Novel therapeutic approaches for hepatitis B virus covalently closed circular DNA

Hepatitis B virus(HBV) infection is a major global health problem. Although current therapies, such as the use of nucleos(t)ide analogs, inhibit HBV replication efficiently, they do not eliminate covalently closed circular DNA(ccc DNA), which persists in hepatocyte nuclei. As HBV ccc DNA is a viral transcription template, novel therapeutic approaches to directly target HBV ccc DNA are necessary to completely eradicate persistent HBV infections. HBV ccc DNA levels in HBV-infected human liver cells are extremely low; thus, more reliable and simple measurement methods are needed to correctly monitor their levels during therapeutic treatment. Although reverse transcription-polymerase chain reaction or Southern blot procedures are currently used in research studies, these methods are not completely reliable and are also time-consuming and labor-intensive. Genome editing technologies, such as zinc finger nucleases, transcription activator-like effector nucleases, and the clustered regularly interspaced short palindromic repeats/Cas9(CRISPR/Cas9) system, which are designed to target specific DNA sequences, represent highly promising potential therapeutic tools. In particular, the CRISPR/Cas9 system is an easily customizable sequencespecific nuclease with high flexibility and may be the most feasible approach to target HBV ccc DNA. Further research to develop easier, safer, and more effective protocols should be pursued.

Nested real-time quantitative polymerase chain reaction assay for detection of hepatitis B virus covalently closed circular DNA

Background Successful treatment of hepatitis B can be achieved only if the template for hepatitis B virus （HBV） DNA replication, the covalently closed circular HBV DNA （cccDNA） can be completely cleared. To date, detecting cccDNA remains clinically challenging. The purpose of this study was to develop a nested real-time quantitative polymerase chain reaction （PCR） assay for detecting HBV cccDNA in peripheral blood mononuclear cells （PBMCs） and bone marrow mononuclear cells （MMNCs）. Methods Based on the structural differences between HBV cccDNA and HBV relaxed circular DNA （rcDNA）, two pairs of primers were synthesized as well as a downstream TaqMan probe. Blood and bone marrow samples were collected from hepatitis B patients and healthy controls. To remove rcDNA, samples were incubated with mung bean nuclease and the resultant purified HBV cccDNA was then amplified by nested real-time fluorescence quantitative PCR. The cccDNA levels were calculated using a positive standard. Results The nested real-time fluorescence quantitative PCR method for HBV cccDNA was successful, with a linear range of 3.0×10^2 copies/ml to 3.9×10^8 copies/ml. Of the 25 PBMC samples and 7 MMNC samples obtained from chronic hepatitis B or liver cirrhosis patients, 3 MMNC samples and 9 PBMC samples were positive for HBV cccDNA, while all of the 21 PBMC samples from healthy controls were negative. Conclusion The nested real-time fluorescence quantitative PCR may be used as an important tool for detecting cccDNA in hepatitis B patients.

Recent advances in the study of hepatitis B virus covalently closed circular DNA

Chronic hepatitis B infection is caused by hepatitis B virus(HBV) and a total cure is yet to be achieved. The viral covalently closed circular DNA(ccc DNA) is the key to establish a persistent infection within hepatocytes. Current antiviral strategies have no effect on the pre-existing ccc DNA reservoir. Therefore, the study of the molecular mechanism of ccc DNA formation is becoming a major focus of HBV research. This review summarizes the current advances in ccc DNA molecular biology and the latest studies on the elimination or inactivation of ccc DNA, including three major areas:(1) epigenetic regulation of ccc DNA by HBV X protein,(2) immune-mediated degradation,and(3) genome-editing nucleases. All these aspects provide clues on how to finally attain a cure for chronic hepatitis B infection.

Role of hepatitis B virus in development of hepatocellular carcinoma:Focus on covalently closed circular DNA

Chronic infection with hepatitis B virus(HBV)remains a major global health problem,especially in developing countries.It may lead to prolonged liver damage,fibrosis,cirrhosis,and hepatocellular carcinoma.Persistent chronic HBV infection is related to host immune response and the stability of the covalently closed circular DNA(cccDNA)in human hepatocytes.In addition to being essential for viral transcription and replication,cccDNA is also suspected to play a role in persistent HBV infections or hepatitis relapses since cccDNA is very stable in non-dividing human hepatocytes.Understanding the pathogenicity and oncogenicity of HBV components would be essential in the development of new diagnostic tools and treatment strategies.This review summarizes the role and molecular mechanisms of HBV cccDNA in hepatocyte transformation and hepatocarcinogenesis and current efforts to its detection and targeting.

