Electron microscopic study of the morphology and morphogenesis of virus of hemorrhagic fever with renal syndrome

The morphology and morphogenesis of the virus of hemorrhagic fever with renal syndrome (HFRS) and the associated ultrastructural changes in neurons of the infected mouse brain were examined by electron microscopy. The primary location of the infection in large neurons was in the Golgi apparatus, which had highly proliferated laminar and vesicular profiles. A small number of matured virus particles were found later individually or in small groups within the distended Golgi cisternae and vesicles. Most of the virus particles were round, oval, or elongated and measured about 70–110 nm in diameter. A lipid bilayered viral envelope with an external fringe of surface projections could be resolved at high magnification. The maturation (budding) of the virus occurred exclusively at smooth membrane vesicles, and predominantly at membranes in, or adjacent to, Golgi cisternae. Viral inclusion bodies containing fine filamentous material were seen frequently in close proximity to sites of virus maturation. The known morphological and morphogenetic characteristics of the virus particles observed in infected mouse brain gave further evidence for taxonomic identification of HFRS virus as a member of the family of Bunyaviridae.     

Ultrastructure of human retroviruses

We compared the ultrastructure of the human retroviruses by thin-section electron microscopy of infected lymphocytes. Virus particles form at the plasma membrane without involvement of a cytoplasmic precursor. Budding forms of human T-cell leukemia virus types I and II (HTLV-I and -II) consist of a crescent-shaped nulceoid separated from the envelope by an intermediate layer. Mature forms of these viruses are about 100 nm in diameter. The nucleoid is electron lucent and almost completely fills the virion. There is about a 10-nm space between the envelope and nucleoid. The envelope has fuzzy surface projections. HTLV-I and -II resemble other type C retroviruses in morphology. Budding forms of human immunodeficiency virus (HIV, LAV, HTLV-III) also have a crescent-shaped nucleoid but not an intermediate layer between the core and envelope. The envelope has rod-shaped surface projections. Mature forms of HIV have an electron-dense nucleoid that is eccentric and bar- or cone-shaped. Particles have the same ultrastructure as retroviruses of the Lentivirus genus. HIV is readily distinguishable from HTLV-I and -II by thin-section electron microscopy. HIV is usually found in extracellular spaces by transmission electron microscopy of thin sections, and scanning electron microscopy of HIV-infected T4 lymphocytes also shows many particles on the surface of these cells. Lymphadenopathy-associated virus type II (LAV-II) has the same internal ultrastructure as HIV, but its surface projections are more prominent, being about three times the length of those of HIV. Human T lymphotropic virus type IV (HTLV-IV) has the same morphology as LAV-II.     

Atomic force microscopy of recombinant adeno-associated virus-2 prepared by centrifugation

In order to decrease salt crystal formation, reduce the aggregation of viruses in atomic force microscopy (AFM) preparations and obtain an acceptable AFM image, the AFM sampling of viruses must be optimized. In this paper, centrifugal AFM sampling of recombinant adeno-associated virus-2 (rAAV-2) was used. The prepared rAAV-2 virus was imaged by AFM using the tapping mode in air. The results indicate that the rAAV-2 viruses prepared by centrifugal AFM sampling methods could be well imaged by AFM. The rAAV-2 viruses exhibited polymorphous particles in the AFM images. The preliminary off-line section analysis of the individual rAAV-2 virus particle indicated that the half-high widths of the large rAAV-2 particles ranged from 18 to 23 nm, while those of the small rAAV-2 particles ranged from 15 to 17 nm, which is almost in agreement with the results obtained by the off-line particle analysis of the rAAV-2 virus particles. Above all, the aggregation of different-sized rAAV-2 particles was directly imaged by AFM, which verifies the previous speculation that the large number (> ~31 nm) distribution of the mean diameter of rAAV-2 virus particles was probably caused by aggregation of rAAV-2 particles.     

Rapid and reliable method for diagnostic electron microscopy of faeces

Four methods are described for examining viruses in faeces by electron microscopy using negative staining. Faeces samples from a total of 180 patients with diarrhoea illnesses were processed for electron microscopy by the direct staining technique, pseudoreplica technique, microsolute concentration technique and by ultracentrifugation. Virus particles (including rotaviruses and adenoviruses) were found in fifty-five (31%) samples when the results of all methods were combined. Bacteriophages were found in thirty of the samples which were read virus negative. Herpesviruses were also found in the faecal samples of two patients with diarrhoea illness. Parvovirus-like particles were identified in one sample which was rotavirus and adeno-virus positive. Calicivirus-like particles were found in a sample which was adenovirus positive. With the direct staining technique thirty-six (20%) samples contain virus particles; with micro-solute concentration technique forty-eight (27%) samples contain virus particles and with ultracentrifugation thirty-eight (21%) contain virus particles. It is believed the microsolute concentration technique is rapid and more reliable than the pseudoreplica technique and ultracentrifugation method, and is a more preferable method for the diagnosis of faecal sample by electron microscopy.     

