Precore/basal core promoter mutants quantification throughout phases of hepatitis B virus infection by Simpleprobe

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.     

ANALYSIS OF POINT MUTATION IN SITE 1896 OF HBV PRECORE AND ITS DETECTION IN THE TISSUES AND SERUM OF HCC PATIENTS

Aim The 3'-base specific polymerase chain reaction (3'- BS- PCR) method was es-tablished to investigate the relationship between the mutation of precore region of Hepatitis B virus(HBV) and the liver damage to the patients caused by HBV and the possibility of HBV precore gene in-tegration in liver cells。 Mdthods According to the DNA sequence of precore region of HBV,themethod of 3'- BS- PCR is applied to analyze the point mutation site 1896 of HBV precore in 126 clini-cal serum specimens and 23 hepatoeellular carcinoma (HCC) patients' tissues and serum whose trmorshave been surgically excised and pathologically diagnosed.Rdsults The point mutation in site 1896 ofHBV precore has been successfully rates of preore gene of HBV in the 23 patients' tissues and serum are52.2 % (12/23) and 30.4 % (7/23) respectively.Conclusion The established method for HBV ore-core mutation analysis is simple and results can well repeated.It has provided a new approach to clinicalHBV research and its relationship to liver damage.The results obtained suggested that HBV precoremutation exists in a wide range among serum and tissue of the patients infected by HBV and HCC pa-tients,and the pre-c gene of HBV can not be detected in the serum of 21.8% of the HCC patients(tissue HBV precore gene positive).We may deduce that there may be the integration of HBV precoregenee in the genome of liver cells,which may play an important role in the carcinogenesis of HCC.     

乙型肝炎病毒前C区基因变异及与干扰素治疗的关系

目的　探讨乙型肝炎病毒前 C区基因变异对慢性乙型肝炎患者的影响及前 C区基因变异与 α-干扰素治疗的关系。方法　采用多聚酶链反应银染技术检测 3 2例 HBV DNA阳性慢性乙型肝炎患者用干扰素治疗前后的 HBV前 C区基因变异。结果　在治疗前有前 C区突变株感染 8例 ,突变株检出率为 2 5 % ;在治疗后有前 C区突变株感染 9例 ,突变株检出率为 2 8.1%。HBV DNA阴转 17例 ,阴转率为 5 3 .1%。结论　 HBV前 C区基因变异不影响对干扰素的应答     

抗HBe阳性乙型肝炎和无症状携带者的HBVC基因启动子和前C基因变异

目的 研究抗HBe阳性乙型肝炎病人和抗HBe阳性无症状携带(AsC)的乙型肝炎病毒(HBV)C基因启动子(CP)和前C基因变异。方法 通过DNA扩增、基因序列分析检测34例抗HBe阳性和55例抗HBe阴性乙型肝炎病人及28例抗HBe阳性AsC的血清HBVCP和前C基因序列。结果 (1)抗HBe阳性肝炎组的前C终止变异(nt1896G→A)的发生率显高于抗HBe阴性肝炎组(61．8％和14．5％)；而CP双变异(nt1762A→T和1764G→A)则在两组中无明显差异(50．0％和45．5％)；(2)同抗HBe阳性AsC组比较，抗HBe阳性肝炎组的HBVDNA定量呈高水平，前C终止变异的发生率也显增高(61．8％和28．6％)；(3)同抗HBe阳性慢性乙型肝炎(CHB)组比较，抗HBe阳性重型乙型肝炎(CSH)病人的前C终止变异发生率和CP双变异发生率无明显差异(70．0％和58．3％，50．0％和50．0％)，HBV DNA定量也无明显差异，而CPnt 1752A→G变异发生率则显增高(80．0％和29．2％)。结论 前C终止变异、nt1846突变和病毒复制可能与抗HBe阳性乙型肝炎发病有关；而Cpnt1752突变则可能与抗HBe阳性CSH发病有关。     

慢性乙型肝炎病毒感染者病毒前C区和基本核心启动子区变异检测及意义

目的 探讨乙型肝炎病毒（HBV）前C区和基本核心启动子（BCP）区变异与基因型及疾病进展间的关系。方法 收集HBV携带者（ASC）、慢性乙型肝炎（CHB）、肝炎肝硬化（LC）、肝细胞肝癌（HCC）患者血清148份，用半巢式聚合酶链反应扩增HBV前C／C基因部分片段，产物纯化后直接测序，检测前C区A1896及BCP区T1762／A1764变异。用S基因聚合酶链反应-限制性片段长度多态性分析（PCR-RFLP）方法确定HBV基因型。结果 有128份血清能够成功分型和测序，其中B基因型60份，C基因型68份。在B基因型感染者中前C区A1896变异检出率（48．33％）明显高于C基因型感染者（29．41％，X^2=4．83，P〈0．05）；而BCP区T1762／A1764变异检出率却明显低于C基因型感染者，差异亦有统计学意义（30．00％：73．54%，X^2=24．25。P〈0．05）。前C区A1896变异在CHB、LC、HCC中的阳性检出率分别为46．88％（15/32）、39．39％（13／33）、51．52％（17／33）。与ASC的13．33％（4／30）相比，P分别〈0．05，差异有统计学意义。BCP区T1762／A1764变异检出率在HCC、LC组分别为87．88％（29／33）和72．73％（24／33）．明显高于CHB组的37．50％（12／32）及ASC组10.00％（3／30）（P〈0．05）。结论 前C区A1896变异常见于B基因型感染者，而BCP区T1762/A1764变异C基因型感染者多见。除ASC外．前C区A1896变异与疾病进展关系不大．而BCP区T1762/A1764变异与乙型肝炎进展及顶后相关。     

