Defining the Assembleome of the Respiratory Syncytial Virus.

During respiratory syncytial virus (RSV) particle assembly, the mature RSV particles form as filamentous projections on the surface of RSV-infected cells. The RSV assembly process occurs at the / on the cell surface that is modified by a virus infection, involving a combination of several different host cell factors and cellular processes. This induces changes in the lipid composition and properties of these lipid microdomains, and the virus-induced activation of associated Rho GTPase signaling networks drives the remodeling of the underlying filamentous actin (F-actin) cytoskeleton network. The modified sites that form on the surface of the infected cells form the nexus point for RSV assembly, and in this review chapter, they are referred to as the RSV assembleome. This is to distinguish these unique membrane microdomains that are formed during virus infection from the corresponding membrane microdomains that are present at the cell surface prior to infection. In this article, an overview of the current understanding of the processes that drive the formation of the assembleome during RSV particle assembly is given.

Ultrastructural and Functional Characterization of Mitochondrial Dynamics Induced by Human Respiratory Syncytial Virus Infection in HEp-2 Cells.

Human respiratory syncytial virus (hRSV) is the leading cause of acute lower respiratory tract infections in children under five years of age and older adults worldwide. During hRSV infection, host cells undergo changes in endomembrane organelles, including mitochondria. This organelle is responsible for energy production in the cell and plays an important role in the antiviral response. The present study focuses on characterizing the ultrastructural and functional changes during hRSV infection using thin-section transmission electron microscopy and RT-qPCR. Here we report that hRSV infection alters mitochondrial morphodynamics by regulating the expression of key genes in the antiviral response process, such as Mfn1, VDAC2, and PINK1. Our results suggest that hRSV alters mitochondrial morphology during infection, producing a mitochondrial phenotype with shortened cristae, swollen matrix, and damaged membrane. We also observed that hRSV infection modulates the expression of the aforementioned genes, possibly as an evasion mechanism in the face of cellular antiviral response. Taken together, these results advance our knowledge of the ultrastructural alterations associated with hRSV infection and might guide future therapeutic efforts to develop effective antiviral drugs for hRSV treatment.

Molten Globule Driven and Self-downmodulated Phase Separation of a Viral Factory Scaffold.

Viral factories of liquid-like nature serve as sites for transcription and replication in most viruses. The respiratory syncytial virus factories include replication proteins, brought together by the phosphoprotein (P) RNA polymerase cofactor, present across non-segmented negative stranded RNA viruses. Homotypic liquid-liquid phase separation of RSV-P is governed by an α-helical molten globule domain, and strongly self-downmodulated by adjacent sequences. Condensation of P with the nucleoprotein N is stoichiometrically tuned, defining aggregate-droplet and droplet-dissolution boundaries. Time course analysis show small N-P nuclei gradually coalescing into large granules in transfected cells. This behavior is recapitulated in infection, with small puncta evolving to large viral factories, strongly suggesting that P-N nucleation-condensation sequentially drives viral factories. Thus, the tendency of P to undergo phase separation is moderate and latent in the full-length protein but unleashed in the presence of N or when neighboring disordered sequences are deleted. This, together with its capacity to rescue nucleoprotein-RNA aggregates suggests a role as a "solvent-protein".

The respiratory syncytial virus SH protein is incorporated into infectious virus particles that form on virus-infected cells.

The association of the SH protein with respiratory syncytial virus (RSV) particles was examined in HEp2 cells and human ciliated nasal epithelial cells. Imaging of infected cells demonstrated the presence of the SH protein in virus filaments, and analysis of purified RSV particles revealed a SH protein species whose size was consistent with the glycosylated SH protein. Although the SH protein was detected in virus filaments it was not required for virus filament formation. Analysis of RSV-infected ciliated cells also revealed that the SH protein was trafficked into the cilia, and this correlated with reduced cilia density on these cells. Reduced cilia loss was not observed on ciliated cells infected with a RSV isolate that failed to express the SH protein. These data provide direct evidence that the SH protein is trafficked into virus particles, and suggests that the SH protein may also promote cilia dysfunction on nasal epithelial cells.

RSV-induced expanded ciliated cells contribute to bronchial wall thickening.

