Increased mortality associated with TCDD exposure in mice infected with influenza A virus is not due to severity of lung injury or alterations in Clara cell protein content

Most studies examining the cause of increased mortality in mice infected with a normally non-lethal dose of influenza A virus after exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) have focused on defects in the immune system. This study examined other possible consequences of TCDD exposure, which could alter pulmonary inflammation during infection. We measured bronchoalveolar lavage (BAL) fluid lactate dehydrogenase (LDH) and protein concentrations and lung wet to dry weight ratios to assess lung damage and edema formation. Immunohistochemistry for Cyp1A1 was used to evaluate the responsiveness of the lung to TCDD. Additionally, we characterized the effects of TCDD on Clara cell secretory protein (CCSP), which plays a regulatory role in pulmonary inflammation. There were no differences in BAL fluid LDH and protein levels, lung wet to dry weight ratios, or the amount of CCSP in the lungs from mice treated with TCDD or vehicle control. The amount of Cyp1A1 in endothelial cells, Clara cells, and Type II pneumocytes was greatly induced after TCDD exposure. Although lung tissue was clearly responsive to TCDD as shown by Cyp1A1 induction, the increased mortality in infected mice exposed to TCDD did not correlate with increased damage to the lung or decreased CCSP concentrations.     

白细胞介素-33抗体治疗呼吸道合胞病毒诱导小鼠哮喘的效果观察

为探讨白细胞介素(interleukin,IL)-33在呼吸道合胞病毒(respiratory syncytial virus,RSV)感染导致哮喘急性加重过程中的作用,选取3周龄雌性BALB/c小鼠28只,随机分为5组,分别为正常对照组、单纯RSV感染组、哮喘组、RSV感染哮喘并对照抗体组、RSV感染哮喘并IL-33...     

呼吸道合胞病毒疫苗增强性疾病小鼠模型的建立与评价

呼吸道合胞病毒(respiratory syncytial virus,RSV)是导致婴幼儿严重下呼吸道感染的最重要病原体,但该病毒的灭活疫苗可引起RSV疫苗增强性疾病(RSV vaccine-enhanced disease,RVED),因此至今仍未研制出安全、有效的疫苗。RVED的发生机制目前仍不清楚。使用能有效模拟RVED的动物模型是探索RVED发生机制的主要手段,而本研究的目的就是建立能稳定模拟RVED表现的小鼠模型。将BALB/c小鼠分成3组,即甲醛灭活RSV疫苗接种组(FV组)、活RSV接种组(VV组)和对照组(BV组),并模拟自然感染对其攻毒。通过小鼠体重监测、肺组织苏木精-伊红染色、荧光定量聚合酶链反应(polymerase chain reaction,PCR)、流式细胞术等,观察FV组小鼠感染RSV后的病毒载量、肺部炎症和T细胞免疫状态。结果显示,与VV组和BV组相比,FV组小鼠RSV攻毒后的体重占初始体重百分比显著降低,肺部炎症最明显,且仅FV组出现支气管和毛细血管周围有大量淋巴细胞浸润及嗜酸性粒细胞浸润。荧光定量PCR显示,FV组小鼠的肺部RSV载量最低。流式细胞术显示,攻毒4d后FV组CD~(4+) T细胞数与其他两组相比显著增加,且CD~(4+) T细胞分泌的白细胞介素4(interleukin 4,IL-4)及IL-4/γ干扰素(interferonγ,IFN-γ)比值显著高于其他两组,而CD~(8+) T细胞分泌的肿瘤坏死因子α(tumor necrosis factorα,TNF-α)和IFN-γ低于VV组。结果提示,RVED小鼠呈现出Th2优势型免疫应答及CD~(8+) T细胞功能不足,模型初步建立成功,为RVED发生机制的探索和RSV疫苗的研发奠定了良好基础。     

