p53 suppresses the inflammatory response following respiratory syncytial virus infection by inhibiting TLR2.

-Respiratory syncytial virus (RSV) is a pivotal virus leading to acute lower respiratory tract infections in children under 5 years old. This study aimed to explore the correlation between p53 and Toll-like receptors (TLRs) post RSV infection. p53 levels exhibited a substantial decrease in nasopharyngeal aspirates (NPAs) from infants with RSV infection compared to control group. Manipulating p53 expression had no significant impact on RSV replication or interferon signaling pathway. Suppression of p53 expression led to heightened inflammation following RSV infection in A549 cells or airways of BALB/c mice. while stabilizing p53 expression using Nutlin-3a mitigated the inflammatory response in A549 cells. Additionally, Inhibiting p53 expression significantly increased Toll-like receptor 2 (TLR2) expression in RSV-infected epithelial cells and BALB/c mice. Furthermore, the TLR2 inhibitor, C29, effectively reduced inflammation mediated by p53 in A549 cells. Collectively, our results indicate that p53 modulates the inflammatory response after RSV infection through TLR2.

Genome and proteomic analysis of risk factors for fatal outcome in children with severe community-acquired pneumonia caused by human adenovirus 7.

INTRODUCTION: Human adenovirus 7 (HAdV-7) is an important viral pathogen of severe pneumonia in children and a serious threat to health. METHODS: A cohort of 45 pediatric patients diagnosed with HAdV-7-associated severe pneumonia and admitted to the Pediatric Intensive Care Unit at the Children's Hospital of Chongqing Medical University from May 2018 to January 2020 were included. Risk factors of death were analyzed by the Cox proportional risk mode with Clinical data, serum, and nasopharyngeal aspirate adenovirus load, Genome analysis, Olink proteomics, and cytokine profile between dead and surviving patients were also analyzed. RESULTS: A total of 45 children with a median age of 12.0 months (interquartile range [IQR]: 6.5, 22.0) were included (female 14), including 14 (31.1%) who died. High serum viral load was an independent risk factor for mortality (hazard ratio [HR] = 2.16, 95% confidence interval [CI], 1.04-4.49, p = 0.039). BTB and CNC homology 1 (BACH1), interleukin-5 (IL-5), and IL-9 levels were significantly correlated with serum viral load (p = 0.0400, 0.0499, and 0.0290; r = 0.4663, 0.3339, and -0.3700, respectively), with significant differences between the dead and survival groups (p = 0.021, 0.001, and 0.021). CONCLUSIONS: Severe cytokine storm-associated high serum viral load after HAdV-7 infection may be the main mechanism responsible for poor prognosis in children.

Identification of functional pathways and potential genes associated with interferon signaling during human adenovirus type 7 infection by weighted gene coexpression network analysis.

Human adenovirus type 7 (HAdV-7) can cause severe pneumonia and complications in children. However, the mechanism of pathogenesis and the genes involved remain largely unknown. We collected HAdV-7-infected and mock-infected A549 cells at 24, 48, and 72 hours postinfection (hpi) for RNA sequencing (RNA-Seq) and identified potential genes and functional pathways associated with HAdV-7 infection using weighted gene coexpression network analysis (WGCNA). Based on bioinformatics analysis, 12 coexpression modules were constructed by WGCNA, with the blue, tan, and brown modules significantly positively correlated with adenovirus infection at 24, 48, and 72 hpi, respectively. Functional enrichment analysis indicated that the blue module was mainly enriched in DNA replication and viral processes, the tan module was largely enriched in metabolic pathways and regulation of superoxide radical removal, and the brown module was predominantly enriched in regulation of cell death. qPCR was used to determine transcript abundance of some identified hub genes, and the results were consistent with those from RNA-Seq. Comprehensively analyzing hub genes and differentially expressed genes in the GSE68004 dataset, we identified SOCS3, OASL, ISG15, and IFIT1 as potential candidate genes for use as biomarkers or drug targets in HAdV-7 infection. We propose a multi-target inhibition of the interferon signaling mechanism to explain the association of HAdV-7 infection with the severity of clinical consequences. This study has allowed us to construct a framework of coexpression gene modules in A549 cells infected with HAdV-7, thus providing a basis for identifying potential genes and pathways involved in adenovirus infection and for investigating the pathogenesis of adenovirus-associated diseases.

