伪狂犬病基因缺失疫苗的研究进展

文章就伪狂犬病主要的毒力基因,以及不同的基因缺失苗的特性进行了综述,并对今后的发展进行了展望。     

利用巢式PCR快速鉴定牛传染性鼻气管炎

为了快速检测牛传染性鼻气管炎,采用SDS-蛋白酶K法,提取病毒模板DNA.根据IBRV gB基因序列设计了1对特异引物,在其上下游引物的内侧又分别设计了1对引物,以这4条引物对IBRV模板进行扩增.结果成功扩增出预期目的片段,建立了巢式PCR检测方法.敏感性、特异性等检测试验结果表明该方法能特异检测IBR病毒.本方法具有快速、灵敏、特异的优点,适用于在牛及其遗传物质的进出口检疫中进行牛传染性鼻气管炎快速病原鉴定.     

利用巢式PCR快速鉴定牛传染性鼻气管炎

为了快速检测牛传染性鼻气管炎,采用SDS-蛋白酶K法,提取病毒模板DNA。根据IBRV gB基因序列设计了1对特异引物,在其上下游引物的内侧又分别设计了1对引物,以这4条引物对IBRV模板进行扩增。结果成功扩增出预期目的片段,建立了巢式PCR检测方法。敏感性、特异性等检测试验结果表明该方法能特异检测IBR病毒。本方法具有快速、灵敏、特异的优点,适用于在牛及其遗传物质的进出口检疫中进行牛传染性鼻气管炎快速病原鉴定。     

携带传染性鼻气管炎病毒牦牛肝功能指标检测

采用生物化学方法对34份携带传染性鼻气管炎病毒（IBRV）牦牛（阳性组）血清和18份健康牦牛（阴性组）血清的肝功能指标进行了测定。 结果表明: 阳性组血清的总蛋白、球蛋白、β 球蛋白显著低于阴性组（P<0.05）;而AST、ALT及AST/ALT 均高于阴性组血清,差异极显著（P<0.01）,且部分阳性牛的肝脏损害严重（11.77%）。     

Cedivac-FMD can be used according to a marker vaccine principle

In this study, we investigated whether Cedivac-FMD, an emergency vaccine against foot-and-mouth disease (FMD), is suitable for use conjointly with a screening program intended to confirm freedom from disease in vaccinated herds based on evidence of virus replication in vaccinates. Different sets of sera were tested using the Ceditest FMDV-NS ELISA for the detection of antibodies against non-structural proteins (NSPs) of FMD virus. During a vaccine safety study, serum samples were collected from 10 calves, 10 lambs and 10 piglets following administration of a double dose and a repeat dose of high payload trivalent Cedivac-FMD vaccine. All serum samples collected both 2 weeks following the administration of a double dose as well as those collected 2 weeks after the single dose booster (given 2 weeks after the double dose) were negative in the Ceditest FMDV-NS ELISA. In a series of vaccine potency experiments, serum samples were collected from 70 vaccinated cattle prior to and following exposure to infectious, homologous FMD virus. When testing cattle sera collected 4 weeks after vaccination with a regular dose of monovalent >6 PD(50) vaccines, 1 of 70 animals tested positive in the NSP antibody ELISA. After infection with FMD virus, antibodies to NSP were detected in 59 of 70 vaccinated cattle and 27 of 28 non-vaccinated control animals within 7 days. Cedivac-FMD vaccines do not induce NSP antibodies in cattle, pigs or sheep following administration of a double dose or a repeat dose. FMD-exposed animals can be detected in a vaccinated group within 7-14 days. Because Cedivac-FMD does not induce NSP antibodies, the principle of 'marker vaccine' applies.     

