Published online Apr 7, 2019. doi: 10.3748/wjg.v25.i13.1566
Peer-review started: December 25, 2018
First decision: January 23, 2019
Revised: February 25, 2019
Accepted: March 1, 2019
Article in press: March 2, 2019
Published online: April 7, 2019
Hepatitis delta virus (HDV) seems to strongly suppress hepatitis B virus (HBV) replication, although little is known about the mechanism of this interaction. Both these viruses show a dynamic distribution of mutants, resulting in viral quasispecies. Next-generation sequencing is a viable approach for analyzing the composition of these mutant spectra. As the regulatory hepatitis B X protein (HBx) is essential for HBV replication, determination of HBV X gene (HBX) quasispecies complexity in HBV/HDV infection compared to HBV mono-infection may provide information on the interactions between these two viruses.
To compare HBV quasispecies complexity in the HBX 5’ region between chronic hepatitis delta (CHD) and chronic HBV mono-infected patients.
Twenty-four untreated patients were included: 7/24 (29.2%) with HBeAg-negative chronic HBV infection (CI, previously termed inactive carriers), 8/24 (33.3%) with HBeAg-negative chronic hepatitis B (CHB) and 9/24 (37.5%) with CHD. A serum sample from each patient was first tested for HBV DNA levels. The HBX 5’ region [nucleotides (nt) 1255-1611] was then PCR-amplified for subsequent next-generation sequencing (MiSeq, Illumina, United States). HBV quasispecies complexity in the region analyzed was evaluated using incidence-based indices (number of haplotypes and number of mutations), abundance-based indices (Hill numbers of order 1 and 2), and functional indices (mutation frequency and nucleotide diversity). We also evaluated the pattern of nucleotide changes to investigate which of them could be the cause of the quasispecies complexity.
CHB patients showed higher median HBV-DNA levels [5.4 logIU/mL, interquartile range (IQR) 3.5-7.9] than CHD (3.4 logIU/mL, IQR 3-7.6) (P = n.s.) or CI (3.2 logIU/mL, IQR 2.3-3.5) (P < 0.01) patients. The incidence and abundance indices indicated that HBV quasispecies complexity was significantly greater in CI than CHB. A similar trend was observed in CHD patients, although only Hill numbers of order 2 showed statistically significant differences (CHB 2.81, IQR 1.11-4.57 vs CHD 8.87, 6.56-11.18, P = 0.038). There were no significant differences in the functional indices, but CI and CHD patients also showed a trend towards greater complexity than CHB. No differences were found for any HBV quasispecies complexity indices between CHD and CI patients. G-to-A and C-to-T nucleotide changes, characteristic of APOBEC3G, were higher in CHD and CI than in CHB in genotype A haplotypes, but not in genotype D. The proportion of nt G-to-A vs A-to-G changes and C-to-T vs T-to-C changes in genotype A and D haplotypes in CHD patients showed no significant differences. In CHB and CI the results of these comparisons were dependent on HBV genotype.
The lower-replication CHD and CI groups show a trend to higher quasispecies complexity than the higher-replication CHB group. The mechanisms associated with this greater complexity require elucidation.
Core tip: Hepatitis B virus (HBV) replication is lower in the presence of hepatitis delta virus (HDV), but little is known about the mechanism of this interaction. HBV X gene quasispecies study in HBV/HDV infection could provide data regarding this interaction. With use of next-generation sequencing, we analyzed HBV quasispecies complexity and found a trend to greater complexity in chronic HDV and chronic HBV infection (CI, previously termed inactive carriers) than in chronic hepatitis B. This suggests that HDV may drive the HBV quasispecies to acquire a situation of diversity similar to that of CI patients.