2008～2009年珠海市H3N2亚型流感病毒HA1基因特性分析

为了解2008~2009年珠海市H3N2亚型流感病毒HA1基因变异情况,选择珠海市2008~2009年期间不同时间点的经狗肾传代细胞(MDCK)培养分离的H3N2亚型流感毒株20株,提取病毒RNA,通过RT-PCR扩增HA1基因片段,将产物纯化并测序,推导氨基酸序列,进行基因进化特性分析。与同时期的疫苗株比较,2008年珠海市流行的H3N2亚型流感毒株HA1区抗原决定簇的氨基酸位点变异数少于4个;2009年珠海市流行的H3N2亚型流感毒株除09-0056外,HA1区存在5个位于抗原决定簇内的变异氨基酸位点。2008年H3N2亚型流感毒株的HA1区的糖基化位点与疫苗株一致;2009年H3N2亚型流感毒株HA1区丢失第144位糖基化位点。2008~2009年H3N2亚型流感毒株RBS氨基酸序列未见明显变异。与2008年H3N2亚型流感毒株比较,2009年H3N2亚型流感毒株HA1区抗原决定簇内存在多个位点的氨基酸替换。这些说明2008年珠海市流行的H3N2亚型流感病毒不是新变种;2009年流行的H3N2亚型流感病毒为新的变异株,这可能是H3N2亚型流感病毒在2009年6-9月为珠海地区季节性流感流行优势株的原因。     

广东地区1996年流感暴发的分子变异基础

1996年广东地区流感毒株发生明显的血清学抗原漂移;引起广东地区流感暴发的分子基础是流感毒株HA基因编码的A、B、C、D和E五个抗原决定簇位点变异,尤其是A、C、E位点发生氨基酸改变;而受体结合位点的氨基酸改变对此流感流行未发挥明显影响。HA基因编码氨基酸的第145号和第193号位点变异导致流行毒株的生物学特性改变,即分离毒株适应于MDCK细胞株生长,而难以适应鸡胚生长环境。     

广东地区1996年流感暴发的分子变异基础

1996年广东地区流感毒株发生明显的血清学抗原漂移;引起广东地区流感暴发的分子基础是流感毒株HA基因编码的A、B、C、D和E五个抗原决定簇位点变异,尤其是A、C、E位点发生氨基酸改变;而受体结合位点的氨基酸改变对此流感流行未发挥明显影响。HA基因编码氨基酸的第145号和第193号位点变异导致流行毒株的生物学特性改变,即分离毒株适应于MDCK细胞株生长,而难以适应鸡胚生长环境。     

A(H3N2)亚型流感病毒血凝素基因特性及蛋白结构分析,以南宁市2009～2012年为研究案例

利用生物信息学软件和数据库研究南宁市2009~2012年度A(H3N2)亚型流感病HA基因的遗传变异规律及蛋白结构变化。通过RT-PCR扩增H3N2病毒HA基因并测序,同源比对统计毒株氨基酸位点的差异、构建系统进化树分析进化规律和同源建模分析蛋白结构的变化。系统进化树表明53株HA基因序列被分为4个类群,呈多侧支流行;所有毒株的二硫键和受体结合位点(RBS)高度保守;部分毒株HA在第45位点增加一个糖基化位点,在第144位点丢失一个糖基化位点;HA抗原决定簇氨基酸累计有30个位点发生变异,涉及5个抗原决定簇;HA晶体结构分析抗原决定簇突变位点主要发生在无规则卷曲处,大多数替换的氨基酸种类和性质相同或相似。南宁市A(H3N2)亚型流感病毒株变异活跃,2012年A(H3N2)亚型流感病毒与当年WHO推荐的疫苗株具有较远的进化距离,多数毒株具备了形成新变种的条件,是否形成新的流行株,有待深入研究。     

广东地区流感H3N2毒株血凝素基因特征、进化和变异分析

为揭示广东地区2007～2010年甲型H3N2毒株血凝素(HA)基因特征和变异,采用时空抽样方法抽样,检测广东2007～2010年甲型H3N2毒株HA基因核苷酸序列,同时检索全球HA基因序列作为对照,采用Lasergene 7.1和Mega 5.05软件对HA基因核苷酸序列进行比对和分析;并结合流行病学资料,对变异毒株进行进化速度分析;同时进行抗原分析。结果发现,广东2007～2010年H3N2毒株HA基因同义进化(Ks)和错义进化(Ka)速度分别为2.06×1E-3～2.23×1E-3核苷酸/年和1.05×1E-3～1.21×1E-3核苷酸/年,HA1较HA2的错义突变速率要高3.13倍。与疫苗株A/Perth/16/2009的HA基因比较,2009年广东毒株同源性达到98.8%～99.7%、2010年同源性达到98.0%～98.4%。在广东2007～2010年毒株中,HA1五个抗原表位均有氨基酸位点变异,尤其是2010年毒株B区(N160K)和D区(K174R/N)的变异;此外,广东2010年毒株受体结合部位(RBS)还发生K189E/N/Q和T228A置换变异;两个糖基化位点变异影响到抗原性;目前使用的H3N2疫苗株与目前流行毒株的抗原性有差异。广东地区2007～2010年的毒株中,血凝抑制抗体的抗原分析结果有差异。结果提示,目前广东乃至全球甲型H3N2毒株HA1B区和D区均有氨基酸位点变异,RBS的两个位点发生置换,糖基化位点变异影响到表位A区和B区抗原性;与WHO推荐2011年流感H3N2毒株疫苗株比较,目前流行毒株HA基因有抗原位点变异。     

