Sequence variations of precore/core and precore promoter regions of hepatitis B virus in patients with or without viral reactivation during cytotoxic chemotherapy

Reactivation of the hepatitis B virus (HBV) is a well-described complication among cancer patients undergoing cytotoxic chemotherapy. Mutations in the preC/C and the preC promoter regions of HBV have been reported in some patients who developed this condition. A G-to-A mutation at nt 1896 in the preC/C region (HBeAg negative/ anti-HBe positive) has been associated with more severe liver disease than that caused by wild type virus. In addition, it has been suggested that patients with these mutations may be more likely to reactivate than those with the wild type virus. Whether or not such mutations were present before the commencement of or developed during the course of cytotoxic chemotherapy is not known. In this study, 28 cancer patients (consisting of 14 consecutive patients who developed HBV reactivation and another 14 who had no reactivation during cytotoxic chemotherapy) are reported. The objectives were firstly, to determine the prechemotherapy HBeAg status and nucleotide sequences of the preC/C and preC promoter regions of HBV in order to determine if these parameters affected the rate of reactivation, and secondly, for those who developed reactivation, to determine whether the mutations were present before chemotherapy or developed during, possibly as a result of, cytotoxic chemotherapy. HBV DNA was amplified by PCR and nucleotide sequencing performed on samples taken prior to chemotherapy and at the time of reactivation. Results revealed that 16 of the 28 patients were HBeAg negative/anti-HBe positive. Of these 16, four (57%) of the seven patients who had nt 1896 mutation, but only one (17%) of the six who had the wild type HBV genome, developed reactivation. Three had no detectable HBV DNA. In the majority of cases, the type of virus, i.e. wild/mutant at preC/C, that was detected during the reactivation was identical to that detected in the pretreatment samples. With respect to the preC promoter region, the two commonest mutations detected were at nt 1762 (A to T) and nt 1764 (G to A). When this region was translated into amino acid sequences, stop codons leading to truncated X protein at carboxyl terminus were found in four patients, three of whom developed HBV reactivation. We conclude that chronic HBV carriers who are HBeAg negative/anti-HBe positive with nt 1896 mutation (G to A) may be more likely to develop HBV reactivation during cytotoxic chemotherapy than those with the wild type virus. Cytotoxic chemotherapy does not appear to select out mutant HBV, or to be consistently mutagenic in patients who develop HBV reactivation. The occurrence of stop codons in the amino acid sequences of the X protein in three patients who developed HBV reactivation, including one who was detected only at the time of reactivation, is of particular interest, as such mutant viruses remain replication competent.     

乙型肝炎病毒调控机制的研究进展

乙型肝炎病毒（HBV）可以从复制、表达、转录等多个环节产生调节，发生变异，从而使病毒的致病力、抗原信息传递、宿主的免疫状态、抗美丽药物的疗效、疾病的预后转归等发生发。因此，有必要从基因水平对此进行深入研究。     

Occult Hepatitis B Virus Infection: An Update

Occult hepatitis B virus (HBV) infection (OBI) refers to a condition in which replication-competent viral DNA is present in the liver (with detectable or undetectable HBV DNA in the serum) of individuals testing negative for the HBV surface antigen (HBsAg). In this peculiar phase of HBV infection, the covalently closed circular DNA (cccDNA) is in a low state of replication. Many advances have been made in clarifying the mechanisms involved in such a suppression of viral activity, which seems to be mainly related to the host’s immune control and epigenetic factors. OBI is diffused worldwide, but its prevalence is highly variable among patient populations. This depends on different geographic areas, risk factors for parenteral infections, and assays used for HBsAg and HBV DNA detection. OBI has an impact in several clinical contexts: (a) it can be transmitted, causing a classic form of hepatitis B, through blood transfusion or liver transplantation; (b) it may reactivate in the case of immunosuppression, leading to the possible development of even fulminant hepatitis; (c) it may accelerate the progression of chronic liver disease due to different causes toward cirrhosis; (d) it maintains the pro-oncogenic properties of the “overt” infection, favoring the development of hepatocellular carcinoma.     

