【作者】 蒋增海；

【导师】 陈焕春； 金升藻；

【作者基本信息】 华中农业大学 ， 预防兽医学， 2004， 硕士

【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引起的一种新的、高度传染性疾病，临床上以母猪发热、厌食、早产、流产、木乃伊胎、死胎、弱仔等繁殖障碍及各种年龄猪的呼吸症状和仔猪的高死亡率为特征。该病最早于1987年报道于美国南部，现已遍及世界各地，给全球养猪业造成巨大的经济损失。猪伪狂犬病是由疱疹病毒科α疱疹病毒亚科的伪狂犬病毒（PRV，亦称猪疱疹病毒Ⅰ型）引起的一种急性传染病，在临床上，主要引起母猪流产、死胎和产木乃伊胎等繁殖障碍，初生仔猪呕吐、拉稀、呼吸困难、体温升高，15日龄内的仔猪死亡率几乎高达100％。近年来，全国各地很多猪场都出现了以母猪繁殖障碍、仔猪呼吸困难为特征的传染病。为了弄清病因，解决生产实际，特此开展了猪繁殖与呼吸综合征病毒和猪伪狂犬病病毒的分离和鉴定工作。 1 猪繁殖与呼吸综合征病毒分离、鉴定 利用MARC-145细胞，从湖北、湖南、江西、河南、浙江等地发病仔猪和流产胎儿的肺脏中分离到疑似PRRSV多株，并对其中一株病毒进行了全面的鉴定。该病毒在MARC-145细胞上增殖滴度为10^-4.39/0.1ml，对氯仿敏感，56℃，1h完全灭活，不耐酸碱，不能被5’-碘脱氧尿核苷（IUDR）所抑制。在电镜下，病毒粒子呈球形，有囊膜，大小约为50～65nm。能被美洲型PRRSV阳性血清所中和，而不能被抗猪瘟病毒、猪伪狂犬病病毒、猪细小病毒、猪乙型脑炎病毒等的阳性血清中和。为了进一步从分子生物学进行鉴定，设计了特异性引物对病毒的囊膜蛋白基因（ORF6）进行了RT-PER扩增，扩增到大小约为500bp的片段，与预期大小相当，而阴性对照则不能扩增出相应大小的片段。根据以上结果，证实所分离的病毒为猪繁殖与呼吸综合征病毒，并且属于美洲型，命名为PRRSV臥株。 2 猪繁殖与呼吸综合征病毒ORF5和ORF7基因的克隆及序列分析 根据GenBank上PRRSV VR-2332株的核苷酸序列，设计两对分别包含ORF5和ORF7基因完整编码区的引物。以本实验室分离的PRRSV HA株为模板，通过RT-PCR扩增出ORF5和ORF7全基因。克隆到pMD18-T载体上，获得重组质粒pMD-ORF5和pMD-ORF7，双脱氧末端终止法测序。结果显示PRRSV HA株ORF5基因大小为603bp，ORF7基因的大小为372bp。使用Blast软件分析发现PRRSV HA株ORF5基因和ORF7基因与美洲型代表株VR-2332株、中国分离的Bj-4株、欧洲型代表株LV株的核苷酸同源性分别是98％、99％、70％，99％、99％、93％。由该毒株的ORF5基因的核苷酸序列推导的氨基酸序列与上述3个毒株的同源性分别猪繁殖与呼吸综合征病毒和猪伪狂犬病毒分离、鉴定是94%、94%、57%。这一结果也进一步证实了所分离的毒株为美洲型。3猪伪狂犬病病毒的分离及初步的鉴定 从湖北宜城市某猪场流产胎儿的内脏组织中分离到一株PRV，并进行了初步的鉴定。该病毒在BHK一21、IBRS一2、PK一15、MARC一145等细胞上均可以引起典型的细胞病变。在PK一巧细胞传代后，测得其TCI Dso为10-6’“9/0 .lml。该分离病毒可以被PRV标准阳性血清中和;以PRV标准阳性血清为一抗，进行间接免疫荧光检测，在荧光显微镜下可以观测到特异性的荧光反应。进一步利用特异性引物以所分离病毒的DNA为模板，扩增PRV gD基因，扩增到大小21 7bp的片段，与PRV鄂A株为模板扩增的gD基因片段大小一致。通过这些实验证实所分离的病毒为PRv，命名为PRv Yc株。用TcID50为10一“9/0 .lml的PRv Yc株病毒液，经腹腔接种昆明小鼠3只，每只0.2ml，3一5天后，所有小鼠死亡，而对照组2只的小鼠没有任何反应。说明所分离病毒对小鼠的毒力较强。更多还原

