A generic screening platform for inhibitors of virus induced cell fusion using cellular electrical impedance.

Fusion of the viral envelope with host cell membranes is an essential step in the life cycle of all enveloped viruses. Despite such a clear target for antiviral drug development, few anti-fusion drugs have progressed to market. One significant hurdle is the absence of a generic, high-throughput, reproducible fusion assay. Here we report that real time, label-free measurement of cellular electrical impedance can quantify cell-cell fusion mediated by either individually expressed recombinant viral fusion proteins, or native virus infection. We validated this approach for all three classes of viral fusion and demonstrated utility in quantifying fusion inhibition using antibodies and small molecule inhibitors specific for dengue virus and respiratory syncytial virus.

The effect of grafted methoxypoly(ethylene glycol) chain length on the inhibition of respiratory syncytial virus (RSV) infection and proliferation.

Respiratory syncytial virus (RSV) is a significant cause of morbidity in humans. To date, no effective treatments exist and current prophylactic therapy access is limited and is only approximately 50% effective. To attenuate the risk of RSV infection, we hypothesized that bioengineering of either the virus particle or host cell via the covalent grafting of methoxypoly(ethylene glycol) [mPEG] would prevent infection. To this end, the anti-viral effects of grafting concentration, linker chemistry and polymer length on RSV infection was assessed. For viral modification, short chain polymers (2 kDa) were significantly more effective than long chain (20 kDa) polymers. In contrast, modification of host cells with small polymers provided no (approximately 0%) protection while long chain polymers effectively prevented infection. For example, at 48 hours post-infection at a multiplicity of infection of 0.5 and grafting concentrations of 5, 7.5, and 15 mm, 20 kDa mPEG decreased infection by 45, 83, and 91%, respectively. Importantly, both viral and host cell PEGylation strategies were able to provide near complete protection against RSV infection of both non-polarized and polarized cells. In conclusion, mPEG-modification of either RSV or the host cell is a highly effective prophylactic strategy for preventing viral infection.

Cambios ultraestructurales del trofoblasto en los casos de hipoxia durante la preeclampsia / Ultraestructural changes of trophoblast in hipoxic cases during

Examinar la ultraestructura del sincitiotrofoblasto en placentas de embarazadas complicadas con preeclampsia con especial referencia al efecto de la hipoxia sobre la estructura fina del tejido. Diez placentas, a término, afectadas por preeclampsia, fueron tomadas inmediatamente después del parto por cesárea y de cada una de ellas tres biopsias de la superficie maternal se disecaron en sala de parto, en especímenes de 2 a 5 mm, y se fijaron por inmersión en glutaraldehido al 4 por ciento, pH 7,4,a 4 ºC. Posteriormente se dividieron en fragmentos de 1 mm y sumergidos en solución fresca fijadora por períodos variables de 2 a 72 horas seguidas por una fijación secundaria en tetraóxido de osmio al 1 por ciento en buffer fosfato 0,1 M durante 1 hora. Las muestras se procesaron siguiendo los procedimientos convencionales de a microscopia electrónica de transmisión para su observación. Laboratorio de microscopia electrónica del Ciadana, Facultad de Ciencias de la Salud, Maracay. Los hallazgos revelan proyecciones de la membrana plasmática del sincitio de diversas formas, que simulan desprenderse de la superficie. La membrana basal del sincitio se mostró engrosada. Mitocondrias en diversos grados de degeneración presentaron partículas electron densas en la matriz mitocondrial. Regiones apicales del citoplasma sincitial parecen desprenderse hacia el espacio intervelloso. Numerosas vacuolas intracitoplasmáticas y ampliaciones de las cisternas del retículo endoplásmico rugoso se destacan en el citoplasma. Interrupciones de la membrana sincitial y regiones citoplásmicas sin membrana plasmática se notaron. Fragmentos del sincitio desprendidos de la superficie del mismo sugieren ser los corpúsculos que dañan los endotelios de la unidad materna – feto – placentaria constituyendo uno de los estímulos para l mantenimiento de a patogénesis de a preeclampsia.

Furin cleavage of the respiratory syncytial virus fusion protein is not a requirement for its transport to the surface of virus-infected cells.

The intracellular cleavage of respiratory syncytial virus (RSV) fusion (F) protein by furin was examined. In RSV-infected LoVo cells, which express an inactive form of furin, and in RSV-infected Vero cells treated with the furin inhibitor decanoyl-Arg-Val-Lys-Arg-chloromethyl ketone (dec-RVKR-cmk), the F protein was expressed as a non-cleaved 73 kDa species. In both cases the F protein was initially expressed as an endoglycosidase H (Endo H)-sensitive precursor (F0(EHs)) which was modified approximately 40 min post-synthesis by the addition of complex carbohydrates to produce the Endo H-resistant form (F0(EHr)). The size and glycosylation state of F0(EHr) were identical to a transient intermediate form of non-cleaved F protein which was detected in RSV-infected Vero cells in the absence of inhibitor. Cell surface biotinylation and surface immunofluorescence staining showed that F0(EHr) was present on the surface of RSV-infected cells. RSV filaments have been shown to be the predominant form of the budding virus that is detected during virus replication. Analysis of the RSV-infected cells using scanning electron microscopy (SEM) showed that, in the presence of dec-RVKR-cmk, virus budding was impaired, producing fewer and much smaller viral filaments than in untreated cells. A comparison of immunofluorescence and SEM data showed that F0(EHr) was routed to the surface of virus-infected cells but not located in these smaller structures. Our findings suggest that activation of the F protein is required for the efficient formation of RSV filaments.

