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treatment of tree shrews in China 15,16 as well For the systematic analysis of various clinical and moas the easy adaptation of these animals to the laboratory lecular aspects of hepatitis B virus (HBV) infection, an environment, 17,18 we systematically analyzed the tree shrew experimental small animal system of HBV infection species tupaia belangeri for the study of HBV infection in would be a great advance. The susceptibility to HBV invitro and in vivo. fection, therefore, of hepatocytes from the tree shrew species tupaia belangeri was studied in vitro and in vivo. MATERIALS AND METHODS Primary hepatocytes isolated from livers of tupaias can be reproducibly infected with HBV. In vitro infectionTupaia Colony. For breeding purposes, animals (tupaia belangeri) were obtained from the German Primate Center, Göttingen, Gerresults in viral DNA and RNA synthesis in hepatocytes many. They were bred and maintained at the animal facilities of and secretion hepatitis B surface antigen (HBsAg) and the University Hospital Zü rich in accordance with institutionally hepatitis B e antigen (HBeAg) into culture medium. Tuapproved protocols. Under optimal conditions (room temperature, paias can also be infected with HBV in vivo, resulting 28ЊC; relative humidity, 70%; day-night light cycle, 12 and 12 hours), in viral DNA replication and gene expression in tupaia the mothers give birth to two to three animals every 8 to 10 weeks. livers. Similar to acute, self-limited hepatitis B in hu-At birth, the animals are about 6 to 7 cm long (without tail) and mans, HBsAg is rapidly cleared from serum, followed weigh 16 to 19 g. Within 10 to 12 weeks, they reach adulthood with by seroconversion to anti-HBe and anti-HBs. These data an average length of 12 to 18 cm (without tail) and 180 to 200 g body weight. All animals were negative for serological markers of HBV clearly show that HBV is infectious to tupaia hepato- problem worldwide and is associated with a wide spectrum of exclusion, was 90% to 95%. The cells were washed and seeded at a clinical presentations, ranging from the asymptomatic HBV density of 3 1 10 6 viable cells/10 mL medium on collagen-coated 100-carrier status to the development of cirrhosis and hepatocel-mm tissue culture plates. Plating efficiency was 85%. Cells were lular carcinoma. 1 HBV represents the prototypic member of maintained in Williams' E medium with Glutamax J, buffered with the hepadnaviruses 2,3 and many molecular and clinical as-15 mmol/L hydroxyethyl piperazine ethane sulfuric acid (Sigma), pH 7.4, and supplemented with 1.5% dimethyl sulfoxide (HybriMax, pects of HBV infection have been defined. 4,5 HBV naturally Sigma), 0.1 mmol/L insulin, 0.1 mmol/L dexamethasone, 100 mg/mL infects only humans and experimentally chimpanzees. While penicillin, and 100 mg/mL streptomycine.various aspects of the pathogenesis of HBV-related liver disTransient Transfection of Primary Tupaia Hepatocytes. Transfecease can be studied in transgenic animals 6-9 and animal modtion of primary tupaia hepatocytes with a replicatio...

chronic HBV infection is thought to be immune-mediated, a Hepatitis B virus (HBV) DNA was cloned from serum direct cytopathic role for HBV is postulated in immunosupof a heart transplant recipient who died from fulminant pressed transplantation recipients, especially in patients hepatitis B transmitted by the donor. Restriction enwith histological features of fibrosing cholestatic hepatitis 1 zyme analyses of the clones obtained by conventional and steatoviral or fibroviral hepatitis B.2 In these patients cloning yielded six HBV variants: a major species (pF-1) high levels of viral nucleic acids and accumulation in the representing 88% and five minor species (pF-2 to pF-6), cytoplasm of hepatocytes of the large hepatitis B surface antieach representing 2% to 4% of the clones. The complete gen (HBsAg) have been observed. 3 Various mutations in the nucleotide sequence of these six variants revealed that HBV genome have been implicated in the severe clinical five of the six viral genomes, including pF-1, carried a course of subfulminant or fulminant HBV infection. 4 Among novel 11 base pair (bp) insertion in the core promoter these, mutations resulting in a hepatitis B e antigen (HBeAg) region as well as an 18 bp and an 108 bp in-frame deletion negative phenotype because of interference with the expresin the pre-S1 region not present in the donor. One gesion of HBeAg at either the translational level, as in precore nome was identical to the sequence of the donor. Funcstop codon mutants, [5][6][7][8][9][10][11][12] or at the transcriptional level, as in tional analyses of HBV clones generated by in vitro mucore promoter mutants, 13,14 have been associated with fulmitagenesis and cassette exchange showed that the 11 bp nant and severe acute hepatitis as well as with recurrent insertion is a strong binding site for hepatocyte nuclear HBV infection after liver transplantation [15][16][17][18][19] or fatal HBV factor 1 (HNF-1). In transient transfection experiments, reactivation following cytotoxic treatment. 20 However, other the novel HNF-1 sequence motif was shown to result in studies suggest that fulminant hepatitis B is not caused by enhanced viral replication. Immunohistochemical anala specific genomic mutation 21 and show that the mutations yses revealed high levels of cytoplasmic and nuclear described above are also found in patients with acute selfhepatitis B core antigen (HBcAg) and only scattered heplimited or chronic hepatitis B as well as in asymptomatic atitis B surface antigen (HBsAg) expression in the liver.HBV infection. 12,22-28The data in our immunosuppressed patient showed that In this study, we identified and characterized a novel HBV HBV variants can rapidly accumulate in severe hepatitis mutant in a heart transplantation recipient who died from B and suggest that the novel HNF-1 binding site may 1993, 2 weeks later, the patient died from fulminant hepatitis B.Received September 24, 1996; accepted February 24, 1997. Sera obtained from the donor at the time of transplantation and from Study was...

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