Hepatitis B viral DNA is methylated in liver tissues

The mechanisms that regulate hepatitis B virus (HBV) replication within the liver are poorly understood. Given that methylation of CpG islands regulates gene expression in human tissues, we sought to identify CpG islands in HBV-DNA and to determine if they are methylated in human tissues. In silico analysis demonstrated three CpG islands in HBV genotype A sequences, two of which were of particular interest because of their proximity to the HBV surface gene start codon (island 1) and to the enhancer 1/X gene promoter region (island 2). Human sera with intact virions that were largely unmethylated were used to transfect HepG2 cells and HBV-DNA became partially methylated at both islands 1 and 2 by day 6 following exposure of HepG2 to virus. Examination of three additional human sera and 10 liver tissues showed no methylation in sera but tissues showed methylation of island 1 in six of 10 cases and of island 2 in five of 10 cases. The cell line Hep3B, with integrated HBV, showed complete methylation of island 1 but no methylation of island 2. In conclusion, HBV-DNA can be methylated in human tissues and methylation may play an important role in regulation of HBV gene expression.     

Hepatitis B virus markers among family contacts of asymptomatic HBsAg carriers.

A study was undertaken to establish the risk of family contacts of HBsAg carriers acquiring a hepatitis B virus (HBV) infection. About one-third of all household contacts of asymptomatic HBsAg carriers had signs of past or ongoing HBV infection. Family contacts of HBsAg carriers with high numbers of circulating Dane particles were shown to have a higher risk of developing HBV infection than family contacts of HBsAg carriers without serological evidence of HBV synthesis. The probability of acquiring HBV infection was not different between spouses, parents, children, and brothers and sisters, respectively of asymptomatic HBsAg carriers.     

Hepatitis B viral factors in HBeAg-negative carriers with persistently normal serum alanine aminotransferase levels

Chronic hepatitis B patients with high-normal serum ALT (levels of 0.5-1x upper limit of normal) are still at risk of liver disease progression. We thus investigated the correlation between serum ALT level and hepatitis B viral factors in HBeAg-negative carriers with persistently normal serum ALT level (PNALT). Baseline clinical and virological features of 414 HBeAg-negative carriers, including 176 (42.5%) with low-normal ALT (levels of less than 0.5x upper limit of normal) and 238 (57.5%) with high-normal ALT, were compared. Compared with HBV carriers with low-normal ALT, those with high-normal ALT were older (41 vs. 37 years, P<0.001) and had a greater frequency of serum HBV DNA level>10(4) copies/ml (63.4% vs. 47.5%, P<0.001) as well as a higher prevalence of basal core promoter T1762/A1764 mutant (36.5% vs. 24.2%, P=0.01). Multivariate analysis showed that factors associated with a high-normal serum ALT level included male sex [odds ratio (OR), 1.82; 95% confidence interval (CI), 1.10-3.01, P=0.019], increasing age (OR, <30 years: 1, reference; 30-39 years: 2.43, 95% CI, 1.18-5.03, P=0.016; 40-49 years: 4.22, 95% CI, 1.99-8.93, P<0.001; >or=50 years: 4.06, 95% CI, 1.69-9.78, P=0.002) and serum HBV DNA level>10(4) copies/ml (OR, 1.83; 95% CI, 1.07-3.13, P=0.027). CONCLUSION: HBeAg-negative patients with persistently normal ALT are not a homogenous group, and those with high-normal ALT share some of the characteristics that have been associated with adverse long-term outcomes.     

Hepatitis B viral DNA in infected human liver and in hepatocellular carcinoma

Blot-hybridization analysis of DNA forms from hepatitis B virus (HBV) in the liver of chronic carriers of hepatitis B surface antigen (HBsAg) has revealed the presence of both relaxed and closed circular viral DNA and of novel viral DNA-RNA hybrid molecules in patients with complete virions, with the hepatitis B e antigen (HBeAg), or with both, in serum. One carrier of HBsAg with no detectable virus or HBeAg in his serum had small amounts of free viral DNA in his liver sample, a finding suggesting the potential for production of complete virus; another such carrier had only HBV DNA integrated in cellular DNA and, thus, may have lost the ability to replicate virus. The liver sample of one of eight patients with antibodies to HBsAg in his serum, but no HBsAg, contained small amounts of free viral DNA. Analysis of tissue from hepatocellular carcinoma revealed evidence for integrated viral DNA sequences in multiple-cellular DNA sites, and stoichiometric analysis suggested that the tumors were monoclonal in origin. These results demonstrate the presence of a new form of HBV in infected human liver and reveal that serological profiles are not always a reliable guide in determining the presence of potentially infectious forms of HBV in the liver.     

