Effects of mutations in the X gene of hepatitis B virus on the virus replication

Previously, we have found a?new mutation at nt 1726-1730 that is associated with lower hepatitis B virus (HBV) DNA levels in the liver, and mutations at nt 1762/1764 that are correlated with higher HBV DNA levels. To confirm the effects of these mutations on the virus replication efficiency, substitutions nt 1726-1730 CTGAG and A1762T/G1764A in the HBV X (HBX) gene region were investigated alone or in combination. Cells Huh-7 or HepG2 were transfected with these constructs. The effects of these mutations on HBV were investigated at the gene and protein levels. The double mutation A1762T/G1764A increased whereas the nt 1726-1730 CTGAG mutations decreased the levels of released virion-associated and intracellular HBV DNA. The combined mutations had no appreciable effect on the replication capacity of the virus. Cells bearing the constructs with double mutations A1762T/G1764A contained the lowest levels of hepatitis B e antigen (HBeAg). Lowest expression of HBV X protein was in constructs that had both A1762T/G1764A and 1726-1730 CTGAG mutations. We think that changes in secondary RNA structure that were caused by these mutations might have been responsible for those results. Keywords: hepatitis B virus; X gene; mutants; replication.     

Effects of GB virus-C/hepatitis G virus on hepatitis B and C viremia in multiple hepatitis virus infections

Summary.  We found that patients with dual HBV and GBV-C/HGV infection had comparable serum HBV DNA positivity and mean virus concentration compared with age-matched HBV carriers, and those with triple infection had a significantly lower HBV DNA positivity. Serum HCV RNA positivity and mean virus titer were similar between HCV carriers with or without GBV-C/HGV co-infection, and those with GBV-C/HGV co-infection seemed to have a lower serum ALT level. These data suggest that GBV-C/HGV infection exerts no significant suppression on levels of chronic hepatitis B or hepatitis C viremia. Accepted December 4, 1997 Received September 8, 1997     

MICA and recovery from hepatitis C virus and hepatitis B virus infections

The polymorphic MHC class I chain-related A (MICA) gene encodes a ligand that has different binding affinities for the NKG2D activating receptor of CD8+ T cells and natural killer (NK) cells. We hypothesized that MICA heterogeneity would affect recovery from hepatitis C virus (HCV) and hepatitis B virus (HBV) infections. To test the hypothesis, we initially typed known MICA polymorphisms for 228 persons who cleared HCV infection and 442 persons with persistent hepatitis C matched on other factors affecting viral persistence. Although MICA(*)015 was detected more than two-fold more often in persons with viral clearance (odds ratio 0.36, 95% confidence interval=0.19, 0.80), it occurred in fewer than 5% of the study population. In a similar analysis of 442 persons with chronic hepatitis B and 768 matched controls who recovered, MICA(*)015 was detected in 2.0% of persons with chronic hepatitis B and only 0.9% of controls. No significant associations were detected with other MICA polymorphisms. While further investigation may reveal a structural basis of the MICA(*)015 associations, these data provide little support for the hypothesis that differential distribution of MICA alleles substantially affects recovery from HCV and HBV infections.     

减毒麻疹病毒在体内外对HBV的影响

目的     

Antiviral effect of polysaccharide from Spirulina platensis （ PSP ） on HSV-2 in vitro

To explore the antiviral effect and mechanism of polysaccharide from Spirulina platensis (PSP) on herpes simplex vims type 2 (HSV-2), a standard strain of HSV-2 (333 strain) was used to investigate the antiviral effect of PSP in vitro . PSP in various concentrations was applied to different stages of HSV-2 replication cycle. Finally, the virus infectivity (TCID50),cytopathic effect (CPE), and MTT staining method for viable cells (MTT assay) were used as markers to evaluate the effect of PSP on HSV-2. The quantity of HSV-DNA was detected by real-time fluorescence quantitative PCR (FQ-PCR). The HSV-2 infected Vero cell ultrastructures were observed by transmission electron microscopy (TEM). The results showed that PSP had little cytotoxic effect on Vero cells, it could not directly inactivate HSV-2 infectivity. PSP not only interfered in adsorption of HSV-2 to Vero cells but also inllibited HSV-2 biosynthesis in the cells. FQ-PCR results showed that the inhibitory rate on HSV-DNA also increased in a dose-dependent and time-dependent manner. TEM also confirmed that PSP exhibited pronounced inhibitory effect on HSV-2. In conclusion, the antiviral effect of PSP on HSV-2 may be attributed to the inhibition of vims adsorption, vims replication and synthesis in cells.     

