RESUMO
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.
Assuntos
Vírus da Dengue/efeitos dos fármacos , Impedância Elétrica , Fusão de Membrana/efeitos dos fármacos , Vírus Sinciciais Respiratórios/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Internalização do Vírus/efeitos dos fármacos , Animais , Benzimidazóis/farmacologia , Células COS , Fusão Celular , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/fisiologia , Membrana Celular/virologia , Chlorocebus aethiops , Vírus da Dengue/fisiologia , Vírus da Dengue/ultraestrutura , Avaliação Pré-Clínica de Medicamentos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/ultraestrutura , Células Epiteliais/virologia , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Humanos , Fusão de Membrana/fisiologia , Microscopia Eletrônica de Varredura , Piridinas/farmacologia , Vírus Sinciciais Respiratórios/fisiologia , Vírus Sinciciais Respiratórios/ultraestrutura , Células VeroRESUMO
Numerous methods have been developed for immunogold labeling of thick, cryo-preserved biological specimens. However, most of the methods are permutations of chemical fixation and sample sectioning, which select and isolate the immunolabeled region of interest. We describe a method for combining immunogold labeling with cryo-electron microscopy (cryo-EM) and cryo-electron tomography (cryo-ET) of the surface proteins of intact mammalian cells or the surface glycoproteins of assembling and budding viruses in the context of virus-infected mammalian cells cultured on EM grids. In this method, the cells were maintained in culture media at physiologically relevant temperatures while sequentially incubated with the primary and secondary antibodies. Subsequently, the immunogold-labeled specimens were vitrified and observed under cryo-conditions in the transmission electron microscope. Cryo-EM and cryo-ET examination of the immunogold-labeled cells revealed the association of immunogold particles with the target antigens. Additionally, the cellular structure was unaltered by pre-immunolabeling chemical fixation and retained well-preserved plasma membranes, cytoskeletal elements, and macromolecular complexes. We think this technique will be of interest to cell biologists for cryo-EM and conventional studies of native cells and pathogen-infected cells.
Assuntos
Microscopia Crioeletrônica/métodos , Tomografia com Microscopia Eletrônica/métodos , Glicoproteínas/análise , Imuno-Histoquímica/métodos , Proteínas de Membrana/análise , Vírus Sinciciais Respiratórios/ultraestrutura , Proteínas Virais/análise , Animais , Linhagem Celular , Humanos , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sinciciais Respiratórios/químicaRESUMO
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.
Assuntos
Polietilenoglicóis/farmacologia , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sinciciais Respiratórios/efeitos dos fármacos , Vírus Sinciciais Respiratórios/fisiologia , Replicação Viral/efeitos dos fármacos , Animais , Linhagem Celular , Polaridade Celular/efeitos dos fármacos , Cães , Fluoresceína/metabolismo , Humanos , Vírus Sinciciais Respiratórios/ultraestrutura , Vírion/efeitos dos fármacos , Vírion/ultraestruturaRESUMO
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.
Assuntos
Humanos , Feminino , Gravidez , Hipóxia/patologia , Placenta , Pré-Eclâmpsia/patologia , Trofoblastos/ultraestrutura , Vírus Sinciciais Respiratórios/ultraestrutura , ObstetríciaRESUMO
THE SIXTH INTERNATIONAL RESPIRATORY SYNCYTIAL VIRUS (RSV) SYMPOSIUM was held from October 25 through October 28, 2007 in Marco Island, Florida. During this conference, over 200 students and investigators representing 16 countries convened to present and discuss recent advancements in RSV research. Presentations ranged from bench to bedside studies encompassing aspects of basic virology, pathogenesis, and immunology, as well as therapeutic and vaccine designs. In total, there were 12 invited speakers and 36 oral presentations divided into six sessions. Additionally, there were two posters sessions displaying more than one hundred abstracts. The following summary provides a review of the proceedings from each session.
Assuntos
Antivirais/uso terapêutico , Infecções por Vírus Respiratório Sincicial/imunologia , Infecções por Vírus Respiratório Sincicial/patologia , Vacinas contra Vírus Sincicial Respiratório/imunologia , Vírus Sinciciais Respiratórios/imunologia , Vírus Sinciciais Respiratórios/fisiologia , Humanos , Infecções por Vírus Respiratório Sincicial/fisiopatologia , Infecções por Vírus Respiratório Sincicial/prevenção & controle , Vírus Sinciciais Respiratórios/patogenicidade , Vírus Sinciciais Respiratórios/ultraestruturaRESUMO
Respiratory syncytial virus (RSV), a nonsegmented, negative-sense RNA-containing virus, is a common cause of lower respiratory tract disease. Expression of RSV nucleocapsid protein (N) in insect cells using the baculovirus expression system leads to the formation of N-RNA complexes that are morphologically indistinguishable from viral nucleocapsids. When imaged in an electron microscope, three distinct types of structures were observed: tightly wound short-pitch helices, highly extended helices, and rings. Negative stain images of N-RNA rings were used to calculate a three-dimensional reconstruction at 24 A resolution, revealing features similar to those observed in nucleocapsids from other viruses of the order Mononegavirales. The reconstructed N-RNA rings comprise 10 N monomers and have an external radius of 83 A and an internal radius of 40 A. Comparison of this structure with crystallographic data from rabies virus and vesicular stomatitis virus N-RNA rings reveals striking morphological similarities.
