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2009年24卷1期

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Research Article

哺乳动物呼肠孤病毒非结构蛋白μNS功能研究
Functional Analyses of Mammalian Reovirus Nonstructural Protein μNS*

范超, 方勤

2009, 24(1): 1 doi: 10.1007/s12250-009-3016-5

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呼肠孤病毒基因组复制发生在细胞质包涵体内。由病毒基因片段M3编码的非结构蛋白uNS为非病毒颗粒组分,但是在病毒感染的细胞内高水平表达,并浓缩在病毒感染细胞的细胞质包涵体内。最新研究表明uNS蛋白不仅在形成包涵体过程中起着重要的作用,而且在病毒侵染过程中与其它病毒组分有着相互作用。
Abstract Genome replication of reovirus occurs in cytoplasmic inclusion bodies called viral factories or viroplasms. The viral nonstructural protein μNS, encoded by genome segment M3, is not a component of mature virions, but is expressed to high levels in infected cells and is concentrated in the infected cell factory matrix. Recent studies have demonstrated that μNS plays a central role in forming the matrix of these structures, as well as in recruiting other components to them for putative roles in genome replication and particle assembly.

湖北省高致病性猪生殖和呼吸综合征病毒变种的基因克隆
及进化分析*
Molecular Characterization of a Highly Pathogenetic Porcine
Reproductive and Respiratory Syndrome Virus Variant in Hubei, China*

黄弋, 张兵, 付振芳, 郑方亮, 梁旺旺, 杨克礼, 徐涤平, 王汉中,

2009, 24(1): 9 doi: 10.1007/s12250-009-3012-9

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猪生殖与呼吸综合征(Porcine Reproductive and Respiratory Syndrome, PRRS)是一种以妊娠母猪的繁殖障碍和仔猪的呼吸道疾病为特征的病毒性传染病,该病的病原,PRRSV,已成为危害全球养猪业的主要病原。2006年,国内十几个省市内爆发了由以发热、皮肤发红、高致病率和高死亡率为主要特征的疑似猪生殖与呼吸综合征的传染病。本实验室自2006年6月至2007年4月对湖北省内采集的近百份临床样本进行了RT-PCR检测,并克隆到了病原体的N、GP5和NSP2等几个主要基因。对上述基因的进化分析显示,一株高致病性的PRRSV美洲株变种是造成2006年湖北省猪病爆发的主要病原体。虽然,该变异株的N基因和GP5基因与一些典型的北美株有90%左右的同一性,但是在GP5基因中发现不少突变的氨基酸位点;另外一个具有重要功能的病毒蛋白NSP2与典型的美洲株仅有65%左右的同源性,并且编码该蛋白的基因中有2个明显的缺失(分别位于480位的一个氨基酸和520-530位的29个氨基酸)。利用GenBank中可以的序列信息构建的进化树显示,与其他毒株相比,该PRRSV变异株与经典美洲株的遗传距离最远,这可能可以用来解释该变异株引起的临床症状不同于中国国内的流行株。
Abstract Porcine reproductive and respiratory syndrome virus (PRRSV) has been recognized as one of the most important pathogens of pigs throughout the world. In 2006, more than 10 provinces of China have experienced an epizootic outbreak of pig diseases characterized by high fever, reddened skin and high morbidity and mortality. From June 2006 to April 2007, we have investigated some clinical samples in Hubei province by RT-PCR and cloned several major genes, N, GP5 and NSP2 gene, shown in this study. Phylogenetic analysis of these genes revealed that the highly pathogenic PRRSV variant, ZB, was responsible for 2006 emergent outbreak of pig disease in Hubei province similar with those variants isolated from other provinces in China in 2006, and belongs to the NA-type PRRSV. In the PRRSV variants, the N and GP5 shear about 90% identity with prototypic ATCC VR-2332 and some typical NA-type Chinese isolates, except the 2850bp NSP2 gene (only shares 65% identity with ATCC VR-2332). But they all shear more than and 97% identity with other highly pathogenetic Chinese PRRSV strains. Additionally, there are extensive amino acid (aa) mutations in the GP5 protein and 2 deletions in the Nsp2 protein when compared with the previous isolates. Most of the variants found in 2006 epizootic outbreak of pig diseases in China were the farthest variants from the typical NA-type PRRSV in phylogenetic distance, and these diversities may be responsible for the differences in the pathogenicity observed between these variants and original Chinese PRRSV strains.

