Hepatitis B viral DNA in liver and serum of asymptomatic carriers.

Cloned DNA probes were used to test for hepatitis B virus (HBV) DNA in the liver and serum of 14 asymptomatic hepatitis B surface antigen (HBsAg) carriers and two former carriers. The results were compared with serological markers of HBV infection and liver histopathology. Three groups of carriers were distinguished. In group I, HBV DNA was present in both liver and serum. Hepatitis B e antigen (HBeAg), a marker of viral replication, was uniformly positive in serum. In one individual in this group, viral DNA was also integrated into liver genomic DNA. In group II, lower levels of nonintegrated HBV DNA were detected in the liver, but no HBV DNA was found in the serum. HBeAg was negative and with one exception there were antibodies to HBeAg. Integrated viral DNA was present in each case. In group III, there was no detectable nonintegrated viral DNA in serum or liver, but one individual had integrated sequences. All carriers lacked antibodies to HBsAg and had antibodies to hepatitis B core antigen and demonstrated nonspecific histological abnormalities in the liver. These findings indicate significant quantitative and qualitative differences among asymptomatic HBsAg carriers and suggest that their infectivity may be highly variable.     

干扰素治疗前后慢性乙型肝炎患者的血清学和组织学观察

目的　探讨干扰素治疗前后 ,慢性乙型肝炎患者的血清学和肝组织学变化。方法　于干扰素治疗前 1周内和治疗后 1周内 ,取 2 4例慢性乙型肝炎患者的血清和肝脏活检组织 ,检测其血清ALT、HBsAg、HBcAg、HBeAg、HBVDNA和金属蛋白酶组织抑制剂 1(TIMP 1) ,评价肝组织学活动指数 ,检测肝脏中的HBsAg、HBcAg、HBeAg、活化的肝脏星状细胞 (HSC)和TIMP 1。 结果　治疗后 ,9/ 2 4 (37.5 % )的患者发生了应答反应。与治疗前相比 ,干扰素治疗后慢性乙型肝炎患者血清中的HBVDNA明显下降 (P <0 .0 5 ) ,血清中的TIMP 1、肝脏的组织学活动指数 (HAI)、HBcAg、HBeAg、活化的HSC和TIMP 1均有明显下降 (P <0 .0 5 )。结论　干扰素治疗慢性乙型肝炎患者 ,可以抑制病毒复制 ,减少肝组织中病毒抗原表达 ,减少血清和肝组织中的TIMP 1,减少肝脏中活化的HSC数量。     

体内与转染细胞中乙型肝炎病毒株复制特性的相关性

目的 比较不同乙型肝炎病毒（HBV）株在体内及转染细胞中复制特性是否相符。方法 以核酸杂交定量和聚合酶链反应-酶联免疫吸附试验（PCR-ELISA）测定5例孕妇血清中HBVDNA含量,并测定乙型肝炎表面抗原（HBsAg)和乙型肝炎e抗原（HBeAg)含量,分别克隆血清中的HBV基因组转染细胞,检测培养上清液中HBsAg和HBeAg表达水平,并以Southern印迹及核酸杂交检测转染细胞内、外HBVDNA复制水平。结果 转染细胞内、外HBVDNA复制水平与相应血清HBVDNA量呈正相关趋势,转染细胞表达的病毒抗原水平与相应血清中病毒抗原含量也呈正相关趋势。结论 感染者血清中HBVDNA和病毒抗原的含量与毒株在细胞中复制和抗原表达水平有相符趋势。HBV不同毒株在转染细胞中的复制可基本反映体内毒株的复制特性。     

Replication of hepatitis B virus in first-degree relatives of patients with hepatocellular carcinoma