伪狂犬病基因缺失疫苗SA215抗强毒潜伏感染能力

通过建立能鉴别野毒和疫苗毒的多重PCR,确定试制的伪狂犬病基因缺失疫苗SA215对强毒潜伏感染的建立及随后被激活的影响。同时模拟了田间条件下对野毒潜伏感染猪接种的安全性和接种对已建立的潜伏感染的影响。结果表明,接种伪狂犬病基因缺失疫苗SA215在一定程度上能阻止强毒潜伏感染的建立和随后的激活。试制的疫苗对潜伏感染猪接种安全,对潜伏感染的消除有一定的作用,但不明显。     

表达绿色荧光蛋白的伪狂犬病毒Bartha-K61株TK-突变株的构建

提取伪狂犬病毒(PRV)Bartha—K61株基因组DNA为PCR扩增模板，并根据GenBank已发表的PRV Ka—plan株UL区UL25、UL24、UL23、UL22基因序列，选择保守序列设计了两对引物，分别扩增出位于UL23(TK)两侧(含部分TK基因)的可用于同源重组的左臂片段(L)和右臂片段(R)，L包括部分UL25、全部UL24、部分TK，R包括部分TK及部分UL22，L和R的拼接片段中TK基因内部缺失270个核苷酸。将L片段和R片段克隆于pBlueseript M13—载体上，获得pSKLR；再将绿色荧光蛋白载体pEGFP—C1上含GFP及其多克隆位点的完整的基因表达盒插人pSKLR的L片段和R片段之间构成转移载体pSKLRG。提取pSKLRG质粒，经单酶切线性化后用脂质体与PRV Bartha—K61株共转染143TK^-细胞，在细胞培养液中有5—溴脱氧尿嘧啶(BrdU)存在的条件下筛选出表达绿色荧光蛋白的重组PRV Bartha—K61毒株：PRV rBGFP。     

伪狂犬病基因缺失疫苗株(SA215)某些生物学特性研究

本试验测定了伪狂犬病ｇＥ－／ｇＩ－／ ＴＫ－／ ＬａｃＺ＋基因缺失疫苗株（ＳＡ２１５）的致细胞病变效应、安全性、免疫原性和免疫期等生物学特性。试验结果显示,该疫苗株能在Ｖｅｒｏ细胞上适应生长,并形成典型的蚀斑。其对１日龄仔猪、怀孕母猪、牛、羊以及家兔安全,无不良接种反应,接种动物不向体外散毒。ＳＡ２１５疫苗接种猪能抵御高剂量（１０７ＰＦＵ）Ｆａ株强毒感染,攻毒后试验猪的发热期、增重受阻天数、散毒滴度均低于Ｂａｒｔｈａ株疫苗接种猪,远远低于对照组猪。ＳＡ２１５接种猪能维持长时间的高水平中和抗体滴度,免疫期可达半年以上。试验结果表明,ＳＡ２１５株是一株安全、免疫原性好、免疫期长的疫苗株。     

6株牛传染性鼻气管炎病毒BICP4基因序列的同源性分析

牛传染性鼻气管炎病毒的立即早期启动基因IER4.2编码感染细胞蛋白BICP4基因。各种疱疹病毒的BICP4基因的Ⅰ，Ⅲ，Ⅴ区变异性较大，并为其特征区。为了比较分离自不同牛种，不同部位的5种病毒株BICP4蛋白I区基因的差异性，本文根据已发表的IBBV K22毒株重复序列IRs中BICP4的基因序列设计了一特异性引物，对各毒株进行PCR扩增，均得到约540bp的片段，将其克隆，测序并连同K22株进行同源性比较。结果表明，6株病毒的核苷酸序列及氨基酸序列同源性均在98%以上，说明这些毒株重复序列中的早期基因高度保守，在一定程度上也显示出IBRV的高度保守性。     

IBRV对牛单核巨噬细胞吞噬鸡红细胞功能的影响

为研究牛传染性鼻气管炎病毒（IBRV）感染对牛单核巨噬细胞吞噬功能的影响,应用淋巴细胞分离液获得牛外周血单个核细胞,通过贴壁培养成为单核巨噬细胞。 IBRV 感染单核巨噬细胞24 h后加入鸡红细胞,测定被感染细胞对鸡红细胞的吞噬率。结果显示,单核巨噬细胞被IBRV 感染后,对鸡红细胞的吞噬率显著降低（P<0.05）。     

Development of a classical swine fever subunit marker vaccine and companion diagnostic test

