牛传染性鼻气管炎病毒gB基因荧光定量PCR检测方法建立

为建立检测牛传染性鼻气管炎病毒(Bovine infectious rhinotracheitis virus,IBRV)gB基因的荧光定量PCR检测方法,本研究根据gB基因序列,设计1对引物和相应的TaqMan探针。建立和优化反应体系后,以10倍稀释的病毒来检测该方法的灵敏度。同时,对伪狂犬病毒(Pseudorabies virus,PRV)、马立克病病毒(Marek’s disease virus,MDV)和鸭瘟病毒(Duck plague virus,DPV)进行特异性检测。结果表明,基于gB基因的荧光PCR检测方法可用于鉴定IBRV,该方法具有较好的灵敏度和特异性,其灵敏度为10 copies/μL,即0.02 TCID_(50),且与其他病毒无交叉反应。     

IBRVgB基因TaqMan探针荧光定量PCR检测方法的建立

根据GenBank登陆的IBRV保守基因gB序列,利用分子生物学软件Primer Express3．0分别设计2对特异引物及其相应的TaqMan探针,优化反应体系后,建立牛传染性鼻气管炎病毒TaqMan探针荧光定量PCR检测方法。本方法利用10倍稀释的标准品进行扩增确定该方法的灵敏性,通过对伪狂犬病病毒（PRV）、马立克病病毒（MDV）等检测验证特异性,并进行临床检验。结果显示,建立的IBRVgB基因TaqMan探针荧光定量PCR检测方法的灵敏度为11个拷贝／μL,而且与PRV等非IBRV无交叉反应。本研究所建立的TaqMan探针荧光定量PCR检测方法具有快速、灵敏、准确、等优点,可用于IBRV的检测。     

伪狂犬病病毒野毒和疫苗毒双重RPA鉴别检测方法的建立和应用

为建立伪狂犬病病毒(PRV)野毒和疫苗毒的快速鉴别检测方法,本研究基于PRV gB和gE基因保守区域设计引物,建立了一种双重重组酶聚合酶扩增方法(RPA)。本研究所建立的双重RPA方法在38℃水浴锅中恒温反应20min,能够在同一个反应体系中实现对PRV野毒和疫苗毒的特异性鉴别检测,而与其它常见的猪病毒没有交叉反应。所建立的双重RPA方法对PRV gB和gE基因的检测限均为102拷贝,并且与荧光定量PCR方法检测限一致。通过对4株不同PRV株和37份临床样品的检测,结果表明双重RPA方法能够实现对不同PRV株的检测,对临床样品中PRV gB和gE基因的检出率均为45.9%(17/37),与荧光定量PCR方法检测的符合率为100%。本研究所建立的双重RPA方法反应快速、操作简便、结果可靠,可有效应用于对PRV野毒和疫苗毒的快速鉴别检测。     

牛传染性鼻气管炎病毒套式PCR检测方法的建立

利用套式PCR技术建立一种快速检测牛传染病鼻气管炎病毒(bovine infectious rhinotracheitis virus,IBRV)并能区分野毒株和gB基因缺失疫苗的方法。根据基因库中牛传染性鼻气管炎病毒的gB和gE基因序列,应用pri mer5.0软件设计了gB/BN和gE/EN的2套套式PCR引物,分别对IBRV标准株进行PCR扩增,结果表明,这2套引物均能扩增出与设计目的片段大小一致的特异性条带;用此引物对同属的伪狂犬病毒、马立克氏病毒、鸭瘟病毒进行扩增,具有良好的特异性。引物gB/BN和gE/EN的检测灵敏度分别为0.01TCID50和0.1TCID50。     

检测伪狂犬病病毒的SYBR Green实时荧光定量PCR方法的建立

伪狂犬病病毒（PRV）是严重影响养猪业发展的重要病毒，病毒粒子具有较强的抵抗力。SYBRGreen是结合于双链DNA的荧光染料，可在定量PCR反应时与双链PCR产物结合放出荧光信号，被仪器系统实时监控并检测。目前SYBRGreen荧光定量PCR方法在遗传性疾病的诊断等方面有重要的应用价值。本研究根据编码PRV最保守的基因之一的gB基因和PRV的主要毒力基因，即标志性疫苗缺失的gE基因核酸序列设计引物，以含有gE基因的重组质粒ppgE作外部参照，建立了gB和gESYBR Green PCR方法，研究了该方法的灵敏度、特异性以及重复性，现将结果报告如下。     