Hepatocellular carcinoma (HCC) may occur in family clusters. No genetic mechanism has been identified as responsible for this familial tendency. We suspected that a longer hepatitis B virus (HBV) replication phase might be the reason for a higher risk of HCC in families with this disease. We performed liver biochemical tests, test for viral hepatitis markers and hepatitis B e antigen (HBeAg), and liver ultrasonography in relatives of patients with HCC. A total of 1,885 first-degree relatives from 688 families participated in this study. Seven hundred fifty-two relatives were found to be carriers of hepatitis B surface antigen (HBsAg) and 675 of them were tested for HBeAg. The prevalence of HBeAg was 27.4% in relatives of those with HCC and 20% in asymptomatic HBsAg carriers. The HBeAg prevalence rate was higher in relatives of those with HCC > or = 40 years old than in asymptomatic HBsAg carriers. Moreover, HBeAg was more likely to persist in men than in women > or = 40 years old. We conclude that families with HCC showed a prolonged HBV replication phase that may be one of the cofactors for a familial tendency for HCC.     

Construction and expression of eukaryotic plasmids containing lamivudine-resistant or wild-type strains of Hepatitis B Virus genotype C

AIM： To construct eukaryotic expression plasmids of full-length Hepatitis B Virus （HBV） genotype C genome, which contain lamivudine-resistant mutants （YIDD, YVDD） or wild-type strain （YMDD）, and to observe the expression of HBV DNA and antigens [hepatitis B surface antigen （HBsAg） and hepatitis B e antigen （HBeAg）] of the recombinant plasmids in HepG2 cells. METHODS： Three HBV full-length genomes were amplified from the plasmids pMD18T-HBV/YIDD, pMD18T-HBV/YVDD and pMD18T-HBV/YMDD, using PCR. Three recombinant plasmids were generated by inserting each of the PCR products into the eukaryotic expression vector pcDNA3.1 （＋）, between the EcoRI and HindⅢ sites. After being characterized by restriction endonuclease digestion, and DNA sequence analysis, the recombinant plasmids were transfected into HepG2 cells. At 48 and 72 h post-transfection, the levels of intracellular viral DNA replication were detected by real-time PCR, and the expression of HBsAg and HBeAg in the cell culture supernatant was determined by ELISA.
RESULTS： Restriction endonuclease digestion and DNA sequence analysis confirmed that the three recombinant plasmids were correctly constructed. After transfecting the plasmids into HepG2 cells, high levels of intracellular viral DNA replication were observed, and HBsAg and HBeAg were secreted into the cell culture supernatant.
CONCLUSION： Eukaryotic expression plasmids pcDNA3.1 （＋）-HBV/YIDD, pcDNA3.1 （＋）-HBV/YVDD or pcDNA3.1 （＋）-HBV/YMDD, which contained HBV genotype C full-length genome, were successfully constructed. After transfection into HepG2 cells, the recombinant plasmids efficiently expressed HBV DNA, HBsAg and HBeAg. Our results provide an experimental basis for the further study of HBV lamivudine-resistant mutants.     

HBeAg immunostaining of liver tissue in various stages of chronic hepatitis B.

AIMS: We studied the tissue expression of hepatitis B e antigen (HBeAg) in 29 liver biopsies from 27 HBV carriers. METHODS: HBeAg expression was assessed in relation to HBeAg in serum, precore mutations, HBV DNA levels and liver damage as measured by histology activity index. RESULTS: HBeAg in liver tissue was detected by immunostaining in 6 of 7 patients positive for HBeAg in serum. In patients negative for HBeAg in serum, HBeAg was detected in none of 11 specimens from patients infected exclusively with a precore mutant that disrupts HBeAg synthesis, as compared with 3 of 11 specimens from patients carrying HBV with an intact precore region. These 3 patients all showed high HBV DNA levels in serum and severe liver damage. CONCLUSIONS: Overall, viral replication was strongly associated with the cytoplasmic HBeAg and nuclear HBcAg staining, but not with tissue staining for HBsAg. Because of the close relationship between tissue HBeAg expression and high viral load, the pathogenetic importance of HBeAg remains unclear.     

