of the F protein as they interacted most efficiently with both RSV subtypes in a fixed cell-ELISA and could be used for diagnostic assays detecting RSV.

Prevention and Treatment Strategies for Respiratory Syncytial Virus (RSV)

Respiratory syncytial virus (RSV) is a leading cause of severe lower respiratory tract disease, especially in young children. Despite its global impact on healthcare, related to its high prevalence and its association with significant morbidity, the current therapy is still mostly supportive. Moreover, while more than 50 years have passed since the first trial of an RSV vaccine (which unfortunately caused enhanced RSV disease), no vaccine has been approved for RSV prevention. In the last two decades, our understanding of the pathogenesis and immunopathology of RSV have continued to evolve, leading to significant advancements in RSV prevention strategies. These include both the development of new potential vaccines and the successful implementation of passive immunization, which, together, will provide coverage from infancy to old age. In this review, we provide an update of the current treatment options for acute disease (RSV-specific and -non-specific) and different therapeutic approaches focusing on RSV prevention.

Acteoside attenuates RSV-induced lung injury by suppressing necroptosis and regulating metabolism

Background: Necroptosis and inflammation are closely related to the pathogenesis of respiratory syncytial virus (RSV). Acteoside (AC), a natural phenylpropanoid glycoside from Kuding Tea, has significant anti-RSV effect. However, the roles of AC on RSV-induced lung necroptosis and inflammation are yet to be elucidated. Methods: The effects of AC were investigated in BALB/c mice and A549 cells. Lung histopathology was observed through H&E staining. The viral titer was assessed via plaque assay. The RSV-F expression was determined by RT-qPCR and immunohistochemistry assay. The levels of cytokines were detected by ELISA and RT-qPCR. The necroptosis rate and mitochondrial membrane potential were evaluated via flow cytometry. The expressions of HMGB1/NF-κB and RIP1/RIP3/MLKL/PGAM5/DRP1 were detected by western blot. Additionally, untargeted metabolomics was conducted to investigate the metabolic profiles and related metabolic pathways via Gas Chromatography-Mass Spectrometry. Results: The results showed that compared with the RSV-infected group, AC treatment significantly attenuated lung pathological damage, virus replication, and cytokines levels. AC also alleviated RSV-induced necroptosis and mitochondrial dysfunction in vitro and in vivo. Moreover, AC treatment down-regulated the expression of HMGB1, p-Iκbα/Iκbα, p-p65/p65, RIP1, RIP3, MLKL, PGAM5, and DRP1. Furthermore, metabolomic analyses suggested that the perturbations in major metabolites of AC therapy were related to variations in amino acid and energy metabolism. Conclusion: Our findings validated the beneficial effects of AC in suppressing necroptosis and regulating metabolism, suggesting AC may be a new drug candidate for RSV infection.

Respiratory Syncytial Virus Nonstructural Protein 1 Promotes 5-Lipoxygenase via miR-19a-3p.

Background Respiratory syncytial virus (RSV) infection can regulate the expression of a wide range of noncoding microRNAs (miRNAs), in which mir-19a-3p can participate in airway inflammatory response by regulating 5-lipoxygenase (5-LO) pathway. RSV nonstructural protein (NS) 1 is involved in the airway hyperresponsiveness during RSV infection. Methods The expression levels of miR-19a-3p and inflammatory signaling-related indicators were detected using quantitative real-time PCR and western blot analyses on the A549 cells transfected with NS1 expression plasmids (pNS1). The 5-LO-mediated inflammatory signaling pathway was assessed when the miR-19a-3p or 5-LO was inhibited. Results The immunofluorescence analysis showed that the plasmid-mediated NS1 protein was observed in both the cytoplasm and nucleus. The expression level of miR-19a-3p was significantly upregulated in the pNS1 or RSV-treated cells, which was reversed by the NS1 small interfering RNA. In addition, pNS1 also upregulated the expression of 5-LO, interleukin-5 (IL-5), and leukotriene B4 (LTB4), which was also significantly inhibited by the miR-19a-3p antagonists. The 5-LO inhibitor MK886 prevented the increase in the expression level of IL-5 induced by pNS1. Conclusions These results suggested that the RSV NS1 might play an important role in the pathogenesis of RSV by activating the 5-LO and subsequent inflammatory cytokines through miR-19a-3p.

