南疆PRRSV ORF3基因克隆与序列分析

旨在掌握南疆PRRSV流行毒株特点,为南疆PRRS的有效预防和控制提供理论依据.利用克隆和测序获得了5个南疆PRRSV ORF3基因全序列,并进行了序列分析.结果显示,5个南疆PRRSV ORF3基因长度均为765 bp,他们之间核苷酸同源性为90.3％-99.5％;与欧洲型代表株LV株、美洲型代表株ATCC VR-2332株、中国代表株CH-1a株和中国HP-PRRS代表株JXA1株核苷酸同源性分别为61.2％-62.2％、88.6％-89.2％、92.5％-95.8％和90.5％-99.2％;与中国猪养殖场常用的几种弱毒疫苗株核苷酸同源性为87.8％-99.6％;与美洲型毒株和欧洲型毒株氨基酸同源性为80.3％-99.2％和53.5％-55.1％;本研究的5株南疆PRRSV均属于美洲型亚群Ⅱ毒株.利用南疆PRRSV分子流行病学调查结果,可为南疆PRRS防制疫苗的选择等做进一步的调整或指导奠定基础.     

我国猪繁殖与呼吸综合征病毒NT0901分离株分子特征

猪繁殖与呼吸综合征病毒(PRRSV)是目前引起国内外养猪业严重经济损失的重要病原之一,病毒基因和毒力变异较大。PRRSV NT0801株分离自我国发病猪群,毒力较强,但NSP2基因不存在高致病性PRRSV 30个氨基酸的缺失。为了进一步阐明该分离株的分子特征,本研究对该毒株全基因序列进行了测定和分析,结果该毒株基因组全长15 439 bp,其中包含29 nt Poly(A)。与高致病性PRRSV毒株JXA1比较,核酸序列同源性为96.7%,推导的GP3和GP5氨基酸序列同源性分别为97.2%和98.5%,但NSP2基因无30个氨基酸的缺失;与传统型毒株ch-1a比较,推导的GP3和GP5氨基酸序列同源性分别为92.9%和91.5%;基因进化树分析结果显示其介于高致病性和传统PRRSV毒株之间。与其它不同毒力PRRSV分离株基因序列比较,未发现明显重组信号。不同毒力毒株氨基酸残基比对分析结果显示,15个位点潜在毒力相关氨基酸残基中,该毒株有9个与高致病性PRRSV毒株一致,3个与高致病性PRRSV毒株不同,但与传统型和JXA1疫苗株相同,1个位点只与JXA1疫苗株相同,2个与其它毒株都不相同。表明该分离株与高致病性PRRSV密切相关,PRRSV流行毒株变异与基因突变有关,从而为该病毒毒力基因定位研究奠定了基础。     

2株与疫苗病毒高度同源的猪繁殖与呼吸综合征病毒的鉴定和致病性分析

为了解河南地区猪繁殖与呼吸综合征病毒(PRRSV)流行毒株的特征及致病性,对从河南省两个免疫过高致病性PRRSV(HP-PRRSV)减毒活疫苗后不久发病猪场分离的两株PRRSV HENZK-1和HENPDS-2进行了全基因组测序、分析和致病性研究。结果表明,两株PRRSV分离株与HP-PRRSV的细胞传代致弱疫苗株JXA1-P80/JXA1-R高度同源。致病性试验表明,感染HENZK-1和HENPDS-2的仔猪出现明显临床症状,包括高热、食欲减退、呼吸困难等,且肺部出现了肉眼和组织学病变;而同源商品疫苗对照组JXA1-R虽未出现明显临床症状,但日增重降低,肺部有肉眼和组织学病变,并检测、回收到病毒。综上所述,有理由认为HENZK-1和HENPDS-2是由HP-PRRSV疫苗株JXA1-R演化而来,其来源猪群临床发病与其毒力返强、疫苗毒株在解剖和组织学上的损伤和排毒能力有一定关系,但具体原因仍需进一步研究。本研究为谨慎使用高致病性PRRSV减毒活疫苗及PRRS控制策略提供了重要依据。     