呼吸道合胞病毒F蛋白抗体的研究进展

呼吸道合胞病毒(respiratory syncytial virus,RSV)感染,是造成婴幼儿、学龄前儿童、免疫缺陷患者、老年人等高危群体住院治疗及死亡的重要病因。目前,多个预防RSV感染的候选疫苗正处于研发中,尚无安全、有效的疫苗面世。对RSV感染的处理仍以治疗为主,使用帕利珠单抗(Palivizumab)是当前仅有的预防药物。在过去数年间出现的新型抗体药物,如多克隆抗体、单克隆抗体、纳米抗体等有些已进入了临床前或I、II、III期临床试验阶段。融合蛋白(fusion protein,F蛋白)在RSV感染过程中是不可或缺的,它介导病毒包膜与宿主细胞膜的融合。在感染过程中,F蛋白从亚稳态的融合前构象状态(prefusion fusion protein,pre F)转变为热力学稳定的融合后状态(postfusion fusionprotein,post F)。近年来,研究人员通过不断筛选,获得了多株针对pre F的抗体。与结合post F的抗体相比,这些抗体具有更强的RSV中和活性。一些更新的抗体药物候选品,在实验中显示出了效力强、药代动力学特征明显、半衰期长等特点,并能以其他途径给药,而且能降低其制备成本。现就抗RSV pre F的抗体研究进展作一概述。     

Development of mRNA vaccines against respiratory syncytial virus (RSV)

Respiratory syncytial virus (RSV) is a single-stranded negative-sense RNA virus that is the primary etiologic pathogen of bronchitis and pneumonia in infants and the elderly. Currently, no preventative vaccine has been approved for RSV infection. However, advances in the characterization, and structural resolution, of the RSV surface fusion glycoprotein have revolutionized RSV vaccine development by providing a new target for preventive interventions. In general, six different approaches have been adopted in the development of preventative RSV therapeutics, namely, particle-based vaccines, vector-based vaccines, live-attenuated or chimeric vaccines, subunit vaccines, mRNA vaccines, and monoclonal antibodies. Among these preventive interventions, MVA-BN-RSV, RSVpreF3, RSVpreF, Ad26. RSV.preF, nirsevimab, clesrovimab and mRNA-1345 is being tested in phase 3 clinical trials, and displays the most promising in infant or elderly populations. Accompanied by the huge success of mRNA vaccines in COVID-19, mRNA vaccines have been rapidly developed, with many having entered clinical studies, in which they have demonstrated encouraging results and acceptable safety profiles. In fact, Moderna has received FDA approval, granting fast-track designation for an investigational single-dose mRNA-1345 vaccine against RSV in adults over 60 years of age. Hence, mRNA vaccines may represent a new, more successful, chapter in the continued battle to develop effective preventative measures against RSV. This review discusses the structure, life cycle, and brief history of RSV, while also presenting the current advancements in RSV preventatives, with a focus on the latest progress in RSV mRNA vaccine development. Finally, future prospects for this field are presented.     

呼吸道合胞病毒感染上调Toll样受体7和3基因的早期表达

【目的】Toll样受体（Toll-like receptor,TLR）7和3是两个重要的模式识别受体,分别通过识别病毒的单股和双股RNA而活化细胞。呼吸道合胞病毒（RSV）能被TLR7和TLR3识别。在RSV感染致病的早期阶段,对肺中TLR7、TLR3的表达动力学和表达丰度进行研究,并探讨其表达与肺部炎症反应的关系。【方法】我们以活RSV滴鼻感染BALB/c鼠诱导急性肺炎,在RSV感染0,1,4,8,16和24h的不同时间点,用半定量RT-PCR方法检测鼠肺TLR7、TLR3的mRNA表达,用western blot法检测核转录因子NF-κB的蛋白表达,HE染色观察肺的病理学改变。【结果】我们发现,RSV感染早期能快速上调TLR7和TLR3的基因表达水平,与正常组相比,其升高有显著性差异,并与RSV感染之间存在时间依赖关系;TLR7的反应（RSV感染1h）早于TLR3（RSV感染4 h）。肺中NF-κB在RSV感染的4 h即可被活化。RSV介导的TLR7和TLR3早期转录反应与RSV肺炎的严重程度是平行的。【结论】TLR7和TLR3确实可通过识别病毒RNA参与RSV肺炎的发生和发展,表明感染的器官在识别病毒感染和激发前炎反应时,可能经由多个TLRs。这将对开发制剂用以调节治疗性TLR配体的活性具有重要意义。     

Direct visualization of the small hydrophobic protein of human respiratory syncytial virus reveals the structural basis for membrane permeability

Human respiratory syncytial virus (HRSV) is the leading cause of lower respiratory tract disease in infants. The HRSV small hydrophobic (SH) protein plays an important role in HRSV pathogenesis, although its mode of action is unclear. Analysis of the ability of SH protein to induce membrane permeability and form homo-oligomers suggests it acts as a viroporin. For the first time, we directly observed functional SH protein using electron microscopy, which revealed SH forms multimeric ring-like objects with a prominent central stained region. Based on current and existing functional data, we propose this region represents the channel that mediates membrane permeability.

