Localization of hepatitis A antigen in liver tissue by peroxidase-conjugated antibody method: light and electron microscopic studies.

Hepatitis A antigen (HAAG) was localized in liver tissue from marmosets inoculated with human hepatitis A virus (HAV) by light and electron microscopy by using a peroxidase-conjugated antibody method. The fine granular peroxidase staining was scattered throughout the cytoplasm of liver cells when viewed with the light microscope. The distribution of HAAg-positive cells was focal. Virus-like particles, 24 to 27 nm in diameter, were observed in the cytoplasm of hepatocytes and smaller cells, resembling Kupffer cells, by standard thin-section electron microscopy (thin section EM). By immunoperoxidase electron microscopy (immunoperoxidase EM), HAAg was detected on the particles, which were aggregated within cytoplasmic vesicles of the hepatocyte. The surrounding membrane of the vesicles was also HAAg- positive. Similar HAAg particles were observed in the cytoplasm of smaller cells adjacent to hepatocytes as well. Thus, immunoperoxidase EM revealed that the 24- to 27-nm virus-like particles in the cytoplasm of liver cells obtained from marmosets were infected with HAV contained HAAg.     

Hepatitis A antigen isolated from liver and stool: immunologic comparison of antisera prepared in guinea pigs.

Morphologically similar hepatitis A antigen particles (HA Ag)3 have been detected in the stools of patients with type A hepatitis and in the livers of marmosets experimentally infected with hepatitis A virus. To investigate the humoral antibody responses to these antigens and to compare the immunologic properties of HA Ag from these two sources, we immunized guinea pigs with either marmoset liver-derived HA Ag or with human stool-derived HA Ag in complete Freund's adjuvant and measured their antibody responses by immune electron microscopy (IEM) and immune adherence hemagglutination (IAHA). Antibodies reacting with both hepatitis A antigens were elicited in both groups. As determined by IEM, no distinction was seen between the reaction of guinea pig antiserum to each HA Ag tested under code when reacted against either liver-derived or stool-derived HA Ag. Antibodies elicited to marmoset liver-derived HA Ag and human stool-derived HA Ag had similar end point dilution titers by IAHA when tested against either "light" density (1.34 g/cm3) or "heavy" density (1.40 g/cm3) stool-derived HA Ag or liver-derived HA Ag. Low levels of antibody to normal liver or stool control antigens were observed transiently but did not obscure the specific response to HA Ag. These data suggest that morphologically similar HA Ag particles from different sources and with different densities are immunologically similar and may be identical. In contrast to the heterogeneity of surface antigens of hepatitis B virus, the comparable immunogenicity and apparent antigenic homogeneity of HA Ags derived from various sources may simplify the approach to development of a vaccine against viral hepatitis, type A.     

Hepatitis B Core Antigen: Immunology and Electron Microscopy

TWO DISTINCT VIRAL ANTIGENS ARE ASSOCIATED WITH THE HEPATITIS B VIRUS: the hepatitis B surface antigen (HB(s)Ag, Australia antigen) and the hepatitis B core antigen (HB(c)Ag). HB(s)Ag, purified from the serum of asymptomatic human HB(s)Ag carriers, and HB(c)Ag, purified from the liver of a chimpanzee acutely infected with hepatitis B virus, were examined by serological and immune electron microscopic methods. Antisera raised against HB(s)Ag reacted with the outer, surface component of the Dane particle and with the 20-nm spherical and tubular particles present in HB(s)Ag-positive serum, but not with the internal component of the Dane particle or with purified HB(c)Ag particles. Antisera raised against purified HB(c)Ag particles reacted with the internal component of the Dane particle and with HB(c)Ag, but not with the surface of the Dane particle or with the 20-nm spherical and tubular particles associated with HB(s)Ag. Purified HB(c)Ag particles, 27 nm in diameter, demonstrated distinct subunits. The infectious form of hepatitis B virus appears to be represented by the 42-nm Dane particle composed of a 27-nm nucleocapsid core component (HB(c)Ag) surrounded by an antigenically and morphologically distinct lipoprotein surface component (HB(s)Ag).     

The human immunodeficiency virus type 1-specific protein vpu is required for efficient virus maturation and release.

A deletion mutation affecting vpu was introduced into an infectious molecular clone of human immunodeficiency virus type 1, and the resultant phenotype was examined after infection of human T lymphocytes. The absence of vpu resulted in an accumulation of cell-associated viral proteins and impaired the release of progeny virions. Both electron microscopic and biochemical analyses indicated that a large proportion of the mutant particles was attached to the surface of infected cells. Significant variation in the size and shape of these progeny virions was observed. In addition, intracytoplasmic particles, some of which formed aberrant budding structures, were visualized in T cells infected with the vpu mutant. Indirect immunofluorescence analyses of cultures inoculated with wild-type virus with use of a vpu-specific antiserum demonstrated that vpu is mainly localized to a perinuclear region in the cytoplasm of virus-producing cells.     

