Respiratory syncytial virus

Human respiratory syncytial virus (RSV) is the most common worldwide cause of lower respiratory tract infections (LRI) in infants less than 6 months of age. The prophylaxis against RSV infection by vaccination has been unsuccessful because of its adverse effects. As antiviral drug, ribavirin spray (aerosol) had been used clinically and reduces the amount of virus load, without reducing the necessity of symptomatic therapy and the duration of hospitalization. Therefore RSV LRI has been treated mainly symptomatically. Recently humanized anti-RSV F protein monoclonal antibody was developed and prescribed for prevention in high-risk infants such as premature ones and those with chronic lung and congenital heart diseases. It reduced the incidence of hospitalization significantly. It has been introduced in clinical use in Japan following to Western countries. On the other hand, a number of anti-RSV drugs have now been investigation; however, no valuable drugs for clinical use have been yet developed.     

Respiratory syncytial virus matrix protein induces lung epithelial cell cycle arrest through a p53 dependent pathway

Respiratory syncytial virus (RSV) is the major cause of viral respiratory infections in children. Our previous study showed that the RSV infection induced lung epithelial cell cycle arrest, which enhanced virus replication. To address the mechanism of RSV-induced cell cycle arrest, we examined the contribution of RSV-matrix (RSV-M) protein. In this report, we show that in both the A549 cell line and primary human bronchial epithelial (PHBE) cells, transfection with RSV-M protein caused the cells to proliferate at a slower rate than in control cells. The cell cycle analysis showed that RSV-M protein induced G1 phase arrest in A549 cells, and G1 and G2/M phase arrest in PHBE cells. Interestingly, RSV-M expression induced p53 and p21 accumulation and decreased phosphorylation of retinoblastoma protein (Rb). Further, induction of cell cycle arrest by RSV-M was not observed in a p53-deficient epithelial cell line (H1299). However, cell cycle arrest was restored after transfection of p53 cDNA into H1299 cells. Taken together, these results indicate that RSV-M protein regulates lung epithelial cell cycle through a p53-dependent pathway, which enhances RSV replication.     

Respiratory syncytial virus infection reduces lung inflammation and fibrosis in mice exposed to vanadium pentoxide

Background

Vanadium pentoxide (V₂O₅) exposure is a cause of occupational bronchitis and airway fibrosis. Respiratory syncytial virus (RSV) is a ubiquitous pathogen that causes airway inflammation. It is unknown whether individuals with pre-existing respiratory viral infection are susceptible to V₂O₅-induced bronchitis. We hypothesized that respiratory viral infection will exacerbate vanadium-induced lung fibrosis.

Methods

In this study we investigated the effect of RSV pre- or post-exposure to V₂O₅ in male AKR mice. Mice were pre-exposed by intranasal aspiration to RSV or media vehicle prior to intranasal aspiration of V₂O₅ or saline vehicle at day 1 or day 7. A parallel group of mice were treated first with V₂O₅ or saline vehicle at day 1 and day 7 then post-exposed to RSV or media vehicle at day 8.

Results

V₂O₅-induced airway inflammation and fibrosis were decreased by RSV pre- or post-exposure. Real time quantitative RT-PCR showed that V₂O₅ significantly increased lung mRNAs encoding pro-fibrogenic growth factors (TGF-β1, CTGF, PDGF-C) and collagen (Col1A2), but also increased mRNAs encoding anti-fibrogenic type I interferons (IFN-α, -β) and IFN-inducible chemokines (CXCL9 and CXCL10). RSV pre- or post-exposure caused a significantly reduced mRNAs of pro-fibrogenic growth factors and collagen, yet reduced RNA levels of anti-fibrogenic interferons and CXC chemokines.

Conclusions

Collectively these data suggest that RSV infection reduces the severity of V₂O₅-induced fibrosis by suppressing growth factors and collagen genes. However, RSV suppression of V₂O₅-induced IFNs and IFN-inducible chemokines suggests that viral infection also suppresses the innate immune response that normally serves to resolve V₂O₅-induced fibrosis.     

Respiratory syncytial virus infection in anti-mu-treated mice.

BALB/c mice were depleted of B cells by anti-mu treatment to investigate the pathogenesis of respiratory syncytial virus (RSV) infection in the absence of antibody. Termination of RSV replication after primary infection occurred with the same kinetics in anti-mu-treated mice as in phosphate-buffered saline (PBS)-treated controls. Yet, when rechallenged, anti-mu-treated mice were more permissive to RSV replication than PBS-treated controls. Anti-mu-treated mice also experienced greater illness than PBS-treated controls during both primary infection and rechallenge. Passive transfer of RSV-specific immune serum to anti-mu-treated mice before rechallenge reconstituted complete protection from RSV replication and diminished illness. Thus, RSV-specific antibody is not required to terminate RSV replication in primary infection, but without antibody, only partial immunity against rechallenge is induced. While it is unknown whether the mechanism is a direct effect on RSV titer or modulation of the illness-causing cellular immune response, the presence of RSV-specific antibody reduces illness in both primary RSV infection and rechallenge of mice.     

