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

Abstract

AIMTo detect hyper-conserved regions in the hepatitis B virus (HBV) X gene (HBX) 5’ region that could be candidates for gene therapy.METHODSThe 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.RESULTSNGS 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.CONCLUSIONTwo 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.

Conservation and variability of hepatitis B core at different chronic hepatitis stages

“…Sequence conservation at nt and aa levels was determined by calculating the information content (IC) of each position in a multiple alignment of all haplotypes detected with a frequency > 0.25. This analysis calculates the mean IC for windows of 25 nt (or 10 aa), starting from the first position in the multiple alignment and moving forward in steps of 1 [9] . The hyper-conserved regions were detected by aligning all haplotypes, regardless of clinical stage.…”

“…HBV reverse transcriptase lacks 3' to 5' proofreading activity, which leads to viral genome variability comparable to that observed in an RNA virus [8] . This genetic variability is further increased by inter-and intra-genotype recombination events [9] . In short, HBV circulates as a complex mixture of closely related genetic variants (haplotypes) known as quasispecies [10] .…”

“…Next-generation sequencing (NGS) is a highly sensitive technique for studying viral quasispecies; it is capable of detecting highly conserved regions of the HBV genome, regardless of genome or clinical stage [9] . Moreover, it supports the identification and quantitative determination [15] of specific variants that could be used as markers to predict prognosis and treatment response in patients with HBV infection.…”

Characterization of hepatitis B virus X gene quasispecies complexity in mono-infection and hepatitis delta virus superinfection

“…This information can explain or predict the response of the virus to specific environmental changes. The HBV quasispecies in chronic mono-infection and the HDV quasispecies have been analyzed individually by NGS[19-23]. However, there are no studies comparing the quasispecies in HBV mono-infection and HBV/HDV infection using this technique.…”

“…The aim of this study was to evaluate and compare the complexity of the HBV quasispecies in serum samples from HBV mono-infected patients in the chronic infection phase (CI, previously termed inactive carrier), patients with chronic hepatitis B (CHB) mono-infection, and patients with chronic hepatitis delta (CHD) superinfection, using a high-throughput NGS-based approach. We focused our study on the 5’ end of the HBX coding region and its upstream non-coding region [nucleotides (nt) 1255-1611], which was also examined in a previous study by our group in patients in different stages of chronic HBV infection, including CHB and CI[23].…”