无偿献血者乙型肝炎病毒酶免检测和核酸检测的结果研究

目的探讨乙型肝炎病毒(HBV)酶免检测和核酸检测对无偿献血者的应用价值。方法选取2017年1-12月采集到的16 787份血液样本,采用HBV酶免两种试剂检测及核酸检测方法进行筛查,筛选乙型肝炎表面抗原(HBs Ag)酶免试剂单边阳性或灰区标本及HBV核酸筛查阳性标本做比较分析。结果共选取609份酶免阳性标本,其中有37份是阳性,阳性标准符合率为100%;通过对479份血清HBs Ag酶免试剂检测单侧阳性或灰区标本进行统计分析,发现大多数单一酶免试剂检测阳性或灰区标本主要存在于B酶免试剂中,酶免结果灰区内的标本数量较多。通过对101份HBV核酸检测阳性标本进行统计分析,除了37份HBs Ag酶免试剂双阳性标本核酸都为阳性外,另外64份HBV核酸检测阳性标本中血清酶免试剂反应大多数为阴性。结论通过对HBV酶免检测及核酸检测结果进行比较判断和观察,HBV酶免弱阳性或灰区并不能减少HBV漏检的发生,联合HBV核酸检测可以有效缩短HBV窗口期。

9089例乙型肝炎血清学标志物检测结果分析

目的：探讨乙型肝炎（乙肝）病毒（HBV）血清学标志的表现摸式和本地区的感染情况.方法：将2009年3月15日～2011年3月15日2年的登记资料进行整理统计.结果：血清病毒学标志模式可分为感然期模式组和恢复期模式组,以及HBsAg阳性的不伴随HBsAg阳性模式组、3组共26组模式.结论：HBV血清免疫学标志的模式较为复杂.根据登记资料显示,有的是复检后发报告的、处个别检验误差外,产生少见模式的原因、可能由低滴度抗HBc/抗HBe;还有抗HBe//HBsAg同时存在,HBsAg与抗HBe的转移、HBsAg/抗HBc同时存在等.

乙型肝炎血清学标志物与HBV-DNA含量关系的探讨

目的：为了进一步探讨乙型肝炎患者血清中HBV-DNA含量与HBV血清学标志物之间的关系，了解荧光定量聚合酶链反应（FQ-PCR）检测乙型肝炎病毒的应用价值。方法：应用酶联免疫吸附试验（ELISA法）检测510例来自我院门诊就诊者和住院患者血清标本中的特异性抗原抗体，并同时应用荧光定量PCR分析系统测定血清标本中HBV-DNA的含量进行分析。结果：经ELISA法与FQPCR法检测的510例血清标本中，51例“大三阳”（HBsAg＋HBeAg＋HBcAb）的血清标本，其HBV—DNA检出数均呈阳性，阳性率为100％，其HBV-DNA的拷贝数范围为10^5～10^9／ml 76例“小三阳”（HBsAg＋HBeAb＋HBcAb）的血清标本，其HBV-DNA检出的阳性率为47．4％，HBV-DNA的拷贝数范围为10^4～10^7／ml。结论：HBV-DNA是乙型肝炎病毒感染和复制的特异性指标。应用FQ-PCR技术检测乙型肝炎病毒，具有较高的灵敏度和特异性，其操作简便，定量准确，结果可靠，它比HBV血清学标志物更能反映乙型肝炎病毒在体内的存在和真实复制状况，在HBV-DNA感染诊断，特别是药物疗效的观察中，具有良好的应用价值。因此，HBV-DNA与血清学标志物的相互关系是本文研究的课题。

拉米夫定耐药患者乙型肝炎病毒聚合酶基因G743C和G743A点突变的检测及分析

目的 探讨拉米夫定耐药患者乙型肝炎病毒聚合酶(HBV P)基因点突变。 方法 聚合酶链反应(PCR)扩增拉米夫定耐药患者的HBV P基因,产物经直接测序检测其YMDD变异;同时,用PCR-限制性片段长度多态性(PCR-RFLP)分析测序后的17例YIDD变异样本,先用3对引物扩增HBV P基因,再分别用3个限制性内切酶Fok I、Ssp I、Alw441酶切扩增产物,酶切产物用8.0％聚丙烯酰胺凝胶电泳分析。 结果 拉米夫定耐药患者HBV P基因PCR产物序列分析结果与GenBank的HBV标准株相比,发现16例存在HBV P基因G743C点突变和1例存在G743A点突变。其核苷酸序列由ATG变为ATC和ATA,YMDD基因序列发生变异,即由YMDD变异为YIDD;但是,PCR-RFLP不能确定这17例样本存在YIDD变异。 结论 拉米夫定耐药患者存在HBV P基因G743C和G743A的点突变,同样引起YIDD变异;而PCR-RFLP法不能准确地检测出G743C和G743A的点突变;该发现对HBV P基因YMDD变异检测和临床均有指导意义。

