大麦黄花叶病毒侵染的大麦叶肉细胞超微结构研究

在自然感染大麦黄花叶病毒的大麦叶肉细胞中可见线条状和杆状的病毒粒体以及风轮状内含体。这些病毒的长度一般为480—920nm,宽为lo—20nm。此外,还观察到一种由许多病毒组成的堆束状结构,这种病毒的直径为13nm 左右,长度可达2000nm 以上。感病叶肉细胞的超微结构变化是相当明显的。在病害严重的细胞中,细胞基质丧失严重;叶绿体膜系统破坏;线粒体的嵴和基质减少;内质网膨大或断裂,小泡大量出现,病毒粒体的一端往往与内质网联结在一起,特征性膜性网络结构在感染的细胞质中形成。细胞核和细胞膜也发生了变化。     

Ultrastructural Studies of Barley Mesophyll Cella lnfected with Barley Yellow Mosaic Viruses

lexuous filamentous, rod-shaped particles, and laminated, pinwheel inclusions were observed in the mesophyll cells of the barley plants naturally infected with barley yellow mosaic viruses. These virus particles had a length of 480–920 nm and a width of 10–20 nm. In addition, bundles of filamentous structures which consisted of many particles with more 2000 nm in length were found in the leaves of the infected barley plants. The ultrastructural alterations of the infected mesophyll cells were rather conspicuous. The cytoplasmic matrix was lost seriously, and the chloroplast membrane system was destroyed. The cristae and matrix of the mitochondrium were decreased and some of them became vacuoles. The endoplasmic reticulum (ER) expanded teristic membranous network structures occurred in the cytoplasm of infected cells. The virus particles were often associated at one end with ER and with the membranes of network structures. The nucleus, membrane and wall of ceils also had somewhat variation.     

Cytopathology of satellite tobacco mosaic virus and its helper virus in tobacco

Ultrastructural responses of tobacco cells infected with a newly discovered satellite virus (STMV) that has an isometric morphology and is associated with rigid rodshaped tobacco mosaic virus (TMV) were studied in situ. In cells infected with TMV alone,TMV particles occurred as crystalline arrays in the cytoplasm and were usually associated with TMV-characteristic X bodies. In cells infected with both TMV and STMV, particles of STMV occurred only in cells that contained TMV particles, which suggests a correlation between the satellite and helper virus presence. However, the replication and/or accumulation sites of STMV appear to be independent from its helper virus. Unlike TMV particles, STMV particles were associated with several cytopathic structures such as granular inclusions, membranous vesicles of 50–80 nm, and myelin-like bodies which were all bounded by a single common membrane, No X bodies occurred in cells containing STMV particles, and the mitochondria possessed abnormal tubular structures containing flocculent material.     

Respiratory syncytial virus matures at the apical surfaces of polarized epithelial cells.

Respiratory syncytial (RS) virus infects the epithelium of the respiratory tract. We examined the replication and maturation of RS virus in two polarized epithelial cell lines, Vero C1008 and MDCK. Electron microscopy of RS virus-infected Vero C1008 cells revealed the presence of pleomorphic viral particles budding exclusively from the apical surface, often in clusters. The predominant type of particle was filamentous, 80 to 100 nm in diameter, and 4 to 8 microns in length, and evidence from filtration studies indicated that the filamentous particles were infectious. Cytopathology produced by RS virus infection of polarized Vero C1008 cells was minimal, and syncytia were not observed, consistent with the maintenance of tight junctions and the exclusively apical maturation of the virus. Infectivity assays with MDCK cells confirmed that in this cell line, RS virus was released into the apical medium but not into the basolateral medium. In addition, the majority of the RS virus transmembrane fusion glycoprotein on the cell surface was localized to the apical surface of the Vero C1008 cells. Taken together, these results demonstrate that RS virus matures at the apical surface of polarized epithelial cell lines.     

