OBJECTIVE: To observe the therapeutic effects of urolithin A (UA) on respiratory syncytial virus (RSV)-induced lung infection in neonatal mice and explore the underlying mechanisms.
METHODS: Babl/c mice (5-7 days old) were subjected to nasal instillation of RSV and received intraperitoneal injection of saline or 2.5, 5 and 10 mg/kg UA 2 h after the infection and then once daily for 2 weeks. Bronchoalveolar lavage fluid (BALF) was then collected for detection of inflammatory cells and mediators, and lung pathology was evaluated with HE staining. RSV-infected BEAS-2B cells were treated with 2.5, 5 or 10 µmol/ L UA. Inflammatory factors, cell viability, apoptosis and autophagy were analyzed using ELISA, CCK-8 assay, TUNEL staining, flow cytometry, Western blotting and immunofluorescence staining. The cellular expressions of miR-136 and Sirt1 mRNAs were detected using qRT-PCR. A dual-luciferase reporter system was used to verify the binding between miR-136 and Sirt1.
RESULTS: In neonatal Babl/c mice, RSV infection caused obvious lung pathologies, promoted pulmonary cell apoptosis and LC3-Ⅱ/Ⅰ, Beclin-1 and miR-136 expressions, and increased the total cell number, inflammatory cells and factors in the BALF and decreased p62 and Sirt1 expressions. All these changes were alleviated dose-dependently by UA. In BEAS-2B cells, RSV infection significantly increased cell apoptosis, LC3B-positive cells and miR-136 expression and reduced Sirt1 expression (P<0.01), which were dose-dependently attenuated by UA. Dual-luciferase reporter assay confirmed the binding between miR-136 and Sirt1. In RSV-infected BEAS-2B cells with UA treatment, overexpression of miR-136 and Ex527 treatment both significantly increased the inflammatory factors and cell apoptosis but decreased LC3B expression, and these changes were further enhanced by their combined treatment.
CONCLUSIONS: UA ameliorates RSV-induced lung infection in neonatal mice by activating miR-136-mediated Sirt1 signaling pathway.

Respiratory syncytial virus is the major cause of respiratory viral infections, particularly in infants, immunocompromised populations, and the elderly (over 65 years old), the prevention of RSV infection has become a priority. In this study, we generated a chimeric influenza virus, termed LAIV/RSV/HA-3F, using reverse genetics technology which contained three repeats of the RSV fusion protein neutralizing epitope site II to the N terminal in the background of the hemagglutinin (HA) gene of cold adapted influenza vaccine A/California/7/2009 ca. LAIV/RSV/HA-3F exhibited cold-adapted (ca) and attenuated (att) phenotype. BALB/c mice immunized intranasally with LAIV/RSV/HA-3F showed robust immunogenicity, inducing viral-specific antibody responses against both influenza and RSV, eliciting RSV-specific humoral, cellular and mucosal immune responses. LAIV/RSV/HA-3F also conferred protection as indicated by reduced viral titers and improved lung histopathological alterations against live RSV virus challenge. Mechanismly, single-cell RNA sequencing (scRNA-seq) and single-cell T cell antigen receptor (TCR) sequencing were employed to characterize the immune responses triggered by chimeric RSV vaccine, displaying that LAIV/RSV/HA-3F provided protection mainly via interferon-γ (IFN-γ). Moreover, we found that LAIV/RSV/HA-3F significantly inhibited viral replication in the challenged lung and protected against subsequent RSV challenge in cotton rats without causing lung disease. Taken together, our findings demonstrated that LAIV/RSV/HA-3F has potential as a promising bivalent vaccine with dual purpose candidate for the prevention of influenza and RSV, and preclinical and clinical studies warrant further investigations.

