The hepatitis B virus-associated , antigen was found in the serum of experimentally infected chimpanzees as an internal component of a discrete subpopulation of hepatitis B surface antigen (HBsAg) particles. The 35-to 37-nm particles banded in CsCl at 1.24-1.25 g/cm3 and sedimented with a mobility intermediate between that of the hepatitis B virion and that of the 22-nm form of HBsAg. The particles contained only indistinct internal structure by electron microscopy and were not unique to 5 agent infection, similar particles without 5-antigen activity being observed in the preinfection serum of HBsAg carrier chimpanzees. A small RNA (Mr, 5 X 105) was temporally associated with 5 antigen in the serum of infected chimpanzees and copurified with the -antigen-associated particles. This RNA is smaller than the genomes of known RNA viruses but larger than the viroids of higher plants. The 3 antigen (6-Ag), a relatively new specificity, first was detected by immunofluorescence in the liver of human subjects with chronic hepatitis B surface antigen (HBsAg) hepatitis (1). Ultrastructural studies have failed to demonstrate components of hepatitis B virus (HBV) in b-Ag-positive nuclei (2) and the b-Ag-anti-b-Ag system is distinct from the known antigenantibody systems of HBV (3). Prevalence studies of b-Ag-anti-3-Ag in human populations (4,5) and transmission experiments in chimpanzees (6) indicate that b-Ag is associated with a transmissible pathogenic agent, 3 agent, that is either a HBV mutant with characteristics of a defective interfering particle or a new agent which requires helper functions of HBV for its expression.After extraction from hepatocyte nuclei with guanidine hydrochloride, b-Ag was characterized as a protein with a molecular weight of approximately 68,000 (2). Although b-Ag has not been detected in the sera of patients with intrahepatic b-Ag, such individuals develop high titers of anti-3-Ag which might interfere with the available solid-phase radioimmunoassay for b-Ag. The analysis of serial specimens from chimpanzees to which 3-agent was transmitted revealed 6-Ag in the sera during the acute phase of infection and prior to the development of anti-3-Ag (6). We report here the association of 3-Ag in serum with a discrete subpopulation of HBsAg and a low molecular weight RNA. MATERIALS AND METHODSSource of 5Ag. Two chronic HBsAg-carrier chimpanzees (nos. 29 and 800) were infected with 3 agent by inoculation with serum from a patient with chronic type B hepatitis and intrahepatic b-Ag. Serum samples and percutaneous liver biopsies were taken from each animal before inoculation and weekly thereafter and analyzed for markers of b-Ag and HBV. TheseThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. 6124 chimpanzees were part of a transmission study of the 3 agent and experimental details are reported elsewhere (6). Serum samples containing b-Ag...

ABSTRACT6 agent of human origin was inoculated into four woodchucks chronically infected with woodchuck hepatitis virus (WHV). The animals developed 6 infections with serologic patterns similar to those previously observed in human and chimpanzee infections. 6 antigen was detected transiently in serum and liver and was followed by seroconversion to anti-6 antibody. Analogous to the chimpanzee model of 6 infection, serum and hepatocyte markers of WHV were suppressed in the woodchuck during acute 6 infection. The suppression of WHV DNA in serum was evident only during the time of 6-antigen positivity, while the inhibition of other WHV markers was more protracted. The 6 antigen in woodchuck sera circulated as an internal component of a particle similar in size to the human 6 particle (36-nm diameter) and was encapsidated by the woodchuck hepatitis virus surface antigen; 6 antigen from infected woodchuck and chimpanzee livers had similar biophysical properties. Histologic analysis showed that experimental 6 infection is associated with a transient acute hepatitis in woodchucks and loss of hepatocytes carrying WHV antigens. The lesions differed from the conspicuous hepatitis associated with reappearance of WHV replication. Hepatitis B-like viruses, therefore, appear to provide the requisite helper functions for 6 replication and the woodchuck represents a useful model for study of the virology and pathology of the 6 agent. (HCC) (8-10). Therefore, we investigated the possibility of using the WHV-infected woodchuck as a surrogate host for propagation of 8 agent of human origin.