不同PRRSV毒株间ORF1a基因密码子偏爱性差异分析

运用CodonW、ClustalX、TreeView软件及EMBOSS（,rIleEuropean MolecularBiologyOpenSoftwareSuite）、CIMMiner在线分析软件对选取的29株PRRSVORFla基因进行密码子偏爱性聚类分析．CAI、CBI、Fop、Nc、GC3s和GC含量、基因长度等相关性分析显示PRRSV各毒株编码的ORFla基因密码子偏爱性各有差异,其中Lelystadvirus、LV4-2．1、VR-2332、RespPRRSMIV与国内分离的高致病性PRRSV变异株之间差异较大．密码子使用概率聚类分析表明CC．1、NVSL．97．7895、CH—1a、RespPRRSMLV、LV4．2．1、Lelystadvirus与高致病性PRRSV变异株距离较远．而国内分离株相互间的聚类距离则较接近。此结果与基于氨基酸序列比对构建的系统进化树图谱基本一致．由此可见．PRRSV病毒ORF1a基因密码子使用偏爱性的差别与病毒的遗传多样性密切相关．     

两株田间重组猪繁殖与呼吸综合征病毒的分离鉴定、进化分析及其致病性研究

为了解2型猪繁殖与呼吸综合征病毒(PRRSV2)田间毒株重组特征,本研究对两株分离物(HENXX-8、HENJY-2)进行全基因测序、进化分析及毒力测定,并对其全基因序列进行重组分析。结果显示,两个毒株均属于PRRSV 2,两者与其代表株VR-2332的核苷酸相似性分别为85.6%、85.7%;与高致病性PRRSV(HPPRRSV)毒株JXA1、TJ和WUH4株分别为85.7%～85.8%、85.4%～85.5%;与经典毒株CH-1a、HB-2(sh)分别为84.7%～85.7%、84.5%～85.7%;而与所有NADC30类毒株均在90.0%以上。全基因、ORF5及Nsp2序列进化分析结果均显示两个分离物与国内报道的类NADC30毒株遗传距离较近,同处于一个分支。全基因组重组分析结果表明,两个分离毒株存在明显重组现象,且重组模式均以类NADC30毒株为骨架病毒,与HP-PRRSV毒株发生重组。但重组对象不同:HENJY-2是由类NADC30毒株与TJ株发生重组;而HENXX-8则是由类NADC30毒株与WUH4、TJ株发生3毒株间重组。由于重组部位序列缺乏特异性分子标志,因此无法确定与类NADC30发生重组的是HP-PRRSV还是其减毒的疫苗毒株。两株分离物的重组部位与早期分离毒株HENANHEPB、JL580等有所不同,均未涉及到ORF5基因,而是集中在Nsp1～Nsp2以及ORF2a～ORF3区域,主要发生在病毒基因组的靠5'端(30～7 000 bp)和3'端(11 000～13 000 bp)处。对部分重要基因的核苷酸相似性分析结果显示,两个分离株的ORF2a、ORF3基因与HP-PRRSV毒株相似性最高,表明重组病毒的这两个基因可能来自HPPRRSV毒株。致病性试验结果表明,参考毒株HENXC-4的毒力略高于分离毒株HENXX-8,主要表现在体温升高、日均增重降低和肺部病变上。但两个分离物均未引起发病猪死亡。以上结果表明,目前PRRSV2在田间的基因重组事件存在随机性,提示不同毒株在田间的存在会加剧PRRSV重组事件的发生和流行、毒株类型更加复杂,从而增加了临床防控难度。因此,慎重使用活疫苗并持续监测活疫苗毒株在田间的变异动态,对更好防控本病具有一定的临床指导意义。     