Assuntos
Substâncias Macromoleculares , Proteínas do Nucleocapsídeo/ultraestrutura , RNA Viral/ultraestrutura , Vírus Sinciciais Respiratórios/ultraestrutura , Processamento de Imagem Assistida por Computador , Microscopia Eletrônica de Transmissão , Modelos Moleculares , Proteínas do Nucleocapsídeo/química , Vírus da Raiva/ultraestrutura , Vírus da Estomatite Vesicular Indiana/ultraestruturaRESUMO
At the start of the 21st century, respiratory syncytial virus (RSV) remains a serious global health concern. Although RSV has traditionally been acknowledged as a leading cause of morbidity and mortality in the paediatric population, the elderly and people with suppressed immune systems are now also recognised as being at risk from serious RSV infection. This problem is currently exacerbated by the lack of an effective vaccine to prevent RSV infection. Although the virus proteins play a variety of roles during the virus replication cycle, in many cases these tasks are performed via specific interactions with host-cell factors, including proteins, carbohydrates and lipids. The way in which RSV interacts with the host cell is currently being examined using a battery of different techniques, which encompass several scientific disciplines. This is providing new and interesting insights into how RSV interacts with the host cell at the molecular level, which in turn is offering the hope of new strategies to prevent RSV infection.
Assuntos
Infecções por Vírus Respiratório Sincicial/prevenção & controle , Vírus Sinciciais Respiratórios/fisiologia , Interações Hospedeiro-Parasita , Humanos , Infecções por Vírus Respiratório Sincicial/patologia , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sinciciais Respiratórios/genética , Vírus Sinciciais Respiratórios/ultraestrutura , Replicação ViralRESUMO
Respiratory syncytial virus (RSV) is an important human pathogen that can cause severe and life-threatening respiratory infections in infants, the elderly, and immunocompromised adults. RSV infection of HEp-2 cells induces the activation of RhoA, a small GTPase. We therefore asked whether RhoA signaling is important for RSV replication or syncytium formation. The treatment of HEp-2 cells with Clostridium botulinum C3, an enzyme that ADP-ribosylates and specifically inactivates RhoA, inhibited RSV-induced syncytium formation and cell-to-cell fusion, although similar levels of PFU were released into the medium and viral protein expression levels were equivalent. Treatment with another inhibitor of RhoA signaling, the Rho kinase inhibitor Y-27632, yielded similar results. Scanning electron microscopy of C3-treated infected cells showed reduced numbers of single blunted filaments, in contrast to the large clumps of long filaments in untreated infected cells. These data suggest that RhoA signaling is associated with filamentous virus morphology, cell-to-cell fusion, and syncytium formation but is dispensable for the efficient infection and production of infectious virus in vitro. Next, we developed a semiquantitative method to measure spherical and filamentous virus particles by using sucrose gradient velocity sedimentation. Fluorescence and transmission electron microscopy confirmed the separation of spherical and filamentous forms of infectious virus into two identifiable peaks. The C3 treatment of RSV-infected cells resulted in a shift to relatively more spherical virions than those from untreated cells. These data suggest that viral filamentous protuberances characteristic of RSV infection are associated with RhoA signaling, are important for filamentous virion morphology, and may play a role in initiating cell-to-cell fusion.
Assuntos
Vírus Sinciciais Respiratórios/fisiologia , Transdução de Sinais , Proteína rhoA de Ligação ao GTP/metabolismo , ADP Ribose Transferases/farmacologia , Amidas/farmacologia , Toxinas Botulínicas/farmacologia , Linhagem Celular , Células Gigantes/ultraestrutura , Humanos , Piridinas/farmacologia , Vírus Sinciciais Respiratórios/efeitos dos fármacos , Vírus Sinciciais Respiratórios/ultraestrutura , Replicação Viral , Proteína rhoA de Ligação ao GTP/antagonistas & inibidoresRESUMO
Field emission scanning electron microscopy (FE SEM) was used to visualize the distribution of virus-associated components, the virus-attachment (G) protein, and the host-cell-derived lipid, GM1, in respiratory syncytial virus (RSV) filaments. RSV-infected cells were labeled in situ with a G protein antibody (MAb30) whose presence was detected using a second antibody conjugated to colloidal gold. No bound MAb30 was detected in mock-infected cells, whereas significant quantities bound to viral filaments revealing G protein clusters throughout the filaments. GM1 was detected using cholera toxin B subunit conjugated to colloidal gold. Mock-infected cells revealed numerous GM1 clusters on the cell surface. In RSV-infected cells, these gold clusters were detected on the filaments in low, but significant, amounts, indicating the incorporation of GM1 within the viral envelope. This report describes the first use of FE SEM to map the distribution of specific structural components within the envelope of a Paramyxovirus.
