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.     

Hepatitis B core and surface antigens in liver tissue. Light and electron microscopic localization by the peroxidase-labeled antibody method.

Hepatitis B core antigen (HBcAg) and hepatitis B surface antigen (HBsAg) were localized in human liver tissues by the peroxidase-labeled antibody method at the light and electron microscopic levels. Several methods of fixation, staining, and inhibition of endogenous peroxidase activity were studied. The periodate-lysine-paraformaldehyde fixative effectively preserved the tissue structure and the antigenicity of both antigens, and the peroxidase-labeled Fab' fraction of IgG penetrated well into hepatocytes. HBcAg was present in nuclei, or cytoplasm of hepatic cells, or both. In nuclei, the antigen was found both in virus-like particles of approximately 20 nm. diameter and in nuclear ground substance. In the cytoplasm, the antigen was found on membrane-bound ribosomes and free polysomes, and also in the ground substance of the cytosol near ribosomes and around nuclear membranes, especially near nuclear pores. HBcAg-positive virus-like particles were also demonstrated sparsely or in clusters in the cytoplasm. HBsAg was not present in nuclei but was found in the perinuclear space and in cisternae of endoplasmic reticulum, and on nuclear, endoplasmic reticulum, and cell membranes of hepatic cells. HBsAg-positive 25- to 30-nm. wide tubular forms, round particles (probably cross-sections of tubular forms), and a few large particles of 40 to 50 nm. diameter were seen in cisternae. Such HBsAg-positive particles were also present in the intercellular space and in Disse's space. These findings suggest that HBcAg produced on the cytoplasmic ribosomes migrates through nuclear pores to the nucleus and is assembled into core particles there. These particles may then move through nuclear pores to the cytoplasm where they are invested with HBsAg-positive membrane in cisternae of endoplasmic reticulum or as they enter the endoplasmic reticulum. These virus particles are then released together with other HBsAg-positive forms into the intercellular space by reversed phagocytosis.     

Incidence and nature of cytoplasmic hepatitis B antigen in hepatocytes.

Hepatitis B antigen (HB Ag) in the hepatocytic cytoplasm is detected by immunofluorescence after reaction with fluoresceinated antiserum to HB Ag or by electron microscopy as numerous 20- to 30-nm. tubular and circular structures in dilated cisternae of excess endoplasmic reticulum. On light microscopy, these hepatocytes can be recognized because their cytoplasm has a ground-glass appearance and stains with Gomori's aldehyde fuchsin. Aldehyde fuchsin-positive ground-glass hepatocytes were detected in all 14 asymptomatic carriers of HB Ag and in 16 of 60 HB Ag-seropositive patients with chronic hepatitis, but not in HB Ag-seropositive acute viral hepatitis or in various other HB Ag-seronegative liver diseases. These cells are helpful in identifying on light microscopy HB Ag carriers and a portion of patients with HB Ag-positive chronic hepatitis. Nuclear HB Ag did not stain with aldehyde fuchsin. Nucleic acids were not detected in the ground-glass cytoplasm by special stains at the light or electron microscopic level. We suggest that the tubular and circular structures in the hepatocytic cytoplasm are coat material of the hepatitis B virus or virally coded host cell reaction product rather than the complete hepatitis B virus.     

Electron Microscopy and Immunoelectronmicroscopy of Cytoplasmic Hepatitis B Antigen in Hepatocytes

The localization of hepatitis B antigen (HB Ag) and the nature of the virus-like particles in hepatocytes of patients with HB antigenemia are controversial. In many reports, numerous virus-like particles have been demonstrated in hepatocytic nuclei; the few reported in the cytoplasm are insufficient in number to explain the intense cytoplasmic fluorescence after staining with fluoresceinated antibody to HB Ag (HB Ab). We found numerous tubular and circular structures, measuring 20 to 30 nm in diameter, in the cisternae of the excess smooth endoplasmic reticulum (ER) of varying numbers of hepatocytes in 13 of 16 HB Ag carriers and in 4 of 9 patients with HB Ag-positive chronic hepatitis corresponding to cytoplasmic HB Ag-specific fluorescence. Direct immunoelectronmicroscopy using peroxidase-labeled HB Ab revealed that the intracisternal bodies and the surrounding membranes contain HB antigenic determinants. These bodies are an ultrastructural correlate of cytoplasmic HB Ag. It is suggested that they are virally coded coat material rather than the mature hepatitis B virus or its core.     

Chimpanzee livers after infection with human hepatitis viruses A and B: Ultrastructural studies.

Electron microscopical studies were carried out on coded liver biopsy specimens from chimpanzees inoculated with human hepatitis A or B virus. Hepatitis B was recognized by the presence of hepatitis B core particles in hepatocellular nuclei. Hepatitis A was characterized by unidentified large, dense, and more irregular heterochromatin-like particles in hepatocellular nuclei coincidental with peak aminotransferase activities. As type A hepatitis illness became manifest in the chimpanzees, mitochondrial cristae were curled and attenuated, and clusters of endoplasmic reticulum were tightly packed. In contrast, the livers in viral hepatitis B showed mainly hypertrophy of tubular smooth endoplasmic reticulum. This suggested different pathogenetic mechanisms in A and B chimpanzee viral hepatitis.     

