Growth and Intracellular Development of a New Respiratory Virus

The multiplication of a new, ether-sensitive, ribonucleic acid virus, 229E, isolated from the human respiratory tract, has been studied in cultures of WI-38 human diploid cells. In thin sections of these cells examined with the electron microscope, particles appeared in vesicles in the cytoplasm of cells at a time corresponding to the initial increase in infectious virus. Antigen was also detected in the cytoplasm of cells by the immunofluorescent technique. Extracellular particles of similar morphology were prominent soon after. These events preceded a detectable cytopathic effect. Later, an electron-dense particle appeared within vacuoles in the cytoplasm but was never found extracellularly. Its role in virus development is not known. Complement-fixing antigen developed along with the increase in infectious virus.     

Ultrastructural analysis of the replication cycle of pseudorabies virus in cell culture: a reassessment.

We reinvestigated major steps in the replicative cycle of pseudorabies virus (PrV) by electron microscopy of infected cultured cells. Virions attached to the cell surface were found in two distinct stages, with a distance of 12 to 14 nm or 6 to 8 nm between virion envelope and cell surface, respectively. After fusion of virion envelope and cell membrane, immunogold labeling using a monoclonal antibody against the envelope glycoprotein gE demonstrated a rapid drift of gE from the fusion site, indicating significant lateral movement of viral glycoproteins during or immediately after the fusion event. Naked nucleocapsids in the cytoplasm frequently appeared close to microtubules prior to transport to nuclear pores. At the nuclear pore, nucleocapsids invariably were oriented with one vertex pointing to the central granulum at a distance of about 40 nm and viral DNA appeared to be released via the vertex region into the nucleoplasm. Intranuclear maturation followed the typical herpesvirus nucleocapsid morphogenesis pathway. Regarding egress, our observations indicate that primary envelopment of nucleocapsids occurred at the inner leaflet of the nuclear membrane by budding into the perinuclear cisterna. This nuclear membrane-derived envelope exhibited a smooth surface which contrasts the envelope obtained by putative reenvelopment at tubular vesicles in the Golgi area which is characterized by distinct surface projections. Loss of the primary envelope and release of the nucleocapsid into the cytoplasm appeared to occur by fusion of envelope and outer leaflet of the nuclear membrane. Nucleocapsids were also found engulfed by both lamella of the nuclear membrane. This vesiculation process released nucleocapsids surrounded by two membranes into the cytoplasm. Our data also indicate that fusion between the two membranes then leads to release of naked nucleocapsids in the Golgi area. Egress of virions appeared to occur via transport vesicles containing one or more virus particles by fusion of vesicle and cell membrane. Our data thus support biochemical data and mutant virus studies of (i) two steps of attachment, (ii) the involvement of microtubules in the transport of nucleocapsids to the nuclear pore, and (iii) secondary envelopment in the trans-Golgi area in PrV infection.     

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.     

RBSDV在玉米叶脉细胞内的侵染状态与灰飞虱传毒活力的关系

根据水稻黑条矮缩病毒(RBSDV)侵染玉米(Zea mays L.)的症状发展过程先后取叶脉做超薄切片,在透射电镜下观察病毒在细胞内的侵染状态,并存取样前用灰飞虱无毒若虫进行饲毒和传毒试验。结果显示RBSDV侵入玉米叶细胞后先出现在细咆壁附近,个别粒子似与胞间连丝相连;细胞质内产生病毒基质,病毒粒子先增殖并分布其周边,后向病毒基质内扩展;当病毒粒子布满病毒基质后在细胞质中出现直径约90nm的管状结构,病毒成串排列在该管状结构中;随后管状结构逐渐消失,最终形成晶格状聚集排列。用灰飞虱无毒若虫在细胞内病毒基质出现和病毒增殖期饲毒的,到成虫时分别有2．93％和7．83％个体传毒率;在细胞内病毒成串分布于管状结构和品格状聚集排列期饲毒的,到成虫时均不能传毒。     

