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.     

Replication of Mayaro virus in Aedes albopictus cells: an electron microscopic study

Summary The replication of Mayaro virus inAedes albopictus cells, was studied by electron microscopy at various times post-infection. In infected cells we observed the presence of cytoplasmic vesicles containing viral nucleocapsids and mature virus particles but at no time did we detect virus budding into such vacuoles. Budding of virus through plasma membrane was rarely observed. Our results are discussed considering the possibility of the release of virus particles to the extracellular space by exocytosis.     

鸭乙型肝炎病毒在鸭胆管上皮细胞内增殖的超微结构研究

Liver specimens from Shanghai Ma ducks infected with duck hepatitis B virus (DHBV) were examined by immunohistochemical technique and electron microscopy. The results showed that DHBV and DHBV antigen were found not only in the hepatocytes but also in the biliary epithelial cells of infected ducks. Electron microscopy also revealed that incomplete spherical particles, 40-50 nm in diameter, were present in the dilated cisternae of rough endoplasmic reticulum (RER), and complete spherical virions, 55-65 nm in diameter, existed in the cytoplasmic vesicles and cytoplasm in small amounts. The results of the present study seem to confirm the possibility of infection and replication of DHBV not only in the liver cells but also in the biliary epithelial cells of ducks.     

Three-dimensional structure of Rubella virus factories

Viral factories are complex structures in the infected cell where viruses compartmentalize their life cycle. Rubella virus (RUBV) assembles factories by recruitment of rough endoplasmic reticulum (RER), mitochondria and Golgi around modified lysosomes known as cytopathic vacuoles or CPVs. These organelles contain active replication complexes that transfer replicated RNA to assembly sites in Golgi membranes. We have studied the structure of RUBV factory in three dimensions by electron tomography and freeze-fracture. CPVs contain stacked membranes, rigid sheets, small vesicles and large vacuoles. These membranes are interconnected and in communication with the endocytic pathway since they incorporate endocytosed BSA-gold. RER and CPVs are coupled through protein bridges and closely apposed membranes. Golgi vesicles attach to the CPVs but no tight contacts with mitochondria were detected. Immunogold labelling confirmed that the mitochondrial protein p32 is an abundant component around and inside CPVs where it could play important roles in factory activities.     

Detection of hepatitis A antigen in human liver.

For the first time, hepatitis A viral antigen (HAAg) was shown in liver biopsy tissue from a patient in the acute phase of hepatitis type A by light and electron microscopy, using the peroxidase-antibody technique. Under light microscopy, the staining for HAAg appeared as a fine, granular reaction product, scattered throughout the cytoplasm of hepatocytes and sinusoidal lining cells. Standard thin-section electron microscopy revealed virus-like particles, 24 to 27 nm in diameter, in cytoplasmic vesicles of hepatocytes and Kupffer cells. By immunoperoxidase electron microscopy, HAAg was detected on particles aggregated within cytoplasmic vesicles of hepatocytes, thus demonstrating that the virus-like particles (24 to 27 nm) are hepatitis A virus. The surrounding membrane of the vesicles was also positive for HAAg. The distribution patterns of HAAg in human liver were virtually identical to those described for experimentally infected marmosets. It is notable that most HAAg was detected within vesicles of liver cell cytoplasm, suggesting the possibility of vesicle-oriented morphogenesis of hepatitis A virus.     

Cytopathology of chick renal epithelial cells experimentally infected with avian infectious bronchitis virus

The renal ducto-tubular epithelial cells of chicks infected with the MA-87 strain of avian infectious bronchitis virus (IBV) were examined ultrastructurally. Infected epithelial cells containing IBV particles were more numerous in the collecting ducts, collecting tubules, distal convoluted tubules and Henle's loops than in the proximal convoluted tubules. Virus particles invaded host cells through endocytotic vesicles. Cytopathologic changes in the infected epithelial cells were manifested by a variety of organlle alterations including swelling of mitochondria, dilation of Golgi vesicles and an increase in the amount of rough endoplasmic reticulum (RER). Virus particles were produced by budding into RER and, rarely, toward the perinuclear space. As virus replication progressed, virus particles were enclosed mainly in the dilated RER, cytoplasmic vesicles or virus-containing electron-dense bodies. Virus particles were also found in vesicles of Golgi complex, the dilated perinuclear space, in some autophagic vacuoles or free in the cytoplasm. Virus particles were released by exocytosis through cytoplasmic vesicles, or appeared to be discharged through disrupted cell membranes. It was concluded that epithelial cells of lower nephron and ducts are the primary target cells in IBV-infected kidneys.     

