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.     

Intracellular localization of human immunodeficiency virus type 1 Gag and GagPol products and virus particle release: relationship with the Gag-to-GagPol ratio

Human immunodeficiency virus (HIV) Gag precursor protein is cleaved by viral protease (PR) within GagPol precursor protein to produce the mature matrix (MA), capsid, nucleocapsid, and p6 domains. This processing is termed maturation and required for HIV infectivity. In order to understand the intracellular sites and mechanisms of HIV maturation, HIV molecular clones in which Gag and GagPol were tagged with FLAG and hemagglutinin epitope sequences at the C-termini, respectively were made. When coexpressed, both Gag and GagPol were incorporated into virus particles. Temporal analysis by confocal microscopy showed that Gag and GagPol were relocated from the cytoplasm to the plasma membrane. Mature cleaved MA was observed only at sites on the plasma membrane where both Gag and GagPol had accumulated, indicating that Gag processing occurs during Gag/GagPol assembly at the plasma membrane, but not during membrane trafficking. Fluorescence resonance energy transfer imaging suggested that these were the primary sites of GagPol dimerization. In contrast, with overexpression of GagPol alone an absence of particle release was observed, and this was associated with diffuse distribution of mature cleaved MA throughout the cytoplasm. Alteration of the Gag-to-GagPol ratio similarly impaired virus particle release with aberrant distributions of mature MA in the cytoplasm. However, when PR was inactive, it seemed that the Gag-to-GagPol ratio was not critical for virus particle release but virus particles encasing unusually large numbers of GagPol molecules were produced, these particles displaying aberrant virion morphology. Taken together, it was concluded that the Gag-to-GagPol ratio has significant impacts on either intracellular distributions of mature cleaved MA or the morphology of virus particles produced.     

Light and electron microscopy of virus inclusions inAmaranthus lividus cells

Summary Amaranthus plants infected with a virus of rod-shaped particles showed under the light microscope intracytoplasmic amorphous and crystalline inclusions.The submicroscopic organization of mesophyll cells from infectedAmaranthus leaves by electron microscopy is described. Besides big crystalline inclusions, long dark inclusions correspondent to needle-like inclusions observed by light microscopy are definable in the cytoplasm. The amorphous inclusion bodies were formed by an overgrown protrusion of vacuolate cytoplasm containing virus particles, long very dark stained inclusions forming dense bands and rings, normal elements of the cytoplasm such as mitochondria, endoplasmic reticulum and ribosomes, and some spherosomes. Inclusions and virus particles were not found in chloroplasts, mitochondria or nuclei of infected cells.     

芝麻花叶病的病毒病原鉴定

从患芝麻花叶病的病叶中提纯了一种线条状病毒颗粒,长700～800nm,宽13nm。经汁液摩擦接种可感染心叶烟、大豆,甜菜等9种植物,不感染西瓜,苋色藜、豇豆等。主要传毒介体是发生在芝麻田的桃蚜。病土、病种均不传病。该病毒与西瓜花叶病毒、芜菁花叶病毒及马铃薯Y病毒的抗血清无反应。超薄切片中可见到风轮形和纸卷形的圆柱状内含体以及结晶状内含体。结晶状内含体分布在细胞质和叶绿体中,其它内含体均见于细胞质中。同时,细胞质中还可见到大量聚集的线状病毒颗粒。初步认为此病毒可能是马铃薯Y病毒群中的一个新成员,暂称为芝麻花叶病毒。国内外均未见报道。     

Growth and Intracellular Development of a New Respiratory Virus

The multiplication of a new, ether-sensitive, ribonucleic acid virus, 229E, isolated from the human respiratory tract, has been studied in cultures of WI-38 human diploid cells. In thin sections of these cells examined with the electron microscope, particles appeared in vesicles in the cytoplasm of cells at a time corresponding to the initial increase in infectious virus. Antigen was also detected in the cytoplasm of cells by the immunofluorescent technique. Extracellular particles of similar morphology were prominent soon after. These events preceded a detectable cytopathic effect. Later, an electron-dense particle appeared within vacuoles in the cytoplasm but was never found extracellularly. Its role in virus development is not known. Complement-fixing antigen developed along with the increase in infectious virus.     