An ultrastructural and autoradiographic study of the immune response in Hyalophora cecropia pupae

Summary Membrane-bounded spherical vesicles found in rat Sertoli cells have been examined quantitatively during the cycle of the seminiferous epithelium. Most of the vesicles were localized to the basal and columnar portions of the Sertoli cell cytoplasm. The thin lateral projections of the Sertoli cells contained very few vesicles. Morphometric analysis of the basal portion of the Sertoli cell cytoplasm revealed that the volume density (V _v ) of the vesicles changed markedly during the cycle. The V _v was at its minimum (0.036) at stage VII and maximum (0.117) at stages XI-I. The vesicles were also smaller at stage VII compared to the vesicles at stages IX-V. The stage-dependent difference in the size of the vesicles was found both in the basal and the columnar portions of the Sertoli cells. At stage VII some of the vesicles appeared to be elongated much like the tubular elements of the smooth endoplasmic reticulum (SER) from which they are probably derived. The stage-dependent differences in volume density and size of the Sertoli cell vesicles may be related to cyclic biochemical variations in the Sertoli cells, and are further indications of a variation in Sertoli cell function during the cycle of the seminiferous epithelium. Whether or not this is due to an internal cycle of the Sertoli cell or to influences from adjacent germ cells remains to be determined.     

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.     

The release of acetylcholine: from a cellular towards a molecular mechanism

The isolation of synaptic vesicles rich in acetylcholine (ACh) from the electric organ of Torpedo has indeed strengthened the hypothesis of transmitter exocytosis, but soon after it was found that non-vesicular free ACh was released and renewed upon stimulation. In contrast, vesicular ACh and the number of vesicles remained stable during physiological stimulations. In addition free ACh variations (representing the cytoplasmic pool) were correlated to the release kinetics as measured by the electroplaque discharge. Consequently, the mechanism releasing ACh from the cytoplasm in a packet form was searched at the presynaptic membrane itself. With synaptosomes isolated from the electric organ of Torpedo, it became possible to freeze them rapidly at the peak of ACh release and study their membrane and contents after cryofracture. A statistical analysis showed that the main structural change was the occurrence of large intramembrane particles at the peak of ACh release and under all release conditions. This impressive change contrasted with the stability in the number of vesicles. Another role for the vesicle was envisaged during intense stimulations when the cytoplasmic ACh and ATP pools become exhausted. The decrease in ATP leads to an increase in calcium and protons in the cytoplasm; this signals the depletion of vesicular ACh and ATP stores in the cytoplasm. Release can go on, while ATP promotes the uptake of calcium by vesicles. At the end of its cycle the vesicle will be full of calcium and will perhaps release it. As far as the mechanism of ACh release is concerned it probably depends on a membrane component (perhaps the large particles) activated by calcium and able to translocate ACh in a quantal or subquantal form. In most recent work we showed that if a lyophilized presynaptic membrane was used to make proteoliposomes filled with ACh, they released ACh upon calcium action.     

C-type virus-lymphocyte interactions in developing mouse thymus.

The appearance of C-type virus particles in thymus cells of Swiss mouse embryos, 11.5 to 15.5 days post-conception age (PCA), was studied with the electron microscope. In thymic rudiments of all specimens examined, virus particles were seen in epithelial cytoplasm, budding from epithelial cell surfaces and in extracellular spaces. Lymphoid cells were first seen in thymic rudiments of 13.5 days PCA, and did not display virus particles at this stage. At 14.5 days PCA, thymic lymphocytes had localized plasmalemmal thickenings of high electron-density which were adjacent to extracellular virus particles. Viruses appeared to be penetrating thymic lymphocytes by viropexis in embryos of 15.5 days PCA. At this stage, many lymphocytes also had cytoplasmic virus-containing vesicles and viral buds at their surfaces. These observations suggest the possibility that, in embryos, C-type viruses are transmitted horizontally from thymic epithelium to early populations of thymic lymphocytes.     

