A novel hepatitis B virus mutant with A-to-G at nt551 in the surface antigen gene

AIM: Hepatitis B surface antigen (HBsAg) mutant of hepatitis B virus (HBV) is one of the important factors that result in immune escape and cause failure of immunization. In this study we reported and characterized a novel HBV mutant with A-to-G at nt551 and intended to provide theoretical data for prevention of HBV infection in China.METHODS: A methodology comprising polymerase chain reaction (PCR) amplifying, M13 bacteriophage cloning and nucleotide sequencing was used to analyze the sera of the pediatric patient who was hepatitis B (HB) immune failure.Expression plasmids containing the mutant S gene and a wild-type (adr) S gene were constructed respectively and the recombinant HBsAg were expressed in COS-7 cells under the regulation of SV40 early promoter. The recombinant proteins were investigated for their immunological reactivity with different monoclonal antibodies (mAb) against ‘a’determinant and vaccine-raised human neutralizing antibodies.RESULTS: It was found that there was a new point mutation at nt551 of the HBV (adr) genome from A to G, leading to a substitution of methionine (Met) to valine (Val) at position 133 in the ‘a’ determinant of HBsAg. Compared to the wildtype HBsAg, the binding activity of the muant HBsAg to mAbs (A6, All and S17) and to vaccine-raised human antihepatitis B surface antibody (anti-HBs) decreased significantly.CONCLUSION: According to the facts that the patient has been immunized with HB vaccine and that the serum is antiHBs positive and HBsAg negative, and based on the nucleotide sequence analysis of the mutant HBV S gene and its alteration of antigenicity, the HBV is considered to be a new vaccine-induced immune escape mutant different from the known ones.     

反向聚合酶链反应检测肝细胞癌中乙型肝炎病毒DNA整合

目的 应用反向聚合酶链反应(inverse polymerase chain reaction,IPCR)法检测肝细胞癌(HCC)中乙型肝炎病毒(HBV)DNA整合。方法 提取手术切除石蜡包埋入肝癌组织中DNA,根据IPCR原理,选用HBV DRl区无酶切位点的限制性内切酶酶切,通过连接反应形成环化DNA分子,以此作为模板进行PCR扩增得到已知序列的旁侧序列。琼脂糖凝胶电泳观察PCR扩增产物片段。结果 19例HCC标本中有14例检出整合型HBV DNA,2例同时存在游离型HBV DNA。结论 IPCR可以准确测定肝细胞中HBVDNA的整合。该方法为研究HBV DNA在肝细胞中的整合机制提供一简单、快速、经济途径。     

Novel assay of competitively differentiated polymerase chain reaction for screening point mutation of hepatitis B virus

AIM: Point mutation, one of the commonest gene mutations,is the most important molecular pathogenesis of cancer and chronic infection. The commonest methods for detection of point mutation are based on polymerase chain reaction (PCR). These techniques, however, cannot be used in large scale screening since they are neither accurate nor simple.For this reason, this study established a novel method of competitively differentiated PCR (CD-PCR) for screening point mutation in clinical practice.METHODS: Two competitively differentiated primers for mutant-type and wild-type templates respectively with an identically complemented region in 3‘ end except for last 2 base pairs and a different non-complemented region in 5‘ end were designed. Thus, competitive amplification might be carded out at a lower annealing temperature at first, and then differentiated amplification at a higher annealing temperature when primers could not combine with initial templates. The amplification was performed in one-tube.The products of CD-PCR were detected using microplate hybridization assay. CD-PCR was evaluated by detecting G1896A variant of hepatitis B virus (HBV) in form of recombinant plasmids and in sera from patients with hepatitis B, and compared with allele-specific PCR (AS-PCR) and competitive AS-PCR.RESULTS: CD-PCR was successfully established, it could clearly distinguish wild-type and mutant-type plasmid DNA of G1896A variant when the amount of plasmid DNA was between 102-108copies/reaction, while for AS-PCR and competitive AS-PCR, the DNA amount was between 10^2-10^4 copies/reaction. CD-PCR could detect one copy of G1896A variant among 10-100 copies of wild-type plasmid DNA. The specificity of CD-PCR was higher than those of AS-PCR and competitive AS-PCR in the detection of HBV G1896A variant in sera from patients with hepatitis B. CD-PCR was independent of the amount of HBV DNA in serum. HBV G1896A variant was more often found in HBeAg (-) patients with a lower level of detectable viremia than that with a higher level of detectable viremia (P=-0.0192).CONCLUSION: CD-PCR is more specific since it is less influenced by the amount of initial templates and the cross amplification between mutant- and wild-type amplified products. It is also simple and time-saving. Thus, CD-PCR might be useful in routine gene typing and point mutation screening. HBV G1896A or other more important mutations have to be routinely detected in patients with a detectable level of viremia after HBeAg/antibody conversion in clinical practice.     