贯叶连翘提取物对甲型流感病毒的抑制作用的研究*
Anti-Influenza A Virus Effect of Hypericum perforatum L. Extract*

蒲秀英, 梁剑平, 王学红, 许涛, 华兰英, 尚若峰, 刘宇,

2009, 24(1): 19 doi: 10.1007/s12250-009-2983-x

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为研究贯叶连翘提取物(HPE)体内外对甲型(H1N1亚型)流感病毒(IAV)活性的影响。以MDCK细胞培养增殖的IAV为研究对象,通过中性红染色(NR)和观察细胞病变(CPE)效应,评价贯叶连翘提取物体外抑制PRRSV活性的效应。结果显示:贯叶连翘提取物体外有效抗H1N1型流感病毒,其半数有效浓度(EC50)为40 μg/mL ,对MDCK细胞的平均半数中毒浓度(CC50)是1.5 mg/mL,而药物对照病毒唑的EC50和CC50分别是5.0 μg/mL和520 μg/mL。滴鼻感染流感病毒给小鼠4小时后,分别经口灌服贯叶连翘提取物和病毒唑,每天2次,连续治疗5 d。可有效的预防小鼠死亡率、减轻动脉血氧饱和度的下降、抑制肺实变和降低肺组织病毒滴度。贯叶连翘提取物和病毒唑的最小有效剂量分别是31.25 mg/kg/day和40 mg/kg/day。药物对小鼠的毒性试验表明:当灌服贯叶连翘提取物低于2000 mg/kg/day剂量以下时,几乎所有小鼠存活,其属低毒药物;病毒唑的半数致死量(LD50)是200 mg/kg/day。经口灌服500 mg/kg/day的贯叶连翘提取物和70 mg/kg/day的病毒唑(1/3LD50)后,对感染流感病毒48小时的小鼠仍起到保护作用。两个药物均显示出了相似的抗流感病毒作用,鉴于贯叶连翘提取物的毒性很小,研究其作为抗流感药物将更有前景。
Abstract To study the antiviral effect of Hypericum perforatum L. extract (HPE) on influenza A virus(IAV)(H1N1) in vitro and in vivo. Cytopathic effect (CPE) and neutral red (NR) dye uptake were used to examine the antiviral effect of HPE on Madin Darby Canine Kidney(MDCK) cells which were infected with IAV in vitro. HPE was effective against influenza A virus (IAV) in vitro, with a 50% effective concentration (EC50) of 40 μg/mL. The mean 50% cytotoxic concentration (CC50) in the MDCK used in these experiments was 1.5 mg/mL. Ribavirin was run in parallel with EC50 values of 5.0 μg/mL; the mean CC50 for ribavirin was 520 μg/mL. Oral gavage administrations of HPE or ribavirin to mice infected with the IAV were highly effective in preventing death, slowing the decline of arterial oxygen saturation, inhibiting lung consolidation and reducing lung virus titers. The minimum effective dose of HPE in these studies was 31.25 mg/kg/day, which was administered twice daily for 5 d beginning 4 h prior to virus exposure. Below a dosage of 2000 mg/kg/day, almost all treated mice survived, which suggests that HPE is of low toxicity. Ribavirin’s minimum effective dose was 40 mg/kg/day with the LD50 determined to be 200 mg/kg/day. Delay of the initiation of either HPE or ribavirin therapy, using approximately 1/3 LD50 dose each time, could still be protective as late as 48 h after exposure to the IAV. While both agents appeared to have similar efficacy against IAV infections, HPE was considered to be less toxic and may warrant further evaluation as a possible therapy for influenza.