猪伪狂犬病毒野毒感染的PCR检测方法建立及应用

根据猪伪狂犬病病毒(PRV)gE、gB基因序列,设计并合成了2对特异性引物,以PRV容A株细胞培养毒为模板,通过对PCR扩增条件的优化,建立了区分PRV野毒株和疫苗毒株的双重PCR方法。该方法能从野毒株基因组中同时扩增出2条大小分别为400bp(gE基因)、582bp(gB基因)的特异性片段,从疫苗毒DNA中仅扩增出1条大小为582bp的片段。试验证明所建立的方法具有良好的特异性和敏感性,对临床上20份PRV感染疑似病料进行检测,双重PCR检测的结果与单重PCR检测结果总体符合率为100%,表明所建立的双重PCR检测方法可用于猪伪狂犬病野毒感染的快速诊断和流行病学调查。     

伪狂犬病病毒gE／gB PCR鉴别方法的建立及其应用

为了检测伪狂犬病病毒(PRV)潜伏感染及确定病毒潜伏的主要部位，建立了能鉴别PRV野毒株和gE基因缺失疫苗株gE／gB的PCR诊断方法。结果表明，所建立的PCR特异性强、敏感性高、稳定性好，能用于病毒潜伏感染的检测；同时还确定PRV潜伏感染的主要部位是三叉神经节、扁桃体、嗅球、脑干、脑桥和咽黏膜。     

应用荧光定量PCR方法检测伪狂犬病病毒弱毒株在猪体组织中的分布

为了解伪狂犬病病毒(PRV)TK/PK/gE三基因缺失弱毒株在猪体组织中的分布情况,针对PRV gB基因保守区设计并合成1对特异性引物,建立并优化了一种可快速、定量检测PRV的SYBR-GreenⅠ荧光定量PCR方法。该检测方法特异性强且敏感度高,最低检测浓度为10¹拷贝/μL。PRV TK/PK/gE三基因缺失弱毒株免疫仔猪后可少量存在于大脑、小脑、肝脏、脾脏、肺脏、扁桃体和颌下淋巴结中,部分仔猪鼻甲骨病毒含量较高。应用本研究建立的荧光定量PCR方法阳性检出率为79.2%,而常规PCR方法阳性检出率仅为45.8%。结果表明,本研究建立的荧光定量PCR方法为了解弱毒株在猪体的分布提供了快速、敏感的检测手段。PRV TK/PK/gE三基因缺失弱毒株在猪组织中的分布情况为进一步揭示其组织嗜性、安全性和免疫机制提供了数据。     

用多重PCR鉴别猪伪狂犬病野毒与疫苗毒的研究

根据基因库中的猪伪狂犬病病毒(PRV)各基因的序列，设计了与PRV的gB、gD、gE基因序列互补的3对引物。对样品中的PRVDNA模板进行了多重PCR扩增及反应条件的优化，结果同时得到与设计相符合的3条特异性条带，分别为549bp(gB)、429bp(gD)、366bp(gE)。用这3对引物对三基因缺失疫苗毒的样品DNA模板进行多次多重PCR扩增，均能稳定得到与设计相符合的2条特异性条带。敏感性试验结果表明，多重PCR可以检测到106Pg三基因缺失疫苗毒或756PgPRV野毒的核酸模板量。特异性试验结果表明，以正常对照细胞及猪圆环病毒和猪细小病毒DNA为模板进行多重PCR扩增，均无任何条带。     

广东某猪场伪狂犬病病毒GD2013株的分离鉴定

本研究于2013年3月在广东某猪场采集疑似伪狂犬病(PR)的猪脑组织。对脑组织进行病毒分离,通过透射电镜观察及PCR扩增gB和gE基因,并进行序列测定。结果表明,透射电镜显示该病毒粒子具有典型的疱疹病毒结构,PCR可以扩增出伪狂犬病病毒(PRV)gB和gE基因,经序列分析和生物学特性鉴定,证实该分离病毒株为我国近几年流行的PRV变异株,并将其命名为PRV-GD2013。小鼠致病性试验表现出典型的PR症状。通过以上实验可以确定该猪场感染病原为PRV。     