To investigate the status and epidemiological characteristics of respiratory pathogens infections in children with influenza-like illnesses (ILI) in Beijing Children\'s Hospital from 2022 to 2023. A dual amplification technique was used to detect nucleic acids of seven common respiratory pathogens, including influenza A virus (Flu A), influenza B virus (Flu B), mycoplasma pneumoniae (MP), respiratory syncytial virus (RSV), parainfluenza virus (PIV), adenovirus (ADV), and Chlamydia pneumoniae (CP), in outpatient and inpatient children (aged 0-18 years) with influenza-like symptoms who sought medical care at Beijing Children\'s Hospital, from January 2022 to March 2023. A total of 43 663 children were included in the study, of which 27 903 tested positive for respiratory pathogens with a total detection rate of 63.91%. Flu A had the highest detection rate of 69.93% (27 332/39 084), followed by MP about 13.22% (380/2 875). The total detection rate of RSV, PIV and ADV was 7.69% (131/1 704). Flu B had a detection rate of 0.16% (64/39 084). No CP was detected in this study. A total of 7 cases of dual infections were detected, with a detection rate of 0.41% (7/1 704). The Chi-square test was used to analyze the differences in detection rates of pathogens among different genders, age groups, and different seasons. Among the seven pathogens, only Flu A had statistically significant differences in gender (χ2=16.712, P<0.001). The detection rates of Flu A and MP showed an increasing trend with age (both P trend<0.001), while the detection rates of RSV and PIV showed a decreasing trend with age (both P trend<0.001). Flu A had its epidemic peak in winter and spring, with detection rates of 61.30% (3 907/6 374) and 77.47% (23 207/29 958) respectively; MP and PIV had higher detection rates in autumn (25.14% and 7.64% respectively); RSV showed a relatively higher detection rate in winter (8.69%); Flu B and ADV had lower detection rates throughout the study period (0.16% and 1.17% respectively). In conclusion, children with ILI in 2022-2023 were mainly infected with a single respiratory pathogen, and occasionally dual pathogen infections were observed. Among them, the detection rate of Flu A was the highest, and only Flu A showed a gender difference in detection rate. As the age of the children patients increased, the detection rate of Flu A and MP showed an increasing trend, while RSV and PIV showed a decreasing trend. The prevalence of Flu A, Flu B, MP, PIV, and RSV were seasonal.
分析2022—2023年北京儿童医院流感样疾病（ILI）患儿呼吸道病原体感染情况及流行病学特征。于2022年1月至2023年3月，采用双扩增技术对就诊于北京儿童医院有流感样症状的门诊和住院患儿（0~18岁）进行甲型流感病毒（Flu A）、乙型流感病毒（Flu B）、肺炎支原体（MP）、呼吸道合胞病毒（RSV）、副流感病毒（PIV）、腺病毒（ADV）和肺炎衣原体（CP）7种常见呼吸道病原体核酸检测。共检测患儿43 663例，其中呼吸道病原体阳性27 903例，总检出率为63.91%。Flu A检出率居首位为69.93%（27 332/39 084）；其后是MP为13.22%（380/2 875）；RSV，PIV和ADV 这3种病原体总检出率为7.69%（131/1 704）；Flu B为0.16%（64/39 084）；未检出CP。双重感染共检出7例，检出率为0.41%（7/1 704）。采用χ2检验对病原体在不同性别、年龄组和不同季节间检出率的差异进行分析显示，7种病原体中，仅Flu A在性别上差异有统计学意义（χ2=16.712，P<0.001）。Flu A和MP病原体检出率呈现随年龄增长而增长的趋势（均P趋势<0.001），而RSV和PIV检出率呈现随年龄增长而降低的趋势（均P趋势<0.001）。Flu A在冬季和春季呈流行高峰，检出率分别为61.30%（3 907/6374）和77.47%（23 207/29 958）；MP和PIV在秋季检出率较高（分别为25.14%和7.64%）；RSV在冬季出现流行高峰（检出率为8.69%）；研究期间Flu B和ADV检出率较低（分别为0.16%和1.17%）。综上，2022—2023年流感样疾病患儿以单种呼吸道病原体感染为主，偶见双重病原体感染；其中 Flu A 检出率最高，仅Flu A的检出率有性别差异；随着患儿年龄的增长，Flu A、MP检出率呈增长趋势，而RSV和PIV呈现降低趋势。Flu A、Flu B、MP、PIV和RSV的流行有季节性。.

OBJECTIVE: The study evaluated the antiviral effect of Verapamil against respiratory syncytial virus (RSV) and investigated its underlying mechanism.
METHODS: RSV-infected BALB/c mice were treated with Verapamil. Body weight, survival rates, viral load, lung damage, inflammatory factors, and the expression of RSV fusion (F) protein were analyzed. In cellular studies, intracellular Ca2+ and viral titers were measured in the presence of Verapamil, Calcium Chloride, and EGTA. A time-of-addition assay assessed the antiviral effect of Verapamil.
RESULTS: Mice infected with RSV and treated with Verapamil exhibited a significant decrease in weight loss, an increase in survival rates, and reductions in viral titers, RSV F protein expression, inflammatory responses, and lung tissue injury. Verapamil reduced intracellular calcium levels, which correlated with reduced viral titers. The addition of calcium chloride reversed the anti-viral effects mediated by Verapamil, while EGTA potentiated them. The antiviral activity of Verapamil was observed during the early phase of RSV infection, likely by blocking Ca2+ channels and inhibiting virus replication.
CONCLUSIONS: Verapamil effectively inhibits RSV infection by blocking calcium channels and reducing intracellular calcium levels, thereby impeding viral replication. Thus, Verapamil shows promise as a treatment for RSV.

