Biological and morphological aspects of the growth of equine abortion virus

Darlington, R. W. (St. Jude Children's Research Hospital, Memphis, Tenn.), and C. James. Biological and morphological aspects of the growth of equine abortion virus. J. Bacteriol. 92:250-257. 1966.-The growth of equine abortion virus (EAV) was studied by bioassay and electron microscopy in L-cell monolayer and suspension cultures, and in HeLa and BHK 21/13 cell monolayers. Results of virus assay (plaque-forming units) indicated that production of cell-associated virus (CAV) began at 6 to 9 hr after infection in all of the cell strains used. Virus release occurred 1 to 2 hr later. By 15 to 20 hr after infection, the amount of released virus (RV) equaled or surpassed that of CAV in all cells other than the HeLa cells, where the amount of RV did not equal CAV until 48 hr after infection. Electron microscopy of infected cells revealed no differences in the morphology of virus development in any of the cells used. Developing virus particles were first detected in cell nuclei at 9 hr after infection. At 12 hr, virus particles could be seen budding from the inner nuclear envelope. Budding into cytoplasmic vacuoles was not seen. Budding virus, virus in cytoplasmic vacuoles, and extracellular virus were all approximately 145 mmu in diameter, and were indistinguishable morphologically. These results indicated that EAV is quite similar to herpes simplex virus with respect to growth and morphology, and that the inner nuclear membrane is the principal site of virus envelopment.     

Morphogenesis of aura virus

Aura virus, a member of the Western equine-encephalitis-Whataroa subgroup of group A arboviruses, was studied by electron microscopy in suckling mouse brain and chick embryo cultured cells. Virus precursors, budding particles, and complete virus particles were first detected 10 hr after infection in chick embryo cells and 24 hr after inoculation in mouse brain. Virus precursors were generally seen aligned along cytomembranes, and were less frequently seen closely associated with viroplasm-like foci, tubular aggregates, or scattered in the cytoplasmic matrix without an apparent connection to any other structure. The assembly of mature virus was observed to take place by a budding process of the virus precursor from the plasma membrane into the extracellular space, and from the cytoplasmic membranes into the lumina of vacuoles and cisternae. It was demonstrated that the endoplasmic reticulum participates in the assembly of intracellular virions. Indirect evidence was found to indicate that the Golgi complex may also form mature virus. Aura virions had a size, shape, and structure similar to those of the previously described group A arboviruses.     

Membrane traffic after inhibition of endocytosis in renal proximal tubules

This study was performed to examine quantitatively the cellular organelles involved in membrane recycling after inhibition of luminal endocytosis in renal proximal tubules. Paraffin oil was microinfused into rat renal proximal convoluted tubules to prevent luminal endocytosis. After 1-2 hr the kidneys were fixed by perfusion and prepared for electron microscopy. Segment 1 proximal tubules infused with paraffin oil and control tubules from the same kidney were studied. In addition we examined proximal tubules from kidneys fixed by immersion 30 sec after removal of the kidney. In the oil-infused tubules the large endocytic vacuoles (greater than 0.5 micron) disappeared, the amount of small endocytic vacuoles (less than 0.5 micron) was reduced to about 10%, and the amount of dense apical tubules was significantly increased. The dense apical tubules were very seldom seen connected to the apical plasma membrane in controls but this was occasionally observed in tubules fixed by immersion and relatively often in oil-infused tubules. An ultrastructural morphometric analysis substantiated and extended the qualitative observations and provided quantitative estimates of volumes and surface areas for large endocytic vacuoles, lysosomes, mitochondria, small endocytic vacuoles, and dense apical tubules in control and experimental tubules. The results strongly support the suggestion that the dense apical tubules located in the apical cytoplasm represent the vehicle for the recycling of membrane from endocytic vacuoles back to the plasma membrane, and show that in renal proximal tubule cells small and large endocytic vacuoles are transformed into dense apical tubules when endocytosis is stopped.     

