中国2000—2004年鸡传染性支气管炎病毒地方分离株核蛋白基因的遗传变异分析

对2000-2004年从中国9个省市分离到的13株IBV的核蛋白基因片段进行序列测定及分析的结果表明,13株IBV分离株核蛋白基因均含有一个长1230bp的ORF,但存在基因突变现象。与GenBank中的42株参考毒株核蛋白基因序列进行比较和分析,系统进化关系显示55株IBV毒株分属于9个群。第Ⅰ-Ⅲ群主要包括美国、日本、荷兰等国家以及中国的部分IBV分离株和疫苗株。其中本研究中的CK/CH/LHN/00I可能为一株分离的疫苗毒,CK/CH/LSD/03I、CK/CH/LDL/01I可能为重组毒。而中国近十年来分离的IBV毒株主要分布在第Ⅵ-Ⅷ群中,此3群内IBV毒株之间N蛋白推导氨基酸同源性为88.3%～100%,与其他各群之间同源性为62.3%～95.1%。因此,此基因型的IBV毒株可能在中国已有较长时期的存在且发生了较大程度的变异。其中第Ⅵ群中两株韩国分离株与中国IBV分离株具有较近的亲缘关系。以上结果表明,中国大多数IBV分离株在N基因进化关系上较为独立,与国外毒株相比,和韩国毒株进化关系密切。此外,中国IBV毒株基因重组现象更加普遍,尤其是疫苗毒和野毒之间的重组。     

中国2000-2004年鸡传染性支气管炎病毒地方分离株核蛋白基因的遗传变异分析

对2000-2004年从中国9个省市分离到的13株IBV的核蛋白基因片段进行序列测定及分析的结果表明,13株IBV分离株核蛋白基因均含有一个长1230bp的ORF,但存在基因突变现象.与GenBank中的42株参考毒株核蛋白基因序列进行比较和分析,系统进化关系显示55株IBV毒株分属于9个群.第Ⅰ-Ⅲ群主要包括美国、日本、荷兰等国家以及中国的部分IBV分离株和疫苗株.其中本研究中的CK/CH/LHN/00I可能为一株分离的疫苗毒,CK/CH/LSD/03I、CK/CH/LDL/01I可能为重组毒.而中国近十年来分离的IBV毒株主要分布在第Ⅵ-Ⅷ群中,此3群内IBV毒株之间N蛋白推导氨基酸同源性为88.3%～100%,与其他各群之间同源性为62.3%～95.1%.因此,此基因型的IBV毒株可能在中国已有较长时期的存在且发生了较大程度的变异.其中第Ⅵ群中两株韩国分离株与中国IBV分离株具有较近的亲缘关系.以上结果表明,中国大多数IBV分离株在N基因进化关系上较为独立,与国外毒株相比,和韩国毒株进化关系密切.此外,中国IBV毒株基因重组现象更加普遍,尤其是疫苗毒和野毒之间的重组.     

应用随机PCR方法鉴定一株真养产碱杆菌

采用随机引物PCR技术从新建细胞培养室空气中获得一段长414bp的片段,通过克隆测序及序列分析,结果表明所测序列与真养产碱杆菌主要参考菌株的同源性分别高达79%-83%,由其推导的氨基酸序列与真养产碱杆菌主要参考菌株的同源性高达86.4%-89.1%,从而确定所分离菌株为真养产碱杆菌。     

