鸽新城疫病毒毒力致弱株的拯救及免疫原性分析  

Rescuing the attenuated Newcastle disease virus of pigeon and analysis of its immunogenicity

作　　者：蒋艳玉 赵莎莎[1] 孙娜娜[1] 孙军峰[1] 孔宪刚[1] 刘胜旺[1] 刘怀然[1] JIANG Yan-yu;ZHAO Sha-sha;SUN Na-na;SUN Jun-feng;KONG Xian-gang;LIU Sheng-wang;LIU Huai-ran(Division of Avian Infectious Diseases,State Key Laboratory of Veterinary Bioteehnology,Harbin Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Harbin 150069,China)

机构地区：[1]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室/禽呼吸道病研究创新团队,黑龙江哈尔滨150069

出　　处：《中国预防兽医学报》2018年第11期1037-1042,共6页Chinese Journal of Preventive Veterinary Medicine

基　　金：公益性行业(农业)科研专项经费项目(201303033);青年科学基金项目(31201884)

摘　　要：为快速获得鸽新城疫(ND)疫苗株,本研究构建了鸽NDV/Pi/CH/LHLJ/110822株反向遗传操作平台,拯救得到2株亲本病毒株r0822-21A、r0822-17A,以及1株F蛋白裂解位点突变为La Sota相应位点的病毒株r0822-FCS。HA/HI试验结果显示r0822-FCS毒力已致弱,将其灭活乳化后免疫SPF鸡可刺激其产生高滴度HI抗体。安全性评价试验结果显示其安全性良好,但接种14 d时SPF鸡的HI抗体滴度仅达3 log2,对基因Ⅶ型鸡源强毒CK/CH/HLJ/01/06攻毒保护率小于50%;免疫效力试验显示,r0822-FCS分别以10~5EID_(50)/0.1 mL、10~6EID_(50)/0.1 mL剂量接种14日龄SPF鸡,未检测到明显的抗体阳转。基因组测序分析结果显示,3株拯救病毒均在L基因的不同位置与新城疫病毒(NDV) V4株高度同源的外源基因片段重组,推测在实验条件下的细胞内拯救病毒过程中,病毒L基因与辅助质粒中的L基因发生了随机同源重组。综上所述,本研究获得的r0822-FCS株作为弱毒疫苗效果不佳,但可作为鸽ND灭活疫苗候选株。此外,本研究首次报道了反向遗传操作实验条件下,NDV基因组与V4株辅助质粒之间发生了基因重组,且L基因是决定病毒复制的关键基因。此次基因重组现象应为实验条件下的偶发事件,其机制和风险尚需后继实验研究分析。To get an avirulent virus apply to vaccine study for pigeon Newcastle disease (ND),the reverse genetic operation system was established with NDV strain Pi/CH/LHLJ/110822(0822).In this study,r0822-21A and r0822-17A were rescued from parent strain 0822.r0822-FCS was rescued from 0822whose F protein amino acid motif were mutant into the same sites of La Sota.The pathogenicity index of r0822-FCS was confirmed and showed that r0822-FCS had been changed into an avirulent strain. r0822-FCS were inactivated and inoculated to 20-day-old SPF chicks,higher HI antibody titer could be detected,which showed that r0822-FCS had the proper immtmogenicity as the activate vaccine in chicks.In safety evaluation tests,3-day-old SPF chickens were inoculated by 0.1mL r0822-FCS allantoic fluid via ED and IN,there were no morbidity and mortality were observed,but the chickens showed a weak immune reaction with the antibody titer less than 3log2,and the protection rate to NDV genotype Ⅶ virulent strain CK/CH/HLJ/01/06was also lower than 50%post 14days immunization.In the immunogenicity test,r0822-FCS was inoculated to 14day-old SPF chickens with the dose of 105EID50/0.1mL and 106EID50/0.1mL respectively,unfortunately,we could not detect the apparent antibody to NDV.To analyze the abnormal immunogenicity of r0822-FCS,the genome of 3rescued viruses were sequenced.The results showed that 3viruses had different size of exogenous gene segments in L gene and these exogenous genes had the highest homology with L gene of NDV V4strain by blast analysis.Further tests were performed to exclude some interference factors such as plasmid contamination and mixture of other virus.In the end we put forward the assumption with caution that L gene of the parent virus had a random homologous recombination with L gene in helper plasmids from V4during rescuing process in experiment.To sum up,r0822-FCS obtained in this study was not effective as attenuated vaccine,but could be used as a candidate strain for pigeon ND inactivated vaccine.In addition,this paper

关 键 词：鸽新城疫病毒 反向遗传 致弱毒株 L基因重组 

分 类 号：S852.65[农业科学—基础兽医学]