Cytoplasmic trafficking of the canine parvovirus capsid and its role in infection and nuclear transport

To begin a successful infection, viruses must first cross the host cell plasma membrane, either by direct fusion with the membrane or by receptor-mediated endocytosis. After release into the cytoplasm those viruses that replicate in the nucleus must target their genome to that location. We examined the role of cytoplasmic transport of the canine parvovirus (CPV) capsid in productive infection by microinjecting two antibodies that recognize the intact CPV capsid into the cytoplasm of cells and also by using intracellular expression of variable domains of a neutralizing antibody fused to green fluorescence protein. The two antibodies tested and the expressed scFv all efficiently blocked virus infection, probably by binding to virus particles while they were in the cytoplasm and before entering the nucleus. The injected antibodies were able to block most infections even when injected 8 h after virus inoculation. In control studies, microinjected capsid antibodies did not interfere with CPV replication when they were coinjected with an infectious plasmid clone of CPV. Cytoplasmically injected full and empty capsids were able to move through the cytosol towards the nuclear membrane in a process that could be blocked by nocodazole treatment of the cells. Nuclear transport of the capsids was slow, with significant amounts being found in the nucleus only 3 to 6 h after injection.     

RBSDV在玉米叶脉细胞内的侵染状态与灰飞虱传毒活力的关系

根据水稻黑条矮缩病毒(RBSDV)侵染玉米(Zea mays L.)的症状发展过程先后取叶脉做超薄切片,在透射电镜下观察病毒在细胞内的侵染状态,并存取样前用灰飞虱无毒若虫进行饲毒和传毒试验。结果显示RBSDV侵入玉米叶细胞后先出现在细咆壁附近,个别粒子似与胞间连丝相连;细胞质内产生病毒基质,病毒粒子先增殖并分布其周边,后向病毒基质内扩展;当病毒粒子布满病毒基质后在细胞质中出现直径约90nm的管状结构,病毒成串排列在该管状结构中;随后管状结构逐渐消失,最终形成晶格状聚集排列。用灰飞虱无毒若虫在细胞内病毒基质出现和病毒增殖期饲毒的,到成虫时分别有2．93％和7．83％个体传毒率;在细胞内病毒成串分布于管状结构和品格状聚集排列期饲毒的,到成虫时均不能传毒。     

Cell-Cycle Dependent Appearance of Murine Leukemia-Sarcoma Virus Antigens

Normal rat kidney (NRK) cells, NRK cells infected with Rauscher murine leukemia virus, and NRK cells infected with Kirsten murine sarcoma-leukemia virus (NRK-K) were synchronized by a double thymidine block. At intervals after release from thymidine blockage, the cells were examined for the presence of viral antigens in the cytoplasm and on the cell surface by immunofluorescent microscopy by using goat anti-Rauscher murine leukemia virus and goat anti-Moloney leukemia virus (Tween-ether disrupted) sera. Detection of viral antigens in the cytoplasm was periodic during the cell cycle. Antigens were detected first during the S phase, increased during the G2 phase, and disappeared during the M and G1 phases. A similar pattern of surface immunofluorescence was observed. Infectious virus was detected in culture fluids from synchronized cells during the M phase. Surface immunofluorescence was detected in NRK-K cells with anti-Rauscher murine leukemia virus and may represent the presence of group-specific antigens on the cell surface. Control, uninfected NRK cells, which did not normally fluoresce, showed weak immunofluorescence during the S and G2 phases after synchronization. Synchronization can be used to amplify latent oncornavirus expression.     

Electron Microscopy of Cells Infected with Semliki Forest Virus Temperature-Sensitive Mutants: Correlation of Ultrastructural and Physiological Observations

Cells infected at the permissive temperature with three temperature-sensitive mutants of Semliki Forest virus were not significantly different in appearance from cells infected, at either the permissive or nonpermissive temperature, with wild-type virus. Virus particles, nucleocapsids, spherules, and tubules were seen in the cytoplasm. But replication of the mutants was inhibited in cells infected at the nonpermissive temperature. This was evidenced by the absence of virus particles and nucleocapsids (except in one case) and the absence or limited production of spherules and tubules. These observations are discussed with reference to the physiological defects of the mutants.     

