Transmission of G145R mutant of HBV to an unrelated contact

Household contacts of HBV-related chronic liver disease patients constitute a high-risk group for acquisition of HBV infection. Some of the HBsAg mutants are associated with liver disease and some are reported to be transmitted vertically. There is limited information on the horizontal transmission of Gly 145 Arg (G145R) mutant to related contacts. Its possible transmission to an unrelated third degree contact is reported in the present study. An HBV related chronic liver disease patient; the index patient, and his 11 household contacts were studied. This included four 1 degrees, three 2 degrees, one 3 degrees, and a sexual contact. Surface gene sequencing including the "a" determinant region was carried out in HBV DNA+ve subjects. The sequences were aligned and compared for the homology. HBV DNA was found to be positive in one 1 degrees, three 2 degrees, and one 3 degrees contact, besides the index patient. Histopathological studies revealed evidence of chronic hepatitis in all these contacts. Mutation T118V was present in all the six subjects. Mutant G145R along with T118V and T143M was identified in three subjects who included one 1 degrees, one 2 degrees, and one 3 degrees contact. Presence of T118V and T143M mutations along with G145R mutation in these subjects provides an indirect evidence for the possible horizontal transmission of G145R HBV variant to a 3 degrees unrelated contact. Of these three contacts with G145R mutation, only one 1 degrees contact was found to be HBsAg-ve. The data also reaffirms the earlier finding of HBsAg positivity in presence of G145R mutation of the S-gene. HBV exists as quasi-species and mixed population in subjects with chronic HBV infection.     

G145R突变后HBsAg"a"决定簇合成肽的免疫学特性分析

目的研究G145R突变后乙肝表面抗原(HBsAg)"a"决定簇合成肽的免疫学特性改变情况. 方法首先合成2条短肽P1-wt和P2-145R,分别代表野毒株和G145R突变后HBsAg"a"决定簇合成肽.然后用β-巯基乙醇(2-ME)变性试验以及用乙肝疫苗标准品制备的小鼠多抗血清研究2条合成肽的空间构象以及抗原性异同.最后用等量合成肽免疫小鼠,用酶免疫法、竞争抑制试验和Western blot试验等研究G145R突变后"a"决定簇合成肽的免疫原性改变.结果合成肽P1-wt和P2-145R用2-ME温和变性后,PAGE结果表现为相对分子质量(Mr)为4×103左右的单一条带,而变性前2条合成肽均表现为Mr是从4×103到30×103的弥漫条带,主带位置在5×103和10×103,分别相当于二聚体和四聚体位置;用HBsAg的多克隆抗体(anti-HBs)检测合成肽的抗原性,结果在固定抗原量的前提下,在抗体1∶32 000稀释时仍可检测到P1-wt的阳性结果,而当同一抗体稀释到1∶8000时,P2-145R检测结果即为阴性.合成肽P2-145R免疫小鼠产生的抗体与P1-wt合成肽的反应滴度比与P2-145R合成肽本身的反应低4～8倍. 结论针对HBsAg"a"决定簇合成肽可自发形成一定的空间构象,G145R突变株的HBsAg"a"决定簇的抗原性和免疫原性与野毒株相比发生了明显改变,为正确评价G145R变异株的流行危害以及现行疫苗的保护效果提供了实验依据.     

Hepatitis B virus DNA in sera of virus carriers positive exclusively for antibodies to the hepatitis B core antigen

The prevalence of serum HBV DNA in individuals positive for anti-HBc alone was determined by the polymerase chain reaction in two groups with endemic HBV infection from Canton (group A) and Hainan (group B), provinces of China. Twenty-one out of 294 individuals in group A (7.2%) and 193 out of 1995 in group B (9.7%) were positive for anti-HBc but negative for other markers of ongoing or past HBV infection (HBsAg and anti-HBs). HBV DNA was detected in 6/21 sera in group A (28.6%) and 68/193 in group B (35.2%) in their initial serum specimen. One of the six HBV-DNA-positive individuals in group A became negative after 6 months and four of the 58 positive in group B became negative at 4 years of follow-up. All of the individuals remained positive for anti-HBc and negative for anti-HBs, but one of them became positive for HBsAg on follow-up. None of the anti-HBc- and HBV-DNA-positive subjects had symptoms of liver diseases. They were, therefore, defined as chronic asymptomatic HBV carriers with undetectable HBsAg. This type of carrier should be added to the typical HBsAg-positive carrier, who constitutes about 10-15% of the general Chinese population, to give a more complete estimate of asymptomatic HBV carriers in China.     

