乙型肝炎病毒核心蛋白在长期培养淋巴细胞中的表达

目的 构建表达乙型肝炎病毒核心蛋白（HBcAg）的真核表达载体，并在外源性人端粒酶逆转录酶基因转导的正常人外周血淋巴细胞中进行稳定表达。方法 用PCR法获得编码乙型肝炎病毒核心蛋白的基因，利用基因重组技术构建表达该蛋白的真核表达载体，用质粒转染表达外源性人端粒酶逆转录酶的外周血淋巴细胞，间接免疫荧光及Western blot检测HBcAg在细胞内的表达。结果 表达外源性人端粒酶逆转录酶的外周血淋巴细胞能够在体外长期生存，增殖旺盛。间接免疫荧光及Western blot均能够检测到HBcAg在细胞内的表达。结论 乙型肝炎病毒核心蛋白基因在长期培养淋巴细胞中表达，建立了一种较理想的乙型肝炎病毒核心蛋白细胞模型。     

Hepatitis C virus core protein fused to hepatitis B virus core antigen for serological diagnosis of both hepatitis C and hepatitis B infections by ELISA

The sequence encoding the truncated core protein (amino acids 1–98) of hepatitis C virus (HCc) was expressed in E. coli for production of HCc_(1–98), or fused with the truncated core antigen (HBcAg) and segments from the preS1 and preS2 regions from hepatitis B virus (HBV) for production of HBcPreS₁PreS₂HCc_(1–98). The HCc_(1–98) and HBcPreS₁PreS₂HCc_(1–98) proteins reacted with sera from HCV-infected individuals by immunoblot analyses, while the latter protein also exhibited HBV core antigenicity. They induced antibodies against HBcAg and/or HCV core protein in rabbits and in mice. Moreover, HBcPreS₁PreS₂HCc_(1–98) is more immunogenic than HCc_(1–98) in terms of anti-HCc induction. An ELISA that employed recombinant HCV core antigens of either HCc_(1–98) or HBcPreS₁PreS₂HCc_(1–98) to detect anti-HCc and/or anti-HBc antibodies was developed. Evaluation of serum samples with different status of HBV and HCV infections suggested that HCc_(1–98) might be suitable for the determination of antibodies against HCV core protein, while HBcPreS₁PreS₂HCc_(1–98) might be of value to detect HCV and/or HBV infection in donated blood in HBV low-prevalence countries. J. Med. Virol. 57:104–110, 1999. © 1999 Wiley-Liss, Inc.     

乙肝病毒感染与乙肝病毒相关性肾炎间的关系分析

目的 探讨研究乙肝病毒(HBV)感染与乙肝病毒相关性肾炎之间的关系.方法 对124例资料完整的HBV相关肾炎(HBV-related glomerulonephritis,HBV-GN,)患者的血清病毒学、肾脏及肝脏病理学和常规实验室检查结果进行回顾分析研究.结果 124例血清HBsAg和/或HBeAg阳性的患者中,HBV-GN的发生率69.49％,男性发生率(45.0％)显著高于女性(24.49％),P＜0.05.血清HBeAg阳性组HBV-GN的发生率显著高于HBeAg阴性组.HBV DNA定量＞105 copies/mL组HBV-GN的发生率亦显著高于定量＜105 copies/mL组.HBV-GN患者血清HBeAg阳性与阴性组之间比较,24小时尿蛋白定量及内生肌醉清除率均无显著差异.血清HBV DNA定量＞105copies/mL组与定量＜ 105 copies/mL组之间比较24小时尿蛋白定量与内生肌酐清除率亦无显著差异.HBV-GN患者肾组织局部HBcAg沉积阳性组与阴性组相比较,24小时尿蛋白定量有显著性差异(P＜0.05),而内生肌酐清除率无显著性差异.结论 HBV感染及其复制状态与HBV-GN的发生密切相关;但HBV的复制状态可能并不明显影响HBV-GN患者肾、肝组织的病变程度.     

