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。     

多重引物聚合酶链反应扩增丙型肝炎病毒基因及基 …

利用聚合酶链反应（ＰＣＲ）技术对丙型肝炎病毒（ＨＣＶ）的５’－非编码区（５’－ＮＣＲ）、Ｃ及ＮＳ４基因区的３对引物分别及同时扩增，检测８０例抗－ＨＣＶ阳性患者的血清ＨＣＶ ＲＮＡ，并进行了ＨＣＶ基因分型研究。各不同引物所介导的ＰＣＲ检出ＨＣＶ ＲＮＡ的结果为：５’－ＮＣＲ基因区６０％（４８／８０），Ｃ基因区３７％（３０／８０），ＮＳ４基因区３０％（２４／８０）。以上３对引物同时扩增仅４２％（３４／     

丁型肝炎病毒基因组RNA中核酶区的cDNA的体外转录

以质粒ｐＳＶＬＤ３为模板，通过聚合酶链反应（ＰＣＲ）扩增得到１条１３９ｂｐ的ｃＤＮＡ片段，它含有丁型肝炎病毒（ＨＤＶ）基因组ＲＮＡ中核酶区的ｃＤＮＡ，将此片段克隆到ｐＧＥＭ－３２中，经筛选、鉴定，得到克隆株ｐＨＤＮＡ１０８，提取质粒后经双脱氧末端终止法测序，发现有２个碱基变异。以该质粒为模板，通过Ｔ７ ＲＮＡ聚合酶转录出ＨＤＶ基因组ＲＮＡ中核酶区的前体，并观察到其自身裂解产物，自裂率达７１％。     

血清学标志阴性的非甲～戊型肝炎的病原学研究

目的对血清学标志阴性的非甲～戊型肝炎进行病原学研究。方法用ＨＢＶＰＣＲ、ＨＣＶＲＴ－ＰＣＲ和ＨＥＶＲＴ－ＰＣＲ分别检测血清学标志阴性的非甲～戊型肝炎患者血清，并对其部分阳性产物进行克隆测序。结果８７例非甲～戊型肝炎血清ＨＢＶＤＮＡ均为阴性，９例（１０．３％）为ＨＣＶＲＮＡ阳性，部分经测序证实为ＨＣＶ１ｂ亚型；余７８例为ＨＢＶＤＮＡ和ＨＣＶＲＮＡ均阴性。该７８例中，１４例因无血清未作ＨＥＶＲＮＡ检测，余６４例中４９例（７６．６％）为ＨＥＶＲＮＡ阴性，１５例（２３．４％）为ＨＥＶＲＮＡ阳性。经序列分析显示，其中９例为典型的中国ＨＥＶ株基因序列，６例变异较大，与典型的中国株基因序列的同源性仅为８０％左右。４９例ＨＢＶＤＮＡ、ＨＣＶＲＮＡ和ＨＥＶＲＮＡ均阴性的血清中１６例（３２．６％）ＨＧＶＲＮＡ阳性。由此可见，该８７例中至少有９例为ＨＣＶ感染，１５例为ＨＥＶ感染，１６例为ＨＧＶ感染。结论对血清学标志阴性的非甲～戊型肝炎的病人应该用ＰＣＲ法进行病原学分型，以明确其诊断     

中国上海丁型肝炎病毒部分基因的克隆与序列分析

从我国上海无症状乙型肝炎病毒表面抗原（ＨＢｓＡｇ）携带者混合血清中提取ＲＮＡ，通过逆转录聚合酶链反应获得丁型肝炎病毒（ＨＤＶ）的长约３７０个碱基对的ｃＤＮＡ片段，将其克隆测序并与已知的ＨＤＶ各基因型代表株序列比较，结果表明，它与意大利株的同源性较高（９８．１％），与台湾株（Ｉａ型）及美国－１（Ｉｂ型）的同源性相近（９１．０％，９２．０％），与日本－１株（Ⅱ型）及秘鲁株（Ⅲ型）的同源性为７９．３％（     

中国4株丁型肝炎病毒抗原编码区基因的cDNA克隆与…

从我国四川、广西、河南的丁型肝炎、病毒抗原（ＨＤＡｇ）或抗体阳性的ＨｂｓＡｇ携带者中，筛选出４株ＨＤＶ ＲＮＡ阳性标本，经逆转录和聚合酶反应（ＲＴ－ＰＣＲ）后，获得包含ＨＤＶ抗原编码区的ｃＤＮＡ片段，并对其进行克隆与序列测定。经计算机分析比较表明：四川、广西株与美国－１株同源性最高，分别为９９．３％、９９．０％；而河南－１、河南－２株与台湾株的同源性较高，分别为９４．３％、９２．１％；上述４株与日     

