Hepatitis D virus dual-infection among Chinese hepatitis B patient related to hepatitis B surface antigen,hepatitis B virus DNA and age

The screening practices for hepatitis D virus(HDV)are diverse and nonstandardized worldwide,and the exact prevalence of HDV is uncertain.AIM To estimate HDV prevalence and investigate viral marker quantity trends in patients with hepatitis D.METHODS We collected 5594 serum samples from patients with hepatitis B in Jilin Province,China(3293 males and 2301 females,age range of 2 to 89 years).We then conducted tests for hepatitis B surface antigen(HBsAg),hepatitis B Virus(HBV)DNA,anti-hepatitis D antigen(HDAg),and HDV RNA.RESULTS We found that the prevalence of anti-HDAg and HDV RNA among hepatitis B patient were 3.6%(3.2-4.2%)and 1.2%(0.9-1.5%),respectively,87.69%of hepatitis D patients were 51-70 years old.HDV infection screening positive rate of patients with HBV DNA levels below 2000 IU/mL(2.0%)was higher than those above 2000 IU/mL(0.2%).Among anti-HDAg positive patients,the HDV RNA positive rate was positively correlated with the HBsAg level and anti-HDAg level.There was a weak correlation between HBsAg and anti-HDAg levels among hepatitis D patients.CONCLUSION Our study highlights the importance of considering multiple factors when assessing the severity of HDV infection,comprehensive evaluation of patients’clinical and laboratory parameters is necessary for proper diagnosis and treatment.

Screening and evaluation of human single-chain fragment variable antibody against hepatitis B virus surface antigen

BACKGROUND: Phage display technology has become a vital tool in studies aimed at identifying molecules binding to a specific target. It enables the rapid generation and selection of high affinity, fully human antibody product candidates to essentially any disease target appropriate for antibody therapy. In this study, we prepared the recombinant single-chain fragment variable ( ScFv) antibody to hepatitis B virus surface antigen (HBsAg) by the phage display technology for obtaining a virus-targeting mediator. METHODS: mRNA was isolated from B-lymphocytes from a healthy volunteer and converted into cDNA. The fragment variables of heavy and light chain were amplified separately and assembled into ScFv DNA with a specially constructed DNA linker by polymerase chain reaction. The ScFv DNA was ligated into the phagmid vector pCANT-AB5E and the ligated sample was transformed into competent E. coli TG1. The transformed cells were infected with M13K07 helper phage to form a human recombinant phage antibody library. The volume and recombinant rate of the library were evaluated by bacterial colony count and restriction analysis. After two rounds of panning with HBsAg. the phage clones displaying ScFv of the antibody were selected by enzyme-linked immunosorbant assay ( ELISA) from the enriched phage clones. The antigen binding affinity of the positive clone was detected by competition ELISA. HB2151 E. coli was transfected with the positive phage clone demonstrated by competition ELISA for production of a soluble form of the anti-HBsAg ScFv. ELISA assay was used to detect the antigen binding affinity of the soluble anti-HBsAg ScFv. Finally, the relative molecular mass of soluble anti-HBsAg ScFv was measured by SDS-PAGE. RESULTS: The variable heavy ( VH ) and variable light (VL) and ScFv DNAs were about 340bp, 320bp and 750bp, respectively. The volume of the library was up to 2 × 106 and 8 of 10 random clones were recombinants. Two phage clones could strongly compete with the original HBsAb for binding to HBsAg. Within 2 strong positive phage clones, the soluble anti-HBsAg ScFv from one clone was found to have the binding activity with HBsAg. SDS-PAGE showed that the relative molecular weight of soluble anti-HBsAg ScFv was 32 kDa. CONCLUSION: The anti-HBsAg ScFv successfully produced by phage antibody technology may be useful for broadening the scope of application of the antibody.

Is there a need for universal double reflex testing of HBsAg-positive individuals for hepatitis D infection?