AN ELECTRON MICROSCOPE STUDY OF THE EARLY ASSOCIATION BETWEEN TWO MAMMALIAN VIRUSES AND THEIR HOSTS

Early interaction between two animal viruses, vaccinia and adenovirus 7, which multiply readily in L strain and HeLa cells, respectively, was examined in both whole mount preparations and in thin sections. To observe the association at the surface, cells carrying adsorbed virus were swelled under controlled conditions and then "stained" with neutral phosphotungstate. Each particle of both virus types becomes attached to the cell by several capsomeres and is then ingested by phagocytosis. Within the cell, near the surface, single particles or small clumps of adenovirus are lodged within vesicles. Deeper in the cytoplasm this virus is packed in large, numerous inclusions, whereas very close to the nuclear envelope only free particles are found. Vaccinia, on the other hand, either free or in vesicles, is always found in the cytoplasm, at some distance from the nucleus (11). Adsorption and intracellular disposition of these two viruses is discussed in relation to the infectious process.     

Visualization of binding and receptor-mediated uptake of low density lipoproteins by human endothelial cells

Low density lipoproteins (LDL) were conjugated to colloidal gold for investigation of the ultrastructural aspects of binding and receptor-mediated internalization of LDL by cultured endothelial cells from the human umbilical artery and vein. The number of LDL receptors was increased by preincubation in lipoprotein-depleted serum. When the cells were incubated with LDL-gold particles for 2 h at 4 degrees C, the complexes were found in coated pits as well as in clusters attached to the plasma membrane. Small vesicles containing a few LDL-gold complexes appeared in the cytoplasm close to the plasma membrane when the cells were incubated with the conjugate for 5 min at 37 degrees C. After 15 min at 37 degrees C, larger vesicles with a pale matrix and membrane-orientated LDL-gold complexes were seen. After incubation for 30 min at 37 degrees C, colloidal gold particles were present in dense bodies. Quantification of the binding of LDL-gold complexes to the plasma membrane at 4 degrees C showed no differences between arterial and venous endothelial cells.     

Electron microscopic studies of the endocytotic process of cationized ferritin in cultured human retinal pigment epithelial cells

Summary The endocytotic process in cultured human RPE cells was observed after 1 min, 20 min, and 2 h incubation with cationized ferritin. Within 1 min the ferritin particles were seen to attach to the cell membrane, especially between microvilli. Uncoated and coated pits could be recognized on the cell membranes, and uncoated and coated endocytotic vesicles were found in the cytoplasm after 20 min of incubation. These vesicles were surrounded by abundant microfilaments and had no visible membranes. Loss of membrane may be an initial step in the process of developing into the irregular clumps of ferritin particles found inside the plasma membrane. With time, more irregular clumps of ferritin, smaller than the particles introduced during incubation, appeared just beneath the cell membrane. Lysosomes were adjacent to the clumps of ferritin particles associated with microtobules and finally degraded these particles. The phagolysosomes containing many particles were surrounded by many microtubules. Small ferritin particles surrounded but had not entered the rough endoplasmic reticulums, and no particles were seen either around or in the Golgi apparatus. Presented at the 7th International Congress of Eye Research, Nagoya, Japan, 27 September 1986.     

An electron microscopic study of the initial stages of infection of isolated tomato fruit protoplasts by tobacco mosaic virus

Summary Protoplasts were isolated from tomato fruit locule tissue and incubated with tobacco mosaic virus. Electron microscope observations on sections of suitably fixed and embedded material revealed that virus particles readily became attached to the plasmalemma, particularly in small invaginations in the surface of the protoplast. Virus particles were later observed in vesicles within the cytoplasm and it was clear that these vesicles were being formed as a result of pinocytic activity at the surface of the protoplast. Later, virus particles were observed near the nucleus. It is suggested that an initial attachment of the virus to the plasmalemma followed by a pinocytic uptake may represent the initial stages of virus infection of plant cells and that the pinocytic vesicle, containing virus, serves as the vehicle of cellular infection.     