Ultrastructural features of the AIDS virus (HIV) and its morphogenesis

HIV particles were usually seen on the surface of established lymphoid cells derived from AIDS patients or on CEM cells infected with HIV, and sometimes in cytoplasmic vacuoles. The virus particles were formed by a budding process from the plasma membrane of an infected cell. The budding particles were of a doughnut form. Various profiles of virus particles were seen extracellularly: type 1 had a bar-shaped, electron-dense core, type 2 had a central and type 3 an eccentric electron-dense round core, type 4 was doughnut-shaped, and type 5 had a layered core. However, projection patterns of the AIDS virus model suggested that type 1, 2 and 3 particles are similar. Therefore, the AIDS virus may be one of three main types: with or without a dense core, and with a layered core. It is thought that a particle with a layered core and a doughnut-type particle may be immature viruses.     

Climbing the steps of viral atomic force microscopy: visualization of Dengue virus particles

In recent years, the application of atomic force microscopy (AFM) to biological systems has highlighted the potential of this technology. AFM provides insights into studies of biological structures and interactions and can also identify and characterize a large panel of pathogens, including viruses. The Flaviviridae family contains a number of viruses that are important human and animal pathogens. Among them, Dengue virus causes epidemics with fatal outcomes mainly in the tropics. In this study, Dengue virus is visualized for the first time using the in air AFM technique. Images were obtained from a potassium-tartrate gradient-purified virus. This study enhances the application of AFM as a novel tool for the visualization and characterization of virus particles. Because flavivirus members are closely related, studies of the morphologic structure of the Dengue virus can reveal strategies that may be useful to identify and study other important viruses in the family, including the West Nile virus.     

Virus present in the reproductive tract of asymptomatic drones of honey bee (Apis mellifera l.), and possible infection of queen during mating

Virus particles and viral inclusions were detected by transmission electron microscopy examination of sections of the seminal vesicles and mucus gland of asymptomatic young drones from colonies of Apis mellifera lightly infested by Varroa mite. In the mucus gland the infection was found in the muscular sheath and epithelium, while in the seminal vesicle in cells of the outer serosa. Isolated viral particles were also observed in the hemolymph occupying the intercellular spaces of the muscular sheath fibers. In the muscle the virus appeared as polygonal crystalloid inclusions, while in the epithelium mainly inside cytoplasmic vesicles. The infected cells apparently are not damaged. The virus particles are present in the hemolymph and forming more mature structures, as crystalloids, in the muscle. This suggests that the virus is liberated in the body fluid and infects the tissues penetrating the cells through endocytosis. The presence of virus in mucus gland epithelial vesicles raise the possibility of its transference to the gland secretion and therefore, to the semen.     

The fine structure and morphogenesis of the envelope and nucleocapsid of goat poxvirus and infectious bovine rhinotracheitis virus: A freeze-fracture study

We studied the morphology and morphogenesis of viral envelopes and nucleocapsids of goat poxvirus (GPV) and infectious bovine rhinotracheitis virus (IBRV) by means of the freeze-fracture technique. The GPV at an early development stage was fractured in its middle, and the envelope was shown to be a bilayer of particles. The mature GPV was fractured between two monolayers of the envelope. These facts suggest that there is little lipid and mainly protein particles in the envelope at the early-development stage, then the lipid inserts into the envelope during viral development. We found that there were still many intramembranous protein particles in protoplasmic fracture face (PF) and extracellular fracture face (EF) of the envelope of mature GPV in the cytoplasm, and fewer particles in the envelope of released GPV. In the envelope of mature IBRV, however, there were many more intramembrane protein particles in the PF face than that in the EF face. Spike-like structures could be seen at the outer edge of the IBRV envelope at times. Protein particles were regularly arranged in the plasmic membranes contacting IBRV. This phenomenon seems to be related to IBRV release. The naked cores, empty capsids, and nucleocapsids of IBRV were assembled in the nucleus of infected cells at the same time. The assembled nucleocapsids could be divided into five types according to their different fracturing positions, whose morphology was observed after deep etching. The morphology of the samples prepared with different methods was compared as well.     

