多种方法检测HBV血清标志物结果分析

ＰＣＲ技术直接检测血清中的ＨＢＶ－ＤＮＡ［１］，并与反向间接血凝法（Ｒ－ＩＨＡ）、乳胶凝集法（ＬＡＴ）和酶联免疫吸附法（ＥＬＩＳＡ）检测乙型肝炎血清标志物进行对比观察，以探讨ＰＣＲ技术在乙型肝炎病原诊断中的应用及ＨＢｖ－ＤＮＡ与乙肝五项标志物（ＨＢＶ－ｍ）之间的关系。材料与方法检测对象：１４６例肝炎患者，其中急性肝炎９１例，慢性肝炎４１例，肝炎后肝硬化１１例，重症肝炎３例，均符合１９９０年上海全国病毒性肝炎会议制订的诊断标准。方法：１４６例均用ＰＣＲ技术俭测血清中ＨＢＶ－ＤＮＡ，用Ｒ－ＩＨＡ检测…     

应用聚合酶链反应技术检测肝组织中的HBV－DNA

采用聚合酶链反应（ＰＣＲ）技术，对４２例肝活切组织石蜡切片中乙型肝炎病毒（ＨＢＶ）ＤＮＡ进行检测，并与乙肝表面抗原（ＨＢｓＡｇ）的免疫组织化学及血清学检测进行比较，ＨＢＶ－ＰＣＲ阳性率为７３．８％，高于组织及血清ＨＢｓＡｇ阳性率（分别为５９．５％和５０．０％）。３例病理形态呈肝炎改变，而血清ＨＢｓＡｇ（─）的肝组织中有２例检出ＨＢＶ－ＤＮＡ，提示ＰＣＲ的高度敏感性和准确性。８３．３％的门脉性肝硬变和８７．５％的肝细胞癌组织中ＨＢＶ－ＰＣＲ呈阳性，进一步证实了上述两病与ＨＢＶ的关系密切。我们还发现肝细胞淤胆患者ＨＢＶ感染率较高，ＨＢＶ－ＤＮＡ及组织ＨＢｓＡｇ阳性比例各为６／９和４／８。     

PCR鉴定HBsAg阴性肝炎HBV DNA感染的意义

ＰＣＲ鉴定ＨＢｓＡｇ阴性肝炎ＨＢＶＤＮＡ感染的意义１李伟１王学春１张金池２刁玉贞收集ＨＢｓＡｇ阴性ＨＢｃＡｂ阳性的慢性肝炎病人血清３０例及恢复期急性乙型肝炎病人血清１００例（共计１３０例），利用聚合酶链反应（ＰＣＲ）检测ＨＢＶＤＮＡ。ＰＣＲ方法及步骤...     

HBcAg/HBeAg对慢性乙型肝炎患者PBMC中Th1/Th2类细胞应答的影响

目的 探讨ＨＢｃＡｇ／ＨＢｅＡｇ对慢性乙型肝炎患者ＰＢＭＣ中Ｔｈ１／Ｔｈ２类细胞应答的影响。方法 用套式ＰＣＲ法检测６４便慢性ＨＢＶ感染者ＰＢＭＣ中ＨＶＢ ＤＮＡ;分别用ＰＨＡ、ＨＢｃＡｇ和ＨＢｅＡｇ体外培养;ＥＬＩＳＡ法检测ＰＢＭＣ产生Ｔｈ１类细胞因子（ＩＬ－２、ＩＦＮ－γ）和Ｔｈ２类细胞因子（ＩＬ－４、ＩＬ－１０）的含量。结果 表明ＨＢＶ ＤＮＡ阳性组和阴性组相比,无论是在ＰＨＡ还是在ＨＢｃＡ     

用聚合酶链反应检测人肝细胞癌乙型肝炎病毒DNA和丙型肝炎病毒RNA

为研究乙型肝炎病毒ＤＮＡ（ＨＢＶＤＮＡ）和丙型肝炎病毒ＲＮＡ（ＨＣＶＤＮＡ）与肝细胞癌的关系，用聚合酶链反应（ＰＣＲ）和巢式ＰＣＲ（ｎｅｓｔｅｄ－ＰＣＲ）分别检测４２例肝肿瘤组织中ＨＢＶＤＮＡ和ＨＣＶＲＮＡ。结果：１例胆管细胞癌组织ＨＢＶＤＮＡ和ＨＣＶＲＮＡ均阳性，１例胆管囊腺瘤ＨＢＶＤＮＡ阳性。４０例肝细胞癌组织中，单纯ＨＢＶＤＮＡ阳性１９例，单纯ＨＣＶＲＮＡ阳性３例，二者均阳性１０例。ＨＢＶＤＮＡ阳性率７２．５％，ＨＣＶＲＮＡ阳性率３２．５％。ＨＢＶＤＮＡ和ＨＣＶＲＮＡ感染与肝癌组织学分型无关；且肝细胞癌中ＨＣＶ感染与ＨＢＶ未见相关。结果提示，我国ＨＢＶ感染仍是引起肝细胞癌的主要原因。但由于肝细胞癌患者中ＨＣＶ的感染率也较高，且有上升趋势，因此ＨＣＶ可能也是肝细胞癌发生的重要原因之一。     