Vasoactive intestinal peptide: a potential target for antiviral therapy.

Viral infection is clinically common and some viral diseases, such as the ongoing global outbreak of coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), have high morbidity and mortality. However, most viral infections are currently lacking in specific therapeutic agents and effective prophylactic vaccines, due to inadequate response, increased rate of drug resistance and severe adverse side effects. Therefore, it is urgent to find new specific therapeutic targets for antiviral defense among which "peptide-based therapeutics" is an emerging field. Peptides may be promising antiviral drugs because of their high efficacy and low toxic side effects. Vasoactive intestinal peptide (VIP) is a prospective antiviral peptide. Since its successful isolation in 1970, VIP has been reported to be involved in infections of SARS-CoV-2, human immune deficiency virus (HIV), vesicular stomatitis virus (VSV), respiratory syncytial virus (RSV), Zika virus (ZIKV) and cytomegalovirus (CMV). Additionally, given that viral attacks sometimes cause severe complications due to overaction of inflammatory and immune responses, the potent anti-inflammatory and immunoregulator properties of VIP facilitate it to be a powerful and promising candidate. This review summarizes the role and mechanisms of VIP in all reported viral infections and suggests its clinical potential as an antiviral therapeutic target.

Establishing a patient registry study database of dust mite allergic asthma in children: design, methodology and preliminary exploration.

BACKGROUND: Asthma is a heterogeneous disease with different phenotypes, endotypes and responses to treatment. Dust mite allergic asthma (DMAA) is the most common type in children. Compared with randomized control trials, a patient registry study (PRS) can reflect the real physical condition and clinical diagnosis more comprehensively. METHODS: Children who visited the asthma clinic of the Children's Hospital of Chongqing Medical University between August 2018 and August 2020, and met the inclusion criteria and also agreed to participate, were enrolled in the registry study. Clinical information, laboratory tests and peripheral blood samples were collected after informed consent was given by guardians. RESULTS: To date, 208 children have been enrolled in the patient registry database of DMAA. They are mainly male, with >50% having a history of allergic rhinitis, cesarean section, positive family history and passive smoking. Eosinophils and total immunoglobulin E levels were all significantly higher than normal. According to results for the childhood asthma control test (c-ACT) and ratio of forced expiratory volume in 1 s to predicted value after inhaled corticosteroid treatment, the uncontrolled group had higher hemoglobin (Hb) levels than the control group. The group exhibiting abnormal pulmonary function was older, and had longer disease duration, higher fractional exhaled nitric oxide and Hb than the group in which pulmonary function was restored. CONCLUSIONS: We have preliminarily established a registered study database of children with DMAA. By cluster analysis and using blood samples, we can further study the different pathophysiological mechanisms in order to provide more individualized and targeted treatments for all children.

Human adenovirus type 7 infection causes a more severe disease than type 3.