HBeAg immunostaining of liver tissue in various stages of chronic hepatitis B

Abstract: Aims: We studied the tissue expression of hepatitis B e antigen (HBeAg) in 29 liver biopsies from 27 HBV carriers. Methods: HBeAg expression was assessed in relation to HBeAg in serum, precore mutations, HBV DNA levels and liver damage as measured by histology activity index. Results: HBeAg in liver tissue was detected by immunostaining in 6 of 7 patients positive for HBeAg in serum. In patients negative for HBeAg in serum, HBeAg was detected in none of 11 specimens from patients infected exclusively with a precore mutant that disrupts HBeAg synthesis, as compared with 3 of 11 specimens from patients carrying HBV with an intact precore region. These 3 patients all showed high HBV DNA levels in serum and severe liver damage. Conclusions: Overall, viral replication was strongly associated with the cytoplasmic HBeAg and nuclear HBeAg staining, but not with tissue staining for HBsAg. Because of the close relationship between tissue HBeAg expression and high viral load, the pathogenetic importance of HBeAg remains unclear.     

乙型肝炎病毒B和C基因型全基因组的克隆与真核细胞表达

目的 构建B和C基因型重组HBV表达载体,检测其在Huh7细胞内的DNA复制和HBsAg、HBeAg的表达.方法 扩增B和C基因型HBV全基因组,并将其连接于真核表达载体pHY106,将这2个载体分别转染Huh7细胞,以pHY106空载体转染作对照.Southern印迹法检测转染72 h后HBV DNA的复制,实时定量PCR检测转染后24、48、72、96和120 h Huh7细胞内HBV DNA水平,ELISA检测转染后24、48、72、96和120 h细胞培养上清液中HBsAg和HBeAg的表达.结果 成功构建了B和C基因型HBV表达载体.转染Huh7细胞后72 h,Southern印迹法检测到细胞内HBV核心颗粒内的HBV复制中间体,包括松弛环状DNA、双链DNA和单链DNA.实时定量PCR检测发现病毒DNA复制水平可达8 lg拷贝/mL、ELISA结果显示HBsAg和HBeAg的表达于转染后72 h达高峰,然后逐渐下降.结论 成功构建B和C基因型重组HBV真核表达载体,并能在Huh7细胞内高水平复制和表达,为进一步研究HBV的结构与功能、基因表达与调控,以及抗HBV药物的筛选等提供了良好的平台.     

Changes in serum and histology of patients with chronic hepatitis B after interferon alpha-2b treatment

AIM: Chronic hepatitis B is a serious health problem. Interferon has long been used to treat Chronic hepatitis B. To evaluate the effects of interferon on chronic hepatitis B better, we designed the study to investigate the changes in sera and liver histology of patients with chronic hepatitis B after interferon alpha-2b treatment. METHODS: Twenty-four patients with chronic hepatitis B were enrolled in this study. They all received interferon alpha-2b treatment as following: 3 million units, i.m. t.i.w., for 18 weeks. Sera of all patients were obtained respectively for evaluation of ALT, HBsAg, HBcAg, HBeAg, HBV DNA and TIMP-1 before and after interferon treatment, also a liver biopsy pre- and post-treatment was performed for comparison of HAI, HBsAg, HBcAg, HBeAg, TIMP-1 and activated HSC in the liver tissue. RESULTS: Patients who had normalization of serum ALT and seroconversion of HBeAg and/or HBV DNA (blot hybridization) after treatment were defined as responders. The response rate in this study group was 37.5 % (7/24). Compared to pretreatment, the serum HBV DNA and TIMP-1 decreased significantly (P<0.05), so did the HAI, HBcAg, HBeAg, TIMP-1 and activated HSC (P<0.05). CONCLUSION: The significant decrease in HBV DNA in sera, the seroconversion of HBeAg, and the decrease of viral expression in liver indicated that interferon alpha-2b treatment can inhibit viral replication. The normalization of ALT in sera and the improvement of HAI in liver showed that interferon alpha-2b can improve the liver histology of patients with chronic hepatitis B. At the same time, interferon alpha-2b treatment can reduce the TIMP-1 in serum and liver and decrease the number of activated HSC, which may alleviate or inhibit hepatic fibrosis. Although the response rate was unsatisfactory, interferon play a beneficial role on patients with chronic hepatitis B in other respects. We still need further studies to improve the therapy effects.     