Interaction between desialylated hepatitis B virus and asialoglycoprotein receptor on hepatocytes may be indispensable for viral binding and entry

The cellular receptor for hepatitis B virus (HBV) infection has not yet been identified. The purpose of this study was to address the possibility of participation by desialylated HBV and the asialoglycoprotein receptor (ASGP-R) exclusively expressed on liver parenchymal cells, in infection. Assays for viral binding and entry were performed by culturing a hepatoblastoma cell line, HepG2, and HBV particles derived from the HBV carrier in the presence or absence of neuraminidase (NA). Viral binding and entry were clearly enhanced in the presence of NA, and the enhancement of the binding could be blocked by asialo-fetuin and ethylenediamine-tetraacetic acid (EDTA). In addition, covalently closed circular (CCC)-DNA, as a marker of infectivity, was detected in the presence of NA, but not in its absence. The optimal concentration of NA raised infectivity more than 1000 times. We concluded that this method makes it feasible to evaluate the infectivity of HBV in vitro and that ASGP-R may be a specific HBV receptor once viral particles are desialylated.     

Comparison of the hepatitis B virus core, surface and polymerase gene substitution rates in chronically infected patients

For phylogenetic comparison of hepatitis B virus (HBV) isolates, often a region of the HBV surface gene is analysed. Because the surface gene completely overlaps the polymerase gene, its evolution is constrained, and it may not be the best choice for genetic comparison of HBV isolates. Analysing serial sample pairs of 33 chronically HBV-infected, untreated patients, with a cumulative follow-up of 184 years, the synonymous and nonsynonymous substitution rates of a part of the overlapping HBV surface and polymerase genes were compared to those of a nonoverlapping part of the HBV core gene. The substitution rate of the HBV core gene was higher (8.15 × 10(-4) vs 4.57 × 10(-4) substitutions/site/year) than that of the surface gene. The difference was mainly due to a significantly lower synonymous substitution rate in the surface gene, with dN/dS ratios of 0.412 in the core gene and 0.986 in the surface gene. Contrary to the core gene, the number of substitutions in the surface gene was higher in low viraemic hosts, who control HBV infection by suppressing replication. The number of substitutions in the core gene correlated more strongly with the duration of follow-up. The overlapping HBV surface and polymerase genes experience strong negative selection, which limits the number of substitutions. Because the HBV core gene reflects the duration of infection more accurately, it is more suitable for the analysis of short-term viral evolution and of hepatitis B transmission chains.     

应用Digoxigenin标记探针定量检测血清HBV DNA

本文报道采用非同位素Ｄｉｇｏｘｉｇｅｎｉｎ标记探针定量检测血清ＨＢＶ ＤＮＡ。显色膜经空气干燥，液体石蜡透明处理后，即可置酶联免疫检测仪上测定各斑点的光密度值，液长６３０ｎｍ。结果发现已各含量的ＨＢＶ ＤＮＡ在２－５００ｐｇ／样点范围内与其相应的ＯＤ值有着良好的线性关系。标本测得ＯＤ值后，便可知其含量，该法重复性试验试验结果良好，１１份ＨＢＶ ＤＮＡ阳性的血清经定量测定，其ＨＢＶ ＤＮＡ含量在２０     

病毒性肝炎血清HBV DNA和HCV RNA同时检测的探索

本文对ＨＢＶ ＤＮＡ和ＨＣＶ ＲＮＡ用ＰＣＲ方法一次性同时检测技术进行了探索，经过９２例患者血清的检测的结果显示，与同份血清单一ＰＣＲ方法所检测的ＨＢＶ和ＨＣＶ感染阳性总鹰率为９８．１６％，说明此种多重套式ＰＣＲ方法对临床诊断ＨＢＶ和ＨＣＶ感染具有实用价值。     

