Molecular and clinical aspects of hepatitis D virus infections

Hepatitis D virus (HDV) is a defective virus with circular, single-stranded genomic RNA which needs hepatitis B virus (HBV) as a helper virus for virion assembly and infectivity. HDV virions are composed of a circular shape HDV RNA and two types of viral proteins, small and large HDAgs, surrounded by HBV surface antigen (HBsAg). The RNA polymerase II from infected hepatocytes is responsible for synthesizing RNAs with positive and negative polarities for HDV, as the virus does not code any enzyme to replicate its genome. HDV occurs as co-infection or super-infection in up to 5% of HBsAg carriers. A recent multi-center study highlighted that pegylated interferon α-2a (PEG-IFN) is currently the only treatment option for delta hepatitis. Nucleotide/nucleoside analogues, which are effective against HBV, have no relevant effects on HDV. However, additional clinical trials combining PEG-IFN and tenofovir are currently ongoing. The molecular interactions between HDV and HBV are incompletely understood. Despite fluctuating patterns of HBV viral load in the presence of HDV in patients, several observations indicate that HDV has suppressive effects on HBV replication, and even in triple infections with HDV, HBV and HCV, replication of both concomitant viruses can be reduced. Additional molecular virology studies are warranted to clarify how HDV interacts with the helper virus and which key cellular pathways are used by both viruses. Further clinical trials are underway to optimize treatment strategies for delta hepatitis.     

肝细胞肝癌和肝硬化组织中丙型肝炎病毒各区段抗 …

目的 研究丙型肝炎病毒（ＨＣＶ）在肝细胞肝癌（ＨＣＣ）和肝硬化（ＬＣ）中所起的作用。结合乙型肝炎病毒（ＨＢＶ）进行分析，并初步探讨了ＨＣＶ与ＨＢＶ感染是否有相互促进作用。方法 采用ＨＣＶ－Ｃ，Ｅ，ＮＳ３，ＮＳ４区单克隆抗体，ＨＢｓＡｇ多克隆抗体用免疫组化方法检测了５９例ＨＣＣ及３５例ＬＣ组织标本。结果 ＨＣＶ阳性反应主要分布在肝细胞及癌细胞的胞浆内，呈细颗粒状。ＨＣＣ中，ＨＣＶ感染率；北京（２９例     

Persistence of intrahepatic hepatitis B virus DNA integration in patients developing hepatocellular carcinoma after hepatitis B surface antigen seroclearance

Background/AimsThe role of hepatitis B virus (HBV) integration into the host genome in hepatocarcinogenesis following hepatitis B surface antigen (HBsAg) seroclearance remains unknown. Our study aimed to investigate and characterize HBV integration events in chronic hepatitis B (CHB) patients who developed hepatocellular carcinoma (HCC) after HBsAg seroclearance.MethodsUsing probe-based HBV capturing followed by next-generation sequencing technology, HBV integration was examined in 10 samples (seven tumors and three non-tumor tissues) from seven chronic carriers who developed HCC after HBsAg loss. Genomic locations and patterns of HBV integration were investigated.ResultsHBV integration was observed in six patients (85.7%) and eight (80.0%) of 10 tested samples. HBV integration breakpoints were detected in all of the non-tumor (3/3, 100%) and five of the seven (71.4%) tumor samples, with an average number of breakpoints of 4.00 and 2.43, respectively. Despite the lower total number of tumoral integration breakpoints, HBV integration sites in the tumors were more enriched within the genic area. In contrast, non-tumor tissues more often showed intergenic integration. Regarding functions of the affected genes, tumoral genes with HBV integration were mostly associated with carcinogenesis. At enrollment, patients who did not remain under regular HCC surveillance after HBsAg seroclearance had a large HCC, while those on regular surveillance had a small HCC.ConclusionsThe biological functions of HBV integration are almost comparable between HBsAg-positive and HBsAg-serocleared HCCs, with continuing pro-oncogenic effects of HBV integration. Thus, ongoing HCC surveillance and clinical management should continue even after HBsAg seroclearance in patients with CHB.     

An appraisal of the relationship between primary hepatocellular carcinoma and hepatitis B virus

The association between hepatitis B virus infection and primary hepatocellular carcinoma is reviewed. On the basis of serological and tissue examination there is a close link between virus infection and the tumour. While there is evidence to favour an oncogenic role for virus this is not conclusive, and other possible explanations for the relationship are discussed.     

