Dane Particles Research Articles

Hepatic lesions in alcoholic HBV carriers

Liver biopsies were taken from 54 alcoholic HBV carriers with liver dysfunction to assess whether HBV infection or habitual drinking was the main cause of their illness. In 28 cases, ultrastructural studies were done. Results showed that 50% of the cases predominantly displayed virus-related histological changes, whereas 13% of them mainly had alcohol-related ones. Both pathological changes were evenly distributed in four cases. The remaining 15 cases showed nonspecific or other histological changes. Electron microscopy revealed that HBV core and Dane particles were seen with Mallory bodies in the same hepatocytes. Thus, we postulate that HBV-related changes are more often encountered than alcoholic ones in alcoholic HBV carriers and that HBV replication can occur even in hepatocytes bearing Mallory bodies.

Three envelope proteins of hepatitis B virus: large S, middle S, and major S proteins needed for the formation of Dane particles

The infectious particles of hepatitis B virus are called Dane particles and consist of viral nucleic acid encapsulated within a core particle that is enveloped by virus-coded surface proteins. The major S protein constitutes a significant fraction of these surface proteins. In addition, there are two other related proteins (large S and middle S), but their role in envelope formation has not yet been elucidated. We modified the translation initiation codon ATG of each of the envelope proteins by site-directed mutagenesis and found that mutant genomes that did not produce one or two of these proteins were unable to form Dane particles. The particles released into the culture medium by such mutants did not carry DNA. Synthesis of virus-coded RNA still occurred normally, and core particles carrying DNA accumulated intracellularly. The DNA in such core particles was mostly in the double-stranded open circular form, in contrast to the normal situation in which the particles contain mostly RNA and its complementary single-stranded DNA or else contain linear DNA that is partially single stranded and otherwise duplex. The role of the large S and middle S proteins in the formation of Dane particles is discussed.

Tissue culture system for infection with human hepatitis delta virus

An in vitro culture system was developed for assaying the infectivity of the human hepatitis delta virus (HDV). Hepatocytes were isolated from chimpanzee liver and grown in a serum-free medium. Cells were shown to be infectible by HDV and to remain susceptible to infection for at least 3 weeks in culture, as evidenced by the appearance of RNA species characteristic of HDV replication as early as 6 days postinfection. When repeated experiments were carried out on cells derived from an animal free of hepatitis B virus (HBV), HDV infection occurred in a consistent fashion but there was no indication of infection with the HBV that was present in the inoculum. Despite numerous attempts with different sources of HBV inocula free of HDV, there was no evidence that indicated susceptibility of these cells to HBV infection. This observation may indicate that HBV and HDV use different modes of entry into hepatocytes. When cells derived from an HBV-infected animal were exposed to HDV, synthesis and release of progeny HDV particles were obtained in addition to HBV replication and production of Dane particles. Although not infectible with HBV, primary cultures of chimpanzee hepatocytes are capable of supporting part of the life cycle of HBV and the entire life cycle of HDV.

Production and Effect of Infectious Dane Particles in Transgenic Mice

We have demonstrated by immunoelectron microscopy that 42‐nm particles with double‐shelled structures characteristic of Dane particles are present in the serum of transgenic mice, 1.2HB‐BS 10, carrying partly duplicated hepatitis B virus (HBV) genome. Furthermore, these particles were shown to infect primary human fetal hepatocytes as demonstrated by the elevation of MBV surface antigen (HBsAg) in the culture medium. HBV DNA is known to be expressed in a liver‐ and kidney‐specific manner in the adult mouse, so we examined the developmental expression of viral antigens. In the liver, viral antigens (HBsAg and HBV e antigen) began to be expressed before birth and the level of expression showed a sharp rise after birth. On the other hand, in the kidney, viral antigens began to be expressed after birth. Serum levels of viral antigens were roughly proportional to the levels of expression in the liver, suggesting that the liver is the main source for viral antigens in the serum. None of these transgenic mice produced anti‐HBs or anti‐HBV core response or showed biochemical or pathological change up to at least 24 months of age. All these results suggest that infectious viral particles can be produced in transgenic mice, and that expression and replication of HBV DNA are not toxic in vivo.