两种免疫分析方法检测乙肝病毒血清标志物的差异分析

目的:比较酶联免疫吸附试验分析方法(ELISA)和化学发光免疫法(CLIA)检测乙型肝炎病毒血清标志物(HBsAg、HBsAb、HBeAg、HBeAb和HBcAb)的差异。方法:采用ELISA法和CLIA法对2011年1～12月在我院传染病门诊首次就诊300例患者的血清样本进行平行检测乙型肝炎病毒血清标志物,分析两种免疫分析方法灵敏度和阳性结果符合性。结果:CLIA法检测血清HBsAg、HBsAb、HBeAg、HBeAb和HBcAb检测的灵敏度分别为0.2ng/ml、10mIU/ml、0.1Ncu/ml、1Ncu/ml和0.5Ncu/ml,均高于ELISA法。CLIA法和ELISA法测定300份血样HB-sAg、HBsAb、HBeAg和HBcAb结果差异无统计学意义(P>0.05),CLIA法测定HBeAb差异有统计学意义(P<0.05)。ELISA法相对于CLIA法测定HBsAg、HBsAb、HBeAg和HBcAb的阳性符合率分别为94.17%、96.23%、97.20%和92.42%,而HBeAb阳性符合率仅为65.52%。结论:采用CLIA检测乙型肝炎血清标志物的灵敏度和符合率优于ELISA法,并且可以直接对血清标志物表达进行定量,值得临床推广应用。

HBV聚合酶活性区变异与临床病情变化及HBsAg阳性相关性

研究乙型肝炎病毒(HBV)聚合酶活性区变异与临床病情变化及乙肝表面抗原(HBsAg)阳性的相关性。290例慢性乙型肝炎患者,HBV聚合酶活性区变异140例,其余无变异;分析HBV聚合酶活性区变异与肝功能及HBsAg阳性表达的关系。变异组患者的天门冬氨酸氨基转移酶(AST)、丙氨酸氨基转移酶(ALT)、碱性磷酸酶(ALB)和总胆红素(TBIL)肝功能指标水平显著高于无变异组(P<0.05);变异组肝细胞内HBsAg阳性率显著低于无变异组(P<0.05);变异组HBsAg以浆核型为主,约占阳性表达患者的47.25%,无变异组以核型为主,约占阳性表达患者的46.15%,两组比较,差异有统计学意义(P<0.05)。HBV聚合酶活性区变异的乙肝患者临床病情更重,HBV表面抗原阳性表达比例低。

聚合酶链反应在检测慢性HBsAg携带者血清HBV-DNA传染性研究中的应用

聚合酶链反应是一种对特定的DNA片段在体外进行快速扩增的新方法 ,操作简便 ,具有高度特异性和敏感性的生物学新技术。我们将其用于检测慢性HBsAg携带者血清HBV DNA ,研究其传染性和酶联免疫吸附技术(ELISA)检测血清HBsAg作对比。实验结果证明 ,聚合酶链反应直接检测血清HBV DNA比ELISA检测血清HBsAg更加敏感特异。在被检测的 1 2 31份血清标本中 ,用ELISA检出 91 9份慢性HBsAg携带者 ,血清HBsAg呈阳性结果。聚合酶链反应检测血清HBV DNA亦阳性 ,两者结果一致。而ELISA检测HBsAg阴性的 31 2份血清中 ,用聚合酶链反应检测又有 2 70份 (86 .5 %)可检测出HBV DNA ,总检出率 96 .6 %。此组研究证实血清HBsAg阴性标本仍有HBV DNA存在 ,具有传染性。琼脂糖凝胶电泳法比较简便 ,结果易观察。

