DHAV-3适应鸡胚毒株的培育及特性测定  

Development and characterization evaluation of chicken embryo-adapted strain of duck hepatitis A virus serotype 3

作　　者：杨云川 刘锦琳 刘泓邑 吴敏[1] 刘海兰[1] 徐兴胜 陈思怀 李阁 李德龙 李继祥[1,2] YANG Yunchuan;LIU Jinlin;LIU Hongyi;WU Min;LIU Hailan;XU Xingsheng;CHEN Sihuai;LI Ge;LI Delong;LI Jixiang(College of Veterinary Medicine,Southwest University,Chongqing 402460,China;Immunology Research Center,Medical Research Institute,Southwest University,Chongqing 402460,China;Chongqing Health Forever Biotechnology Co.,Ltd,Chongqing 402460,China)

机构地区：[1]西南大学、动物医学学院,重庆402460 [2]西南大学、医学研究院免疫学研究中心,重庆402460 [3]重庆永健生物技术有限公司,重庆402460

出　　处：《中国兽医学报》2023年第1期41-48,共8页Chinese Journal of Veterinary Science

基　　金：中央高校基本科研业务费专项资金资助项目(XDJK2018C055);西南大学教育教学改革研究资助项目(2020JY013)。

摘　　要：旨在培育鸭甲型肝炎病毒血清3型(DHAV-3)适应鸡胚毒株并对其特性进行研究。利用鸭胚从临床病料中分离得到的DHAV-3 LC强毒株经卵黄囊接种鸡胚传代、纯化获得适应鸡胚毒株,并对毒株的致病性、毒力稳定性、免疫原性和基因组特征进行测定。结果显示,利用鸭胚接种成功从病料中分离鉴定DHAV-3 LC强毒株,对鸭胚和雏鸭的半数致死量分别为10-6.20/0.2 mL和10^(-4.25)/0.2 mL。经卵黄囊接种鸡胚69代,获得适应鸡胚毒株DHAV-3 CA。CA株的F4、F16、F21、F31、F41和F51代病毒液的ELD50分别为10^(-6.44)/0.3 mL,10^(-6.60)/0.3 mL,10^(-7.44)/0.3 mL,10^(-7.80)/0.3 mL,10-7.43/0.3 mL和10^(7.25)/0.3 mL;各代病毒对1日龄雏鸭不致病,但在体内连续传代出现接种雏鸭死亡。F21代病毒制备的油包水灭活疫苗免疫蛋鸡,血清中和抗体效价达1∶500以上;以2.5×10^(4.8)ELD50及以上剂量的F31代病毒经颈部皮下接种1日龄雏鸭,攻毒保护率均在90%以上。F21代病毒全基因组序列分析显示,与DHAV-3 LC相比,基因组内无碱基插入或缺失,但有32个位点发生碱基突变;VP0基因的C1410T和A1411G突变与病毒在鸡胚中的传代次数有相关性。本试验成功培育出适应鸡胚毒株DHAV-3 CA,具有良好的免疫原性,但存在致病性返祖现象。The aim of this study is to develop one duck hepatitis A virus serotype 3(DHAV-3)chicken embryo-adapted strain and study its characteristics.The virulent DHAV-3 LC strain isolated clinically was serially passaged and purified through chicken embryo yolk sac to cultivate chicken embryo-adapted strain.The pathogenicity,virulence stability,immunogenicity and genomic characteristics of the chicken embryo-adapted strain were determined.The results showed that the virulent strain of DHAV-3 LC was successfully isolated and identified from the diseased material by inoculating the duck embryo,and the 50%lethal dose(LD50)to duck embryos and ducklings were 10-6.20/0.2 mL and 10^(-4.25)/0.2 mL,respectively.The chicken embryo-adapted strain DHAV-3 CA was obtained by chicken embryo yolk sac inoculation for 69 consecutive passages.The ELD50of the F4,F16,F21,F31,F41 and F51 viruses were 10^(-6.44)/0.3 mL,10^(-6.60)/0.3 mL,10^(-7.44)/0.3 mL,10^(-7.80)/0.3 mL,respectively.The viruses of each generation were not pathogenic to 1-day-old ducklings,but the ducklings died after 2-7 consecutive passages in the body.The water-in-oil inactivated vaccine prepared by the F21 generation virus was used to immunize layer chicken,and the serum neutralizing antibody titer reached over 1:500.1-day-old ducklings were subcutaneously inoculated with F31 virus at the dose of 2.5×10^(4.8)ELD50 and above,the protection rates against DHAV-3 LC strain were all over 90%.The whole genome sequence analysis of the F21 virus showed that there were no base insertions or deletions in the genome but 32 site mutations,compared with DHAV-3 LC.The C1410T and A1411G mutations of the VP0 gene were correlated with the number of virus passages in chicken embryos.The study successfully cultivated the chicken embryo-adapted strain DHAV-3 CA22.The virus had good immunogenicity,but existed pathogenic atavism.

关 键 词：鸭甲型肝炎病毒血清3型 适应鸡胚毒株 卵黄囊接种 致病性返祖 

分 类 号：S852.65[农业科学—基础兽医学]