Defining RSV epidemic season in southwest China and assessing the relationship between birth month and RSV infection: A 10-year retrospective study from June 2009 to May 2019.

Respiratory syncytial virus (RSV) is one of the most common causes of lower respiratory tract infections (LRTI). However, only limited information is available regarding its seasonality and its relationship with birth month. A retrospective hospital-based study was carried out from June 2009 to May 2019 in Chongqing, southwest of China. LRTI cases under 5 years were enrolled in this study and PCR was used to detect 8 respiratory viruses. RSV seasonality was determined using "average annual percentage" (AAP) and "percent positivity" method. A total of 6991 cases were enrolled in this study, with an RSV positivity of 34.5%. From June 2009 to May 2019, we analyzed RSV epidemic season during 10 RSV epidemic years in Chongqing using two methods. The result of AAP method was similar to that of percent positivity method with a 30% threshold, which showed an epidemic season of roughly October to March in the subsequent year, with a small peak in June. On average, the RSV epidemic season in RSV-A dominant years typically started earlier (week 42 for RSV-A vs. week 46 for RSV-B), ended earlier (week 12 for RSV-A vs. week 14 for RSV-B), lasted longer (24 weeks for RSV-A vs. 22 weeks for RSV-B), and reached its peak earlier (week 2 for RSV-A vs. week 3 for RSV-B) than in RSV-B dominant years. The proportion of severe LRTI was higher in cases of single infection with RSV-A compared to those of single infection with RSV-B (26.3% vs. 22.3%, p = 0.024). Among infants under 1 year, those born in May and August through December were more likely to be infected with RSV. Infants born 1-2 months before the epidemic season were relatively more susceptible to RSV infection. In Chongqing, the RSV epidemic was seasonal and usually lasted from October to March of next year with a small peak in summer. Infants born 1-2 months before the epidemic season were relatively more susceptible to RSV infection and this population should be targeted while developing RSV immunization strategies.

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.

Respiratory syncytial virus activates Rab5a to suppress IRF1-dependent IFN-λ production, subverting the antiviral defense of airway epithelial cells.

The limited antiviral options and lack of an effective vaccine against human respiratory syncytial virus (RSV) highlight the need for a novel antiviral therapy. One alternative is to identify and target the host factors required for viral infection. Here, using RNA interference to knock down Rab proteins, we provide multiple lines of evidence that Rab5a is required for RSV infection: (a) Rab5a is upregulated both in RSV-A2-infected A549 cells and RSV-A2-challenged BALB/c mice's airway epithelial cells at early infection phase; (b) shRNA-mediated knockdown of Rab5a is associated with reduced lung pathology in RSV A2 challenged mice; (c) Rab5a expression is correlated with disease severity of RSV infection of infants. Knockdown of Rab5a increases IFN-λ (lambda) production by mediating IRF1 nuclear translocation. Our results highlight a new role for Rab5a in RSV infection, such that its depletion inhibits RSV infection by stimulating the endogenous respiratory epithelial antiviral immunity, which suggests that Rab5a is a potential target for novel therapeutics against RSV infection.Importance This study highlights the important role of Rab5a in RSV infection, such that its depletion inhibits RSV infection by stimulating the endogenous respiratory epithelial antiviral immunity and attenuates inflammation of the airway, which suggests that Rab5a is a powerful potential target for novel therapeutics against RSV infection.

Energy acquisition strategy for reproduction in a semelparous squid.

BACKGROUND: Energy demand for reproduction leads to a wide diversity of foraging and life-history strategy among wild animals, linking to a common objective to maximize reproductive success. Semelparous squid species in particular can use up to 50% of the total energy intake for reproduction. However, the energy acquisition strategy for reproduction is still a controversial issue regarding whether the squid shift in diet ontogenetically. Here we used Argentinean shortfin squid (Illex argentinus) as a case study to investigate the strategy of energy acquisition for reproduction, by analyzing energy density of the squid's reproductive tissues including ovary, nidamental glands and oviduct eggs, and stable isotopes and fatty acids of the squid's ovary. RESULTS: The reproductive energy (the sum of the energy accumulated in ovary, nidamental glands and oviduct eggs) increased significantly with maturation. The ovary nitrogen stable isotopes (δ15N) showed a significant increase with maturation, but the increase by maturity stage was not equal to the typical enrichment of about 3‰ per trophic level. Isotopic niche width showed an increasing trend with maturation, and isotopic niche space exhibited greater overlap at advanced maturity stages. The relative amounts of 16:0, 20:5n3 and 20:4n6 in the ovary, tracing for carnivores and top predators, increased after the onset of maturation. The overall fatty acid profiles of the ovary showed significant differences among maturity stages, but obvious overlaps were found for mature squids. Mixed-effects model results revealed that reproductive energy was positively correlated with δ15N values. The reproductive energy was also positively related to the relative amounts of 18:0 and 20:4n6, respectively tracing for herbivores and top predators. CONCLUSIONS: Our results validate that the squid shifts to feed on higher trophic prey for reproduction as energy demand increases once maturation commences. However, the squid does not shift feeding habits at a trophic level but instead broadens prey spectrum, coupled with increasing intake of higher trophic prey items, to meet the energy demand for reproduction. Such energy acquisition strategy may be selected by the squid to maximize reproductive success by balancing energy intake and expenditure from foraging, warranting future studies that aim to clarify such strategy for reproduction among semelparous species.