Hepatitis B virus DNA in children's liver diseases: detection by blot hybridisation in liver and serum.

Molecular hybridisation using cloned hepatitis B virus DNA (HBV DNA) was applied to liver and serum samples from 46 children (39 with liver diseases and seven controls) for detection of HBV DNA sequences, free and integrated into the liver cell genome. HBV DNA integration was observed in 10 children. The young age of some of these cases indicates that such integration can occur early in liver disease and is not related to the duration of viral infection. Thirteen children exhibited serological evidence of active viral multiplication. All but one had free HBV DNA in liver tissue and integrated HBV DNA sequences were found in four cases. Integrated HBV DNA sequences alone were also detected in three children with neither HBV-antigens nor HBV DNA in serum. One had inactive cirrhosis, and the two others, chronic active hepatitis. Consequently DNA hybridisation may be useful for diagnosis, in the absence of serological signs of HBV infection; its specificity was enhanced in the present investigation by negative results in six children with autoimmune chronic active hepatitis. Taken together, the above results imply that HBV-DNA integration can occur in both active and inactive liver disease. Integrated HBV DNA was also observed in the liver of three children with fatal hepatic failure who presented with antibodies to HBsAg and/or to hepatitis B core antigen in the serum. This finding raises the question of the relationship between the host immune factors and the state of HBV DNA.     

原发性肝癌患者血清HBV标志物和HBV DNA测定的临床意义

目的观察原发性肝癌患者血清HBV标志物模式及HBV DNA水平。方法采用化学发光法检测151例PHC、132例慢性乙型肝炎和140例乙型肝炎肝硬化患者血清乙型肝炎病毒标记物;采用聚合酶链反应检测血清HBV DNA水平。结果在151例原发性肝癌患者中,HBV感染率达97.4%（147/151）,HBV DNA阳性率为74.8%（113/151）,平均水平为5.6±1.1 lgcopies/ml;HBsAg（＋）、HBeAb（＋）、HBcAb（＋）90例（59.6%）,其血清HBVDNA检出率为76.7%,平均水平为5.1±0.9lgcopies/ml;HBsAg（＋）、HBeAg（＋）、HBcAb（＋）38例（25.2%）,血清HBVDNA检出率100.0%,平均水平为6.4±0.9 lgcopies/ml。结论 PHC患者HBV感染率高,HBV与PHC发生有十分密切的关系。随着慢性乙型肝炎和乙型肝炎肝硬化病情的进展,血清HBeAg自发性发生血清学转换和HBV DNA载量下降,要警防原发性肝癌的发生。     

Duck hepatitis B virus DNA in liver and serum of Chinese ducks: integration of viral DNA in a hepatocellular carcinoma.

The presence of duck hepatitis B virus (DHBV) DNA in liver and serum and its state (integrated vs. free) were studied in 23 ducks from Chi-tung county in China by spot hybridization and Southern blot hybridization, respectively. In 16 of 23 (70%), DHBV DNA was detected in serum and/or in liver tissue. These infected ducks showed a variety of pathological changes including advanced chronic disease in the liver. In contrast, none of the virus-negative ducks had advanced hepatic changes. One DHBV DNA-seropositive duck had a large hepatocellular carcinoma. Southern blot analysis demonstrated integrated DHBV DNA in neoplastic tissue and abundant episomal DHBV DNA in non-neoplastic tissue of the liver. In one noninfected duck with a small adenoma, no viral DNA was detected in tumor or non-neoplastic tissue. The detection of integrated DHBV DNA in hepatocellular carcinoma suggests that DHBV behaves like human and woodchuck hepatitis viruses in relation to chronic liver disease and hepatocarcinogenesis.     

Hepatitis B virus DNA in chronic HBsAg carriers: correlation with HBeAg/anti-HBe status, anti-HD and liver histology.

