庚型肝炎病毒5‘非编码区基因分析及基因型的初步划分

为了分析我国庚型肝炎病毒（ＨＧＶ）的基因结构特点，对２名个体供血者、２例慢性肝炎患者及２例肝硬化患者的血清标本进行了庚型肝炎病毒基因组５’非编码区２７７个核苷酸的基因扩增、分子克隆和序列分析，并利用基因分析软件处理结果。结果表明，分离出的６株ＨＧＶ５’非编码区基因序列（Ｇ００１～Ｇ００６）之间同源性在９６．２％以上。而与国外报道的３株ＨＧＶ序列比较同源性在８６．９％～９１．６％。通过对６株ＨＧＶ（Ｇ００１～Ｇ００６）及国外报道的３株ＨＧＶ基因序列变异性的比较，将ＨＧＶ基因型初步划分为不同的３个组。结果提示我国ＨＧＶ株５’非编码区序列有较高的同源性，而与国外报道的有较大差异，但不同毒株在此区域均可能形成稳定而相似的二级结构模式。     

中国大陆柯萨奇病毒A组16型全基因参照序列的初步建立和分析

目的建立中国大陆柯萨奇病毒A组16型（CoxA16）流行株的全基因参照序列。方法从GenBank中获得CoxA16中国大陆分离株的所有全基因序列和氨基酸序列,用DNAstar/MegAlign软件对所得序列进行多序列比对和进化分析,按照参照序列标准拟定CoxA16的全基因参照序列和氨基酸序列,并与参考序列进行比对分析。结果中国大陆分离的CoxA16毒株全基因序列共8株,分离于2005-2009年,其中2008年5株,广东省5株;通过序列比对获得了大陆CoxA16全基因和氨基酸参照序列;参考毒株间核苷酸序列同源性介于79.0%～98.8%,氨基酸序列同源性为94.3%～99.9%,与参照序列的核苷酸同源性为79.7%～97.0%,氨基酸同源性介于95.1%～99.5%之间,同源性最低的为2008年安徽阜阳的FY18株。结论比对分析了中国大陆CoxA16毒株全序列,成功建立了中国大陆CoxA16全基因和氨基酸参照序列。     

从我国5城市分离的10株GBV-CHGV基因型分析

最近研究表明〔１,２〕,世界各地分离的ＧＢＶＣ／ＨＧＶ株间存在不同的基因型,即１型、２型和３型。上述分型所采用的核酸序列大多数来自非洲、欧美和日本等地,我国ＧＢＶＣ／ＨＧＶ分离株的基因型及其分布则报道较少。为此,本研究应用分子进化学方法对从我国５城市分离的１０株ＧＢＶＣ／ＨＧＶ５′ＵＴＲ部分序列进行基因分型。结果报道如下：材料与方法１－血清标本：ＨＧＶＲＮＡ阳性的血清标本共１０份,其中ＢＪ１和ＳＪＺ１～２分别采于北京市和石家庄市的献血员血清;ＢＪ２和ＮＣ１为北京市和南昌市急性乙型肝炎病人…     

乙型肝炎病毒D基因型系统进化树分析

目的：研究HBV D基因型不同毒株全基因进化关系。方法：用聚合酶链式反应（PCR）、克隆及核酸序列测定的方法，测定了1例中国人慢性无症状携带者感染的乙型肝炎病毒D基因型全基因序列。此D基因型毒株全基因序列GenBank Accession为AF280817，将GenBank中已发表的HBV D基因型30株的全序列进行了系统进化树分析。结果：中国株HBV D基因型与源于瑞典（Sweden）的4株HBV D基因型全基因的进化距离最近；地中海地区及欧洲国家系HBV D基因型分布的优势地域，亚洲并非HBV D基因型分布的优势地域。结论：HBV D基因型病毒株传入来源、迁移的方向不同以及病毒株在具有不同遗传和免疫特质的宿主中的长期选择是形成HBV D基因序列病毒进化差异的原因。     

