Cytopathology caused by the AC-1 isolate of Marek's disease virus in the feather follicle epidermis

The AC-1 strain of Marek's disease herpesvirus (MDV), recently isolated from an outbreak of Marek's disease in vaccinated chickens, was used for intraperitoneal inoculation of one-day-old Single Comb White Leghorns. Chicks were necropsied 12 to 32 days post inoculation and skin was collected for electron microscopic studies. This paper focuses on early stages of MDHV development in feather follicle epithelial cells and on cytoplasmic changes that occur in formation and release of virus particles. In some nuclei we observed helical threads of DNA which were partially encircled by capsid membranes. This was interpreted to be an early stage of nucleocapsid development. In the infected epidermal cells, the smooth endoplasmic reticulum vesiculated and appeared to be hyperplastic. Immature virus particles released into cytoplasm acquired their outer envelope while budding into cytoplasmic vesicles. Vesicles containing mature viruses were referred to as cytoplasmic vesicular inclusions. It appeared that the degenerative process in cells facilitated the release of virions from the feather follicle epithelium.     

Growth of an autonomously replicating parvovirus (feline panleukopenia): Kinetics and morphogenesis

Summary Feline embryo (FEmb) cell cultures, in which 90 percent of cells were dividing (cycling), were synchronized, by serum deprivation, to the degree that 88 per cent of the cells divided within a 12 hour period. When such cultures were infected with feline panleukopenia virus (FPV) at a multiplicity of infection of 5.7, a maximum level of cell associated virus was attained 28 hours post-infection (p.i.). There was a tendency for virus to remain cell associated in that cell lysis did not begin until 40 hours p.i.The genesis of FPV inclusion bodies was studied by light microscopy. Inclusions were intranuclear, weakly basophilic and Feulgen positive; they were first observed 8 hours p.i., and increased to be present in 90 percent of cells by 40 hours. Mitosis was markedly inhibited in FPV infected monolayers.The earliest changes observed by electron microscopy of infected cells were the presence of virus particles within nuclei, progressive chromatin margination, and nucleolar changes involving apparent segregation of the fibrillar and granular components. Virus particles measured 20 nm in diameter, and appeared either uniformly electron dense or possessed a dense margin and a pale center; many of the latter contained a single, central, dark spot. Virions ultimately became closely packed in all areas unoccupied by other nuclear components. In some nuclei a linear arrangement of virions was noted, but paracrystalline arrays were not seen.Other changes observed in infected nuclei included the presence of nucleolar remnants sometimes in the form of solid or hollow bodies comprised of nucleolar granules or filaments; distension of the space between the two membranes of the nuclear envelope; and the presence of aggregates of abnormal, electron dense material within the nucleus. Discontinuities of the plasma membrane and swelling of cytoplasmic organelles were commonly seen in cells showing advanced nuclear changes, but at least the inner membrane of the nuclear envelope generally remained intact. The characteristic, well defined inclusions of light microscopy were not observed by electron microscopy, and thus probably represented a preparation (shrinkage) artifact.With 11 Figures     

Ultrastructural studies of the envelopment and release of guinea pig herpes-like virus in cultured cells

The process of envelopment and release of guinea pig herpes-like virus was examined in both infected guinea pig kidney and thymus tissue culture cells by electron microscopy. The majority of the nucleocapsids were enveloped by budding into nuclear vacuoles; some were enveloped by budding from the inner nuclear membrane. Budding into cytoplasmic vacuoles was also seen. Many enveloped virus particles inside the nuclear vacuoles were pear shaped with a tail-like structure. Approximately 23% of pear-shaped virus particles were seen in the infected thymus fibroblastic cells, but only 6% were found in the infected epithelial cells. The envelopes of all nuclear enveloped virus particles appeared as smooth membranes, while the majority of particles exhibiting fuzzy and thick dense envelopes were seen in the cytoplasm or extracellular space. The average diameter of the cytoplasmic or extracellular enveloped virus particles was approximately 167 nm, and the average diameter of the nuclear enveloped virus particles was about 146 nm.Data also showed that mature nuclear virus particles were first released into perinuclear cisterna and then traveled through cytoplasmic channels to the extracellular space.     

