New Antigen-Antibody System in Australia-Antigen-Positive Hepatitis

Almeida, June D.; Rubenstein, David; Stott, E. J.

doi:10.1016/s0140-6736(71)90543-5

Cited by 326 publications

(111 citation statements)

References 4 publications

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“…Dane particles share hepatitis B surface antigen (HBAg) with 20-nm spherical and tubular forms, but their cores, hepatitis B core antigen (HB,Ag), have a distinct antigenicity (Almeida, Rubenstein and Stott, 1971). We have developed a method for the isolation of Dane particles from the plasma of asymptomatic carriers of HBAg on a large scale, and demonstrated a double-stranded DNA molecule extruding directly from their cores (Takahashi, T. et al, 1976).…”

Section: Plate Xi1mentioning

confidence: 99%

Isolation of Dane Particles Containing a Dna Strand by Metrizamide Density Gradient

Takahashi

Kaga

Akahane

et al. 1980

Journal of Medical Microbiology

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PLATE XI1DANE, CAMERON and BRIGGS (1970) identified 42-nm "Dane" particles in the serum of individuals infected with hepatitis B virus (HBV), and they have been accepted as representing HBV. Dane particles share hepatitis B surface antigen (HBAg) with 20-nm spherical and tubular forms, but their cores, hepatitis B core antigen (HB,Ag), have a distinct antigenicity (Almeida, Rubenstein and Stott, 1971). We have developed a method for the isolation of Dane particles from the plasma of asymptomatic carriers of HBAg on a large scale, and demonstrated a double-stranded DNA molecule extruding directly from their cores (Takahashi, T. et al., 1976).In the course of this study, we realised that the cores of Dane particles shedding a DNA strand were surprisingly few; more than 90% of Dane particles were thought to be "empty" because they did not seem to contain any DNA strand. The separation of the Dane particles containing DNA from such defective ones, to create a homogenous population of complete HBV, would be a prerequisite for the study and understanding of their physicochemical properties, such as the DNA structure.Birnie, Rickwood and Hell (1973) observed that the density of DNA decreased remarkably in the presence of metrizamide due to its hydration. They noted that DNA had a density of 1.1 g/cm3 in metrizamide solution, as against 1.7 g/cm3 in CsCl solution. Taking advantage of this phenomenon, we tried to separate the cores of Dane particles containing DNA from those without DNA. MATERIALS AND METHODSIsolation of Dane particles. Blood-plasma units of asymptomatic carriers containing hepatitis B e antigen (HBeAg) (Magnius and Espmark, 1972) were pooled, and Dane particles were isolated by a succession of steps including continuous flow zonal centrifugation, floating-type centrifugation, and rate zonal centrifugation . When the purified preparation was observed in an electron microscope, more than 98% of all the particulate elements were identified as Dane particles. From 1.3 litres of plasma the yield of Dane particles was approximately 2 x lo'*.Ultracentrifugation in metrizamide density gradient. The Dane-particle preparation was labelled with tritiated thymidine triphosphate ([3H]TTP) in the presence of Nonidet P-40 (NP-40) by a modification (Moritsugu et al., 1975) of the method originally described by Kaplan et al. (1973), and a mixture of Dane-particle cores with and without radioactivity was obtained. Dane-particle core preparation (1 ml) was brought to a density of 1 -35 g/cm3 by the addition of metrizamide (Nyegaard & Co., AS, Oslo), and applied to the bottom of a Beckman SW-65 tube. A linear gradient of metrizamide, density 1.30-1 .lo g/cm3 (volume 4.5 ml) was prepared on the surface of the sample, and the tube was centrifuged at 50000 r.p.m. for 16 h. Metrizamide solution with a density of 1.40 g/cm3 was applied at the bottom, and 0.2-ml

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Section: Plate Xi1mentioning

confidence: 99%

Isolation of Dane Particles Containing a Dna Strand by Metrizamide Density Gradient

Takahashi

Kaga

Akahane

et al. 1980

Journal of Medical Microbiology

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“…The virion (also known as the Dane particle) has a complex structure (1, 2) with hepatitis B surface antigen (HBsAg) on its outer lipid-containing envelope (1,2) and the distinct hepatitis B core antigen (HBcAg) on its internal core or nucleocapsid (2). Substantial evidence indicates that the HBsAg is specified by the viral genome (3).…”

mentioning

confidence: 99%

Restriction endonuclease cleavage map and location of unique features of the DNA of hepatitis B virus, subtype adw2.

