广东省登革病毒的分子流行病学研究

目的 通过对广东省90年代以来流行的登革病毒分子流行病学研究,初步了解我国毒株的可能来源,方法 采用RT－PCR方法扩增广东省不同年份分离的登革病毒（DEN）1型和2型NS1部分基因片段,然后进行克隆测序,测序后的基因片段与国际上已知的多个DEN1和DEN2相应的序列进行比较,并以核苷酸的差异在6％以上作为基因亚型分型的标准。结果 ①广州1991年分离的DEN1（GD03／91）与潮洲995年分离的DEN1（GD23／95）同源性很高,为97％,属同一基因亚型,而两者与从潮洲997年分离的DEN1（GD14／97）同源性均为93％,分属不同的基因亚型。基因系统树分析提示：GD03／91和GD23／95可能来自东南亚或瑙鲁等地,GD14／97可能来自新加坡。②佛山19934昕分离的DEN2（GD06／93）和南海市1998年分离的DEN2（GD01／98）同源性为93％,分属不同的基因亚型。基因系统树分析提示：GD01／98与泰国流行株ThNH-P28/93株共享序列非常接近,核苷酸同源性为98%,氨基酸同源性为100％,因此推测GD01／98可能来源于泰国。结论 广东省90年代以来流行的登革热来源于不同的传染源。     

四川省流行性乙型脑炎病毒优势基因型的研究

目的了解四川省乙脑主要流行区乙脑病毒的分子生物学特性,为防治提供依据。方法对2007-2010年间分离到的13株乙脑病毒进行PreM和E基因区扩增,采用MEGA5生物学软件完成氨基酸序列和病毒进化树分析。结果基因分型显示13株均属于基因I型。13株病毒之间比较,PreM基因核苷酸和氨基酸同源性为97％-100％和98．7％-100％,E基因核苷酸和氨基酸同源性为97．8％～99．9％和99．6％～100％,其同源性极高。13株病毒与2004年四川分离株比较E基因核苷酸同源性在97．7％～99．6％之间,氨基酸同源性在98．6％-100％之间;PreM基因的核苷酸同源性在96．2％-99．1％之间,氨基酸同源性在97．5％-98．7％之间;与疫苗株P3和SA14—14—2比较E基因核苷酸和氨基酸同源性分别为87．6％～88．3％和97％～97．8％;PreM基因核苷酸和氨基酸同源性分别为84．1％～85．8％和93．7％-96．2％。13株病毒E基因的8个氨基酸毒力位点均没有发生改变。结论四川省乙脑病毒已呈现基因I型为优势型别的态势,其PreM和E区核苷酸和氨基酸高度保守,关键的氨基酸毒力位点没有变化,提示目前使用的疫苗对流行株的感染具有保护作用。     

中国分离乙脑病毒与灭活疫苗株(P3株)E基因差异分析

目的 分析我国近年来从蚊虫及患者标本中分离的乙脑病毒与灭活疫苗株（P3株）之间在E基因区段核苷酸及氨基酸差异。方法 从GenBank中获取相应乙脑病毒株E基因区段核苷酸序列，通过Clustal X（1．8）、DNASTAR、GENEDOC（3．2）等生物学软件进行分析。结果 P3株与福建分离株之间核苷酸同源性在98．3％-98．5％之间、氨基酸同源性在98．2％-98．6％之间；P3株与上海分离株之间核苷酸同源性在88．0％-88．5％之间、氨基酸同源性在98．0％-98．4％之间。E基因区段500个氨基酸中P3株与所有新分离乙脑病毒株之间共存在19个位点的差异，其中在E-76、E-306、E-408处所有新分离毒株与P3株存在共同的差异；在E-160、E-487处福建分离株与P3株存在共同差异；上海分离株与P3株在E-129、E-222、E-227、E-366存在共同差异。结论 上海蚊虫中分离的Ⅰ型乙脑病毒和福建省脑炎患者中分离的Ⅲ型乙脑病毒与P3株在E基因的部分氨基酸位点存在差异，但均不处在影响病毒生物学特性的关键位点。     