Mucosal Immune Responses to Respiratory Syncytial Virus.

Despite over half a century of research, respiratory syncytial virus (RSV)-induced bronchiolitis remains a major cause of hospitalisation in infancy, while vaccines and specific therapies still await development. Our understanding of mucosal immune responses to RSV continues to evolve, but recent studies again highlight the role of Type-2 immune responses in RSV disease and hint at the possibility that it dampens Type-1 antiviral immunity. Other immunoregulatory pathways implicated in RSV disease highlight the importance of focussing on localised mucosal responses in the respiratory mucosa, as befits a virus that is essentially confined to the ciliated respiratory epithelium. In this review, we discuss studies of mucosal immune cell infiltration and production of inflammatory mediators in RSV bronchiolitis and relate these studies to observations from peripheral blood. We also discuss the advantages and limitations of studying the nasal mucosa in a disease that is most severe in the lower airway. A fresh focus on studies of RSV pathogenesis in the airway mucosa is set to revolutionise our understanding of this common and important infection.

1186. Increased Respiratory Syncytial Virus (RSV) Viral Replication Leads to Increased Cytokine Production and Polarized Interferon Response in Infant Mucosal Epithelium

BackgroundRSV is the most frequent etiology of pediatric lower respiratory tract infection. Most children hospitalized for RSV are previously healthy without known risk factors. Children with mild disease managed as outpatients (OP) have higher viral loads than those hospitalized with severe disease. OP children have higher concentrations of the mucosal interferon (IFN), IFNλ2/3, and IP-10, but no differences in IFNλ1. We examined how RSV replication impacts cytokine production kinetics in the nasal mucosa.MethodsPrimary infant human nasal epithelial (iHNE) cells were collected from nasopharyngeal swabs and cultured on an air-liquid interface. Cultures were infected with 0.1 or 0.001 multiplicity of infection (MOI) of RSV-A, or mock infected. Concentrations of IFN-related (IFNα2, β, γ, λ1, λ2/3, and IP-10) and inflammatory (IL-1β, -6, -12, and TNFα) cytokines secreted to the apical and basolateral surfaces were quantified via immunoassay. Kinetics according to viral inocula were compared by ANOVA with Dunn post-hoc testing of the area under the curve (AUC) for each cytokine. Peak concentrations were compared according to MOI and secretion surface by 2-way ANOVA.ResultsAUC of IFNs in both surfaces of RSV infected cells were significantly higher than those of mock infected. The 0.1 MOI RSV inoculum resulted in significantly higher AUCs for all IFN cytokines on both surfaces than the 0.001 MOI. Peak IFNλ1 concentrations were higher on the apical than basolateral side; peak IFNλ2/3 concentrations were higher on the basolateral side than apical. AUCs of inflammatory cytokines in RSV infection were significantly higher on the basolateral, but not apical, surfaces than mock; all basolateral inflammatory cytokines were higher in the 0.1 MOI than the 0.001 MOI.ConclusionHigher RSV inoculum induces higher concentrations of IFN-related cytokines on both sides of epithelial cells, and higher concentrations of inflammatory cytokines on the basolateral side. Differential secretion of IFNλ1 and IFNλ2/3 to the apical and basolateral surfaces suggests they may play different roles in immune response during RSV infection. These data support viral replication as an important factor influencing RSV pathogenesis and severity through cytokine production.Disclosures Asuncion Mejias, MD, PhD, MsCS, Janssen (Grant/Research Support, Advisor or Review Panel member)Merck (Grant/Research Support, Advisor or Review Panel member)Roche (Advisor or Review Panel member)Sanofi (Advisor or Review Panel member) Octavio Ramilo, MD, Adagio (Consultant)Bill & Melinda Gates Foundation (Grant/Research Support)Janssen (Grant/Research Support)Lilly (Consultant)Merck (Consultant, Grant/Research Support)NIH (Grant/Research Support)Pfizer (Consultant)SANOFI (Board Member)

Respiratory syncytial virus infection induces the release of transglutaminase 2 from human airway epithelial cells.