荧光标记杂交双探针PCR融解曲线法在临床的应用评价

目的:对国产荧光标记杂交双探针PCR融解曲线法(FH—PCR—MC)检测乙型肝炎病毒聚合酶酪氨酸-蛋氨酸-天门冬氨酸-天门冬氨酸基序(HBVYMDD)变异的试剂进行临床应用评价.方法:慢性乙型肝炎患者外周血浆标本217份,分别采用荧光标记Taqman探针定量PCR法(FT—PCR)和荧光标记杂交双探针PCR融解曲线法(FH—PCR—MC)检测HBVDNA含量和YMDD及其变异;其中78份HBVDNA阳性标本采用基因型特异性多引物对巢式PCR法进行基因分型(A—F),30份标本用PCR产物克隆测序法检测HBVYMDD及其变异.结果:血浆标本HBVDNA阳性率(≥1.0×106copies/L)为75.6%(164/217).YMDD及其变异检出率为67.7%(147/217).其中YMDD44.9%(66/147),YIDD22.5%(33/147).YVDD17.7%(26/147),YIDD/YVDD混合株10.9%(16/147),其他混合株4.0%(6/147);结合HBVDNA含量,HBVYMDD及其变异的检出下限为HBVDNA=5.0×106copies/L,但在106copies/L时检出率较低,仅为36.4%,随着HBVDNA含量增高,检出率显著增高(>80%),且在107copies/L时即有显著增高(χ2=7.177,P<0.01).在基因分型的78份标本中,C基因型占89.8%,B和D基因型分别占8.9%和1.3%,YMDD及其变异总检出率为100%,变异总检出率为59.0%;1份D基因型YMDD及其变异检测为YIDD/YVDD混合变异;B,C两种基因型变异检出率分别为71.4%和55.7%,但二者无统计学差异.以测序法的检测结果为相对标准,则FH—PCR—MC法检测HBVYMDD及其变异的相对敏感性、特异性和符合率分别为96.3%(26/27),100%(3/3)和96.7%(29/30);对于HBVYMDD及其变异的类型,两种方法检测结果一致.结论:FH—PCR—MC法是一种快速、特异的HBVYMDD及其变异检测方法,具有较高的检出率,且能区分YMDD及其变异的类型.

Effective inhibition of expression of hepatitis B virus genes by DNAzymes

AIM: To evaluate the inhibitory effects of DNAzymes on the expressions of hepatitis B virus (HBV) s (HBsAg) and e (HBeAg) in 2.2.15 cells, and to explore the potential therapeutic effects of DNAzymes on replication of HBV genome. METHODS: DNAzymes DrzBS and DrzBC specific to HBV (aywsubtype) s gene ORF A^157UG and e gene ORF A^1816UG, were designed and synthesized. Inhibitory effects of DrzBS or DrzBC on the expressions of HBV s and e genes as well as HBV DNA levels in culture supernatants were observed in 2.2.15 cells. RESULTS: After being treated with DrzBS or DrzBC, the expression of HBV s or e genes in 2.2.15 cells was depressed dramatically. The maximum inhibition rate was 94.2% and 91.8% for DrzBS and DrzBC, respectively. The concentration for effective inhibition of both DrzBS and DrzBC was within 0.1-2.5 μmol/L, showing a dosedependence. The efficiency of inhibiting HBsAg, HBeAg in 2.2.15 cells by DrzBS or DrzBC was higher than that of the same target genes by antisense oligonucleotides (ASON). The concentration for effective inhibition of DNAzymes was at least 10-fold lower compared with ASON controls. Neither inhibition on the replication of HBV DNA nor toxicity to 2.2.15 cells was observed. CONCLUSION: DrzBS and DrzBC can block the expression of HBV s- and e-genes in 2.2.15 cells and provide a specific and effective anti-HBV gene therapeutic means.

Rapid quantification of semen hepatitis B virus DNA by real-time polymerase chain reaction

AIM： To examine the sensitivity and accuracy of real-time polymerase chain reaction （PCR） for the quantification of hepatitis B virus （HBV） DNA in semen. METHODS： Hepatitis B viral DNA was isolated from HBV carriers＇ semen and sera using phenol extraction method and QIAamp DNA blood mini kit （Qiagen, Germany）. HBV DNA was detected by conventional PCR and quantified by TaqMan technology-based real-time PCR （quantitative polymerase chain reaction （qPCR））. The detection threshold was 200 copies of HBV DNA for conventional PCR and 10 copies of HBV DNA for real time PCR per reaction. RESULTS： Both methods of phenol extraction and QIAamp DNA blood mini kit were suitable for isolating HBV DNA from semen. The value of the detection thresholds was 500 copies of HBV DNA per mL in the semen. The viral loads were 7.5×10^7 and 1.67×10^7 copies of HBV DNA per mL in two HBV infected patients＇ sera, while 2.14×10^5 and 3.02×10^5 copies of HBV DNA per mL in the semen. CONCLUSION： Real-time PCR is a more sensitive and accurate method to detect and quantify HBV DNA in the semen.

