【作者】 朱来华；

【导师】 陆承平；

【作者基本信息】 南京农业大学 ， 预防兽医学， 2007， 博士

【摘要】 1．在分析EHV-1、EHV-2和EHV₄等3种马疱疹病毒基因序列的基础上，设计、合成了3对针对各自糖蛋白B基因编码区的型特异性引物进行多重PCR，不仅可以在数小时内分别检测这3种马疱疹病毒，而且在同一反应系统内可以清晰地区分EHV-1、EHV-2和EHV-4，其PCR产物大小分别为226bp、333bp、570bp，符合预期的片段大小，序列分析证实与文献发表的序列一致，但不能扩增其他动物的疱疹病毒（BHV-1、PHV-1、GHV-1、AHV-1）的DNA，说明本方法特异性强。这种检测方法的灵敏度高，检测限可以达到10³TCID₅₀，并分别从血清学阳性但病毒分离阴性的1匹进口马组织样品和一些出口前检疫马的鼻咽样品检测到EHV-1和EHV-4病毒特异性核酸。2．为进一步提高检测技术的速度和灵敏度，应用一种新型荧光染料——Eva Green在荧光PCR中，通过设计特异性的引物来检测和区分4个型的马疱疹病毒（EHV-1、EHV-2、EHV-3、EHV-4）。结果显示，EHV-1、EHV-2、EHV-3和EHV-4各自特异性的溶解曲线峰值分别出现在79℃、86℃、84℃、81℃，而其他动物的疱疹病毒无溶解曲线出现，证明了本方法的特异性。本技术的灵敏度更高，检测限为1fg的病毒DNA，即约16个拷贝的EHV，并分别从血清学阳性但病毒分离阴性的1匹进口马组织样品和15匹出口前检疫马的鼻咽样品检测到EHV-1和EHV-4病毒特异性核酸。攻毒试验结果显示马驹在攻毒后，外周血白细胞（PBL）出现短暂的低拷贝EHV-4阳性，但通过鼻腔长时间大量排毒，达3-21天。3．为在同一系统内快速、同步检测多种病原，通过分子克隆技术获得马疱疹病毒1型、马动脉炎病毒、马流感病毒、马传染性贫血病毒和东部马脑脊髓炎病毒等5种病毒各一段高度保守的特异性基因片段，用芯片点样仪逐点分配到处理过的玻片上，制备成检测芯片。提取样品中的RNA，进行反转录和荧光标记后滴加到芯片上进行特异性杂交，对杂交结果进行扫描检测和计算机软件分析。结果显示，制备的基因芯片可同时检测和鉴别上述5种病毒，可检测到阳性杂交信号的最高稀释度为10^-6的病毒液，约25个病毒DNA拷贝，但其他病毒材料未见红色荧光信号，证明了本方法的特异性。在进口马的隔离检疫期间，采集马鼻肺炎、马动脉炎中和抗体阳性但病毒分离阴性马匹的白细胞悬液，分别在EHV-1、EAV位点处可检测到阳性杂交信号。结果证明基因芯片技术不但快速、准确、灵敏，而且可同时进行多种病毒的检测。4．在建立马鼻肺炎病原检测技术的基础上，进一步建立马鼻肺炎快速、灵敏的抗体检测技术，以马疱疹病毒1型（EHV-1）为模板，利用PcR技术扩增EHV-1／4 gD基因中具有交叉反应的、保守的主要中和抗原编码区片段（456bp），将其克隆到原核表达载体pGEX-6p-1中，获得的重组质粒pGEX-EHV-gD转化大肠杆菌后用IPTG诱导表达。SDS-PAGE电泳分析显示，EHV-1 gD蛋白以GST-EHVgD融合蛋白形式高效表达，符合预期大小（43kDa），其含量约占菌体总蛋白的32．7％；Western-blot分析表明，表达产物均能与EHV-1和EHV-4抗血清发生反应，证实其具有良好的型共同反应原性。用重组蛋白免疫家兔获得的高免血清能中和EHV-l和EHV-4，且中和抗体效价较高，说明原核表达产物具有良好的型交叉免疫原性。原核表达产物经过变性、复性、纯化以及GST的切除等处理后，GST-gD融合蛋白和无GST的纯gD蛋白，仍可以被马抗EHV-1、EHV-4阳性血清所特异性识别。分别用纯化的GST-EHvgD或gD包被酶标板，优化反应条件，建立了型共同EHVgD-E LISA，二者在等摩尔浓度下无明显的差异。本方法仅可检出EHV-1／4抗体，不与马动脉炎阳性血清、马流感阳性血清和马传贫阳性血清发生交叉反应．与病毒中和试验比较，EHVgD-ELIsA具有更高的灵敏度，符合率在实验阶段和出入境检验检疫应用阶段分别为92．8％和94．8％，与从西班牙INGENASA公司进口的马鼻肺炎ELIsA试剂盒符合率分别为95．1％（428／450）。利用EHVgD-ELJSA对新疆、内蒙古、黑龙江、吉林和河北等中国北部5省区马群进行了血清流行病调查，共采集血清22 198份，其中阳性为3 821份，阳性率为17．2％。统计分析发现血清学阳性率从每年的6月份开始下降，10月份达到最低水平，而后从12月份开始上升，第二年4月份达到最高水平，符合马鼻肺炎在冬春季节常发的流行病学特点。血清流行病调查中还发现，新疆、内蒙古等以自由放牧为主的地区马群血清学阳性率普遍低于黑龙江、吉林和河北等以集中饲养为主的地区马群血清学，说明密切接触似乎为马鼻肺炎的传播提供了方便。因此，EHVgD-ELISA具有灵敏、特异、快速、简便和可靠等特点，为国内血清流行病调查和检测以及出入境检验检疫提供了一种非常实用的检测工具。5．为完善马鼻肺炎抗体检测体系，本研究以马鼻肺炎病毒（马疱疹病毒1型，EHV-1 RacH株）为标准种毒，在RK-13细胞上培养后获得了最高滴度的工作用病毒，制备了标准的EHV-1补体结合抗原，建立了特异、灵敏、可靠的微量补体结合试验（CFT）、微量血清中和试验（SNT）；通过克隆EHV-1 Rac H株gD主要抗原区域于大肠杆菌表达，以原核表达产物为包被抗原，建立了灵敏、特异的EHVgD-ELISA方法。与常见的其他马病抗血清不发生交叉反应，证明这3种血清学试验具有较强的特异性。CFT、EHVgD-ELISA与经典的SNT符合率分别为85．6％、94．8％，CFT与SNT相对灵敏度和特异性分别为63．3％、91．5％，EHVgD．ELISA与SNT相对灵敏度和特异性分别为100％、93．4％。线性回归数据显示，与sNT相比，CFT具有高度相关性（R2=0．7008．p＜＜0．0005，t-检验），EHVGD-ELISA具有更高的相关性（R²=0．741 5，p＜＜0．0005，t-检验）。迄今为止，接受检测我国部分地区流行病学调查、全国口岸检验检疫机构以及大陆境外（土库曼斯坦、香港、澳门）委托的出入境马、驴、斑马、羊驼等马属动物血清样品共8750份，马鼻肺炎检测情况为：补体结合抗体阳性1 749份，中和抗体阳性1829份，EHVGD-ELISA抗体阳性2285份。应用EHVgD-ELISA在我国新疆、内蒙古等部分地区进行流行病学调查、出口马属动物检验检疫过程中，证实我国马鼻肺炎血清学阳性率17．2％（3 821／22 198）。更多还原