Specific deletions in the hepatitis B virus core open reading frame in patients with chronic active hepatitis B

The polymerase chain reaction (PCR) and DNA sequencing were used to examine genomic variation in the pre-core/core open reading frame of the hepatitis B virus (HBV) in chronically infected patients. Gel electrophoresis of amplification products showed the presence of shortened forms of the core gene in addition to the full length product. These shortened forms were seen only in patients with chronic active hepatitis (CAH) seropositive for HBeAg and not in patients with chronic persistent hepatitis (CPH) or HBeAg minus CAH. Cloning and DNA sequencing revealed the presence of a number of overlapping deletions within the core gene, the majority being in-frame, which were clustered within aa 81-114 of the core gene product. These deletions were found in patients with CAH from different racial and geographical backgrounds, whereas PCR analysis of the surface and ×open reading frames showed no shortened forms suggesting deletions to be specific to the core gene in these patients. Because the product of the core gene-the HBV core antigen–is believed to be the major target for T-cell-mediated liver damage, it seems likely that the products of core genes carrying deletions will alter immune recognition and may be of importance in the progression of inflammatory liver damage.     

Detection of hepatitis B viral DNA by polymerase chain reaction in patients with hepatitis B surface antigen

Hepatitis B virus (HBV) DNA was assayed using the polymerase chain reaction in serum samples of 116 hepatitis B surface antigen (HBsAg) carriers, including 30 positive for hepatitis B e antigen (HBeAg) and 86 negative for HBeAg. In the HBeAg-positive group, all were positive for HBV DNA. In the HBeAg-negative group, 80.2% were positive for HBV DNA (80.0% in the healthy carrier group, 90.0% in the chronic active liver disease group, and 69.2% in patients with cirrhosis). This study indicated that every HBeAg-positive carrier as well as the majority of HBeAg-negative carriers were infectious and, in the latter group, that viral replication is most active in patients with chronic active liver disease.     

慢性乙型肝炎患者血清和肝组织中HBV DNA含量与庚型肝炎病毒感染的关系

目的：观察慢性乙型肝炎（CH－B）患者血清、肝组织中HBV DNA含量与庚型肝炎病毒（HGV）感染的关系，探讨HGV感染对CH－B患者乙型肝炎病毒（HBV）复制的影响。方法：应用逆转录－聚合酶链反应（RT－PCR）、免疫组织化学法、荧光定量PCR（FQ－PCR）技术方法对56份CH－B患者血清HGV RNA、肝组织HGV Ag、血清及肝组织中HBV DNA含量分别进行了检测，并将血清HGV RNA与肝组织HGV Ag的表达、HGV RNA、HGV Ag阳性与阴性患者HBV DNA含量分别进行了对比研究。结果：血清HGV RNA、肝组织HGV Ag阳性分别为10份（17．9％）、8份（14．3％）。血清HGV RNA阳性与肝组织HGV Ag表达显著相关（P＜0．01），但部分肝组织HGV Ag阴性患者亦有血清HGV RNA表达。血清HGV RNA、肝组织HGV Ag阳性与阴性患者血清及肝组织中HBV DNA含量差异无显著性（P＞0．05）。结论：HGV感染对CH－B患者HBV复制无影响。HGV可在肝脏中复制，但致病性可能较微弱。     

Elimination of hepatitis delta virus infection after loss of hepatitis B surface antigen in patients with chronic delta hepatitis

