ELECTRON MICROSCOPIC STUDY OF THE CYTOPATHOLOGY OF ECHO VIRUS INFECTION IN CULTIVATED CELLS

The cultivated monkey kidney cell is subject to changes when infected with ECHO viruses 6, 9, and 19. The electron microscope reveals three stages of infection: (a) initial stage. The nucleus appears granular with chromatin condensation on the nuclear envelope. The cytoplasm contains electron transparent vesicles and vacuoles forming nests. (b) Intermediate stage. The nucleus seems to diminish, appearing more pycnotic and displaced toward the periphery. The cytoplasm is filled with electron transparent vacuoles and vesicles, and dense masses as well as some spiral bodies are seen. The mitochondria retain their shape. Dense particles are seen, which are possibly of viral nature. (c) Final stage. The nucleus is contracted to a narrow strip close to the cellular membrane or is completely destroyed. The cytoplasm shows no apparent changes. Crystals are frequently observed in cells infected with ECHO viruses 6 and 19, consisting of dense particles with an average diameter of 14.4 mµ ranging from approximately 13.2 to 15.6 mµ for ECHO virus 6, and 14.5 mµ ranging from approximately 12.5 to 16.5 mµ for ECHO virus 19. These particles are clustered in hexagonal packages forming angles of 75° and 105°. The particles in most crystals are arranged in rows separated by a constant distance, the latter varying from one crystal to another and being approximately 1.5 and 2.5 times the distance between particles. Other particles were observed which, however, are not considered to be of viral nature.     

Ultrastructure of an Unusual Erythrocytic Form of Plasmodium berghei*

SYNOPSIS. Unusual dense forms were discovered in ultrathin sections of Plasmodium berghei-infected rat erythrocytes. These parasites frequently occurred with one or more typical trophozoites in a single blood cell. They appeared darker than both the neighboring trophozoites and the host erythrocyte. Ribosomes were visible in clusters in their compact cytoplasm. The endoplasmic reticulum, when present, had dilated cisternae often containing a material of low density. Large food vacuoles werecommonly seen along with the small vesicles harboring pigment granules. The single large nucleus had dense nucleoplasm. Multilaminated membraned bodies and sausage-shaped vacuoles were, seen in some of the parasites. The exact identity of this form of P. berghei is not known. Its possible significance is discussed with particular reference to the differentiation of gametocytes.     

Fine Structure of Microgametocytes and Macrogametes of Eimeria nieschulzi

SYNOPSIS. An electron microscope study of microgametocytes and macrogametes of Eimeria nieschulzi Dieben, 1924 revealed that they lie within vacuoles bounded by a host unit membrane. The vacuole surrounding the microgametocyte contains granular material. The vacuole around the macrogamete is narrower and contains vesicles and membranes. Micropores were seen on the surface of the plasma membrane of microgametocytes and macrogametes. Microtubules were seen in macrogametes. Young microgametocytes and macrogametes have a similar cytoplasmic matrix, mitochondria and nuclei. Glycogen granules apparently develop around vacuoles in both microgametocytes and macrogametes. Glycogen granules were also seen along the margins of parallel bundles of fibers in microgametocytes. As nuclei of the microgametocyte divide, they move to the periphery of the parasite. Three basal bodies, each with 9 fibers in triplet form, develop in association with each nucleus. Microgametes have 2 free flagella and a central short, attached flagellum. Basal granules lie along the outer fibers of the central flagellum. Each microgamete has an elongate mitochondrion in close contact with the nucleus. In macrogametes wall-forming bodies develop in lacunae in the cytoplasm. Smaller dark bodies with areas of low density were also seen. Wall-forming bodies and dark bodies move to the periphery of mature macrogametes.     

