乙型肝炎病毒前S1蛋白与病毒复制的关系

目的探讨乙型肝炎病毒前S1蛋白(preS1)与乙型肝炎病毒血清标志物(HBV-M)以及HBV-DNA之间的关系;了解preS1在乙肝病毒感染及复制中的应用价值。方法应用ELISA法检测1254例preS1和HBV-M;应用荧光定量PCR法检测372例HBV-DNA。结果1254例中有573例preS1阳性,阳性率45.7%并且preS1阳性只存在于HBsAgHBeAgHBcAb(大三阳)、HBsAgHBeAbHBcAb(小三阳)、HBsAgHBcAb(小二阳)3种血清模式中,阳性率分别为96.0%、68.6%、81.4%,经χ2检验,3者之间差异极显著。分析372例HBV-DNA,有292例>1×104,其中preS1阳性268例,阳性符合率为91.8%,而HBeAg阳性符合率为53.4%;另外80例HBV-DNA<1×104病例中,preS1阳性率仍为50.0%,而HBeAg阳性率仅为2.5%。结论通过对preS1、HBeAg和HBV-DNA的检测分析,证实了preS1作为判断HBV病毒感染和复制的指标比单纯用HBeAg和HBV-DNA定量更敏感,更确切,更方便,是一项非常好的检测指标。

乙型肝炎病毒SYBR GreenⅠ实时荧光定量PCR方法的建立及临床应用

目的建立 SYBR Green I实时荧光定量 PCR检测乙型肝炎病毒 DNA的快速方法，并探讨其临床应用价值。方法根据GenBank公布的Hepatitis B virus gp1基因序列设计引物，建立SYBR Green I实时荧光定量PCR，并对反应体系和扩增程序进行优化。定量标准品通过基因克隆方法获得，同时以浙江省夸克公司 HBV DNA定量检测试剂盒作对照，应用于随机选取的100份乙型肝炎患者血清检测。结果 SYBR Green I 实时荧光定量 PCR 检出限范围为5＆#215;102 copies／ml～5＆#215;108 copies／ml，HBV DNA浓度与CT值有良好线性关系，无交叉反应，整个过程仅需2.5 h。在随机抽取的100份临床标本应用中，与浙江省夸克公司的 HBV荧光定量 PCR检测试剂相比，建立的 SYBR Green I实时荧光定量PCR体系灵敏度100％，特异度92.5％。结论 SYBR Green实时荧光定量PCR快速、简便、灵敏度高、特异度强，可用于乙型肝炎患者病情监测，有效指导临床用药，准确评价 HBV感染者的病情。

改良聚合酶链反应检测HBV共价闭合环状DNA

目的:建立一种基于聚合酶链反应(PCR)的简便快速、具有较高敏感性和特异性的检测乙型肝炎病毒(HBV)共价闭合环状DNA(cccDNA)的方法.方法:分别提取HepG2.2.15细胞内的cccDNA及培养上清中的松驰环DNA(rcDNA)样品,试剂盒纯化;设计2对特异性引物,其扩增区域跨越rcDNA单链区;设计2对非特异性引物,扩增区域位于rcDNA双链区.经单链特异性绿豆芽核酸酶(MBN)分别消化cccDNA及rcDNA样品;以特异性引物和非特异性引物对消化前后的两种样品分别进行PCR扩增,并改变PCR扩增参数如底物数量、循环次数等,观察特异性引物能否顺利扩增消化后的cccDNA,同时又不扩增消化后的rcDNA.HBV基因组质粒样品作为对照.此外还采用实际乙型肝炎患者体内病毒样本检验此策略的实用性.结果:分别以非特异性引物和特异性引物扩增不同模板数的HBVrcDNA样品,2对非特异性引物可扩增出模板数在102以上的HBVrcDNA样品,2对cccDNA特异性引物也可以扩增出模板数在104以上的样品.特异性引物在PCR反应模板数较多时将不能区分消化前的rcDNA和cccDNA.不同数量HBVcccDNA和rcDNA模板在MBN消化前后,分别应用非特异性引物和特异性引物进行PCR扩增,发现不同数量的cccDNA模板分子经过MBN消化后,仍可用特异性引物和非特异性引物扩增出相应条带;rcDNA样品经过MBN消化后,非特异性引物可扩增出产物条带,而特异性引物无法扩增出条带.采用此种策略,我们发现慢性乙肝患者血清HBV核酸样品主要成份为rcDNA,并带有少量cccDNA,而肝细胞内HBV核酸样品富含cccDNA,与实际情况一致.结论:联合应用MBN选择性消化和cccDNA特异性引物的PCR检测法简便快速,敏感性和特异性均较满意.