Viruses of Entamoeba histolytica II. Morphogenesis of the Polyhedral Particle (ABRM2→HK-9)→HB-301 and the Filamentous Agent (ABRM)2→HK-9

The intracellular development of two morphologically different amoebal viruses has been studied by electron microscopy. One is a polyhedral agent which was observed as early as 24 hr after infection in the perinuclear cytoplasm. Subsequently, cell lysis occurred and particles were found in large number bound to membranes of disrupted amoebae. Other particles were found in phagocytic vacuoles suggesting a possible portal of entry into amoebae. The other virus is a filamentous particle which is first seen in small clusters in the nucleus after 24 hr of infection. The number of particles increases such that by 72 hr massive whorls of particles occupy a substantial part of the nucleus. After rupture of the nuclear membrane, clusters of filaments are widely dispersed throughout the cytoplasm. Still later, the cytoplasmic membrane disintegrates and clusters of filaments are found extracellularly, but free of cell membranes. The morphology of these agents is discussed in comparison with a variety of plant, animal, and bacterial viruses.     

Inhibition of arbovirus assembly by cycloheximide

Addition of cycloheximide (100 μg/ml) to cultures of chick cells infected with Semliki Forest virus (SFV) halted subsequent increase in virus titers. When added after 4 hr of infection, the drug had no effect on the rate of viral ribonucleic acid (RNA) synthesis, although marked inhibition of protein synthesis was seen. All of the previously identified forms of SFV RNA were seen in the drug-treated cells at higher concentrations than were present in untreated controls. The latter observation appeared to result from a failure to form viral “cores” or nucleocapsids in the cycloheximide-treated cells, resulting in sequestration of viral RNA intracellularly. The failure to form new virus cores was correlated with the failure of type II cytopathic vacuoles to appear in thin sections. Virus budding from the cell surface and the formation of type I cytopathic vacuoles persisted in cycloheximide-treated cells. The cellular pool of the major protein present in the virus core appeared to be small. None of this protein was found in a free pool in cytoplasm. The results indicated that, in the presence of cycloheximide, virus assembly was impaired because of the small size of the cellular pool of the major protein required for virus core formation.     

Structure and Development of Rabies Virus in Tissue Culture

Structure and development of two fixed rabies virus strains in baby hamster kidney cells (BHK/21) were investigated by electron microscopy. The morphological development was correlated with fluorescent-antibody staining and infectivity titration. The uptake of virus was enhanced by addition of diethylaminoethyl dextran, and structural changes became apparent in the cytoplasm 8 to 9 hr after infection, when fluorescent-antibody staining was first discernible. These changes consisted of matrices containing fibers replacing normal cytoplasmic structures. Virus particles appeared at the edges of these matrices and inside them at 24 to 48 hr. This corresponded to significant rises in intracellular infectious virus. Formation of virus particles by budding from cell membranes was seen at 72 hr. Further incubation of the infected cells resulted in synthesis of bizarre structural elements. The complete virus particle was bullet-shaped with an average size of 180 by 75 mmu. It consisted of an inner core of filamentous material surrounded by two membranes of different densities. The surface showed a honeycomb arrangement with surface protrusions 60 to 70 A long having a knoblike structure at their distal end. These surface protrusions were absent at the flat end of the virus particle.     

Penetration of Semliki Forest virus from acidic prelysosomal vacuoles

To identify and characterize the intracellular site from which the penetration of Semliki Forest virus (SFV) to the cytosolic compartment of the host cell occurs, we determined the time course and temperature dependence of nucleocapsid uncoating and infection in BHK-21 cells. At 37 degrees C the genome release to the cytosol was detected within 5-7 min after virus endocytosis, whereas delivery of the virus particles to secondary lysosomes occurred within 15-20 min. At temperatures of 15 degrees -20 degrees C virus particles were internalized by endocytosis, but they were not delivered to the secondary lysosomes. Nevertheless, at 20 degrees C nucleocapsid uncoating and infection occurred, indicating that secondary lysosomes are not required for SFV penetration. We conclude that the penetration reaction occurs in prelysosomal endocytic vacuoles (endosomes). As SFV penetration by membrane fusion requires a pH less than 6 and the presence of cholesterol in the target membrane, the data indicate that endosomes are acidic and contain cholesterol.     