Myristylation of a duck hepatitis B virus envelope protein is essential for infectivity but not for virus assembly

In addition to the major surface (S) protein, the envelope of the duck hepatitis B virus (DHBV) contains a related presurface (preS) protein whose N-terminus bears a covalently attached myristate group. We have explored the functional significance of this modification by examining the replicative potential of a mutant viral genome whose myristylation signal has been inactivated. Following transfection into permissive hepatoma cells, the mutant expresses an unmyristylated preS protein of normal size, immunoreactivity and stability. Cytoplasmic cores containing viral DNA are synthesized, and Dane particles are assembled and exported into the medium. However, the mutant is noninfectious when inoculated into susceptible ducklings. We conclude that myristylation of preS proteins is essential for hepadnaviral infectivity but not for viral assembly; myristylation is most likely required for an early step of the life cycle involving the entry or uncoating of virus particles.

Development and application of anin vitro model for screening anti-hepatitis B virus therapeutics

The development of effective anti-hepatitis B virus agents has been hampered by the lack of reliable in vitro systems for the screening of new therapeutics. In an effort to circumvent this problem, we have developed an in vitro system for screening anti-hepatitis B virus drugs using hepatitis B virus DNA-transfected Hep G2 cells. The cell line designated 2.2.15 produces replicative viral DNA intermediates, mature Dane particles and high levels of viral antigens. Subconfluent 2.2.15 cells were treated with a variety of commonly used anti-hepatitis B virus therapeutics, and their efficacy was determined by analyzing changes in the replicative cellular or extracellular hepatitis B virus DNA content by Southern blotting or slot-blot hybridization. The slot-blot method was sensitive, reproducible and rapid and correlated well with Southern blotting. Analysis of the media for hepatitis B virus DNA was indicative of changes in intracellular, replicative hepatitis B virus DNA, permitting sampling of the media. Therefore 2.2.15 cells may provide a valuable method for identifying and monitoring effective anti-hepatitis B virus therapeutics. Using this system to test various agents, we confirm that 2'-deoxyguanosine strongly inhibited viral replication, whereas others tested were less effective. Correlation with in vivo systems is now needed.

HBV production in transgenic mice

We produced transgenic mice by microinjecting a partially duplicated copies of hepatitis B virus (HBV) gene into fertilized eggs of C57BL/6 mice. One mouse was a high producer of HBV surface antigen (HBsAg) and HBV e antigen (HBeAg) in the serum. All offspring carrying HBV DNA were positive for both antigens in the serum. The HBV DNA was expressed in liver- and kidney-specific manner. The normal process of HBV replication, including the packaging of the pregenome 3.5-kb RNA into a nucleocapsid, the reverse-transcription of the complete minus strand DNA, and the release of Dane particles into the serum before the completion of synthesis of plus strand, occurred in the liver of these transgenic mice. These results suggest that the species specificity of HBV infection is not due to the inability to replicate in nonnatural host but to the lack of receptors or factors needed for virus adsorption and internalization. The founder mouse is now 19 months of age but shows no clinical or pathological change, suggesting that HBV itself is not cytopathic.

In vitro inhibition of hemopoietic cell line growth by hepatitis B virus

The effects of hepatitis B virus (HBV) on established human cell lines of various tissue origins were evaluated by clonal or colorimetric assays in methylcellulose culture. HBV exposure inhibited the growth of six hemopoietic cell lines, while similar incubation did not affect the growth of seven nonhemopoietic carcinoma cell lines of breast, colon, liver, and stomach origin. The inhibition of hemopoietic cell line colony formation was dependent on the presence of intact viral (Dane) particles and the ratio of exposure of virions to cells and was reversible with antibodies to pre-S1, pre-S2, and S envelope protein epitopes. Purified HBV DNA, surface antigen pre-S antigens, and core antigen did not inhibit cell line growth. These results further demonstrate the tropism of HBV for cells of hemopoietic origin, confirming our previous findings on the effects of HBV on the growth of normal bone marrow progenitor cells in vitro. Established human tissue culture cell lines may be used to study the interactions of hemopoietic cells with HBV.