Assuntos
Gangliosídeo G(M1)/metabolismo , Vírus Sinciciais Respiratórios/metabolismo , Proteínas Virais/metabolismo , Animais , Anticorpos Monoclonais , Anticorpos Antivirais , Membrana Celular/metabolismo , Membrana Celular/virologia , Chlorocebus aethiops , Microscopia Eletrônica de Varredura/métodos , Vírus Sinciciais Respiratórios/crescimento & desenvolvimento , Vírus Sinciciais Respiratórios/ultraestrutura , Células VeroRESUMO
This paper presents the first isolation of bovine respiratory syncytial virus in Brazil and its physicochemical, morphological and molecular characterization. The virus was isolated from 33 samples of nasotracheal secretions, successively inoculated into a Madin-Darby bovine kidney cell culture, which was characterized by physicochemical tests and morphological observation by electron microscopy. The Brazilian sample is an RNA pleomorphic, enveloped, thermolabile and non-hemagglutinating spicular virus. Reverse transcription, followed by nested polymerase chain reaction (nRT-PCR) assay was carried out using oligonucleotides B1, B2A, B3 and B4 for the fusion proteins (F) and B5A, B6A, B7A and B8 for the attachment protein (G). The nRT-PCR-F amplified a fragment of 481 bp corresponding to part of the gene that codes for protein F, whereas nRT-PCR-G amplified a fragment of 371 bp, in agreement with part of the G gene. The virus isolated from Brazilian samples in this study corresponded to the bovine respiratory syncytial virus, and RT-PCR proved to be useful for the diagnosis of bovine clinical samples.
Assuntos
Vírus Sinciciais Respiratórios/isolamento & purificação , Animais , Brasil , Bovinos , Células Cultivadas , Microscopia Eletrônica , RNA Viral/análise , Vírus Sinciciais Respiratórios/genética , Vírus Sinciciais Respiratórios/ultraestrutura , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Mink lung epithelial cells (Mv-1-Lu) were tested for their ability to support the growth and serial passage of respiratory syncytial virus (RSV) in vitro. Indian isolates of RSV induced distinctive cytopathic effect with typical rounding of cells followed by detachment with more than 50 per cent cells showing bright fluorescence using anti-RSV monoclonal antibodies in immunofluorescence test. Serial passage of RSV was possible in Mv-1-Lu cells without loss of sensitivity of the cells for virus growth. Titration of cell associated virus and virus released in the supernatant indicated that 60 per cent of the virus was released in the supernatant, and 40 per cent remained cell associated. Transmission electron microscopic studies of negatively stained RSV particles and ultra-thin sections of RSV infected Mv-1-Lu cells showed roughly spherical particles with club shaped projections, budding from the cytoplasmic membrane. These results indicate that Mv-1-Lu cell line is suitable for the growth and propagation of RSV.
Assuntos
Mucosa Respiratória/virologia , Vírus Sinciciais Respiratórios/crescimento & desenvolvimento , Animais , Linhagem Celular , Tamanho Celular , Pré-Escolar , Efeito Citopatogênico Viral , Feminino , Humanos , Índia , Lactente , Masculino , Vison , Mucosa Respiratória/citologia , Mucosa Respiratória/metabolismo , Vírus Sinciciais Respiratórios/metabolismo , Vírus Sinciciais Respiratórios/ultraestrutura , Cultura de VírusRESUMO
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.
Assuntos
Membrana Celular/metabolismo , Processamento de Proteína Pós-Traducional , Vírus Sinciciais Respiratórios/metabolismo , Subtilisinas/metabolismo , Proteínas Virais de Fusão/metabolismo , Animais , Biotinilação , Linhagem Celular , Membrana Celular/ultraestrutura , Membrana Celular/virologia , Chlorocebus aethiops , Imunofluorescência , Furina , Glicosilação , Cinética , Microscopia Eletrônica de Varredura , Peso Molecular , Mutação/genética , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Vírus Sinciciais Respiratórios/efeitos dos fármacos , Vírus Sinciciais Respiratórios/crescimento & desenvolvimento , Vírus Sinciciais Respiratórios/ultraestrutura , Inibidores de Serina Proteinase/farmacologia , Subtilisinas/antagonistas & inibidores , Subtilisinas/genética , Células Vero , Proteínas Virais de Fusão/química , Proteínas Virais de Fusão/ultraestrutura , Montagem de Vírus/efeitos dos fármacosRESUMO
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.
