猪流行性腹泻病毒变异株传代致弱株的免疫效率研究  

Study on the immune efficiency of porcine epidemic diarrhea virus attenuation strain by serial passage

作　　者：丁小燕 张海涛 尹丽萍 常长琳 钱杰 刘岳龙 彭大新 DING Xiao-yan;ZHANG Hai-tao;YIN Li-ping;CHANG Zhang-lin;QIAN Jie;LIU Yue-long;PENG Da-xin(Jiangsu Lihua Animal Husbandry Co.,Ltd.,Changzhou 213168,China;Yangzhou University,Yangzhou 225100,China)

机构地区：[1]江苏立华牧业股份有限公司,江苏常州213168 [2]扬州大学,江苏扬州225100

出　　处：《中国预防兽医学报》2024年第11期1159-1165,共7页Chinese Journal of Preventive Veterinary Medicine

基　　金：常州市科技计划(CJ20220012)。

摘　　要：为探究猪流行性腹泻病毒(PEDV)变异株传代致弱株对仔猪免疫效率的影响,本研究将GII基因型PEDV JS2018株在Vero细胞中连续传至120代,对第10代(P10)、P40、P60、P90和P120 PEDV的S、M、N和ORF3基因进行PCR扩增和测序分析,结果显示,与P10代PEDV相比,P120代PEDV的S蛋白存在S^(27)L、P^(776)S、N^(1009)D、E^(1015)G和S^(1078)A变异,M蛋白存在S^(219)R变异,而N和ORF3蛋白未发生变异。对未饮用初乳的健康仔猪口服不同代次PEDV(106.0TCID50/mL)后检测各代次PEDV对仔猪的致病性,结果显示P10组和P40组仔猪100%发病,P60组仔猪50%发病,而P90组、P120组和对照组仔猪均未发病;P10、P40组仔猪均检测到排毒,P60组50%仔猪检测到排毒,其他组仔猪均未检测到排毒现象;P10组、P40组和P60组仔猪剖检可见肠道特征性病变,而P90组、P120组和对照组仔猪肠道均未见明显病变;肠道组织病理切片观察结果显示,P40组仔猪存在明显的空肠和十二指肠组织损伤,而P90组和对照组仔猪肠道未见损伤;免疫组化试验结果显示,90代病毒在仔猪肠道上皮细胞中的定植能力较P40组病毒明显减弱。将后备母猪分别口服、肌注免疫P40、P60、P90和P120病毒,免疫21 d后的中和抗体效价检测结果显示,各组母猪中和抗体效价均明显上升,且高于1:37,口服免疫组母猪中和抗体效价明显高于肌肉注射组,P120组母猪中和抗体效价略低于P90组。上述各组后备母猪在免疫21 d后攻击PEDV强毒株JS2018 P10,观察临床症状,检测排毒情况,结果显示免疫组母猪在观察期内未出现任何不良症状,且未检测到排毒。上述结果表明PEDV在传代过程中致病性逐渐减弱,90和120代PEDV完全致弱,但90代PEDV诱导猪产生的免疫反应优于120代PEDV。本研究揭示了不同代次PEDV免疫效率的变化规律,为筛选免疫效果最佳的PEDV弱毒株提供了参考依据。In order to study the effect of immune efficiency of porcine epidemic diarrhea virus(PEDV)attenuation strain by serial passage,the JS2018 strain of GII genotype PEDV that isolated and preserved in our laboratory was continuously passaged for 120 generations in Vero cells.The S,M,N and ORF3 genes of 10th,40th,60th,90th and 120th passage were amplified by PCR and sequenced.The amino acid sequence alignment showed that compared with P10,P120 PEDV had S^(27)L,P^(776)S,N^(1009)D,E^(1015)G and S^(1078)A mutations in S protein and the S^(219)R mutation in M protein,while no mutation was found in N and ORF3 protein.The pathogenicity of PEDV of different passages to piglets was tested after oral administration of PEDV(106.0TCID50/mL),and the results showed that the morbidity caused by P10 and P40 was 100%,while 50%by P60 and 0 by P90,P120 and control group.The anatomy of piglet intestine showed typical pathological changes of PEDV infection in P10,P40 and P60 virus-infected piglets,while no pathological changes were found in P90,P120 and control group.The results of intestinal pathological sections showed that the jejunum and duodenum of P40 virus infected piglets were significantly damaged,while no obvious damage to intestine was detected in P90 virus infection group and control group.Immunohistochemical results showed that the colonization ability of P90 virus in intestinal epithelial cells was significantly weaker than that of P40 virus.Gilts were immunized with P40,P60,P90 and P120 viruses orally or intramuscularly,and the results of neutralizing antibody level at 21 days after the immunization were showed as follows:neutralizing antibody levels in all groups increased significantly and were higher than 137.The antibody levels in oral immunization groups were obviously higher than those of muscle immunization groups,while those of in P120 group were slightly lower than those of in P90 group.Meanwhile,the gilts in the above groups were challenged with PEDV JS2018 P10 virus at 21 days after immunization,and the results

关 键 词：猪流行性腹泻病毒 致病性 免疫效率 

分 类 号：S852.65[农业科学—基础兽医学]