用PCR技术建立CD23全基因的无性繁殖系

参照国外已发表的ＣＤ２３ｃＤＮＡ全基因序列，自行设计并合成一对ＤＮＡ引物（２５ｍｅｒ和１８ｍｅｒ），以ＣＤ２３阳性细胞株ＨＦＢｄ２／３ＤＮＡ为模板，应用ＰＣＲ技术，成功地扩增了ＣＤ２３ｃＤＮＡ全基因（９８７ｂｐ，含人工设计的酶切点），并定向克隆入原核表达载体ｐＢＶ２２０，构建了ｐＢｖ２２０－ＣＤ２３全基因重组体，经６种限制性内切酶酶切鉴定，结果与已发表序列的酶切图谱完全一致，初步证实为ＣＤ２３全基因。这将为今后ＣＤ２３基因的序列测定和表达，进一步在基因水平上探讨ＣＤ２３的生物学功能提供了物质基础。     

线探针核酸杂交分析贵州地区HCV感染株基因型

目的　分析贵州地区丙型肝炎病毒（ＨＣＶ）感染株的基因型。方法　用第二代线探针核酸杂交方法,对９８ 例ＨＣＶ感染者血清进行基因分型。结果　１２ 例献血员血清,均为ＨＣＶ１ｂ 型;１５ 例血液病患者中,１４ 例（９３－３％ ）为ＨＣＶ１ｂ ,１ 例（６－７％ ）为ＨＣＶ１ｂ ＋ ２ 型。慢性丙肝患者３７ 例中,ＨＣＶ１ｂ 型３４例（９１－９％ ） ,混合感染基因型３ 例（ＨＣＶ１ａ ＋ １ｂ 、ＨＣＶ１ｂ ＋ ２ 、ＨＣＶ１ａ ．１ｂ ＋ ２ａ２ｃ 各１ 例）。静脉药瘾者３４ 例,其中ＨＣＶ１ｂ 感染１３ 例（３８－２％ ） ,ＨＣＶ６ａ１０ 例（２９－４％ ） ,ＨＣＶ３ｂ４ 例（１１－８％ ） ,ＨＣＶ３ａ１ 例（２－９ ％） ,混合感染６ 例（１７－６ ％） ,大多为ＨＣＶ２ 复合其他基因型。结论　贵州地区ＨＣＶ感染者中,以ＨＣＶ１ｂ 基因型为主,其次为ＨＣＶ６ａ ,尚存在ＨＣＶ３ａ 和ＨＣＶ３ｂ 。其中ＨＣＶ３ａ、３ｂ 、６ａ ,在本地区系首次报告,但主要存在于静脉药瘾者中。虽然有混合感染基因型的存在,但其基因基础需测序分析     

丙型肝炎病毒非结构基因5b(NS 5b)区一级结构的变异

目的　分析中国丙型肝炎病毒非结构基因５ ｂ 区核苷酸序列变异。方法　以逆转录套式聚合酶链反应（ＲＴｎｅｓｔｅｄＰＣＲ） 从４９ 例中国病人的血清中获得互补的ＤＮＡ片段,产物克隆后测序。结果　３３ 株系基因Ⅱ１ ｂ 型,１６ 株系Ⅲ２ ａ 型,中国ＨＣＶ株型与日本ＨＣＶ株型同属１ 个基因亚型,但在一些核苷酸保守位点上有一定的差异,对３３ 株Ⅱ１ ｂ 型和１６ 株Ⅲ２ ａ 型间的同源性进行比较,发现１６ 株Ⅲ２ ａ 型的同源性低于３３ 株Ⅱ１ ｂ 型的同源性。首次在ＨＣＶＮＳ５ ｂ 区发现１ 个新的３ 个核苷酸的缺失突变及１ 个移码突变。结论　同一基因亚型之内不同ＨＣＶ株的核苷酸序列可能具有一定的地理分布特征,每个地区有一定的流行株。     