Viral infection, particularly respiratory syncytial virus (RSV), causes inflammation in the bronchiolar airways (bronchial wall thickening, also known as bronchiolitis). This bronchial wall thickening is a common pathophysiological feature in RSV infection, but it causes more fatalities in infants than in children and adults. However, the molecular mechanism of RSV-induced bronchial wall thickening remains unknown, particularly in healthy adults. Using highly differentiated pseudostratified airway epithelium generated from primary human bronchial epithelial cells, we revealed RSV-infects primarily ciliated cells. The infected ciliated cells expanded substantially without compromising epithelial membrane integrity and ciliary functions and contributed to the increased height of the airway epithelium. Furthermore, we identified multiple factors, e.g., cytoskeletal (ARP2/3-complex-driven actin polymerization), immunological (IP10/CXCL10), and viral (NS2), contributing to RSV-induced uneven epithelium height increase in vitro. Thus, RSV-infected expanded cells contribute to a noncanonical inflammatory phenotype, which contributes to bronchial wall thickening in the airway, and is termed cytoskeletal inflammation.

PIK-24 Inhibits RSV-Induced Syncytium Formation via Direct Interaction with the p85α Subunit of PI3K.

Phosphoinositide-3 kinase (PI3K) signaling regulates many cellular processes, including cell survival, differentiation, proliferation, cytoskeleton reorganization, and apoptosis. The actin cytoskeleton regulated by PI3K signaling plays an important role in plasma membrane rearrangement. Currently, it is known that respiratory syncytial virus (RSV) infection requires PI3K signaling. However, the regulatory pattern or corresponding molecular mechanism of PI3K signaling on cell-to-cell fusion during syncytium formation remains unclear. This study synthesized a novel PI3K inhibitor PIK-24 designed with PI3K as a target and used it as a molecular probe to investigate the involvement of PI3K signaling in syncytium formation during RSV infection. The results of the antiviral mechanism revealed that syncytium formation required PI3K signaling to activate RHO family GTPases Cdc42, to upregulate the inactive form of cofilin, and to increase the amount of F-actin in cells, thereby causing actin cytoskeleton reorganization and membrane fusion between adjacent cells. PIK-24 treatment significantly abolished the generation of these events by blocking the activation of PI3K signaling. Moreover, PIK-24 had an obvious binding activity with the p85α regulatory subunit of PI3K. The anti-RSV effect similar to PIK-24 was obtained after knockdown of p85α in vitro or knockout of p85α in vivo, suggesting that PIK-24 inhibited RSV infection by targeting PI3K p85α. Most importantly, PIK-24 exerted a potent anti-RSV activity, and its antiviral effect was stronger than that of the classic PI3K inhibitor LY294002, PI-103, and broad-spectrum antiviral drug ribavirin. Thus, PIK-24 has the potential to be developed into a novel anti-RSV agent targeting cellular PI3K signaling. IMPORTANCE PI3K protein has many functions and regulates various cellular processes. As an important regulatory subunit of PI3K, p85α can regulate the activity of PI3K signaling. Therefore, it serves as the key target for virus infection. Indeed, p85α-regulated PI3K signaling facilitates various intracellular plasma membrane rearrangement events by modulating the actin cytoskeleton, which may be critical for RSV-induced syncytium formation. In this study, we show that a novel PI3K inhibitor inhibits RSV-induced PI3K signaling activation and actin cytoskeleton reorganization by targeting the p85α protein, thereby inhibiting syncytium formation and exerting a potent antiviral effect. Respiratory syncytial virus (RSV) is one of the most common respiratory pathogens, causing enormous morbidity, mortality, and economic burden. Currently, no effective antiviral drugs or vaccines exist for RSV infection. This study contributes to understanding the molecular mechanism by which PI3K signaling regulates syncytium formation and provides a leading compound for anti-RSV infection drug development.

Activation of the Chemokine Receptor CCR1 and Preferential Recruitment of Gαi Suppress RSV Replication: Implications for Developing Novel Respiratory Syncytial Virus Treatment Strategies.

