Replication of hepatitis B virus with corticosteroid therapy in hepatitis B virus related membranous nephropathy

Summary The therapeutic effect of corticosteroid in hepatitis B virus (HBV) related membranous nephropathy was investigated in a 29-year-old chronic HBV carrier. Prednisolone (60 mg/day) was given for eight weeks and gradually reduced over the subsequent four months. In the renal biopsies taken before and after corticosteroid therapy, light microscopy revealed progression of sclerosis. Immunofluorescent staining showed glomerular capillary deposition of hepatitis B core antigen (HBcAg) by polyclonal antisera and hepatitis B e antigen (HBeAg) by monoclonal antibodies. Electron microscopy revealed 40–50 nm diameter virus-like particles in the glomeruli only from the biopsy performed after corticosteroid therapy. The serum concentrations of alanine aminotransferase, HBeAg, and HBV DNA increased with corticosteroid therapy suggesting active viral replication despite the absence of overt clinical hepatitis. Renal function did not improve and corticosteroid therapy was apparently not helpful in this patient. Our results conflict with the earlier notion that short-term corticosteroid does not interfere with a favorable outcome of the infection of the related renal disease.     

人类乙肝病毒体外感染树鼩肝原代细胞的研究

目的 为进一步研究人类乙肝病毒(HBV)提供接近自然感染状态的理想细胞模型.方法 体外二步灌流法分离树鼩原代肝细胞,纯化后的乙肝患者血清感染上述肝细胞,Southern blot和Noahem blot检测感染后细胞内的DNA和RNA,ELISA方法 检测细胞上清的HBsAg,免疫组化检测细胞内HBsAg的表达.结果 可检测出肝细胞内cccDNA(共价闭合环状DNA)、pgRNA(前基因组RNA)和sgRNA(亚基因组mRNA),感染后第7天,信号开始增强,持续到实验结束的第14天.细胞上清中HBsAg自第1天到第5天S/CO值逐渐下降,随后S/CO值逐渐升高.结论 HBV可在原代树鼩肝细胞中复制和表达.     

血清乙肝病毒外膜大蛋白检测及其与病毒复制的关系

目的探讨血清乙型肝炎病毒外膜大蛋白（LHBs）检测对于判定乙型肝炎病毒（HBV）复制的临床意义。方法分别采用ELISA法、时间分辨免疫荧光分析法和实时荧光定量PCR法检测340份慢性乙型肝炎患者血清中LHBs、Pre-S1蛋白、HBV-M和HBVDNA，并进行相关性分析。结果340份慢性乙型肝炎患者血清中LHBs水平与HBVDNA拷贝数变化相一致，两者呈正相关（r=0．899。P=0．038）；在不同模式的HBeAg血清中，LHBs与HBVDNA的阳性率差异均无统计学意义（P均〉0．05）；HBV DNA阳性血清中LHBs阳性率（83．15％）明显高于Pre-S1蛋白和HBeAg的阳性率（50．54％和54．48％），差异有统计学意义（P均〈0．05）。结论血清LHBs水平能反映HBV感染者体内HBV复制程度，其灵敏度高于Pre-S1蛋白和HBeAg，可作为判断HBV复制新的血清学指标。     