Viruses of Entamoeba histolytica II. Morphogenesis of the Polyhedral Particle (ABRM2→HK-9)→HB-301 and the Filamentous Agent (ABRM)2→HK-9

The intracellular development of two morphologically different amoebal viruses has been studied by electron microscopy. One is a polyhedral agent which was observed as early as 24 hr after infection in the perinuclear cytoplasm. Subsequently, cell lysis occurred and particles were found in large number bound to membranes of disrupted amoebae. Other particles were found in phagocytic vacuoles suggesting a possible portal of entry into amoebae. The other virus is a filamentous particle which is first seen in small clusters in the nucleus after 24 hr of infection. The number of particles increases such that by 72 hr massive whorls of particles occupy a substantial part of the nucleus. After rupture of the nuclear membrane, clusters of filaments are widely dispersed throughout the cytoplasm. Still later, the cytoplasmic membrane disintegrates and clusters of filaments are found extracellularly, but free of cell membranes. The morphology of these agents is discussed in comparison with a variety of plant, animal, and bacterial viruses.     

The fine structure of blood cells in the ascidian Perophora viridis

The fine structure of each of the blood cell types of Perophora viridis has been characterized and strong evidence for localization of vanadium in two of these types is given. There are eight cell types; phagocytes which may contain completely engulfed cells, lymphocytes with a prominant nucleolus and scanty cytoplasm packed with clustered ribosomes, and six other cell types each with distinctive granules. Morula cells contain a central nucleus and cytoplasm filled by wedged bodies, about five of which are seen in section. These bodies contain regularly spaced electron dense foci. Green cells have the same organization but contain bodies which are electron dense throughout. Granular amoebocytes contain many smaller lightly staining oval bodies and much glycogen. Another cell type (probably orange cells of light microscopy) contains numerous granular rounded bodies. Compartment cells have vacuoles containing electron dense particles and signet ring cells have usually one large vacuole which is electron dense lined and may contain electron dense particles. Developmental stages of these cell types show involvement of endoplasmic reticulum and Golgi bodies in granule formation. After glutaraldehyde fixation alone the only extremely electron dense components are particles in the compartment cells and signet ring cells implicating these as sites of vanadium localization, although not excluding other cell types.     

Phagotrophy and two new structures in the malaria parasite Plasmodium berghei

Blood collected from rats infected with Plasmodium berghei was centrifuged and the pellet was fixed for 1 hour in 1 per cent buffered OsO(4) with 4.9 per cent sucrose. The material was embedded in n-butyl methacrylate and the resulting blocks sectioned for electron microscopy. The parasites were found to contain, in almost all sections, oval bodies of the same density and structure as the host cytoplasm. Continuity between these bodies and the host cytoplasm was found in a number of electron micrographs, showing that the bodies are formed by invagination of the double plasma membrane of the parasite. In this way the host cell is incorporated by phagotrophy into food vacuoles within the parasite. Hematin, the residue of hemoglobin digestion, was never observed inside the food vacuole but in small vesicles lying around it and sometimes connected with it. The vesicles are pinched off from the food vacuole proper and are the site of hemoglobin digestion. The active double limiting membrane is responsible not only for the formation of food vacuoles but also for the presence of two new structures. One is composed of two to six concentric double wavy membranes originating from the plasma membrane. Since no typical mitochondria were found in P. berghei, it is assumed that the concentric structure performs mitochondrial functions. The other structure appears as a sausage-shaped vacuole surrounded by two membranes of the same thickness, density, and spacing as the limiting membrane of the body. The cytoplasm of the parasite is rich in vesicles of endoplasmic reticulum and Palade's small particles. Its nucleus is of low density and encased in a double membrane. The host cells (reticulocytes) have mitochondria with numerous cristae mitochondriales. In many infected and intact reticulocytes ferritin was found in vacuoles, mitochondria, canaliculi, or scattered in the cytoplasm.     

Ultrastructure of erythrocytes parasitized by Haemobartonella felis.

