Observations of Measles Virus Infection of Cultured Human Cells : I. A Study of Development and Spread of Virus Antigen by Means of Immunofluorescence

The development of measles virus in cultures of both primary human amnion cells and H.Ep.-2 cells has been followed by means of the indirect fluorescent antibody technic and concurrent light and electron microscope observations. The immunofluorescence studies revealed that there is a latent period for development of demonstrable measles virus antigen. In amnion cells the latent period lasted for at least 3 days. In contrast, virus antigen could be detected in H.Ep.-2 cells as early as 12 hours following inoculation. In each cell system virus antigen was seen in either nucleus or cytoplasm of infected cells, or both. Early localization tended to be perinuclear. Intranuclear fluorescence was generally less bright and less widespread than cytoplasmic fluorescence. Giant cells and long cytoplasmic spindle-shaped processes appeared regularly in infected cultures. Infectious virus was liberated into the nutrient fluid but when extracellular virus was inhibited by antibody, spread of infection from cell to cell in the monolayer still continued. Results obtained in concurrent electron microscope studies will be presented separately. Correlation of the results of the immunofluorescence and electron microscope studies suggests the possibility that much of the immunofluorescence observed might be due to antigen in virus precursors or components.     

Minimizing false negatives in electron microscope searches for virus or other specific features of cancer cells.

Electron microscopic searches for virus particles and other specific features of cancer cells involve exceptionally poor sampling statistics. It is probable that most searches conducted in the past were of limited value in establishing whether such viruses or special features are associated with one or more tumor types. On the other hand, in a few cases, the electron microscope has provided the first, or even the only, evidence of virus particles. A radical change in the way the electron microscope is applied to this problem is suggested whereby a high-voltage microscope is used to examine thicker sections and an image-processing arrangement is used to focus and select images and to search the images for virus particles. It is suggested that it is important to distinguish virus producers amongst oncogenic viral genome carriers since these may be in a more advanced state of tumor induction and may be capable of transmitting the virus to others.     

Fine Structure of Cellular Inclusions in Measles Virus Infections

Cells which are infected with measles virus have been known for some time to contain inclusion material that is distinguishable from normal cellular components by application of traditional staining methods and observation in the light microscope. The fine structure of the inclusion material contained in HeLa cells infected with Edmonston strain of measles virus has been examined in the electron microscope. Two steps have been found necessary in this study: (1) the recognition by phase-contrast microscopy of the living cell of bodies that are defined as inclusion material when the cells are classically stained; and (2) the recognition in the electron microscope of inclusion-body material that had previously been identified in the living cell. The fine structure of the nuclear and cytoplasmic inclusion material in osmium-treated cells was found to consist mainly of randomly arrayed filaments of low electron density. Dense, highly ordered arrays of filaments were found near the center of the nuclear inclusions, sometimes as a two-dimensional, nearly orthogonal arrangement. If the size of the measles virus is taken to be around 100 mµ in diameter, the strands seen in the inclusions cannot be fully formed virus.     

Same serial section correlative light and energy-filtered transmission electron microscopy.

Correlative imaging of a specific cell with both the light microscope and the electron microscope has proved to be a difficult task, requiring enormous amounts of patience and technical skill. We describe a technique with a high rate of success, which can be used to identify a particular cell in the light microscope and then to embed and thin-section it for electron microscopy. The technique also includes a method to obtain many uninterrupted, thin serial sections for imaging by conventional or energy-filtered transmission electron microscopy, to obtain images for 3D analysis of detail at the suborganelle level.     

Linear, Single-Stranded Deoxyribonucleic Acid Isolated from Kilham Rat Virus

Kilham rat virus (KRV) was grown in a rat nephroma cell line and was purified by two isopycnic centrifugations in cesium chloride. The virus contains single-stranded deoxyribonucleic acid (DNA) with a molecular weight of approximately 1.6 x 10(6). The DNA was extracted from the virion by both phenol extraction and by 2% sodium dodecyl sulfate at 50 C. KRV DNA, extracted by both procedures, was observed in an electron microscope by using a cytochrome c or diethylaminoethyldextran monolayer. The DNA was also exposed to exonuclease I, an enzyme which hydrolyzes specifically linear, single-stranded DNA. Hydrolysis of 70 to 80% of the DNA was observed. Both the enzymatic and the electron microscope studies support the conclusion that extracted KRV DNA is a single-stranded, linear molecule. The length of the DNA was measured in the electron microscope and determined to be 1.505 +/- 0.206 mum.     