Respiratory syncytial virus (RSV) is an important human pathogen that causes severe lower respiratory tract infections in young children, the elderly, and the immunocompromised, yet no effective treatments or vaccines are available. The precise mechanism underlying RSV-induced acute airway disease and associated sequelae are not fully understood; however, early lung inflammatory and immune events are thought to play a major role in the outcome of the disease. Moreover, oxidative stress responses in the airways play a key role in the pathogenesis of RSV. Oxidative stress has been shown to elevate cytosolic calcium (Ca2+) levels, which in turn activate Ca2+-dependent enzymes, including transglutaminase 2 (TG2). Transglutaminase 2 is a multifunctional cross-linking enzyme implicated in various physiological and pathological conditions; however, its involvement in respiratory virus-induced airway inflammation is largely unknown. In this study, we demonstrated that RSV-induced oxidative stress promotes enhanced activation and release of TG2 from human lung epithelial cells as a result of its translocation from the cytoplasm and subsequent release into the extracellular space, which was mediated by Toll-like receptor (TLR)-4 and NF-κB pathways. Antioxidant treatment significantly inhibited RSV-induced TG2 extracellular release and activation via blocking viral replication. Also, treatment of RSV-infected lung epithelial cells with TG2 inhibitor significantly reduced RSV-induced matrix metalloprotease activities. These results suggested that RSV-induced oxidative stress activates innate immune receptors in the airways, such as TLRs, that can activate TG2 via the NF-κB pathway to promote cross-linking of extracellular matrix proteins, resulting in enhanced inflammation.

Nuclear-localized human respiratory syncytial virus NS1 protein modulates host gene transcription.

SUMMARYHuman respiratory syncytial virus (RSV) is a common cause of lower respiratory tract infections in the pediatric, elderly, and immunocompromised individuals. RSV non-structural protein NS1 is a known cytosolic immune antagonist, but how NS1 modulates host responses remains poorly defined. Here, we observe NS1 partitioning into the nucleus of RSV-infected cells, including the human airway epithelium. Nuclear NS1 coimmunoprecipitates with Mediator complex and is chromatin associated. Chromatin-immunoprecipitation demonstrates enrichment of NS1 that overlaps Mediator and transcription factor binding within the promoters and enhancers of differentially expressed genes during RSV infection. Mutation of the NS1 C-terminal helix reduces NS1 impact on host gene expression. These data suggest that nuclear NS1 alters host responses to RSV infection by binding at regulatory elements of immune response genes and modulating host gene transcription. Our study identifies another layer of regulation by virally encoded proteins that shapes host response and impacts immunity to RSV.

#17: Disease and Immunoprophylaxis Model of Human Nose Organoids to Study SARS-CoV-2 and RSV Infection

BackgroundThere is a significant and unmet need for pre-clinical models to predict responsiveness of immunotherapies to both severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and respiratory syncytial virus (RSV) infection. Airway organoid models have been recently developed to study respiratory viruses; however, the current methods rely on invasive or biopsy derived samples to generate lung or airway organoids.ObjectiveTo establish human nose organoids (HNOs) as a model to study SARS-CoV-2 and RSV pathogenesis and test therapeutics.MethodsWe developed a non-invasive method to establish HNOs using stem cells isolated from nasal-wash and mid-turbinate samples. We made air liquid interface (ALI) cultures from undifferentiated 3-dimensional HNOs and differentiated for 21 days to form differentiated nasal epithelium. We inoculated the apical epithelium and assessed SARS-CoV-2 and RSV infection on the apical compartment using real time-polymerase chain reaction, plaque assays and immunofluorescence techniques. We then evaluated the feasibility of HNO-ALI model system to test the efficacy of serum antibodies to prevent SARS-CoV-2 infection and palivizumab monoclonal antibodies to prevent infection using palivizumab sensitive and resistant RSV strains. We introduced the antibodies in the basolateral compartment and monitored its neutralizing capacity on the apical side mimicking the neutralizing effects of antibodies in circulation.ResultsOur HNO-ALI cultures consist of well-differentiated, pseudostratified, ciliated, and mucosal respiratory epithelial cells and are susceptible to SARS-CoV-2, RSV A and B infection. SARS-CoV-2 and RSV replicates in the apical ciliated cells of the HNO-ALI cultures, peaks at 4 days, and plateaus at 8 days post infection. Infected HNO-ALI recapitulates aspects of SARS-CoV-2 and RSV disease, including viral shedding, asynchronous cilia beating/ciliary damage, and mucus hyper-secretion. Our model effectively showed protection to infection in a concentration dependent manner of the antibodies used.ConclusionWe established a non-invasive method to generate HNO-ALI epithelial model as an authentic and an alternative model to 1-D cell culture systems. Our ex-vivo HNO-ALI infection model provides a novel approach for testing therapeutic interventions.