Hepatitis B virus DNA was determined in the sera of 198 chronic hepatitis B surface antigen (HBsAg) carriers by the spot hybridization technique. The results were correlated with hepatitis Be antigen (HBeAg) and antibody (anti-HBe), delta antibody (anti-HD) and liver histology. All subjects had a liver biopsy. The prevalence of HBV DNA was 63% in HBeAg-positive subjects and 8.8% in anti-HBe positives. HBV DNA was not found more frequently in chronic HBsAg carriers who had histological evidence of liver disease than in carriers without such evidence. Anti-HD was detected in 48.5% of subjects, with an increasing trend (p less than 0.001) according to the severity of liver disease. Among patients with more severe liver disease (CAH and cirrhosis), HBV DNA and HBeAg were detected less frequently in anti-HD-positive than in anti-HD-negative subjects (7% vs. 42.3%, p less than 0.001 and 7% vs. 34.4%, p less than 0.005, respectively). These findings indicate that HDV infection jointly affects both HBeAg status and HBV DNA.     

Hepatitis C virus RNA in serum and liver histology in asymptomatic anti-HCV positive subjects

Summary This study was designed to evaluate serum HCV-RNA, liver histology, and RIBA-II pattern in asymptomatic anti-HCV positive subjects with persistently normal or slightly (i.e. 1.5 times the upper limit of the normal range) elevated serum ALT levels. To this purpose, 22 asymptomatic anti-HCV positive subjects (11 men and 11 women, median age 40, range 21–70 years) underwent liver biopsy and determination of serum HCV-RNA. Positivity for anti-HCV was determined by ELISA-2 and by RIBA-II. Serum HCV-RNA was determined by PCR. Our data show that: 1) 9/22 symptom-free, anti-HCV positive subjects had histological features of chronic liver disease associated with ongoing HCV infection; 2) four subjects had no histological signs of chronic hepatitis and normal serum ALT levels despite positivity for serum HCV-RNA; 3) serum ALT levels did not discriminate HCV-RNA positive subjects with from those without chronic hepatitis; 4) in anti-HCV positive subjects with normal serum ALT levels, a positive RIBA-II pattern was not always predictive of HCV viraemia or chronic hepatitis while an indeterminate RIBA-II pattern was frequently associated with nonspecific liver changes or normal histology. In conclusion, based on these findings, true healthy carriers of HCV (i.e. subjects with normal serum ALT levels and no histological features of chronic hepatitis despite HCV viraemia) may exist.

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Hepatitis C Virus-RNA in Serum und Leberhistologie bei asymptomatischen, anti-HCV positiven Personen

Zusammenfassung Bei asymptomatischen, anti-HCV positiven Personen mit andauernd normalen oder leicht (das heißt 1,5 fach über der oberen Normgrenze) liegenden Serum ALT-Spiegeln wurden die HCV-RNA im Serum, Leberhistologie und das RIBA-II-Muster untersucht. Bei 22 asymptomatischen, anti-HCV positiven Personen (11 Männern und 11 Frauen im mittleren Alter von 40; Bereich 21–70 Jahren) wurde eine Leberbiopsie durchgeführt und HCV-RNA im Serum bestimmt. Der Nachweis von anti-HCV wurde mit ELISA-2 und RIBA-II-Tests geführt. Die HCV-RNA im Serum wurde mittels PCR bestimmt. Unsere Untersuchungen zeigten, 1.) daß 9/22 asymptomatischen, anti-HCV-positiven Personen histologische Zeichen einer chronischen Lebererkrankung mit fortbestehender HCV-Infektion hatten. 2.) Vier Personen hatten keine histologischen Zeichen einer chronischen Hepatitis und normale Serum-ALT-Spiegel trotz Nachweises von HCV-RNA im Serum. 3.) Die ALT-Spiegel im Serum unterschieden HCV-RNA positive Personen mit chronischer Hepatitis nicht von denjenigen ohne chronische Hepatitis. 4.) Bei anti-HCV positiven Personen mit normalen ALT-Spiegeln ist ein positiver RIBA-II Test nicht immer mit einer HCV-Virämie oder einer chronischen Hepatitis assoziiert. Bei unklarem RIBA-II-Muster bestanden häufig unspezifische Leberveränderungen oder ein normaler histologischer Befund. Wirklich gesunde Träger von HCV (das heißt, Personen mit normalem Serum ALT-Spiegel, ohne histologische Zeichen einer chronischen Hepatitis trotz HCV Virämie) können folglich vorkommen.

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Data from this paper have been presented in part at the 1991 Italian Society of Gastroenterology annual meeting in Catania, Italy and published in abstract form (Ital. J. Gastroenterol. 23 [1991] 511).     