Tenofovir rescue therapy for chronic hepatitis B patients after multiple treatment failures

AIM:To evaluate the efficacy and safety of tenofovir disoproxil fumarate(TDF) for chronic hepatitis B(CHB) patients after multiple failures.METHODS:A total of 29 CHB patients who had a suboptimal response or developed resistance to two or more previous nucleoside/nucleotide analogue(NA) treatments were included.Study subjects were treated with TDF alone(n = 13) or in combination with lamivudine(LAM,n = 12) or entecavir(ETV,n = 4) for ≥ 6 mo.Complete virologic response(CVR) was defined as an achievement of serum hepatitis B virus(HBV) DNA level ≤ 60 IU/mL by real-time polymerase chain reaction method during treatment.Safety assessment was based on serum creatinine and phosphorus level.Eleven patients had histories of LAM and adefovir dipivoxil(ADV) treatment and 18 patients were exposed to LAM,ADV,and ETV.Twenty-seven patients(93.1%) were hepatitis B e antigen(HBeAg) positive and the mean value of the baseline serum HBV DNA level was 5.5 log IU/mL ± 1.7 log IU/mL.The median treatment duration was 16 mo(range 7 to 29 mo).RESULTS:All the patients had been treated with LAM and developed genotypic and phenotypic resistance to it.Resistance to ADV was present in 7 patients and 10 subjects had a resistance to ETV.One patient had a resistance to both ADV and ETV.The cumulative probabilities of CVR at 12 and 24 mo of TDF containing treatment regimen calculated by the Kaplan Meier method were 86.2% and 96.6%,respectively.Although one patient failed to achieve CVR,serum HBV DNA level decreased by 3.9 log IU/mL from the baseline and the last serum HBV DNA level during treatment was 85 IU/mL,achieving near CVR.No patients in this study showed viral breakthrough or primary non-response during the follow-up period.The cumulative probability of HBeAg clearance in the 27 HBeAg positive patients was 7.4%,12%,and 27% at 6,12,and 18 mo of treatment,respectively.Treatment efficacy of TDF containing regimen was not statistically different according to the presence of specific HBV mutations.History of prior exposure to specific antiviral agents did not make a difference to treatment outcome.Treatment efficacy of TDF was not affected by combination therapy with LAM or ETV.No patient developed renal toxicity and no cases of hypophosphatemia associated with TDF therapy were observed.There were no other adverse events related to TDF therapy observed in the study subjects.CONCLUSION:TDF can be an effective and safe rescue therapy in CHB patients after multiple NA therapy failures.

Intramuscular vs intradermal route for hepatitis B booster vaccine in celiac children

AIM： To compare intradermal （ID） and intramuscular （IM） booster doses, which have been used in healthy and high risk subjects, such as healthcare workers, haemodialysis patients, human immunodeficiency virus patients, and renal transplant recipients unresponsive to initial hepatitis B vaccination, in celiac individuals. METHODS： We conducted our study on 58 celiac pa- tients, vaccinated in the first year of life, whose blood analysis had showed the absence of protective hepati- tis B virus （HBV） antibodies. All patients had received the last vaccine injection at least one year before study enrolment and they had been on a gluten free diet for at least 1 year. In all patients we randomly performed an HBV vaccine booster dose by ID or IM route. Thirty celiac patients were revaccinated with recombinant hepatitis B vaccine （Engerix B） 2 μg by the ID route, while 28 celiac patients were revaccinated with Engerix B 10 μg by the IM route. Four weeks after every boost- er dose, the anti-hepatitis B surface （HBs） antibody titer was measured by an enzyme-linked immune- adsorbent assay. We performed a maximum of three booster doses in patients with no anti-HBs antibodies after the first or the second vaccine dose. The cut off value for a negative anti-HBs antibody titer was 10 IU/L.Patients with values between 10 and 100 IU/L were considered ＂low responders＂ while patients with an antibody titer higher than 1000 IU/L were considered ＂high responders＂. RESULTS： No significant difference in age, gender, du- ration of illness, and years of gluten intake was found between the two groups. We found a high percent- age of ＂responders＂ after the first booster dose （ID = 76.7%, IM = 78.6%） and a greater increase after the third dose （ID = 90%, IM = 96.4%） of vaccine in both groups. Mloreover we found a significantly higher num- ber of high responders （with an anti-HBs antibody titer 〉 1000 IU/L） in the ID （40%） than in the IM （7.1%） group, and this difference was evident after the first booster dose of vaccination （P 〈 0.01）. No side effects were recorded in performing delivery of the vaccine by either the ID or IM route. CONCLUSION： Our study suggests that both ID and IM routes are effective and safe options to administer a booster dose of HBV vaccine in celiac patients. Howev- er the ID route seems to achieve a greater number of high responders and to have a better cost/benefit ratio.