我国2002−2016年间鸡传染性支气管炎病毒基因组序列重组分析

【目的】传染性支气管炎病毒(Infectious bronchitis virus,IBV)主要引起鸡群呼吸道与肾脏疾病,是影响养禽业重要的病毒性病原之一。了解我国IBV的流行及基因重组情况。【方法】收集GenBank中我国2002?2016年间分离的92株IBV S1基因及55株IBV基因组序列,并对这些序列进行比对分析。【结果】IBV S1基因序列分析结果表明,2002?2016年间我国流行的92株IBV可以分为13个基因型,包括QX、4/91、Mass、tl/CH/LDT3/03、CK/CH/LSC/99I、TW-I、TW-II、TC07-2、Ck/CH/LDL/97I、N1/62-associated、Arkansas、New-I及一个新鉴定的基因分支New-II。值得注意的是,属于美国相关基因分支的ck/CH/LSD/110712已在我国出现。RDP4方法进行重组分析显示,新出现的基因分支New-II病毒S1基因来源于tl/CH/LDT3/03型与QX型毒株重组,我国2002?2016年间流行的55株IBV中有52株IBV基因组存在重组事件,其中25个IBV分离株基因组中发现有疫苗型(Mass,tl/CH/LDT3/03及4/91型等)病毒基因组片段的重组,这一结果在SimPlot分析中进一步得到确认。【结论】根据生物信息学分析结果,证明流行于我国的IBV基因型众多,疫苗毒株基因频繁参与了IBV基因重组,导致IBV新的基因型或变异株出现,提示在防控IB时要注意合理使用IBV疫苗。     

致胰腺泛黄鸭1型甲肝病毒全基因组分子特征

【目的】揭示致胰腺泛黄鸭1型甲肝病毒(duck hepatitis A virus 1,DHAV-1)全基因组的分子特征。【方法】运用RT-PCR技术,扩增出致胰腺泛黄DHAV-1 MPZJ1206株全基因组序列,并与鸭甲肝病毒参考毒株基因组序列进行比对分析。【结果】致胰腺泛黄DHAV-1 MPZJ1206株基因组全长为7703 nt,(G+C)%为43.05%,包含一个大的开放阅读框,编码2249个氨基酸残基的多聚蛋白,基因组结构与其他DHAV-1参考毒株一致。序列比对结果显示,MPZJ1206株全基因组序列与GenBank数据库中DHAV-1参考毒株核苷酸序列同源性在93.5%-99.6%之间,氨基酸同源性在97.9%-99.6%之间,遗传距离均低于7%;分子系统进化分析显示与2011年分离的2株DHAV-1(Du/CH/LGD/111238和Du/CH/LGD/111239)亲缘关系最近。【结论】尽管MPZJ1206毒株感染雏番鸭引起的临床病变与传统DHAV-1差异明显,但其基因组分子特征与传统的DHAV-1毒株相似,病毒致病型的改变可能与其组织嗜性改变相关。     

几种鸡传染性支气管炎病毒活疫苗对中国流行毒株CK/CH/LDL/97I的保护作用评价

选择我国应用的五株鸡传染性支气管炎活疫苗毒株(JAAS、IBN、Jlin、J9和H120)和当地流行毒株(CK/CH/LDL/97 Ⅰ)作为研究对象,对其S1基因进行序列比对分析,结果表明疫苗株与流行毒株的核昔酸序列及推导的氨基酸序列同源性分别不超过76.4%和78.7%.S1基因的核苷酸系统发育树显示,疫苗株与流行毒株分属不同进化群,亲缘关系较远,属于不同的基因型.用这五株活疫苗进行针对强毒株CK/CH/LDL/97Ⅰ株的免疫保护实验,可见临床发病率为30%～100%;攻毒5d后每组随机扑杀10只鸡,采集器官,应用RT-PCR法检测病毒,气管样品病毒检出率为50%～90%,肾脏样品病毒检出率为10%～30%.由此可见:我国目前使用的主要活疫苗对异种IBV分离株的感染不能提供完全的保护作用.     

