PRRSV诊断方法的建立及鲁豫冀地区PRRSV的流行与遗传变异研究
|
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。
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。
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。
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。
引文
[1]蔡宝祥，姜平。我国PRRS诊断技术与疫苗研究进展[J]。畜牧与兽医，2007，39（8）：1-4。
[2] Plana Duran, Collins J E, Mengling W L, et al. Efficacy of an in-activated vaccine forprevention of reproductive failure induced by porcine reproductive and respiratorysyndrome virus. Vet Microbiol,1997,55:361-370.
[3] Wensvoort G. Lelystad virus and the porcine epidemicabortion and respiratorysyndrome[J].Vet Res,1993,24:117-124.
[4]．殷震,刘景华.动物病毒学（第二版）[M].北京:中国科学出版社,1997:1019-1029.
[5]王艳艳.山东省猪繁殖与呼吸综合征、猪流感流行病学调查[D].泰安：山东农业大学，2010.
[6]童武,周艳君,肖少波,等.2010年国内部分省份猪场常见病毒性疾病的病原学调查[J].中国动物传染病学报,2011,5:1～7.
[7]卢洪芬,蒋启荣,董振光,等.猪繁殖与呼吸道综合征的诊断与防制[J].中国兽医科技,1999,29(3):33-34
[8]韩明远,沈青春,张志刚,等。影响猪繁殖与呼吸综合征病毒感染与增殖的因素[J]。动物医学进展,2010,31(1):87-91。
[9]陈溥言。如何应对猪瘟病毒―变脸‖——透视猪瘟免疫失败现象分析和防治措施[J]。中国动物保健，2007，9：65-66，68。
[10] I. Toplak, D. Rihtaric, P. Hostnik, et al. Identification of a genetically diversesequence of porcine reproductive and respiratory syndrome virus in Slovenia and theimpact on the sensitivity of four molecular tests[J]. J Virol Meth.2012(179):51-56.
[11] J Christopher-Hennings. Porcine reproductive and respiratory syndrome virus (PRRSV)dagnostics in the United States: past, present and future.2013[C].Beijing: InternationalPRRS Symposium.32.
[12] Ryo Inoue, Takamitsu Tsukahara, Chinatsu Sunaba, et al. Simple and rapid detectionof the porcine reproductive and respiratory syndrome virus from pig whole blood usingfilter paper[J]. Journal of Virological Methods.2007,141:102-106.
[13] Seong-Hee Kim, In-Soon Roh, Eun-Jin Choi, et al. A molecular analysis of Europeanporcine reproductive and respiratory syndrome virus isolated in SouthKorea[J].Veterinary Microbiology,2010,143,2–4,394-400.
[14] Zhi Zhou, Jianqiang Ni, Zhen Cao, et al. The epidemic status and genetic diversity of14highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV)isolates from China in2009[J]. Vet Microbiol.2011(150):257-269.
[15] H.Yang. Prcine reproductive and respiratory syndrome situation in China: the past,present and future.2013[C].Beijing: International PRRS Symposium.48.
[16]李儒曙、李静、江鹏华等，华南地区规模化猪场猪瘟抗体水平的血清学调查及免疫水平低下原因分析[J]。中国动物检疫，2007，24（11）：30-31。
[17]杨宗照，方维焕。猪瘟合并猪繁殖与呼吸综合征病毒（PRRSV）和猪圆环病毒2型（PCV2）感染[J]。中国兽医学报，2006，26（3）：240-242。.
[18] Basavaraj Binjawadagi. Intranasal delivery of an adjuvanted modified live PRRSVvaccine reduces the ROS induced lung pathology.2011[C].US: International PRRSSymposium.
[19] J. Ellis, E. Clark, D. Haines,et al. Porcine circovirus-2and concurrent infections in thefield[J]. Veterinary Microbiology,2004,98:159-163.
[20] Yijun Du, Dongwan Yoo, Marie Anne Paradis, et al. Antiviral activity of tilmicosinfor type1and type2PRRSV in cultured PAMs[J]. J Antivir Antiretrovir.2011,3(3):028-033.
[21]丁壮，杨松涛，乔红伟.动物疫病流行病学[M]．北京：金盾出版，2007
[22] C.provost, Y. Hernandez Reyes, C.Levesque, et al. Co-infection study betweenPRRSV and a bacteria led to the discovery of a possible new bacteria antiviral.2013[C].Beijing: International PRRS Symposium.157.
[23] Ying Fang, Emmely E Treffers, Yanhua Li, et al. Efficient-2frameshifting bymammalian ribosomes to synthesize an additional arterivirus protein[J]. Proc Natl AcadSci U S A.2012,109(43):2920-8.
[24]黄文林.分子病毒学（第二版）[M].北京：人民卫生出版社，2006.
[25] J.Q.Wu, J Yu, S N Liu, et al. PRRS in hybrd wild boars, China.2013[C].Beijing:International PRRS Symposium.77.
[26]刘永宏，赵丽，刘俊峰，等。从猪繁殖与呼吸综合征病毒的起源与进化谈猪繁殖与呼吸综合征的防制[J].中国兽医杂志，2012.48（3）：56-58.
[27] C.K.Godell, P.Lopez, A.Kittawornrat, et al. Reflection of sow serology in piglet littersoral fluids-a sow farm surveillance field study.2013[C].Beijing: International PRRSSymposium.36.
[28]唐娜,王金良,管宇,等．2006-2013年鲁豫冀地区18株PRRSV分离鉴定与GP5蛋白的遗传变异分析[A].国际猪繁殖与呼吸综合征学术研讨会论文集[C]．北京,2013:67.
[29] Cancel-Tirado S M, Evans R B, Yoon K J. Monoclonal antibody analysis of PorcineReproductive and Respiratory Syndrome virus epitopes associated withantibody-dependent enhancement and neutralization of virus infection [J]. Vet Immunoland Immunop,2004,102,3(8):249-262.
[30] Q Y Yang, Y J Qin, Y N Zhang, et al. The role of antibody in PRRSV infection PAM.2013[C]. Beijing: International PRRS Symposium.143.
[31] Dokland. T. The structural biology of PRRSV[J].Vrius Research,2010,154:86～97.
[32] Asit k. Pattnaik, Hiep L.X. Vu, Byungjoon Kwon,et al. PRRSV proteins in infectionand immunity.2013[C].Beijing: International PRRS Symposium.
[33] M.Han, The regulatory role of the zinc finger motif of the PRRSV nsp1a protein forIFN modulation.2013[C].Beijing: International PRRS Symposium.143.
[34] Chen Z., Lawson S., Sun Z., et al. Identinication of two auto-cleavage products ofnonstructural protein1(nsp1) in porcine reproductive and respiratory syndrome virusinfected cells: nsp1function as interferon antagonist [J]. Virology,2010,398:87-97.
[35] Fei Xue, Yuna Sun, Liming Yan, et al. The Crystal Structure of Porcine Reproductiveand Respiratory Syndrome Virus Nonstructural Protein Nsp1β Reveals a NovelMetal-Dependent Nuclease[J]. J Virol,2010.6461-6471.
[36] Johnson C.R., Yu W., Murtaugh M.P. Cross-reactive antibody responses to nsp1andnsp2of Porcine reproductive and respiratory syndrome virus[J]. J Gen Virol.2007,88:1184-1195.
[37] Zhi Sun, Zhenhai Chen, Steven R Lawson, et al. The Cysteine Protease Domain ofPorcine Reproductive and Respiratory Syndrome Virus Nonstructural Protein2Possesses Deubiquitinating and Interferon Antagonism Functions[J]. J Virol,2010:7832-7846.
[38] Wang F X, Song N, Chen L Z, et al. Non-structural protein2of the porcinereproductive and respiratory syndrome (PRRS) virus: A crucial protein in viralpathogenesis, immunity and diagnosis[J]. Research in Veterinary Science,2013,95(1):1-7.
[39]刘飞，王开功，周碧君，等.贵州省PRRSV分离株Nsp2基因的克隆及序列分析[J].中国预防兽医学报，2010，32(12)：924～928.
[40] Lei Zhou, Jialong Zhang, Jingwen Zeng, et al. The30-Amino-Acid Deletion in theNsp2of Highly Pathogenic Porcine Reproductive and Respiratory Syndrome VirusEmerging in China Is Not Related to Its Virulence[J]. Journal of Virology,2009:5156-5167
[41] Ying Fang, Eric J Snijder. The PRRSV replicase Exploring the multifunctionality ofan intriguing set of nonstructural proteins[J]. Virus Research,2010,154:617.
[42]付云云,单悦,郎广平,等.猪繁殖与呼吸综合征病毒蛋白质研究进展[J].中国畜牧兽医,2012,04:60-64.
[43] Roongroje Thanawongnuwech, Sanipa Suradhat. Taming PRRSV Revisiting thecontrol strategies and vaccine design[J]. Virus Research,2010,154:133-140.
[44] Lalit K. Beura, Sakthivel Subramaniam, Hiep L.X. Vu, et al. Identification of aminoacid residues important for anti-IFN activity of porcine reproductive and respiratorysyndrome virus non-structural protein1[J] Virology,2012,433(2):431-439.
[45] Dong Li, Establishment of a stable cell line expressing non-structural protein11ofPRRSV.2011[C]. Chicago: International PRRS Symposium.63.
[46] Ying Fang, Emmely E Treffers, Yanhua Li, et al. Efficient-2frameshifting bymammalian ribosomes to synthesize an additional arterivirus protein[J]. Proc Natl AcadSci U S A.2012,109(43):2920-8.
[47] E.H.J.Wissink, M.V.Kroese, J.G.Maneschijn-Bonsing, et al. Significance of theoligosaccharides of the porcine reproductive and respiratory syndrome virusglycoproteins GP2a and GP5for infectious virus production[J]. Journal of GeneralVirology,2004,85,3715-3723
[48] Phani B Das, Phat X Dinh, Israrul H Ansari, et al. The Minor Envelope GlycoproteinsGP2a and GP4of Porcine Reproductive and Respiratory Syndrome Virus Interact withthe Receptor CD163[J]. Journal of Virology.2010,1731-1740.
