【作者】 刘永宏；

【导师】 王凤龙；

【作者基本信息】 内蒙古农业大学 ， 基础兽医学， 2010， 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)引起猪的一种传染病,表现为母猪生殖衰竭和仔猪、成年猪呼吸困难,是影响养猪业最大的传染病之一。PRRSV属于动脉炎病毒科成员,基因组长度约为15kb～15.5kb,至少含有9个开放阅读框架,编码20种以上成熟的蛋白质。根据基因型和抗原性的差异将PRRSV分为北美洲型和欧洲型两种,各自代表株分别为VR-2332株和LV株。2006年在中国暴发了空前规模巨大的猪高热症,感染猪达200万头以上,至少40万头死亡,这给中国经济带来巨大的损失,也严重威胁了全球养猪业和世界公共卫生。本研究为了分析猪高热症的真正原因,通过对北京各郊区县和周边地区15个猪养殖场(户)55个病例进行发病情况、流行病学调查和临床症状观察,以及通过对发病猪和病死猪剖检、病理组织学观察、免疫组化染色和电镜观察,同时对冰冻病料和血清做了核酸和抗体检测。研究结果表明:PRRSV核酸阳性率为60%,猪圆环病毒2型(Porcine Circovirus Type 2, PCV-2)核酸阳性率为32.7%,PRRSV和PCV-2混合感染率为18.2%;此次猪高热症的主要病原是PRRSV,发病特点不同于以往典型的PRRS,同时有PCV-2的混合感染;PRRS病理组织学病变群主要为间质性肺炎、非化脓性脑炎、出血性坏死性淋巴结炎、急性炎性脾肿、变质性心肌炎、急性间质性肾炎伴随肾病和出血、坏死性扁桃体炎、母猪子宫内膜炎、变质性肝炎和肝细胞可见胞浆内嗜酸性包涵体;PRRSV阳性脏器为肺脏、脾脏、淋巴结、扁桃体、脑、肝脏、肾脏、肾上腺、胃肠和心脏;PRRSV阳性细胞为单核-巨噬细胞、黏膜上皮细胞、淋巴细胞、血管内皮细胞、腺上皮细胞(肺上皮细胞、肝细胞、肾小管上皮细胞等)、杯状细胞、纤维细胞、成纤维细胞、心肌细胞、神经细胞、胶质细胞和软骨细胞。为了分析此次疫情高发病率和高死亡率的原因,本研究将4份没有接种PRRS疫苗RT-PCR鉴定为单一PRRSV阳性的病料,接种MARC-145细胞进行病毒分离,经RT-PCR、IFA和电镜鉴定,表明分离到4株病毒且均为PRRSV,其TCID50均为10-6.5/0.1mL,命名为BJSY-1、BJPG、BJSD和BJBLZ株。进一步设计了相互重叠的16段引物,RT-PCR产物直接纯化、测序和拼接,得到4株PRRSV的全基因序列,运用分子生物学软件进行比对和遗传演化分析,结果显示:本研究分离的BJSY-1、BJPG、BJSD和BJBLZ株基因全长分别为15319nt、15315nt、15345nt和15316nt,通过美国国立生物技术信息中心(NCBI)的审核,GenBank登录号分别为FJ950744、FJ950746、FJ950747和FJ950745;本研究的4株与北美洲型毒株核苷酸同源性在83.3%～99.4%之间,与欧洲型毒株核苷酸同源性只有58.3%左右,均属于北美洲型亚群Ⅱ,且属于HP-PRRSV;ORF6基因与北美洲株同源性和欧洲株同源性均最高,其次是ORF2基因,再次是ORF7、ORF4和ORF5基因,同源性最低的是ORF3基因。本研究选择了HP-PRRSV BJSY-1株变异性最小的ORF6基因,设计合成了一对特异性引物,扩增ORF6基因片段克隆入原核表达载体pET-32a(+),重组质粒pET-ORF6转化BL21宿主菌后,鉴定构建成功后摸索合适条件诱导表达M蛋白,纯化回收得到了可溶性的具有免疫学反应活性的融合蛋白HIS-M,分子量约为39kDa。本研究得到了一种稳定的、免疫原性好的抗原,为制备单抗或作为检测抗原奠定了基础。考虑到PRRSV抗体和抗原双阴性猪不易获得,试图寻找一种标准的且对PRRSV易感的实验动物代替猪来研究PRRSV,同时为了验证致病力增强的HP-PRRSV BJSY-1毒株是否可以感染其它动物。本研究对BALB/c小白鼠和日本大耳白兔进行了不同剂量的人工感染实验,攻毒后观察体温、体重等变化,按照实验设计定期剖检取材进行PRRSV核酸检测和病理学检查。结果显示:小白鼠和兔体温、体重等无异常变化,PRRSV核酸检测阴性,没有出现PRRSV特异的病理组织学变化。本研究证实了HP-PRRSV不感染BALB/c小白鼠和日本大耳白兔。更多还原