Structured summary

MINT-7890792, MINT-7890805: SH (uniprotkb:P04852) and SH (uniprotkb:P04852) bind (MI:0407) by chromatography technology (MI:0091)MINT-7890784, MINT-7890776: SH (uniprotkb:P04852) and SH (uniprotkb:P04852) bind (MI:0407) by electron microscopy (MI:0040)     

Innate immune recognition of respiratory syncytial virus infection

Respiratory syncytial virus (RSV) is the leading cause of respiratory infection in infants and young children. Severe clinical manifestation of RSV infection is a bronchiolitis, which is common in infants under six months of age. Recently, RSV has been recognized as an important cause of respiratory infection in older populations with cardiovascular morbidity or immunocompromised patients. However, neither a vaccine nor an effective antiviral therapy is currently available. Moreover, the interaction between the host immune system and the RSV pathogen during an infection is not well understood. The innate immune system recognizes RSV through multiple mechanisms. The first innate immune RSV detectors are the pattern recognition receptors (PRRs), including toll-like receptors (TLRs), retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs), and nucleotide-biding oligomerization domain (NOD)-like receptors (NLRs). The following is a review of studies associated with various PRRs that are responsible for RSV virion recognition and subsequent induction of the antiviral immune response during RSV infection. [BMB Reports 2014; 47(4): 184-191]     

Identification of nyasol and structurally related compounds as the active principles from Anemarrhena asphodeloides against respiratory syncytial virus (RSV)

Three known phenolic compounds, (-)-(R)-nyasol (= 4,4'-(1Z,3R)-Penta-1,4-diene-1,3-diyldiphenol; 1), its derivative 2, and broussonin A (3)--isolated from the rhizomes of Anemarrhena asphodeloides--were for the first time identified as the active principles capable of efficient respiratory-syncytial-virus (RSV) inhibition. The IC50 values of 1-3 against the RSV-A2 strain, propagated in HEp-2 cells, were determined, their activities being higher than that of the standard antiviral drug ribavirin (IC50 = 1.15 microM). In addition, the known, but inactive, compound 'trans-N-(para-coumaroyl)tyramine' (= (2E)-3-(4-hydroxyphenyl)-N-[2-(4-hydroxyphenyl)ethyl]prop-2-enamide; 4) was isolated from this plant for the first time.     

Co-infection of human metapneumovirus with adenovirus or respiratory syncytial virus among children in Japan

Human metapneumovirus (hMPV) is one of the etiological agents of acute respiratory tract infections. From June 2005 to May 2006, we collected 185 clinical specimens from children in Osaka City, Japan, and detected 41 hMPV RNA. Of the 41 specimens, four (9.8%) also contained other viruses (3 with adenovirus [AdV] and 1 with respiratory syncytial virus [RSV]). The clinical symptoms of patients coinfected with AdV were indistinct from those of patients mono-infected with hMPV. The symptoms of the one patient co-infected with RSV were clinically severe. Further research is needed to clarify the effect of hMPV on other respiratory viruses or vice versa.     

Vitamin D3 protects against respiratory syncytial virus-induced barrier dysfunction in airway epithelial cells via PKA signaling pathway

Respiratory syncytial virus (RSV) is the leading cause of acute lower respiratory tract infection in infants and young children globally and is responsible for hospitalization and mortality in the elderly population. Virus-induced airway epithelial barrier damage is a critical step during RSV infection, and emerging studies suggest that RSV disrupts the tight junctions (TJs) and adherens junctions (AJs) between epithelial cells, increasing the permeability of the airway epithelial barrier. The lack of commercially available vaccines and effective antiviral drugs for RSV emphasizes the need for new management strategies. Vitamin D₃ is a promising intervention for viral infection due to its critical role in modulating innate immune responses. However, there is limited evidence on the effect of vitamin D₃ on RSV pathogenies. Here, we investigated the impact of vitamin D₃ on RSV-induced epithelial barrier dysfunction and the underlying mechanisms. We found that pre-incubation with 1,25(OH)₂D₃, the active form of vitamin D₃, alleviated RSV-induced epithelial barrier disruption in a dose-dependent manner without affecting viability in 16HBE cells. 1,25(OH)₂D₃ induced minor changes in the protein expression level of TJ/AJ proteins in RSV-infected cells. We observed increased CREB phosphorylation at Ser133 during 1,25(OH)₂D₃ exposure, indicating that vitamin D₃ triggered protein kinase A (PKA) activity in 16HBE. PKA inhibitors modified the restoration of barrier function by 1,25(OH)₂D₃ in RSV-infected cells, implying that PKA signaling is responsible for the protective effects of vitamin D₃ against RSV-induced barrier dysfunction in airway epithelial cells. Our findings suggest vitamin D₃ as a prophylactic intervention to protect the respiratory epithelium during RSV infections.     

