Neutrophil extracellular traps cause airway obstruction during respiratory syncytial virus disease

Human respiratory syncytial virus (RSV) is the most important cause of severe lower respiratory tract disease (LRTD) in young children worldwide. Extensive neutrophil accumulation in the lungs and occlusion of small airways by DNA‐rich mucus plugs are characteristic features of severe RSV–LRTD. Activated neutrophils can release neutrophil extracellular traps (NETs), extracellular networks of DNA covered with antimicrobial proteins, as part of the first‐line defence against pathogens. NETs can trap and eliminate microbes; however, abundant NET formation may also contribute to airway occlusion. In this study, we investigated whether NETs are induced by RSV and explored their potential anti‐viral effect in vitro. Second, we studied NET formation in vivo during severe RSV–LRTD in infants and bovine RSV–LRTD in calves, by examining bronchoalveolar lavage fluid and lung tissue sections, respectively. NETs were visualized in lung cytology and tissue samples by DNA and immunostaining, using antibodies against citrullinated histone H3, elastase and myeloperoxidase. RSV was able to induce NET formation by human neutrophils in vitro. Furthermore, NETs were able to capture RSV, thereby precluding binding of viral particles to target cells and preventing infection. Evidence for the formation of NETs in the airways and lungs was confirmed in children with severe RSV–LRTD. Detailed histopathological examination of calves with RSV–LRTD showed extensive NET formation in dense plugs occluding the airways, either with or without captured viral antigen. Together, these results suggest that, although NETs trap viral particles, their exaggerated formation during severe RSV–LRTD contributes to airway obstruction. Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Complement activation by respiratory syncytial virus-infected cells

Summary Respiratory syncytial virus (RSV), a major respiratory pathogen of children, has been speculated to cause disease by immunologic mechanisms. Although circulating levels of complement (C) are normal during RSV infections, the role of C in respiratory tract secretion is unclear. Since epithelial cells of the respiratory tract of children infected with RSV express viral surface antigens, the ability of RSV infected human cells to activate C was studied. RSV infected human cells (HeLa) were found to activate both the classical and alternative C pathways as measured by the cleavage of native C3 into its breakdown products. Increased C activation occurred in the presence of antibody. Cytolysis of RSV infected cells was then studied using a chromium release assay. Both the classical and alternative C pathways in the presence of antibody participated in the lysis of RSV infected cells. The combined effects of activation of C and the lysis of RSV infected cells by C and antibody may contribute to the pathogenesis of disease.With 1 FigureSupported in part by the Development and Applications Branch, National Institute of Allergy and Infectious Diseases, National Institutes of Health (Contract N01 AI 02645).     

Detection of bovine respiratory syncytial virus by RT-PCR

The paper presents the results of studying the diagnostic efficiency of RT-PCR for the detection of respiratory syncytial virus in cattle of different ages. Glycoprotein F gene sequences were used as a target for amplification. The sensitivity of the reaction was 10 TCD50/ml and the virus detection rate in biomaterials averaged 19%. samples. That in RT-PCR correlated with the presence of clinical signs in sick animals.     

Immunofluorescence with respiratory syncytial virus

The growth of respiratory syncytial (RS) virus in tissue cultures of HEp-2 cells at a low multiplicity of infection has been studied by means of several staining procedures including immunofluorescence, as well as by serial infectivity titrations.Specific fluorescent staining was first noted 10 hours after infection and was restricted entirely to the cytoplasm throughout the growth cycle.A significant increase in the amount of infectious virus present in the system was first detected 10 hours after inoculation, and peak values were achieved after 48 hours.The pattern of development of RS antigen has been compared with that of other medium-sized ether-sensitive agents and possible taxonomic implications are discussed.     