Development of real-time polymerase chain reaction assays for rapid detection and differentiation of wild-type pseudorabies and gene-deleted vaccine viruses

The successful eradication of pseudorabies in U.S. domestic swine was accomplished through the use of glycoprotein E (gE) deleted modified live virus vaccines and an accompanying gE differential enzyme-linked immunosorbent assay (ELISA). Yet, pseudorabies virus (PRV) was established in feral swine in the United States, becoming a potential reservoir of PRV for infection of domestic swine and other native wildlife. A critical need for the current PRV surveillance program in the United States is the rapid detection of PRV infection. For this reason, a set of 2 real-time polymerase chain reaction (PCR) assays by using TaqMan chemistry was developed and evaluated for their capability in the detection and differentiation of field and vaccine strains of PRV. PCR primers and probes were designed for gB and gE genes of PRV, respectively. The newly developed PRV-specific real-time PCR assays could detect all wild-type PRV isolates from diagnostic submissions and differentiate them from vaccine strains. The analytical sensitivity of the assays was approximately 0.1 plaque-forming units per reaction. The assays were highly specific for PRV, because no positive results were obtained from testing other common swine viral pathogens and other animal herpesviruses. The results of testing samples from domestic and feral swine and from bovine showed that the real-time PCR assays are more sensitive than gel-based PCR. These results demonstrated the potential application of the developed real-time PCR assays as a differential test for rapid and specific detection of PRV in domestic and feral swine, as well as nonporcine species that can be infected with PRV and serve as carriers.     

猪伪狂犬病病毒灭活疫苗对猪伪狂犬病病毒流行毒株和经典毒株的免疫保护效果评估

为评估猪伪狂犬病病毒（Pseudorabies virus,PRV）灭活疫苗（HN1201-ΔgE株）免疫后对PRV流行毒株和经典毒株的保护效果,本研究对试验猪分别免疫PRV灭活疫苗（HN1201-ΔgE株）和PRV活疫苗（Bartha-K61）,免疫后第0、7、10、14、17、21、24和28天采血测定PRV gB抗体,并分别使用PRV流行毒株HN1201株和经典毒株闽A株测定免疫后第0、7、14、21和28天血清的中和抗体水平,于免疫后第28天分别使用HN1201株和闽A株攻毒并观察,之后测定体温,测定攻毒后第7和14天PRV gE抗体,及攻毒后0~8 d的排毒情况。结果显示,HN1201-ΔgE免疫组较Bartha-K61免疫组gB抗体和中和抗体产生早,且抗体水平较高。两个免疫组试验猪在攻毒后虽然均无明显临床症状,且免疫组织化学检测（IHC）组织中的病毒抗原均为阴性,但HN1201-ΔgE免疫组试验猪脏器未见任何病理损伤,Bartha-K61免疫组试验猪部分脏器具有病理损伤。与未免疫对照组相比,2个免疫组试验猪在HN1201株和闽A株攻毒后,gE抗体转阳时间晚且排毒率低,HN1201-ΔgE免疫组gE抗体水平整体均低于Bartha-K61免疫组,攻毒后排毒检测中,Bartha-K61免疫组于2个毒株攻毒后第3~5天可检测到排毒,而HN1201-ΔgE免疫组全程未检测到排毒。研究结果表明,灭活疫苗（HN1201-ΔgE株）对PRV流行毒株和经典毒株均可提供完全保护。     

伪狂犬病病毒gB和gE蛋白重组表达及抗体间接ELISA检测方法的建立

本研究利用PCR扩增伪狂犬病病毒(PRV)Bartha-K61株gB基因核心抗原区和闵A株gE基因核心抗原区,构建了重组质粒pET-32a-gB和pET-32a-gE,并转化至大肠杆菌BL21(DE3)感受态细胞中,经IPTG诱导进行表达.SDS-PAGE分析表明,pET32a-gB蛋白分子量约为44 kDa,pET3...     