Comparison of Subacute Sclerosing Panencephalitis and Measles Viruses: an Electron Microscope Study

The ultrastructure of CV-1 cells infected with subacute sclerosing panencephalitis (SSPE) viruses was compared with that of CV-1 cells infected with the wild or Edmonston strain of measles virus. Both SSPE viruses and the measles viruses produced two types of nucleocapsid structures: smooth filaments, 15 to 17 nm in diameter, and granular filaments, 22 to 25 nm. The smooth and granular filaments produced by SSPE and measles virus did not differ in appearance. In CV-1 cells infected with SSPE viruses, smooth filaments formed large intranuclear inclusions and granular filaments occupied a large area of the cytoplasm, but always spared the area under the cell membrane. Particles budding from the surface of these cells contained no nucleocapsids. In CV-1 cells infected with measles virus, only small aggregates of smooth filaments were seen in the nuclei. Granular filaments in the cytoplasm predominantly occupied the area under the cell membrane, and were aligned beneath the cell membrane in a parallel fashion and assembled into budding particles. These differences between SSPE and measles virus may be regarded as quantitative, but they do distinguish SSPE viruses from measles virus. Moreover, the formation of large nuclear inclusions filled with smooth filaments appears to be a characteristic process of SSPE, but not of measles, since this type of inclusion is invariably seen in SSPE brain tissues, brain cultures derived from them, and CV-1 cells infected with SSPE viruses.     

Filamentous structures in dengue type 3 virus infected mouse neurones

Dengue type 3 (H-87) virus was inoculated into suckling mouse brain and animals were sacrificed at 24 hr intervals. Parallel filamentous structures were found in infected neurones in close association with virus particles in the distended endoplasmic cisternae. They were usually arranged in a crystalloid pattern, oriented in different directions within the cisternae. Faint helical features were sometimes observed. These filamentous structures measured 15-25 nm in width and varied in length. Their possible involvement with viral material or a viral core is postulated.     

Morphogenesis and Ultrastructure of Respiratory Syncytial Virus

Respiratory syncytial (RS) virus was grown in Vero cells and fixed for electron microscopy at various stages of maturation. Both filamentous and round or kidney-shaped particles, either with (complete) or without (incomplete) internal structure, were observed. All four morphological forms were identical with respect to their reactivity with ferritin-labeled antibody to RS virus. Freezeetching revealed a structural feature apparently unique for RS virus, namely helical striations around the core on the internal aspect of the envelope. This specific configuration was already detectable during the early stages of viral differentiation of the host cell membrane. Concentration of free virus by zonal ultracentrifugation of culture fluids onto sucrose cushions yielded predominantly filamentous forms up to 10 mum in length.     

Ebola virus VP40 drives the formation of virus-like filamentous particles along with GP

Using biochemical assays, it has been demonstrated that expression of Ebola virus VP40 alone in mammalian cells induced production of particles with a density similar to that of virions. To determine the morphological properties of these particles, cells expressing VP40 and the particles released from the cells were examined by electron microscopy. VP40 induced budding from the plasma membrane of filamentous particles, which differed in length but had uniform diameters of approximately 65 nm. When the Ebola virus glycoprotein (GP) responsible for receptor binding and membrane fusion was expressed in cells, we found pleomorphic particles budding from the plasma membrane. By contrast, when GP was coexpressed with VP40, GP was found on the filamentous particles induced by VP40. These results demonstrated the central role of VP40 in formation of the filamentous structure of Ebola virions and may suggest an interaction between VP40 and GP in morphogenesis.     

Phage-like infectious virus of the green alga Chlorococcum

A pure culture of the green eukaryotic alga Chlorococcum sp. and a virus infectious for it were isolated. Infection of the culture leads to the destruction of most of the cells, although complete destruction of the culture is not usually observed. The virus particles are similar in morphology to bacteriophage and consist of a hexagonal head 220 nm long and 180 nm wide and a tail which is inside the head in a majority of the particles, but which is on the outside in some. The virus is not adsorbed on the cell membranes, but evidently penetrates into the zoospores through the flagellar channels, where particles of it are regularly found. Destruction of the nucleus and membrane structures of the cytoplasm occurs during growth of the virus, but the chloroplasts and mitochondria are retained. The membranes of infected cells are partially destroyed and the virus particles are released into the medium. The virus is designated as PLVCH (L)--page-like virus of Chlorococcum.     