Localization of rubella virus core particles in vero cells

Rubella virus (RV) infection induces a variety of morphological changes in the host cell including the modification of lysosomes to produce "replication complexes" and the alteration of mitochondrial morphology and distribution. The morphogenesis of RV was further characterized with particular emphasis on the localization of RV core particles. Thin-section electron microscopy (TSEM) studies indicated that RV core-like particles, measuring approximately 33 nm in diameter, were found associated with RV replication complexes. Immunogold-labeling electron microscopy (EM) using monoclonal antibodies to RV capsid proteins confirmed that these particles were viral cores. RV core particles were also detected in association with mitochondria as observed by TSEM and immunogold-labeling EM using monoclonal antibodies to capsid or polyclonal antibodies to RV virions. The results of this study indicate that the localization of RV core particles in relation to replication complexes is similar to that found for the alphaviruses. However, the association of RV core particles with mitochondria appears unique within the family Togaviridae.     

Morphogenesis of porcine rotavirus in porcine kidney cell cultures and intestinal epithelial cells.

The morphogenesis of porcine rotavirus was similar in vitro in porcine kidney (PK) cell cultures and in vivo in porcine epithelial cells as examined by electron microscopy. Infected cells contained cytoplasmic, non-membrane-bound viroplasm and accumulations of virus particles within cisternae of the rough endoplasmic reticulum (RER). Three types of virus particles were noted: double-shelled or complete particles which averaged 77 nm in diam.; single-shelled or naked particles which ranged from 50 to 55 nm in diam.; and electron-dense nucleoids, or cores, 31 to 38 nm in diam. Virus particles acquired outer shells by budding through either matrices of granular, electron-dense viroplasm or membranes of distended RER. Accumulation of numerous single-shelled particles was observed only in PK cell cultures containing a high percentage of infected cells. In these cells, virus release occurred through disruption of the plasma membrane. Tubules, similar in diameter to the single-shelled particles, were observed in the nuclei of a few infected PK cells.     

Structural maturation of rubella virus in the Golgi complex

Rubella virus is a small enveloped virus that assembles in association with Golgi membranes. Freeze-substitution electron microscopy of rubella virus-infected cells revealed a previously unrecognized virion polymorphism inside the Golgi stacks: homogeneously dense particles without a defined core coexisting with less dense, mature virions that contained assembled cores. The homogeneous particles appear to be a precursor form during the virion morphogenesis process as the forms with mature morphology were the only ones detected inside secretory vesicles and on the exterior of cells. In mature virions potential remnants of C protein membrane insertion were visualized as dense strips connecting the envelope with the internal core. In infected cells Golgi stacks were frequently seen close to cytopathic vacuoles, structures identified as the sites for viral RNA replication, along with the rough endoplasmic reticulum and mitochondria. These associations could facilitate the transfer of viral genomes from the cytopathic vacuoles to the areas of rubella assembly in Golgi membranes.     

Ultrastructural study of Mayaro virus replication in BHK-21 cells

Summary The replication of Mayaro virus in BHK-21 cells was studied by electron microscopy. The infected cells show an intense vacuolization and proliferation of membranous structures. At 5 h post-infection, precursor virus particles were seen in the cytoplasm of infected cells. Later, mature virus particles were found outside the cells and budding from the plasma membrane. Enveloped virus particles were also observed inside the vesicles and budding across their membrane. The release of virus particles into the extracellular space by exocytosis was also observed. In a later stage of the infection, inclusion bodies were sometimes present in the cytoplasm of infected cells. We conclude that in BHK-21 cells, budding from the plasma membrane is the main process of Mayaro virus maturation, and in this kind of cell replication differs significantly from that observed inAedes albopictus cells.     

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.     

Detection of rabbit haemorrhagic disease virus particles in the rabbit liver tissues.

Liver tissues from rabbits experimentally infected with rabbit haemorrhagic disease virus (RHDV) were studied electron microscopically. The earliest change in hepatocytes of the rabbits infected with RHDV was hydropic degeneration. Rough endoplasmic reticulum was dilated with a mild increase in polysomes and cytoplasmic cisternae in degenerated hepatocytes. Characteristic cytopathological changes of necrotic hepatocytes included shrinkage of the cell body, formation of cytoplasmic vesicles, vacuoles or cisternae and karyolysis. A large number of viral particles resembling a calicivirus in size and morphology was demonstrated in the cytoplasm of many necrotic hepatocytes. The particles had accumulated mainly in the membrane-bound cisternae or scattered around the membrane-bound vacuoles of the necrotic hepatocytes. Western blot analysis demonstrated that RHDV antigen was present in the infected hepatocytes. RHDV particles were also detected by immunoelectron microscopy. Replicating patterns of RHDV particles and subsequent cytopathology resembled those in other calicivirus infections.     

Morphogenesis of avian infectious bronchitis virus in primary chick kidney cells

Summary Primary chick kidney cells were infected with avian infectious bronchitis virus (IBV) and examined by electron microscopy. Virus particles entered the cells by viropexis and distinction could be made between engulfment by cell processes (phagocytosis) and entry by micropinocytosis in coated transport vesicles.Virus maturation occurred by budding into either the cisternae of the endoplasmic reticulum or cytoplasmic vacuoles, and evidence was obtained to suggest that the viral surface projections could be attached during the budding process. Late in infection large numbers of virus particles were present, mainly in cytoplasmic vacuoles, and the majority were released by cell lysis. Release by fusion of vacuoles with the plasma membrane was also observed, and individual virions could be transported from the endoplasmic reticulum to the surface within coated vesicles.With 10 Figures     

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.     