慢性乙肝发生致死性重型肝炎与重叠嗜肝病毒感染及HBV e系统状态关系的研究

目的　为了查清慢性乙型肝炎 (慢性乙肝 )病程中发生致死性重型肝炎与重叠感染甲、丙、丁或戊型肝炎病毒以及乙肝病毒 (HBV)e系统状态的关系 ,以便采取相应措施降低慢性乙肝的死亡率。方法　用ELISA法检测慢性乙肝病程中发生致死性重型肝炎时重叠甲、丙、丁或戊型肝炎病毒的感染情况以及HBVe系统的状态。结果　在慢性乙肝病程中发生致死性重型肝炎的 2 19例患者中 ,甲、丙、丁或戊型肝炎病毒的重叠感染率分别为 1. 4 % (3 /2 19)、9.6 % (2 1 2 19)、1.8% (4 2 19)和30.1% (6 6 / 2 19) ,重叠嗜肝病毒的感染率之和为 4 2.9% (94 2 19)。其中 ,以戊型肝炎病毒为主 ,并且近10年来感染率基本没有改变。原因未明者为 5 7.1% (12 5 2 19)。HBeAg和抗 HBe的阳性率在甲、丙、丁或戊型肝炎病毒重叠感染组分别为 17.0 % (16 94 )和 5 4.0 2 % (5 1 94 ) ;原因未明组分别为 2 7.2 %(34 /12 5 )和 4 7.2 % (5 9/12 5 ) ,两组之间HBeAg和抗 HBe阳性率的比较差异无统计学意义 (P >0 .0 5 )。结论　研究结果提示 ,重叠嗜肝病毒感染是发生致死性重型肝炎的重要原因 ;严格饮食卫生和使用安全的血液制品可降低慢性乙肝的死亡率。未能发现HBVe抗原的血清转换与慢性乙肝患者病程中发生致死性重型肝炎有明确的关系     

乙型肝炎表面抗原定量与肝组织病理关系的临床研究

目的 探讨慢性乙型肝炎病毒感染者血清乙肝表面抗原定量水平与肝脏病理之间的关系.方法 收集我院272例乙肝表面抗原阳性超过6个月并接受肝脏穿刺活检的患者的临床资料.检测血清乙肝表面抗原定量、乙肝病毒基因型、ALT、HBV DNA、血常规、肝脏组织病理等各项资料,分别按照肝脏炎症活动程度(G0、G1、G2、G3、G4)和纤维化程度(S0、S1、S2、S3、S4)分组.观察分析乙肝表面抗原定量、HBV DNA与肝组织炎症活动程度和纤维化程度之间的关系.分析乙肝表面抗原定量与HBV DNA之间的相关性.结果 乙肝表面抗原定量与HBV DNA之间的相关性是显著的,表面抗原定量对数是对肝脏纤维化有显著影响的变量.结论 乙肝表面抗原对数是较DNA定量对数特异度和灵敏度更高的诊断肝脏纤维化程度的指标.     

Molecular epidemiology of hepatitis B virus in Spain: identification of viral genotypes and prediction of antigenic subtypes by limited sequencing

The hepatitis B virus (HBV) genotypes were studied by a line probe assay (LiPA) and by direct sequencing of a 339 nucleotide fragment from the S region of the viral genome in samples from 269 carriers living in Spain, either native to Spain (231) or immigrants from Africa, Asia, and Eastern Europe (38). The sequences were also used to predict the HBV surface antigen (HBsAg) subtype on the basis of the amino acids specified at selected positions of the HBsAg molecule. Agreement between the two genotyping methods was found in most cases (98.1%) and a HBV genotype could be assigned to all samples. The viral groups D/ayw2 (30.1%), D/ayw3 (28.6%), and A/adw2 (21.2%) were prevalent, with an additional participation of the groups D/ayw4 (4.8%), F/adw4q- (1.9%), A/ayw1 (1.9%), and D/adw3 (0.7%), all of them present among the autochthonous carriers. Strains from genotypes B and C were found exclusively among Chinese immigrants. Genotype E strains were found in immigrants from Central Africa and in one patient native of Spain. Point mutations leading to amino acid changes of residues involved in the expression of the HBsAg subtype determinants were found in 12 samples (4.5%). Some mutations would predict the putative novel genotype-subtype associations A/adw4q+, A/ayr, D/ayr, and E/ayw1, while others would suggest the loss of subtype-specific determinants. The finding of HBV strains characteristic for Africa among the autochthonous carriers confirms the emergence of African HBV strains in Spain.     

