伪狂犬病病毒广西B、W株gE基因的扩增、克隆及序列分析

在已鉴定了的伪狂犬病病毒（Pseudorabies Virus，PRV）11种糖蛋白中，gE是其中十分重要的一种非必需糖蛋白，是伪狂犬病病毒重要的毒力基因，它在介导感染细胞的融合，病毒在细胞间的扩散，病毒粒子的释放等方面均起着十分重要的作用，尤其是在影响伪狂犬病病毒神经嗜性和毒力方面的作用。利用PRV gE基因缺失疫苗，结合血清学方法可以区分疫苗接种猪和野毒感染猪。     

伪狂犬病病毒Bartha株gC膜外区部分基因的原核表达及纯化

构建伪狂犬病病毒(PRV)gC膜外区部分基因的重组质粒,原核表达并纯化目的蛋白.根据GenBank已发表的伪狂犬病病毒Bartha(PRV Ba)株gC基因的序列(NC EU719641),设计并合成了1对引物,以PRV Ba株为模板,PCR扩增出PRV gC膜外区部分基因片段;将该基因片段克隆到原核表达载体pET-28a上,转化大肠埃希菌BL21(DE3),经IPTG诱导,获得大小为35 ku的重组蛋白,命名为pET-gCN813.按照His-Bind纯化试剂盒说明书纯化表达产物,获得融合蛋白的纯化产物.     

抗伪狂犬病病毒gC抗体制备及其gC蛋白表达

用纯化的重组蛋白His-gCN813制备抗PRV-gC抗体,并分析伪狂犬病病毒（pseudorabies virus,PRV）gC蛋白在真核细胞的表达情况。本研究以提取的病毒基因组为模板,PCR克隆PRV gC全长基因,构建gC真核表达质粒p3xFLAG-gC,在真核细胞中表达gC基因;纯化蛋白His-gCN813制备的抗体,Western blotting和间接免疫荧光（IFA）检测到p3xFLAG-gC转染真核细胞和PRV感染细胞中的gC蛋白。结果表明本试验成功构建了PRV gC基因真核表达系统,获得了特异性抗PRV gC抗体,重组gC真核表达质粒p3xFLAG-gC转染Vero细胞,表达的重组蛋白gC为72 ku,PRV感染Vero细胞,表达的gC蛋白为55、72和94 ku,主要定位在细胞浆,gC蛋白可以作为PRV感染的指示分子,为下一步研究PRV和宿主相互作用及PRV的复制奠定了基础。     

猪伪狂犬病的诊断与控制

正1伪狂犬病的病原学及流行病学(1)伪狂犬病的病原学。伪狂犬病又称Aujeszky氏病,是一种急性高度接触性传染病。目前,PR已经被我国列为2类传染病。PR的致病病毒为猪疱疹病毒I型伪狂犬病病毒(PRV),该病毒属于疱疹病毒,是一个封闭的核衣壳,属于双股DNA病毒,有囊膜。PRV感染的发生与该病毒的囊膜有着直接的关系,而     

一株猪伪狂犬病病毒变异株的分离鉴定

为了解广东省猪伪狂犬病病毒（pseudorabies virus，PRV）野毒株的基因变异及遗传演化的情况，本试验对广东佛山疑似暴发伪狂犬病的猪场采集的病料（脑、肺脏、扁桃体、肝脏、脾脏）进行了PCR鉴定，初步鉴定为PRV毒株后，将阳性病料接种非洲绿猴肾细胞（Vero），进行毒株传代培养，对分离毒株进行PCR检测及小鼠感染试验，证实该病毒为PRV，并命名为PRV FS-2015株；并对该毒株进行细胞病变观察、病毒TCID₅₀测定、毒株gC和TK基因扩增及序列分析。结果显示，PRV FS-2015株TCID₅₀为10^-7.5/0.1 mL。PRV FS-2015株的gC和TK基因序列与国内外PRV参考毒株进行同源性比对分析发现，其核苷酸序列同源性分别为95.8%~100.0%和99.4%~100.0%，氨基酸同源性分别为92.3%~100.0%和98.7%~100.0%。遗传进化分析表明，PRV FS-2015株与国内近几年分离的PRV变异株GY、ZJ01、HB1201、HN1201、JS2012、BJ/YT和BP属于同一分支，同源性较高，亲缘关系更近；但与PRV经典株Kaplan、Becker、NIA3、Kolchis、Bartha、Yangsan、Min-A、SS和SL株的同源性较低，基因变异较大，表明PRV FS-2015毒株属于近几年流行的变异株。本研究结果可为广东省伪狂犬病的防控工作和疫苗株的选择提供科学依据。     