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.     

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。     

Inducible system in human hepatoma cell lines for hepatitis C virus production

We cloned the complete complementary DNA of an isolate of the hepatitis C virus, HCV-S1, into a tetracycline-inducible expression vector and stably transfected it into two human hepatoma cell lines, Huh7 and HepG2. Twenty-six Huh7 and two HepG2-positive clones were obtained after preliminary screening. Two Huh7 (SH-7 and -9) and one HepG2 (G-19) clones were chosen for further characterisation. Expression of HCV proteins in these cells accumulated from 6 h to 4 days posttreatment. Full-length viral plus-strand RNA was detected by Northern analyses. Using RT-PCR and ribonuclease protection assay, we also detected the synthesis of minus-strand HCV RNA. Plus- and minus-strand viral RNA was still detected after treatment with actinomycin D. Indirect immunofluorescence staining with anti-E2, NS4B, and NS5A revealed that these proteins were mostly localised to the endoplasmic reticulum (ER). Culture media from tet-induced SH-9 cells was separated on sucrose density gradients and analysed for the presence of HCV RNA. Viral RNA levels peaked at two separate ranges, one with a buoyant density of 1.08 g/ml and another from 1.17 to 1.39 g/ml. Electron microscopy demonstrated the presence of subviral-like particles (approximately 20-25 nm in diameter) in the cytoplasm of SH-9 and G-19 cells, which were positively labelled by anti-HCV core antibodies. Anti-E2 antibodies strongly labelled cytoplasmic vesicular structures and some viral-like particles. Complete viral particles of about 50 nm which reacted with anti-E2 antibodies were observed in the culture media of tet-induced SH-9 cells following negative staining. Supernatant from tet-treated SH-9 cells was found to infect nai;ve Huh7 and stable Huh7-human CD81 cells.     

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.     

Core and coat of the hepatitis b-virus and cytoplasmic viruslike particles in chronic hepatitis. An electron microscopic study (author's transl)]

In the second liver biopsy material of a seropositive patient receiving immunosuppressive therapy (Corticosteroid + Imuran) for chronic active hepatitis (CAH), intranuclear, ring-shaped, 20--25 nm in diameter non-coated particles (core) of liver cells and intracisternal filaments, 23 nm in diameter (coat) of the soft ER in the "ground-glass" hepatocytes were demonstrated by electron microscopy. Core-particles in the cytoplasm were seen occasionally. Dane-particles could not be visualized. Cytoplasmic spherical and ring-shaped virus-like structures (circ. 80 nm in diameter) different from B-virus components and Dane-particle were also found (second virus-infection?). The role of immuno-suppression in the appaerance of viral structures is considered because such particles could not be detected in the first biopsy before therapy when CAH and antigenaemia were already present.     

Acute glandular fever-like illness in a patient with HTLV-III antibody

A lymph node biopsy obtained from a patient with human T-cell lymphocytotropic virus III/lymphadenopathy-associated virus (HTLV-III/LAV) antibody, presenting with an acute glandular fever-like illness, was examined by electron microscopy. Numerous pathological changes were present in the biopsy, including hypertrophy of smooth endoplasmic reticulum, intracytoplasmic rod-like inclusions within the cisternae of endoplasmic reticulum, multivesicular bodies, test-tube and ring-shaped forms, and tubulo-reticular structures. Intranuclear and intracytoplasmic viral-like particles measuring 105-120 nm in diameter and small cytoplasmic particles measuring 50-70 nm in diameter were found in some degenerating lymph node cells. These pathological findings may reflect a host cell response to various pathological and viral stimuli resulting from immune deficiency owing to infection with HTLV-III/LAV.     

Characterization of infectious and defective cloned avian hepadnavirus genomes

The infectivity in vivo, replication competence in vitro, and expression of viral genes of several molecularly cloned duck hepatitis B virus (DHBV) genomes were investigated. In addition, replication competence, core protein expression, and secretion of viral proteins were investigated for a grey heron hepatitis B virus genome. Except two, all DHBV isolates tested induced a systemic infection in Pekin ducks when injected as cloned viral DNA into the liver. After transfection of chicken hepatoma cells, both defective DHBV genomes expressed intracellular nucleocapsid and pre-S envelope proteins and secreted DHBs/pre-S particles into the medium. One of the defective DHBV genomes and HHBV produced within the cells replicative intermediates encapsidated in core particles and secreted virions, whereas the other defective DHBV genome did not and was unable to efficiently encapsidate the RNA pregenome. Comparative sequence analysis was performed to identify potential amino acid changes in viral proteins of both defective DHBV genomes. The data obtained demonstrate that most cloned avian hepadnaviruses are infectious or replication competent and suggest defects in envelope, polymerase or encapsidation function, respectively, in two cloned DHBV genomes.     

