热带医学杂志2010年第10卷总目次

【硕博专栏论著】 细粒棘球绦虫重组Bb-Eg95-EgA31疫苗诱导小鼠脾细胞增殖变化的研究，周必英，陈雅棠，李文桂，等（1）：1三峡库区上、下游血吸虫病流行区钉螺线粒体cox1基因遗传变异研究，陈琳，张锡林，何谐，等（1）：5三种旋毛虫肌肉期幼虫染色方法的比较研究，陈莹，杨晓燕，郑焕钦，等（1）：9     

湖南长沙及湘西泡状带绦虫分离株线粒体cox1基因的克隆及序列分析

目的研究湖南省长沙及湘西泡状带绦虫分离株线粒体细胞色素C氧化酶第1亚基（cox1）基因部分序列（pcox1）的遗传变异情况,并用peox1序列重构泡状带绦虫与其他带科绦虫的种群遗传关系。方法应用聚合酶链反应（PCR）扩增泡状带绦虫虫株的pcox1,应用Clusta1X1．81程序对序列进行比对。然后用Phylip3．67程序MP法和Mage4．0程序NJ法绘制种系发育树,并用Puzzle5．2程序构建最大似然树;同时利用DNAstar5．0中的Megalign程序进行同源性分析,并与GenBank^TM中已知泡状带绦虫相应基因序列进行比较分析。结果所获得的pcox1序列长度一致,均为343bp,与GenBank^TM公布的带科绦虫序列进行比较分析表明,湖南长沙分离株1和湘西分离株5与已知泡状带绦虫相应基因的相似性分别为99．4％和99．7％,与其它带科绦虫的相似性均小于90．0％;湖南长沙1和湘西分离株5与已知泡状带绦虫相应基因遗传距离分别为0．006和0．003,与其他带科绦虫的遗传距离均大于0．100。种系发育树表明,2个分离株与已知泡状带绦虫位于同一分枝,Bootstrap值在97％以上。结论由于泡状带绦虫pcox1序列种内相对保守,种问差异较大,故可作为种间遗传变异研究的标记,研究结果为进一步研究泡状带绦虫的群体遗传结构奠定了基础。     

我国6个弓形虫虫株线粒体cox1部分序列的比较分析

目的对采自我国的6个弓形虫虫株线粒体细胞色素c氧化酶第Ⅰ亚基（cox1）基因部分序列（pcox1）进行比较分析，并与RH株的相应序列进行比较，研究弓形虫线粒体cox1基因与弓形虫的种群遗传关系。方法应用聚合酶链反应（PCR）扩增弓形虫不同虫株的pcox1，并进行序列分析比较。结果每个虫株都获得599bp的pcox1序列，比较分析表明，弓形虫不同虫株的pcox1序列之间没有差异。结论弓形虫pcox1序列不能作为种群遗传变异研究的标记，也与弓形虫毒力无关。研究结果为进一步研究弓形虫的群体遗传结构奠定了一定的基础。     

中国日本血吸虫DNA遗传变异及聚类分析

目的 :探讨中国日本血吸虫的遗传变异及相互进化关系。方法 :采用随机扩增多态性DNA技术(RAPD)对中国大陆及台湾不同地域的日本血吸虫进行RAPD扩增 ,计算地域株间的遗传距离 ,并以非加权配对聚类分析 (UPGMA)法绘制中国日本血吸虫的聚类图。结果 :中国大陆与台湾各聚一类 ;大陆的四川山区与湘、鄂、赣湖区分别聚为一类 ;湖区的湖北武汉、湖北钟祥与湖南岳阳聚为一类 ,湖北阳新与江西新建聚为一类 ,湖北松滋自成一类。结论 :中国日本血吸虫亲缘关系密切 ,存在共同的起源 ,同时在基因型上存在不同程度的遗传分化 ,分化的程度同地理生态环境差异相关     

