Subcellular localization of the coat protein in tobacco cells infected by cucumber mosaic virus isolated from Catharanthus roseus

Electron microscopy and immunolabelling with antiserum specific to cucumber mosaic virus coat protein were used to examine tobacco leaf cells infected by cucumber mosaic virus isolated from Catharanthus roseus (CMV-Cr). Crystalline and amorphous inclusions in the vacuoles were the most obvious cytological modifications seen. Immunogold labelling indicated that the crystalline inclusion was made up of virus particles and amorphous inclusions contained coat protein. Rows of CMV-Cr particles were found between membranes of dictyosomes, but membranous bodies and tonoplast-associated vesicles were not evident. Virus particles and/or free coat protein were easily detected in the cytoplasm by immunolabelling. No gold labelling was found within nuclei, chloroplasts and mitochondria.     

蚕豆萎焉病毒2感染豌豆叶细胞后引起的线粒体增生和聚集

应用超薄切片和免疫金标记电镜技术,结合体视学分析研究了受蚕豆萎焉病毒2(BBWV 2) 中国分离物B935侵染的豌豆(Pisum sativum)叶细胞中线粒体的异常变化。结果表明,感病细胞线粒体增生并聚集于细胞质的膜增生区周围,体积增大,形状畸变,一些线粒体内含有类结晶包涵体。病叶细胞与健康对照之间线粒体的体积密度(VV)、表面积密度(SV)、数密度(NV)等参数存在显著差异(P<0.01),而形状因子(PE)、周长指数(CI)、比表面积(RSV)等参数随不同病变阶段而有变化。在线粒体周围及线粒体之间的网格结构可被BBWV 2金标记抗体特异性标记．推断为正在组装的病毒粒子。子代病毒形成结晶体和管状体,有高密度的免疫金颗粒标记。上述研究结果提示BBWV 2 引起的细胞线粒体异常变化与病毒复制组装有关,聚集线粒体的外膜粘连面可能是病毒粒子组装部位,一些线粒体内的类结晶包涵体可能代表了某种蛋白质异常积累。     

蚕豆萎焉病毒2感染豌豆叶细胞后引起的线粒体增生和聚集

应用超薄切片和免疫金标记电镜技术,结合体视学分析研究了受蚕豆萎焉病毒2（BBWV2）中国分离物B935侵染的豌豆（Pisumsativum）叶细胞中线粒体的异常变化。结果表明,感病细胞线粒体增生并聚集于细胞质的膜增生区周围。体积增大,形状畸变,一些线粒体内含有类结晶包涵体。病叶细胞与健康对照之间线粒体的体积密度（Vv）、表面积密度（Sv）、数密度（Nv）等参数存在显著差异（P〈0．01）,而形状因子（PE）、周长指数（CI）、比表面积（Rsv）等参数随不同病变阶段而有变化。在线粒体周围及线粒体之间的网格结构可被BBWV2金标记抗体特异性标记。推断为正在组装的病毒粒子。子代病毒形成结晶体和管状体,有高密度的免疫金颗粒标记。上述研究结果提示BBWV2引起的细胞线粒体异常变化与病毒复制组装有关。聚集线粒体的外膜粘连面可能是病毒粒子组装部位。一些线粒体内的类结晶包涵体可能代表了某种蛋白质异常积累。     

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.     

Ultrastructural Alteration of Host Plants Infected with Tomato Mosaic Virus

The ultrastructural aheration of two host plants infected with tomato mosaic virus (ToMV) were studies with transmission electron microscopy. A large number of virus particles were found being accumulated in different cells such as epidermis, parenchyma cells and vascular bundle cells of Lycopersicon esculentum Mill. grown at 25℃ Crystalline inclusions and paracrystal inclusions composed of ToMV particles were observed in the cytoplasm or vacuoles. Some muhivesicular bodies and myeloid bodies protming into the vacuole and vires-specific vesicles associated with the tonoplast were also observed. The ultrastructuml alteration of Nicotiana tabacum L. tv. Xanthinn was similar to that in tomato infected by ToMV grown at 25 cE. In addition to the aggregate inclusions described above, some cytoplasmic angularly-layered aggregates and abnormal chloroplasts with small peripheral vesicles were observed in the parenchyma cells. The densely stained amorphous material was seen in the cytoplasm of N. tabacum L. cv. Xanthiun grown at 35℃. No X- body was observed in the cytoplasm of the ToMV infected tomato and tobacco grown at 25℃ or 35℃. The authors' results suggest a significant difference between the cytopathological effects of ToMV and tobacco mosaic virus (TMV). These characteristic difference may be useful in the virus diagnosis and identification virus infections in plants.     

