HBV DNA was extracted from 400 mL of serum by QIAamp UltraSens Virus Kit (Qiagen GmbH, Germany), then resuspended in 50 mL water and stored at 220uC until analysis. HBV genotypes, including C/D recombinant, were initially assigned using the PCR based restriction fragment length polymorphism (RFLP) methods described previously [9] , [8] .