海南岛两株甲病毒基因组3′末端核苷酸序列的克隆与分析

目的　从分子水平鉴定海南省分离的两株甲病毒 (HBb17和M1病毒 )。方法　采用RT-PCR方法 ,分别扩增两株病毒基因组的 3′末端核苷酸序列 ,扩增产物经亚克隆 ,筛选重组子并测定核苷酸序列对序列进行分析。结果　HBb17和M1病毒分别扩增出约 1 6kb和 1 3kb的特异片段。序列分析表明 ,在 3′末端非翻译区HBb17病毒与罗斯河病毒 (国际标准株T48病毒 )核苷酸同源性为99 % ,M1病毒与鹭山病毒 (SAG)同源性为 98% ;两病毒结构基因的E1区序列 ,HBb17病毒与罗斯河病毒核苷酸 (氨基酸 )同源性为 99% (99% ) ,M1病毒与鹭山病毒核苷酸 (氨基酸 )同源性为 97% (99% ) ,M1病毒与盖塔病毒同源性为 94% (98% )。结论　序列分析表明 ,海南岛分离的HBb17病毒属于罗斯河病毒 ,M1病毒可能是鹭山病毒或盖塔病毒的变异株     

肠道病毒71型中国分离株基因组3区的分析

目的 测定我国分离的肠道病毒71型(EV71)SHZH98株3C基因的核苷酸序列，进行遗传进化途径的分析。方法 用逆转录聚合酶链反应(RT-PCR)方法扩增我国分离的EV71-SHZH98株3C基因cDNA，测定PCR产物的核苷酸序列。结果 SHZH98株3C基因为549bp，和已发表的肠道病毒71型3C基因序列比较，SHZH98株与MS株同源性为78．7％，与BrCr株的同源性为76．7％，与台湾分离株POLY、NCKU、TW2086、TW2272株的同源性分别为74．0％、73．8％、71．9％、69．8％。由其核苷酸序列推导的氨基酸序列同源性比较显示与MS的同源性为93．45％，与BrCr、POLY、NCKU、TW2086、TW2272株的同源性分别为89．1％、90．7％、90．2％、84．2％、82．5％。遗传进化分析显示SHZH98株3C片段在亲缘关系上与Coxsackievirus A16 G-10株及欧美分离株MS、BrCr株较密切，而与台湾分离株相差较远。结论 推测EV71病毒中国分离株非结构蛋白的进化途径可能与台湾流行株不同。     

中国基因3型乙型脑炎病毒E基因分子特征

目的 以减毒活疫苗(SA14-14-2株)为对照,分析我国分离的基因3型乙脑病毒E基因区段核苷酸及氨基酸序列分子特征.方法 从GenBank中获取相应乙脑病毒株E基因区段核苷酸序列,通过Clustal X(1.81)、DNAStar、GENEDOC(3.2)等生物学软件进行核苷酸和氨基酸位点差异分析.以蜱传脑炎病毒可溶性蛋白晶体结构为模板进行乙脑病毒E蛋白氨基酸位点分析.结果 我国不同地域、不同宿主分离的基因3型乙脑病毒与SA14-14-2株核苷酸同源性分别在96%和95%以上,氨基酸同源性在95%和94%以上.在同一地域、同一宿主类型分离的毒株之间核苷酸和氨基酸同源性非常高.在E基因区段存在10处共同的氨基酸位点差异,在结构域Ⅰ(E160)、结构域Ⅱ(E123和E227)和两个未在结构域中的氨基酸位点(E441和E487)等5个位点在部分基因3型乙脑病毒中存在差异.结论 我国分离的基因3型乙脑病毒与减毒活疫苗株(SA14-14-2株)E基因区段同源性高,存在5处基因3型乙脑病毒特异的氨基酸位点差异,但现行减毒活疫苗株理论上可以保护我国分离的基因3型乙脑病毒野毒株.     