BACKGROUND: Human adenovirus type 3 (HAdV-3) and 7 (HAdV-7) cause significant morbidity and develop severe complications and long-term pulmonary sequelae in children. However, epidemiologic reports have suggested that nearly all highly severe or fatal adenoviral diseases in children are associated with HAdV-7 rather than HAdV-3. Here, we conduct in-depth investigations to confirm and extend these findings through a comprehensive series of assays in vitro and in vivo as well as clinical correlates. METHODS: A total of 8248 nasopharyngeal aspirate (NPA) samples were collected from hospitalized children with acute respiratory infections in Children's Hospital of Chongqing Medical University from June 2009 to May 2015. Among 289 samples that tested positive for HAdVs, clinical data of 258 cases of HAdV-3 (127) and HAdV-7 (131) infections were analyzed. All HAdV-positive samples were classified by sequencing the hexon and fiber genes, and compared with clinical data and virological assays. We also performed in vitro assays of virus quantification, viral growth kinetics, competitive fitness, cytotoxicity and C3a assay of the two strains. Mouse adenovirus model was used to evaluate acute inflammatory responses. RESULTS: Clinical characteristics revealed that HAdV-7 infection caused more severe pneumonia, toxic encephalopathy, respiratory failure, longer mean hospitalization, significantly lower white blood cell (WBC) and platelet counts, compared to those of HAdV-3. In cell culture, HAdV-7 replicated at a higher level than HAdV-3, and viral fitness showed significant differences as well. HAdV-7 also exhibited higher C3a production and cytotoxic effects, and HAdV-7-infected mice showed aggravated pathology and higher pulmonary virus loads, compared to HAdV-3-infected mice. Macrophages in BALF remained markedly high during infection, with concomitant increase in pro-inflammatory cytokines (TNF-α, IL-1ß, IFN-γ, and IL-6), compared HAdV-3 infection. CONCLUSIONS: These results document that HAdV-7 replicates more robustly than HAdV-3, and promotes an exacerbated cytokine response, causing a more severe airway inflammation. The findings merit further mechanistic studies that offer the pediatricians an informed decision to proceed with early diagnosis and treatment of HAdV-7 infection.

The Role of Industrial Parks in Mitigating Greenhouse Gas Emissions from China.

This study uncovered the direct and indirect energy-related greenhouse gas (GHG) emissions of 213 Chinese national-level industrial parks, providing 11% of China's gross domestic product, from a life-cycle perspective. Direct emissions are sourced from fuel combustion, and indirect emissions are embodied in energy production. The results indicated that in 2015, the direct and indirect GHG emissions of the parks were 1042 and 181 million tonne CO2 equiv, respectively, totally accounting for 11% of national GHG emissions. The total energy consumption of the parks accounted for 10% of national energy consumption. Coal constituted 74% of total energy consumption in these parks. Baseline and low-carbon scenarios are established for 2030, and five GHG mitigation measures targeting energy consumption are modeled. The GHG mitigation potential for these parks in 2030 is quantified as 111 million tonne, equivalent to 9.1% of the parks' total emission in 2015. The measures that increase the share of natural gas consumption, reduce the GHG emission factor of electricity grid, and improve the average efficiency of industrial coal-fired boilers, will totally contribute 94% and 98% in direct and indirect GHG emissions reductions, respectively. These findings will provide a solid foundation for the low carbon development of Chinese industrial parks.

HMGB1 mediates HAdV-7 infection-induced pulmonary inflammation in mice.

Human adenovirus (HAdV) is a common respiratory pathogen in children, with no safe and effective treatment currently available. HAdV type 7 (HAdV-7), in particular, causes severe pediatric pneumonia with a high incidence of sequelae and mortality. Clinical data and animal experiments suggest that HAdV-7-induced pneumonia promotes cell necrosis, releasing a large number of inflammatory mediators. In recent years, the high mobility group box-1 (HMGB1) protein, released by necrotic cells, has been shown to play important roles in several viral infections. Here, we show that HMGB1 levels gradually increased in the media supernatants of HAdV-7 infected A549 cells, starting at 12 h post-infection. In vivo, HMGB1 levels in BALF and mRNA levels in lung tissues significantly increased after 3 days of HAdV-7 infection. Among the HMGB1 receptor genes, TLR-4 and TLR-9 expression increased, and so did the receptor for advanced glycation end-products (RAGE). Interestingly, NF-κB levels also increased concomitantly. Conversely, when HMGB1 was blocked, the pathological scores from lung tissues, inflammatory mediator levels, and viral copy number all were reduced significantly; in addition, HMGB1-related signaling pathway molecules, namely TLR-4, TLR-9, RAGE, and NF-κB were also reduced. We conclude that HMGB1 promotes HAdV-7 replication and signals through TLR-4, TLR-9, and RAGE receptors to activate NF-κB, stimulating the release of inflammatory mediators and contributing to adenoviral pathology. Thus, HMGB1 could be used as a therapeutic target in HAdV-7 infection.