Systemic eosinophil response induced by respiratory syncytial virus

Respiratory syncytial virus (RSV) is a common cause of lower respiratory tract disease (LRTD) in infants. Eosinophils have been suggested to play a role in the disease pathogenesis of LRTD. Inflammation can induce functional and morphological alterations of peripheral blood granulocytes. In patients with RSV LRTD, we aimed to investigate the eosinophil activation status by analysing surface markers. In vitro stimulation of eosinophils with cytokines leads to up-regulation of CD11b and priming markers recognized by the recently developed priming markers A17 and A27, whereas interleukin (IL)-5Ralpha is being down-regulated. In 51 patients and 10 controls we examined the expression of these surface markers on eosinophils in moderate to severe RSV-induced LRTD patients at the time of admission and 6 weeks later during the convalescence phase. RSV-patients were characterized by a higher eosinophil CD11b expression compared to controls. Although basal A17 and A27 expression was not increased, we observed a significantly higher expression of these priming epitopes on N-formyl-methionyl-leucyl-phenylalanine (fMLP)-stimulated cells of RSV patients compared with cells of controls, indicative of prior in vivo priming. Furthermore, IL-5Ralpha expression was down-regulated on peripheral blood eosinophils of these patients. Follow-up blood samples showed normalization of all markers but CD11b, which was persistently increased. Utilizing cellular markers, we observed that peripheral blood eosinophils from infants with RSV LRTD are in a more activated state compared to eosinophils of controls, which normalizes only partially during convalescence.     

Virus-induced complement activation and neutrophil-mediated cytotoxicity against respiratory syncytial virus (RSV).

Complement-dependent neutrophil-mediated cytotoxicity (CDNC) was determined by specific release of 51-chromium (51Cr) from respiratory syncytial virus infected HEp2 cells in a microcytotoxicity assay. There was significant release of 51Cr from RSV infected cells as compared to uninfected cells in the presence of complement (C) and neutrophils (PMN). The degree of cytotoxicity was dependent upon the concentration of C used in the assay. Such cytotoxicity was effectively abolished after heat-inactivation of complement. Complement deficient in C4 did not induce cytotoxicity. Similarly, inhibitors of C1 or C3 blocked CDNC. The maximal CDNC was observed at 37 degrees C with little or no response at 4 degrees C. Lymphocytes and monocytes mediated complement-dependent cytotoxicity very poorly in comparison to PMN. Evidence of complement activation by infected cells was demonstrated by the detection of C3 fixed to RSV infected cells by indirect immunofluorescence. Treatment of C with EDTA or heat prevented subsequent attachment of C3 to the infected cells. These in vitro observations suggest an initial activation of complement by RSV infected cells and subsequent lysis by PMN. It is proposed that this process may play a role in the elimination of virus in the early phase of infection in the absence of specific antibody or sensitized lymphocytes.     

Eosinophil activation and cysteinyl leukotriene production in infants with respiratory syncytial virus bronchiolitis

BACKGROUND: It has been suggested that acute infantile bronchiolitis associated with respiratory syncytial virus (RSV) may share some pathogenic features with atopic asthma in that virus-specific IgE is produced and cysteinyl leukotrienes (cLTs) and eosinophil cationic protein (ECP) have been detected in airway secretions. ECP is a specific marker of eosinophil activation although leukotrienes can be released from a variety of cells including mast cells, eosinophils and monocytes. OBJECTIVE: To test the association between eosinophil activation and cysteinyl leukotriene production in the upper airway secretions of infants with RSV positive (RSV+ve) bronchiolitis. METHODS: Nasal lavage samples were performed in 78 infants (0.0-11.5 months) admitted to hospital with RSV+ve bronchiolitis soon after admission (0-48 h). Leukotriene C4 (LTC4) was assayed by enzyme immunoassay (EIA) and eosinophil cationic protein (ECP) by fluoroimmunoassay (FIA). RESULTS: LTC4 was detectable in 51 and ECP in 57 of 78 samples with a significant positive relationship between LTC4 and ECP (r=0.557, P<0.001). CONCLUSION: In the majority of our subjects with RSV+ve bronchiolitis ECP and LTC4 were detectable in upper airway secretions and were significantly associated with each other. In this clinical setting much of the detected LTC4 within upper airway secretions is likely to originate from the eosinophil, an observation that may have implications for clinical management and for delineation of the underlying mechanisms associated with this illness.     