[49] MaorongYu, Xiaoling Liu, Lei Sun, et al. Subcellular localization and topology ofporcine reproductive and respiratory syndrome virus E protein[J]. Virus Research,2010,152(1–2):104-114.
[50] Das P B, Dinh P X, Ansari I H, et al. The minor envelope glycoproteins GP2a andGP4of porcine reproductive and respiratory syndrome virus interact with the receptorCD163[J]. J Virol,2010,84:1731-1740.
[51]田志军，安同庆，周艳君，等。致弱过程中高致病性猪繁殖与呼吸综合征病毒HuN4株部分生物学特性的研究[J]。中国预防兽医学报，2009,31（6）：424-426
[52] J. Janková, V. Celer. Expression and serological reactivity of Nsp7protein of PRRSgenotype I virus[J]. Research in Veterinary Science,2012,93(3):1537-1542.
[53] Yijun Du,Asit K. Pattnaik,Cheng Song, et al. Glycosyl-phosphatidylinositol(GPI)-anchored membrane association of the porcine reproductive and respiratorysyndrome virus GP4glycoprotein and its co-localization with CD163in lipid rafts[J].Virology.2012(424):18-32.
[54]姚茂平，陈雄庭.质膜脂筏的研究进展[J]。安徽农业科学，2007,35（26）:8106-8108.
[55]赵琳，王中明，夏平安，等。猪繁殖与呼吸综合征病毒河南分离株GP4蛋白免疫活性研究[J]。中国畜牧兽医，2010，37（1）:57-62。
[56] Breedam W V, Costers S, Vanhee M, et al. Porcine reproductive and respiratorysyndrome virus (PRRSV)-specific mAbs: supporting diagnostics and providing newinsights into the antigenic properties of the virus[J]. Vet Immunol and Immunop,2011,141,(3-4):246-257.
[57]蔡锦顺，李珈莹，马永和，等。PRRSVGP5与GP4蛋白重组腺病毒的构建及其免疫特性研究[J]。安徽农业科学，2010，38(10)：5155-5157，5193。
[58] Min-Yuan Chia, Shih-Hsuan Hsiao, Hui-Ting Chan, et al. The immunogenicity ofDNA constructs co-expressing GP5and M proteins of porcine reproductive andrespiratory syndrome virus conjugated by GPGP linker in pigs[J]. VeterinaryMicrobiology.2010,146:189-199.
[59] Sally R. Robinson, Marina C. Figueiredo, Juan E. Abrahante, et al. novel PRRSVORF5a protein is not immunprotective but drives GP5glycosylation[A].2013International PRRS symposium[C].2013.
[60] Johnson C R, Griggs T F, Gnanandarajah J, et al. Novel structural protein in porcinereproductive and respiratory syndrome virus encoded by an alternative ORF5present inall arteriviruses[J]. J Gene Virol,2011,92:1107～1116.
[61] Y Fang, E J Snijder. The PRRSV replicate: structure, function and implications
[A].Beijing:2013International PRRS symposium[C].2013.
[62] Michael P. Murtaugh, Tomasz Stadejek, Juan E. Abrahante, et al. The ever-expandingdiversity of porcine reproductive and respiratory syndrome virus[J].Virus Research,2010,154,1–2:18-30.
[63] L.Ferrari, P. Borghetti, M. Morganti, et al. Immune reactivity is associated withclinical protection upon vaccination with a modified-live PRRSV-1vaccine andsubsequent exposure to natural infection by a field strain.2011[C]. Chicago:International PRRS Symposium.31.
[64]黄梅清,郑敏,陈仕龙,等。猪繁殖与呼吸综合征病毒(PRRSV)欧洲株和美洲株的理化特性及致病性比较[J].2012,27(1):03-07.
[65]仇化吉，郭宝清，童光志，等.猪繁殖与呼吸道综合症病CH-1A株的基因型鉴定[J]。中国兽医学报，1996，18(2)：118-121。
[66]仇华吉，童光志，卢景良.猪生殖-呼吸道综合征病毒的分子生物学[J]，国外兽医学-畜禽传染病，1997，17（3）：7-9
[67] Ranjni Chand, Yu Wang, Raymond R.R. Rowland. Investigation of model systems forthe study of recombination in PRRSV.2011[C]. Chicago: International PRRSSymposium.28.
[68] M Shi, F C Leung. Recombination leads to the re-emergence of highly pathogenicPRRSV in China.2013[C].Beijing: International PRRS Symposium.62.
[69] Shijuan Dong, Yanbo Yin, Shiyuan Shen, et al. Inhibitory effects of recombinantporcine interferon-a on high-and low-virulence porcine reproductive and respiratorysyndrome viruses[J]. Research in Veterinary Science,2012,93,(2):1060-1065.
[70] Liu D, Zhou R, Zhang J L, et al. Recombination analyses between two strains ofporcine reproductive and respiratory syndrome virus in vivo[J]. Virus Research,2011,155(2):473～486.
[71]李彬，中国大陆欧洲型PRRSV N蛋白的遗传变异分析[A].2013国际猪繁殖与呼吸综合征大会[C].2013，北京.
[72]薛青红，张彦明，刘湘涛，等。中国部分地区2005-2007年猪繁殖与呼吸综合征病毒分离株ORF5基因和NSP2基因遗传变异分析[J].中国农业科学，2009,42（5）：1805-1812
[73] Zhou Z, Ni J Q, Cao Z, et al. The epidemic status and genetic diversity of14highlypathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) isolatesfrom China in2009[J]. Vet Microbiol,2011,150:257～269.
[74] Z. Jiang, L.F. Zhang, J.J. Michal, et al. Pathway-based approach revealed candidategenes significantly associated with genetic resistance to PRRSV infection in pigs[A].2011International PRRS symposium[C].30.
[75] B.Thaa, B.C.Sinhadri, C.Tielesch, et al. signal peptide cleavage fro GP5of PRRSV:only a minor subfraction of molecules retains the decoy epitope, a presumed ofmolecular cause for viral persistence.2013[C].Beijing: International PRRSSymposium.131.
[76] Bin Li, Liurong Fang, Suyan Liu, et al. The genomic diversity of Chinese porcinereproductive and respiratory syndrome virus isolates from1996to2009[J]. VetMicrobiol,2010,146:226-237
[77] Cheon D S, Chae C. Comparision of the pathogenicity of two strains(wild type and vaccine-like) of porcine reproductive and respiratory syndrome virus(PRRSV) in experimentally infected sows[J]. J Comp Pathol,2004,130:105-111.
[78] Christopher C.Overend, Joseph Ambrogio. The antiviral state induced by IFNβ differsin MARC-145cells and PAMs as demonstrated by infection outcomes with differentPRRSV isolates[J]. J Vetimm.2008,10:248
[79] Chenglan Jiang, Feng Xing, Jinyi Xing, et al. Different expression patterns of PRRSVmediator genes in the lung tissues of PRRSV resistant and susceptible pigs [J]. Develop&Compara Immunol,2013,39(1–2):127-131.
[80] L.Ferrari, P. Borghetti, M. Morganti, et al. Different cytokine patterns in ex vivostimulated PBMC are related to the PRRSV isolate.2011[C]. Chicago: InternationalPRRS Symposium.31.
[81] Darwich L, Da I, Mateu E. Certainties, doubts and hypotheses in porcine reproductiveand respiratory syndrome virus immunobiology[J]. Virus Research,2010,154:123～130.
[82] K.P.C.Araujo, S. Abrams, A. Kittawornrat, et al. Differences in cytokine expression insera from pigs infected with type1and2PRRSV using a multiplex FMIA.2011[C].Chicago: International PRRS Symposium.21.
[83] Lee S M, Schommer S K, Kleiboeker S B, et al. Porcine reproductive and respiratorysyndrome virus field isolates differ in in vitro interferon phenotypes[J]. Vet ImmunolImmunopathol.2004,102:217-231。
[84] Laila Darwich, Ivan Da, Enric Mateu. Certainties, doubts and hypotheses in porcinereproductive and respiratory syndrome virus immunobiology[J]. Virus Research.2010,154:123-132.
[85] Laura Batista, Carlos Pijoan, Scott Dee, et al. Virological and immunologicalresponses to porcine reproductive and respiratory syndrome virus in a large populationof gilts[J]. Can J Vet Res,2004,68:267-273.
[86] Yongming Sang, Raymond RR Rowland, Frank Blecha. Porcine type I interferons:polymorphic sequences and activity against PRRSV[J]. BMC Proc.2011,5(Suppl4):S8.
[87] H. J. Nauwynck, H. Van Gorp, M. Vanhee,et al. Micro-Dissecting the Pathogenesisand Immune Response of PRRSV Infection Paves the Way for More Efficient PRRSVVaccines[J]. Transboundary and Emerging Diseases.2012,59:50–54.
[88] Gorp H V, Breedam W V, Delputte P L, et al. Sialoadhesin and CD163join forcesduring entry of the porcine reproductive and respiratory syndrome virus[J]. Journal ofGeneral Virology.2008,89:2943-2953
[89] Joan K. Lunney, Derek Petry, Rodger Johnson, et al. Swine immunity and geneticresistance to porcine reproductive and respiratory syndrome virus (PRRSV)infection[J].J Vetimm.2008,10:026.
[90] Kim J K, Fahad A M, Shanmukhappa K, et al. Defining the cellular target(s) ofporcine reproductive and respiratory syndrome virus blocking monoclonal antibody7G10[J]. J Virol.2006,80,689-696.
[91] R.M. Molina, S.-H. Cha, W. Chittick, S. Lawson, et al. Immune response againstporcine reproductive and respiratory syndrome virus during acute and chronicinfection[J].Veterinary Immunology and Immunopathology,2008,126,3–4:283-292.