1．2倍基因组长度C基因型乙型肝炎病毒重组体构建及其在HepG2细胞中表达和复制

目的构建基于pBlueBac4．5质粒的1．2倍基因组长度C基因型乙型肝炎病毒（HBV）重组体，并研究其在HepG2细胞中的表达和复制。方法以重组质粒pWT上的1．2倍基因组长度C基因型HBVDNA序列和pBB4．5HBV1．3（D基因型）上的pBlueBac4．5载体序列为模板，构建重组质粒pBB4．5HBV1．2（C基因型）。用FuGENEHD瞬时转染法，将pBB4．5HBV1．2导人HepG2细胞。用化学发光免疫分析法、Southern印迹杂交法、荧光定量PCR法，分别检测转染后不同时间点HBsAg和HBeAg、HBV复制中间体及HBVDNA水平。此外，对转染时重组质粒用量进行优化。结果酶切和序列分析证实，pBB4．5HBV1．2重组质粒构建成功。初步转染实验证实，在转染细胞培养上清中可检测到HBsAg和HBeAg。优化后转染条件为：使用60mm细胞培养皿，8～11tLgpBB4．5HBV1．2，质粒与转染试剂5：9（μg：μl）。在此条件下，5d实验周期内可检测到HBsAg和HBeAg持续表达（峰值一般出现在转染后第3天）、HBVDNA持续复制（10^6～10^8拷贝／ml）及HBVDNA复制中间体形成。结论在HepG2细胞中，建立了以杆状病毒转移载体pBlueBac4．5为基础的1．2倍基因组长度C基因型HBV体外培养体系，有望为研究HBV耐药、筛选新抗病毒药物等提供新技术平台。     

乙肝病毒感染者血清HBeAg模式与HBVDNA定量关系的研究

探讨乙肝病毒感染者血清HBeAg模式与HBV DNA定量之间的关系。采用酶联免疫技术和实时荧光定 量PCR法,分别检测随机选择的80例乙肝病毒感染者血清HBeAg模式及HBV DNA定量。结果显示,当乙肝病毒 感染者血清HBV DNA含量为A、C级时,血清HBeAg模式与HBV DNA定量之间亦无相关性(P>0．05)。当乙肝病 毒携带者血清HBV DNA含量为B级时,HBeAg阳性模式HBV DNA含量显著高于HBeAg阴性者,血清HBeAg模式 与HBV DNA定量之间有显著相关性(r=0．28,t=1．19,P<0．05)。由此可推测,只有当机体内乙型肝炎病毒水平在 一定的范围内,机体的免疫应答能力才与乙型肝炎病毒水平相呼应。     

广西肝癌中HBV感染与N_ras基因突变的研究

AIM To observe the roles of N-ras gene mutation and hepatitis B virus (HBV) infection in the carcinogenesis of hepatocellular carcinoma (HCC) in Guangxi, China.METHODS The polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and immunohistochemistry were used to detect N-ras gene mutation and HBV infection in 29 cases of HCC.RESULTS The aberration rates at codon 2-37 of N-ras were 79.31% in HCCs and 80.77% in adjacent non-tumorous liver tissues. More than 2 point mutations of N-ras gene were observed in 22 (75.86%) cases. HBsAg and HBxAg positive rates were 86.2% and 79.3%. There was a parallel tendency between HBV marker detections and the mutation rate of N-ras gene.CONCLUSION HBV infection and N-ras gene mutation may be involved in the carcinogenesis and development of HCC in Guangxi. Since the aflatoxin B1 contamination is one of risk factors for HCC in this area, it may contribute to the mutation of N-ras gene in carcinogenesis of HCC.INTRODUCTIONHepatocellular carcinoma (HCC) is one of common malignant tumors in People′s Republic of China. Guangxi is a high incidence area of HCC. Many factors are involved in hepatocarcinogenesis. Many studies revealed that hepatitis B virus (HBV) infection might be a risk factor for hepatocellular carcinogenesis. One theory for hepatocarcinogenesis is that the oncogene(s) may be transactivated by hepatitis B x antigen (HBxAg)[1]. It is found recently that activation of N-ras gene may be the molecular basis for the carcinogenesis and development of HCC[2,3]. There have been reports about overexpression of N-ras oncogene in human HCC[4], but a few dealt with the roles of N-ras gene mutation and HBV infection, and their relationship with HCC. We analyzed the N-ras gene mutation and HBV infection in HCC by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and immunohistochemistry in 29 cases of human HCC.     