丙型肝炎病毒血清学分型与基因分型研究

为了解某农村单采浆供血员人群所感染丙型肝炎病毒（ＨＣＶ）的血清型和基因型构成，并对两种分型方法进行比较，本工作以ＨＣＶＣ区型特异性多肽对抗－ＨＣＶ进行血清学分型，对已确定血清型的血清进行５′非编码区（ＮＣＲ）逆转录套式聚合酶链反应（ＲＴ－ｎＰＣＲ）和限制性片段长度多态性（ＲＦＬＰ）分析以确定基因型，并对６份扩增产物进行序列测定。结果显示１４０份抗－ＨＣＶ阳性血清中，血清Ⅰ型和Ⅱ型分别为４４份（３１．４３％）和１２份（８．５７％），余８４份未能分型。４４株已知血清型的ＨＣＶ中，１ｂ、２ａ和３ｂ等３种基因型分别为３４株（７７．２７％）、９株（２０．４５％）和１株（２．２７％）。两种分型方法一致率为９３．１８％（４１／４４）。对６株ＨＣＶ５′ＮＣＲ的序列分析证实了ＲＦＬＰ分型的正确性。结论认为该人群ＨＣＶ感染以血清Ⅰ型或基因１ｂ型为主；Ｃ区型特异性多肽血清学分型法与ＲＦＬＰ基因分型法符合率高，具有一定应用价值。     

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     

我国急性散发性戊型肝炎病毒部分核苷酸序列分析

用逆转录套式聚合酶链反应（ＲＴ－ｎＰＣＲ）自深圳、长春、杭州等地４１份急性散发性戊型肝炎病人血清中获得２８株ＨＥＶｃＤＮＡ，对其中３株ＨＥＶｃＤＮＡ的ＯＲＦ２基因片段，用荧光法直接测定其核苷酸序列，并与戊型肝炎病毒墨西哥株（Ｍ）、缅甸株（Ｂ）和新疆流行株（ＣＨ１·１）进行了比较，结果该３株散发性ＨＥＶ与Ｍ株的核苷酸序列同源性分别为８０．２％、７９．９％和７９．４％；与Ｂ流行株的同源性为９５．５％、９３．９％和９５．１％；与散发株的同源性为９３．４％、９２．３％和９３．８％；与ＣＨ１·１的同源性为９７．０％、９６．５％和９５．９；表明该３株散发性ＨＥＶ与ＨＥＶ（Ｂ）和ＣＨ１·１的核酸序列同源性较高，可能属同一亚型。     

乙型肝炎病毒基因组前-前-S和前-X区基因分布的研究

目的研究不同基因型乙型肝炎病毒(HBV)前-前-S和前-X区核苷酸和氨基酸序列分布。方法应用DNAstar软件中的MegAlign程序分析GenBank登记的35株和3株由本室测定的不同基因型HBV全序列中前-前-S和前-X区基因分布情况，并用Blast软件对GenBank中598株HBV全基因组序列进行前-前-S和前-X区的核苷酸和氨基酸相似性分析。结果除D基因型外，在各型HBV基因组中均存在前-前-S区核苷酸序列，但仅在C、F和H型HBV基因组中存在前-前-S区开放读码框架(ORF)。但前-X区核苷酸序列及其ORF仪见于C基因型HBV。在GenBank登记的598株不同基因型HBV中，与推导的前-前-S区相似氨基酸序列有36株，而与推导的前-X区相似的氨基酸序列有47株。结论前-前-S区序列存在于除D基因型以外的所有HBV基因型，而前-X区基因仅存在于C基因型HBV基因组中。     

HBVB／C混合基因型对慢性乙型肝炎干扰素α抗病毒疗效的影响

目的探讨慢性乙型肝炎（CHB）患者B／C混合基因型乙型肝炎病毒（HBV）感染与干扰素α（IFN—α）治疗效果的相关性。方法经IFN—α治疗的HBeAg阳性B／C混合基因型CHB患者,采集治疗基线及治疗12周后血清,扩增HBV全基因组并进行克隆,每份标本17—20个克隆测序并进行序列分析及分子进化分析。结果2例HBeAg阳性B／C混合基因型感染CHB患者,优势病毒株均为C型毒株。经IFN-α治疗后,1例发生完全病毒学应答,1例发生部分病毒学应答并且治疗后B基因型病毒株完全清除。完全应答者与部分应答者治疗前C基因型准种复杂度为0．5562VS0．6305,部分病毒学应答者治疗后全基因组准种复杂度高于治疗前C基因型准种复杂度（0．6305VS0．7200）,全基因组核苷酸水平的平均遗传距离高于治疗前（0．00454VS0．00648核苷酸替换／位点）。结论（1）B／C混合基因型感染CHB患者优势病毒株为C型毒株。（2）B基因型病毒株对干扰素的敏感性高于C基因型,B／C混合基因型感染患者治疗后病毒准种复杂度及准种多样性升高。     