Respiratory syncytial virus (RSV) is a major pathogen that can cause acute respiratory infectious diseases of the upper and lower respiratory tract, especially in children, elderly individuals, and immunocompromised people. Generally, following viral infection, respiratory epithelial cells secrete cytokines and chemokines to recruit immune cells and initiate innate and/or adaptive immune responses. However, whether chemokines affect viral replication in nonimmune cells is rarely clear. In this study, we detected that chemokine CCL5 was highly expressed, while expression of its receptor, CCR1, was downregulated in respiratory epithelial cells after RSV infection. When we overexpressed CCR1 on respiratory epithelial cells in vivo or in vitro, viral load was significantly suppressed, which can be restored by the neutralizing antibody for CCR1. Interestingly, the antiviral effect of CCR1 was not related to type I interferon (IFN-I), apoptosis induction, or viral adhesion or entry inhibition. In contrast, it was related to the preferential recruitment and activation of the adaptor Gαi, which promoted inositol 1,4,5-triphosphate receptor type 3 (ITPR3) expression, leading to inhibited STAT3 phosphorylation; explicitly, phosphorylated STAT3 (p-STAT3) was verified to be among the important factors regulating the activity of HSP90, which has been previously reported to be a chaperone of RSV RNA polymerase. In summary, we are the first to reveal that CCR1 on the surface of nonimmune cells regulates RSV replication through a previously unknown mechanism that does not involve IFN-I induction. IMPORTANCE Our results revealed a novel mechanism by which RSV escapes innate immunity. That is, although it induces high CCL5 expression, RSV might attenuate the binding of CCL5 by downregulating the expression of CCR1 in respiratory epithelial cells to weaken the inhibitory effect of CCR1 on HSP90 activity and thereby facilitate RSV replication in nonimmune cells. This study provides a new target for the development of co-antiviral inhibitors against other components of the host and co-molecular chaperone/HSP90 and provides a scientific basis for the search for effective broad-spectrum antiviral drugs.

Biophysical studies of the interaction of hRSV Non-Structural 1 protein with natural flavonoids and their acetylated derivatives by spectroscopic techniques and computational simulations.

Human respiratory syncytial virus (hRSV) infections are one of the most causes of acute lower respiratory tract infections in children and elderly. The development of effective antiviral therapies or preventive vaccines against hRSV is not available yet. Thus, it is necessary to search for protein targets to combat this viral infection, as well as potential ways to block them. Non-Structural 1 (NS1) protein is an important factor for viral replication success since reduces the immune response by interacting with proteins in the type I interferon pathway. The influence of NS1 on the cell's immune response denotes the potential of its inhibition, being a possible target of treatment against hRSV infection. Here, it was studied the interaction of hRSV NS1 with natural flavonoids chrysin, morin, kaempferol, and myricetin and their mono-acetylated chrysin and penta-acetylated morin derivatives using spectroscopic techniques and computational simulations. The fluorescence data indicate that the binding affinities are on the order of 105 M-1, which are directly related to the partition coefficient of each flavonoid with Pearson's correlation coefficients of 0.76-0.80. The thermodynamic analysis suggests that hydrophobic interactions play a key role in the formation of the NS1/flavonoid complexes, with positive values of enthalpy and entropy changes. The computational approach proposes that flavonoids bind in a region of NS1 formed between the C-terminal α3-helix and the protein core, important for its biological function, and corroborate with experimental data revealing that hydrophobic contacts are important for the binding. Therefore, the present study provides relevant molecular details for the development of a possible new strategy to fight infections caused by hRSV.

Importance of RNA length for in vitro encapsidation by the nucleoprotein of human respiratory syncytial virus.

Respiratory syncytial virus has a negative-sense single-stranded RNA genome constitutively encapsidated by the viral nucleoprotein N, forming a helical nucleocapsid which is the template for viral transcription and replication by the viral polymerase L. Recruitment of L onto the nucleocapsid depends on the viral phosphoprotein P, which is an essential L cofactor. A prerequisite for genome and antigenome encapsidation is the presence of the monomeric, RNA-free, neosynthesized N protein, named N0. Stabilization of N0 depends on the binding of the N-terminal residues of P to its surface, which prevents N oligomerization. However, the mechanism involved in the transition from N0-P to nucleocapsid assembly, and thus in the specificity of viral genome encapsidation, is still unknown. Furthermore, the specific role of N oligomerization and RNA in the morphogenesis of viral factories, where viral transcription and replication occur, have not been elucidated although the interaction between P and N complexed to RNA has been shown to be responsible for this process. Here, using a chimeric protein comprising N and the first 40 N-terminal residues of P, we succeeded in purifying a recombinant N0-like protein competent for RNA encapsidation in vitro. Our results showed the importance of RNA length for stable encapsidation and revealed that the nature of the 5' end of RNA does not explain the specificity of encapsidation. Finally, we showed that RNA encapsidation is crucial for the in vitro reconstitution of pseudo-viral factories. Together, our findings provide insight into respiratory syncytial virus viral genome encapsidation specificity.