Sera and liver biopsies from 30 Italian patients, carriers of HBsAg for at least 3 years, were examined for markers of hepatitis B virus (HBV) infection by serological assays and immunofluorescence. Biopsies were analyzed for HBcAg, HBsAg, and delta antigen by immunofluorescence; sera were assayed for HBsAg/anti-HBs, HBcAg/anti-HBc, HBeAg/anti-HBe, delta/anti-delta, HBV-specific DNA polymerase activity and the presence of HBV DNA. HBcAg, HBeAg, and DNA polymerase tests were positive in the sera of 71, 86, and 57%, respectively, of carriers with intrahepatic HBcAg. HBV DNA was detected in 100% of patients expressing HBcAg in the liver with a strong correlation between the concentration of serum DNA and the intensity of HBcAg immunofluorescence in the liver. HBV DNA was detected in the sera of 63% of carriers with intrahepatic delta where the other markers of HBV replication (HBeAg, DNA polymerase) were undetectable. The assay for serum HBV DNA appears to be an excellent noninvasive method for detecting active replication of HBV in HBsAg carriers.

An assay based on nucleic acid hybridization detects and quantitates hepatitis B virus (HBV) DNA in particles present in serum. This assay allows rapid examination of multiple samples and is sensitive and reproducible; serum samples are treated with proteolytic enzyme and detergent and then extracted with phenol and chloroform. The deproteinized extracts which may contain HBV DNA are made alkaline to denature the DNA, neutralized, and bound to nitrocellulose filters in 3-mm in diameter "spots" in a special 96-well filtration apparatus. HBV DNA is detected by its ability to hybridize with 32P-labeled DNA prepared from recombinant plasmids containing the complete HBV genome. After hybridization, the nitrocellulose is washed and autoradiographed; samples containing HBV produce spots on X-ray film whose intensity (in the linear exposure range of the film) is proportional to the amount of HBV DNA in the serum sample. The assay is specific and sensitive, correlates with infectivity of sera titered in chimpanzees as well as biophysical parameters, and is in agreement with serological indicators of HBV presence.

We have constructed a simian virus 40 recombinant carrying a fragment of DNA from hepatitis B virus. Cultured monkey kidney cells infected with this recombinant produce hepatitis B surface antigen. The antigen is excreted into the culture medium as 22-nm particles with the same physical properties, antigenic composition, and constituent polypeptides as those found in the sera of patients with type B hepatitis.At least half of the world population shows evidence of past or present infection by hepatitis B virus (HBV), and the approximately 200 million carriers in the world are at serious risk of chronic liver disease and, possibly, primary liver cancer. The classic marker for chronic infection by this virus is the surface antigen HBsAg which circulates in the serum of HBV carriers in three forms: 22-nm spherical particles, 22-nm filaments of various lengths, and the 42-nm spherical form known as the Dane particle. The 22-nm particles and filaments are subviral forms containing two predominant polypeptides, with apparent molecular weights of about 23,000 and 29,000, together with several minor polypeptides of larger size (1, 2). The two predominant species, which are probably identical except that the larger is glycosylated, carry both the group (a) and the subtype (d/y) antigenic determinants of HBsAg (3). The Dane particle, which represents the infectious virion, consists of a lipoprotein coat (HBsAg) surrounding an internal core particle which contains a DNA polymerase and the 3200-base pair (bp) circular DNA genome. The 22-nm particle is the predominant form in the sera of chronic carriers and circulates at concentrations as high as 100-200 ,ug/ml.Characterization ofthe life cycle and biology ofHBV has been hampered by its narrow host range, which is restricted to humans and a few other primates, and by its inability to grow in cultured cells. Recently, however, several groups have succeeded in cloning the viral genome in Escherichia coli phage A (4) and plasmid vectors (5, 6) and in determining its primary structure (7)(8)(9). This has allowed the identification of a continuous 892-bp sequence that could encode surface antigen (7), a 549-bp sequence that may specify the core antigen (8), and several additional open sequences of unknown function (9).Although the DNA sequence provides crucial structural information, it clearly is not sufficient to establish all of the HBV gene products or to indicate how these products interact during infection of the target cell. For this purpose it would be useful to develop a system for introducing defined portions ofthe viral genome into cultured cells. Simian virus 40 (SV40), a small DNA tumor virus that can lytically infect cultured monkey cells, provides a useful vector for this purpose. In this paper we describe the construction and propagation of a SV40 recombinant carrying a 1350-bp fragment of HBV DNA that includes the structural sequences for surface antigen. We show that monkey kidney cells infected with this recombinant synthesize surface antigen that is ...