阿昔洛韦与IFN序贯抗HBV感染时血清HBVDNA含量变化

目的为探讨HBV感染者单用IFN或阿昔洛韦（Acy）联合IFN序贯治疗时，血中HBVDNA水平变化与疗效关系．方法血清HBsAg，HBeAg，抗HBc：和HBVDNA皆阳性HBV感染者33例．IFN组15例，用重组IFNa-1b300万U，im，1次／d×14，1次／2d×36，共 50支；合用组 18例，用 Acy25 mg／（kg·d），iv计×15，接着 IFNa-1b300万 U，用法同 IFN组，IFN治疗20 d时再用 Acy 15 d，方法同前采用信号引物能量转移定量PCR，测定患者治疗前。中、治疗结束时血清HBVDNA水平并判断疗效．结果完全反应老干扰素治疗前血清HBVDNA含量（拷贝／L）范围 5．03×107～4．58×109；部分反应和无反应者范围 5.68×107～2．37×1015．Acy能降低血中HBVDNA含量，Acy联合IFN序贯治疗组完全反应率（72．2％）明显高于单用IFN组（46．6％）．结论先用Acy降低患者血中HBVDNA含量。

High level of hepatitis B virus DNA after HBeAg-to-anti-HBe seroconversion is related to coexistence of mutations in its precore and basal core promoter

AIM: G1896A mutation in precore or A1762T/G1764Amutations in basal core promoter are suspected to be responsible for patients with detectable level of HBV DNA in serum after seroconversion from HBeAg to anti-Hbe. However, G1896A variant has impaired, while A1762T/ G1764A variant may have intact replication ability. They themselves or their coexistence status may play different roles in such meaningless seroconversion. For these reasons, the significances of these two types of mutations were comparatively investigated in this study. METHODS: One hundred and sixty-five sera with positive anti-Hbe and HBV DNA were collected from different patients. Mutations of G1896A and A1762T/G1764A among these serum samples were detected using competitively differentiated PCR. HBV DNA was demonstrated using real-time quantitative PCR. RESULTS: G1896A and/or A1762T/G1764A mutations were detected in 89.1% (147/165) out of patients with detectable HBV DNA in serum after HBeAg-to-anti-Hbe seroconversion. The positive rate of G1896A variants was significantly higher than that of A1762T/G1764A mutations (77.6% vs 50.3%, X2 = 26.61, P＜0.01). The coexistence positive rate of these two types of mutations was 38.8% (64/165). Coexistence mutations were found in 77.1% (64/83) out of sera with A1762T/G1764A mutations, and in 50.0% (64/128) out of sera with G1896A mutation. Compared with variants with G1896A mutation only, the coexistence mutations were predominant in patients with high level of serum HBV DNA, and related to higher total bilirubin, lower serum albumin and progressive liver diseases. CONCLUSION: The coexistence of G1896A mutation and A1762T/G1764A mutations is very common, and responsible for the major cases with high level of HBV DNA in serum and progressive liver diseases after HBeAg-to-anti-Hbe seroconversion. This coexistence mutation variant may have higher pathogenicity and replication ability.

TT virus infection in patients with chronic hepatitis B and response of TTV to lamivudine

AIM: To investigate the responses of TT virus (TTV) and hepatitis B virus (HBV) to a long-term lamivudine therapy.METHODS: Sixteen patients infected with both TTV and HBV were treated with lamivudine 100 mg daily for 30 months. Blood samples were drawn at the beginning of the therapy and subsequently at month 3, 6, 9, 12 and 30.Serum TTV was quantified by real time PCR and serum HBV was detected by hybridization assay and nested polymerase chain reaction.RESULTS: TTV infection was detected in 100 % of HBV-infected patients. Loss of serum TTV DNA after one year of treatment occurred in 1/16 (6 %) patients. At the end of therapy, TTV DNA was positive in 94 % of them. The decline of HBV viremia was evident at 3 months after therapy and the response rate was 31%, 44 %, 63 %, 50 % and 50 %at month 3, 6, 9, 12 and 30, respectively.CONCLUSION: TTV replication is not sensitive to lamivudine and is highly prevalent in HBV-infected patients.