The development of a classical swine fever (CSF) subunit marker vaccine, based on viral envelope glycoprotein E2, and a companion diagnostic test, based on a second viral envelope glycoprotein E(RNS), will be described. Important properties of the vaccine, such as onset and duration of immunity, and prevention of horizontal and vertical transmission of virus were evaluated. A single dose of the vaccine protected pigs against clinical signs of CSF, following intranasal challenge with 100LD(50) of virulent classical swine fever virus (CSFV) at 2 weeks after vaccination. However, challenge virus transmission to unvaccinated sentinels was not always completely inhibited at this time point. From 3 weeks up to 6 months after vaccination, pigs were protected against clinical signs of CSF, and no longer transmitted challenge virus to unvaccinated sentinels. In contrast, unvaccinated control pigs died within 2 weeks after challenge. We also evaluated transmission of challenge virus in a setup enabling determination of the reproduction ratio (R value) of the virus. In such an experiment, transmission of challenge virus is determined in a fully vaccinated population at different time points after vaccination. Pigs challenged at 1 week after immunization died of CSF, whereas the vaccinated sentinels became infected, seroconverted for E(RNS) antibodies, but survived. At 2 weeks after vaccination, the challenged pigs seroconverted for E(RNS) antibodies, but none of the vaccinated sentinels did. Thus, at 1 week after vaccination, R1, and at 2 weeks, R=0, implying no control or control of an outbreak, respectively. Vertical transmission of CSFV to the immune-incompetent fetus may lead to the birth of highly viraemic, persistently infected piglets which are one of the major sources of virus spread. Protection against transplacental transmission of CSFV in vaccinated sows was, therefore, tested in once and twice vaccinated sows. Only one out of nine once-vaccinated sows transmitted challenge virus to the fetus, whereas none of the nine twice-vaccinated sows did. Finally, our data show that the E(RNS) test detects CSFV-specific antibodies in vaccinated or unvaccinated pigs as early as 14 days after infection with a virulent CSF strain. This indicates that the E2 vaccine and companion test fully comply with the marker vaccine concept. This concept implies the possibility of detecting infected animals within a vaccinated population.     

A live bovine herpesvirus-1 marker vaccine is not shed after intramuscular vaccination

It should be established, whether animals vaccinated intramuscularly (IM) with a live Bovine herpesvirus type 1 (BHV-1) marker vaccine become viremic and/or excrete vaccine virus with nasal discharge. Five cattle, seronegative for BHV-1, were vaccinated with an overdose of the vaccine (Bovilis IBR marker live) via the IM route. Nasal swabs and blood samples were taken at regular intervals and tested for BHV-1 in a virus infectivity assay. In addition, a polymerase chain reaction (PCR) specific for BHV-1 DNA was performed on the blood samples. BHV-1 neutralizing antibody titres were determined in the sera taken prior to the vaccination and four weeks after immunisation. AIl animals were successfully vaccinated as judged by the development of BHV-1 neutralising antibodies. However, all nasal swab samples were tested negative for vaccine virus, and all blood samples were found negative for BHV-1 vaccine virus and BHV-1 specific DNA. From these data it can be concluded that the vaccine virus was not excreted with nasal discharge after IM vaccination and that the vaccinated animals did not have a detectable viremia. Therefore, it is recommended to apply the tested BHV-1 marker live vaccine by the IM route in situations where it is undesirable that the vaccine virus is excreted.     

蚌埠市某奶牛场IBRV和BVDV的血清学调查

本试验对蚌埠市一家未进行牛传染性鼻气管炎及病毒性腹泻免疫的规模化奶牛场的94份奶牛血清样品,分别使用牛传染性鼻气管炎及病毒性腹泻抗体ELISA试剂盒进行检测,共检出牛传染性鼻气管炎阳性血清65份,阳性检出率为69．15％;病毒性腹泻阳性血清83份,阳性检出率为88．30％。结果表明,该规模化奶牛场存在牛传染性鼻气管炎及病毒性腹泻的感染和接触史,应采取净化措施进行控制。     