孕产妇和新生儿血清乙肝免疫标志物与HBV—DNA检测结果的比较…

利用ＨＢＶ－ＤＮＡ出现先于血清其它血清学指标理论依据。采用聚合酶链式反应（ＰＣＲ）对乙肝免疫标志物至少一项改变的２００例孕产妇、新生儿血清进行ＰＣＲ扩增，结果有１０８份标本ＨＢＶ ＤＮＡ阳性、总阳性率为５４％。其中ＨＢｅＡｇ阳性率８３．３％（７０／８４），ＨＢｓＡｇ阳性率４８％（２４／５０），ＨＢｅＡｂ阳性率１０．５％（２／１９），ＨＢｓＡｂ９．８％（２／２２），可见ＰＣＲ早期诊断，判断其传染性，     

肝硬变内HBV DNA及其五种抗原的表达及意义

取２２５例人肝硬变活检组织石蜡切片，检测了ＨＢＶＤＮＡ及其５种抗原。分别用免疫组化ＡＢＣ法检测ＨＢｘＡｇ、ｐｒｅ－Ｓ＿１和ｐｒｅ－Ｓ＿２抗原；用ＰＡＰ法检测ＨＢｓＡｇ和ＨＢｃＡｇ；用原位杂交方法检测ＨＢＶＤＮＡ；用免疫组化、原位杂交双标记方法检测ＨＢＶＤＮＡ和ＨＢｓＡｇ、ＨＢｘＡｇ或ＨＢｃＡｇ。结果显示，阳性检出率ＨＢｓＡｇ为７０．０％（１２８／１８３例），ｐｒｅ－Ｓ＿１抗原为６４．４％（８５／１３２例）、ｐｒｅ－Ｓ＿２抗原为６１．４％（８１／１３２例），ＨＢｘＡｇ为７５．３％（１１３／１５０例），ＨＢｃＡｇ为２２．４％（３９／１７４例），ＨＢＶＤＮＡ为６２．４％（５８／９３例）。双标阳性检出率ＨＢＶＤＮＡ和ＨＢｓＡｇ为３７．３％（１９／５１例），ＨＢＶＤＮＡ和ＨＢｘ－Ａｇ为８６．３％（４４／５１例），ＨＢＶＤＮＡ和ＨＢｃＡｇ为３９．２％（２０／５１例）。ＨＢＶＤＮＡ和ＨＢＶ５种抗原阳性病例中８０％以上均伴有肝细胞不典型增生。这一结果表明，在我国肝硬变的发生发展与ＨＢＶ慢性感染有密切的关系。     

PCR和ELISA检测单纯疱疹病毒的比较

依据ＨＳＶ ＤＮＡ多聚酶基因核苷酸序列设计并合成一对引物，建立了ＰＣＲ方法。用ＰＣＲ和ＥＬＩＳＡ平行检测ＨＳＶ－１，ＨＳＶ－２标准毒株感染的细胞，拟诊ＨＳＶ脑炎患者脑脊液（ＣＳＦ）及多种对照标本。结果表明，两方法特异性完全一致；ＰＣＲ检测阳性率高，比ＥＬＩＳＡ敏感近２０倍，更适用于ＨＳＶ脑炎的早期诊断。     

抗HSV糖蛋白单克隆抗体可变区基因的克隆和序列测定

选用具有动物体内高保护作用的抗ＨＳＶ－１／ＨＳＶ－２型共同性糖蛋白Ｃ（ｇＣ）单克隆抗体１Ａ１２，从其杂交瘤细胞中提取总ＲＮＡ，以Ｏｌｉｇｏ（ｄＴ）１５为引物反转录合成ｃＤＮＡ，用一对鼠抗体通用ＶＨ引物和一对Ｖ引物进行ＰＣＲ，扩增出１Ａ１２ＶＨ和１Ａ１２ＶＬ基因，并分别克隆入ｐＵＣ１８质粒中，序列分析结果表明，１Ａ１２ＶＨ基因由３３６ｂｐ组成，编码１１２个氨基酸，隶属于小鼠重链第３组亚组（Ｄ）；１Ａ     

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。     

Hepatitis B surface antigen particles with all four subtypic determinants: point mutations of hepatitis B virus DNA inducing phenotypic changes or double infection with viruses of different subtypes.