传染性法氏囊病病毒野毒株的致病性及其vp2基因比较

对4个IBDV野毒株的致病性和它们的vp2基因高变区序列同时做了比较分析。结果表明,4个IBDV毒株在致病性程度上存在较大差异。其中有一个是真正的超强毒,即GX8／99,其他几个虽然达不到真正超强毒的毒力,但也比经典的标准毒的致死性高得多。在vp2基因高变区,这4个IBDV野毒株与超强毒参考株HK46同源性很高,在DNA水平为96．8％～99．5％,在氨基酸(aa)水平为96．6％～100％o而与疫苗毒D78有很大差异,分别只有91．7％～93．6％和91．8％～93．2％。说明IBDV毒株的vp2基因高变区确实与其致病性有一定关系。特别是SD-1／97、SD-3／98、JS-30／99株之间及其与HK46在DNA和aa水平的同源性高达98．4％和98．6％以上,SD-3／99、JS-30／99株之间及其与HK46的氨基酸同源性为100％。然而,致病性特别高的GX8／99株病毒与其他3个野毒株及HK46在DNA和氨基酸水平的同源性相对较低,在DNA和aa水平的同源性只有96．8％～9712％和96．6％～97．9％。相对于国内的流行毒株和香港超强毒参考株HK46,GX8／99株在致病性和VP2高变区都已发生了一定的变异。     

环介导间接PCR鉴别高、低致病性猪繁殖与呼吸综合征病毒方法的建立

建立猪繁殖与呼吸综合征病毒(Porcine reproductive and respiratory syndrome virus,PRRSV)环介导间接PCR检测方法,用于该病毒的感染检测和高致病性毒株与低致病性毒株的鉴别诊断。根据GenBank数据库中PRRSV中国流行株ORF7和ORF1a基因序列的保守性,设计2对特异性探针,分别标记于大豆Lectin基因两端,作为报告基因,经过一步链置换反应后,采用反向PCR扩增报告基因,建立PRRSV环介导间接PCR检测方法,该方法扩增HP-PRRSV可得大小为193bp和355bp的特异片段,扩增LP-PRRSV可得大小为193bp和442bp的特异片段。试验结果表明,该方法能成功鉴别HP/LP-PRRSV,可检测出5.6TCID50/mL LP-PRRSV和18TCID50/mL HP-PRRSV的病毒RNA,HP/LP-PRRSV混合感染不影响检测灵敏度,与CSFV、PPV、PRV、PCV2、ETEC和Haemophilus parasui等常见猪源性病原的检测无明显交叉反应;对20份临床样本进行比较检测,环介导间接PCR检测出14份PRRSV阳性样本,其中4份LP-PRRSV、9份HP-PRRSV和1份LP/HP-PRRSV,与常规PCR检测结果一致。环介导间接PCR是一种简便快速、灵敏、特异的病原学诊断工具,适合PRRSV感染的快速检测和HP/LP-PRRSV鉴别诊断,尤其适合HP/LP-PRRSV混合感染的鉴别。     

高致病性猪繁殖与呼吸综合征病RT-PCR快速鉴别诊断方法的建立与临床应用

根据高致病性猪繁殖与呼吸综合征病毒(PRRSV)Nsp2基因的缺失信息,设计了3条特异性引物,以含高致病性PRRSV Nsp2基因的质粒pMDNSP2及普通PRRSV VR2332株RNA为模板,建立了快速诊断高致病性PRRSV RT-PCR方法.通过对临床组织病料的总RNA进行不同稀释倍数检测,结果表明该方法能从0.265 pg的总RNA中检测到PRRSV的基因,说明敏感性高.用该方法对猪瘟病毒(CSFV)、Ⅱ型圆环病毒(PCV-2)、伪狂犬病病毒(PRV),链球菌(Streptococcus)、副猪嗜血杆菌(Haemophilus parasuis)和大肠杆菌(Escherichia coli)同条件检测,结果都为阴性.进一步对36份疑似高致病性PRRSV临床组织病料细胞培养物、2株PRRSV商品活疫苗以及52个猪场所送检的184份临床样品进行了检测应用,结果36份疑似高致病性PRRSV临床组织病料的细胞培养物有5份样品为阳性且都为高致病性PRRSV,2株PRRSV商品活疫苗为普通PRRSV,52个猪场中有42个猪场(123份样品)呈阳性,其中只有1份为普通PRRSV.实验表明该方法能够准确地鉴别诊断高致病性PRRSV和普通PRRSV,且具有快速,敏感和特异的特点,具有临床实用性.     