Experimental Haemobartonella felis infections were studied in 3 mature, intact cats by examining peripheral blood, lung, and spleen by electron microscopy. Coccoid, rod, or ring forms of the organism were found on or close to the erythrocytic membrane, and adjacent parasitized erythrocytes often were attached. Intracytoplasmic crystalloid inclusions occupying most of erythrocytic cytoplasm were seen in the 3 infected cats. The cat with the highest parasitemia had inclusions in about 10% of the erythrocytes. Less than 0.01% of the erythrocytes of a control cat contained inclusions. Parasitized erythrocytes, with and without inclusions, were seen in capillaries of the lung and spleen of infected cats. Macrophages in the lung and spleen of infected cats contained parasitized erythrocytes, either with or without inclusions. Some macrophages contained erythrocyte-free organisms in phagocytic vacuoles.     

Giardia muris: ultrastructural analysis of in vitro excystation

Giardia muris cysts were examined by transmission electron microscopy before treatment, after induction, and at timed intervals during the incubation phase of in vitro excystation. Untreated G. muris cysts had a thick cyst wall composed of a fibrous outer wall and a thin, electron-dense inner membrane which extended from the trophozoite plasma membrane. The cytoplasm was devoid of endoplasmic reticulum, Golgi bodies,and mitochondria. Numerous large vacuoles were present within the ectoplasm just beneath the plasma membrane in untreated cysts. Following induction these cysts lacked ectoplasmic vacuoles. Concurrently, numerous membrane bound vesicles were seen in the peritrophic space closely adhering to the surface of the trophozoite. These vesicles appear to be of cytoplasmic origin. The cytoplasm of fully excysted trophozoites lacked ectoplasmic vacuoles but displayed well-developed ribbons of microtubular bodies, probably precursors of ventral disk, lateral flange, and median bodies and also contained extensive granular endoplasmic reticulum. No more than two nuclei were observed within each organism. The earliest excysted organisms were observed 0-5 min after incubation had begun and most organisms had excysted within 10 min. Cytokinesis occurred only after excystation was complete.     

Phagotrophy and Two New Structures in the Malaria Parasite Plasmodium berghei

Blood collected from rats infected with Plasmodium berghei was centrifuged and the pellet was fixed for 1 hour in 1 per cent buffered OsO₄ with 4.9 per cent sucrose. The material was embedded in n-butyl methacrylate and the resulting blocks sectioned for electron microscopy. The parasites were found to contain, in almost all sections, oval bodies of the same density and structure as the host cytoplasm. Continuity between these bodies and the host cytoplasm was found in a number of electron micrographs, showing that the bodies are formed by invagination of the double plasma membrane of the parasite. In this way the host cell is incorporated by phagotrophy into food vacuoles within the parasite. Hematin, the residue of hemoglobin digestion, was never observed inside the food vacuole but in small vesicles lying around it and sometimes connected with it. The vesicles are pinched off from the food vacuole proper and are the site of hemoglobin digestion. The active double limiting membrane is responsible not only for the formation of food vacuoles but also for the presence of two new structures. One is composed of two to six concentric double wavy membranes originating from the plasma membrane. Since no typical mitochondria were found in P. berghei, it is assumed that the concentric structure performs mitochondrial functions. The other structure appears as a sausage-shaped vacuole surrounded by two membranes of the same thickness, density, and spacing as the limiting membrane of the body. The cytoplasm of the parasite is rich in vesicles of endoplasmic reticulum and Palade's small particles. Its nucleus is of low density and encased in a double membrane. The host cells (reticulocytes) have mitochondria with numerous cristae mitochondriales. In many infected and intact reticulocytes ferritin was found in vacuoles, mitochondria, canaliculi, or scattered in the cytoplasm.     

Bacteriophage-associated spherical bodies in Bacteroides fragilis.

