Retrospective Study
Copyright ©The Author(s) 2015. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Jun 7, 2015; 21(21): 6639-6648
Published online Jun 7, 2015. doi: 10.3748/wjg.v21.i21.6639
Precore/basal core promoter mutants quantification throughout phases of hepatitis B virus infection by Simpleprobe
Wen-Hui Tu, Ying Lv, Yong-Mei Zhang, Wei Hou, Jin-Yu Wang, Yi-Jun Zhang, Hong-Yan Liu, Hao-Xiang Zhu, Yan-Li Qin, Ri-Cheng Mao, Ji-Ming Zhang
Wen-Hui Tu, Ying Lv, Yong-Mei Zhang, Jin-Yu Wang, Yi-Jun Zhang, Hong-Yan Liu, Hao-Xiang Zhu, Yan-Li Qin, Ri-Cheng Mao, Ji-Ming Zhang, Department of Infectious Disease, Huashan Hospital, Fudan University, Shanghai 200040, China
Wen-Hui Tu, Wei Hou, Department of Infectious Disease, Taizhou Municipal Hospital, Taizhou 318000, Zhejiang Province, China
Ying Lv, Department of Infectious Disease, Public Health Clinical Center of Shanghai, Fudan University, Shanghai 201508, China
Author contributions: Tu WH and Lv Y contributed equally to this work; Tu WH, Lv Y and Zhang JM designed the study; Tu WH and Lv Y performed the majority of the study; Tu WH and Hou W collected the serum specimens and clinical data; Zhang YM, Wang JY, Zhang YJ, Liu HY, Zhu HX, Qin YL and Mao RC also participated in the experiments; Tu WH, Lv Y and Zhang YM wrote the manuscript; Zhang JM revised the manuscript.
Supported by National Science and Technology Major Project of China, No. 2012ZX10002007-001-002 and No. 2013ZX10002001 (to Zhang JM); the National Natural Science Foundation of China, No. 81271833 and No. 81471933 (to Zhang JM); the Science and Technology Plan Project of Taizhou, Zhejiang province, No. 1402ky19 (to Tu WH and Hou W); and the Scientific Research Project of Taizhou University, Zhejiang province, No: 2014PY054 (to Tu WH and Hou W).
Ethics approval: The study was reviewed and approved by the Huashan Hospital and Taizhou Municipal Hospital Institutional Review Board.
Informed consent: All study participants, or their legal guardian, provided informed written consent prior to study enrollment.
Conflict-of-interest: All participating authors have declared no conflicts of interest.
Data sharing: Technical appendix, statistical code, and dataset available from the corresponding author at jmzhang@fudan.edu.cn. Participants gave informed consent for data sharing.
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/
Correspondence to: Ji-Ming Zhang, MD, Department of Infectious Disease, Huashan Hospital, Fudan University, No. 12 Wulumuqi Zhong Road, Shanghai 200040, China. jmzhang@fudan.edu.cn
Telephone: +86-21-52888125 Fax: +86-21-62489015
Received: November 2, 2014
Peer-review started: November 3, 2014
First decision: December 26, 2014
Revised: February 3, 2015
Accepted: February 11, 2015
Article in press: February 11, 2015
Published online: June 7, 2015
Abstract

AIM: To investigate precore/basal core promoter (PC/BCP) mutants throughout hepatitis B virus (HBV) infection and to determine their relationship to hepatitis B early antigen (HBeAg) titers.

METHODS: We enrolled 191 patients in various stages of HBV infection at the Huashan Hospital and the Taizhou Municipal Hospital from 2010 to 2012. None of the patients received antiviral therapy. HBV DNA from serum, was quantified by real-time PCR. The HBV genotype was determined by direct sequencing of the S gene. We used the Simpleprobe ultrasensitive quantitative method to detect PC/BCP mutants in each patient. We compared the strain number, percentage, and the changes in PC/BCP mutants in different phases, and analyzed the relationship between PC/BCP mutants and HBeAg by multiple linear regression and logistic regression.

RESULTS: Patients with HBV infection (n = 191) were assigned to groups by phase: Immune tolerance (IT) = 55, Immune clearance (IC) = 67, Low-replicative (LR) = 49, and HBeAg-negative hepatitis (ENH) = 20. Of the patients (male, 112; female, 79) enrolled, 122 were HBeAg-positive and 69 were HBeAg-negative. The median age was 33 years (range: 18-78 years). PC and BCP mutation detection rates were 84.82% (162/191) and 96.86% (185/191), respectively. In five HBeAg-negative cases, we detected double mutation G1896A/G1899A. The logarithm value of PC mutant quantities (log10 PC) significantly differed in IT, IC, and LR phases, as well as in the ENH phase (F = 49.350, P < 0.001). The logarithm value of BCP mutant quantities (log10 BCP) also differed during the four phases (F = 25.530, P < 0.001). Log10 PC and log10 BCP values were high in the IT and IC phases, decreased in the LR phase, and increased in the ENH phase, although the absolute value at this point remained lower than that in the IT and IC phases. PC mutant quantity per total viral load (PC%) and BCP mutant quantity per total viral load (BCP%) differed between phases (F = 20.040, P < 0.001; F = 10.830, P < 0.001), with PC% and BCP% gradually increasing in successive phases. HBeAg titers negatively correlated with PC% (Spearman’s rho = -0.354, P < 0.001) and BCP% (Spearman’s rho = -0.395, P < 0.001). The negative correlation between PC% and HBeAg status was significant (B = -5.281, P = 0.001), but there was no such correlation between BCP% and HBeAg status (B = -0.523, P = 0.552).

CONCLUSION: PC/BCP mutants become predominant in a dynamic and continuous process. Log10 PC, log10 BCP, PC% and BCP% might be combined to evaluate disease progression. PC% determines HBeAg status.

Keywords: Precore mutant, Basal core promoter mutant, Hepatitis B virus, Quantification, Hepatitis B early antigen titers

Core tip: During the natural history of hepatitis B virus infection, no evidence for the correlation between the dynamic alteration of precore/basal core promoter (PC/BCP) mutated strains and hepatitis B early antigen titers has been obtained by qualitative analysis. Using Simpleprobe ultrasensitive quantification of the wild-type and mutated hepatitis B virus (HBV) strains, we provided new insights into the process by which PC/BCP-mutated strains become dominant during the natural course of infection. Thus, we provide important clues for the evaluation of HBV infection status and the corresponding host immune responses.