Hepatitis D virus(HDV)can infect HBsAg-positive individuals,causing rapid fibrosis progression,early decompensation,increased hepatocellular carcinoma risk,and higher mortality than hepatitis B virus(HBV)mono-infection.Most countries lack high-quality HDV prevalence data,and the collection techniques employed often bias published data.In recent meta-analyses,HDV prevalence in HBsAg-positive patients reaches 5%-15%and is even significantly higher in endemic areas.Since HBV vaccination programs were implemented,HDV prevalence has decreased among younger populations.However,owing to immigrant influx,it has increased in some Western countries.The current practice of HDV screening in HBsAg-positive individuals is stepwise,based on physician’s discretion,and limited to at-risk populations and may require numerous visits.Double reflex testing,which includes anti-HDV testing in all HBsAg-positive individuals and then HDV RNA testing for anti-HDV-positive ones,is uncommon.Reflex testing can identify more HDV infection cases and link identified patients to further care and follow-up.Moreover,laboratory-based double reflex screening is less biased than physician-led testing.Therefore,health-care providers should learn about reflex testing,and federal and provincial hepatitis control programs should implement laboratory-based double reflex testing to obtain reliable HDV prevalence estimates.The test’s cost-effectiveness depends on the number of HBV-positive patients screened to identify one HDV-positive patient.Such testing may be viable in areas with low HBsAg but high HDV prevalence.However,its economic impact on areas with low HDV prevalence needs further study.

Development of Fok-I based nested polymerase chain reaction-restriction fragment length polymorphism analysis for detection of hepatitis B virus X region V5M mutation

AIM: To develop a Fok-I nested polymerase chain reaction(PCR)-restriction fragment length polymorphism analysis(PRA) method for the detection of hepatitis B virus X region(HBx) V5 M mutation.METHODS: Nested PCR was applied into DNAs from 198 chronic patients at 2 different stages [121 patients with hepatocellular carcinoma(HCC) and 77 carrier patients]. To identify V5 M mutants, digestion of nested PCR amplicons by the restriction enzyme Fok-I(GGA TGN9↓) was done. For size comparison, the enzymetreated products were analyzed by electrophoresis on 2.5% agarose gels, stained with ethidium bromide, and visualized on a UV transilluminator.RESULTS: The assay enabled the identification of 69 patients(sensitivity of 34.8%; 46 HCC patients and 23 carrier patients). Our data also showed that V5 M prevalence in HCC patients was significantly higher than in carrier patients(47.8%, 22/46 patients vs 0%, 0/23 patients, P < 0.001), suggesting that HBx Ag V5 M mutation may play a pivotal role in HCC generation in chronic patients with genotype C infections.CONCLUSION: The Fok-I nested PRA developed in this study is a reliable and cost-effective method to detect HBx Ag V5 M mutation in chronic patients with genotype C2 infection.

Hepatitis B virus markers in hepatitis B surface antigen negative patients with pancreatic cancer:Two case reports

BACKGROUND Hepatitis B virus(HBV)is a known carcinogen that may be involved in pancreatic cancer development.Detection of HBV biomarkers[especially expression of HBV regulatory X protein(HBx)]within the tumor tissue may provide direct support for this.However,there is still a lack of such reports,particularly in non-endemic regions for HBV infection.Here we present two cases of patients with pancreatic ductal adenocarcinoma,without a history of viral hepatitis,in whom the markers of HBV infection were detected in blood and in the resected pancreatic tissue.CASE SUMMARY The results of examination of two patients with pancreatic cancer,who gave informed consent for participation and publication,were the source for this study.Besides standards of care,special examination to reveal occult HBV infection was performed.This included blood tests for HBsAg,anti-HBc,anti-HBs,HBV DNA,and pancreatic tissue examinations with polymerase chain reaction for HBV DNA,pregenomic HBV RNA(pgRNA HBV),and covalently closed circular DNA HBV(cccDNA)and immunohistochemistry staining for HBxAg and Ki-67.Both subjects were operated on due to pancreatic ductal adenocarcinoma and serum HBsAg was not detected.However,in both of them anti-HBc antibodies were detected in blood,although HBV DNA was not found.Examination of the resected pancreatic tissue gave positive results for HBV DNA,expression of HBx,and active cellular proliferation by Ki-67 index in both cases.However,HBV pgRNA and cccDNA were detected only in case 1.CONCLUSION These cases may reflect potential involvement of HBV infection in the development of pancreatic cancer.

