H9N2亚型AIV鼠肺适应株的获得及其氨基酸变异分析

【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10^3.5EID₅₀，10⁶EID₅₀、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。

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

【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 the spreading 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-M1eight 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 10^3.5EID₅₀, 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 inoculated with 10⁶EID₅₀ JN virus, the mice body weight change rates were similar to the negative group. JN-P5-2-M1, JN-P9-2-M1, and JN could binding to SAa-2,6Gal receptor, like B influenza virus. JN virus was detected only in the lungs of mice, but JN-P9-2-M1 not only in the lungs of mice but also could be detected in liver, spleen, kidney and brain. 【Conclusion】JN-P9-2-M1’s pathogenicity in mice significantly improved, compared with the original drug increased by at least 1 000 times. The three amino acid sites PB2 E627K, HA N313D, and HA N496S might be the reasons of preliminary improvement of the virus virulence in mice, and the two acid sites PA L342I and NA N218T might be possible to further improve the virus virulence in mice.