深圳5例肠道病毒71型手足口病病毒株遗传衍化分析

目的调查来源于2008年深圳5例EV71（Enterovirus71,EV71）型手足口病患者的5株肠道病毒71型的分子流行病学特点及其分子进化关系。方法通过病毒分离培养得到5株肠道病毒71型分离株,并对其进行全基因组核苷酸序：列测定,通过生物信息学软件分析,与既往其他国家地区分离株进行全基因序列分析比较,以及进化树分析。结果通过对VP1和VP4基因序列分析,5株病毒株均属于C4基因亚型。5株EV71分离株在全长核苷酸和氨基酸水平上差异都较小,其核苷酸和氨基酸同源性分别高于93．0％和98．0％,进化树分析提示2008深圳分离株与2004深圳株同源性最为接近,与2008安徽阜阳流行株和1998深圳株亦相距很近,死亡病例株与2008安徽阜阳株距离同源性最近（同源性为99．1％和96％）。结论推测这起疫情是由同一个病毒传播链引起的,本次流行株可能系1998深圳株演变而来。     

三峡库区生态环境变化对血吸虫病流行的影响及防治对策研究

目的研究三峡成库后生态环境变化对血吸虫病流行的影响，为制定防治方案提供科学依据。方法在三峡库区中段和尾端江岸海拔160m和180m高度，模拟成库后灌溉区和淤积区类似条件，投放湖北肋壳钉螺和四川光壳钉螺，观察其生长繁殖1年的情况；用IHA抽样筛查来往于疫区流动人群中潜在的传染源．粪便检查血吸虫虫卵；了解耕牛等生畜引进情况；调查重庆市历史疫情报告和三峡医院、重庆医科大学附属第一医院诊断的血吸虫病病例；调查影响钉螺输入的因素。结果肋壳钉螺和光壳钉螺成活率仅少数月份在50％以下，多数月份在80％左右，各试验区发现钉螺交配数量较少，相对以3-6月为多，同时发现活幼螺；来往疫区流动人员血清抗体检查1075人，血吸虫抗体阳性率为1．77％，疫区返乡人员1030人，血清阳性率为1．07％；1989—2006年，发现有记载的输人性血吸虫病病例14例；血吸虫病区有大量造纸原料和花草树木进入库区。结论三峡成库后，生态环境发生变化，存在血吸虫病流行的潜在危险。     

2008年三峡库区流动人口血吸虫感染分析

目的探讨三峡库区流动人口血吸虫感染特征,为制定三峡库区血吸虫病防治对策和措施提供科学依据。方法采用流行病学调查和酶联免疫吸附试验（ELISA）在三峡库区调查流动人口的血吸虫感染情况。结果调查流动人口1416人,其中返乡人员792人,疫区来渝人员624人。返乡人员流动原因以外出务工为主,疫区来渝人员以经商为主;流动人口血清学阳性率为3．04％,其中返乡人员、疫区来渝人员分别为4．04％、1．76％;来自流行省、传播阻断省分别为3．60％、1．33％,来自非流行省未发现阳性。流动人员主要来自湖北、湖南、四川,分别占33．40％、17．44％、10．52％;血清阳性率以江西、江苏、湖北最高,均超过4％。多因素Logistic回归分析显示。流动人口来源、可疑感染地区与血清学感染差异有统计学意义（P〈0．01）;返乡人员血清学阳性率显著高于疫区来渝人员（x^2=9．00,P〈0．01）,来自血吸虫病流行区的阳性率高于其它地区（x^2=6．99,P〈0．01）。结论三峡库区流动人口存在血吸虫感染情况,主要来自血吸虫病流行省份,重点监测来自库区周边省份的流动人员。特别是返乡人员。     