Establishment and characterization of four human hepatocellular carcinoma cell lines containing hepatitis B virus DNA

AIM To investigate the characteristics of newly established four hepatocellular carcinoma cell lines (SNU 739, SNU 761, SNU 878 and SNU 886) from Korean hepatocellular cancer patients. METHODS Morphologic and genetic studies were done. RESULTS All four lines grew as a monolayer with an adherent pattern, and their doubling times ranged from 20 to 29 hours. The viability rate was relatively high (88%-94%). Neither mycoplasmal nor bacterial contamination was present. The lines showed different patterns in fingerprinting analysis. The hepatitis B virus (HBV) DNA was integrated in the genomes of all four lines, and in all of them HBx, HBc and HBs transcripts were detected by reverse transcriptase PCR methods. Among the three cell lines used as control (Hep 3B, SK Hep1 and Hep G2), only Hep 3B showed HBx expression, and this line was used as a HBV integrated control. The RNA of albumin was detected in three lines (SNU 761, SNU 878 and SNU 886), that of transferrin in two lines (SNU 878, SNU 886), and that of IGF Ⅱ was detected in none of the cell lines. CONCLUSION These well characterized cell lines may be very useful for studying the biology of hepatocellular carcinoma in association with the hepatitis B virus.

Hepatitis B virus reactivation during immunosuppressive therapy: Appropriate risk stratification

Our understanding of hepatitis B virus(HBV) reactivation during immunosuppresive therapy has increased remarkably during recent years. HBV reactivation in hepatitis B surface antigen(HBs Ag)-positive individuals has been well-described in certain immunosuppressive regimens, including therapies containing corticosteroids, anthracyclines, rituximab, antibody to tumor necrosisfactor(anti-TNF) and hematopoietic stem cell transplantation(HSCT). HBV reactivation could also occur in HBs Ag-negative, antibody to hepatitis B core antigen(anti-HBc) positive individuals during therapies containing rituximab, anti-TNF or HSCT.For HBs Ag-positive patients, prophylactic antiviral therapy is proven to the effective in preventing HBV reactivation. Recent evidence also demonstrated entecavir to be more effective than lamivudine in this aspect. For HBs Ag-negative, antiHBc positive individuals, the risk of reactivations differs with the type of immunosuppression. For rituximab, a prospective study demonstrated the 2-year cumulative risk of reactivation to be 41.5%, but prospective data is still lacking for other immunosupressive regimes. The optimal management in preventing HBV reactivation would involve appropriate risk stratification for different immunosuppressive regimes in both HBs Ag-positive and HBs Ag-negative, anti-HBc positive individuals.

Establishment of transgenic mouse harboring hepatitis B virus (adr subtype) genomes

INTRODUCTIONHepatitis B virus (HBV) belongs to the group ofhepatovirus, a major pathogen of human acute andchronic hepatitis B[1 4], which has a very closeassociation with human hepatocellular carcinoma(HCC)[5-8], For example, a statistical data from ahospital in Shanghai showed that 80% of HCCpatients were positive for HBsAg ( personalcommunication).

Differential reactivity of mouse monoclonal anti-HBs antibodies with recombinant mutant HBs antigens

AIM: To investigate the reactivity of a panel of 8 mouse anti-hepatitis B surface antigen (HBsAg) monoclonal antibodies (mAbs) using a collection of 9 recombinant HBsAg mutants with a variety of amino acid substitutions mostly located within the “a” region.METHODS: The entire HBs genes previously cloned into a mammalian expression vector were transiently transfected into COS7 cells. Two standard unmutated sequences of the ayw and adw subtypes served as controls. Secreted mutant proteins were collected and measured by three commercial diagnostic immunoassays to assess transfection efficiency. Reactivity of anti-HBs mAbs with mutated HBsAgs was determined by sandwich enzyme-linked immunosorbent assay (ELISA).RESULTS: Reactivity of anti-HBs mAbs with mutated HBsAgs revealed different patterns. While three mutants reacted strongly with all mAbs, two mutants reacted weakly with only two mAbs and the remaining proteins displayed variable degrees of reactivity towards different mAbs. Accordingly, four groups of mAbs with different but overlapping reactivity patterns could be envisaged. One group consisting of two mAbs (37C5-S7 and 35C6-S11) was found to recognize stable linear epitopes conserved in all mutants. Mutations outside the “a” determinant at positions 120 (P→S), 123(T→N) and 161(M→T) were found to affect reactivity of these mAbs.CONCLUSION: Our findings could have important implications for biophysical studies, vaccination strategies and immunotherapy of hepatitis B virus (HBV) mutants.

