Feasible and effective cell models for hepatitis B virus (HBV) infection are required for investigating the complete lifecycle of this virus,including the early steps of viral entry.Resistance to dimethyl sulfoxide/polyethylene glycol (DMSO/PEG),hNTCP expression,and a differentiated state are the limiting factors for successful HBV infection models.In the present study,we used a hepatoma cell line (Huh7DhNTCP) to overcome these limiting factors so that it exhibits excellent susceptibility to HBV infection.To achieve this goal,different hepatoma cell lines were tested with 2.5％ DMSO / 4％ PEG8000,and one resistant cell line (Huh7D) was used to construct a stable hNTCP-expressing cell line (Huh7DhNTCP) using a recombinant lentivirus system.Then,the morphological characteristics and differentiation molecular markers of Huh7DhNTCP cells with or without DMSO treatment were characterized.Finally,the susceptibility of Huh7DhNTCP cells to HBV infection was assessed.Our results showed that Huh7D cells were resistant to 2.5％ DMSO / 4％ PEG8000,whereas the others were not.Huh7DhNTCP cells were established to express a high level of hNTCP compared to liver extracts,and Huh7DhNTCP cells rapidly transformed into a non-dividing,well-differentiated polarized phenotype under DMSO treatment.Huh7DhNTCP cells fully supported the entire lifecycle of HBV infection.This cell culture system will be useful for the analysis of host-virus interactions,which should facilitate the discovery of antiviral drugs and vaccines.