2018年伪狂犬病病毒的流行特征及其遗传变异分析

为了了解当前伪狂犬病病毒（PRV）在我国猪群中的流行现状及其遗传变异情况,本研究利用PCR的方法对2018年1-12月来源于我国28个省、市、自治区的1 328份疑似猪伪狂犬病发病猪的组织样品开展了PRV-gE的检测及病毒的分离鉴定,同时针对所分离的病毒的gB、gC和gE基因进行PCR扩增和DNA测序,并展开遗传变异分析。结果显示,共有92份样品为PRV-gE基因检测阳性,阳性检出率为6.93%。从92份PRV-gE阳性样品分离到13株PRV。分别基于gB基因部分片段、gC和gE基因进行遗传变异分析发现13株PRV与我国2012年以后流行的毒株（如HeN1等变异株）亲缘关系较近,而与2012年以前所分离的毒株（如Ea等经典毒株）的亲缘关系较远;此外,绝大多数中国分离株与国外分离毒株（如NIA-3、Bartha、Kaplan等）在遗传进化树中位于不同的进化分支;相对于国外分离株及国内早期流行的经典毒株而言,13株PRV分离株的gB、gC和gE蛋白中均存在许多特征性的氨基酸位点变异。本研究对于了解我国PRV流行现状及当前流行的PRV毒株的生物学特征具有十分重要的意义。     

猪源伪狂犬病病毒AH02LA株糖蛋白的基因序列测定与对比分析

为了分析猪源伪狂犬病病毒AH02LA株糖蛋白的基因序列特征,本研究设计了17对特异性引物,通过PCR扩增AH02LA株的11个糖蛋白基因并进行序列测定,然后将其与PRV变异株(TJ株与HeN1株)和PRV经典株(Bartha株与Kaplan株)作比对分析。结果:成功扩增了11个糖蛋白基因序列,序列比对发现,AH02LA株的gB、gD、gG、gK和gM基因与PRV变异株的同源性为100%,而与PRV经典株的同源性为98%~99%;AH02LA株的gC、gE和gI基因与PRV变异株的同源性为99%,而与经典株的同源性为95%~97%,而且AH02LA株的gB、gC、gD、gE、gI和gN基因的插入或缺失也与变异株完全一致。研究表明,AH02LA株的主要糖蛋白基因序列与变异株高度同源,该毒株是一株典型的变异株。     

猪伪狂犬病病毒LC株的分离鉴定及其重要功能基因的进化分析

为确诊广东某猪场母猪流产发病原因,本研究收集该猪场流产死胎的脑、淋巴结、肺脏混合液,进行PCR鉴定、病毒分离培养、半数组织培养感染剂量（tissue culture infective dose,TCID₅₀）测定、猪伪狂犬病病毒（pseudorabies virus,PRV）重要功能基因（gB、gC、gD、gE）序列测定和进化分析及动物回归试验。结果显示,病料混合液为PRV阳性,接种Vero细胞传至第3代即出现稳定的细胞病变（CPE）,第5代TCID₅₀达到10^-6.8/0.1 mL,PRV gB、gC、gD、gE基因序列测定、同源性及进化树分析显示为,PRV中国变异株,命名为LC株。动物试验显示,LC株对12周龄猪具有一定致病性,可形成PRV典型临床症状及病理变化。本研究分离到一株PRV流行毒株,推测当前使用疫苗Bartha-K61株尚无法完全控制新毒株的流行。     

山东省部分地区猪伪狂犬病病毒分离株gC抗原基因的变异分析

为了解当前山东省猪伪狂犬病病毒(PRV)流行特点及病毒gC抗原基因的变异情况,本研究对2018年来自山东省不同地区的23份猪伪狂犬病疑似病料进行PRV的分离鉴定,并对分离株的gC抗原基因进行扩增及序列分析。结果表明,共分离到9株PRV;gC基因同源性比对结果显示,分离株与2012年之后国内PRV变异株的核苷酸及氨基酸序列的同源性均高于欧美毒株;遗传进化树及推导氨基酸序列分析结果显示,分离株与国内2012年后分离到的变异毒株同属于GenotypeⅡ;因此,推测目前山东省流行的PRV以变异毒株为主。9株分离株与Bartha-K61疫苗株相比,糖基化蛋白gC在潜在抗原位点和O-糖基化位点的重要位置(aa23～aa453)发生改变,这或许会影响分离株的抗原性,从而有助于其逃避宿主免疫防御,最终造成疫苗诱导产生的中和抗体不能有效地中和目前流行的变异PRV。本研究为变异PRV的防控及相关疫苗研制提供了一定的理论依据。     