Apoptosis and its pathway in X gene-transfected HepG_2 cells

AIM： To investigate the effect of hepatitis B virus （HBV） X gene on apoptosis and expressions of apoptosis factors in X gene-transfected HepG2 cells.METHODS： The HBV X gene eukaryon expression vector pcDNA3-X was transiently transfected into HepG2 cells by lipid-media transfection. Untransfected HepG2 and HepG2 transfected with pcDNA3 were used as controls. Expression of HBx in HepG2 was identified by RT-PCR. MTT and TUNEL were employed to measure proliferation and apoptosis of cells in.three groups. Semi-quantified RT-PCR was used to evaluate the expression levels of Fas/FasL, Bax/Bcl-xL, and c-myc in each group.RESULTS： HBV X gene was transfected into HepG2 cells successfully. RT-PCR showed that HBx was only expressed in HepG2/pcDNA3-X cells, but not expressed in HepG2 and HepG2/pcDNA3 cells. Analyzed by MTT, cell proliferationcapacity was obviously lower in HepG2/pcDNA3-X cells （0.08910±0.003164） than in HepG2 （0.14410±0.004927） and HepG2/pcDNA3 cells （0.12150±0.007159） （P〈0.05 and P〈0.01）. Analyzed by TUNEL, cell apoptosis was much more in HepG2/pcDNA3-X cells （980/2 000） than HepG2 （420/2 000）, HepG2/pcDNA3 cells （520/2 000） （P〈0.05 and P〈0.01）. Evaluated by semi-quantified RT-PCR, the expression level of Fas/FasL was significantly higher in HepG2 cells transfected with HBx than in HepG2 and HepG2/pcDNA3 cells （P〈0.05 and P〈0.01）. Bax/Bcl-xL expression level was also elevated in HepG2/pcDNA3-X cells （P〈0.05 and P〈0.01）. Expression of c-myc was markedly higher in HepG2/pcDNA3-X cells than in HepG2 and HepG2/pcDNA3 cells （P〈0.05 and P〈0.01）.CONCLUSION： HBV X gene can impair cell proliferation capacity, improve cell apoptosis, and upregulate expression of apoptosis factors. The intervention of HBV X gene on the expression of apoptosis factors may be a possible mechanism responsible for the change in cell apoptosis and proliferation.

Durability of viral response after off-treatment in HBeAg positive chronic hepatitis B

AIM:To evaluate the durability in hepatitis B e antigen (HBeAg) positive chronic hepatitis B patients who discontinued antiviral treatment. METHODS:A total of 48 HBeAg positive chronic hepatitis B patients who were administered nucleoside analogues and maintained virological response for ≥ 6 mo [hepatitis B virus (HBV) DNA < 300 copies/mL and HBeAg seroconversion] before cessation of treatment were enrolled between February 2007 and January 2010. The criteria for the cessation of the antiviral treatment were defined as follows:(1) achievement of virological response; and (2) duration of consolidation therapy (≥ 6 mo). After treatment cessation, the patients were followed up at 3-6 mo intervals. The primary endpoint was serologic and virologic recurrence rates after withdrawal of antiviral treatment. Serologic recurrence was defined as reappearance of HBeAg positivity after HBeAg seroconversion. Virologic recurrence was defined as an increase in HBV-DNA level > 104 copies/mL after HBeAg seroconversion with previously undetectable HBV-DNA level. RESULTS:During the median follow-up period of 18.2 mo (range:5.1-47.5 mo) after cessation of antiviral treatment, the cumulative serological recurrence rate was 15 % at 12 mo. The median duration between the cessation of antiviral treatment and serologic recurrence was 7.2 mo (range:1.2-10.9 mo). Of the 48 patients with HBeAg positive chronic hepatitis, 20 (41.6%) showed virological recurrence. The cumulative virologic recurrence rates at 12 mo after discontinuing the antiviral agent were 41%. The median duration between off-treatment and virologic recurrence was 7.6 mo (range:4.3-27.1 mo). The mean age of the virological recurrence group was older than that of the non-recurrence group (46.7 ± 12.1 years vs 38.8 ± 12.7 years, respectively; P = 0.022). Age (> 40 years) and the duration of consolidation treatment (≥ 15 mo) were significant predictive factors for offtreatment durability in the multivariate analysis [P = 0.049, relative risk (RR) 0.31, 95% CI (0.096-0.998) and P = 0.005, RR 11.29, 95% CI (2.054-65.12), respectively]. Patients with age (≤ 40 years) who received consolidation treatment (≥ 15 mo) significantly showed durability in HBeAg positive chronic hepatitis B patients (P = 0.014). These results suggest that additional treatment for more than 15 mo after HBeAg seroconversion in patients who are ≤ 40 years old may be beneficial in providing a sustained virological response. CONCLUSION:Our data suggest that HBeAg seroconversion is an imperfect end point in antiviral treatment. Long-term consolidation treatment (≥ 15 mo) in younger patients is important for producing better prognosis in HBeAg positive chronic hepatitis B.