Detection of the covalently closed circular DNA of duck hepatitis B virus by Taq-Man fluorescent quantitative PCR assay

To develop a fluorescent quantitative PCR assay based on Taq-Man chemistry to detect the covalenfly closed circular DNA （eccDNA） of duck hepatitis B virus （DHBV）, a pair of primers was designed from both sides of the nick in the minus strand of DHBV and a Taq-Man probes between the primers, modified with 6-Fam at 5＇ end and Tamra at its 3＇ end was designed to detect the PCR products during PCR cycles. The DHBV DNA fragment was cloned into vector PUCm-T, and the recombinant plasmid was purified and subsequently qualified as the HBV DNA standard. The experimental conditions and reagents used in PCR assay for amplification were sophisticatedly optimized in order to yield a perfect amplification efficacy and reduce the possibility to produce non-specific amplification. It was demonstrated that the detect limit of assay was 10^3 copies/ml, and a linear standard curve was obtained between 10^5 -10^9 copies/ml [ C1 =-2.8361 ln（x） ＋ 41.45, r =-0.9985]. The coefficient of variation was 0.2%-3.14% and 2.22%-4.43% for intra- and inter-assay respectively. After a dynamic survey on the contents of DHBV DNA in serum of ducks, it was found that its peak value appeared at the second week of birth in ducks. It is evident that this method of Taq-Man fluorescent quantitative PCR assay appears to be simple, sensitive and specific.

Intracellular interferon signalling pathways as potential regulators of covalently closed circular DNA in the treatment of chronic hepatitis B

250 million people worldwide continue to be chronically infected with the virus.While patients may be treated with nucleoside/nucleotide analogues,this only suppresses HBV titre to sub-detection levels without eliminating the persistent HBV covalently closed circular DNA(cccDNA)genome.As a result,HBV infection cannot be cured,and the virus reactivates when conditions are favorable.Interferons(IFNs)are cytokines known to induce powerful antiviral mechanisms that clear viruses from infected cells.They have been shown to induce cccDNA clearance,but their use in the treatment of HBV infection is limited as HBVtargeting immune cells are exhausted and HBV has evolved multiple mechanisms to evade and suppress IFN signalling.Thus,to fully utilize IFN-mediated intracellular mechanisms to effectively eliminate HBV,instead of direct IFN administration,novel strategies to sustain IFN-mediated anti-cccDNA and antiviral mechanisms need to be developed.This review will consolidate what is known about how IFNs act to achieve its intracellular antiviral effects and highlight the critical interferon-stimulated gene targets and effector mechanisms with potent anti-cccDNA functions.These include cccDNA degradation by APOBECs and cccDNA silencing and transcription repression by epigenetic modifications.In addition,the mechanisms that HBV employs to disrupt IFN signalling will be discussed.Drugs that have been developed or are in the pipeline for components of the IFN signalling pathway and HBV targets that detract IFN signalling mechanisms will also be identified and discussed for utility in the treatment of HBV infections.Together,these will provide useful insights into design strategies that specifically target cccDNA for the eradication of HBV.

Effects of antiviral agents and HBV genotypes on intrahepaticcovalently closed circular DNA in HBeAg-positive chronichepatitis B patients