Expression of hepatitis B virus core and precore antigens in insect cells and characterization of a core-associated kinase activity

The hepatitis B virus core open reading frame with and without the precore domain was expressed in insect cells using a baculovirus expression system. Precore antigen was not properly processed in insect cells and was present in highly insoluble cytoplasmic aggregates. Core antigen without the precore domain formed core particles with a diameter of 28 nm that were secreted into the medium. Both core and precore antigens were phosphorylated in insect cells. The immune response in mice to both antigens yielded antibodies with a high degree of preferential reactivity for the homologous immunizing polypeptide. A kinase activity that phosphorylated core antigen was associated with highly purified core particles. The kinase activity resembled that previously demonstrated for core particles purified from the cytoplasm of infected hepatocytes and detergent-treated Dane particles. Partial resistance of the phosphate-label to phosphatase treatment suggested that some of the phosphorylated sites are in the interior of the particle. The presence of kinase activity in recombinant core particles demonstrated that this activity is not derived from another hepatitis B virus-encoded polypeptide, and the lack of a kinase consensus sequence in the core open reading frame suggests that the kinase is of cellular origin.

In vitro Virucidal Effectiveness of a 0.12%-Chlorhexidine Gluconate Mouthrinse

The purpose of this work was to assess the in vitro antiviral effectiveness of a mouthrinse (Peridex) containing 0.12% chlorhexidine gluconate (CH) on several viruses that are associated with the oral cavity. These included herpes simplex virus (HSV), cytomegalovirus (CMV), influenza A, parainfluenza, polio, and hepatitis B (HBV). Virucidal assays in tissue cultures were performed on all viruses except HBV. The virucidal effect on HBV was assessed by inactivation of the DNA polymerase contained within the Dane particle of HBV. The CH mouthrinse had virucidal activity against all of the viruses, except polio, in as little as 30 s. The virucidal activity increased with time. However, there were differences in the responses of these viruses to the challenge of the CH mouthrinse, probably due to subtle differences in the physical/chemical structures of the virus envelopes. Results on DNA polymerase of the HBV virus were similar to those on the other viruses, except polio, suggesting a common mechanism. With respect to this mechanism, it was proposed that CH exerted its antiviral effect on the envelopes of these viruses, and that the absence of an envelope on polio precluded effectiveness against this virus.

A nude mouse model for the in vivo production of hepatitis B virus

Hepatitis B virus genome-transfected HepG2 cells (2.2.15 cells) inoculated into nude mice produced tumors within 2–8 wk. Dane particles, hepatitis B virus deoxyribonucleic acid polymerase activity, hepatitis B surface antigen, and hepatitis B e antigen were detected in the serum, and 36% of mice developed antibodies to hepatitis B core antigen. In the tumors, hepatitis B surface, core, and e antigens were observed by electron microscopy and immunoenzymatic techniques. In-situ hybridization and Southern blot analysis showed hepatitis B virus deoxyribonucleic acid in the tumor. Tumors could be propagated by injection of minced tumor tissue or of a tumor-derived cell line. Liver of tumor-bearing mice as well as sera and tissues of mice inoculated with control cell lines did not show hepatitis B virus genome or viral markers. Tumors induced by both 2.2.15 and nontransfected HepG2 cells exhibited myc oncogene protein and various hepatoma-associated antigens (α-fetoprotein, α-1-antitrypsin, α-1-antichymotrypsin, carcinoembryonic antigen, cytokeratin), suggesting that viral formation does not interfere with expression of these antigens. This experimental model will be helpful to study the effect of drugs on in-vivo hepatitis B virus replication and viral antigen expression.