[92] Mingeun Sagong, Choi-Kyu Park, Seong-Hee Kim, et al. Human telomerase reversetranscriptase-immortalized porcine monomyeloid cell lines for the production of porcinereproductive and respiratory syndrome virus[J]. Journal of Virological Methods.2012,179:26-32.
[93] Barald K F, Wessells N K. Differential antigen adhesivity used to select spleen cellsfor the production of monoclonal antibodies to embryonic neurons[J]. J Immunol Meth,1984,73(1):1-15.
[94] Posthuma C C, Nedialkova D D, Zevenhoven-Dobbe J C, et al. Site-directedmutagenesis of the nidovirus replicative endoribonuclease nendou exerts pleiotropiceffects on the arterivirus life cycle[J]. Journalof Virology,2006,1653-1661.
[95] Hso-Chi Chaung, Chien-Wei Chen, Bing-Lun Hsieh, et al. Toll-like receptorexpressions in porcine alveolar macrophages and dendritic cells in responding to poly ICstimulation and porcine reproductive and respiratory syndrome virus (PRRSV)infection[J]. Comp Immunol Microbiol Infect Dis,2010,33(3):197-213.
[96] Liu C H, Chaung H C, Chang H L, et al. Expression of Toll-like receptor mRNA andcytokines in pigs infected with porcine reproductive and respiratory syndrome virus[J].Vet. Microbiol.2009,136,266-276.
[97] Miguel J C, Chen J, Van Alstine, et al. Expression of inmmatory cytokines andToll-like receptors in the brain and respiratory tract of pigs infected with porcinereproductive and respiratory syndrome virus[J]. Vet Immunol Immunopathol.2010,135(3–4):314-319.
[98] Calzada-Nova G.,Schnitzlein W.,Husmann R.,et al. Characterization of the cytokineand maturation responses of pure populations of porcine plasma cytoid dendriticcells toporcine viruses and toll-like receptor agonists[J]. Vet Immunol Immunop.2010,135:20-33.
[99] Miller L C, Lager K M, Kehrli Jr. M E, et al. Role of Toll-like receptors in activationof porcine alveolar macrophages by porcine reproductive and respiratory syndromevirus[J]. Clin Vaccine Immunol.2009,16:360-365.
[100] Loving C L, Brockmeier S L, Sacco R E. Differential type I interfere on activationand susceptibility of dendritic cell populations to porcine arterivirus[J]. Immunology,2007,120:217-229.
[101] Lemke C D, Haynes J S, Spaete R, et al. Lymphoid hyperplasia resulting in immunedisregulation is caused by porcine reproductive and respiratory syndrome virus infectionin neonatal pigs[J]. J Immunol,2004,172:1916-1925.
[102] Wu Q, Brum M C, Caron L, et a1.Adonovirus-mediated type I interferon expressiondelays and reduces disease signs in cattle challonsed with font-and-mouth diseasevirus[J].Interferon Cytokine Res,2003,23(7):359-368.
[103] Adam E, Nasz I. Significance of recombinant adenovirus in experimental genetherapy[J]. Orv Hetil.1995,136(15):755-761.
[104] Beura,L.K., Sarkar,S.N., Kwon,B., et al. Porcine reproductive and respiratorysyndrome virus nonstructural Protein1beta modulates host innate immune response byantagonizing IRF3activation[J]. J Virol.2010,84:1574-1584
[105] J.H.Lee, I.J. Kang, N. Shabir, et al. Application of high molecularpoly-gamma-glutamic acid on pigs infected with PRRS virus.2013[C].Beijing:International PRRS Symposium.60.
[106] Varun Dwivedi, Cordelia Manickam, Basavaraj Binjawadagi, et al. PLGAnanoparticle entrapped killed porcine reproductive and respiratory syndrome virusvaccine helps in viral clearance in pigs[J]. Vet Microbiol,2013,166(1–2):47-58.
[107] Sem Genini, Thomas Paternoster, Alessia Costa, et al. Identification of serumproteomic biomarkers for early porcine reproductive and respiratory syndrome (PRRS)infection[J].Proteome Science.2012,10:48-53.
[108] Silva-Campa E, Mata-Haro V, Mateu E, et al. Porcine reproductive and respiratorysyndrome virus induces CD4+CD8+CD25+Foxp3+regulatory T cells(Tregs)[J].Virology.2012,430(1):73-80.
[109] Yi Fu, Rong Quan, Hexiao Zhang, et al. Porcine Reproductive and RespiratorySyndrome Virus (PRRSV) Induces Interleukin-15through the NF-κB SignalingPathway[J]. Journal of Virology.2012,80,18:1128.
[110] Shijuan Dong, Yanbo Yin, Shiyuan Shen, et al. Inhibitory effects of recombinantporcine interferon-a on high-and low-virulence porcine reproductive and respiratorysyndrome viruses[J]. Research in Veterinary Science,2012,93,(2):1060-1065.
[111] R.J.Langenhorst, S. Lawson, A. Kittawornrat, et al. Simultaneous detection ofantibodies against PRRSV nsp7and nucleocapsid protein in swine oral fluid and serausing a fluorescence microsphere immunoassay.2011[C]. Chicago: International PRRSSymposium.40.
[112] Wang Z, Liu H, Xu J, Bao J, Zheng D, Sun C. Genotyping of Newcastle diseaseviruses isolated from2002to2004in China. Ann N Y Acad Sci,2006,1081:228–239.
[113] Sara G. Nezami, DongSun，Ontogeny of PRRSV nucleocapsid-specific antibodyisotypes in pigs after experimental infection[C].2011International PRRS Symposium,2011, Chicago.
[114] Takano T, Tomiyama Y, Katoh Y, et al. Mutation of neutralizing/antibody-dependentenhancing epitope on spike protein and7b gene of feline infectious peritonitis virus:Influences of viral replication in monocytes/macrophages and virulence in cats[J]. VirusRes.2011,156(1-2):72-80.
[115] Kim W I, Kim J J, Cha S H, et al. Significance of genetic variation of PRRSVORF5in virus neutralization and molecular determinants corresponding to crossneutralization among PRRS viruses[J]. Vet Microbiol.2013,162(1):10-22.
[116] Gonin P，Pirzadeh B，Gagnon CA，et al. Seroneutralization of porcine reproductiveand respiratory syndrome virus correlates with antibody response to ORF5majorenvelope glycoprotein[J].J Vet Diagn Invest.1999,11(1):20-26.
[117] Fang L,Jiang Y, Xiao S, et al. Enhanced immunogenicity of the modified GP5ofporcine reproductive and respiratory syndrome virus[J]. Virus Genes.2006,32,5-11.
[118] Chao-Liang Leng, Tong-Qing An, Jia-Zeng Chen, et al. Highly pathogenic porcinereproductive and respiratory syndrome virus GP5B antigenic region is not aneutralizing antigenic region [J]. Vet Microbiol,2012,159(3–4):273-281.
[119] Kay S. Faaberg, Siba K. Samal. Induction of type I interferons by a novel porcinereproductive and respiratory syndrome virus isolate[J]. Virology.2012,432(2),261-270.
[120] Ansari IH, Kwon B, Osorio FA, et al. Influence of N-linked glycosylation ofporcine reproductive and respiratory syndrome virus GP5on virus infectivity,antigenicity, and ability to induce neutralizing antibodies[J]. J Virol.2006,80(8):3994-4004.
[121] Z.Z.Wei, F Gao, Y F Jiang, et al. The effect of the position of N-link glycosylationsites within hypervariable region of GP5on virus neutralization[A]. Beijing:2013International PRRS symposium[C].163.
[122] Costers S, Lefebvre DJ, Goddeeris B, et al. Functional impairment ofPRRSV-specific peripheral CD3+CD8high cells[J]. Vet Res.2009,40(5):46-53.
[123] John Schwartz, Michael Murtaugh, and Juan Abrahante. The pig heavy and lightchain immunoglobulin repertoires provide insights into the adaptive immune responseagainst porcine reproductive and respiratory syndrome virus (PRRSV)[J]. The Journalof Immunology,2012,188,160.4.
[124] Vashisht K,Goldberg T L,Husmann R J,et al.Identi-fication of immunodominantT-cell epitopes present inglycoprotein5of the North American genotype of por-cinereproductive and respiratory syndrome virus[J].Vaccine,2008,26(36):4747-4753.
[125] Díaz I, Gimeno M, Darwich L, et al. Characterization of homologous andheterologous adaptive immune responses in porcine reproductive and respiratorysyndrome virus infection[J]. Vet Res.2012,43:30
[126] Raymond R. R. Rowland, Joan Lunney, Jack Dekkers. Control of porcinereproductive and respiratory syndrome (PRRS) through genetic improvements in diseaseresistance and tolerance[J]. Front Genet.2012;3:260.
[127] Joan K Lunney, Juan Pedro Steibel, James M Reecy, et al.Probing genetic control ofswiner esponses to PRRSV infection: current progress of the PRRS host geneticsconsortium[J]. BMC Proceedings.2011,5(Suppl4):30-35.
[128] Robert Parker, K. Nechvatalova, L. Dupuis, et al. Adjutants for live PRRS vaccines.2011[C]. Chicago: International PRRS Symposium.34.
[129] Xiangdong Li, Amy Galliher-Beckley, Hongzhou Huang, et al. Peptide nanofiberhydrogel adjuvanted live virus vaccine enhances cross-protective immunity to porcinereproductive and respiratory syndrome virus [J]. Vaccine,2013,31(41):4508-4515.
[130] Caiwei Chen, Jing Li, Yuhai Bi, et al. Synthetic B-and T-cell epitope peptides ofporcine reproductive and respiratory syndrome virus with Gp96as adjuvant inducedhumoral and cell-mediated immunity[J]. Vaccine,2013,31(14):1838-1847
[131] Nilubol D, Platt K B, Halbur P G, et al. The effect of a killed porcine reproductiveand respiratory syndrome virus (PRRSV) vaccine treatment on virus sheddingin previously PRRSV infected pigs[J]. Vet Microbiol,2004,102(1-2):11-18.