The aim of this study was to evaluate whether patients with chronic hepatitis delta virus (HDV) infection treated with alpha interferon and subsequent loss of hepatitis B surface antigen (HBsAg) eliminate HDV. HDV RNA was detected in 26 of 28 patients with chronic delta hepatitis using the polymerase chain reaction. Seventeen patients in whom HDV RNA was detected were treated with alpha interferon; in 65%, HDV RNA remained detectable during treatment or reappeared after stopping therapy whereas in three patients HDV RNA remained absent (17.5%). HDV RNA became and remained undetectable in serum and liver of two of these three patients who lost HBsAg from serum and in one patient who was intermittently HBsAg negative during therapy. After loss of HBsAg, hepatitis B virus (HBV) DNA was still detectable in the liver, but not HBV RNA, indicating absent or very low HBV replication. Three patients were lost to follow up (17.5%). Two nontreated patients with chronic HDV infection also lost HBsAg during follow up; HDV RNA also became undetectable in their serum. Thus, HDV replication does not persist after the loss of HBsAg. Clearance of HBsAg may be a useful guide to when therapy can be stopped. © 1994 Wiley-Liss, Inc.     

Hepatitis B virus DNA in liver, serum, and peripheral blood mononuclear cells after the clearance of serum hepatitis B virus surface antigen

The integration of hepatitis B virus (HBV) DNA in the liver of chronic HBV carriers has been documented extensively. However, the status of the viral genome during acute infection has not been assessed conclusively. While HBV DNA sequences are detected often in serum, liver, and peripheral blood mononuclear cells (PBMCs) after the clearance of serum the hepatitis B virus surface antigen (HBsAg), the precise status of the viral genome, and in particular the possible persistence of integrated genomes in PBMCs, has not been established. A highly sensitive PCR-derived assay (Alu-PCR) was employed to re-examine liver and PBMC specimens obtained from patients with acute (n = 19) and chronic (n = 22) hepatitis in whom serum HBsAg was present (n = 12) (HBV-related chronic active hepatitis) or absent with anti-HCV (n = 10) (HCV-related chronic active hepatitis). Viral integration was demonstrated in 3 out of 19 liver specimens from patients with acute hepatitis and 12 out of 12 specimens from patients with chronic hepatitis. Viral integration was also observed in 4 out of 7 PBMC samples from HBV-related chronic active hepatitis patients and 2 out of 10 liver and PBMC samples from HCV-related chronic active hepatitis patients. In one liver specimen from an acute hepatitis patient, HBV DNA was found integrated in the intronic sequence of the tumour necrosis factor (TNF)-induced protein gene; viral integration into cellular sequences was also found in the PBMCs of four HBV-related chronic active hepatitis and two HCV-related chronic active hepatitis. The results demonstrate the early integration of HBV genome during acute viral infections and the persistence of the viral genome in an integrated form in PBMCs.     

套式PCR和原位杂交技术检测肝病患者单个核细胞内TTV DNA

目的　了解慢性乙型肝炎(中度)和原发性肝癌(HBsAg阳性)患者PBMC内TTVDNA存在情况。方法　采用套式PCR以及原位杂交技术检测外周血单个核细胞(PBMC)内TTVDNA。结果　套式PCR检测26例慢性乙型肝炎(中度)患者PBMC内TTVDNA阳性7例,阳性率269%,非常显著高于健康对照(χ2=143,P<0.001);21例原发性肝癌(HBsAg阳性)患者PBMC内TTVDNA阳性4例,阳性率190%,显著高于健康对照(χ2=486,P<0.05)。7例PBMC内TTVDNA阳性的慢性乙型肝炎(中度)患者PBMC原位杂交,其中4例细胞内阳性。结论　慢性乙型肝炎(中度)和原发性肝癌(HBsAg阳性)患者PBMC细胞浆内存在TTVDNA,且阳性率相当高。     

