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A new cell line derived from a woodchuck hepatocellular carcinoma serially transplanted in athymic nude mice has been established and named WH257GE10. The original tumor in the nude mouse system produces woodchuck hepatitis surface antigen and albumin. In addition, woodchuck hepatitis virus DNA is integrated into cellular DNA. Adaptation of the cells to the in vitro culture condition was completed after 15 months with the doubling time of 40 hr. The morphologic features of the cell by light microscopy are of an epithelial type. The modal chromosome number is 36 and the karyotype is mainly metacentric, similar to that observed in normal woodchuck liver cells. Ornithine and tyrosine aminotransferase activities were detected. Production of albumin was demonstrated in the cytoplasm by indirect immunofluorescence. Integration of woodchuck hepatitis virus DNA was shown by Southern blot analysis, although the secretion of woodchuck hepatitis surface antigen was not detected. This cell line provides an excellent in vitro model to study human hepatocellular carcinoma related to hepatitis B virus.

Solid-phase radioimmunoassays for woodchuck hepatitis virus core antigen (WHcAg) and antibody (anti-WHc) were developed. WHcAg in woodchuck liver homogenates was characterized by ultracentrifugation in CsCl gradients; both heavy (1.35 g/cm3) and light (1.31 g/cm3) cores were obtained from the liver of an animal during acute WHV infection, which is consistent with observations in hepatitis B virus infection in man. Endpoint titers of anti-WHc were higher in chronic WHV carriers than in animals recovered from acute infections. Both IgM and IgG anti-WHc antibodies were produced by infected woodchucks. A survey of colony woodchucks demonstrated that 88/89 animals having one or more markers of past or ongoing WHV infection were positive for anti-WHc. Thus, serum anti-WHc appears to be a sensitive marker of WHV infection.

Five out of seven American woodchucks inoculated with woodchuck hepatitis virus developed antigenemia after 2 to 13 weeks followed by an antibody response. One animal became a carrier, and another animal exhibited a primary antibody response. Clinical disease was not obvious and aminotransferase elevation could not be demonstrated. Liver biopsy showed mononuclear portal infiltration and little parenchymal cell necrosis.

Solid-phase radioimmunoassays for woodchuck hepatitis virus (WHV) surface antigen (WHsAg) and antibody to it (anti-WHs) were developed. The test for WHsAg could detect as little as 10 ng/ml. In both tests it was necessary to employ radiolabeled WHsAg instead of anti-WHs as the probe because the latter appeared to be labile to the conditions of labeling. The tests were used to characterize naturally acquired and experimental WHV infections of woodchucks. Forty-three of 72 wild-caught woodchucks had serological evidence of WHV infections; 16 of these resulted in chronic infection, and the remainder were self-limiting. All chronically infected animals were positive for WHsAg and DNA polymerase activity. During 3 years of observation, 11 of the 16 WHsAg-positive animals and 3 of the 27 anti-WHs-positive animals, but none of the 21 uninfected animals developed hepatocellular carcinoma. Seroconversion, possibly resulting from infection with WHV, was documented in a chimpanzee inoculated with WHV. An immune adherence hemagglutination test for WHsAg was also developed by using anti-WHs of chimpanzee origin as a reagent, but the test was not useful for detecting anti-WHs of woodchuck origin because of the lability of the latter.

'Ausria II' polystyrene beads (Abbott Labs, N. Chicago) are reacted with woodchuck serum positive for WHsAg in a dilution predetermined by titration. This modified bead is used in a blocking assay to detect the presence of antibody to the surface antigen of woodchuck hepatitis virus (anti-WHs). Serum containing woodchuck anti-WHs and commercial horseradish peroxidase (HRP) labeled anti-HBs are sequentially added. A drop in optical density at 492 nm of 50% or more due to the blocking of HRP conjugated anti-HBs by anti-WHs compared with a control (negative woodchuck serum) is a measure of anti-WHs. The ease and simplicity of converting readily available 'Ausria II' beads to specific reagents for detecting anti-WHs should be welcomed by investigators studying WHV. The method described is both sensitive and reproducible.

Woodchuck sera were tested for antigens and antibodies with tests which detect human hepatitis virus antigens and antibodies. Data on 264 woodchuck sera are presented.

Hepatic imaging with 99mTc-sulfur colloid was used to diagnose primary hepatocellular carcinoma (PHC) in woodchucks infected with woodchuck hepatitis virus (WHV). Based on imaging results, 6 of 12 WHV-infected woodchucks had space-occupying hepatic lesions, and all 6 had PHC. Of the remaining woodchucks, 2 did not have PHC, 2 had discrete tumors (less than 1 cm diameter), 1 had miliary small tumors, and 1 had tumors located near the great vessels. Hepatic imaging was a valuable technique for diagnosis of PHC in WHV-infected woodchucks.

The extent of association between woodchuck hepatitis virus surface antigen and host hepatocyte plasma membrane in chronic hepatitis was studied. Purified membranes containing the antigen were treated with various agents which perturb plasma membrane constituents to elute woodchuck hepatitis virus surface antigen. The products from disrupted membranes were analyzed by sedimentation in sucrose gradients and tested to identify the antigen reactivity. The results indicated that membrane-bound woodchuck hepatitis virus surface antigen was partially released by 4 M potassium chloride, potassium thiocyanate and guanidine, 6 M urea or 0.1 N sodium hydroxide (pH 13.5), but not in the presence of low concentrations of these reagents or by 10% 2-mercaptoethanol and 1% sodium dodecyl sulfate. No more than 15% of the total membrane-associated woodchuck hepatitis virus surface antigen was eluted by 0.1 N NaOH, which was found to be the most effective eluent among tested agents at the antigen removal. The remaining woodchuck hepatitis virus surface antigen was resistant to further extraction with sodium hydroxide, as expected for an integral membrane protein. Treatment of the infected membranes with 1% Triton X-100 or 50 mM deoxycholic acid, that solubilize the membrane lipid bilayer releasing most of the integral membrane proteins, resulted in the sedimentation of almost all detectable woodchuck hepatitis virus surface antigen reactivity with the detergent-insoluble membrane residues, suggesting a firm interaction of the antigen with the plasma membrane matrix.(ABSTRACT TRUNCATED AT 250 WORDS)