Electron microscopy of herpes simplex virus. IV. Studies with ferritin-conjugated antibodies.

Small aggregates of viral antigen were encountered in the nuclear matrix. The capsids did not tag with antibodies specific for the virus or for the host cell. This observation remains unexplained. Nuclear and cytoplasmic membranes, as well as the envelope of the virus, reacted with both types of antibodies and appear, therefore, to contain host cell and viral protein. Large amounts of viral antigen are synthesized within the cytoplasm. This antigen was either diffusely spread or localized at the surface of membranes. The surface of infected cells contains viral antigen, which accumulates as infection progresses. At circumscribed sites, the cell wall becomes altered antigenically and structurally so as to resemble the envelope of the virus. Hypotheses are presented regarding the manner in which cell fusion occurs.

Electron microscopy of herpes simplex virus. 3. Effect of hydroxyurea.

The effect of hydroxyurea on the development of herpes virus is mediated through its inhibitory action on deoxyribonucleic acid (DNA) synthesis. Concentrations of the drug that suppress the production of infectious virus cause typical developmental anomalies: failure in formation of the normally dense cores or "complete" viral particles, and either faulty or no envelopment of viral capsids by membranes. The synthesis of viral capsids and virus-stimulated nuclear and cytoplasmic membranes, however, is not interrupted. Combining these results with those of time sequence experiments, the following hypotheses can be presented regarding viral development. Protein synthesis, which is characterized by capsids enclosing cores of low density, precedes DNA synthesis, which is characterized by the appearance of dense cores. Capsids with dense cores are selectively transported to the cytoplasm. Envelopment generally takes place as capsids pass from the nucleus to the cytoplasm. The process of envelopment is also selective, with the result that the majority of particles that have an envelope contain a full quota of DNA.

Structure and development of viruses as observed in the electron microscope. 8. Entry of influenza virus.

It is suggested that, after attachment of influenza virus to the cell, the viral coat disintegrates. With fusion of virus to the cytoplasmic membrane, the latter undergoes dissolution. Rupture of the viral core permits release of nucleoprotein directly into the cytoplasm. Stages in the process are illustrated.

Electron microscopy of herpes simplex virus. I. Entry.

Although capsids of herpes simplex virus were encountered within phagocytic vesicles, they were more commonly observed free within the cytoplasm. Stages in the release of virus from vesicles were not seen. There appeared to be five distinct steps in the process whereby the virus initiates infection: attachment, digestion of the viral envelope, digestion of the cell wall, passage of the capsid directly into the cytoplasm, and digestion of the capsid with release of the core. Antibody probably interferes with the first two stages.

Electron microscopy of herpes simplex virus. II. Sequence of development.

Examination of infected cells at sequential intervals after infection revealed that the first viral forms to appear were capsids enclosing cores of low density. Not until the 6th hr were dense cores encountered, and at approximately the same time enveloped virus was seen. Envelopment occurred most frequently in close proximity to the nuclear surface, although the process was also encountered within the nuclear matrix and in the cytoplasm. There was often extensive proliferation of the nuclear membrane. Envelopment of the virus by budding from the cell surface was not observed. It was concluded that enveloped virus consitutes the infectious particle and that the unenveloped capsid is unstable outside the cell. Nevertheless, it is likely that capsids enclosing infectious nucleic acid can pass directly from one cell to another after fusion has taken place.

Immunofluorescence and cytochemical studies of visna virus in cell culture.

Sequential morphological changes occurring in sheep choroid plexus cells infected with visna virus were studied by direct immunofluorescence, acridine orange, and hematoxylin and eosin staining methods. Specific immunofluorescence was first detected in the perinuclear cytoplasm of solitary cells 24 hr after infection. As the infection progressed, viral antigen appeared in an increasing number of cells, and rounded globular cells with long slender processes harboring intense fluorescence were seen. Nuclear fluorescence was not observed in infected monolayers. Polykaryocytes formed within 6 hr after inoculation due to the direct cell-fusing effect of the virus inoculum did not show specific fluorescence. Viral antigen was found, however, in the cytoplasm of multinucleated giant cells in cover slips harvested after new infective virus had been released, and later in the course of infection circular fluorescent inclusions were seen in the cytoplasm of polykaryocytes. Comparable eosinophilic inclusions were observed in hematoxylin and eosin preparations, and acridine orange staining of infected monolayers demonstrated similar inclusions which fluoresced with the color characteristic of single-stranded nucleic acid and were susceptible to digestion with ribonuclease. Visna virus appears to be a ribonucleic acid virus which replicates in the cytoplasm.

Electron microscopy of chloramphenicol-treated Escherichia coli.

Thin sections of Escherichia coli were examined by electron microscopy at sequential intervals after addition and then removal of chloramphenicol. The first changes, occurring at 1 hr after exposure to the drug, were disappearance of the ribosomes and aggregation of the nuclear material toward the center of the bacteria. At 2 hr, aggregates of abnormal cytoplasmic granules first appeared and subsequently increased in size. By 23 hr, amorphous, electron-dense material had accumulated within, and at the periphery of, the nuclear matrix. With the removal of chloramphenicol, the bacteria became normal in appearance, passing through a series of stages that were sequential but not synchronous. At 145 min after removal of chloramphenicol, bacteria were encountered in the process of abnormal division. The influence of deoxyribonucleic acid and ribonucleic acid synthesis, and of energy metabolism, upon the changes seen electron microscopically in chloramphenicol-treated cells, was investigated by selectively inhibiting these functions with hydroxyurea, azauracil, and sodium azide, respectively.

Electron microscopic study of the development of simian virus 40 by use of ferritin-labeled antibodies.

An electron microscopic study of simian virus 40 has revealed a number of structural changes that are related to the development of the virus. The presence of viral antigens in both the nucleus and the cytoplasm has been demonstrated by means of ferritin-labeled antibodies. Although cytoplasmic virions are readily tagged, the lack of tagging of nuclear particles presents a perplexing problem. Presumably, the virus, after release from the nucleus, acquires a new antigenic reactivity in the cytoplasm.

Morphogenesis of type 2 parainfluenza virus examined by light and electron microscopy.

The development of type 2 parainfluenza virus in HeLa and stable human amnion cells was examined by use of antisera labeled with fluorescein and ferritin. Serum containing antibody predominantly to soluble viral antigen gave specific fluorescence which was first detectable in small cytoplasmic foci 8 to 10 hr after initiation of infection. By 20 to 24 hr, when the production of infective virus and hemagglutinin was maximal, large perinuclear aggregates of fluorescence were observed which corresponded in distribution and time of appearance to the eosinophilic inclusions seen in similar preparations stained with azure eosin. The inclusions, examined by electron microscopy, were composed of fibrils, presumably viral ribonucleoprotein, which specifically bound the antibody labeled with ferritin. With antiserum to concentrated virus, on the other hand, specific fluorescence was most marked at the surface of infected cells. Foci of fluorescence at the surface represented segments of membrane which had become differentiated morphologically and antigenically to resemble the viral envelope. These were the sites where mature virions appeared. The latter exhibited marked pleomorphism; in some instances, particles were formed which lacked recognizable internal fibrils but which possessed an enclosing membrane bearing viral antigen. Filamentous forms showing an organized internal structure were also observed at the cell surface, but were never encountered in negatively stained preparations. No clear relationship between these filaments and the spherical or oval forms could be established. In negatively stained preparations, nucleocapsid released by rupture of viral particles was similar in appearance to that reported for other paramyxoviruses. It seems probable that this component has a helical configuration.