[132] Daniel C.L. Linhares, Jean Paul Cano, Thomas Wetzell, et al. Effect ofmodified-live porcine reproductive and respiratory syndrome virus (PRRSv) vaccine onthe shedding of wild-type virus from an infected population of growing pigs[J]. Vaccine,2012,30:407-413.
[133] E. Pileri, E. Gibert, F.Soldevila, et al. Quantificaton of PRRSV transmission: effectof pig vaccination.2013[C].Beijing: International PRRS Symposium.67.
[134] M.Roca, M.Gimeno, S.Bruguera, et al. Effects of challenge with a virulentgenotype II strain of porcine reproductive and respiratory syndrome virus on pigletsvaccinated with an attenuated genotype I strain vaccine[J] The Veterinary Journal,2012,193(1):92-96.
[135] Wu J, Lj JQ, SunM S, et a1. The Immune Response Induced by RecombinantAdenovirus with Administration of Interaperitonea Injection[J]. Virologica Sinica,2002,17:51-55．
[136] Meng X J. Heterogeneity of porcine reproductive and respiratory syndrome virus:implications for current vaccine efficacy and future vaccine development[J].VetMicrobiol.2000,12:309-329．
[137] Ha M.Truong, Z.Lu, GeraldF.Kutish, et al. A highly pathogenic porcinereproductive and respiratory syndrome virus generated from an infectious cDNA cloneretains the in vivo virulence and transmissibility properties of the parental virus[J].Virology.2004,325,308-319.
[138] L. Enjuanes, vaccine vectors based on coronavirus genomes to protect againstPRRSV and SARS-CoV.2011[C]. Chicago: International PRRS Symposium.6.
[139] G.Z.Tong, Y.Z.Yu, Y.J.Zhou, et al. Development and application of a dual-markervaccine for highly pathogenic PRRS.2013[C].Beijing: International PRRSSymposium.47.
[140] JazminaL.G.Cruz, SoniaZniga, MartinaBcare, et al. Vectored vaccines to protectagainst PRRSV[J]. Virus Research.2010,154:150-160.
[141] Hiep L.X. Vu, Byungjoon Kwon, Marcelo de Lima, et al. Characterization of aserologic marker candidate for development of a live-attenuated DIVA vaccine againstporcine reproductive and respiratory syndrome virus [J].Vaccine,2013,31(40):4330-4337
[142]王朝文，吴润，王一成，等。大肠杆菌LTB增强PRRSV-GP5蛋白免疫反应的研究[J].甘肃农业大学学报，2010,45（6）:12-17.
[143] CHEN Hong-ying, WEI Zhan-yong, ZHANG Hong-ying, et al. Use of a MultiplexRT-PCR Assay for Simultaneous Detection of the North American Genotype PorcineReproductive and Respiratory Syndrome Virus, Swine Influenza Virus and JapaneseEncephalitis Virus[J].Agricultural Sciences in China,2010,9(7):1050-1057.
[144] R Sina, V Lazar, R Pogranichniy. Real time PCR detection of the PRRSV genomeusing a single primer set.2011[C]. Chicago: International PRRS Symposium.27.
[145] E. Redondo, I. Gil, M. Garcia-duran, et al. Development of real timeRT-PCR(RRT-PCR) commercial kit for reliable detection and typing of PRRSV.2013[C].Beijing: International PRRS Symposium.54.
[146] Sautto G, Mancini N, Diotti R A, et al. Anti-hepatitis C virus E2(HCV/E2)glycoprotein monoclonal antibodies and neutralization interference[J]. Antivir Res,2012,96(1):82-89.
[147] Rockman S, Camuglia S, Vandenberg K, et al. Reverse engineering the antigenicarchitecture of the haemagglutinin from influenza H5N1clade1and2.2viruses withfine epitope mapping using monoclonal antibodies[J]. Mol Immunol,2013,53(4):435-442.
[148] Yang M, Holland H, Clavijo A, et al. Production of monoclonal antibodies againstwhole virus particles of foot-and-mouth disease virus serotype O and A and theirpotential use in quantification of intact virus for vaccine manufacture[J]. Vaccine,2008,26,(27–28):3377-3382.
[149] H. Liu, L. Mu, M.Yang, et al. The establishment of the PRRS detection liquidblocking ELISA method.2013[C].Beijing: International PRRS Symposium.57.
[150] J.Y.Song, E.J.Choi, E.J.Kim,et al. Detection of PRRSV using the light s catteringassay based on the mcrofluidic chip.2013[C].Beijing: International PRRSSymposium.71.
[151] C.N.Lin comparison of virema of type2PRRSV infected pigs by zip nucleic acidprobes based real-time PCR.2013[C].Beijing: International PRRS Symposium.64.
[152] Patrick Gonin, Boroushan Pirzadeh, Carl A. et al. Seroneutralization of porcinereproductive and respiratory syndrome virus correlates with antibody response to theGP5major envelope glycoprotein. J Vet Diagn Invest.1999,11:20-26.
[153] Z.Q.Huang, H.Liu, M.Yang,et al. preparation of monoclonal antibody againstPRRSV nsp9gene establishment of block-ELISA method[A]. Beijing:2013InternationalPRRS symposium[C].84.
[154] Xu X G, Wang Z S, Zhang Q, et al. Baculovirus as a PRRSV and PCV2bivalentvaccine vector: Baculovirus virions displaying simultaneously GP5glycoprotein ofPRRSV and capsid protein of PCV2[J]. J Virol Methods,2012,179:359～366.
[155] M.Achacha，A. Khyar, A. Bensari. High level protein expression of a syntheticPRRSV nuclecapsid gene optimized in E.coli host.2011[C]. Chicago: InternationalPRRS Symposium.23.
[156] O J Lopez, F A Osorio. Role of neutralizing antibodies in PRRSV protectiveimmunity[J]. Veterinary Immunology and Immunopathology,2004,102:155–163.
[157] Pirzadeh B, Gagnon C A, Dea S. Genomic and antigenic vatiations of porcinereproductive and respiratory syndrome major envelope ORF5glycoprotein. Can J VetRes,1998,62(3):170-177.
[158] K.Ouyang, B.Binjawadagi, J.Hiremath, et al. Development and validation ofPRRSV specific neutralizing antibody detection assay in pig oral fluid samples.2013[C].Beijing: International PRRS Symposium.98.
[159] R.J.Langenhorst, S. Lawson, A. Kittawornrat, et al. Simultaneous detection ofantibodies against PRRSV nsp7and nucleocapsid protein in swine oral fluid and serausing a fluorescence microsphere immunoassay.2011[C]. Chicago: International PRRS.
[160] Prather RS, Rowland RR, Ewen C, et al. An intact sialoadhesin(Sn/SIGLEC1/CD169) is not required for attachment/internalization of the porcinereproductive and respiratory syndrome virus (PRRSV)[J]. J Virol.2013,87(17):9538-46.
[161] Stadejek T, Oleksiewicz MB, Potapchuk D,et al. Porcine reproductive andrespiratory syndrome virus strains of exceptional diversity in eastern Europe support thedefinition of new genetic subtypes[J]. J Gen Virol.2006,87(7):1835-41.
[162] M Frias, J Schmid, F Kuhn, et al. discrimination of PRRSV type1and type2by anindirect ELISA.2013[C].Beijing: International PRRS Symposium.70.
[163]郎广平,郭敏,韩盈盈,等。猪繁殖与呼吸综合征诊断方法与疫苗研究进展[J].中国畜牧兽医，2013，40(6):195～198.
[164]李玉峰，王先炜，陈闻，等。猪繁殖与呼吸综合征病毒nsp2基因缺失株RT-PCR检测方法的建立[J]。南京农业大学学报，2009,32（3）：169-171.
[165] Dan Cheng,Jian-Jun Zhao, Na Li, et al. Simultaneous detection of Classical swinefever virus and North American genotype Porcine reproductive and respiratorysyndrome virus using a duplex real-time RT-PCR[J]. J Virol Meths.2008,151,194-199.
[166] E.Martnez, P.Riera, M.Sitja, et al. Simultaneous detection and genotyping ofporcine reproductive and respiratory syndrome virus (PRRSV) by real-time RT-PCRand amplicon melting curve analysis using SYBR Green[J]. Research in VeterinaryScience.2008,85:184-193.
[167]刘秀梵.单克隆抗体在农业上的应用[M].合肥：安徽科学技术出版社.1994:21-24.32-36.
[168]王世若,王兴龙,韩文瑜.现代动物免疫学[M].2版.长春:吉林科学技术出版社,1997.
[169]徐志凯.实用单克隆抗体技术[M].西安：陕西科学技术出版社,1991:125-126.
[170]孙晨,王丽,孙秀萍,等.水泡性口炎病毒单克隆抗体的制备与鉴定[J].中国兽医学报,2012,32(3):411-414.
[171]万博，乔松林，张改平，等。猪繁殖与呼吸综合征病毒不同毒株感染对猪外周血免疫细胞含量的影响.畜牧兽医学报，2009,40（11），1695-1698.
[172]孙建富，郝建伟，荣福龙，等。猪繁殖与呼吸综合征病毒的分离与鉴定及ORF5基因的序列分析[J]。中国畜牧兽医，2011，38（9）：138-142。
[173] T. Opriessnig, M. Fenaux, S. Yu,et al. Effect of porcine parvovirus vaccination onthe development of PMWS in segregated early weaned pigs coinfected with type2porcine circovirus and porcine parvovirus[J]. Veterinary Microbiology,2004,98:209-220.
[174] Martin-Valls G E, Burgara-Estrella A J, Darwich L, et al. ecombination events arecommon in genotype1and2PRRS virus worldwide and may generate mosaic
viruses[A].2013International PRRS symposium[C].13.