ASSEMBLY AND AGGREGATION OF TOBACCO MOSAIC VIRUS IN TOMATO LEAFLETS

Cells of tomato leaflets (Lycopersicum esculentum Mill.) were studied by phase and electron microscopy at various intervals after inoculation with a common strain of tobacco mosaic virus (TMV). Forty-eight hours after inoculation, prior to the development of assayable virus, individual TMV particles, and also particle aggregates, were observed in the ground cytoplasm of mesophyll cells. The most rapid synthesis of virus occurred between 80 and 300 hours after inoculation. Cytological changes during this time were characterized by an increased number of individual particles in the cytoplasm, growth of some aggregates, distortion and vacuolation of chloroplasts, and formation of filaments in the cytoplasm which were approximately four times the size of TMV. These filaments were interpreted as possible developmental forms of the TMV particle. Vacuoles in chloroplasts commonly contained virus particles. Evidence indicated that TMV was assembled in the ground cytoplasm and, in some cases, subsequently was enveloped by distorted chloroplasts.     

不同CMV分离物侵染寄主的超微结构变化

应用电镜观察了黄瓜花叶病毒ＣＭＶ不同分离物侵染寄主的细胞超微结构变化。来自一患红（Ｓａｌｖｉａｓｐｌｅｎｄｅｎｓ）的不含卫星ＲＮＡ分离物Ｍ－２２侵染心叶烟,病毒粒子散布于细胞质,在液泡中形成大片病毒粒子结果,液泡膜边缘产生小泡结构,完整的病毒粒子穿过胞间连丝在细胞间运转,胞间连丝中央部分有扩张现象。     

Hyperproduction of adenovirus type 12 E1B gene product in monkey cells, using a simian virus 40 vector.

Simian virus 40 recombinant DNAs carrying the adenovirus type 12 E1B gene were constructed, propagated, and packaged in monkey cells. Monkey cells infected with the resulting virus stocks hyperproduced the E1B gene products in more than 80% of the cells as revealed by immunofluorescence. The products were distributed in both the nuclei and the cytoplasm, and a condensed form of fleck structure was observed in the cytoplasm. Polyacrylamide gel electrophoresis of the cell extracts and their immunoprecipitates detected the E1B-coded 19,000-molecular-weight protein but not the 50,000-molecular-weight protein. The 19,000-molecular-weight protein and the simian virus 40 VP1 protein were synthesized in nearly equal amounts.     

Entry and Release of Poliovirus as Observed by Electron Microscopy of Cultured Cells

Cells of differing culture types were inoculated with poliovirus at 37 C, sampled at intervals during the replicative cycle, and examined in thin sections by electron microscopy. The earliest samples, taken at 2 and 5 min postinoculation, showed virus particles adjacent to the exterior of the plasma membrane and others that had apparently penetrated it directly; later samples showed fewer such particles or none. Particles lying in the peripheral cytoplasm frequently appeared swollen and distorted in shape. No sign of virus entry by a pinocytotic process was found at any time. At 3 hr, and subsequently during the replication cycle, particles of progeny virus appeared in the cytoplasm. They were found free in the cytoplasmic matrix, aligned along the elements of filamentous complexes, and enclosed within vesicles. Some of the vesicles were found to be open to the extracellular space, indicating a likely mechanism of virus release.     

Analysis of Nuclear Accumulation of Influenza Nucleoprotein Antigen in the Presence of p-Fluorophenylalanine

When p-fluorophenylalanine (FPA) was added to influenza virus RI/5+-infected cells 4 hr after infection, virus-specific proteins were synthesized but infectious progeny virus was not produced. In these cells, synthesis of viral RNA was strongly inhibited and nucleoprotein (NP) antigen was found predominantly in the nucleus in contrast to untreated cells in which NP antigen was distributed throughout the whole cell. The intracellular location and migration of NP were examined by isotope labeling followed by fractionation of infected cells. In untreated cells, a large portion of the NP was present in the cytoplasm and most of it was detected in the form of ribonucleoprotein (RNP). In contrast, in FPA-treated cells little viral RNP was detectable and NP was present predominantly in the nucleus in a nonassembled, soluble form. When FPA was removed from the culture, synthesis of viral RNA was soon restored and a large amount of viral RNP appeared in the cytoplasm; this was followed by the production of infectious virus. The results of the experiments suggest that the NP synthesized in the presence of FPA is not assembled into viral RNP because of the lack of available RNA, and such NP migrates readily into the nucleus and accumulates there.     

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.     

Structure and Development of Viruses as Observed in the Electron Microscope IX. Entry of Parainfluenza I (Sendai) Virus

After attachment, the uncoating of Sendai virus, which was accompanied by dissolution of the plasma membrane and fusion of virus to cell, proceeded quickly. Nucleoprotein filaments were found at stages of transit from virus to cytoplasm.     