中国腮腺炎病毒分离株SP 株的全序列的测定及分析*
Complete Nucleotide Sequence of a Mumps Virus SP
Strain Isolated in China*

马绍辉, 刘建生, 施海晶, 王丽春, 王晶晶, 刘龙丁, 李琦涵*Shao-hui MA

2009, 24(1): 28 doi: 10.1007/s12250-009-2984-9

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对中国腮腺炎病毒分离株SP株的全序列的测定及分析。方法为提取病毒RNA,利用一步法进行RT-PCR,然后克隆、鉴定及测序。结果该毒株与其他腮腺炎病毒一样,长为15,384bp,编码7个蛋白。但在核苷酸水平有4%-6.8%的差异。由于氨基酸上的差异,特别是HN 和N蛋白,可以认为该分离株在抗原上有明显的变异。该结果是第一次对腮腺炎病毒F 基因型的全基因进行测序以及分析了流行株的遗传特征。
Abstract The complete nucleotide sequence of the mumps virus SP, which was isolated in China, was determined. As with other mumps viruses, its genome was 15 384 nucleotides (nts) in length and encoded seven proteins. The full-length nucleotide sequence of the SP isolate differed from other strains by 4% –6.8% at the nucleotide sequence level. Due to variations of amino acids over the full genome (including the HN and N genes), this isolate exhibited significant variations in the antigenic sites. This report is the first to describe the full-length genome of a genotype F strain and provide an overview of the diversity of genetic characteristics of a circulating mumps virus.

3株H9N2亚型禽流感病毒广西分离株全基因组序列的测定与分析*
Genome sequencing and phylogenetic analysis of three avian influenza H9N2 subtypes in Guangxi*

谢芝勋, 董建宝, 唐小飞, 刘加波, 庞耀珊, 邓显文, 谢志勤, 谢丽基

2009, 24(1): 37 doi: 10.1007/s12250-009-2985-8

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本研究根据GenBank公布的H9N2亚型禽流感病毒(AIV)的全基因序列,设计了8对引物,运用RT-PCR方法获取了3株H9N2亚型AIV广西地方分离株A/Chick-en/Guangxi/1/00、A/Chicken/Guangxi/14/00和A/Chicken/Guangxi/17/00的全基因序列,并对所得序列进行了比较分析。同源性分析及遗传进化分析发现,所分离的3个毒株的各基因与A/Chicken/Guangdong/4/00和A/Chicken/Jiangsu/1/00的相应基因的核苷酸序列有着较高的同源性和较近的亲缘关系,可能起源于同一种系。所分离毒株中每个毒株的8个基因,在遗传进化树上的分支不具有统一性,说明此3个毒株可能为不同基因亚系间发生自然重排的产物。氨基酸序列比较发现,A/Chicken/Guangxi/1/00、A/Chicken/Guangxi/14/00和A/Chicken/Guangxi/17/00 3株AIV的na基因核苷酸序列在开放性阅读框架的187~195位均缺失了ACAGAGATA共9个核苷酸,它们所编码的氨基酸为T、E、I。3个分离株的HA蛋白第226位氨基酸均为Gln,证明它们为人类非易感性的H9N2亚型AIV,此3株H9N2亚型流感病毒为基因自然重排的产物,其na基因均存在缺失,且从分子水平上证明它们对人类不具有易感性
Abstract Three isolates of H9N2 Avian Influenza viruses (AIV) were isolated from chickens in Guangxi province. Eight pairs of specific primers were designed and synthesized according to the sequences of H9N2 at GenBank. phylogenetic analysis showed a high degree of homology between the Guangxi isolates and isolates from Guangdong and Jiangsu provinces, suggesting that the Guangxi isolates originated from the same source. However, the eight genes of the three isolates from Guangxi were not in the same sublineages in their respective phylogenetic trees, which suggests that they were products of natural reassortment between H9N2 avian influenza viruses from different sublineages. The 9 nucleotides ACAGAGATA which encode amino acids T, G, I were absent between nucleotide 205 and 214 in the open reading frame of the NA gene in the Guangxi isolates. AIV strains that infect human have, in their HA proteins, leucine at position 226. The analysis of deduced amino acid sequence of HA proteins showed that position 226 of these isolates contained glycine instead of leucine, suggesting that these three isolates differ from H9N2 AIV strains isolated from human infections

双抗原夹心法检测肝炎患者血清中抗丁肝病毒(HDV)抗体
Detection of Antibody to Hepatitis Delta Virus in Human Serum
by Double Antigen Sandwich ELISA

