Elsevier

Virus Research

Volume 155, Issue 1, January 2011, Pages 254-258
Virus Research

Characterization of hepatitis virus B isolated from a multi-drug refractory patient

https://doi.org/10.1016/j.virusres.2010.10.018Get rights and content

Abstract

Prolonged treatment of chronic hepatitis B (CHB) with nucleoside analogues (NAs) almost invariably engenders viral resistance, and sequential NAs monotherapy can promote multi-drug resistance. This study aimed to investigate the molecular characteristics and the mutation profile of multi-drug resistant hepatitis B virus (HBV). The complete genome of HBV isolated from a multi-drug refractory patient was amplified and cloned, and 22 clones were selected for sequencing. The homology of the full-length genome between clones ranged from 98.7% to 99.9%. A precore stop codon mutation of G1896A and basic core promoter (BCP) mutations A1762T/G1764A were detected in a majority of clones. A phylogenetic analysis showed that all clones were classified as subgenotype B2. Three mutations in the surface (S) antigen region, sC76Y, sP120T and sI195M, were detected in 100%, 100% and 77.3% of the clones, respectively. In the core (C) antigen region, a mutation at codon 135 (cP135Q) was detected in 100% of clones. Lamivudine (LAM)-resistant mutations, rtL180M and rtM204V/I were detected in 86.4% of clones. Adefovir (ADV) or entecavir (ETV)-resistant mutations were not detected. Several novel mutations, such as rtT128N, rtA222T, rtS256G, rtL271M, rtS332R, and rtN/T337D, were present in a majority of clones. Furthermore, six pairs of mutations in the overlapping reverse transcriptase (RT) gene and S gene were detected. In conclusion, the complex HBV mutation profile detected in the multi-drug refractory patient highlights the problems associated with the ongoing selection of mutations, including further compensatory mutations as well as potential cross-resistance mutations.

Introduction

Hepatitis B virus (HBV) is estimated to infect more than 2 billion populations currently worldwide and among them, around 360 million are suffered from chronic hepatitis, cirrhosis and hepatocellular carcinoma (WHO, 2010, Shepard et al., 2006). Despite being a DNA virus, HBV uses pre-genomic RNA as a replicative intermediate. Moreover, HBV reverse transcriptase (RT) lacks proofreading capacity, thus leading to mutation and genetic variation during replication. Consequently, HBV circulates in vivo as a viral population with a spectrum of genetically distinct but closely related variants, known as quasispecies (Domingo and Gomez, 2007). Mutations have been reported in each gene and in their promoter regions, and these mutations can change the efficiency of HBV replication and the clinical course of patients.

In recent years, treatment of chronic HBV has been improved by the availability of the nucleoside analogues (NAs), such as lamivudine (LAM), adefovir dipivoxil (ADV), telbivudine (LDT), and entecavir (ETV). Unfortunately, the efficacy of NAs is limited by the selection of resistant viruses during the course of therapy. Drug resistance has been associated with the emergence of polymerase gene mutations, followed by viral breakthrough, subsequent increasing alanine aminotransferase (ALT) levels, and, in some instances, liver failure (Liu et al., 2001, Wang et al., 2002, Fung et al., 2005). In the case of LAM-resistance, rates are estimated to reach over 70% in monoinfected individuals and up to 90% in HIV–HBV coinfected individuals after 4 years of treatment (Benhamou et al., 1999). The main mutation associated with LAM resistance has been well characterized, and it occurs within the tyrosine–methionine–aspartate–aspartate (YMDD) motif of the RT region, rtM204V/I/S (Ling et al., 1996, Niesters et al., 2002). More recently, it has been recognized that there are at least six major genotypic patterns associated with LAM-resistance (Locarnini, 2003), including a triple mutation pattern (rtV173L + rtL180M + rtM204V). In patients treated continuously with adefovir at 10 mg/d as a monotherapy, drug-resistant mutants emerged in 2%, 5.9%, 18%, and 29% of patients after 2–5 years, respectively. Resistance to adefovir is most frequently conferred by the selection of a rtN236T mutation or a rtA181V mutation (Angus et al., 2003, Villeneuve et al., 2003, Brunelle et al., 2005). Entecavir resistance was observed primarily in the therapy of lamivudine refractory patients. The resistance rate appears to be approximately 10% after 2 years and 25% after 3 years in patients with lamivudine failure and 0.8% in naive patients over 3 years (Colonno et al., 2006). The main ETV-resistance mutations are rtT184G, rtS202I, rtM250V on a background of LAM-resistant mutations (Tenney et al., 2004). In addition to the RT region, variants in other regions may influence antiviral efficacy. However, previous studies have focused primarily on the mutation within the RT region. In this study, a detailed clonal HBV complete genome analysis was conducted on a serum sample from a multi-drug refractory patient to investigate the molecular characteristics and the mutation profile of HBV.