不同慢性肝病患者血清中乙型肝炎病毒DNA定量检测及其临床意义

目的探讨乙型肝炎病毒ＤＮＡ含量的临床意义。了解乙型肝炎病毒（ＨＢＶ）免疫标志不同状态的慢性肝病患者血清ＨＢＶＤＮＡ浓度及其临床意义。方法应用建立的竞争性聚合酶链反应（ＰＣＲ）方法定量检测慢性肝炎（ＣＨ）５１例、肝硬化（ＬＣ）３６例、原发性肝癌（ＰＨＣ）３８例的血清ＨＢＶＤＮＡ浓度。结果ＨＢＶＤＮＡ阳性的ＣＨ患者血清ＨＢＶＤＮＡ浓度为４．３６ｌｏｇ１０ＨＢＶＤＮＡ拷贝５０μｌ（下同），ＬＣ为４．５５，ＰＨＣ为４．４３，三组间无显著性差异（Ｐ＞０．０５）；血清ＨＢＶ五项免疫标志均阴性或抗－ＨＢｓ阳性的慢性肝病患者中，有３７．５％患者存在低水平ＨＢＶ复制；ＨＢｅＡｇ阳性患者的ＨＢＶＤＮＡ浓度总体上明显高于抗－ＨＢｅ阳性组，但其中部分患者的ＨＢＶＤＮＡ浓度也很高。结论提示ＨＢＶ的复制状态与慢性肝病的病期无明显关系；在抗－ＨＢｅ阳性的患者中存在个体差异，故不能仅依据抗－ＨＢｅ阳转来判断ＨＢＶ复制减少或停止。     

Clinical implications of hepatitis B surface antigen quantitation in the natural history of chronic hepatitis B virus infection

BackgroundHBsAg quantitation may be useful for managing patients with hepatitis B virus (HBV) infection.ObjectivesWe explored the clinical implications of HBsAg quantitation for patients with HBsAg levels >250 IU/ml (Abbott Diagnostics).Study designTwo hundred and thirty-three HBV-infected patients comprising 29 immune tolerance cases, 49 treatment-naïve HBeAg-positive chronic hepatitis B (CHB) cases, 91 inactive HBV carrier cases, and 64 treatment-naïve HBeAg-negative CHB cases were analyzed. HBsAg was quantified by the Architect HBsAg assay (Abbott Diagnostics) after a 1:500 automated dilution.Results and conclusionsHBsAg (log 10 IU/ml) was established for immune tolerance (4.50 ± 0.43), HBeAg-positive CHB (4.17 ± 0.66), inactive HBV carrier (3.32 ± 0.44), and HBeAg-negative CHB (3.23 ± 0.40); (p = 4.92 × 10⁻³⁵). No significant difference was observed between inactive HBV carrier and HBeAg-negative CHB (p = 0.247). The proportions of HBsAg <2000 IU/ml for inactive HBV carrier and HBeAg-negative CHB were 51.6% and 59.3%, respectively (p = 0.341). Positive correlations between HBsAg and HBV DNA were observed for immune tolerance (p = 1.23 × 10⁻⁴) and HBeAg-positive CHB (p = 0.003), but not for HBeAg-negative CHB (p = 0.432). A negative correlation between HBsAg and age was observed for immune tolerance (p = 0.030), HBeAg-positive CHB (p = 0.016), and inactive HBV carrier (p = 0.001), but not in HBeAg-negative CHB (p = 0.249). No significant differences between HBsAg and ALT for HBeAg-positive (p = 0.338) or HBeAg-negative CHB (p = 0.564) were observed. For patients with HBsAg quantitation >250 IU/ml, HBsAg may reflect HBV DNA replication for HBeAg-positive cases. HBsAg is not a suitable marker for evaluating hepatitis activity and distinguishing between cases of HBeAg-negative CHB and inactive HBV carrier state.     