无症状HBV携带者血清HBV DNA水平与肝组织炎症活动度的关系

目的探讨无症状HBV携带者(AsC)肝脏病理损害程度与血清HBV DNA水平的关系.方法对139例AsC于超声引导下采用美国生产的DARD自动活检枪经皮肝穿刺行病理学检查;HBV DNA用PCR测定.结果139例AsC中G1者94例,G2者26例,G3者19例,无G0和G4者;血清HBV DNA水平与肝组织炎症活动度间无显著性差异(P＞0.05);HBeAg阴性组G3者所占比例大于HBeAg(+)组(P=0.019).结论AsC肝脏均有不同程度损害,其损害程度与HBV DNA水平无相关性.     

HBV携带者T细胞亚群状况与肝组织病理学对比

目的:研究HBV携带者的细胞免疫功能变化及其与血清HBVDNA,HBeAg的关系,并对T细胞亚群与肝组织病理学改变进行对比分析,探讨HBV携带及其所导致肝组织病理改变的机制.方法:应用流式细胞仪检测109例HBV携带者和40例健康对照外周血T细胞亚群百分率,并对其中28例行肝组织病理学检查,ELISA法检测HBV标志物,PCR法检测HBVDNA.结果:HBV携带者外周血CD3 、CD4 细胞百分率及CD4 /CD8 比值较正常对照组显著降低(67.2±9.0%vs71.0±3.5%,P<0.05;33.1%±6.6vs40.3±2.8%,P<0.001;1.10±0.36vs2.01±0.19,P<0.001),CD8 细胞百分率明显升高(33.8±8.4%vs20.2±1.9%,P<0.001).HBVDNA( )组及HBeAg( )组分别与HBVDNA(-)组及HBeAg(-)组比较,CD3 细胞无统计学差异,CD4 细胞显著降低(31.2±6.3%vs37.2±5.4%,P<0.001;31.0±6.0%vs35.8±6.5%,P<0.001),CD8 细胞明显升高(36.7±8.4%vs27.9±4.2%,P<0.001;37.3±8.4%vs29.5±6.0%,P<0.001),CD4 /CD8 比值明显降低(0.91±0.32vs1.35±0.26,P<0.001;0.89±0.30vs1.26±0.33,P<0.001).HBVDNA( )组肝病理组织学改变68.8%达到G1S1及以上程度,而HBVDNA(-)组仅为16.7%,二组间差异显著(c2=5.57,P<0.01).G1S1组CD3 细胞较G1S0组明显降低(F=2.919,P=0.047),CD4 细胞降低(P>0.05).G2S1组与G1S0组相比,CD3 细胞及CD4 细胞百分率有降低趋势,CD8 细胞百分率有升高趋势,CD4 /CD8 比值有明显降低趋势.结论:HBV感染可导致HBV携带者细胞免疫功能改变,HBVDNA复制增加可进一步加重HBV携带者的细胞免疫功能紊乱,并加重肝组织损害.     

慢乙肝患者血清及肝组织HBVDNA表达及基因芯片检测研究

用点样仪将 PCR扩增的 HBVDNA探针制成基因芯片 ,对 15例慢性乙型肝炎 (下称慢乙肝 )患者的血清及肝活检组织 ,分别用基因芯片、原位分子杂交法、免疫组织化学法、雅培试剂检测 HBVDNA、HBc Ag、HBs Ag、HBe Ag。结果 15例患者的血清 HBs Ag、HBe Ag及基因芯片检测均阳性。15例肝组织标本中 ,免疫组化法HBc Ag阳性 15例 ,HBVDNA原位分子杂交法阳性 14例 ,基因芯片检测阳性 14例。认为肝炎基因诊断可同时检测乙肝患者血清及肝组织中的 HBVDNA。     

乙型肝炎病毒cccDNA

目前慢性乙型肝炎已成为危害人类健康的主要疾病.分子生物学的研究表明,乙型肝炎病毒(HBV)的共价环状闭合DNA(covalently closed circular DNA,cccDNA)在病毒持续感染、抗病毒治疗后病毒的再度活跃复制以及药物耐受方面起关键作用.     

