RhoA interacts with the fusion glycoprotein of respiratory syncytial virus and facilitates virus-induced syncytium formation.
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The fusion glycoprotein (F) of respiratory syncytial virus (RSV), which mediates membrane fusion and virus entry, was shown to bind RhoA, a small GTPase, in yeast two-hybrid interaction studies. The interaction was confirmed in vivo by mammalian two-hybrid assay and in RSV-infected HEp-2 cells by coimmunoprecipitation. Furthermore, the interaction of F with RhoA was confirmed in vitro by enzyme-linked immunosorbent assay and biomolecular interaction analysis. Yeast two-hybrid interaction studies with various deletion mutants of F and with RhoA indicate that the key binding domains of these proteins are contained within, or overlap, amino acids 146 to 155 and 67 to 110, respectively. The biological significance of this interaction was studied in RSV-infected HEp-2 cells that were stably transfected to overexpress RhoA. There was a positive correlation between RhoA expression and RSV syncytium formation, indicating that RhoA can facilitate RSV-induced syncytium formation. (+info)
Antigenic and genetic characterization of twenty-six strains of human respiratory syncytial virus (subgroup A) isolated during three consecutive outbreaks in Havana city, Cuba.
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Twenty-six human respiratory syncytial virus strains (subgroup A) isolated from three outbreaks in Havana City during the period 1994/95, 1995/96 and 1996/97 were analyzed to determine their antigenic and genetic relationships. Analyses were performed by monoclonal antibodies and restriction mapping (N gene) following amplification of the select region of the virus genome by polymerase chain reaction. All isolated strains were classified as subgroup A by monoclonal antibodies and they showed a restriction pattern NP4 that belonged to subgroup A. Thus the results obtained in this work, showed a close relation (100%) between antigenic and genetic characterization of the isolated strains in our laboratory. These methods permit the examination of large numbers of isolates by molecular techniques, simplifying the researchs into the molecular epidemiology of the virus. (+info)
Association of fever and severe clinical course in bronchiolitis.
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Little attention has been given to the relation between fever and the severity of bronchiolitis. Therefore, the relation between fever and the clinical course of 90 infants (59 boys, 31 girls) hospitalised during one season with bronchiolitis was studied prospectively. Fever (defined as a single recording > 38.0 degrees C or two successive recording > 37.8 degrees C) was present in 28 infants. These infants were older (mean age, 5.3 v 4.0 months), had a longer mean hospital stay (4.2 v 2.7 days), and a more severe clinical course (71.0% v 29.0%) than those infants without fever. Radiological abnormalities (collapse/consolidation) were found in 60. 7% of the febrile group compared with 14.8% of the afebrile infants. These results suggest that monitoring of body temperature is important in bronchiolitis and that fever is likely to be associated with a more severe clinical course and radiological abnormalities. (+info)
Respiratory syncytial virus stimulates neutrophil degranulation and chemokine release.
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Neutrophil infiltration of the airways is a common finding in respiratory syncytial virus (RSV) bronchiolitis. Neutrophil-derived chemokines and neutrophil granule contents can cause further inflammation, hyperresponsiveness, and damage of the airways. In this study, peripheral blood neutrophils incubated with RSV (multiplicity of infection (MOI) = 10) induced IL-8, macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, and myeloperoxidase (MPO) release. In contrast, LPS induced only chemokine but not MPO release. RSV-induced chemokine and MPO release was noncytotoxic as assessed by trypan blue exclusion. The mechanism of RSV-induced chemokine release was shown to be transcription dependent since cytokine mRNA synthesis was increased with RSV stimulation and the process was inhibited by actinomycin-D. In addition, the effect of dexamethasone (dex) on mediator release was also studied. Dex significantly inhibited chemokine release but did not inhibit MPO release. The mechanism of inhibition of the release of these chemokines is probably posttranscriptional since the mRNA synthesis was not inhibited by dex. We conclude that the release of chemokines (IL-8, MIP-1alpha, MIP-1beta) and granule enzymes (MPO) by RSV-stimulated neutrophils may contribute to the pulmonary pathology in RSV bronchiolitis. These in vitro findings showing that dex failed to consistently inhibit all the RSV-induced release of neutrophil inflammatory mediators may explain the variable efficacy of corticosteroids in the treatment of RSV bronchiolitis. (+info)
Respiratory viral antigens in autopsy lung tissue specimens from patients with cancer or myocardial infarction.
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Using immunoenzyme histochemical analysis, we retrospectively examined lung tissue specimens obtained at autopsy from 118 patients with cancer who had received chemotherapy and 20 patients who had died after myocardial infarction. Respiratory viral antigens were demonstrated in lung tissue specimens from eight of 118 cancer patients and two of 20 myocardial infarction patients. Most of the patients with demonstrable viral antigens were febrile and had signs of pulmonary infection, but in no case was pulmonary viral infection considered clinically. The following viral antigens were demonstrated: influenza A virus (6 patients), respiratory syncytial virus (2), influenza B virus (1), and parainfluenza virus type 1 (1). (+info)
Passive IgA monoclonal antibody is no more effective than IgG at protecting mice from mucosal challenge with respiratory syncytial virus.