Electron microscopy of cells showing viral cytopathic effects in Papanicolaou smears

A technique is described whereby cells showing cytologic changes suggestive of virus infection by light microscopy can be processed further for examination in the electron microscope so that virus particles present in the cell can be visualized directly. We present the results of electron microscopy of over 100 Papanicolaou-stained smears processed this way. The morphologic changes in the cells and the ultrastructural appearances of the virus particles are demonstrated. This technique is particularly valuable for retrospective studies of mounted cytologic or histologic material. It has also proven to be a valuable research tool in the study of human polyomavirus and human wart virus infection.     

Effect of poly-l-ornithine on isolated tobacco mesophyll protoplasts: Evidence against stimulated pinocytosis

Summary The effects of poly-l-ornithine on the surface membrane of isolated tobacco protoplasts have been examined in the electron microscope using a colloidal metal oxide and a spherical virus as marker substances. No evidence was found to suggest that isolated protoplasts take up either of these markers by a pinocytotic process. Poly-l-ornithine increased the degree of damage observed in fixed preparations, and specifically caused lesions of the plasmalemma which were favoured sites for the binding of both external marker substances. It is suggested that the function of poly-l-ornithine and other treatments used to obtain virus infection of protoplasts is to stress the cell membrane to allow a non-physiological entry of high molecular weight materials. Pinocytosis appears not to occur nor to be necessary for uptake of these materials under conditions of membrane stress.     

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.     

Colloidal gold probes for the identification of virus particles: An appraisal

Electron microscope examination of negatively stained preparations continues to be the method of choice for the diagnosis of virus particles although in some instances an immunological test is necessary. Colloidal gold immunocytochemical probes are becoming increasingly popular for electron microscopy and their suitability for the identification of virus particles is assessed.Virus particles were immunolabelled in situ on plastic/carbon coated electron microscope grids with specific antibody and colloidal gold probes. The labelling obtained was specific, definite and with very little background. The technique is very sensitive, very quick, and since a minimum of preparation is needed it appears to possess considerable potential for virus diagnosis.     

Electron microscopic studies of viruses labeled with magnetite

We were able to develop a method with which to successfully and specifically detect virus particles under the electron microscope by using magnetite. This method was devised on the principle that magnetite-labeled antibody or magnetite coupled with protein A selectively bind virus or antibody-treated virus particles on the electron microscope grid by the action of an electromagnet. Another advantage characterizing the technique is the possibility of detection of a small number of virus particles. This is done through a process of concentration and purification of the reaction complexes trapped rigidly by magnetic force.     

Are Animal Tumor Viruses Always Virus-Like?

Three tumors initiated by well characterized viruses, but in which virus is not detectable by ordinary virological techniques, are discussed. The question of the possible state of the virus within these seemingly non-infectious tumors is considered, largely from the standpoint of findings with the rabbit papilloma virus. This agent in its natural host, the cottontail rabbit, is infective, can be seen as virus bodies with the electron microscope, and can be visualized with fluorescent antibody only in the upper keratinizing cells of individual papillomas. At the growing bases of such papillomas, where neoplasia is in active progress, no infective virus is demonstrable and viral bodies cannot be visualized by either the electron microscope or fluorescent antibody. A hypothesis is presented that rabbit papilloma virus exists in cottontail papillomas in two forms—one, the complete mature virus, composed of nucleic acid and protein, and the other, immature virus, composed of naked viral nucleic acid without its protein coating. The function of the mature papilloma virus is to initiate tumor formation,—that of the immature virus, to maintain neoplasia. In the non-infective domestic rabbit papilloma, the viral nucleic acid and protein fail to combine to form mature infective virus and, as in the cottontail papilloma, neoplasia is maintained by the activity of the viral nucleic acid alone.     