谢立, 黄德庄, 贺立香, 罗朝霞, 周育森, 吴晓东

2009, 24(1): 45 doi: 10.1007/s12250-009-2981-2

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本文采用RT-PCR法从HDV阳性血清中扩增HDV基因表达片断,构建高纯度、高效价HD-PQE31表达株,用Ni-NTA层析法获得高纯度、高滴度的纯化丁型肝炎病毒抗原蛋白,并采用Western blot和ELISA进行鉴定,建立了检测人血清中抗HDV的双抗原夹心ELISA方法,并对该项试验的敏感性、特异性进行鉴定。鉴定结果为:(1)纯化的HDV抗原蛋白纯度达90%,ELISA滴度为1×10-5。(2)对42份抗HD 阳性血清进行测定,并与ELISA竞争法作比较,结果显示其敏感度明显优于竞争法(t=2.44,p<0.01)。(3)对2份抗HD阳性血清作中和试验,经特异抗原HDAg处理后,抑制率为74-93%。(4)特异性检测表明,60例其他各类型肝炎病毒阳性血清样品和30例正常人血清样品检测抗HD的结果均为阴性,只有2例作为质控血清的样品抗HD结果呈阳性。因此,本研究以高效价、高纯度重组HDAg蛋白为主要试剂建立的双抗原夹心ELISA法用于测定人血清抗HDV,具有良好的特异性和敏感性,在丁肝感染的临床诊断中可以推广应用。
Abstract A simple rapid detection of antibody to hepatitis delta virus (anti-HDV) in human serum was developed by using double antigen sandwich ELISA. HDV gene fragment encoding HDAg was isolated from a Chinese patient infected with HDV by RT-PCR, and a high-efficient expression HD-PQE31 strain was constructed with the fragment. We obtained high titer and good quality hepatitis delta virus protein purified by Ni-NTA metal-affinity chromatography, which was identified by Western blot and ELISA, then we set up the double antigen sandwich ELISA for detection of anti-HDV in human serum, and the performance of the sandwich ELISA was evaluated in terms of specificity and sensitivity. Results were: 1) The purified HDAg protein’s purity was 90%, and its ELISA titer was 1/100 000. 2) 42 anti-HDV positive sera were detected and showed that the sensitivity of sandwich ELISA was higher than that of competitive ELISA (t=2.44, p<0.01). 3) The inhibitory rates for 2 anti-HDV positive sera by the specific HDAg were 74% and 93% respectively. 4) For the assay of specificity, all 60 samples infected by other hepatitis viruses and 30 normal samples were negative for anti-HDV. These results suggested that the double antigen sandwich ELISA with purified recombinant HDAg showed higher specificity and sensitivity, It can be used in routine laboratories to diagnose the HDV infection.

一株三源基因重配的H3N2亚型流感病毒的拯救和遗传进化分析*
Genetic Analysis and Rescue of a Triple-reassortant H3N2 Influenza A Virus Isolated From Swine in Eastern China*