Unique spherical bodies with multilayered walls were observed by electron microscopy in cells of a single strain of Bacteroides fragilis subsp. fragilis. Phage-like particles were present in the same cells, both free in the cytoplasm and within the spheres. The proportion of cells containing the phage-associated spherical structures ranged from less than 0.01% to about 7% depending on the culture conditions. Phage particles of morphological type B and spherical bodies were also found free in the medium surrounding the cells. Spherical bodies with discontinuities in their walls, through which phage-like particles sometimes appeared to be escaping, were also found both intra- and extracellularly. The biological significance of these distinctive spherical structures is a matter of conjecture.     

Herpesvirus Envelopment

The growth and envelopment processes of three representative herpesviruses, equine abortion, pseudorabies, and herpes simplex, were examined in baby hamster kidney (BHK 21/13) cells by bioassay (plaque-forming units) and electron microscopy. The envelopment process was identical for all three viruses. After assembly in the nucleus, the nucleocapsid acquired an envelope by budding from the inner nuclear membrane. This membrane was reduplicated as the enveloped particle was released so that the budding process did not result in disruption of the continuity of the nuclear membrane. That portion of the nuclear membrane which comprised the viral envelope was appreciably thicker than the remainder of the membrane and exhibited numerous projections on its surface. Once enveloped, the viral particles were seen in vesicles and vacuoles in the cell cytoplasm. These appeared to open at the cytoplasmic membrane, releasing the virus from the cell. There was no detectable difference in the size or appearance of enveloped particles in intra- or extracellular locations.     

Studies on vinblastine-induced autophagocytosis in mouse liver. III. A quantitative study

The microtubule inhibitor vinblastine (25 mg/kg, i.p.) induces autophagocytosis in mouse hepatocytes. The formation of autophagic vacuoles, their contents, and other cellular changes after vinblastine injection in hepatocytes, were studied by light and electron microscopic morphometric analysis. The volume density of autophagic vacuoles increased significantly during the experimental period (24 h). This increase was due to the significant increase in their number, which was approximately 5-fold 4 h, 12 h and 24 h after vinblastine injection. The mean volume of the autophagic vacuoles increased significantly 1 h after vinblastine injection, at which time the formation of new autophagic vacuoles was at its greatest. There was an accumulation of single membrane-limited, obviously older autophagic vacuoles in the cytoplasm. Their volume density was at its maximum 12 h after injection, suggesting a retarded turnover of autophagic vacuoles. The segregation of cytoplasmic components into autophagic vacuoles may not be selective after vinblastine injection. The injurious effects of vinblastine were evident both in light and electron microscopic studies. In the parenchymal cells the Golgi cisternae were dilated and disorganized and the volume density of the Golgi apparatus was significantly decreased 12 h after vinblastine injection. The volume density of lysosomes was increased during the 12 h after vinblastine injection. Vesicles containing very low density lipoprotein particles accumulated in the cytoplasm so that their volume density was significantly increased during the entire experimental period. Vinblastine apparently interfered with the transport and secretion of the very low density lipoproteins from the parenchymal cells.     

Micromorphological Aspects of the Development of Rubella Virus in BHK-21 Cells

Sequential effects of rubella virus infection in BHK-21 cells were studied by electron microscopy of thin sections of control and infected cells, 2 to 7 days after infection. Vacuolization of cytoplasm in Golgi areas apparently preceded budding of virions from vacuole membranes and involvement of the endoplasmic reticulum. Newly formed endoplasmic reticulum cisternae encircled and segregated virionforming vacuoles together with other cellular elements. Large vacuolar complexes with numerous virus particles developed, and virus release from these areas occurred with disruption at the cell periphery. The viral particles, with a mean diameter of about 56 nm, consisted of an electron-dense core surrounded by a less dense capsid, enveloped by a typical unit membrane derived from the vacuole membrane.     

