Treatment of chronic hepatitis B virus (HBV) infections with the reverse transcriptase inhibitor lamivudine leads to a rapiddecline in plasma viremia and provides estimates for crucial kinetic constants of HBV replication. We find that in persistentlyinfected patients, HBV particles are cleared from the plasma with a half-life of approximately 1.0 day, which implies a 50%daily turnover of the free virus population. Total viral release into the periphery is approximately 10(11) virus particlesper day. Although we have no direct measurement of the infected cell mass, we can estimate the turnover rate of these cellsin two ways: (i) by comparing the rate of viral production before and after therapy or (ii) from the decline of hepatitisB antigen during treatment. These two independent methods give equivalent results: we find a wide distribution of half-livesfor virus-producing cells, ranging from 10 to 100 days in different patients, which may reflect differences in rates of lysisof infected cells by immune responses. Our analysis provides a quantitative understanding of HBV replication dynamics in vivoand has implications for the optimal timing of drug treatment and immunotherapy in chronic HBV infection. This study alsorepresents a comparison for recent findings on the dynamics of human immunodeficiency virus (HIV) infection. The total dailyproduction of plasma virus is, on average, higher in chronic HBV carriers than in HIV-infected patients, but the half-lifeof virus-producing cells is much shorter in HIV. Most strikingly, there is no indication of drug resistance in HBV-infectedpatients treated for up to 24 weeks.

> There is currently no universally accepted numbering convention for the antiviral drug-related resistance mutations in the reverse transcriptase (rt) domain of the human hepatitis B virus (HBV) polymerase. The published inconsistencies have resulted from different HBV genotypes. A standardized numbering system for HBV polymerase is proposed. The new system is based on functional observations of HBV surface gene proteins (preS1, preS2, and HBsAg) and on the current convention used for human immunodeficiency virus type 1 (HIV-1) polymerase proteins (protease, rt, and integrase), in which the amino acid numbering restarts at the first codon position of each domain. The HBV polymerase protein can be divided into 4 domains (terminal protein, spacer, rt, ribonuclease H) and each of these can be numbered separately. In this proposal, the HBV rt domain starts with the highly conserved EDWGPCDEHG motif, contains 344 amino acids, and the lamivudine-related resistance mutations are found at amino acid rtL180M (previously amino acid 528, 526, 515, or 525) and rtM204V/I (previously 552, 550, 539, or 549). The new consensus rt domain numbering system is genotype independent and allows investigators to number any previously and newly discovered antiviral-related amino acid change in a standardized manner.

Abstract Background/Aims Mutations in hepatitis B virus (HBV) to lamivudine resistance that arise during prolonged treatment frequently cause amino acid substitutions in the YMDD motif of HBV DNA polymerase. Current methods of detecting such variants are time-consuming, labor intensive, and unsuitable for screening large numbers of samples. Here, we describe the development of a matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) genotyping assay suitable for detecting HBV variants in a sensitive and specific manner. Methods The assay is based on PCR amplification and mass measurement of oligonucleotides containing sites of mutation of the YMDD motif. Results The MALDI-TOF MS-based genotyping assay is sufficiently sensitive to detect as few as 100 copies of HBV genome per millilitre of serum, with superior specificity for determining mixtures of wild-type and variant viruses. When sera from 40 patients were analyzed, the MALDI-TOF MS-based assay correctly identified known viral variants and additional viral quasi-species not detected by previous methods, as well as their relative abundance. Conclusions The sensitivity, accuracy and amenability to high-throughput analysis makes the MALDI-TOF MS-based assay suitable for mass screening of HBV infected patients receiving lamivudine, and can help provide further understanding of disease progression and response to therapy.