Ultrastructure of Soybean Leaf Cells Infected with Cowpea Mild Mottle Virus

Cowpea mild mottle virus (CMMV), a whitefly-transmitted, rod-shaped virus isolated in Thailand, induced feather-like structures in the cytoplasm of infected soybean cells. These structures were the results of a complex arrangement of virus particles and occurred in all types of cells observed. An organized arrangement of virus particles in the form of layers was also observed in the cytoplasm of the infected cells. In ultrathin sections, the particles measured about 10 nm wide and more than 600 nm long, which corresponded to the size reported for the purified preparations of CMMV. No feather-like structures or virus particles were observed in the comparable healthy tissues.     

Electron-microscopic study of a mycoplasmatales virus,strain MV-Lg-pS2-L 172

Morphology and adsorption of a globular virus, lysing Acholeplasma laidlawii were studied in ultrathin sections of plaques in a lawn of the host strain. The virus was globular, about 50 to 90 nm in diameter, with a clearly defined membrane, 6.5 to 8 nm thick. A protuberance about 25 to 35 nm long and 12 to 20 nm thick was observed on numerous virus particles. The evenly granulated, electron-optically dense content of the cells became clearer in cells affected by the viruses. Fibrillar structures of different thickness and small dense areas appeared in cells assumed to be in the preliminary stages of lysis. The interactions in the virus-host system and possible development stages of the virsu are discussed.     

Morphological and Cytochemical Studies on Lymphocytic Choriomeningitis Virus

Lymphocytic choriomeningitis (LCM) virus was observed by electron microscopy in thin sections of infected tissue cultures. The particles were pleomorphic and varied greatly in size. The smaller particles (50 to 200 nm) appeared to be spherical, whereas the largest (over 200 nm) were often cup-shaped. All particles contained one to eight or more electron-dense granules which were removed by ribonuclease. The particles were formed by budding from the plasma membrane and appeared to have spikes. The morphological evidence suggests that LCM should be considered as belonging to the presently unclassified group of lipoprotein-enveloped ribonucleic acid viruses.     

Semliki Forest Virus in HEp-2 Cell Cultures

The growth and development of Semliki Forest virus (SFV), an arbovirus of serological group A, in HEp-2 cells in tissue culture was examined by various techniques at frequent intervals. Infectivity and fluorescent-antibody studies demonstrated the presence of infective virus and viral antigens within the cells at 8 hr after infection. The antigen was particulate and distributed throughout the cytoplasm. Thereafter, there was rapid progression of virus production and cell destruction. By electron microscopy, tubular structures bounded by a fine membrane were observed in cytoplasm at 12 hr. Rows of small (25 mmu) virus particles were often present on the outer surface of these membranes, and at later times they became progressively more encrusted with the small virus particles. These structures subsequently increased rapidly in number, size, and complexity, and the space between the membrane and the tubules increased, thus forming vacuoles which contained tubules and were covered with the small particles. At later times (24 hr and later) larger (42 to 50 mmu) particles were observed, usually inside of the vacuoles. These larger particles (and occasionally the smaller ones) were also seen at the cell periphery and in the extracellular space. The large SFV particles appear to form by three distinct processes: (i) from the smaller particles, (ii) by development on an intravacuolar membrane, and (iii) at the ends of the tubules. The mode of development of SFV is unique among viruses studied to date, but in some characteristics it resembles that of other group A arboviruses. Its development differs from that of most arboviruses of group B and other serological groups.     

Fine structure of influenza A virus observed by electron cryo-microscopy.

A rapidly frozen vitrified aqueous suspension of influenza A virus was observed by high resolution electron cryomicroscopy. The influenza particles were grouped into small (diameter < 150 nm) spherical particles with well organized interiors, large spherical ones with less internal organization, and filamentous ones. Envelopes of most of the large virus particles were phospholipid bilayers, and the chromatography fraction containing these large particles was largely devoid of viral activity. The envelopes of most of the filamentous and small spherical virus particles, on the other hand, gave a strange contrast which could be ascribed to a combination of a thin outer lipid monolayer and a 7.2 nm thick protein-containing inner layer. These latter particles represented most of the viral activity in the preparation. Densitometric traces of the near in-focus images confirmed these structural differences. Some viral envelope structures apparently intermediate between these two distinct types of membrane were also detected. A structural model of intact biologically active influenza virus particles was formulated from these results, together with computer simulations.     