丙肝病毒(HCV)及乙肝病毒(HBV)双表达载体的构建及表达

目的 :为探索HCV/HBV高效联合基因免疫策略 ,构建具有 2套独立表达单元的HCV/HBV真核表达载体。方法 :分别将与HCV核心区基因互补的cDNA和HBV核心区基因克隆于具有 2套独立表达单元的真核表达载体pRSC的巨细胞病毒启动子和RSV启动子下游 ,称为pRSC HBV/HCV ,转染SP2 / 0细胞 ,通过免疫荧光和Western印迹法观测蛋白的表达 ,免疫Balb/c小鼠 ,用酶联免疫小鼠体液免疫应答。结果 :pRSC HBV/HCV转染SP2 / 0细胞可见HBcAg及HCV核蛋白染色阳性荧光细胞 ,SDS Page电泳显示在 14kD及 2 1kD处均可见蛋白条带 ,与HBcAg及HCV核蛋白的的理论预期值一致 ,Western印迹分析显示在 14kD及 2 1kD处可见特异性的蛋白条带。 5只免疫鼠中全部出现抗 HCV及抗 HBV抗体 ,而对照组全阴性。结论 :pRSC HBV/HCV可分别表达HBcAg及HCV核蛋白 ,免疫Balb/c小鼠后可诱导其体液免疫应答 ,为进一步开展HCV/HBV联合基因免疫奠定了实验基础。     

Suppression of hepatitis B virus replication by SRPK1 and SRPK2 via a pathway independent of the phosphorylation of the viral core protein

The SR-domain protein kinase (SRPK) 1 and 2 are two important kinases involved in cellular RNA splicing. Recently, it was suggested that these two kinases, which could bind to the hepatitis B virus (HBV) core protein, might be the major cellular kinases that phosphorylate the core protein to regulate HBV replication. In this report, we tested the role of SRPK1 and SRPK2 in HBV replication and found that both of them could suppress HBV replication by reducing the packaging efficiency of the pgRNA without affecting the formation of the viral core particles. This suppressive effect of SRPK1 and SRPK2 on HBV replication cannot be explained by their phosphorylation activities on the HBV core protein as the over-expression of these two kinases had no detectable effects on HBV core protein phosphorylation in vivo and their mutants that lacked the kinase activity could still suppress HBV DNA replication. Thus, these findings demonstrate a negative role of SRPK1 and SRPK2 in the regulation of HBV replication through a mechanism not involving the phosphorylation of the core protein.     

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.     

Clinical significance of occult hepatitis B virus infection in chronic hepatitis C patients

Background/Aims

We investigated the frequency of occult hepatitis B virus (HBV) infection in anti-hepatitis C virus (HCV)-positive individuals and the effects of occult HBV infection on the severity of liver disease.

Methods

Seventy-one hepatitis B virus surface-antigen (HBsAg)-negative patients were divided according to their HBV serological status into groups A (anti-HBc positive, anti-HBs negative; n=18), B (anti-HBc positive, anti-HBs positive; n=34), and C (anti-HBc negative, anti-HBs positive/negative; n=19), and by anti-HCV positivity (anti-HCV positive; n=32 vs. anti-HCV negative; n=39). Liver biopsy samples were taken, and HBV DNA was quantified by real-time PCR.

Results

Intrahepatic HBV DNA was detected in 32.4% (23/71) of the entire cohort, and HBV DNA levels were invariably low in the different groups. Occult HBV infection was detected more frequently in the anti-HBc-positive patients. Intrahepatic HBV DNA was detected in 28.1% (9/32) of the anti-HCV-positive and 35.9% (14/39) of the anti-HCV-negative subjects. The HCV genotype did not affect the detection rate of intrahepatic HBV DNA. In anti-HCV-positive cases, occult HBV infection did not affect liver disease severity.

Conclusions

Low levels of intrahepatic HBV DNA were detected frequently in both HBsAg-negative and anti-HCV-positive cases. However, the frequency of occult HBV infection was not affected by the presence of hepatitis C, and occult HBV infection did not have a significant effect on the disease severity of hepatitis C.     

乙型肝炎分子流行病学研究进展

乙肝的分子流行病学是运用先进的分子生物学技术研究乙肝流行病学的诸多问题,能够进一步从分子水平阐明乙型肝炎病毒感染的病因、致病过程及发病机制,对于疾病早期诊断、传染性的评估、病情的预测、抗病毒药物疗效的观察、疾病发展进程的监测和乙肝感染自然史的研究等方面均有重要的指导意义.针对近几年乙肝分子流行病学研究的几个热点即乙型肝炎病毒的检测、基因分型、基因系统进化分析、基因变异、基因多态性的研究进展作一回顾.虽然近年来该领域的研究迅速增多,但多数仍处于探索阶段,乙肝病毒感染、清除等诸多机制的研究仍任重而道远.     