HBV NAT positive [corrected] blood donors in the early and late stages of HBV infection: analyses of the window period and kinetics of HBV DNA

BACKGROUND AND OBJECTIVES: The Japanese Red Cross (JRC) carries out nucleic acid amplification testing (NAT) for hepatitis B virus (HBV), hepatitis C virus (HCV) and human immunodeficiency virus-1 (HIV-1) by using a multiplex (MPX) reagent. Screening is undertaken on serologically negative units. In this study we characterized HBV NAT-positive donations individually and analysed the window period and kinetics of HBV DNA, during acute infection, in follow-up studies. MATERIALS AND METHODS: Two hundred and seventy-seven HBV DNA-positive donations have been identified in Japan since the introduction of NAT screening of 50-donation minipools. The viral loads and genotypes of these HBV DNA-positive donations were characterized. The doubling time and half-life of HBV was estimated from the data of 123 follow-up donors. The sensitivity of the NAT system (based on 50-donation minipools) was compared with the sensitivities of the enzyme immunoassay (EIA) and the chemiluminescence immunoassay (CLIA). Samples that were CLIA negative, but with > 10(4) copies/ml of HBV DNA, were analysed by sequencing the hepatitis B surface antigen (HBsAg) region. RESULTS: Out of 277 HBV NAT-positive samples, 125 (45%) were found to have an increasing viral load and 45 (16%) a decreasing viral load. Forty per cent of HBV NAT-positive samples with an increasing viral load, and 33% of those with a decreasing viral load, were negative when tested by using the CLIA. No mutations related to escape mutants were found in the samples that were CLIA negative but with HBV DNA loads of > 10(4) copies/ml. The median HBV doubling time was 2.6 days (n = 93, 1.3-15.2 days) and the half-life was 1.6 days (n = 55, 0.9-6.3 days). Some kinetic difference was observed between genotypes A and B. CONCLUSIONS: HBV NAT screening detected HBV DNA in both early (the so-called serological window period) and late stages of acute HBV infection.     

preS2S/adw真核表达质粒构建及其表达的初步研究

目的：针对我国乙型肝炎（乙肝）病毒流行的血清型，采用美国药品及食物鉴定委员会承认的应用于疫苗临床使用的载体pVAX1，构建了肝病毒adw血清型核酸疫苗preS2S的真核表达质粒，对其在真核细胞中的表达进行初步研究。方法：采用PCR法扩增preS2S片段，插入pVAX1载体中，测定DNA序列后，转染SP2／0细胞，抽提转染后SP2／0细胞的总RNA，再用RT－PCR法扩增其中的preS2S片段，以检测其表达的基础，结果：测序证实了该片段为乙肝病毒adw型preS2S基因。PCR扩增法检测出的转染细胞中存在preS2S基因，结论：获得了adw血清型乙肝病毒preS2S的真核表达质粒，提示其能够在真核细胞中表达目的基因蛋白。     

乙型肝炎患者血清HBV标志物与HBV DNA相关性分析

目的探讨乙型肝炎患者血清学标志(HBVM)与HBV DNA的关系。方法对257例乙型肝炎患者同时检测HBVM与HBV DNA。HBVM检测用EHSA法，HBV DNA检测用PCR法。根据不同检测结果进行对比分析。结果在HBsAg HBeAg HBcAb阳性的血清中HBV DNA阳性率和含量最高，血清HBeAg与HBV DNA含量密切相关，但部分HBeAg阴性或抗-HBe阳性患者也有较高的HBV DNA阳性率及含量。结论PCR定量检测HBV DNA含量更有助于判断体内HBV复制的情况及传染性强弱，在临床上有重要意义。     