ALT正常的HBV慢性携带者肝组织病理结果分析

了解ALT正常的HBV慢性携带者（ASC）的肝组织病理改变状况,探讨其临床意义及其与HBVDNA、HBeAg的关系。对32例ALT正常的HBV慢性携带者行快速经皮肝穿刺术取肝组织,研究肝组织炎症活动度及纤维化程度分级分期;ELISA法检测血清HBVM,PCR法检测血清HBVDNA。结果没有真正的健康ASC（病理状态为G0S0）,32例ASC中,G1S0有15例,G1S1有13例,G2S1有4例;肝组织学的炎症活动及纤维化改变程度HBVDNA阳性组明显重于HBVDNA阴性组,男、女之间、HBeAg定性检测及HBVDNA水平的比较,无明显差异。     

Hepatitis B virus DNA in serum from patients with acute hepatitis B

Sera from 77 consecutive patients with acute type B hepatitis were examined for hepatitis B virus DNA (HBV DNA) by a spot hybridization method. The median follow-up time was 8 months (range, 1 week to 3 years). HBV DNA was detected in 26 (34%) patients on admission to the hospital. A significant positive correlation was found between short duration of symptoms and the presence of HBV DNA (p less than 0.025). Twenty-four (46%) of 52 HBeAg-positive patients were HBV DNA positive compared to 2 HBV DNA-positive patients of 25 HBeAg-negative patients (8%) (p less than 0.001). Four HBeAg-negative patients had serum HBV DNA initially or during follow-up; three had anti-HBe. Six of 77 patients with acute type B hepatitis (8%) became chronic HBsAg carriers, and HBV DNA was detectable from 5 months to more than 3 years after onset of symptoms. The presence of serum HBV DNA for more than 8 weeks after initial symptoms may predict development of a chronic HBsAg carrier state. In none of the chronic carriers was serum HBV DNA present after clearing of HBeAg.     

Hepatitis C viral infection in liver transplant recipients.

In this study we examined multiple serial liver biopsy specimens from liver transplant recipients to determine the pathological features of hepatitis C virus-induced hepatitis. Hepatitis C virus infections acquired after transplantation and previous infections that recurred in patients after transplantation were confirmed by the results of the polymerase chain reaction. Of 43 patients infected with the hepatitis C virus, 18 had a mild form of chronic hepatitis. Four patients had hepatitis that progressed to focal bridging fibrosis or cirrhosis. There were no significant clinical or pathological differences between infections acquired after transplantation and recurrent infections (as determined by polymerase chain reaction) except that acquired infections more often developed into hepatitis. Findings indicative of hepatitis C infection included portal and parenchymal mononuclear infiltrates of varying degrees, acidophilic necrosis and swollen hepatocytes. Other common findings included lymphoid aggregates, bile duct damage and fatty change. Atypical pathological conditions included extensive hepatocyte swelling or acidophilic necrosis with minimal inflammation mimicking ischemia and ductal or ductular damage and proliferation with mixed portal infiltrates mimicking rejection or obstruction. We conclude that in transplant recipients infection by the hepatitis C virus usually produces a mild disease state, but the diagnosis of hepatitis can be difficult to make because indicators of hepatitis may mimic those of rejection, ischemia, obstruction or other hepatic infections. Serial biopsy specimens with persistent pathology and polymerase chain reaction may be necessary to define the presence of a hepatitis C virus lesion.     

Hepatitis B virus deoxyribonucleic acid in liver of chronic carriers. Correlation with serum markers and changes associated with loss of hepatitis B e antigen after antiviral therapy

Patients with chronic hepatitis B receiving antiviral or immunomodulatory therapy were prospectively studied to determine the relationship between the state of hepatitis B virus deoxyribonucleic acid in liver and the levels of serum markers of hepatitis B virus infection. Hepatitis B virus deoxyribonucleic acid was quantitated and the molecular forms determined using molecular hybridization in two separate liver specimens taken at least 1 yr apart. All 30 patients initially had hepatitis B surface antigen and e antigen in serum and all had hepatitis B virus deoxyribonucleic acid, including replicative intermediate forms, present in liver. The amount of viral deoxyribonucleic acid in liver correlated significantly with the e antigen titer in serum. At the time of the second liver biopsy, e antigen was no longer detectable in the serum of 12 patients although all except 1 patient still had detectable hepatitis B surface antigen. In this group, the amount of hepatitis B virus deoxyribonucleic acid in liver had decreased significantly and replicative viral intermediates had disappeared. In contrast, among patients who remained e antigen-positive, the amount of viral deoxyribonucleic acid did not change appreciably and replicative intermediate forms were still detectable. These findings imply that in chronic hepatitis B, loss of e antigen is usually associated with resolution of hepatitis B virus replication.