On the entry of semliki forest virus into BHK-21 cells

The pathway by which semliki forest virus (SFV), a membrane-containing animal virus, enters BHK-21 cells was studied morphologically and biochemically. After attaching to the cell surface, the majority of viruses was rapidly trapped into coated pits, internalized by endocytosis in coated vesicles, and sequestered into intracellular vacuoles and lysosomes. Direct penetration of viruses through the plasma membrane was never observed. To assess the possible involvement of lysosomes in the release of the genome into the cytoplasm, the effect of five lysosomotropic agents, known to increase the lysosomal pH, was tested. All of these agents inhibited SFV infectivity and one, chloroquine (the agent studied in most detail), inhibited a very early step in the infection but had no effect on binding, endocytosis, or intracellular distribution of SFV. Thus, the inhibitory effect was concluded to be either on penetration of the nucelocapsid into the cytoplasm or on uncoating of the viral RNA. Possible mechanisms for the penetration of the genome into the cytoplasm were studied in vitro, using phospholipids-cholesterol liposomes and isolated SFV. When the pH was 6.0 or lower, efficient fusion of the viral membranes and the liposomal membranes occurred, resulting in the transfer of the nucleocapsid into the liposomes. Infection of cells could also be induced by brief low pH treatment of cells with bound SFV under conditions where the normal infection route was blocked. The results suggest that the penetration of the viral genome into the cytosol takes place intracellularly through fusion between the limiting membrane of intracellular vacuoles and the membrane of viruses contained within them. The low pH required for fusion together with the inhibitory effect of lysosomotropic agents implicate lysosomes, or other intracellular vacuoles with sufficiently low pH, as the main sites of penetration.     

Invaginated apical vacuoles in the cells of the proximal convoluted tubule in the rat kidney

Summary Following perfusion fixation of the rat kidney with glutaraldehyde the proximal tubule cells display small apical vacuoles, large apical vacuoles, and apical vacuoles in which a part of the limiting membrane is invaginated into the vacuole. These invaginated apical vacuoles occur more frequently in proximal convoluted tubules than in proximal straight tubules. One tubular cell may contain apical vacuoles of different sizes and stages of invagination, ranging from larger vacuoles with a wide lumen and a small area of invaginated membrane to smaller elements with no apparent lumen and a large area of invaginated membrane. Invaginated apical vacuoles lie either singly in the cytoplasm or close to the membranes of other apical vacuoles, but never in contact with the cell membrane or the membranes of lysosomes, endoplasmic reticulum, Golgi apparatus, mitochondria and peroxisomes.These findings suggest that the invaginated apical vacuoles are not fixation artifacts, but rather develop in living state in cells of the proximal tubule from spherical endocytotic elements.Supported by the Deutsche Forschungsgemeinschaft (SFB 105)     

Biogenesis of type I cytopathic vacuoles in Semliki Forest virus-infected BHK cells.

We investigated the biogenesis of type I cytopathic vacuoles (CPVIs) in Semliki Forest virus (SFV)-infected cells by immunofluorescence and electron microscopy. By using the ts1 mutant of SFV at the restrictive temperature to avoid superinfection, we showed that the multiplicity of infection affects the time of appearance but not the number of CPVIs in a cell. Formation of CPVIs did not require incoming virus particles, because they were found in BHK cells transfected with infectious RNA from the SFV prototype strain or ts1 mutant. When the SFV gene for nsP3 was expressed alone in BHK cells, the nsP3 protein was localized to numerous vesiclelike structures and large vacuoles. The nsP3 protein may function as an anchoring protein for the RNA replication complex of SFV.     

Preformed cytoplasmic nucleocapsids are not necessary for alphavirus budding.

According to the present model for assembly of alphaviruses, e.g. Semliki Forest virus (SFV), the viral genome is first encapsidated into a nucleocapsid (NC) in cytoplasm and this is then used for budding at plasma membrane (PM). The preformed NC is thought to act as a template on which the viral envelope can be organized. In the present work we have characterized two SFV deletion mutants which did not assemble NCs in the cytoplasm but which instead appeared to form NCs at the PM simultaneously with virus budding. The deletions were introduced in a conserved 14 residue long linker peptide that joins the amino-terminal RNA-binding domain with the carboxy-terminal serine-protease domain of the capsid protein. Despite the deletions and the change in morphogenesis, wild-type (wt)-like particles were produced with almost wt efficiency. It is suggested that the NC assembly defect of the mutants is rescued through spike-capsid interactions at PM. The results show that the preassembly of NCs in the cytoplasm is not a prerequisite for alphavirus budding. The apparent similarities of the morphogenesis pathways of wt and mutant SFV with those of type D and type C retroviruses are discussed.     

Electron Microscopic Study of the Morphogenesis of Vesicular Stomatitis Virus

Except for the rate, vesicular stomatitis virus (VSV) grows as well at 25 C as at 37 C in primary chick embryo fibroblast cells and in a pig kidney cell line [PK(H13)]. Maximal yields were reached at about 28 hr at 25 C and 10 hr at 37 C in these cells. Morphogenesis, as observed by electron microscopy, was similar at the two temperatures. The main feature was accumulation of virus in intracytoplasmic vacuoles. Mode of release of VSV has been controversial; both budding (as displayed by myxoviruses) and maturation at membranes of cytoplasmic vacuoles (as with arboviruses) have been claimed. Our observations support the latter view, and the apparent dichotomy in interpretation is discussed.     