Hyperglycemia results in decreased immune cell infiltration and increased viral load in the lung in a mouse model of RSV infection

Respiratory Syncytial virus (RSV) infection is a feared disease in vulnerable populations with impaired immune responses. There is currently no vaccine against RSV and young children along with elderly people are at increased risk of severe or sometimes life-threatening RSV infection. Hyperglycemia with immunomodulatory patterns can impact on infectious disease outcomes and immune system responses in diabetic patients. Even though research continues to uncover the complex mechanisms underlying RSV immunopathogenesis and diabetes mellitus disease separately, limited information is available about interaction between these two phenomena. Here, we evaluated the influence of hyperglycemia as the hallmark of diabetes mellitus disease on the pathogenesis and immunopathogenesis of RSV in a mouse model. In this experiment, hyperglycemia was induced by intraperitoneal injection of Streptozotocin (STZ), and after diabetes confirmation, mice were infected with RSV-A2, and the immune responses were followed for 5 days until the mice were sacrificed. Analyses on airway immune cell influx, T-Lymphocyte subtypes, cytokines secretion, lung histopathology, and viral load were conducted. Our results showed that hyperglycemia resulted in reduced lung immune cells infiltration totally and it was associated with decreased pathological damage of the lung. Following RSV infection in hyperglycemic mice, the ratio of CD4/CD8 T-Lymphocytes due to CD8+ depletion, increased. Furthermore, the level of IFN-γ and IL-17A cytokines decreased, whereas IL-10 showed an upward trend and the viral load increased in hyperglycemic mice compared with normoglycemic mice. In conclusion, these findings indicate that hyperglycemia can ameliorate and downregulate RSV-induced inflammatory and antiviral responses, and result in increment of viral load.

Molecular and Cellular Mechanisms of Respiratory Syncytial Viral Infection: Using Murine Models to Understand Human Pathology.

Respiratory syncytial virus (RSV) causes severe pathology of the lower respiratory tract in infants, immunocompromised people, and elderly. Despite decades of research, there is no licensed vaccine against RSV, and many therapeutic drugs are still under development. Detailed understanding of molecular and cellular mechanisms of the RSV infection pathology can accelerate the development of efficacious treatment. Current studies on the RSV pathogenesis are based on the analysis of biopsies from the infected patients; however deeper understanding of molecular and cellular mechanisms of the RSV pathology could be achieved using animal models. Mice are the most often used model for RSV infection because they exhibit manifestations similar to those observed in humans (bronchial obstruction, mucous hypersecretion, and pulmonary inflammation mediated by lymphocytes, macrophages, and neutrophils). Additionally, the use of mice is economically feasible, and many molecular tools are available for studying RSV infection pathogenesis at the molecular and cellular levels. This review summarizes new data on the pathogenesis of RSV infection obtained in mouse models, which demonstrated the role of T cells in both the antiviral defense and the development of lung immunopathology. T cells not only eliminate the infected cells, but also produce significant amounts of the proinflammatory cytokines TNFα and IFNγ. Recently, a new subset of tissue-resident memory T cells (TRM) was identified that provide a strong antiviral defense without induction of lung immunopathology. These cells accumulate in the lungs after local rather than systemic administration of RSV antigens, which suggests new approaches to vaccination. The studies in mouse models have revealed a minor role of interferons in the anti-RSV protection, as RSV possesses mechanisms to escape the antiviral action of type I and III interferons, which may explain the low efficacy of interferon-containing drugs. Using knockout mice, a significant breakthrough has been achieved in understanding the role of many pro-inflammatory cytokines in lung immunopathology. It was found that in addition to TNFα and IFNγ, the cytokines IL-4, IL-5, IL-13, IL-17A, IL-33, and TSLP mediate the major manifestations of the RSV pathogenesis, such as bronchial obstruction, mucus hyperproduction, and lung infiltration by pro-inflammatory cells, while IL-6, IL-10, and IL-27 exhibit the anti-inflammatory effect. Despite significant differences between the mouse and human immune systems, mouse models have made a significant contribution to the understanding of molecular and cellular mechanisms of the pathology of human RSV infection.