非甲-非戊型慢性病毒性肝炎患者隐匿性HBV感染的研究

目的 观察非甲-非戊型慢性病毒性肝炎患者隐匿性HBV感染的状况,探讨荧光定量聚合酶链反应(FQ-PCR)技术对隐匿性HBV感染的诊断价值.方法 应用FQ-PCR技术对57例非甲-非戊型慢性病毒性肝炎患者进行了血清、肝组织HBV-DNA定量检测,并将肝组织HBV DNA定量水平与肝脏炎症活动度的关系进行了分析.结果 血清、肝组织HBV DrqA定量阳性分别为13例(22.81%)、22例(38.60%).13例血清HBV DNA定量阳性患者其肝组织定量亦均阳性,但9例肝组织HBV DrqA定量阳性患者其血清定量为阴性,差异有统计学意义(P<0.01);同时13例血清与肝组织定量均阳性患者比较.显示肝组织HBV DNA定量水平显著高于血清定量水平[(6.62±1.21)拷贝,gvs.(4.03±1.06)拷贝/ml,(P<0.01)].肝组织HBV DNA水平与肝脏炎症活动度并无相关性,10例G2,7例G3,5例G4患者HB'q DNA定量分别为(6.13±1.65)拷贝/g、(5.92±1.81)拷贝,g、(5.83±1.89)拷贝/g,(P0.05),但HBV DNA定量阳性患者均为活动性肝脏病变.结论 HBV隐匿性感染是部分非甲-非戊型慢性病毒性肝炎患者的病因.单纯检测血清免疫学标志物对HBV感染诊断存在漏诊,对非甲-非戊型慢性病毒性肝炎患者应用FQ-PCR技术开展血清定量尤其是肝组织中HBV DNA定量检测可提高HBV感染的诊断.对隐匿性HBV感染的慢性病毒性肝炎亦应给予有效的抗病毒治疗.     

乙型肝炎病毒preS/S蛋白对人PCNA基因启动子激活作用的研究

目的：研究乙型肝炎病毒preS/S蛋白对人增殖细胞核抗原（PCNA）基因启动子的激活作用。方法：利用荧光素酶报告基因，用共转染方法研究该整合片段对人PCNA启动子的作用。用免疫组织化学法确定preS/S蛋白细胞中的定位。结果：在人肝细胞L02中，含有上述HBV整合片段的质粒pKSH7C-Hpa I能以剂量依赖方式激活PCNA启动子，使PCNA启动子活性提高0.5倍；免疫组化分析表明，其蛋白定位于细胞质中。结论：preS/S蛋白对PCNA启动子有激活作用，但作用并非直接，可能是通过细胞信号传导途径来影响PCNA活性。     

Calcium ions affect the hepatitis B virus core assembly

Previous report showed that cytosolic Ca2+ induced by hepatitis B virus X protein (HBx) promotes HBV replication. In this study, in vitro experiments showed that (i) HBV core assembly in vitro was promoted by Ca2+ through the sucrose density gradient and the analytical ultracentrifuge analysis. Also, (ii) transmission electron microscope analysis demonstrated these assembled HBV core particles were the capsids. Ex vivo experiments showed that the treatment of BAPTA-AM and cyclosporine A (CsA) reduced HBV capsids in the transfected HepG2 cells. In addition to that, the treatment of Thapsigargin (TG) increased HBV capsids in the transfected HepG2 cells. Furthermore, we investigated the increased HBV core assembly by HBx. The results show that the increased cytosolic calcium ions by HBx promote the HBV core assembly.     

肾小球肾炎肾组织中HBV感染的标志

为了解肾组织中ＨＢＶＤＮＡ与ＨＢＶ抗原存在的关系，探讨肾组织中ＨＢＶ抗原的来源及其与病理变化的关系，我们检测２４６例肾炎肾组织中三种ＨＢＶ抗原。发现除肾小球沉积外，肾小管常有阳性表达。肾小管ＨＢｃＡｇ阳性率达２１．５４％，高于肾小球（１０．９８％）。有１８例肾组织经Ｓｏｕｔｈｅｒｎ印迹杂交检测ＨＢＶＤＮＡ，１５例阳性者中１４例肾组织ＨＢＶ抗原同时阳性，１２例血清感染标志亦阳性。结果提示某些肾炎可能与肾组织感染ＨＢＶ相关，肾组织中出现的ＨＢＶ抗原抗体免疫复合物除来自血循环外，有肾源性──原位形成的可能。     

Hepatitis B virus regulatory HBx protein binding to DDB1 is required but is not sufficient for maximal HBV replication