乙型肝炎病毒在丁型肝炎病毒包装中的作用机理研究

用含ＨＢＶ全基因和ＨＢＶ大、中、小分子表面蛋白基因的真核细胞表达质粒（ＣＭＶ－ＨＢＶ、ＣＭＶ－ＬＳ、ＣＭＶ－ＭＳ、ＣＭＶ－Ｓ）分别与含ＨＤＶ ｃＤＮＡ三聚体的重组质粒共转染ＣＨＯ细胞。转染后３天在上述４种转染细胞内及培养上清中均检出了ＨＤＶ ＲＮＡ和ＨＤＡｇ表明上述４种转染细胞的培养上清中均有ＨＤＶ病毒颗粒的包装和分泌。提示：ＨＤＶ病毒的包装可能仅需ＨＢＶ Ｓ 基因及其小分子表面蛋白的辅助。     

DNA cloned from the ayw subtype of hepatitis B virus

DNA from the ayw subtype of hepatitis B virus (HBV) was ligated into the EcorI site of DNA from plasmid pBR322 and propagated in E coli chi 776. A plasmid with a 3200 base pair insert (pHBV-1) was isolated and the cloned HBV DNA was mapped with restriction endonucleases. Differences were found in restriction endonuclease cleavage sites for DNAs from HBV of subtype ayw and adr.     

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.     

Occult hepatitis B virus and hepatitis C virus infections

Occult HBV infection is a well-recognised clinical entity characterised by the detection of HBV-DNA in serum and/or in liver in the absence of detectable hepatitis B surface antigen (HBsAg). Occult HBV infection has been described not only in patients who have resolved an acute or chronic HBV infection but also in patients without any serological markers of a past HBV infection. Occult HBV infection in patients with chronic HCV infection may induce more severe liver disease and lower response rate to interferon treatment. The existence of occult HCV infections has been also reported more recently. Occult HCV infection is characterised by the presence of HCV-RNA in liver and peripheral blood mononuclear cells in the absence of detectable serum HCV-RNA. Occult HCV infection may occur under two different clinical situations: in hepatitis C antibody-(anti-HCV) negative and serum HCV-RNA-negative patients with abnormal liver function tests and in anti-HCV-positive patients who have no detectable serum HCV-RNA and who have normal liver enzymes. The clinical relevance of occult HCV infections is still under investigation.     

乙型肝炎病毒剪接特异性蛋白对病毒自身调控序列的影响

目的 研究双剪接型2.2 kb乙型肝炎病毒(HBV)剪接变异体特异性蛋白TPds对病毒自身调控序列的影响.方法 PCR扩增6种HBV启动子/增强子序列,以Kpn Ⅰ及Xho Ⅰ位点克隆于pGL3-basic,分别构建萤火虫荧光素酶重组报告载体pGL3-BCP(含HBV基本核心启动子)、pGL3-CP1601(含增强子Ⅱ的核心启动子)、pGL3-XP(X基因最小启动子)、pGL3-XP1071(含增强子Ⅰ的X基因启动子)、pGL3-SP1(表面抗原大蛋白基因启动子)、pGL3-SP2(表面抗原中蛋白基因启动子).以FuGENE6将TPds表达载体pcDNA3.1/HisC-TPds或空白载体pcDNA3.1/HisC分别与6种HBV启动子/增强子报告载体共转染Huh7细胞,转染后48 h裂解细胞并检测胞内萤火虫荧光素酶活性,实验重复5次,数据以SPSS11.5软件分析.结果 在一定的质量比值范围内,与pcDNA3.1/HisC空白载体对照相比,pcDNA3.1/HisC-TPds分别与pGL3-CP1601、pGL3-XP1071、pGL3-SP1、pGL3-SP2共转染后,Huh7细胞内萤火虫荧光素酶活性增高,而pcDNA3.1/HisC-TPds分别与pGL3-BCP或pGL3-XP共转染后,胞内荧光素酶活性无变化.结论 TPds蛋白可反式激活HBV启动子SP1、SP2和增强子Ⅰ、Ⅱ,对基本核心启动子和X基因最小启动子无影响.     