AIM： To evaluate the effects of antiviral agents and HBV genotypes on intrahepatic covalently closed circular DNA （ccc DNA） in HBeAg-positive chronic hepatitis B patients.METHODS： Seventy-one patients received lamivudine （n = 35）, or sequential therapy with lamivudine- interferon alpha 2b （IFN-α 2b, n = 24） for 48 wk, or IFN-α 2b （n = 12） for 24 wk. All subjects were followed up for 24 wk. Intrahepatic ccc DNA was measured quantitatively by PCR. HBV genotypes were analyzed by PCR-RFLP.RESULTS： Sequential lamivudine- INF-α therapy, lamivudine and INF-α monotherapy reduced ccc DNA of 1.7 log, 1.4 log and 0.8 log, respectively （P 〈 0.05）. Seventeen out of the 71 patieots developed HBeAg seroconversion, the reduction of ccc DNA in the HBeAg seroconversion patients was more significant than that in the HBeAg positive patients （3.0 log vs 1.6 log, P = 0.0407）. Twenty-four weeks after antiviral therapy withdrawal, 16 patients had a sustained virological response, the baseline intrahepatic ccc DNA in the patients with a sustained virological response was significantly lower than that in the patients with virological rebound （4.6 log vs 5.4 log, P = 0.0472）. HBV genotype C accounted for 85.9% （n = 61）, and genotype B for 14.1% （n = 10）, respectively, in the 71 patients. There was no significant difference in the change of ccc DNA level between HBV genotypes C and B （2.1 log vs 1.9 log）.CONCLUSION： Forty-eight week sequential lamivudine- INF-α therapy and lamivudine monotherapy reduce ccc DNA more significantly than 24-wk INF-α monotherapy. Low baseline intrahepatic ccc DNA level may predict the long-term efficacy of antiviral treatment. HBV genotypes C and B have no obvious influence on ccc DNA load.

无锡地区HBsAg-/HBV DNA+献血人群HBcrAg检出特点分析

目的分析新型血清标志物乙型肝炎核心相关抗原(HBcrAg)在无锡地区HBsAg-/HBV DNA+献血人群中的检出特点。方法通过电话追踪随访了37名既往HBsAg-/HBV DNA+献血者并获得其血清,采用电化学发光法和实时荧光定量PCR核酸筛检出22例HBsAg-/HBV DNA+献血者血清作为OBI组进行HBcrAg酶联免疫吸附法检测。挑选出20名经2遍酶免和1遍核酸筛检的健康献血者的血清作为健康对照组,20例经无锡第五人民医院临床诊断为慢性乙型肝炎患者血清作为实验的CHB组,分别进行HBcrAg酶联免疫吸附法检测;并对OBI组进行HBcrAg与HBeAb、HBcAb、ALT、HBV DNA的相关性分析。结果37份献血者标本经化学发光法检测HBsAg和核酸筛查,检出22份HBsAg-/HBV DNA+标本即OBI组,检出率59.46%。OBI组与健康对照组、CHB组血清的HBcrAg表达含量分别是(0.92±0.13)ng/mL、(0.47±0.09)ng/mL、(1.14±0.23)ng/mL(P<0.05),OBI组与CHB组的HBcrAg表达均高于健康对照组(P<0.05)。OBI组的HBcrAg与HBeAb、HBcAb、ALT、HBV DNA指标均无相关性(P>0.05)。结论OBI组与CHB组的HBcrAg表达均高于健康对照组,其血清HBcrAg在一定程度上与HBeAb、HBcAb、ALT、HBV DNA无相关性,HBcrAg在筛查HBsAg-/HBV DNA+献血者中具有较好的应用前景。

HBV pgRNA联合cccDNA对慢性乙型肝炎患者抗病毒疗效的预测价值

目的探究乙型肝炎(HBV)前基因组RNA(pgRNA)联合共价闭合环状DNA(cccDNA)对慢性乙型肝炎(CHB)患者抗病毒疗效的预测价值。方法收集2019年8月至2022年8月期间于本院进行抗病毒治疗的96例CHB患者的临床资料,根据患者治疗48周后是否获得完全应答分为完全应答组(78例)及非完全应答组(15例)。检测患者不同时间HBV cccDNA和HBV pgRNA水平,Logistic回归分析影响CHB患者获得完全应答的因素,并应用受试者工作特征(ROC)曲线分析pgRNA与cccDNA联合检测在抗病毒疗效的预测价值。结果96例患者中78例抗病毒治疗后获得非完全应答;完全应答组治疗24、48周HBV pgRNA、HBV cccDNA水平均低于非完全应答组(P<0.05);Logistic回归分析显示,治疗24周HBV pgRNA、HBV cccDNA高水平及治疗前ALT低水平是影响CHB患者抗病毒治疗无效的独立危险因素(P<0.05);经ROC分析显示,HBV pgRNA预测CHB患者抗病毒疗效的AUC值为0.618,95%CI为0.513~0.716,HBV cccDNA预测的AUC值为0.667,95%CI为0.561~0.760,二者联合预测的AUC值为0.881,95%CI为0.799~0.938。结论HBV pgRNA与cccDNA在CHB患者抗病毒治疗中下调,且治疗24周HBV pgRNA联合cccDNA检测对CHB抗病毒疗效具有更高的预测价值。