Generation of the regulatory protein rtTA transgenic mice

AIM: To translate Tet-on system into a conditional mouse model, in which hepatitis B or C virus (HBV or HCV) gene could be spatiotemporally expressed to overcome 'immune tolerance' formed during the embryonic development and 'immune escape' against hepatitis virus antigen(s), an effector mouse, carrying the reverse tetracycline-responsive transcriptional activator (rtTA) gene under the tight control of liver-specific human apoE promoter, is required to be generated. METHODS: To address this end, rtTA fragment amplified by PCR was effectively inserted into the vector of pLiv.7 containing apoE promoter to create the rtTA expressing vector, I.e., pApoE-rtTA. ApoE-rtTA transgenic fragment (-6.9 kb) released from pApoE-rtTA was transferred into mice by pronucleus injection, followed by obtaining one transgene (+) founder animal from microinjection through PCR and Southern blot analysis.RESULTS: rtTA transgene which could be transmitted to subsequent generation (F1) derived from founder was expressed in a liver-specific fashion. CONCLUSION: Taken together, these findings demonstrate that rtTA transgenic mice, in which rtTA expression is appropriately targeted to the murine liver, are successfully produced, which lays a solid foundation to 'off-on-off' regulate expression of target gene (s) (e.g., HBV and/or HCV) in transgenic mice mediated by Tet-on system.

Liver stiffness in the hepatitis B virus carrier:A non-invasive marker of liver disease influenced by the pattern of transaminases

AIM： To investigate the usefulness of transient elastography by Fibroscan （FS）, a rapid non-invasive technique to evaluate liver fibrosis, in the management of chronic hepatitis B virus （HBV） carriers. METHODS： In 297 consecutive HBV carriers, we studied the correlation between liver stiffness （LS）, stage of liver disease and other factors potentially influencing FS measurements. In 87 chronic hepatitis B （CriB） patients, we monitored the FS variations according to the spontaneous or treatment-induced variations of biochemical activity during follow-up. RESULTS： FS values were 12.3 ± 3.3 kPa in acute hepatitis, 10.3 ± 8.8 kPa in chronic hepatitis, 4.3 ± 1.0 kPa in inactive carriers and 4.6 ± 1.2 kPa in blood donors. We identified the cut-offs of 7.5 and 11.8 kPa for the diagnosis of fibrosis ≥S3 and cirrhosis respectively, showing 93.9% and 86.5% sensitivity, 88.5% and 96.3% specificity, 76.7% and 86.7% positive predictive value （PPV）, 97.3% and 96.3% negative predictive value （NPV） and 90.1% and 94.2% diagnostic accuracy. At multivariate analysis in 171 untreated carriers, fibrosis stage （t = 13.187,P 〈 0.001）, active vs inactive HBV infection （t = 6.437, P 〈 0.001）, alanine aminotransferase （ALT） （t = 4.740, P 〈 0.001） and HBV-DNA levels （t = -2.046, P = 0.042） were independently associated with FS. Necroinflammation score （t = 2.158, 〉 10/18 vs ≤ 10/18, P = 0.035） and ALT levels （t = 3.566, P =0.001） were independently associated with LS in 83 untreated patients without cirrhosis and long-term biochemical remission （t = 4.662, P 〈 0.001） in 80 treated patients. During FS monitoring （mean followup 19.9 ± 7.1 mo） FS values paralleled those of ALT in patients with hepatitis exacerbation （with 1.2 to 4.4-fold increases in Crib patients） and showed a progressive decrease during antiviral therapy. CONCLUSION： FS is a non-invasive tool to monitor liver disease in chronic HBV carriers, provided that the pattern of biochemical activity is taken into account. In the inactive carrier, it identifies non-HBV-related causes of liver damage and transient reactivations. In CHB patients, it may warrant a more appropriate timing of control liver biopsies.