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.     

引物差示PCR法快速检测乙型肝炎病毒前C区点突变

目的 乙型肝炎病毒（HBV）前C区第1896位核苷酸（nt)G1896→A1896的突变是HBeAg阴性HBV感染主要原因。方法 利用PCR引物3′末端碱基选择性配对的原理建立了引物差示PCR快速检测HBV前C区G1896→A1896点突变的方法。结果 实验结果表明,引物差示PCR系统对相应模板均能高效扩增。该系统对HBV检测具有较高的特异性,对野生型模板与突变型模板的选择性均达到了预期要求,可以用于对临床标本的分型检测。结论 该方法的建立,对于e抗原阴性的慢性乙型肝炎病人体内病毒复制状况监测以及e抗原阴转抗理的研究均具有一定的指导意义。     

Incidence of HBV variants with a mutation at nt551 among hepatitis B patients in Nanjing and its neighbourhood

AIM: To investigate the epidemiology of hepatitis B virus (HBV) strains with a mutation at nt551 in surface gene among hepatitis B patients in Nanjing and its neighbourhood. METHODS: By using mutation-specific polymerase chain reaction (msPCR) established by our laboratory for amplifying HBV DNAs with a mutation at nt551, 117 serum samples taken from hepatitis B patients were detected. RESULTS: The results showed that 112 samples were positive for nt551A, 4 samples were positive for nt551G. One sample was positive for nt551T. No nt551C of HBV DNA was found. The incidence of HBsAg mutants with G, C, T, A at nt551 among 117 samples was 3.42%, 0%, 0.85%, 95.73%, respectively. CONCLUSION: In Nanjing and its neighbourhood, hepatitis B patients are mainly infected with wild genotype HBV. The incidence of mutants with a mutation at nt551 in HBV genome is significantly lower than that in wild genotype HBV DNA (P<0.01). The necessity of adding components of HBsAg mutants to HBV vaccine needs further investigation.     

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 7.5x10~7 and 1.67x10~7 copies of HBV DNA per mL in two HBV infected patients' sera, while 2.14x10~5 and 3.02x10~5 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.     

应用竞争性荧光定量聚合酶链反应检测乙型肝炎病毒DNA

目的:建立竞争性荧光定量聚合酶链反应(CFQ-PCR),并探讨CFQ-PCR在乙型肝炎病毒(HBV)临床检测中的意义.方法:根据HBV病毒adr亚型基因组序列合成一对HBV特异的引物,和一条特异的TaqMan探针;根据上述引物序列,采用分子克隆技术制备内对照DNA;再根据内对照序列合成一条内对照DNA特异的与上述TaqMan探针不同标记的TaqMan探针;将适量的内对照DNA加入到PCR反应体系中,使其与HBV靶序列共扩增.结果:在30μL CFQ-PCR反应体系中,加入约20拷贝内对照DNA能够稳定地获得共扩增曲线;经琼脂糖凝胶电泳分析,加入约100-500拷贝内对照DNA能够有效地获得共扩增产物条带信号;在210个临床HBsAg阳性血清标本的CFQ-PCR扩增中识别出8个未能有效扩增的标本,60份HBsAg阴性血清标本中识别出2个内对照未能有效扩增的标本,后经DNA纯化处理,上述全部标本的内对照均获得阳性扩增结果,其中有7个HBsAg阳性血清标本获得HBV DNA扩增阳性结果.结论:CFQ-PCR能够有效地提示临床标本HBV DNA体外扩增时由于扩增失败导致的假阴性,适合临床推广应用.     

