乙型肝炎病毒外膜大蛋白检测的临床意义

目的：通过检测患者血清乙肝病毒外膜大蛋白（HBV-LP）、HBV-DNA、乙肝病毒标志物（HBV-M）,探讨HBV-LP对于反映体内乙肝病毒复制的意义。方法：采用荧光定量PCR方法对180份HBV感染血清的HBV-DNA进行检测,并采用酶联免疫吸附试验（ELISA）对HBV-LP、HBV-M及HBV-DNA进行检测。结果：大蛋白的检出结果与HBV-DNA的检出结果无明显差异。不同HBV-M模式的HBV-DNA与HBV-LP的检出结果均无显著性差异。HBV-DNA拷贝数的对数值与HBV-LP表达具有相关关系（r=0.95,P〈0.05）。结论：HBV-LP能够反映HBV的复制情况。血清中HBV-LP的含量与HBV-DNA的拷贝数具有较好的相关性。     

Characterization of Full-Length Genomes of Hepatitis B Virus Quasispecies in Sera of Patients at Different Phases of Infection

Hepatitis B virus (HBV) infection results in different clinical presentation due to different levels of immune response. Our study aimed to characterize HBV full-length genome quasispecies (QS) in patients with different phases of infection to better understand its pathogenesis. Forty treatment-naive HBV-infected patients were enrolled, including 10 cases of acute hepatitis B (AHB), 9 cases of immunotolerant (IT) HBV carriers, 11 cases of chronic hepatitis B (CHB), and 10 cases of acute-on-chronic liver failure (ACLF). The present study was conducted by clone-based sequencing. QS heterogeneity within each open reading frame was calculated. The mutation frequency index (MFI) and amino acid variations within the large HBsAg, HBcAg, and HBxAg regions were analyzed based on the different infection phases. In total, 606 HBV full-length sequences were obtained. HBV QS had higher heterogeneity in ACLF and CHB than that in IT among chronically infected individuals. AHB patients had the lower QS heterogeneity at onset than those with chronic infection. ACLF patients had the highest frequency of mutations in the core promoter and precore region. A triple mutation (A1762T/G1764A/G1896A) was observed more frequently in genotype C than in genotype B. The MFI indicated that specific peptides of the studied regions had more frequent mutations in ACLF. Furthermore, several amino acid variations, known as T- and B-cell epitopes, were potentially associated with the immunoactive phase of infection. More HBV genome mutations and deletions were observed in patients with more severe diseases, particularly in specific regions of the core and preS regions, the clinical significance and mechanism of which need to be further investigated.     

Universal Primers for Detection and Sequencing of Hepatitis B Virus Genomes across Genotypes A to G

Hepatitis B virus (HBV) has been divided into 10 genotypes, A to J, based on an 8% nucleotide sequence divergence between genotypes. The conventional practice of using a single set of primers to amplify a near-complete HBV genome is hampered by its low analytical sensitivity. The current practice of using overlapping conserved primer sets to amplify a complete HBV genome in a clinical sample is limited by the lack of pan-primers to detect all HBV genotypes. In this study, we designed six highly conserved, overlapping primer sets to cover the complete HBV genome. We based our design on the sequences of 5,154 HBV genomes of genotypes A to I downloaded from the GenBank nucleotide database. These primer sets were tested on 126 plasma samples from Malaysia, containing genotypes A to D and with viral loads ranging from 20 to >79,780,000 IU/ml. The overall success rates for PCR amplification and sequencing were >96% and >94%, respectively. Similarly, there was 100% amplification and sequencing success when the primer sets were tested on an HBV reference panel of genotypes A to G. Thus, we have established primer sets that gave a high analytical sensitivity for PCR-based detection of HBV and a high rate of sequencing success for HBV genomes in most of the viral genotypes, if not all, without prior known sequence data for the particular genotype/genome.     