云南大理猪戊型肝炎病毒分子流行病学调查

目的：了解云南大理农村的猪群中戊型肝炎病毒（ hepatitis E virus, HEV）感染情况,为HEV的防控提供理论依据。方法利用逆转录巢式聚合酶链反应（ RT?nested PCR）技术,对所采集大理41份猪粪便样品进行HEVORF2基因部分片段扩增,经琼脂糖凝胶电泳后对目的条带进行回收纯化及克隆测序,序列利用DNAStar和MEGA4．0软件进行同源性比较和进化树构建。结果 RT?nested PCR方法扩增出348 bp目的基因序列,系统进化树显示该病毒属于基因4型h亚型HEV。此次实验共检测41份猪粪便样品,其中23份为阳性,阳性率为56．10％。结论大理猪群存在较高的HEV感染率,存在HEV跨种间感染人的风险,应该加强防控以免HEV爆发流行。     

云南彝族九个短串联重复序列位点遗传多态性

目的研究中国云南彝族人群的群体遗传结构。方法选择9个短串联重复序列（short tandem repeats，STR）位点（D3S1358、vWA、FGA、TH01、TPOX、CSFlPO、D5S818、D13S317、D7S820），采用STR复合扩增及荧光标记STR基因扫描技术，检测84名彝族无关个体血液样本。结果9个STR位点在84名云南彝族共检出69种等位片段，频率分布在0．0060-0．5060之间；检出164种基因型，频率分布在0．0119-0．4167之间。9个STR位点的基因型分布符合Hardy．Weinberg平衡定律（P〉0．05）。9个位点多态信息量分布在0．5804-0．8777之间，杂合度分布在0．6507～0．8002之间，个体识别力分布在0．7976-0．9558之间，除TPOX，TH01两个位点低于0．5外，其余7个位点的非父排除率分布在0．5207～0．8386之间。Neighbor joining（NJ）法构建彝族与云南地区其他11个少数民族的系统进化树显示：彝族首先与白族、普米族、德昂族、阿昌族、独龙族、怒族聚在一起，然后与傈僳族、傣族相聚，最后与景颇族、苗族、纳西族相聚。绪论获得了云南彝族9个STR位点的遗传多态数据，在人类群体遗传数据库建设、法医学亲权鉴定和个体识别研究及应用领域有重要价值。     

微卫星锚定PCR技术研究云南微小按蚊群体遗传结构

目的研究云南不同地理来源微小按蚊的群体遗传结构,探讨不同群体间的遗传结构和分化现象。方法在云南省东、西、南、北及中部各选择1～2个自然村,用紫外诱蚊灯于每晚17时至次日7时诱蚊,收集雌成蚊以氯仿麻醉,经形态学鉴别为微小按蚊的样本取单蚊蚊腿,再经复合PCR方法鉴别微小按蚊A或C。采用微卫星锚定PCR技术（SSR-PCR）扩增微小按蚊单蚊基因组DNA,用BIOSIS,RAPDFST,RAPDDIST及PHILIP等软件统计分析基因位点多态性、固定指数FST及θ、种群间的迁移率（Nm）以及遗传距离、聚类分析构建系统树。结果以多态位点比例衡量各种群的遗传多态性,云南不同地区微小按蚊均共享较高多态性,其中元江（C）的变异程度较低,为43.3%,潞西（A）的变异程度较高,为78.6%。FST和θ结果提示,微小按蚊的遗传变异主要存在于种群内部。微小按蚊A与C分别或两者合并分析,Nm均大于1。系统树主要分为两支,元阳（C）、大关（C）和勐腊（C）聚为一支;另一支分为元江（C）与潞西（A）,新平（A）和临沧（C）,以及勐腊（A）共3层。结论各群体间遗传距离部分与亲缘种分类有关,未发现与地理距离相关。     