Detection and localisation of picorna-like virus particles in tissues of Varroa destructor, an ectoparasite of the honey bee, Apis mellifera

Virus-like particles, 27 nm in diameter, were observed in extracts of individual Varroa destructor mites and in sections of mite tissue. Application of a purification procedure resulted in virus preparations that were used to prepare an antiserum to detect the virus in individual mites. Immunohistology studies showed that the gastric caecae were heavily infected, whereas no immunostaining could be detected in other mite tissues or organs, like the salivary glands, brain, rectum or reproductive organs. By electron microscopy large aggregates of virus-like particles in para-crystalline lattices were found in cells of the gastric caecae. The particles, reminiscent to picorna-like viruses, occurred mainly in the cytoplasm, whereas some virus particles were sparsely scattered in vacuoles. Occasionally, particles were observed in membrane-bound vesicles or in long tubular membrane structures in the cytoplasm. The accumulation of the picorna-like virus particles in the cytoplasm and the presence of the virus in membrane structures give a strong indication that the virus replicates in the mite.     

Vaccinia Virus Intracellular Mature Virions Contain only One Lipid Membrane

Vaccinia virus (VV) morphogenesis commences with the formation of lipid crescents that grow into spherical immature virus (IV) and then infectious intracellular mature virus (IMV) particles. Early studies proposed that the lipid crescents were synthesized de novo and matured into IMV particles that contained a single lipid bilayer (S. Dales and E. H. Mosbach, Virology 35:564–583, 1968), but a more recent study reported that the lipid crescent was derived from membranes of the intermediate compartment (IC) and contained a double lipid bilayer (B. Sodiek et al., J. Cell Biol. 121:521–541, 1993). In the present study, we used high-resolution electron microscopy to reinvestigate the structures of the lipid crescents, IV, and IMV particles in order to determine if they contain one or two membranes. Examination of thin sections of Epon-embedded, VV-infected cells by use of a high-angular-tilt series of single sections, serial-section analysis, and high-resolution digital-image analysis detected only a single, 5-nm-thick lipid bilayer in virus crescents, IV, and IMV particles that is covered by a 8-nm-thick protein coat. In contrast, it was possible to discern tightly apposed cellular membranes, each 5 nm thick, in junctions between cells and in the myelin sheath of Schwann cells around neurons. Serial-section analysis and angular tilt analysis of sections detected no continuity between virus lipid crescents or IV particles and cellular membrane cisternae. Moreover, crescents were found to form at sites remote from IC membranes—namely, within the center of virus factories and within the nucleus—demonstrating that crescent formation can occur independently of IC membranes. These data leave unexplained the mechanism of single-membrane formation, but they have important implications with regard to the mechanism of entry of IMV and extracellular enveloped virus into cells; topologically, a one-to-one membrane fusion suffices for delivery of the IMV core into the cytoplasm. Consistent with this, we have demonstrated previously by confocal microscopy that uncoated virus cores within the cytoplasm lack the IMV surface protein D8L, and we show here that intracellular cores lack the surface protein coat and lipid membrane.     

Electron Microscopy of Equine Infectious Anemia Virus

Equine infectious anemia (EIA) virus was observed in thin sections of infected cultured horse leukocytes by electron microscopy. The virus particles had a spherical shape and were between 80 and 120 nm in diameter. Most of them contained an electron-dense nucleoid 40 to 60 nm in diameter. They were observed to form by a process of budding from the plasma membrane and appeared to have thin surface projections. The particles described were not detected in uninfected cultured cells, and their appearance could be prevented by adding EIA immune serum to the inoculum. The implications of these findings in the classification of EIA virus are discussed.     

Ultrastructural and immunocytochemical evidences of core-particle formation in the methylotrophic Pichia pastoris yeast when expressing HCV structural proteins (core-E1).

