乙型肝炎病毒逆转录酶区基因序列准种与变异特点

乙型肝炎病毒(Hepatitis B Virus,HBV)P基因编码产物从功能上分为末端蛋白(1～178aa)、间隔区(179～336aa)、逆转录酶区(337～682aa)和RNA酶H区(683～816aa),各区有相应的生物学功能;逆转录酶区包含S基因主蛋白编码区.近年来的研究提出HBV感染者体内存在有准种[1,2]的假说.我们以逆转录酶区序列为研究靶区域,应用聚合酶链反应(PCR)技术扩增慢性乙型肝炎患者血清中的靶基因序列,随机选择克隆测序,比较其结果,证明了HBV准种特点的存在,并发现多种基因突变形式.     

山东省潍坊市慢性乙型肝炎患者HBVP区基因耐药突变检测及相关因素分析

探讨慢性乙型肝炎(Chronic hepatitis B,CHB)患者多聚酶基因逆转录保守区(P区)位点突变情况。选择212例行核苷(酸)类似物抗病毒治疗的CHB患者,采用PCR产物直接测序法检测HBV P基因区耐药变异位点,同时检测其HBV基因型。结果表明,HBV的P基因区突变位点有173、180、18l、184、204、236和250,主要的耐药位点为204和180,分别占35.8%和23.5%。180位点在不同年龄组之间比较均有显著差异,204位点在30岁以下组与41~50岁组、51~60岁组间比较有显著差异(P0.05,P0.01);180位点联合204位点突变率为66.6%,l81位点联合236位点突变率为23.3%;HBV C基因型患者年龄明显大于B基因型患者(P0.01)。M204V/I多以联合L180M突变的形式存在,突变率与年龄有关,HBV基因型和HBV P区耐药位点的检测对CHB患者的治疗和病情预后具有重要的临床意义。     

乙型肝炎病毒X基因准种特点的研究

以乙型肝炎病毒(HBV)X基因序列的异质性表现来探讨HBV准种在慢性感染者中的存在状态.以中国株HBV基因序列为依据,设计特异性多聚酶链反应引物,自9例慢性HBV感染患者血清中扩增HBV X基因,克隆入pGEM Teasv质粒,随机挑选克隆进行DNA测序以确定病毒的变异程度.37例测序结果提示来源于不同患者HBV X基因序列高度保守,但每个序列均不一致.X区除了存在广泛的碱基点替换突变外,序列的缺失突变占测序克隆总数的51.4%(19/37);氨基酸缺失及移框突变多发生于123位氨基酸残基之后,可导致X蛋白多种羧基端形式.结果提示HBV长期携带者体内有HBV准种共存,X区内存在热点缺失突变区,该区突变结果可能影响X蛋白反式激活功能.     

采用熔点曲线法研究乙肝病毒准种与拉米夫定临床耐药相关性

为了研究乙型肝炎病毒(HBV)准种与拉米夫定耐药的关系以指导临床用药,随机选取拉米夫定治疗后发生YMDD变异的慢性乙型肝炎(CHB)患者30例,以及停用拉米夫定后发生病毒反弹的CHB患者30例作为研究对象,同时以未经拉米夫定治疗的CHB患者30例为对照,采用聚合酶链反应(PCR)扩增这些病人体内HBV的P区,再用熔点曲线法分析3组患者体内的HBV准种情况。另外,采用相同方法对HBV C区、S区准种也进行了对比。结果显示,病毒变异组患者体内HBV P区准种数量为2.50±0.86个,病毒反弹组为5.30±0.95个,未治疗组为8.37±0.93个,3组间准种数量两两比较均有显著性差异(P<0.05)。HBV C区病毒变异组准种数量为6.10±1.86个,病毒反弹组为6.37±1.81个,未治疗组为6.33±1.64个,3组准种数量无显著性差异(P>0.05)。HBV S区病毒变异组准种数量为5.23±1.85个,病毒反弹组为6.17±1.93个,未治疗组为5.77±2.11个,3组准种数量无显著性差异(P>0.05)。HBV P区熔点曲线图显示,病毒变异组优势病毒群的熔点与病毒反弹组和未治疗组相比,已发生明显的偏移,而在HBV C区和S区熔点曲线图中,3组的波峰数和优势病毒群的熔点均没有明显变化。可见,在拉米夫定的作用下HBV P区准种数量减少,准种的性质也发生变化,发生变异后劣势耐药病毒株变为优势病毒株易被检测到。拉米夫定对HBV C区、S区作用不明显。     