24个群体11个Y-STR基因座单倍型遗传关系的聚类分析

目的 选择具有高度遗传多态性与稳定性的11个Y-STR基因座,从父系遗传角度探讨24个群体的分子遗传学关系.方法 应用PowerPlex(R)Y System荧光标记复合扩增系统检测204名回族、280名锡伯族、203名满族、215名重庆土家族无关男性个体血样,用ABI310遗传分析仪进行基因分型,计算等位基因和单倍型频率,并结合已公开发表的国内外其他20个群体相同基因座的单倍型数据,计算各群体间的遗传距离Rst,进行聚类分析,并采用Neighbor-Joining法重建系统发生树.结果 不同群体特定Y-STR的单倍型频率和遗传距离存在差异;聚类分析与系统发生树结果一致,12个汉族群体主要分为南北两类,云南和四川汉族与北方汉族遗传距离较近(0.002 3、0.000 6);沈阳回族群体自成一类,其他少数民族彼此独立分支,其中朝鲜族与韩国、日本遗传距离较近(0.013 3、0.041 3).结论 24个群体之间存在一定的基因交流,个别少数民族相对独立;研究特定Y-STR单倍型数据的遗传距离对了解各群体的起源、迁移以及相互关系有重要的意义.     

24个群体11个Y-STR基因座单倍型遗传关系的聚类分析

目的 选择具有高度遗传多态性与稳定性的11个Y-STR基因座,从父系遗传角度探讨24个群体的分子遗传学关系.方法 应用PowerPlex(R)Y System荧光标记复合扩增系统检测204名回族、280名锡伯族、203名满族、215名重庆土家族无关男性个体血样,用ABI310遗传分析仪进行基因分型,计算等位基因和单倍型频率,并结合已公开发表的国内外其他20个群体相同基因座的单倍型数据,计算各群体间的遗传距离Rst,进行聚类分析,并采用Neighbor-Joining法重建系统发生树.结果 不同群体特定Y-STR的单倍型频率和遗传距离存在差异;聚类分析与系统发生树结果一致,12个汉族群体主要分为南北两类,云南和四川汉族与北方汉族遗传距离较近(0.002 3、0.000 6);沈阳回族群体自成一类,其他少数民族彼此独立分支,其中朝鲜族与韩国、日本遗传距离较近(0.013 3、0.041 3).结论 24个群体之间存在一定的基因交流,个别少数民族相对独立;研究特定Y-STR单倍型数据的遗传距离对了解各群体的起源、迁移以及相互关系有重要的意义.     

Diversity and evolution of a newly emerged North American Type 1 porcine arterivirus: analysis of isolates collected between 1999 and 2004

Summary. European-like Type 1 porcine reproductive and respiratory syndrome virus (PRRSV) isolates, known as North American (NA) Type 1 PRRSV, appeared in United States (U.S.) swine herds in 1999. Their diversity and evolution were studied over a five-year period by constructing phylogenetic trees using nsp2 and ORF5 sequences of 20 NA Type 1 isolates, including the only known isolate from Hawaii. All but two of the isolates possessed the same 51-nt deletion in nsp2, suggesting a clonal origin. Parsimony and distance analysis showed that viruses could be placed into two distinct sub-clades, which were similar for both nsp2 and ORF5. An incongruity between the two trees identified one isolate, 04-41, as the product of recombination. Recombination analysis using SimPlot identified a break point located downstream of the nsp2/3 junction. Results from this study suggest that NA Type 1 PRRSV in the U.S. is a well-established and rapidly evolving group. However, the forces driving genetic diversity and separation are complex and remain to be elucidated.     

Phylogenies and the forces of evolution

The construction of phylogenetic trees from gene frequency data assumes that a history of binary fissioning of populations has been the major cause of genetic variation. However, in many areas of the world human populations have been relatively stable with local gene flow. This population history is closer to an isolation by distance model. It was modelled by a simulation of gene frequency changes in a linear sequence of 50 stable populations with gene flow among neighboring populations. Phylogenetic trees were constructed from the gene frequencies after the simulation was run for 500 generations. Using only a few loci there is little correlation between genetic and geographic distance, but with 40 or more loci, there was a perfect correlation with geographic distance. A different population model can thus result in a phylogenetic tree comparable to those assumed to be produced by binary fission.     

