A Comparative Study on the Presence of C-Type Viral Particles in Placentas from Primates and Other Animals

A herpes-like virus has been isolated from duck embryo fibroblast (DEF) cultures inoculated with blood from Marek's disease (MD) infected birds. Cultures which contained this virus produced MD in susceptible chickens while virus negative cultures and control cultures failed to do so. This and other circumstantial evidence including similarities in properties of the virus and the MD agent implicate this virus in the etiology of MD.Histochemical studies demonstrated the presence of DNA-staining intranuclear inclusion bodies in polykarocytes in infected cultures. Distinct nucleo-plasmic aggregates were also seen in sections of similar multinucleated cells examined with the electron microscope. These aggregates are probably the same as the inclusion bodies seen with the light microscope. Naked viral particles were observed in the nucleus of infected cells within or on the edges of the nucleoplasmic aggregates. These particles measured 95-100mμ, in diameter and rarely escaped into the cytoplasm or nuclear vesicles by budding through the nuclear membrane (Fig. 1). The enveloped particles (Fig. 2) formed in this manner measured 150-170mμ in diameter and always had a densely stained nucleoid. The virus in supernatant fluids consisted of naked capsids with 162 hollow, cylindrical capsomeres (Fig. 3). Enveloped particles were not seen in such preparations.

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Detection of viruses by electron microscopy: Increased sensitivity by direct micro-ultracentrifugation of samples

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100128821 ◽

1987 ◽

Vol 45 ◽

pp. 908-909

Author(s):

Alain R. Trudel ◽

M. Trudel

Keyword(s):

Electron Microscopy ◽

Fold Increase ◽

Observation Time ◽

Clinical Samples ◽

Maximum Speed ◽

Viral Particles ◽

Structural Details ◽

Latex Spheres ◽

Increased Sensitivity ◽

Size Of Particles

AirfugeR (Beckman) direct ultracentrifugation of viral samples on electron microscopy grids offers a rapid way to concentrate viral particles or subunits and facilitate their detection and study. Using the A-100 fixed angle rotor (30°) with a K factor of 19 at maximum speed (95 000 rpm), samples up to 240 μl can be prepared for electron microscopy observation in a few minutes: observation time is decreased and structural details are highlighted. Using latex spheres to calculate the increase in sensitivity compared to the inverted drop procedure, we obtained a 10 to 40 fold increase in sensitivity depending on the size of particles. This technique also permits quantification of viral particles in samples if an aliquot is mixed with latex spheres of known concentration.Direct ultracentrifugation for electron microscopy can be performed on laboratory samples such as gradient or column fractions, infected cell supernatant, or on clinical samples such as urine, tears, cephalo-rachidian liquid, etc..

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A rapid method for the direct identification of paramyxovirus in canine CSF by ultracentrifugation and negative staining as a rule out for distemper

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100160522 ◽

1990 ◽

Vol 48 (3) ◽

pp. 594-595

Author(s):

W.L. Steffens ◽

M.B. Ard ◽

C.E. Greene ◽

A. Jaggy

Keyword(s):

Subacute Sclerosing Panencephalitis ◽

Clinical Signs ◽

Viral Disease ◽

Etiologic Agent ◽

Mucosal Inflammation ◽

Worldwide Distribution ◽

Negative Stain ◽

Viral Particles ◽

Systemic Manifestations ◽

Rule Out

Canine distemper is a multisystemic contagious viral disease having a worldwide distribution, a high mortality rate, and significant central neurologic system (CNS) complications. In its systemic manifestations, it is often presumptively diagnosed on the basis of clinical signs and history. Few definitive antemortem diagnostic tests exist, and most are limited to the detection of viral antigen by immunofluorescence techniques on tissues or cytologic specimens or high immunoglobulin levels in CSF (cerebrospinal fluid). Diagnosis of CNS distemper is often unreliable due to the relatively low cell count in CSF (<50 cells/μl) and the binding of blocking immunoglobulins in CSF to cell surfaces. A more reliable and definitive test might be possible utilizing direct morphologic detection of the etiologic agent. Distemper is the canine equivalent of human measles, in that both involve a closely related member of the Paramyxoviridae, both produce mucosal inflammation, and may produce CNS complications. In humans, diagnosis of measles-induced subacute sclerosing panencephalitis is through negative stain identification of whole or incomplete viral particles in patient CSF.

