Deep sequencing of hepatitis B virus basal core promoter and precore mutants in HBeAg-positive chronic hepatitis B patients.

Linlin Yan,Linlin Yan,Linlin Yan,Xingwang Xie,Hongsong Chen,Gaixia He,Yulin Kang,Ruifeng Yang,Ruifeng Yang,Henghui Zhang,Hongsong Chen,Henghui Zhang,Wei Li,Wei Li,Hui Ma,Hui Ma,Xingwang Xie,Di Liu,Di Liu,Qixiang Shao,Jianghua Wang,Jianghua Wang,Hao Wang,Hao Wang,Lai Wei,Lai Wei,Gaixia He,Zuhong Lu

doi:10.1038/srep17950

Abstract

Mutants in the basal core promoter (BCP) and precore (PC) regions of hepatitis B virus (HBV) genome are associated with the progression of chronic hepatitis B (CHB) infection. However, quasispecies characteristics of naturally occurring mutants in those regions in HBeAg-positive CHB patients has not been well described, partly limited by quantitative assay. This study aimed to develop an Ion Torrent deep sequencing assay to determine BCP and PC mutant percentages in HBeAg-positive CHB patients who were treatment naïve and correlate them with different viral and host factors. Our results showed that Ion Torrent deep sequencing could achieve high accuracy (R2>0.99) within a dynamic range between 1% and 100%. Twelve hotspots with prevalence of greater than 20% were observed in EnhII/BCP/PC regions. G1719T, T1753V, A1762T and G1764A were genotype C related. BCP A1762T/G1764A double mutants were generally accompanied with PC 1896 wild type or lower PC G1896A mutant percentage. Lower serum HBeAg and HBsAg levels were associated with higher BCP A1762T/G1764A mutant percentages (≥50%). ALT levels were higher in patients with PC G1896A mutant percentage greater than 10%. In conclusion, deep sequencing such as Ion Torrent sequencing could accurately quantify HBV mutants for providing clinical relevant information during HBV infection.

Highlights

Hepatitis B virus (HBV) is a 3.2 kb circular, partially double-stranded DNA virus, but replicates through reverse transcription of an RNA intermediate[1]
basal core promoter (BCP) A1762T/G1764A double mutants increase the risk of liver cirrhosis and hepatocellular carcinoma (HCC)[5,6,7,8,9], PC G1896A mutant decreases the risk of HCC6, the impact of PC G1896A mutant on HCC development remain controversial
To confirm the accuracy of Ion Torrent Personal Genome Machine (PGM) sequencing for quantification of hepatitis B virus (HBV) mutants, two reference plasmids containing A1762/G1764/G1896 and T1762/A1764/A1896 were constructed, which serve as BCP/PC wild type and mutant type plasmids respectively

Summary

Introduction

Hepatitis B virus (HBV) is a 3.2 kb circular, partially double-stranded DNA virus, but replicates through reverse transcription of an RNA intermediate[1]. Most previous studies analyzed HBV mutants using qualitative assay, such as direct sequencing, which limited the quantitative detection of mutants with low frequencies. Ion Torrent PGM sequencing is based on semiconductor technology, which allowing for low-cost, high sensitivity, large-scale production and high throughput[11]. It has been broadly applied in determining microbial community diversity and cancer unique mutations[12,13]. We developed a fast and sensitive quantitative method to analyze HBV mutants based on Ion Torrent PGM sequencer platform. We took advantage of this assay to analyze the percentages of HBV EnhII/BCP/PC mutants in 58 HBeAg-positive CHB patients and correlate them with different viral and host factors

Objectives

Methods

Results

Conclusion