重组禽流感病毒(H5N2)的拯救及其生物学特性分析  

RESCUE AND CHARACTERIZATION OF RECOMBINANT AVIAN INFLUENZA VIRUS (H5N2)

作　　者：杨禹[1] 邵卫星[2] 徐天刚[2] 王志亮[2] 乔薪瑗[1] 唐丽杰[1] 葛俊伟[1] 李一经[1] 

机构地区：[1]东北农业大学动物医学院,哈尔滨150030 [2]中国动物卫生与流行病学中心,青岛266000

出　　处：《中国动物传染病学报》2013年第1期48-52,共5页Chinese Journal of Animal Infectious Diseases

基　　金："十二五"农村领域国家科技计划课题(2012AA101304-3)

摘　　要：为控制在我国流行的H5N1高致病性禽流感(Avian influenza virus,AIV)和筛选具有标记的疫苗毒株,用流行的H5N1亚型AIV的HA基因和H9N2亚型AIV的NA基因及H1N1亚型AIV(A/PR/8/34毒株)的6个内部基因通过流感8质粒反向遗传操作系统拯救了重组病毒rH5N2/PR8株。为了降低重组病毒的毒力,对H5N1亚型AIV的HA基因进行了修饰,使其裂解模式由PLRERRRKR↓GL修饰为PLIETR↓GL。将获得的rH5N2/PR8株在9日龄SPF鸡胚上连续传10代。该重组病毒的血凝效价稳定在1:2048,其半数感染量(EID50)可达10-8.77/0.1 mL。该病毒的毒力显著降低,对鸡胚的半数致死量(ELD50)为10-5/0.1 mL。将该病毒灭活与油佐剂乳化,制成灭活疫苗,给6周龄SFP鸡接种不同剂量,接种21 d后,用H5N1流行野毒A/Chicken/SD/2010(H5N1)攻击,结果显示:接种剂量为0.1 mL/只的试验组,10只鸡中有5只获得保护;接种0.3 mL/只的试验组可获得100%保护。以上说明,本实验获得的重组病毒具有疫苗标记、繁殖滴度高、毒力低、免疫原性好等特点,非常适合作为"标记疫苗"候选株,为AIV(H5N1)的标记疫苗研发奠定了坚实基础。In response to the emergence of highly pathogenic avian influenza virus of H5N1 subtype and lack of＂marker＂ vaccine viruses for used in DIVA, an recombinant avian influenza virus rH5N2/PR8 strain was generated with 8-plasmids reverse genetic system. rH5N2/PR8 consisted of HA gene of H5N1, NA gene of H9N2 and 6 internal genes of A/PR8/34（H1N1）. To reduce high pathogenecity due to a series of basic residues in HA, the motifs of the cleavage site （ PLRERRRKR↓,GL ） was replaced with PLIETR↓GL. rH5N2/ PR8 was serially passed in 9-day-old SPF chicken embryos 10 times. The highest hemagglutination titer was 1：2048 and EIDs0 was 10 877/0.1 mL. The virulence ofrH5N2/PR8 was greatly reduced and ELD50 was 10^-5/0.1 mL. Three groups of 10 six-week-old chickens were inoculated with 0.1, 0.2 and 0.3 mL of inactivated rH5N2/PR8 emulsified with oil adjuvant. After 21 days, each of inoculated chickens and 5 non-vaccinated control chickens were oronasally challenged with 0.1 mL of A/Chichen/SD/2010 （H5NI）. Five out of 10 chickens inoculated with 0.1 mL vaccine survived while all 10 chickens inoculated with 0.3 mL vaccine were protected. In clonclusion, rH5N2/PR8 possessed the characteristic features with ＂molecular marker＂, high propagation, low virulence and good immunogenicity, therefore it can be used as a candidate for development of a marker vaccine.

关 键 词：反向遗传 禽流感病毒 重组病毒 标记疫苗 

分 类 号：S852.659.5[农业科学—基础兽医学]