NS5ATP9与HBx相互作用促进HBV cccDNA的形成与转录

目的探讨丙型肝炎病毒NS5A反式调节蛋白9(hepatitis C virus NS5Atransactivated protein 9,NS5ATP9)在乙型肝炎病毒(hepatitis B virus,HBV)共价闭合环状DNA(covalently closed circular DNA,cccDNA)形成与转录中的作用机制。方法利用1.3拷贝HBV表达质粒转染Huh7和HepG2细胞、整合有4拷贝HBV基因组的HepG2.2.15细胞、在诱导型四环素启动子控制下表达HBV的HepAD38细胞构建NS5ATP9过表达或干扰的HBV细胞模型,收集样品和细胞上清液,提取RNA、HBV核心DNA(coreDNA)、cccDNA和蛋白,利用酶联免疫吸附试验、实时荧光定量聚合酶链反应(polymerase chain reaction,PCR)、Southern blot和Western blot技术检测HBV总RNA、前基因组RNA(pregenomic RNA,pgRNA)、乙型肝炎病毒s抗原(hepatitis B virus s antigene,HBsAg)、乙型肝炎病毒e抗原(hepatitis B virus e antigene,HBeAg)、松弛环状DNA(relax circular DNA,rcDNA)以及cccDNA水平。在HepG2细胞中转染乙型肝炎病毒x蛋白(hepatitis B virus x protein,HBx),通过免疫荧光成像及免疫共沉淀方法检测NS5ATP9与HBx的结合情况。双荧光素酶报告基因实验检测NS5ATP9对HBx启动子活性的影响。利用Huh7细胞转染HBV1.3及HBV稳定表达细胞株HepG2.2.15和HepAD38转染NS5ATP9过表达/干扰质粒,通过Western blot技术检测DDB1和SMC6的蛋白水平。结果在HBV病毒活跃的细胞中,NS5ATP9 mRNA水平[HepG2.2.15细胞:1.891±0.567比1.00±0.034,t=2.87,P=0.0351;HepAD38 tet+细胞:1.978±0.399比1.00±0.034,t=4.131,P=0.0091;HepAD38 tet-细胞:2.642±0.672比1.00±0.034,t=4.127,P=0.0091]和蛋白水平均显著增加。过表达NS5ATP9后可显著增加HBeAg[(5.402±0.327)S/COV比(2.68±0.552)S/COV,t=7.35,P=0.0018]、HBsAg[(2.846±0.185)S/COV比(1.512±0.221)S/COV,t=8.02,P=0.0013]、HBV pgRNA及rcDNA的表达水平,而干扰NS5ATP9后此增加作用消失[HBeAg:(2.029±0.09)S/COV比(3.733±0.445)S/COV,t=6.501,P=0.0029;HBsAg:(1.501±0.105)S/COV比(1.878±0.174)S/COV,t=3.216,P=0.0324)]。机制研究显示,NS5ATP9和HBx蛋白主要位于细胞核核仁内,并具有共定位信号,且NS5ATP9可显著提高HBx启动子(1071.06±79.44比488.47±40.12,t=13.09,P=0.00012)的转录活性。另外,过表达NS5ATP9可显著降低DDB1和SMC6的蛋白水平,而沉默NS5ATP9则可显著提高DDB1和SMC6的蛋白水平。结论HBV上调NS5ATP9的表达,形成HBV-NS5ATP9-HBV cccDNA-HBV的正反馈环路,NS5ATP9通过与HBx相互作用上调肝细胞中HBV cccDNA的形成与转录,进而促进慢性乙型肝炎的发生发展。

Curcumin inhibits hepatitis B virus infection by downregulating ccc DNA-bound histone acetylation