雄黄纳米微粒在细胞水平上抑制呼吸道合胞病毒复制的初步研究

建立呼吸道合胞病毒A型(RSV-A型)感染Hep-2细胞模型,通过预防、治疗及直接灭活三种不同给药方式,观察中药雄黄对RSV-A型感染Hep-2细胞病变(CPE)的抑制作用。用高能球磨机研磨双蒸水水飞处理制备雄黄纳米微粒,应用砷钼蓝染色法测定雄黄纳米微粒浓度并在Nano Series粒度测定仪上测定其粒度。以MTT法计算药物的半数中毒剂量(TC50)。通过三种不同给药方式即预防给药、治疗给药及直接灭活给药方式进行体外实验,以利巴韦林为阳性对照药,观察雄黄纳米微粒对RSV-A型感染Hep-2细胞病变所起的作用,并对药物的量效关系进行分析。雄黄纳米微粒TC50值为0.649μg/mL。预防、治疗及直接灭活给药方式均可减轻RSV-A感染Hep-2细胞的CPE程度,其抑制RSV-A型感染Hep-2细胞病变的半数有效浓度(IC50)分别为0.20μg/mL、0.13μg/mL、0.16μg/mL,治疗指数(TI)分别为3.18、4.99和4.11,雄黄纳米微粒对RSV-A型感染Hep-2细胞病变的抑制作用存在着明显的量效关系。雄黄纳米微粒按预防、治疗及直接灭活给药方式给药时,其中治疗给药方式更有利于减轻RSV-A感染Hep-2细胞引起的病变。     

Both heptad repeats of human respiratory syncytial virus fusion protein are potent inhibitors of viral fusion

Heptad repeat regions (HR1 and HR2) are highly conserved peptides located in F(1) of paramyxovirus envelope proteins. They are important in the process of virus fusion and form six-helix bundle structure (trimer of HR1 and HR2 heterodimer) post-fusion, similar to those found in the fusion proteins of other enveloped viruses, such as retrovirus HIV. Both HR1 and HR2 show potent inhibition for virus fusion in some members of paramyxovirus. However, in other members, only HR2 gives strong inhibition whereas HR1 does not. Human respiratory syncytial virus (hRSV) is a member of paramyxovirus and its crystal structure of HR1 and HR2 six-helix bundle was solved lately. Although hRSV HR2 inhibition was reported, nevertheless the effect of HR1 on virus fusion is not known. In this study, hRSV HR1 and HR2 were expressed as fusion protein separately in Escherichia coli system and their complex assembly and virus fusion inhibition effect have been analysed. It shows that both HR1 and HR2 (in the fusion form with 50-amino-acid fusion partner) of hRSV F protein give strong inhibition on virus fusion (IC(50) values are 1.68 and 2.93 microM, respectively) and they form stable six-helix bundle in vitro with both in the fusion protein form.     

Determination of the disulfide bond arrangement of human respiratory syncytial virus attachment (G) protein by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

The attachment protein or G protein of the A2 strain of human respiratory syncytial virus (RSV) was digested with trypsin and the resultant peptides separated by reverse-phase high-performance liquid chromatography (HPLC). One tryptic peptide produced a mass by matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) corresponding to residues 152-187 with the four Cys residues of the ectodomain (residues 173, 176, 182, and 186) in disulfide linkage and absence of glycosylation. Sub-digestion of this tryptic peptide with pepsin and thermolysin produced peptides consistent with disulfide bonds between Cys173 and Cys186 and between Cys176 and Cys182. Analysis of ions produced by post-source decay of a peptic peptide during MALDI-TOF-MS revealed fragmentation of peptide bonds with minimal fission of an inter-chain disulfide bond. Ions produced by this unprecedented MALDI-induced post-source fragmentation corroborated the existence of the disulfide arrangement deduced from mass analysis of proteolysis products. These findings indicate that the ectodomain of the G protein has a non-glycosylated subdomain containing a "cystine noose."     