新分离的无乳鼠神经毒力的登革2型病毒福建株基因组结构特征的研究

目的 测定对乳鼠不致病登革2型病毒福建株（DEN2－FJ11）全基因组序列，探讨其基因组结构与功能关系。方法 采用RT－PCR和5′、3′端RACE方法测定病毒基因组的全序列，并运用DNASTAR软件的Clustal法将该毒株的序列与其它6株登革2型病毒进行比较，分析其进化关系。结果 DEN2－FJ11株基因组全长10723个核苷酸，含有1个单一的读码框架，编码3391个氨基酸。5′、3′端非编码区（NCR）长度分别为96和454个核苷酸。DEN2－FJ11与同时分离的对乳鼠致病的FJ10株之共有32个核苷酸不同及13个氨基酸的变化，其中8个氨基酸是其极性或电荷的改变。3′端非编码区134位的单个核苷酸变化导致其二级结构改变。DEN2－FJ11与FJ10株的核苷酸和氨基酸的同源性分别为99.7%和99.6%，这2株病毒与印度尼西亚株亲缘关系最近，同为Ⅳ基因型。结论 DEN2－FJ11株基因组与FJ10株及其它登革2型病毒株类似。位于病毒编码区的8个氨基酸差异及3′端非编码区134位的核苷酸可能与病毒的乳鼠神经毒力有关。     

北京地区2000-2004年分离的呼吸道合胞病毒B亚型株G蛋白基因的序列分析

目的 了解近年来北京地区流行的B亚型呼吸道合胞病毒（BSV）G蛋白的基因特征。方法 2000年冬季至2004年冬季自首都儿科研究所附属儿童医院收集的急性呼吸道感染患儿呼吸道标本，从中分离到B亚型RSV毒株，每年随机选取1至2株毒株，用RT-PCR扩增其G蛋白全基因后克隆至pBS-T载体中，筛选出阳性克隆后测序，并与B亚型标准株（CH18537）和文献报道的毒株序列进行比较分析。结果 所测毒株G蛋白全基因核苷酸长度分别为915、921和981bp，推导的氨基酸长度分别为292、293、312和315从。分离株与B亚型标准株CH18537间存在着明显的差异，CH18537株同分离株间的核苷酸的同源性为91．9％～93．7％，氨基酸的同源性只有85．0％～89．0％。分离株间核苷酸的同源性为93．4％～98．8％，氨基酸的同源性为88．2％～98．6％。氨基酸的变异主要集中在胞外区一个高度保守区的两端，而胞内区和跨膜区相对保守。此外，在进行G蛋白基因分析的8株B亚型分离毒株中，我们发现有3株BSVG蛋白在490～495位核苷酸缺失，3株RSVG蛋白在c末端791位后出现了60个核苷酸的插入，导致C末端259位后出现20个氨基酸的插入。这60个核苷酸与相邻的前60个核苷酸高度重复，只出现3至4个核苷酸的差异。该3株分离株与文献报道的有60个核苷酸插入的两株（S00-4和BA4128／99B）核苷酸和氨基酸同源性分别为97．5％～98．6％和95．5～98．1％，在插入的20个氨基酸中，有高达50％（9～10个氨基酸）左右的丝氨酸和苏氨酸氧连接的糖基化位点。结论 RSVB亚型G蛋白基因变异存在着多样性，分离株既有核苷酸替代，又有基因缺失和插入，还有糖基化位点的改变和氨基酸长度的改变。这种变异使G蛋白高度糖基化，提示最终可能导致病毒抗原性的改变。北京分离的有60个核苷酸插入的变异株与日本及西班牙报道的变异株有非常近的亲缘关系，提示这种G蛋白基因中有60个核苷酸插入的B亚型变异株已经在子代病毒中形成稳定遗传并在世界范围内传播。     