雌激素受体α(ESR1)基因PvuⅡ和XbaⅠ位点基因多态性与HBV慢性感染的相关性研究

目的:探讨雌激素受体ESR1(Estrogen Receptor alpha gene)基因的PvuⅡ(rs2234693)和XbaⅠ (rs9340799)两个单核苷酸多态性(single nucleotide polymorphisms, SNPs)位点的基因多态性与乙型肝炎病毒HBV(Hepatitis B Virus)慢性感染的相关性,为控制HBV持续感染提供新的思路和科学依据。方法:选择107例慢性乙型病毒性肝炎患者为病例组及107例同期体检的健康人群为对照组,基于高分辨熔解曲线技术(High Resolution Melting,HRM)建立PCR-HRM分子诊断方法,检测其雌激素受体ESR1基因两个SNP位点rs2234693(TC)和rs9340799(AG)的基因多态性,并通过基因测序进一步验证,探讨上述两个SNP位点与HBV慢性感染的相关性。结果:病例组和健康对照组ESR1基因rs2234693(TC)位点的基因型频率比较差异具有统计学意义(P0.05),而两组间rs2234693位点等位基因频率比较差异没有统计学意义(P0.05);病例组和健康对照组间ESR1基因rs9340799(AG)位点的各基因型频率差异具有统计学意义(P0.05),慢性乙肝病例组GG基因型明显升高,两组间rs9340799位点等位基因频率差异亦具有统计学意义(P0.05)。Logistic回归分析显示rs9340799位点的G基因可增加HBV慢性感染的发病风险,A基因可降低HBV慢性感染的发病风险。结论:雌激素受体基因ESR1的rs9340799 (AG)位点的GG基因型和G等位基因可能是HBV感染慢性化的遗传易感基因,GG基因型与HBV的慢性感染具有一定的相关性。     

雌激素受体α(ESR1)基因PvuⅡ和XbaI位点基因多态性与HBV慢性感染的相关性研究

摘要 目的：探讨雌激素受体ESR1(Estrogen Receptor alpha gene)基因的PvuⅡ(rs2234693)和XbaI (rs9340799)两个单核苷酸多态性(single nucleotide polymorphisms, SNPs)位点的基因多态性与乙型肝炎病毒HBV(Hepatitis B Virus)慢性感染的相关性,为控制HBV持续感染提供新的思路和科学依据。方法：选择107例慢性乙型病毒性肝炎患者为病例组及107例同期体检的健康人群为对照组,基于高分辨熔解曲线技术(High Resolution Melting,HRM)建立PCR-HRM分子诊断方法,检测其雌激素受体ESR1基因两个SNP位点rs2234693(T>C)和rs9340799(A>G)的基因多态性,并通过基因测序进一步验证,探讨上述两个SNP位点与HBV慢性感染的相关性。结果：病例组和健康对照组ESR1基因rs2234693(T>C)位点的基因型频率比较差异具有统计学意义(P<0.05),而两组间rs2234693位点等位基因频率比较差异没有统计学意义(P>0.05);病例组和健康对照组间ESR1基因rs9340799(A>G)位点的各基因型频率差异具有统计学意义(P<0.05),慢性乙肝病例组GG基因型明显升高,两组间rs9340799位点等位基因频率差异亦具有统计学意义(P<0.05)。Logistic回归分析显示rs9340799位点的G基因可增加HBV慢性感染的发病风险,A基因可降低HBV慢性感染的发病风险。结论：雌激素受体基因ESR1的rs9340799 (A>G)位点的GG基因型和G等位基因可能是HBV感染慢性化的遗传易感基因,GG基因型与HBV的慢性感染具有一定的相关性。     

