Infection with hepatitis B virus (HBV) e-antigen (HBeAg)-negative strains is increasingly prevalent. Currently, detailed information of the obtained natural HBV strain is not available except for the B genotype and HBeAg-negative. The aim of the present study was to characterize the natural genetic variation of the HBeAg-negative strain and investigate its function. The genic sequence was determined using Sanger sequencing, and compared to related sequences using alignment and phylogenetic analysis. In vivo, virus-specific serum markers were investigated in CBA/CaJ mice. The sequence had a full genome length of 3215 nucleotides. Sites 122, 125, 127, and 160 in S regions were identified as lysine, threonine, proline, and lysine respectively. The main four point variants including A1762T, G1764A, G1896A, and G1899A were detected in the full-length genome. The genotype of the sequence was B, with sub genotype B2 and serological subtype adw2. The characterize of the natural genetic variation strain showed no reported drug-resistant variant in P region and no reported immune escape site in S region. The strain will increase viral replication and infection for mutations A1762T and G1764A in the basal core promoter region, and mutations G1896A and G1899A in the pre-core region. The G1896A variant resulted in a premature stop codon and abolished HBeAg expression. HBsAg persisted for 26 weeks and HBeAg was still negative in CBA/CaJ mice. The present sequence is representative of the HBeAg-negative genome and may serve as a valuable reference for studying HBeAg-negative strains. The present findings were successfully verified in CBA/CaJ mice, demonstrating good applicability of the sequence. (C) 2018 Elsevier Inc. All rights reserved.