Mutations Of The Hemagglutinin Gene Of H9N2 Subtype Avian Influenza Viruses Under The Selective Pressure Of Antibody

The LG1 strain which was isolated in our lab was proliferated in the SPF embryos.Then,the cDNA of HA gene of H9N2 avian influenza isolate was amplified , cloned and sequenced. The result of its sequence analysis shows that cDNA of HA gene is composed of 1 683 bp nucleotides ,encoding for 560 amino acids. The sequence of the amino acids is R - S - S - R , typical of lowly pathogenic avian influenza. The results of the comparative sequence analysis indicated 91.7 %～96.3 % of nucleotide homology and 93.4 %～96.1 % of amino acid homology between the isolated H9N2 and the other from Chain . The genetic relationship between them was close. However, The genetic relationship between the strain from Hokkaido and the LG1 strain was remote.They belonged to the eurasia system.The leghorn chickens sourced SPF chicken were inoculated inactivated elai-vaccine (LG1 strain of H9N2 subtype) at the beginning of egg laying ,Their serum for the hemagglutination inhibition H9 antibody titer was 29 .We collected fertilized eggs and hatched after egg laying, as the embryo with moternal antibodies. At the same time, we got SPF chicken eggs directly from the same strain as the embryos without moternal antibodies.We continuously passed a H9N2-AIV strain LG1 in embryos with or without maternal antibody to LG1 in 6 separate serials. At the 10th, 20th, 30th, 40th and 50th passages in each serial, H9 hemagglutinatin gene (HA) sequences were determined and compared to the original LG1 strain. Only unstable random mutations happened in 29 sites with nonsynonymous vs synonymous (N/S) mutation ratio of 1.42 during 50 passages in 2 serials without maternal antibodies. However, multiple stable nonsynonymous mutations were detected gradually during 50 passages of 4 serials in embryos with maternal antibody to LG1 strain, and the NS/S ratio was as high as 3.46 among 45 mutated sites. There are 12 base sites began to stabilize among the 45 variation sites which variated in the 10-40 sites,and nian variation base sites were nonsynonymousamong the 12 base sites,they were 17,313,477,496,592,593,672,796,806.The results suggested that the antibody selective pressure influenced mutations of H9N2 AIV HA gene during the passages in embryos with maternal antibody. Embryos with maternal antibody to certain viruses could be used as experimental models to study the immune selective pressures on viral mutations.