H9N2亚型AIV鼠肺适应株的获得及其氨基酸变异分析

发布时间：2018-09-12 18:45

【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,具有人样流感病毒的受体结合特性;JN病毒仅能在小鼠肺部检测到,病毒滴度很高,而JN-P9-2-M1不仅在小鼠肺部有很高的滴度,在小鼠的肝脏、脾脏、肾脏和脑部都能检测到。【结论】JN-P9-2-M1对小鼠的致病性明显提高,比原毒提高了至少1 000倍;PB2 E627K、HA N313D和HA N496S等3个位点可能是病毒对小鼠致病力初步提高的原因,而PA L342I和NA N218T这两个点突变,就有可能是进一步提高病毒对小鼠致病力的关键位点。
[Abstract]:[objective] after the continuous transmission of H9N2 subtype avian influenza virus in guinea pigs for 9 generations, the virus could be transmitted steadily between guinea pigs. It can be seen that the infection ability of H9N2 subtype avian influenza virus to mammals and the ability of transmission among mammals are still very strong. Therefore, the mouse model of A/Chicken/Jinan/Li-2/2010 (H9N2) was used to study the pathogenicity of H9N2 subtype influenza virus in the lung of mice and the variation of influenza virus in the course of passage. To detect and screen the key amino acid sites of influenza virus pathogenicity variation, to explore the molecular mechanism of influenza virus variation of H9N2 subtype. [methods] A strain of H9N2 subtype avian influenza virus A/Chicken/Jinan/Li-2/2010 (H9N2) was subcultured in the lungs of mice and dissected in mice. The lungs were removed, and the next generation mice were inoculated by nasal drip after grinding and centrifugation. After nine generations of transmission, the MDCK cells were used to spread the virus. The JN-P9-2-M1; amplification of mouse lung adaptation strain was obtained. The whole gene of JN-P5-2-M1 and JN-P9-2-M1 was cloned and sequenced, and the amino acids encoded by each gene were deduced. In comparison with nucleotide and amino acid pairs of JN primary virus (P0), the sites of nucleotide and amino acid changes in each passage of virus were obtained, and the viral titers in lung, liver, spleen, kidney, brain and intestine were obtained by dissecting the mice. After anaesthetized mice with ether, 3 mice were inoculated with each virus diluted nose to detect the survival rate and morbidity of the mice, and the lungs of the mice were collected and fixed for pathological sections and immunohistochemistry. [results] the pathogenicity of JN-P9-2-M1 on mice was significantly improved by comparing the damage of primary JN virus and JN-P9-2-M1 to the lungs of mice. The survival rate of mice whose MLD50 was at the dose of 103.5EID50106EID50105EID50104EID50 attack was 0, and the pathogenicity of JN to mice was at least 1 000 times higher than that of the original virus, and the weight and clinical symptoms of the mice with the dose of JN-P9-2-M1 virus 106-103EID50 were obviously decreased, and the clinical symptoms were also obvious. At 3-8 days after poisoning, the mice showed symptoms such as listlessness, disorder of hair, shortness of breath, back of bow, etc. In 106EID50, the change rate of body weight of JN was the same as that of the negative control group, JN-P5-2-M1 and JN-P9-2-M1 had the same receptor binding characteristics with JN. JN virus can only be detected in the lungs of mice, the titer of JN virus is very high, and JN-P9-2-M1 has very high titer not only in the lungs of mice, but also in the liver and spleen of mice. [conclusion] the pathogenicity of JN-P9-2-M1 in mice was significantly increased, and the pathogenicity of JN-P9-2-M1 was increased by at least 1 000 times than that of the original virus. The three sites, such as HA N313D and HA N496S, may be the reasons for the initial increase of the virulence of the virus to mice. The point mutations of PA L342I and NA N218T may be the key sites to further improve the virulence of the virus to mice.
【作者单位】：东北林业大学野生动物资源学院;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;
【基金】：国家科技支撑计划(2012ZX10004301-008)
【分类号】：S855.3

【共引文献】