Total nucleic acid was extracted from 1000 µl of subjects’ sera using the Qiagen UltraSense kit (QIAGEN, Hilden, Germany). An elution volume of 50 µl was used and extracts were stored at −80ºC until use. For HBV DNA amplification, the master mix was prepared from the Superscript III Platinum One-Step kit (Invitrogen, USA) with a modification of the number of cycles to 35 and template volume of 10 µl per each reaction. We amplified a 2100 base pair fragment covering nucleotides 2400 – 1150 according to the numbering of the EcoRI that included the full S gene overlapping with part of Pol using HBV primers Core-F and Werle-AS as previously described [15 (link)]. The PCR conditions included preheating at 95°C for 2 minutes, denaturing at 95°C for 30 seconds, annealing at 62.5°C for 30 seconds, and extension at 72°C for 4 minutes. Amplicons were confirmed on 1% agarose gel and purified using QIAquick (Qiagen, Hilden, Germany). The BigDye Terminator v3.0 kit (Applied Biosystems; Foster City, CA, USA) was used for sequencing. Six overlapping primers were used to prepare separate master-mixes for PCR. The thermocycling conditions were denaturing at 96°C for 10 seconds, annealing at 50°C for 5 seconds, and final extension at 60°C for 4 minutes for 25 cycles. Direct sequencing was performed using the automated Sequencer (ABI PRISM 3130xl; Applied Biosystems).