Robust hepatitis B virus (HBV) replication is stimulated by the regulatory HBx protein. HBx binds the cellular protein DDB1; however, the importance of this interaction for HBV replication remains unknown. We tested whether HBx binding to DDB1 was required for HBV replication using a plasmid based replication assay in HepG2 cells. Three DDB1 binding-deficient HBx point mutants (HBx⁶⁹, HBx^90/91, HBx^R96E) failed to restore wildtype levels of replication from an HBx-deficient plasmid, which established the importance of the HBx-DDB1 interaction for maximal HBV replication. Analysis of overlapping HBx truncation mutants revealed that both the HBx-DDB1 binding domain and the carboxyl region are required for maximal HBV replication both in vitro and in vivo, suggesting the HBx-DDB1 interaction recruits regulatory functions critical for replication. Finally we demonstrate that HBx localizes to the Cul4A-DDB1 complex, and discuss the possible implications for models of HBV replication.     

血清乙型肝炎病毒前S1抗原检测及其与病毒复制的关系

用抗Ｓ和抗前Ｓ１单抗的双抗体夹心ＥＬＩＳＡ法检测１５０例慢性乙型肝炎患者、乙型肝炎病毒表面抗原（ＨＢｓＡｇ）携带者和健康人血清中的ＨＢＶ前Ｓ１抗原，其结果和ＨＢＶＤＮＡ聚合酶链反应（ＰＣＲ）、乙型肝炎血清标志的检测结果进行比较。结果表明：前Ｓ１抗原在乙型肝炎病毒ｅ抗原（ＨＢｅＡｇ）阳性组中的检出率和相对滴度显著高于ＨＢｅＡｇ阴性组（Ｐ＜０．０１）；在ＨＢｅＡｇ阴性组中，抗－ＨＢｅ阴性人群前Ｓ１抗原的检出率和相对滴度也显著高于抗－ＨＢｅ阳性人群（Ｐ＜０．０１）。前Ｓ１抗原和ＨＢＶＤＮＡ检测结果的符合率达８０％，两者检出率的相关系数ｒ＝０．９８２６（Ｐ＜０．０１）。结论：血清前Ｓ１抗原和乙型肝炎病毒的存在关系密切。     

Transmissible gastroenteritis virus infection induces cell cycle arrest at S and G2/M phases via p53-dependent pathway

p53 signaling pathway plays an important role in the regulation of cell cycle. Our previous studies have demonstrated that TGEV infection induces the activation of p53 signaling pathway. In this study we investigated the effects of TGEV infection on the cell cycle of host cells and the roles of p53 activation in this process. The results showed that TGEV infection induced cell cycle arrest at S and G2/M phases in both asynchronous and synchronized PK-15 and ST cells, while UV-inactivated TGEV lost the ability of induction of cell cycle arrest. TGEV infection promoted p21 accumulation, down-regulated cell cycle-regulatory proteins cyclins B1, cdc2, cdk2 and PCNA. Further studies showed that inhibition of p53 signaling could attenuate the TGEV-induced S- and G2/M-phase arrest by reversing the expression of p21 and corresponding cyclin/cdk. In addition, TGEV infection of the cells synchronized in various stages of cell cycle showed that viral genomic RNA and subgenomic RNA, and virus titer were higher in the cells released from S-phase- or G2/M phase-synchronized cells than that in the cells released from the G0/G1 phase-synchronized or asynchronous cells after 18 h p.i. Taken together, our data suggested that TGEV infection induced S and G2/M phase arrest in host cells, which might provide a favorable condition for viral replication.     