AIM To investigate the potential effect of curcumin on hepatitis B virus(HBV) covalently closed circular DNA(ccc DNA) and the underlying mechanism.METHODS A Hep G2.2.15 cell line stably transfected with HBV was treated with curcumin, and HBV surface antigen(HBs Ag) and e antigen(HBe Ag) expression levels were assessed by ELISA. Intracellular HBV DNA replication intermediates and ccc DNA were detected by Southern blot and real-time PCR, respectively. The acetylation levels of histones H3 and H4 were measured by Western blot. H3/H4-bound ccc DNA was detected by chromatin immunoprecipitation(Ch IP) assays. The deacetylase inhibitors trichostatin A and sodium butyrate were used to study the mechanism of action for curcumin. Additionally, short interfering RNAs(si RNAs) targeting HBV were tested along with curcumin.RESULTS Curcumin treatment led to time-and dose-dependent reductions in HBs Ag and HBe Ag expression and significant reductions in intracellular HBV DNA replication intermediates and HBV ccc DNA. After treatment with 20 μmol/L curcumin for 2 d, HBs Ag and ccc DNA levels in Hep G2.2.15 cells were reduced by up to 57.7%(P < 0.01) and 75.5%(P < 0.01), respectively, compared with levels in non-treated cells. Meanwhile, time-and dose-dependent reductions in the histone H3 acetylation levels were also detected upon treatment with curcumin, accompanied by reductions in H3-and H4-bound ccc DNA. Furthermore, the deacetylase inhibitors trichostatin A and sodium butyrate could block the effects of curcumin. Additionally, transfection of si RNAs targeting HBV enhanced the inhibitory effects of curcumin.CONCLUSION Curcumin inhibits HBV gene replication via downregulation of ccc DNA-bound histone acetylation and has the potential to be developed as a ccc DNA-targeting antiviral agent for hepatitis B.

Combination of small interfering RNAs mediates greater suppression on hepatitis B virus cccDNA in HepG2.2.15 cells

AIM： To observe the inhibition of hepatitis B virus （HBV） replication and expression in HepG2.2.15 cells by combination of small interfering RNAs （siRNAs）. METHODS： Recombinant plasmid psiI-HBV was constructed and transfected into HepG2.2.15 cells. At 48 h, 72 h and 96 h after transfection, culture media were collected and cells were harvested for HBV replication assay. HBsAg and HBeAg in the cell culture medium were detected by enzyme-linked immunoadsorbent assay （ELISA）. Intracellular viral DNA and covalently closed circular DNA （cccDNA） were quantified by real-time polymerase chain reaction （PCR）. HBV viral mRNA was reverse transcribed and quantified by reverse-transcript PCR （RT-PCR）. RESULTS： siRNAs showed marked anti-HBV effects. siRNAs could specifically inhibit the expression of HBsAg and the replication of HBV DNA in a dosedependent manner. Furthermore, combination of siRNAs, compared with individual use of each siRNA, exerted a stronger inhibition on antigen expression and viral replication. More importantlycombination of siRNAs significantly suppressed HBV cccDNA amplification. CONCLUSION： Combination of siRNAs mediates a stronger inhibition on viral replication and antigenexpression in HepG2.2.15 cells, especially on cccDNA amplification.

Effect of oxymatrine on the replication cycle of hepatitis B virus in vitro

AIM:To determine the antiviral mechanism or target of oxymatrine against hepatitis B virus(HBV).METHODS:HepG2.2.15 cells were incubated with culture medium containing 500 μg/mL of oxymatrine for 2 and 5 d.The surface antigen of HBV(HBsAg) and e antigen of HBV(HBeAg) in supernatant were determined by ELISA.HBV DNA in supernatant,and intracellular covalently closed circular DNA(cccDNA),relaxed circular DNA(rcDNA) and pregenomic RNA(pgRNA) were quantif ied by specif ic real-time polymerase chain reaction(PCR) or reverse transcription(RT)-PCR.RESULTS:Treatment with oxymatrine for 2 d and 5 d reduced the production of HBV by the cell line,as indicated by the decline of HBsAg(22.67%,t = 5.439,P = 0.0322 and 22.39%,t = 5.376,P = 0.0329,respectively),HBeAg(55.34%,t = 9.859,P = 0.0101 and 43.97%,t = 14.080,P = 0.0050) and HBV DNA(40.75%,t = 4.570,P = 0.0447 and 75.32%,t = 14.460,P = 0.0047) in the supernatant.Intracellular cccDNA was also markedly reduced by 63.98%(t = 6.152,P = 0.0254) and 80.83%(t = 10.270,P = 0.0093),and intracellular rcDNA by 34.35%(t = 4.776,P = 0.0413) and 39.24%(t = 10.050,P = 0.0097).In contrast,intracellular pgRNA increased by 6.90-fold(t = 8.941,P = 0.0123) and 3.18-fold(t = 7.432,P = 0.0176) after 500 μg/mL of oxymatrine treatment for 2 d and 5 d,respectively.CONCLUSION:Oxymatrine may inhibit the replication of HBV by interfering with the process of packaging pgRNA into the nucleocapsid,or inhibiting the activity of the viral DNA polymerase.