Serum hepatitis B virus DNA in acute hepatitis B

With the use of the dot blot hybridization technic, sera from 30 consecutive patients with acute hepatitis B were examined for the presence of hepatitis B virus (HBV)-DNA. In an additional five patients with acute hepatitis B, serial serum samples, beginning before the serum alanine amino transferase elevation to the clearance of hepatitis B surface antigen, also were tested for various hepatitis B virus markers, including HBV-DNA. Thirteen of the 30 patients (43%) had circulating HBV-DNA and HBeAg at the time of their first clinic visit. However, 11 additional patients with HBeAg did not have HBV-DNA in their sera. In the 13 patients with HBV-DNA, there was no correlation between the titers of HBeAg and the levels of HBV-DNA. Examination of the serial serum samples from the additional five patients showed HBV-DNA and HBeAg to be present several days before the peak of serum alanine amino transferase. In all five patients, HBV-DNA was present in the serum before the appearance of IgM anti-HBc. However, HBsAg was present in all these patients' sera at the time of HBV-DNA positivity. None of the patients in this study became chronic carriers of hepatitis B virus.     

Distribution of hepatitis B virus in the liver of chronic hepatitis C patients with occult hepatitis B virus infection

Although occult hepatitis B virus (HBV) infection (HBV-DNA in serum in the absence of hepatitis B surface antigen [HBsAg]) is common in chronic hepatitis C, its characteristics are not well known. In this work, the presence of HBV-DNA (by polymerase chain reaction; PCR) and its distribution (by in situ hybridization) in liver biopsies and peripheral blood mononuclear cells (PBMCs) from 32 patients with chronic hepatitis C and occult HBV infection and in 20 HBsAg chronic carriers were determined. The results showed that serum HBV-DNA levels were statistically lower (P = 0.001) in patients with occult HBV infection than in HBsAg chronic carriers. The HBV infection pattern in liver cells was identical between patients with occult HBV infection and those with chronic hepatitis B. However, the mean percentage of HBV-infected hepatocytes was significantly lower (P = 0.001) in patients with occult HBV infection (5 +/- 4.44%) than in HBsAg chronic carriers (17.99 +/- 11.58%). All patients with chronic hepatitis B have HBV-DNA in their PBMCs while this occurred in 50% of the cases with occult HBV infection. In conclusion, patients with occult HBV infection have a low number of HBV-infected hepatocytes and this fact could explain the lack of HBsAg detection and low viremia levels found in these cases.     

Leucocyte hepatitis B virus DNA in acute and chronic hepatitis B virus infection

In the present study we have investigated 53 patients with a spectrum of acute and chronic hepatitis B virus (HBV) infection for the presence of leucocyte HBV-DNA with the aid of molecular techniques. HBV-DNA was detected in peripheral blood mononuclear cells of 31 of 45 (69%) of chronic HBsAg carriers and 2 of 8 (25%) patients with acute hepatitis B. Although HBV-DNA was detected more frequently in leucocytes from those HBsAg carriers seropositive for HBeAg (79%), 50% of those with anti-HBe in serum had leucocytes positive for HBV-DNA independent of the presence of serum HBV-DNA. Examination of various leucocyte subpopulations showed the presence of HBV-DNA in polymorphonuclear leucocytes as well as T- and non-T-enriched mononuclear cell fractions. The HBV-DNA identified was predominantly 3.2-kilobase (kb), while higher molecular weight sequences were rarely detected, and lower molecular weight sequences indicative of active viral replication were not observed. These data indicate that although leucocytes do not actively support viral replication, they frequently harbour 3.2-kb HBV-DNA and may act as a reservoir for infection and, more importantly, since leucocytes contaminate several body secretions, may be involved in virus transmission.     