Origin of unenveloped capsids in the cytoplasm of cells infected with herpes simplex virus 1.

In cells infected with herpes simplex viruses the capsids acquire an envelope at the nuclear membrane and are usually found in the cytoplasm in structures bound by membranes. Infected cells also accumulate unenveloped capsids alone or juxtaposed to cytoplasmic membranes. The juxtaposed capsids have been variously interpreted as either undergoing terminal deenvelopment resulting from fusion of the envelope with the membrane of the cytoplasmic vesicles or undergoing sequential envelopment and deenvelopment as capsids transit the cytoplasm into the extracellular space. Recent reports have shown that (i) wild-type virus attaches to but does not penetrate cells expressing glycoprotein D (G. Campadelli-Fiume, M. Arsenakis, F. Farabegoli, and B. Roizman, J. Virol. 62:159-167, 1988) and that (ii) a mutation in glycoprotein D enables the mutant virus to productively infect cells expressing the wild-type glycoprotein (G. Campadelli-Fiume, S. Qi, E. Avitabile, L. Foa-Tomasi, R. Brandimarti, and B. Roizman, J. Virol. 64:6070-6079, 1990). If the unenveloped capsids in the cytoplasm result from fusion of the cytoplasmic membranes with the envelopes of viruses transiting the cytoplasm, cells infected with virus carrying the mutation in glycoprotein D should contain many more unenveloped capsids in the cytoplasm inasmuch as there would be little or no restriction in the fusion of the envelope with cytoplasmic membranes. Comparison of thin sections of baby hamster kidney cells infected with wild-type and mutant viruses indicated that this was the case. Moreover, in contrast to the wild-type parent, the mutant virus was not released efficiently from infected cells. The conclusion that the unenveloped capsids are arrested forms of deenveloped capsids is supported by the observation that the unenveloped capsids were unstable in that they exhibited partially extruded DNA.     

Virological, Immunochemical, and Cytochemical Studies of Four HeLa Cell Lines Infected with Two Strains of Influenza Virus

The production of infectious virus, hemagglutinin, and viral (V) antigens and the changes in ribonucleoprotein (RNP) and lipoprotein metabolism have been studied in four sublines of HeLa cells infected with the PR8 and a PR8 recombinant strain of influenza virus. Much greater amounts of infectious virus and much less hemagglutinin were produced by the PR8 recombinant than by PR8 virus in all four cell lines. Different amounts of infectious virus per infected cell were produced by the recombinant in the four cell lines, whereas very little infectious virus was produced by the PR8 strain in any of the HeLa cells. In all cell lines infected with both strains of virus, "soluble" (S) antigen appeared early in the nucleolus. In cells infected with PR8 recombinant, S antigen subsequently filled the nucleus and later appeared in the cytoplasm. In most cells infected with PR8 virus, nuclear S antigen did not fuse to fill the nucleus, and S antigen was not detected in the cytoplasm. V antigen was observed in the cytoplasm of cells when diffuse nuclear S antigen had formed. The earliest and most frequent change in the RNP of the infected cells was a decrease in stainable RNP spherules (nucleolini) in the nucleolus. This was followed, in a smaller proportion of cells, by the appearance of nuclear and cytoplasmic inclusions containing RNP. There was a characteristic difference in the morphology of the cytoplasmic inclusions produced by the two strains of virus, but the same types of inclusions were observed in all four HeLa lines. A significant increase in lipoprotein was observed only in association with the cytoplasmic inclusions produced by PR8 recombinant virus. There was a striking difference in the proportion of cells with cytochemical changes in RNP in the four cell lines. A significant cytopathic effect (CPE) was observed only in three virus-cell systems in which a high proportion of cells exhibited changes in nucleolinar RNP. It is suggested that disappearance of RNP in the nucleolini may be an indication of shutdown of host ribonucleic acid synthesis and that this in turn results in a CPE. Virus infection resulted in a C-mitotic block that was followed by karyorrhexis. Infection of the cell did not always result in the production of infectious virus, in changes in the RNP of the nucleolini, in the development of nuclear or cytoplasmic RNP inclusions, or in CPE. The results suggest that production of infectious virus, shutdown of cellular RNP synthesis with accompanying CPE, and the formation of inclusions appear to be independent events.