Dengue virus infection: A review of advances in the emerging rapid detection methods

Dengue virus infections are increasing worldwide generally and in Asia, Central and South America and Africa, particularly. It poses a serious threat to the children population. The rapid and accurate diagnostic systems are essentially required due to lack of effective vaccine against dengue virus and the progressive spread of the dengue virus infection. The recent progress in developing micro- and nano-fabrication techniques has led to low cost and scale down the biomedical point-of-care devices. Starting from the conventional and modern available methods for the diagnosis of dengue infection, this review examines several emerging rapid and point-of-care diagnostic devices that hold significant potential for the progress in smart diagnosis tools. The given review revealed that an effective vaccine is required urgently against all the dengue virus serotypes. However, the rapid detection methods of dengue virus help in early treatment and significantly reduce the dengue virus outbreak.     

High-pressure freezing in the study of animal pathogens

High‐pressure freezing is applicable to both morphological and immunocytochemical studies. We are investigating the morphogenesis of foot‐and‐mouth disease virus and African swine fever virus by the use of high‐pressure freezing of infected cells. Foot‐and‐mouth disease virus particles are not detected in sections of conventionally immersion‐fixed infected cells, but when the cells are prepared by high‐pressure freezing, newly formed virions are readily seen throughout the cell. We report two methods for high‐pressure freezing of virally infected cells: first, two sapphire discs frozen ‘face to face’ with a narrow spacer to prevent cell damage and, second, a fibrous filter substrate that can be easily cut into discs to fit into the freezing planchettes. Cells readily adhere to the fibres in vitro, and the complete disc can be rapidly transferred to the planchettes for freezing. Immunolabelling studies of the microneme proteins of the parasite Eimeria tenella indicate that high‐pressure freezing followed by freeze‐substitution in acetone with uranyl acetate allows high‐sensitivity immunolabelling for these proteins.     

Imaging and nano-dissection of tobacco mosaic virus by atomic force microscopy

Tobacco mosaic virus (TMV) has been deposited on freshly cleaved mica substrates. The topography was investigated by contact, non-contact and lateral-force microscopy under ambient conditions in air. The results were in accord with known dimensions of TMV (i.e. 18 nm in diameter and 300 nm in length). However, convolution of tip shape with TMV morphology resulted in an apparent width of 80–140 nm in the lateral plane, a factor of 4–7 greater than the known diameter. Other artefacts - broadening and double images - were observed and ascribed to tip anomalies. High force loadings and slow repetitive scanning resulted in controlled removal of parts of the TMV structure. Accordingly, it was possible to reveal and image the central core channel of the TMV. The precision and resolution of dissection induced by AFM is currently limited by the shape of the tip, having a 40-nm radius of curvature for standard Si₃N₄ tips. It is estimated that sharper tips, with a radius of curvature of less than 10 nm, should be able to resolve, non-destructively, the protein subunits in the non-contact mode, and selectively remove single subunits in the contact mode.     

Improving the contrast of topographical AFM images by a simple averaging filter

New image-processing methods were applied to atomic force microscopy images in order to visualize small details on the surface of virus particles and living cells. Polynomial line flattening and plane fitting of topographical images were performed as first step of the image processing. In a second step, a sliding window approach was used for low-pass filtering and data smoothing. The size of the filtering window was adjusted to the size of the small details of interest. Subtraction of the smoothed data from the original data resulted in images with enhanced contrast. Topographical features which are usually not visible can be easily discerned in the processed images. The method developed in this study rendered possible the detection of small patterns on viral particles as well as thin cytoskeleton fibers of living cells. It is shown that the sliding window approach gives better results than Fourier-filtering. Our method can be generally applied to increase the contrast of topographical images, especially when small features are to be highlighted on relatively high objects.     

General morphology and ultrastructure of the female reproductive apparatus of Trichomalopsis shirakii crawford (Hymenoptera,Pteromalidae)

The morphology and ultrastructure of the female reproductive system were examined for a larval–pupal parasitoid Trichomalopsis shirakii Crawford of Oulema oryzae Kuwayama using light and electron microscopes. The reproductive system includes two ovaries, two pairs of accessory glands, an unbranched venom gland, a large venom reservoir and a Dufour gland. Each ovariole contains follicles and oocytes at different stages of maturation. A fibrous layer covers the surface of mature egg. The accessory glands are made up of a layer of secretory cells surrounded by muscle fibers. In these secretory cells, numerous mitochondria, electron‐dense secretory granules and vesicles filled with dense granular particles are present. These granular particles appear as virus‐like particles (VLPs). The venom gland consists of a single layer of secretory cells which are organelle rich with abundant rough endoplasmic reticulum, mitochondria and vesicular organelles, a layer of duct cells and an inner intima. The reservoir consists of a muscular sheath, epidermal cells with few organelles and an intima layer. The Dufour gland has a relatively large lumen surrounded by a single layer of columnar epithelial cells which are characterized by clusters of smooth endoplasmic reticulum and lipid droplets. Aside from the venom, the fibrous layer coating the egg and the granular particles which may be VLPs have been discovered in our study. They may serve as one of the parasitoid‐associated factors in their host–parasitoid relationship and play a role in host immune suppression. Microsc. Res. Tech. 79:625–636, 2016. © 2016 Wiley Periodicals, Inc.     