Long-term remission of chronic hepatitis B after alpha-interferon therapy

OBJECTIVE: To evaluate whether remissions of chronic hepatitis B induced by alpha-interferon therapy are of long duration. DESIGN: Cohort study. SETTING: Clinical Center of the National Institutes of Health, a tertiary referral center. PATIENTS: Sixty-four patients with chronic hepatitis B were treated with alpha-interferon between 1984 and 1986. MAIN OUTCOME MEASURES: Patients were followed with frequent examinations and determinations of serum liver biochemical tests and hepatitis B virus (HBV) markers including hepatitis B surface antigen (HBsAg), hepatitis B e antigen (HBeAg), and HBV DNA using blot hybridization and polymerase chain reaction. RESULTS: Among 64 patients with chronic hepatitis B who were treated with alpha-interferon, 23 (36%) responded to treatment with loss of HBeAg and improvement in serum aminotransferases. All 23 have been followed for 3 to 7 years (mean, 4.3 years). During follow-up, 3 of 23 patients relapsed, with reappearance of HBeAg and abnormal serum aminotransferases, all within 1 year of therapy. The remaining 20 patients continued to have no detectable HBeAg or HBV DNA (using blot hybridization) in serum and to be asymptomatic for liver disease, although 3 had minimal elevations in serum aminotransferases. Thirteen patients (65%) became negative for HBsAg between 0.2 and 6 years (mean, 3 years) after loss of HBeAg. Although no patient had HBV DNA that was detectable by blot hybridization, the 7 patients who remained HBsAg positive all had HBV DNA in serum detected by polymerase chain reaction, but only 2 of 13 HBsAg-negative patients had viral genome using this method. Testing sequential samples indicated that HBV DNA detected by polymerase chain reaction usually disappeared at or around the time that test results for HBsAg became negative. CONCLUSIONS: Remissions in chronic hepatitis B induced by alpha-interferon are of long duration and are followed, in most patients, by the loss of HBsAg and all evidence of residual virus replication.     

Biologic and prognostic significance of hepatocyte hepatitis B core antigen expressions in the natural course of chronic hepatitis B virus infection

To elucidate the biologic significance of hepatocyte hepatitis B core antigen (HBcAg) expression and its relation to the natural course of hepatitis B virus (HBV) infection, the patterns of HBcAg were correlated with HBV virus replication state and the disease activity in 598 needle liver biopsies performed on 569 hepatitis B surface antigen (HBsAg) carriers aged 1-81 years. A good correlation of liver HBcAg with serum HBeAg and HBV DNA status was demonstrated. HBcAg was present in the hepatocyte nuclei (nHBcAg) or cytoplasm (cHBcAg), or in both (mixed). Pure nHBcAg was seen mainly in children and young adults; 86% of the patients had non-aggressive disease, but rare cases of chronic active hepatitis (CAH) and HBeAg seroconversion were observed. In contrast, cHBcAg was predominantly associated with CAH (52%) and accompanied by a significantly higher HBeAg seroconversion rate (27%). The HBeAg-negative group, particularly the liver HBcAg-negative subgroup, had a lower frequency of CAH, but an increased incidence of non-aggressive disease as well as cirrhosis and/or hepatocellular carcinoma, indicating that HBeAg seroconversion to anti-HBe does not necessarily mean a favorable prognosis. The results suggest that expression of HBcAg correlates with the liver pathology and the three phases of chronic HBV infection: (1) the early immune tolerance phase is characterized by nHBcAg, mild disease and low HBeAg seroconversion rate; (2) the virus replication/elimination phase by cHBcAg or negative HBcAg, frequent CAH, and high HBeAg seroconversion rate; and (3) the inactive virus replication phase by negative HBcAg and a bipolar disease spectrum.     