Respiratory syncytial virus and neutrophil activation

Respiratory syncytial virus infects almost all children by 2 years of age. Neutrophils are the predominant airway leucocytes in RSV bronchiolitis and they are activated in the presence of infection. However it is not clear whether RSV can directly signal to activate neutrophil cytotoxic function. To investigate this we have used a preparation of RSV washed using a new centrifugal diafiltration method to rapidly remove inflammatory molecules produced by the epithelial cells used to propagate the RSV stock. Human neutrophils were isolated from peripheral blood and activated with either the unwashed crude RSV preparations or the purified intact RSV. Neutrophils were also challenged with purified RSV G-glycoprotein. The effect of challenging human neutrophils with these preparations of intact RSV, or the RSV G-glycoprotein, was assessed by measuring the cell surface expression of CD11b and CD18b, the phagocytic oxidative burst, and intracellular release of calcium pools. Neutrophils challenged with the washed RSV exhibited significantly lower activation of surface marker expression (P < 0.001) and oxidative burst (P < 0.001) than those challenged with unwashed virus or with virus free supernatant. There was no increase in intracellular calcium release on exposure to the washed RSV. Purified G glycoprotein did not stimulate neutrophils, whilst the use of a blocking antibody to the F protein did not prevent unwashed RSV from activating cytotoxic responses. These results suggest that neutrophils have no innate signalling system that recognizes RSV but they are activated at sites of RSV infection as a result of the cytokines and inflammatory molecules released by virally infected cells.     

静脉注射用丙种球蛋白对呼吸道合胞病毒治疗的作用

目的:研究静脉注射用丙种球蛋白(IVγG)体内外抗呼吸道合胞病毒(RSV)的作用。方法:①采用微量细胞病变抑制法试验(MCIA),检测不同浓度的IVγG抑制RSV的作用。②建立RSV感染的BALB/c小鼠模型,将40只感染RSV的小鼠分为4组,即IVγG治疗组、病毒唑治疗组、Palivizumab治疗组、生理盐水对照组,及10只未感染RSV的小鼠作为正常对照组,每组10只。于治疗后第5及第7天,分2批处死小鼠,分离病毒并观察肺组织的病理积分。结果:①IVγG具有抑制RSV的作用,其治疗指数(TI)为275,较病毒唑高6倍,较Palivizumab低2倍。②IVγG治疗组小鼠肺组织的病理积分较生理盐水对照组明显减低(P<0.01),但其5d的治疗作用低于Palivizumab治疗组(P<0.05)。结论:IVγG在体内外均有一定的抗RSV的作用,但效果均低于Palivizumab。     

Molecular epidemiology of respiratory syncytial virus

Respiratory syncytial virus (RSV) is a major cause of viral acute respiratory tract infections in young children. The virus is characterised by distinct seasonality that is dependent upon the latitude and its ability to cause reinfection. Respiratory syncytial virus demonstrates a complex molecular epidemiology pattern as multiple strains and/or genotypes cocirculate during a single epidemic. Previous studies have investigated the relationship between RSV genetic diversity, reinfection, and clinical features. Here, we review the evidence behind this relationship together with the impact that the advancement of whole genome sequencing will have upon our understanding and the need for reconsidering the classification of RSV genotypes.     

Pathogenesis of respiratory syncytial virus infection

Respiratory syncytial virus (RSV) is recognized as the most important cause of serious lower respiratory tract illness in infants and young children worldwide causing repeat infections throughout life with serious complications occurring in the elderly and immune compromised patient. The level of disease pathogenesis associated with RSV infection is balanced between virus elimination and the nature of the immune response to infection. The innate and adaptive immune responses to RSV infection are not fully elucidated; however, significant progress has been made in understanding the virus-host relationship and mechanisms associated with disease pathogenesis. This review summarizes important aspects of these findings, and provides current perspective on processes that may contribute to RSV disease pathogenesis.     

