Elsevier

Antiviral Research

Volume 90, Issue 3, June 2011, Pages 116-125
Antiviral Research

Evolutionary pattern of full hepatitis B virus genome during sequential nucleos(t)ide analog therapy

https://doi.org/10.1016/j.antiviral.2011.03.183Get rights and content

Abstract

The evolutionary and mutational pattern of full hepatitis B virus (HBV) quasispecies during sequential nucleos(t)ide analog (NUC) therapy remains unclear. In this study, full-length HBV clones were generated from serial serum samples of five chronic hepatitis B patients who received sequential NUC therapies (treated patients) and two untreated patients with acute flares. The evolutionary and mutational patterns of full HBV quasispecies were studied. In the three treated patients who received lamivudine as initial antiviral therapy, nucleotide polymorphism and nonsynonymous divergence all decreased at lamivudine breakthrough but increased after rescue therapies. Conversely, two other treated patients showed a distinct change in divergence during adefovir–telbivudine sequential therapies. Untreated subjects exhibited increased polymorphism and divergence in the preC/C region at ALT flare. Four of the treated patients presented amino acid changes in the “a” determinant during NUC therapy. All of the treated subjects showed amino acid changes within the known T-cell or B-cell epitopes in the surface or core antigen, most of which were accompanied by mutations in reverse transcriptase (RT) region. Co-variations in the core promoter, the preC region and in the known epitopes of the preS gene accompanied by RT mutations, were common. In untreated patients, most of these co-variations located in the preC/C gene. In conclusion, the distribution of genetic variability of HBV shows remarkably different patterns between the treated and untreated subjects and the quasispecies divergence of different regions of HBV may vary remarkably even within a single host.

Introduction

Chronic hepatitis B virus (HBV) infection is a major public health problem worldwide (Lee, 1997). In recent years, treatment of chronic hepatitis B (CHB) has been improved with the availability of nucleoside/nucleotide analogs (NUCs) such as lamivudine (LAM), adefovir dipivoxil (ADV), telbivudine (LDT), and entecavir (ETV). NUCs target the HBV reverse transcriptase (RT), thus inhibiting viral replication and leading to virologic, biochemical, and histological improvement in most patients. However, the emergence of drug-resistant mutations has become an increasing problem during the treatment with NUCs. Drug resistance has been associated with the emergence of polymerase gene mutations and is often followed by viral breakthrough, subsequent increase in alanine amino transferase (ALT) levels, and, in some circumstances, liver failure.

Most studies of HBV drug resistance have focused on the analysis of the A to D domain of the HBV DNA polymerase gene after detection of antiviral-resistant HBV mutants and the drug-resistant mutational pattern that occurs in this region has been well characterized. The HBV genome contains four partially overlapping open reading frames (ORFs) [Surface (S), Polymerase (P), Core (C), and X proteins (X)], with no noncoding regions and about 50% of its genome involved in two overlapping ORFs. Mutations in the RT domain can affect the amino acid sequence of the surface protein, especially the “a” determinant or T-cell epitope, leading to alterations of immunogenicity (Tai et al., 1997, Chisari and Ferrari, 1995). HBV-related hepatitis activity is HLA class I restricted and T-cell mediated (Chisari and Ferrari, 1995), and previous studies have found that the immune-escape variants probably appeared after antiviral-resistant variants emerge; this might be responsible for the exacerbation of chronic hepatitis B hepatitis (Ehata et al., 1993, Liu et al., 2003). Based on these findings, dynamic changes within other regions (especially in the immune-targeted surface and core antigen) might also be informative, and investigation of the mutational pattern of the HBV full genome is necessary.

Although HBV is a DNA virus, it replicates through RNA intermediates which require reverse transcriptase. Because the proof-reading function of the reverse transcriptase is insufficient, mutations occur at a higher rate than in other DNA viruses (Nowak et al., 1996). As a result, like other RNA viruses, HBV shows quasispecies distribution in infected patients. Each HBV-infected person harbors a group of viral quasispecies, or a swarm of genetically distinct but related variants. Evolution of the HBV genome has proven to be an informative marker of host–virus interaction. Quasispecies distributions of HBV facilitate the selection of variants that possess survival advantages against host immune responses and antiviral therapeutic agents (Pawlotsky, 2005), thus its evolution and distribution should be monitored to allow chronic infection management.

Pallier et al. (2006) showed that quasispecies variants bearing drug-resistant mutations could be detected at several months before virologic breakthrough and gradually became predominant. The dynamic evolution of HBV quasispecies during the commonly used antiviral therapies have also been investigated in other studies (Ji et al., 2009, Feng et al., 2008, Villet et al., 2007, Guo et al., 2009, Yim et al., 2006). These studies focused on the HBV DNA polymerase gene and did not investigate the mutational pattern of the HBV full genome. Several recent studies have reported the mutational pattern of the HBV full genome. Enomoto et al. (2007) suggested that mutational patterns of HBV DNA at the time of emergence of YMDD variants were unrelated to clinical outcomes during lamivudine therapy. Horiike et al. (2007) found no significant difference of mutations between breakthrough hepatitis and non-breakthrough hepatitis patients. Chen et al. (2010) found in LAM/ADV-treated patients that amino acid changes within the known T-cell or B-cell epitopes of the HBV surface and core antigens might emerge at the LAM and/or ADV resistance. In most of these studies, the HBV full genome sequences were examined by direct sequencing, not by cloning. Thus, the evolutionary pattern of complete HBV quasispecies remains unclear.

In this study, we used full genome sequences generated from patient sera to assess the quasispecies evolution dynamics of five CHB patients with sequential NUC therapies, as well as of two untreated patients with acute flares. The aim of this study is to elucidate the mutational pattern and quasispecies evolution of full-length HBV sequences from CHB patients with sequential NUC therapies.

Section snippets

Source of samples

Sera were obtained from seven patients who were persistently positive for hepatitis B surface antigen (HBsAg) and who were followed at the Institute for Infectious Diseases, Southwest Hospital of the Third Military Medical University. The seven patients included five patients with NUCs therapy and two untreated patients with acute flare. The untreated patients showed ALT levels that were within the normal range on regular examinations performed every 3–6 months for more than 2 years before ALT

Clinical characteristics of study patients

The clinical course of the study patients is shown in Fig. 1. Treated subjects 1, 2 and 5 received LAM as their initial antiviral therapy; another NUC was administered when virologic breakthrough occurred. All three patients showed virologic breakthrough to the rescue therapy. Treated subject 3 showed no response to LAM therapy, and a virologic breakthrough was observed during both the subsequent rescue therapies, one with ADV and one with LDT. Treated subject 4 received ADV-LDT sequential

Discussion

This report describes the evolution of the HBV full genome sequences in CHB patients receiving sequential NUC therapies during chronic infection. Two untreated patients with acute flares were also included in the study.

The divergence of HBV quasispecies in treated patients is interesting. For treated patients 1, 2 and 5 who received LAM initial therapy, divergence in four ORFs all decreased at LAM breakthrough but became elevated after rescue therapies. This pattern of clustering to a new clade

Conflict of interest statement

None declared.

Acknowledgements

This study was supported by the State Key Project of National Natural Science Foundation of China (30830090) and the State Key Project specialized for Infectious Diseases, PR China (2008ZX10002-007).

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