Replication of hepatitis B in HBsAg-positive siblings

Persistent hepatitis B virus (HBV) replication is important for progression of chronic liver diseases. To understand whether there is a trend of HBV replication in siblings or not, 1850 relatives of patients with hepatocellular carcinoma (HCC) were examined prospectively for liver function test, viral markers and HBV DNA. The prevalence of HBsAg in the parents', siblings', children's and grandchildren's generations were 43.4%, 57.2%, 35.5% and 32.1%, respectively. The prevalence of hepatitis B e antigen (HBeAg) in sibling's generation (mean age 44.4 years) was 19%, which is higher than that of asymptomatic HBsAg carriers. For siblings in the children's generation, the prevalence of HBeAg in hepatitis B surface antigen (HBsAg) carriers declined from 40% in the eldest siblings to 19% in the youngest siblings. In 75 families clustered with three or more HBsAg carrier siblings, the mean age for seven families of which all siblings remained HBeAg + was younger, whereas the mean age for 35 families of which all siblings had cleared HBeAg was older. For the remaining 33 families, in only 10 families had the older siblings cleared the HBeAg earlier than the younger siblings. Twenty families showed that younger siblings cleared the HBeAg earlier than the older or middle siblings. We concluded that HBV replication in HCC relatives cannot be explained by familial tendency alone. A significant number of younger siblings appeared to have a shorter HBV replication phase than their older siblings. The possible role of this in maternal–fetal transmission is discussed.     

Inhibition of hepatitis B virus expression and replication by RNA interference in HepG2.2.15

INTRODUCTION Hepatitis B is a severe infectious disease threatening peoples’ health all over the world. There is still no efficient therapy to control HBV persistent replication, which may lead to the development of liver cirrhosis and hepatocellualar ca…     

Expression and replication of hepatitis B virus genome in transgenic mice.

We produced transgenic mice by microinjecting a partial tandem duplication of the complete hepatitis B virus (HBV) genome into fertilized eggs of C57BL/6 mice. One of eight transgenic mice was a high producer for HBV surface antigen (HBsAg) and HBV e antigen (HBeAg) in the serum. The HBV genomes were transmitted to the next generation and these F1 mice also produced HBsAg and HBeAg. mRNAs of 3.5, 2.1, and 0.8 kilobases were detected in the livers and the kidneys of these mice. In addition, a 0.8-kilobase RNA was detected in the testis. Single-stranded and partially double-stranded HBV DNAs were shown to be produced in the cytoplasm of the liver and kidneys. These HBV DNAs were associated with the core particles, indistinguishable from nucleocapsid produced in an infected human liver. Viral genome DNA was detected in the serum. These results demonstrate that the HBV genome integrated into the mouse chromosome acted as a template for viral gene expression, allowing viral replication. Thus, these transgenic mice should be useful for detailed studies of the replication and expression of HBV and for pathological studies of hepatitis, including the development of hepatocellular carcinoma.     

Hepatitis B virus markers in peripheral blood mononuclear cells]