Particulate antigens of the Hepatitis C virus (HCV) are reported for the first time by transmission electron microscopy in Pichia pastoris. The yeast was cloned to express the first 339 NH2-terminal amino acids of the HCV polyprotein (C-E1.339 polypeptide). The C-E1.339 polypeptide covers the putative 191 aa of the core protein (aa 1-191) and 148 aa of the E1 envelope antigen (aa 192-339). Virus-like particles (VLP) with diameters ranging from 20 nm to 30 nm were specifically observed in those cells expressing the HCV polyprotein. The VLP appeared along the membrane of the endoplasmic reticulum, but were fundamentally localized in vacuoles, either free or inside autophagic bodies. Clustered particles, chains of particles, high-density reticular structures, and crystalloid bodies were also detected, the last one being an orderly arrangement of particles with 20 nm diameters. The crystal-associated particles are well differentiated from the intracellular VLP because of their uniform size and shape. We argue that membrane components are retained in the architecture of the VLP, conferring to this particle certain heterogeneity. Both kinds of particles, the VLP formed after treatment with NP-40 and the crystal-associated particles, were core protein-positives. Whether they reflect mature HCV nucleocapsid or intermediary states in the viral nucleocapsid morphogenesis remains unknown. We conclude that, like mammalian cell lines, the P. pastoris yeast could be an appropriate host for the analysis of HCV polyprotein processing and, eventually, virus assembly.     

Initiation and maintenance of persistent infection by respiratory syncytial virus.

Propagation of cells infected with temperature-sensitive (ts) mutants of respiratory syncytial (RS) virus at nonpermissive temperature (39 degrees C) resulted in cytolytic, abortive, or persistent infection, depending on the mutant used to initiate infection. Five mutants from complementation group B produced cytolytic or abortive infections, whereas a single mutant (ts1) from group D and a noncomplbmenting mutant produced persistent infections. The persistently infected culture initiated by mutant ts1 (RS ts1/BS-C-1) has been maintained in serial culture for greater than 100 transfers, and infectious-center assays and immunofluorescent staining indicated that all cells harbored the RS virus genome. RS ts1/BS-C-1 cultures were resistant to superinfection by homologous and some heterologous viruses, and interferon-like activity against some heterologous viruses was present in the culture medium. Small amounts (0.002 to 0.2 PFU/cell) of infectious virus were present in the culture fluid, but autointerfering defective particles were not detected. This released virus formed small plaques and produced persistent infection of BS-C-1 cells at 37 degrees C. The RS ts1/BS-C-1 cells contained abundant RS virus antigen internally, but little at the surface, although the cells showed enhanced agglutinability by concanavalin A. Nucleocapsids and the 41,000-molecular-weight nucleoprotein were present in extracts of both nucleated and enucleated cells. No infectious RS virus was obtained by transfection of DNA from RS tsl/BS-C-1 cells to susceptible BS-C-1 or feline embryo cells under conditions allowing efficient transfection of a foamy virus proviral DNA. It was concluded that persistent infection was maintained in part by a non-ts variant of RS virus partially defective in maturation. The karyotype of the RS ts1/BS-C-1 culture differed from that of unifected cells.     

Comparison, by freeze-fracture electron microscopy, of chromatophores, spheroplast-derived membrane vesicles, and whole cells of Rhodopseudomonas sphaeroides.

By using freeze-fracture electron microscopy, chromatophores and spheroplast-derived membrane vesicles from photosynthetically grown Rhodopseudomonas sphaeroides were compared with cytoplasmic membrane and intracellular vesicles of whole cells. In whole cells, the extracellular fracture faces of both cytoplasmic membrane and vesicles contained particles of 11-nm diameter at a density of about 5 particles per 10(4) nm2. The protoplasmic fracture faces contained particles of 11 to 12-nm diameter at a density of 14.6 particles per 10(4) nm2 on the cytoplasmic membrane and a density of 31.3 particles per 10(4) nm2 on the vesicle membranes. The spheroplast-derived membrane fraction consisted of large vesicles of irregular shape and varied size, often enclosing other vesicles. Sixty-six percent of the spheroplast-derived vesicles were oriented in the opposite way from the intracellular vesicle membranes of whole cells. Eighty percent of the total vesicle surface area that was exposed to the external medium (unenclosed vesicles) showed this opposite orientation. The chromatophore fractions contained spherical vesicles of uniform size approximately equal to the size of the vesicles in whole cells. The majority (79%) of the chromatophores purified on sucrose gradients were oriented in the same way as vesicles in whole cells, whereas after agarose filtration almost all (97%) were oriented in this way. Thus, on the basis of morphological criteria, most spheroplast-derived vesicles were oriented oppositely from most chromatophores.