1996-2004年中国福建省甲3亚型流感病毒（H3N2）的分子进化

对1996～2004年甲3亚型流感病毒的HA1基因在中国南方福建省的变异特点与流行特征进行研究．对25株（其中11株基因序列来自GenBank）甲3亚型流感病毒HA1基因进行种系发生学分析．研究表明在约8个流感流行季节中,HA1基因在不断进行着点突变,期间可能发生了4次抗原性漂移．氨基酸突变的位点主要位于HA1分子抗原决定簇A～E内及附近和某些受体结合位点．其中,抗原位点B,A和受体结合位点226位的突变对于抗原漂移至关重要,但随着流感病毒的进化,抗原位点和与抗原漂移有关的关键密码子也会发生改变．因此,在亚洲南部监测甲3亚型流感病毒HA1基因阳性选择密码子的突变是很重要的．     

奥司他韦对我国A(H3N2)亚型流感病毒红细胞凝集和抑制试验的影响

比较加入神经氨酸酶抑制剂奥司他韦(Oseltamivir)对我国A(H3N2)亚型流感病毒红细胞凝集(Hemagglutination,HA)和凝集抑制(Hemagglutinin inhibition,HI)试验结果的影响,以期获得病毒更真实的HA和抗原性变异分析结果。选择2014年10月-2015年5月在中国大陆分离的395株A(H3N2)亚型流感病毒,在HA及HI试验中加入神经氨酸酶抑制剂Oseltamivir,对实验结果进行比较分析,根据HA试验,挑选其中部分毒株进行基因组测序,比较NA氨基酸位点变异情况。在HA试验中加入神经氨酸酶抑制剂Oseltamivir后,44.8%的毒株HA滴度未改变;43.8%的毒株HA滴度下降,仅有11.4%的毒株HA滴度升高。加入Oseltamivir后,与A/TX/50/2012鸡胚株抗原性类似的毒株的比例高于未加入Oseltamivir时,与A/SZ/9715293/13细胞株抗原性类似的毒株的比例低于未加入Oseltamivir时类似株的比例,统计学分析有显著性差异。以A/TX/50/2012细胞分离株和A/SZ/9715293/133鸡胚分离株作为参考病毒,Oseltamivir对实验结果的影响无显著性差异。挑选19株A(H3N2)亚型流感毒株进行基因组测序,进行NA蛋白氨基酸位点分析,与A/TX/50/2012鸡胚分离株相比加入20nM Oseltamivir后,滴度降低超过4倍的5株毒株,没有共同氨基酸位点变异,但A/山东莱城/119/2015流感毒株有D151G氨基酸位点突变;A/吉林铁西/1194/2015流感毒株有V412I和T434A氨基酸位点变异。加入20nM Oseltamivir后,滴度降低为2~4倍的毒株,具有I26T、G93S、V149I、N234D、T267K、S416G等位点突变。在对我国近年流行的A(H3N2)亚型流感病毒进行血凝滴度和抗原性分析中,加入Oseltamivir可获得更为真实的HA和HI结果,分析病毒的变异情况,评价疫苗的匹配性。     

1996～2005年中国H3N2亚型人流感病毒神经氨酸酶基因特性分析

测定了1996~2005年间在中国分离并保存的395株H3N2亚型人流感病毒神经氨酸酶(NA)基因序列,应用生物信息学工具进行了分析。结果表明:NA基因序列的进化树表现为一主要进化主干伴随多侧分支进化,同一年份的毒株可以分为几个分支存在;疫苗株在NA基因序列进化树上存在明显的滞后现象;NA没有氨基酸的丢失与插入;抗原决定簇大部分位点保守且各抗原决定簇之间的变异情况各有特点,其中197~199位、431~434位和339~347位点的变异频率最高,而153位、328~336位、367~370位、400~403位抗原决定簇的氨基酸变异频率相对比较小;除了抗原决定簇外还有些氨基酸位点的变异频率很高,它们分别是18、23、30、93、143、208、216、221、249、265、267、307、385、437位氨基酸,其中143位和267位这两个位点的变异频率高于抗原决定簇位点,具体生物学意义还需要进一步研究;NA蛋白的酶活性中心位点高度保守;二硫键和糖基化位点保守。NA基因的这些特点为流感的预防、控制以及NA抑制剂药物的应用提供了一定的参考依据。     