Roles of hepatocyte nuclear factors in hepatitis B virus infection

Approximately 350 million people are estimated to be persistently infected with hepatitis B virus(HBV) worldwide. HBV maintains persistent infection by employing covalently closed circular DNA(ccc DNA), a template for all HBV RNAs. Chronic hepatitis B(CHB) patients are currently treated with nucleos(t)ide analogs such as lamivudine, adefovir, entecavir, and tenofovir. However, these treatments rarely cure CHB because they are unable to inhibit ccc DNA transcription and inhibit only a late stage in the HBV life cycle(the reverse transcription step in the nucleocapsid). Therefore, an understanding of the factors regulating ccc DNA transcription is required to stop this process. Among numerous factors, hepatocyte nuclear factors(HNFs) play the most important roles in ccc DNA transcription, especially in the generation of viral genomic RNA, a template for HBV replication. Therefore, proper control of HNF function could lead to the inhibition of HBV replication. In this review, we summarize and discuss the current understanding of the roles of HNFs in the HBV life cycle and the upstream factors that regulate HNFs. This knowledge will enable the identification of new therapeutic targets to cure CHB.

Occult hepatitis B—the result of the host immune response interaction with different genomic expressions of the virus

With over 40 years of history,occult hepatitis B infection(OBI)continues to remain an important and challenging public health problem.Defined as the presence of replication-competent hepatitis B virus(HBV)DNA(i.e.,episomal HBV covalently closed circular DNA)in the liver and/or HBV DNA in the blood of people who test negative for hepatitis B surface antigen(HBsAg)in currently available assays,OBI is currently diagnosed using polymerase chain reaction(PCR)and real-time PCR assays.However,all efforts should be made to exclude a false negative HBsAg in order to completely follow the definition of OBI.In recent years,significant advances have been made in understanding the HBV lifecycle and the molecular mechanisms that lead to the persistence of the virus in the occult form.These factors are mainly related to the host immune system and,to a smaller proportion,to the virus.Both innate and adaptive immune responses are important in HBV infection management,and epigenetic changes driven by host mechanisms(acetylation,methylation,and microRNA implication)are added to such actions.Although greater genetic variability in the S gene of HBV isolated from OBIs was found compared with overt infection,the mechanisms of OBI are not mainly viral mutations.

Effect of interferon-γ and tumor necrosis factor-α on hepatitis B virus following lamivudine treatment

AIM： To evaluate anti-hepatitis B virus （HBV） activity and cytotoxicity of interferon-α, （IFN-3,） and tumor ne- crosis factor-α （TNF-α） following lamivudine treatment of HepG2.2.15 cells. METHODS： HepG2.2.15 cells were treated with 2 pmol/L lamivudine for 16 d （lamivudine group）, cultured for 10 d, followed by 5 ng/ml TNF-α and 1000 U/mL IFN-γ, for 6 d （cytokine group）, or treated with 2 ~tmol/L lami- vudine for 10 d followed by 5 ng/mL TNF-α and 1000 U/mL IFN-γ, for 6 d （sequential group）, or cultured without additions for 16 d （control group）. Intracellular DNA was extracted from 3 ×10^ HepG2.2.15 cells from each group. The extracted DNA was further purified with mung bean nuclease to remove HBV relaxed circu- lar DNA that may have remained. Both HBV covalently closed circular DNA （cccDNA） and HBV DNA were exam- ined with real-time polymerase chain reaction. The titers of hepatitis B surface antigen （HBsAg） and hepatitis B e antigen （HBeAg） were quantified with enzyme-linkedimmunosorbent assay. Cell viability was measured with the cell counting kit-8 assay. RESULTS： Compared to lamivudine alone （22.63%±0.12%）, both sequential （51.50% ± 0.17%, P = 0.034） and cytokine treatment （49.66% ± 0.06%, P = 0.041） showed a stronger inhibition of HBV cccDNA; the dif- ference between the.sequential and cytokine groups was not statistically significant （51.50% ± 0.17% vs 49.66% ± 0.06%, P = 0.88）. The sequential group showed less inhibition of HBV DNA replication than the lamivudine group （67.47% ±0.02% vs 82.48% ± 0.05%, P = 0.014）; the difference between the sequen- tial and cytokine groups was not statistically significant （67.47% ± 0.02% vs 57.45% ± 0.07%, P = 0.071）. The levels of HBsAg and HBeAg were significantly de- creased in the sequential treatment group compared to the other groups [HBsAg： 3.48 ± 0.04 （control）, 3.09 ± 0.08 （lamivudine）, 2.55± 0.13 （cytokine）, 2.32 ± 0.08 （sequential）, P = 0.042 for each between-group comparison; HBeAg 3.48 ± 0.01 （control）, 3.08 ± 0.08 （lamivudine）, 2.57 ± 0.15 （cytokine）, 2.34 ± 0.12 （se- quential）, P = 0.048 for each between-group compari- son]. Cell viability in the cytokine group was reduced to 58.03% ± 8.03% compared with control cells （58.03% ± 8.03% vs 100%, P = 0.000）. Lamivudine pretreat- ment significantly reduced IFN-γ, ± TNF-αmediated toxicity of HepG2.2.15 cells [85.82% =1= 5.43% （sequen- tial） vs 58.03% ± 8.03% （cytokine）, P = 0.002]. CONCLUSION： Sequential treatment overcame the lower ability of lamivudine alone to inhibit cccDNA and precluded the aggressive cytotoxicity involving IFN-y and TNF-α by decreasing the viral load.