2015－2020年天津地区猪伪狂犬病病毒gB、gC、gE基因遗传变异分析

为掌握天津地区猪伪狂犬病病毒（Pseudorabies virus,PRV）的流行及遗传变异情况,本研究对2015－2020年天津地区分离的20个分离株的gB、gC和gE基因进行扩增、测序,并与参考毒株序列进行比对分析。相似性分析结果显示,分离株与2012年后中国变异株相比,3种基因核苷酸及编码氨基酸序列相似性分别为：gB基因均为99.9%~100%;gC基因为99.7%~100%和99.4%~100%;gE基因为99.7%~100%和99.6%~100%。遗传进化和序列比对分析结果显示,依据gB、gC、gE基因绘制遗传进化树均可将PRV毒株分为GⅠ型和GⅡ型,天津分离株属于GⅡ型;其中19个分离株与PRV变异株遗传关系较近,属于同一亚分支,并存在相同的氨基酸变异位点;另外1个分离株（TJBD6株）gB和gE基因与PRV变异株遗传关系较近,氨基酸变异位置与PRV变异株一致,但其gC基因与经典株Ea株遗传关系较近,且核苷酸和氨基酸序列相似性为100%。上述结果表明,2015年以来天津地区流行的PRV毒株存在经典株和变异株2种类型,其中变异株为主要流行株。本次研究初步调查了天津地区PRV分子流行特征,可为猪伪狂犬病防控提供依据。     

Efficacy of a killed gpX deleted pseudorabies virus vaccine.

Ten inactivated vaccines containing one of four adjuvants and varying concentrations of pseudorabies virus (PRV) antigens were compared in order to select a vaccine suitable for commercial production. A genetically engineered strain of PRV lacking the gene coding for glycoprotein X (gpX) was used in these vaccines. Vaccinated pigs were challenged intranasally with virulent PRV to determine the efficacy of vaccines. Vaccination of pigs with one dose of experimental vaccines adjuvanted with 50% Montanide ISA 50 or 20% Syntrogen induced a protective immunity at least equal to that induced by two commercially available killed PRV vaccines also evaluated. An experimental vaccine containing 20% Syntrogen was selected and further evaluated according to United States Department of Agriculture licensing requirements. None of the pigs vaccinated with this vaccine produced gpX antibodies detectable by the HerdChek: Anti-PRV-gpX assay. Therefore, this assay could differentiate PRV vaccine induced antibodies from antibodies induced by natural exposure when used in conjunction with this killed gpX deleted PRV vaccine.     

The detection of antibodies to the glycoprotein X antigen of pseudorabies virus

The persistence of antibodies to glycoprotein X (gpX) in the serum of pigs experimentally infected with pseudorabies virus (PRV) was determined using an anti-gpX enzyme-linked immunosorbent assay (ELISA). Antibodies to gpX were detected for at least 365 days postchallenge in nonvaccinated pigs. Previous sensitization of pigs by vaccination with S/PRV had no apparent effect on the antibody response of pigs to gpX postchallenge. In determining previous exposure of pigs to PRV strains containing the gpX gene, the anti-gpX ELISA was highly specific, but its sensitivity was lower than the standard serological procedures currently used for detecting PRV antibodies.     

猪伪狂犬病病毒JSSQ2013株的分离鉴定及重要功能基因序列分析

为了解江苏省猪伪狂犬病病毒(Pseudorabies virus,PRV)野毒株的特点,本研究从2013年采自江苏省宿迁市的疑似PRV感染病料中分离纯化了一株PRV病毒,对其进行了PCR和间接免疫荧光法(IFA)鉴定,并进一步在Vero细胞上测定该分离株的病毒滴度TCID50和一步生长曲线,扩增其gB、gC、gD和gE基因进行序列比对及分子遗传进化分析,并将该分离株分别接种新西兰白兔和15日龄仔猪研究其致病性。结果显示,该病毒为一株PRV,命名为PRV JSSQ2013株,纯化后的病毒滴度为10^7.8 TCID50/ml;生长曲线测定显示在感染20h后病毒滴度即达到最高,为10^8.6 TCID50/ml。与我国近几年分离的PRV变异株序列相比,PRV JSSQ2013株的gB、gC、gD和gE基因核苷酸序列同源性分别为99.5~99.6%、99.5~99.6%、99.5~99.6%和98.7~99.7%,氨基酸序列同源性分别为98.9~99.0%、99.5~99.7%、99.0~99.2%和98.1~99.3%,均高于其与经典毒株(Ea、Fa和SC株)和欧美毒株(Becker、Kaplan、Bartha、Kolchis和NIA3)的同源性;基于gB、gC、gD和gE基因的遗传进化树分析均显示PRV JSSQ2013株与国内近几年分离的PRV变异株属同一分支。该病毒接种新西兰白兔后均出现典型的PR症状,如厌食、兴奋、啃咬或用爪挠接种部位等典型症状,且在48h内全部死亡;接种仔猪后第1天开始出现典型的PR症状,第5天全部死亡。以上结果证实,从江苏省宿迁市采集的疑似PRV感染病料中分离到一株强毒力的PRV变异株。本研究为了解江苏PRV分子流行特征、丰富我国PRV分子流行病学资料及新型疫苗的研制奠定了基础。     