Long-term effects of lamivudine treatment in Japanese chronic hepatitis B patients

AIM： To analyze the association between the emergence of tyrosine-methionine-asparatate-asparatate （YMDD） mutants （reverse transcription; rtM204I/V） and deterioration of liver function during long-term lamivudine treatment of Japanese patients with chronic hepatitis B virus （HBV） infection. METHODS： The data of 61 consecutive Japanese pa- tients with chronic hepatitis B who underwent continu- ous lamivudine treatment for more than 24 mo and had a virological response were analyzed. Analysis of YMDD mutants was done by real-time polymerase chain reaction with LightCycler probe hybridization assay for up to 90 mo （mean, 50.8 too; range, 24-90 too）.RESULTS： A mixed mutant-type （YMDD ＋ tyrosine-iso- leucine-asparatate-asparatate： YIDD or tyrosine-valineasparatate-asparatate： YVDD） or a mutant-type （YIDD or YVDD） were found in 57.4% of 61 patients at i year, 78.7% of 61 patients at 2 years, 79.6% of 49 patients at 3 years, 70.5% of 34 patients at 4 years, 68.4% of 19 patients at 5 years, 57.1% of 14 patients at 6 years, and 33.3% of 6 patients at 7 years. Of the 61 patients, 56 （92%） had mixed mutant- or a mutant-type. Only 5 （8%） had no mutants at each observation point. Vi- rological breakthrough was found in 26 （46.4%） of 56 patients with YMDD mutants, 20 of whom had a hepa- titis flare-up： the remaining 30 （53.6%） had neither a virological breakthrough nor a flare-up. All 20 patients who developed a hepatitis flare-up had a biochemical and virological response after adefovir was added to the lamivudine treatment. CONCLUSION： Our results suggest that it is possible to continue lamivudine treatment, even after the emergence of YMDD mutants, up to the time that the patients develop a hepatitis flare-up.

Hepatitis B virus genotypes in southwest Iran: Molecular, serological and clinical outcomes

AIM: To investigate the associations of hepatitis B virus (HBV) genotype with HBeAg and anti-HBe status, alanine aminotransferase (ALT) levels and HBV-DNA detection in different groups of HBV-infected patients in southwest Iran. METHODS: A total of 89 HBsAg-positive serum samples were collected from the same number of patients. All sera were then investigated to determine HBV DNA and serological markers. For all the polymerase chain reaction (PCR)-positive samples, biochemical, histopathological assays and genotyping were also performed. RESULTS: Genotype D was the only type of HBV foundin different clinical forms of acute and chronic infections. There was a high prevalence of HBeAg-negative HBV- infected patients with chronic hepatitis (52.7%). Out of 55 patients with chronic hepatitis, seven (12.7%) were diagnosed with cirrhosis. A significant association between the presence of anti-HBe antibody and an increase in ALT level, among either HBeAg-negative (P = 0.01) or HBeAg-positive (P = 0.026) patients, was demonstrated. No significant differences were observed between the clinical outcomes of HBeAg-positive and -negative individuals (P = 0.24). CONCLUSION: Genotype D has been recognized as the only type of HBV found in different clinical forms of HBV infections, including cirrhosis, among the residents of southwest Iran. Anti-HBe possibly plays a role in disease progression in some patients with chronic hepatitis, at least for a period of disease.