感染病毒的小麦全蚀菌的超微结构

将感染病毒的小麦全蚀菌山东烟台株培养 20天的菌体细胞,进行超微结构的研究。于电镜下观察到球状病毒颗粒,平均直径23—30nm,多是无规则松散的分布于胞质中;或紧密聚集于液泡、线粒体周围;或排列成线状;或7—8个颗粒排列成环状。病毒仅分布于细胞质中,细胞核、脂肪体内均未见病毒颗粒。病毒浓度在较老的菌体内有增加的趋势。全蚀菌的菌丝细胞壁有三层,外层电子致密内含纤维状物,内层电子较为透明,中层为一电子致密度很深的狭窄夹层。壁的厚度不均,外缘不规则;在菌丝体产生隔膜的早期阶段,于隔膜附近有1—3个外被膜结构的沃罗宁体 Woronin body,隔膜形成的后期,见电子致密物质沉积在核膜孔上,形成中的隔膜顶端为尖状突起向基部逐渐增宽略成金字塔形。     

Ultrastructural observations in hepatitis C virus-infected lymphoid cells

It is currently unclear whether the hepatocellular damage in chronic hepatitis C virus (HCV) infection is produced through the intrahepatic action of the anti-HCV immune response or through a direct cytopathic effect. In order to investigate the features of HCV replication (morphogenesis and cytopathic effect), we studied the infection of a permissive lymphocytic B cell line, Daudi cells, which were infected with sera of HCV-positive patients, and were examined after various time points under electron microscope. Viral genomic RNA was detected by in situ hybridization, and apoptosis with the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method. The amount of viral genomic RNA was observed to increase during infection. HCV replicated rapidly, since characteristics of viral morphogenesis resembling those of yellow fever virus in a hepatoma cell line could be found 2 days after infection. These included the following: a) several viral particles identical in size (about 42 nm) and structure (a spherical 30-nm-sized electron-dense nucleocapsid surrounded by a membrane) to yellow fever virus were present in the cytoplasm of cells displaying already typical signs of the early stage of apoptosis; b) numerous membrane-bound organelles and in particular the endoplasmic reticulum and vacuoles were observed; c) proliferation of membranes was apparent; and d) intracytoplasmic electron-dense inclusion bodies which have been demonstrated to correspond to nucleocapsids for other flaviviruses were detected. Several cells presented electron-dense areas in the endoplasmic reticulum displaying 30-nm circular structures lying among an amorphous material. Striking cytopathic features with ballooning, extremely enlarged vacuoles and signs of apoptosis were found in cells often containing sequestered aggregates of virus-like particles. By in situ hybridization we found that such enlarged cells contained HCV RNA. Our results thus indicate that the ultrastructural features of HCV viral particles and their morphogenesis resemble that of yellow fever virus and dengue virus. In Daudi cells, HCV infection seems to rapidly trigger apoptotic cell death, and efficient release of viral particles does not seem to take place.     

Cytopathology of soybean tissue culture cells infected with southern bean mosaic virus

Summary Virus distribution patterns and ultrastructural changes in soybean callus cells after infection with the type, or bean strain, of southern bean mosaic virus (SBMV) were observed. Calli grown in liquid Linsmaier and Skoog (LS) medium were inoculated with SBMV and incubated in fresh LS medium. Calli were sampled at 5, 10, 15, and 20 d after inoculation and examined by transmission electron microscopy. Five days after inoculation, viruslike particles (VLP) measuring 22 to 27 nm in d were observed in the cytoplasm. The particles formed loose aggregates with some tendency to associate in regular patterns. By the 10th d after infection, particles were observed in the vacuoles in similar loose arrangements. Viruslike particles were readily identified in vacuoles because of the absence of ribosomes. Crystalline aggregations of VLP were found from Day 10 to Day 20 in the cytoplasm only. Five days after inoculation particles similar to the VLP observed in the cytoplasm also were present in nuclei. Other cytopathic effects were noted, particularly several types of inclusion bodies. These observations differ considerably from reports of the type strain in intact bean plant tissues in the frequent occurrence of VLP in vacuoles and virus crystals in cytoplasm of soybean callus infected with the same strain of virus. Michigan Agricultural Experiment Station Journal Article Number 10020. We thank Dr. Karen Baker for useful suggestions.     