A DNA vaccine coding for glycoprotein B of pseudorabies virus induces cell-mediated immunity in pigs and reduces virus excretion early after infection

Glycoproteins B (gB), gC and gD of pseudorabies virus (PRV) have been implicated as important antigens in protective immunity against PRV infection. As cell-mediated immunity plays a major role in this protective immunity, we determined the significance of these glycoproteins in the actual induction of cell-mediated immunity. We vaccinated pigs with plasmid DNA constructs coding for gB, gC or gD and challenged them with the virulent NIA-3 strain of pseudorabies virus. Vaccination with plasmid DNA coding for gB induced the strongest cell-mediated immune responses including cytotoxic T cell responses, whereas plasmid DNA coding for gD induced the strongest virus neutralising antibody responses. Interestingly, vaccination with gB-DNA reduced virus excretion early after challenge infection while vaccination with gC-DNA or gD-DNA did not.This is the first study to demonstrate that DNA vaccination induces cytotoxic T cell responses in pigs and that cell-mediated immunity induced by vaccination with gB-DNA is important for the reduction of virus excretion early after challenge infection.     

猪伪狂犬病病毒gE蛋白在野毒诊断中的应用进展

With mass immunization application of pseudorabies virus （PRV） gE gene deleted attenuated vaccine in China, it played an important role in prevention and control of porcine pseudorabies, meanwhile it also needed to establish a differential diagnosis technology for gE gene deleted vaccine matching to eliminate virus affected swine and reduce immune pigs to be killed, and gradually realized the purification of PRV. With the gradual progress of molecular biology and immunology, molecular biological and serological diagnosis methods based on PRV gE protein continuously developed and improved. In this paper, recent study on various molecular and serological diagnosis methods of gE protein based on the current situation and development prospect were reviewed, in order to provide reasonable scientific basis for diagnosis and prevention of PRV in China.     

PCR技术检测猪伪狂犬病毒及其潜伏感染部位的研究

本研究合成了伪狂犬毒糖蛋白ｇｐ５０基因引笺，该引物能够扩增糖蛋白ｇｐ５０基因中４３４－６５１之间的２１７ｂｐＤＮＡ片段，该片段含 ＳａｌＩ酶切位点，应用引物对几种不同的伪狂犬病毒株多聚酶链式反应扩增结果全为阳性。     

Detection of pseudorabies virus infection in subunit-vaccinated and nonvaccinated pigs using a nucleocapsid-based enzyme-linked immunosorbent assay.

The potential of a pseudorabies virus (PRV) nucleocapsid protein (NC)-based enzyme-linked immunosorbent assay (ELISA) as a screening assay for PRV infection in subunit-vaccinated and nonvaccinated pigs was studied. The NC-ELISA compared favorably to a commercial ELISA for detecting PRV infection in nonvaccinated pigs. Virus-specific antibody was first detected by the NC-ELISA between days 14 and 21 in 5 pigs challenged intranasally with 10(4) PFU of virus. Antibody continued to be detected in these pigs through day 42, when the experiment was terminated. The NC-ELISA also detected antibody in 23 of 24 pigs from PRV-infected herds. In contrast, the commercial ELISA detected antibody 1 week earlier than the NC-ELISA in experimentally infected pigs but failed to detect antibody in 3 naturally exposed pigs that were identified by the NC-ELISA. Infection in these animals was confirmed by radioimmunoprecipitation analysis. The potential usefulness of the NC-ELISA for detecting infection in vaccinated pigs was also evaluated. The nucleocapsid-specific antibody responses of 10 PRV envelope glycoprotein subunit-vaccinated pigs were monitored prior to and following nasal exposure to a low dose (10(2.3) PFU) of PRV. Sera were collected periodically for 113 days after infection. Nucleocapsid-specific antibody responses measured by the NC-ELISA remained below the positive threshold before challenge but increased dramatically following virus exposure. Maximum ELISA responses were obtained on day 32 postchallenge (p.c.). Mean ELISA responses decreased thereafter but remained well above the positive threshold on day 113 p.c. PRV nucleocapsid protein can be used effectively as antigen in the ELISA for detecting PRV infection in both nonvaccinated and subunit-vaccinated pigs.     