Cytopathology of the soybean looper,Pseudoplusia includens,infected with the Pseudoplusia includens icosahedral virus

Ultrastructural responses of soybean looper cells of various tissues infected with Pseudoplusia includens icosahedral virus (PIIV), a newly characterized RNA virus [Y. C. Chao, H. A. Scott, and S. Y. Young (1983)J. Gen. Virol.64, 1835–1838], were studied in situ. Most cells of fat body and epidermis consistently contained virus particles and associated cytopathic structures. Virus particles, corresponding to those of purified PIIV in morphology and size, always occurred in the cytoplasm either in membrane-bound virogenic stroma and/or freely in the ground cytoplasm. Virogenic stroma, which appeared to be distinct from those induced by other insect viruses, consisted of electron-dense matrix material, in which virus particles were embedded, and membranous vesicles, 70 or 80 nm in diameter, containing nucleic acid-like fibrils. Virus particles in virogenic stroma occurred as hexagonally arranged crystalline arrays made up primarily of homogeneously dense particles, while those in the ground cytoplasm were dispersed randomly and had an electron-lucent central core. Extremely large numbers of virus particles were also located in noncellular cuticle layers of the integument.     

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.     

Morphogenesis of Sindbis virus in cultured Aedes albopictus cells.

Cultured mosquito cells were found to produce Sindbis virus nearly as efficiently as BHK-21 cells at 28 C. In virtually all of the cells observed in the electron microscope, virus morphogenesis was found to occur within complex vesicular structures which developed after viral infection. Viral nucleocapsids were first seen in these vesicles and appeared to be enveloped within these structures. The process of envelopment within these inclusions differed in some respects from the process previously described for the envelopment of nucleocapsids at the plasma membrane of vertebrae cells. Free nucleocapsids were only rarely seen in the cytoplasm of infected mosquito cells, and budding of virus from the cell surface was detected so infrequently that this process of virus production could not account for the amount of virus produced by the infected cells. The vast majority of extracellular virus was produced by the fusion of the virus-containing vesicles with the plasma membrane releasing mature virions and membrane nucleocapsid complexes in various stages of development.     

L-particle production during primary replication of pseudorabies virus in the nasal mucosa of swine

Different tissue culture cell lines infected with a number of alphaherpesviruses produce, in addition to virions, light particles (L particles). L particles are composed of the envelope and tegument components of the virion but totally lack the proteins of the capsid and the virus genome; therefore, they are noninfectious. In this electron microscopy report, we show that L particles are produced during primary replication of the alphaherpesvirus pseudorabies virus (PRV) in the nasal mucosa of experimentally infected swine, its natural host. Although PRV infected different types of cells of the respiratory and olfactory mucosae, PRV L particles were found to be produced exclusively by epithelial cells and fibroblasts. We observed that formation of noninfectious particles occurred by budding of condensed tegument at the inner nuclear membrane and at membranes of cytoplasmic vesicles, resulting in intracisternal and intravesicular L particles, respectively. Both forms of capsidless particles were clearly distinguishable by the presence of prominent surface projections on the envelope and the higher electron density of the tegument, morphological features which were only observed in intravesicular L particles. Moreover, intravesicular but not intracisternal L particles were found to be released by exocytosis and were also identified extracellularly. Comparative analysis between PRV virion and L-particle morphogenesis indicates that both types of virus particles share a common intracellular pathway of assembly and egress but that they show different production patterns during the replication cycle of PRV.     