Retinoic Acid-Inducible Gene I Activation Inhibits Human Respiratory Syncytial Virus Replication in Mammalian Cells and in Mouse and Ferret Models of Infection.

Infections caused by human respiratory syncytial virus (RSV) are associated with substantial rates of morbidity and mortality. Treatment options are limited, and there is urgent need for the development of efficient antivirals. Pattern recognition receptors such as the cytoplasmic helicase retinoic acid-inducible gene (RIG) I can be activated by viral nucleic acids, leading to activation of interferon-stimulated genes and generation of an "antiviral state." In the current study, we activated RIG-I with synthetic RNA agonists (3pRNA) to induce resistance to RSV infection in vitro and in vivo. In vitro, pretreatment of human, mouse, and ferret airway cell lines with RIG-I agonist before RSV exposure inhibited virus infection and replication. Moreover, a single intravenous injection of 3pRNA 1 day before RSV infection resulted in potent inhibition of virus replication in the lungs of mice and ferrets, but not in nasal tissues. These studies provide evidence that RIG-I agonists represent a promising antiviral drug for RSV prophylaxis.

Neutrophil Extracellular Traps Do Not Induce Injury and Inflammation in Well-Differentiated RSV-Infected Airway Epithelium.

Respiratory syncytial virus (RSV) lower respiratory tract infection (LRTI) causes a major burden of disease. The host response in RSV-LRTI is characterized by airway epithelial injury, inflammation and neutrophil influx, with the formation of neutrophil extracellular traps (NETs). However, the precise role of NETs in the pathophysiology of RSV-LRTI remains to be elucidated. Here, we used well-differentiated human airway epithelial cultures (HAE) of a pediatric and adult donor to study whether NETs cause airway epithelial injury and inflammation in the setting of RSV infection. The exposure of uninfected and RSV-infected HAE cultures to NETs, as produced by stimulation of neutrophils by a low dose of phorbol 12-myristate 13-acetate (PMA), did not induce or aggravate cell injury or inflammation. RSV infection of HAE cultures caused release of pro-inflammatory cytokines such as IL-6 and RANTES in both adult and pediatric cultures, but the differential gene expression for regulated cell death differed between culture donors. In this in vitro airway epithelial model, NETs in the setting of RSV infection did not cause or aggravate epithelial injury or inflammation.

RSV-induced changes in a 3-dimensional organoid model of human fetal lungs.

We have shown that respiratory syncytial virus (RSV) can spread hematogenously from infected airways of a pregnant woman to the developing fetal lungs in utero. This study sought to measure RSV replication, cytopathic effects, and protein expression in human lung organoids (HLOs) reproducing architecture and transcriptional profiles of human fetal lungs during the 1st trimester of gestation. HLOs derived from human pluripotent stem cells were microinjected after 50 or 100 days in culture with medium or recombinant RSV-A2 expressing the red fluorescent protein gene (rrRSV). Infection was monitored by fluorescent microscopy and PCR. Immunohistochemistry and proteomic analysis were performed. RSV infected HLOs in a dose- and time-dependent manner. RSV-infected HLOs increased expression of CC10 (Club cells), but had sparse FOXJ1 (ciliated cells). Disruption of F-actin cytoskeleton was consistent with proteomic data showing a significant increase in Rho GTPases proteins. RSV upregulated the transient receptor potential vanilloid 1 (TRPV1) channel and, while ß2 adrenergic receptor (ß2AR) expression was decreased overall, its phosphorylated form increased. Our data suggest that prenatal RSV infection produces profound changes in fetal lungs' architecture and expression profiles and maybe an essential precursor of chronic airway dysfunction. expression profiles, and possibly be an important precursor of chronic airway dysfunction.

Short-chain fatty acid acetate triggers antiviral response mediated by RIG-I in cells from infants with respiratory syncytial virus bronchiolitis.