祁贤, 焦永军, 潘浩, 崔仑标, 范伟兴, 黄保续, 史智扬, 汪华

2009, 24(1): 52 doi: 10.1007/s12250-009-3006-7

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从猪体内分离一株H3N2亚型流感病毒(A/Swine/Shangdong/3/2005, Sw/SD/3/2005)。全基因组序列分析表明Sw/SD/3/2005是一株三源基因重配病毒,其中HA, NA, NP, M, PB1和PA基因来源于人H3N2病毒,PB2基因来源于人H1N1病毒,而NS基因则来源于古典猪H1N1病毒。Sw/SD/3/2005的分离进一步为猪是A型流感病毒“基因混合器”观点提供了证据,同时也提示应该加强A型流感病毒在猪体内的生态分布和分子进化的监测研究。将经RT-PCR得到的A/swine/Shandong/3/2005八个基因的cDNA片段分别克隆到含有pol Ⅰ和pol Ⅱ启动子和终止子序列的PHW2000质粒,得到了8 个可在真核细胞中复制和表达的质粒。将这8个质粒共转染293T 细胞,培养6h 后吸取上清液,转接入SPF鸡胚和MDCK细胞中,再经过72h 培养,得到病毒血凝滴度为1:8 。继续接种鸡胚,经过3次传代后,病毒血凝价稳定在1:256。Sw/SD/3/2005的成功拯救为进一步深入研究H3N2亚型流感病毒跨物种传播的分子机制奠定了基础
Abstract One influenza H3N2 virus, A/swine/Shandong/3/2005 (Sw/SD/3/2005), was isolated from pigs with respiratory disease on a farm in eastern China. Genetic analysis revealed that Sw/SD/3/2005 was a triple-reassortant virus with a PB2 gene from human-like H1N1, NS from classical swine H1N1, and the remaining genes from human-like H3N2 virus. These findings further support the concept that swine can serve as reservoir or mixing vessels of influenza virus strains and maintain genetic and antigenic stability of viruses. Furthermore, we have successfully established a reverse genetics system based on eight plasmids and rescued Sw/SD/3/2005 through cell transfection. HI tests and RT-PCR confirmed that the rescued virus maintained the biological properties of the wild type Sw/SD/3/2005. The successful establishment of the reverse genetics system of Sw/SD/3/2005 will enable us to conduct extensive studies of the molecular evolution of H3N2 influenza viruses in swine.

人肠道病毒EV71VP1基因正选择压力分析
Positive Selection Analysis of VP1 Genes of Worldwide Human Enterovirus 71 Viruses

史卫峰, 张忠, 顿爱社, 张彦周, 于广福, 庄东明, 朱朝东

2009, 24(1): 59 doi: 10.1007/s12250-009-2976-9

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人肠道71病毒在全世界范围内长期流行。本文对肠道71病毒核衣壳蛋白VP1基因序列进行了正选择压力分析。结果表明,尽管绝大多数位点受到负选择压力作用或者处于中性进化,仍有VP1基因的四个位点受到正选择压力影响。这可能解释肠道71病毒的扩散和频繁爆发,以及增强的神经毒性。特别地,位点98可能与中和抗体、调节病毒与受体的相互作用以及增加病毒的毒力相关。而位点145和241也可能参与决定受体的特异性。但是,这些位点的氨基酸多态性并没有表现出太大的差异。另外,取自中国的病毒的VP1基因没有位点受到正选择压力的作用。
Abstract Human enterovirus 71 viruses have been long circulating throughout the world. In this study, we performed a positive selection analysis of the VP1 genes of capsid proteins from Enterovirus 71 viruses. Our results showed that although most sites were under negative or neutral evolution, four positions of the VP1 genes were under positive selection pressure. This might account for the spread and frequent outbreaks of the viruses and the enhanced neurovirulence. In particular, position 98 might be involved in neutralizing antibodies, modulating the virus-receptor interaction and enhancing the virulence of the viruses. Moreover, both positions 145 and 241 might correlate to determine the receptor specificity. However, these positions did not display much difference in amino acid polymorphism. In addition, no position in the VP1 genes of viruses isolated from China was under positive selection.

不同寄主来源H9N2禽流感病毒血凝素基因选择压力分析
Selection Pressure on Haemagglutinin Genes of H9N2 Influenza Viruses from Different Hosts

史卫峰, 顿爱社, 张忠, 张彦周, 于广福, 庄东明, 朱朝东

2009, 24(1): 65 doi: 10.1007/s12250-009-2988-5

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本文研究了不同寄主来源H9N2禽流感病毒血凝素基因正选择压力,以及比较了这些病毒所受选择压力的差异。结果表明,尽管血凝素基因大多数的位点处于中性或者清除进化,但是位于抗原和受体结合区域的部分位点受到正选择压力作用。进一步研究表明,这其中一些位点在种群水平受到正选择压力影响。对于来自不同宿主的H9N2病毒总是存在一些氨基酸位点受到不同的选择压力作用。这些受到正选择压力作用的位点以及受到不同选择的位点可能解释H9N2禽流感病毒宿主范围扩大的原因和对不同宿主的不同适应。
Abstract Positive selection and differential selective pressure analyses were carried out to study Haemagglutinin (HA) genes of H9N2 influenza viruses from different hosts in this paper. Results showed that, although most positions in HAs were under neutral or purifying evolution, a few positions located in the antigenic regions and receptor binding sites were subject to positive selection and some of them were even positively selected at the population level. In addition, there were always some positions differentially selected for viruses from different hosts. Both selection pressure working on HA codons and positions differentially selected might account for the extension of the host range and adaptations to different hosts of H9N2 influenza viruses.