Immunocytochemical and Electron Microscopic Study of Hepatitis B Virus Antigen and Complete Particle Production in Hepatitis B Virus Dna Transfected Hepg2 Cells

The relationship between the presence of hepatitis B virus antigens, their localization and hepatitis B virus replication was studied in different clones of cultured HepG2 hepatoblastoma cells transfected with cloned hepatitis B virus DNA. Intracellular hepatitis B virus antigens were detected by immunofluorescence. The production of these antigens was evaluated in the culture media by enzyme-linked immunoassay. Hepatitis B virus DNA was detected using dot-blot hybridization. Three types of HBcAg staining were observed in transfected HepG2 cells: (a) cells with nuclear HBcAg, (b) cells with cytoplasmic HBcAg and (c) cells with both nuclear and cytoplasmic HBcAg. Cell types b and c also expressed hepatitis B virus DNA in their culture media. Our results suggest that cytoplasmic HBcAg may be more involved than nuclear HBcAg in hepatitis B virus replication. The site of hepatitis B virus formation in hepatocytes was studied by electron microscopic examination of a specific hepatitis B virus producer clone, thereby allowing detection of intracellular Dane particles more easily than liver biopsy samples from infected patients. Dane particles and HBsAg filaments were found in large, dilated structures probably related to the endoplasmic reticulum. Budding of core particles into cisternae of endoplasmic reticulum-related structures appears to be a possible mechanism for hepatitis B virus formation; our results suggest that the exocytosis of cisternae to extracellular spaces may be a mechanism for release of hepatitis B virus particles.

Replication of hepatitis B virus in a hepatocellular carcinoma

Hepatitis B virus transcripts and DNA from paired samples of neoplastic and nonneoplastic liver tissue of HBsAg seropositive patients were analyzed. The data obtained support the view that transcription of integrated DNA is frequent, both in neoplastic as well as in nonneoplastic liver tissue. In the case of one patient, integrated and free forms of hepatitis B virus DNA were detected in the tumor. Complete cycles of viral replication in this tumor were suggested by the following markers: (i) DNA and RNA intermediates expected to occur during replication of the viral genome, (ii) HBcAg and HBsAg, (iii) core and Dane particles. Viral DNA cloned from tumor tissue was proven to be replication competent in a transient replication assay. Five independent clones of viral DNA were established and found to be closely related at the nucleotide level. A preX open reading frame and a stop codon within preC were common features. In tissue surrounding the tumor, a nonreplicative state of virus infection prevailed, characterized by free viral DNA exclusively of the covalently closed, circular form. The replication of the viral DNA appeared to be blocked at the level of transcription.

Detection of hepatitis B virus DNA in serum by spot hybridization technique: Sensitivity and specificity of radiolabeled and biotin-labeled probes

The detection of serum hepatitis B virus (HBV) DNA in 32 chronic HBsAg carriers was performed by spot hybridization technique using both biotinylated and radiolabeled probes, in order to compare their specificity and sensitivity. Our results show that both assays are specific since neither evidence of cross-hybridization between HBV DNA and sera from patients with chronic non-A, non-B (NANB) hepatitis was found, nor HBV DNA was detected both in patients with chronic anti-HBs/anti-HBc-positive hepatitis and in patients negative for all HBV markers. An agreement between the two assays was observed in 94% of the tested sera. Even though in 5 serum samples (6%) low levels of HBV DNA (0.1-1 pg/100 microliter) remained undetected using the biotin-labeled probe, the lower detection limit of the two assays (0.1 pg of HBV DNA) was the same using purified Dane particles as control. This study indicates that the enzymatic detection of HBV DNA is suitable for routine and rapid monitoring of HBV replication in both HBeAg- and anti-HBe-positive patients, as well as for a semiquantitative analysis of serum HBV DNA.

HBcAg expressed on the surface of circulating Dane particles in patients with hepatitis B virus infection without evidence of anti-HBc formation.