新分离登革2型病毒福建株基因组全序列的测定

目的 对新近分离的导致1999年福建省登热流行的登革2型病毒FJ－10株进行基因组全序列测定及系统发生树分析。方法 利用RT－PCR和5′、3′RACE法扩增FJ－10株cDNA,并进行克隆测序,利用DNASTAR折Clustal方法绘制系统发生树。结果 JF－10株基因组全长10723个核苷酸,有1个单一开放读码框架（ORF,第97－10269nt）,编码3391个氨基酸,5′和3′非编码区长度分别为96和454个核苷酸,通过与标准株NGC株和我国其他地区分离株DEN2－04、43、44株比较,核苷酸同源性分别为94.0%、92.8%、93.9%和93.9%,氨基酸同源性分别为97.9%、97.2%、97.7%和97.9%。以47株登革2型病毒E／NS1连接区240个核苷酸序列进行发生树分析,福建株与印度尼西亚和斯里兰卡分离株亲缘关系较近,同属第Ⅳ基因型。结论 FJ－10株基因组全序列一级结构与其他登革2型病毒类似,其基因型不同于我国其他地区分离株DEN2－04、43和44株。     

两株登革2型病毒E、NS1蛋白基因序列测定及其系统发生分析

目的　对从登革出血热 (denguehemorrhagicfever,DHF)病人血清分离到的一株登革 2型病毒 (denguevirustype 2 ,DEN 2B株 )和DEN 2NGC株作E、NS1蛋白部分基因序列测定并进行系统发生树分析 ,了解其变异及分子进化特征。方法　利用RT PCR法扩增B株和NGC株E、NS1部分基因片段 ,PCR产物直接测序 ;利用DNAssist软件预测其氨基酸序列 ,以及PHYLIP软件的CLUSTAL方法绘制系统发生树。结果　B株与NGC株E蛋白基因区第 1～ 4 76nt( 4 76bp)碱基序列存在 8个位点的差异 ;编码氨基酸存在 4个位置的不同 ;NS1基因区第 5 89～ 10 0 2nt( 4 13bp)碱基序列存在 7个位点的差异 ,编码氨基酸有 2个位置的改变 ;B株与NGC株和DEN 2 4 4株E区部分片段核苷酸同源性分别为99 .1%和 98.7% ;与NS1区部分片段核苷酸同源性分别为 98.3%和 98.1%。B株E基因、NS1基因序列已登录GenBank ,注册号分别为AY179733和AY2 384 71。结论　B株E、NS1基因序列和氨基酸序列与NGC株存在差异 ;B株与我国海南 1989年流行的DEN 2 4 4株和NGC株系统进化关系较近。     

Baculovirus structural polypeptides

The structural polypeptides of eight insect baculoviruses were studied using vertical slab polyacrylamide gel electrophoresis. All viruses revealed a complex but unique composition of 15 to 25 bands with molecular weights ranging from 15,000 to 160,000. Since certain baculoviruses have more than one nucleocapsid per viral envelope (multiples), comparisons were made of the multiples and singles (enveloped single nucleocapsids) for each virus. Quantitative and qualitative differences were documented to exist in polypeptide composition. Where possible, the envelopes of certain baculoviruses were selectively removed in order to identify the major capsid proteins. Autographa californica MNPV (NPV: nuclear polyhedrosis virus) capsids contained two major polypeptides, VP18.5 and VP37. Rachiplusia ou MNPV capsids contained several polypeptides of which VP16, VP17, VP18, VP30, and VP36 were considered major constituents. Anticarsa gemmatalis MNPV contained one major capsid protein, VP29, and several minor polypeptides. Major capsid proteins of Heliothis zea SNPV were VP16, VP28, and VP63; as were VP16, VP17, and VP31 of Trichoplusia ni granulosis virus (GV).     