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.

[Neuro-immune interactions in respiratory diseases].

The nervous system and the immune system are relatively independent but interactional, and neuro-immune regulation is very important for the respiratory system to resist external harmful stimuli and to maintain homeostasis. Neuro-immune interaction is involved in the occurrence and development of respiratory diseases, and is essential for monitoring and modulating inflammation and tissue repair. This article summaries the neuro-immune regulation of respiratory system and discusses its role in respiratory diseases, aiming to provide a theoretical basis for further understanding the crosstalk between the nervous and immune systems, to explore the underlying mechanism in respiratory diseases, and to provide new thoughts for the prevention and treatment of respiratory diseases.

[Epidemiological characteristics of respiratory syncytial virus in hospitalized children with acute lower respiratory tract infection in Chongqing, China, from 2013 to 2018: an analysis of 2 066 cases].

OBJECTIVE: To study the detection rate, epidemic pattern, and clinical features of respiratory syncytial virus (RSV) in hospitalized children with acute lower respiratory infection (ALRI). METHODS: Nasopharyngeal aspirates were collected from children with ALRI, aged < 2 years, who were hospitalized in Children's Hospital of Chongqing Medical University from June 2013 to May 2018. Multiplex PCR was used to detect 16 common respiratory viruses. The epidemiological characteristics of RSV were analyzed. RESULTS: A total of 2 066 hospitalized children with ALRI were enrolled. Among the children, 1 595 (77.20%) tested positive for virus and 826 (39.98%) tested positive for RSV [410(49.6%) positive for RSV-A, 414 (50.1%) positive for RSV-B, and 2 (0.2%) positive for both RSV-A and RSV-B]. RSV-B was the main subtype detected in 2013-2014 and 2016-2017, while RSV-A was the main subtype in 2014-2015 and 2017-2018, and these two subtypes were prevalent in 2015-2016. The highest detection rate of RSV was noted in winter. RSV + human rhinovirus was the most common combination of viruses and was detected in 123 children. These children were more likely to develop wheezing than those with single RSV detected (P=0.030). A total of 298 samples were detected with single RSV, 148 were detected with RSV mixed with other viruses, 389 were detected with other viruses, and 241 were detected negative for viruses. Compared with the other viruses and negative virus groups, the single RSV group had a significantly younger age and significantly higher incidence rates of dyspnea, respiratory failure, and severe lower respiratory tract infection (P < 0.0083). The RSV-A positive group had a significantly higher proportion of boys than the RSV-B positive group (P=0.004), but there were no significant differences in clinical manifestations between the two groups. CONCLUSIONS: In Chongqing in 2013-2018, RSV-A and RSV-B not only can predominate alternately, but also can co-circulate during a season. RSV is the major viral pathogen of hospitalized children with ALRI and can cause severe lower respiratory tract infection. There are no differences in clinical manifestations between children with RSV-A infection and those with RSV-B infection, but boys are more susceptible to RSV-A infection.

Lethal avian influenza A (H5N1) virus replicates in pontomedullary chemosensitive neurons and depresses hypercapnic ventilatory response in mice.