Studies by immune electron microscopy of hepatitis B surface antigen in PLC/PRF/5 cells

Electron microscopic studies of the morphology of hepatitis B surface antigen (HBsAg) produced by PLC/PRF/5 cells in vitro were carried out. Aggregates of 20-nm spherical particles in 3-day culture supernatants were observed by immune electron microscopy (IEM). Aggregates of tubular structures were found with IEM in the extracts of the cells. Tubular structures 18 to 22 nm in diameter were seen by electron microscopy (EM) in the cisternae of the endoplasmic reticulum in 2-3% of the cells. The tubular structures in the cytoplasm and extracts of PLC/PRF/5 cells resembled those observed in the hepatocytes of human carriers of hepatitis B virus (HBV). Intracellular localization of HBsAg in PLC/PRF/5 cells by direct peroxidase-conjugated antibody staining was observed on the tubular structures and the cisternal wall, which contained these structures. Rotation technique analysis indicated that the tubular structures were composed of 11 or 12 subunits.     

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.     

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.     

Electron and immunoelectron microscopic study on liver tissues of marmosets infected with hepatitis A virus.

Electron and immunoelectron microscopic studies were carried out on liver tissues from three marmosets, experimentally infected with hepatitis A virus and sacrificed during the acute phase of illness. Ultrastructurally, the liver cells demonstrated marked cisternal dilation of endoplasmic reticulum and vesicular transformation and contortion of endoplasmic reticulum profiles. Clusters of virus-like particles of 24 to 27 nm. in diameter, both "solid" and "empty" forms, were found in membrane-bound cytoplasmic vesicles. In one animal, the virus-like particles were significantly smaller, measuring 17 to 22 nm. in size, and almost all were solid forms embedded in an amorphous matrix. Clusters of virus-like particles were found in the bile canaliculi of liver cell cords and in lysosomal structures of monocytes or Kupffer cells in the hepatic sinusoids. The latter correlated with the immunofluorescent microscopic finding. Indirect immunoferritin staining was carried out on fresh and formalin-fixed liver tissues, using convalescent phase serum from patients recovered from hepatitis A virus infection as the primary antibody, and the ferritin-labeled rabbit anti-human IgG or ferritin-labeled staphylococcal protein A as the secondary antibody. Specific stainings were observed with the virus-like particles, indicating that the particles were probably antigenically related to hepatitis A virus. Our findings are in agreement with the immunofluorescent and immunoelectron microscopic studies reported by others and support the concept that hepatitis A virus is produced in the liver. The infection seems to produce cytopathic effect especially to the endoplasmic reticulum organelle of hepatocytes.     

Occurrence of Tubuloreticular Inclusions in Acute Viral Hepatitis

Light and electron microscopy were used to study the liver needle-biopsy material of 20 patients with acute viral hepatitis. According to clinical and serologic data, 5 cases proved to be acute viral hepatitis type A, 13 were type B, and 2 were type non-A/non-B. In 2 of the hepatitis type A, in 6 of the hepatitis type B, and in one of the type non-A/non-B cases, tubuloreticular inclusions (TRI) were found in the endoplasmic reticulum of the sinusoidal endothelial cells of the liver.

These data support the possible presumption that the inclusions represent a characteristic reaction of the endoplasmic reticulum to different influences, as for example to viral infection, rather than to the virus itself.     

An ultrastructural study of liver explants from infants with vertically transmitted hepatitis

Liver biopsy specimens were taken from three infants whose mothers had acute hepatitis-B-antigen-positive (HB-Ag-positive) viral hepatitis within 2 mo of delivery. The biopsy specimens were taken at 11, 28, and 27 mo of age, all three infants having developed chronic HB-antigenemia within 3 mo after birth. Part of the liver tissue from each biopsy specimen was immediately processed and examined by light and electron microscopy while the remaining portions were maintained in explant culture media and examined by electron microscopy at intervals ranging from a few hours to 6 wk. Intranuclear virus-like particles, with a diameter of approximately 24 nmeters, were located in hepatocytes of the initial biopsy tissues. Examination of the explanted hepatocytes revealed evidence of proliferation of these intranuclear particles. In one of the explants, cytoplasmic membrane-bound virus-like particles, with a diameter of approximately 33 nmeters, were located. It was concluded that the hepatocyte intranuclear particles were viral in nature and that they proliferate within the nucleus. The possibility of further development of these particles within the hepatocyte cytoplasm, resulting in membrane-bound particles, is discussed in the light of current concepts regarding the origin of a mature hepatitis virus.     

中国流行性出血热脏器标本病毒样颗粒属性的探讨

本文应用电镜观察了12例流行性出血热(EHF)尸体标本,其中7例(58%)出现了泡状病毒样颗粒。它为圆形或椭圆形,直径72～116nm,平均92nm;有包膜,没有核样体,表面粗糙或有微突。颗粒主要分布于网织细胞及心、肝实质细胞的核膜及线粒体,偶尔见于内质网和胞浆。泡状病毒样颗粒在大小和形态上,与洪涛等在培养细胞见到的EHF病毒以及其他学者观察的汉坦病毒和布尼亚病毒基本一致,为EHF病毒在人体脏器细胞的形态表现。