【Abstract】 1. A multiplex PCR method was established to simultaneously identify and distinguishequine herpesviruses EHV-1, EHV-2 and EHV-4 in the same reaction within several hours,using 3 pairs of type-specific primers chosen from the glycoprotein B （gB） coding region ofeach serotype. The PCR amplicons of EHV-1, EHV-2 and EHV-4, as predicted 226, 333,570 base pairs, respectively, could be readily separated by gel electrophoresis. Whencompared to the absence of speific DNA bands in herpesviruses （BHV-1、PHV-1、GHV-1、AHV-1） from other animals, the specificity of these amplifications was confirmed bydetermining their nucleotide sequences of the PCR products, consisting with the publishedsequence of these gB genes. And the assay was sensitive, indicating a detection limit of 103TCID₅₀. Tissues from one seropositive horse of post-arrival quarantine was positive forEHV-1 by PCR whereas some nasaopharyngeal swabs from seropositive horses ofpre-export quarantine were positive for EHV-4. Infectious viruses were not recovered inany tested specimens from which the animals did not show any clinical signal of EHV-1 orEHV-4 infections. It is showed that multiplex PCR assay, which is quick, specific, sensitive,and reliable, seems to be a practical alternative to identify the low level of non-infectiousequine herpesviruses or the presence of latent forms in horse tissue.2. To enhance the sensitivity of inspection for equine herpesviruses, a novel Eva Green dyewas applied in fluorescence PCR to detect and distinguish 4 types of Equine Herpesviruses（EHV-1, EHV-2, EHV-3 and EHV-4） by specific primers. This assay was more sensitive inthat it indicated a detection limit of 1 fg of viral DNA （16 molecules of EHV）. And itsspecificity was confirmed by the presence of dissociation curves of the PCR products ofEHV-1, EHV-2, EHV-3 and EHV-4 at 79℃, 86℃, 84℃and 81℃, respectively. Tissuesamples from one seropositive horse of post-arrival quarantine was positive for EHV-1 byfluorescence PCR but negative by virus isolation, whereas fifteen nasaopharyngeal swabsfrom seropositive horses of pre-export quarantine were positive for EHV-4 by fluorescencePCR but negative by virus isolation. EHV-4 DNA detected by fluorescence PCR was lowand of short duration in Peripheral blood leucocytes （PBLs）, but high and of long duration in nasopharyngeal secretions （NS） after experimental challenge. It is showed thatfluorescence PCR assay, which is quick, specific, sensitive, and reliable, seems to be apractical alternative to identify the low level of non-infectious equine herpesviruses or thepresence of latent forms in horse tissue.3. To identify various equine pathogens, the highly conservative DNAs of Equineherpesvirus 1, Equine arteritis virus, Equine influenza virus, Equine infectious anaemiavirus and Eastern equine encephalomyelitis virus were acquired by molecular cloning, andthen spotted on the treated glass slides as the diagnostic gene-chip. And the cDNAsreverse-transcripted from RNAs of samples were labeled with Cy5 fluorescence asprobes. Following specific hybridization of deposited gene chip