慢加急性肝衰竭患者外周血肿瘤坏死因子-α基因启动子甲基化状态的研究

目的探讨肿瘤坏死因子-仅及表观遗传调控在慢加急性肝衰竭发病机制中的可能作用。方法ELISA法检测外周血TNF-α的表达,并与患者的肝功能进行相关性分析。同时提取外周血单个核细胞DNA,重亚硫酸盐处理后,用针对改变后的DNA序列设计特异性引物并进行聚合酶链反应（PCR）。根据修饰后DNA序列的不同,运用在线MethPrimer软件设计引物,甲基化特异性聚合酶链反应（Methylationspecificpolymerasechainreaction,MSP）检测标本中TNF-α扩增产物,分析TNF-α启动子甲基化与血清TNF-α表达的相关性,并分析甲基化与病情严重程度的相关性。采用SPSS16．0软件进行统计分析。结果慢加急性肝衰竭患者组外周血TNF-α蛋白水平（44．9260±26．48523）均高于慢性乙型病毒性肝炎患者组（18．92505±9．04461）和健康对照组（11．9172±5．04612）,且各组间比较差异均具有统计学意义（P〈0．05）。慢加急性肝衰竭患者组外周血清TNF—a水平与血清TBIL及终末期肝脏病模型（MELD）评分之间呈明显的正相关,与PTA呈明显的负相关。慢加急性肝衰竭组、慢性乙型病毒性肝炎患者组、健康对照组TNF-α启动子甲基化状态的差别有统计学意义。慢加急性肝衰竭患者与慢性乙型病毒性肝炎患者及健康对照甲基化组血清TNF-α水平均明显低于非甲基化组（P〈0．05）。结论TNF-α与肝炎活动及肝细胞损害有密切关系,DNA甲基化在慢加急肝衰竭患者中参与调控细胞因子的表达中。     

慢性乙型肝炎外周血单个核细胞内HBV-DNA与血清HBV-DNA及HBeAg表达关系的研究

目的研究慢性乙型肝炎患者外周血单个核细胞(PBMC)内HBV-DNA和血清中HBV-DNA表达量、e抗原表达的关系。方法采用聚合酶链反应(PCR)检测208例慢性乙型肝炎患者PBMC内HBV-DNA,应用荧光定量聚合酶链反应(FQ-PCR)检测血清中HBV DNA含量,应用酶联免疫吸附(ELISA)法检测乙肝血清标志物。结果208例慢性乙型肝炎患者PBMC内HBV-DNA阳性106例、阴性102例。HBV-DNA(PBMC)阳性组、阴性组血清HBV-DNA定量≥1.0E5患者比例分别为91.5%(97例)、45.1%(46例)(χ2=52.12,P<0.01);HBeAg阳性率分别为76.4%(81例)、50.9%(52例)(χ2=21.55,P<0.01)。结论PBMC内HBV-DNA的检测与血清中HBV-DNA定量检测及HBeAg阳性率存在明显的正相关。提示血清HBV-DNA高载量的HBeAg阳性患者外周血单个核细胞感染HBV-DNA明显增加。     

锁核酸捕获TaqMan探针实时聚合酶链反应构建及检测乙型肝炎病毒DNA

建立和检测新型乙型肝炎病毒(HBV)DNA检测技术锁核酸(LNA)捕获TaqMan探针实时聚合酶链反应(PCR)。方法 2009年8月至2010年3月选取本院20例健康献血者和肝病专科住院的75例经COBAS Amplicor检测HBV DNA阳性慢性乙型肝炎(CHB)患者,分别采集血液样本进行LNA捕获TaqMan探针实时PCR特异性试验。同时对LNA-实时PCR方法的重复性、特异性和线性范围等进行分析。结果 其线性范围为10～2.3×109 IU/ml。将10 IU/ml作为检测下限,具有95％可信区间。线性回归分析显示其斜率接近1.0 (R2=0.999)。批内变异值为3.88％～4.36％,批间变异值为8.5％～11.7％。20例健康献血者的阴性对照血清HBV-DNA检测均为阴性,而75例经COBAS Amplicor检测HBV DNA阳性患者的血清样本除1例阴性外,其余都为阳性。结论 LNA捕获TaqMan探针实时PCR方法为HBV-DNA检测的一种快捷灵敏、准确度高的新方法,值得临床实验室推广应用。     