人呼吸道合胞病毒减毒活疫苗的研究进展

人呼吸道合胞病毒(Respiratory syncytial virus,RSV)是一种可引起严重下呼吸道疾病的病原体,易感人群为婴幼儿、老年人及免疫力低下者。目前尚无针对RSV的疫苗和特异而有效的抗病毒药物。研究发现,减毒活疫苗不产生疾病增强效应,在母传抗体的存在下仍可复制,且有较强的免疫原性,被认为是最有希望的RSV疫苗。就RSV减毒活疫苗的研究进展作一综述。     

Respiratory syncytial virus mRNA coding assignments.

The polypeptide coding assignments for six of the respiratory syncytial virus-specific mRNAs were determined by translation of the individual mRNAs in vitro. The coding assignments of the RNAs are as follows. RNA band 1 is complex and can be separated into at least two components on the basis of electrophoretic mobility (molecular weights [MWs] approximately equal to 0.21 X 10(6) and 0.31 X 10(6), respectively) that code for three polypeptides of 9.5, 11, and 14 kilodaltons (K). RNA 2 (MW, 0.39 X 10(6)) codes for a 34K polypeptide; RNA 3 (MW, 0.40 X 10(6)) codes for a 26K polypeptide; RNA 4 (MW, 0.47 X 10(6)) codes for a 42K polypeptide; and RNA 5 (MW, 0.74 X 10(6)) codes for a 59K polypeptide. By limited-digest peptide mapping, the 34, 26, and 42K polypeptides synthesized in vitro appeared to be unique. Additionally, peptide mapping showed that the 34, 26, and 42K polypeptides synthesized in vitro were indistinguishable from their counterparts synthesized in infected cells. Thus, the 34, 26, and 42K polypeptides coded for by mRNAs 2, 3, and 4, respectively, were identified as the respiratory syncytial virus phosphoprotein (34K), matrix protein (26K), and nucleocapsid protein (42K), respectively. RNA 5 was shown to code for a 59K polypeptide. The 59K polypeptide synthesized in vitro did not comigrate with any polypeptide specific to infected cells, suggesting that it is a candidate for co- or post-translational modification.     

Influence of endogenous catecholamines on responses of rat lung to beta-adrenergic agonists

Relaxation responses to the beta-adrenoceptor agonists: isoprenaline (non selective), salbutamol (beta 2-selective) and noradrenaline (plus phentolamine 10(-5) M) (beta 1-selective) have been obtained on rat lung parenchymal strips in the absence and presence of pargyline and tropolone (monoamino-oxidase and catechol-O-methyltransferase inhibitors), cocaine (neuronal uptake blocking agent), corticosterone (extraneuronal uptake inhibitor) as well as in reserpinized rat. Responses to these beta-adrenergic agonists were not potentiated in the presence of any of these inhibitors. This indicates that endogenous catecholamines, enzymatic or uptake processes, do not modulate beta-adrenoceptor mediated responses of rat lung strip and demonstrates that there is no correlation between neuronal uptake/beta 1-adrenoceptors and extraneuronal uptake/beta 2-adrenoceptor mediated responses, as had previously been suggested.     

Respiratory syncytial virus (RSV) infection induces cyclooxygenase 2: a potential target for RSV therapy

Cyclooxygenases (COXs) are rate-limiting enzymes that initiate the conversion of arachidonic acid to prostanoids. COX-2 is the inducible isoform that is up-regulated by proinflammatory agents, initiating many prostanoid-mediated pathological aspects of inflammation. The roles of cyclooxygenases and their products, PGs, have not been evaluated during respiratory syncytial virus (RSV) infection. In this study we demonstrate that COX-2 is induced by RSV infection of human lung alveolar epithelial cells with the concomitant production of PGs. COX-2 induction was dependent on the dose of virus and the time postinfection. PG production was inhibited preferentially by NS-398, a COX-2-specific inhibitor, and indomethacin, a pan-COX inhibitor, but not by SC-560, a COX-1-specific inhibitor. In vivo, COX-2 mRNA expression and protein production were strongly induced in the lungs and cells derived from bronchioalveolar lavage of cotton rats infected with RSV. The pattern of COX-2 expression in vivo in lungs is cyclical, with a final peak on day 5 that correlates with maximal histopathology. Treatment of cotton rats with indomethacin significantly mitigated lung histopathology produced by RSV. The studies described in this study provide the first evidence that COX-2 is a potential therapeutic target in RSV-induced disease.     