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。     

Helical structure in the apical tubules of several absorbing epithelia

Summary Unique and highly ordered structures were discovered in the so-called apical tubules of several absorbing epithelia (kidney proximal tubule, visceral yolk sac and ductuli efferentes) fixed in situ with a mixture of formaldehyde, glutaraldehyde and osmium tetroxide. The apical tubules were especially numerous in the apical cytoplasm, in addition to the invaginations of the apical plasma membrane, newly formed endocytic vesicles and large endocytic vacuoles. They showed a cylindrical structure (80 nm in diameter) limited by a smooth membrane. Helically wound parallel rows of particles (11 nm in diameter) were found in the apical tubules in close proximity to their limiting membrane. The structure of the helix was determined by following the rows through serial sections and semithin sections, and was found to be a left-handed quadruple helix. These particles surround an electron-lucent cylinder (35 nm in diameter), containing at its center a single row of particles (9 nm in diameter). The apical tubules with the luminal specializations were not seen in continuity with the apical plasma membrane, but were frequently connected with the large endocytic vacuoles, which were present in the deeper levels of the apical cytoplasm. From these observations, it is suggested that the apical tubules are not derivatives of the apical plasma membrane; rather, they represent an intracellular compartment, which is morphologically related to the large endocytic vacuoles.     

Morphogenesis of Bittner Virus

The morphogenesis of Bittner virus (mouse mammary tumor virus) was studied in sectioned mammary tumor cells. Internal components of the virus (type A particles) were seen being assembled in virus factories close to the nucleus and were also seen forming at the plasma membrane. The particles in virus factories became enveloped by budding through the membrane of cytoplasmic vacuoles which were derived from dilated endoplasmic reticulum. Complete virus particles were liberated from these vacuoles by cell lysis. Particles budding at the plasma membrane were released into intercellular spaces. Maturation of enveloped virus occurred after release, but mature internal components were rarely seen in the cytoplasm before envelopment. Direct cell-to-cell transfer of virus by pinocytosis of budding particles by an adjacent cell was observed, and unusual forms of budding virus which participated in this process are illustrated and described. There was evidence that some virus particles contained cytoplasmic constituents, including ribosomes. Certain features of the structure of internal components are discussed in relation to a recently proposed model for the internal component of the mouse leukemia virus. Intracisternal virus-like particles were occasionally seen in tumor cells, but there was no evidence that these structures were developmentally related to Bittner virus.     

Basis for Variable Response of Arboviruses to Guanidine Treatment

The effect of guanidine on the replication of the group A arboviruses, Sindbis virus, and Semliki Forest virus (SFV) was studied. Guanidine rapidly, but reversibly, inhibited SFV ribonucleic acid (RNA) synthesis. The synthesis of all species of viral RNA was inhibited, but that of ribonuclease-resistant forms was least affected. This inhibition occurred when the drug was added at any point during the log phase of virus growth. The growth of SFV was also markedly inhibited, but Sindbis virus growth was unimpaired. Infection of guanidine-treated cells with the viruses together resulted in a significant inhibition of the yields of both. It appears that, in the case of Sindbis virus, viral RNA is ordinarily produced in such excess that inhibition of its synthesis does not reduce virus yields. In the case of SFV, guanidine also markedly distorts the pattern of RNA synthesis by greatly decreasing the production of the 26S interjacent RNA form. This may account for the observed inhibition of SFV growth in the presence of guanidine.     

OBSERVATIONS ON THE MODE OF RELEASE OF HERPES VIRUS FROM INFECTED HELA CELLS

The development of a well adapted strain of herpes virus has been studied in HeLa cells using thin sectioning techniques for electron microscopy. Particular attention was directed to events in the cytoplasm and certain new features were observed. Profuse immature particles with a nucleoid and single limiting membrane were present in the nuclei of infected cells, often in crystalline array; morphologically indistinguishable immature particles were also found very frequently in the cytoplasm. Cells with such particles were intact and well preserved, and contained smooth vacuoles apparently derived from the Golgi component of the endoplasmic reticulum. The cytoplasmic particles escaped from the cells by bulging out as buds through the cell membrane or through that of the cytoplasmic vacuoles until they were attached only by a pedicle and then became free. During this process the particles were gradually enclosed by the membrane through which they passed and carried a coat of it with them as they matured. After permanganate fixation the triple-layered structure of the cell membrane and vacuolar membranes was evident and was identical with that of the outer coat of the mature virus. These findings are discussed both in relation to different types of virus structure and to function in the endoplasmic reticulum and cell membrane.     