中国4株丁型肝炎病毒抗原编码区基因的cDNA克隆与序列分析

从我国四川、广西、河南的丁型肝炎病毒抗原（ＨＤＡｇ）或抗体（ａｎｔｉ－ＨＤ）阳性的ＨＢｓＡｇ携带者中，筛选出４株ＨＤＶＲＮＡ阳性标本，经逆转录和聚合酶反应（ＲＴ－ＰＣＲ）后，获得包含ＨＤＶ抗原编码区的ＣＤＮＡ片段，并对其进行克隆与序列测定。经计算机分析比较表明：四川、广西株与美国－１株同源性最高，分别为９９．３％、９９．０％；而河南－１、河南－２株与台湾株的同源性较高。分别为９４．３％、９２．１％，上述４株与日本－１、秘鲁－１的同源性在６６．１％－７７．８％之间。推导的ＨＤＡｇ的氨基酸序列的同源性为：四川、广西株与美国株分别为９９．５％、９６．４％；河南－１、河南－２株与台湾株分别为８９．７％、８９．３％，故我国至少存在基因型Ｉ型的两种亚型，其中四川、广西株为ＩＡ型；河南２株皆为ＩＢ型。同时，实验结果还证实了ＨＤＶ毒株的异质性呈地区分布特征。     

CLONING AND SEQUENCE CHARACTERIZATION OF THE RE-ARRANDED VH GENE FROM HYBRIDOMA CELLS SECRETING ANTI-HUMAN C1-INHIBITOR McAb

由分泌抗人Ｃ１－ＩＮＨＭｃＡｂ的杂交瘤Ｆ７细胞株提取总ＲＮＡ，合成与ＶＨ基因ＦＲ１和ＦＲ４互补的通用引物，以ＲＮＡ反转录合成的第一链ｃＤＮＡ为模板，ＰＣＲ法克隆出Ｆ７ＶＨ基因的ＤＮＡ片段。将分离获得的目的ＤＮＡ片段亚克隆入ｐＵＣ１８／１９测序载体，从两头进行双脱氧核苷酸随机终止法的ＤＮＡ序列测定。结果显示：ＶＨ基因是由３３３个碱基组成，编码１１１个氨基酸残基。通过国际联机检索进行ＥＭＢＬ和Ｋａｂａｔ基因库扫描发现，Ｆ７ＶＨＤＮＡ仅与Ｉｇ同源，符合小鼠Ｉｇ的ＶＨ基因特征；同源性为６０％～８５％，应归属于Ｉｇ的ＶＨ基因。根据Ｋａｂａｔ分类方法，Ｆ７ＶＨ基因推导的氨基酸顺序属于小鼠Ｉｇ的ＶＨ基因的Ⅱ（Ａ）亚组，是由ＶＨ－Ｄ－ＪＨ３重排产生；其框架区的９和６７位点为脯氨酸（Ｐｒｏ）和赖氨酸（Ｌｙｓ）（非芳香族氨氨酸），符合Ⅱ（Ａ）亚组框架残基结构模式；其ＣＤＲ３区含有７个氨氨酸残基，表明Ｃ１－ＩＮＨ抗原表面结构并不复杂；ＦＲ２和ＦＲ３的２２和８９位点为半胱氨酸残基（Ｃｙｓ），与ＶＨ片段二硫键形成有关。成功获得Ｆ７ＶＨ基因为进一步构建和表达单链Ｆｖ（ｓｃＦｖ）抗体打下良好的基础。     

肝病患者中庚型肝炎病毒感染的检测

目的探讨临床肝病病人中庚型肝炎病毒（ＧＢＶ－Ｃ／ＨＧＶ）感染情况及临床特点。方法应用庚型肝炎病毒基因组５’ＵＴＲ两对寡核苷酸作为引物，建立逆转录套式聚合酶链反应，检测１６９例不同肝病患者血清标本中ＧＢＶ－Ｃ／ＨＧＶＲＮＡ，并对其中１例ＰＣＲ扩增产物进行克隆及测序。结果１６９例各型肝病病人ＧＢＶ－Ｃ／ＨＧＶＲＮＡ总的检出率为９５％（１６／１６９）。在２９例有手术输血史患者中，３１０％（９／２９）ＧＢＶ－Ｃ／ＨＧＶＲＮＡ呈阳性，明显高于无手术输血史组（５％，Ｐ＜００１）。序列分析显示１株庚肝病毒５’ＵＴＲ部分基因片段与已知庚肝病毒株核苷酸同源性在８９１４％～９８９１％之间。结论ＧＢＶ－Ｃ／ＨＧＶ感染普遍存在于临床肝病患者中，病人感染ＧＢＶ－Ｃ／ＨＧＶ的临床表现未发现有特殊性，ＧＢＶ－Ｃ／ＨＧＶ可能不是非甲～戊型肝炎的主要致病因子。     