【Abstract】 The Porcine Reproductive and Respiratory Syndrome (PRRS) is one of the economically most important swine diseases worldwide, is one of the most economically influential infectious diseases in the industry of swine cultivation. Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative agent, a member of the family Arteriviridae in the order Nidovirales, PRRS is characterised by reproductive failure of sows and respiratory problems of piglets and growing pigs. Genomic analysis of PRRSV has shown that the virus genome varies from 15kb to 15.5 kb and comprises at least 9 open reading frames (ORFs) that encode nearly 20 mature proteins. PRRSV with different geograpHic origins and antigenic can be classified into 2 major genotypes, the European type (type I, EU-type) and North American type (type II, NA-type), Lelystad virus and isolation of VR2332 is the European prototype and the North American prototype PRRSV, respectively. The unprecedented large-scale porcine high fever disease (PHFD) outbreaks in 2006 swept over nearly half of the People’s Republic of China and infected >2,000,000 pigs, dead >40,000 pigs, which posed great concern to the global swine industry and to public health.This study is for analying the true cause of porcine high fever disease, through to investigate 55 cases of observing the morbidity situation, the epidemiology and the clinical symptoms in the suburban country of Beijing and to15 pig farms in the surrounding areas, and through anatomical examine, the histopathological observe, immunohistochemical stains and observing under electron microscope to the disease pig and death pig, meanwhile making a nucleicacid and antibody test to the frozen samples and serum, respectively. The study result indicates that the positive rate of PRRSV nucleicacid is 60%, PCV-2 nucleicacid is 32.7%, and the rate of PRRSV and PCV-2 mixed infection is 18.2%. In this porcine high fever disease, the main pathogen is PRRSV. The invasion characteristics are different with the typical PRRS which formerly appeared, meanwhile the PCV-2 has mixed infection. The PRRS histopathological groups mostly are interstitial pneumonia, nonsuppurative encepHalitis, hemorrhage necrotizing lympHadenitis and acute inflammatory splenectasis, degenerate myocarditis, acute interstitial nepHritis accompanying to bleed, necrotic amygdalitis, endometritis of sow, alterative hepatitis, eosinopHilic inclusion in cytoplasm of hepatocytes. The positive organ of PRRSV are lung, spleen, lympHaden, amygdala, brain, liver, kidney, suprarene, stomach, intestines and heart. The positive cells of PRRSV are monocaryon-macropHage, epithelial cells, mucosa cellula epithelialis, lympH-cells, vascular endothelial cells, glandular epithelium cells (pulmonary epithelial cells, hepatocyte, enal tubular epithelial cells, and so on), caliciform cell, fibrocyte, fibroblast, cadiocyte, neurocyte, gliocyte and chondrocyte. In order to analyze the outbreak of high morbidity and mortality, In this study 4 tissue samples which without inoculated PRRS vaccine and were positive by RT-PCR identifying inoculated to MARC-145 cells for virus isolation. By RT-PCR, IFA and electron microscopy analysis proved that all the 4 strains of virus were PRRSV, the TCID50 were 10-6.5/0.1mL, named BJSY-1, BJPG, BJSD and BJBLZ strains. Further designed 16 overlapping primers, RT-PCR products were directly purified, sequenced, and spliced, we had got 4 complete genome sequence of PRRSV. Using molecular biology software gene comparison and evolution analysis, the results showed: The separation BJSY-1, BJPG, BJSD and BJBLZ strain complete genome sequences were 15319nt, 15315nt, 15345nt and 15316nt, respectively, through the audit by National Center for Biotechnology Information (NCBI), GenBank accession number were FJ950744, FJ950746, FJ950747 and FJ950745. The nucleotide homology of this Studiy 4 and the North American type strain were 83.3% to 99.4%, and the European-type strains is about only 58.3%. All the strains belong to the North America-based subgroupⅡ, also belong to HP-PRRSV. ORF6 gene homology with the North American and European strains were the highest, followed by the ORF2 gene, is again ORF7, ORF4 and ORF5 gene, the lowest was ORF3 gene.In this syudy, we chose the lowest variability ORF6 gene sequences of HP-PRRSV BJSY-1 strain, designed and synthesized a pair of specific primers. The amplified ORF6 gene fragment was then cloned into the prokaryotic expression vector pET-32a(+),the recombinant plasmid pET-ORF6 was transformed into host cell BL21, after the successful construction, we grope appropriate conditions to induce M protein, and gained the solublefusion protein with immunocompetence in the HIS-Taq?,which was about 39kDa by purification and recycle. We gained an antigen which is stable and have better immunogenicity in this study which lay on foundation for preparation monoclonal antibody or as detect antigen.We considered that it is difficult to find the swine which antibody and antigen were all negative, so, tried to select the experimental animal which susceptive for PRRSV instead of porcine on research PRRSV, simultaneously another reason was verify if HP-PRRSV strain which have increased pathogenicity could infected other animals. In this study, we did the artificial infection experiment on BALB/c mice and Japanese big ear rabbits with different doses, then observed the variation of temperature and weight, According to the experimental design regular necroscopy and drawn materials for detecting nucleic acid and pathological examination on HP-PRRSV BJSY-1 strain. The results showed that: the change of the temperature and weight of the mice and the rabbits was no abnormalities, it was negative to detecting nucleicacid and not show distinctive histopathological changes on PRRSV. This study indicated that the HP-PRRSV strain did not infected BALB/c mice and Japanese big ear rabbits.更多还原