H9N2亚型AIV鼠肺适应株的获得及其氨基酸变异分析  

The Adaptation of H9N2 Subtype AIV in Mouse and Analysis of Amino Acid Mutation

作　　者：丁洁[1,2] 高玉伟[2] 桑晓宇[2] 程凯慧[2] 于志君[2] 张坤[2] 柴洪亮[1] 王铁成[2] 夏咸柱[2] 华育平[1] 

机构地区：[1]东北林业大学野生动物资源学院,哈尔滨150040 [2]军事医学科学院军事兽医研究所/吉林省人兽共患病预防与控制重点实验室,长春130062

出　　处：《中国农业科学》2015年第15期3056-3063,共8页Scientia Agricultura Sinica

基　　金：国家科技支撑计划(2012ZX10004301-008)

摘　　要：【目的】将H9N2亚型禽流感病毒在豚鼠体内连续性传播9代后,能够稳定的在豚鼠间传播,可见H9N2亚型禽流感病毒对哺乳动物的感染能力以及在哺乳动物间传播的能力还是很强的。因此,使用此株病毒A/Chicken/Jinan/Li-2/2010(H9N2)简称JN,应用小鼠动物模型,研究H9N2亚型流感病毒在小鼠肺内适应性传代后对小鼠的致病力,以及传代过程中流感病毒的变异。检测和筛选流感病毒致病性变异的关键氨基酸位点,探索H9N2亚型流感病毒变异的分子机制。【方法】将一株H9N2亚型禽流感病毒A/Chicken/Jinan/Li-2/2010(H9N2)在小鼠的肺脏进行传代,将小鼠解剖、摘除肺部、研磨离心后经滴鼻接种下一代小鼠,传播九代后,用MDCK细胞扩繁病毒,得到鼠肺适应株JN-P9-2-M1;扩增、克隆传代病毒JN-P5-2-M1和JN-P9-2-M1的全基因测序,推断各基因编码的氨基酸,与JN原代病毒(P0)的核苷酸和氨基酸相比对,得到各代次病毒核苷酸与氨基酸变化的位点;解剖小鼠,获得小鼠的肺脏、肝脏、脾脏、肾脏、脑和肠,滴定各组织中的病毒滴度,检测病毒的组织噬性;乙醚麻醉小鼠后,每个病毒稀释度鼻内接种3只小鼠,检测小鼠的幸存率和发病率;采集攻毒小鼠的肺部,固定后进行病理切片和免疫组化,比较JN原代病毒与JN-P9-2-M1对小鼠肺部的损害。【结果】JN-P9-2-M1对小鼠的致病性明显提高,其MLD50为103.5EID50,106EID50、105EID50、104EID50攻毒剂量的小鼠幸存率是0,而原毒JN对小鼠不致死,JN-P9-2-M1对小鼠的致病性比原毒提高了至少1 000倍;攻入JN-P9-2-M1病毒剂量为106—103EID50的小鼠,体重明显减少,临床症状也很明显,攻毒后3—8 d,小鼠表现精神萎靡、被毛零乱、呼吸急促、弓背等症状,而原毒JN在106EID50时,小鼠的体重变化率都跟阴性对照组相似;JN-P5-2-M1和JN-P9-2-M1同原毒JN的受体结合特性相同,都是只能特异性结合SAa-2,6Gal受体,具有人样流感病毒【Objective】H9N2 subtype avian influenza virus can spread between the guinea pig after passages in guinea pigs continuously for 9 generations, showing that the capacity of H9N2 subtype avian influenza virus infection in mammals as well as thespreading capacity between mammals are still very strong. Thus, this experiment using this strain virus A/Chicken/Jinan/Li-2/2010（H9N2）, JN, to study the molecular basis of H9N2 subtype influenza virus variation, and screen the pathogenicity related amino acid sites.【Method】An H9N2 subtype AIV was serial passaged in the lung of mice to acquire the variant strains JN-P9-2-M1, the mice were dissected, the lungs were removed, after grinding and centrifugation, the mice of next generation were inoculated intranasally, after nine generations of spreading, MDCK cells were used for virus multiplication. Then the full-length sequences of JN-P5-2-M1 and JN-P9-2-M1 eight segments were amplified, cloned and analyzed. Amino acids encoded by each gene were deduced, compared to JN primary virus（P0）, and the nucleotide and amino acid changes in virus passages were obtained. The mice were dissected, the lung, liver, spleen, kidneys, brain and intestines were obtained, and the virus titer of tissues was titrated. Mice were anesthetized, and each virus dilution was used to inoculate intranasally three mice for detecting the survival rate and morbidity of mice. Lungs of mice were collected, then the pathological and immunohistochemical staining was made to compare the JN and JN-P9-2-M1 virus. 【Result】 JN-P9-2-M1 had high pathogenicity to mice, its MLD50 was 103.5EID50, when had a 106EID50, 105EID50, 104EID50 dose in mice, its survival rate was 0, at least 1000 times higher than JN, the JN in mice was not lethal. When the dose of JN-P9-2-M1 was 106-103EID50 for mice, the body weight was significantly reduced, and the clinical symptoms were obvious. After inoculation for 3-8 d the mice showed listless, hair messy, shortness of breath, arched, etc., but when the mice were inocul

关 键 词：禽流感 H9N2 小鼠 致病力 

分 类 号：S855.3[农业科学—临床兽医学]