丙型肝炎病毒基因型与血清HCV RNA含量关系及与干扰素应答的研究

目的探讨丙型肝炎病毒（ＨＣＶ）基因型与血清ＨＣＶＲＮＡ含量的关系及其对干扰素应答的影响。方法应用定量荧光ＰＣＲ（Ａｍｐｌｉｓｅｎｓｏｒ－ＰＣＲ）技术检测了１３５例不同基因型ＨＣＶ感染的慢性丙型肝炎患者血清ＨＣＶＲＮＡ含量，另对其中７７例进行干扰素治疗并随访１２个月以上。结果ＨＣＶ－Ⅱ型感染血清ＨＣＶＲＮＡ水平（１０７．８±３．４拷贝／ｍｌ）显著高于ＨＣＶ－Ⅲ型感染（１０６．３±２．５拷贝／ｍｌ）（Ｐ＜０．０１），Ⅲ型感染的应答率（７／１３，５３．８％）显著高于Ⅱ型感染（２０／６４，３１．３％）（Ｐ＜０．０５）。应答组治疗前血清ＨＣＶＲＮＡ含量（１０６．８±２．７拷贝／ｍｌ）显著低于无应答组（１０８．３±３．２拷贝／ｍｌ）（Ｐ＜０．０１）。ＨＣＶＲＮＡ含量低于１０６．５拷贝／ｍｌ者，无论何种型别ＨＣＶ感染均应答较好，而ＨＣＶＲＮＡ高于１０８．０拷贝／ｍｌ者则应答极差。结论ＨＣＶ基因型及病毒血症水平是预测干扰素疗效的重要因素，且后者比前者意义更大。Ⅱ型感染病毒血症水平较高可能是影响其疗效的原因之一。     

Molecular Phylogenetic Analysis of Iranian HDV Complete Genome

Hepatitis D (delta) virus (HDV) is a subviral pathogen agent and a satellite of Hepatitis B virus. Three distinct genotypes are described for HDV; genotype I is distributed worldwide but other genotypes appear to be more restricted geographically. In the present study, the entire nucleotide sequence of an HDV isolate from an Iranian patient (IR-1) was obtained using twelve pairs of primers to amplify six overlapping fragments covering the whole HDV genome by RT-nested PCR. Phylogenetic and pairwise alignments were done on this new isolate to determine IR-1 position among other isolates. Our results indicate that IR-1 contains 1676 nucleotides encoding 214 a.a. of the hepatitis delta antigen (HDAg). This new isolate belongs to genotype I with most sequence similarity to an Italian HDV isolate (92.6%). At amino acid level, predicted HDAg sequence of IR-1 revealed the most homology with those of Italian and Lebanese isolates. Data analysis confirmed genetic variability and heterogeneity of the HDV species isolated from different geographical areas.     

丁型肝炎病毒中国湖南株高变区部分序列分析

用耐高温逆转录以及套式聚合酶链反应方法,对HDV中国湖南株基因组高变区1642～417片段(D1),以及保守区681～895片段(D3)进行扩增,用荧光法直接测序,对其进行序列分析。D1片段除去引物外可判读222nt(1662～197),D3片段除去引物还有176nt(701～875)。两段序列分别与美国株、意大利株、日本株以及中国河南株进行了比较。结果显示,中国湖南株高变区1662～197片段(D1)与日本株的同源性仅为44.06％,与意大利株的同源性相对较高,但也仅为72.57％。日本株在该片段的同源性特别低,而中国河南株在该片段的同源性并不特别低。各株之间在该片段的同源性仅为44.06％～72.56％,说明HDV基因组1662～197片段是高变区。中国湖南株D1片段与其它各株的同源性为44.06％～72.57％,且与中国河南株同源性也仅为62.61％,提示中国湖南株可能系一新的亚型。各株之间在701～875片段(D3)的同源性较高,中国湖南株与意大利株更为接近(93.03％),而与中国河南株(91.82％)、美国株(89.54％)和日本株(89.54％)的同源性相对较低。各株之间的同源性比较接近,提示HDV基因组701～875片段是相对保守区。同源性的高低与地理位置分布不一致,其原因有待进一步研究。     