Ten C-geranylated flavonoids, along with three known analogues, were isolated from the leaves of Artocarpus communis. The chemical structures of these compounds were unambiguously determined via comprehensive spectroscopic analysis, single-crystal X-ray diffraction experiments, and quantum chemical electronic circular dichroism calculations. Structurally, artocarones A-I (1-9) represent a group of unusual, highly modified C-geranylated flavonoids, in which the geranyl chain is cyclised with the ortho-hydroxy group of flavonoids to form various heterocyclic scaffolds. Notably, artocarones E and G-I (5 and 7-9) feature a 6H-benzo[c]chromene core that is hitherto undescribed in C-geranylated flavonoids. Artocarone J (10) is the first example of C-9-C-16 connected C-geranylated aurone. Meanwhile, the plausible biosynthetic pathways for these rare C-geranylated flavonoids were also proposed. Notably, compounds 1, 2, 4, 8, 11, and 12 exhibited promising in vitro inhibitory activities against respiratory syncytial virus and herpes simplex virus type 1.

BACKGROUND: WuHuTang (WHT) is a traditional Chinese medicine compound for treating asthma, and the evidence supports that it has a good effect on acute asthma attacks in children and adults. Respiratory syncytial virus (RSV) is an important factor in the pathogenesis of acute asthma attacks, and the effect on dendritic cells is the key to its pathogenesis. Previous studies have confirmed that the pathogenesis of viruses is related to exosomes. However, there are few studies on the exosomes induced by RSV. Whether WHT can improve the changes caused by RSV-induced exosomes or not is worthy of further exploration.
OBJECTIVE: We aim to study the effects of RSV-induced exosomes on the function and autophagy of dendritic cells, and to observe the intervention effect of WHT serum on the above effects.
METHODS: The co-culture model of exosomes derived from bone marrow mesenchymal stem cells induced by RSV (BMSCs-Exo-RSV) and dendritic cells was established, and then WHT serum was used to intervene. After 24 h of intervention, the CCK-8 method, flow cytometry, Elisa, RT-qCPR, and Western blot were used to detect the above-mentioned culture model.
RESULTS: RSV-induced exosomes had certain effects on viability, apoptosis, and costimulatory molecules generation of dendritic cells. At the same time, the levels of IL-6, IL-12, TNF-α, and autophagy increased, while the levels of IL-4, IL-10, and TGF-β decreased, and the AKT/TSC/mTOR pathway was inhibited. WHT serum could activate this pathway and reverse the above changes in dendritic cells.
CONCLUSIONS: This study reveals that the pathogenic effect of RSV is related to the exosomes induced by RSV. The exosomes induced by RSV affect the function of dendritic cells by inhibiting the AKT/TSC/mTOR pathway, which can be activated by WHT to reverse the effects caused by RSV-induced exosomes.

BACKGROUND: Quercetin has received extensive attention for its therapeutic potential treating respiratory syncytial virus (RSV) infection diseases. Recent studies have highlighted quercetin\'s ability of suppressing alveolar macrophages (AMs)-derived lung inflammation. However, the anti-inflammatory mechanism of quercetin against RSV infection still remains elusive.
OBJECTIVE: This study aims to elucidate the mechanism about quercetin anti-inflammatory effect on RSV infection.
METHODS: BALB/c mice were intranasally infected with RSV and received quercetin (30, 60, 120 mg/kg/d) orally for 3 days. Additionally, an in vitro infection model utilizing mouse alveolar macrophages (MH-S cells) was employed to validate the proposed mechanism.
RESULTS: Quercetin exhibited a downregulatory effect on glycolysis and tricarboxylic acid (TCA) cycle metabolism in RSV-infected AMs. However, it increased itaconic acid production, a metabolite derived from citrate through activating immune responsive gene 1 (IRG1), and further inhibiting succinate dehydrogenase (SDH) activity. While the suppression of SDH activity orchestrated a cascading downregulation of Hif-1α/NLRP3 signaling, ultimately causing AMs polarization from M1 to M2 phenotypes.
CONCLUSIONS: Our study demonstrated quercetin stimulated IRG1-mediated itaconic acid anabolism and further inhibited SDH/Hif-1α/NLRP3 signaling pathway, which led to M1 to M2 polarization of AMs so as to ameliorate RSV-induced lung inflammation.