and labeled probes,fluorescence signals were scanned by laser scanner and the obtained imagewas analyzed by QiamtArray software with the digital computer. The resultsshowed that the prepared gene chip could detect and distinguish the five equine viruses.And its sensitivity was about 25 copies of viral genomes. The hybridization specificity wasconfirmed by the presence of red fluorescence signals on the corresponding sites withsamples from the five relevant viruses in horses and by the absence of positive signals withthe specimens from irrelevant viruses in other animals. Peripheral blood leucocyte （PBL）from some seropositive horses of post-arrival quarantine was negative by virus isolation butpositive for EHV-1 and EAV by gene chip. The evidence suggests that gene chip, which isquick, specific, sensitive, and reliable, can provide a practical alternative to screen andquarantine a large number of animal samples within a very short period of time.4. To establish a rapid, sensitive ELISA to inspect antibodies to EHV-1/4, the equineherpesviruses 1 （EHV-1） gD gene encoding major neutralizing antigenic domain withcross-reactitve, conservative epitopes of both EHV-1 and EHV-4 was amplified by PCRassay and cloned into prokaryotic expression vector pGEX-6p-1. After induction oftransformed E. coli with the recombinant plasmid designated as pGEX-EHV-gD by IPTG,EHV gD was highly expressed in the form of GST-EHVgD fusion protein, as predicted43kDa in SDS-PAGE, which was about 32.7% of total proteins in bacteria. The sharedantigenicity of the recombinant protein was confirmed by Western-blot with antiseraagainst EHV-1/4. Hyperimmune sera generated in rabbits immunized with the expressedproduct could neutralize EHV-1/4 with high titers, indicating its good type-commonimmunogenicity. Following a serial treatment of denaturation, refolding, purification andPreScission Protease cleavage and re-purification of fusion protein with GSTrap FF affinity chromatography column, highly purified GST-gD fusion protein and GST-free gD protein（about 99%） were recognized specifically by both EHV-1/4 antisera in Western-blot. Theoptical dilution of antigen and test sera were determined by checkerboard titration before anindirect type-common EHVgD-ELISA was established and improved using theserecombinant gD fusion proteins （GST-gD） or GST-free gD （gD） as coating antigens,without significant divergence if at the same mol concentration. Horse antisera to EHV-1/4were revealed as positive reaction whereas horse antisera against equine arteritis, equineinfluenza and equine infectious anaemia were exhibited as negative reaction.EHVgD-ELISA was more sensitive with a detection rate of 41.1% compared to virusnueralization with a detection rate of 34.1%, but their conformance ratio was 93.1% and95.2%, respectively for tentative serum samples and clinical serum samples collected fromhorses for entry-exit inspection and quarantine. The agreement rate