Circulating immune complexes composed of HBcAg and anti-HBc have been demonstrated recently in patients with hepatitis B virus replication. After dissociation of immune complexes by chaotropic ions, HBcAg was quantified radioimmunologically. In the present study, we describe 10 patients with hepatitis B virus replication, absent or delayed anti-HBc formation and exposed HBcAg in serum. Four of the 10 patients had acute hepatitis, and six patients had chronic persistent hepatitis. In seven of 10 patients, a secondary immune defect was apparent due to acquired immunodeficiency syndrome, leukemia, histiocytosis X, sarcoidosis or end-stage renal disease. Electron microscopy demonstrated that Dane particles from anti-HBc-negative patients were agglutinated after addition of monoclonal anti-HBc antibodies, whereas Dane particles from anti-HBc-positive sera did not show agglutination. Monoclonal HBsAg-specific antibodies aggregated Dane particles independent of the presence of anti-HBc. Circulating HBcAg was always associated with the Dane particle fraction after density gradient separation. Hepatitis B virus core proteins from patients with and without anti-HBc studied by immunoblotting after sodium dodecyl sulfate-gel electrophoresis showed identical patterns. Hepatocytes from anti-HBc-negative patients were positive for HBcAg but negative for immunoglobulin G by immunofluorescence technique. The data indicate that HBcAg may also be expressed on the surface of Dane particles, where it is commonly masked by anti-HBc.

Distribution of HBcAg in hepatitis B detected by immunoperoxidase staining with three different preparations of anti-HBc antibodies.

To evaluate the role of the expression of hepatitis B core antigen (HBcAg) in liver cell damage the immunoperoxidase staining pattern of cryostat liver biopsy specimens from 16 chronic carriers of hepatitis B surface antigen (HBsAg) was investigated using three different kinds of anti-HBc antibodies. Polyclonal antibody prepared from recombinant HBcAg seemed to be more sensitive in detecting HBcAg than did monoclonal antibody from the same antigen. The topographical distribution of HBcAg detected by these two antibodies was similar, showing a close correlation to the histological activity of disease. Furthermore, the predominant localisation of cytoplasmic HBcAg usually reflected an active and severe ongoing hepatitis. On the other hand, monoclonal antibody prepared from purified Dane particles resulted in the prominent cytoplasmic staining for HBcAg regardless of histological severity of the hepatitis. The quantitative expression and topographical distribution of HBcAg depended on the type of anti-HBc antibodies used.

ウイルス除去膜としての多孔性再生セルロース中空糸を用いた血しょうろ過特性

The Bemberg Microporous Membrane (BMM) is composed of microporous regenerated cellulose hollow fiber and has been developed for virus removal from plasma. When fresh plasma was appiled on the BMM-30 (average pore size 30nm), 93 percent of albumin was passed through. However, more than 50 percent of IgM and Factor VIII were trapped in the hollow fiber. On the other hand with the BMM-50 filtration, 70 percent of IgM and Factor VIII were recovered in the filtrate. The double filtration by BMM-50 modules was carried out for HB virus positive fresh frozen plasma. Trapping of plasma components occurred only at the first BMM filtration and there was virtually no trapping at the second BMM filtration. HBV-DNA in Dane particles could be effectively removed by the double BMM-50 filtration. The residual amount of HBV-DNA in the first filtrate became under the detectable level after the second filtration. Thus, HBV-free plasma with sufficient amounts of plasma components could be obtained by the double filtration with the BMM-50 modules.

Hepatitis B virus (HBV) infections in turtles.