伪狂犬病病毒鄂A株TK-/gE-/gI-基因缺失疫苗的安全性和保护力研究

对PrV Ea TK^-/gE^-/gI^-基因缺失疫苗的安全性和保护力进行了系统的研究。试验表明，该基因缺失疫苗10^5.0TCID50和10^6.0TCID50病毒剂量对妊娠母猪、新生仔猪和育肥猪均是安全的，并可保护妊娠母猪抵抗10^7.1 TCID50强毒的攻击。新生仔猪免疫30d后，gE鉴刖ELISA试验表明，PrV Ea TK^-/gE^-/gI^-免疫猪不产生针对gE的抗体。育肥猪在二次免疫后中和抗体水平显著升高。以10^5.0 TCID50和10^6.0 TCID50疫苗病毒接种家兔、猫和奶山羊等非靶动物，结果非靶动物未出现精神异常或死亡现象，说明该基因缺失疫苗具有极高的生物安全性。     

猪伪狂犬病三基因缺失疫苗(SA215株)的免疫效果观察

进一步验证伪狂犬病三基因缺失疫苗SA215株通过肌肉注射接种后对妊娠母猪、育肥猪、仔猪的安全性和生产成绩。试验证实：仔猪免疫后生长良好，未观测到仔猪出现体温升高：呼吸道症状和消化道症状；母猪免疫后能提高产仔成绩（窝平均成活数高2．98头）和成活率平均为97．36％；对70日龄商品猪免疫接种后，经过近100d育肥，其出栏重增加20．83％；发生伪狂犬病的猪场猪肌注本品48～72h后能控制疾病的发生。     

不同毒力牛传染性鼻气管炎病毒感染MDBK细胞的差异蛋白组学

毒力不同的中传染性鼻气管炎病毒(IBRV)株感染宿主细胞后,会产生不同的临床免疫反应,目前关于强弱IBRV毒株感染宿主细胞后的差异蛋白质组学研究尚未报道。本研究使用基于双向电泳与MALDI-TOF/MS的蛋白质组学技术,对比分析了MDBK细胞感染IBRV强毒株LN01/08与弱毒株LNM前后蛋白质组变化,共鉴定了8个差异蛋白。结果显示,丙酮酸激酶(PKM2)、肌切蛋白(SCIN)、转化生长因子(TGFBR1)及热应激蛋白90(Hsp90)在IBRV LN01/08株与IBRV LNM株感染组中表达量发生显著差异,其中PKM2和TGFBR1在IBRV LN01/08株组中高表达,Hsp90在IBRV LNM株感染组和MDBK细胞对照组中高表达,在IBRV LN01/08感染组中无表达。     

鉴别猪伪狂犬病病毒强弱毒的高效纳米PCR 检测试剂盒及初步应用

试验建立了鉴别猪伪狂犬病病毒（PRV）强弱毒高效纳米PCR检测方法,并对相关条件进行优化,组装了试剂盒。根据PRV保守序列设计3对引物分别扩增PRV基因组的gB（431 bp）、gE（316 bp）和gG（202 bp）3个基因,用于区分PRV强毒与基因缺失毒株,优化反应条件后建立了纳米PCR检测PRV强弱毒的方法并组装试剂盒,对试剂盒进行了特异性、敏感性、批内批间可重复性及保存期评估试验,并对临床样品进行检测。特异性试验表明,此试剂盒对于PRV能够扩增出431（gB）、316（gE）和202 bp（gG）的目的片段,对于PRV-Bartha-K61能够扩增出431和202 bp的目的片段,对于猪捷申病毒、非洲猪瘟病毒、猪圆环病毒2型、猪繁殖与呼吸综合征病毒、猪细小病毒及大肠杆菌等DNA或cDNA均未扩增出条带。敏感性试验表明,此试剂盒的方法比常规PCR方法敏感100-1000倍,最低核酸拷贝数检出量可以达到101 copy/μL数量级。试剂盒的批内、批间检测结果无明显差异,稳定性良好。-20℃至少可保存12个月。在对中国黑龙江、吉林等7个省市的临床送检的117份样品进行检测,结果显示,PRV强毒阳性率为51%,阴性率为49%,未发现有弱毒感染。鉴别PRV强弱毒纳米PCR试剂盒的研制,对PRV感染的早期检测、野毒株和疫苗株的鉴别、疾病控制等都有重要意义,而且也可以用于其他动物PRV感染的检测和早期诊断。