Characterization of a new genotype II hepatitis delta virus from Taiwan

Three genotypes of HDV, which may be associated with different clinical pictures and epidemiological patterns, have been identified. In contrast to Type I and Type III HDV, both of which have multiple isolates, Type II HDV so far includes only a single isolate (Japan-1) from a low prevalence area (Japan). Recently, Type II has been reported to be the predominant genotype in Taiwan, which is also a low prevalence area, and is associated with less aggressive disease than Type I. However, the sequence and structure of these viruses have not been characterized. The complete characterization of a second member (Taiwan-3 isolate) of the Type II HDV from Taiwan is reported. These two Type II HDV isolates (Taiwan-3 and Japan-1) have 93.8% nucleotide homology and 89.3% amino acid homology, respectively. These shared sequences establish the common characteristics of Type II viruses. Sequence comparisons of various HDV genotypes show that the autocatalytic region of the RNA is relatively conserved between Type I and Type II (88.5–95.6% homology) but is significantly divergent in Type III (76.8–80.3% homology). The hypervariable region (nucleotides 1602–658) of RNA, however, is heterogeneous (64.9–73.0%) among all three genotypes. The delta antigen sequence is also very heterogeneous (64.9–73.0%). Most strikingly, the C-terminal sequence (19 amino acids) of the large delta antigen is almost completely different in each of the three genotypes. The heterogeneity in this region of three HDV genotypes may be a basis for their different biological properties, and the nucleotide sequences of this region can be used to differentiate the different genotypes of HDV. The consensus sequence in the four previously identified conserved domains of HDV RNA is defined more precisely. © 1996 Wiley-Liss, Inc.     

Introduction of a new hepatitis agent in retrospect: genetic studies of Swedish hepatitis D virus strains.

The supposedly first outbreak of hepatitis D virus (HDV) infection in Sweden occurred among intravenous drug addicts in the Malmö area in the mid-1970s. Stored sera from this outbreak were used for viral RNA extraction and analysis. By sequence comparisons, the HDV genomes from those Swedish patients fell into two separate clusters, within which the RNA sequences were closely related. These two HDV groups genetically resembled the French and US-1 isolates of genotype I, respectively, indicating that there had been at least two separate sources of HDV infection. The genetic alterations of the HDV RNA were investigated by sequence analysis of nine annually drawn serum samples from one patient and paired samples collected between 2 and more than 10 years apart from six patients with chronic HDV infection. Only mutational changes were observed, and no insertion or deletion appeared throughout the periods observed. It was found that the Swedish HDV isolates mutationally evolved at an average rate of 1.1 x 10(-3) substitutions per nucleotide per year over a long time course of chronic HDV infection, which is of the same magnitude as that of other RNA viruses.     

Phylogenetic analysis of rabbit haemorrhagic disease virus in France between 1993 and 2000, and the characterisation of RHDV antigenic variants

Summary.  The first molecular epidemiological study of Rabbit haemorrhagic disease virus undertaken in France between 1988 and 1995, identified three genogroups, two of which (G1, G2) disappeared quickly. We used immunocapture-RT-PCR and sequencing to analyse 104 new RHDV isolates collected between 1993 and 2000. One isolate was obtained in 2000 from a French overseas territory, the Reunion Island. The nucleotide sequences of these isolates were aligned with those of some French RHDV isolates representative of the three genogroups previously identified, of some reference strains and German and American RHDV antigenic variants. Despite the low degree of nucleotide sequence variation, three new genogroups (G4 to G6) were identified with significant bootstrap values. Two of these genogroups (G4 and G5) were related to the year in which the RHDV isolates were collected. Genogroup G4 emerged from genogroup G3, which has now disappeared. Genogroup G5 is a new independent group. The genogroup G6 contained an isolate collected in mainland France in 1999 and the isolate collected from the Reunion Island, as well as German and American RHDV variants. Multiple sequence alignments of the VP60 gene and antigenic analysis with monoclonal antibodies demonstrated that these French isolates are two new isolates of the RHDV variant. Received June 7, 2002; accepted August 12, 2002     

Complete genome sequences and phylogenetic analysis of hepatitis delta viruses isolated from nine Turkish patients

Hepatitis delta virus (HDV) is a subviral agent of hepatitis B virus (HBV), and its life cycle is dependent on HBV. It is commonly accepted that HDV has eight distinct genotypes. In this study, the complete nucleotide sequences of HDV genomes isolated from nine Turkish patients were obtained by RT-PCR using two pairs of primers that cover the entire HDV genome. PCR products were sequenced directly. The results showed that these 9 isolates were approximately 1680 base pairs in length and clustered in the genotype HDV-1 branch when phylogenetic analysis was done with the sequences together with the complete sequences of HDV genomes representing each genotype retrieved from GenBank. Analysis of a portion of the large hepatitis D antigen (L-HDAg) gene showed that sequence similarity among these Turkish isolates is between 87.4 and 97.1%, and the Turkish isolates have the most sequence similarity to HDV-1 (90.5%), while they have the least sequence similarity to HDV-3 (64.1%). Full-genome analysis indicates that the sequence similarity is between 80.7 and 95.4%, and the highest sequence similarity is 84.8% (between the Turkish isolates and HDV-1). The lowest sequence similarity is 56.4% (between the Turkish isolates and HDV-3). In conclusion, phylogenetic analysis shows that the Turkish HDV isolates belong to HDV-1.     