“Anti-HBc alone” in human immunodefi ciency virus-positive and immuno-suppressed lymphoma patients

Hepatitis B virus (HBV) infection is endemic in various parts of the world. A proportion of patients have resolved prior exposure to HBV, as evidenced by the clearance of circulating hepatitis B surface antigen and the appearance of antibody to hepatitis B core antigen (anti-HBc), which could produce protective antibody to hepatitis B surface antigen (anti-HBs). With time, anti-HBs in some patients may become negative. Such patients are described as having occult HBV infection or "anti-HBc alone". In the context of immunodef icient patients, such as HIV patients or lymphoma patients undergoing immunosuppressive immunotherapy, the lack of protective anti-HBs may increase the risk of hepatitis B reactivation. Serum HBV DNA testing may be necessary in "anti-HBc alone" patients, to detect patients at a high risk of developing HBV infection allowing appropriate prophylactic management.

A comparative study on serologic profiles of virus hepatitis B

INTRODUCTIONHepatitis B viral infection, one of the most-prevalent liver disorders in China and Korea, is aserious infectious disease as it has the potential ofprogressing into liver cirrhosis and primary hepaticcarcinoma. China and Korea both belong to high-risk endemic regions of viral hepatitis[1]. TheHBsAg positive rates in China ranged from 6.9% -17.9% by age, race and test methods[2-5].

Association of core promoter mutations of hepatitis B virus and viral load is different in HBeAg(+) and HBeAg(-) patients

AIM:To identify the prevalence of hepatitis B e antigen (HBeAg) and to assess the association of hepatitis B virus (HBV) core promoter mutations and viral load in Indonesian patients.METHODS:Sixty-four patients with chronic hepatitis,65 with liver cirrhosis and 50 with hepatocellular carcinoma were included in this study.HBeAg and hepatitis B e antibody (HBeAb) tests were performed using enzyme-linked immunosorbent assay and the mutations were analyzed by sequencing.Viral load was measured by real-time polymerase chain reaction.RESULTS:Of 179 patients,108 (60.3%) were HBeAg(-) and 86 (79.6%) of these HBeAg(-) patients had been seroconverted.The A1896 mutation was not found in HBeAg(+) patients,however,this mutation was detected in 70.7% of HBeAg(-) patients.This mutation was frequently found when HBeAg was not expressed (87.7%),compared to that found in HBeAg seroconverted patients (65.1%).The A1899 mutation was also more prevalent in HBeAg(-) than in HBeAg(+) patients (P=0.004).The T1762/A1764 mutation was frequently found in both HBeAg(+) and HBeAg(-) patients,however,the prevalence of this mutation did not significantly differ among the two groups (P=0.054).In HBeAg(+) patients,the T1762/A1764 mutation was correlated with lower HBV DNA (P < 0.001).The A1899 mutation did not correlate with HBV DNA (P=0.609).In HBeAg(-) patients,the T1762/A1764 mutation alone was not correlated with HBV DNA (P=0.095),however,the presence of either the T1762/A1764 or A1896 mutations was associated with increased HBV DNA (P < 0.001).CONCLUSION:The percentage of HBeAg(-) patients is high in Indonesia,and most of the HBeAg(-) patients had been seroconverted.The A1896 mutation was most likely the major cause of HBeAg loss.The T1762/A1764 mutation alone was associated with lower viral loads in HBeAg(+) patients,but not in HBeAg(-) patients.

