STRUCTURE AND DEVELOPMENT OF VIRUSES AS OBSERVED IN THE ELECTRON MICROSCOPE : V. WESTERN EQUINE ENCEPHALOMYELITIS VIRUS

Stages in the development and release of Western equine encephalomyelitis virus are illustrated and described. It is suggested that precursor particles 22 mµ in diameter differentiate at template sites close to membranes bordering cytoplasmic vacuoles and that these particles either pass into the lumen of the vacuole, acquiring in the process a coat and peripheral membrane, or are dispersed in the cytoplasm and extruded through the cellular wall, emerging as viral particles on the surface. Although necrosis and dissolution of the cell with release of contents, including virus, may intervene at any stage of infection, ejection of virus from the vacuoles presumably can occur without rupture of the cell. The virus consists of a 30 mµ core separated by a zone of lesser density from a sharply defined peripheral membrane 45 to 48 mµ in diameter. Precursor particles, as well as viral particles, occasionally crystallize, the former in the cytoplasm, the latter in vacuoles and probably on the cellular surface

USE OF SERIAL SECTIONS TO DELINEATE THE STRUCTURE OF PORTHETRIA DISPAR VIRUS IN THE ELECTRON MICROSCOPE

Consecutive serial sections of polyhedra obtained from gipsy moth larvae infected with P. dispar virus revealed bundles of viral rods scattered and oriented at random within the polyhedral body. Each bundle was entirely surrounded by a dense, sharply defined membrane. The rods measured 18 to 22 mµ in diameter and averaged 280 mµ in length. No spherical viral particles were encountered. The effects of variable compression and periodic distortion of the sections on the appearance of the virus are discussed

STRUCTURE AND DEVELOPMENT OF VIRUSES OBSERVED IN THE ELECTRON MICROSCOPE : III. INFLUENZA VIRUS

Rods and spheres believed to represent viral particles were observed at the free surface of entodermal cells of the chorioallantoic membrane 6 to 44 hours after infection. Although occasional short rods revealed poorly defined internal bodies, the majority, as well as all the longer rods (filaments), exhibited no visible internal structure. The spheres presumed to lie central to the plane of section contained an inner body 20 to 22 mµ in diameter. Both forms possessed a dense, sharply defined limiting membrane 30 A thick and a diffuse external coat of lesser density. Where superimposition within the section was minimal, the viral particles were separated by a relatively constant distance. Measured to include this spacing, on the assumption that it reflected the presence of a component of the outer coat, the diameters of a majority of the rods were 50 to 60 mµ, whereas the spheres averaged 60 to 70 mµ. The rods appeared to form by a process of extrusion from the cell wall and became detached either singly or in bundles of variable length. The spheres seemed to differentiate at the cell surface and to acquire the inner body, limiting membrane, and outer coat as they migrated through the membrane of the host cell. No characteristic changes were seen in the nuclei or adjacent cytoplasm, and recognizable viral particles were never encountered in these areas of the cell. No support was obtained for the assumption that the spheres developed primarily by segmentation of the rods. It is suggested that the spherical form of the virus is the elemental infectious unit and that the filamentous form is largely or completely non-infective

SOME EFFECTS OF THE MICROTOME KNIFE AND ELECTRON BEAM ON METHACRYLATE-EMBEDDED THIN SECTIONS

A technique for the examination of specimens at low electron beam intensity has been presented. Sections micrographed with this technique showed numerous knife scratches and frequently contained bands running parallel to the knife edge. Banding with an average spacing of 0.2 µ appeared to result from periodic distortion produced by impact of the knife. At the beam intensities customarily employed, differential sublimation and probably flow of the methacrylate resulted in obliteration of the bands and all but the deepest knife scratches. In addition, changes in the size, shape, and orientation of certain structures were noted. Artifacts resulting from incineration or sublimation of tissue components fixed in formalin were illustrated, and the suggestion was made that such instability to the electron beam accounted in part for the differences observed in osmium- and formalin-fixed tissues. The deformation revealed in serial sections was discussed, and it was pointed out that shortening in the axis perpendicular to the knife edge was associated with elongation in the axis parallel to the cutting edge, the elongation usually occurring locally without change in the width of the section. It was noted that the material causing contamination of the surface of sections during examination exhibited no structure but caused progressive loss of contrast

STRUCTURE AND DEVELOPMENT OF VIRUSES AS OBSERVED IN THE ELECTRON MICROSCOPE : VII. INCOMPLETE INFLUENZA VIRUS

Chicken embryos were infected by the chorioallantoic route with influenza virus, PR8 strain, in the form of undiluted chorioallantoic fluid. Electron microscopic examination 24 hours after infection revealed that membrane-bound fragments of cytoplasm appeared to be in process of release from entodermal cells of the chorioallantois. The number of such fragments was greatly increased in proportion to the number of typical viral particles after the third serial passage, which was accompanied by a reduction of the infectivity-hemagglutinin ratio (von Magnus effect). The lack of recognizable internal components, together with the presence of surface structure which closely resembled that of the virus and frequently contained viral antigen, suggested that many of these fragments were incomplete viral particles. It is proposed that concentrated inocula damage the cells and interfere with differentiation of the virus, but do not inhibit formation and detachment of cytoplasmic processes. Under these circumstances the accumulation of viral antigen at the surface of the cell will result in the predominant formation of incomplete virus