Prenatal nicotinic exposure prolongs superior laryngeal C-fiber-mediated apnea and bradycardia through enhancing neuronal TRPV1 expression and excitation.

Maternal cigarette smoke, including prenatal nicotinic exposure (PNE), is responsible for sudden infant death syndrome (SIDS). The fatal events of SIDS are characterized by severe bradycardia and life-threatening apneas. Although activation of transient receptor potential vanilloid 1 (TRPV1) of superior laryngeal C fibers (SLCFs) could induce bradycardia and apnea and has been implicated in SIDS pathogenesis, how PNE affects the SLCF-mediated cardiorespiratory responses remains unexplored. Here, we tested the hypothesis that PNE would aggravate the SLCF-mediated apnea and bradycardia via up-regulating TRPV1 expression and excitation of laryngeal C neurons in the nodose/jugular (N/J) ganglia. To this end, we compared the following outcomes between control and PNE rat pups at postnatal days 11-14: 1) the cardiorespiratory responses to intralaryngeal application of capsaicin (10 µg/ml, 50 µl), a selective stimulant for TRPV1 receptors, in anesthetized preparation; 2) immunoreactivity and mRNA of TRPV1 receptors of laryngeal sensory C neurons in the N/J ganglia retrogradely traced by 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate; and 3) TRPV1 currents and electrophysiological characteristics of these neurons by using whole-cell patch-clamp technique in vitro Our results showed that PNE markedly prolonged the apneic response and exacerbated the bradycardic response to intralaryngeal perfusion of capsaicin, which was associated with up-regulation of TRPV1 expression in laryngeal C neurons. In addition, PNE increased the TRPV1 currents, depressed the slow delayed rectifier potassium currents, and increased the resting membrane potential of these neurons. Our results suggest that PNE is capable of aggravating the SLCF-mediated apnea and bradycardia through TRPV1 sensitization and neuronal excitation, which may contribute to the pathogenesis of SIDS.-Gao, X., Zhao, L., Zhuang, J., Zang, N., Xu, F. Prenatal nicotinic exposure prolongs superior laryngeal C-fiber-mediated apnea and bradycardia through enhancing neuronal TRPV1 expression and excitation.

Prenatal nicotinic exposure upregulates pulmonary C-fiber NK1R expression to prolong pulmonary C-fiber-mediated apneic response.

Prenatal nicotinic exposure (PNE) prolongs bronchopulmonary C-fiber (PCF)-mediated apneic response to intra-atrial bolus injection of capsaicin in rat pups. The relevant mechanisms remain unclear. Pulmonary substance P and adenosine and their receptors (neurokinin-A receptor, NK1R and ADA1 receptor, ADA1R) and transient receptor potential cation channel subfamily V member 1 (TRPV1) expressed on PCFs are critical for PCF sensitization and/or activation. Here, we compared substance P and adenosine in BALF and NK1R, ADA1R, and TRPV1 expression in the nodose/jugular (N/J) ganglia (vagal pulmonary C-neurons retrogradely labeled) between Ctrl and PNE pups. We found that PNE failed to change BALF substance P and adenosine content, but significantly upregulated both mRNA and protein TRPV1 and NK1R in the N/J ganglia and only NK1R mRNA in pulmonary C-neurons. To define the role of NK1R in the PNE-induced PCF sensitization, the apneic response to capsaicin (i.v.) without or with pretreatment of SR140333 (a peripheral and selective NK1R antagonist) was compared and the prolonged apnea by PNE significantly shortened by SR140333. To clarify if the PNE-evoked responses depended on action of nicotinic acetylcholine receptors (nAChRs), particularly α7nAChR, mecamylamine or methyllycaconitine (a general nAChR or a selective α7nAChR antagonist) was administrated via another mini-pump over the PNE period. Mecamylamine or methyllycaconitine eliminated the PNE-evoked mRNA and protein responses. Our data suggest that PNE is able to elevate PCF NK1R expression via activation of nAChRs, especially α7nAChR, which likely contributes to sensitize PCFs and prolong the PCF-mediated apneic response to capsaicin.

