A 47-year-old woman (weight: 62 kg) was diagnosed with chronic active HBeAg-positive hepatitis B in 1996. Viral parameters such as HBeAg, antiHBe IgG and HBV plasma viral load from frozen samples were simultaneously measured by a unique operator to reduce intra and inter-assay variations. HBeAg and anti-HBe were determined by electrochemiluminescence (ELECSYS 2010, Roche Diagnostic). HBV viral load levels were determined by real time PCR (COBAS TaqMan Roche Molecular Systems, dynamic range 30 IU/ml to 110,000,000 IU/ml) []. Liver inflammation was measured by serum alanine aminotransferase (ALT) levels. For sequence analysis, HBV pol and preC-core genes were sequentially sequenced with primers as previously described []. The analysis was performed using an ABI3100 instrument (Applied Biosystems), and the sequences introduced in this work as well as those obtained from the GenBank database were aligned with ClustalX v1.83 [] and edited with Bioedit v7.0.9.0 []. The HBV genotype was assessed by phylogenetic inference using the Neighbor-joining algorithm with the Kimura-two-parameter model of molecular evolution in the MEGA v3.1 software []. Sequences from both genes were obtained at different time-points during antiviral therapy. A more detailed HBV pol gene clonal analysis was performed on seven selected viral isolates from serum samples harvested during sequential therapies. Each selected PCR product of pol gene was cloned into pGEM-T Easy vector according to the manufacturer's instructions (Promega, Wisconsin, USA) and at least 15 clones were further analyzed by sequencing. This allowed analysing the evolution of viral quasispecies under the successive antiviral pressures based on analysis of the pol gene. The anti-HBV treatment interventions accompanied by the HBV viral load and ALT level kinetics are depicted in figure. Therapy was initiated when HBV viral load rose to 6.5 × 106 IU/ml. In 1998 when the patient was treated with IFN-[alpha] 5 MU thrice weekly for 30 weeks, the HBV DNA level was lower than the detection limit. This therapy was interrupted and replaced in 1999 by lamivudine (150 mg once a day). After 2 years of continuous treatment, HBV DNA was detectable (15.5 × 106 IU/ml). HBeAg remained negative with detectable anti-HBe during this period of treatment. In 2001, after a 6-month discontinuation, HBV DNA level evidenced a post-treatment flare (1 × 108 IU/ml). At the end of 2003 lamivudine therapy was reintroduced (150 mg once daily). It was accompanied by a rare event of reversion to an HBeAg-positive/anti-HBe negative profile, which remained detectable for two years. The viral load reached 3.1 × 106 IU/ml at this point. Ten months later, in 2006, the lamivudine regimen was replaced by adefovir monotherapy (10 mg/day). Initially, viral load levels were low (8.5 × 104 IU/ml) but after 48 weeks of therapy these levels reached 7.15 × 106 IU/ml. ALT levels were normal during this interval. Reactivation of HBV replication was assumed considering the simultaneous detection of HBeAg/anti-HBe [] and the high replication detected. The concurrence of HBeAg and anti-HBe does not appear to be uncommon among antiviral treatment-naïve patients, but its prevalence in therapy-experienced patients is unknown. This serological pattern has been related to extensive hepatocyte damage and severe immune-mediated liver injury and consequent dysfunction []. Six months later, ALT level slightly increased (1.5× upper-normal limit - UNL -) and entecavir monotherapy was instituted (1 mg daily). After a 5-month entecavir monotherapy, tenofovir (300 mg once daily) was added to the treatment regimen for dual therapy. The HBV DNA declined to 1 × 105 IU/ml for four months, but three months later the viral load reached > 1.1 × 108 IU/ml. Additionally, an increase in the ALT level (20× UNL) denoted liver inflammation. During the following 48 weeks, the viral replication slightly decreased to 6.1 × 106 IU/ml but rebounded first to 3.6 × 107 IU/ml and later to > 1.1 × 108 IU/ml. More recently (second semester 2010), HBV DNA levels fluctuated between 4.3 × 104 IU/ml and 4.9 × 107 IU/ml. By the end of the study follow-up, we were able to measure tenofovir concentration in plasma by high performance-liquid chromatography in three consecutive samples 20 hours after administration reaching 71.6 ng/ml ± 47.6 (mean ± SD). The ALT-AST levels remained slightly elevated (2× UNL) during the last two years of follow-up. HBV isolates from the patient were ascribed to genotype A2 based on phylogenetic relatedness among partial HBV genomic sequences from both pol and preC-C genes. A further longitudinal genotypic analysis, including quasispecies composition of HBV strains isolated from the patient revealed the presence of resistance mutations based on pol gene sequences. At the beginning of lamivudine treatment, HBV was wild-type in all clones. After 2 years under such therapy, this viral population was completely replaced by mutation rtM204I -homogenously exhibiting lamivudine resistance. When this therapy was interrupted for 6 months, quasispecies composition almost exclusively consisted of wild-type pol nucleotide sequences, except for a minor contribution (< 10%) showing the adefovir resistance-associated mutation I233V. Once lamivudine therapy was reintroduced, and until entecavir plus tenofovir were administered, the dual lamivudine resistance-associated mutations rtL180M and rtM204I were invariably detected. Under the latter therapeutic scheme, only ephemerally and with minor contribution (~5%), the A181T and T184L lamivudine-adefovir and entecavir resistance-associated mutations were detected, respectively. Additionally, another two variations were detected in the reverse transcriptase catalytic domain at the time of lamivudine resistance. These rtA200V and rtI253V variants remained present during entecavir therapy, and were also detected in samples sequenced after breakthrough during entecavir therapy. The HBV genomic characterization at preC-C level showed the presence of A1762T and G1764A double mutation that was early detected when the patient was untreated, remaining in this condition during the entire follow-up. The T1753C mutation was also consistently detected. The presence of these core promoter mutations are also related to the above-mentioned HBeAg-antiHBe concurrent profile found in this patient, as recently reported [].