Mutations in surface and polymerase gene of chronic hepatitis B patients with coexisting HBsAg and anti-HBs

AIM： To investigate the clinical significance and presence of mutations in the surface （S） and overlapping polymerase gene of hepatitis B patients with coexisting HBsAg and anti-HBs. METHODS： Twenty-three patients with chronic hepatitis B were studied. Of the 23 patients, i i were both positive for hepatitis B virus （HBV） surface antigen （HBsAg） and antibody to HBV surface antigen （anti-HBs）, 12 were negative for anti-HBs while positive for HBsAg. DNA was extracted from 200 μL serum of the patients. Nucleotide of the surface and overlapping polymerase gene from HBV-infected patients was amplified by PCR, and the PCR products were sequenced. RESULTS： Forty-one mutations were found within the surface gene protein of HBV in 15 patients （10 with coexisting HBsAg and anti-HBs）. Six （14.6%） out of 41 mutations were located at ＂α＂ determinant region in 5 patients （4 positive for HBsAg and anti-HBs）. Eleven mutations （26.8%） occurred in the downstream or upstream of ＂α＂ determinant region. Lamivudine （LMV）- selected mutations were found in three patients who developed anti-HBs, which occurred in amino acid positions （196, 198, 199） of the surface protein and in YMDD motif （M204I/V） of the polymerase protein simultaneously. Presence of these mutations did not relate to changes in ALT and HBV DNA levels.CONCLUSION： Besides mutations in the ＂α＂ determinant region, mutations at downstream or upstream of the ＂α＂ determinant region may contribute to the development of anti-HBs. These mutations do not block the replicating competency of HBV in the presence of high titer of anti-HBs.

Optimization of competitively differentiated poiymerase chain reaction in detection of HBV basal core promoter mutation

AIM:To improve competitively differentiated polymerase chain reaction (CD-PCR) in detection of HBV basal core promoter mutation. METHODS: Recombinant plasmid of double point mutation A1762T/G1764A in basal core promoter of HBV constructed by site-directed mutagenesis was used as mutant control. To reveal the deficiency mechanism of CD-PCR, relationship between the circle number of PCR and the increased speed of products of each competitive primer was comparatively studied. Diversified amount of dNTPs and mutual primer of the competitive primers were tried to optimize CD-PCR. Optimized CD-PCR was evaluated by detecting A1762T/G1764A mutation in recombinant plasmids and clinical sera from patients with HBV infection. RESULTS: The deficiency mechanism of CD-PCR was that the products of mismatched competitive primer grew fast when the amplification of matched primer entered into plateau stage, which led to decrease in or disappearance of the difference in the amount of their products. This phenomenon could be eliminated by reducing dNTPs to 10μmol/L and mutual primer to about 100μmol/L Optimized CD-PCR could detect both mutant and wild strain independent of the amount of templates and the number of PCR cycles. Its detection limit was 103 copies/mL, about 50 copies/reaction. About 10% of mutant DNAs among wild type DNAs could be detected. A1762T/G1764A mutant was detected in 41.8% (51/122) of patients with HBV infection, but not detected in controls with negative HBsAg. CONCLUSION: Optimized CD-PCR can detect mutation independent of the amount of initial templates and the number of PCR cycles.

Detection of YMDD.mutants using universal template real-time PCR

AIM： To establish a rapid and accurate method for the detection of lamivudine-resistant mutations in hepatitis B virus and monitor of lamivudine resistance during lamivudine treatment in patients with chronic hepatitis B virus infection. METHODS： We established a real-time PCR method using a universal template and TaqMan probe to detect YMDD mutants. Variants of YVDD and YIDD were tested by individual reactions （reaction Ⅴ and reaction Ⅰ） and total hepatitis B viruses were detected in another reaction for control （reaction C）. Results were determined by △Ct〈3.5 （△Ct = Ct of reaction Ⅴ or Ⅰ - Ct of reaction C）. Clones of the HBV polymerase gene containing different YMDD mutations were tested. Serum samples from 163 lamivudine-treated patients with chronic hepatitis B virus infection were detected using this method and the results were confirmed by DNA sequencing. RESULTS： As many as 1000 copies per milliliter of widetype plasmid were detected and nonspecific priming was excluded. In the 163 samples from patients treated with lamivudine, lamivudine-resistant mutations were detected in 51 samples. CONCLUSION： This universal real-time PCR is a rapid and accurate method for quantification of YMDD mutants of HBV virus in lamivudine-treated patients and can be used to monitor lamivudine-resistant mutations before and during lamivudine therapy.