IN VIVO AND IN VITRO OBSERVATIONS OF LEPTOSPIRA POMONA BY ELECTRON MICROSCOPY.

Miller, Norman G. (University of Nebraska College of Medicine, Omaha) and Richard B. Wilson. In vivo and in vitro observations of Leptospira pomona by electron microscopy. J. Bacteriol. 84:569-576. 1962.-Leptospira pomona 3341 was observed by electron microscopy, after the preparation of thin sections from culture material and from infected hamster tissue. The external membrane of low electron density envelops the entire leptospire and appears to be quite flexible, as suggested by its many folds. The spiral protoplasmic body is tubular in structure with a relatively dense wall and a central area of low electron density. Occasionally, very dark circumscribed bodies were seen imbedded in the protoplasmic wall. Detailed morphology is presented of a knoblike structure located at the end of the axial filament. Bifurcation of the axial filament could be demonstrated in leptospires from cultures. Leptospires were observed free or enclosed in vesicles within the cytoplasm of liver parenchymal and renal tubule cells. Erythrocytes located in kidney tissue also contained leptospires within the cytoplasm. The appearance of intracellular leptospires is much the same as those seen extracellularly or from culture.     

Encystment ofBodo caudatus

Summary Before formation of the cyst wall, the food vacuoles are lost, the cell rounds up and the flagella lie close against the body in a flagellar groove. At this early stage, the contractile vacuole is very active, the Golgi apparatus is prominent and the basophilic cytoplasm is composed of closely packed ribosomes. As the cyst wall is secreted, layer by layer, the large Golgi apparatus is replaced by several smaller membrane stacks and mitochondrial changes occur involving local loss and modification of the cristae. Some parts of the mitochondrion undergo degenerative changes and may become surrounded by bacilliform bodies. These same bodies are also associated with small particles of sequestered cytoplasm which are present throughout the encystment process and are believed to be autophagic vacuoles. As the cyst wall thickens, cell shrinkage is manifest as a number of membrane invaginations. The final cyst wall is of uneven thickness and possesses a single operculum which is visible only by electron microscopy. Probable cyst wall precursor is found in small vesicles scattered throughout the cytoplasm.     

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.     

Cytoplasmic assembly and accumulation of human immunodeficiency virus types 1 and 2 in recombinant human colony-stimulating factor-1-treated human monocytes: an ultrastructural study.

Recombinant human colony-stimulating factor-1-treated human peripheral blood-derived monocytes-macrophages are efficient host cells for recovery of the human immunodeficiency virus (HIV) from blood leukocytes of patients with acquired immunodeficiency syndrome. These cells can be maintained as viable monolayers for intervals exceeding 3 months. Infection with HIV resulted in virus-induced cytopathic effects, accompanied by relatively high levels of released progeny virus, followed by a prolonged low-level release of virus from morphologically normal cells. In both acutely and chronically infected monocytes, viral particles were seen budding into and accumulating within cytoplasmic vacuoles. The number of intravacuolar virions far exceeded those associated with the plasma membrane, especially in the chronic phase, and were concentrated in the perinuclear Golgi zone. In many instances, the vacuoles were identified as Golgi elements. Fusion of virus-laden vacuoles with primary lysosomes were rare. The pattern of cytoplasmic assembly of virus was observed with both HIV types 1 and 2 and in brain macrophages of an individual with acquired immunodeficiency syndrome encephalopathy. Immunoglobulin-coated gold beads added to acutely infected cultures were segregated from the vacuoles containing virus; relatively few beads and viral particles colocalized. The assembly of HIV virions within vacuoles of macrophages is in contrast to the exclusive surface assembly of HIV by T lymphocytes. Intracytoplasmic virus hidden from immune surveillance in monocytes-macrophages may explain, in part, the persistence of HIV in the infected human host.