Demonstration of hepatitis A virus in cell culture by electron microscopy with immunoperoxidase staining

Virus-like particles were demonstrated by electron microscopy in BS-C-1 cells infected with hepatitis A virus (HAV). Particles were usually enclosed within vesicles and accompanied by myelin-like membranous structures. Less often they were seen free in the cytoplasm. They were never observed in the nucleus. By immunoperoxidase staining particles were found to contain HAV antigens. These antigens were also found in the membrane of the vesicles surrounding the masses of particles and adjacent parts of the mitochondrial membranes. Our results demonstrate the usefulness of an electron microscopic immunocytochemical technique to study replication of HAV.     

Intracellular localisation of dengue-2 RNA in mosquito cell culture using electron microscopic in situ hybridisation

Summary.  Non-isotopic in situ hybridisation was used at the electron microscope level to determine the localisation of viral RNA in dengue-2 infected mosquito cells at 14, 24, 48 and 72 h post-infection. In situ hybridisation was carried out on sections of dengue-2 infected mosquito cells using a digoxigenin-labelled DNA probe to the envelope protein gene sequence of the virus. Viral RNA was consistently localised over the rough endoplasmic reticulum and the virus-induced smooth membrane structures which form within the endoplasmic reticulum. During the later stages of infection electron-dense areas were observed to develop in close proximity to the smooth membrane structures. Electron microscopic in situ hybridisation showed that these denser areas contained both viral RNA and virus particles. Our results show that in dengue-2 infected mosquito cells the smooth membrane structures are an important site for the concentration of dengue viral RNA and its possible subsequent encapsidation into virus particles. Accepted July 31, 1997 Received June 6, 1997     

Ultrastructural characterization of a new member of the Rhabdovirus group-Klamath virus

Summary Klamath virus was observed by negative contrast and thin section electron microscopy. Bullet-shaped particles in infected cell cultures had mean dimensions of 167×80 nm and were typical of members of the Rhabdovirus group. In infected mouse brain, virus particles budded from intracytoplasmic membranes in association with a prominent matrix. Although the virus is serologically unrelated to any of the known Rhabdoviruses of animals, this intracytoplasmic accumulation of virus particles and matrix with little frank cytopathology, most closely resemblesin vivo rabies virus infection.     

Ultrastructural studies of the development of feline calicivirus in a feline embryo cell line

The ultrastructural changes in a feline embryo continuous cell line infected with feline calicivirus at a multiplicity of infection of approximately 1 were studied. Virus was found only in the cytoplasm and was observed as single particles, as extensive, non-regular accumulations, as paracrystalline arrays, and as single or multiple linear arrays associated with microfbrils. Mature virus particles were readily distinguished from ribosomes in that they were larger (35nm diameter) and consisted of a central, electron-dense core 20 nm diameter surrounded by a less electron-dense coat. Other changes ovserved in infected cells included rounding of the cell and nucleus and loss of pseudopodia. There was extensive production of smooth-membrane bound vesicles in the cytoplasm. Virus accumulations of each type, but especially paracrystalline arrays, were frequently closely associated with collections of these vesicles. The cisternae of the endoplasmic reticulum and the space between the two layers of the nuclear membrane was distended. By Feulgen staining and light microscopy, as well as electron microscopy, it was established that nuclear chromatin undergoes profound changes consisting of condensation usually into a single, rounded, central mass.     

Differences in the morphology of herpes simplex virus infected cells

The two types of herpes simplex virus (HSV-1, HSV-2) induced significantly different alterations in the morphology and permeability of infected cells. HEp-2 cells infected with HSV-1 (strain THEA) were characterized by the formation of polynuclear syncytia. In contrast, after infection with HSV-2 (strain D316, DD), the cells were rounded up. The HSV-1 strains KOS and LS5039 and the HSV-2 strain 196 induced both types of cytopathic effect. As shown by comparative scanning and transmission electron microscopy newly synthesized virus particles of the various strains of HSV-1 were generally found to be restricted to smooth areas of the cell surface. In these areas the number of microvilli was reduced in comparison to uninfected cells. However, the progeny viruses of the strains of HSV-2 were mainly connected with protrusions of the cell membrane (microvilli and filopodia). The morphological changes in cells infected with either type of HSV were associated with different functional alterations of the cell membrane. The membranes of HEp-w cells became more stable after infection with HSV-1. This is characterized by a reduced permeability for 51Cr as well as by a decreased sensitivity to the detergent Triton-X-100. HSV-2 induced opposite effects on the stability of the membrane in infected cells. In contrast to these findings with HEp-2 cells, opposite results were obtained with primary chick embryo fibroblasts: Infection with HSV-1 rendered the cell membrane more permeable for 51Cr and a reduction of the 51Cr-release was achieved by infection with HSV-2. The results show that HSV-cell interactions depend on the type of the virus as well as on the type of the infected cell.     