HBV基因型与前C/C启动子变异及抗病毒疗效的关系

目的 了解深圳市乙型肝炎病毒（HBV）基因分型情况，探讨HBV基因型与前C／C启动子变异、乙肝的病程进展及抗病毒疗效的关系。方法 用单克隆抗体ELISA法（mAbs ELISA）对深圳市165例HBV感染者进行HBV基因分型；随机抽取24例慢性乙型肝炎（CUB）患者，用基因芯片技术检测HBV前C／C启动子变异；回顾性分析HBV基因型与干扰素、贺普丁抗HBV疗效的关系。结果 ①165例患者中，以B型106例（64.2％）和C型48例（29．1％）为主。慢性无症状乙肝病毒携带者（ASC）组B型占95．4％，肝硬化（LC）组C型占64．7％（P〈0．05）。②24例CHB患者中，16例（10例B型，6例C型）发生HBV前C／C启动子变异：前C区变异（nt1896、1862）者10例（B型9例，C型1例）。基本C区启动子变异（BCP）变异（nt1762、1764）者6例（B型1例，C型5例）。③用干扰素治疗的27例HBeAg（＋）CHB患者，达到完全应答者B型11例（62．5％）较C型1例（9．1％）多见（P〈0．05）。用贺普丁治疗的29例HBeAg（＋）CHB患者，持续应答者B型15例（78．9％）较C型3例（30．0％）多见（P〈0．05）。结论 ①深圳市HBV基因分型以B型为主，C型次之。②C型较B型易发生BCP变异，发生肝硬化机会较高，且对于扰素及贺普丁疗效较差。     

原发性肝细胞肝癌患者癌组织和血清中HBV前C区基因的检测

目的 乙型肝炎炎病毒（ＨＢＶ）是原发性肝细胞肝癌（ＨＣＣ）的主要诱导因素之一。为探讨ＨＢＶ在ＨＣＣ中的重要性，我们检测了ＨＣＣ患者癌组织及血清中的ＨＢＶ前Ｃ区基因。方法 根据ＨＢＶ前Ｃ区ＤＮＡ序列，采用聚合酶链式反应（ＰＣＲ）对２３例手术切除后病理确诊有ＨＣＣ患者癌组织以及血清进行了检测。结果 ＰＣＲ显示２３例ＨＣＣ患者癌组织和血清中ＨＢＶ前Ｃ区基因的阳性率分别为５２．２％（１２／２３）和３０．４     

Precore/core region mutations of hepatitis B virus related to clinical severity

Despite the availability of an effective vaccine, hepatitis B virus(HBV) infection remains a major health problem, with more than 350 million chronically infected people worldwide and over 1 million annual deaths due to cirrhosis and liver cancer. HBV mutations are primarily generated due both to a lack of proofreading capacity by HBV polymerase and to host immune pressure, which is a very important factor for predicting disease progression and therapeutic outcomes. Several types of HBV precore/core(preC/C) mutations have been described to date. The host immune response against T cells drives mutation in the pre C/C region. Specifically, pre C/C mutations in the MHC class Ⅱ restricted region are more common than in other regions and are significantly related to hepatocellular carcinoma. Certain mutations, including preC G1896 A, are also significantly related to HBe Ag-negative chronic infection. This review article mainly focuses on the HBV pre C/C mutations that are related to disease severity and on the HBe Ag serostatus of chronically infected patients.     

Targeting the hepatitis B virus precore antigen with a novel IgNAR single variable domain intrabody

The Hepatitis B virus precore protein is processed in the endoplasmic reticulum (ER) into secreted hepatitis B e antigen (HBeAg), which acts as an immune tolerogen to establish chronic infection. Downregulation of secreted HBeAg should improve clinical outcome, as patients who effectively respond to current treatments (IFN-α) have significantly lower serum HBeAg levels. Here, we describe a novel reagent, a single variable domain (V_NAR) of the shark immunoglobulin new antigen receptor (IgNAR) antibodies. V_NARs possess advantages in stability, size (~ 14 kDa) and cryptic epitope recognition compared to conventional antibodies. The V_NAR domain displayed biologically useful affinity for recombinant and native HBeAg, and recognised a unique conformational epitope. To assess therapeutic potential in targeting intracellular precore protein to reduce secreted HBeAg, the V_NAR was engineered for ER-targeted in vitro delivery to function as an intracellular antibody (intrabody). In vitro data from HBV/precore hepatocyte cell lines demonstrated effective intrabody regulation of precore/HBeAg.