环孢素A促进HepG2.2.15细胞中乙型肝炎病毒核心蛋白入核的研究

目的观察磷酸脂酶抑制剂环孢素A（CSA）对HBcAg表达和定位的影响，了解HBV生活的周期。方法以30μg/ml的CSA处理HepG2．2．15细胞2d或4d；共聚集显微镜观察细胞内HBcAg和HBsAg亚细胞定位；TUNEL方法检测细胞凋亡。结果对照组HepG2．2．15细胞中HBcAg主要定位于胞质。CSA处理2d后可使胞质中HBcAg和HBsAg水平下降，细胞核内HBcAg水平升高，可见约5％细胞发生凋亡。CSA处理4d后细胞核内HBcAg水平进一步提高，约25％细胞核内表达强度明显高于胞质，并约有30％细胞发生凋亡。结论CSA可促使HepG2．2．15细胞内HBcAg进入细胞核。HBcAg的核内表达定位增强可能与蛋白磷酸化水平增高，及细胞衰老或者凋亡有关。     

The immune response induced by hepatitis B virus principal antigens

Hepatitis B virus （HBV） infection occurs primarily in hepatocytes in the liver with release of infectious virions and non-infectious empty surface antigen particles into the bloodstream. HBV replication is non-cytopathic. Transient infections run a course of several months, and chronic infections are often life-long. Chronic infections can lead to liver failure with cirrhosis and hepatocellniar carcinoma. It is generally accepted that neutralizing anti-HBs antibodies plays a key role in recovery from HBV infection by containing the spread of infection in the infected host and facilitating the removal and destruction of viral particles. However, the immune response initiated by the T-cell response to viral antigens is also important for viral clearance and disease pathogenesis in HBV infection. The three structural forms of the viral proteins, the HBsAg, the particulate HBcAg, and the nonparticulate HBeAg, may preferentially elicit different Th cell subsets. The different IgG subclass profiles of anti-HBs, anti-HBc, and anti-HBe in different HBV infection status were revealed. Moreover, the different IgG subclass profiles in chronic carriers did not change with different ALT and AST levels and may reflect the difference between stimulating antigens, immune response, and the stages of viral disease and provide the basis for the use of vaccines and prophylactic treatments for individuals at high risk of human HBV infection. This review elucidates the detailed understanding of the immune responses induced during transient and persistent infection, and the development of immunotherapy and immunodiagnosis in patients with HBV infection, and possible means of reducing the liver damage.     

Cost-effectiveness and long-term outcomes of liver transplantation using hepatitis B core antibody-positive grafts with hepatitis B immunoglobulin prophylaxis in Korea

Background/AimsHepatitis B core antibody (anti-HBc)-positive donors are used as an extended donor pool, and current guidelines recommend the usage of nucleos(t)ide analogues (NAs) as prophylaxis for preventing de novo hepatitis B virus infection (DNH). We analyzed the long-term outcomes of a large cohort of liver transplantation (LT) patients receiving anti-HBc-positive grafts and evaluated the risk of DNH when hepatitis B immunoglobulin (HBIG) monotherapy was used as prophylaxis. We also compared the cost-effectiveness of HBIG and NAs.MethodsWe retrospectively reviewed 457 patients with anti-HBc-positive grafts and 898 patients with anti-HBc-negative grafts who underwent LT between January 2001 and December 2018. We compared recipient characteristics according to the anti-HBc status of the donor, and compared the costs of using NAs for the rest of the patient’s life and using HBIG to maintain hepatitis B surface antibody titers above 200 IU/L.ResultsThe 1-, 5-, and 10-year patient survival rates were 87.7%, 73.5%, and 67.7%, respectively, in patients with anti-HBc-positive grafts, and 88.5%, 77.4%, and 70.3%, respectively, in patients with anti-HBc-negative grafts (P=0.113). Among 457 recipients with anti-HBc-positive grafts, 117 (25.6%) were non-HBV recipients. The overall incidence of DNH was 0.9%. When using HBIG under insurance coverage, the cumulative cost was lower compared with using NA continuously without insurance coverage in Korea.ConclusionsAnti-HBc-positive grafts alone do not affect patient survival or graft survival. HBIG monoprophylaxis has good outcomes for preventing DNH, and the patient’s long-term cost burden is low in Korea because of the national insurance system in this cohort.     