ELECTRON MICROSCOPIC OBSERVATIONS ON THE DEVELOPMENT OF HERPES SIMPLEX VIRUS

Study of the J.M. strain of herpes simplex virus in human amnionic and HeLa cell tissue cultures revealed the presence of intranuclear crystals composed of viral particles with a single membrane enclosing a central body. Randomly dispersed virus with double coats was seen in the nuclear matrix and between multiple membranes at the nuclear periphery. The majority of intracytoplasmic viral particles were within walled vacuoles. It is suggested that this strain of virus differentiates and frequently crystallizes at template sites which are characterized by aggregates of granules near the nuclear margin; that particles, either singly or occasionally in small groups, become enclosed by a second peripheral membrane while still within the nucleus; that the virus can pass into the cytoplasm through reduplications of the nuclear membrane which are deposited behind the virus in such a manner as to prevent rupture of the nucleus; that most of the intracytoplasmic virus is contained within sacks formed by nuclear membranes; and that rupture of these sacks at the cell surface results in extrusion of virus without disruption of the cell. No evidence was obtained to support the hypothesis that virus develops in the cytoplasm. Examination of the H.R. and C.G. strains of herpes simplex virus in identical cell lines grown under similar conditions failed to show viral crystals, but reduplication of the nuclear membranes was evident. Study of the J.M. strain in cells of chicken embryo chorioallantoic membranes indicated that the basic mechanisms of viral development and release did not differ from those operating in HeLa and human amnionic cells

A CORRELATIVE STUDY BY ELECTRON AND LIGHT MICROSCOPY OF THE DEVELOPMENT OF TYPE 5 ADENOVIRUS : I. ELECTRON MICROSCOPY

Stages in the nuclear changes consequent to infection with type 5 adenovirus are shown and described. Viral development seems to be confined to the nucleus where characteristic particles are found. The shape of the intracellular virus depends upon the method of preservation employed, appearing spherical after osmium tetroxide or freezing-substitution, occasionally exhibiting angulated faces after formalin and often assuming an hexagonal profile after potassium permanganate. The non-viral crystals are encountered in zones of low density, and it is suggested that crystallization results from the accumulation of protein in these areas. An hypothesis is presented to explain why these crystals, in contrast to the insect polyhedra, contain few viral particles

A CORRELATIVE STUDY BY ELECTRON AND LIGHT MICROSCOPY OF THE DEVELOPMENT OF TYPE 5 ADENOVIRUS : II. LIGHT MICROSCOPY

The evolution of the intranuclear lesion produced by type 5 adenovirus in HEp-2 and HeLa cells is described as seen in the light microscope and the bodies formed in the course of the infection characterized histochemically. Some 12 hours after infection acidophilic protein bodies, without appreciable nucleic acid, first appear in the nucleus and coalesce into a network. Within or in association with this material, DNA-containing masses (viral aggregates) are formed which rapidly increase in amount and then coalesce. At the same time, a protein is produced, histochemically different from that of the acidophilic or basophilic structures mentioned, within the infected nucleus, which constitutes a matrix within which regular cytstals of a protein, (presumably non-viral) materialize. These structural and histochemical features are correlated with details which have been observed in parallel studies with the electron microscope

STRUCTURE AND DEVELOPMENT OF VIRUSES OBSERVED IN THE ELECTRON MICROSCOPE : IV. VIRUSES OF THE RI-APC GROUP

Representative viruses of the RI-APC group were observed with the electron microscope in thin sections of infected HeLa cells. The viral particles varied in density, were approximately 60 mµ in diameter and had a center to center spacing when close packed of about 65 mµ. Many of the less dense particles exhibited an internal body averaging 24 mµ in diameter. It was suggested that within the nucleus the virus differentiated from dense granular and reticular material and formed crystals. Disintegration of the crystals and disruption of the nuclear membrane with release of virus into the cytoplasm appeared to occur at any stage. No evidence to suggest development of the virus in the cytoplasm was obtained. It was possible to deduce the structure of the viral crystal from the electron micrographs. The viral particles are packed in a cubic body—centered lattice. Correlative histochemical observations in the light microscope which are now in progress revealed that the crystals and non-crystalline aggregates of virus were strongly Feulgen-positive

STRUCTURE AND DEVELOPMENT OF VIRUSES OBSERVED IN THE ELECTRON MICROSCOPE : II. VACCINIA AND FOWL POX VIRUSES

Vaccinia and fowl pox viruses were visualized by the electron microscope in sections of infected chorioallantoic membrane of chicken embryos. The viruses were of similar structure and size, averaging 200 x 300 mµ with considerable individual variation. Intracytoplasmic viral particles contained a dense, nucleus-like body (nucleoid) separated from granular material (viroplasm) by a zone of lesser density. They were enclosed by a single membrane. Near the surface of the host cell and in the extracellular space the particles consisted of a central body of variable shape and density enclosed by a double membrane. The initial sites of development were confined to the cytoplasm of the host cell. Before release from the host cell the viral nucleoids appeared to enlarge and to occupy a central position within the particle, which became enclosed by a double limiting membrane. The brick-shaped forms found after removal of the embedding plastic from thick sections indicated that drying caused characteristic distortion of certain viral particles