伪狂犬病病毒基因缺失疫苗制苗用毒种特性研究

本试验通过对PRV基因缺失株SA215(gE-/gI-/TK- )细胞培养特性、理化特性及形态发生过程进行研究,来确定gE、gI和TK 基因缺失对病毒特性的影响。结果表明,基因缺失对该毒株在培养细胞的吸附和穿入过程没有影响,但与亲本株相比,表现为生长掩蔽期延长,增殖速度减缓,但能达到相似的增殖滴度,并且基因缺失对病毒的理化特性影响很小。形态发生过程观察结果表明,PRV SA215 株在细胞培养上形态发生正常,能形成感染性病毒粒子,但在由核膜出芽和囊膜形成上受到一定程度的阻碍。本研究为疫苗研制和生产提供指导和依据。     

伪狂犬病病毒上海株gE和gI基因的克隆及缺失转移载体的构建

根据已发表的伪狂犬病病毒 (PseudorabiesVirus,PRV)的 gI和 gE 基因序列 ,设计两对引物用PCR方法扩增出PRV SH gI和 gE 基因 ,将其克隆入 pUC18载体中 ,获得了缺失部分gI基因的转移载体质粒 ,命名为 pgEI。     

伪狂犬病病毒上海株gE和gI基因的克隆及缺失转移载体的构建

根据已发表的伪狂犬病病毒(Pseudorabies Virus,PRV)的gI和gE基因序列,设计两对引物用PCR方法扩增出PRV-SH gI和gE基因,将其克隆入pUC18载体中,获得了缺失部分gI基因的转移载体质粒,命名为pgEI.     

伪狂犬病病毒上海株(PRV-SH)gE-/gI/GFP+缺失株的构建

在构建了含伪狂犬病病毒（Pseudorabies Virus,PRV）上海株gI基因和gE基因克隆鉴定的基础上,采用酶切的方法构建了载体pgEI。然后用限制性内切酶BamHI和BstpI缺失掉gE基因5'端363bp,同时把绿色荧光蛋白（GFP）基因表达盒插入到缺失部分,并在下游引入一个多克隆位点,构建了缺失转移载体pgEIGFP。用DOTAP转染试剂盒将pgEIGFP转染了感染PRVSH的BHK21细胞,待出现80%病变后收获病毒,并以蚀斑法得到纯化的缺失了gE/gI重组病毒株。小鼠试验证实了缺失株的毒力有所下降。     

伪狂犬病病毒上海株（PRV—SH）gE^—／gI^—／GFP^＋缺失株的构建

在构建了含伪狂犬病病毒（Pseudorabies Virus,PRV）上海株gI基因和gE基因克隆鉴定的基础上,采用酶切的方法构建了载体pgEI。然后用限制性内切酶BamHI和BstPI缺失掉gE基因5‘端363bp,同时把绿色荧光蛋白（GFP）基因表达盒插入到缺失部分,并在下游 引入一个多克隆位点,构建了缺失转移载体pgEI-GFP。用DOTAP转染试剂盒将pgEI-GFP转染了感染PRV-SH的BHK-21细胞,待出现80％病变后收获病毒,并以蚀斑法得到纯化的缺失了gE/gI重组病毒株。小鼠试验证实了缺失株的毒力有所下降。     

伪狂犬病毒gI基因的克隆表达及其对病毒增殖的影响

从伪狂犬病毒(PRV)国内地方分离Ea株基因组DNA片段中克隆了完整的gI基因,序列分析结果表明,gI基因编码区全长1101bp,可编码366个氨基酸残基,二级结构预测具有典型I型膜蛋白特征。与GenBank中收录的国外Rice株的同源比较发现,Ea株gI在核苷酸和氨基酸水平上均存在多处突变,尤其是潜在胞浆区中连续两个碱基的缺失导致移码突变,致使gI基因的读码框架后移,从而导致Ea株gI较rice株长16个氨基酸残基。将gI基因克隆到真核表达载体pcDNA31+中的人巨细胞病毒早期启动子下游,构建的真核表达质粒转染PK15细胞,间接免疫荧光检测证实gI获得正确表达。进一步测定天然缺失gI的PRV弱毒Bartha株在表达gI细胞系和空白载体转染的对照细胞系中的蚀斑形成单位(pfu)和组织细胞培养半数感染量(TCID50),结果显示：Bartha株在表达gI细胞系中的pfu和TCID\-\{50\}分别为对照细胞系的164%和200%。说明gI具有促进病毒增殖的功能。     