Siddiqui¹,

Sattler²,

Robinson³

1979

Proc. Natl. Acad. Sci. U.S.A.

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Hepatitis B virus (HBV) has several interesting features. The virion (also known as the Dane particle) has a complex structure (1, 2) with hepatitis B surface antigen (HBsAg) on its outer lipid-containing envelope (1, 2) and the distinct hepatitis B core antigen (HBcAg) on its internal core or nucleocapsid (2). Substantial evidence indicates that the HBsAg is specified by the viral genome (3). A group-specific determinant a found in all HBsAg preparations and two pairs of subtype determinants (d, y and w, r), which are for the most part mutually exclusive and thus behave as alleles, have been described (4, 5). Antigenic heterogeneity of the w determinant and additional determinants such as q and x or g have been found (6). Virus strains of the eight HBsAg subtypes aywi, ayw2, ayw3, ayw4, ayr, adw2, adw4, and adr as well as more complex and less common subtypes such as adyw and adywr ha've been identified (7). Both group-specific and type-specific HBsAg determinants appear to reside in the major 22,00dalton polypeptide, which can be isolated from purified HBsAg particles (8).Hepatitis B virions contain a circular DNA molecule (9) We have constructed a map of restriction endonuclease cleavage sites of HBV DNA (HBsAg subtype adw2) and have localized certain physical features of the DNA within this map, including the single-stranded region, the 5' end of the short strand, and the nick in the long strand. We have also found systematic differences in the restriction endonuclease cleavage patterns for DNA of HBV of HBsAg subtypes adw2, ayw3, and adrq+. MATERIALS AND METHODSPlasma rich in Dane particles was obtained by plasmaphoresis from patients persistently infected with HBV (HBsAg carriers). HBsAg subtyping was kindly performed by A. M. CouroucePauty. The detailed methods of Dane particle preparation and DNA extraction have been described (9). The single-stranded region of HBV DNA was always made double-stranded by an extensive virion DNA polymerase reaction in the presence of high deoxynucleoside triphosphate (dNTP) concentrations as described (12)

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“…Immunogold electron microscopy was carried out as previously described (16)(17)(18). We used goat polyclonal anti-HBs antibody (Biostride, USA) as the primary antibody, and colloidal gold-conjugated rabbit anti-goat IgG as the secondary antibody.…”

Section: Methodsmentioning

confidence: 99%

Ultrastructural demonstration of hepatitis B virus production in a mouse model produced by hydrodynamic transfection

Konishi

Tanaka²,

Kaito³

et al. 2007

Int J Mol Med

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Abstract. A mouse model of hepatitis B virus (HBV)infection produced by hydrodynamic injection of HBV DNA has been recently established. However, the ultrastructural demonstration of HBV particles in this mouse model has not as yet been reported. In our study, plasmid DNA containing wild-type HBV DNA was rapidly injected into 8-week-old female SCID mice via the tail vein. Serum levels of HBsAg were measured by ELISA kit. Intrahepatic HBV protein expression was detected by immunohistochemistry of HBcAg. Ultrastructural study of the serum samples was performed by transmission electron microscopy and immunogold electron microscopy. Serum HBsAg and intrahepatic HBcAg were detected in HBV DNA-injected mice for at least 14 days. Spherical and filamentous particles 22 nm in diameter and double-shelled Dane-like particles 42 nm in diameter were detected in the sera of mice. The ultrastructural features of these particles were identical to HBV particles observed in serum from chronic hepatitis B patients. These particles were confirmed to be HBV particles by immunogold electron microscopy. We conclude that our present HBV mouse model using hydrodynamic transfection of HBV DNA is appropriate for production of HBV virions including Dane particles. This mouse model may be useful for screening in vivo the efficacy of antiviral drugs.

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New Antigen-Antibody System in Australia-Antigen-Positive Hepatitis

Cited by 326 publications

References 4 publications

Isolation of Dane Particles Containing a Dna Strand by Metrizamide Density Gradient

Isolation of Dane Particles Containing a Dna Strand by Metrizamide Density Gradient

Restriction endonuclease cleavage map and location of unique features of the DNA of hepatitis B virus, subtype adw2.

Ultrastructural demonstration of hepatitis B virus production in a mouse model produced by hydrodynamic transfection

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