Hepatitis B virus X protein promotes liver cell proliferation via a positive cascade loop involving arachidonic acid metabolism and p-ERK1/2

Hepatitis B virus X protein （HBx） plays a crucial role in the development of hepatocellular carcinoma. Here, we sought to identify the mechanisms by which HBx mediates liver cell proliferation. We found that HBx upregulated the levels of cyclooxygenase-2 （COX-2）, 5-1ipoxygenase （5-LOX） and phosphorylated extracellular signal-regulated protein kinases 1/2 （p-ERK1/2） in liver cells. HBx-induced p-ERK1/2 was abolished by inhibition of Gi/o proteins, COX or LOX. In addition, HBx increased the amounts of prostaglandin E2 （PGE2） and leukotriene B4 （LTB4） released from cell lines derived from hepatocytes. Moreover, these released arachidonic acid metabolites were able to activate ERK1/2. Interestingly, activated ERK1/2 could upregulate the expression of COX-2 and 5-LOX in a positive feedback manner. In conclusion, HBx enhances and maintains liver cell proliferation via a positive feedback loop involving COX-2, 5-LOX, released arachidonic acid metabolites, Gi/o proteins and p-ERK1/2.

N-terminal and C-terminal cytosine deaminase domain of APOBEC3G inhibit hepatitis B virus replication

AIM: To investigate the effect of human apolipoprotein B mRNA-editing enzyme catalytic-polypeptide 3G (APOBEC3G) and its N-terminal or C-terminal cytosine deaminase domain-mediated antiviral activity against hepatitis B virus (HBV) in vitro and in vivo. METHODS: The mammalian hepatoma cells HepG2 and HuH7 were cotransfected with APOBEC3G and its N-terminal or C-terminal cytosine deaminase domain expression vector and 1.3-fold-overlength HBV DNA as well as the linear monomeric HBV of genotype B and C. For in vivo study, an HBV vector-based mouse model was used in which APOBEC3G and its N-terminal or C-terminal cytosine deaminase domain expression vectors were co-delivered with 1.3-fold-overlength HBV DNA via high-volume tail vein injection. Levels of hepatitis B virus surface antigen (HBsAg) and hepatitis B virus e antigen (HBeAg) in the media of the transfected cells and in the sera of mice were determined by ELISA.The expression of hepatitis B virus core antigen (HBcAg) in the transfected cells was determined by Western blot analysis. Core-associated HBV DNA was examined by Southern blot analysis. Levels of HBV DNA in the sera of mice as well as HBV core-associated RNA in the liver of mice were determined by quantitative PCR and quantitative RT-PCR analysis, respectively. RESULTS: Human APOBEC3G exerted an anti-HBV activity in a dose-dependent manner in HepG2 cells, and comparable suppressive effects were observed on genotype B and C as that of genotype A. Interestingly, the N-terminal or C-terminal cytosine deaminase domain alone could also inhibit HBV replication in HepG2 cells as well as Huh7 cells. Consistent with in vitro results, the levels of HBsAg in the sera of mice were dramatically decreased, with more than 50 times decrease in the levels of serum HBV DNA and core-associated RNA in the liver of mice treated with APOBEC3G and its N-terminal or C-terminal cytosine deaminase domain as compared to the controls. CONCLUSION: Our findings provide probably the first evidence showing that APOBEC3G and its N-terminal or C-terminal cytosine deaminase domain could suppress HBV replication in vitro and in vivo.

YMDD variants of HBV DNA polymerase gene: Rapid detection and clinicopathoiogical analysis with long-term Iamivudine therapy after liver transplantation

AIM: To look for a rapid low-cost technique for the detection of HBV variants.METHODS: Two patients who underwent orthotopic liver transplantation (OLT) for HBV infection were treated with lamivudine (100 mg daily) and HBV infection recurred in the grafted livers. The patients were monitored intensively for liver enzymes, hepatitis B surface antigen (HBsAg) and HBV DNA in serum. Liver biopsy was performed regularly. HBV DNA in a conserved polymerase domain (the YMDD locus) was amplified from serum of each patient by PCR and sequenced. HBV genotypes were analyzed by restriction fragment length polymorphism (RFLP) of the PCR products generated from a fragment of the polymerase gene.RESULTS: YMDD wild-type HBV was detected in one patient by PCR-RFLP and DNA sequencing 19 mo after OLT, and YIDD mutant-type HBV in the other patient, 16 mo after OLT.CONCLUSION: PCR-RFLP assay is an accurate and simple method for genotyping lamivudine-resistant HBV variants.