Morphogenesis of sandfly viruses (Bunyaviridae family)

The events occurring in the morphogenesis of sandfly fever viruses have been examined by thin-section electron microscopy and by an analysis of the association of virus-specific polypeptides with membranes of infected cells. Two representative sandfly fever viruses have been studied, Karimabad virus (KV) and Punta Toro virus (PTV), which appeared indistinguishable both in terms of virion structure, as monitored by negative staining, and morphogenesis, as observed in thin sections of infected Vero cells. Ammonium molybdate negative staining of purified, glutaraldehyde-fixed virions revealed essentially spherical particles, 87 nm in diameter, in which the surface proteins were constructed into closely packed, hollow, cylindrical subunits measuring 10–11 nm in diameter and 9–10 nm in length. These surface units are located peripherally to a 7-nm membrane bilayer which surrounds a nucleoid of variable electron density. As seen in thin sections of infected cells, the assembly of these particles was first detected at 12 hr after infection, occurred exclusively at smooth membrane vesicles, and predominantly at membranes in, or adjacent to, Golgi cisternae. Morphologically mature particles were formed by continuous involution (budding) of modified membrane segments into the lumen of these vesicles. Viral ribonucleoprotein (RNP), which was not observed free in the cytoplasm, condensed at the cytoplasmic face of these vesicles at areas at which viral spike structures could be observed at the contralateral (luminal) face. Neither RNP nor spike structures could be observed on adjacent sections of the vesicular membrane, or other membranes, which were not directly involved in the assembly of a budding virion. Analysis of the viral polypeptides in unfractionated membrane vesicles prepared from infected cells demonstrated that KV-specific envelope and nucleocapsid proteins rapidly became membrane bound, whereas a nonstructural polypeptide was found only in cytoplasmic fractions. Chymotrypsin treatment of these vesicles has indicated that at least one viral glycoprotein is inserted into cellular membranes such that approximately 12% of its sequence remains at the cytoplasmic membrane face. Proteolytic removal of this sequence generated an immunoprecipitable, glycosylated fragment which was protected from further proteolysis by its orientation within the vesicle. These morphologic and biochemical data have been used to construct a model for the assembly of these viruses. Virus particles are released from infected cells by exocytosis, a process which does not appear to result in significant modification of cell surface membranes with viral antigens.     

Replication and morphogenesis of avian coronavirus in Vero cells and their inhibition by monensin

Avian infectious bronchitis virus (IBV) was adapted to Vero cells by serial passage. No significant inhibition of IBV replication was observed when infected Vero cells were treated with α-amanitin or actinomycin D. In thin sections of infected cells, assembly of IBV was observed at the rough endoplasmic reticulum (RER), and mature IBV particles were located in dilated cisternae of the RER as well as in smooth cytoplasmic vesicles. In addition to typical IBV particles, enveloped particles containing numerous ribosomes were identified at later times postinfection. Monensin, a sodium ionophore which blocks glycoprotein transport to plasma membranes at the level of the Golgi complex, was found to inhibit the formation of infectious IBV. In thin sections of infected Vero cells treated with the ionophore, IBV particles were located in dilated cytoplasmic vesicles, but fewer particles were found when compared to controls. A similar pattern of virus-specific proteins was detected in control or monensin-treated IBV-infected cells, which included two glycoproteins (170000 and 24000 daltons) and a polypeptide of 52000 daltons. These results suggesl lhal the ionophore inhibits assembly of a virus which malures at intracellular membranes.     

The cytopathology and development of a human polyoma virus (B.K.)