Fifty years of structural glycoproteins

During decades preceding and following the last war, a favourite subject of biochemists was to study glycoproteins. One class of these substances, found in connective tissues were characterised as polysaccharides, most of them found to be linked to proteins, designated later as glycosaminoglycans and proteoglycans. Another family of glycoconjugates represented epithelial mucins as found in the gastro-intestinal and respiratory tracts and conduits. A third family of glycoconjugates is represented by circulating glycoproteins isolated from the blood plasma, mostly studied by medical biochemists in relation to pathological conditions comprising those increasing during the inflammatory reaction: acute phase glycoproteins. Their study suggested that they might be derived from connective tissues. Although inflammatory glycoproteins derive mostly from the liver, the possibility of connective tissue origin of glycoproteins remained open. Using cornea, an avascular tissue, we could show that connective tissues also synthesize glycoproteins. We proposed to designate them "structural glycoproteins" (SGP-s) to distinguish them from circulating, blood-born glycoproteins coming from the liver. They play locally "structural" roles in connective tissues where they are synthesized. Soon after fibronectin was identified and shown to mediate cell-matrix interactions. A large family of glycoproteins were then isolated from a variety of sources, cells, tissues others than liver, confirming our original hypothesis. The first experiments on these glycoproteins were published from 1961/1962 giving the opportunity to recapitulate this biochemical adventure 50 years later, together with the celebration of the foundation of the first connective tissue society in Europe, as described in the first article in this issue.     

HTLV-1 structural proteins

HTLV-1 structural proteins do not appear to ensure virus transmission as efficiently as most other retrovirus structural proteins do, whereas all other retroviruses can be transmitted via either free virions or cell-to-cell contacts, infection by HTLV-1 by free virions is very inefficient, and effective infection requires the presence of HTLV-1 infected cells. This characteristic feature of HTLV-1 provides a unique tool which can be used to analyse retrovirus cellular transmission in the absence of simultaneous cell-free infection. Here we summarise what is known about HTLV-1 structural proteins and identify the questions about these proteins which remain to be answered.     

Some structural features of vital dyes

Lymphocytes and macrophages of healthy rats, cells of lymphosarcoma and ovarian tumors (strain OYa) of rats, and cells of sarcoma 37 and lymphosarcoma Nk/Ly of mice were stained intravitally with 1·10^–5–3·10^–2 M solutions of 86 dyes. The presence of alkylating amino groups in the molecule of the dye substantially increases its ability to penetrate to living cells and to be deposited in their cytoplasm. Acid radicals considerably reduce the ability of the dye to stain cells intravitally. This has been shown for thiazine, oxazine, triphenylmethane, acridine, diazine, and xanthene compounds. The degree of basicity of the dye molecule and also of its amino group does not play a decisive role in the process of vital staining.Laboratory of Chemical Carcinogenic Agents, N. N. Petrov Scientific-Research Institute of Oncology, Ministry of Health of the USSR, Leningrad. (Fresented by Academician of the Academy of Medical Sciences of the USSR L. M. Shabad.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 83, No. 4, pp. 499–503, April, 1977.     

The structural basis of T-cell allorecognition

Foreign allogeneic major histocompatibility complex (MHC) class I and class II molecules elicit an exceptionally vigorous T-cell response. A small component of the alloresponse comprises CD4+ T cells that recognize allogeneic MHC indirectly after processing into peptide fragments that are bound and presented by self-MHC class II. The majority of alloreactive T cells directly recognize intact allogeneic MHC molecules expressed on foreign cells. Some alloreactive T-cell interactions with allogeneic MHC molecules are indifferent to the bound peptide, but evidence suggests that most show specificity to peptide. The vigor and diversity of the direct alloreactive T-cell response can therefore be explained by summation of numerous responses to each of the peptides in the novel set bound by allogeneic MHC molecules. Structural studies definitively show that the overall mechanism of T-cell receptor (TCR) recognition of self-MHC and allogeneic MHC molecules is similar. Many alloreactive T cells recognize several different combinations of MHC and bound peptide that do not necessarily possess structural homology. Flexibility within the TCR structure allows adaptation to the different contact surfaces. Crossreactivity seems to be an intrinsic property of the TCR required, because a single TCR must possess the ability to interact with both self-MHC during positive selection and at least one combination of foreign antigenic peptide presented by self-MHC. Recognition of allogeneic MHC molecules is an inadvertent consequence of the need for TCR crossreactivity.     

Immunological cross-reactivity of adenovirus structural proteins

The interserotypic cross-reactivity of adenovirus-specific antisera was examined on Western blots of virion proteins. The pattern of cross-reactivity was very complex. Virion polypeptides III, VI and the core VII were the major reactants. Generally, the degree of reactivity correlated with type and subgroup specificity.