Hepatitis B surface antigen (HBsAg) particles carry the common determinant, a, as well as d or y and w or r subtype determinants, and are classified into the four major subtypes, i.e., adw, adr, ayw and ayr. Rare sera contain HBsAg particles with all four subtype determinants (adywr). Target sequences (nucleotides 38-550) in the S gene of hepatitis B virus (HBV) DNA in two such sera were amplified by the polymerase chain reaction. Individual amplification products were cloned in an M13 phage vector. The HBV DNA clones obtained were subtyped by determining the second letters of codon 122 and 160 for lysine (AAA/AAG) or arginine (AGA/AGG), which specify the d or y and w or r determinants, respectively. From one serum (S-63), two adw, 10 adr and 58 ayr clones were obtained. When the two adw clones and two representatives each of the adr and ayr clones were compared against each other, for the sequence of 235 base pairs representing nucleotides 295-529 in the S gene, they differed only by 0.4-2.1% (average 1.2%). These results indicated multiple point mutations of a single HBV strain of subtype ayr and co-infection of hepatocytes with the original HBV strain and its mutant of subtype adw as the mechanism for the production of HBsAg/adywr particles. From the other serum (K-45), 1 adw, 73 adr and 4 ayw clones were obtained. The adw clone and two representative adr clones differed only by 0-1.7% in the S gene sequences, but they differed by 8.5% or greater from two representative ayw clones. HBsAg/adywr particles in this serum, therefore, could be explained by double infection of hepatocytes with two HBV strains of different subtypes (adr and ayw).     

Hepatitis B virus genotypes and HBsAg subtypes in refugees and injection drug users in the United States determined by LiPA and monoclonal EIA.

Hepatitis B virus (HBV) genotyping and hepatitis B surface antigen (HBsAg) subtyping were carried out on sera from 196 HBsAg-positive patients, including 151 refugees entering the United States and 45 injection drug users in Seattle. HBsAg subtyping was performed by enzyme immunoassay (EIA) using a panel of monoclonal antibodies and the HBV genotype was determined by polymerase chain reaction (PCR) followed by detection of amplified HBV DNA by a reverse-phase hybridization line probe assay (LiPA) using genotype-specific probes. HBV DNA was detected by PCR in 155 (79%) of the 196 sera and all 155 were genotyped by LiPA. Samples from Southeast Asia were predominantly genotype B/subtype ayw1 and genotype C/adr; samples from the former Soviet Union and eastern Europe were mostly genotype D/ayw2 and genotype D/ayw3; samples from east Africa were mainly genotype A/adw2 and genotype D/ayw2; and samples from injection drug users were mostly genotype D/ayw3 and genotype A/adw2. Some strains of ayw3 gave atypical monoclonal antibody reactivity patterns in the subtyping assay due to a Val/Ala instead of a Thr at amino acid residue 118 and a Thr instead of a Met at residue 125. A strain of ayw2 also gave an atypical monoclonal antibody reactivity pattern due to an Ala instead of a Thr at amino acid residue 123. LiPA genotyping and monoclonal EIA subtyping can provide useful information for epidemiological studies.     

Discrimination of hepatitis B virus (HBV) subtypes using monoclonal antibodies to the PreS1 and PreS2 domains of the viral envelope.

We report the production and characterization of murine anti-PreS2 and anti-PreS1 monoclonal antibodies (mAb) and demonstrate their utility in discriminating hepatitis B virus (HBV) subtypes. On the basis of Western blotting and reciprocal competition binding to HBV virions, at least five distinct epitopes have been identified in the PreS domain: two within the PreS1 region and three within the PreS2 region. All PreS2 mAb bind M protein (gp33 and gp36) but only one group binds strongly to M and L proteins (p39 and gp42). This group determinant was mapped to peptide residues 120-145. The second group bound to an endoglycosidase F-sensitive epitope which is defined by a mannose-rich glycan at ASN 123 in the PreS2 region. The third group was mapped to peptide residues 150-174 and was reactive with the M envelope proteins but not L or S proteins on Western blots. All PreS1 mAb bind L protein but not M protein on Western blots. Using these mAb, HBV subtype assays were developed allowing evaluation of the Paris (1975) HBsAg subtype panel members along with other HBsAg-positive specimens. All Paris subtype members (except ayw2 and ayw3) could be easily distinguished by differential PreS2 mAb reactivity. The Paris subtypes, adw2, adw4, and adr, could be classified as distinct groups by PreS2 and PreS1 mAb binding. Specimens from Hong Kong and the United States classified as adw2 in the S region fell into two groups based on PreS2 mAb binding: one having reactivity similar to Paris adw2 subtype and the other having identical reactivity to Paris ayw1 subtype. Furthermore, some specimens classified as adr in the S region gave similar reactivity to the Paris ayr subtype in the PreS2 and PreS1 regions. One complicating factor in this approach toward subtyping was the discovery that some HBsAg positive sera may contain factors which block PreS epitopes. Grouping of HBV subtypes by PreS1, PreS2, and S mAb reactivity may allow better correlation with groupings based on HBV DNA sequence homology.     