HBV感染者血清中HBVcccDNA、HBeAg及HBV DNA的关系

探讨HBV感染者血清HBVcccDNA与血清HBV DNA及HBeAg的关系。分别以PCR分子信标技术和ELISA方法对非HBV相关肝炎、HBV健康携带者、急性乙型肝炎(AHB)、慢性乙型肝炎(CHB)、乙肝肝硬化、乙肝患者血清中HBVcccDNA HBV DNA含量及HBeAg进行了检测。HBVcccDNA仅见于HBVDNA阳性血清中;HBeAg阳性组的HBVcccDNA阳性率显著高于HBeAg阴性组(P<0.05):145例HBVDNA阳性患者中,HBVcccDNA阳性组HBVD- NA水平显著高于HBVcccDNA阴性组(P<0.01)。血清HBVcccDNA可能是乙肝病毒在患者体内大量复制的血清标志。

乙型肝炎患者血清中HBV cccDNA与HBV DNA、HBsAg和HBeAg定量关系的分析

目的定量检测乙型肝炎患者血清中乙型肝炎病毒(HBV)共价闭合环状DNA(cccDNA)、HBV DNA、乙型肝炎表面抗原(HBsAg)和乙型肝炎e抗原(HBeAg)并分析其相互关系,探讨定量检测血清中HBV cccDNA的临床应用价值。方法随机选取HBV感染者83例,采用实时荧光定量聚合酶链反应(FQ-PCR)检测血清中HBVcccDNA及HBV DNA,同时采用化学发光法定量检测血清中HBsAg和HBeAg。结果 83例患者中HBV DNA阳性62例,其中HBV DNA载量≥105拷贝/mL者40例,血清HBV cccDNA阳性28例(70.00%);HBV DNA载量<105拷贝/mL者22例,血清HBV cccDNA阳性2例(9.09%),不同DNA载量组HBV cccDNA差异有统计学意义(P<0.01)。HBeAg阳性组和阴性组HBV cccDNA阳性率分别为70.00%(28/40)和4.65%(2/43),两组差异有统计学意义(P<0.01)。HBV cccDNA定量值与HBV DNA、HBsAg呈正相关(P<0.01)。结论血清中HBV cccDNA水平可同时反应血清HBV DNA和HBsAg水平,血清HBV cccDNA含量可作为观察HBV复制情况和评价抗病毒疗效的相关血清指标。