BACKGROUND: Gut microbiota-derived short-chain fatty-acid (SFCA) acetate protects mice against RSV A2 strain infection by increasing interferon-ß production and expression of interferon-stimulated genes (ISGs). However, the role of SFCA in RSV infection using strains isolated from patients is unknown. METHODS: We first used RSV clinical strains isolated from infants hospitalized with RSV bronchiolitis to investigate the effects of in vitro SCFA-acetate treatment of human pulmonary epithelial cells. We next examined whether SCFA-acetate treatment is beneficial in a mouse model of RSV infection using clinical isolates. We sought to investigate the relationship of gut microbiota and fecal acetate with disease severity among infants hospitalized with RSV bronchiolitis, and whether treating their respiratory epithelial cells with SCFA-acetate ex-vivo impacts viral load and ISG expression. We further treated epithelial cells from SARS-CoV-2 infected patients with SCFA-acetate. FINDINGS: In vitro pre-treatment of A549 cells with SCFA-acetate reduced RSV infection with clinical isolates and increased the expression of RIG-I and ISG15. Animals treated with SCFA-acetate intranasally recovered significantly faster, with reduction in the RSV clinical isolates viral load, and increased lung expression of IFNB1 and the RIG-I. Experiments in RIG-I knockout A549 cells demonstrated that the protection relies on RIG-I presence. Gut microbial profile was associated with bronchiolitis severity and with acetate in stool. Increased SCFA-acetate levels were associated with increasing oxygen saturation at admission, and shorter duration of fever. Ex-vivo treatment of patients' respiratory cells with SCFA-acetate reduced RSV load and increased expression of ISGs OAS1 and ISG15, and virus recognition receptors MAVS and RIG-I, but not IFNB1. These SCFA-acetate effects were not found on cells from SARS-CoV-2 infected patients. INTERPRETATION: SCFA-acetate reduces the severity of RSV infection and RSV viral load through modulation of RIG-I expression. FUNDING: FAPERGS (FAPERGS/MS/CNPq/SESRS no. 03/2017 - PPSUS 17/2551-0001380-8 and COVID-19 20/2551-0000258-6); CNPq 312504/2017-9; CAPES) - Finance Code 001.

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.

Chitinase 3-like 1 is involved in the induction of IL-8 expression by double-stranded RNA in airway epithelial cells.

Viral respiratory infection causes inflammatory lung disease. Chitinase 3-like 1 (CHI3L1) contributes to airway inflammation, but its role in human airway epithelial cells following viral infection is unclear. Thus, we investigated whether CHI3L1 regulates inflammatory responses caused by viral infections in airway epithelial cells. Human bronchial epithelial cells, BEAS-2B, were stimulated with a synthetic analog of viral double-stranded RNA, polyinosinic:polycytidylic acid (poly(I:C)). To confirm the specific role of CHI3L1, CHI3L1 was knocked down in BEAS-2B cells using shRNA lentivirus. The expression of CHI3L1 and proinflammatory cytokines such as IL-8 and phosphorylation of mitogen-activated protein kinase (MAPK) pathways were analyzed. In addition to poly(I:C), BEAS-2B cells were infected with the human respiratory syncytial virus (RSV) A2 strain, and CHI3L1 and IL-8 expression was analyzed. Stimulating the cells with poly(I:C) increased CHI3L1 and IL-8 expression, whereas IL-8 expression was abrogated in CHI3L1 knockdown BEAS-2B cells. Poly(I:C) stimulation of BEAS-2B cells resulted in phosphorylation of MAPK pathways, and inhibition of MAPK pathways significantly abolished IL-8 secretion. Phosphorylation of MAPK pathways was diminished in CHI3L1 knockdown BEAS-2B cells. Infection with RSV increased CHI3L1 and IL-8 expression. IL-8 expression induced by RSV infection was abrogated in CHI3L1 knockdown cells. In conclusion, CHI3L1 may be involved in IL-8 secretion by regulating MAPK pathways during respiratory viral infections in airway epithelial cells.

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.

4'-Fluorouridine is an oral antiviral that blocks respiratory syncytial virus and SARS-CoV-2 replication.

The COVID-19 pandemic has underscored the critical need for broad-spectrum therapeutics against respiratory viruses. Respiratory syncytial virus (RSV) is a major threat to pediatric patients and older adults. We describe 4'-fluorouridine (4'-FlU, EIDD-2749), a ribonucleoside analog that inhibits RSV, related RNA viruses, and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), with high selectivity index in cells and human airway epithelia organoids. Polymerase inhibition within in vitro RNA-dependent RNA polymerase assays established for RSV and SARS-CoV-2 revealed transcriptional stalling after incorporation. Once-daily oral treatment was highly efficacious at 5 milligrams per kilogram (mg/kg) in RSV-infected mice or 20 mg/kg in ferrets infected with different SARS-CoV-2 variants of concern, initiated 24 or 12 hours after infection, respectively. These properties define 4'-FlU as a broad-spectrum candidate for the treatment of RSV, SARS-CoV-2, and related RNA virus infections.