Detection of hepatitis B virus DNA in sera from patients with chronic hepatitis B virus infection by DNA microarray method

We have developed a sensitive and quantitative assay using a DNA microarray for the detection of hepatitis B virus (HBV) DNA in serum. Fluorescently labeled target cDNA prepared from cloned HBV DNA or serum HBV DNA was hybridized to capture DNA on a slide. A linear relationship was obtained between the intensities of the array spot and the amount of the cloned or serum HBV DNA, indicating the quantitative accuracy of this assay system. In addition, there was a significant correlation between the number of molecules of serum HBV DNA determined by the DNA microarray and that determined by a branched-DNA assay (n = 21, r = 0.89). Given these results, we conclude that the DNA microarray assay system may be useful as a diagnostic technique in the clinical laboratory.     

Molecular analysis of hepatitis B virus genomes isolated from black African patients with fulminant hepatitis B

To investigate further the possible role of mutant hepatitis B viruses in the pathogenesis of fulminant hepatitis B, the genomic sequence of hepatitis B virus isolates from 9 South African blacks with this disease, including 5 entire genomes, was analysed. Seven of the isolates were genotype A. The mutation most often reported in patients with fulminant hepatitis B, the G1896A precore stop-codon substitution, was, as expected, not present in the genotype A isolates with the exception of one in which it was accompanied by a compensatory C1858T substitution. G1896A was, however, present in the one genotype D isolate. No other precore-defective mutants were detected. The other mutation commonly found in patients with fulminant hepatitis B, the paired A1762T, G1764A substitution in the basic core promoter, was present in only one patient and G1764A in one other. The pre-surface initiation-codon mutation documented in a number of patients with fulminant hepatitis B was not found in our isolates. An 18-amino acid deletion present in the pre-surface region of one isolate has not previously been described in fulminant hepatitis B. Variations within the surface region were mainly genotype specific and not previously described. A relatively large number of mutations were present in the middle region of the core gene in those isolates without G1896A or A1762T, G1764A mutations, although the pattern was not consistent with those in published studies. Thus, as in other published series in which the entire genome of hepatitis B virus responsible for fulminant hepatitis was sequenced, we detected many mutations in different genes, but none was common to all the reported isolates.     

Inhibition of enterovirus 71-induced apoptosis by allophycocyanin isolated from a blue-green alga Spirulina platensis

Enterovirus 71 infection causes significant morbidity and mortality in children, yet there is no effective treatment. In this study, a protein-bound pigment, allophycocyanin purified from blue-green algae is first reported to exhibit anti-enterovirus 71 activity. Allophycocyanin neutralized the enterovirus 71-induced cytopathic effect in both human rhabdomyosarcoma cells and African green monkey kidney cells. The 50% inhibitory concentration of allophycocyanin for neutralizing the enterovirus 71-induced cytopathic effect was approximately 0.045 +/- 0.012 microM in green monkey kidney cells. The cytotoxic concentrations of allophycocyanin for rhabdomyosarcoma cells and African green monkey kidney cells were 1.653 +/- 0.003 microM and 1.521 +/- 0.012 microM, respectively. A plaque reduction assay showed that the concentrations of allophycocyanin for reducing plaque formation by 50% were approximately 0.056 +/- 0.007 microM and 0.101 +/- 0.032 microM, when allophycocyanin were added at the state of viral adsorption and post-adsorption, respectively. Antiviral activity was more efficient in cultures treated with allophycocyanin before viral infection compared with that in the cultures treated after infection. Allophycocyanin was also able to delay viral RNA synthesis in the infected cells and to abate the apoptotic process in enterovirus 71-infected rhabdomyosarcoma cells with evidence of characteristic DNA fragmentation, decreasing membrane damage and declining cell sub-G1 phase. It is concluded that allophycocyanin possesses antiviral activity and has a potential for development as an anti-enterovirus 71 agent.