[2] Plana Duran, Collins J E, Mengling W L, et al. Efficacy of an in-activated vaccine forprevention of reproductive failure induced by porcine reproductive and respiratorysyndrome virus. Vet Microbiol,1997,55:361-370.
[3] Wensvoort G. Lelystad virus and the porcine epidemicabortion and respiratorysyndrome[J].Vet Res,1993,24:117-124.
[4]．殷震,刘景华.动物病毒学（第二版）[M].北京:中国科学出版社,1997:1019-1029.
[5]王艳艳.山东省猪繁殖与呼吸综合征、猪流感流行病学调查[D].泰安：山东农业大学，2010.
[6]童武,周艳君,肖少波,等.2010年国内部分省份猪场常见病毒性疾病的病原学调查[J].中国动物传染病学报,2011,5:1～7.
[7]卢洪芬,蒋启荣,董振光,等.猪繁殖与呼吸道综合征的诊断与防制[J].中国兽医科技,1999,29(3):33-34
[8]韩明远,沈青春,张志刚,等。影响猪繁殖与呼吸综合征病毒感染与增殖的因素[J]。动物医学进展,2010,31(1):87-91。
[9]陈溥言。如何应对猪瘟病毒―变脸‖——透视猪瘟免疫失败现象分析和防治措施[J]。中国动物保健，2007，9：65-66，68。
[10] I. Toplak, D. Rihtaric, P. Hostnik, et al. Identification of a genetically diversesequence of porcine reproductive and respiratory syndrome virus in Slovenia and theimpact on the sensitivity of four molecular tests[J]. J Virol Meth.2012(179):51-56.
[11] J Christopher-Hennings. Porcine reproductive and respiratory syndrome virus (PRRSV)dagnostics in the United States: past, present and future.2013[C].Beijing: InternationalPRRS Symposium.32.
[12] Ryo Inoue, Takamitsu Tsukahara, Chinatsu Sunaba, et al. Simple and rapid detectionof the porcine reproductive and respiratory syndrome virus from pig whole blood usingfilter paper[J]. Journal of Virological Methods.2007,141:102-106.
[13] Seong-Hee Kim, In-Soon Roh, Eun-Jin Choi, et al. A molecular analysis of Europeanporcine reproductive and respiratory syndrome virus isolated in SouthKorea[J].Veterinary Microbiology,2010,143,2–4,394-400.
[14] Zhi Zhou, Jianqiang Ni, Zhen Cao, et al. The epidemic status and genetic diversity of14highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV)isolates from China in2009[J]. Vet Microbiol.2011(150):257-269.
[15] H.Yang. Prcine reproductive and respiratory syndrome situation in China: the past,present and future.2013[C].Beijing: International PRRS Symposium.48.
[16]李儒曙、李静、江鹏华等，华南地区规模化猪场猪瘟抗体水平的血清学调查及免疫水平低下原因分析[J]。中国动物检疫，2007，24（11）：30-31。
[17]杨宗照，方维焕。猪瘟合并猪繁殖与呼吸综合征病毒（PRRSV）和猪圆环病毒2型（PCV2）感染[J]。中国兽医学报，2006，26（3）：240-242。.
[18] Basavaraj Binjawadagi. Intranasal delivery of an adjuvanted modified live PRRSVvaccine reduces the ROS induced lung pathology.2011[C].US: International PRRSSymposium.
[19] J. Ellis, E. Clark, D. Haines,et al. Porcine circovirus-2and concurrent infections in thefield[J]. Veterinary Microbiology,2004,98:159-163.
[20] Yijun Du, Dongwan Yoo, Marie Anne Paradis, et al. Antiviral activity of tilmicosinfor type1and type2PRRSV in cultured PAMs[J]. J Antivir Antiretrovir.2011,3(3):028-033.
[21]丁壮，杨松涛，乔红伟.动物疫病流行病学[M]．北京：金盾出版，2007
[22] C.provost, Y. Hernandez Reyes, C.Levesque, et al. Co-infection study betweenPRRSV and a bacteria led to the discovery of a possible new bacteria antiviral.2013[C].Beijing: International PRRS Symposium.157.
[23] Ying Fang, Emmely E Treffers, Yanhua Li, et al. Efficient-2frameshifting bymammalian ribosomes to synthesize an additional arterivirus protein[J]. Proc Natl AcadSci U S A.2012,109(43):2920-8.
[24]黄文林.分子病毒学（第二版）[M].北京：人民卫生出版社，2006.
[25] J.Q.Wu, J Yu, S N Liu, et al. PRRS in hybrd wild boars, China.2013[C].Beijing:International PRRS Symposium.77.
[26]刘永宏，赵丽，刘俊峰，等。从猪繁殖与呼吸综合征病毒的起源与进化谈猪繁殖与呼吸综合征的防制[J].中国兽医杂志，2012.48（3）：56-58.
[27] C.K.Godell, P.Lopez, A.Kittawornrat, et al. Reflection of sow serology in piglet littersoral fluids-a sow farm surveillance field study.2013[C].Beijing: International PRRSSymposium.36.
[28]唐娜,王金良,管宇,等．2006-2013年鲁豫冀地区18株PRRSV分离鉴定与GP5蛋白的遗传变异分析[A].国际猪繁殖与呼吸综合征学术研讨会论文集[C]．北京,2013:67.
[29] Cancel-Tirado S M, Evans R B, Yoon K J. Monoclonal antibody analysis of PorcineReproductive and Respiratory Syndrome virus epitopes associated withantibody-dependent enhancement and neutralization of virus infection [J]. Vet Immunoland Immunop,2004,102,3(8):249-262.
[30] Q Y Yang, Y J Qin, Y N Zhang, et al. The role of antibody in PRRSV infection PAM.2013[C]. Beijing: International PRRS Symposium.143.
[31] Dokland. T. The structural biology of PRRSV[J].Vrius Research,2010,154:86～97.
[32] Asit k. Pattnaik, Hiep L.X. Vu, Byungjoon Kwon,et al. PRRSV proteins in infectionand immunity.2013[C].Beijing: International PRRS Symposium.
[33] M.Han, The regulatory role of the zinc finger motif of the PRRSV nsp1a protein forIFN modulation.2013[C].Beijing: International PRRS Symposium.143.
[34] Chen Z., Lawson S., Sun Z., et al. Identinication of two auto-cleavage products ofnonstructural protein1(nsp1) in porcine reproductive and respiratory syndrome virusinfected cells: nsp1function as interferon antagonist [J]. Virology,2010,398:87-97.
[35] Fei Xue, Yuna Sun, Liming Yan, et al. The Crystal Structure of Porcine Reproductiveand Respiratory Syndrome Virus Nonstructural Protein Nsp1β Reveals a NovelMetal-Dependent Nuclease[J]. J Virol,2010.6461-6471.
[36] Johnson C.R., Yu W., Murtaugh M.P. Cross-reactive antibody responses to nsp1andnsp2of Porcine reproductive and respiratory syndrome virus[J]. J Gen Virol.2007,88:1184-1195.
[37] Zhi Sun, Zhenhai Chen, Steven R Lawson, et al. The Cysteine Protease Domain ofPorcine Reproductive and Respiratory Syndrome Virus Nonstructural Protein2Possesses Deubiquitinating and Interferon Antagonism Functions[J]. J Virol,2010:7832-7846.
[38] Wang F X, Song N, Chen L Z, et al. Non-structural protein2of the porcinereproductive and respiratory syndrome (PRRS) virus: A crucial protein in viralpathogenesis, immunity and diagnosis[J]. Research in Veterinary Science,2013,95(1):1-7.
[39]刘飞，王开功，周碧君，等.贵州省PRRSV分离株Nsp2基因的克隆及序列分析[J].中国预防兽医学报，2010，32(12)：924～928.
[40] Lei Zhou, Jialong Zhang, Jingwen Zeng, et al. The30-Amino-Acid Deletion in theNsp2of Highly Pathogenic Porcine Reproductive and Respiratory Syndrome VirusEmerging in China Is Not Related to Its Virulence[J]. Journal of Virology,2009:5156-5167
[41] Ying Fang, Eric J Snijder. The PRRSV replicase Exploring the multifunctionality ofan intriguing set of nonstructural proteins[J]. Virus Research,2010,154:617.
[42]付云云,单悦,郎广平,等.猪繁殖与呼吸综合征病毒蛋白质研究进展[J].中国畜牧兽医,2012,04:60-64.
[43] Roongroje Thanawongnuwech, Sanipa Suradhat. Taming PRRSV Revisiting thecontrol strategies and vaccine design[J]. Virus Research,2010,154:133-140.
[44] Lalit K. Beura, Sakthivel Subramaniam, Hiep L.X. Vu, et al. Identification of aminoacid residues important for anti-IFN activity of porcine reproductive and respiratorysyndrome virus non-structural protein1[J] Virology,2012,433(2):431-439.
[45] Dong Li, Establishment of a stable cell line expressing non-structural protein11ofPRRSV.2011[C]. Chicago: International PRRS Symposium.63.
[46] Ying Fang, Emmely E Treffers, Yanhua Li, et al. Efficient-2frameshifting bymammalian ribosomes to synthesize an additional arterivirus protein[J]. Proc Natl AcadSci U S A.2012,109(43):2920-8.
[47] E.H.J.Wissink, M.V.Kroese, J.G.Maneschijn-Bonsing, et al. Significance of theoligosaccharides of the porcine reproductive and respiratory syndrome virusglycoproteins GP2a and GP5for infectious virus production[J]. Journal of GeneralVirology,2004,85,3715-3723
[48] Phani B Das, Phat X Dinh, Israrul H Ansari, et al. The Minor Envelope GlycoproteinsGP2a and GP4of Porcine Reproductive and Respiratory Syndrome Virus Interact withthe Receptor CD163[J]. Journal of Virology.2010,1731-1740.