Thirty turtles (15 Clemys mutica and 15 Geoclemys reevesii) which were inoculated with human sera those were positive for hepatitis B surface antigen (HBsAg) and hepatitis B "e" antigen (HBeAg) were found to be infected with hepatitis B virus (HBV). The levels of HBV infection markers, such as HBsAg and antibody to HBsAg (anti-HBsAg), were retinely monitored in the turtles' serum for 46 weeks. Within two weeks of the inoculation, 42% of the turtles tested were positive for HBsAg, and their reciprocal titers as measured by reverse passive hemagglutination (RPHA) and enzyme linked immunoabsorbance assay (ELISA) ranged from 16 to 96. Within 20 weeks, the remaining turtles tested HBsAg positive, as confirmed by ELISA. At 20 weeks, all but one of the turtles exhibited changes in HBV blood marker from HBsAg to anti-HBs; the one exception was positive for both HBsAg and anti-HBs. At the 47th week, 7 animals were killed and their organs were examined for HBV infected cells utilizing an immunofluorescent technique. Numerous fluorescent cells which reacted with human anti-HBs nad anti-HBc were observed in the following organs: pancreas, liver, kidney, and brain. Histopathologically, edematous changes in hepatocytes and minor cellular infiltration attributed to an inflammatory response were noted. Liver and kidney cells from the infected animals were cultured, and HBV antigen positive cells for HBsAg and HBcAg were detected in the cultures. Throughout the experiment, HBsAg was detected in the supernatant by ELISA. Virus particles which were indistinguishable from Dane particles were seen in the cytoplasmic vacuoles of the cultured cells by electron microscopy. Finally, the presence of HBV DNA was established by molecular hybridization techniques in the culture supernatants of kidney cells from the infected turtles.

In Vitro Hepatitis B virus suppression of erythropoiesis is dependent on the multiplicity of infection and is reversible with anti-HBs antibodies

Exposure of human bone marrow mononuclear cells to hepatitis B virus results in the suppression of the in vitro growth of several hematopoietic progenitor cells. We studied the degree of inhibition of erythroid progenitor cells that results as a function of the time of exposure of mononuclear cells to hepatitis B virus and the ratio of virus to mononuclear cells, the multiplicity of infection. With an overnight incubation of mononuclear cells with hepatitis B virus-containing sera, a multiplicity of infection of greater than one virus per mononuclear cell is required to observe significant inhibition of erythroid colony formation. This multiplicity of infection effect is also observed with purified Dane particles. Exposure of mononuclear cells to an increasing number of Dane particles results in a dose-dependent suppression of erythroid colony formation with significant inhibition observed with a multiplicity of infection of virus to mononuclear cells as low as 5:1. Murine monoclonal antibodies to HBsAg completely neutralize the hepatitis B virus-mediated inhibition of CFU-E while control antibodies do not. Purified HBsAg has no effect on colony formation. In conclusion, the hepatitis B virus-mediated inhibition of erythrogenesis in vitro provides a basis for understanding the bone marrow depression observed during hepatitis B virus infections and may provide an in vitro model for examining hepatitis B virus infection.

Intranuclear inclusion bodies in liver of chronic hepatitis B with delta infection.

Seventy-two patients with HBsAg-positive chronic hepatitis were studied for serum antibody to delta antigen (anti-HD) by radioimmunoassay. Delta antigen (HDAg) in liver of the 72 patients was also studied immunohistochemically using peroxidase-labeled anti-HD. In one of the 72 patients, high titers of anti-HD and intrahepatic HDAg were detected. In this patient, HDAg was localized mainly in nuclei and occasionally in the cytoplasm of hepatocytes. By immunoelectron microscopy, HDAg was observed diffusely in some hepatocytic nuclei, but their nucleoli, perichromatin and heterochromatin were devoid of HDAg. Occasionally, inclusion body-like ring-shaped or irregularly shaped aggregates of HDAg were observed in nuclei. At higher magnification, these aggregates consisted of an HDAg-positive amorphous substance and microtubular structures. In their vicinity, scattered HDAg-positive granules were also present. By conventional electron microscopy, similar inclusion bodies composed of a clustered amorphous substance and microtubular structures 10-20 nm in diameter, together with 20-30 nm irregular granules were observed in hepatocyte nuclei, but no core of Dane particles were found. These novel intranuclear structures were not observed in other patients with chronic hepatitis B. It is suggested that they may represent the characteristic ultrastructure associated with delta infection.