Pathogenesis of hepatitis B virus infection

Infection with hepatitis B virus (HBV) leads to a wide spectrum of clinical presentations ranging from an asymptomatic carrier state to self-limited acute or fulminant hepatitis to chronic hepatitis with progression to cirrhosis and hepatocellular carcinoma. Infection with HBV is one of the most common viral diseases affecting man. Both viral factors as well as the host immune response have been implicated in the pathogenesis and clinical outcome of HBV infection. In this review, we will discuss the impact of virus-host interactions for the pathogenesis of HBV infection and liver disease. These interactions include the relevance of naturally occurring viral variants for clinical disease, the role of virus-induced apoptosis for HBV-induced liver cell injury and the impact of antiviral immune responses for outcome of infection.

Antiviral treatment of hepatitis B virus-transgenic mice by a marine organism, Styela plicata

AIM： To evaluate the antiviral effect of the effective ingredient of Styela plicata in a murine model of hepatitis B virus carrier. METHODS： HBV-transgenic mice were divided into 3 groups （control group, lamivudine treatment group and the effective ingredient of Styela plicata treatment group） and assigned to receive normal diet, lamivudine or the effective ingredient of Styela plicata for consecutive weeks. Serum hepatitis B surface antigen was detected by enzyme-linked immunosorbent assay （ELISA） method. Serum HBV DNA was detected by real-time polymerase chain reaction （RT-PCR）. Serum T helper （h） 1 cytokine interleukin （IL）-2 and Th2 cytokine IL-6 were detected by the quantitative sandwich enzyme immunoassay technique. Another group of HBV-transgenic mice was assigned to receive the effective ingredient of Styela plicata for consecutive weeks. The histology of liver tissue was evaluated before and after treatment. RESULTS： Twelve weeks after starting the therapy, serum hepatitis B surface antigen was significantly lowered in Styela plicata -treated mice and lamivudine-treated mice compared with the mice receiving normal diet （F12wk = 88.81, P12wk = 0.000 〈 0.01）. Serum HBV DNA was significantly lowered in Styela plicata -treated mice and lamivudine-treated mice compared with the mice receiving normal diet （F12wk = 20.71, P12wk = 0.000 〈 0.01）. However, like lamivudine, the effective ingredient of Styela plicata could not inhibit the replication of HBV completely. A rebound phenomenon of hepatitis B surface antigen and HBV DNA in sera could be found 4 wk after withdrawal of medication. Eight weeks after starting the therapy, serum levels before and after Styela plicata treatment of IL-2 were 2.41 ± 0.38 and 10.56 ± 0.78 ng/L, respectively （t8wk = -16.51, P8wk = 0.000 〈 0.01）. Compared with the serum levels of IL-2 in the normal diet-treated mice （2.48 ± 0.17 ng/L; t8wk = 13.23, P8wk = 0.000 〈 0.01）. Serum levels before and after Styela plicata treatment of IL-6 were 63.62 ± 6.31 and 54.52 ± 6.22 ng/L, respectively, compared with the serum levels of IL-6 in the normal diet-treated mice （60.84 ± 4.21 ng/L）. Histological analysis of liver from Styela plicata-treated HBV-transgenic mice also showed catabatic status in inflammation and hepatitis B surface antigen. CONCLUSION： Styela plicata may be an effective anviral medicine in treating chronic hepatitis B.

Interplay between hepatitis B virus and the innate immune responses:implications for new therapeutic strategies

Hepatitis B virus(HBV) infection is still a worldwide health problem;however,the current antiviral therapies for chronic hepatitis B are limited in efficacy.The outcome of HBV infection is thought to be the result of complex interactions between the HBV and the host immune system.While the role of the adaptive immune responses in the resolution of HBV infection has been well characterized,the contribution of innate immune mechanisms remains elusive until recent evidence implicates that HBV appears to activate the innate immune response and this response is important for controlling HBV infection.Here,we review our current understanding of innate immune responses to HBV infection and the multifaceted evasion by the virus and discuss the potential strategies to combat chronic HBV infection via induction and restoration of host innate antiviral responses.

