Detection of hyper-conserved regions in hepatitis B virus X gene potentially useful for gene therapy

World J Gastroenterol. 2018 May 21;24(19):2095-2107. doi: 10.3748/wjg.v24.i19.2095.


Aim: To detect hyper-conserved regions in the hepatitis B virus (HBV) X gene (HBX) 5' region that could be candidates for gene therapy.

Methods: The study included 27 chronic hepatitis B treatment-naive patients in various clinical stages (from chronic infection to cirrhosis and hepatocellular carcinoma, both HBeAg-negative and HBeAg-positive), and infected with HBV genotypes A-F and H. In a serum sample from each patient with viremia > 3.5 log IU/mL, the HBX 5' end region [nucleotide (nt) 1255-1611] was PCR-amplified and submitted to next-generation sequencing (NGS). We assessed genotype variants by phylogenetic analysis, and evaluated conservation of this region by calculating the information content of each nucleotide position in a multiple alignment of all unique sequences (haplotypes) obtained by NGS. Conservation at the HBx protein amino acid (aa) level was also analyzed.

Results: NGS yielded 1333069 sequences from the 27 samples, with a median of 4578 sequences/sample (2487-9279, IQR 2817). In 14/27 patients (51.8%), phylogenetic analysis of viral nucleotide haplotypes showed a complex mixture of genotypic variants. Analysis of the information content in the haplotype multiple alignments detected 2 hyper-conserved nucleotide regions, one in the HBX upstream non-coding region (nt 1255-1286) and the other in the 5' end coding region (nt 1519-1603). This last region coded for a conserved amino acid region (aa 63-76) that partially overlaps a Kunitz-like domain.

Conclusion: Two hyper-conserved regions detected in the HBX 5' end may be of value for targeted gene therapy, regardless of the patients' clinical stage or HBV genotype.

Keywords: Gene therapy; HBV conserved regions; Hepatitis B X gene; Hepatitis B X protein; Hepatitis B virus; Next-generation sequencing; Small interference RNA.

MeSH terms

  • 5' Untranslated Regions / genetics
  • Adult
  • Aged
  • Aged, 80 and over
  • DNA, Viral / genetics
  • DNA, Viral / isolation & purification
  • Female
  • Genetic Therapy / methods*
  • Hepatitis B e Antigens / immunology
  • Hepatitis B e Antigens / isolation & purification
  • Hepatitis B virus / genetics*
  • Hepatitis B virus / immunology
  • Hepatitis B virus / isolation & purification
  • Hepatitis B, Chronic / blood
  • Hepatitis B, Chronic / immunology
  • Hepatitis B, Chronic / therapy*
  • Hepatitis B, Chronic / virology
  • High-Throughput Nucleotide Sequencing
  • Humans
  • Male
  • Middle Aged
  • Phylogeny
  • RNA, Small Interfering / therapeutic use
  • Sequence Alignment
  • Sequence Analysis, DNA
  • Trans-Activators / genetics*
  • Trans-Activators / isolation & purification
  • Viral Regulatory and Accessory Proteins


  • 5' Untranslated Regions
  • DNA, Viral
  • Hepatitis B e Antigens
  • RNA, Small Interfering
  • Trans-Activators
  • Viral Regulatory and Accessory Proteins
  • hepatitis B virus X protein