African swine fever virus interaction with microtubules

The role of microtubules in intracellular transport of African swine fever virus (ASFV) and virus-induced inclusions was studied by immunofluorescence using anti-ASFV and anti-tubulin antibodies, by electron microscopy of infected Vero cells and by in vitro binding of virions to purified microtubules. MTC, a reversible colchicine analogue, was used to depolymerize microtubules. In cells treated with MTC multiple large inclusions containing ASFV antigens and particles were observed in the cytoplasm. Removal of the drug lead to migration and fusion of the inclusions at a perinuclear location. To study the effect of microtubule repolymerization on virus particle distribution, the particles were counted in thin sections of MTC treated cells and at different times after removal of the drug. In cells treated with MTC 6.8% and 3.6% of the virus particles were found respectively in the cytoplasm and at the cell membrane while 38% of the particles were located around the virosome. With reversal of the drug effect the number of virus particles around the virosomes progressively decreased to 10% at 2 h while the number of particles in the cytoplasm and at the cell membrane increased. At 2 h after removal of the drug 33.5% of the particles were found budding from the cell membrane. Virus particles were found closely associated with microtubules in cytoskeletons obtained by Triton X-100 extraction of taxol treated cells. The association of virus particles with microtubules was also observed in vitro using purified microtubules and virus particles. The results show that microtubules are involved in the transport of African swine fever virus particles from the assembly site to the cell surface and in the movement and fusion of the virus inclusions.     

芝麻花叶病的病毒病原鉴定

从患芝麻花叶病的病叶中提纯了一种线条状病毒颗粒,长700～800nm,宽13nm。经汁液摩擦接种可感染心叶烟、大豆,甜菜等9种植物,不感染西瓜,苋色藜、豇豆等。主要传毒介体是发生在芝麻田的桃蚜。病土、病种均不传病。该病毒与西瓜花叶病毒、芜菁花叶病毒及马铃薯Y病毒的抗血清无反应。超薄切片中可见到风轮形和纸卷形的圆柱状内含体以及结晶状内含体。结晶状内含体分布在细胞质和叶绿体中,其它内含体均见于细胞质中。同时,细胞质中还可见到大量聚集的线状病毒颗粒。初步认为此病毒可能是马铃薯Y病毒群中的一个新成员,暂称为芝麻花叶病毒。国内外均未见报道。     

Electron microscopy of twoPelargonium viruses

Summary Samples ofPelargonium zonale with different virus symptoms were collected from several gardens in Madrid. Inoculation to test plants and electron microscopy of the samples were made.2 viruses were isolate from the samples; by symptomatology, size of the virus particles, and distribution of the virions in the host cells, one of them (P₁) was identified withPelargonium leaf curl and the other (P₂) seems to be a previously undescribed virus. The virus P₂ forms crystalline inclusions composed of virus particles in the vacuoles of the infected cells.     

Ultrastructure of crystalline inclusions in the thylakoids of dodder<Emphasis Type="Italic">(cuscuta japonica)</Emphasis> plastids

The plastids from seedlings of the parasitic angiospermCuscuta japonica were ultrastructurally investigated. In shoot subapical cells from 3-d-old seedlings grown in the dark, the etioplasts contained prolamellar bodies and amorphous and dense inclusions. In the shoot subapical cells obtained from 6-d-old seedlings grown under light conditions for the last 3 d, the underdeveloped chloroplasts contained phytoferritin within the stroma as well as amorphous and dense inclusions that were limited by the thylakoid membranes. In the developing chloroplasts, electron-dense materials were detected within the transversely sectioned thylakoid lumens. This dense material presented two different images, depending upon the sectional plane. When transversely prepared, the materials appeared as somewhat thick, linear structures, whereas longitudinally sectioned thylakoids revealed very large crystalline inclusions. In the developed chloroplasts, the amounts of electron-dense material or crystalline inclusions were remarkably reduced in the thylakoid lumens, which were electron-translucent. Far fewer crystalline inclusions were observed in the developed chloroplasts of seedlings than in the developing chloroplasts. These results suggest that the crystalline inclusions may be temporarily reserved within the thylakoid lumens of chloroplasts in the Gjaponica seedlings.     