Helicobacter species sequences in liver samples from patients with and without hepatocellular carcinoma

AIM:Only a minority of patients carrying a defined viral aetiologic agent develop cirrhosis and ultimately hepatocellular carcinoma(HCC),the mechanism underlying the worsening is still undefined.Experimental infection by Helicobacter hepaticus in mice causes chronic hepatitis and HCC and recently,more Helicobacterspecies(Helicobacter spp.)have been detected in the liver of patients suffering from cholestatic diseases and HCC arising from non-cirrhotic liver.We investigated whether Helicobacterspp.sequences could be detected in the liver of patients with cirrhosis and HCC compared to subjects with metastasis to liver from colon cancer. METHODS:Twenty-three liver samples from patients operated upon for HCC superimposed on hepatitis C virus (HCV)-related cirrhosis and 6 from patients with resected metastases from colorectal cancer,were tested by polymerase chain reaction for presence of genomic 16S rRNA of Helicobacter genus using specific primers.DNA sequencing and cag A gene analysis were also performed. RESULTS:Genornic sequences of Helicobacter spp.were found in 17 of 20(85%)liver samples from patients with HCC and in 2 of 6 samples from patients with liver metastasis. In three samples of the first group the result was uncertain. Hpyloriwas revealed in 16 out of 17 positive samples and Helicobacter pullorum in the other. CONCLUSION:Helicobacter spp.,carcinogenic in mice, were found at a higher frequency in the liver of patients with HCV-related cirrhosis and HCC than those in patients without primary liver disease.

Development of Novel Therapeutics for Chronic Hepatitis B

Chronic infection of hepatitis B virus (HBV) presents one of the serious public health challenges worldwide. Current treatment of chronic hepatitis B (CHB) is limited, and is composed of interferon and nucleoside/nucleotide reverse transcriptase inhibitors (NRTI). Interferon is poorly tolerated and is only responsive in a small fraction of CHB patients and NRTIs often face the problem of emergence of drug resistance during long-term treatment. The current treatment of CHB can be improved in several ways including genotyping mutations associated with drug resistance before treatment to guide the choice of NRTIs and suitable combinations among NRTIs and interferon. It is important to continue research in the identification of novel therapeutic targets in the life cycle of HBV or in the host immune system to stimulate the development of new antiviral agents and immunotherapies. Several antiviral agents targeting HBV entry, cccDNA, capsid formation, viral morphogenesis and virion secretion, as well as two therapeutic vaccines are currently being evaluated in preclinical studies or in clinical trials to assess their anti-HBV efficacy.

Relationship between hepatitis B virus DNA levels and liver histology in patients with chronic hepatitis B

AIM： To study the relationship between hepatitis B virus （HBV） DNA levels and liver histology in patients with chronic hepatitis B （CHB） and to determine the prevalence and characteristics of hepatitis B e antigen （HBeAg） negative patients.METHODS： A total of 213 patients with CHB were studied, and serum HBV DNA levels were measured by the COBAS Amplicor HBV Monitor test. All patients were divided into two groups according to the HBeAg status.The correlation between serum HBV DNA levels and liver damage （liver histology and biochemistry） was explored.RESULTS： Of the 213 patients with serum HBV DNA levels higher than 10^5 copies/mL, 178 （83.6%） were HBeAg positive, 35 （16.4%） were HBeAg negative. The serum HBV DNA levels were not correlated to the age,history of CHB, histological grade and stage of liver disease in either HBeAg negative or HBeAg positive patients. There was no correlation between serum levels of HBV DNA and alanine aminotransferanse （ALT）,aspartate aminotrans-ferase （AST） in HBeAg positive patients. In HBeAg negative patients, there was no correlation between serum levels of HBV DNA and AST,while serum DNA levels correlated with ALT （r = 0.351, P = 0.042）. The grade （G） of liver disease correlated with ALT and AST （P 〈 0.05, r = 0.205, 0.327 respectively）in HBeAg positive patients. In HBeAg negative patients,correlations were shown between ALT, AST and the G （P 〈 0.01, and r = 0.862, 0.802 respectively）. HBeAg negative patients were older （35 ± 9 years vs 30 ±9 years, P 〈 0.05 ） and had a longer history of HBV infection （8 ± 4 years vs 6 ± 4 years, P 〈 0.05） and a lower HBV DNA level than HBeAg positive patients （8.4± 1.7 Log HBV DNA vs 9.8 ± 1.3 Log HBV DNA, P 〈0.001）. There were no significant differences in sex ratio,ALT and AST levels and liver histology between the two groups.CONCLUSION： Serum HBV DNA level is not correlated to histological grade or stage of liver disease in CHB patients with HBV DNA more than 10^5 copies/mL.Compared to HBeAg positive patients, HBeAg negative patients are older and have a lower HBV DNA level and a longer HBV infection history. There is no significant difference in sex ratio, ALT and AST levels and liver histology between the two groups.

