【作者】 唐娜；

【导师】 柳增善；

【作者基本信息】 吉林大学 ， 兽医， 2013， 博士

【摘要】 猪繁殖与呼吸综合征（Porcine reproductive and respiratory syndrome, PRRS）是由猪繁殖与呼吸综合征病毒（Porcine reproductive and respiratory syndrome virus, PRRSV）引起的猪的严重呼吸系统与繁殖障碍疾病，目前仍然是影响养猪业健康发展的最重要的疾病之一。上世纪末至今，世界各国多个领域的人员，尤其是养殖场、兽医部门以及研究人员已经在猪繁殖与呼吸综合征的防控中投入了大量精力并取得了一定的成果。但截止目前，人们仍尚未破解PRRSV的致病机理、遗传变异等难题,该病仍在持续流行中，给全球养猪业造成巨大损失。综合开发PRRS病原学、血清学诊断方法，开展PRRS流行情况调查与流行毒株分离鉴定，分析PRRSV流行株的遗传变异趋势，对于正确开展防控技术研究，控制和减少PRRS的发生具有非常重要的意义。本论文以山东及周边（河南、河北）地区为研究范围，在开展流行毒株分离鉴定，建立PCR鉴别诊断方法、抗体诊断方法，研制单克隆抗体的基础上，开展了PRRSV流行病学调查，并分析了2007-2012年间山东及周边地区PRRSV流行株的遗传变异趋势。分别以Nsp2基因、ORF7基因为靶基因，设计了2对引物，建立了鉴别检测PRRSV的RT-PCR诊断方法并进行了初步优化，可以特异灵敏地鉴别诊断缺失株与经典毒株、美洲株与欧洲株，分别能够检测到浓度低至0.01TCID50的美洲型（Ⅱ型）疫苗毒和0.1TCID50浓度的欧洲型（Ⅰ型）疫苗毒，重复性较好。应用克隆表达的重组N蛋白为包被抗原，初步构建了抗体间接ELISA检测方法，该方法能够特异性的检测病毒N蛋白抗体，与国际商品化试剂盒的检测符合率良好。以高致病性PRRSV病毒株经密度梯度离心纯化后免疫BALB/C小鼠，通过细胞融合、阳性克隆的筛选鉴定，获得3株特异性识别PRRSV的单克隆抗体，其中7F2能够特异性识别N蛋白，6A4能够特异性识别GP5蛋白。病毒中和试验和抗体依赖性增殖作用试验表明，3株抗体均无促进病毒增殖作用，但6A4和3F1有病毒中和作用。3株抗体均具有较高的效价与特异性，在PRRSV的诊断与免疫学研究中具有很好的应用价值。以MARC-145细胞为研究工具进行山东及周边地区流行PRRSV毒株的分离与鉴定，并对其中5株代表性毒株进行生物学特性等研究。其中1株为类似疫苗株（SDDY2007），4株为致病性毒株（SDBZ2006、HN2010、SDWF2010、HBXT2009），1株（HN2010）在MARC-145细胞上具有较低增殖能力，对其中SDBZ2006株进行了动物回归实验鉴定，确定其为高致病性毒株。以建立的PCR与ELISA诊断方法开展山东省及周边地区PRRS流行情况调查，检测了957份血清样品中的PRRSV抗体和272份疑似病例组织、血液样本中的PRRSV病毒，各猪场PRRSV抗体平均阳性率49.8％，抗体阳性率从2007年至2012年间上下波动明显，但2012年有显著上升趋势，不同地区间抗体阳性率差异较大，PRRSV中和抗体平均为25.1％，不同地区间差异较大；PCR检测发现各地区猪场均存在PRRSV（阳性率26.84％），部分猪场出现一定程度混合感染；检测到1例Ⅰ型病毒感染，6例Ⅱ型传统病毒，1例传统株与缺失株共感染；不同病料组织器官，其病毒分离阳性率有所差异。对通过MARC-145细胞分离到的15株病毒株，并分别采用RT-PCR扩增其ORF5基因和部分Nsp2基因并测序，推导氨基酸序列与GenBank中68个ORF5推导序列和41个Nsp2序列进行比对表明，15株分离株均属于美洲型毒株，其中13个毒株与JXA1株有较高的同源性（95.5%～97.5%）；1株（DY2007株）与疫苗株MLV和VR-2332株亲缘关系相近，而1株可能存在基因重组。总之，试验所建立的PCR、ELISA和单克隆抗体具有较好的应用性，病毒分离、血清学调查、病原检测和遗传变异分析结果表明，2007～2012年间高致病性PRRSV是山东及周边地区的优势流行毒株，在该地区流行较为严重，存在一定比例混合感染，且可能存在基因重组毒株，而疫苗防疫尚未能发挥显著的作用；除高致病性毒株外，山东及周边地区仍然存在一定经典毒株如疫苗毒株和欧洲毒株，三省区流行毒株间有一定遗传差异，但没有明显地域特征。本研究所建立的诊断方法、制备的抗体、分离的毒株以及取得的数据信息为开展PRRS防控研究提供了有效的技术工具和可靠的数据参考。更多还原