猪圆环病毒2型－细小病毒－伪狂犬重组病毒免疫小鼠试验研究

为了研究表达猪2型圆环病毒ORF2基因和猪细小病毒VP2基因的重组伪狂犬病毒疫苗株SA215(D)的疫苗价值,对该病毒株安全性、免疫原性、保护力及对PRV Fa株定值进行了研究。研究结果表明重组病毒SA215（D）毒力显著低于PRV Fa强毒株,可以抵御伪狂犬病强毒Fa株的攻击,并能抵御其在小鼠体内的定植;抗体检测显示SA215（D）免疫小鼠后第2周抗PRV、PCV2和PPV的抗体水平均很低,加强免疫后第4周抗体水平大幅上升,且显著高于阴性对照组,而与阳性对照组无明显差异,表明SA215（D）可诱导小鼠产生体液免疫反应。淋巴细胞增殖试验显示SA215（D）可诱导小鼠产生较强的淋巴细胞增殖反应,与亲本株SA215组相比,差异显著(0.01     

猪伪狂犬病综合诊断及分离毒株gE和TK基因遗传变异分析

2012年以来,许多使用gE基因缺失活疫苗免疫过的猪场广泛性出现伪狂犬病毒(PRV)感染,gE抗体阳性率不断升高,伪狂犬典型病例不断增加。2018年3月鲁南地区几个种猪场先后发生疑似伪狂犬病疫情,怀孕母猪流产、产死胎和木乃伊胎,仔猪出现神经症状且死亡率高。通过对病死猪及死胚剖检进行初步诊断,取病料进一步进行病理组织学诊断及病毒分离鉴定。结果显示,病死猪均可见病毒性脑炎、肝细胞变性坏死及淋巴组织坏死等病理变化,在病变的神经元、肝细胞、扁桃体隐窝上皮细胞等细胞核内见红染包涵体。对分离到的4株PRV进行了gE和TK基因的序列测定及遗传变异分析发现,4株PRV的gE和TK核苷酸序列的同源性分别为98.8%~99.3%和98.9%~99.6%,与国内流行毒株其核苷酸序列的同源性分别99.1%~99.7%和98.6%~99.8%,与匈牙利和美国等流行毒株核苷酸序列的同源性分别为97.3%~97.8%和98.8%~99.5%,表明4株分离株高度同源,与国内PRV变异株处在同一分支,而与匈牙利和美国等毒株遗传距离较远。传统疫苗对PRV变异毒株不能提供有效保护,给猪场伪狂犬病的防控和净化工作带来了新的挑战。     

伪狂犬病病毒鄂A株TK－／gG－／LacZ＋突变株的构建

为了以国内地方分离株鄂A株为亲本构建伪狂犬病病毒双基因缺失株,采用外切酶Ⅲ和绿豆核酸酶酶切,构建了缺失主要毒力基因TK基因部分编码区的重组质粒pSTK1-4,进一步改造成为转移质粒pUCPB4。用HindⅢ将质粒pUCPB4线性化,然后与用EcoRI消化的伪狂犬病病毒鄂A株TK^-/LacZ^ 突变株基因组DNA共转染PK－15细胞,等完全病变后,收毒作空斑试验,PCR筛选TK缺失的重组病毒。重组病毒空斑纯化3次,随机挑取空斑进行PCR扩增,证实所获得的病毒为均一的无TK^-/LacZ^ 突变株污染的TK缺失的重组病毒,分别以TK缺失株病毒与鄂A野毒株为模板对TK基因进行PCR扩增,扩增产物经酶切分析和测序后发现：TK缺失重组病毒的TK基因缺失了205个碱基,XhoⅠ和SalⅠ位点消失,SmaⅠ酶切片段发生变化,两种病毒在PK－15细胞上形成空斑的大小和增殖 滴度无明显的差别。进一步提取TK缺失突变株基因组DNA,与含gG-LacZ的转移质粒pUSKZ通过磷酸钙法共转染PK－15细胞,待完全病变后,在X-gal存在下筛选蓝斑,将桃取的蓝斑纯化3次后,对纯化的重组病毒进行TK、LacZ扩增,结果既能扩增出较以鄂A野毒株为模板扩增的要小的TK基因片段,同时又能扩增出LacZ基因,证实所得到的重组病毒为TK^-/gG^-/LacZ^ 突变株。此双缺失突变株的构建成功,为在我国最终根除伪狂犬病提供了有用的工具。     

伪狂犬病毒(PRV) GZ-JS2017株的分离鉴定及主要毒力基因分子特征分析

为查明贵州省遵义地区某规模化养殖场猪只发生急性死亡的病因,以及明确病原主要毒力基因的分子特征。本试验采用PCR技术、细胞接种试验、动物试验及透射电子显微镜观察等方法进行病原的分离鉴定,并对该病原的主要毒力基因进行克隆、测序及生物信息学分析。结果显示:PCR检测可扩增出PRV特异性核酸阳性条带,接种Vero细胞后可见明显CPE现象,在透射电子显微镜下可见囊泡中呈圆形、160 nm左右的成熟病毒粒子,胞核中可见病毒包涵体和实心、空心2种无囊膜病毒粒子;接种健康猪可引起神经症状,最后麻痹死亡;通过生物信息学分析显示该毒株的gE、gC、gD及TK基因序列均与参考毒株中湖北HNX株和河南HN1201株相对应的核苷酸同源性最高,且在gE基因的48位和496位都有1个天冬氨酸(Asp)的插入;根据gE、gC、gD及TK基因序列的遗传进化树结果显示本次分离毒株与2011年以后所分离鉴定的变异毒株属同一分支。说明本试验成功分离鉴定一株猪伪狂犬病病毒变异毒株,以期为贵州省猪伪狂犬病病毒的流行及变异提供一些参考。     

