Cloning and Sequence Analysis of gE and gI Genes of Pseudorabies Virus NP Isolate

According to published gE and gI gene sequences of pseudorabies virus (PRV) in GenBank, we designed two pairs of primers for PCR amplification of gE and gI genes of PRV NP isolate, after PCR products recycling, cloning and sequencing, the sequencing results were consistent with expectations of PRV gE and gI genes.Homology comparison analysis results revealed that compared with the domestic PRV strains, the homologies of gE and gI amino acids of PRV NP isolate were 95.7% to 99.8% and 89.9% to 99.5%, respectively.Phyogenetic tree analysis and amino acid sequence alignment results found that the gE amino acid sequence site changes of PRV NP isolate were the same with the PRV strains which were isolated from domestic in 2012, thus we could speculate that PRV NP isolate had mutanted.This study had laid the foundation for the epidemiological investigation and analysis of PRV, also provided the scientific basis for the development of scientific, effective and new pseudorabies vaccine.     

大中型猪场猪伪狂犬病抗体检测与分析

为了解大中型猪场猪伪狂犬病免疫抗体水平,采用ELISA方法对辽宁3个区域大中型猪场共7个场采集猪血清样品共270份进行猪伪狂犬病gE抗体和gB抗体检测。结果显示:猪伪狂犬病gE抗体和gB抗体的总阳性率分别为15.9%和85.9%。其中区域一、区域二、区域三猪伪狂犬病gE抗体阳性率分别为21.8%、0和23.8%,猪伪狂犬病g B抗体阳性率分别为85.5%、91.3%和81.3%。结果表明:大中型猪场的免疫措施得当,免疫效果明显,但还存在感染的猪只。区域一和区域三所采样的大中型场中均有不同程度的野毒感染,区域二的野毒感染率和免疫抗体水平均优于其它两个地区。     

山东省一例山羊伪狂犬病的病原分离鉴定

2017年4月,山东省某羊场饲养的山羊发生疑似伪狂犬病疫情。为确诊引发疫情的病原,采集死亡山羊组织样品,匀浆处理后接种家兔,并进行病原分离、PCR鉴定、效价测定、电镜观察及主要毒力基因测序分析。结果显示:家兔接种病料上清液后出现奇痒、麻痹,最后死亡;Marc145细胞接种病例样品后,产生变亮、变大、破裂,呈"包涵体"样等PRV特征性细胞病变,细胞分离物伪狂犬病病毒(PRV)g E PCR检测结果为阳性,病毒效价可达10-7.50 TCID50/0.1 m L,电镜观察可见病毒粒子呈圆形,有囊膜,直径约150 nm,将其命名为SDPD-17株。测序发现该毒株g E糖蛋白48、497位各插入了1个天冬氨酸,有12个氨基酸位点发生点突变;g C蛋白氨基酸序列64～70位出现了7个氨基酸(AASTPAA)插入;TK基因无碱基插入或缺失。分离鉴定结果证实,该病例由PRV野毒感染所致。     

大理地区散养户猪伪狂犬病病毒野毒株感染的血清学分析

为了解云南大理地区散养户猪伪狂犬病病毒(PRV)野毒感染的情况,从大理地区30多个散养户6月龄及6月龄以上猪群中随机采集了共112份血清,采用间接ELISA方法对血清样本进行PRVgE抗体检测。结果显示,112份血清中抗PRVgE抗体阳性率为8.04%。结果表明,大理地区散养户6月龄及6月龄以上猪只猪伪狂犬病病毒野毒株感染率较低。     

Molecular Comparisons of Marek＇s disease virus strains of different pathotypes for their gⅠ, gE, pp38 and meq genes

Five to ten serotype I Marek‘ s disease virus (MDV1) strains of different pathotypes were compared for their DNA sequences of gⅠ, gE, pp38 and meq genes. The reference strains were vMDV GA and JM, vvMDV RB1B and Mdll(pl6), vv MDV strains 648A and 584A, vaccine strain CVI988/Rispens; Chinese strains were: v MDV strain N, vvMDV strain G2, vaccine strain 814. Only random aa changes were found in 12 positions within gE of 497 aa among 10 analyzed strains and in 10 positions within gⅠ of 355 aa among 5 strains. There was no relationship found between virus pathotypes and aa changes of both glycoproteins. The aa changes happened in 14 positions within meq of 339 aa among 5 compared strains. But a proline deletion at aa # 194 in a proline- rich domain of meq were shared by two vaccine strains CVI988/Rispens and 814, the latter was a non - pathogenic vaccine strain of serotype 1 isolated in 1983 in China. In another hand, vv MDV strains 648A demonstrated 5 unique aa changes and 4 of them also located in the proline - rich region. The pp38 was very conservative among sequenced 10 strains, there were only two positions at # 107 and # 109 with an altered in its 290 aa. All tested MDV1 strains had glufamine at # 107, instead, only vaccine CVI988 had arginine at the position and lost its epitope reactive with Mab H19, to which all ether tested MDV1 strains were positive. However, CVI988 and vMDV GA shared a Mab T65 - recognized epitope when there was glyeine at aa # 109. It was glutamie acid at aa # 109 in all other MDV1 strains which were not reactive with Mab T65. It seems like that there is some relationship between pathotypes and sequences of pp38 and meq, but more strains need to be compared.     

