192 Anti-HBV activity of B. nivea extract in vivo Results Tumor Formation Caused by HepG2 and HepG2 2.2.15 Cells in SCID Mice Human hepatoma cells, HepG2 and HepG2 2.2.15, were implanted subcutaneously into the flank of SCID mice, and tumor size was measured and calculated in terms of mass (Fig. 2A). Both hepatoma cells produced tumors of 1130–1250 mg within 28 days. The tumor-take rate of each cell line was 100%. HepG2 2.2.15–derived tumors grew at a faster rate than HepG2-derived tumors. Secretion of Serum HBsAg in HepG2 2.2.15 Tumor-bearing Mice On Days 14 and 28 after tumor cell inoculation, sera from tumor-bearing mice were collected and analyzed for HBV DNA and the HBV-related maker, HBsAg. HepG2 2.2.15 tumor-bearing mice had high levels of serum HBsAg, whereas the HepG2 tumor-bearing mice did not (HepG2 is a hepatocellular carcinoma without HBV infection; Fig. 2B). Production of HBV DNA in Serum from HepG2 2.2.15 Tumor-bearing Mice To investigate whether the HepG2 2.2.15 cells inoculated into the SCID mice produced HBV particles in circulat- ing blood, serum was analyzed for HBV DNA using quantitative real-time PCR. In the HepG2 2.2.15 group, the level of serum HBV DNA was 6 104 copies ml 1 on Day 14 and increased to 1.4 105 copies ml 1 on Day 28 (Fig. 2C). These data indicate that HBV virema was produced in SCID animals upon injection of HBV- producing HepG2 2.2.15 cells. Additionally, serum HBV DNA level was proportional to the size of the HepG2 2.2.15 tumor, thereby simulating the increase in virus load in chronic hepatitis B patients. These results suggest that our HepG2 2.2.15 SCID mouse system constitutes an animal model of HBV-viremia. Variation in Tumor Weight and Body Weight Among the Experimental Groups We used our HBV-viremia model to evaluate the effects of anti-HBV drugs (see below). In the absence of drugs, all HepG2 2.2.15 tumor-bearing mice had lower body weight compared with the sham group (injected with PBS but no cells) (Fig. 3A). The loss of body weight caused by HepG2 2.2.15 tumor was associated with tumor forma- tion. Thus, body weight loss was associated with tumor formation. Serum HBsAg and HBV DNA in the HBV-viremia SCID mice were elevated by Day 13, thus allowing us to evaluate the anti-HBV efficacy of BNE. HBV-viremia Figure 2. Tumor weight, serum HBsAg level and HBV DNA level in the HBV-viremia animal model. HepG2 or HepG2 2.2.15 cells (107) were inoculated subcutaneously into the flank of SCID mice. (A) Tumor size was measured every 4 days as shown. Blood was withdrawn from animals on post-inoculation Days 14 and 28. (B) Serum HBsAg and (C) serum HBV DNA were measured by ELISA and quantitative real-time PCR, respectively. Data represent mean SE. mice were treated with oral 3TC, oral BNE and intraperitoneal BNE administration for 10 consecutive days. Tumor growth in all HBV-viremia animals was not affected by drug administration (Fig. 3B), suggesting that