Association between apolipoprotein E genotype,chronic liver disease,and hepatitis B virus

Background/Aims

Apolipoprotein E (ApoE) plays an important role in regulating lipid and lipoprotein metabolism, and ApoE genotypes are known to affect plasma lipoprotein concentrations. We investigated whether ApoE genotype determines the disease outcome in hepatitis B virus (HBV)-infected individuals, and verified the association between ApoE genotype and the occurrence of hepatocellular carcinoma (HCC) in patients with chronic liver diseases of various etiologies.

Methods

This hospital-based, case-controlled study enrolled 156 subjects (47 healthy controls, 50 HBV-related liver cirrhosis patients, and 59 HCC patients). ApoE genotypes were determined using PCR-based ApoE genotyping kits. The biological significance of ApoE genotype was verified by measuring serum ApoE levels using an ELISA kits.

Results

The ε3 allele was the most common allele, with allele frequencies among the entire cohort of 5.8%, 84.3%, and 9.9% for the ε2, ε3, and ε4 alleles, respectively. Significantly more of those patients carrying the ε3/3 genotype had developed liver cirrhosis compared to the control subjects. Being an ApoE4 carrier was associated with a lower probability of developing liver cirrhosis. The allele frequencies and genotype distribution of ApoE did not differ significantly between the liver cirrhosis and HCC patients. The serum level of ApoE was significantly higher in patients with liver cirrhosis than in the healthy controls, but did not differ significantly with the ApoE genotype.

Conclusions

The ApoE ε3/3 genotype frequency was higher in patients with HBV-associated liver cirrhosis than in the controls.     

Persistent delta antigenaemia in chronic delta hepatitis and its relation with human immunodeficiency virus infection.

The prevalence of persistent hepatitis delta (HD) antigenaemia and associated factors in patients with chronic infection with the hepatitis delta virus (HDV) were investigated. Among 157 consecutive patients known to be carriers of hepatitis B surface antigen (HBsAg), 36 (23%) had one serum marker of HDV infection (anti-HD and/or HDAg). Nine of the patients with an HDV marker were HDAg positive, including three who were anti-HD negative. A follow-up over a mean period of 13 months showed that five of five patients had a persistent HD antigenaemia. This serological profile was associated with the presence of antibody to the human immunodeficiency virus (anti-HIV) (P < 0.01), serum HIV antigen (HIVAg) (P < 0.2), and the female sex (P < 0.05). Persistent HD antigenaemia could be the consequence of the suppression of T cell cytotoxic activity against hepatocytes expressing HDAg, a lower humoral response, and/or hormonal factors.     

慢性乙型肝炎患者肝组织中HBV抗原表达特征及其临床意义

目的探讨慢性乙型病毒性肝炎肝活检组织中检测乙肝表面抗原(HBsAg)和乙肝核心抗原(HBcAg)表达强度及表达方式的必要性。方法采用EnVision免疫组织化学法检测196例慢性乙型肝炎患者肝穿组织中HBsAg和HBcAg的表达水平,并用荧光定量PCR检测其血清中的HBV DNA的含量。对肝组织进行炎症活动度分级和纤维化分期。结果肝组织中的HBsAg表达强度和表达方式与炎症分级、纤维化分期和血清乙肝病毒载量均无相关性(P>0.05)。HBcAg表达强度与炎症分级无相关性(r=-0.02,P>0.05);与纤维化分期呈负相关(r=-0.28,P<0.01);与血清乙肝病毒载量呈正相关(r=0.53,P<0.01)。HBcAg表达方式与炎症分级为负相关(r=-0.27,P<0.01),其中浆型组炎症活动度分级高于核型组和混合型组(P<0.01),混合型组高于核型组(P<0.01)。HBcAg表达方式与纤维化分期亦呈较弱的负相关(r=-0.23,P<0.01),其中浆型组纤维化分期高于核型组和混合型组(P<0.05)。HBcAg表达方式与血清乙肝病毒载量呈正相关(r=0.22,P<0.01)。结论区分肝组织中的HBsAg表达强度和表达方式无益于了解慢性乙型肝炎患者肝损害的程度,而检测肝组织中的HBcAg则有助于临床抗病毒治疗。     

