猪轮状病毒流行毒株的分离及部分特性鉴定
发布时间：2019-06-18 20:14
【摘要】：猪轮状病毒(Porcine rotavirus,PRV)属呼肠病毒科,轮状病毒属。猪轮状病毒是引起仔猪病毒性腹泻常见的病原之一,主要临床症状表现为黄色水样腹泻并伴随着呕吐。因该病流行范围广,发病率高,常与其他疾病混合感染而使病情加重,导致高死亡率,给畜牧业造成严重经济损失。轮状病毒血清群和血清型众多,且不同毒株的生物学特性不同,故对轮状病毒的分离鉴定及相关特性的研究,无论是对该病毒的基础性研究亦或是对该病的应用性研究均具有十分重要的意义。本研究对2012年从河北某发病猪场采集的仔猪腹泻病料进行了轮状病毒的分离鉴定。首先对疑似病料,进行了PRV VP6部分基因的PCR检测,对扩增产物测序证明,获得的275 bp的PRV VP6部分基因序列,同时对TGEV和PEDV进行了RT-PCR扩增,未见针对这两种病毒的特异条带。结果表明该病料中含有轮状病毒。将PRV PCR检测阳性粪便10倍稀释,经病毒释放和无菌处理后,分别接种于猪小肠上皮细胞IEC-2、恒河猴胎肾细胞MA-104和Marc-145,经过一定代次的盲传,在以上三种细胞基质中均可观察到细胞膜融合,细胞核固缩、空泡化,细胞脱落等细胞病变(CPE),且随着传代次数的增加,CPE也越发明显;对该轮状病毒地方流行株的细胞培养物进行电镜观察,可观察到典型的车轮状病毒结构,病毒呈双层衣壳,直径大小约70~80 nm;免疫电镜检测,可见有大量的典型的轮状病毒粒子聚集在一起;病毒感染MA-104细胞后不同时间超薄切片电镜观察显示,病毒接种30h在胞质中可见典型的病毒粒子,60h时病毒明显增多,90h后细胞失去固有形态;间接免疫荧光检测表明,接种该轮状病毒分离株的IEC-2细胞、MA-104细胞和Marc-145细胞中均有特异性免疫荧光出现;病毒中和实验表明,分离病毒所致病变,可被特异性猪轮状病毒抗血清所抑制。为进一步纯化该病毒,将病毒感染的细胞培养物与氯仿连续作用5代后在MA-104细胞上扩大培养,利用双层琼脂法进行蚀斑试验,通过多次挑取蚀斑在细胞上培养,达到了病毒的纯化。本实验对所分离的毒株进行了部分理化特性的测定。测定结果表明,该轮状病毒地方流行分离株对脂溶性试剂表现出极度不敏感,pH3.0处理病毒1h后其感染能力略微下降,经50℃水浴处理30min后,感染能力明显下降。将分离毒细胞培养物灌服新生仔猪后,30h开始轻微腹泻,70h后出现呕吐和水样腹泻,感染后96h死亡。剖检肠壁变薄,松弛胃内充满凝乳块,对所收集的粪便样品进行PCR检测和免疫荧光检测,均为阳性,表明所分离的病毒对猪有一定致病性。该猪轮状病毒的分离纯化,为该病病原学、流行病学、及其免疫防治提供了重要的物质基础。
[Abstract]:Pig rotavirus (Porcine rotavirus,PRV) belongs to reenteroviridae and rotavirus. Pig rotavirus is one of the common pathogens causing viral diarrhea in piglets. The main clinical symptoms are yellow watery diarrhea accompanied by vomiting. Because of its wide epidemic range and high incidence, it is often mixed with other diseases to aggravate the disease, resulting in high mortality and serious economic losses to animal husbandry. There are many serotypes and serotypes of rotavirus, and the biological characteristics of different strains are different. Therefore, the isolation, identification and related characteristics of rotavirus are of great significance both for the basic research of rotavirus and for the application of the disease. In this study, rotavirus was isolated and identified from piglets with diarrhea collected from a pig farm in Hebei in 2012. Firstly, the PRV VP6 partial genes of suspected disease materials were detected by PCR, and the PRV VP6 partial gene sequences of 275 bp were confirmed by sequencing the amplified products. At the same time, TGEV and PEDV were amplified by RT-PCR, and no specific bands for these two viruses were found. The results showed that rotavirus was found in the diseased material. The positive feces detected by PRV PCR were diluted 10 times and inoculated into pig intestinal epithelial cells IEC-2, rhesus monkey fetal kidney cells MA-104 and Marc-145, after 10 times dilution and aseptic treatment, respectively. cell membrane fusion, nuclear pyknosis, vacuolation, cell shedding and other cytopathic (CPE), were observed in the above three cell matrices, and CPE became more and more obvious with the increase of passage times. The cell culture of the rotavirus local epidemic strain was observed by electron microscope. The typical rotavirus structure was observed. The virus was double capsid, the diameter was about 70 脳 80 nm; immunoelectron microscope, and a large number of typical rotavirus particles gathered together. Electron microscopic observation of ultra-thin sections of MA-104 cells at different time after virus infection showed that typical virus particles could be seen in cytoplasm at 30 h, the virus increased significantly at 60 h, and the cells lost their inherent morphology after 90 h. Indirect immunofluorescence assay showed that specific immunofluorescence appeared in IEC-2 cells, MA-104 cells and Marc-145 cells inoculated with the rotavirus isolate. Virus neutralization test showed that the disease caused by isolation of virus could be inhibited by specific swine rotavirus antiserum. In order to further purify the virus, the cell culture infected by the virus was cultured on MA-104 cells after 5 generations of continuous action with chloroform. The plaque test was carried out by double-layer Agar method. The plaque was cultured on the cell many times, and the purification of the virus was achieved. In this experiment, some physical and chemical properties of the isolated strains were determined. The results showed that the rotavirus isolates were extremely insensitive to fat-soluble reagents. The infection ability of the rotavirus was slightly decreased after 1 hour of pH3.0 treatment, and significantly decreased after 50 鈩，