IFI27 may predict and evaluate the severity of respiratory syncytial virus infection in preterm infants

BackgroundPreterm infants are a special population that vulnerable to respiratory syncytial virus (RSV) infection and the lower respiratory tract infections (LRTIs) caused by RSV could be severe and even life-threating. The purpose of the present study was to identify candidate genes of preterm infants who are susceptible to RSV infection and provide a new insight into the pathogenesis of RSV infection.MethodsThree datasets (GSE77087, GSE69606 and GSE41374) containing 183 blood samples of RSV infected patients and 33 blood samples of healthy controls from Gene Expression Omnibus (GEO) database were downloaded and the differentially expressed genes (DEGs) were screened out. The function and pathway enrichments were analyzed through Database for Annotation, Visualization and Integrated Discovery (DAVID) website. The protein-protein interaction (PPI) network for DEGs was constructed through Search Tool for the Retrieval of Interacting Genes (STRING). The module analysis was performed by Cytoscape software and hub genes were identified. Clinical verification was employed to verify the expression level of top five hub genes among 72 infants including 50 RSV infected patients and 22 non-RSV-infected patients hospitalized in our center. Further, the RSV infected infants with high-expression IFI27 and those with low-expression IFI27 were compared (defined as higher or lower than the median mRNA level). Finally, the gene set enrichment analysis (GSEA) focusing on IFI27 was carried out.ResultsTotally, 4028 DEGs were screened out and among which, 131 most significant DEGs were selected. Subsequently, 13 hub genes were identified, and function and pathway enrichments of hub genes mainly were: response to virus, defense response to virus, regulation of viral genome replication and regulation of viral life cycle. Furthermore, IFI27 was confirmed to be the most significantly expressed in clinical verification. Gene sets associated with calcium signaling pathway, arachidonic acid metabolism, extracellular matrix receptor interaction and so on were significantly enriched when IFI27 was highly expressed. Moreover, high-expression IFI27 was associated with more severe cases (p = 0.041), more requirements of mechanical ventilation (p = 0.034), more frequent hospitalization (p < 0.001) and longer cumulative hospital stay (p = 0.012).ConclusionIFI27 might serve to predict RSV infection and evaluate the severity of RSV infection in preterm infants.

Respiratory syncytial virus: from pathogenesis to potential therapeutic strategies

Respiratory syncytial virus (RSV) is one of the most important viral pathogens causing respiratory tract infection in infants, the elderly and people with poor immune function, which causes a huge disease burden worldwide every year. It has been more than 60 years since RSV was discovered, and the palivizumab monoclonal antibody, the only approved specific treatment, is limited to use for passive immunoprophylaxis in high-risk infants; no other intervention has been approved to date. However, in the past decade, substantial progress has been made in characterizing the structure and function of RSV components, their interactions with host surface molecules, and the host innate and adaptive immune response to infection. In addition, basic and important findings have also piqued widespread interest among researchers and pharmaceutical companies searching for effective interventions for RSV infection. A large number of promising monoclonal antibodies and inhibitors have been screened, and new vaccine candidates have been designed for clinical evaluation. In this review, we first briefly introduce the structural composition, host cell surface receptors and life cycle of RSV virions. Then, we discuss the latest findings related to the pathogenesis of RSV. We also focus on the latest clinical progress in the prevention and treatment of RSV infection through the development of monoclonal antibodies, vaccines and small-molecule inhibitors. Finally, we look forward to the prospects and challenges of future RSV research and clinical intervention.

Fcγ Receptor IIa (FCGR2A) Polymorphism Is Associated With Severe Respiratory Syncytial Virus Disease in Argentinian Infants.