新生儿乙型肝炎疫苗免疫后13年效果观察

目的 研究乙型肝炎的远期免疫效果。方法 采用单纯随机方法连续13年对1986年出生并接种乙型肝炎疫苗的儿童进行隔年随访,采血检测HBsAg、抗-HBs、抗-HBc。结果 13年间HBsAg阳性率在0.46%-0.97%之间,未随免疫时间的延长而上升,乙型肝炎疫苗的远期保护效果为81.67%,与近期免疫效果相当。结论 免疫后13年仍无需加强免疫。     

慢性乙型肝炎患者血清HBV DNA水平与肝纤维化标志物的关系

目的研究慢性乙型肝炎患者血清HBV复制水平和肝纤维化血清学标志物的关系。方法入选临床确诊为慢性乙型肝炎的157例患者，其中49例为早期肝硬化，采用荧光定量PCR检测血清HBVDNA水平，放射免疫法和酶免疫法检测肝纤维化血清标志物：透明质酸、层黏蛋白、Ⅲ型前胶原N端肽和Ⅳ型胶原，对血清HBVDNA水平和肝纤维化标志物的关系进行研究分析，并对49例早期肝硬化患者和108例无肝硬化患者的血清HBVDNA及肝纤维化血清标志物水平进行比较。结果慢性乙型肝炎患者的血清HBVDNA水平和肝纤维化标志物水平无显著相关性（P〉0．05），早期肝硬化患者的血清肝纤维化标志物水平显著高于无肝硬化的患者，而HBVDNA水平却低于无肝硬化的患者（P〈0．05）。结论慢性乙型肝炎患者的血清HBVDNA水平和肝纤维化标志物水平无显著相关性。     

Primary hepatocellular carcinoma and hepatitis B virus infection in korea

Serological evidence of hepatitis B virus (HBV) infection and serum alphafetoprotein (AFP) were assayed in sera from 112 Korean patients with primary hepatocellular carcinoma (PHC) and from 63 age- and sex-matched controls. Serological evidence of HBV infection was found in 100% of PHC patients and in 97% of controls. The majority of PHC patients (87%) were positive for hepatitis B surface antigen (HBsAg). In contrast, only 14% of control individuals were positive for HBsAg, but 82% were positive for antibody to HBsAg (anti-HBs). Hepatitis B e antigen (HBeAg) was detected in a high percentage (38%) of HBsAg-positive PHC patients, but in none of the nine HBsAg-positive control individuals. Serum AFP was detectable in 83% of PHC patients but in only one of 63 controls (1.5%). These results document that HBV infection may be the mjor factor in the development of PHC in this country.     

Concurrent hepatitis C virus and hepatitis delta virus superinfection in patients with chronic hepatitis B virus infection.

Since hepatitis C virus (HCV) and hepatitis delta virus (HDV) are transmitted by the same routes as hepatitis B virus (HBV), simultaneous or concurrent HCV and HDV infection in patients with chronic HBV infection may occur. To test this hypothesis and to examine the clinicohistological and immunopathological presentations of such multiple hepatitis virus infections, acute and/or convalescent serum specimens from 86 patients with acute HDV superinfection were tested by enzyme immunoassay for antibodies to HCV. Of the 86 patients, 18 (20.9%) were associated with HCV infection. Although patients with early mortality cannot be evaluated by the HCV markers used in this study, the results showed that the clinical and histologic features were similar except that patients with HCV infection were older than those without HCV infection (P less than 0.01). Immunopathological studies carried out within 2 months after the onset of acute HDV superinfection demonstrated that hepatitis B core antigen (HBcAg) was not detected in any patient and HDV antigen was detected in 18.2% of the patients with HCV infection whereas HBcAg and HDAg were found in 7% and 65.1%, respectively, of those without HCV coinfection (P less than 0.02). It is concluded that concurrent HCV and HDV superinfections can and do occur in patients with chronic HBV infection. In these triple viral infections, HCV may even transiently suppress HDV and HBV.     