丙型肝炎病毒体外感染原代人胎肝细胞的研究

目的　研究丙型肝炎病毒 (HCV)体外细胞培养方法。方法　原代人胎肝细胞与HCV感染血清共孵育后 ,用逆转录 聚合酶链反应、原位杂交、免疫组化分别检测细胞和培养上清中的HCVRNA和HCV抗原表达。结果　从感染血清和细胞共同孵育后的 2～ 2 5d ,细胞内和 /或培养上清中可间断检出HCV正、负链RNA ;HCVNS3抗原在细胞内能稳定表达 ;原位杂交显示HCV负链RNA阳性物质多位于细胞浆。结论　原代人胎肝细胞对HCV易感 ,可用作HCV体外感染和复制的靶细胞     

中国乙肝病毒B基因亚型的分布

目的调查乙肝病毒B基因亚型在我国的分布情况。方法采用聚合酶链反应-限制性片段长度多态性(PCR-RFLP)法并结合测序法,对来自全国7个中心的共511份B基因型感染血清样本进行了检测。结果511份B基因型样本经亚型分析,确定全部为Ba亚型,未发现Bj亚型的存在。结论流行于我国南方及北方地区的乙肝病毒B基因型毒株绝大部分为Ba亚型,Bj亚型非常罕见。     

Hepatitis B virus HBx protein localized to the nucleus restores HBx-deficient virus replication in HepG2 cells and in vivo in hydrodynamically-injected mice

Identifying the requirements for the regulatory HBx protein in hepatitis B virus (HBV) replication is an important goal. A plasmid-based HBV replication assay was used to evaluate whether HBx subcellular localization influences its ability to promote virus replication, as measured by real time PCR quantitation of viral capsid-associated DNA. HBx targeted to the nucleus by a nuclear localization signal (NLS-HBx) was able to restore HBx-deficient HBV replication, while HBx containing a nuclear export signal (NES-HBx) was not. Both NLS-HBx and NES-HBx were expressed at similar levels (by immunoprecipitation and Western blotting), and proper localization of the signal sequence-tagged proteins was confirmed by deconvolution microscopy using HBx, NLS-HBx, and NES-HBx proteins fused to GFP. Importantly, these findings were confirmed in vivo by hydrodynamic injection into mice. Our results demonstrate that in these HBV replication assays, at least one function of HBx requires its localization to the nucleus.     

乙型肝炎病毒表面大蛋白检测用于筛查隐匿性HBV感染

目的 用血清学方法检测乙型肝炎病毒表面大蛋白(HBLP)来筛查隐匿性HBV感染,探讨临床隐匿性HBV感染的血清学检测策略.方法 在日常工作中从临床榆测备份管随机收集2000份用国产ELISA试剂检测HBsAg阴性结果的血清标本,双份分装-20℃冻存,单份标本实施HBLP检测,HBLP阳性标本增加另外两种共三种国产ELISA试剂双份复查HBsAg;HBLP阳性标本再经美国超灵敏MONOLISA HBsAg ULTRA试剂双份检测HBsAg,并行双份DNA定量分析、结果取均值.结果 2000份HBsAg阴性标本共检出15例HBLP阳性:HBLP阳性标本用三种国产ELISA试剂双份复查HBsAg结果一致阴性;通过美国超灵敏MONOLISA HBsAS ULTRA试剂复查HBsAg结果全部阳性;前述15份标本HBV DNA定量分析结果显示均小于500拷贝/ml,其中400～500拷贝/ml 1例,300～400拷贝/ml 3例,200～300拷贝/ml 5例,100～200拷贝/ml 4例,小于100拷贝/ml 2例.结论 用国产常规ELISA试剂检测HBsAg普遍存在漏检,漏检标本HBLP结果可能阳性,检测HBLP有利于筛查出隐匿性H BV感染,为积极寻找临床隐匿性HBV感染的检测策略提供了血清学参考.     