Summary Light and electron microscopic studies on foetal human fibroblast and glial cells infected with B. K. virus show a cytopathogenic effect similar to but not identical with that caused by polyoma and SV 40 viruses in murine and monkey cells, respectively. The first cytopathogenic effects appear at approximately 14 days and are complete at about 30 to 40 days. Light microscopy of unstained monolayers demonstrates roughening of the cell surface, liberation of exudate, cytoplasmic vacuolization and rounding of cells. Stained preparations show the same features and a series of nuclear changes consisting of light eosinophilic patches in the nucleoplasm, chromatin clumping and the development of a typical inclusion body. The nucleolus is not involved.Electron microscopy of the cells reveals virus crystals in intact nuclei followed by breakdown of the nuclear membrane, clumping of chromatin into dense masses and liberation of the viruses which often become associated with cytoplasmic membranes. Mass liberation of virus crystals occurs by disruption of the cell membrane. Liberation of virus particles from cells with intact nuclei occurs occasionally. Budding from the nucleus or cell membrane was not observed.     

Viral polypeptide composition of Japanese encephalitis virus-infected cell membranes

Chick embryo cells infected with Japanese encephalitis virus were separated into smooth and rough membrane fractions, as judged by electron microscopy. Polyacrylamide gel electrophoretic analysis of the membrane fractions showed them to contain all of the virus specific polypeptides; however, the relative proportions of the polypeptides varied among smooth and rough membranes. The relationship of these observations to current concepts of group B arbovirus morphogenesis is discussed.     

Morphology and morphogenesis of a coronavirus infecting intestinal epithelial cells of newborn calves.

The morphology and morphogenesis of virus strain LY-138 recovered from neonatal diarrheic calves were investigated by electron microscopy using negativestaining techniques and ultrathin sectioning. Purified viral particles were spherical in shape and measured 90 nm in average diameter in negatively stained preparations. Pleomorphic forms were also present. The virions had envelopes with petal-shaped projections characteristic of coronaviruses. In ultrathin sections, cores in viral factories were round with a diameter of 50–60 nm. Most of these cores were electron dense but some had an electron-lucent center. In cytoplasmic vacuoles, Golgi vesicles, and on the apical plasmalemma of intestinal epithelial cells, the virions were round or ellipsoidal in shape, measuring 70–80 nm in diameter, and had fine thread-like projections on their surfaces. Uptake of virus occurred through fusion of viral envelopes with the plasmalemma of the microvillous border or by entry into intercellular spaces and interaction with the lateral cell membranes of adjacent intestinal epithelial cells. As a result of this interaction, the lateral cell membranes became altered and ill-defined. During the early stage of infection, the rough andasmooth elements of the endoplasmic reticulum became distended with electron-dense granulofibrillar material. This material accumulated subsequently as well-defined, smooth membrane-bound areas mainly in the apical cytoplasm of infected cells. These structures were considered to be viral factories. The morphogenesis of virus occurred mainly through condensation of the electron-dense, granulo-fibrillar material into viral cores in cytoplasmic viral factories or within the distended cisternes of the rough endoplasmic reticulum. Viral envelopment occurred on membranes of cytoplasmic vacuoles, Golgi vesicles, or in association with membranes of viral factories. Release of virus from infected cells occurred by lysis and fragmentation of the apical plasmalemma and flow of the cytoplasm with its contents into the gut lumen. Release also occurred by digestion and lysis of extruded infected cells or by fusion of virus-containing cytoplasmic vacuoles with the apical plasmalemma and liberation of their contents.     

Replication of human adenoviruses in guinea pig embryo cells: an ultrastructural study

Summary Guinea pig embryo (GPE) cells showed different degrees of susceptibility to human adenovirus types as determined by virus infectivity assay and electron microscopic examination. Adenovirus 2 and 5 induced extensive cellular changes and produced high titers of infectious virus in GPE cells as in human cells. Mature progeny virus and protein crystals were observed in both cell types. Adenovirus 7 induced some cellular changes in GPE cells but only a small number of cells yielded progeny virus as determined by electron microscopy. Adenovirus 3, 8 and 31 induced some cellular changes but no progeny virus was found under electron microscopic examination. Characteristic fibers were observed in nuclei of adenovirus 31 infected cells. The ability of human adenovirus 2 and 5 to replicate in GPE cells is an example of an unusual cross-species biological property of certain adenovirus types. This property may be useful as a biological marker for these virus types.With 8 Figures     

Visceral target organs in systemic St. Louis encephalitis virus infection of hamsters

Ultrastructural aspects of St. Louis encephalitis virus infection of the major extraneutral organs and tissues of suckling hamsters were examined. In the pancreas, both the exocrine and endocrine portions were equally affected by the virus. A feature apparently unique to flaviviruses was the accumulation of virus particles in all types of secretory granules in this organ. Virus particles were seen within myocardial fibers and within the smooth muscle cells and endothelial cells of small blood vessels of the heart. In the intestines, the lamina propria was the most severely infected, with virus particles accumulated in all cell types.     