Respiratory syncytial virus: its transmission in the hospital environment

Respiratory syncytial virus (RSV) over the past two decades has been recognized as the most important cause of lower respiratory tract disease in infants and young children. Recently, it has also been identified as a major nosocomial hazard on pediatric wards. The potential for RSV to spread on such wards is underlined by several singular characteristics of RSV. It arrives in yearly epidemics and is highly contagious in all age groups. Immunity is of short duration, allowing repeated infections to occur. Thus, during an epidemic 20--40 percent of infants admitted for other conditions may acquire nosocomial RSV infection, as well as 50 percent of the ward personnel. The usual infection control procedures for respiratory illnesses have had limited success in controlling the spread of RSV. This may be due in part to the modes of transmission of RSV. Inoculation occurs mainly through the eye and nose, rather than the mouth. This may be via large-particle aerosols or droplets, requiring close contact. The virus, however, does not seem capable of traversing distances by small-particle aerosols. Nevertheless, it is able to remain infectious on various environmental surfaces, suggesting fomites as a source of spread. Indeed, inoculation after touching such contaminated surfaces can occur, and may be a major second means of spread, in hospitals as well as in families.     

Respiratory syncytial virus ts mutants and nuclear immunofluorescence.

A replicated sector-plating procedure was used to isolate 35 induced temperature-sensitive (ts) mutants and one spontaneous ts mutant from a wild-type stock of respiratory syncytial (RS) virus cloned from recent clinical material. Seven of these mutants were ts for plaque formation at 37 degrees C as well as at the restrictive temperature of 39 degrees C. The wild-type strain did not differ markedly from standard laboratory strains of RS virus. It was dependent on exogenous arginine (84 mug/ml) for optimal growth, and was not significantly inhibited by mitomycin C (10 mug/ml). It was sensitive to actinomycin D (2.5 mug/ml) during the early part of the growth phase. A characteristic focal cytopathic effect was obtained in BS-C-1 cells. Staining of infected monolayers by an indirect immunofluorescence procedure revealed a profusion of filamentous processes extending from the plasma membrane, and a similar modification of the surface of infected cells could be visualized by scanning electron microscopy. Filament production was inhibited when certain ts mutants were incubated at 39 degrees C, confirming the virus-specific nature of the phenomenon. Thirty-four of the mutants were classified into three groups by immunofluorescence. Complementation was observed in mixed infection with a single mutant from each group. Nuclear, as well as cytoplasmic, immunofluorescence was detected in RS virus-infected cells using a high-titer bovine anti-bovine RS virus serum. Visualization of nuclear antigen was dependent on the inhibition of cytoplasmic fluorescence obtained when ts mutants in groups I and III were incubated at restrictive temperature.     

Human metapneumovirus induces a profile of lung cytokines distinct from that of respiratory syncytial virus

Lung cytokine and chemokine production by BALB/c mice infected with human metapneumovirus (hMPV) was compared to respiratory syncytial virus (RSV)-infected mice. hMPV infection induced lower levels of the inflammatory cytokines interleukin-1 (IL-1), IL-6 and tumor necrosis factor alpha but was a more potent inducer of granulocyte-macrophage colony-stimulating factor and triggered a more sustained production of the CXC chemokine KC compared to RSV. hMPV was a stronger inducer of both alpha interferon (IFN-alpha) and IFN-gamma responses than RSV. In regard to immunomodulatory cytokines, hMPV failed to induce detectable IL-10 or IL-12p70 but was a potent inducer of IL-12 p40 subunit. The implications for hMPV pathogenesis are discussed.     

Respiratory syncytial virus proteins: identification by immunoprecipitation.

The proteins of respiratory syncytial virus have not been clearly identified due to the lability of the virus and difficulties in its purification. We have pulse-labeled respiratory syncytial virus with [35S]methionine and [35S]cysteine and analyzed cell lysates by polyacrylamide gel electrophoresis. Five 35S-labeled viral proteins ranging in molecular weight from 21,000 to 73,000 (VP73, VP44, VP35, VP28, and VP21) were easily discernable above background cellular proteins. Treatment of the infected cells with 0.15 M NaCl before labeling suppressed host cell protein synthesis and allowed clearer visualization of the five viral proteins by polyacrylamide gel electrophoresis. Three glycoproteins (VGP 92, VGP 50, and VGP 17) were also identified after labeling with [3H]glucosamine. Five of these polypeptides (VP51, VP44, VP35, VP28, and VGP92) were shown to be antigenically active because they could be immunoprecipitated with anti-respiratory syncytial virus antibody produced in New Zealand white rabbits, cotton rats, and humans before analysis by polyacrylamide gel electrophoresis.     