肠道传播非甲非乙肝炎在恒河猴中的传代研究

用ET-NANBH感染的两只猴(R5和R6)含病毒颗粒的粪便悬液和肝组织悬液分别接种7只和4只恒河猴,分别有5只猴和4只猴在攻毒后20—49天内ALT开始升高,持续时间为7—10天,肝组织学的特征性改变为肝细胞的嗜酸性变和嗜酸小体的形成。在猴ALT升高前2—3天和升高后一周内均检查到大量的27—34nm的病毒样颗粒,这些颗粒只与ET-NANBH病人血清、黑猩猩和猴感染后急性期和恢复期血清发生特异性聚集。在猴感染后血清中未检出抗-HAV、抗-HAV-IgM、HBsAg和抗-HBe-IgM。结果提示:恒河猴是研究ET-NANBH较适宜的动物模型;病人和猴粪便中的27—34nm的病毒样颗粒是ET-NANBH的病原因子。     

Isolation of the novel agent from human stool samples that is associated with sporadic non-A, non-B hepatitis.

The agent(s) responsible for sporadic non-A, non-B hepatitis in humans was serially transmitted in rhesus monkeys by intravenous inoculation of the stool extract from a patient. A novel agent called HFV (hepatitis French [origin] virus) was present as 27- to 37-nm particles in the infectious stool extract. Hepatopathic lesions were noticed in infected monkeys during the acute phase of illness. The purified viral 27- to 37-nm particles consist of a double-stranded DNA of approximately 20 kb and are detected in infected monkey liver. Analysis of cell culture detects the approximately 20-kb-long viral DNA in stool samples from infected monkeys and sporadic enteric non-A, non-B hepatitis patients. Furthermore, the 27- to 37-nm viral particles were able to protect monkeys challenged with infectious stool extract. Our results indicate that 27- to 37-nm virus like particles are responsible for sporadic non-A, non-B hepatitis in rhesus monkeys.     

An electron microscopic study of the structural polymorphism of hepatitis B antigen from human sera.

The physical features of hepatitis B antigen (HBAg) particles from human sera are investigated with electron microscopy and immune electron microscopic technique. All the virus-like particles are pleomorphic in structure; although they are classified into two categories: HBsAg and HBcAg by immunological technique. The small, spherical particles measured in a range of 16 to 30 nm in diameter, mostly 20 to 22 nm,are populous in the positive serum. The tubular particles have the width of 18 to 22 nm and the length of 50 to 230 nm or even longer. Sometimes these particles contain a larger end and become the tadpole shape. The large particles or Dane particles measured mainly 42 nm in diameter have an inner core and the outer coats. The inner core of 27 nm in diameter can expose spontaneously. It can be released from the coats by heating at 56 degrees C for 30 min or by treatment of Tween 80 (1%) at room temperature. When specific antibody is added in the positive sample, the aggregate of the antigen-antibody clumping can be revealed with electron microscope. The core antigen of the large particles may attach to the molecular protein of the antibody and show the spike-like structure. This polymorphism of HBAg particles seems unique in animal virology. The roles of these particles played in medical and virological fields are discussed.     

Cloned hepatitis delta virus cDNA is infectious in the chimpanzee.

A head-to-tail trimer of a full-length cDNA clone of the hepatitis delta virus (HDV) genome was examined for infectivity by direct inoculation into the liver of a chimpanzee that was already infected with hepatitis B virus. Five weeks after inoculation, a marked elevation of serum alanine aminotransferase activity was observed, followed by the appearance of high levels of HDV RNA and antigen in both liver and serum and a high level of viral particles in the serum. A transient suppression of hepatitis B virus replication was evident during the acute phase of HDV infection. Seroconversion for antibodies to delta antigen occurred 3 weeks after the onset of the disease. These results demonstrate that a typical HDV infection can be initiated by inoculation of a susceptible animal with recombinant HDV cDNA.     

Virus of Pekin ducks with structural and biological relatedness to human hepatitis B virus.