乙型肝炎病毒感染肾炎患者肾组织病毒抗原和复制中间体的检测及其意义

目的：观察乙型肝炎病毒(HBV)感染者肾组织中三种病毒抗原成分的分布特点及其与HBV感染状态和临床病理之间的联系，探讨在肾组织局部是否存在HBV的复制。方法：免疫组化法检测合并HBV感染的30例膜性肾病和12例膜增生性肾炎病例的肾活检组织切片中的HBsAg、HBcAg和HBeAg，同时检测肾小球和循环中的HBV基因组DNA及其复制中间体——闭合环状双链DNA(cccDNA)。结果：膜性肾病肾组织中病毒抗原的检出率(83．3％)显著高于膜增生性肾炎(33％)；膜性肾病肾组织检出的抗原以HBc舷和HBeAg多见，其中，血清HBeAg阳性病例肾组织HBeAg的检出率显著高于HBeAg阴性的病例。膜增生性肾炎肾组织检出的抗原主要是HBeAg。肾组织HBeAg的检出与循环中HBeAg的存在明显相关。伴血清转氨酶升高者肾组织HBV抗原的检出率较转氨酶正常者有升高的趋势。肾小球HBVDNA和cccDNA的检出均与循环中的检测结果高度一致，并以伴活动性HBV感染者检出率为高。结论：在合并HBV感染的肾炎患者中，肾组织HBV抗原的检出率在膜性肾病患者明显高于膜增生性肾炎。肾小球中检出的HBV抗原成分以HBeAg和HBcAg最多见，肾小球HBe他的检出与血清中是否存在HBeAg明显相关。合并肝功能损害者肾组织HBV抗原的检出率较肝功能正常者有增高趋势。在乙肝相关性肾炎患者的肾小球中确实能检测到HBV复制中间体的存在，它的出现与循环中HBV复制中间体检出的高度一致性，不能排除循环中HBV感染细胞在肾组织潴留对结果的影响，其意义还有待进一步阐明。     

Risk stratification for hepatitis B virus related hepatocellular carcinoma

Hepatitis B virus (HBV) infection is the major cause of chronic hepatitis, cirrhosis, and hepatocellular carcinoma (HCC) worldwide, especially in the Asia–Pacific region. Several hepatitis B viral factors predictive of clinical outcomes in HBV carriers have been identified. The Risk Evaluation of Viral Load Elevation and Associated Liver Disease/Cancer‐HBV (REVEAL‐HBV) study from Taiwan illustrated the strong association between HBV‐DNA level at study entry and risk of HCC over time. In this community‐based cohort study, male gender, older age, high serum alanine aminotransferase level, positive hepatitis B e antigen, higher HBV‐DNA level, HBV genotype C infection, and core promoter mutation are independently associated with a higher risk of HCC. Another large hospital‐based Elucidation of Risk Factors for Disease Control or Advancement in Taiwanese Hepatitis B Carriers cohort of Taiwanese patients further validated the findings of REVEAL‐HBV. The risk of HCC started to increase when HBV‐DNA level was higher than 2000 IU/mL. Both HBV‐DNA and HBsAg levels were shown to be associated with HCC development. While HBV‐DNA level had better predictive accuracy than HBsAg level, when investigating the overall cohort in patients with HBV‐DNA level < 2000 IU/mL, HBsAg level ≥ 1000 IU/mL was identified as a new independent risk factor for HCC. With the results from REVEAL‐HBV, a risk calculation for predicting HCC in non‐cirrhotic patients has been developed and validated by independent cohorts (Risk Estimation for Hepatocellular Carcinoma in Chronic Hepatitis B).Taken together, ample evidence indicates that HBsAg level can complement HBV‐DNA level in predicting HCC development, especially in HBV carriers with low viral load. In conclusion, HBV treatment guidelines should include the risk stratification of HCC to individualize the management of HBV carriers with different levels of HCC risk.     

乙型肝炎患者检测肝组织HBV-DNA的意义

为了探讨提高乙型肝炎病毒微量检出率的有效途径。采用PCR分别检测血清及肝穿刺组织HBVD-NA，血清检出总阳性率为41.7%，肝组织检出总阳性率为72.9%。在血清HBsAg阴性的病例中PCR血清检出阳性率为26.7%。肝组织检出阳性率为66.7%。早此可见PCR检测灵敏。准确、特别对血清HBVM难以检测的微量HBVDNA更有价值。两种标本来源相比肝组织较血清为优。     