Assuntos
Técnicas Imunoenzimáticas/normas , Técnicas Microbiológicas , Vírus Sinciciais Respiratórios/imunologia , Vírus Sinciciais Respiratórios/isolamento & purificação , Anticorpos Monoclonais/análise , Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/análise , Anticorpos Antivirais/imunologia , Humanos , Técnicas Imunoenzimáticas/métodos , Microscopia Eletrônica , Vírus Sinciciais Respiratórios/ultraestruturaRESUMO
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).
Assuntos
Proteína HN , Vírus Sinciciais Respiratórios/crescimento & desenvolvimento , Anticorpos Monoclonais , Citoplasma/virologia , Humanos , Microscopia Imunoeletrônica , Nucleocapsídeo/metabolismo , Nucleocapsídeo/ultraestrutura , Vírus Sinciciais Respiratórios/ultraestrutura , Células Tumorais Cultivadas , Proteínas do Envelope Viral , Proteínas Virais/metabolismo , Proteínas Virais/ultraestrutura , Vírion/ultraestruturaRESUMO
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.
Assuntos
Proteína HN , Corpos de Inclusão Viral/metabolismo , Vírus Sinciciais Respiratórios/metabolismo , Proteínas Virais/biossíntese , Linhagem Celular , Microscopia Imunoeletrônica , Nucleoproteínas/biossíntese , Fosfoproteínas/biossíntese , Vírus Sinciciais Respiratórios/ultraestrutura , Transfecção , Proteínas do Envelope Viral , Proteínas Virais/genética , Replicação ViralRESUMO
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.
Assuntos
Transplante de Medula Óssea/patologia , Líquido da Lavagem Broncoalveolar/citologia , Vírus Sinciciais Respiratórios , Infecções por Respirovirus/diagnóstico , Autopsia , Transplante de Medula Óssea/efeitos adversos , Líquido da Lavagem Broncoalveolar/microbiologia , Criança , Citoplasma/microbiologia , Citoplasma/ultraestrutura , Imunofluorescência , Humanos , Pulmão/microbiologia , Pulmão/patologia , Pulmão/ultraestrutura , Masculino , Microscopia Eletrônica , Vírus Sinciciais Respiratórios/isolamento & purificação , Vírus Sinciciais Respiratórios/ultraestruturaRESUMO
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.
Assuntos
Doenças dos Bovinos/patologia , Alvéolos Pulmonares/ultraestrutura , Vírus Sinciciais Respiratórios/ultraestrutura , Infecções por Respirovirus/veterinária , Animais , Bovinos , Células Gigantes/ultraestrutura , Hiperplasia , Hipertrofia , Macrófagos/ultraestrutura , Masculino , Microscopia Eletrônica , Necrose , Neutrófilos/ultraestrutura , Fagocitose , Alvéolos Pulmonares/microbiologia , Vírus Sinciciais Respiratórios/fisiologia , Infecções por Respirovirus/patologia , Replicação ViralRESUMO
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.
Assuntos
Brônquios/ultraestrutura , Doenças dos Bovinos/patologia , Vírus Sinciciais Respiratórios/ultraestrutura , Infecções por Respirovirus/veterinária , Idoso , Animais , Brônquios/microbiologia , Bovinos , Cílios/ultraestrutura , Epitélio/ultraestrutura , Células Gigantes/ultraestrutura , Humanos , Hiperplasia , Hipertrofia , Imuno-Histoquímica , Macrófagos/ultraestrutura , Masculino , Microscopia Eletrônica , Necrose , Neutrófilos/ultraestrutura , Infecções por Respirovirus/patologiaRESUMO
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.
Assuntos
Vírus da Parainfluenza 3 Humana/fisiologia , Alvéolos Pulmonares/microbiologia , Vírus Sinciciais Respiratórios/fisiologia , Replicação Viral , Animais , Bovinos , Separação Celular , Células Cultivadas , Centrifugação com Gradiente de Concentração , Células Epiteliais , Epitélio/microbiologia , Epitélio/ultraestrutura , Filtração , Imunoglobulina G , Lectinas , Masculino , Microscopia Eletrônica , Vírus da Parainfluenza 3 Humana/ultraestrutura , Alvéolos Pulmonares/citologia , Alvéolos Pulmonares/ultraestrutura , Vírus Sinciciais Respiratórios/ultraestruturaRESUMO
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.