Electron microscopic observation of feline kidney cells infected with a feline calicivirus

An electron microscopic study of kitten kidney cells infected with a feline calicivirus (a member of the family Picornaviridae) has been carried out. Although cells appeared to be synchronised by the light microscope, electron microscopic changes were extremely variable. The first observable and consistent changes occurred in the nucleus followed by the formation of membrane bound vesicles in the cytoplasm. A variety of arrangements of virus particle accumulation were observed in infected cells. These included crystalline arrays, membranous cisternae and accumulation of particles in fine fibrillar material. The finding of accumulations of virus particles in association with smooth membranes is of importance in respect of the recent biochemical evidence of poliovirus assembly in relation to smooth membranes.     

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     

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.     

Antigenicity of mouse hepatitis virus strain 3 subcomponents in C57 strain mice

Summary Morphogenesis of human rotavirus type 2 Wa strain in MA 104 cells was observed. The virus antigen in the cytoplasm was detected by indirect immunofluorescence twelve hours after infection. The cytopathic effect occurred 24 hours after infection when virus particles were detected by EM in the culture fluid as well as in thin sections of the infected cells. Virus particles were observed in the dilated RER, nuclear envelope (perinuclear space), viroplasm, and a lysosome-like body. Three types of virus particles were noted: double-shelled particles 75–85 nm in diameter, single-shelled particles 64–68 nm in diameter and electron-dense nucleoids or cores 32–40 nm in diameter. The outer shell of virus particles was acquired by budding through the membrane of the dilated RER. Tubular structures, similar in diameter to the single-shelled particles, were found in the cytoplasm and nucleus of infected MA 104 cells. Bundles of filaments or the leaflet-like inclusion bodies of membrane-bounded bundles of filaments were found in the cytoplasm and seemed to be associated with virus particles.With 9 Figures     

Electron microscope study of mosquito densonucleosis virus maturation.

Maturation of mosquito densonucleosis virus (MDV) was studied in its natural host, Aedes aegypti L. larvae. First ultrastructural changes were observed in the cytoplasm of virus-infected cells. They consisted in the formation of paracrystalline structures containing particles 18--20 nm in diameter as well as microtubules 20 nm in diameter. Virogenic stromas and paracrystalline virion arrays were found in the nuclei of virus-infected cells.     

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.     

Virus-like particles in an antarctic Aggregata sp.: I. Sporogonial stages

An unknown Aggregata sp. was found in the renal organ of an antarctic Benthoctopus sp., when it was inspected for the presence of dicyemid mesozoans. Merozoites invaded the renal epithelium, whereas sporogonial stages resided in the submucosal connective tissue. Interestingly, individuals of all developmental stages were found to be infected with hitherto unknown virus-like particles. These virus-like particles, spherical in shape, nonenveloped and measuring approximately 30 nm in diameter, could be observed in the nuclei as well as within the cytoplasm. Greater amounts of virus-like particles tended to be arranged in paracrystalline arrays. Although the infection was extensive, it obviously had no pathogenic effect on the parasites themselves. On the basis of the host specificity, size, morphology and histochemical analysis, which suggested the putative viral genome as RNA, a relationship with Totoviridae is assumed.     

Ultrastructural changes in small intestinal epithelium of neonatal pigs infected with pig rotavirus

Summary The small intestine of piglets orally infected with rotavirus was examined by electron microscopy 18, 24, 48 and 60 hours after infection. At 18 and 24 hours after infection columnar epithelial cells covered the villi. Infected epithelial cells tended to be less electron-dense than uninfected cells and were more numerous at 24 hours after infection. Two types of rotavirus particle were seen, usually within dilated cisternae of the RER: non-enveloped particles measuring 50 to 60 nm and enveloped particles measuring 65 to 75 nm. Non-membrane bound viroplasm containing electron-dense cores was encountered outside the cisternae 18 and 24 hours after infection. Tubular structures measuring 44 to 56 nm were often found in nuclei of infected cells. Single-membraned (44 to 55 nm) and double-membraned tubules (70 to 80 nm) associated with viral manufacture were found in the cytoplasm of infected cells.At 48 and 60 hours after infection a proportion of villous epithelial cells were cuboidal. Virus particles were detected in only a few epithelial cells and nuclear and cytoplasmic tubules were not seen.At all times after infection some infected cells showed a reduction in the number and size of the microvilli comprising the brush border.With 7 Figures     