乙型肝炎病毒RT 区变异及其与基因型分布的关系

目的:探讨大样本乙型肝炎病毒(HBV)感染患者RT区耐药位点变异的流行情况,及各耐药位点变异与HBV基因型的关系。方法:采用P区测序法对1117例慢性乙型肝炎患者的血清病毒进行P区测序、进化树分型。结果:RT区耐药位点变异发生率与基因型关系密切,在基因型C患者中的变异发生率远远高于基因型B患者(P=0.000)。Rt180、rtM204V、rtM204I、rt181、rt213位点变异均与基因型C有关(P<0.05)。主要的三种变异类型rt180+rtM204V、rtM204I、rt180+rtM204I间基因型分布存在显著差异(P=0.003)。不同HBeAg状态下,耐药变异的发生有显著差异(P=0.020),特别是rt181和rt236位点变异。结论:HBV基因型影响RT区耐药变异发生率及变异类型,且耐药变异发生率也与HBeAg状态有关。     

乙型肝炎病毒RT区变异及其与基因型分布的关系

目的：探讨大样本乙型肝炎病毒（HBV）感染患者RT区耐药位点变异的流行情况,及各耐药位点变异与HBV基因型的关系。方法：采用P区测序法对1117例慢性乙型肝炎患者的血清病毒进行P区测序、进化树分型。结果：RT区耐药位点变异发生率与基因型关系密切,在基因型C患者中的变异发生率远远高于基因型B患者（P=O．000）。Rt180、rtM204V、rtM204I、rt181、rt213位点变异均与基因型c有关（P〈O．05）。主要的三种变异类型rt180＋rtM204V、rtM204I、rt180＋rtM204I间基因型分布存在显薯差异（P=0．003）。不同HBeAg状态下,耐药变并的发生有显著差并（P=O．020）,特别是rt181和rt236位点变畀。结论：HBV基因型影响RT区耐药变异发生率及变异类型。且耐药变异发生率也与HBeAg状态有关。     

IL28B基因在乙型肝炎病毒感染中的作用

乙型肝炎病毒(Hepatitis B virus,HBV)感染是引发乙型肝炎的病因,慢性化程度较高,后期可诱发肝硬化甚至肝癌。IL28B基因属于新型干扰素λ家族,已有研究报道其遗传多态性与HBV感染者的治疗效果和病毒自然清除率具有相关性。通过探讨IL28B基因遗传易感性与HBV感染、患者治疗应答和病毒清除的关系,可以进一步了解宿主基因多态性在HBV感染治疗和病毒自然清除中的作用机制,为乙肝患者个体化医疗提供一定的理论基础。     

乙型肝炎病毒表面抗原-抗体复合物治疗慢性乙型肝炎患者病毒S基因变异研究

目的 研究应用乙型肝炎病毒表面抗原(HBsAg)-抗体复合物治疗性疫苗(YIC)治疗慢性乙型肝炎患者是否会诱生S基因免疫逃逸变异株的出现。方法 选取5例用30μg或60μgYIC治疗后血清乙型肝炎病毒(HBV)DNA水平下降>2log10、伴有乙型肝炎病毒e抗原(HBeAg)转阴应答,但在随访6个月后病毒DNA水平重复升高的患者作为研究对象,另选取1例对YIC治疗始终无应答患者和1例注射安慰剂患者作为对照,用聚合酶链反应(PCR)方法扩增治疗前(0周)及治疗后(44周)血清中HBV DNA的S基因、前-核心基因、核心基因启动子片段,并进行序列比对分析。结果 S基因“a”决定簇及前-核心基因均未发生变异,但YIC治疗后有3例HBV的核心基因启动子1762/1764位点序列有变异,另有2例在核心基因启动子的其他位点有核苷酸变异。结论 研究显示5例出现病毒重新复制的患者并非由于发生了病毒S基因逃逸变异所致。     

The relationship between core promoter mutation of hepatitis B virus, viral load and hepatitis B e antigen status in chronic hepatitis B patients