Relationship between hepatitis B virus associated primary hepatocellular carcinoma and alteration of tumor suppressor gene p53

Objective: To explore the changes and significance of tumor suppressor gene p53 in primary hepatocellu-lar carcinoma (PHC ) with hepatitis B virus (HBV ) infection. Methods: Tumor tissues and surrounding nontumortissues of sixteen PHC cases were studied by Southern hybridization to detect the state of HBV-DNA in tissues, byimmunohistochemical staining to determine HBsAg, HBxAg and p53 protein, and by PCR directed sequencing toanalyse the point mutation of p53 gene exons 5 to 8. Results: Among the 16 cases. 13 cases were HBV-DNA posi-tive, 10 tumor cases and 13 nontumor tissues cases HBxAg positive, and 9 cases posltive for p53 protein. The se-quencing of p53 gene point mutation was found in 5 cases, only one of which was sited at codon 249 G to T. Con-clusion: The mutation of p53 gene codon 249 is infrequent in HBV related PHC,indicating the accumulation of p53protein in cells may be associated with expression of HBxAg. HBxAg binding to p53 protein and inactivation of p53function play important roles in the development of PHC.

乙型肝炎病毒x抗原在HBV宫内感染中的作用机制

目的研究高复制水平HBV血清感染人绒毛膜滋养细胞株JEG-3后细胞内乙型肝炎病毒x抗原(HBxAg)及其下游蛋白在细胞内的表达情况,探讨HBxAg在HBV宫内感染中的作用。方法对人早孕绒毛膜滋养细胞株JEG-3进行传代培养,将高复制水平乙型肝炎病毒血清(HBV-DNA定量为107～108拷贝/mL)与传代培养的JEG-3细胞共同孵育48～72h;逆转录-聚合酶链式反应检测细胞内HBx-mRNA水平;Western blot检测细胞内HBxAg的表达;免疫组化检测细胞内HBxAg表达以及细胞内磷脂酰肌醇激酶3(PI3K)的表达;流式细胞仪检测细胞内pAKT的表达。结果 HBV感染JEG-3细胞后,在细胞中检测到HBx-mRNA,并检测到HBxAg的表达,同时发现细胞内PI3K及pAKT的表达水平明显增加,经统计学分析,差异有统计学意义(P<0.05)。结论 HBxAg/PI3K/pAKT抗细胞凋亡途径是高复制水平HBV宫内感染的可能机制。

HBx和癌胚抗原相关细胞黏附分子1在乙型肝炎相关性肝癌中的作用及病理特征

目的探讨HBx和癌胚抗原相关细胞黏附分子1(CEACAM1)在乙型肝炎相关性肝癌中的表达及意义。方法采用免疫组织化学方法(SP法)检测81份乙肝相关性肝癌组织中HBx及CEACAM1的表达情况,分析HBx和CEACAM1与乙肝相关性肝癌的临床病理特征。Western印迹检测人正常肝细胞系QZG、肝癌细胞HepG2及稳定转染HBx的肝癌细胞HepG_2(HepG_2-X)中CEACAM1的表达。结果 81份乙肝相关性肝癌组织中HBx表达阳性率为74.07%(60/81),CEACAM1表达阳性率为71.60%(58/81),二者与门静脉侵袭、淋巴结转移和TNM分期存在显著相关,HBx与CEACAM1的表达呈负相关(r_s=-0.310,P<0.01);在HepG_2-X中,CEACAM1蛋白表达水平与肝癌细胞HepG_2及人正常肝细胞系QZG相比显著降低。结论 HBx的高表达和CEACAM1的低表达与乙肝相关性肝癌的侵袭、转移密切相关,HBx有可能通过抑制CEACAM1的表达而诱导乙肝相关性肝癌的侵袭与转移。