The aim of this study was to prepare a mouse monoclonal antibody against the nonstructural protein 1 (NS1) of respiratory syncytial virus (RSV) to analyze its expression and distribution during transfection and infection. Additionally, we aimed to evaluate the antibody\'s application in immunoprecipitation assay. Firstly, the NS1 gene fragment was cloned into a prokaryotic plasmid and expressed in Escherichia coli. The resulting NS1 protein was then purified by affinity chromatography, and used to immunize the BALB/c mice. Subsequently, hybridoma cells capable of stably secreting the NS1 monoclonal antibody were selected using indirect enzyme linked immunosorbent assay (ELISA). This monoclonal antibody was employed in both indirect immunofluorescence assay (IFA) and Western blotting to analyze the expression and distribution of RSV NS1 in overexpressed and infected cells. Finally, the reliability of this monoclonal antibody was evaluated through the immunoprecipitation assay. The results showed that the RSV NS1 protein was successfully expressed and purified. Following immunization of mice with this protein, we obtained a highly specific RSV NS1 monoclonal antibody, which belonged to the IgG1 subtype with an antibody titer of 1:15 360 000. Using this monoclonal antibody, the RSV NS1 protein was identified in both transfected and infected cells. The IFA results revealed predominant distribution of NS1 in the cytoplasm and nucleus. Moreover, we confirmed that this monoclonal antibody could effectively bind specifically to NS1 protein in cell lysates, making it suitable as a capture antibody in immunoprecipitation assay. In conclusion, our study successfully achieved production of the RSV NS1 protein through a prokaryotic expression system and prepared a specific monoclonal antibody against NS1. This antibody demonstrates the ability to specifically identify the NS1 protein and can be used in the immunoprecipitation assay, thereby laying a foundation for the functional studies of the NS1 protein.

Respiratory syncytial virus (RSV) is the primary cause of bronchiolitis-related hospitalizations among children under 5 years of age, with reinfection being common throughout life. Maternal vaccination has emerged as a promising strategy, delivering elevated antibody levels to newborns for immediate protection. However, limited research has explored the protective efficacy of maternal antibodies (matAbs) against secondary RSV infections in offspring. To address this gap, we employed a mouse model of maternal RSV vaccination and secondary infection of offspring to evaluate lung pathology following RSV reinfection in mice with varying levels of maternal antibody (matAb). Additionally, we aimed to investigate the potential causes of exacerbated lung inflammation in offspring with high matAb levels following secondary RSV exposure. Our findings revealed that offspring with elevated levels of maternal pre-F antibody demonstrated effective protection against lung pathology following the initial RSV infection. However, this protection was compromised upon reinfection, manifesting as heightened weight loss, exacerbated lung pathology, increased expression of RSV-A N genes, eosinophilia, enhanced IL-5, IL-13, MUC5AC, and eosinophils Major Basic Protein (MBP) production in lung tissue compared to offspring lacking matAbs. Importantly, these unexpected outcomes were not attributed to antibody-dependent enhancement (ADE) resulting from declining matAb levels over time. Notably, our findings showed a decline in secretory IgA (sIgA), mucosal IgA, and mucosal IgG levels in offspring with high matAb levels post-primary RSV challenge. We propose that this decline may be a critical factor contributing to the ineffective protection observed during secondary RSV exposure. Overall, these findings offer valuable insights into maternal vaccination against RSV, contributing to a comprehensive understanding and mitigation of potential risks associated with maternal RSV vaccination.

Rapid and accurate detection of respiratory virus aerosols is highlighted for virus surveillance and infection control. Here, we report a wireless immunoassay technology for fast (within 10 min), on-site (wireless and battery-free), and sensitive (limit of detection down to fg/L) detection of virus antigens in aerosols. The wireless immunoassay leverages the immuno-responsive hydrogel-modulated radio frequency resonant sensor to capture and amplify the recognition of virus antigen, and flexible readout network to transduce the immuno bindings into electrical signals. The wireless immunoassay achieves simultaneous detection of respiratory viruses such as severe acute respiratory syndrome coronavirus 2, influenza A H1N1 virus, and respiratory syncytial virus for community infection surveillance. Direct detection of unpretreated clinical samples further demonstrates high accuracy for diagnosis of respiratory virus infection. This work provides a sensitive and accurate immunoassay technology for on-site virus detection and disease diagnosis compatible with wearable integration.