[49] MaorongYu, Xiaoling Liu, Lei Sun, et al. Subcellular localization and topology ofporcine reproductive and respiratory syndrome virus E protein[J]. Virus Research,2010,152(1–2):104-114.
[50] Das P B, Dinh P X, Ansari I H, et al. The minor envelope glycoproteins GP2a andGP4of porcine reproductive and respiratory syndrome virus interact with the receptorCD163[J]. J Virol,2010,84:1731-1740.
[51]田志军，安同庆，周艳君，等。致弱过程中高致病性猪繁殖与呼吸综合征病毒HuN4株部分生物学特性的研究[J]。中国预防兽医学报，2009,31（6）：424-426
[52] J. Janková, V. Celer. Expression and serological reactivity of Nsp7protein of PRRSgenotype I virus[J]. Research in Veterinary Science,2012,93(3):1537-1542.
[53] Yijun Du,Asit K. Pattnaik,Cheng Song, et al. Glycosyl-phosphatidylinositol(GPI)-anchored membrane association of the porcine reproductive and respiratorysyndrome virus GP4glycoprotein and its co-localization with CD163in lipid rafts[J].Virology.2012(424):18-32.
[54]姚茂平，陈雄庭.质膜脂筏的研究进展[J]。安徽农业科学，2007,35（26）:8106-8108.
[55]赵琳，王中明，夏平安，等。猪繁殖与呼吸综合征病毒河南分离株GP4蛋白免疫活性研究[J]。中国畜牧兽医，2010，37（1）:57-62。
[56] Breedam W V, Costers S, Vanhee M, et al. Porcine reproductive and respiratorysyndrome virus (PRRSV)-specific mAbs: supporting diagnostics and providing newinsights into the antigenic properties of the virus[J]. Vet Immunol and Immunop,2011,141,(3-4):246-257.
[57]蔡锦顺，李珈莹，马永和，等。PRRSVGP5与GP4蛋白重组腺病毒的构建及其免疫特性研究[J]。安徽农业科学，2010，38(10)：5155-5157，5193。
[58] Min-Yuan Chia, Shih-Hsuan Hsiao, Hui-Ting Chan, et al. The immunogenicity ofDNA constructs co-expressing GP5and M proteins of porcine reproductive andrespiratory syndrome virus conjugated by GPGP linker in pigs[J]. VeterinaryMicrobiology.2010,146:189-199.
[59] Sally R. Robinson, Marina C. Figueiredo, Juan E. Abrahante, et al. novel PRRSVORF5a protein is not immunprotective but drives GP5glycosylation[A].2013International PRRS symposium[C].2013.
[60] Johnson C R, Griggs T F, Gnanandarajah J, et al. Novel structural protein in porcinereproductive and respiratory syndrome virus encoded by an alternative ORF5present inall arteriviruses[J]. J Gene Virol,2011,92:1107～1116.
[61] Y Fang, E J Snijder. The PRRSV replicate: structure, function and implications
[A].Beijing:2013International PRRS symposium[C].2013.
[62] Michael P. Murtaugh, Tomasz Stadejek, Juan E. Abrahante, et al. The ever-expandingdiversity of porcine reproductive and respiratory syndrome virus[J].Virus Research,2010,154,1–2:18-30.
[63] L.Ferrari, P. Borghetti, M. Morganti, et al. Immune reactivity is associated withclinical protection upon vaccination with a modified-live PRRSV-1vaccine andsubsequent exposure to natural infection by a field strain.2011[C]. Chicago:International PRRS Symposium.31.
[64]黄梅清,郑敏,陈仕龙,等。猪繁殖与呼吸综合征病毒(PRRSV)欧洲株和美洲株的理化特性及致病性比较[J].2012,27(1):03-07.
[65]仇化吉，郭宝清，童光志，等.猪繁殖与呼吸道综合症病CH-1A株的基因型鉴定[J]。中国兽医学报，1996，18(2)：118-121。
[66]仇华吉，童光志，卢景良.猪生殖-呼吸道综合征病毒的分子生物学[J]，国外兽医学-畜禽传染病，1997，17（3）：7-9
[67] Ranjni Chand, Yu Wang, Raymond R.R. Rowland. Investigation of model systems forthe study of recombination in PRRSV.2011[C]. Chicago: International PRRSSymposium.28.
[68] M Shi, F C Leung. Recombination leads to the re-emergence of highly pathogenicPRRSV in China.2013[C].Beijing: International PRRS Symposium.62.
[69] Shijuan Dong, Yanbo Yin, Shiyuan Shen, et al. Inhibitory effects of recombinantporcine interferon-a on high-and low-virulence porcine reproductive and respiratorysyndrome viruses[J]. Research in Veterinary Science,2012,93,(2):1060-1065.
[70] Liu D, Zhou R, Zhang J L, et al. Recombination analyses between two strains ofporcine reproductive and respiratory syndrome virus in vivo[J]. Virus Research,2011,155(2):473～486.
[71]李彬，中国大陆欧洲型PRRSV N蛋白的遗传变异分析[A].2013国际猪繁殖与呼吸综合征大会[C].2013，北京.
[72]薛青红，张彦明，刘湘涛，等。中国部分地区2005-2007年猪繁殖与呼吸综合征病毒分离株ORF5基因和NSP2基因遗传变异分析[J].中国农业科学，2009,42（5）：1805-1812
[73] Zhou Z, Ni J Q, Cao Z, et al. The epidemic status and genetic diversity of14highlypathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) isolatesfrom China in2009[J]. Vet Microbiol,2011,150:257～269.
[74] Z. Jiang, L.F. Zhang, J.J. Michal, et al. Pathway-based approach revealed candidategenes significantly associated with genetic resistance to PRRSV infection in pigs[A].2011International PRRS symposium[C].30.
[75] B.Thaa, B.C.Sinhadri, C.Tielesch, et al. signal peptide cleavage fro GP5of PRRSV:only a minor subfraction of molecules retains the decoy epitope, a presumed ofmolecular cause for viral persistence.2013[C].Beijing: International PRRSSymposium.131.
[76] Bin Li, Liurong Fang, Suyan Liu, et al. The genomic diversity of Chinese porcinereproductive and respiratory syndrome virus isolates from1996to2009[J]. VetMicrobiol,2010,146:226-237
[77] Cheon D S, Chae C. Comparision of the pathogenicity of two strains(wild type and vaccine-like) of porcine reproductive and respiratory syndrome virus(PRRSV) in experimentally infected sows[J]. J Comp Pathol,2004,130:105-111.
[78] Christopher C.Overend, Joseph Ambrogio. The antiviral state induced by IFNβ differsin MARC-145cells and PAMs as demonstrated by infection outcomes with differentPRRSV isolates[J]. J Vetimm.2008,10:248
[79] Chenglan Jiang, Feng Xing, Jinyi Xing, et al. Different expression patterns of PRRSVmediator genes in the lung tissues of PRRSV resistant and susceptible pigs [J]. Develop&Compara Immunol,2013,39(1–2):127-131.
[80] L.Ferrari, P. Borghetti, M. Morganti, et al. Different cytokine patterns in ex vivostimulated PBMC are related to the PRRSV isolate.2011[C]. Chicago: InternationalPRRS Symposium.31.
[81] Darwich L, Da I, Mateu E. Certainties, doubts and hypotheses in porcine reproductiveand respiratory syndrome virus immunobiology[J]. Virus Research,2010,154:123～130.
[82] K.P.C.Araujo, S. Abrams, A. Kittawornrat, et al. Differences in cytokine expression insera from pigs infected with type1and2PRRSV using a multiplex FMIA.2011[C].Chicago: International PRRS Symposium.21.
[83] Lee S M, Schommer S K, Kleiboeker S B, et al. Porcine reproductive and respiratorysyndrome virus field isolates differ in in vitro interferon phenotypes[J]. Vet ImmunolImmunopathol.2004,102:217-231。
[84] Laila Darwich, Ivan Da, Enric Mateu. Certainties, doubts and hypotheses in porcinereproductive and respiratory syndrome virus immunobiology[J]. Virus Research.2010,154:123-132.
[85] Laura Batista, Carlos Pijoan, Scott Dee, et al. Virological and immunologicalresponses to porcine reproductive and respiratory syndrome virus in a large populationof gilts[J]. Can J Vet Res,2004,68:267-273.
[86] Yongming Sang, Raymond RR Rowland, Frank Blecha. Porcine type I interferons:polymorphic sequences and activity against PRRSV[J]. BMC Proc.2011,5(Suppl4):S8.
[87] H. J. Nauwynck, H. Van Gorp, M. Vanhee,et al. Micro-Dissecting the Pathogenesisand Immune Response of PRRSV Infection Paves the Way for More Efficient PRRSVVaccines[J]. Transboundary and Emerging Diseases.2012,59:50–54.
[88] Gorp H V, Breedam W V, Delputte P L, et al. Sialoadhesin and CD163join forcesduring entry of the porcine reproductive and respiratory syndrome virus[J]. Journal ofGeneral Virology.2008,89:2943-2953
[89] Joan K. Lunney, Derek Petry, Rodger Johnson, et al. Swine immunity and geneticresistance to porcine reproductive and respiratory syndrome virus (PRRSV)infection[J].J Vetimm.2008,10:026.
[90] Kim J K, Fahad A M, Shanmukhappa K, et al. Defining the cellular target(s) ofporcine reproductive and respiratory syndrome virus blocking monoclonal antibody7G10[J]. J Virol.2006,80,689-696.
[91] R.M. Molina, S.-H. Cha, W. Chittick, S. Lawson, et al. Immune response againstporcine reproductive and respiratory syndrome virus during acute and chronicinfection[J].Veterinary Immunology and Immunopathology,2008,126,3–4:283-292.