Recent studies have shown tropism of the hepatitis B virus (HBV) by peripheral blood mononuclear cells (PBMC). The consequences of this phenomenon and their clinical use are not yet clear, however. Seventy-nine patients were studied between March 1989 and October 1990. Sixty-nine patients had chronic liver disease with histological evaluations, and 10 were vaccinated for HBV. The following markers were determined: serum: HBsAg, HBeAg, anti-HBe, antitotal-HBc, anti-HBs, anti-HCV, HBV-DNA; lysated PMBC cells: HBsAg, HBeAg. Hepatic tissue: HBsAg, HBcAg. Four groups were formed according to serology. Group I--positive HBsAg patients (n = 25) HBsAg was observed in the lysated of PBMC in 19 (76%) of the patients. HBeAg in PBMC was detected in 8 (32%), all of them showed evidence of viral replication (presence of HBcAg and/or HBV-DNA in the serum HBcAg in the tissue). Group II--antitotal HBc/anti-HBs positive (n = 14), HBsAg in PBMC was found in 5 (36%) and HBeAg in 1 (7.0%). In this patient replication markers in the serum and in the tissue (HBV-DNA, HBcAg) was also present. Three patients out of 9 anti-HBs positive had HBsAg in PBMC. Group III--seronegative patients for HBV. HBsAg was present in PBMC in 2 (6.6%) of the patients, but was absent in all of them. There was concomitant presence of HBsAg in MN and the hepatic tissue in 1 patient. Replication markers were not observed in the group. Group IV--10 asymptomatic individuals vaccinated for HBV. Except anti-HBs in serum, no other HBV marker could be identified in serum or in PBMC.(ABSTRACT TRUNCATED AT 250 WORDS)     

Correlation of quantitative assay of hepatitis B surface antigen and HBV DNA levels in asymptomatic hepatitis B virus carriers

OBJECTIVE: This study was to elucidate the correlation between quantity of hepatitis B surface antigen (HBsAg) and hepatitis B virus (HBV) DNA levels in asymptomatic carriers. METHODS: Based on the presence of the hepatitis B e antigen (HBeAg) and HBV DNA levels, 67 asymptomatic carriers were divided into four groups. HBV DNA was determined by hybridization (sensitivity 141 500 copies/ml) and polymerase chain reaction (PCR, sensitivity < 10 copies/ml). Cases of groups I (n = 18), II (n = 17) and III (n = 16) were negative for HBeAg and had HBV DNA levels of < 10 (PCR undetectable), 10 to 10 (PCR detectable) and > 10 copies/ml (hybridization detectable), respectively. Cases of group IV (n = 16) were positive for HBeAg and high HBV DNA levels (> 2 x 10 copies/ml). HBsAg was determined quantitatively by the ARCHITECT HBsAg assay. RESULTS: Our data showed HBsAg levels were correlated with HBV DNA (r = 0.709; P < 0.001) on a log scale. The mean log HBsAg (IU/ml) of groups I, II, III and IV were 2.68 +/- 0.8, 2.93 +/- 1.03, 3.22 +/- 0.45, 4.83 +/- 0.19, respectively. That of group IV was significantly higher than the mean log HBsAg of any other group (P < 0.001). The best cut-off for HBsAg in differentiating group IV from other groups was 15 000 IU/ml with both sensitivity and specificity of 100%. That of group I was significantly lower than those of group III (P = 0.035) and IV (P < 0.001). The best cut-off in differentiating group I from the other groups was 1600 IU/ml with a sensitivity of 69.4% and a specificity of 66.7%. CONCLUSION: Quantitative measurement of HBsAg titres may be an easy and economical reference for HBV replication in HBV carriers.     

Serum protein expression patterns in detecting a new viral protein in HBeAg‐negative chronic hepatitis B

We analysed the changes in viral protein expression in HBeAg‐negative chronic hepatitis B (CHB). In total, 160 samples were obtained from individuals infected by hepatitis B virus (HBV) and divided into four groups. Group A included 71 cases of hepatitis B e antigen (HBeAg)‐negative CHB, Group B included 58 cases of inactive seroconverters and Group C included 31 cases of HBeAg‐positive CHB. Group D included 22 normal healthy individuals as a control. All serum samples were examined using surface enhance laser desorption/ionization time of flight‐mass spectrometry (SELDI‐TOF‐MS). The results indicated that a peak with 4140 m/z increased markedly in Group A at 1295.55 ± 745.87, which was significantly different from that in Group B at 896.99 ± 534.86 (P = 0.013). This peak indicated a close relationship with HBV DNA replication and may contribute to pathogenesis of HBeAg‐negative chronic hepatitis.