本文编号：2501760
[Abstract]:Pig rotavirus (Porcine rotavirus,PRV) belongs to reenteroviridae and rotavirus. Pig rotavirus is one of the common pathogens causing viral diarrhea in piglets. The main clinical symptoms are yellow watery diarrhea accompanied by vomiting. Because of its wide epidemic range and high incidence, it is often mixed with other diseases to aggravate the disease, resulting in high mortality and serious economic losses to animal husbandry. There are many serotypes and serotypes of rotavirus, and the biological characteristics of different strains are different. Therefore, the isolation, identification and related characteristics of rotavirus are of great significance both for the basic research of rotavirus and for the application of the disease. In this study, rotavirus was isolated and identified from piglets with diarrhea collected from a pig farm in Hebei in 2012. Firstly, the PRV VP6 partial genes of suspected disease materials were detected by PCR, and the PRV VP6 partial gene sequences of 275 bp were confirmed by sequencing the amplified products. At the same time, TGEV and PEDV were amplified by RT-PCR, and no specific bands for these two viruses were found. The results showed that rotavirus was found in the diseased material. The positive feces detected by PRV PCR were diluted 10 times and inoculated into pig intestinal epithelial cells IEC-2, rhesus monkey fetal kidney cells MA-104 and Marc-145, after 10 times dilution and aseptic treatment, respectively. cell membrane fusion, nuclear pyknosis, vacuolation, cell shedding and other cytopathic (CPE), were observed in the above three cell matrices, and CPE became more and more obvious with the increase of passage times. The cell culture of the rotavirus local epidemic strain was observed by electron microscope. The typical rotavirus structure was observed. The virus was double capsid, the diameter was about 70 脳 80 nm; immunoelectron microscope, and a large number of typical rotavirus particles gathered together. Electron microscopic observation of ultra-thin sections of MA-104 cells at different time after virus infection showed that typical virus particles could be seen in cytoplasm at 30 h, the virus increased significantly at 60 h, and the cells lost their inherent morphology after 90 h. Indirect immunofluorescence assay showed that specific immunofluorescence appeared in IEC-2 cells, MA-104 cells and Marc-145 cells inoculated with the rotavirus isolate. Virus neutralization test showed that the disease caused by isolation of virus could be inhibited by specific swine rotavirus antiserum. In order to further purify the virus, the cell culture infected by the virus was cultured on MA-104 cells after 5 generations of continuous action with chloroform. The plaque test was carried out by double-layer Agar method. The plaque was cultured on the cell many times, and the purification of the virus was achieved. In this experiment, some physical and chemical properties of the isolated strains were determined. The results showed that the rotavirus isolates were extremely insensitive to fat-soluble reagents. The infection ability of the rotavirus was slightly decreased after 1 hour of pH3.0 treatment, and significantly decreased after 50 鈩，
本文编号：2501760
本文链接：https://www.wllwen.com/yixuelunwen/dongwuyixue/2501760.html