乙型肝炎病毒聚合酶链反应基因分型法的建立及应用

目前应用基因序列测定法分析乙型肝炎病毒(HBV)基因型虽然结果可靠,但操作较复杂,且费用较高,不适用于大规模的临床和流行病学调查。旨在建立一种简便的HBV基因分型法,即基因型特异引物PCR法,并用此法对2001年北京地坛医院110例慢性乙型肝炎患者的HBV基因型进行了分析。     

Screening blood donations for hepatitis C virus by polymerase chain reaction

BACKGROUND AND OBJECTIVES: The contamination of blood components by bacteria is an adverse event, which, although very uncommon, has an exceptionally high mortality rate. CASE REPORT: A patient suffering from terminal adenocarcinoma of the ovary received a red blood cell unit. During the transfusion, the patient developed fever. Cultures of both the patient's blood and the blood unit were done, and she was treated with antibiotics. Forty-eight and seventy-two hours after the transfusion, Candida parapsilosis grew in the blood cultures of the red blood cell bag and of the patient. The infection was controlled with amphotericin. The patient died from cancer progression. CONCLUSIONS We describe the first case of transfusion-associated sepsis caused by C. parapsilosis.     

Failure to detect hepatitis C virus genome in human secretions with the polymerase chain reaction.

Although hepatitis C infection has been clearly demonstrated to be transmitted through blood products or blood contamination, most cases of sporadic hepatitis C infection are unassociated with parenteral risk factors, and it is unclear how infection might be acquired by nonparenteral means. One potential mode of nonparenteral transmission is through body secretions. We used a highly sensitive and specific polymerase chain reaction assay to determine whether hepatitis C viral genomic RNA could be detected in secretions obtained from nineteen individuals with chronic hepatitis C virus infection. Although hepatitis C genomic RNA was found in all 19 sera, hepatitis C virus RNA was not detected in any samples of saliva, semen, urine, stool or vaginal secretions from these patients. Viral titers in serum ranged from 10(2) to 10(7) polymerase chain reaction units/ml. The sensitivity of our polymerase chain reaction assay indicates that, if hepatitis C virus were in secretions, it would be present in amounts less than 1 to 4 polymerase chain reaction units/ml. This contrasts with hepatitis B virus infection, in which serum titers frequently are in excess of 10(9) copies of hepatitis B genomes/ml. Body secretions have been found to contain up to 10(6) copies of hepatitis B genomes/ml. Our findings support seroepidemiological studies indicating that nonparenteral transmission of hepatitis C through secretions is uncommon and probably much less efficient than hepatitis B virus infection.     

Analysis of DNA polymerase reaction products for detecting hepatitis B virus in serum--comparison with spot hybridization technique

An assay for DNA polymerase reaction products using slab gel electrophoresis and autoradiography was compared with the spot hybridization technique for the detection of hepatitis B virus DNA in 317 blood samples. The former could identify the nature and size of DNA on electrophoresis, and reduce potentially false-positive results due to artifacts. Discordant results between the two methods occurred in 36 of 317 samples; 22 were positive by the spot technique alone, and 14 were positive by the analysis of DNA polymerase reaction products alone. However, the samples positive with the spot test alone showed weak radioactive signals on electrophoresis/autoradiography that were often interpreted as "inconclusive" by blind observations. Correlation of hepatitis B e antigen/antibody with hepatitis B virus DNA was studied in 91 patients with various chronic liver diseases. Discordant results, i.e., presence of the DNA in antibody positive sera, or its absence in the antigen positive sera, were obtained in 15 (19%) cases. Such patients tended to have advanced liver disease with fluctuating serum aminotransferase levels. Analysis of DNA polymerase reaction products by slab gel electrophoresis and autoradiography is not only sensitive, but is also as specific as the Southern blot technique in the detection of hepatitis B virus DNA in serum, and may prove useful in selected samples, especially where no cloned hepatitis B virus DNA is available, or in search of new hepatitis B virus-like viruses.     