ULTRASTRUCTURAL FEATURES OF BETA LEAVES INFECTED WITH BEET YELLOWS VIRUS

A cytochemical and electron microscope study has been made of leaves of sugar beet infected with beet yellows virus. Inclusions of particles, which agree in size with beet yellows virus particles isolated by other investigators, have been localized in the ground cytoplasm, in the chloroplasts, and in the nuclei. These particles are circa 100 A in diameter and have an electron-transparent core of 30 to 40 A. Use of acridine orange, azure B, and pyronine Y has revealed that the cytoplasmic inclusion bodies, which consist wholly of the elongate particles, have a strong RNA reaction removable by RNase pretreatment. Particles observed in the chloroplasts may or may not be associated with lipid spheres. If they are, the particles are confined to the periphery of the spheres. In this position the particles are arranged tangentially and are further arranged parallel into groups which lie at various angles to one another. Within the groups the particles are regularly spaced in a three dimensional lattice. Particles located free in the stromal regions are often arranged regularly in curved rows which lie parallel to one another so that a three dimensional lattice is formed. The dispersed and compact forms of virus inclusions are described and related to the condition of the associated cytoplasm. The ground cytoplasm of cells associated with the sieve elements contains numerous ribosomes. A decrease in the number of ribosomes is concomitant with the increase in size of virus aggregations in a cell. Vesiculation of some component of the cytoplasm occurs during the period of virus replication. The vesicles are approximately 100 mµ in diameter and could be derived from the dictyosomes. At later stages of infection these vesicles collapse and convoluted membranous material appears.     

Pathology and Development of the Grasshopper Inclusion Body Virus in Melanoplus sanguinipes

Grasshoppers, Melanoplus sanguinipes (F.), infected with the grasshopper inclusion body virus (GIBV) showed a general torpor, took longer to develop, and had abnormally high rates of mortality. Infection was found only in the fat body, and developing viruses and inclusion bodies were observed in the nuclei and cytoplasm of infected cells. Although the size of the inclusion bodies in cells varied at different stages of infection, the inclusion bodies appeared to grow during the infection. Electron microscopic investigations of viral replication showed that at about 8 days after inoculation presumptive viral particles had developed as buds or protrusions from precursor granular masses; thereafter, these particles underwent internal differentiation and were incorporated into developing inclusion bodies. The GIBV was similar to insect viruses in the genus Vagoiavirus Weiser and to pox viruses, particularly vaccinia.     

Electron Microscopy of Herpes Simplex Virus: I. Entry

Although capsids of herpes simplex virus were encountered within phagocytic vesicles, they were more commonly observed free within the cytoplasm. Stages in the release of virus from vesicles were not seen. There appeared to be five distinct steps in the process whereby the virus initiates infection: attachment, digestion of the viral envelope, digestion of the cell wall, passage of the capsid directly into the cytoplasm, and digestion of the capsid with release of the core. Antibody probably interferes with the first two stages.     

ELECTRON MICROSCOPE OBSERVATIONS ON INTRACELLULAR VIRUS-LIKE PARTICLES ASSOCIATED WITH THE CELLS OF THE LUCKÉ RENAL ADENOCARCINOMA

The common renal adenocarcinoma of the leopard frog was studied in thin sections with the electron microscope. Approximately a third of the tumors examined were found to contain spheroidal bodies of uniform size and distinctive morphology that are believed to be virus particles. These consist of hollow spheres (90 to 100 mµ) having a thick capsule and a dense inner body (35 to 40 mµ) that is eccentrically placed within the central cavity (70 to 80 mµ). Virus particles of this kind occur principally in the cytoplasm but occasionally they are also found in the nucleus and in the extracellular spaces of the tumor. The intranuclear inclusion bodies that are visible with the light microscope are largely comprised of hollow, spherical vesicles with thin limiting membranes. These are embedded in a finely granular matrix. A few of the thin walled vesicles contain a dense inner body like that of the cytoplasmic virus particles. This suggests that they may be immature virus particles. The inclusion bodies are believed to be formed in the course of virus multiplication but they usually contain very few mature virus particles. Bundles of dense filaments and peculiar vacuolar inclusions also occur in the cytoplasm of the tumor cells. These seem to be related in some way to the presence of virus but their origin and significance remain obscure. These findings are discussed in relation to previous work suggesting that the Lucké adenocarcinoma is caused by an organ-specific filtrable agent. It is concluded that the "virus particles" found in electron micrographs of the tumor cells may be the postulated tumor agent. On the other hand, the possibility remains that the particles described here are not those that are causally related to the tumors.