Prenatal nicotinic exposure augments cardiorespiratory responses to activation of bronchopulmonary C-fibers.

Rat pups prenatally exposed to nicotine (PNE) present apneic (lethal ventilatory arrest) responses during severe hypoxia. To clarify whether these responses are of central origin, we tested PNE effects on ventilation and diaphragm electromyography (EMGdi) during hypoxia in conscious rat pups. PNE produced apnea (lethal ventilatory arrest) identical to EMGdi silencing during hypoxia, indicating a central origin of this apneic response. We further asked whether PNE would sensitize bronchopulmonary C-fibers (PCFs), a key player in generating central apnea, with increase of the density and transient receptor potential cation channel subfamily V member 1 (TRPV1) expression of C-fibers/neurons in the nodose/jugular (N/J) ganglia and neurotrophic factors in the airways and lungs. We compared 1) ventilatory and pulmonary C-neural responses to right atrial bolus injection of capsaicin (CAP, 0.5 µg/kg), 2) bronchial substance P-immunoreactive (SP-IR) fiber density, 3) gene and protein expressions of TRPV1 in the ganglia, and 4) nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) protein in bronchoalveolar lavage fluid (BALF) and TrkA and TrkB genes in the ganglia between control and PNE pups. PNE markedly strengthened the PCF-mediated apneic response to CAP via increasing pulmonary C-neural sensitivity. PNE also enhanced bronchial SP-IR fiber density and N/J ganglia neural TRPV1 expression associated with increased gene expression of TrkA in the N/G ganglia and decreased NGF and BDNF in BALF. Our results suggest that PNE enhances PCF sensitivity likely through increasing PCF density and TRPV1 expression via upregulation of neural TrkA and downregulation of pulmonary BDNF, which may contribute to the PNE-promoted central apnea (lethal ventilatory arrest) during hypoxia.

Congenital diaphragmatic eventration in children: 12 years' experience with 177 cases in a single institution.

OBJECTIVE: This study sought to summarize the diagnostic and treatment aspects of congenital diaphragmatic eventration (CDE) in children by retrospectively analyzing their medical records to identify and understand the complications of CDE, its treatment, and to evaluate the long-term outcomes of diaphragmatic plication. METHODS: The medical records of children who received treatment for CDE from January 2000 to December 2011 at the Children's Hospital of Chongqing Medical University, China were analyzed. Data analyzed included the following: age, sex, symptom, location of eventration, associated anomalies, surgical procedures, complications, and survival and follow up details after diaphragmatic plication. RESULTS: The medical records of 177 children (boys: 128, girls: 49, mean age: 10.28±2.35 months) with CDE were included in this study. Specific symptoms of eventration of the diaphragm were reported for 86 cases; and the typical symptoms included rapid breathing, vomiting, and recurrent respiratory infections. Except for a bilateral case, all the other patients had unilateral CDE. Associated malformations were observed in 31 cases (17.5%), hypoplastic lung (10 cases) was the most common followed by congenital heart disease (9 cases), and cryptorchidism (3 cases). Interestingly, 91 patients were asymptomatic. Diaphragmatic plication was performed in all symptomatic patients (86 cases, 48.5%) and none had recurrence. CONCLUSIONS: Clinical symptoms of CDE varied in severity, ranging from asymptomatic conditions to life-threatening respiratory distress. Timely accurate diagnosis and treatment of symptomatic CDE could effectively resolve respiratory morbidity and reduce complications. The diaphragm plication surgery provided good results among the study population with no recurrence.

The genetic variability of glycoproteins among respiratory syncytial virus subtype A in China between 2009 and 2013.

