摘要
为了解新疆博乐地区某牛场牛病毒性腹泻病毒(bovine viral diarrhea virus,BVDV)的流行情况及致病性,采用反转录PCR(reverse transcription PCR,RT-PCR)检测粪便样品,将阳性病料接种至胎牛肾细胞(madin-darby bovine kidney,MDBK),盲传15代后通过5轮蚀斑纯化试验得到1株致细胞病变型(cytopathic type,CP)BVDV毒株;通过理化性质检测、间接免疫荧光试验、电镜观察、基因序列分析及小鼠致病性试验对该毒株进行鉴定。结果显示:成功分离纯化得到1株CP BVDV毒株,将其命名为BVDV-BL314株;盲传15代BVDV毒株接种至MDBK细胞培养引起明显的细胞皱缩、变圆、碎裂、脱落、产生合胞体等致细胞病变效应(cytopathogenic effect,CPE);测定该毒株的滴度为2×10^(8) TCID50/0.1mL;理化性质检测发现该病毒对乙醚、氯仿等有机溶剂敏感,对酸碱敏感;56℃处理30min可显著性降低病毒活性;间接免疫荧光试验检测胞浆中可见特异性荧光;电镜观察该病毒呈球形,直径为40~60nm;遗传进化分析显示该分离株与BVDV-125c株有较近的亲缘关系,属于BVDV-2a基因亚型;免疫BALB/c小鼠后,剖检表现为多器官肿大、出血、质地变脆,边缘钝圆,无光泽等症状;镜检可见炎性细胞浸润、出血等变化。结果表明,分离纯化得到1株致细胞病变型新疆地方性BVDV基因2型毒株,且具有较强的致病性,将为新疆BVDV流行病学调查及致病机制研究提供依据。
In order to investigate the epidemic situation and pathogenicity of bovine viral diarrhea virus(BVDV)in a cattle farm in Bole area,Xinjiang,reverse transcription PCR(RT-PCR)was used to detect stool samples,positive disease material was inoculated into MDBK(madin-darby bovine kidney)cells.After 15passages of blind passage,a cytopathic type(CP)BVDV strain was obtained through five rounds of plaque purification tests.The isolated strain was identified by physical and chemical properties detection,indirect immunofluorescence test,electron microscope observation,sequence analysis and pathogenicity test on mice.The results showed that one CP BVDV strain was successfully isolated and purified,it was named as BVDV-BL314strain.The BVDV strain was inoculated into MDBK cells and it caused obvious cytopathogenic effect(CPE)such as cell shrinkage,rounding,fragmentation,shedding,and syncytial formation.The titer of the strain was determined to be 2×10^(8) TCID50/0.1mL.Physical and chemical properties detection revealed that the isolated strain was sensitive to organic solvents such as ether and chloroform,and was sensitive to acid and alkali.Treatment at 56℃for 30min significantly reduced virus activity.The result of indirect immunofluorescence assay showed that the specific fluorescence could be seen in the cytoplasm.Electron microscope observation indicated that the particle of isolated strain was spherical,40-60nm in diameter.The genetic evolution analysis showed the isolate strain was homologous to the BVDV-125cstrain and belonged to the BVDV-2agene subtype.After immunizing BALB/c mice,post-mortem examination showed multiple organ enlargement,bleeding,brittle texture,and obtuse edges,no gloss and other symptoms.Microscopic examination showed inflammatory symptoms such as inflammatory cell infiltration and bleeding.In summary,these results showed that a genotype 2,cytopathic Xinjiang endemic BVDV strain was isolated and purified,and it has a serious pathogenicity,which will provide the basis for BVDV epidemiological investigation and pathogenic mechanism research in Xinjiang.
作者
李祯
郭妍婷
陈俊贞
胡新艳
赵新艳
董文丽
贺渊秀
王万顺
李泽宇
姚刚
冉多良
付强
史慧君
LI Zhen;GUO Yanting;CHEN Junzhen;HU Xinyan;ZHAO Xinyan;DONG Wenli;HE Yuanxiu;WANG Wanshun;LI Zeyu;YAO Gang;RAN Duoliang;FU Qiang;SHI Huijun(College of Veterinary Medicine,Xinjiang Agricultural University,Urumqi 830052,China)
出处
《中国兽医学报》
CAS
CSCD
北大核心
2021年第4期646-654,共9页
Chinese Journal of Veterinary Science
基金
新疆维吾尔自治区自然科学基金面上资助项目(2017D01A35)。
