猪传染性胃肠炎病毒分离及部分特性鉴定
发布时间：2018-11-02 15:52
【摘要】：猪传染性胃肠炎病毒(Transmissible gastroenteritis virus of swine,TGEV)属冠状病毒科,冠状病毒属,是猪病毒性腹泻的重要病原体之一。该病毒引起的猪传染性胃肠炎(Transmissible gastroenteritis,TGE)以严重腹泻、呕吐和脱水为主要临床特征。TGE发病急,流行范围广,且经常与猪轮状病毒(Porcine rotavirus,PRV)和猪流行性腹泻病毒(Porcine epidemic diarrhea virus,PEDV)等其他病毒或细菌混合感染,从而加重了其发病和死亡,给畜牧业造成严重的损失。对TGEV流行毒株的分离与鉴定,无论是对该病的基础性研究或病毒相关生物产品研制及疾病的科学防控都有重要意义。本研究以TGEV粪便病料为材料,比较了TGEV在猪原代睾丸细胞,传代细胞系ST细胞以及PK15细胞上的增殖情况。病毒在三种细胞上连续传代,传至25代左右,提取核酸检测,通过RT-PCR测定病毒在三种细胞的增殖情况,测定结果显示,病毒在这三种细胞上均可扩增出病毒特异条带。通过对不同代次细胞毒的TCID50测定比较,在ST传代细胞病毒滴度与其他两种细胞滴度相当,且在ST细胞出现病变最早,故本实验选择ST细胞来进行TGEV病毒的连续培养。当病毒适应ST细胞系后,用TGEV,PED,PRV特异性引物对病毒进行检测。经过核酸检测证明除TGEV外,细胞培养物中还可检测到PEDV和PRV,表明在进行TGEV传代过程中,存在有三种病毒的细胞混合感染。为使TGEV得到纯化,本研究采用蚀斑纯化技术对病毒进行了纯化。通过对TGEV蚀斑形成试验的摸索,确定了蚀斑形成条件为:琼脂糖浓度为1.6%,培养液-琼脂糖混合培养液的最适温度约30℃,琼脂糖厚度3mm,中性红浓度为0.02%。通过核酸检测为TGEV阳性的蚀斑毒继续进行蚀斑试验,直致蚀斑大小形态一致,且核酸检测TGEV阳性。对纯化后的病毒在ST细胞上连续传代,利用核酸检测,证明病毒可在在细胞上的稳定增殖。间接免疫荧光检测接种TGEV病毒,可产生特异性荧光,电镜观察观察表明。病毒直径约150 nm左右,病毒粒子具有典型的冠状结构;病毒感染ST细胞后进行超薄切片电镜观察,可看到病毒弥散的存在于细胞质中,直径在60-100 nm。本实验对纯化后病毒的部分理化特性进行了测定,结果显示,病毒对热敏感,60℃水浴30min即可完全失活;病毒不耐酸碱,在PH为3和PH为9的条件下病毒滴度明显降低。该病毒对有机试剂如乙醚氯仿等较敏感。以纯化后的细胞培养毒30 ml口服感染新生仔猪,仔猪在感染后30 h出现呕吐,腹泻,消瘦和脱水,表现为行动迟缓,腿部僵直等症状,仔猪在感染后8 d死亡,仔猪感染后不同时间收集粪便样品,进行RT-PCR检测,感染仔猪72 h开始排毒。病死猪剖检发现肠管扩张,充满液体,小肠绒毛变短,脱落。猪传染性胃肠炎病毒的成功分离纯化,为该病的流行病学、实验室诊断及和病毒的生物学特征以及疫苗的制备等奠定了物质基础。
[Abstract]:Porcine infectious gastroenteritis virus (Transmissible gastroenteritis virus of swine,TGEV) belongs to coronavirus family and coronavirus genus, which is one of the important pathogens of porcine viral diarrhea. The infectious gastroenteritis (Transmissible gastroenteritis,TGE) caused by the virus is characterized by severe diarrhea, vomiting and dehydration. TGE is acute, widespread and often associated with porcine rotavirus (Porcine rotavirus,. PRV) and other viruses or bacteria, such as porcine epidemic diarrhea virus (Porcine epidemic diarrhea virus,PEDV), caused serious damage to animal husbandry. It is of great significance to isolate and identify the epidemic strains of TGEV, whether it is the basic research of the disease, the development of virus-related biological products, and the scientific prevention and control of the disease. In this study, TGEV fecal material was used to compare the proliferation of TGEV on primary testicular cells, ST cells and PK15 cells. The virus was subcultured on three kinds of cells and passed to about 25 generations. The nucleic acid was extracted to detect the nucleic acid, and the proliferation of virus in the three kinds of cells was determined by RT-PCR. The results showed that the virus could amplify the virus specific bands on the three kinds of cells. The titer of the virus in the ST passaged cells was similar to that of the other two cell lines, and the lesion was the earliest in the ST cells, so we chose the ST cells for the continuous culture of the TGEV virus. When the virus adapted to ST cell line, TGEV,PED,PRV specific primers were used to detect the virus. In addition to TGEV, PEDV and PRV, could also be detected in cell cultures, which indicated that there were three kinds of virus mixed infection in the course of TGEV passage. In order to purify TGEV, the virus was purified by plaque purification technique. The conditions of plaque formation of TGEV were as follows: agarose concentration 1.6, agarose mixed culture medium 30 鈩，
本文编号：2306242
[Abstract]:Porcine infectious gastroenteritis virus (Transmissible gastroenteritis virus of swine,TGEV) belongs to coronavirus family and coronavirus genus, which is one of the important pathogens of porcine viral diarrhea. The infectious gastroenteritis (Transmissible gastroenteritis,TGE) caused by the virus is characterized by severe diarrhea, vomiting and dehydration. TGE is acute, widespread and often associated with porcine rotavirus (Porcine rotavirus,. PRV) and other viruses or bacteria, such as porcine epidemic diarrhea virus (Porcine epidemic diarrhea virus,PEDV), caused serious damage to animal husbandry. It is of great significance to isolate and identify the epidemic strains of TGEV, whether it is the basic research of the disease, the development of virus-related biological products, and the scientific prevention and control of the disease. In this study, TGEV fecal material was used to compare the proliferation of TGEV on primary testicular cells, ST cells and PK15 cells. The virus was subcultured on three kinds of cells and passed to about 25 generations. The nucleic acid was extracted to detect the nucleic acid, and the proliferation of virus in the three kinds of cells was determined by RT-PCR. The results showed that the virus could amplify the virus specific bands on the three kinds of cells. The titer of the virus in the ST passaged cells was similar to that of the other two cell lines, and the lesion was the earliest in the ST cells, so we chose the ST cells for the continuous culture of the TGEV virus. When the virus adapted to ST cell line, TGEV,PED,PRV specific primers were used to detect the virus. In addition to TGEV, PEDV and PRV, could also be detected in cell cultures, which indicated that there were three kinds of virus mixed infection in the course of TGEV passage. In order to purify TGEV, the virus was purified by plaque purification technique. The conditions of plaque formation of TGEV were as follows: agarose concentration 1.6, agarose mixed culture medium 30 鈩，
本文编号：2306242
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