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A combined pseudoreplica-immunocytochemical technique for research and diagnostic virology

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100162065 ◽

1990 ◽

Vol 48 (3) ◽

pp. 900-901

Author(s):

Blayne Fritz ◽

Stanley J. Naides ◽

Kenneth Moore

Keyword(s):

Phosphotungstic Acid ◽

Immunogold Labelling ◽

Uranyl Acetate ◽

Distilled Water ◽

Clinical Specimens ◽

Tissue Sections ◽

Specimen Retrieval ◽

Transmission Electron ◽

Viral Particles ◽

Diagnostic Virology

The pseudoreplica method of staining viral particles for visualization by transmission electron microscopy is a very popular technique. The ability to concentrate clinical specimens while semi-embedding viral particles makes it especially well suited for morphologic and diagnostic virology. Immunolabelling viral particles with colloidal gold is a technique frequently employed by both research and diagnostic virologists. We have characterized a procedure which provides the advantage of both by modifying and combining pseudoreplica staining and immunogold labelling.Modification of specimen retrieval and delay of staining allows us to utilize pseudoreplica processed specimens within our standard immunogold labelling protocol. In brief, we absorbed samples onto 2% agarose, added.25% Formvar and wicked dry. We then floated the Formvar-virus film onto double distilled water, added grids and retrieved with parafilm. The Formvar-virus specimens were then treated as thin tissue sections within our standard two stage immunolabelling protocol. Following completion of immunogold labelling; each grid was negatively stained with phosphotungstic acid or uranyl acetate contrast stains.

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Progress in high-resolution CRYO-SEM imaging of viral particles

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100166555 ◽

1996 ◽

Vol 54 ◽

pp. 818-819

Author(s):

Ya Chen ◽

Geoffrey Letchworth ◽

John White

Keyword(s):

Electron Microscopy ◽

Scanning Electron Microscopy ◽

High Resolution ◽

Structural Information ◽

Signal To Noise Ratio ◽

Flexible Structures ◽

Simian Virus ◽

Topographic Features ◽

Viral Particles ◽

Scanning Electron

Low-temperature high-resolution scanning electron microscopy (cryo-HRSEM) has been successfully utilized to image biological macromolecular complexes at nanometer resolution. Recently, imaging of individual viral particles such as reovirus using cryo-HRSEM or simian virus (SIV) using HRSEM, HV-STEM and AFM have been reported. Although conventional electron microscopy (e.g., negative staining, replica, embedding and section), or cryo-TEM technique are widely used in studying of the architectures of viral particles, scanning electron microscopy presents two major advantages. First, secondary electron signal of SEM represents mostly surface topographic features. The topographic details of a biological assembly can be viewed directly and will not be obscured by signals from the opposite surface or from internal structures. Second, SEM may produce high contrast and signal-to-noise ratio images. As a result of this important feature, it is capable of visualizing not only individual virus particles, but also asymmetric or flexible structures. The 2-3 nm resolution obtained using high resolution cryo-SEM made it possible to provide useful surface structural information of macromolecule complexes within cells and tissues. In this study, cryo-HRSEM is utilized to visualize the distribution of glycoproteins of a herpesvirus.

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Identification of a baculovirus polyhedron formation mutant with a novel phenotype

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100166440 ◽

1996 ◽

Vol 54 ◽

pp. 796-797

Author(s):

James M. Slavicek ◽

Melissa J. Mercer ◽

Mary Ellen Kelly

Keyword(s):

Viral Gene ◽

Gene Products ◽

Type Number ◽

Infection Cycle ◽

Wild Type ◽

Protein Matrix ◽

Insect Midgut ◽

Viral Particles ◽

Budded Virus ◽

Viral Form

Nucleopolyhedroviruses (NPV, family Baculoviridae) produce two morphological forms, a budded virus form and a viral form that is occluded into a paracrystalline protein matrix. This structure is termed a polyhedron and is composed primarily of the protein polyhedrin. Insects are infected by NPVs after ingestion of the polyhedron and release of the occluded virions through dissolution of the polyhedron in the alkaline environment of the insect midgut. Early after infection the budded virus form is produced. It buds through the plasma membrane and then infects other cells. Later in the infection cycle the occluded form of the virus is generated (reviewed by Blissard and Rohrmann, 1990).The processes of polyhedron formation and virion occlusion are likely to involve a number of viral gene products. However, only two genes, the polyhedrin gene and 25K FP gene, have been identified to date that are necessary for the wild type number of polyhedra to be formed and viral particles occluded.

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