Genetic analysis of attachment glycoprotein (G) gene in new genotype ON1 of human respiratory syncytial virus detected in Japan

We studied the evolution of the G gene in the new genotype ON1 of RSV detected from patients with acute respiratory infection in Japan. Phylogenetic analyses and the evolutionary timescale were obtained by the Bayesian MCMC method. We also analyzed p‐distance and positive selection sites. A new genotype ON1 emerged around 2001. The evolution rate was rapid (3.57 × 10^?3 substitutions/site per year). The p‐distance was short and no positive selection site was found in the present strains. These results suggested that a new genotype ON1 of RSV‐A emerged approximately10 years ago and spread to some countries with a high evolution rate.

     

白细胞介素23在呼吸道合胞病毒感染致Th1、Th2及Th17细胞分化中的作用及机制

本研究旨在探讨白细胞介素23(interleukin 23,IL-23)在呼吸道合胞病毒(respiratory syncytial virus,RSV)感染支气管上皮细胞BEAS-2B后对Th1、Th2和Th17细胞分化的影响及作用机制。将RSV感染BEAS-2B后的上清液与淋巴细胞共孵育,并分别阻断IL-23受体(IL-23 receptor,IL-23R)、IL-23p19亚基及p38丝裂原活化蛋白激酶(p38 mitogen-activated protein kinase,p38 MAPK)信号通路。应用酶联免疫吸附试验(enzyme-linked immunosorbent assay,ELISA)检测上清液中细胞因子γ干扰素(interferon γ,IFN-γ)、IL-4、IL-17的浓度。同时,应用实时聚合酶链反应(polymerase chain reaction,PCR)检测相关转录因子(t-bet、gata3、rorγt)和信号转导子(stat4、stat6、stat3)的表达。结果显示,RSV感染后IFN-γ、IL-4和IL-17蛋白表达上调,转录因子及信号转导子的表达也有所增加。阻断IL-23和p38 MAPK信号通路后,Th1、Th2和Th7细胞分泌的细胞因子及转录因子表达均明显下降。结果提示,阻断IL-23后可在基因转导层面抑制RSV感染上皮细胞后诱导的Th1、Th2和Th17细胞分化,此过程可能与p38 MAPK信号通路有关。     

Proliferating cell nuclear antigen (PCNA/cyclin) immunocytochemistry as a labeling index in mouse lung tissues

Summary Proliferating cell nuclear antigen is expressed in cells from late G₁ through the S-phase of the cell cycle. Therefore, antibodies directed against this molecule should provide a probe for labeling immunocytochemically the nuclei of proliferating cells. Herein we demonstrate the feasibility and reliability of this technique by quantifying immunostained pulmonary nuclei. We applied polyclonal and monoclonal antisera to alveolar and bronchiolar pulmonary epithelial cells in various proliferative states in tissue-sections and in vitro. A/J mice had a slightly higher labeling index than C57BL/6J mice, and proliferation in both strains increased dramatically after butylated hydroxytoluene treatment produced compensatory hyperplasia of Type-II pneumocytes. Immunostaining in fetal and neonatal lung samples from mice was higher than in adults. Spontaneous lung adenomas had a higher labeling index than the surrounding normal lung tissue. In addition, new data contained herein demonstrate a strain difference in proliferation of bronchiolar epithelial cells, and quantify the extent to which BHT-induced lung damage increases these proliferative rates. This mammalian nuclear antigen did not cross-react with antiserum to a functionally related bacterial protein, the beta subunit of E. coli DNA polymerase-III holoenzyme.     

Stochastic modeling of the transmission of respiratory syncytial virus (RSV) in the region of Valencia,Spain

In this paper, we study the dynamics of the transmission of respiratory syncytial virus (RSV) in the population using stochastic models. The stochastic models are developed introducing stochastic perturbations on the demographic parameter as well as on the transmission rate of the RSV. Numerical simulations of the deterministic and stochastic models are performed in order to understand the effect of fluctuating birth rate and transmission rate of the RSV on the population dynamics. The numerical solutions of stochastic models are calculated using Euler-Maruyama and Milstein schemes, and confidence intervals for stochastic solutions are given using Monte-Carlo method. Analysis of the numerical results reveals that perturbations on the transmission rate are more decisive in the dynamics of RSV than perturbations on demographic parameters. In addition, the stochastic models show the advantage of reproducing more effectively the noisy RSV hospitalization data. It is concluded that these stochastic models are a viable option to provide a realistic modeling of the RSV dynamics on the population.