Ultrastructure of human nasal epithelium during an episode of coronavirus infection

The nasal epithelium from a young girl was examined by electron microscopy and found to be infected by coronavirus. Virions are seen within and outside the ciliated cells, but not outside or within the goblet cells or other cells of the nasal mucosa. Some virions are located near the microvilli, others in pockets in the apical cell membrane. The cytoplasm contains many small vesicles with a single virion, large apical vesicles containing hundreds of virions, and lysosome-like cytosomes with a moderate number of virions. Some viruslike particles devoid of an electron-dense interior are seen both in the cytosomes and extracellularly. Virus budding was observed in the Golgi apparatus but nowhere else in the cell. The ciliated cells seem not to be destroyed by the viruses, although in many cases the cilia are withdrawn into the cell body. The loss of cilia is likely to cause rhinorrhoea.     

Replication of porcine enteric adenovirusesin vivo

Summary The replication of an enteropathogenic porcine adenovirus in the intestinal epithelial cells of naturally and experimentally piglets has been studied by transmission electron microscopy (TEM) and by immunoperoxidase (Ip) staining of paraffin sections.Three types of intranuclear inclusion bodies were observed. Viral particles appeared to be assembled from electron dense crescents which seemed to originate from type II intranuclear inclusion bodies. Virus accumulated in the nucleus of infected cells. It formed paracrystalline arrays in the electron dense centre of the nucleus but was dispersed in the peripheral electron translucent zone. Virus was released from the cells after rupture of the nuclear and plasma membranes.The results are compared with published data on the replication of adenoviruses in cell cultures.With 6 Figures     

戊型肝炎病毒归科的形态学证据

采用超簿切片制样及负染色免疫电镜方法，对经细胞培养自戊型肝炎病人分离的８７Ａ株病毒进行了形态及形态发生的研究。结果显示，感染细胞胞浆局灶性空泡液泡病变区明显而普遍；病毒呈晶格样排列，在胞浆内装配成熟，与粗面内质网、病毒性包涵体、微丝等密切相关；病毒毒粒无囊膜，大小约３０ｎｍ呈圆形，表面形态不规整，电致密度不均一，偶见“凹杯形”、“刺突”等表面超微结构；感染细胞核呈现扭曲变形，异染色质趋边等显著受累及病变。其形态及形态发生特征表明其应为嵌杯病毒科新属—嗜肝ＲＮＡ病毒属的成员，而部分核苷酸序列分析也支持其为戊型肝炎病毒。     

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.     

A thin-section study of the morphogenesis of Aujeszky's disease virus in synchronously infected cell cultures

Summary The morphogenesis of Aujeszky's Disease Virus in synchronously infected pig kidney cells was studied at hourly intervals for 24 hours. Naked capsids were present in the nucleus from 3 hours and in the cytoplasm from 4 hours after infection. Capsids acquired their envelope not only at the inner nuclear membrane but also in the cytoplasm at smooth-membraned vesicles, the outer nuclear membrane, rough endoplasmic reticulum or the Golgi complex. Extracellular mature virus first appeared at 5 hours and after this time most enveloped particles were extracellular. Virus was probably released by fusion of vesicles with the plasma membrane. These results extend previous work on Aujeszky's Disease Virus and in particular, establish the time required for virus morphogenesis.     

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.     