Two Sensitive PCR-Based Methods for Detection of Hepatitis B Virus Variants Associated with Reduced Susceptibility to Lamivudine

Two novel assays, a restriction fragment length polymorphism (RFLP) assay and an assay based on the 5'-nuclease activity of Taq DNA polymerase, were developed for screening viral variants in lamivudine-treated patients' sera containing <1,000 copies of the hepatitis B virus (HBV) genome per ml. Both assays were designed to detect single-nucleotide changes within the HBV DNA polymerase gene that are associated with lamivudine resistance in vitro and have been used to screen a number of patients' sera for variant virus. Results obtained with these assays and standard sequencing technology were compared with regard to throughput, ability to detect individual virus species present at low concentrations, and ability to detect, distinguish, and quantitate wild-type (wt) and HBV tyrosine methionine(552) aspartate aspartate motif variants in mixed viral populations. Unlike DNA sequencing, both assays are amenable to high-throughput screening and were shown to be able to quantitatively detect variant virus in the presence of a background of wt virus. As with DNA sequencing, both new assays incorporate a PCR amplification step and are able to detect the relatively low amounts of virus found in lamivudine-treated patients' sera. However, these assays are far less labor intensive than the DNA-sequencing techniques presently in use. Overall, the RFLP assay was more sensitive than DNA sequencing in detecting and determining the ratios of wt to variant virus. Furthermore, the RFLP assay and 5'-nuclease assay were equally sensitive in the detection of mixed viral species, but the RFLP assay was superior to the 5'-nuclease assay in the quantitation of mixed viral species. These assays should prove useful for further understanding of virological response to therapy and disease progression.     

人源性抗HBsAg单抗重链可变区基因序列分析

应用生物工程菌表达法，本文建立了抗ＨＢｓＡｇ单克隆抗体片段组合文库。并筛选阳性株制备了相应的抗体活性片段。人工法对５个阳性株的抗体编码ＤＮＡ重链可变区序列进行了分析，结果表明，５株中有２株ＤＮＡ序列相同，与国外学者所作同类抗体的序列不同，提示是具有抗体活性的不同菌株。同时比较ＤＮＡ、序列发现，氨基酸排列的异同决定抗体的结合活性。ＤＮＡ序列相同的２株其抗体片段的表达能力也相似，表现为酶免疫测定时Ａ值近似。抗体编码基因的序列分析有助于深入探讨抗体的表达能力、亲和力等特性。     

Dynamics of Defective Hepatitis C Virus Clones in Reinfected Liver Grafts in Liver Transplant Recipients: Ultradeep Sequencing Analysis

Hepatitis C virus (HCV) reinfects liver allografts in transplant recipients by replicating immediately after transplantation, causing a rapid increase in blood serum HCV RNA levels. We evaluated dynamic changes in the viral genetic complexity after HCV reinfection of the graft liver; we also identified the characteristics of replicating HCV clones using a massively parallel ultradeep sequencing technique to determine the full-genome HCV sequences in the liver and serum specimens of five transplant recipients with genotype 1b HCV infection before and after liver transplantation. The recipients showed extremely high genetic heterogeneity before transplantation, and the HCV population makeup was not significantly different between the liver and blood serum specimens of the individuals. Viral quasispecies complexity in serum was significantly lower after liver transplantation than before it, suggesting that certain HCV clones selectively proliferated after transplantation. Defective HCV clones lacking the structural region of the HCV genome did not increase in number, and full-genome HCV clones selectively increased in number immediately after liver transplantation. A re-increase in the same defective clone existing before transplantation was detected 22 months after transplantation in one patient. Ultradeep sequencing technology revealed that the genetic heterogeneity of HCV was reduced after liver transplantation. Dynamic changes in defective HCV clones after liver transplantation indicate that these clones have important roles in the HCV life cycle.     

丁型肝炎病毒在乙肝病毒感染者中分布状况及其分子生物学研究

通过对丁型肝炎病毒（ＨＤＶ）的放免法检测和巢式ＰＣＲ技术检测，在１２０例乙肝病毒感染者中，检测到１３例ＨＤＶ标记阳性（阳性率为１０．８％），５０例乙肝病毒携带者，３例抗ＨＤＶ－ＩｇＭ阳性。经对照所得乙肝患者同乙肝病毒携带者的ＨＤＶ阳性差异结果支持ＨＤＶ的存在常伴随着谷丙转氨酶（ＡＬＴ）活性上升的观点。实验结果还提示了ＨＤＶ和乙型肝炎病毒（ＨＢＶ）重叠感染易发生重症或慢性肝炎。地ＨＤＶ的检测方法进行     

Use of the Novel INNO-LiPA Line Probe Assay for Detection of Hepatitis B Virus Variants That Confer Resistance to Entecavir Therapy

A line probe assay (INNO-LiPA DR, version 3) for the detection of hepatitis B virus mutations that confer resistance to entecavir therapy was evaluated. The INNO-LiPA DR assay is a highly sensitive assay that is easily applicable for the detection and monitoring of entecavir resistance-conferring mutations and is more sensitive than sequencing for the detection of mixed sequences.     