Detection of hepatitis B virus DNA in serum by polymerase chain reaction. Application for clinical diagnosis

Standard methods of virus DNA detection using the polymerase chain reaction can be time consuming and can involve multiple steps in which contamination with exogenous DNA can occur. Therefore, we developed a simplified method for detecting hepatitis B virus DNA in serum. The main advantages of this method are that it can be performed rapidly, consists of only several steps, and has a false positive rate of less than 0.1% in our laboratory. In testing serial samples from five chimpanzees experimentally infected with hepatitis B virus, we found that hepatitis B virus DNA was detected 2-3 weeks before the appearance of hepatitis B surface antigen, and it continued to be detectable 1-3 weeks after the production of antibody to hepatitis B surface antigen. In testing serum from 84 human patients, we found hepatitis B virus DNA in all patients who had hepatitis B surface antigen and hepatitis B e antigen in serum and in 64% of the patients with hepatitis B surface antigen with antibody to hepatitis B e antigen in serum. Also, 3 out of 11 patients who were chronic hepatitis B carriers and who subsequently lost hepatitis B surface antigen were found to test positive for hepatitis B virus in serum. In contrast, all patients who lost hepatitis B surface antigen after acute hepatitis B virus infection or those classified as having non-A, non-B hepatitis tested negative for hepatitis B virus DNA. Thus, the modified PCR technique is a sensitive and rapid method for detecting hepatitis B virus DNA-containing virions in serum.     

The determination of serum hepatitis B virus DNA by polymerase chain reaction in hepatitis B patients treated with alpha-interferon]

To clarify the status of HBV in serum of chronic hepatitis B (CHB) patients who were treated with alpha-interferon, we determined the serum HBV DNA before and after treatment in 15 CHB patients with polymerase chain reaction (PCR). Before treatment all the 15 patients were HBsAg and HBeAg positive. HBV DNA was also positive with dot-blot hybridization (DB), PCR, ethidium bromide (PCR-EB) and PCR Southern-blot hybridization (PCR-SBH). HBeAb was negative in all the 15 patients. 12 patients has the determination repeated 2 to 39 weeks after treatment, 7 out of the 12 patients became HBeAg negative and HBV DNA also negative with DB and PCR-EB. However, in 5 of the 7 patients HBV DNA was still positive with PCR-SBH. Seroconversion of HBeAb from negative to positive occurred in 4 of the 12, but HBV DNA of the 4 patients remained positive with PCR-SBH. After an interval of one and half year or more following treatment 12 patients repeated the examination, only 5 of the 12 patients became seronegative for HBeAg and HBV DNA with DB and PCR-EB, but in 4 of the 5 HBV DNA was positive with PCR-SBH. Two of the 5 were seropositive for HBeAb and HBV DNA with PCR-SBH. The mechanism of residual viraemia after alpha-interferon treatment in CHB patients is uncertain.     

Evaluation of liver tissue by polymerase chain reaction for hepatitis B virus in patients with negative viremia

AIM: To assess the clinical significance of Hepatitis B virus (HBV) DNA localization in the liver tissue of patients with positive HBsAg and negative viremia. METHODS: HBV virological parameters of 33 HBsAg positive chronic hepatitis patients, including seromarkers and HBV DNA amplification in both sera and liver biopsies, were evaluated. RESULTS: Ten patients had negative viremia and positive HBV DNA in their liver biopsies. Most of them had HBeAg-negative/HBeAb-positive chronic hepatitis. Their liver biochemical and histopathological profiles were different from the viremic patients. Their disease pattern was designated as "hepatitis B in situ". CONCLUSION: Hepatitis B in situ is a consequential entity which can be missed in clinical practice. It is a new clinical pattern of chronic HBV infection that considers HBV in liver biopsy and adds a new indication for antiviral therapy.