The Putative Recombination of Hepatitis B Virus Genotype B with Pre-C/C Region of Genotype C

Among hepatitis B virus (HBV) genotypes the B and C are most prevalent in China. To further study on the inside story of the intertypes, the genotype of 136 sequences from Chinese patients were analyzed either by restriction fragment length polymorphism on fragments or by phylogenetic analysis and bootscanning on full genome. The 22 complete sequences of genotype B clustered with different genotypes depending on gene fragments analyzed, which indicated that recombinant events occurred during HBV evolutionary history. To locate the recombinant regions, the sequences of HBV entire genome were analyzed by SimPlot program. The recombinant regions of B genotype with recombination were mapped in the pre-C/C region with relatively less varied size. Besides, three sequences of genotype C have recombination with genotype B or D in different regions. However, among all of the 136 sequences, none of authentic genotype B was identified. To investigate the possible mechanism responsible for intertype recombination, the selection pressure on the recombinant region was estimated by using CODEML program. All models allow for positively selected sites suggest existence of positive selection pressure. In conclusion, the genotype B with recombination was exclusive subgroup of genotype B in China. The mosaic genotype B might result from immune pressure on the pre-C/C gene.     

我国藏族居民乙型肝炎病毒基因型的初步研究

目的　初步确定流行于西藏和青海藏族居民中乙型肝炎病毒(HBV)基因型的基本情况,为制定肝炎防治策略提供依据。方法　利用PCR扩增西藏和青海藏族居民HBVS基因和C基因测序,并用MEGA3软件进行核苷酸序列的同源性比较及构建系统发生树,分析基因型。结果　绝大多数样本HBV的S基因序列聚集于系统发生树中的基因型D ,所有样本HBV的C基因序列聚集于系统发生树中的基因型C结论　西藏和青海HBV基因型绝大多数为C D重组基因型。     

Complete genomic sequence and phylogenetic relatedness of hepatitis B virus isolates in Cambodia

Although it is known that Cambodia is one of the high endemic area of hepatitis B virus (HBV) infection, molecular characterization of HBV circulating in this country has not been reported. In this study, pre-S gene of HBV from 12 Cambodian patients was sequenced. Phylogenetic analysis based on the pre-S gene sequence revealed that 8 out of 12 isolates (66.7%) belonged to HBV/C1 and remaining four (33.3%) were HBV/B4. Furthermore, complete genomic sequences were also determined for three Cambodian HBV isolates. They all comprised of 3,215 bp long and two of them belonged to subgenotype B4, which had recombination event with genotype C in the precore/core gene confirmed by SimPlot and BootScanning analyses. Our results showed that both HBV strains belonged to subgenotypes B4 and C1, which are circulating in this country. This is the first report on molecular characterization of the HBV prevalent in Cambodia.     

Recent high incidence of fulminant hepatitis in Samara, Russia: molecular analysis of prevailing hepatitis B and D virus strains

Until 1991, the Russian city of Samara was largely isolated from other parts of Russia and the rest of the world. Very recently, Samara has seen an alarming increase in the incidence of hepatitis. The proportion of fulminant cases is unusually high. We wanted to assess the roles of hepatitis B virus (HBV) and hepatitis D virus (HDV) in acute viral hepatitis in this region by analyzing the prevailing strains of both and by determining their genotypes and possible origin. Serum samples were screened for different serological markers and by PCR followed by direct sequencing. Of the 94 HBV-positive samples (80% of which were acute infections), 37 (39%) were also HDV positive. Sixty-seven percent of the patients had anti-HCV antibodies. Twenty-five percent of all patients in the study had fulminant hepatitis. Statistically significant sex differences were found among fulminant cases. For HBV, the core promoter sequences of 62 strains were determined and all but one were found to be of genotype D. None of these had any deletions. Only one strain, from a patient with fulminant fatal hepatitis, showed multiple mutations. The pre-S2 region sequences of 31 HBV strains were also compared. Phylogenetically, these fell into two distinct groups within genotype D, suggesting different origins. For HDV, part of the region encoding the delta-antigen was sequenced from four strains. All proved to be of genotype I and were similar to Far Eastern and Eastern European strains. The contribution of intravenous drug use to the sharp increase in viral hepatitis in this unique setting is discussed.     

