【作者】 侯振中；

【导师】 王林安；

【作者基本信息】 东北农业大学 ， 兽医外科学， 2003， 博士

【摘要】 猪繁殖与呼吸综合征(Porcine Reproductive and Respiratory Syndrome,PRRS)自1987年首次于美国爆发，主要引起母猪的流产，如死胎和木乃伊胎以及各种年龄猪的呼吸道疾病。其病原为猪繁殖与呼吸综合征病毒(Porcine Reproductive and Respiratory Syndrome Virus,PRRSV)，该病毒首先与1991年由荷兰Wensvoort发现，命名为Lalystad病毒。该病毒系动脉炎病毒家族成员之一，其基因组含有9个开放阅读框，总长度为15kb，其中ORF1a和ORF1b编码病毒的复制酶，ORF2～ORF7编码病毒的结构基因。 1995年郭宝清等首次从国内疑似PRRS的猪群分离到PRRSV，从而证实我国也有PRRSV的流行，随后在我国大部分地区相继检出PRRSV。为查明我国不同地区PRRSV的来源及遗传学差异，为研制有效疫苗提供分子生物学根据，本研究在获得CH-1a株完整的基因组序列以后，根据Genebank发表的序列，设计了不同的引物对，利用PCR技术，对对流行于我国山东(SD株)和大庆地区(DQ株)的PRRSV结构基因ORF2～ORF7各结构基因扩增条件进行优化，获得了不同的目的基因片段然后进行序列分析，并将其与CH-1a株、BJ-4株、LV株以及VR-2332株进行了比较分析。结果表明：流行于我国不同地区的PRRSV代表株碱基及核苷酸组成基本相同，与美洲株的同源性为90％以上，而与欧洲株相差较远，同源性仅为54.1％。在发生突变的核苷酸序列中，80％以上是A-G，T-C之间的转换。除已经确定的OH-1a株已经认定为美洲型外，我国所流行的SD毒株和DQ毒株也均为美洲型，但又存在着地域性的差异。 为深入研究PRRS的发病机理和PRRSV对断奶仔猪致病情况，本试验利用美洲型代表毒株VR-2332接种妊娠后期母猪和断奶仔猪，寻找病理变化特征和确定病毒组织分布，以便为有效预防、诊断和有针对性治疗提供理论依据。应用VR-2332强毒人工感染4头妊娠末期母猪和5头断奶仔猪，复制典型病例，对死胎和弱仔及感染断奶仔猪进行组织病理学检查，并用间接免疫荧光试验检测病毒组织分布。结果显示PRRS患猪具有典型的病变：病理组织学显示出肮脏有以肺泡膈增宽并有巨噬细胞增生为特点的增生性间质性肺炎，以及发生广泛性微循环障碍所引起的病理变化，这在其他呼吸道疾病中不是同时存在的，有望作为PRRS的示病症状，通过间接免疫荧光试验发现，病毒侵害最重的是免疫系统。 用美洲型代表毒株VR-2332强毒株和弱毒株接种断奶仔猪，通过流式细胞仪检测了接种后的仔猪CD4~+和CD8~+T淋巴细胞的变化，发现PRRSV强毒感染后，CD4~+和CD8~+ T淋巴细胞含量没有明显变化，表明PRRSV强毒的细胞免疫调节功能较弱。PRRSV弱毒感染后，则引起迅速而强烈的CN4~+和CD8~+ T淋巴细胞数量动态变化，表明所使用的弱毒株对仔猪细胞免疫具有较强的调节作用，是用于预防PRRS疫苗的较佳候选毒株。 通过上述的实验研究，实现了对国内不同地区PRRSV代表株的结构基因解析，确定了国内流行的SD毒株和囚毒株毒株均属子美洲型，与美洲型毒株具有高度的同源性。通过确定病毒侵害细胞和组织的种类和部位，为有针对性诊断指明采样方向。并通过监测对PRRSV感染猪细胞免疫和体液免疫，了解了PffeV感染时猪体免疫功能的变化规律。这不仅为疫苗研制提供分子生物学基础，而且对建立快速的Its＄病理学诊断和合理的兔疫方案以及高效灵敏的诊断方法忆I］造良好条件。更多还原

【Abstract】 Porcine Reproductive and Respiratory Syndrome (PRRS) broke out first in America in 1987, and then went through worldwide quickly with clinical signs of abortion, such as death, mummy and respiratory diseases in pigs with various age. The pathogen of PRRS, porcine reproductive and respiratory syndrome virus (PRRSV), was first detected by Wensvoort in 1991 in Holland, and named Lalystad virus. The virus is one of the members of the artivirus family. The genome of PRRSV is 15kb in length, encoding 9 ORFs. ORFla and ORFlb, the longest ORF, encode the polymerase of PRRSV, ORF2-ORF7 encode the structural gene of PRRSV.The first PRRSV in China was isolated in swine with suspected PRRS in 1995 by Baoqing Guo, confirming that PRRSV had spreaded in China, and then followed by other PRRSV found in most of China. This paper acquired the target gene fragments of the different structural genes by PCR after designing different pairs of primers which were based on the published sequences from Genebank and optimizing the amplifying conditions after acquiring the whole sequence of CH-la strain in order to clarify the source and genetic background of PRRSV from different parts of China, thus providing molecular basis for vaccine research. The sequences of the structural genes of PRRSV from Shandong province and Daqing area were analyzed and compared with those of CH-la strain, BJ-4 strain, VR-2332 and LV strain. Result: The base composition was basically the same among different PRRSV strains in China Among those nucleosides that changed, more than 80% were transformed between A-G and T-C. The results show that all PRRSV strains in China are closely related to the strain VR-2332. Among different isolates in China, ORF6 displayed the most significant changes, while the ORF7 was the most conservative. The obvious CPE could not be seen until the fourth passage when Mark-145 cells were vaccinated with PRRSV field strain, and 70% - 80% cells showed CPE in the sixth passage in only 2~3 days, indicating that PRRSV litres could increase.Localization of PRRSV in vivo and the pathology of PRRSV-infected porcine have been studied extensively, however, no exact criterion has been achieved. Therefore, this study used VR-2332 to inoculate the pregnant swine and piglets. The result indicates that the patient had the symptoms of pulmonary cirrhosis and wide microcirculation malfunction, which may be used as an index for the diagnosis of PRRS. With the indirect immune fluenscence we found that VR-2332 located mainly in immune tissue, such as spleen, and stayed in macrophages and vascular endothelial cells determined by IFA.The dynamic changes of CD4+ and CD8+ T lymphocytes after vaccinated with rivulent and weak VR-2332 strains were studied using flow cytometry. The results was that the virulent strain could not cause significant changes of CD4+ and CD8+ lymphocytes, indicating that rivulent virus did not have obvious effects on the cellular inmmunity. But after the weak virus strain infection the CD4+ and CD8+ T lymphocytes significant changes. It indicated that weak virus had very strong effects on the cellular immunity. The results indicated that this weakIIIVR-2332 strain had distinct immune-regulation to piglets, so the weak virus was the best vaccine against PRRS.In summary, mis experiment finished the analysis of structural genes of PRRSV from different areas of China The determination of the site of PRRSV infection in vivo gives a new way to sample the patient and the immunological changes of the patient were snowed through the detection of cell immunity and humoral immunity after infected by PRRSV, which all belong to American strain. Through monitoring the changes of cell immunity and humoral immunity after infected with PRRSV, we have obtained the immune regular patterns. The results can set biological foundation for vaccine researching, and provide excellent requirement for establishing immediately reasonable immune program and high efficiency, accurately diagnosis methods.更多还原