Intracellular appearance of Penicillium chrysogenum virus

An electron microscopic study of intracellular Penicillium chrysogenum virus showed virus particles with a dense core distributed as individuals or aggregates in cytoplasm and/or in vacuoles of mycelial cells. In cytoplasm the virus particles were often near, but not in, the nucleus. Electron-dense regions of granular appearance enclosed more complete virus particles in some cells. Lipid bodies, vacuoles, and membranous structures were often prominent in infected cells. Sometimes cylindrical arrays of virus particles occurred in vacuoles and also in degenerated cells. Aberrations consisting of various degrees of degeneration or disappearance of cytoplasm and cellular organelles were often observed in aged cells with large quantities of virus. The virus particles sometimes released fibrous material forming networks from their core region.     

Aspects of the developmental morphology of California encephalitis virus in cultured vertebrae and arthropod cells and in mouse brain

An electron microscopic study was made of the replication of California encephalitis (La Crosse strain) in cultured vertebrate cells (Vero, African green monkey kidney), in a line of cultured mosquito cells (Aedes albopictus), and in brain tissue of suckling mice. Morphologically similar virus particles, approximately 95 nm in diameter, were encountered in all three systems, and a common mode of virus assembly and maturation appeared to obtain. Virus assembly was shown to occur exclusively at internal cytomembrane interfaces, the Golgi complex appearing as the initial assembly site, which site became less focal as infection progressed due to the proliferation of Golgi smooth membranes and the dilation of cisternae and vesicles. The assembly process involved viral budding into cisternal and vesicular lumina, the virion thereby acquiring its limiting membrane. The envelope of such intracellular virions exhibited a poorly defined fringe, approximately 8 nm in width, which appeared to undergo a maturational change as the virions were discharged from the cell, such that, extracellularly, virions displayed a well-developed fringe, approximately 12 nm wide—a change especially noteworthy in the case of virions in infected mouse brain.The presence was noted in infected Vero cells—and in one instance in an infected mosquito cell—of crescent-shaped segments of thickened cisternal membrane, which possibly represented an early phase of viral assembly in which nucleocapsid aligned itself in close apposition to a membrane segment preparatory to the initiation of budding.In areas of the cytoplasm adjacent to sites of viral assembly in neurons, a fine granulofibrillar matrix was frequently found, enmeshed in which were numbers of 50–60 nm spherical structures. In a low proportion of cells from infected mosquito cultures, dense granulofibrillar masses were found in the cytoplasm.In Vero cells, infection with CE virus was cytolytic, while in A. albopictus cells, no gross cytopathic effects were manifest, and persistently infected cultures developed upon subcultivation. However, less than 10% of challenged mosquito cells became productively infected and for a proportion of these, at least as determined by electron microscopy, the infection was lethal.     

Replication of Theiler's murine encephalomyelitis viruses in BHK21 cells: an electron microscopic study

The replication of two different Theiler's murine encephalomyelitis viruses (GDVII and DA) in BHK21 cells was studied by electron microscopy. During the early stages of infection, the changes observed in infected cells were identical for both viruses, and typical of those described in the literature for other picornaviruses. However, fundamental differences in the intracellular development of GDVII and DA viruses were observed late in the infection. Well-developed crystalline arrays of virions were observed in the cytoplasm of GDVII virus-infected cells, and virus was readily released upon cell lysis. In contrast, DA virus did not form similar crystals. Instead, in DA virus-infected cells unique membranous structures consisting of two membrane units enclosing a layer of virions one particle deep were regularly observed in the cytoplasm at late times in the infection. In addition, DA virus did not appear to be freely released upon cell lysis. Single cell growth kinetics of GDVII and DA viruses in BHK21 cells showed a close correlation with the electron microscopic observations. While GDVII virus was released from cells DA virus remained cell associated. The proliferation of similar membranous structures in parallel with maturation of virus particles has not been described previously in electron microscopic studies of other picornaviruses.