Gene expression profiles of normal human lung cells affected by adenoviral E1B

Adenoviruses with deletion of E1b gene can selectively replicate in cancer cells. The underlying mechanisms in tumor-selective replication of E1b-deleted adenoviruses are insufficiently understood. Identifying genes with altered expression patterns caused by the E1B proteins in virus-infected cells will further increase our understanding of E1B functions and provide insight into the tumor-selective replication of E1b-mutated adenoviruses on the molecular level. An approach based on large-scale gene array was applied to analyze molecular changes affected by viral E1B. We identified a total of 345 genes with expression changes of two-fold or greater affected by wild-type adenovirus compared with its E1b-deleted counterpart. The gene array data were confirmed by quantitative real-time PCR and Western blot. E1B proteins affect the expression of a diverse range of genes involved in cell cycle regulation, apoptosis, stress responses and angiogenesis. This is the first study of the global profile of gene expression altered by the viral E1B proteins in human lung cells, and the majority of the genes were previously not known to be affected by the viral proteins. The data presented in this study will lead to more detailed analysis of E1B functions and may also lead to development of new agents and approaches for oncolytic therapy.     

Differences in the patterns and outcomes of enhanced viral replication between hepatitis C virus and hepatitis B virus in patients with hepatocellular carcinoma during transarterial chemolipiodolization

Background/Aims

Enhanced replication of hepatitis C virus (HCV) is well described in the setting of moderate to severe immunosuppression. The aims of this retrospective study were to determine the incidence of enhanced HCV replication in hepatocellular carcinoma (HCC) patients undergoing transarterial chemolipiodolization (TACL) and to identify the factors associated with enhanced replication of HCV. The clinical pattern of enhanced HCV replication was compared with hepatitis B virus (HBV) reactivation during TACL.

Methods

This study enrolled 49 anti-HCV-seropositive patients who were diagnosed with HCC between January 2005 and December 2010 and who underwent TACL using epirubicin and/or cisplatin with consecutive HCV RNA copies checked. For comparison, 46 hepatitis B surface antigen¹-positive patients with HCC who were treated with TACL were also enrolled. The frequency, associated factors, and clinical outcomes of enhanced HCV replication were analyzed and compared with those of HBV reactivation during TACL.

Results

Enhanced replication of HCV occurred in 13 (26.5%) of the 49 anti-HCV-seropositive patients during TACL. Of these 13 patients, 4 developed hepatitis, but none of the subjects developed decompensation due to the hepatitis. No significant clinical factors for enhanced HCV replication during TACL were found. Compared with HBV reactivation, the frequency of hepatitis attributed to enhanced HCV replication was significantly lower than that for HBV reactivation (8.2% vs. 23.9%, P=0.036).

Conclusions

TACL can enhance HCV replication; however, the likelihood of hepatitis and decompensation stemming from enhanced HCV replication was lower than that for HBV reactivation in patients undergoing TACL.     

Precore and core promoter mutations of the hepatitis B virus gene in chronic genotype C-infected children

The precore (G1896A) and core promoter (A1762T, G1764A) mutations of the hepatitis B virus gene are known to be associated with changes in immunologic phase or the progression to complicated liver disease in adults. We analyzed these mutations in chronically HBV-infected children. Serum was collected from 37 children with chronic HBV infection from March 2005 to September 2008. HBV DNA extraction and nested PCR were followed by sequencing of the PCR products. The children were 6.7 ± 4.6 yr old. All of 37 children had HBV genotype C. Of the cohort, 31 (83.8%) were HBeAg-positive and 6 (16.2%) were HBeAg-negative; the former group comprised 18 (48.6%) who were in the immune-tolerance phase (ITP) and 13 (35.2%) in the immune-clearance phase (ICP). Most of the patients had HBV DNA levels of > 1.0 × 10(8) copies/mL. In the ITP group, only 1 (5.5%) had core promoter mutations, and none had the precore mutation. In the ICP group, only 2 (15.4%) had core promoter mutations; the remaining 6 patients had HBV DNA levels of < 2.0 × 10(3) copies/mL and no core promoter/precore mutations. The very low incidence of the precore/core promoter gene mutation, in children, suggests that these mutations may be the result of life-long chronic HBV infection.