The highly pathogenic H5N1 (HK483) viral infection causes a depressed hypercapnic ventilatory response (dHCVR, 20%↓) at 2 days postinfection (dpi) and death at 7 dpi in mice, but the relevant mechanisms are not fully understood. Glomus cells in the carotid body and catecholaminergic neurons in locus coeruleus (LC), neurokinin 1 receptor (NK1R)-expressing neurons in the retrotrapezoid nucleus (RTN), and serotonergic neurons in the raphe are chemosensitive and responsible for HCVR. We asked whether the dHCVR became worse over the infection period with viral replication in these cells/neurons. Mice intranasally inoculated with saline or the HK483 virus were exposed to hypercapnia for 5 min at 0, 2, 4, or 6 dpi, followed by immunohistochemistry to determine the expression of nucleoprotein of H5N1 influenza A (NP) alone and coupled with 1) tyrosine hydroxylase (TH) in the carotid body and LC, 2) NK1R in the RTN, and 3) tryptophan hydroxylase (TPH) in the raphe. HK483 viral infection blunted HCVR by ∼20, 50, and 65% at 2, 4, and 6 dpi. The NP was observed in the pontomedullary respiratory-related nuclei (but not in the carotid body) at 4 and 6 dpi, especially in 20% of RTN NK1R, 35% of LC TH, and ∼10% raphe TPH neurons. The infection significantly reduced the local NK1R or TPH immunoreactivity and population of neurons expressing NK1R or TPH. We conclude that the HK483 virus infects the pontomedullary respiratory nuclei, particularly chemosensitive neurons in the RTN, LC, and raphe, contributing to the severe depression of HCVR and respiratory failure at 6 dpi. NEW & NOTEWORTHY The H5N1 virus infection is lethal due to respiratory failure, but the relevant mechanisms remain unclear. In this study, we demonstrated a gradual diminution of hypercapnic ventilatory response to a degree, leading to respiratory failure over a 6-day infection. Death was associated with viral replication in the pontomedullary respiratory-related nuclei, especially the central chemosensitive neurons. These results not only provide insight into the mechanisms of the lethality of H5N1 viral infection but also offer clues in the development of corresponding treatments to minimize and prevent respiratory failure.

Pathogenic difference of respiratory syncytial virus infection in cotton rats of different ages.

Human respiratory syncytial virus (RSV) is the most common viral pathogen of lower respiratory tract infection worldwide. The virus selectively infects the respiratory epithelium, and causes diseases of variable severity in infants and the elderly. However, the differences in pathogenesis in the age groups remain poorly studied. Age is a major determinant of RSV disease, and the most severe morbidity and mortality occur in the infants and the elderly, because of the immature immunity in infants and declining immunity in old age. The cotton rat is a good model of RSV infection as it is naturally susceptible to RSV. In this study, we established an infant/adult/elderly RSV infection model in 3-week, 8-week and 30-week-old cotton rats and infected them with equal dose of RSV. This model exhibited airway neutrophils infiltration. In the 3-week-old group, higher viral load was observed in the lungs and noses, may due to low IFN-α/Mx2 levels. In contrast, the 8-week-old group had adequate IFN-α/Mx2 but exhibited the most obvious pulmonary inflammation and peribronchiolitis. Interestingly, the most severe pathology and delayed viral clearance in the lungs were observed in the 30-week-old group, may related to the increase of mucus induced by TNF-α and the lower antiviral effect of IFN-α. These results clearly revealed that an age-dependent severity of RSV disease and antiviral defense in the cotton rats, which may provide an effective model for personalized vaccine research and specific treatment strategies for different RSV age groups.

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.

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.

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.

Respiratory Syncytial Virus Nonstructural Protein 1 Blocks Glucocorticoid Receptor Nuclear Translocation by Targeting IPO13 and May Account for Glucocorticoid Insensitivity.

Despite their powerful antiinflammatory effect, glucocorticoids have shown no significant clinical benefit in respiratory syncytial virus (RSV)-induced bronchiolitis, the reason for which remains unclear. Upon glucocorticoid binding, the cytoplasmic glucocorticoid receptor (GR) translocates to the nucleus with the help of importin 13 (IPO13). Here, we report that RSV infection reduced GR nuclear translocation in nasopharyngeal aspirates from RSV-infected infants, lungs of infected mice, and A549 cells, which coincided with decreased IPO13 expression. This led to repression of GR-induced antiinflammatory genes, such that dexamethasone failed to suppress airway inflammation and airway hyperresponsiveness in the infected mice. The anti-GR effect of RSV was mediated by viral nonstructural protein 1 , which likely functioned by competing with IPO13 for GR binding. Our findings provide a mechanism for the ineffectiveness of glucocorticoids in RSV-related disease and highlight the potential to target the IPO13-GR axis as a treatment for multiple glucocorticoid-related diseases.

Lethal avian influenza A (H5N1) virus induces ataxic breathing in mice with apoptosis of pre-Botzinger complex neurons expressing neurokinin-1 receptor.