[Development of new approaches to standartization of enzyme immunoassay test systems for detection of antibodies to respiratory syncytial virus using electron microscopy and monoclonal antibodies]. / Razrabotka novykh podkhodov k standartizatsii immunofermentnykh test-sistem dlia detektsii antitel k respiratorno-sintsitial'nomu virusu s ispol'zovaniem élektronnoi mikroskopii i monoklonal'nykh antitel.

Respiratory syncytial virus (RSV), strain Long, was purified through 20-60% sucrose gradient. The virions from different sucrose density zones were tested by ELISA for reactivity with monoclonal antibodies (MAB) to F- (MAB 9C5) and N- (MAB 8B10) proteins of RSV. Comparative study of the same patterns of RSV by electron microscopy after negative staining showed a close relationship between the virion morphology and MAB binding in ELISA. MAB 9C5 were highly reactive with the surface domains of both mature RSV virions and "empty" virion envelopes without formed inner nucleocapsid structures. MAB 8B10 reacted well only with mature virions with completely assembled nucleocapsids. These MAB failed to reorganize the N-protein epitope of immature and destroyed virions, which indicated a conformation dependence of the 8B10 binding site. For practical purposes, MAB tests can be used to determine the RSV patterns, which can be used in ELISA for serologic diagnosis of RSV infection. Testing with these MAB demonstrate the stability of RSV to extreme exposures (lyophilization, storage, heating), which is important for creation of sensitive ELISA test systems and their standardization.

Maturation of respiratory syncytial virus within HEp-2 cell cytoplasm.

Electron microscopy of HEp-2 cells infected with respiratory syncytial virus (RSV) strain Long revealed the maturation of RSV on an ultrastructural level. The results showed that the virus maturated by two different pathways. In one of them, the virus assembled and matured before reaching the plasma membrane on the internal vesicle membrane within cytoplasm. The mature virus was delivered to the plasma membrane and to the extracellular space most likely by the transport vesicles and exocytosis. In the other pathway, the virus matured on the plasma membrane as described with other members of the family Paramyxoviridae. Using monoclonal antibodies (MoAbs), we localized viral nucleoprotein (NP) and envelope proteins in cytoplasm by immunoelectron microscopy (IEM).

Cytoplasmic inclusions of respiratory syncytial virus-infected cells: formation of inclusion bodies in transfected cells that coexpress the nucleoprotein, the phosphoprotein, and the 22K protein.

Immunofluorescence staining and immunoelectron microscopy of respiratory syncytial (RS) virus-infected cells revealed the presence of cytoplasmic inclusions that were specifically labeled with monoclonal antibodies directed against the nucleoprotein (NP), the phosphoprotein (P), or the 22K protein. Transient expression of these three proteins with the vaccinia-T7 system, either individually or in different combinations, demonstrated that coexpression of NP and P was sufficient to induce the formation of cytoplasmic inclusions similar to those found in RS virus-infected cells. In addition, the 22K protein was also incorporated to the inclusions when coexpressed with both NP and P proteins. Immunobinding assays revealed the presence of NP-P and NP-22K complexes in extracts of RS virus-infected cells. These complexes were also detected in extracts of transfected cells that coexpressed the corresponding proteins. The implications of these results for the RS virus replicative cycle are discussed.

Cytologic diagnosis of respiratory syncytial virus infection in a bronchoalveolar lavage specimen from a bone marrow transplant recipient.

Cytologic examination of a bronchoalveolar lavage specimen from a 6-year-old bone marrow transplant recipient revealed pulmonary infiltrates and occasional cells containing discrete pink cytoplasmic inclusions on a May-Grunwald-Giemsa stain. Direct immunofluorescence stains of cytospins prepared from the same specimen were positive for respiratory syncytial virus (RSV). Electron microscopy revealed occasional epithelial cells with cytoplasmic inclusions composed of filamentous virions. The patient died 6 months after the specimen was taken. An autopsy showed ongoing bronchiolitis with diffuse alveolar damage. Occasional bronchiolar epithelial cells contained discrete eosinophilic cytoplasmic inclusions, which on ultrastructural examination proved to be compatible with RSV. Examination of bronchoalveolar lavage fluid from bone marrow transplant recipients should include a search for cytopathic changes compatible with RSV infection. Electron microscopy can be helpful in confirming this diagnosis.

Ultrastructural features of alveolar lesions in induced respiratory syncytial virus pneumonia of calves.