The aim of this study is to detect the possible association of hepatitis B virus (HBV) core mutation, hepatitis B e antigen (HBeAg) status and the viral load in chronic hepatitis B (CHB) patients. Sixty-six patients with CHB were enrolled. Hepatitis markers and hepatitis C virus antibody (HCV-Ab) were tested using micro particle enzyme immunoassay kits. Viral load was measured by real-time polymerase chain reaction (PCR) and the mutation was analyzed by nested PCR followed by restriction fragment length polymorphism. Most of CHB patients were HBeAg (-ve). The HBeAg status did not have an influence on the presence or absence of T1762/A1764 mutation. HBV-DNA serum level was not significantly different in patients with core mutation and patients without core mutation in HBeAg (-ve) group, while in HBeAg (+ve) group HBV-DNA serum level was significantly higher in patients with core mutation. This study reports the predominance of HBeAg (-ve) and HBV core promoter mutation.     

Levels of hepatitis B virus replicative intermediate in serum samples of chronic hepatitis B patients

Hepatitis B virus (HBV) cccDNA levels is an absolute marker of HBV replication in the liver of HBV infected patients. This study aimed to quantify the HBV cccDNA levels in sera and liver tissue samples of treatment naïve patients with chronic hepatitis B. Eighty one chronic hepatitis B (CHB) treatment naïve patients were enrolled from January 2009 to June 2011. Total HBV DNA and HBV cccDNA levels were quantified using sensitive real time PCR assay. The mean age of recruited patients was 34 ± 11.5 years. Fifty four (66.7 %) patients were HBeAg negative. Liver tissue samples were available from 2 HBeAg positive and 21 HBeAg negative CHB patients. The amount of total intrahepatic HBV DNA ranged from 0.09 to 1508.92 copies/cell. The median intrahepatic HBV cccDNA was 0.31 and 0.20 copies/cell in HBeAg positive and HBeAg negative cases, respectively. Serum HBV cccDNA was detectable in 85.2 % HBeAg positive and 48.1 % HBeAg negative CHB patients. Median serum HBV cccDNA was 46,000 and 26,350 copies/mL in HBeAg positive and HBeAg negative subjects, respectively. There was a significant positive correlation between the levels of intrahepatic total HBV DNA and intrahepatic HBV cccDNA (r = 0.533, p = 0.009). A positive correlation was also seen between serum HBV cccDNA levels and serum HBV DNA levels (r = 0.871, p < 0.001). It was concluded that serum HBV cccDNA could be detectable in higher proportion of HBeAg positive patients compared to HBeAg negative patients. Moreover, the median level of serum HBV cccDNA was significantly higher in HBeAg positive patients in contrast to HBeAg negative subjects.     

Lower Mutation Frequency of BCP/Precore Regions in e Antigen-Negative Chronic HBV-Infected Children instead of Adults Patients

To describe the Hepatitis B e antigen(HBeAg) seroconversion related mutation profiles of the basal core promoter(BCP)/precore regions in e antigen seroconverted child patients, a cohort of 245 child patients with CHB and a control patients group of 92 adult patients with CHB were recruited. The mutation frequencies of six nucleotides or nucleotide combinations including nucleotide (nt)1896, nt1762/1764, nt1752, nt1846, nt1899 and nt1753 showed significant differences between HBeAg positive and HBeAg-negative child patients groups. The frequencies of these HBeAg seroconversion-related mutations were significantly lower in HBeAg-negative children with CHB than in HBeAg-negative adults with CHB, especially for the mutation G1896A (41.1% vs 91.7%, P<0.001), and the average number of BCP/precore region mutations in samples from HBeAg-negative child patients was also obviously lower than in HBeAg-negative adult patients(3.62±3.03 vs 4.89±2.09, P<0.001), suggesting less impact of mutations in the BCP/precore region on HBeAg seroconversion in child patients than adult patients.     