A virus found in the sera of Pekin ducks appears to be a new member of the human hepatitis B-like family of viruses. This virus had a diameter of 40 nm and an appearance in the electron microscope similar to that of human hepatitis B virus. The DNA genome of the virus was circular and partially single stranded, and an endogenous DNA polymerase associated with the virus was capable of converting the genome to a double-stranded circle with a size of ca. 3,000 base pairs. An analysis for viral DNA in the organs of infected birds indicated preferential localization in the liver, implicating this organ as the site of virus replication. In all of these aspects, the virus bears a striking resemblance to human hepatitis B virus and appears to be a new member of this family, which also includes ground squirrel hepatitis virus and woodchuck hepatitis virus.     

Buoyant Density of the Hepatitis A Virus-Like Particle in Cesium Chloride

We recently visualized by immune electron microscopy a virus-like particle in the stools of patients with hepatitis A. The particle measured approximately 27 nm in diameter and morphologically resembled a picornavirus or parvovirus. To further characterize this particle, we have determined its buoyant density in cesium chloride (CsCl) by ultracentrifugation. Hepatitis A particles from three positive stool specimens were isopycnically banded in separate experiments, and the gradient fractions were examined for particles by immune electron microscopy by using hepatitis A convalescent sera. In each experiment, the particles were observed in a normal distribution about a peak fraction with a mean density of approximately 1.4 g/cm(3). The buoyant density of 1.4 g/cm(3) in CsCl together with its morphology and the reported resistance of hepatitis virus to acid, ether, and heat suggest that this particle is parvovirus-like.     

Electron microscopy of hepatitis B virus core antigen expressing yeast cells by freeze-substitution fixation

We have used the freeze-substitution fixation technique for electron microscopy of yeast cells that express the hepatitis B virus core antigen (HBcAg) following transformation with the cloned gene. Abundant spherical particles were found within the transformed cells. These particles had a uniform size and shape, measured about 21 nm in diameter, had electron-lucent centers, and consisted of many subunits. They were localized in both the cytoplasm and the nucleus. None of these particles was found in the cells of the parent strain. Comparison of the HBcAg particles isolated from the yeast cells and the particles within the yeast cells demonstrated that the 21-nm particles were in fact ultrastructurally superimposable on HBcAg. Thus, the HBcAg particles within the yeast cells were similar to the HBcAg particles in human liver tissues infected with hepatitis B virus, not only in their size and appearance, but also in their intracellular localization. These results suggest that the yeast cell has the same machinery for synthesis and intracellular translocation of the HBcAg polypeptides as the human cell.     

Production of infectious hepatitis C virus in tissue culture from a cloned viral genome

Hepatitis C virus (HCV) infection causes chronic liver diseases and is a global public health problem. Detailed analyses of HCV have been hampered by the lack of viral culture systems. Subgenomic replicons of the JFH1 genotype 2a strain cloned from an individual with fulminant hepatitis replicate efficiently in cell culture. Here we show that the JFH1 genome replicates efficiently and supports secretion of viral particles after transfection into a human hepatoma cell line (Huh7). Particles have a density of about 1.15-1.17 g/ml and a spherical morphology with an average diameter of about 55 nm. Secreted virus is infectious for Huh7 cells and infectivity can be neutralized by CD81-specific antibodies and by immunoglobulins from chronically infected individuals. The cell culture-generated HCV is infectious for chimpanzee. This system provides a powerful tool for studying the viral life cycle and developing antiviral strategies.     

Noncytopathic hepatitis A virus induces surface alterations in LLC-MK2 cells revealed by thin sections,negative staining,and scanning electron microscopy

Previous electron microscope studies of ultrastructural events during hepatitis A virus replication in experimentally infected cells have used only ultrathin section techniques. Nevertheless, no important differences were observed between infected and uninfected cells. This study was carried out using scanning electron microscopy and negative staining of whole LLC-MK2 cells grown directly on grids covered with support membranes, and then infected with an hepatitis A virus strain. Thin sections of infected and uninfected controls were also analyzed. An intricate web of projections forming a net between cell interfaces was observed only in infected cells. Some of these projections were more than 700 nm long and had ballooning tips. Nevertheless, HAV particles were not visualized in the infected cells.     

Purification of infectious pancreatic necrosis (IPN) virus and comparison of polypeptide composition of different isolates.

Infectious pancreatic necrosis (IPN) virus was partially purified by freon extraction of infected CHSE-214 cells and concentrated by polyethylene glycol (PEG) precipitation of virus from the medium. Both methods resulted in virus concentrates that could be further purified by two CsCl gradient centrifugations with little loss of infectivity. A Recovery of 80 to 100% of the virus infectivity was obtained and over 100-fold concentration of viral infectivity was achieved by these methods. This purification was used to compare 10 isolates of IPN virus with regard to their physiochemical properties by electron microscopy, buoyant density in CsCl, and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of the purified virions. Electron-microscopic observations showed that the virus isolates were identical in that they were isometric, hexagonal in profile, and had a particle diameter of 71 nm. The buoyant densities of the virus isolates in CsCl were found to be 1.33 g/ml. SDS-gel electrophoresis of the virus isolates revealed the presence of three polypeptides of molecular weight 50, 30, and 27 x 10(3) designated as VP50, VP30, and VP27, respectively.     