Crystalline Structures Associated with the Infection of the Potexvirus Argentine Plantago Virus (APla V)

Abstract An electron microscopy study of Argentine plantago virus (APlaV)-infected plants has revealed that the virus induces crystal-like inclusions in both the cytoplasm and the nucleus of infected cells, in addition to the charcteristic aggregates of virus particles formed by most potexviruses. These crystals are produced in different plants and in tissue at different stage of development and they can be readily detected by leaf dip preparations. We conclude that the crystalline inclusions are virus-specific, and therefore can be regarded as a characteristic feature of APlaV infections.     

Immunogold labelling indicates high catalase concentrations in amorphous and crystalline inclusions of sunflower (Helianthus annuus L.) peroxisomes

Summary Immunogold labelling and electron microscopy were used to investigate whether catalase was present in peroxisomal inclusions, the composition of which has not yet been determined in plant cells. In the mesophyll cells of sunflower (Helianthus annuus L.) cotyledons, the catalase gold label was confined to peroxisomes. At day 2 of postgerminative growth in darkness, peroxisomes were free of inclusions, and the matrix was homogeneously labelled with gold particles. Thereafter, amorphous inclusions appeared, but by day 5 of growth, conspicuous crystalline inclusions (cores) were the predominant type. This developmental change, first observed in cotyledons grown in continuous light between day 2.5 and 5, also took place in cotyledons kept in permanent darkness. Both amorphous and crystalline inclusions showed a much higher immunogold label than did the peroxisomal matrix, indicating that catalase was a component of both types of peroxisomal inclusions. In contrast to catalase, the immunogold label of glycolate oxidase was almost completely absent from cores and was confined to the peroxisomal matrix. Together with reports on the absence of other enzymes from peroxisomal inclusions in sunflower and other species (Vaughn, 1989) our results suggest that catalase is a major constituent of amorphous and crystalline peroxisomal inclusions in plants.     

Ultrastructural investigation on a strain of a cytoplasmic-polyhedrosis virus with nuclear inclusions

A strain of a cytoplasmic-polyhedrosis virus causes the formation of crystalline inclusions almost entirely in the nucleus, and only rarely in the cytoplasm, of the midgut epithelial cells of the silkworm Bombyx mori. It also differs from the typical strain in causing the hypertrophy of the nucleoli and the formation of dense reticulum and spherical bodies in the nucleus. The virus particles and the virogenic stromata of the new strain resemble those of the typical strain. The cytoplasmic inclusions contain virus particles, while the nuclear inclusions do not. When the infected larvae are kept at 30°C for 15 hr or at 35°C for 3–15 hr, the nuclear inclusions break up into particles of 70–250 nm in diameter. The particles are dispersed in the cells but not present in the spaces previously occupied by the decomposed inclusions.     

Ultrastructural pathology of leaf cells of ryegrass (Lolium multiflorum) infected with ryegrass mosaic virus.

Ryegrass mosaic virus particles and virus induced lamellar inclusions were found in mesophyll and epidermal cells of virus infected ryegrass leaves. The lamellar inclusions were occasionally found in phloem cells also. Virus particles occurred in cytoplasm, inside plasmodesmata and often in membrane bound sacs embedded in a matrix between plasmalemma and cell wall at or near plasmodesmata. Electron dense plugs protruding from plasmodesmata, finger-like cell wall outgrowths and cell wall deposits usually at plasmodesmata were also observed. Cytopathological changes in organelles in infected cells included dense deposits in the cisternae of endosplasmic reticulum and Golgi apparatus, mitochondria with electron-dense or opaque matrix, proliferating cristae and deteriorating unit membrane; and disintegrating chloroplasts.     

