摘要
2006年夏秋季节,我国南方部分地区猪群出现了以高热、厌食或不食、眼结膜炎、咳嗽、喘等呼吸道症状、后躯无力不能站立或摇摆等神经症状以及高死亡率为主要临床特征的一种新的传染病,当时由于具体病因不明,所以被称为猪的“高热病”。鉴于其对我国经济和国民生活产生的巨大影响,诊断、分离和鉴定该病原以发现更为有效的监测方法和控制策略是非常必要的。
本研究用在湖北地区采集的组织病料在仔猪体上进行病例复制,采集产生典型病变猪的组织毒分别用各种原代细胞和传代细胞系进行分离、鉴定,测定TCID50,RT-PCR扩增其高变区和全部蛋白编码区,电镜观察各组织器官病变情况。
1、将肺部病料处理后分别用猪肺泡巨噬细胞、猪原代肾细胞、Marc-145、PK-15、BHK-21等原代细胞和传代细胞系进行病毒的分离与鉴定,结果只在Marc-145细胞上有典型病变,连续传代3次后病变稳定,接毒48h～72h后病变可达70%左右,传至第7代时TCID50可达10-5.51/ml。由此证实本研究成功地分离到一株高致病性蓝耳病病毒(将其命名为HBGS)。所分离到的毒株致病力强,目前已传至第14代,可通过进一步有限传代为疫苗研制提供一个良好的候选毒株。
2、对病毒高变区和全部的蛋白编码区的核酸序列比对发现,HBGS分离株与欧洲型标准株LV株只有66.2%的序列一致性,与美洲型标准株ATCC VR-2332株的核苷酸一致性可达91.8%,和先前的CH1a、HB-1、BJ-4等中国分离株的核苷酸的一致性分别为96.2%、97.1%和91.6%。
3、采集复制病例猪的肺、肾、肝、心、脾和淋巴结等主要病变脏器组织进行电镜组织学观察:肺脏出现典型的支气管肺炎症状,肝脏淤血并有不同程度的颗粒变性,脾脏广泛出血、淤血和大量细胞坏死,肾小管上皮细胞广泛坏死并伴有一定程度的肾小球肾炎,心肌纤维广泛坏死并可见少量颗粒变性。
4、本研究通过条件优化建立了一个针对ORF7和Nsp2阅读框的较为敏感的检测方法,为本病的监测和分子流行病学的调查奠定了坚实的基础。
An unparalleled large-scale of an originally unknown so-called“high fever”disease in China outbreak in 2006, which characterized by high fever, eye conjunctivitis, cough, asthma, neurological symptoms and high mortality. Because of serious influence of this disease to the national economy, it is very essential to diagnose, isolate and identify the causative agent.
Artificial case in 30-day-aged pigs was made using the disease materials collected from Hubei province, isolated and identified was by some kinds of primary cells and cell lines,and tested TCID50, RT-PCR amplifyed the sequence of Nsp2 and the structural protein area.The pathological changes of the organs or tissues was observed under the microscope..
1. Isolate on of the virus using the PAMs, pig kidney primary cells, Marc-145, PK-15 and BHK-21:it turned out that only the Marc-145 cells line had the clear CPE, and the CPE could reach 70% at 48h-72h after 3 passages of the virus, as well as TCID50 can reach 10-5.51/ml at 7th passage. So a high pathogenic PRRSV stain was abtained successfully (named HBGS). This isolation strain has a high pathogenicity which has passaged for 14th generation by now, and may be a good candidate strain for vaccine.
2. Comparison of sequence of Nsp2 and structural protein area: A conclusion can be got that the homology between HBGS and the European standard strain LV stain, the American standard strain ATCC VR-2332 stain were 66.2% and 91.8% respectively, the homology between HBGS and CH1a, HB-1, BJ-4 were 96.2%, 97.1%, 91.6% respectively. The result above proves that the real causative agent was probably Chinese local varied PRRSV strain.
3. Histological observation on lung, kidney, liver, heart, spleen and lymph:It showed that bronchopneumonia in lung cells, congestion and different degree of granular degeneration in liver cells, hemorrhage, congestion and large-scaled cells necrosis in spleen cells, glomerulonephritis in kidney cells, large-scaled cells necrosis and granular degeneration in heart cells. All of them manifested that the high pathogenic PRRSV can cause multisystem and multi-organ failure, of which the main lesion is in lung.
In this research a sensitive detection assay have been designed based on ORF7 and Nsp2 which can set a concrete foundation for PRRS monitoring and molecular epidemiology investigation.
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