Detection of hepatitis B virus DNA in hepatocytes, bile duct epithelium, and vascular elements by in situ hybridization.

Abstract

A radiolabeled probe specific for hepatitis B virus (HBV) nucleotide sequences was hybridized in situ to liver tissue from three patients with chronic hepatitis B. The HBV genome was detected not only in infected hepatocytes but also in bile duct epithelial cells, endothelial cells, and smooth muscle cells. These findings extend the known host cell range for HBV, suggest new mechanisms of viral dissemination, and illustrate the usefiuness of in situ hybridization in the study of pathogenesis of HBV infection.HBV infection is a major public health problem, with an estimated 200 million persistently infected people worldwide (1)(2)(3). HBV is a DNA virus that infects only humans and certain nonhuman primates and causes a wide spectrum of acute and chronic liver disease (4). Recent evidence further suggests that this virus is involved in the etiology of hepatocellular carcinoma (4-13). In infected individuals, the virion and DNA-free particles of hepatitis B surface antigen (HBsAg) circulate in blood, and viral antigen has been found in most body fluids, including bile (4, 14-16). However, the study of the pathobiology of HBV infection is impeded by the current inability to propagate HBV in cell culture. The availability of cloned HBV DNA now permits us to investigate by in situ hybridization the presence and quantity of viral copies at the cellular level and their correlation with histologic lesions. This paper presents findings indicating that HBV DNA in virus-infected liver is detectable not only in hepatocytes, as previously reported by Gowans et al. (17), but also in bile duct epithelium and vascular elements, thereby identifying new and unsuspected target cells for HBV. Cellular DNA Extraction. PLC/PRF/5 cells in a confluent monolayer were trypsinized and washed once with phosphatebuffered saline. The cells were lysed at room temperature by incubation in 10 mM Tris HCI, pH 7.4/1 mM EDTA/100 mM NaCl/1% sodium dodecyl sulfate. After addition of proteinase K (Boehringer Mannheim) to a final concentration of 100 ,g/ ml, the lysate was incubated at 37°C for 12-16 hr. The solution was then extracted twice with buffer-saturated phenol/chloroform/isoamyl alcohol, 25:24:1 (vol/vol), and once with chloroform/isoamyl alcohol, 24:1. The nucleic acids were precipitated with 2 vol of ethanol. The precipitate was dissolved in 10 mM Tris'HCl, pH 7.4/1 mM EDTA/100 mM NaCl, treated with DNase-free RNase (Millipore) at 100 ,ug/ml for 3 hr at 370C, and extracted and precipitated as described above. The number of HBV DNA copies per Alexander cell was determined by dot blot analysis (22) with 32P-labeled HBV DNA as a probe (specific activity, 1-2 x 109 cpm/,ug) and a known amount of cloned HBV DNA or of DNA extracted from PLC/ PRF/5 cells. The hybridization was quantitated by liquid scintillation counting. The number of four copies of HBV DNA per Alexander cell was calculated by assuming a cellular DNA content of 6 pg. MATERIALS AND METHODSIn 6685The publication costs of this article were defrayed in part by page charge ...

Hepatitis B virus transcription in the infected liver.

“…From the intensity of this band relative to standards of known concentration we estimated that on average -50 molecules of HBV DNA per cell were present in the piece of liver analysed. The number of HBV DNA molecules in hepatocytes supporting HBV replication has been estimated by in situ hybridization to be several hundreds to several thousands (Burrell et al, 1982;Blum et al, 1983), but it has also been observed that not all hepatocytes supported HBV replication (Gowans et al, 1983). Thus it is likely that only a fraction of the hepatocytes in the liver biopsy used for DNA and RNA extraction was productively infected, and that the number of HBV genomes (-50) and HBV transcripts (20-50) per infected cell is in fact much higher.…”

Frequency of hepatic HBV-DNA in patients with cirrhosis and hepatocellular carcinoma: relation to serum HBV markers

“…A similar situation occurred when using immuno-histochemistry Tur-Kaspa et al, 1986) and in situ hybridisation in combination with molecular hybridisation (Blum et al, 1983). Liver HBV-DNA sequences have been detected in patients with putative NANB hepatitis (Figus et al, 1984), but we, like Harrison et al (1986), were unable to confirm these findings in any of our nine presumed NANB patients.…”

Comparative Expression of the Hepatitis B Surface Antigen Gene in Biochemically Transformed Human, Simian and Murine Cells

“…The human hepatitis B virus (HBV) causes polymorphic liver diseases in man and certain non-human primates (Szmuness, 1975). The host cell range of HBV is more extensive than had been previously thought, since HBV DNA has been detected not only in hepatocytes but also in bile duct epithelium, endothelial cells, smooth muscle cells of blood vessel walls (Blum et al, 1983), and lymphoblastoid cells from bone marrow (Romet-Lemonne et al, 1983). There is no in vitro cellular system available in which the replication of HBV can be studied.…”