鸡传染性支气管炎病毒变异株全基因组序列分析

为了明确传染性性支气管炎病毒(Infectious bronchitis virus,IBV)分离株CK/CH/SD09/005的分子特征,以进一步丰富国内IBV的分子流行病学信息。本研究设计了25对引物对其全基因组进行了序列测定,并与参考株进行了同源性比较和S1基因遗传进化分析。结果显示CK/CH/SD09/005基因组为27 691bp(不包括5′端Cap和3′端Poly A)。全基因组同源性比对发现,CK/CH/SD09/005仅与GenBank中广西2009年分离株GX-NN09032各基因高度同源(97%~99%)。除GX-NN09032外,CK/CH/SD09/005基因组5′端复制酶基因(Gene 1)和3′端非转录区(Untranslated region,UTR)与2个QX基因型参考株ck/CH/LDL/091022和SDIB821/2012同源性最高,分别为97%和98%,但是3′端结构蛋白和非结构蛋白基因(S-3a-3b-3c/E-M-5a-5b-N)与这两个毒株同源性较低,仅为72%~90%。其ORF3c/E、5a、5b和N分别与韩国分离株1011、国内分离株CK/CH/LXJ/02I、DK/CH/HN/ZZ2004和YX10同源性最高,分别为97%、96%、99%和96%,而其ORF3a、3b和M与参考株同源性均低于90%。S1基因遗传进化分析发现,CK/CH/SD09/005和国内外39个参考株形成7个进化分支(基因型),CK/CH/SD09/005和2007以来几个分离株属于基因Ⅳ型,与其它6个基因型参考株S1和S2基因同源性为66%~69%和72%~81%,S1基因不仅表现广泛性点突变,而且有多处碱基插入和缺失,S2仅表现点突变。本研究结果表明CK/CH/SD09/005是一个变异株,可能是QX基因型IBV流行株与其它毒株重组进化而来,此外还涉及基因突变、插入和缺失等多种变异机制。     

新城疫分离毒HN基因的分子特性和片段同源相关性

选取国内1997-2005年分离的新城疫病毒(Newcastle disease virus,NDV)24株,经蚀斑纯化克隆其血凝素-神经氨酸酶(HN)基因,与在GenBank发表的36株国内外不同时期的NDV毒株,进行氨基酸遗传变异分析,并利用SPSS8.0软件对其不同片段的氨基酸进行同源相关比较。结果显示:国内所有NDV分离毒株氨基酸高度同源,同源性为94.4%-99.4%;与LaSota、Clone30疫苗株等的氨基酸同源性为86.9%-89%;与强毒株F48E9的氨基酸同源性为87.9%-89.9%;与国外NDV的氨基酸同源性为87.2%-96.2%。系统发育分析表明:国内NDV分离毒HN遗传距离较近,而与LaSota、Clone30和F48E9遗传距离较远。国内NDV分离毒均缺乏538-540位糖基化位点。不同片段与全长的氨基酸同源性高度相关,且与前80个氨基酸相关最密切。     

肾综合征出血热疫苗候选毒株A16株的分子特性

为研究肾综合征出血热疫苗候选毒株A16株的分子基础,应用RT－PCR方法扩增并测定了A16株的M和S片段的序列,结果A16株M片段的全基因序列共3615个核苷酸,编码1133个氨基酸。S片段的全基因序列共1770个核苷酸,编码429个氨基酸。A16株M片段核苷酸全基因序列及其的氨基酸与HTN型毒株同源率为75.5%-90.3%和85.9%-97.1%,即A16与HTN同型毒株间的差异高达9.7%-24.3%和2.9%-14.1%,而与SEO型的同源率比较,核苷酸和氨基酸分别仅为70.2%-70.8%和73.4%-76.7%.S片段的序列同源率与M片段的结果相类似,即核苷酸和氨基酸的同源率与HTN型毒株分别为76.0%－905和92.1%-97.7%,与SEO型为67.0%-67.7%和81.7%-82.6%。结果显示,A16株虽然属于HTN型,但该毒株为HTN型病毒的新亚型病毒株。     