Hepatitis B surface antigen particles of subtypes adw and adr, and compound subtype (adwr) in symptom-free carriers in Japan.

Of sera from 1,878 Japanese blood donors who carried hepatitis B surface antigen (HBsAg), 420 were subtyped as adw (22.4%) and 1,443 as adr (76.8%); only 15 (0.8%) contained HBsAg of subtype ayw or ayr. Sera with HBsAg/adr had higher HBsAg titres than those with HBsAg/adw (geometric mean of haemagglutination titre: 10.1 +/- 2.4 vs. 9.7 +/- 2.4, p less than 0.01), and a higher prevalence of hepatitis B e antigen (24% vs. 13%, p less than 0.001). Carriers of HBsAg/adr progressively predominated over those of HBsAg/adw with increasing age. Of sera from 1,863 carriers of HBsAg/adw or HBsAg/adr, 182 (9.8%) contained HBsAg particles with both subtypic determinants in the w/r allele. The presence of w and r determinants on the same particles was ascertained by sandwiching them between monoclonal antibody with the specificity for w and that with the specificity for r. HBsAg particles of compound subtype (adwr) were found more often in sera with hepatitis B e antigen than those without it (145/403 [36.0%] vs. 37/1,460 [2.5%], p less than 0.001). Sera with HBsAg/adwr particles had HBsAg titres higher than those without them (12.4 +/- 1.9 vs. 9.7 +/- 2.3, p less than 0.001). HBsAg/adwr particles arise from phenotypic mixing of the S-gene product of wild-type virus and that of mutants with point mutations for subtypic changes. The results obtained indicated that HBV strains of subtype adr have a higher replicative activity than those of adw, and suggested that mutations in the S gene for subtypic changes would be associated with an active replication of hepatitis B virus.     

Molecular epidemiology of hepatitis B virus in Spain: identification of viral genotypes and prediction of antigenic subtypes by limited sequencing

The hepatitis B virus (HBV) genotypes were studied by a line probe assay (LiPA) and by direct sequencing of a 339 nucleotide fragment from the S region of the viral genome in samples from 269 carriers living in Spain, either native to Spain (231) or immigrants from Africa, Asia, and Eastern Europe (38). The sequences were also used to predict the HBV surface antigen (HBsAg) subtype on the basis of the amino acids specified at selected positions of the HBsAg molecule. Agreement between the two genotyping methods was found in most cases (98.1%) and a HBV genotype could be assigned to all samples. The viral groups D/ayw2 (30.1%), D/ayw3 (28.6%), and A/adw2 (21.2%) were prevalent, with an additional participation of the groups D/ayw4 (4.8%), F/adw4q- (1.9%), A/ayw1 (1.9%), and D/adw3 (0.7%), all of them present among the autochthonous carriers. Strains from genotypes B and C were found exclusively among Chinese immigrants. Genotype E strains were found in immigrants from Central Africa and in one patient native of Spain. Point mutations leading to amino acid changes of residues involved in the expression of the HBsAg subtype determinants were found in 12 samples (4.5%). Some mutations would predict the putative novel genotype-subtype associations A/adw4q+, A/ayr, D/ayr, and E/ayw1, while others would suggest the loss of subtype-specific determinants. The finding of HBV strains characteristic for Africa among the autochthonous carriers confirms the emergence of African HBV strains in Spain.     