Comparative features of a coronavirus isolated from a cheetah with feline infectious peritonitis

A coronavirus which was isolated from a cheetah (Acinonyx jubatus) that succumbed to feline infectious peritonitis was characterized in vitro. The virus was determined to be highly cell-associated with Crandell feline kidney (CrFK) cells and was routinely maintained as a persistent infection (CrFK 83-4497). The cheetah coronavirus was compared with other members of the feline coronavirus group including the feline enteric coronavirus (FECV) 79-1683 and the feline infectious peritonitis viruses (FIPV), 79-1146, and UCD-1. The cheetah coronavirus was demonstrated to have a restricted host-cell range with limited cytopathic effect. Indirect immunofluorescence with antisera to FIPV UCD-1 revealed the concentration of viral antigens in the perinuclear region of cells infected with the cheetah coronavirus. Ultrastructural studies of the cheetah coronavirus indicated a limited number of immature viral particles within cytoplasmic vesicles and at the cell surface. This was in contrast to electron microscopy results of FECV 79-1683 and FIPV 79-1146, which had numerous mature virus particles within the cytoplasmic vesicles, as well as at the cell surface. The cheetah coronavirus was tentatively placed in the feline coronavirus family based upon its antigenic reactivity by immunofluorescence; however, the possibility that it represents a unique coronavirus of cheetahs should not be dismissed without further analyses at the host and genomic levels.     

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.     

Immunoelectron Microscopic Identification of Human Parvovirus B19

A mildly macerated, hydropic fetus was delivered spontaneously at 25 weeks gestation by a multigravidous black mother. At autopsy, gross and microscopic evidence suggested fetal anemia, and excessive extramedullary erythroblastosis was present generally. Erythroid precursor cells in the pulmonary capillary circulation contained eosinophilic nuclear inclusions consistent with human parvovirus B19 infection. Transmission electron microscopy on osmicated Epon lung sections showed lucent, granular chromatin corresponding to the inclusions. In rare foci only these sections contained paracrystalline arrays of 20-nm virions. In the same cells, similar virions were seen within the cytoplasm, both randomly distributed and in paracrystalline aggregates. Postembedding immunoelectron microscopy, done on formalin-fixed lung embedded in a hydrophilic resin, showed positive labelling over the nuclear inclusions, and also localized the viral capsid antigen to cytoplasmic virion aggregates. The nuclear aggregates suggest that the virus would reach the circulation after cell lysis whereas those in cytoplasm suggest that parvovirus also may be excreted from infected cells before they lyse.     

Immunoelectron Microscopic Identification of Human Parvovirus B19

A mildly macerated, hydropic fetus was delivered spontaneously at 25 weeks gestation by a multigravidous black mother. At autopsy, gross and microscopic evidence suggested fetal anemia, and excessive extramedullary erythroblastosis was present generally. Erythroid precursor cells in the pulmonary capillary circulation contained eosinophilic nuclear inclusions consistent with human parvovirus B19 infection. Transmission electron microscopy on osmicated Epon lung sections showed lucent, granular chromatin corresponding to the inclusions. In rare foci only these sections contained paracrystalline arrays of 20-nm virions. In the same cells, similar virions were seen within the cytoplasm, both randomly distributed and in paracrystalline aggregates. Postembedding immunoelectron microscopy, done on formalin-fixed lung embedded in a hydrophilic resin, showed positive labelling over the nuclear inclusions, and also localized the viral capsid antigen to cytoplasmic virion aggregates. The nuclear aggregates suggest that the virus would reach the circulation after cell lysis whereas those in cytoplasm suggest that parvovirus also may be excreted from infected cells before they lyse.     

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.     

Study of the morphological aspects of Okhotski? virus replication in a chick fibroblast culture]

Electron microscopic studies of chick embryo fibroblast cells 24 hours after infection with Okhotsky virus revealed changes typical orbivirus infection in the cytoplasm and the nuclei of the cells. The nuclei of the infected cells contained fine-granular matrices with forming virus particles and tubular structures. The cell cytoplasm also contained tubular structures, crystal arrays, fine-granular matrices and virus particles 50-60 nm in diameter.