Human respiratory syncytial virus (RSV) is the leading cause of acute lower respiratory tract infections in infants and children under 5years of age. The novel genotype ON1 has a 72-nucleotide duplication, which is the largest duplicated genome portion of RSV. Whether the ON1 genotype will follow the pattern of the BA genotype, which has a 60-nucleotide duplication, and become the predominant RSV-A strain is a global concern. To obtain information regarding the prevalence of the ON1 genotype in Chongqing in Southwestern China, we examined the circulation pattern of RSV-A identified over four consecutive years (June 2009 to August 2013). In this study, 312 (12%) RSV-A strains were isolated from 2601 nasopharyngeal aspirates, and partial G gene was sequenced successfully in 250 isolates. Of the sequenced Chongqing RSV-A isolates, 237 (94.8%) strains were the NA1 genotype, 4 (1.6%) strains were the NA3 genotype, 4 (1.6%) strains were the NA4 genotype, 1 (0.4%) strain was the GA1 genotype, and 4 (1.6%) strains were identified as the ON1 genotype. Analysis of the distribution, phylogeny, and evolution of the ON1 strains that were collected globally until December 2013 revealed that the ON1 genotype has rapidly disseminated across the world under positive selection pressures. Future studies will determine whether this new genotype will continue to spread and become the dominant strain of RSV-A worldwide. These findings may contribute to the understanding of RSV evolution and to the potential development of a vaccine against RSV.

Detection of respiratory syncytial virus fusion protein variants between 2009 and 2012 in China.

Respiratory syncytial virus (RSV) causes respiratory tract infection, particularly acute lower respiratory tract infection (ALRTI), in early childhood. The RSV fusion protein (F protein) is an important surface protein, and it is the target of both cytotoxic T lymphocytes (CTL) and neutralizing antibodies; thus, it may be useful as a candidate for vaccine research. This study investigated the genetic diversity of the RSV F protein. To this end, a total of 1800 nasopharyngeal aspirates from hospitalized children with ALRTI were collected for virus isolation between June 2009 and March 2012. There were 333 RSV-positive cases (277 cases of RSV A, 55 of RSV B, and 1 with both RSV A and RSV B), accounting for 18.5 % of the total cases. Next, 130 clinical strains (107 of RSV A, 23 of RSV B) were selected for F gene sequencing. Phylogenetic analysis revealed that the F gene sequence is highly conserved, with significant amino acid changes at residues 16, 25, 45, 102, 122, 124, 209, and 447. Mutations in human histocompatibility leukocyte antigen (HLA)-restricted CTL epitopes were also observed. Variations in RSV A F protein at the palivizumab binding site 276 (N→S) increased between 2009 and 2012 and became predominant. Western blot analysis and microneutralization data showed a substitution at residue 276 (N→S) in RSV A that did not cause resistance to palivizumab. In conclusion, the RSV F gene is geographically and temporally conserved, but limited genetic variations were still observed. These data could be helpful for the development of vaccines against RSV infection.

Resveratrol Inhibits respiratory syncytial virus-induced IL-6 production, decreases viral replication, and downregulates TRIF expression in airway epithelial cells.

Respiratory syncytial virus (RSV) is the most common pathogen responsible for lower respiratory diseases in children. So far, there is no effective treatment or preventative vaccine available for RSV infection, although ribavirin and dexamethasone are commonly prescribed. Resveratrol has been shown to inhibit the replication of several other viruses, thus the effect of resveratrol on RSV-induced inflammatory mediators in 9HTEo cell cultures was evaluated, and possible mechanisms of action were explored and compared with dexamethasone and ribavirin. Incubation with resveratrol resulted in decreased IL-6 production and partial inhibition of RSV replication. Resveratrol treatment also inhibited virus-induced TIR-domain-containing adapter-inducing interferon-ß (TRIF) and TANK binding kinase 1 (TBK1) protein expression. These data demonstrate the ability of resveratrol to inhibit cytokine production by RSV in airway epithelial cells, indicating that it might be a therapeutic agent with both anti-inflammatory and antiviral potential for the treatment of RSV infection.