Molecular evolution of hepatitis B virus over 25 years

Determining the longitudinal molecular evolution of hepatitis B virus (HBV) is difficult due to HBV's genomic complexity and the need to study paired samples collected over long periods of time. In this study, serial samples were collected from eight hepatitis B virus e antigen-negative asymptomatic carriers of HBV genotype B in 1979 and 2004, thus providing a 25-year period to document the long-term molecular evolution of HBV. The rate, nature, and distribution of mutations that emerged over 25 years were determined by phylogenetic and linear regression analysis of full-length HBV genome sequences. Nucleotide hypervariability was observed within the polymerase and pre-S/S overlap region and within the core gene. The calculated mean number of nucleotide substitutions/site/year (7.9 x 10(-5)) was slightly higher than published estimates (1.5 x 10(-5) to 5 x 10(-5)). Nucleotide changes in the quasispecies population did not significantly alter the molecular evolutionary rate, based on linear regression analysis of evolutionary distances among serial clone pre-S region sequences. Therefore, the directly amplified or dominant sequence was sufficient to estimate the putative molecular evolutionary rate for these long-term serial samples. On average, the ratio of synonymous (dS) to nonsynonymous (dN) substitutions was highest for the polymerase-coding region and lowest for the core-coding region. The low dS/dN ratios observed within the core suggest that selection occurs within this gene region, possibly as an immune evasion strategy. The results of this study suggest that HBV sequence divergence may occur more rapidly than previously estimated, in a host immune phase-dependent manner.     

新疆维吾尔族与汉族乙型肝炎患者HBVDNA 及肝功能检测结果的比较

目的:探讨新疆乌鲁木齐地区伴有肝功能指标:丙氨酸氨基转移酶(ALT)浓度异常的维吾尔族(维族)及汉族HBeAg阳性乙型肝炎初次就诊患者,乙型肝炎病毒DNA复制载量及ALT浓度是否存在差异及其对患者诊断、预后的意义。方法:回顾性选取门诊伴有ALT浓度异常的汉族、维族初次就诊患者并筛选出HBeAg阳性患者汉族、维族共373例。采用实时荧光定量聚合酶链反应、生化测定及酶联免疫吸附试验法分别测定HBV DNA、ALT浓度及乙肝HBeAg。结果:(1)汉族HBV DNA组秩和8869,维族HBV DNA组秩和10359.36,经Mann-Whitney Test检验两组间尚不能肯定HBVDNA分布有统计学意义,即伴有肝功能损害的汉族、维族初次就诊HBeAg阳性患者HBV DNA复制程度没有差异。(2)汉族ALT组秩和26818.50,维族ALT组秩和22009.50,经Mann-Whitney Test检验两组间ALT分布有统计学意义,即伴有肝功能损害的初次就诊HBeAg阳性患者汉族肝功能损害程度高于维族。(3)HBVDNA低复制组(103-104copy/mL):汉族秩和3771.46,维族秩和4993.2;中复制组(104-106copy/mL):汉族秩和6412.4,维族秩和5088.2;高复制组(>106copy/mL):汉族秩和929.04,维族秩和666.96,经Mann-Whitney Test检验在低复制组两民族间ALT分布无统计学意义,在中、高复制组两民族间ALT具有统计学意义。即:伴有肝功能损害的初次就诊HBeAg阳性患者在HBV DNA低复制组两民族间肝功能损害程度无差异,但在中、高复制组汉族肝功能损害程度高于维族。结论:新疆乌鲁木齐地区伴有肝功能损害的初次就诊的HBeAg阳性的汉族与维族之间HBV DNA的病毒复制无统计学意义(P>0.05),但两民族间的ALT具有统计学意义,可能跟维族的民俗、饮食习惯及生存环境、免疫相关基因HLA基因频率分布差异等因素有关。     

Virological Characteristics of Acute Hepatitis B in Eastern India: Critical Differences with Chronic Infection

