Clinical application of estimating hepatitis B virus quasispecies complexity by massive sequencing: correlation between natural evolution and on-treatment evolution.
OBJECTIVE
To evaluate HBV quasispecies (QA) complexity in the preCore/Core regions in relation to HBeAg status, and explore QA changes under natural evolution and nucleoside analogue (NUC) treatment.


METHODS
Ultra-deep pyrosequencing of HBV preCore/Core regions in 30 sequential samples (baseline [diagnosis], treatment-free, and treatment-nonresponse) from 10 retrospectively selected patients grouped according to HBeAg status over time: HBeAg+ (N = 4), HBeAg- (N = 2), and fluctuating HBeAg (transient seroreversion/seroconversion pattern) (N = 4). QA complexity was defined by Shannon entropy, mutation frequency, nucleotide diversity, and mutation frequency of amino acids (MfAA) in preCore and Core.


RESULTS
The QA was less complex in HBeAg+ than in HBeAg- or fluctuating HBeAg. High complexity in preCore was associated with decreased viral replication (preCore MfAA negatively correlated with HBV-DNA, p = 0.005). QA complexity in the treatment-free period negatively correlated with values seen during treatment. Specific variants were mainly selected in the Core region in HBeAg- and fluctuating HBeAg patients, suggesting higher immune pressure than in HBeAg+.


CONCLUSIONS
The negative correlation between QA natural evolution and on-treatment evolution indicates the importance of pre-treatment QA study to predict QA changes in NUC nonresponders. Study of QA complexity could be useful for managing HBV infection.
Centro de investigación biomédica en red: enfermedades hepáticas y digestivas (CIBERehd), Instituto de Salud Carlos III, Barcelona, Spain; Liver Pathology Unit, Departments of Biochemistry and Microbiology, Hospital Vall d'Hebron, Universitat Autònoma de Barcelona, Barcelona, Spain; Virology Unit, Department of Microbiology, Hospital Vall d'Hebron, Universitat Autònoma de Barcelona, Barcelona, Spain.
Maria
M
Homs
Liver Pathology Unit, Departments of Biochemistry and Microbiology, Hospital Vall d'Hebron, Universitat Autònoma de Barcelona, Barcelona, Spain.
Andrea
A
Caballero
Centro de investigación biomédica en red: enfermedades hepáticas y digestivas (CIBERehd), Instituto de Salud Carlos III, Barcelona, Spain; Liver Diseases, Research Institute Hospital Vall d'Hebron, Barcelona, Spain.
Josep
J
Gregori
Centro de investigación biomédica en red: enfermedades hepáticas y digestivas (CIBERehd), Instituto de Salud Carlos III, Barcelona, Spain; Liver Pathology Unit, Departments of Biochemistry and Microbiology, Hospital Vall d'Hebron, Universitat Autònoma de Barcelona, Barcelona, Spain; Virology Unit, Department of Microbiology, Hospital Vall d'Hebron, Universitat Autònoma de Barcelona, Barcelona, Spain.
David
D
Tabernero
Centro de investigación biomédica en red: enfermedades hepáticas y digestivas (CIBERehd), Instituto de Salud Carlos III, Barcelona, Spain; Liver Diseases, Research Institute Hospital Vall d'Hebron, Barcelona, Spain.
Josep
J
Quer
Liver Pathology Unit, Departments of Biochemistry and Microbiology, Hospital Vall d'Hebron, Universitat Autònoma de Barcelona, Barcelona, Spain; Virology Unit, Department of Microbiology, Hospital Vall d'Hebron, Universitat Autònoma de Barcelona, Barcelona, Spain.
Leonardo
L
Nieto
Centro de investigación biomédica en red: enfermedades hepáticas y digestivas (CIBERehd), Instituto de Salud Carlos III, Barcelona, Spain; Liver Unit, Department of Internal Medicine, Hospital Vall d'Hebron, Universitat Autònoma de Barcelona, Barcelona, Spain.
Rafael
R
Esteban
Centro de investigación biomédica en red: enfermedades hepáticas y digestivas (CIBERehd), Instituto de Salud Carlos III, Barcelona, Spain; Liver Unit, Department of Internal Medicine, Hospital Vall d'Hebron, Universitat Autònoma de Barcelona, Barcelona, Spain.
Maria
M
Buti
Centro de investigación biomédica en red: enfermedades hepáticas y digestivas (CIBERehd), Instituto de Salud Carlos III, Barcelona, Spain; Liver Pathology Unit, Departments of Biochemistry and Microbiology, Hospital Vall d'Hebron, Universitat Autònoma de Barcelona, Barcelona, Spain; Virology Unit, Department of Microbiology, Hospital Vall d'Hebron, Universitat Autònoma de Barcelona, Barcelona, Spain.
Francisco
F
Rodriguez-Frias
D000328:Adult; D000368:Aged; D004279:DNA, Viral; D019143:Evolution, Molecular; D005260:Female; D006509:Hepatitis B / Q000188:drug therapy / Q000821:virology*; D006513:Hepatitis B e Antigens / Q000737:chemistry; D006515:Hepatitis B virus / Q000145:classification / Q000235:genetics*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D008137:Longitudinal Studies; D008297:Male; D008875:Middle Aged; D009154:Mutation; D009711:Nucleotides / Q000737:chemistry; D015203:Reproducibility of Results; D012189:Retrospective Studies; D016896:Treatment Outcome
D004279:DNA, Viral; D006513:Hepatitis B e Antigens; D009711:Nucleotides