194 Anti-HBV activity of B. nivea extract in vivo Figure 5. Effects of BNE on the activities of serum ALT and NK cells in HBV-viremia mice. HepG2 2.2.15 cells (107) were subcutaneously inoculated into the flank of SCID mice. (A) On post-inoculation Days 13 and 24, blood was withdrawn from test animals, and the activity of serum ALT was measured. (B) On Day 24, drug-treated HBV-viremia animals were sacrificed and the spleen was homogenized into a single- cell suspension; the activity of NK cells was subsequently assayed as described in Subjects and methods section. Data represent mean SE **P < 0.01 by one-way ANOVA and Dunnett’s test. Assessment of ALT Activity in the Experimental Groups To evaluate the biochemical and immunological effects by BNE, we measured the levels of serum ALT. There was no significant difference in the serum ALT level of the sham group between Days 13 and 24 (Fig. 5A). In contrast, the serum ALT level was elevated in all HBV-viremia animals, suggesting that these animals responded biochemically to the implanted tumors. However, this elevated ALT activity was not suppressed by 3TC or BNE administration (Fig. 5A). Inhibition of NK Cell Activity by BNE To investigate the effect of BNE on innate immunity, animals were sacrificed and the spleens were taken for NK cell activity analysis. NK activity was increased by 60% in HBV-viremia animals (the vehicle group) com- pared to the sham group (no tumor) (Fig. 5B). This elevated NK activity—set as 100% with respect to the difference between the sham group and the vehicle group (distilled water)—was inhibited by 26% by 3TC. NK cell activity also was inhibited by 31% in animals treated orally with BNE. Dramatically, NK activity was inhib- ited by 75% in animals injected intraperitoneally with BNE (195 mg kg 1—the same dose given to the gavage group), suggesting that intraperitoneally administered BNE is more effective than by gavage. These data suggest that serum HBV DNA level is inhibited by BNE, which in turn results in a decrease in NK activity. Discussion Extensive efforts have been put forth in the development of an HBV animal model to mimic the natural infection (15). However, these HBV animal models can only explain a part of the mechanism of anti-HBV drugs; moreover, the maintenance of animals in these models, such as HBV transgenic mice (23–25) and the Peking duck model (26), is very expensive. In this study, we implanted the HBV-producing cell line, HepG2 2.2.15, in SCID mice to simulate the phenomenon of HBV viremia, because viruses generated from HepG2 2.2.15 cells have been reported to be infectious (27). HBV particles and HBsAg were detected in the circulation of HepG2 2.2.15 tumor-bearing mice, and the serum HBV DNA level correlated with tumor growth. Therefore, this HBV- viremia model might provide information not only on the absorption, distribution, and metabolism of anti-HBV drugs but also their efficacy, and these aspects may be applicable to the treatment of HBV in humans. Clinical evaluation of the levels of HBsAg, HBeAg and HBV DNA in serum represents the different stages of HBV infection (28). Although high levels of serum HBsAg represent the acute phase of HBV infection, HBV DNA is one of the most important factors for diagnosis of different stages of hepatitis B (29). In this study, elevated serum HBsAg and HBV DNA were detected in the vehicle group because of HBV production from HepG2 2.2.15 tumors, and the elevated HBV DNA in serum was sufficient to allow us to evaluate the anti-HBV activities of 3TC and BNE. Serum HBsAg and HBV DNA levels were suppressed by BNE, but tumor growth was unaffected. BNE caused a decrease in both serum HBsAg and HBV DNA levels. Furthermore, the intra- peritoneal administration of BNE was more effective than by gavage. These findings are consistent with our previous in vitro study, in which HBV DNA level was reduced to a greater extent than HBsAg level (13). 3TC was also effective at suppressing HBV replication (i.e. 50% reduction in serum HBV DNA level) in