乙型肝炎病毒感染相关性肝癌中HBV cccDNA水平与突变关系的研究

目的:研究乙型肝炎病毒(hepatitis B virus,HBV)感染相关性肝癌(hepatocellular carcinoma,HCC)中HBV共价闭合环状DNA(covalently closed circular DNA,ccc DNA)水平与突变关系。方法:运用特异性荧光定量PCR定量分析了12例肝癌组织和癌旁组织中乙肝病毒复制水平的表征因子HBV ccc DNA,HBV total DNA及前基因组RNA(pregenome RNA,pg RNA)的表达水平,再用滚环扩增(rolling circle amplification,RCA)特异性扩增癌组织中HBV ccc DNA,测序分析了病毒基因组4个编码调控区域pre C/C、RT、X、pre S中ccc DNA序列的突变。最后检测了肿瘤组织ccc DNA的AP位点。结果:HBV ccc DNA在癌组织的表达水平明显低于癌旁组织(3.32 copies/cell vs.9.64 copies/cell,P=0.029),但是HBV总DNA和pg RNA的表达没有明显差异(16.94 copies/cell vs.12.18 copies/cell,P=0.325,75.54 copies/cell vs.22.46 copies/cell,P=0.128);在肝癌组织中,HBV ccc DNA pre C/C和X区间,G-A突变是主要的突变类型;其次,pre C/C和X区域发生G-A突变的位置上,均更倾向于GA二联核苷酸组合方式,在肝癌中所占比例为37.1%和40%。最后,肿瘤组织中APE1酶处理后,HBV ccc DNA水平明显下降(1 011 copies/μl vs.501.8 copies/μl,P=0.058)。结论:HBV ccc DNA水平在癌组织中明显低于癌旁组织,可能与癌组织中HBV ccc DNA发生大量GA突变及形成的AP位点有关。

乙型肝炎病毒相关性肾小球肾炎患儿血清中HBV-cccDNA水平检测及其临床意义

目的:分析血清中乙肝病毒共价闭合环状双链DNA (HBV-cccDNA)水平对乙型肝炎病毒相关性肾小球肾炎(HBV-GN)患儿肝肾功能、肾组织中HBV抗原的检出、肝组织病理分级和分期的影响,评价HBVcccDNA水平检测对HBV-GN患儿诊断的临床应用价值。方法:选取39例HBV-GN初治患儿作为研究对象(观察组),所有患儿均接受肝脏和肾脏穿刺;选取肝功能正常HBV携带患儿40例作为对照组。检测2组患儿丙氨酸氨基转移酶(ALT)和天门冬氨酸氨基转移酶(AST)水平,采用PCR荧光分子信标技术检测患儿血清中HBV-cccDNA水平,HE染色检测患儿肝脏和肾脏组织的形态表现,免疫荧光法检测患儿肾组织中HBsAg、HBeAg和HBcAg检出率,采用受试者工作特征(ROC)曲线及曲线下面积(AUC)评价HBV-cccDNA水平检测在HBV-GN诊断中的价值。以HBV-cccDNA的Ax荧光值>21判定为阳性,反之判定为阴性,将HBV-GN患儿分为HBV-cccDNA阳性组(n=24)和HBV-cccDNA阴性组(n=15)。在抗病毒治疗后第2、4、8和12周时检测患儿血清中HBV DNA及HBV-cccDNA水平。结果:HBV-cccDNA阳性组患儿ALT和AST水平异常率、HBeAg和尿蛋白阳性率均高于HBV-cccDNA阴性组(χ~2=4.454,P=0.035;χ~2=5.912,P=0.022;χ~2=8.770,P=0.007)。HBV-cccDNA阳性和阴性组HBV-GN患儿肝组织炎症和纤维化程度比较差异无统计学意义(P>0.05),HBV-GN患儿肾脏活检病理类型以膜性肾病(MN)为主,HBV抗原成分以HBeAg及HBcAg为主,HBV-cccDNA阳性组患儿HBeAg及HBcAg检出率明显高于HBV-cccDNA阴性组(χ~2=5.652,P=0.027;χ~2=12.523,P=0.001)。ROC曲线评价,HBV-cccDNA水平检测能有效鉴别观察组HBV-GN患儿和对照组患儿,AUC=0.804 (95%CI:0.709~0.883)。10例规范治疗且有完整追踪治疗资料的HBV-GN患儿在治疗第2周时其HBV-cccDNA水平明显降低,治疗至第12周时其中7例患儿HBeAg转阴,无蛋白尿、血尿症状,ALT和AST水平均正常;治疗无效的3例患儿血清中HBV-cccDNA水平均高于其余7例标本。结论:HBV-cccDNA高表达与HBV-GN患儿肝功能、蛋白尿及肾脏HBV抗原检出有密切关联,检测HBVcccDNA水平对儿童HBV-GN患儿的辅助诊断及疗效评估具有潜在的临床应用价值。