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.     

The insertion domain of the duck hepatitis B virus core protein plays a role in nucleocapsid assembly

Synthesis of hepadnaviral DNA is dependent upon both the viral DNA polymerase and the viral core protein, the subunit of the nucleocapsids in which viral DNA synthesis takes place. In a study of natural isolates of duck hepatitis B virus (DHBV), we cloned full-length viral genomes from a puna teal. One of the clones failed to direct viral DNA replication in transfected cells, apparently as a result of a 3 nt inframe deletion of histidine 107 in the core protein. Histidine 107 is located in the center of a predicted helical region of the "insertion domain", a stretch of 45 amino acids which appears to be at the tip of a spike on the surface of the nucleocapsid. The mutation was introduced into a well-characterized strain of DHBV for further analysis. Core protein accumulated in cells transfected with the mutant DHBV but was partially degraded, suggesting that it was unstable. Assembled nucleocapsids were not detected by capsid gel electrophoresis. Interestingly, the mutant protein appeared to form chimeric nucleocapsids with wild-type core protein. The chimeric nucleocapsids supported viral DNA replication. These results suggest that the insertion domain of the spike may play a role either in assembly of stable nucleocapsids, possibly in formation of the dimer subunits, or in triggering nucleocapsid disintegration, required during initiation of new rounds of infection.     

乙型肝炎病毒C基因启动子区准种与变异特点的研究

目的 研究乙型肝炎病毒(HBV)C基因启动子区难种与变异的特点。方法 以中国株HBV基因序列为依据，设计特异性聚合酶链反应(PCR)引物，自3例慢性HBV感染患者外周血血清中扩增HBVCP序列，克隆入pGEM Teasy质粒，随机挑选克隆进行DNA测序以确定病毒的变异程度。结果 测序结果发现HBV CP序列高度保守，但在TATA样盒(1—3)可发生多种突变，其中184nt(T→C)位替换突变员为常见；在直接重复序列(DR)I上游存有一缺失突变高发区33．3％(5／15)。结论 CP区内有一缺失高变区，TATA样盒3的变异可能影响前C蛋白的表达。结果 提示HBV长期携带者体内有HBV准种共存。     

重组乙肝病毒核心基因DNA与表面抗原-抗体共免疫应答研究

目的:了解小鼠对乙肝病毒核心抗原(HBcAg)基因免疫及与乙肝表面抗原-抗体复合物(HBsAg-抗HBs)联合免疫的应答。方法:用表达乙肝病毒核心抗原N端144个氨基酸的重组质粒DNA(简称pHBc144)100μg及含1μgHBsAg的重组乙肝表面抗原-鼠抗体组建的复合物(简称sIC)免疫Balb/c小鼠。用ELISA法检测血清抗体IgG和IgG1、IgG2a亚类的效价。用半定量PCR法分别检测鼠脾细胞IFN-γ及IL-4的mRNA。结果:pHBc144及sIC联合免疫鼠的抗HBs IgG、IgG1、IgG2a效价均显著高于sIC组(P＜0．05)。同时小鼠还可产生抗HBc及由HBsAg、HBcAg特异诱生的IFN-γ及IL-4。但与sIC共免疫,小鼠对HBcAg诱生的IFN-γ mRNA有所降低。结论:可用HBcAg基因与sIC共免疫,以获得针对乙肝病毒2种结构蛋白的免疫应答。     

乙型肝炎病毒基本核心启动子及前C区突变与基因型的关系

目的　探讨乙型肝炎病毒 (HBV)基本核心启动子 (BCP)及前C区突变与基因型之间的关系。方法　随机选取 113例慢性HBV感染者外周血 ,采用INNO LiPA法测定BCPT176 2 A176 4双突变及前C区A1896突变 ,测定HBVS基因序列明确基因型。结果　在C基因型感染者中BCPT176 2 A176 4双突变率明显高于B基因型感染者 ,差异有显著性 (34.2 %∶10 % ,χ2 =6 .74 ,P <0 .0 1) ,而前C区A1896突变率在B基因型和C基因型感染者中差异无显著性 (2 .5 %∶4 .1% ,χ2 =0 .0 0 ,P >0 .0 5 )。结论　与B基因型相比 ,C基因型感染者更易发生BCPT176 2 A176 4双突变。     