Section snippets

Samples and DNA extraction

The serum sample was collected from a Chinese patient with CHB (male, 41 years old) who had failed antiviral therapy with anti-HBV drugs including IFN + LAM, ADV, and ETV as described (Shaw et al., 2006). At the time of serum sample collection, the ALT level of this patient was 53 IU/L. The patient was seropositive for hepatitis B surface antigen (HBsAg) and anti-HBe, but was seronegative for anti-HBs, and hepatitis B e antigen (HBeAg). HBV DNA was 4.98 × 106 copy/ml. HBsAg, anti-HBs, HBeAg, and

Homogeneity of the nucleotide sequences

The percent identities of sequence between clones from the patient of the C gene, polymerase region, whole S region, X gene and whole genome were 98.2–100%, 99.1–99.8%, 98.4–100%, 98.2–100% and 98.7–99.9%, respectively.

Phylogenetic analysis

A phylogenetic tree based on the entire genome sequences, compared with isolates retrieved from GenBank, showed that all clones clustered with genotype B2 (Fig. 2), which is one of the predominant genotypes in China.

Mutation patterns of the 22 clones

The nucleotide and deduced amino acid sequences of the C, S, and

Discussion

Multi-drug resistant HBV mutants are becoming increasingly prevalent with sequential NA monotherapy, but the report of multi-drug resistant HBV based on the entire genome analysis is rare. In the present study, we analyzed the full-length genome of HBV isolated from a multi-drug refractory patient.

Because of the existence of quasispecies (Blum, 1993, Domingo and Gomez, 2007), long distance PCR was employed to obtain HBV haplotype sequences instead of assembling the PCR sequences of several

Acknowledgment

This work was supported in part by National Natural Science Foundation of China (Project No.: 30571665).

References (36)

  • C.J. Liu et al.

    Hepatitis B virus variants in patients receiving lamivudine treatment with breakthrough hepatitis evaluated by serial viral loads and full-length viral sequences

    Hepatology

    (2001)
  • T. Shaw et al.

    HBV drug resistance: mechanisms, detection and interpretation

    J. Hepatol.

    (2006)
  • J. Torresi et al.

    Restoration of replication phenotype of lamivudineresistant hepatitis B virus mutants by compensatory changes in the “fingers” subdomain of the viral polymerase selected as a consequence of mutations in the overlapping S gene

    Virology

    (2002)
  • J.P. Villeneuve et al.

    Selection of a hepatitis B virus strain resistant to adefovir in a liver transplantation patient

    J. Hepatol.

    (2003)
  • Q. Zhang et al.

    Universal primers for HBV genome DNA amplification across subtypes: a case study for designing more effective viral primers

    Virol. J.

    (2007)
  • Z. Awan et al.

    Hepatitis B virus YMDD-motif mutations with emergence of lamivudine-resistant mutants: a threat to recovery

    Gastroenterol. Hepatol. Bed Bench

    (2010)
  • Y. Benhamou et al.

    Long-term incidence of hepatitis B virus resistance to lamivudine in human immunodeficiency virus-infected patients

    Hepatology

    (1999)
  • A. Bertoletti et al.

    Natural variants of cytotoxic epitopes are T-cell receptor antagonists for antiviral cytotoxic T cells

    Nature

    (1994)
  • Cited by (5)

    View full text