【Abstract】 Porcine reproductive and respiratory syndrome (PRRS) is one of the most seriousdiseases affected the development of pig industry, which is caused by porcine reproductiveand respiratory syndrome virus (PRRSV). PRRSV can cause severe respiratory andreproductive disorders in pigs. Since the last century, people around the world in many fields,especially farmers, veterinary authorities and researchers have invested a lot of effort andachieved certain result in PRRS. But until now, many questions around PRRSV, such aspathogenesis and genetic variation are still not known. PRRS is still prevalent and causesenormous damage to global pig industry.For the carried out of PRRS prevention and control technology research, with aims tocontrol and reduce the disease incidence, it is very important to comprehensively developepathogenic and serological diagnostic methods of PRRSV, investigate the epidemicpandemic, isolate and identify the epidemic virus strains and analysis the genetic variationtrend of PRRSV strains, and so on. In this study, samples were collected from pig farms in9areas of Shandong, Henan and Hebei provinces. Using these samples, several PRRSVisolates were isolated and identified; RT-PCR detection methods for virus RNA andindirect-ELISA method for virus antibodies were found. Based on the establishment of PCRmethod and antibody diagnostic method, monoclonal antibodies were developed for studyand diagnostic uses; antibody and etiology epidemiological investigations during2007-2012were carried out; ORF5and partical nsp2gene squences of isolated virus and GenBankassesion strains were identified and analyzed to find the genetic variation trend.Using partical nsp2gene and ORF7gene as target genes, two pairs of primers weredesigned to establish differential detection RT-PCR diagnostic methods of PRRSV andconducted a preliminary optimized. The methods had specifically sensitivities to thedifferential diagnosis with classical strains and deficient strains, Americas strains andEuropean strains, which were able to detect the virus concentration of lower than0.1TCID50(EO subtype virus) and0.01TCID50(US subtype virus).Recombinant N protein, which was cloned, expressed, identified and purified by otherresearchers in the lab, was applied as the coating antigen to construct an indirect ELISAmethod, to detect the N protein-specific antibodies in pigs. The tests showed that the methodhad great sensivity, specificity, and stability, and was well coincided to IDEXX ELISA kit.An isolate of Highly pathogenic PRRSV was collected and purified by density gradientcentrifugation and immunized to BALB/c mice. Following immunication, cell fusion, cellselection and several identifications, three positive cell strains, named3F1,7F2and6A4,which could induce anti-PRRSV monoclonal antibodies, were obtained. During them, 7F2could specifically recognize some epitope on N protein, and6A4could recognize someepitope on GP5protein. By the ADE and antibody neutralization tests,6A4and3F1showedneutralization activity, and7F2showed no ADE activity or neutralization activity, but it canbe potential applicated in N protein detection by ELISA. All the three antibodies were higheffecency and specificity, and could be well used in PRRSV diagnology, immunologystudied.Using MARC-145cells as a research tool, representative five PRRSV isolates wereisolated and identified for biological characteristics. One of five isolates (SDDY2007) wassimilar to a vaccine strain (US subtype), and the other four were more similar to highpathogenic strains, in which an isolate of the four (SDBZ2006) was identified by pigexperiments infection, and another one (HN2010) showed a lower proliferative capacity inMARC-145cells.PRRS prevalence survey was carried out in Shandong Province and surrounding areasbased on established PCR and ELISA.957serum samples and272suspected tissues andblood samples were examined. The average positive rate of PRRSV antibody was49.8%.From2007to2012, though the antibody positive rate obviously fluctuated, a significantupward trend was found during2012. Antibody-positive rate of different regions are quitedifference. The average positive rate of PRRSV neutralizing antibody was25.1%(using cellneutralization test), with outstanding differences in different regions. PRRS pathgens in the272samples were detected by RT-PCR, which showed that73samples were virus positive(positive rate of26.84%), and several farms had co-infection in them. A sample of genetypeⅠ,6samples of classical genetype Ⅱ were found,66sample of nsp2deleted, and1samplewith both classical and deleted subtype viruses were detected. Virus isolation positive ratesvaried in different diseased tissues and organs. During2007-2012,15strains were isolatedand identified by MARC-145cells, the ORF5and partial nsp2gene were amplified, clonedand sequencing. The sequences were blasted with68ORF5sequences and41nsp2sequencein GenBank, showed that all the15isolates were belong to American genetype strain, ofwhich13strains showed high homology with JXA1strain(95.5%~97.5%), one (DY2007strain) was closed to the vaccine strain MLV and VR-2332strain, and one might be a straincoming from genetic recombination.The study established good PCR and ELISA methods and applicated well. Virusisolation, serological surveys, pathogen detection and genetic variation analysis showed thathigh pathogenic PRRSV is the advantage strain in Shandong and surrounding areas from2007to2012. High pathogenic PRRSV is more serious prevalent in the region and canco-infection with other viruses. There may be some recombinant strains, too. It seemed thatvaccine immunization has not been able to play an enough prevention acctions in PRRScontrol. Addition to high pathogenic strains, there are also classic US strains liked vaccinestrains and European strain in Shandong and the surrounding areas. Pandemic strains in the three provinces have some genetic differences, but no obvious regional characteristics.In clusion, the established diagnostic methods, antibodies, isolates and epidemiology,genetic diversity information provide us effective technical tools and reliable data referencefor PRRS prevention and control。更多还原