Viral burden and diversity in acute respiratory tract infections in hospitalized children in wet and dry zones of Sri Lanka.

The present study was done to identify the viral diversity, seasonality and burden associated with childhood acute respiratory tract infection (ARTI) in Sri Lanka. Nasopharyngeal aspirates (NPA) of hospitalized children (1 month-5 years) with ARTI were collected in 2 centers (wet and dry zones) from March 2013 to August 2014. Respiratory viral antigen detection by immunofluorescence assay (IFA) was used to identify the infecting viruses. IFA negative 100 NPA samples were tested for human metapeumovirus (hMPV), human bocavirus and corona viruses by polymerase chain reaction. Of the 443 and 418 NPAs, 37.2% and 39.4% were positive for any of the 8 different respiratory viruses tested from two centers studied. Viral co-infection was detected with respiratory syncytial virus (RSV) in both centers. Peak viral detection was noted in the wet zone from May-July 2013 and 2014 and in the dry zone from December-January 2014 suggesting a local seasonality for viral ARTI. RSV showed a clear seasonality with a direct correlation of monthly RSV infections with rainy days in the wet zone and an inverse correlation with temperature in both centers. The case fatality rate was 2.7% for RSV associated ARTI. The overall disability adjusted life years was 335.9 and for RSV associated ARTI it was 241.8. RSV was the commonly detected respiratory virus with an annual seasonality and distribution in rainy seasons in the dry and wet zones of Sri Lanka. Identifying the virus and seasonality will contribute to employ preventive measures and reduce the empirical use of antibiotics in resource limited settings.

The Cellular and Viral circRNAome Induced by Respiratory Syncytial Virus Infection.

Circular RNAs (circRNAs) are a new class of noncoding RNAs that have gained increased attention. DNA virus infections have been reported to induce modifications in cellular circRNA transcriptomes and express viral circRNAs. However, the identification and expression of cellular and viral circRNAs are unknown in the context of respiratory syncytial virus (RSV), a human RNA virus with no effective treatments or vaccines. Here, we report a comprehensive identification of the cellular and viral circRNAs induced by RSV infection in A549 cells with high-throughput sequencing. In total, 53,719 cellular circRNAs and 2,280 differentially expressed cellular circRNAs were identified. Trend analysis further identified three significant expression pattern clusters, which were related to the antiviral immune response according to gene enrichment analysis. Subsequent results showed that not only RSV infection but also poly(I·C) treatment and another RNA virus infection induced the upregulation of the top 10 circRNAs from the focused cluster. The top 10 circRNAs generally inhibit RSV replication in turn. Moreover, 1,254 viral circRNAs were identified by the same circRNA sequencing. The induced expression of viral circRNAs by RSV infection was found not only in A549 cells but also in HEp-2 cells. Additionally, we profiled the general characteristics of both cellular and viral circRNAs such as back-splicing signals, etc. Collectively, RSV infection induced the differential expression of cellular circRNAs, some of which affected RSV infection, and RSV also expressed viral circRNAs. Our study reveals novel layers of host-RSV interactions and identifies cellular or viral circRNAs that may be novel therapeutic targets or biomarkers. IMPORTANCE Noncoding RNAs (ncRNAs) demonstrate substantial roles in cell-virus interactions. Circular RNAs (circRNAs) are a newly identified class of ncRNAs that have gained increased attention recently. DNA virus infections have been reported to induce modifications in cellular circRNA transcriptomes and express viral circRNAs. However, the identification and expression of cellular and viral circRNAs are unknown in the context of respiratory syncytial virus (RSV), a human RNA virus with no effective treatments or vaccines. Here, we report a comprehensive identification of the cellular and viral circRNAs induced by RSV infection by high-throughput sequencing. We revealed that RSV infection induces the differential expression of cellular circRNAs, some of which affected RSV infection, and that RSV also expresses viral circRNAs. Our study reveals novel layers of host-RSV interactions and identifies cellular or viral circRNAs that may be novel therapeutic targets or biomarkers.