Structure and Infectivity of Picornaviral RNA Encapsidated by Cowpea Chlorotic Mottle Virus Protein

Poliovirus and Mengo virus RNA were shown to associate efficiently with cowpea chlorotic mottle virus protein to form pseudovirions. The sedimentation coefficient for the pseudovirions was similar to that of poliovirus, and electron microscope observations showed the Mengo pseudovirions to be similar in size to Mengo virus. Such pseudovirions were infectious and were more resistant to ribonuclease than viral RNA; however, under our assay conditions, their infectivity was about equal to that of viral RNA.     

DNA Configuration in the Core of Marek''s Disease Virus

Marek's disease virus DNA appears to be wound around a central structure connecting the two inner poles of the capsid. Based on electron micrographs of Marek's disease virions, a diagram of spatial configuration of virus DNA is presented. This diagram may explain the pleomorphic character of the herpesvirus core observed with the electron microscope.     

A CORRELATED HISTOCHEMICAL AND ELECTRON MICROSCOPIC STUDY OF THE INTRANUCLEAR CRYSTALLINE AGGREGATES OF ADENOVIRUS (RI-APC VIRUS) IN HELA CELLS

HeLa cells in tissue cultures infected with types 3, 4, or 7 of adenovirus (RI-APC virus) were studied in order to correlate certain histochemical and electron microscopic findings. Adjacent thin (ca. 0.05 µ) and thick (2–4 µ) sections of osmium-fixed, methacrylate-embedded cells were cut; by mapping the sections the same cells could be identified with both the electron and the light microscope. Intranuclear crystalline aggregates seen with the electron microscope to be composed of ordered arrays of viral particles were found by means of the Feulgen reaction to contain DNA. DNA is therefore assumed to be a constituent of the viral particle. The virus appeared to develop from an osmiophilic Feulgen-negative matrix. Displacement of nuclear chromatin occurred during this process. A Feulgen-azure staining method was found to permit clear distinction between viral and nuclear (host) DNA in thick sections.     

包含体——流行性出血热病毒形态学的重要特征

用普通电镜和免疫酶电镜方法观察了流行性出血热病毒A9株和R22株感染的VeroE-6细胞中的病毒包含体,发现这些包含体具有以下三种基本形态:1.丝状或丝状颗粒包含体,2.松散颗粒包含体,3.致密包含体。在免疫酶法中,包含体均呈特异性酶染色阳性反应。描述和讨论了包含体与细胞及病毒发育成熟的关系。     

辛德毕斯病毒YN87448株与宿主细胞凋亡的研究

为了了解云南辛德毕斯病毒在BHK2 1细胞上的生长特性 ,并观察该病毒在BHK2 1细胞、3d龄和 2周龄小白鼠中是否有凋亡反应出现。在接种后不同时间取样 ,检测病毒的滴度 ,电镜检测凋亡细胞 ,检测细胞的DNA阶梯。一步生长曲线结果表明病毒在出现CPE之前就有大量繁殖。秋水仙素对该病毒的成熟释放没有抑制作用。该病毒对BHK2 1、小白鼠均可产生细胞凋亡     

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 mmicro) having a thick capsule and a dense inner body (35 to 40 mmicro) that is eccentrically placed within the central cavity (70 to 80 mmicro). 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.     

A Classification of Virus Particles Based on Morphology

Recent improvements in electron microscope techniques which allow the study of virus fine structure have permitted the grouping of many viruses on a purely morphological basis. Briefly the techniques used in electron microscopy for the study of viruses are reviewed and the symmetry properties of virus particles as revealed by negative staining are discussed somewhat more fully.Finally, virus particles are grouped on two bases, firstly the site of formation of the virus within the cell as seen by thin sectioning techniques, and secondly the symmetry property of the virus as seen by negative staining. Consideration of the groupings obtained in this way reveals that the biochemical and physical properties of a virus can be deduced from the readily established morphological characteristics.     

The development and application of negative staining to the study of isolated virus particles and their components: A personal account

The subsequent development and application of the negative staining technique to isolated virus particles and their components, following the early studies on the structure of T2 bacteriophage proteins, is described. The use of the method to prepare particles covering a wide range of different types of virus for examination in the electron microscope is reviewed, together with more recent advances in image analysis of electron micrographs obtained from negatively stained virus specimens.