2010?2011年全球季节性H3N2流感病毒表面蛋白基因的分子遗传特性分析

[目的]分析2010年1月至2011年9月间全球季节性H3N2流感病毒血凝素(Hemagglutinin,HA)和神经氨酸酶(Neuraminidase,NA)基因的演变和分子特征,为流感病毒的防制提供分子信息依据.[方法]搜集期间季节性H3N2流感病毒HA和NA基因的完整核苷酸序列,分别绘制两基因编码序列的进化树;推导出相应的氨基酸序列,统计不同毒株间氨基酸位点差异并分析重要功能位点的变化.[结果]在136条完整的片段4和131条片段6中,2条HA和l条NA序列源自猪群流感病毒,剩余的序列根据进化特征可被分为两群.相比疫苗毒株,发生在HA和NA蛋白抗原位点的平均差异数分别为5.33和2.01个,3个毒株分别在HA宿主受体结合位点和二硫键及NA耐药位点出现突变,多数毒株的糖基化位点增多.江苏毒株和广东毒株分别属于群l和群2,且两省毒株间在HA蛋白抗原位点的差异数从7到13个不等.[结论]2010年1月至2011年9月间的全球季节性H3N2病毒主要呈现两种基因进化特征.因抗原性差异对疫苗开发具有指导作用,而多数毒株的抗原性检测信息仍然未知,但从抗原位点和糖基化位点的变异情况来看,多数毒株的抗原性可能已经变化,为判断是否形成新的流行株,应开展进一步的抗原性检测;并且各地区卫生行政部门应根据耐药位点的变化,制定相应的抗病毒治疗措施.     

新发甲型H1N1流感病毒HA分子的变异分析

目的:从分子进化水平上分析流感的起源及发展问题,研究目前爆发的H1N1病毒的HA分子的变异行为.方法:以GenBank公布的甲型流感H1N1病毒血凝素(hemagglutinin,HA)核酸序列和我国及世界范围内近几年来报告的H1N1流感病毒HA的核酸及氨基酸序列为研究对象,利用CLUSTAL 1.83和NetNGlyc 1.0等生物信息学软件对HA核酸和氨基酸序列进行了比对分析;将其糖基化位点、氨基酸序列和抗原决定簇与以往流感病毒进行了比较.同时,还将人源和猪源甲型H1N1流感病毒的HA氨基酸序列进行了序列比对和系统发育分析.结果:最新爆发的甲型H1N1流感病毒的HA除了在60,259,453,512位点高度保守区域与之前爆发的流感病毒一致外,在249位点新出现1个"-NTT-"的糖基化位点.发现所有的甲型病毒的氨基酸序列在8个氨基酸位点均发生改变,而8个氨基酸位点位于6个抗原抗原决定簇上.结论:糖基化位点的增加,氨基酸位点的改变导致抗原决定簇的改变,即抗原性漂移现象,都成为引起其传染性改变的重要原因.     

Definitions,measurements, and classifications of stimuli,situations, and environments

A review is presented of issues relevant to the definition, measurement, and classification of stimuli, situations, and environments. Problems such as the lack of adequate definitions of concepts, error and bias in measurement procedures, confusion between measurement of a concept and measurement of its behavioral effects, and the lack of agreement among alternative measures are emphasized. It is suggested that concepts be defined in terms of objective characteristics while allowing for the study of the transactional relationship between organism and environment. The work of the ethologists in defining stimuli while studying their relationship to different organismic states and situational contexts is emphasized in this regard. Following Brunswik, it is also suggested that wherever possible there be a representative sampling of variables in natural settings. Note from the editors: From time to time, Human Ecology will publish a review article. Our first in this series is a review by a psychologist of basic definitional and conceptual problems in environmental studies.This paper was prepared while the author was a Visiting Research Fellow at the Educational Testing Service. The support of ETS and my colleagues in the Division of Psychological Studies is gratefully acknowledged. The review was also supported in part by a grant from the Rutgers University Research Council.     

Synthesis,tautomerism, and antimicrobial,anti-HCV,anti-SSPE,antioxidant, and antitumor activities of arylazobenzosuberones

2-Dimethylaminomethylene-1-benzosuberone 1 was coupled with diazotized aniline derivatives to afford a series of the hitherto unreported 2-arylazo-1-benzosuberones 3a–i. The tautomeric structure and the effect of substituents on the tautomeric form (s) of the products 3a–i were discussed. Similar coupling of the enaminone 1 with diazonium salts of heterocyclic amines gave the respective fused azolotriazino-benzosuberones. Some of the newly synthesized compounds showed potent antimicrobial, anti-HCV, antioxidant, antitumor (as topoisomerase I inhibitors), and antimicrobial activities.     