Ultrastructure and cytochemistry ofPhaseolus leaf tissues infected with an isolate of tobacco necrosis virus inducing localized wilting

Summary The cytopathology induced by an isolate of tobacco necrosis virus (TNV-W) causing wilted, non-self-limiting lesions inPhaseolus bean was compared with that of abiotically-induced wilting. The main cell alterations specific to TNV-W infection were ER proliferation and vesiculation, plasmolysis and plasmalemma proliferation. Later there was lysis of most cell membranes, formation of crystalline inclusions in the chloroplasts and development of fibrous structures in the cytoplasm. It is suggested that the chloroplast inclusions consist of ribulose-1,5-bisphosphate carboxylase (RuBisCo). TNV-W replicated extensively in infected cells, often forming large crystalline aggregates of virus particles. Tissue wilting, as well as crystallization of the virus and RuBisCo, may have been caused by cell dehydration due to the loss of plasmalemma integrity.Abbreviations CA chromic acid - CI crystalline inclusion - DG dense granule - E ethanol - ER endoplasmic reticulum - FS fibrous structure - H₂O₂ hydrogen peroxyde - MVB multivesicular body - Pb lead citrate - PTA phosphotungstic acid - RuBisCo ribulose-1,5-bisphosphate carboxylase - TNV-W tobacco necrosis virus-wilting - UA uranyl acetate     

A nonoccluded virus of the army cutworm

A nonoccluded virus was isolated from larvae of the army cutworm, Euxoa auxiliaris. Infected larvae became lethargic and shrunken, and death usually occurred 12–20 days after infection. The primary site of viral infection and replication appeared to be the nuclei of midgut epithelial cells; however, virus replication also occurred in cells of the tracheal matrix and in muscle. Nuclei in early stages of the infection contained large granular areas with the chromatin scattered near the nuclear membrane. These areas differentiated into viral particles that measured 24 nm and formed crystalline arrays, occasionally 10 μm long. Disruption of the nuclear membrane liberated these arrays of particles into the cytoplasm. Fluorescence microscopy studies indicated that the viral particles contained DNA. The crystalline arrays were Feulgen positive. The virus also infected larvae of the armyworm, Pseudaletia unipuncta, and corn carworm, Heliothis zea, in laboratory tests.     

Electron Microscopy of an Isometric Caulimo-Like Virus from Sweet Potato (Ipomoea batatas)

A newly recognised virus with isometric particles 50 nm in diameter was detected in Ipomoea batatas (sweet potato) by graft-transmission to I. setosa. Virus particles and intracellular inclusions, although not seen in infected I. batatus, were found in the cytoplasm of most cell types of I. setosa. The caulimo-like particles were most abundant with vesicles immediately adjacent to inclusions. The ovoid or spherical inclusions, which differ markedly from those of caulimovi, ruses, have a large central lacuna and usually several smaller peripheral lacunae. Infected vascular parenchyma cells sometimes protrude into, and occasionally completely occlude, adjacent xylem vessels, observations possibly explaining the sudden wilting and premature senescence of infected leaves.     

Interactions between pepper ringspot virus and cylindrical inclusions of two potyviruses

Immunoelectron microscopy showed that cylindrical inclusions (CI) of the potyvirus potato virus Y (PVY) bound in addition to their homologous virions those of a co-infecting rod-shaped virus, pepper ringspot virus (PRV), in infected Nicotiana benthamiana leaf cells. The latter virus does not code for cylindrical inclusions and is cl assified as a Tobravirus. Virions of PRV were scattered throughout the cell cytoplasm and not associated with mitochondria in PVY + PRV double infections. Binding of PRV to mitochondria was disrupted in PVY + PRV infected cells. In double infections with a second potyvirus, tobacco etch virus (TEV), and PRV in N. benthamiana cells, TEV-CI bound homologous TEV virions but did not bind PRV. In contrast to PVY + PRV infections, virions of PRV attached end-on to mitochondrial limiting membranes in PRV-only and in TEV + PRV double infections. The results are interpreted to mean that there are differences in the PRV virion binding sites of PVY-CI and TEV-CI. In previous reports, potyviral CI have been nondiscriminating in binding virions or capsid proteins of other co-infecting rod-shaped viruses.     