重型病毒性肝炎中丁型肝炎病毒的检测

为了探讨丁型肝炎病毒（ＨＤＶ）感染在重型病毒性肝炎中的作用，对北京佑安医院１９８０年至１９８９年收治的５４例急性和亚急性重型肝炎和３８例急性乙肝患者血清，应用国产ＨＤＶＥＬＩＳＡ试剂测定抗－ＨＤ、抗－ＨＤＩｇＭ和ＨＤＡｇ，应用斑点杂交技术测定ＨＤＶＲＮＡ。结果发现重型肝炎组ＨＯＶ－Ｍ检出率明显高于急性乙肝组（２７．８％比５．３％．Ｐ＜０．０５）。单独ＨＢＶ感染和ＨＤＶ／ＨＢＶ混合感染的重型肝炎患者均有较高的病死率。提示ＨＤＶ感染是重型肝炎中重要的病原学因素之一，ＨＤＶ与ＨＢＶ具有协同作用加重肝损害，导致肝衰竭。     

乙型肝炎病毒基因分型检测试剂临床研究要求探讨

结合体外诊断试剂相关法规和此类试剂临床用途,分析其临床研究的主要要求.对该类试剂的伦理要求、临床试验方案、临床研究单位的选择、临床对象的选择、对比方法的选择、统计学分析、临床试验总结报告撰写等方面进行了详细解析.阐述乙型肝炎病毒基因分型检测类体外诊断试剂的临床研究要点.为从事上市前临床研究的行业人员提供参考.     

应用实时荧光PCR技术检测HBV YMDD变异

目的　应用YMDD变异荧光PCR检测试剂盒检测乙型肝炎病毒 (hepatitisBvirus,HBV)聚合酶基因区的酪氨酸 蛋氨酸 天门冬氨酸 天门冬氨酸基序 (tyrosine methionine asparticacid asparticacid ,YMDD)变异 ,希望能够替代目前使用的测序方法。方法　对 4 7例血清标本使用荧光PCR技术检测YMDD变异 ,其YMDD变异结果与DNA测序结果进行比较。结果　荧光PCR方法测得YMDD野生株 2 8例 ,变异株 19例 ;与DNA测序结果完全一致 ,符合率为 10 0 %。结论　荧光PCR技术检测YMDD变异具有方便、快速、准确的特点。     

Cloning and Sequencing of Hepatitis B Virus Pre-S and S Gene Regions

The polymerase chain reaction (PCR) was used to amplify the pre-S1, pre-S2 and S gene regions of hepatitis B virus (HBV). Sera from three different patients were used as the source of HBV DNA. The resulting 1.2-kb amplification product was cloned into the plasmid pI B130. Restriction enzyme analysis revealed that there are two BamHI sites located about 300 bp apart within the S gene. DNA sequencing revealed a greatest homology to the HBV adw subtype.     

慢性乙肝患者血清前Pre-S1抗原检测及其与肝功能关系的研究

目的 检测慢性乙型肝炎病毒患者前S1(Pre-S1)抗原,探讨其与肝功能的关系.方法 收集270例慢性乙型肝炎病毒患者血清,采用ELISA方法检测Pre-S1抗原;采用日立7170全自动生化分析仪检测丙氨酸氨基转移酶(ALT)和天冬氨酸氨基转移酶(AST).结果 270例慢性乙型肝炎病毒患者中,Pre-S1抗原阳性中ALT异常检出率为91.1%,AST异常检出率为93.0%,Pre-S1抗原中阴性中ALT异常检出率为28.3%,AST异常检出率为35.4%,两组结果有显著性差异(P<0.05).结论慢性乙型肝炎患者中,Pre-S1抗原可作为判断乙型肝炎病毒(HBV)感染,复制及对肝细胞损伤的另一个指标之一.     

HBV基因分型检测试剂性能评估研究要点

依据《体外诊断试剂注册管理办法》和乙型肝炎病毒核酸分型试剂自身特点,提出对HBV基因分型检测试剂性能研究的特殊要求.对该类试剂的参考品和质控品研制、阳性/阴性参考品符合率、最低检测限、分析特异性、精密度、基因混合型研究、携带污染率等方面进行了详细解析.阐述了乙型肝炎病毒基因分型类体外诊断试剂的性能研究要点.为从事该类产品注册申报前性能研究的行业人员提供参考.     