乙型肝炎病毒X基因的原核表达及血清抗HBx抗体的检测

目的应用表达蛋白检测乙型肝炎患者血清抗-HBx抗体水平并探讨其临床意义。方法通过PCR扩增获得HBV X基因,与原核表达载体Pet32a+连接构建PET32a-HBX原核表达载体,转化E.coliBL21表达获得重组融合蛋白。经切胶透析纯化后,应用重组蛋白HBx建立检测血清中抗-HBx抗体的间接ELISA方法,分别检测正常人组、急性肝炎组、慢性肝炎组、肝硬化组和肝细胞癌组患者血清中的抗-HBx抗体。结果获得具有免疫原性的HBx融合蛋白;ELISA检测表明,慢性肝炎组、肝硬化组和肝细胞癌组的抗-HBx抗体的水平均高于急性肝炎组,差异具有显著性;在三组之间,慢性肝炎组高于肝硬化组和肝细胞癌组,差异具有显著性,肝硬化组和肝细胞癌组的抗-HBx抗体水平无显著性差异。结论HBV患者血清中抗-HBX抗体是乙型肝炎病毒感染的一种特异性抗体,是HBV感染的血清学指标之一,可以反映乙型肝炎肝炎患者病情的变化。

肝细胞癌组织内HBxAg的表达及其与p21ras的关系

应用免疫组织化学技术ＡＢＣ与ＰＡＰ法检测６６例肝细胞癌（其中５７例含有癌周组织）与１５例肝硬化组织内ＨＢｘＡｇ，ＨＢｓＡｇ，ＨＢｃＡｇ及ｐ２１ｒａｓ的表达，分析了ＨＢｘＡｇ与ｐ２１的关系。结果：ＨＢｘＡｇ在癌周与癌组织内均有高频率表达，癌内ＨＢｘＡｇ阳性组ｐ２１的阳性率显著高于阴性组（Ｐ＜０．０１），但癌周组织ＨＢｘＡｇ阳性组与阴性组间ｐ２１阳性率无显著差异。提示：ＨＢｘＡｇ与肝细胞癌变有密切关系，在控制、调节ｒａｓ癌基因表达上起重要作用。

乙型肝炎病毒X蛋白截断变异对HBeAg含量和HBV DNA定量的影响

目的研究乙型肝炎病毒(HBV)X蛋白截断变异对HBeAg含量和HBV DNA定量的影响。方法通过DNA扩增、基因序列分析检测75例慢性乙肝和14例慢性重型乙肝和34例肝硬化病人血清的HBV X基因和前C基因序列,通过微粒子发光法定量检测血清中HBeAg的含量,以及通过荧光定量PCR技术定量检测血清中的HBVDNA。结果(1)9例检出X蛋白截断变异。(2)同无变异组比较,X蛋白截断变异组、CP双变异(nt 1762A→T,1764G→A)组和前C基因终止变异(nt 1896G→A)组的HBeAg含量均显著下降,其中前C终止变异组HBeAg血清转换最为明显。(3)同无变异组比较,X蛋白截断变异组的HBV DNA含量无明显差异。结论X蛋白截断变异影响HBeAg表达,但影响程度不及前C基因终止变异;X蛋白截断变异对HBV DNA定量无明显影响。

乙型肝炎患者血清抗-HBx抗体的检测及临床意义研究

目的获得重组HBx蛋白,检测乙型肝炎患者血清抗HBx抗体并探讨其临床意义。方法利用分子克隆技术克隆HBVX基因构建PET32aHBX原核表达载体,表达和纯化HBx蛋白,用该重组蛋白建立检测抗HBx抗体的间接ELISA方法;采用该ELISA分别检测正常人组、急性肝炎组、慢性肝炎组、肝硬化组和肝细胞癌组患者血清中的抗HBx抗体。结果获得具有免疫原性的HBx融合蛋白;ELISA检测表明,慢性肝炎组、肝硬化组和肝细胞癌组的抗HBx抗体的水平均高于急性肝炎组,差异具有显著性;在这三组之间,慢性肝炎组高于肝硬化组和肝细胞癌组,差异具有显著性,肝硬化组和肝细胞癌组的抗HBx抗体水平无显著性差异。结论HBV患者血清中抗HBX抗体是乙型肝炎病毒感染的一种特异性抗体,是HBV感染的血清学指标之一,可以反映乙型肝炎肝炎患者病情的变化。