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Respiratory syncytial virus (RSV) is a mucosally restricted pathogen that can cause severe respiratory disease. Although parenteral administration of sufficient RSV-specific IgG can reduce severity of lower respiratory tract infection in high-risk infants, delivery of antibody by direct airway administration is an attractive alternative. Topical and parenteral administration of an IgA monoclonal antibody (MAb) specific for the RSV F glycoprotein was compared with an IgG MAb, specific for the same antigenic site, for ability to protect mice against RSV infection. Administration of RSV-specific IgG was more effective in reducing RSV titers in lung (4.6 log10 pfu/g) than IgA MAb (3.6 log10 pfu/g) when given intranasally immediately prior to infection (P=.005). RSV titers in the nose were reduced only by prophylactic administration of IgG parenterally. Therefore, topical administration of IgA is no more effective than topically administered IgG and is less effective than systemically administered IgG for protecting against RSV infection. (+info)
The M2-2 protein of human respiratory syncytial virus is a regulatory factor involved in the balance between RNA replication and transcription.
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The M2 mRNA of human respiratory syncytial virus (RSV) contains two overlapping ORFs, encoding the transcription antitermination protein (M2-1) and the 90-aa M2-2 protein of unknown function. Viable recombinant RSV was recovered in which expression of M2-2 was ablated, identifying it as an accessory factor dispensable for growth in vitro. Virus lacking M2-2 grew less efficiently than did the wild-type parent in vitro, with titers that were reduced 1, 000-fold during the initial 2-5 days and 10-fold by days 7-8. Compared with wild-type virus, the intracellular accumulation of RNA by M2-2 knockout virus was reduced 3- to 4-fold or more for genomic RNA and increased 2- to 4-fold or more for mRNA. Synthesis of the F and G glycoproteins, the major RSV neutralization and protective antigens, was increased in proportion with that of mRNA. In cells infected with wild-type RSV, mRNA accumulation increased dramatically up to approximately 12-15 hr after infection and then leveled off, whereas accumulation continued to increase in cells infected with the M2-2 knockout viruses. These findings suggest that M2-2 mediates a regulatory "switch" from transcription to RNA replication, one that provides an initial high level of mRNA synthesis followed by a shift in the RNA synthetic program in favor of genomic RNA for virion assembly. With regard to vaccine development, the M2-2 knockout has a highly desirable phenotype in which virus growth is attenuated while gene expression is concomitantly increased. (+info)
Replacement of the F and G proteins of respiratory syncytial virus (RSV) subgroup A with those of subgroup B generates chimeric live attenuated RSV subgroup B vaccine candidates.
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Human respiratory syncytial virus (RSV) exists as two antigenic subgroups, A and B, both of which should be represented in a vaccine. The F and G glycoproteins are the major neutralization and protective antigens, and the G protein in particular is highly divergent between the subgroups. The existing system for reverse genetics is based on the A2 strain of RSV subgroup A, and most efforts to develop a live attenuated RSV vaccine have focused on strain A2 or other subgroup A viruses. In the present study, the development of a live attenuated subgroup B component was expedited by the replacement of the F and G glycoproteins of recombinant A2 virus with their counterparts from the RSV subgroup B strain B1. This gene replacement was initially done for wild-type (wt) recombinant A2 virus to create a wt AB chimeric virus and then for a series of A2 derivatives which contain various combinations of A2-derived attenuating mutations located in genes other than F and G. The wt AB virus replicated in cell culture with an efficiency which was comparable to that of the wt A2 and B1 parents. AB viruses containing temperature-sensitive mutations in the A2 background exhibited levels of temperature sensitivity in vitro which were similar to those of A2 viruses bearing the same mutations. In chimpanzees, the replication of the wt AB chimera was intermediate between that of the A2 and B1 wt viruses and was accompanied by moderate rhinorrhea, as previously seen in this species. An AB chimeric virus, rABcp248/404/1030, which was constructed to contain a mixture of attenuating mutations derived from two different biologically attenuated A2 viruses, was highly attenuated in both the upper and lower respiratory tracts of chimpanzees. This attenuated AB chimeric virus was immunogenic and conferred a high level of resistance on chimpanzees to challenge with wt AB virus. The rABcp248/404/1030 chimeric virus is a promising vaccine candidate for RSV subgroup B and will be evaluated next in humans. Furthermore, these results suggest that additional attenuating mutations derived from strain A2 can be inserted into the A2 background of the recombinant chimeric AB virus as necessary to modify the attenuation phenotype in a reasonably predictable manner to achieve an optimal balance between attenuation and immunogenicity in a virus bearing the subgroup B antigenic determinants. (+info)