慢性乙型肝炎患者外周血单核细胞与血清HBV DNA定量对比研究

目的　探讨慢性乙肝患者外周血单个核细胞 (PBMC)内HBV -DNA的出现与血清HBV -DNA浓度之间关系 .方法　应用荧光定量PCR技术检测 5 7例慢性乙肝患者血清和PBMC中HBV -DNA含量、对血清中不同的病毒浓度进行分组比较分析 .结果　① 5 7例PBMC内HBV DNA总检测出率为 4 2 .1% (2 4 / 5 7) ,两者检测结果一致占 88.6 % .②根据血清内HBV -DNA浓度分成三组 ,三组PBMC内HBV -DNA浓度及阳性率比较p <0 .0 1,存在显著差异 ;③血清HBV -DNA阴性而PBMCHBV -DNA阳性只有 1例 (1.7% ) ,其浓度为 1.5 0× 10 8/L .结论　①血清HBV -DNA浓度与PBMC内HBV -DNA的出现及HBV -DNA浓度存在明显相关性 .②临床上检测PMBC中HBV -DNA是对血清HBV -DNA的一个重要补充 ;③PBMC内HBV -DNA检测对观察病毒在非血清内的状态、间接反映肝细胞病毒复制情况以及进一步指导抗病毒治疗有一定意义     

慢性乙型肝炎患者外周血单核细胞与血清HBV DNA定量对比研究

目的探讨慢性乙肝患者外周血单个核细胞(PBMC)内 HBV-DNA的出现与血清HBV-DNA浓度之间关系.方法应用荧光定量PCR技术检测57例慢性乙肝患者血清和PBMC中HBV-DNA含量、对血清中不同的病毒浓度进行分组比较分析.结果①57例PBMC内HBV-DNA总检测出率为42.1%(24/57),两者检测结果一致占88.6%.②根据血清内HBV-DNA浓度分成三组,三组PBMC内HBV-DNA浓度及阳性率比较p＜0.01,存在显著差异;③血清HBV-DNA阴性而 PBMC HBV-DNA阳性只有1例(1.7%),其浓度为1.50×108/L.结论①血清HBV-DNA浓度与PBMC内HBV-DNA的出现及HBV-DNA浓度存在明显相关性.②临床上检测PMBC中HBV-DNA是对血清HBV-DNA的一个重要补充;③PBMC内HBV-DNA检测对观察病毒在非血清内的状态、间接反映肝细胞病毒复制情况以及进一步指导抗病毒治疗有一定意义.     

Clinical Implications of Evolutionary Patterns of Homologous,Full-Length Hepatitis B Virus Quasispecies in Different Hosts after Perinatal Infection

Chronic hepatitis B virus (HBV) infection via perinatal transmission is common in the Asia-Pacific region, but related quasispecies (QS) characteristics are not yet defined. To investigate the homologous, full-length HBV QS after perinatal infection and their clinical implications, five pairs of mother-daughter patients with chronic HBV infection (one patient with liver cirrhosis, one with immune tolerance, and eight with chronic hepatitis) were included. Full-length HBV were amplified by PCR from serum samples before antiviral treatment and cloned; an average of 17 clones per sample were sequenced, and the QS complexities, diversities, and evolution patterns were analyzed. Full-length HBV sequence similarities within mother-daughter pairs were 91.3 to 98.3%. The QS complexities of full-length HBV were similar between mothers and daughters (median of 0.9734 compared to 0.9688, P > 0.05), as were the diversities (median of 3.396 × 10⁻³ compared to 4.617 × 10⁻³substitutions/site, P > 0.05). However, the distribution patterns of QS complexities and diversities within specific genes were different, and QS genetic distances of the mothers were higher than those of daughters, both more evident in pairs with different antiviral responses and different immune phases or stages. The nucleotide substitution rate of full-length HBV was 14.388 × 10⁻⁵ substitutions/site/year, whereas the preC/C gene rate was the highest. Mutations and indels were more common in clones from the mothers, which decreased the affinity of epitopes by 6- to 89-fold. The various genes from homologous HBV genomes evolved in different patterns despite numerically comparable full-length QS complexities and diversities. The different patterns may correlate with the immune stages of chronic HBV infection, which warrants further study.