The effect of malathion,diazinon, and various concentrations of zinc,copper, nickel,lead, iron,and mercury on fish

Acute and chronic toxicity tests for malathion, diazinon, copper (Cu), mercury (Hg), lead (Pb), zinc (Zn), nickel (Ni), and iron (Fe) were conducted. Mortalities ofBarilius vagra andCyprinus carpio (common carp) were variable but LC50-96 hr were similar for pesticides. AdultB. vagra seem to be more sensitive to malathion than juvenile carp. Both juvenile carp and adultB. vagra were extremely sensitive to diazinon. Long-term exposure to pesticides modified morphology and behavior. The LC50-96 values for Cu, Hg, and Pb were 0.3, 0.16, and 0.44, respectively, for smaller fish and 1.0, 0.77, and 1.33, respectively, for larger fish. Replicate LC50 values for Zn, Ni, and Fe were somewhat variable, and for these metals, the size of the fish seemed to affect response because LC50 values increased as fish size increased. Cooper, Pb, Zn, and Fe residues following exposure to sublethal concentrations of these metals for 15 d were significantly greater in whole juvenile common carp than in controls.     

Low-frequency dynamics and Raman scattering of crystals, of B-, A-, and Z-DNA, and fibers of C-DNA

Normal modes of vibration of DNA in the low-frequency region (10-300 cm-1 interval) have been identified from Raman spectra of crystals of B-DNA [d(CGCAAATTTGCG)], A-DNA [r(GCG)d(CGC) and d(CCCCGGGG)], and Z-DNA [d(CGCGCG) and d(CGCGTG)]. The lowest vibrational frequencies detected in the canonical DNA structures--at 18 +/- 2 cm-1 in the B-DNA crystal, near 24 +/- 2 cm-1 in A-DNA crystals, and near 30 +/- 2 cm-1 in Z-DNA crystals--are shown to correlate well with the degree of DNA hydration in the crystal structures, as well as with the level of hydration in calf thymus DNA fibers. These findings support the assignment [H. Urabe et al. (1985) J. Chem. Phys. 82, 531-535; C. Demarco et al. (1985) Biopolymers 24, 2035-2040] of the lowest frequency Raman band of each DNA to a helix mode, which is dependent primarily upon the degree of helix hydration, rather than upon the intrahelical conformation. The present results show also that B-, A-, C-, and Z-DNA structures can be distinguished from one another on the basis of their characteristic Raman intensity profiles in the region of 40-140 cm-1, even though all structures display two rather similar and complex bands centered within the intervals of 66-72 and 90-120 cm-1. The similarity of Raman frequencies for B-, A-, C-, and Z-DNA suggests that these modes originate from concerted motions of the bases (librations), which are not strongly dependent upon helix backbone geometry or handedness. Correlation of the Raman frequencies and intensities with the DNA base compositions suggests that the complex band near 90-120 cm-1 in all double-helix structures is due to in-plane librational motions of the bases, which involve stretching of the purine-pyrimidine hydrogen bonds. This would explain the centering of the band at higher frequencies in structures containing G.C pairs (greater than 100 cm-1) than in structures containing A.T pairs (less than 100 cm-1), consistent with the strengths of G.C and A.T hydrogen bonding.     

Synthesis and properties of N-, O-, and S-phospho derivatives of amino acids, peptides, and proteins

The literature on chemical (i.e., nonenzymic) phosphorylation of amino acids, peptides, and proteins is reviewed through 1982. The review covers synthetic methods, chemical reactions, and physical properties, with emphasis on the techniques used for separation and characterization of the products. Synthetic methods are classified by reagent rather than product, and are illustrated by experimental procedures for the most important methods. Chemical reactions are classified into four groups depending on whether the reaction site is the phospho group, the amino group, the carboxyl group, or in the case of serine the hydroxyl group. Physical data are given for all of the known N-, O-, and S-phospho derivatives of the amino acids, peptides, and proteins, within certain limitations, and are discussed in detail in the section on physical properties. Emphasis is given to the techniques used for separation of the products, such as chromatography and electrophoresis, and for characterization of the products, particularly spectroscopy. Medical and other uses of the products are mentioned.     

Cultural, Biochemical, and Immunological Properties of Mima, Herellea, and Flavobacterium Species

From study of cultural and biochemical characteristics of 40 strains of Herellea, Mima, or Flavobacterium species, a proposed schema for identification was developed. The reactions observed by agglutination, gel diffusion, and immunofluorescence suggest antigenic heterogeneity of this group of organisms.