between home-madeEHVgD-ELISA and imported ELISA kit reched to 95.1%. To investigate the disseminationof Equine rhinopneumonitis in major horse populations from 5 provinces of North China,including Xinjiang, Neimenggu, Helongjiang, Jilin and Hebei, EHVgD-ELISA was used todetect antibodies to EHV-1 and EHV-4 in 22 198 horse serum samples, of which 3 821samples （17.2%） were identified as positive. Statistic analysis has shown thatseroprevalence was declining in June to the lowest level in October, and then climbing inDecember to the highest level in next April. This obervation may be explained byepizootiological characteristics of frequent infection or reactivation of EHV-1 and EHV-4 incold seasons of late winter and early spring. Morever, during epidemioiogical survey,seroprevalence in pasture raised horses in Xinjiang and Neimenggu was lower than that inintensively managed horses in Helongjiang, Jilin and Hebei, illustrating colse contactfacilitates virus transmission in horse population. Take together, the overall results suggestthat EHVgD-ELISA, which is rapid, specific, sensitive, convinient and reliable, can providea practical tool for serological diagnosis, epidemiological survey and entry-exit inspectionand quarantine of both EHV-1 and EHV-4.5. To develop a inspection system for EHV-1/4, standardized procedures for micro-serumcomplement fixation test （CFT）, micro-serum neutralization test （VN） and EHVgD-ELISAhad been established to detect antibodies against equine rhinopneumonitis （ER）, usingEuropean vaccine strain Rac H of Equine herpsevirus-1 （EHV-1 Rac H） as VN workingvirus, crude prepation of EHV-1 Rac H cultured in RK-13 as CF antigen andprokaryotically recombinant antigens within EHV-1 gD expressed in Escherichia coli as ELISA coating antigen. No cross-reactivity was observed in three above-mentionedserological methods with serum against other equine viruses, confirming their strongspecificity. Compared with the reference method （VN）, CFT and EHVGD-ELISAdemonstrated 85.6% and 94.8% agreement, respectively, and CFT gave a relativesensitivity of 63.3% and a relative specificity of 91.5%, and EHVGD-ELISA gave arelative sensitivity of 100% and a relative specificity 93.4%. Linear regression of these VNand CFT titers as well as VN and EHVGD-ELISA titers revealed a highly significantcorrelation （R²=0.7008, p＜＜0.0005, student’s t-test） and a more highly significantcorrelation （R²=0.7415, p＜＜0.0005, student’s t-test）, respectively. Of 8750 sera collected inhorses for epidemiological survey, in equid species for entry-exit quarantine frominspection and quarantine organs and some institutions outside the Chinese mainland（Kazakhstan, Hong Kong, Macao）, seropositive reactivity were demonstrated in 1749samples in CFT, 1829 in VN, 2285 in ELISA. Using EHVGD-ELISA, seroprevalence wassubstantiated about 17.2% （3 821/22 198） in domestic horses from Xinjiang andNeimenggu in China and exported equid species.更多还原