Microscopic approach of powder compaction using finite element method

An approach for simulating microscopic densification behaviour of powder particles in compaction using a finite element method is proposed. In this method, the contacts between powder particles during the compaction are detected, and plastic deformation of the particles is calculated by the finite element method for a porous metal. The finite element mesh is generated by connecting the centres of the particles in contact. It is assumed that the finite elements are porous metals having an average relative density calculated from the volumes of the powder and pore inside the element. The elements are classified into the triangular and quadrilateral ones used in the conventional finite element methods and a linear one for the simple compression. The accuracy of the stiffness for plastic deformation of the particles is improved by applying the finite element method. The calculated plastic deformation of powder particles in plane-strain compaction is compared with that for a model experiment using aluminium rods.     

The computation of particle size-shape distributions in transmission microscopy

A method has been developed, and a computer program written, to correct measurements of particle length and width for the effects of section thickness and swelling in the preparation of sectioned samples for transmission microscopy. The method is applied to the system of silver particles in gelatin, and the resulting calculated extinction spectra are compared with those observed and calculated without correction. Improved correlations show that the method is useful.     

Bias in bacteriophage morphological classification by transmission electron microscopy due to breakage or loss of tail structures

Virtually every study that has used transmission electron microscopy (TEM) to estimate viral diversity has acknowledged that loss of phage tails during sample preparation may have biased the results. However, the magnitude of this potential bias has yet to be constrained. To characterize biases in virus morphological diversity due to tail loss, six phage strains representing the order Caudovirales were inoculated into sterile sediments and soils. Phage particles were then extracted using standard methods. Morphologies of extracted phage particles were compared to those of unmanipulated control samples to determine the extent of tail breakage incurred by extraction procedures. Podoviruses exhibited the smallest frequency of tail loss during extraction (1.2-14%), myoviruses were moderately susceptible to tail breakage (15-40%), and siphoviruses were highly susceptible (32-76%). Thus, TEM assessments of viral diversity in soils or sediments by distribution of tail morphologies may be biased toward podoviruses and virions lacking tails, while simultaneously underestimating the abundance of siphoviruses. However, since the majority of viral capsids observed under TEM were intact, estimates of viral diversity based on the distribution of capsid diameters may provide a more reliable basis for morphological comparisons within and across ecosystems.     

探寻企业的计算机病毒查杀机制与病毒防治

从计算机病毒查杀的机制出发,探讨如何更好地预防企业网络中的计算机被病毒入侵,并说明如何查杀计算机病毒.     

Pattern analysis of irregularly shaped particles

Methodical considerations are presented on the application of a commercial pattern-recognition instrument like the Quantimet 720 for the computer-supported analysis of irregularly shaped particles. To discriminate between particles of different shape on the basis of some simple geometrical models proposed, effective analytical criteria are suggested and adequate expressions are derived for the numerical calculation of length and width of particles irrespective of their actual shape as well as for such properties as convexity, clustering or eccentricity. A slightly enhanced k-factor compared to that in common use is discussed. Based on the results so far obtained with single particles, formulae for the determination of an average orientation factor and an autocorrelation function are provided. All considerations were extensively tested by the numerical evaluation of proper drawing models.     

Laser cutting of 7050 Al alloy reinforced with Al2O3 and B4C composites

In the present study, laser cutting of 7050 aluminum alloy sheets reinforced with Al₂O₃ and B₄C particles are carried out. The cut geometry is examined using scanning electron microscope and the optical microscope. The lump parameter analysis technique is used to formulate and determine the kerf width size. The predictions for kerf width are compared with experimental data. The percentage kerf width size variation along the cut edges is determined and the influence of the laser power level and duty cycle of the laser pulses on the percentage kerf width size variation is examined. It is found that 7050 aluminum alloy reinforced with 20% Al₂O₃ composite results in relatively large kerf width size as compared to its counter parts that corresponding to 7050 aluminum alloy reinforced with 20% B₄C composite. The kerf width size predicted agreed with the experimental data for both composites.     

基于线性插补加工零刃带螺旋回转面刀具的精度研究

针对多轴线性插补方法加工零刃带回转面刀具建立了刃带宽度误差的分析模型,兼顾分析了两个几何体——砂轮大圆和锥面,在工件表面按一定规律运动时对刀刃宽度的精度影响,给出了减小误差的方法。同时提出了刀刃节点交错的刀位(CL)轨迹计算原理,可以大大改善刀具加工工件的表面质量,此方法简单可靠,具有通用性。