[92] Mingeun Sagong, Choi-Kyu Park, Seong-Hee Kim, et al. Human telomerase reversetranscriptase-immortalized porcine monomyeloid cell lines for the production of porcinereproductive and respiratory syndrome virus[J]. Journal of Virological Methods.2012,179:26-32.
[93] Barald K F, Wessells N K. Differential antigen adhesivity used to select spleen cellsfor the production of monoclonal antibodies to embryonic neurons[J]. J Immunol Meth,1984,73(1):1-15.
[94] Posthuma C C, Nedialkova D D, Zevenhoven-Dobbe J C, et al. Site-directedmutagenesis of the nidovirus replicative endoribonuclease nendou exerts pleiotropiceffects on the arterivirus life cycle[J]. Journalof Virology,2006,1653-1661.
[95] Hso-Chi Chaung, Chien-Wei Chen, Bing-Lun Hsieh, et al. Toll-like receptorexpressions in porcine alveolar macrophages and dendritic cells in responding to poly ICstimulation and porcine reproductive and respiratory syndrome virus (PRRSV)infection[J]. Comp Immunol Microbiol Infect Dis,2010,33(3):197-213.
[96] Liu C H, Chaung H C, Chang H L, et al. Expression of Toll-like receptor mRNA andcytokines in pigs infected with porcine reproductive and respiratory syndrome virus[J].Vet. Microbiol.2009,136,266-276.
[97] Miguel J C, Chen J, Van Alstine, et al. Expression of inmmatory cytokines andToll-like receptors in the brain and respiratory tract of pigs infected with porcinereproductive and respiratory syndrome virus[J]. Vet Immunol Immunopathol.2010,135(3–4):314-319.
[98] Calzada-Nova G.,Schnitzlein W.,Husmann R.,et al. Characterization of the cytokineand maturation responses of pure populations of porcine plasma cytoid dendriticcells toporcine viruses and toll-like receptor agonists[J]. Vet Immunol Immunop.2010,135:20-33.
[99] Miller L C, Lager K M, Kehrli Jr. M E, et al. Role of Toll-like receptors in activationof porcine alveolar macrophages by porcine reproductive and respiratory syndromevirus[J]. Clin Vaccine Immunol.2009,16:360-365.
[100] Loving C L, Brockmeier S L, Sacco R E. Differential type I interfere on activationand susceptibility of dendritic cell populations to porcine arterivirus[J]. Immunology,2007,120:217-229.
[101] Lemke C D, Haynes J S, Spaete R, et al. Lymphoid hyperplasia resulting in immunedisregulation is caused by porcine reproductive and respiratory syndrome virus infectionin neonatal pigs[J]. J Immunol,2004,172:1916-1925.
[102] Wu Q, Brum M C, Caron L, et a1.Adonovirus-mediated type I interferon expressiondelays and reduces disease signs in cattle challonsed with font-and-mouth diseasevirus[J].Interferon Cytokine Res,2003,23(7):359-368.
[103] Adam E, Nasz I. Significance of recombinant adenovirus in experimental genetherapy[J]. Orv Hetil.1995,136(15):755-761.
[104] Beura,L.K., Sarkar,S.N., Kwon,B., et al. Porcine reproductive and respiratorysyndrome virus nonstructural Protein1beta modulates host innate immune response byantagonizing IRF3activation[J]. J Virol.2010,84:1574-1584
[105] J.H.Lee, I.J. Kang, N. Shabir, et al. Application of high molecularpoly-gamma-glutamic acid on pigs infected with PRRS virus.2013[C].Beijing:International PRRS Symposium.60.
[106] Varun Dwivedi, Cordelia Manickam, Basavaraj Binjawadagi, et al. PLGAnanoparticle entrapped killed porcine reproductive and respiratory syndrome virusvaccine helps in viral clearance in pigs[J]. Vet Microbiol,2013,166(1–2):47-58.
[107] Sem Genini, Thomas Paternoster, Alessia Costa, et al. Identification of serumproteomic biomarkers for early porcine reproductive and respiratory syndrome (PRRS)infection[J].Proteome Science.2012,10:48-53.
[108] Silva-Campa E, Mata-Haro V, Mateu E, et al. Porcine reproductive and respiratorysyndrome virus induces CD4+CD8+CD25+Foxp3+regulatory T cells(Tregs)[J].Virology.2012,430(1):73-80.
[109] Yi Fu, Rong Quan, Hexiao Zhang, et al. Porcine Reproductive and RespiratorySyndrome Virus (PRRSV) Induces Interleukin-15through the NF-κB SignalingPathway[J]. Journal of Virology.2012,80,18:1128.
[110] Shijuan Dong, Yanbo Yin, Shiyuan Shen, et al. Inhibitory effects of recombinantporcine interferon-a on high-and low-virulence porcine reproductive and respiratorysyndrome viruses[J]. Research in Veterinary Science,2012,93,(2):1060-1065.
[111] R.J.Langenhorst, S. Lawson, A. Kittawornrat, et al. Simultaneous detection ofantibodies against PRRSV nsp7and nucleocapsid protein in swine oral fluid and serausing a fluorescence microsphere immunoassay.2011[C]. Chicago: International PRRSSymposium.40.
[112] Wang Z, Liu H, Xu J, Bao J, Zheng D, Sun C. Genotyping of Newcastle diseaseviruses isolated from2002to2004in China. Ann N Y Acad Sci,2006,1081:228–239.
[113] Sara G. Nezami, DongSun，Ontogeny of PRRSV nucleocapsid-specific antibodyisotypes in pigs after experimental infection[C].2011International PRRS Symposium,2011, Chicago.
[114] Takano T, Tomiyama Y, Katoh Y, et al. Mutation of neutralizing/antibody-dependentenhancing epitope on spike protein and7b gene of feline infectious peritonitis virus:Influences of viral replication in monocytes/macrophages and virulence in cats[J]. VirusRes.2011,156(1-2):72-80.
[115] Kim W I, Kim J J, Cha S H, et al. Significance of genetic variation of PRRSVORF5in virus neutralization and molecular determinants corresponding to crossneutralization among PRRS viruses[J]. Vet Microbiol.2013,162(1):10-22.
[116] Gonin P，Pirzadeh B，Gagnon CA，et al. Seroneutralization of porcine reproductiveand respiratory syndrome virus correlates with antibody response to ORF5majorenvelope glycoprotein[J].J Vet Diagn Invest.1999,11(1):20-26.
[117] Fang L,Jiang Y, Xiao S, et al. Enhanced immunogenicity of the modified GP5ofporcine reproductive and respiratory syndrome virus[J]. Virus Genes.2006,32,5-11.
[118] Chao-Liang Leng, Tong-Qing An, Jia-Zeng Chen, et al. Highly pathogenic porcinereproductive and respiratory syndrome virus GP5B antigenic region is not aneutralizing antigenic region [J]. Vet Microbiol,2012,159(3–4):273-281.
[119] Kay S. Faaberg, Siba K. Samal. Induction of type I interferons by a novel porcinereproductive and respiratory syndrome virus isolate[J]. Virology.2012,432(2),261-270.
[120] Ansari IH, Kwon B, Osorio FA, et al. Influence of N-linked glycosylation ofporcine reproductive and respiratory syndrome virus GP5on virus infectivity,antigenicity, and ability to induce neutralizing antibodies[J]. J Virol.2006,80(8):3994-4004.
[121] Z.Z.Wei, F Gao, Y F Jiang, et al. The effect of the position of N-link glycosylationsites within hypervariable region of GP5on virus neutralization[A]. Beijing:2013International PRRS symposium[C].163.
[122] Costers S, Lefebvre DJ, Goddeeris B, et al. Functional impairment ofPRRSV-specific peripheral CD3+CD8high cells[J]. Vet Res.2009,40(5):46-53.
[123] John Schwartz, Michael Murtaugh, and Juan Abrahante. The pig heavy and lightchain immunoglobulin repertoires provide insights into the adaptive immune responseagainst porcine reproductive and respiratory syndrome virus (PRRSV)[J]. The Journalof Immunology,2012,188,160.4.
[124] Vashisht K,Goldberg T L,Husmann R J,et al.Identi-fication of immunodominantT-cell epitopes present inglycoprotein5of the North American genotype of por-cinereproductive and respiratory syndrome virus[J].Vaccine,2008,26(36):4747-4753.
[125] Díaz I, Gimeno M, Darwich L, et al. Characterization of homologous andheterologous adaptive immune responses in porcine reproductive and respiratorysyndrome virus infection[J]. Vet Res.2012,43:30
[126] Raymond R. R. Rowland, Joan Lunney, Jack Dekkers. Control of porcinereproductive and respiratory syndrome (PRRS) through genetic improvements in diseaseresistance and tolerance[J]. Front Genet.2012;3:260.
[127] Joan K Lunney, Juan Pedro Steibel, James M Reecy, et al.Probing genetic control ofswiner esponses to PRRSV infection: current progress of the PRRS host geneticsconsortium[J]. BMC Proceedings.2011,5(Suppl4):30-35.
[128] Robert Parker, K. Nechvatalova, L. Dupuis, et al. Adjutants for live PRRS vaccines.2011[C]. Chicago: International PRRS Symposium.34.
[129] Xiangdong Li, Amy Galliher-Beckley, Hongzhou Huang, et al. Peptide nanofiberhydrogel adjuvanted live virus vaccine enhances cross-protective immunity to porcinereproductive and respiratory syndrome virus [J]. Vaccine,2013,31(41):4508-4515.
[130] Caiwei Chen, Jing Li, Yuhai Bi, et al. Synthetic B-and T-cell epitope peptides ofporcine reproductive and respiratory syndrome virus with Gp96as adjuvant inducedhumoral and cell-mediated immunity[J]. Vaccine,2013,31(14):1838-1847
[131] Nilubol D, Platt K B, Halbur P G, et al. The effect of a killed porcine reproductiveand respiratory syndrome virus (PRRSV) vaccine treatment on virus sheddingin previously PRRSV infected pigs[J]. Vet Microbiol,2004,102(1-2):11-18.