Hepatitis delta virus cDNA sequence from an acutely HBV-infected chimpanzee: sequence conservation in experimental animals

Hepatitis delta virus (HDV) RNA was isolated from the serum of a chimpanzee acutely infected with hepatitis B virus (HBV) and superinfected with HDV. Interference of HDV with HBV resulted in decreased HBV DNA levels in the serum. This interference did not change the size of the two HBV specific RNAs present in the liver of the chimpanzee. The complete cDNA sequence of the HDV RNA (5th passage) was determined. Comparison of this cDNA sequence with our previously published sequence (4th passage), located in the variable domain of HDV, was highly conserved. The HDV strain used for these infections originated from a human HDV isolate also used for five to seven HDV passages in chronic HBV carrier chimpanzees (subtypes adw and ayw) or woodchucks chronically infected with woodchuck hepatitis virus (WHV). The complete HDV cDNA sequence showed an extreme conservation (up to 99.8% homology) with the previously published animal-derived HDV cDNA sequences irrespective of passage number and animal species. In contrast a markedly lower homology (85-89%) was found when compared with 3 human-derived HDV cDNA sequences. Comparison of our complete cDNA sequence with the human-derived cDNA sequences showed that the nucleotide changes in the human-derived isolates were restricted to specific regions on the genome and to specific basepair substitutions. The hepatitis Delta antigen (HDAg) is highly conserved both in the human- and animal-derived cDNA sequences showing mainly conservative amino acid changes.     

Phylogenetic analysis of hepatitis D viruses indicating a new genotype I subgroup among African isolates.

Genetic analysis was performed on 13 hepatitis D virus (HDV) isolates from Ethiopia, Somalia, Jordan, Kuwait, Bulgaria, Moldavia, and Sweden. The complete nucleotide sequence and genomic organization are described for the first time for two African HDV isolates. Phylogenetic analysis showed all the African isolates to be intrarelated and to form a novel group within HDV genotype I; the suggested designation for this group is IC. The genetic distance to previously described type I isolates was about 0.15. The HDV genotype I isolates (total of 22 examined) phylogenetically formed three clusters, each of them corresponding to certain geographic regions; the "western" group consisted of six HDV isolates from western Europe and the United States plus one from Kuwait; the "eastern" group consisted of two isolates from Moldavia and one each from Bulgaria, Nauru, mainland China, and Taiwan; and the "African-Middle East" group consisted of six HDV isolates from Ethiopia and one from Somalia, Jordan, and Lebanon.     

Evolution rate of hepatitis delta virus RNA isolated in Taiwan

The complete RNA sequences of hepatitis delta viruses (HDV) isolated at 3 years apart from a chronic delta hepatitis patient in Taiwan were determined. The sequence analysis showed an overall evolution rate of 3.18 × 10^?3 substitutions/nucleotide/year. The evolution rates in different parts of HDV RNA varied. The hypervariable region evolved faster (4.55 × 10^?3 substi-tutions/nucleotide/year) than the hepatitis delta antigen (HDAg)-coding region (2.60 × 10^?3 substitutions/nucleotide/year) and the autocata-lytic region (1.11 × 10^?3 substitutions/nucleotide/ year). These data are compatible with the previous finding that the hypervariable region is more divergent than the HDAg-coding region and the au-tocatalytic regions among the HDV isolates from different geographic areas. No substitution was found in the four previously identified conserved domains of HDV RNA, further confirming their functional importance in viral replication. The evolution rate of this HDV RNA is higher than that determined from the partial RNA sequences of two Japanese HDV isolates and similar to that found in a Lebanon isolate. Further, it was found that this HDV RNA retained the same microheterogeneities at 15 nucleotide positions detected in the RNA 3 years earlier. It is concluded that HDV RNA in patients' serum is extremely heterogeneous, and that the nucleotide substitutions in certain nucleotide positions likely have conferred evolutionary advantages for HDV. Viral sequence evolution is a possible mechanism for chronic HDV infection. © 1994 Wiley-Liss, Inc.