猪流行性腹泻病毒陕西株的分离鉴定及S基因的遗传变异分析

发布时间：2018-04-13 20:38

本文选题：猪流行性腹泻病毒 + 分离鉴定　；参考：《西北农林科技大学》2017年硕士论文

【摘要】：猪流行性腹泻(Porcine epidemic diarrhea,PED)是由猪流行性腹泻病毒(Porcine epidemic diarrhea virus,PEDV)引起的一种高度接触性肠道传染病,发病特征为腹泻、呕吐、脱水,新生仔猪的死亡率高达100%,各种年龄猪和不同品系的猪群均易感。该病于1971年在欧洲首次被报道后,不久相继在亚洲及南美相继暴发,给全球养猪业造成严重的经济损失。自2010年10月开始,PED在中国东南部10余个养猪大省暴发并迅速波及到全国,呈现出高发病率、高死亡率的态势,死亡仔猪超过100万头,给中国养猪业造成了沉重的打击。近几年PED的流行范围仍在不停的扩大,成为严重危害中国养猪业的传染病之一,直至目前仍没有削减的趋势。为进一步了解猪流行性腹泻病毒的流行特点及遗传变异情况,加强对猪流行性腹泻的防控,本研究对疑似PED的仔猪小肠和粪便样品进行了PEDV的分离与鉴定,并对分离到的毒株的S基因进行了序列测定与遗传变异分析,其主要结果如下:1.PEDV SX-YL株的分离与鉴定:将经RT-PCR鉴定为PEDV阳性的病料样品处理后,接种于Vero细胞,盲传至8代时,部分细胞开始出现皱缩、变圆、颗粒物质增多、折光性增强等CPE,对其进行TCID50测定,TCID50为10-6.125/mL。为证明该病变是由PEDV所引起,采用RT-PCR对细胞培养物进行了PEDV N基因的扩增,结果均为阳性。进一步进行了间接免疫荧光检测(IFA),结果表明,用PEDV阳性小鼠血清在PEDV感染24h后的Vero细胞中检测到了病毒抗原的存在,可见到红色荧光,而未被PEDV感染的Vero细胞,观察不到荧光。最终确定该病毒为PEDV,将其命名为PEDV SX-YL株。2.PEDV SX-YL株S基因的遗传变异分析:对前面分离到的PEDV SX-YL株的S基因进行测序,结果表明,该毒株S基因全长为4149bp,编码1382个氨基酸,与欧洲经典毒株CV777、韩国分离株DR13、欧洲近年流行株以及中国近年流行株的S基因进行比对,该毒株与经典毒株CV777、DR13、SC1402毒株同源性高达99%以上,处于同一分支,表明其与经典毒株的亲缘关系较近。综上所述,该研究从疑似PED的仔猪病料成功分离出一株PEDV毒株,与经典毒株有较近的亲缘关系,为陕西规模化猪场PED科学合理的免疫程序制定与有效防控提供了参考信息。
[Abstract]:Porcine epidemic diarrhea (PED) is a highly contact intestinal infectious disease caused by porcine epidemic diarrhea virus (PEDVV), characterized by diarrhea, vomiting, dehydration.The mortality rate of newborn piglets is as high as 100, and pigs of all ages and strains are susceptible.The disease was reported for the first time in Europe in 1971, and soon after outbreak in Asia and South America, which caused serious economic losses to the global pig industry.The outbreak of PED in more than 10 major pig raising provinces in southeast China began in October 2010 and spread rapidly to the whole country, showing a trend of high morbidity and high mortality. More than 1 million piglets died, which caused a heavy blow to the pig industry in China.In recent years, the epidemic scope of PED is still expanding, and it has become one of the infectious diseases that seriously harms the pig industry in China. Up to now, there is still no trend of reduction.In order to further understand the epidemic characteristics and genetic variation of porcine epidemic diarrhea virus and strengthen the prevention and control of porcine epidemic diarrhea, the small intestine and fecal samples of piglets suspected of PED were isolated and identified by PEDV.The S gene of the isolated strain was sequenced and genetic variation was analyzed. The main results were as follows: 1. The isolates of PEDV SX-YL strain were isolated and identified as follows: the samples identified by RT-PCR as PEDV positive were treated and inoculated into Vero cells for 8 passages.Some cells began to crinkle, round, increase in particle matter, increase in refraction, and other CPEs. The TCID50 of these cells was 10 ~ 6.125% mL / mL by TCID50.In order to prove that the lesion was caused by PEDV, RT-PCR was used to amplify the PEDV N gene of cell culture, and the results were all positive.Further indirect immunofluorescence assay was performed. The results showed that the presence of virus antigen was detected in Vero cells 24 hours after PEDV infection with the serum of PEDV positive mice. Red fluorescence could be seen in Vero cells without PEDV infection, but no fluorescence could be observed in Vero cells infected with PEDV.Finally, the virus was named PEDV SX-YL strain. 2.The genetic variation analysis of S gene of PEDV SX-YL strain: the S gene of PEDV SX-YL strain was sequenced. The result showed that the S gene of the virus strain was 4149 BP, encoding 1382 amino acids.Compared with the classical European strain CV777, the Korean isolate DR13, the European strain of recent epidemic and the Chinese strain of recent years, the S gene of this strain is more than 99% homology with the classical strain CV777, DR13, SC1402, and is in the same branch.The results showed that the relationship between the strain and the classical strain was close.To sum up, a strain of PEDV virus was successfully isolated from the piglets infected with suspected PED, which was closely related to the classical strain, which provided reference information for the establishment of scientific and reasonable immunization procedures and effective prevention and control of PED in large-scale pig farms in Shaanxi Province.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.651

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