The contamination of wild watercress with Fasciola hepatica in central France depends on the ability of several lymnaeid snails to migrate upstream towards the beds

As most natural watercress beds in central France are located upstream of the permanent habitats of two lymnaeid species, Galba truncatula and Omphiscola glabra, field investigations were made from 1999 to 2004 on 67 beds to determine why the contamination of watercress with Fasciola hepatica is irregular over time in these sites, while definitive hosts, especially lagomorphs, are regularly found infected around them. Snails are able to migrate upstream in winter and spring towards the beds, and a 4-year survey demonstrated the existence of annual variation in the colonization of these sites by snails. In the 45 beds irregularly contaminated with F. hepatica over time, 37.7–62.2%, according to the year, were not populated by lymnaeids, in spite of the presence of snail populations downstream. G. truncatula was found alone in 8.8–13.3% of sites and was the first colonizing snail in 24.3–33.3% when the two lymnaeid species successively settled in these waterholes. The colonizing ability of O. glabra was more limited, as it was observed alone in 2.2% of beds and was the first colonizing snail in only 2.2–20% of them. The distances covered by these snails significantly increased with increasing migration time. After migration, a few overwintering snails (3.8% of G. truncatula and 6.8% of O. glabra) are able to colonize the beds, but their numbers decreased when the distance of migration was greater. O. glabra migrated more quickly and reached more watercress beds than G. truncatula. However, this did not influence the natural infections of snails, which were more frequent in the F1 of G. truncatula. The variability observed in the colonization of beds by snails might explain the regular or irregular contamination of wild watercress by metacercariae of F. hepatica. One of the factors which may explain this variability is the occurrence of showers in spring, so that a few snails are able to reach watercress beds after their upstream migration.     

Population genetic analysis of grapevine fanleaf virus

Population genetic analysis of grapevine fanleaf virus (GFLV) was done on the basis of the virus movement protein (MP) gene sequences from the isolates detected and identified in this study and those of all previously reported GFLV strains/isolates. These revealed that the GFLV populations of Iran and Slovenia were highly distinct, whereas those of France, Germany, Italy and the USA were composed of multiple lineages. All populations were significantly differentiated from each other. However, two GFLV isolates from Tunisia, the only recorded GFLVs from that country, were not statistically distinct from the French, German and Italian populations. The ratio of non-synonymous nucleotide diversity to synonymous nucleotide diversity (Pi(a)/Pi(s)) was less than 1, suggesting that the MP gene has been under purifying selection. The neutrality tests were indicative of a balancing selection that is operating within Iranian and USA GFLV isolates, but they show a purifying selection within the other populations. Eleven recombination events were detected in a total of 50 isolates from France, Germany, Iran, Italy, Slovenia and the USA. The results from the recombination analysis were in agreement with those of the phylogenetic analysis. This study suggests that diversity among GFLV geographical populations resulted from possible host adaptation, recombination and founder effects.     

Genetic diversity and recombination analysis in the coat protein gene of Banana bract mosaic virus

Banana bract mosaic virus (BBrMV), a member of the genus Potyvirus, family Potyviridae, is the causal agent of the bract mosaic disease (BBrMD) that causes serious yield losses in banana and plantain in India and the Philippines. In this study, global genetic diversity and molecular evolution of BBrMV based on the capsid protein (CP) gene were investigated. Multiple alignments of CP gene of 49 BBrMV isolates showed nucleotide (nt) and amino acid (aa) identity of 79–100 and 80–100 %, respectively. Phylogenetic analysis revealed that except two Indians isolates (TN14 and TN16), all isolates clustered together. Eleven recombination events were detected using Recombination Detection Program. Codon-based maximum-likelihood methods revealed that most of the codons in the CP gene were under negative or neutral selection except for codons 28, 43, and 92 which were under positive selection. Gene flow between BBrMV populations of banana and cardamom was relatively frequent but not between two different populations of banana infecting isolates identified in this study. This is the first report on genetic diversity, and evolution of BBrMV isolates based on recombination and phylogenetic analysis in India.     