HBV cccDNA在2.2.15细胞表达的动态观察

目的研究2.2.15细胞中是否存在HBVcccDNA,探讨2.2.15细胞内HBV产物的动态表达规律。方法采用PCR方法检测2.2.15细胞内cccDNA,Taqman定量PCR技术检测2.2.15细胞内及培养上清中HBVDNA含量,EIA方法检测培养上清中HBsAg、HBeAg的动态表达,并进行定量资料相关性统计学分析。结果2.2.15细胞及培养上清中存在cccDNA,2.2.15细胞培养上清中HBVDNA与HBsAg、HBeAg之间存在相关性(r=0.833,P<0.05和r=0.939,P<0.01),而细胞内HBVDNA与培养上清HBVDNA及HBsAg、HBeAg之间无相关性(r=0.024,P>0.05和r=0.177,P>0.05)。结论为阐明2.2.15细胞内HBV的复制规律提供一定依据。     

乙型肝炎病毒感染者癌基因蛋白的表达及与HBeAg的关系

目的为了阐明癌基因蛋白异常表达与乙型肝炎病毒（ＨＢＶ）复制的关系。方法用链霉菌素－生物素（ＳＬＡＢ）免疫组织化学染色和酶联免疫吸附试验（ＥＬＩＳＡ）方法，检测了６４例慢性ＨＢＶ感染者肝细胞中Ｃ－ｅｒｂＢ－２Ｐ１８５、ｒａｓＰ２１和Ｐ５３等癌基因蛋白和血清乙型肝炎病毒ｅ抗原（ＨＢｅＡｇ）。结果Ｃ－ｅｒｂＢ－２Ｐ１８５和ｒａｓＰ２１阳性组中，血清ＨＢｅＡｇ的阳性检出率分别为８９４％和８４６％，而阴性组ＨＢｅＡｇ的检出率分别为２０％和４８％，两组差异显著。结论表明癌基因蛋白Ｃ－ｅｒｂＢ－２Ｐ１８５和ｒａｓＰ２１的异常表达与ＨＢＶ复制密切相关。     

Hepatitis B and D virus infection among drug abusers in Taiwan

Approximately 15 to 20% of the general population in Taiwan are chronic hepatitis B surface antigen (HBsAg) carriers. However, the incidence of hepatitis D virus (HDV) infection is low (5-8%) in patients with HBsAg-positive chronic liver diseases in this area. To evaluate the prevalence of hepatitis B virus (HBV) and HDV infection among drug abusers in Taiwan, serum samples were collected from 152 drug abusers at the Taipei Municipal Anti-Narcotic Institute and test for HBV and HDV markers. Of these, 24 (15.8%) were HBsAg positive, and only 15 (9.9%) were seronegative for all HBV markers. Of the 115 intravenous drug abusers, serum antibody to hepatitis D antigen (anti-HD) was positive in 78.9% of 19 persons who were HBsAg positive, and in 7.5% of 80 persons who were positive for antibody to HBsAg (anti-HBs). Anti-HD was not detected in the sera from all 37 nonintravenous drug abusers regardless of the status of their HBV markers. Also, none of 63 asymptomatic HBsAg carrier pregnant women or 23 patients with acute type B viral hepatitis had measurable anti-HD in their sera. Thus, the high frequency of HDV detected among Chinese HBsAg carrier intravenous drug abusers in Taiwan is similar to that reported in Western countries.     

Detection of hepatitis B surface antigen (HBsAg) in peripheral blood mononuclear cells of patients with acute and chronic active hepatitis B

Seventy five patients with acute and chronic active hepatitis (CAH) were studied by indirect immunofluorescence with monoclonal antibodies for the presence of hepatitis B surface antigen (HBsAg) on peripheral blood mononuclear cells (PBMC). The viral surface antigen was detected in the PBMC of all the patients with hepatitis B virus (HBV)-induced CAH and in acute patients with more than 2 months of evolution. No HBsAg was detected in the samples obtained from 12 normal controls or from 14 non-A, non-B CAH patients. Analysis of PBMC subsets revealed that HBsAg was present in non-T cells; dual fluorescence studies showed HBsAg on surface Ig-positive lymphocytes. The binding of anti-HBs monoclonal antibodies was higher than that of a goat anti-HBs serum, and the highest reactivity was observed with an antibody against the pre-S(2)-region sequence. Both HBsAg and hepatitis B core antigen (HBcAg) were also detected in lysates of PBMC by dot blot analysis.