BackgroundMost patients with respiratory syncytial virus (RSV) infection requiring hospitalization have no risk factors for severe disease. Genetic variation in the receptor for the Fc portion of IgG (FcγR) determines their affinity for IgG subclasses driving innate and adaptive antiviral immunity. We investigated the relationship between FcγRIIa-H131R polymorphism and RSV disease.MethodsBlood samples were collected from 182 infants ≤24-month-old (50 uninfected, 114 RSV-infected with moderate course and 18 suffering severe disease). FcγRIIa-H131R SNP genotypic frequencies (HH, HR, RR) and anti-RSV IgG1, IgG2 and IgG3 levels were studied.ResultsGenotypic frequencies for FcγRIIa-H131R SNP were comparable between uninfected and RSV-infected infants. In contrast, we found a significant higher frequency of HH genotype in severe RSV-infected children compared to moderate patients. Among severe group, HH infants presented more factors associated to severity than HR or RR patients did. Furthermore, compared to moderate RSV-infected infants, severe patients showed higher levels of anti-RSV IgG1 and IgG3.ConclusionsWe found an association between an FcγRIIa (H131) polymorphism and severe RSV disease, which points towards a critical role for interactions between FcγRs and immune complexes in RSV pathogenesis. This genetic factor could also predict the worse outcome and identify those infants at risk during hospitalization.

Imiquimod suppresses respiratory syncytial virus (RSV) replication via PKA pathway and reduces RSV induced-inflammation and viral load in mice lungs

Respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract disease and bronchiolitis in children, as well as an important cause of morbidity and mortality in elderly and immunocompromised individuals. However, there is no safe and efficacious RSV vaccine or antiviral treatment. Toll Like Receptors (TLR) are important molecular mediators linking innate and adaptive immunity, and their stimulation by cognate agonists has been explored as antiviral agents. Imiquimod is known as a TLR7 agonist, but additionally acts as an antagonist for adenosine receptors. In this study, we demonstrate that imiquimod, but not resiquimod, has direct anti-RSV activity via PKA pathway in HEp-2 and A549 cells, independently of an innate response. Imiquimod restricts RSV infection after viral entry into the host cell, interfering with viral RNA and protein synthesis. Probably as a consequence of these anti-RSV properties, imiquimod displays cytokine modulating activity in RSV infected epithelial cells. Moreover, in a murine model of RSV infection, imiquimod treatment improves the course of acute disease, evidenced by decreased weight loss, reduced RSV lung titers, and attenuated airway inflammation. Consequently, imiquimod represents a promising therapeutic alternative against RSV infection and may inform the development of novel therapeutic targets to control RSV pathogenesis.

Effect of tobacco smoke on respiratory syncytial virus-induced high-mobility group box 1-mediated innate antiviral signaling

Abstract Exposure to tobacco smoke (TS), either direct or secondhand, is associated with an increase in the severity of lower respiratory tract infections, including bronchiolitis, mainly caused by respiratory syncytial virus (RSV). No vaccine or effective treatment is available for RSV. Although the mechanism of TS-induced RSV severity remains largely unknown, lung immune responses are key. High Mobility Group Box-1 (HMGB1) is a multifunctional nuclear protein that is released to the extracellular space during infection and regulates inflammatory responses as damage-associated molecular pattern. The goal of this study is to dissect the role of HMGB1 in the activation of antiviral signaling in RSV and TS-induced airway inflammation. Studies were conducted in airway epithelial cells, including A549 and normal small alveolar epithelial cells. We show that RSV and TS-induced oxidative stress post-translationally modifies HMGB1 including hyperacetylation, phosphorylation, and oxidation, leading to its enhanced extracellular secretion from lung epithelial cells. Secreted HMGB1 activates signaling cascades to activate p38 MAPK and NF-κB to induce proinflammatory mediator release from immune cells and induce inflammation. We also show that hmgb1 knockdown exacerbates inflammation in RSV-infected and TS-exposed cells and that treatment with recombinant HMGB1 activates immune cells to induce an inflammatory response, promotes the activation of antiviral pathways and inhibits viral replication. Our studies suggest an important role for HMGB1 in TS-induced RSV pathogenesis and guide the development of novel therapeutic strategies against respiratory infections that are caused or exacerbated by environmental toxicants such as TS.