The degrees of hepatocyte cytoplasmic expression of hepatitis B core antigen correlate with histologic activity of liver disease in the young patients with chronic hepatitis B infection

Subcellular localizaton of HBcAg have been found to be related to the activity of liver disease and HBV replication. The aim of this study was to determine whether the degree of expression of HBcAg in the hepatocyte nucleus and cytoplasm reflects the level of viral replication and histological activity in chronic HBV infection. A total of 102 patients with biopsy proven chronic hepatitis B were included. There was a highly significant correlation between the levels of HBV DNA in serum and the degree of expression of HBcAg in the nucleus for HBeAg-positive(p=0.000) and negative patients(p=0.04). There was a highly significant, correlation between the degrees of expression of HBcAg in hepatocyte cytoplasm and histologic activities (p<0.01) for HBeAg-positive patients. The degrees of expression of HBcAg in the hepatocyte cytoplasm correlated positively with the lobular activities (p<0.01), but not correlated with the portal activity and fibrosis for HBeAg-negative patients. In conclusion, in the young patients with chronic B viral hepatitis, the degree of expression of HBcAg in the hepatocyte nucleus may affect viral load, and the degree of expression of HBcAg in the hepatocyte cytoplasm may affect histologic activities of liver disease.     

乙型肝炎病毒C基因启动子及前C变异的动态研究

目的明确乙型肝炎病毒（ＨＢＶ）Ｃ基因启动子（ＢＣＰ）与前Ｃ变异株在慢性乙型肝炎患者中的动态消长,及与ＨＢｅＡｇ血清学状态的关系。方法对３２例慢性乙型肝炎患者的１０５份系列血清进行错配聚合酶链反应（ＰＣＲ）－限制性片段长度多态性分析,并对其中１５例患者系列血清标本的ＰＣＲ产物进行直接测序。结果ＢＣＰ和Ａ８３变异株感染率均明显增高（６２．５％＞４６．９％;３１．３％＞１２．５％）;ＢＰＣ变异株更多地（１４／１６）表现为优势积累,且先于ＨＢｅＡｇ血清阴转;ＢＣＰ野株／变异株比例变化影响ＨＢｅＡｇ血清学的稳定性。结论两种类型的变异株在炎症活动中均具有选择优势,最终造成ＨＢｅＡｇ（－）的ＨＢＶ感染;可能与ＨＢＶ感染之持续有关。     

重型乙型肝炎病人中乙型肝炎病毒C基因启动子变异的研究

目的　了解重型乙型肝炎（ 乙肝） 病人血清乙肝病毒（ＨＢＶ）ＤＮＡＣ基因启动子（ＣＰ） 的变异。方法　对用聚合酶链反应（ＰＣＲ） 法扩增的血清ＨＢＶＤＮＡ 直接测序。结果　７ 例亚急性重型肝炎病人的ＨＢＶ 分离株ＣＰ区分别有２～１２ 个替代变异，１ 例病人有１１ｂｐ 的碱基插入。ＣＰ变异主要发生于ＣＰ的第１ 和第２ 个ＡＴ丰富区，ｎｔ１ ７６２ 和ｎｔ１ ７６４ 的替代变异见于７ 例亚急性重型肝炎病人的４ 例中，是ＣＰ变异的热点，其中３ 例ＨＢｅＡｇ 阴性，说明和ＨＢｅＡｇ 阴性表型相关。ＣＰ的第３ 个ＡＴ丰富区、ＨＢＶ逆转录起始位点（ＤＲ１） 和前Ｃ基因、前基因组转录起始位点未见变异。结论　重型肝炎病人的ＨＢＶＣＰ区存在较多的变异，ＣＰ变异主要发生于和前Ｃ基因相关的第１ 和第２ 个ＡＴ丰富区，可能和ＨＢｅＡｇ 阴性表型相关