A preliminary study on the molecular evolution of the two routes of intrauterine transmission of HBV

Intrauterine transmission of hepatitis B virus (HBV) is one of the main reasons for the failure of vaccination and plays an important role in areas with high HBV prevalence. In the present study, the quasispecies isolated from eight pairs of HBsAg-positive mothers and their neonates, who were infected with HBV by intrauterine transmission, were selected as study subjects. Phylogenetic trees of the HBV strains of each pair of mother and neonate were constructed, the topological structures were compared, and the distance between and within the quasispecies was calculated. The eight phylogenetic trees included four types. In the first type, the maternal and neonatal sequences clustered into one clade. In the second type, the sequences of the mothers and neonates formed separate monophyletic clusters, and the two clades were sister groups. In the third type, the strains of mother were the ancestors of the neonatal strains. In the fourth type, the strains of the mothers clustered with only some of the sequences of the neonate, and the other strains of the neonate formed another monophyletic group. Combined with the genetic distance, possible transmission routes of the eight cases are proposed.     

Relationships between intrapopulational and interpopulational genetic diversity in man

The bottleneck effect (or extended period of reduced population size) is known to increase genetic distance (D) substantially, and this can be a serious factor that disturbs the phylogenetic relationships of populations inferred from genetic distance estimates. The bottleneck effect is also known to be a factor that disturbs the hierarchial relationships of the fixation indices (FST) or the coefficients of gene differentiation (GST) in subdivided populations. To examine the extent of the bottleneck effect on D and GST in human populations, the D and GST values were computed for various groups of populations from around the world, and their relationships with within-population heterozygosities were examined by using gene frequency data for protein and immunological loci. The results obtained indicate that the D value between a pair of populations is negatively correlated with the average within-population heterozygosity. This suggests that genetic distance estimates for small populations are seriously affected by the bottleneck effect, and that phylogenetic trees should be studied by taking into account this factor. The bottleneck effect on GST was also revealed from examination of the total gene diversity HT and its components, interpopulational genetic variation (DST) and intrapopulational genetic variation (HS). That is, a large value of GST in small populations was sometimes associated with the decrease of HS rather than the increase of DST. Generally speaking, however, GST was larger when geographically distant populations were considered than when closely located populations were considered. When there is any trace of bottleneck effects, phylogenetic trees should be constructed by a method in which the rate of evoluationary change is allowed to vary from branch to branch.     

Compatibility of Schistosoma japonicum from the hilly region and Oncomelania hupensis hupensis from the marshland region within Anhui,China

Schistosome japonicum remains one main public concern in China. This is exemplified in the hilly region in Anhui Province, where rodents have served as reservoirs for the parasite and no effective intervention could target such wild animals. The closer relationship between the hilly region and the near marshland induces the worry of spread of the hill parasite to the marshland region. Therefore, the level of snail-parasite compatibility between the hill parasite and snail populations from the Yangtze River valley was investigated. The results of this study demonstrated that both the hill (Shitai, Anhui) and the marshland (Wuxi, Jiangsu) strains of parasite were more infective to the marshland strains of snail (Zongyang and Hexian, Anhui) than to the hill strain of snail (Shitai, Anhui). When snails were individually exposed to one single miracidium, the longest prepatent period for cercarial development was observed in the combination of Shitai schistosome/Shitai snail. A nocturnal cercarial emergence pattern was observed for the hill parasite, either harbored in the hill or the marshland strain of snails. The results suggested a high compatibility between the marshland strains of snail and both the hill and the marshland strains of parasite. This would have practical implications. Moreover, the fact of the lower compatible relationship between the hill parasite and its local intermediate hosts warranted more studies.