Molecular epidemiology of respiratory syncytial virus

Human respiratory syncytial virus (RSV) is the major cause of lower respiratory tract disease in infants. It is unusual in that it causes repeated infections throughout life. Despite considerable efforts there is as yet no satisfactory vaccine available. This paper reviews the molecular epidemiology of the RSV and describes the complex genotypic structure of RSV epidemics. The evolution of the virus is discussed, with particular reference to the antigenic and genetic variability of the attachment glycoprotein.     

呼吸道合胞病毒感染的研究进展

呼吸道合胞病毒是在世界范围内引起婴幼儿呼吸道感染的最常见病原微生物,它不仅可以引起呼吸系统的一些常见症状和体征,而且在肺外器官如中枢神经系统、心血管系统、内分泌系统等各器官也可引起一些尚未被人们普遍认识的肺外表现,而这些临床特点的重要性也愈来愈受到人们的重视.     

Structure of bovine respiratory syncytial virus

Summary Bovine respiratory syncytial (BRS) virus propagated in calf kidney (BK) cell cultures was examined by negative contrast and thin section electron microscopy.In negative stained preparations the virus proved to be of great pleomorphism. Many particles appeared roughly spherical with an overall diamter of 80–450 m. On an average, enveloped viruses covered with spikes measured 200 m in diameter.In ultrathin sections the budding of mature virus particles from the cytoplasmic membrane was clearly visualized. The size of mature viruses was 80–130 m and that of the internal component varied from 11 to 15 m.The similarity with the ultrastructure of human respiratory syncytial (RS) virus as well as of pneumonia virus of mice (PVM) has been pointed out. It is therefore proposed to classify these three viruses together in the metamyxovirus subgroup.     

Serological studies with respiratory syncytial virus

Summary Cross-neutralization tests with 9 strains of RS virus and antisera prepared against them by intra-nasal infection of ferrets, showed that antigenic variation does occur among RS virus strains. One strain, 8/60, differed from all the other strains tested, and there were smaller differences between some other strains.     

Nosocomial respiratory syncytial virus infections.

We studied the frequency and severity of respiratory syncytial virus infections acquired nosocomially on an infants' ward during a community outbreak. Every three or four days all infants and staff were examined, and specimens were obtained for viral isolation. During two months, 14 of 44 contact infants acquired the virus. All were ill, and four had pneumonia. Infected infants had a significantly longer mean hospital stay (21.5 days) than uninfected ones (9.2 days, P less than 0.001). Risk of nosocomial infection could not be related to age or to underlying disease, but was linked to length of hospitalization: 45 per cent of infants hospitalized for one week or more became infected, and the percentage increased with length of stay. Ten of 24 staff members also acquired the virus and appeared to play a major role as virus carriers. Nosocomial respiratory syncytial virus infection poses a major risk for hospitalized infants and adds to hospital costs.     

呼吸道合胞病毒抗原酶联免疫吸附测定检测方法的建立及应用

目的建它呼吸道合胞病毒抗原酶联免疫吸附测定(ELISA)检测方法,应用其对来源于本院的临床样本进行检测.方法建立ELISA方法,检测标本中的呼吸道合胞病毒,与呼吸道合胞病毒细胞培养检测方法进行对照,并利用该方法进行该病毒2007年在广州市的流行性分析.结果本方法的灵敏度略高于呼吸道合胞病毒的细胞培养检测方法,对甲型流感病毒(H3N2亚型、H1N1亚型)、乙型流感病毒、副流感病毒(Ⅰ型、Ⅲ型)、呼吸道腺病毒(Ⅲ型、Ⅶ型)无特异性反应.结论本呼吸道合胞病毒抗原ELISA检测方法可用于呼吸道合胞病毒感染的临床诊断和鉴别诊断.