22株猪瘟病毒E2基因部分编码序列的序列分析

RT－PCR方法获得了13株猪瘟病毒分离株、石门系强毒、中国C株及法国温度敏感株Thiverval株的E2基因部分编码序列的拉增片段,并对其进行了测序,得到了251bp的E2基因部分编码序列。利用DNAStar软件对其中224bp的片段进行了序列分析,并与已发表的Alfort、Brescia等毒株进行比较,结果13株猪瘟分离株所测片段均为猪瘟病毒E2基因的序列,与石门系强毒的序列相比所有毒株的碱基替换随机地分布于整个序列,无碱基缺失和碱基插入。其中变化较大的区域位于序列的3′端。2 2株HCV E2基因部分编码序列的核苷酸及氨基酸同源性范围分别为78.1%-100%、78.4%-100%,其中13株猪瘟病毒流行毒株的核苷酸及氨基酸同源性范围分别为:78.1%-100%、78.4%-100%,4株70－80年代分离的毒株的核苷酸及氨基酸同源性范围分别为：79.0%-88.3%、81.1%-87.8%,9株90年代分离的毒株的核苷酸及氨基酸同源性范围分别为：90.3%-100%、83.8%-100％;说明猪病毒流行株的变异呈现一定的多样性。     

Synthesis and biological activity of 3 beta-fluorovitamin D3: comparison of the biological activity of 3 beta-fluorovitamin D3 and 3-deoxyvitamin D3

The alteration in the biologic activity of the vitamin D3 molecule resulting from the replacement of a hydrogen atom with a fluorine atom is a subject of fundamental interest. To investigate this problem we synthesized 3 beta-fluorovitamin D3 6 and its hydrogen analog, 3-deoxyvitamin D3 7, and tested the biologic activity of each by in vitro and in vivo methods. Contrary to previous reports which showed that 3 beta-fluorovitamin D3 was as active as vitamin D3 in vivo, we found that the fluoro-analog was less active than vitamin D3. With regard to stimulation of intestinal calcium transport and bone calcium mobilization in the D-deficient hypocalcemic rat, 3 beta-fluorovitamin D3 showed significantly greater biologic activity than its hydrogen analog, 3-deoxyvitamin D3. In the organ-cultured, embryonic chick duodenum, 3 beta-fluorovitamin D3 was approx 1/1000th as active as the native hormone, 1,25-dihydroxyvitamin D3, while 3-deoxyvitamin D3 was inactive even at microM concentrations, in the induction of the vitamin D-dependent, calcium-binding protein. With regard to in vitro activity in displacing radiolabeled 25-hydroxyvitamin D3 from vitamin D binding protein and radiolabelled 1,25-dihydroxyvitamin D3 from a chick intestinal cytosol receptor, 3 beta-fluorovitamin D3 and 3 beta-deoxyvitamin D3 both showed very poor binding efficiencies when compared with vitamin D3. Our results show that the substitution of a fluorine atom for a hydrogen atom at the C-3 position of the vitamin D3 molecule results in a fluorovitamin 6 with significantly more biological activity than its hydrogen analog, 3-deoxyvitamin D3 7.     

3-Bromo-3-deazaneplanocin and 3-bromo-3-deazaaristeromycin: Synthesis and antiviral activity

As an outgrowth of our program to explore 3-deazaadenine carbocyclic nucleosides, 3-bromo-3-deazaneplanocin (5) and 3-bromo-3-deazaaristeromycin (6) have been synthesized from a readily available cyclopentenol and cyclopentanone and either 4-amino- or 4-chloro-1H-imidazo[4,5-c]pyridine (6-amino- or 6-chloro-3-deazaadenine) in 5 steps and 7 steps, respectively. Antiviral analysis found 5 to display significant activity towards a number of (-)-ssRNA and a few dsDNA viruses. Compound 6 was less active than 5 against selected examples of those viruses affected by 5.     