Morphogenesis of the assembly and release of bovine enterovirus

Fluorescent antibody (FA) studies of cells infected with bovine enterovirus showed cytoplasmic blebs with specific fluorescence to the virus. These structures were also found extracellularly in the debris of lysed cells and were RNA-positive by acridine orange (AO) staining. The morphology of virus-infected cells was further studied by scanning electron microscopy (SEM). Transmission electron microscopy (TEM) with immunoferritin tagging showed the development of long sacs with bilaminated and multilaminated membranes. These sacs had multiple twists at different intervals along their length forming a chain of vesicles. The development and maturation of the virus were observed in these vesicles. A number of virus-containing vesicles were also present extracellularly in the debris of lysed cells. In addition, virus was observed in layers of membranous cisternae closely associated with vacuoles and plasma membrane. Some of the cisternae opened to the extracellular space and appeared to allow the release of the virus. Virus particles were also found in patches and in crystals within the cytoplasmic matrix. Many lysed cells contained fibrils often associated with patches of ferritin-tagged virus. This study presents morphological evidence for the release of the virus in vesicles after cell lysis, via cisternae with openings to the extracellular space, and in cytoplasmic blebs.     

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.     

Ultrastructural studies on dengue virus infection of human lymphoblasts

Ultrastructural studies of dengue-2 virus-infected lymphoblastoid Raji cells showed that the virus induced an increase in the size of the rough endoplasmic reticula (RER) and that the replication of the virus was confined to the cisternae of these RER. The proliferating RER formed cytoplasmic inclusions that could be seen by light microscopy. This observation could be used as evidence of a cytopathogenic effect of dengue virus on infected Rajii cells in routine cultures. Accumulation of virions in the infected cells was minimal in comparison with other cell systems, however. Sporadic clusters of mature virions were often seen on the plasma membrane. These extracellular virions were distributed adjacent to the virus-bearing RER and were presumably released virions. Vertical transmission of the virus was evident in mitotic lymphoblasts. The replication pattern of dengue virus in lymphoblastoid cells suggests that efforts should be made to determine whether blast-transformed lymphocytes, numerous in secondary dengue infections, support dengue virus replication in vivo.     

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.     

Development of an Equine Herpesvirus in Two Cell Culture Systems: Light and Electron Microscopy

Development of equine herpesvirus strain 82A was studied in cells from primary horse kidney (HOK) cultures and an equine dermis (ED) cell strain. HOK and ED cells are equally susceptible to the 82A virus infection and yield about the same amount of infectious virus. Intranuclear inclusions were present in both cell systems, but a ring-shaped syncytial formation was observed only in infected ED cells. Ultrastructural studies revealed the presence of dense granules 30 nm in diameter and characteristic star-like clusters of granules in the infected HOK cells, but these granules were rarely seen in the infected ED cells. Viral nucleocapsids were associated with homogenous nuclear matrices, with moderate electron density in both cell systems. Viral nucleocapsids acquired envelopes by budding into the nuclear vacuoles in both HOK and ED cells. Budding from inner nuclear membranes into perinuclear cisterna or into cytoplasmic vacuoles also was observed frequently in HOK cells but was not seen often in infected ED cells. Multiple, membrane-bound intranuclear inclusions of fibrillar material which may be associated with virus envelopes were seen only in infected ED cells. Enveloped virus particles seen in nuclear vacuoles or perinuclear cisterna were more regular in shape and had a 130-nm diameter, whereas the enveloped virus particles seen in the cytoplasm and extracellular space were more irregular in shape and had a 130- to 160-nm diameter.     

Ultrastructure of potato leaf phloem infected with potato leafroll virus

Electron microscopy of Russet Burbank potato (Solanum tuberosum L.) leaf midveins infected with the potato leafroll virus (PLRV) revealed the pathological effects induced by the virus in the phloem. Virus particles were observed in mature sieve elements, companion cells, and plasmodesmata connecting the two kinds of cells. An early indication of virally induced cellular disturbance in companion cells was dilation of mitochondrial cristae followed closely by the appearance of vesicles in the parietal cytoplasm. One type of vesicle contained fibrils suggestive of nucleic acids and the second type carried an electron opaque material. Both types of vesicles were surrounded by two membranes. Rough endoplasmic reticulum (ER) formed the outer membrane. Both vesicle types fused with the nuclear envelope and were taken up into the nucleus. PLRV particles appeared mainly in the cytoplasm, where they were either scattered or localized along the membranes of mitochondria, chloroplasts, and vacuoles. In some cells, groups of particles were enclosed in the vacuole. The relation of virus particles to the nucleus remains problematic: there was no evidence that uptake of vesicles into the nucleus resulted in the formation of particles in this organelle. Cells in which particles formed became degenerated. Mitochondria swelled and cristae separated from the envelope and clumped. Most organelles including the nucleus, ribosomes, rough ER, and chloroplasts broke down leaving a fibrillar material interspersed with virus particles.