Dane Particles, DNA Polymerase, and e-Antigen in Two Different Categories of Hepatitis B Antigen Carriers

Nordenfelt, Erik; Kjellén, Lars

doi:10.1159/000149918

Cited by 163 publications

(62 citation statements)

References 14 publications

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“…We have purified Dane particles from plasma containing HBAg and HBeAg. HBeAg is regarded as an indicator of the presence of Dane particles in a high concentration because it is closely associated with HB,Ag and HBV-specific DNA polymerase (Nordenfeld and Kjellen, 1975;Imai et al, 1976;. In the present report, equilibrium ultracentrifugation in metrizamide density gradient has been successfully applied to the separation of the cores of Dane particles containing a DNA strand, taking advantage of the fact that the density of DNA decreases markedly in metrizamide solution because it becomes hydrated (Birnie et al, 1973).…”

Section: Discussionmentioning

confidence: 85%

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: Discussionmentioning

confidence: 85%

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 following connections have been found. (i) When hepatitis B e antigen (HBeAg), which is part of HBcAg (Ohori et al, 1979; Mackay et al, 1981), is detectable in serum, Dane particles, HBV-specific DNA polymerase and DNA are demonstrable in the serum (Nordenfelt & Kjellen, 1975;Imai et al, 1976;Ohori et al, 1980) and HBcAg and HBV DNA in liver tissues (Bonino et al, 1981;Hadziyannis et al, 1983). (ii) The localization of HBcAg in hepatocytes varies even in the same tissue (Gudat et al, 1975;Yamada & Nakane, 1977; Huang & Neurath, 1979).…”

Section: Discussionmentioning

confidence: 99%

Relationship between the Replication of Hepatitis B Virus and the Localization of Virus Nucleocapsid Antigen (HBcAg) in Hepatocytes

Akiba¹,

Nakayama

Miyazaki

et al. 1987

Journal of General Virology

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SUMMARYAccording to the localization of hepatitis B virus (HBV) core antigen (HBcAg), detected by the avidin biotin complex method, infected hepatocytes were classified into three types, i.e. those having nuclear (type I), nuclear and cytoplasmic (type II) or only cytoplasmic (type III) antigen. HBcAg-positive hepatocytes of all specimens (three) from non-specific reactive hepatitis and of most (five of seven) from chronic persistent hepatitis (CPH) patients were only type I; the other two CPH samples and all (seven) chronic active hepatitis samples were composed of a mixture of types I, II and III. Linear correlations between the frequency of type I, as well as that of all types (I, II and III) of the HBcAg-positive hepatocytes, and the amount of HBV DNA in serum were found. The relative HBV production of HBcAg-positive hepatocytes (serum HBV DNA amount/frequency of HBcAg-positive cells) was 0.11 in type I and 0-07 in all hepatocytes including types I, II and III. HBV core particles and complete HBV particles were found in type I hepatocytes. On the other hand, these particles were not found in a predominantly type III liver specimen. These results suggest that type I hepatocytes are more involved in the propagation of HBV than types II and III.Several immunological, histological and biochemical studies on the relationship between the expression of hepatitis B virus (HBV)-related antigens, especially of HBV core antigen (HBcAg), and viral replication have been performed using serum and liver tissues from patients with type B hepatitis. The following connections have been found. (i) When hepatitis B e antigen (HBeAg), which is part of HBcAg (Ohori et al., 1979; Mackay et al., 1981), is detectable in serum, Dane particles, HBV-specific DNA polymerase and DNA are demonstrable in the serum (Nordenfelt & Kjellen, 1975;Imai et al., 1976;Ohori et al., 1980) and HBcAg and HBV DNA in liver tissues (Bonino et al., 1981;Hadziyannis et al., 1983). (ii) The localization of HBcAg in hepatocytes varies even in the same tissue (Gudat et al., 1975;Yamada & Nakane, 1977; Huang & Neurath, 1979). However, because conflicting results have been obtained in studies of the localization of HBcAg and the presence of HBV DNA in hepatocytes, valid conclusions about the correlation between HBV replication and HBcAg expression in hepatocytes can not yet be drawn (Blum et al., 1984;Burrell et al., 1985).In the present study, we have examined staining conditions for the detection of HBcAg, and classified the HBcAg-positive hepatocytes with respect to the localization of this antigen in liver tissues from patients whose sera were positive for HBeAg. The amount of HBV DNA in serum was determined and electron microscopic observations were made. These results are discussed with regard to virus replication in the three types of hepatocyte. t Present address:

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“…A close physical correlation between HBeAg and HBV particles, however, was later shown Yoshizawa et al, 1979) and HBeAg is now regarded as an integral component, in a cryptic form, of core particles (Yoshizawa et al, 1979;Budkowska et al, 1979;Ohori et al, 1979). There is a strong correlation between the detection in patients' sera of HBeAg and the presence of either HBV particles (Nordenfelt & Kjellen, 1975;Imai et al, 1976;Maynard et al, 1976) or the HBV particle-associated HBcAg Trepo et al, 1976).…”

Section: Introductionmentioning

confidence: 98%

Immunological and Morphological Properties of HBeAg Subtypes (HBeAg/1 and HBeAg/2) in Hepatitis B Virus Core Particles

Ohori

Shimizu

Yamada

et al. 1984

Journal of General Virology

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Dane Particles, DNA Polymerase, and e-Antigen in Two Different Categories of Hepatitis B Antigen Carriers

Cited by 163 publications

References 14 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

Relationship between the Replication of Hepatitis B Virus and the Localization of Virus Nucleocapsid Antigen (HBcAg) in Hepatocytes

Immunological and Morphological Properties of HBeAg Subtypes (HBeAg/1 and HBeAg/2) in Hepatitis B Virus Core Particles

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