Kinetic comparison of procaspase-3 and caspase-3

Caspases, the key enzymes in apoptosis, are synthesized as proenzymes and converted into active form by proteolytic cleavage. The residues on active site reorganize during the activation process as shown in the comparative studies of crystallographic structures of procaspase-7 and its mature form. On the other hand, the proenzyme itself has some activity. Aiming to characterize the activation process, the comparative kinetic study for the pro- and mature caspase-3 was performed. In 1/K(M) versus pH study, a residue with pKa of 6.89+/-0.13 was detected only in caspase-3. While Vmax versus pH kinetic results were consistent with the existence of a residue with pKa of 6.21+/-0.06 in procaspase-3 mutant (D9A/D28A/D175A) but not in caspase-3. In the inactivation assays with diethylpyrocarbonate, a residue (pKa, 6.61+/-0.05) could be determined only for caspase-3 whereas with iodoacetamide a residue with pKa value (6.01+/-0.05) could be assigned only for procaspase-3. Considering that those residues could be protected by caspase-3-specific inhibitor from the inactivation, the modifiers are histidine- and cysteine-specific, respectively, and the involvement of these residues in the characteristic catalytic dyad of caspases, the results indicate that the pKa values of the catalytic histidine and cysteine residues are changed during the activation process.     

Beta-eliminative cleavage of 3-ketocerebroside and 3-ketosphingomyelin

3-Ketosphingolipids derived from cerebroside and sphingomyelin were converted to a less polar compound in as weak alkali as 0.0005 M Na₂CO₃ in chloroform-methanol 2 : 1 (v/v). The product was identified as 2-acylamido-3-keto-1,4-octadecadiene by several spectroscopic procedures and cleavage was concluded to occur by beta-elimination. The beta-elimination occurred at above pH 11.5 in aqueous solution. At higher pH values deacylation also occurred. The influence of solvent effects on beta-elimination was studied. This reaction can be used to obtain d oligosaccharides, phosphorycholine and other hydrophilic moieties from sphingolipids.     

Anaerobic degradation of poly-3-hydroxybutyrate and poly-3-hydroxybutyrate-co-3-hydroxyvalerate

The anaerobic degradation of the polyesterspoly-3-hydroxybutyrate (PHB) andpoly-3-hydroxybutyrate-co-3-hydroxyvalerate (PHBV) wasinvestigated with special regard to intermediateproducts, kinetics, and yields. During the degradationof PHBV acetate, propionate, n-butyrate, andn-valerate were detected. Additionally,3-hydroxybutyrate and 3-hydroxyvalerate and fourdimeric esters of these two molecules were identifiedby GC-MS measurements. Three different test systemsfor the anaerobic degradation of polyesters werestudied. It was not possible to get reproducibleresults by means of the Anaerobic Sturm-test, a simplesystem based on carbon dioxide measurement. Secondly,a system based on the GC measurement of accumulatedorganic acids was investigated. A degradation of 90%in two days was calculated by a carbon balance. Bestresults were reached with the third test system basedon the measurement of methane with a gas meter. Adegradation of 99% was observed within 30 days.     

Anaerobic degradation of poly-3-hydroxybutyrate and poly-3-hydroxybutyrate-co-3-hydroxyvalerate

The anaerobic degradation of the polyesters poly-3-hydroxybutyrate (PHB) and poly-3-hydroxybutyrate-co-3-hydroxyvalerate (PHBV) was investigated with special regard to intermediate products, kinetics, and yields. During the degradation of PHBV acetate, propionate, n-butyrate, and n-valerate were detected. Additionally, 3-hydroxybutyrate and 3-hydroxyvalerate and four dimeric esters of these two molecules were identified by GC-MS measurements. Three different test systems for the anaerobic degradation of polyesters were studied. It was not possible to get reproducible results by means of the Anaerobic Sturm-test, a simple system based on carbon dioxide measurement. Secondly, a system based on the GC measurement of accumulated organic acids was investigated. A degradation of 90% in two days was calculated by a carbon balance. Best results were reached with the third test system based on the measurement of methane with a gas meter. A degradation of 99% was observed within 30 days.