2018-2019年福州腹泻住院儿童A组轮状病毒全基因组分析

了解2018-2019年福州市五岁以下腹泻住院儿童标本中A组轮状病毒(RVA)基因组特征.通过反转录-聚合酶链式反应(RT-PCR)对49份RVA阳性标本进行核酸扩增,对扩增到的40份标本进行全基因组二代测序,获得G1P[8]型RVA毒株7株,G9P[8]型RVA毒株33株.根据VP7节段分型,40株RVA毒株中有30株G9-Ⅵ亚型、3株G9-Ⅲ亚型和7株G1-Ⅰ亚型;根据VP4节段分型,40株RVA毒株均属于P[8]-3亚型.全基因组核苷酸序列分析表明,除了VP7节段外,序列差异比较大的有NSP4和NSP1两个节段.本研究获得11株G9P[8]型Waa-ike株(G9-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1),22株NSP4节段重配的G9P[8]-E2株(G9-P[8]-I1-R1-C1-M1-A1-N1-T1-E2-H1),在测序的33株G9P[8]型RVA毒株中G9P[8]-E2重配株占66.7％.7株G1P[8]型RVA毒株均为Wa-like株.研究结果表明,2018-2019年福州地区流行的RVA毒株中G9P[8]-E2重配株已经成为优势株,为了更全面了解福州地区RVA毒株的遗传变异情况,后续还需加大标本量持续进行监测.     

传染性法氏囊病病毒野毒株的致病性及其vp2基因比较

对4个IBDV野毒株的致病性和它们的vp2基因高变区序列同时做了比较分析.结果表明,4个IBDV毒株在致病性程度上存在较大差异.其中有一个是真正的超强毒,即GX8/99,其他几个虽然达不到真正超强毒的毒力,但也比经典的标准毒的致死性高得多.在vp2基因高变区,这4个IBDV野毒株与超强毒参考株HK46同源性很高,在DNA水平为96.8%～99.5%,在氨基酸(aa)水平为96.6%～100%.而与疫苗毒D78有很大差异,分别只有91.7%～93.6%和91.8%～93.2%.说明IBDV毒株的vp2基因高变区确实与其致病性有一定关系.特别是SD-1/97、SD-3/98、JS-30/99株之间及其与HK46在DNA和aa水平的同源性高达98.4%和98.6%以上,SD-3/99、JS-30/99株之间及其与HK46的氨基酸同源性为100%.然而,致病性特别高的GX8/99株病毒与其他3个野毒株及HK46在DNA和氨基酸水平的同源性相对较低,在DNA和aa水平的同源性只有96.8%～97.2%和96.6%～97.9%.相对于国内的流行毒株和香港超强毒参考株HK46,GX8/99株在致病性和VP2高变区都已发生了一定的变异.     

Anthropology and genetics of the caucasus peoples and the problem of the origin of the caucasoids

The electrophoretical polymorphisms of some blood proteins were studied in the Talysh population of Pirasora situated in South-East Azerbaidjan. We calculated the gene frequencies of these polymorphisms and determined the genetic distances between the Talyshes and some Iranian populations of North, Central and South Iran, Afghans, and three populations of Azerbaijan. The Talyshes are very close to Iranians of Shiraz, whereas they are distant from the Azerbaijanians. Anthropological investigations showed that the Caucasoids and Mongoloids lived in the Aragvi Basin since the Eneolithic period. This was stated by Alexeev (1974), who emphasized the mixture of the Caucasus populations from ancient times on. We calculated the genetic distances between the Caucasus populations and numerous populations of other geographic regions, considering 28 alleles of 12 loci of blood group, serum protein and red cell enzyme polymorphisms and constructed the dendrogram of these populations. The position of the Caucasus populations in the dendrogram corresponds on principle to the earlier anthropological observations. The clustering of the Caucasoid populations corresponds completely with anthropological and historical data, and supports our earlier hypothesis (Nazarova 1999) concerning the differentiation of Caucasoids, Northern Mongoloids and Amerinds from the populations, which inhabitated Asia in palaeolithic times.     