Respiratory syncytial virus infection of human lung endothelial cells enhances selectively intercellular adhesion molecule-1 expression

Respiratory syncytial virus (RSV) is worldwide the most frequent cause of bronchiolitis and pneumonia in infants requiring hospitalization. In the present study, we supply evidence that human lung microvascular endothelial cells, human pulmonary lung aorta endothelial cells, and HUVEC are target cells for productive RSV infection. All three RSV-infected endothelial cell types showed an enhanced cell surface expression of ICAM-1 (CD54), which increased in a time- and RSV-dose-dependent manner. By using noninfectious RSV particles we verified that replication of RSV is a prerequisite for the increase of ICAM-1 cell surface expression. The up-regulated ICAM-1 expression pattern correlated with an increased cellular ICAM-1 mRNA amount. In contrast to ICAM-1, a de novo expression of VCAM-1 (CD106) was only observed on RSV-infected HUVEC. Neither P-selectin (CD62P) nor E-selectin (CD62E) was up-regulated by RSV on human endothelial cells. Additional experiments performed with neutralizing Abs specific for IL-1alpha, IL-1beta, IL-6, and TNF-alpha, respectively, excluded an autocrine mechanism responsible for the observed ICAM-1 up-regulation. The virus-induced ICAM-1 up-regulation was dependent on protein kinase C and A, PI3K, and p38 MAPK activity. Adhesion experiments using polymorphonuclear neutrophil granulocytes (PMN) verified an increased ICAM-1-dependent adhesion rate of PMN cocultured with RSV-infected endothelial cells. Furthermore, the increased adhesiveness resulted in an enhanced transmigration rate of PMN. Our in vitro data suggest that human lung endothelial cells are target cells for RSV infection and that ICAM-1 up-regulated on RSV-infected endothelial cells might contribute to the enhanced accumulation of PMN into the bronchoalveolar space.     

Respiratory syncytial virus induces TLR3 protein and protein kinase R, leading to increased double-stranded RNA responsiveness in airway epithelial cells

Respiratory syncytial virus (RSV) preferentially infects airway epithelial cells, causing bronchiolitis, upper respiratory infections, asthma exacerbations, chronic obstructive pulmonary disease exacerbations, and pneumonia in immunocompromised hosts. A replication intermediate of RSV is dsRNA. This is an important ligand for both the innate immune receptor, TLR3, and protein kinase R (PKR). One known effect of RSV infection is the increased responsiveness of airway epithelial cells to subsequent bacterial ligands (i.e., LPS). In this study, we examined a possible role for RSV infection in increasing amounts and responsiveness of another TLR, TLR3. These studies demonstrate that RSV infection of A549 and human tracheobronchial epithelial cells increases the amounts of TLR3 and PKR in a time-dependent manner. This leads to increased NF-kappaB activity and production of the inflammatory cytokine IL-8 following a later exposure to dsRNA. Importantly, TLR3 was not detected on the cell surface at baseline but was detected on the cell surface after RSV infection. The data demonstrate that RSV, via an effect on TLR3 and PKR, sensitizes airway epithelial cells to subsequent dsRNA exposure. These findings are consistent with the hypothesis that RSV infection sensitizes the airway epithelium to subsequent viral and bacterial exposures by up-regulating TLRs and increasing their membrane localization.     

Characterization of myometrial desensitization to beta-adrenergic agonists

Continuous exposure of ovine myometrial strips exposed to isoproterenol (10 microM) resulted in only transient inhibition with contractions returning within 60 min. Rechallenging these strips with isoproterenol failed to induce inhibition, confirming the occurrence of desensitization. In contrast, exposure of myometrial tissue to isoproterenol for only 5 min did not result in desensitization. Myometrial strips exposed to isoproterenol demonstrated a significant increase in cAMP content associated with inhibition of contractile activity and a subsequent fall in cAMP content upon desensitization. Elevation of endogenous cAMP levels by either inhibition of cAMP-dependent phosphodiesterase activity (0.5 mM isobutylmethylxanthine, in ovine strips) or direct activation of adenylyl cyclase (10 microM forskolin, in rat strips) induced a rapid and significant inhibition of myometrial contractile activity in desensitized tissue. Scatchard analysis of the binding of the beta-adrenoceptor antagonist, [125I]iodocyanopindolol, revealed a significant reduction in the concentration of beta-adrenergic receptors (but no change in binding affinity) in desensitized myometrial tissue. Incubation of desensitized tissue with fresh buffer for 3 h induced only a partial recovery in responsiveness to isoproterenol. These data suggest that prolonged, but not acute, exposure of the myometrium to beta-adrenergic agonists induces a state of desensitization that is associated with a down-regulation of beta-adrenoceptors but maintenance of postreceptor function.