Electron Microscopic Observations of Mouse Brain Infected with Bunyamwera Group Arboviruses

Ultrathin sections of brain tissue from mice infected with three Bunyamwera group arboviruses, Bunyamwera, Tensaw, and Maguari, were examined by electron microscopy. In all three cases, virus was observed budding from membranes into intracytoplasmic cisternae. Mature virus was found accumulated within these distended cisternae and in extracellular spaces. The three viruses were morphologically indistinguishable; particles were round or oval, 98 nm in diameter, and had a closely adherent irregular envelope.     

Arenoviruses in Vero Cells: Ultrastructural Studies

Thin-section electron microscopy was carried out on Vero green monkey kidney cell cultures infected with some viruses of the newly constituted arenovirus group. Junin, Machupo, Amapari, Pichinde, Parana, Tamiami, and Latino viruses were morphologically identical and indistinguishable from lymphocytic choriomeningitis virus, the prototype virus of the group. Virus particles were round, oval, or pleomorphic, 60 to 280 nm in diameter, and matured via budding from plasma membranes. Most characteristically, particles contained various amounts of homogeneous, 20- to 25-nm, dense granules; these granules in large masses also formed distinctive intracytoplasmic inclusions. In negative-contrast preparations from infected Vero cell culture supernatant fluids, several of the viruses appeared as pleomorphic membrane-bound forms with rather pronounced surface projections. Most particles were between 90 and 220 nm in diameter, although some reached 350 nm in their longest dimension. Internal structure was not resolved by negative-contrast electron microscopy. All observations supported the current delineation of a distinct arenovirus group.     

Isolation of a lethal rhabdovirus from the cultured Chinese sucker Myxocyprinus asiaticus

A rhabdovirus was found to be associated with a lethal hemorrhagic disease in the cultured Chinese sucker Myxocyprinus asiaticus Bleeker. The rhabdovirus was amplified and isolated from the infected GCO (grass carp ovary) cells. In ultrathin sections of liver cells from the diseased fish, the virus particles exhibited the characteristic bacilliform morphology, and budded through vesicle membranes of the infected cells. The isolated rhabdovirus particles were found to have a bacilliform morphology with 2 rounded ends rather than a typical flat base. The virus particles were measured and ranged in size from 150 to 200 nm in length and 50 to 60 nm in diameter. Most other characteristics, including their size, extensive virus infectivity to fish cell lines, strong cytopathogenic effects, stability at high temperatures, vesicle formation in infected cells, structure protein electrophoretic patterns and the presence of an RNA genome, very closely resembled those of other fish rhabdoviruses. At present it is not known if this is a novel virus species or if it is an isolate of a known fish rhabdovirus. Until a confirmed identification can be made, we will temporarily refer to this virus as Chinese sucker rhabdovirus (CSRV).     

Inactivation kinetics of model and relevant blood-borne viruses by treatment with sodium hydroxide and heat.

To determine the efficacy of a clean-in-place system for the inactivation of viruses present in human plasma, the effect of 0.1 M sodium hydroxide at 60 degrees C on viral infectivity was investigated. Inactivation of the following model and relevant viruses were followed as a function of time: human hepatitis A virus (HAV), canine parvovirus (CPV; a model for human parvovirus B-19) pseudorabies virus (PRV, a model for hepatitis B virus), and bovine viral diarrhoea virus (BVDV, a model for hepatitis C virus and human immunodeficiency virus). Infectivity of CPV was determined by a novel in situ EIA method which will prove useful for studies to validate parvovirus inactivation or removal. Infectivity of BVDV, PRV and CPV were shown to be reproducibly inactivated below the limit of detection by 0.1 M NaOH at 60 degrees C within 30 s. HAV was inactivated to below the limit of detection within 2 min. Treatment with heat alone also resulted in some log reduction for all viruses tested except for CPV which remained unaffected after heating at 60 degrees C for 16 min. Treatment of HAV with hydroxide alone (up to 1.0 m) at 15 degrees C did not lead to rapid inactivation. Collectively, these data suggest that 0.1 M NaOH at 60 degrees C for two min should be sufficient to inactivate viruses present in process residues.