Antigenic Diversity of Hepatitis B Virus Strains of Genotype F in Amerindians and Other Population Groups from Venezuela

The adw4 subtype of hepatitis B virus (HBV) belongs to a unique genomic group (genotype F) representing the original HBV strains from the New World. Data regarding the prevalence of this subtype among HBV carriers in South America are, however, scarce, and those concerning HBV genotype F are based on only a few samples from Latin America. In this study, serum samples were obtained from 141 hepatitis B surface antigen (HBsAg) carriers from Amerindians and urban populations from Venezuela. The HBsAg subtype was identified with monoclonal antibodies in 105 samples, and the HBV genotype was identified by reverse-phase hybridization with DNA fragments in 58 samples. The adw4 subtype was highly prevalent in the population studied (75%); among the Amerindians, the prevalence was 97%. The adw2 subtype was also present (10%), while other subtypes (ayw3 and ayw4) were only occasionally found. The HBV subtype was associated with the expected genotype in most cases (80%), and thus genotype F was highly prevalent. Sequencing of viral strains that gave genotypes unpredicted by the HBsAg subtyping confirmed seven of them as belonging to not previously described genotype-subtype associations: namely, adw2 and ayw4 within genotype F.     

A solid-phase enzyme immunoassay for the common and subtypic determinants of hepatitis B surface antigen with monoclonal antibodies

Monoclonal antibodies were raised against the common (a) as well as subtypic determinants (d, y, w and r) of hepatitis B surface antigen (HBsAg). They were applied to subtyping HBsAg by sandwiching it between antibody against a fixed on a solid-phase support and antibody against one or other of d, y, w and r, linked to horseradish peroxidase. The assay was applied to evaluate antigenic specificities of the NIH and Japanese panels composed of 44 sera containing HBsAg particles of various subtypes. HBsAg particles of a hybrid subtype, adyr, were sandwiched between monoclonal antibody against d and that against y, thereby indicating that they possessed both d and y determinants on the selfsame particle. The expression of d and y determinants on hybrid HBsAg particles was much less than that on ordinary particles of adw, adr, ayw or ayr subtype.     

Determination of HBsAg subtypes in Western Siberian part of Russia

A set of monoclonal antibodies with specificity for hepatitis B surface antigen (HBsAg) was used for subtyping this antigen in sera from indigenous natives, blood donors, and drug users in Western Siberia with a modified commercial enzyme immunoassay kit for HBsAg detection. Three subtypes of HBsAg in a ratio of 36 (78%) ayw2:8 ayw3varB (18%):2 (4%) adw2 were found in 46 (100%) HBsAg-positive sera of different aboriginal populations of Western Siberia: the Tundra Nenets, Northern Khanty, Southern Altaians, and Kazakhs. Four subtypes of HBsAg in a ratio of 81 (57%) ayw2:58 (15 ayw3varA and 43 ayw3varB; 44%):2 (1%) adw2 were detected in 141 (100%) samples of blood donors from ten cities of Western Siberia. Three subtypes of HBsAg in a ratio of 34 ayw3:(both variants, 33 ayw3varA and 1 ayw3varB; 97.1%):1 (2.9%) ayw2 were found in blood of 35 injection drug users in Novosibirsk.     

Characteristics of the X gene of hepatitis B virus

The 465-nucleotide sequence of the X gene from our cloned hepatitis B virus (HBV) DNA (subtype adw) was determined and compared to the same gene of 10 other published HBV sequences (3 of adw, 4 of adr, 2 of ayw, and 1 of ayr). We found (i) a total of 56 base differences among the 11 sequences (without counting the 27-base deletion in one adr) which resulted in 88% nucleotide homology, and (ii) 5 pairs of repeated sequence (3 direct repeats and 2 inverted repeats) that were highly conserved. Comparison of the protein amino acid sequences indicated that (i) there is 80% amino acid homology in total, and (ii) there are four highly conserved cysteine residues. In addition, the X gene of the adw subtype is more conserved than that of adr.     

粤东客家地区乙型肝炎病毒S基因序列多态性分析

 目的：探讨广东客家地区乙型肝炎病毒（HBV）S基因序列的多态性，了解该地区S基因的流行病学特征。 方法： 运用基因扩增技术对40例来自广东客家地区的乙肝表面抗原阳性及HBVDNA阳性的HBV感染者S基因进行扩增，再对扩增产物进行DNA测序，将该地区S基因序列与标准序列对比分析。 结果： 40例样本中，adw型36例，adr型4例。36例adw型中，第131位天冬酰胺（Asn）均被苏氨酸（Thr）替代，其它位点也不同程度存在着氨基酸密码的差异：第127位脯氨酸（Pro）被Thr替代，第133位蛋氨酸(Met)被亮氨酸(Leu)或Thr替代, 第134位苯丙氨酸（Phe）被酪氨酸（Tyr）替代, 第143位Thr被Met替代，更多位点的遗传密码存在着简并性。而4例adr型的氨基酸序列高度同源。 结论： HBV亚型的分布具有明显的域性，广东客家人感染的HBV亚型主要为adw型，只有少数为adr型。与参照序列相比，adw型存在较明显的多态性。