Abstract: 【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. 

Key words: avian influenza, H9N2, mice, pathogenicity