Vaccine genotype and route of administration affect pseudorabies field virus latency load after challenge

The influence of vaccine genotype and route of administration on the efficacy of pseudorabies virus (PRV) vaccines against virulent PRV challenge was evaluated in a controlled experiment using five genotypically distinct modified live vaccines (MLVs) for PRV. Several of these MLVs share deletions in specific genes, however, each has its deletion in a different locus within that gene. Pigs were vaccinated with each vaccine, either via the intramuscular or intranasal route, and subsequently challenged with a highly virulent PRV field strain. During a 2-week period following challenge with virulent PRV, each of the vaccine strains used in this study was evaluated for its effectiveness in the reduction of clinical signs, prevention of growth retardation and virulent virus shedding. One month after challenge, tissues were collected and analyzed for virulent PRV latency load by a recently developed method for the electrochemiluminescent quantitation of latent herpesvirus DNA in animal tissues after PCR amplification. It was determined that all vaccination protocols provided protection against clinical signs resulting from field virus challenge and reduced both field virus shedding and latency load after field virus challenge. Our results indicated that vaccine efficacy was significantly influenced by the modified live vaccine strain and route of administration. Compared to unvaccinated pigs, vaccination reduced field virus latency load in trigeminal ganglia, but significant differences were found between vaccines and routes of administration. We conclude that vaccine genotype plays a role in the effectiveness of PRV MLVs.     

Potency of an experimental DNA vaccine against Aujeszky's disease in pigs

Intradermal vaccination with plasmid DNA encoding envelope glycoprotein C (gC) of pseudorabies virus (PrV) conferred protection of pigs against Aujeszky's disease when challenged with strain 75V19, but proved to be inadequate for protection against the highly virulent strain NIA-3. To improve the performance of the DNA vaccine, animals were vaccinated intradermally with a combination of plasmids expressing PrV glycoproteins gB, gC, gD, or gE under control of the major immediate-early promotor/enhancer of human cytomegalovirus. 12.5 microg per plasmid were used per immunization of 5-week old piglets which were injected three times at biweekly intervals. Five out of six animals survived a lethal challenge with strain NIA-3 without exhibiting central nervous signs, whereas all the control animals succumbed to the disease. This result shows the increased protection afforded by administration of the plasmid mixture over vaccination with a gC expressing plasmid alone. A comparative trial was performed using commercially available inactivated and modified-live vaccines and a mixture of plasmids expressing gB, gC, and gD. gE was omitted to conform with current eradication strategies based on gE-deleted vaccines. All six animals vaccinated with the live vaccine survived the lethal NIA-3 challenge without showing severe clinical signs. In contrast, five of six animals immunized with the inactivated vaccine died, as did two non-vaccinated controls. In this test, three of six animals vaccinated with the DNA vaccine survived without severe clinical signs, whereas three succumbed to the disease. Comparing weight reduction and virus excretion, the DNA vaccine also ranged between the inactivated and modified-live vaccines. Thus, administration of DNA constructs expressing different PrV glycoproteins was superior to an adjuvanted inactivated vaccine but less effective than an attenuated live vaccine in protection of pigs against PrV infection. Our data suggest a potential use of DNA vaccination in circumstances which do not allow administration of live attenuated vaccines.     

安徽部分地区猪伪狂犬病PCR诊断及流行病学调查

针对安徽省部分地区猪伪狂犬病发病率较高,给全省的养猪业造成较大危害的状况,应用聚合酶链反应(PCR)对安徽省7个地区21个养猪场的187头疑似发病猪的血清进行伪狂犬病病毒(PRV)的检测,并对阳性猪的流行病学分布情况进行统计分析。结果显示,安徽省猪PRV平均阳性率为34.2%;其中安徽省南部地区PRV阳性率最高,北部地区最低,规模养殖场PRV阳性率明显低于散养户,其结果为安徽省猪伪狂犬病流行病学调查及防控提供参考资料。