Lethal influenza A (H5N1) induces respiratory failure in humans. Although it also causes death at 7 days postinfection (dpi) in mice, the development of the respiratory failure and the viral impact on pre-Botzinger complex (PBC) neurons expressing neurokinin 1 receptor (NK1R), which is the respiratory rhythm generator, have not been explored. Body temperature, weight, ventilation, and arterial blood pH and gases were measured at 0, 2, 4, and 6 dpi in control, lethal HK483, and nonlethal HK486 viral-infected mice. Immunoreactivities (IR) of PBC NK1R, H5N1 viral nucleoprotein (NP), and active caspase-3 (CASP3; a marker for apoptosis) were detected at 6 dpi. HK483, but not HK486, mice showed the following abnormalities: 1) gradual body weight loss and hypothermia; 2) tachypnea at 2-4 dpi and ataxic breathing with long-lasting apneas and hypercapnic hypoxemia at 6 dpi; and 3) viral replication in PBC NK1R neurons with NK1R-IR reduced by 75% and CASP3-IR colabeled at 6 dpi. Lethal H5N1 viral infection causes tachypnea at the early stage and ataxic breathing and apneas (hypercapnic hypoxemia) leading to death at the late stage. Its replication in the PBC induces apoptosis of local NK1R neurons, contributing to ataxic breathing and respiratory failure.

Pulmonary C Fibers Modulate MMP-12 Production via PAR2 and Are Involved in the Long-Term Airway Inflammation and Airway Hyperresponsiveness Induced by Respiratory Syncytial Virus Infection.

UNLABELLED: Children with acute respiratory syncytial virus (RSV) infection often develop sequelae of persistent airway inflammation and wheezing. Pulmonary C fibers (PCFs) are involved in the generation of airway inflammation and resistance; however, their role in persistent airway diseases after RSV is unexplored. Here, we elucidated the pathogenesis of PCF activation in RSV-induced persistent airway disorders. PCF-degenerated and intact mice were used in the current study. Airway inflammation and airway resistance were evaluated. MMP408 and FSLLRY-NH2 were the selective antagonists for MMP-12 and PAR2, respectively, to investigate the roles of MMP-12 and PAR2 in PCFs mediating airway diseases. As a result, PCF degeneration significantly reduced the following responses to RSV infection: augmenting of inflammatory cells, especially macrophages, and infiltrating of inflammatory cells in lung tissues; specific airway resistance (sRaw) response to methacholine; and upregulation of MMP-12 and PAR2 expression. Moreover, the inhibition of MMP-12 reduced the total number of cells and macrophages in bronchiolar lavage fluid (BALF), as well infiltrating inflammatory cells, and decreased the sRaw response to methacholine. In addition, PAR2 was upregulated especially at the later stage of RSV infection. Downregulation of PAR2 ameliorated airway inflammation and resistance following RSV infection and suppressed the level of MMP-12. In all, the results suggest that PCF involvement in long-term airway inflammation and airway hyperresponsiveness occurred at least partially via modulating MMP-12, and the activation of PAR2 might be related to PCF-modulated MMP-12 production. Our initial findings indicated that the inhibition of PCF activity would be targeted therapeutically for virus infection-induced long-term airway disorders. IMPORTANCE: The current study is critical to understanding that PCFs are involved in long-term airway inflammation and airway resistance after RSV infection through mediating MMP-12 production via PAR2, indicating that the inhibition of PCF activity can be targeted therapeutically for virus infection-induced long-term airway disorders.

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.

NK cells contribute to persistent airway inflammation and AHR during the later stage of RSV infection in mice.

RSV can lead to persistent airway inflammation and AHR and is intimately associated with childhood recurrent wheezing and asthma, but the underlying mechanisms remain unclear. There are high numbers of NK cells in the lung, which not only play important roles in the acute stage of RSV infection, but also are pivotal in regulating the pathogenesis of asthma. Therefore, in this study, we assumed that NK cells might contribute to persistent airway disease during the later stage of RSV infection. Mice were killed at serial time points after RSV infection to collect samples. Leukocytes in bronchoalveolar lavage fluid (BALF) were counted, lung histopathology was examined, and airway hyperresponsiveness (AHR) was measured by whole-body plethysmography. Cytokines were detected by ELISA, and NK cells were determined by flow cytometry. Rabbit anti-mouse asialo-GM-1 antibodies and resveratrol were used to deplete or suppress NK cells. Inflammatory cells in BALF, lung tissue damage and AHR were persistent for 60 days post-RSV infection. Type 2 cytokines and NK cells were significantly increased during the later stage of infection. When NK cells were decreased by the antibodies or resveratrol, type 2 cytokines, the persistent airway inflammation and AHR were all markedly reduced. NK cells can contribute to the RSV-associated persistent airway inflammation and AHR at least partially by promoting type 2 cytokines. Therefore, therapeutic targeting of NK cells may provide a novel approach to alleviating the recurrent wheezing subsequent to RSV infection.