H epatitis B virus (HBV) is a small enveloped DNA virus and causes chronic infection of the liver that often leads to chronic hepatitis, cirrhosis, and hepatocellular carcinoma. [1][2][3][4] The lack of a practical small animal model has impeded the study of the biology of this virus and the development of effective antiviral therapies. Chimpanzee is the only natural host that allows active replication of HBV. [5][6][7] Although this animal is a valuable model for the study of hepatitis viruses, 8 the practical use of chimpanzees is severely limited both ethically and economically.Several small animal models of HBV infection have been reported. The HBV transgenic mouse is a very useful model for the study of virology and evaluation of antiviral drugs. [9][10][11][12] However, the liver cells of this model are not permissive for HBV infection; therefore, studying virus-cell interactions such as receptor binding and entry is not possible. The HBVtrimera mouse is another useful mouse model. 13 In this model, ex vivo HBV-infected human liver fragments are implanted into lethally irradiated mice after SCID mouse bone marrow transplantation. Approximately 80% of the mice develop viremia 2 to 3 weeks after infection. However, the rate of positivity subsequently decreases to less than 20% 6 weeks after infection. The level viremia is approximately 10 5 copies/mL. More recently, HBV-containing human serum samples were used to infect human hepatocyte repopulated mice. 14 A high-level viremia (4.5 and 10 ϫ 10 8 copy/ mL) and HBs antigenemia are observed 8 weeks after injection. This mouse model is promising because HBV replicates in natural host cells, human hepatocytes. However,

G to A hypermutation of the human immunodeficiency virus type 1 (HIV-1) is induced by a deaminase APOBEC3G and is related to host antiviral defense. APOBEC3G has also been found to reduce the replication of HIV-1 by an unknown mechanism. This enzyme also reduces the production of hepatitis B virus, although the mechanism for this action has not been clearly elucidated. The hypermutated hepatitis B virus (HBV) is rarely found in usual sequencing analyses. Using peptide nucleic acid mediated by polymerase chain reaction clamping, we detected the hypermutated HBV DNA in 1 of 8 patients with acute HBV infection and 4 of 10 with chronic HBV infection. In the latter group, hypermutated genomes were found only in eAb-positive patients. As much as 72.5% of G residues were mutated in the hypermutated clones. G to A substitutions were predominant in almost all clones sequenced compared with other substitutions. G to A mutated viral genomes also were found in HepG2-derived cell lines that continuously produced HBV into the supernatant. Both alpha and gamma interferon reduced virus production in these cell lines, but they did not alter the frequency of the hypermutation. Transcripts of APOBEC3G, as well as some other deaminases, were found in these cell lines. In conclusion, our results show that part of the minus strand DNA of HBV is hypermutated both in vitro (HepG2 cell lines) and in vivo. The role and mechanism of hypermutation in reducing HBV replication should be further investigated to understand the anti-HBV defense system. (HEPATOLOGY 2005;41:626-633.)

G to A hypermutation of Hepatitis B virus (HBV) and retroviruses appears as a result of deamination activities of host APOBEC proteins and is thought to play a role in innate antiviral immunity. Alpha and gamma interferons (IFN-a and -c) have been reported to upregulate the transcription of APOBEC3G, which is known to reduce the replication of HBV. We investigated the number of hypermutated genomes under various conditions by developing a quantitative measurement. The level of hypermutated HBV in a HepG2 cell line, which is semi-permissive for retrovirus, was 2.

Lamivudine (LAM) is a nucleoside analogue widely used for the treatment of chronic hepatitis B virus (HBV) infection. Emergence of resistant strains with amino acid substitutions in the tyrosine-methionine-aspartate-aspartate (YMDD) motif of reverse transcriptase is a serious problem in patients on LAM therapy. The amount of covalently closed circular DNA in the serum is reported to be higher in patients who develop YMDD mutants than in those without mutants. However, there is no useful serum marker that can predict early emergence of mutants during LAM therapy. Analysis of patients who were treated with entecavir (n ‫؍‬ 7) and LAM (n ‫؍‬ 36) showed some patients had high serum levels of HBV RNA. Median serum levels of HBV RNA were significantly higher in patients in whom the YMDD mutant had emerged within 1 year (n ‫؍‬ 6, 1.688 log copies/ml) than in those in whom the YMDD mutant emerged more than 1 year after treatment (n ‫؍‬ 12, 0.456 log copies/ml, P ‫؍‬ 0.0125) or in whom the YMDD mutant never emerged (n ‫؍‬ 18, 0.688 log copies/ml, P ‫؍‬ 0.039). Our results suggest that HBV RNA is a valuable predictor of early occurrence of viral mutation during LAM therapy. (HEPATOLOGY 2007;45:1179-1186 T he hepatitis B virus (HBV) is a member of the hepadnaviridae family. Worldwide, approximately 350 million people are estimated to be chronically infected with HBV. 1 Patients with chronic HBV infection develop chronic hepatitis, cirrhosis, and hepatocellular carcinoma, accounting for approximately 1 million deaths per year. 2 Recently, inhibitors of reverse transcriptase have been developed and widely used for patients with chronic HBV infection. Lamivudine (LAM), a cytosine nucleoside analogue, was first developed as an antiviral agent against HIV and later was used effectively against HBV because HBV also uses reverse transcriptase for replication. 3,4 Because LAM suppresses HBV replication, patients who are treated with LAM show a decreased level or disappearance of HBV DNA in serum and hepatitis B e antigen, normalization of serum alanine aminotransferase (ALT) level, and histological improvement. [5][6][7][8][9][10][11][12] However, discontinuation of therapy often leads to reactivation of HBV. 6,8,13,14 Therefore, long-term therapy is necessary for many patients with chronic HBV infection. During long-term LAM therapy, drug-resistant mutants with amino acid substitutions in the tyrosine-methionine-aspartate-aspartate (YMDD) motif emerge, resulting in expression of HBV DNA increasing again and in worsening of hepatitis. 6,10,[15][16][17][18] Moreover, some patients develop a severe flare-up of hepatitis that could lead to fatal hepatic failure. Therefore, prediction of the emergence of YMDD mutants is an important issue.In our hunt for useful serum markers to detect the early emergence of YMDD mutants, we noticed some patients who showed a discrepancy in the expression of HBV DNA measured by the transcription-mediated amplifica-

We developed a reverse genetics system of hepatitis C virus (HCV) genotypes 1a and 2a using infectious clones and human hepatocyte chimeric mice. We inoculated cell cultureproduced genotype 2a (JFH-1) HCV intravenously. We also injected genotype 1a CV-H77C clone RNA intrahepatically. Mice inoculated with HCV by both procedures developed measurable and transmissible viremia. Interferon (IFN) alpha treatment resulted in greater reduction of genotype 2a HCV levels than genotype 1a, as seen in clinical practice. Genetically engineered HCV infection system should be useful for analysis of the mechanisms of resistance of HCV to IFN and other drugs.