猪轮状病毒江西株AY01的分离鉴定

Isolation and Identification of Porcine Rotavirus Jiangxi Strain AY01

【目的】确定江西省某猪场哺乳仔猪发生腹泻的病因。【方法】对送检的仔猪小肠样品进行猪流行性腹泻病毒(Porcine epidemic diarrhea virus,PEDV)、猪传染性胃肠炎病毒(Transmissible gastroenteritis virus,TGEV)和猪轮状病毒(Porcine rotavirus,PoRV)的RT-PCR检测,将阳性样品接种MA104细胞传代进行PoRV的分离;对分离毒株进行电镜观察、间接免疫荧光试验、PoRV VP4和VP7基因序列测序和动物回归等试验。【结果】猪小肠病料样品经终浓度15μg/mL的胰酶处理37℃孵育2 h,接种MA104细胞,能在细胞上增殖传代,第6代开始表现稳定的细胞病变;电镜观察可见病毒粒子直径大小为61~70 nm,平均大小为65 nm,呈带有短纤突且外缘光滑的形似车轮状的粒子,具有轮状病毒粒子典型的形态特征;间接免疫荧光试验和RT-PCR检测均为PoRV阳性,确定该分离株为PoRV。分离株VP4和VP7基因序列分析显示,VP4基因型与P[23]基因型相似性最高,VP7基因型与G5基因型相似性最高,根据A群轮状病毒最新分类方法,分离株属于G5P[23]型。动物回归试验结果显示,经口感染该分离株的1日龄初生仔猪于感染后24 h左右陆续出现水样腹泻、呕吐等临床症状,并能在粪便中检测到PoRV。【结论】通过MA104细胞连续传代,从江西某猪场的腹泻仔猪小肠样品成功分离到1株PoRV,该分离株属于G5P[23]型PoRV,为哺乳仔猪发生腹泻的病原。

【Objective】The study was aimed to determine the etiology of diarrhea piglets in a pig farm in Jiangxi province.【Method】The small intestine samples of piglets were tested for Porcine epidemic diarrhea virus(PEDV),porcine Transmissible gastroenteritis virus(TGEV)and Porcine rotavirus(PoRV)by RT-PCR.Positive sample was inoculated into MA104 cells for passage to isolate PoRV.The isolated strain was detected by electron microscope observation,indirect immunofluorescence test,PoRV VP4 and VP7 genes sequencing and animal regression test.【Result】The results showed that porcine intestinal disease samples were incubated at 37℃for 2 h with final concentration of 15μg/mL trypsin and inoculated with MA104 cells,which could proliferate and passage on cells,and the 6 th-generation began to show stable cytopathic effect.Electron microscopic observation showed that the virus particles were 61-70 nm in diameter,the average size was 65 nm,with short fibrils and smooth outer edges,similar to wheel-like particles,which had typical morphological characteristics of PoRV virus particles.Both indirect immunofluorescence assay and RT-PCR were positive for PoRV,and the isolated strain was determined to be PoRV.Sequence analysis of VP4 and VP7 genes revealed that VP4 genotype had the highest homology with P[23]genotype,and VP7 genotype had the highest homology with G5 genotype,according to the latest classification method of group A Rotavirus,the isolate was classified as G5 P[23]genotype.The results of animal regression test showed that the one-day-old newborn piglets with oral infection of the isolate had watery diarrhea,vomiting and other clinical symptoms about 24 h after infection,and could detect PoRV in the feces.【Conclusion】Through continuous passage of MA104 cells,a strain of PoRV was successfully isolated from the small intestine samples of diarrhea piglets in a pig farm in Jiangxi province,the isolated strain belonged to G5 P[23]genotype PoRV,which was the pathogen of diarrhea in piglets.