Membrane fusion mutants of Semliki Forest virus

Previous reports have indicated that the entry of Semliki Forest virus (SFV) into cells depends on a membrane fusion reaction catalyzed by the viral spike glycoproteins and triggered by the low pH prevailing in the endosomal compartment. In this study the in vitro pH-dependent fusion of SFV with nuclease-filled liposomes has been used to select for a new class of virus mutants that have a pH-conditional defect. The mutants obtained had a threshold for fusion of pH 5.5 as compared with the wild- type threshold of 6.2, when assayed by polykaryon formation, fusion with liposomes, or fusion at the plasma membrane. They were fully capable of infecting cells under standard infection conditions but were more sensitive to lysosomotropic agents that increase the pH in acidic vacuoles of the endocytic pathway. The mutants were, moreover, able to penetrate and infect baby hamster kidney-21 cells at 20 degrees C, indicating that the endosomes have a pH below 5.5. The results confirm the involvement of pH-triggered fusion in SFV entry, emphasize the central role played by acidic endosomal vacuoles in this reaction, shed further light on the mechanism of SFV inhibition by lysosomotropic weak bases, and demonstrate the usefulness of mutant viruses as biological pH probes of the endocytic pathway.     

AN ELECTRON MICROSCOPE STUDY OF THE DEVELOPMENT OF A MOUSE HEPATITIS VIRUS IN TISSUE CULTURE CELLS

Samples taken at different intervals of time from suspension cultures of the NCTC 1469 line of mouse liver—derived (ML) cells infected with a mouse hepatitis virus have been studied with the electron microscope. The experiments revealed that the viruses are incorporated into the cells by viropexis within 1 hour after being added to the culture. An increasing number of particles are found later inside dense cytoplasmic corpuscles similar to lysosomes. In the cytoplasm of the cells from the samples taken 7 hours after inoculation, two organized structures generally associated and never seen in the controls are observed: one consists of dense material arranged in a reticular disposition (reticular inclusion); the other is formed by small tubules organized in a complex pattern (tubular body). No evidence has been found concerning their origin. Their significance is discussed. With the progression of the infection a system of membrane-bounded tubules and cisternae is differentiated in the cytoplasm of the ML cells. In the lumen of these tubules or cisternae, which are occupied by a dense material, numerous virus particles are observed. The virus particles which originate in association with the limiting membranes of tubules and cisternae are released into their lumen by a "budding" process. The virus particles are 75 mµ in diameter and possess a nucleoid constituted of dense particles or rods limiting an electron transparent core. The virus limiting membrane is sometimes covered by an outer layer of a dense material. In the cells from the samples taken 14 to 20 hours after inoculation, larger zones of the cell cytoplasm are occupied by inclusion bodies formed by channels or cisternae with their lumens containing numerous virus particles. In the samples taken 20 hours or more after the inoculation numerous cells show evident signs of degeneration.     

A possible iridovirus in erythrocytes of Bufo marinus in Costa Rica

Icosahedral viral particles were found in the cytoplasm of erythrocytes and splenic reticular cells of a marine toad (Bufo marinus) collected from Costa Rica. Capsids had a maximum diameter of 312 nm and a spherical core with biphasic electron density. Viruses in erythrocytes were associated with cytoplasmic assembly areas and vacuoles in cytoplasm. Nuclei had finely granular material of decreased electron density located centrally, but contained no viral particles. A group of unenveloped viral particles was seen extracellularly in a splenic vessel. The virus was consistent with an iridovirus. In a blood smear stained with Giemsa round basophilic bodies with average diameters of 1.70 microns and morphologically similar to Pirhemocyton sp. were seen in the cytoplasm of erythrocytes and occasionally in the cytoplasm of monocytes or extracellularly. Erythrocytes containing these bodies had vacuoles and irregular pale-staining areas in the cytoplasm and pale-staining areas in the nucleus. These changes corresponded to the viral particles, assembly areas, vacuoles and nuclear changes at the ultrastructural level.