Clinicopathologic characteristics of intrahepatic cholangiocarcinoma in patients with positive serum a-fetoprotein

AIM:To explore clinicopathologic characteristics of intrahepatic cholangiocarcinoma (ICC) in patients with positive serum a-fetoprotein (AFP). METHODS:One hundred and thirty one patients who underwent surgical dissection for pathologically confirmed ICC were divided into a positive AFP (> 20 ng/mL) group (n = 32) and a negative AFP group (n = 99), whose clinicopathologic features were analyzed and compared. RESULTS:The positive rate of HBsAg and liver cirrhosis of the positive AFP group was higher than that of the negative AFP group, while the positive rate of CA19-9 (> 37 U/mL) and the lymph node metastasis rate was lower. CONCLUSION:ICC patients with positive AFP share many clinicopathologic similarities with hepatocellular carcinoma.

Programmed death-1 expression is associated with the disease status in hepatitis B virus infection

AIM： TO define the potential role of programmed death-i/programmed death-ligand （PD-1/PD-L） pathway in different hepatitis B virus （HBV） infection disease status; we examined the expression of PD-1 on antigen specific CD8＋T cells in peripheral blood of patients with chronic hepatitis B （CriB） and acute exacerbation of hepatitis B （AEHB） infection. METHODS： The PD-1 level on CD8＋ T lymphocytes and the number of HBV specific CD8＋ T lymphocytes in patients and healthy controls （HCs） were analyzed by staining with pentameric peptide-human leukocyte antigen2 （HLA2） complexes combined with flow cytometry. Real-time quantitative polymerase chain reaction （PCR） was used to measure the serum HBV- DNA levels. RESULTS： The level of PD-1 expression on total CD8＋ T cells in CHB patients （13.86% ± 3.38%） was significantly higher than that in AEHB patients （6.80%± 2.19%, P 〈 0.01） and healthy individuals （4.63% ± 1.23%, P 〈 0.01）. Compared to AEHB patients （0.81% ± 0.73%）, lower frequency of HBV-specific CD8＋ T cells was detected in chronic hepatitis B patients （0.37% ± 0.43%, P 〈 0.05）. There was an inverse correlation between the strength of HBV-specific CD8＋ T-cell response and the level of PD-1 expression. Besides, there was a significant positive correlation between HBV viral load and the percentage of PD-1 expression on CD8＋ T cells in CriB and AEHB subjects （R = 0.541, P 〈 0.01）. However, PD-1 expression was not associated with disease flare-ups as indicated by alanine aminotransferase （ALT） levels （R = 0.066, P 〉 0.05）. CONCLUSION： Our results confirm previous reports that HBV specific CD8＋T-cell response in the peripheral blood is more intense in patients with AEHB than in chronic hepatitis B wlth persistent viral infection. Moreover, there is a negative correlation between the level of PD-1 and the intensity of virus specific CD8＋ T cell response.

Hepatitis B virus replication

Hepadnaviruses, including human hepatitis B virus （HBV）, replicate through reverse transcription of an RNA intermediate, the pregenomic RNA （pgRNA）. Despite this kinship to retroviruses, there are fundamental differences beyond the fact that hepadnavirions contain DNA instead of RNA. Most peculiar is the initiation of reverse transcription： it occurs by protein-priming, is strictly committed to using an RNA hairpin on the pgRNA, ε, as template, and depends on cellular chaperones; moreover, proper replication can apparently occur only in the specialized environment of intact nucleocapsids. This complexity has hampered an in-depth mechanistic understanding. The recent successful reconstitution in the test tube of active replication initiation complexes from purified components, for duck HBV （DHBV）, now allows for the analysis of the biochemistry of hepadnaviral replication at the molecular level. Here we review the current state of knowledge at all steps of the hepadnaviral genome replication cycle, with emphasis on new insights that turned up by the use of such cellfree systems. At this time, they can, unfortunately, not be complemented by three-dimensional structural information on the involved components. However, at least for the ~ RNA element such information is emerging, raising expectations that combining biophysics with biochemistry and genetics will soon provide a powerful integrated approach for solving the many outstanding questions. The ultimate, though most challenging goal, will be to visualize the hepadnaviral reverse transcriptase in the act of synthesizing DNA, which will also have strong implications for drug development.

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.