对虾白斑综合症病毒核衣壳蛋白VP15核定位信号的鉴定
Identification and Characterization of Nuclear Localization Signals within the Nucleocapsid Protein VP15 of White Spot Syndrome Virus*

李莉娟*, 张化俊, 张聪, 石正丽*Li-juan LI#

2009, 24(1): 71 doi: 10.1007/s12250-009-3013-8

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对虾白斑综合症病毒核衣壳蛋白VP15是一个富含碱性氨基酸的DNA结合蛋白。生物信息学分析发现,在VP15蛋白序列中有三个核定位信号,命名为NLS1 (aa 11-27), NLS2 (aa 33-49)和NLS3 (44-60)。为了确定VP15的核定位功能,我们将全长VP15基因以及三个不同的核定位信号序列分别克隆到带有荧光蛋白报告基因的瞬时表达载体,并在昆虫细胞中进行表达。在转染全长VP15基因的细胞里,荧光信号只分布在细胞核内。NLS1也能使荧光蛋白分布到核内,但效率没有全长蛋白高。NLS2 和3 单独融合荧光蛋白表达,荧光信号在细胞内呈分散式的分布,但如果NLS2 和3 作为一个肽段与荧光蛋白融合表达,大部分荧光信号也只出现在细胞核内。此外,VP15的两个氨基酸11RR12如果突变成两个丙氨酸 11AA12, 荧光信号则只出现在细胞质内。这项结果说明VP15是一个核定位蛋白,它的核定位功能需要三个核定位信号共同作用, 其中氨基酸位点11RR12在蛋白从细胞质运输到细胞核的过程中起关键作用。
Abstract The nucleocapsid protein VP15 of white spot syndrome virus (WSSV) is a basic DNA-binding protein. Three canonical bipartite nuclear localization signals (NLSs), called NLS1 (aa 11-27), NLS2 (aa 33-49) and NLS3 (44-60), have been detected in this protein, using the ScanProsite computer program. To determine the nuclear localization sequence of VP15, the full-length open reading frame, or the sequence of one of the three NLSs, was fused to the green fluorescent protein (GFP) gene, and transiently expressed in insect Sf9 cells. Transfection with full-length VP15 resulted in GFP fluorescence being distributed exclusively in the nucleus. NLS1 alone could also direct GFP to the nucleus, but less efficiently. Neither of the other two NLSs (NLS2 and 3) was functional when expressed alone, but exhibited similar activity to NLS1 when they were expressed as a fusion peptide. Furthermore, a mutated VP15, in which the two basic amino acids (11RR12) of NLS1 were changed to two alanines (11AA12), caused GFP to be localized only in the cytoplasm of Sf9 cells. These results demonstrated that VP15, as a nuclear localization protein, needs cooperation between its three NLSs, and that the two residues (11RR12) of NLS1 play a key role in transporting the protein to the nucleus.

云南西双版纳傣族人群首次发现两例HBV E基因型感染*
The HBV E Genotype Discover in Dai Nationality in Xishuangbanna,
Yunnan Province*

赵海平, 申元英, 沈茹, 李强, 王远懿

2009, 24(1): 77 doi: 10.1007/s12250-009-2992-r

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采用型特异性引物巢式PCR结合聚合酶链反应-限制性片段长度多态性法(PCR-RFLP) 对感染者基因型鉴定.在云南西双版纳傣族人群中国内首次发现B+E型,为我国HBV 基因型分布提供了新的资料,并为建立中国人HBV 基因库提供依据。
Abstract To investigate the distribution of Hepatitis B virus (HBV) genotypes among the population of Dai nationality in Xishuangbanna, Yunnan Province HBV genotypes of the Serum samples were tested by PCR-RFLP. This is the first time to discover the B+E genotypes in China. This finding provides new information for understanding the distribution of HBV genotype in China and a provides a basis for establishing a Chinese gene bank.