RELATION OF TOBACCO MOSAIC VIRUS TO THE HOST CELLS

The relation of tobacco mosaic virus (TMV) to host cells was studied in leaves of Nicotiana tabacum L. systemically infected with the virus. The typical TMV inclusions, striate or crystalline material and ameboid or X-bodies, which are discernible with the light microscope, and/or particles of virus, which are identifiable with the electron microscope, were observed in epidermal cells, mesophyll cells, parenchyma cells of the vascular bundles, differentiating and mature tracheary elements, and immature and mature sieve elements. Virus particles were observed in the nuclei and the chloroplasts of parenchyma cells as well as in the ground cytoplasm, the vacuole, and between the plasma membrane and the cell wall. The nature of the conformations of the particle aggregates in the chloroplasts was compatible with the concept that some virus particles may be assembled in these organelles. The virus particles in the nuclei appeared to be complete particles. Under the electron microscope the X-body constitutes a membraneless assemblage of endoplasmic reticulum, ribosomes, virus particles, and of virus-related material in the form of wide filaments indistinctly resolvable as bundles of tubules. Some parenchyma cells contained aggregates of discrete tubules in parallel arrangement. These groups of tubules were relatively free from components of host protoplasts.     

CYTOLOGICAL REACTIONS OF NORMAL AND TMV-INFECTED TOBACCO LEAF CELLS TO ACID AND ALKALINE SOLUTIONS

Acid and basic buffer solutions were applied on slides under cover glasses to thin, hand sections of leaf tissue from normal tobacco plants and equivalent plants infected with tobacco mosaic virus (TMV). The effects on living cells were observed and photographed through phase optics. First, reversible changes and, later, irreversible changes were produced in the cells. Movement of the cytoplasm in a cell could be stopped completely and started again by replacing the unfavorable buffer solution with a favorable medium. Of the organelles, plastids became static first, then mitochondria, and lastly spherosomes. Spherosomes often moved actively when all other organelles were still. Translucent virus monolayers, consisting of particles aggregated side by side, provided markers in the parietal cytoplasm of recently infected cells. Mitochondria generally moved across their surface on the side adjacent to the tonoplast, spherosomes across the surface facing the cell wall. Alkaline buffer solution caused little change of texture in nucleus and cytoplasm or change of form in mitochondria. Clear areas appeared in some plastids. The acid buffer emphasized a cytoplasmic network representing flow lines and eliminated many cytoplasmic vesicles; mitochondria became shorter or spherical in outline, grana of chloroplasts more obvious. Virus inclusions, even the fragile monolayers, were not greatly altered until irreversible changes began. In pH 11–treated cells, the final changes included violent bubbling of cytoplasm, and in pH 2.2–treated cells, snapping of flow lines and coagulation of the cytoplasm. In either case, disintegration of cell structure and virus inclusions was rapid.     

Cytopathology and Ultrastructure of Some African Isolates of Maize Streak and Sugarcane Streak Viruses

Samples of maize leaves naturally infected with maize streak virus (MSV) from Malawi and South Africa, as well as sugarcane leaves naturally infected with sugarcane streak virus (SSV) from Egypt, were examined by light (LM) and transmission electron, microscopy (TEM). Intranuclear inclusions, detectable by both methods, were found mainly in mesophyll and bundle sheath cells, and less frequently in vascular parenchyma and immature phloem cells. At higher TEM magnifications, these inclusions consisted, of crystalline or noncrystalline aggregates of isometric geminivirus–like particles (VLP) that occurred either singly or in geminate arrays. Cytopathological changes in these cells were confined to the nuclei, which were usually larger than normal, with peripheral chromatin and nucleoli. The nuclear envelope of some inclusion–containing nuclei was ruptured, and occasionally a crystal of VLP was found in the cytoplasm of cells in which no intact nuclei were detected. No differences in cytopathology were found between MSV and SSV, or between the two MSV isolates examined.