Recent advances in the study of Kaposi's sarcoma-associated herpesvirus replication and pathogenesis

It has now been over twenty years since a novel herpesviral genome was identified in Kaposi's sarcoma biopsies. Since then, the cumulative research effort by molecular biologists, virologists, clinicians, and epidemiologists alike has led to the extensive characterization of this tumor virus, Kaposi's sarcoma-associated herpesvirus(KSHV; also known as human herpesvirus 8(HHV-8)), and its associated diseases. Here we review the current knowledge of KSHV biology and pathogenesis, with a particular emphasis on new and exciting advances in the field of epigenetics. We also discuss the development and practicality of various cell culture and animal model systems to study KSHV replication and pathogenesis.     

The structure, reproduction and taxonomy of Vidalia and Osmundaria (Rhodophyta, Rhodomelaceae)

Comprises species occurring mostly in subtidal habitats in tropical, subtropical and warm-temperate areas of the world. An analysis of the type species, V. spiralis (Sonder) Lamouroux ex J. Agardh, a species from Australia, establishes basic characters for distinguishing species in the genus. These characters are (1) branching patterns of thalli, (2) flat blades that may be spiralled on their axis, (3) width of the blade, (4) primary or secondary derivation of sterile and fertile branchlets and (5) position of sterile and fertile branchlets on the thalli. Application of the latter two characters provides an important basic method for separation of species into three major groups. Osmundaria , a genus known only in southern Australia, was studied in relation to Vidalia , and its separation from the Vidalia assemblage is not accepted. Species of Vidalia therefore are transferred to the older genus name, Osmundaria. Two new species, Osmundaria papenfussii and Osmundaria oliveae are described from Natal. Confusion in the usage of the epithet, Vidalia fimbriala Brown ex Turner has been clarified, and Vidalia gregaria Falkenberg, described as an epiphyte on Osmundaria pro/ifera Lamouroux, is revealed to be young branches of the host, Osmundaria prolifera.     

New or rare chromosome counts in Artemisia L. (Asteraceae, Anthemideae) and related genera from Kazakhstan

Fifteen chromosome counts of six Artemisia taxa and one species of each of the genera Brachanthemum, Hippolytia, Kaschgaria, Lepidolopsis and Turaniphytum are reported from Kazakhstan. Three of them are new reports, two are not consistent with previous counts and the remainder are confirmations of very scarce (one to four) earlier records. All the populations studied have the same basic chromosome number, x = 9, with ploidy levels ranging from 2x to 6x. Some correlations between ploidy level, morphological characters and distribution are noted.     

肝癌中HBV和HCV基因和抗原的分布及意义

采用原位分子杂交方法检测HCV RNA及HBV X基因;采用免疫组织化学方法研究HCV核心抗原,非结构区C33c抗原及HBxAg在肝细胞肝癌中的定位及分布.结果表明(1)HCV RNA、HBV X基因在肝细胞肝癌组织检出率分别为40%(55/136)和82%(112/136).HCV RNA定位于癌细胞的胞浆内,阳性细胞呈散在、灶状及弥漫分布三种形式;HBV X基因在肝癌细胞中的分布呈胞浆型、核型及核浆型,阳性细胞也呈上述三种分布形式;(2)HCV C33c抗原、核心抗原在肝细胞肝癌中的阳性率为81%(133/164)及86%(141/164).C33c抗原定位于癌细胞及肝细胞的胞浆内;核心抗原既定位于癌细胞核中,又可定位于胞浆中.C33c抗原阳性细胞以灶状分布为主;而核心抗原阳性细     

Selection with two alleles and complete dominance

For a plant selection model with frequency-independent viabilities, fertilities and selfing rates, it is shown that apart from global fixation, for certain parameter combinations a protected polymorphism and facultative fixation (either allele may become fixed according to initial frequencies) may both occur. Facultative fixation requires different selling rates for the dominant and recessive type. Protection of the polymorphism requires resource allocation for male and female function. In this connection the problem of purely genetically caused population extinction is discussed.
For general frequency dependence and regular segregation, the chances for establishment of a completely recessive gene are compared to those of a completely dominant gene. It is proven that the process of establishment of the recessive gene, despite a fitness advantage, may be considerably endangered by drift effects if random mating prevails. The recessive gene may reach the same effectivity in establishment as a dominant gene, only if the recessive homozygote mates exclusively with its own type during the period of establishment.