Sequence variations of full-length hepatitis B virus genomes in Chinese patients with HBsAg-negative hepatitis B infection

PLoS One. 2014 Jun 5;9(6):e99028. doi: 10.1371/journal.pone.0099028. eCollection 2014.

Abstract

Background: 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.

Methods: 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.

Results: 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.

Conclusions: These data suggest an accumulation of multiple mutations constraining viral transcriptional activities contribute to HBsAg-negativity in HBV infection.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adult
  • Aged
  • China
  • DNA Mutational Analysis
  • DNA, Viral / chemistry
  • DNA, Viral / metabolism
  • Female
  • Genetic Variation*
  • Genome, Viral*
  • Genotype
  • Hepatitis B Surface Antigens / genetics
  • Hepatitis B virus / classification
  • Hepatitis B virus / genetics*
  • Hepatitis B virus / metabolism
  • Hepatitis B, Chronic / diagnosis
  • Hepatitis B, Chronic / virology*
  • Humans
  • Liver / pathology
  • Liver / virology
  • Male
  • Middle Aged
  • Nucleic Acid Conformation
  • Phylogeny
  • Promoter Regions, Genetic
  • RNA, Viral / chemistry
  • Sequence Analysis, DNA

Substances

  • DNA, Viral
  • Hepatitis B Surface Antigens
  • RNA, Viral

Grants and funding

The study was supported by Research Grants Council General Research Fund (HKU 782809M). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.