Ultrastructural changes occurred in alveolar epithelium in the acute and repair stages of induced respiratory syncytial virus pneumonia induced in eight calves (calf Nos. 1-7, 3 to 6 days old and calf No. 8, 2 weeks old), using a bovine strain of respiratory syncytial virus. Five of the calves were Friesians, three were Hereford x Friesians, and all were male. Tissues from three mock-infected control calves (two Friesian, one Hereford x Friesian) were also examined. Evidence of respiratory syncytial virus infection was observed in both type I and type II pneumocytes from day 4 to day 8 after infection. Infection of type I pneumocytes frequently resulted in necrosis. The response of type II pneumocytes to respiratory syncytial virus infection varied and included hypertrophy, hyperplasia, and syncytial formation. In some infected type II pneumocytes, there were numerous irregular projections of the cell surface, associated with viral budding. Hypertrophy and hyperplasia of type II pneumocytes, epithelial syncytium formation, and irregular cytoplasmic projections from epithelial cells caused considerable thickening of respiratory membrane and occlusion of alveolar lumina. Neutrophils were frequently observed in close association with virus-infected epithelial cells, but evidence of respiratory syncytial virus infection and replication was not observed in alveolar macrophages or neutrophils. Proliferation of type II pneumocytes appeared to play a major role in maintaining the integrity of the alveolar epithelium during the acute stage of the experimental pneumonia. Increased numbers of type II pneumocytes were present on alveolar walls, particularly from 4 to 8 days after infection, and some alveoli were lined entirely by this cell type. In some areas, however, squamous epithelial cells were also involved in covering exposed alveolar basement membrane.

Ultrastructural features of lesions in bronchiolar epithelium in induced respiratory syncytial virus pneumonia of calves.

Ultrastructural changes were observed in bronchioles in acute and repair stages of respiratory syncytial virus pneumonia induced in eight young calves (calf Nos. 1-8) using a bovine strain of respiratory syncytial virus. Five of the calves were Friesians and three were Hereford x Friesians and all were male. Tissues from three mock-infected control calves (two Friesian, one Hereford x Friesian) were also examined. Calves were from 3 to 6 days old at the time of first inoculation, with the exception of calf No. 8, which was 2 weeks old. In the acute stage of the induced pneumonia, evidence of respiratory syncytial virus replication and release was demonstrable in both ciliated and non-ciliated bronchiolar epithelial cells, with the virus-releasing process most obvious at 4 and 5 days after infection. Respiratory syncytial virus infection of bronchiolar epithelium was associated with various changes, including hypertrophy, hyperplasia, and formation of syncytia. Necrosis of epithelial cell structures usually appeared to be preceded by their desquamation from bronchiolar walls. Respiratory syncytial virus infection resulted in considerable damage to the bronchiolar ciliary apparatus. Such damage was seen as early as 1 day post-infection and was still obvious at 10 days post-infection. Neutrophils were closely associated with respiratory syncytial virus infected epithelial cells and evidence of neutrophil fusion with infected epithelial cells was seen. These observations suggest that neutrophils may be involved in killing respiratory syncytial virus infected cells and that neutrophils might play an important role in early antiviral defense against respiratory syncytial virus at a time when antibody levels are low and other cellular defenses are not fully in play. Bronchiolar repair was evident from 6 days after infection and was well advanced at 10 and 13 days after infection.

Replication of parainfluenza type-3 virus and bovine respiratory syncytial virus in isolated bovine type-II alveolar epithelial cells.

The objectives of our research were to determine whether bovine pulmonary type-II alveolar epithelial cells could be isolated from bovine lung and maintained in tissue culture and to determine whether isolated bovine type-II alveolar epithelial cells would support productive viral replication of bovine parainfluenza type-3 virus and bovine respiratory syncytial virus. Type-II alveolar epithelial cells were isolated from lungs of 4- to 7-day-old male Holstein calves by enzymatic dissociation of pulmonary tissue with trypsin and by separation of cells with the use of filtration and centrifugation on continuous Percoll gradients. Cells were further separated by panning on IgG-coated plastic plates and by lectin binding. Isolated type-II alveolar cells were maintained on basement membrane-coated tissue cultured plates. In culture, type-II cells formed alveolar structures and maintained other cytologic features of type-II cells, including osmiophilic lamellar inclusions. Cell cultures were inoculated with and supported productive replication of bovine parainfluenza type-3 virus and bovine respiratory syncytial virus. This was determined by recovery of infectious viruses from inoculated cell cultures and by identification of viral structures in type-II alveolar epithelial cells by transmission electron microscopy.

Comparison of caprine, human and bovine strains of respiratory syncytial virus.

A new continuous ovine kidney cell line allowing the growth of caprine, human and bovine respiratory syncytial virus was used to minimize host cell related variations for the direct comparison of the viral ultrastructures, serological relationships and structural protein profiles. Results show that all three strains are closely related although a closer relationship was found between bovine and caprine RS.