Hepatitis B Virus (HBV) manifests high genetic variability and is classifiable into ten genotypes (A-J). HBV infection can lead to variable clinical outcomes, ranging from self-limiting acute hepatitis to active chronic hepatitis, cirrhosis and hepatocellular carcinoma. The present study characterizes HBV strains circulating among patients with acute (AHB) and chronic HBV infection (CHB). Among a total of 653 HBsAg positive cases, 40 manifested acute infection. After sequencing the surface(S), basal core promoter/pre-core(BCP/PC) and the X gene regions, phylogenetic tree was constructed using MEGA4 by neighbor-joining method. Statistical robustness was established with bootstrap analysis. Nucleotide diversity was determined by Shannon entropy per site using the Entropy program of the Los Alamos National Laboratories. Analyses of acute patients revealed that HBV/D2 is the major circulating sub-genotype and commonly associated with sexual promiscuity and the age group between15-30 years. Comparison of AHB and CHB patients revealed that HBeAg positivity, ALT levels and genotype D were significantly high in AHB, whereas CHB patients were predominantly male, had a high viral load, and were commonly associated with genotype C. The frequencies of mutations in the S, BCP/PC, and X gene were low in AHB as compared to CHB. Drug resistant mutations were not detectable in the polymerase gene of AHB. Average nucleotide diversity in AHB was considerably low as compared to CHB. Further, the highest average ΔH (average difference in entropy between chronic and acute infection) was observed in the BCP/PC region implying that this region was most vulnerable to mutations upon HBV persistence, especially in case of genotype C. Additionally, among all substitutions, the A1762T and G1764A BCP mutations were the strongest indicators of chronicity. In conclusion, the study exhibits a general portrait of HBV strains circulating among acute hepatitis B patients in Eastern India and their intricate differences with chronic patients which should be useful from the clinical point of view.     

Compartmentalization of hepatitis C virus quasispecies in blood mononuclear cells of patients with mixed cryoglobulinemic syndrome

The aim of this study was to investigate the quasispecies heterogeneity of hepatitis C virus (HCV) in the plasma, cryoprecipitate, and peripheral lymphocytes of chronically infected HCV patients with mixed cryoglobulinemia (MC). We studied 360 clones from 10 HCV-positive patients with MC and 8 age-, gender- and HCV genotype-matched subjects with chronic HCV infection but without MC. A partial nucleotide sequence encompassing the E1/E2 region, including hypervariable region 1 (HVR1), was amplified and cloned from plasma, cryoprecipitates, and peripheral blood mononuclear cells (PBMC), and the genetic diversity and complexity and synonymous and nonsynonymous substitution rates were determined. Heterogeneous selection pressure at codon sites was evaluated. Compartmentalization was estimated by phylogenetic and phenetic (Mantel's test) approaches. The patients with MC had 3.3 times lower nonsynonymous substitution rates (1.7 versus 5.7 substitutions/100 sites). Among the subjects with HCV genotype 1, the MC patients had significantly less complexity than the controls, whereas the diversity and complexity were similar in the genotype 2 patients and controls. Site-specific selection analysis confirmed the low frequency of MC patients showing positive selection. There was a significant correlation between positive selection and the infecting HCV genotype. The quasispecies were less heterogeneous in PBMC than in plasma. Significant compartmentalization of HCV quasispecies was observed in the PBMC of four of nine subjects (three with MC) and seven of nine cryoprecipitates. In one subject with MC, we detected a 5-amino-acid insertion at codons 385 to 389 of HVR1. Our results suggest reduced quasispecies heterogeneity in MC patients that is related to a low selection pressure which is probably due to an impaired immune response, the HCV genotype, and/or the duration of the infection. The frequent HCV quasispecies compartmentalization in patients' PBMC suggests a possible pathogenetic significance.     

抗—HBe阳性慢性乙肝临床研究

报告34例抗-HRe阳性慢性乙型肝炎(CHB),占同期住院抗-HBe阳性者的38％。这些患者于3—9年内肝炎再活动2—4次,累计80次。肝炎再活动时的临床表现及肝功损害与同期住院的HBeAg阳性CHB相似,但抗-HBe阳性CHB者肝硬化及肝癌发生比例显著高于HBeAg阳性者(P<0.05)。血清乙肝病毒标志(HBVM)检测发现,80次肝炎再活动中,38次(47.5％)有HBV活跃复制,提示HBV活跃复制是部分抗-HBe阳性CHB肝炎再活动的根本原因,另一部要考虑是其它肝炎病毒重叠感染的结果。