On the structure and dynamics of the complex of the nucleosome and the linker histone

Several different models of the linker histone (LH)–nucleosome complex have been proposed, but none of them has unambiguously revealed the position and binding sites of the LH on the nucleosome. Using Brownian dynamics-based docking together with normal mode analysis of the nucleosome to account for the flexibility of two flanking 10 bp long linker DNAs (L-DNA), we identified binding modes of the H5-LH globular domain (GH5) to the nucleosome. For a wide range of nucleosomal conformations with the L-DNA ends less than 65 Å apart, one dominant binding mode was identified for GH5 and found to be consistent with fluorescence recovery after photobleaching (FRAP) experiments. GH5 binds asymmetrically with respect to the nucleosomal dyad axis, fitting between the nucleosomal DNA and one of the L-DNAs. For greater distances between L-DNA ends, docking of GH5 to the L-DNA that is more restrained and less open becomes favored. These results suggest a selection mechanism by which GH5 preferentially binds one of the L-DNAs and thereby affects DNA dynamics and accessibility and contributes to formation of a particular chromatin fiber structure. The two binding modes identified would, respectively, favor a tight zigzag chromatin structure or a loose solenoid chromatin fiber.     

Kinematics and dynamics of the temporomandibular joint and of the movements of the mandible

In order to analyze the complicated movements of the mandible as the open-closing movement and the protrusio are, it is useful to evaluate the basic kinematic principles and reduce them to simple technical constructions. Both the open-closing movement and the protrusio could be reduced to 4-bar links, which were used to simulate the movements with help of a computer. Besides, the polodes and the curves of points in the muscular attachments could be constructed. The 2 entirely different 4-bar links have 3 things in common: The resting system - cranium, the moving system - mandibula, and 1 of the 2 arms connecting these 2 systems - the ligamentum laterale. As this ligament is taut during movements it can be considered a "guiding ligament" representing 1 of the 3 determining components of the mandibular movements. The other of the 2 arms has no anatomical equivalent; this arm, however, is "replaced" by the 2 other determining components of the mandibular movements: the joint and the muscles. The curves, which the Caput mandibulae describes, are practically identical for the open-closing movement and the protrusio despite of the different 4-bar links and these curves exactly correspond to the Discus articularis, taut by the upper part of the M. pterygoideus lateralis. The muscles do not only just move the mandibula, but they are also the component, which can choose between the different mandibular movements. By means of the curves, which points in the muscular attachments describe, the function of the masticatory muscles could be analyzed exactly.     

Biophysics and Structure of the Patch and the Gigaseal

Interpreting channel behavior in patches requires an understanding of patch structure and dynamics, especially in studies of mechanosensitive channels. High resolution optical studies show that patch formation occurs via blebbing that disrupts normal membrane structure and redistributes in situ components including ion channels. There is a 1-2 μm region of the seal below the patch where proteins are excluded and this may consist of extracted lipids that form the gigaseal. Patch domes often have complex geometries with inhomogeneous stresses due to the membrane-glass adhesion energy (E_a), cytoskeletal forces, and possible lipid subdomains. The resting tension in the patch dome ranges from 1-4 mN/m, a significant fraction of the lytic tension of a bilayer (∼10 mN/m). Thus, all patch experiments are conducted under substantial, and uneven, resting tension that may alter the kinetics of many channels. E_a seems dominated by van der Waals attraction overlaid with a normally repulsive Coulombic force. High ionic strength pipette saline increased E_a and, surprisingly, increased cytoskeletal rigidity in cell-attached patches. Low pH pipette saline also increased E_a and reduced the seal selectivity for cations, presumably by neutralizing the membrane surface charge. The seal is a negatively charged, cation selective, space with a resistance of ∼7 gigohm/μm in 100 mM KCl, and the high resistivity of the space may result from the presence of high viscosity glycoproteins. Patches creep up the pipette over time with voltage independent and voltage dependent components. Voltage-independent creep is expected from the capillary attraction of E_a and the flow of fresh lipids from the cell. Voltage-dependent creep seems to arise from electroosmosis in the seal. Neutralization of negative charges on the seal membrane with low pH decreased the creep rate and reversed the direction of creep at positive pipette potentials.     