Protection and synergism by recombinant fowl pox vaccines expressing multiple genes from Marek's disease virus

Recombinant fowl poxviruses (rFPVs) were constructed to express genes from serotype 1 Marek's disease virus (MDV) coding for glycoproteins B, E, I, H, and UL32 (gB1, gE, gI, gH, and UL32). An additional rFPV was constructed to contain four MDV genes (gB1, gE, gI, and UL32). These rFPVs were evaluated for their ability to protect maternal antibody-positive chickens against challenge with highly virulent MDV isolates. The protection induced by a single rFPV/gB1 (42%) confirmed our previous finding. The protection induced by rFPV/gI (43%), rFPV/gB1UL32 (46%), rFPV/gB1gEgI (72%), and rFPV/gB1gEgIUL32 (70%) contributed to additional knowledge on MDV genes involved in protective immunity. In contrast, the rFPV containing gE, gH, or UL32 did not induce significant protection compared with turkey herpesvirus (HVT). Levels of protection by rFPV/gB1 and rFPV/gl were comparable with that of HVT. Only gB1 and gI conferred synergism in rFPV containing these two genes. Protection by both rFPV/gB1gEgI (72%) and rFPV/gB1gEgIUL32(70%) against Marek's disease was significantly enhanced compared with a single gB1 or gI gene (40%). This protective synergism between gB1 and gI in rFPVs may be the basis for better protection when bivalent vaccines between serotypes 2 and 3 were used. When rFPV/gB1gIgEUL32 + HVT were used as vaccine against Md5 challenge, the protection was significantly enhanced (94%). This synergism between rFPV/gB1gIgEUL32 and HVT indicates additional genes yet to be discovered in HVT may be responsible for the enhancement.     

应用复合多聚酶链反应方法快速鉴别伪狂犬病弱毒疫苗与野毒

本研究根据伪狂犬病病毒（ＰｒＶ）共有ｇＢ和疫苗株缺失的ｇＥ基因序列分别设计合成１对通用（ＰＢ１／ＰＢ２）和１对鉴别引物（ＰＥ１／ＰＥ２）,以在我国广泛使用的疫苗株Ｂａｒｔｈａ－Ｋ６１及从国内外收集的野毒株Ｓ、ＳＵ、Ｆ、Ｌ、Ｙ、Ｍｉｎ－Ａ、Ｓｈｏｐｅ、Ｓ（川）、ＳＬ１、１０＃、ＥＡ（鄂Ａ）ＤＮＡ为模板在同一反应管中同时扩增ｇＢ和ｇＥ基因序列建立了复合多聚酶链反应（ＰＣＲ）方法。ＰＣＲ产物经２．０％琼     

伪狂犬病病毒FS-2015株gE和gB基因序列分析

为了解猪伪狂犬病病毒（porcine pesudorabies virus,PRV）gB和gE基因变异及遗传演化情况,本研究针对PRV FS-2015野毒株,应用"蚀斑法"对组织病料中病毒进行三轮纯化,应用全长扩增引物对FS-2015株gB和gE基因进行全基因扩增,并对PCR产物进行测序和序列分析。结果显示,PRV FS-2015株的gB、gE基因与国内外PRV参考毒株的核苷酸同源性分别为97.0%~100.0%和97.5%~99.7%,氨基酸同源性分别为96.4%~100.0%和95.3%~99.7%。氨基酸变异位点分析表明,FS-2015株的gB和gE基因均有位点突变和缺失。遗传进化分析表明,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,亲缘关系较近;与PRV经典株Kaplan、Becker、NIA3、Kolchis、Bartha和Yangsan株属于不同分支,同源性较低,亲缘关系较远。从PRV FS-2015毒株与国内外经典毒株和当前国内流行的变异毒株的分析结果可知,PRV FS-2015毒株发生了一定的变异,属于当前国内流行变异毒株。本研究结果为广东省伪狂犬病分子流行病学调查、伪狂犬病的防控和疫苗株挑选工作提供参考数据。     