【Abstract】 Porcine reproductive and respiratory syndrome (PRRS) is a new and highly infectious swine disease. The disease was characterized by inappetence, fever and respiratory disease in pigs of any age , abortion or increased proportions of stillbirn and infirm piglets in pregnant sows and high mortality in pre-weaning piglets. Although porcine reproductive and respiratory syndrome (PRRS) was first reported in swine herds in the United States in 1987, it spread rapidly and became endemic in most of the major swine-producing areas throughout the world during the 1980, bringing an enormous loss in swine production in the world. Today, it is one of the most economically important diseases of the global pork-producing community. Pseudorabies is an acutely infected disease caused by a swine herpesvirus belonging to the sub-family Alphaherpesvirinae. Typical clinical signs of pseudorabies virus(PRV) infection in susceptible animals include abortion,dead fetuses and mummified fetuses in pregnant sows, fearing cold, quivering, having loose bowes and vomitting in shortly born piglets and 100% mortality in pre-15-day pigs. In recent years, most of cases from many farms indicated respiratory problems in piglets and reproductive failaure in pregnant sows . In order to investigate the causative agent of these cases, PRRSV and PRV was isolated and identificated.1. Isolation and identification of PRRSVSeveral doubted PRRSV srains were isolated from visceral organs of dead fetuses and piglets from the farms in Hubei, Hunan, Jiangxi, Henan and Zhejiang ect. One of the strains was identified. After the isolate was inocaluted on MARC-145 for several genenrations, the result of its’TCID50 test was 10-4.39/0.1ml.The isolate was sensitive to chloroform, completely inactivated when exposed to 56 for one hour, and could not be inhibited by IUDR when it replicated on MARC-145 cells. The diameter of virus particle was about 50~65nm. The isolated virus was spherical and has envelope. Virus neutralization test showed that the isolate was neutralized by the serum to American genotype of PRRSV, not by the sera separately agaist HCV PPV PRV JEV. The fragment of about 500bp ORF6 gene of PRRSV was amplified from the isolated virus by RT-PCR. However this 500bp fragment was not amplified from the negative control. According to the above results, the isolated virus was identificated as American geotype PRRSV and named PRRSV HA strain.2. Cloning and sequence analysis of ORF5 and ORFTgenes ofPRRSV strain HAAccording to Nucleotide acids sequence of ORF5 and ORF7 genes of porcine reproductive and respiratory syndrome virus (PRRSV) strain VR-2332, two pairs of primers were designed , which contain a complete encoding region of ORF5 and ORF7 gene, respectively. Using the pairs of special primers,the ORF5 and ORF7 of PRRSV strain HA were amplified by RT-PCR. After agarose gel electrophoresis,the fragments of about 600bp and that of 400bp were collected , respectively ,then inserted into pMD18-T,building pMD-ORF5 and pMD-ORF7. After recombinant plasmids, pMD-ORF5 and pMD-ORF7,were cloned largely in E.coli DH5a,the ORF5 gene and ORF7 gene in two recombinant plasmid was sequenced respectively.The nucleotide and deduced amino acids sequences indicated that there are 603bp and 200 amino acids in the ORF5 gene of PRRSV strain HA , When compared with PRRSV VR-2332,PRRSV strain Bj-4,and PRRSV strain LV , the homology at nucleotide acid level were 98% 99% 70%; the homology at deduced amino acid level were 94% 94%, 57%,respectively . The sequenced result of ORF7 was 372bp. When the sequence of ORF7 was used to compare with the above PRRSV strains , the homology values were 99% 99% 93%,respectively.3. Isolation and identification of PRVIn a large-scale farm, located in YiCheng city of Hubei province, reproductive failure was observed in 90% of pregnant sows, showing abortion, dead futuses, ect. We isolated a strain of PRV from visceral organs of dead fetuses, and carried out preliminary identification . Cuturing the virus on BHK-21 cells, PK-15 cells, IBRS-2更多还原