190 Anti-HBV activity of B. nivea extract in vivo hepatic inflammation, progression of liver fibrosis, and hepatoma (8). Approaches for the treatment of chronic HBV infection have been in development for the past decade, and when combined with a number of anti-viral drugs these treatments may have potential to improve the effective- ness of anti-HBV therapy. Chinese medicinal herbs have been used for centuries to treat liver disease (9). Recently, the flavonoid ellagic acid from woody dicotyledonous plants has been found to block hepatitis B e antigen (HBeAg) secretion either in an HBV-infected cell line or in HBeAg transgenic mice (10). In addition, sesquiterpene lactones from Senecio species suppress the expression of hepatitis B s antigen (HBsAg) and HBeAg (11), and an ethanol extract of Polygonum cuspidatum inhibits HBV production in an HBV-producing cell line (12). An extract of the genus Phyllanthus has a positive effect on the clearance of HBsAg in clinical trials conducted on chronic HBV infections, and the extract has a synergistic effect when administered with IFN-a (9). Our previous studies demonstrated that crude root extract of Boehmeria nivea had anti-HBV activity via the inhibition of HBV production in HepG2 2.2.15 cells (13). This B. nivea extract (BNE) suppressed the production of infectious virus but not intracellular HBV DNA replica- tion, and its anti-HBV mechanism appears to differ from that of the nucleoside analogs. Thus, it is conceivable that BNE might work synergistically with other anti-viral compounds for the treatment of HBV infection. HBV replicates through an RNA intermediate by its reverse transcriptase/polymerase. The viral reverse tran- scriptase/polymerase lacks a proofreading function, how- ever, thus leading to the formation of drug-resistant mutants that in turn cause serious problems with current management of hepatitis B infection (14). Therefore, effective treatment strategies with different pharmacolo- gical modes are urgently needed to lessen the enormous burden of viral hepatitis on health care worldwide. A major obstacle to the development of anti-HBV drugs is the lack of an efficient in vitro cell model or an easy-to-use conventional animal model that is promising for natural viral infection and replication (15). Avail- ability of laboratory animal models of HBV infection is imperative for the development of effective methods to treat these diseases. One of the few animal models for HBV infection is the chimpanzee, which is extensively used to evaluate the safety and immunogenicity of HBV vaccines (16). However, the limited availability and the high cost of these primates severely restrict their use for such purposes. Investigators have attempted to estab- lish conventional animal models to mimic human HBV replication in rats (17), nude mice (18) and transgenic mice (19). Other animal models, such as woodchuck hepatitis virus in woodchuck and duck hepatitis virus in duck, have also been developed to assess anti-viral drugs (20). However, testing of anti-viral agents, including nucleoside analogs, in these non-human HBV models may produce aberrant results as a consequence of virus- specific differential susceptibility of the viral polymerase to these agents (21). Thus, establishment of a rapid, convenient and less expensive animal model would greatly facilitate this area of research. In this study, we established an HBV-viremia animal model and studied the efficacy of BNE in vivo. Subjects and Methods Experimental Animals and Cell Culture Male severe combined immunodeficiency (SCID) mice 4–6 weeks of age, C.B17/icr-scid, were purchased from the experimental animal center of National Taiwan University (Taipei, Taiwan, R.O.C.). The human hepa- toma HepG2 cell line was purchased from American Type Culture Center (ATCC, USA), and the HepG2 2.2.15 cell line was kindly given by Dr Ho, MS (Academia Sinica, Taipei, Taiwan, R.O.C.). These hepatoma cells were maintained in minimum essential medium (Gibco, USA) containing 10% fetal bovine serum (Hyclone, USA), 1.5 g l 1 sodium bicarbonate, 0.1 mM non-essential amino acids, 1.0 mM sodium pyruvate, and 100 U ml 1 penicillin G (Gibco, USA) and 100 mg ml 1 streptomycin (Gibco, USA). A final concentration of 200 mg ml 1 G418 (Gibco, USA) was included in the medium for the maintenance of HepG2 2.2.15 cells. YAC-1 cells (ATCC, USA) for the natural killer (NK) cell activity assay were cultured in RPMI 1640 medium containing 2 mM L-glutamine, 1.5 g l 1 sodium bicarbonate, 10 mM HEPES and 10% fetal bovine serum. Anti-HBV Drugs and BNE To prepare the B. nivea (L.) Gaudich plant extract (i.e. BNE) utilized in our experiments, the roots of the plants were collected and dried (13). Briefly, 100 g of the dried roots was cut into pieces 0.5 cm in length before boiling in 1 l of 20% ethanol under boiling reflux for 3 h. The decoction was filtered through a 0.22 mm filter and lyophilized into powder. 3TC was purchased from GlaxoSmithKline (Holland). Establishment of an HBV-Viremia Animal Model To establish HBV-viremia animals, the 20 experimental SCID mice were divided into three groups: HepG2 group (n = 8), HepG2 2.2.15 group (n = 8) and one vehicle control group (n = 4). The SCID mice were inoculated with 107 HepG2 or HepG2 2.2.15 cells in 100 ml phosphate-buffered saline (PBS) into the flank subcuta- neously. The control group was inoculated likewise with