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 ...
Family members of 13 patients with hepatitis B surface antigen (HB,Ag) positive primary hepatocellular carcinoma (PHC) were tested for the presence of hepatitis B virus-associated antigens and antibodies. Of the 122 members examined, circulating HB,Ag was detected in 47 (39%), antibody to HB,Ag (anti-HB,) was found in 37 (30%), and antibody to hepatitis B core antigen (anti-HB,) alone was present in 13 (11%). The relatives with the highest frequency of HB,Ag positivity were the offspring of the propositus, followed by the nieces and nephews and the grandchildren. Anti-HB, and anti-HB, were detected most often in the spouses and non-blood relatives. Evidence for past and present hepatitis B virus (HBV) infection was more frequently found in the Asian family members when compared to the non-Asians. The e antigen (HB,Ag) was present in 38% of the HB,Ag positive individuals, including four with PHC; antibody to HB,Ag (anti-HB,) was rarely detected. These results indicate that clustering of HBV infection was commonly present in family members of patients with PHC. The HB,Ag positive individuals may be major contributors to the endemic pool of the virus, and may themselves be potential cases of chronic active type B hepatitis, cirrhosis, and PHC.Cancer 44:2338-2342, 1979.HC IS A CONDITION that has been asso-P ciated with the presence of the HBV.Recent studies from various parts of the world have shown that HB,Ag has been detected in the circulation of 40% to 80% of patients has not been shown to be directly oncogenic in man or animals, the high frequency of HB,Ag positivity in individuals with PHC strongly suggests that chronic HBV infection may contribute in some way toward the eventual development of primary liver cell carcinoma.The clustering of HB,Ag within families of patients with PHC was first reported by Ohbayashi et al. from Japan.Is In a study of three kindred, six members had the diagnosis with pHC.Z3l0~1l ,18,23,25,26,30 Although this virus
Blood donations are routinely screened by multiple serologic assays for antigens/antibodies associated with infection by blood-borne viruses, including hepatitis B virus (HBV), hep- MATERIALS AND METHODSTwo pedigreed human plasma samples containing HBVladw and HBVlayw, respectively, were re-Sources of plasma with HBV.
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