Sequence Variations of Full-Length Hepatitis B Virus Genomes in Chinese Patients with HBsAg-Negative Hepatitis B Infection

• Published in: PloS one Vol. 9; no. 6; p. e99028
• Main Authors: Huang, Fung-Yu, Wong, Danny Ka-Ho, Seto, Wai-Kay, Zhang, An-Ye, Lee, Cheuk-Kwong, Lin, Che-Kit, Fung, James, Lai, Ching-Lung, Yuen, Man-Fung
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 05.06.2014; Public Library of Science (PLoS)
• Subjects: Adult; Aged; Amino acids; Amplification; Antigens; Biology and Life Sciences; Biopsy; Blood transfusions; China; Defects; Deoxyribonucleic acid; DNA; DNA Mutational Analysis; DNA sequencing; DNA, Viral - chemistry; DNA, Viral - metabolism; Female; Gene expression; Gene sequencing; Genetic Variation; Genome, Viral; Genomes; Genomics; Genotype; Genotype & phenotype; Health aspects; Hepatitis; Hepatitis B; Hepatitis B surface antigen; Hepatitis B Surface Antigens - genetics; Hepatitis B virus; Hepatitis B virus - classification; Hepatitis B virus - genetics; Hepatitis B virus - metabolism; Hepatitis B, Chronic - diagnosis; Hepatitis B, Chronic - virology; Hepatology; Hospitals; Humans; Infection; Infections; Laboratories; Liver; Liver - pathology; Liver - virology; Liver cancer; Male; Medicine; Medicine and Health Sciences; Middle Aged; Missense mutation; Mutation; Nucleic Acid Conformation; Patients; Phylogeny; Promoter Regions, Genetic; Proteins; Regulatory sequences; RNA; RNA, Viral - chemistry; Sequence Analysis, DNA; Splicing; Transcription; Viruses; China; Hong Kong China
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0099028

The underlying mechanism of HBsAg-negative hepatitis B virus (HBV) infection is notoriously difficult to elucidate because of the extremely low DNA levels which define the condition. We used a highly efficient amplification method to overcome this obstacle and achieved our aim which was to identify specific mutations or sequence variations associated with this entity. A total of 185 sera and 60 liver biopsies from HBsAg-negative, HBV DNA-positive subjects or known chronic hepatitis B (CHB) subjects with HBsAg seroclearance were amplified by rolling circle amplification followed by full-length HBV genome sequencing. Eleven HBsAg-positive CHB subjects were included as controls. The effects of pivotal mutations identified on regulatory regions on promoter activities were analyzed. 22 and 11 full-length HBV genomes were amplified from HBsAg-negative and control subjects respectively. HBV genotype C was the dominant strain. A higher mutation frequency was observed in HBsAg-negative subjects than controls, irrespective of genotype. The nucleotide diversity over the entire HBV genome was significantly higher in HBsAg-negative subjects compared with controls (p = 0.008) and compared with 49 reference sequences from CHB patients (p = 0.025). In addition, HBsAg-negative subjects had significantly higher amino acid substitutions in the four viral genes than controls (all p<0.001). Many mutations were uniquely found in HBsAg-negative subjects, including deletions in promoter regions (13.6%), abolishment of pre-S2/S start codon (18.2%), disruption of pre-S2/S mRNA splicing site (4.5%), nucleotide duplications (9.1%), and missense mutations in "α" determinant region, contributing to defects in HBsAg production. These data suggest an accumulation of multiple mutations constraining viral transcriptional activities contribute to HBsAg-negativity in HBV infection.