伪狂犬病病毒gE基因荧光定量PCR检测方法的建立

伪狂犬病病毒(PRV)野毒有gE基因,而其疫苗毒株无gE基因,这为检测有无PRV野毒感染提供参考。用PCR方法扩增PRV的gE基因片段,构建含有PRV gE基因片段的重组质粒。以系列稀释后的重组质粒作为模板,应用SYBR GreenⅠ来进行检测PRV gE基因的实时荧光定量PCR扩增。结果显示:在(6.9×107~6.9×101)copies/μL范围内,相邻扩增曲线之间间距均匀,所有产物具有单一整齐的熔解曲线峰,标准曲线具有优良的线性关系。该方法最低可准确检测69copies/μL的核酸模板,重复性试验的变异系数小于2%,对PRV-gE基因缺失疫苗毒株和常见猪源DNA病毒的检测结果均为阴性,对临床样品的检出率高于常规PCR方法。研究结果表明,建立了1种特异性强、灵敏度高、重复性好的检测PRV gE基因的SYBR GreenⅠ荧光定量PCR方法,可用于PRV野毒感染的定量检测。     

伪狂犬病毒Sa株gI^-／gE^-／YFP^＋基因缺失载体构建及突变株筛选

根据伪狂犬病病毒sA株的gI和gE基因序列,设计两对引物,缺失掉gE基因5’端738bp,在克隆测序的基础上,采用酶切方法构建了含部分gE基因的转移载体pBgIE,同时将pBLYFP载体上含CMV启动子、多克隆位点、黄色荧光蛋白（YFP）基因和polyA尾巴的表达盒双酶切后插入到缺失位置,构建转移载体,命名为pBgIE—YFP,为下一步开发以伪狂犬病病毒为载体的基因工程疫苗提供了基础。     

猪伪狂犬病病毒囊膜糖蛋白E基因的序列结构分析

对猪伪狂犬病毒鲁A株(PRVLA株)gE基因进行了克隆和序列测定。应用DNAStar程序分析了PRV国内外各分离株以及PRV与HSV-1、BHV-1、EHV-1、CHV-1、MDV-1、SHV-SA8、SVV、ILTV的gE基因。PRV各分离株的gE基因有很高的同源性。不同疱疹病毒的gE核苷酸和氨基酸的同源性很低,但具有相似的结构。对上述α疱疹病毒的gE氨基酸序列进化树分析可以将其分为四个特定的组:单纯疱疹病毒组,水痘疱疹病毒组,类马立克氏病病毒组,传染性喉气管炎病毒组,推测α疱疹病毒的gE基因在进化上可能起源于ILTV的gE基因的或其前体基因。而且gE基因的进化趋势与其宿主的进化情况一致。     

牛传染性鼻气管炎病毒gE基因的截短克隆与表达

以牛传染性鼻气管炎病毒Baaha Nu/67株的DNA作为模板，用PCR扩增gE基N并克隆至pGEM-T Easy裁体，再以此质粒作为模板将gE基因分成6个片段，分别插入原核表达载体pET32a并在大肠杆菌中进行了表达。蛋白电泳结果表明6个片段中有2个片段以可溶形式表达，1个片段以包涵体形式表达，另外3个片段没有表达。采用固定化金属离子亲和层析法在非变性条件下对两个可溶性片段进行了纯化。经免疫印迹试验，间接ELISA和交叉试验证明，两个纯化的重组蛋白均与牛传染性鼻气管炎阳性血清样品发生反应，而与牛传染性鼻气管炎阴性血清无任何反应，显示其具有良好的抗原性和特异性，可用于牛传染性鼻气管炎gE-ELISA诊断方法的建立。     

某规模化猪场伪狂犬病抗体检测与分析

目的:为了掌握规模化猪场伪狂犬病野毒感染情况,减少伪狂犬病所造成的经济损失,以及规模化猪场应当采取的有效防疫措施。方法:采用ELISA法对某规模化猪场7~12月份的经产母猪和后备猪进行伪狂犬病抗体检测。结果:该猪场的伪狂犬病阳性率为21.26%,是伪狂犬病野毒阳性场。结论:该猪场在检测出伪狂犬病抗体呈阳性后,采取了一系列措施,从而控制了疫情的发生。