[132] Daniel C.L. Linhares, Jean Paul Cano, Thomas Wetzell, et al. Effect ofmodified-live porcine reproductive and respiratory syndrome virus (PRRSv) vaccine onthe shedding of wild-type virus from an infected population of growing pigs[J]. Vaccine,2012,30:407-413.
[133] E. Pileri, E. Gibert, F.Soldevila, et al. Quantificaton of PRRSV transmission: effectof pig vaccination.2013[C].Beijing: International PRRS Symposium.67.
[134] M.Roca, M.Gimeno, S.Bruguera, et al. Effects of challenge with a virulentgenotype II strain of porcine reproductive and respiratory syndrome virus on pigletsvaccinated with an attenuated genotype I strain vaccine[J] The Veterinary Journal,2012,193(1):92-96.
[135] Wu J, Lj JQ, SunM S, et a1. The Immune Response Induced by RecombinantAdenovirus with Administration of Interaperitonea Injection[J]. Virologica Sinica,2002,17:51-55．
[136] Meng X J. Heterogeneity of porcine reproductive and respiratory syndrome virus:implications for current vaccine efficacy and future vaccine development[J].VetMicrobiol.2000,12:309-329．
[137] Ha M.Truong, Z.Lu, GeraldF.Kutish, et al. A highly pathogenic porcinereproductive and respiratory syndrome virus generated from an infectious cDNA cloneretains the in vivo virulence and transmissibility properties of the parental virus[J].Virology.2004,325,308-319.
[138] L. Enjuanes, vaccine vectors based on coronavirus genomes to protect againstPRRSV and SARS-CoV.2011[C]. Chicago: International PRRS Symposium.6.
[139] G.Z.Tong, Y.Z.Yu, Y.J.Zhou, et al. Development and application of a dual-markervaccine for highly pathogenic PRRS.2013[C].Beijing: International PRRSSymposium.47.
[140] JazminaL.G.Cruz, SoniaZniga, MartinaBcare, et al. Vectored vaccines to protectagainst PRRSV[J]. Virus Research.2010,154:150-160.
[141] Hiep L.X. Vu, Byungjoon Kwon, Marcelo de Lima, et al. Characterization of aserologic marker candidate for development of a live-attenuated DIVA vaccine againstporcine reproductive and respiratory syndrome virus [J].Vaccine,2013,31(40):4330-4337
[142]王朝文，吴润，王一成，等。大肠杆菌LTB增强PRRSV-GP5蛋白免疫反应的研究[J].甘肃农业大学学报，2010,45（6）:12-17.
[143] CHEN Hong-ying, WEI Zhan-yong, ZHANG Hong-ying, et al. Use of a MultiplexRT-PCR Assay for Simultaneous Detection of the North American Genotype PorcineReproductive and Respiratory Syndrome Virus, Swine Influenza Virus and JapaneseEncephalitis Virus[J].Agricultural Sciences in China,2010,9(7):1050-1057.
[144] R Sina, V Lazar, R Pogranichniy. Real time PCR detection of the PRRSV genomeusing a single primer set.2011[C]. Chicago: International PRRS Symposium.27.
[145] E. Redondo, I. Gil, M. Garcia-duran, et al. Development of real timeRT-PCR(RRT-PCR) commercial kit for reliable detection and typing of PRRSV.2013[C].Beijing: International PRRS Symposium.54.
[146] Sautto G, Mancini N, Diotti R A, et al. Anti-hepatitis C virus E2(HCV/E2)glycoprotein monoclonal antibodies and neutralization interference[J]. Antivir Res,2012,96(1):82-89.
[147] Rockman S, Camuglia S, Vandenberg K, et al. Reverse engineering the antigenicarchitecture of the haemagglutinin from influenza H5N1clade1and2.2viruses withfine epitope mapping using monoclonal antibodies[J]. Mol Immunol,2013,53(4):435-442.
[148] Yang M, Holland H, Clavijo A, et al. Production of monoclonal antibodies againstwhole virus particles of foot-and-mouth disease virus serotype O and A and theirpotential use in quantification of intact virus for vaccine manufacture[J]. Vaccine,2008,26,(27–28):3377-3382.
[149] H. Liu, L. Mu, M.Yang, et al. The establishment of the PRRS detection liquidblocking ELISA method.2013[C].Beijing: International PRRS Symposium.57.
[150] J.Y.Song, E.J.Choi, E.J.Kim,et al. Detection of PRRSV using the light s catteringassay based on the mcrofluidic chip.2013[C].Beijing: International PRRSSymposium.71.
[151] C.N.Lin comparison of virema of type2PRRSV infected pigs by zip nucleic acidprobes based real-time PCR.2013[C].Beijing: International PRRS Symposium.64.
[152] Patrick Gonin, Boroushan Pirzadeh, Carl A. et al. Seroneutralization of porcinereproductive and respiratory syndrome virus correlates with antibody response to theGP5major envelope glycoprotein. J Vet Diagn Invest.1999,11:20-26.
[153] Z.Q.Huang, H.Liu, M.Yang,et al. preparation of monoclonal antibody againstPRRSV nsp9gene establishment of block-ELISA method[A]. Beijing:2013InternationalPRRS symposium[C].84.
[154] Xu X G, Wang Z S, Zhang Q, et al. Baculovirus as a PRRSV and PCV2bivalentvaccine vector: Baculovirus virions displaying simultaneously GP5glycoprotein ofPRRSV and capsid protein of PCV2[J]. J Virol Methods,2012,179:359～366.
[155] M.Achacha，A. Khyar, A. Bensari. High level protein expression of a syntheticPRRSV nuclecapsid gene optimized in E.coli host.2011[C]. Chicago: InternationalPRRS Symposium.23.
[156] O J Lopez, F A Osorio. Role of neutralizing antibodies in PRRSV protectiveimmunity[J]. Veterinary Immunology and Immunopathology,2004,102:155–163.
[157] Pirzadeh B, Gagnon C A, Dea S. Genomic and antigenic vatiations of porcinereproductive and respiratory syndrome major envelope ORF5glycoprotein. Can J VetRes,1998,62(3):170-177.
[158] K.Ouyang, B.Binjawadagi, J.Hiremath, et al. Development and validation ofPRRSV specific neutralizing antibody detection assay in pig oral fluid samples.2013[C].Beijing: International PRRS Symposium.98.
[159] R.J.Langenhorst, S. Lawson, A. Kittawornrat, et al. Simultaneous detection ofantibodies against PRRSV nsp7and nucleocapsid protein in swine oral fluid and serausing a fluorescence microsphere immunoassay.2011[C]. Chicago: International PRRS.
[160] Prather RS, Rowland RR, Ewen C, et al. An intact sialoadhesin(Sn/SIGLEC1/CD169) is not required for attachment/internalization of the porcinereproductive and respiratory syndrome virus (PRRSV)[J]. J Virol.2013,87(17):9538-46.
[161] Stadejek T, Oleksiewicz MB, Potapchuk D,et al. Porcine reproductive andrespiratory syndrome virus strains of exceptional diversity in eastern Europe support thedefinition of new genetic subtypes[J]. J Gen Virol.2006,87(7):1835-41.
[162] M Frias, J Schmid, F Kuhn, et al. discrimination of PRRSV type1and type2by anindirect ELISA.2013[C].Beijing: International PRRS Symposium.70.
[163]郎广平,郭敏,韩盈盈,等。猪繁殖与呼吸综合征诊断方法与疫苗研究进展[J].中国畜牧兽医，2013，40(6):195～198.
[164]李玉峰，王先炜，陈闻，等。猪繁殖与呼吸综合征病毒nsp2基因缺失株RT-PCR检测方法的建立[J]。南京农业大学学报，2009,32（3）：169-171.
[165] Dan Cheng,Jian-Jun Zhao, Na Li, et al. Simultaneous detection of Classical swinefever virus and North American genotype Porcine reproductive and respiratorysyndrome virus using a duplex real-time RT-PCR[J]. J Virol Meths.2008,151,194-199.
[166] E.Martnez, P.Riera, M.Sitja, et al. Simultaneous detection and genotyping ofporcine reproductive and respiratory syndrome virus (PRRSV) by real-time RT-PCRand amplicon melting curve analysis using SYBR Green[J]. Research in VeterinaryScience.2008,85:184-193.
[167]刘秀梵.单克隆抗体在农业上的应用[M].合肥：安徽科学技术出版社.1994:21-24.32-36.
[168]王世若,王兴龙,韩文瑜.现代动物免疫学[M].2版.长春:吉林科学技术出版社,1997.
[169]徐志凯.实用单克隆抗体技术[M].西安：陕西科学技术出版社,1991:125-126.
[170]孙晨,王丽,孙秀萍,等.水泡性口炎病毒单克隆抗体的制备与鉴定[J].中国兽医学报,2012,32(3):411-414.
[171]万博，乔松林，张改平，等。猪繁殖与呼吸综合征病毒不同毒株感染对猪外周血免疫细胞含量的影响.畜牧兽医学报，2009,40（11），1695-1698.
[172]孙建富，郝建伟，荣福龙，等。猪繁殖与呼吸综合征病毒的分离与鉴定及ORF5基因的序列分析[J]。中国畜牧兽医，2011，38（9）：138-142。
[173] T. Opriessnig, M. Fenaux, S. Yu,et al. Effect of porcine parvovirus vaccination onthe development of PMWS in segregated early weaned pigs coinfected with type2porcine circovirus and porcine parvovirus[J]. Veterinary Microbiology,2004,98:209-220.
[174] Martin-Valls G E, Burgara-Estrella A J, Darwich L, et al. ecombination events arecommon in genotype1and2PRRS virus worldwide and may generate mosaic
viruses[A].2013International PRRS symposium[C].13.
© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号 地址：北京市海淀区学院路29号 邮编：100083 电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700 |