Function and Modulation of Type I Interferons during Respiratory Syncytial Virus Infection.

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory infections in infants and young children, accounting for an estimated 3 million hospitalizations annually worldwide. Despite the major health burden, there is currently no licensed RSV vaccine. RSV is recognized by a range of cellular receptors including both toll-like receptors (TLR) and retinoic acid-inducible gene-I-like receptors (RIG-I). This interaction initiates signaling through mitochondrial antiviral signaling (MAVS) and interferon regulatory factor (IRF) proteins, resulting in the induction of type I interferons (IFN). Early viral control is mediated by either IFN-α or IFN-β signaling through the IFN receptor (IFNAR), inducing the production of antiviral interferon-stimulating genes (ISGs). Type I IFNs also initiate the early production of proinflammatory cytokines including interleukin 6 (IL-6), tumor necrosis factor (TNF), and IFN-γ. Type I IFN levels correlate with age, and inadequate production may be a critical factor in facilitating the increased RSV disease severity observed in infants. Here, we review the current literature on the function of type I IFNs in RSV pathogenesis, as well as their involvement in the differential immune responses observed in infants and adults.

Toll-like receptor 4-mediated respiratory syncytial virus disease and lung transcriptomics in differentially susceptible inbred mouse strains.

Respiratory syncytial virus (RSV) causes severe lower respiratory tract disease in infants, young children, and susceptible adults. The pathogenesis of RSV disease is not fully understood, although toll-like receptor 4 (TLR4)-related innate immune response is known to play a role. The present study was designed to determine TLR4-mediated disease phenotypes and lung transcriptomics and to elucidate transcriptional mechanisms underlying differential RSV susceptibility in inbred strains of mice. Dominant negative Tlr4 mutant (C3H/HeJ, HeJ, Tlr4Lps-d) and its wild-type (C3H/HeOuJ, OuJ, Tlr4Lps-n) mice and five genetically diverse, differentially responsive strains bearing the wild-type Tlr4Lps-n allele were infected with RSV. Bronchoalveolar lavage, histopathology, and genome-wide transcriptomics were used to characterize the pulmonary response to RSV. RSV-induced lung neutrophilia [1 day postinfection (pi)], epithelial proliferation (1 day pi), and lymphocytic infiltration (5 days pi) were significantly lower in HeJ compared with OuJ mice. Pulmonary RSV expression was also significantly suppressed in HeJ than in OuJ. Upregulation of immune/inflammatory (Cxcl3, Saa1) and heat shock protein (Hspa1a, Hsph1) genes was characteristic of OuJ mice, while cell cycle and cell death/survival genes were modulated in HeJ mice following RSV infection. Strain-specific transcriptomics suggested virus-responsive (Oasl1, Irg1, Mx1) and epidermal differentiation complex (Krt4, Lce3a) genes may contribute to TLR4-independent defense against RSV in resistant strains including C57BL/6J. The data indicate that TLR4 contributes to pulmonary RSV pathogenesis and activation of cellular immunity, the inflammasome complex, and vascular damage underlies it. Distinct transcriptomics in differentially responsive Tlr4-wild-type strains provide new insights into the mechanism of RSV disease and potential therapeutic targets.

Viral N6-methyladenosine upregulates replication and pathogenesis of human respiratory syncytial virus

N6-methyladenosine (m6A) is the most prevalent internal modification of mRNAs in most eukaryotes. Here we show that RNAs of human respiratory syncytial virus (RSV) are modified by m6A within discreet regions and that these modifications enhance viral replication and pathogenesis. Knockdown of m6A methyltransferases decreases RSV replication and gene expression whereas knockdown of m6A demethylases has the opposite effect. The G gene transcript contains the most m6A modifications. Recombinant RSV variants expressing G transcripts that lack particular clusters of m6A display reduced replication in A549 cells, primary well differentiated human airway epithelial cultures, and respiratory tracts of cotton rats. One of the m6A-deficient variants is highly attenuated yet retains high immunogenicity in cotton rats. Collectively, our results demonstrate that viral m6A methylation upregulates RSV replication and pathogenesis and identify viral m6A methylation as a target for rational design of live attenuated vaccine candidates for RSV and perhaps other pneumoviruses.