Identification of Pseudorabies Virus Variant and Control of Pseudorabies

Due to variant strain,pseudorabies virus (PRV) has broken out again and spread in China since 2011.A swine farm in Guangdong province was found pseudorabies (PR) symptoms-like miscarriage after introduction.The study was carried out to identify and control the PR.Serum of sows with and without miscarriage were randomly collected and the PRV gB and gE were detected by ELISA method,and brain tissues of sick piglets were sampled and the PRV gH gene was tested by PCR.All the sows in the farm were emergently inoculated PRV variant strainin activated vaccine.Serum before and after immunization were collected and detected by ELISA and micro-serum neutralization test.ELISA results showed that gE antibody of all the breeding sows with miscarriage were positive,and that of sows without miscarriage showed weekly positive;The average gB ELISA S/P value of sows with miscarriage was as high as 4.0,while that of sows without miscarriage was over 3.0.PCR of 3 sick piglets were all positive and the sequence of gB gene was 100% identical to BJ-YT-2012,a wide variant stain in 2012. The result of detection of the sows serum at before and after immunization showed that the S/P value of gB rose up from 1.603 before immunization to 2.88 at four weeks after immunization,and the neutralizing antibody rose up from 1:24 to 1:213.This agreed with the results that the sows showed less probability of miscarriage since the first week after immunization and almost no miscarriage after two weeks after immunization.This study suggested that classical PRV vaccine was not effective in this case,while the vaccine made from the variant PRV strain was.     

牛疱疹病毒Ⅰ型gB、gE基因的克隆及其杆状病毒表达载体的构建

本试验用PCR方法扩增了牛疱疹病毒Ⅰ型(bovine herpesvirus-1,BHV-1)Bartha Nu/67株gB、gE基因片段,将其克隆到pGEM-T-easy载体。经转化、筛选、鉴定后将重组质粒经BamHⅠ和EcoRⅠ双酶切后,与经相同方法处理的杆状病毒转移载体pFastBacHTb连接,得到了重组质粒pFBHgB、pFBHgE。经酶切和测序鉴定后,将其转化入含穿梭载体Bacmid的感受态细胞DH10Bac,经抗性、蓝白斑筛选和PCR鉴定,得到了含gB、gE基因的重组穿梭载体。     

伪狂犬病病毒变异株的鉴定及伪狂犬病的防控

2011年以来伪狂犬病病毒（PRV）变异株在中国大范围流行致伪狂犬病（PR）再次暴发。广东某猪场发生疑似PR引起母猪较大范围的流产,为此本试验展开对该病诊断和防控方法的研究。随机抽取流产和未流产母猪血清,应用ELISA检测PRV gE和gB抗体;同时采集发病仔猪脑组织PCR检测PRV gH片段。对全场母猪紧急接种PRV变异株灭活苗,分别应用ELISA和中和试验检测免疫前后的血清抗体。结果显示,已发生流产母猪血清PR gE抗体均为阳性,而未流产母猪血清抗体见弱阳性;流产母猪PRV gB抗体的S/P值高达4.0,未流产母猪也达3.3。PCR检测3头病仔的脑组织均为阳性,测序表明其gB基因与2012年流行毒株BJ-YT-2012序列相似性为100%。ELISA检测免疫灭活疫苗前母猪血清PRV gB抗体S/P值为1.603,免疫4周后升高到2.88;特别是中和抗体从1：24升高到1：213。这与免疫疫苗1周后母猪流产开始减少,2周后母猪少见流产的结果吻合。研究结果提示,PRV经典株疫苗产生的PRV gB抗体对变异株的保护作用不佳,而变异株疫苗的保护效果显著。     

Isolation,Identification and Phylogenetic Analysis of Important Functional Genes of Porcine Pseudorabies Virus LC Strain

To find out the reason of the reproductive failure in pregnant sows in a hoggery in Guangdong, the mixture with brain, lymph nodes and lungs of farm abortion stillbirth were identified by PCR, virus isolation and culture, tissue culture infective dose (TCID₅₀) assay, homology and phylogenetic analysis of the important functional genes (gB, gC, gD and gE) and animal test. The results showed that the mixture was proved to be porcine pseudorabies virus (PRV) positive samples. The typical cytopathogenic effect was induced in the third passage of Vero cell and the titer of the fifth passage was 10^-6.8/0.1 mL. The sequence analysis and phylogenetic relationship of gB, gC, gD and gE genes showed that it was a Chinese PRV variant, which was named as LC strain. The typical pseudorabies clinical and pathological symptoms were presented in 12-week-old piglets inoculated with LC strain. The results demonstrated that a local pseudorabies virus had been isolated, suggesting that the Bartha-K61 vaccine was not fully effective for controlling the current epidemic of pseudorabies in China.