Serology and systematics of the Ebenales and the Theales

The systematic position ofthe Ebenaceae, Sapotaceae, Styracaceae, Ochnaceae, Stachyuraceae, Dipterocarpaceae, Clusiaceae and Hypericaceae has been investigated using serological comparisons of sets of antigenic determinants. The results show that the Sytracaceae and Sapotaceae are undoubtedly more closely associated with the Actinidiaeceae and Theaceae, respectively, than with each other. We found no corresponding determinants betnween antigen systems from the Ebenaceae and systems from any other family whose relations to this family have been proposed. As discovered previously, investigations of antigen systems from the Ochnaceae, Dipterocarpaceae, Stachyuraceae, Clusiaceae and Hypericaceae are against the idea of a natural order “Theales” in which these families, or at least some of them, are combined with the Theaceae and Actinidiaceae. This paper completes our previous investigations which largely support a superorder Ericanae sensu Ehrendorfer and Takhtajan. We propose to include the Actinidiaceae and Theaceae in this superorder, assigning them a central position laong with the Sapotaceae and Sytracaeae on one side and the Primulales and Ericales on the other. Another most interesting finding is that there are corresponding determinants between antigen systems from the members of the Ericanae and representatives of the Polemoniaceae and Loasaceae.     

人类基因组及后基因组研究进展及其应用与开发研究现状

人类对自身基因组的研究,随着人类基因组工作草图的绘制完成和对基因功能研究的深入已加快进入了实质性、关键性的开发利用阶段。本文概述了人类基因组及后基因组的研究进展及依此开展基因治疗及基因（组）药物研制等应用开发研究的现状。     

Comparison of the Effects of Flexion and Extension of the Thumb and Fingers on the Position and Cross-Sectional Area of the Median Nerve

Objective

To assess the separate effects of thumb and finger extension/flexion on median nerve position and cross-sectional area.

Methods

Ultrasonography was used to assess median nerve transverse position and cross-sectional area within the carpal tunnel at rest and its movement during volitional flexion of the individual digits of the hand. Both wrists of 165 normal subjects (11 men, 4 women, mean age, 28.6, range, 22 to 38) were studied.

Results

Thumb flexion resulted in transverse movement of the median nerve in radial direction (1.2±0.6 mm), whereas flexion of the fingers produced transverse movement in ulnar direction, which was most pronounced during flexion of the index and middle fingers (3.2±0.9 and 3.1±1.0 mm, respectively). Lesser but still statistically significant movements were noted with flexion of the ring finger (2.0±0.8 mm) and little finger (1.2±0.5 mm). Flexion of the thumb or individual fingers did not change median nerve cross-sectional area (8.5±1.1 mm²).

Conclusions

Volitional flexion of the thumb and individual fingers, particularly the index and middle fingers, produced significant transverse movement of the median nerve within the carpal tunnel but did not alter the cross-sectional area of the nerve. The importance of these findings on the understanding of the pathogenesis of the carpal tunnel syndrome and its treatment remains to be investigated.     

On the Microanatomy of the Brain and the Innervation and Homologues of the Cephalic Appendages of Hesionidae and Syllidae (Polychaeta)

Abstract Earlier papers dealing with the anatomy of the hesionids and syllids were studied. Thereby it was found that information about the structure of the central nervous system was meagre. As a result, the anterior end appendages, especially the large, laterloventral ones of the Syllidae, have been differently interpreted. This prompted a re-investigation. The circum-oesophageal connectives, the brain commissures and the innervation of the alimentary canal and the cephalic appendages of a number of hesionid and syllid species were studied. The results, summarized in schematic diagrams, were compared with corresponding observations in other polychaete families. Among other things, it was concluded that not only the latero-ventral cephalic appendages of the hesionids but also those of the syllids are homologous with the palps of the nereids and of many other “errant” and “sedentary” families.