乙型肝炎患者血清前S1抗原和HBV DNA与肝功能的关系探讨

目的探讨乙型肝炎病毒前S1抗原(PreS1)及HBV-M和HBV DNA与肝功能检测间的关系。方法对576例血清标本进行PreS1及HBV-M和HBV DNA及丙氨酸氨基转移酶(ALT)、门冬氨酸氨基转移酶(AST)同步检测。结果PreS1、HBV DNA阳性率分别为72.05%和61.81%,两者符合率达74.65%;在HBsAg(-)和正常对照组PreS1、HBV DNA均为阴性;在HBsAg、HBeAg与HBsAg、抗-HBe、抗-HBc和HBsAg、抗-HBc三种阳性模式组中,PreS1阳性率(87.25%、55.71%、68.21%)、HBV DNA阳性率(94.02%、29.29%、45.66%)在各组中相互比较差异均有显著性(P均<0.005);PreS1与HBV DNA阳性符合率为92.03%、53.57%、68.21%,差异有显著性(P<0.05);PreS1阳性组和PreS1阴性组ALT、AST异常率相比,差异有显著性(P<0.05)。结论PreS1主要存在于HbsAg阳性携带者中,且与HBeAg、HBV DNA关系密切,是反映HBV复制及传染性的又一血清学指标,同时PreS1阳性还与肝功能损害有关。PreS1联合HBV-M及HBV DNA和肝功能同步检测具有重要的临床意义。     

肝细胞癌和肝硬变组织中突变型P53蛋白的表达及与HBV DNA的关系

应用LSAB免疫组化技术,检测肝内突变型P53蛋白的表达;用地高辛探针原位杂交检测HBV DNA。结果显示,31例肝细胞癌(HCC)及其癌旁组织中突变型P53蛋白阳性率分别为45％和48％,而11例肝硬变中检出4例(36.4％)。HBV DNA阳性率分别为46％和86％。癌旁组织中突变型P53蛋白表达与HBV DNA存在相关关系(P<0.05)。提示P53基因突变与HBV慢性感染有关,可能是诱发肝癌的机制之一。癌旁组织和肝硬变组织中也存在P53蛋白异常表达,表明肝癌形成早期就发生了53基因的突变。     

表达HBV preS2S基因重组MVA假病毒颗粒的构建及抗原性鉴定

目的构建携带HBV preS2S基因的重组MVA假病毒颗粒,评价其抗原性。方法将HBV preS2S基因通过基因重组构建入穿梭载体pSC11,得到质粒pSC11-preS2S,将此质粒转染MVA病毒通过同源重组得到MVA—preS2S,通过免疫印迹法鉴定其抗原性。结果利用基因测序,PCR鉴定证实所得假病毒颗粒可以表达HBV preS2S基因,并且具有良好的抗原性,经过9次传代得到单克隆重组病毒。结论本试验成功地构建了表达HBV preS2S基因的重组MVA假病毒颗粒MVA-preS2S,为基因治疗HBV慢性感染做了一项实验性奠基工作。     

肝移植术后肝组织中HBV cccDNA的表达及临床意义

目的探讨肝移植术后患者肝组织内乙型肝炎病毒共价闭合环状DNA(HBV cccDNA)的表达及其临床意义。方法应用原位聚合酶链反应及免疫组织化学法检测2004年4月至2007年4月在北京地坛医院肝移植术后出现HBV再感染的25例患者及10例肝移植术后未出现HBV再感染者肝组织中HBV cccDNA及乙型肝炎表面抗原(HBsAg)、乙型肝炎核心抗原(HBcAg)、前-S1的表达。结果在35例标本中检测到cccDNA阳性的有22例(62.9%),阳性信号为紫蓝色呈块状或颗粒状,定位于细胞核。25例术后出现HBV再感染的患者其HBV cccDNA阳性率显著高于未出现HBV再感染者(84%对10%,P<0.01)。HBV cccDNA和HBcAg均为阳性的有20例,有2例患者肝组织中HBcAg阴性,但HBV cccDNA检测为阳性。肝组织中HBV cccDNA的表达阳性率与HBcAg具有高度一致性(Kappa=0.755,P<0.01)。结论肝移植患者肝组织中HBV cccDNA阳性表达可能与乙型肝炎再感染具有密切关系。