乙型肝炎病毒(HBV)相关性肾炎患者肾活检组织中HBV DNA分布的分子生物学检测

为探讨乙型肝炎病毒（ＨＢＶ）相关性肾炎的发病机理，应用地高辛素标记ＨＢＶＤＮＡ探针原位分子杂交（ＩＳＨ）和直接原位聚合酶链反应（ＩＳ－ＰＣＲ）技术，对２０例临床诊断为ＨＢＶ相关性肾炎患者肾活检石蜡包埋组织切片检查。在ＩＳＨ中采用ＨＢＶＤＮＡ两种探针：用全长段探针，８５％（１７／２０）ＨＢＶＤＮＡ阳性，这１７例阳性肾组织切片再用ＨＢＶＤＮＡＳ加Ｃ段探针检测，１４例阳性（８２．３５％）。在ＩＳ－ＰＣＲ中本组病例８５％（１７／２０）亦阳性。发现ＨＢＶＤＮＡ阳性颗粒弥漫沉积于肾小球毛细血管袢、系膜区、肾小管，表现形式以浆核型、核型为主。同组病例肾组织免疫组化染色法（ＬＳＡＢ）显示，ＨＢｓＡｇ与ＨＢＶＤＮＡ的存在部位基本一致。提示ＨＢＶ引起的肾脏病变不仅是免疫介导的损害，亦可能有病毒侵犯参加免疫反应。     

慢性乙肝患者HBV核心基因一组单核苷酸多态性与血清HBV DNA水平的相关性研究

目的 研究慢性乙肝患者HBV核心基因的单核苷酸多态性(SNP)与血清HBV DNA水平的相关关系.方法 采用PCR-RFLP和限制性内切酶Tsp509I榆测HBV核心基因的SNP,采用双脱氧终止法对核心基因进行序列测定,实时荧光PCR技术用于HBVDNA水平的定量检测.结果 慢性乙肝人群中发现5种典型的PCR-RFLP图谱,分别是RFLP-C、RFLP-D、RFLP-E、RFLP-G和RFLP-C/G,各种RFLP图谱的分布频率依次为61.5%、2.6%、9.6%、16.7%和9.6%.A165T、A336C、A336T、T337C和T385C等5种SNP与RFLP图谱的变化有关,但是只有SNP A336C和A336T会导致HBcAg第83位的Glu被Asp所替代.RFLP-C组患者的血清HBV DNA水平显著高于RFLP-G组(P=0.02)和RFLP-C/G组(P=0 006),而且RFLP-C/G组患者血清ALT的阳性率显著低于RFLP-C、RFLP-E和RFLP-G组;当HBeAg阳性时,RFLP-C组患者的血清HBV DNA水平显著高于RFLP-G组(P=0.015)和RFLP-C/G组(P=0.008).结论 本研究中使用的PCR-RFLP能够用于检测核心基因的SNP,RFLP-C组患者的血清HBV DNA水平显著高于RFLP-G组和RFLP-C/G组,可能与Glu83 Asp突变有关.

Abstract：

Objective To investigate the relation between a set of single nucleotide polymorphisms (SNP) in core gene of HBV in chronic hepatitis B patients and HBV DNA levels. Methods PCR restriction fragment length polymorphism(PCR-RFLP) assay and restriction enzyme Tsp509I were adopted to determine HBV SNP in HBV core gene. Nucleotide sequences of core gene were determined using the dideoxy chain termination method. HBV DNA levels were quantitated with real-time PCR. Results Five typical RFLP patterns, RFLP-C, RFLP-D, RFLP-E, RFLP-G and RFLP-C/G mixture were found and the distribution of HBV RFLP patterns was as follows: C, 61. 5% ; D, 2. 6% ; E, 9.6%; G, 16.7%; C/G mixture, 9.6%. Five SNPs, A165T, A336C, A336T, T337C and T385C, were found to be associated with RFLP patterns change and only SNP A336C or A336T caused the substitution of Glu-83 with Asp in HBcAg. The serum HBV DNA levels in RFLP-C group were higher than that in RFLP-G (P =0. 02) and RFLP-C/G group(P = 0. 006) , respectively, furthermore, the positive rate of serum ALT in RFLP-C/G group was lower than that in RFLP-C, RFLP-E and RFLP-G group, respectively. Under the condition of HBeAg-positive, the serum HBV DNA levels in RFLP-C group were higher than that in RFLP-G (P = 0. 015) and RFLP-C/G group(P =0.008) , respectively. Conclusion PCR-RFLP used in this study can be adopted to determine HBV SNPs, not genotypes in Chinese patients with chronic hepatitis B. The serum HBV DNA level in RFLP-C group higher than that in RFLP-G or RFLP-C/G group maybe associated with amino acid mutation, Glu83 Asp.     

