Detection of hepatitis B virus-DNA in liver tissues in chronic type B liver diseases and its relevancy to viral antigens. A study by in situ hybridization using a biotinylated probe and streptavidin-alkaline phosphatase.

Abstract

In situ hybridization using a biotinylated probe and streptavidin-alkaline phosphatase was applied to the detection of hepatitis B virus(HBV)-DNA in formaim-fixed and paraffin-embedded liver-biopsied tissues from 24 patients with chronic type B liver diseases (all carriers of serum HBsAg, and the results were compared with those of the immunohistochemical detection of HBsAg and HBcAg.The specificity of in situ hybridization reactions was confirmed by negative staining of the control tests. HBV-DNA was detected in the sections of 8 cases with serum HBeAg and one case without serum HBeAg, and was located predominantly in the cytoplasm of hepatocytes showing various staining patterns such as diffuse, regional or peripheral.The lobular or pseudolobular distribution of HBV-DNA in the liver tissues was divided into scattered, clustered and diffuse type. The scattered type was observed predominantly in the sections of cases with active liver cirrhosis.As regards HBsAg and HBcAg in the liver tissues, HBsAg was detected in the sections of 18 cases with or without serum HBeAg; 8 were detectable for HBV-DNA by in situ hybridization and 10 were not detectable. HBcAg was detected in the sections of 9 cases all with serum HBeAg; 6 were detectable for HBV-DNA by in situ hybridization and 3 were not detectable. The sections of 5 out of the 6 detectable cases for HBV-DNA revealed the cytoplasmic HBcAg in hepatocytes.These findings suggested that HBV-DNA in hepatocytes detected by in situ hybridization correlated with the presence of serum HBeAg and cytoplasmic HBcAg rather than nuclear HBcAg in hepatocytes in chronic type B liver diseases.Examinations of hepatitis B (HB) surface antigen (HBsAg) and core antigen (HBcAg) in liver tissues as well as serum HBeAg, anti-HBe antibody and anti-HBc antibody have provided useful information for diagnosis and prognosis in chronic type B liver diseases. Of these antigens or antibodies, HBcAg in hepatocytes and serum HBeAg are considered to be markers of active replication of the HB virus (HBV). Recently, HBV-DNA, as a direct marker of HBV-replication in chronic type B liver diseases, has been detected in serum or liver tissues by nucleic acid hybridizations including in situ hybridization (1-5, 7, 8, 10, 11, 14, 17, 18, 20, 23). The technique of in situ hybridization has allowed for identification of viral infected cells and observa-489

Hepatitis B virus core antigen has two nuclear localization sequences in the arginine-rich carboxyl terminus

“…This could direct the nucleocapsid and HBV DNA from an infecting virion to the nucleus, where the next steps in the viral life cycle, including the transcription of the viral genome, occur. The observation that immature nucleocapsids containing pregenomic RNA and minus-strand DNA are localized to the cytoplasm (2,18,19,30,31) suggests that the nuclear localization sequence can be inactivated under certain circumstances. This might occur by a conformational change in the HBcAg polypeptide or as a consequence of the phosphorylation of the serine residues in the carboxylterminal region of the HBcAg polypeptide (3,16,45).…”

Southern-blot analysis and simultaneous In Situ detection of hepatitis B virus-associated DNA and antigens in patients with end-stage liver disease

“…In early studies, events in the virus life cycle were correlated with histopathological findings by examining the physical state of viral DNA extracted from whole tissue or by assaying for the presence of viral antigens in infected tissues (11)(12)(13)(14)(15). The majority of studies performing in situ analysis of both viral-specific DNA and antigens evaluated sequential serial sections from needle biopsy specimens of diseased tissue and therefore provided only limited information about histopathology or the relationship between HBV DNA replication and gene expression at the single-cell level (11)(12)(13)(14)(15)(16)(17)(18). Although the simultaneous detection of HBV DNA and antigens has 1036 HANETAL.…”

In situ hybridizaiton in Viral hepatitis

“…Since &he first report of Gowans et al (9), several investigators have described the detection of hepatitis B virus (HBV) sequences by ISH in naturally infected cells from human specimens (8, [10][11][12][13][14][15][16][17][18][19], in the HBsAg-producing hepatoma cell line PLC / PRF / 5 (1 4,20) and in isolated metaphase chromosomes obtained from the same cell line (21). Similar analyses were performed in the woodchuck animal model experimentally infected with the woodchuck hepatitis virus (WHV) (22,23) and in the Pekin duck infected with the duck hepatitis B virus (DHBV) (24), two hepadnaviruses closely related to HBV.…”

“…At the cellular level, the presence of cytoplasmic HBV-replicative DNA intermediates appears to be associated with the cytoplasmic expression of the HBV nucleocapsid protein (HBcAg) (15,25). The nuclear expression of HBcAg seems unrelated to the presence of viral nucleic acid, and it has been proposed that it may represent an excess of void core particles migrating to the nucleus (25).…”