Complete genomic sequence and phylogenetic relatedness of hepatitis B virus isolates from Iran

Hepatitis B virus (HBV) is one of the main etiological agents of acute and chronic liver disease that is still a major public health problem in the world. Numerous HBV isolates have grouped into eight genotypes, A to H, based on the complete genome sequence. To date, no study has been carried out on the complete HBV genome sequence in Iran. The objective of this study was to investigate the complete genome sequence organization and phylogenetic analysis of the five HBV strains, which obtained from Iranian chronic infected patients. Results showed that Iranian strains were closely related to each other, with 97-100% nucleotide similarity. Phylogenetic analysis based on the complete genome sequences and the precore/core gene sequences revealed that all strains were of genotype D, sub-genotype D1 with bootstrap value 100 and 99%, respectively. The S gene encoded Arg122, Pro127, and Lys160 corresponding to subtype ayw2. Iranian HBV isolates had closely related with Turkish HBV strains. All strains had a nucleotide length of 3,182 base pair (bp) except IR-P4 strain, with a 3,185 bp in length and with a unique Phe89 insertion in the X gene. The intragenotypic divergence of the complete genome sequence of Iranian strains was 1.8% and the intergenotypic in genotype D was 3.8% and with the other genotypes was 7.9-15.4%. In conclusion, this study revealed that the HBV genotype D, sub-genotype D1, subtype ayw2 dominates in the Iranian infected patients. A single Phe89 insertion in the X gene of the one Iranian strain with an unforeseen length of 3185 bp was identified.     

Primate hepatitis B viruses - genetic diversity, geography and evolution

There are six well characterised genotypes (A-F) of human hepatitis B virus that have distinct geographic ranges which generally relate to chronic HBV infection. A seventh human genotype (G) has recently been described, but there is limited information on ethnic and geographic distribution. Despite the fact that early studies indicated that HBV antigens were present in other primates, the prevailing dogma that HBV was a human disease precluded alternative explanations. Within the past 5 years, hepatitis B viruses have been characterised from all the Old World great apes (orangutan, gibbons, gorillas and chimpanzees) and from a New World woolly monkey. Each group of non-human primates appears to have a distinct strain of hepatitis B virus that can be distinguished from human sequences based upon the nucleotide sequence and selected amino acid changes in the viral proteins. The woolly monkey HBV is most divergent from other primate and human sequences, while the great ape HBV sequences cluster together with separate branches for each group.     

Central African Republic is part of the West-African hepatitis B virus genotype E crescent.

BACKGROUND: Recent studies have shown that Hepatitis B virus (HBV) genotype E predominates in a vast crescent in West-Africa spanning from Senegal to Angola. OBJECTIVES: To determine whether HBV strains in the Central African Republic (CAR) belong predominately to the homogeneous West-African genotype E or whether they are more closely related to genotypes found in East Africa. STUDY DESIGN: Serum samples were randomly collected from 196 patients admitted with symptoms of acute or chronic hepatitis to the Central Hospital in Bangui. Thirty complete and 36 partial sequences of HBV strains were obtained. RESULTS: Ninety-four percent (62/66) of the strains belonged to genotype E, while genotype A1, most closely related to a strain from Tanzania and genotype D were detected in only one and three samples, respectively. One strain presented a recombination between the S and X gene of a genotype E precursor and a partial PreC/C gene of a genotype D precursor. CONCLUSIONS: Genotype E is predominant in CAR with little overlap with genotypes from Eastern Africa, extending the West-African HBV genotype E crescent further to the East.