Copyright
©The Author(s) 2019. Published by Baishideng Publishing Group Inc. All rights reserved.
Characterization of hepatitis B virus X gene quasispecies complexity in mono-infection and hepatitis delta virus superinfection
Cristina Godoy, David Tabernero, Sara Sopena, Josep Gregori, Maria Francesca Cortese, Carolina González, Rosario Casillas, Marçal Yll, Ariadna Rando, Rosa López-Martínez, Josep Quer, Gloria González-Aseguinolaza, Rafael Esteban, Mar Riveiro-Barciela, Maria Buti, Francisco Rodríguez-Frías
Cristina Godoy, David Tabernero, Sara Sopena, Maria Francesca Cortese, Carolina González, Rosario Casillas, Marçal Yll, Ariadna Rando, Rosa López-Martínez, Francisco Rodríguez-Frías, Liver Pathology Unit, Departments of Biochemistry and Microbiology, Hospital Universitari Vall d’Hebron, Universitat Autònoma de Barcelona, Barcelona 08035, Spain
Cristina Godoy, Josep Gregori, Maria Francesca Cortese, Marçal Yll, Josep Quer, Liver Unit, Liver Disease Laboratory-Viral Hepatitis, Vall d’Hebron Institut Recerca-Hospital Universitari Vall d'Hebron, Universitat Autònoma de Barcelona, Barcelona 08035, Spain
David Tabernero, Sara Sopena, Josep Gregori, Josep Quer, Rafael Esteban, Mar Riveiro-Barciela, Maria Buti, Francisco Rodríguez-Frías, Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Instituto de Salud Carlos III, Madrid 28029, Spain
Josep Gregori, Roche Diagnostics SL, Sant Cugat del Vallès 08174, Spain
Gloria González-Aseguinolaza, Centro de Investigación Médica Aplicada (CIMA), Universidad de Navarra, Pamplona 31008, Spain
Rafael Esteban, Mar Riveiro-Barciela, Maria Buti, Liver Unit, Department of Internal Medicine, Hospital Universitari Vall d’Hebron, Universitat Autònoma de Barcelona (UAB), Barcelona 08035, Spain
Author contributions: Rodríguez-Frías F designed the research; Tabernero D coordinated the research; Godoy C and Tabernero D equally contributed to design the experiments; Godoy C, Sopena S, Casillas R, Yll M, González C and Rando A performed the experiments; Godoy C, Gregori J, Tabernero D, Cortese MF and Riveiro-Barciela M analyzed data acquired during the experiments and interpreted the results; Godoy C and Tabernero D drafted the manuscript; Cortese MF, Lopez-Martinez R, Buti M, Quer J, González-Aseguinolaza G, Esteban R, Riveiro-Barciela M and Rodríguez-Frías F critically reviewed the manuscript.
Supported by the Instituto de Salud Carlos III, grants PI15/00856 and PI17/02233; co-financed by the European Regional Development Fund (ERDF).
Institutional review board statement: The study was reviewed and approved by the Clinical Research Ethics Committee (CEIC) of Hospital Universitari Vall d’Hebron.
Institutional animal care and use committee statement: No animal models were used in this study.
Conflict-of-interest statement: Josep Gregori is an employee of Roche Diagnostics, SL.
Data sharing statement: No additional data are available.
ARRIVE guidelines statement: The authors have read the ARRIVE guidelines and have prepared the manuscript accordingly.
Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/
Corresponding author: David Tabernero, PhD, Postdoc, Research Scientist, Liver Pathology Unit, Departments of Biochemistry and Microbiology, Hospital Universitari Vall d’Hebron, Universitat Autònoma de Barcelona, Passeig Vall d’Hebron 119-129, Barcelona 08035, Spain.
david.tabernero@ciberehd.org
Telephone: +34-932746897
Received: December 24, 2018
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
Processing time: 103 Days and 16.5 Hours
BACKGROUND
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.
AIM
To compare HBV quasispecies complexity in the HBX 5’ region between chronic hepatitis delta (CHD) and chronic HBV mono-infected patients.
METHODS
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.
RESULTS
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.
CONCLUSION
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.
