©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, 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. email@example.com
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
Hepatitis delta virus (HDV) causes the most severe form of chronic viral hepatitis in persons simultaneously infected with hepatitis B virus (HBV). In longitudinal clinical studies, HDV infection has been associated with a considerable temporary or permanent reduction in HBV viral load, whereas HBV surface antigen levels are usually high. Thus, beyond the interaction with HBV envelope proteins, there are other mechanisms by which HDV inhibits HBV-DNA replication.
To date, little information has emerged on the interaction between HDV and HBV. In this study, we investigated whether HDV can affect the complexity of the HBV quasispecies, and proposed possible mechanisms by which it may do so, to further characterize the interaction between these two viruses.
Considering the essential role of the HBV X protein (HBx) on viral replication, the aim of this study was to analyze the 5’ end of the hepatitis B X gene (HBX) coding region and its upstream non-coding region (nt 1255-1611) by next-generation sequencing (NGS) to evaluate HBV quasispecies complexity between chronic hepatitis delta (CHD)-infected patients and chronic HBV mono-infected patients [HBV chronic infection (CI) and chronic hepatitis B (CHB)].
The HBX 5’ end region, nucleotide (nt) 1255-1611, was PCR-amplified for subsequent NGS (MiSeq, Illumina, United States) in 7 CI, 8 CHB, and 9 CHD patients. HBV quasispecies complexity in the region analyzed was evaluated using incidence-based indices [number of haplotypes (nHpl) and number of mutations (nMuts)], abundance-based indices (Hill numbers of order, q = 1 and q = 2) and functional indices [mutation frequency (Mf) and nt diversity (Pi)]. The pattern of nt changes was evaluated to investigate the cause of HBV quasispecies complexity.
HBV quasispecies complexity was significantly higher in the CI group than in CHB for abundance (Hill numbers q = 1 and q = 2) and incidence (nHpl and nMuts). In CHD, the HBV quasispecies showed a trend towards higher complexity similar to that of CI patients. No significant differences were observed in Mf or Pi between the groups, although CI and CHD showed a trend towards greater quasispecies complexity than CHB patients. The proportion of G-to-A vs. A-to-G and C-to-T vs. T-to-C nt changes in genotype A and D haplotypes by group did not provide conclusive evidence of a hyper-mutation pattern associated with the innate immune system enzyme APOBEC3G.
The HBV quasispecies showed a trend to higher complexity in groups with lower viral replication (CHD and CI) than in the higher-replicating CHB patients. This could indicate that HDV has an effect on the 5’ HBX sequence, increasing HBV quasispecies complexity. Two different mechanisms are proposed to explain how HDV can change the HBV quasispecies: hypermutation by activation of the innate system through HDV stimulation or loss of RNA pol II fidelity due to its interaction with hepatitis delta antigen. Further studies are needed to determine the clinical impact of the increased HBV quasispecies complexity in CHD patients, which may be of help to devise new therapy strategies.
CHD drives the HBV quasispecies to a situation similar to that found in HBV CI: Lower replication level and higher HBX quasispecies complexity than in CHB patients. Further studies are needed to characterize the mechanisms by which HDV acts on the HBV quasispecies, which may include the innate immune system or RNA pol II fidelity.