Analysis of the Hepatitis B virus precore and ORF-X sequences in patients with antibody to hepatitis B e antigen with and without normal ALT levels

Serum samples from 20 anti-hepatitis B e antigen-positive patients with and without normal alanine aminotransferase (ALT) levels who had serum hepatitis B virus (HBV) DNA detectable only by polymerase chain reation (PCR) were examined. Viral DNA was amplified by PCR, using primers that encompassed precore and ORF-X regions and sequenced directly, to investigate whether mutations in the nucleotide sequences of X and precore gene regions of HBV-DNA might be responsible for the difference in the activity of disease and in the levels of viral replication. The HBV-DNA concentration in patients with abnormal ALT levels was higher than in those with normal ALT. The amount of HBV-DNA correlated with the ALT levels (P < 0.05). Seventy-two percent of patients had HBV-DNA harboring the 1896 precore stop mutation, and there was a negative correlation between the percentage of precore mutant genotype and the HBV-DNA concentration (P < 0.05). Thirty percent of patients had mutations in ORF-X. Patients with ORF-X mutations had lower levels of HBV-DNA than those who had wild-type virus. The presence of mutations in precore and X regions may be related to a low HBV-DNA concentration and reduced biochemical activity in patients with anti-HBe. J. Med. Virol. 56:294–299, 1998 . © 1998 Wiley-Liss, Inc.     

Vaccination with a fusion DNA vaccine encoding hepatitis B surface antigen fused to the extracellular domain of CTLA4 enhances HBV-specific immune responses in mice: Implication of its potential use as a therapeutic vaccine

Fusion of specific antigens to extracellular domain of cytotoxic-T-lymphocyte-associated antigen 4 (CTLA4) represents a promising approach to increase the immunogenicity of DNA vaccines. We evaluated this interesting approach for its enhancement on HBV-specific immune responses and its antiviral effects in HBV transgenic mice. A fusion plasmid encoding the extracellular domain of CTLA4 linked with HBsAg was constructed. Mice were immunized by this fusion plasmid. Vaccination with the CTLA4-fused DNA not only induced much higher level of anti-HBs antibody, but also increased HBsAg-specific CD8+ response as well as CTL response in BALB/c mice. Furthermore, both Th1 and Th2 responses were augmented. In HBV transgenic mice, the levels of circulating HBsAg and HBV DNA replication were down-regulated by induction of higher anti-HBs antibody and HBsAg-specific CD8+ response after vaccination with the fusion plasmid. Thus, the CTLA4-fused DNA vaccine led to breakdown of immune tolerance to viral infection in HBV transgenic mice, which might be used as a therapeutic vaccine in HBV infection.     

高灵敏度乙肝HBV DNA检测在乙肝诊疗中的应用及临床意义

目的探讨高灵敏度乙肝HBV DNA检测在乙肝诊疗中的应用及临床意义。方法收集23例普通荧光定量PCR(常规)检测HBV DNA为阴性患者的血清,采用高灵敏度乙肝HBV DNA检测,将检测数据进行整理,与其临床实验室资料相结合,进行综合分析,归纳总结。结果23例国产乙肝荧光定量 PCR检测均为阴性患者,使用高灵敏度乙肝HBV DNA检测结果为13例阳性（最低检测限20IU/mL）,阴性10例,阳性检出率为56.52%,显然高灵敏度乙肝 HBV DNA检测的灵敏度高于常规检测方法。结论高灵敏度乙肝HBV DNA检测,其检测敏感性远高于常规检测方法,能检测到体内低水平的 HBV DNA,结合患者其他实验室资料及用药史综合分析,对乙型肝炎诊断和治疗具有重要的指导意义。