The Diversity Study Of Antigenic Epitope Of Different H9N2 Subtype AIV Strains And Sequence Analysis Of NS Gene During 1998-2005

According to the variation and epidemic status of H9N2 subtype AIV in our country, in order to study the variability and multiformity of HA antigenic epitope of different H9N2 subtype strains,in this reseach we purified 25 H9N2 AIV isolated during 1998-2005,then identified by speciality and RT-PCR methods. Five HA MoAb(A4,F6,H6,C9,C2,G4) that searched and saved were used to do the cross HI test and double layer ELISA test, in HI test ,25 H9N2 AIV strains don't have reactivity to G4 which is not aim at HA,according to the result of HI test of five HA MoAb,we classified them to two kinds,the first kind is having strong virulence: A/Chicken/Henan /A3/98(H9N2)(3#) and A/ Chicken / Henan /C4/02(H9N2)(18#), they don't have reactivity to five HA MoAb;the other kind is 25 strains except 3# and 18# which having reactivity to five HA MoAb,the HI value is between 2¹⁵ and 2¹⁷.And the conclusion of the double layer ELISA test is identical,3# and 18# don't have reactivity to five HA MoAb(P/N value is between 1 and 2),have low reactivity to G4(P/N value is 3);The other 23 strains have reactivity to 6 MoAb,but have difference among them,l# have low reactivity(P/N value is between 2.1 and 3.3),most strains have more highly reactivity (P/N value is between 5.3 and 9.3).Five HA MoAb don't have reactivity to 3# and 18#,it shows that the HA epitope of 3# and 18# have site loss aim at the five MoAb,or have bigger variation. The variable characteristic and obvious different virulence confirm that there is variation in antigenity and multiformity of H9N2 AIV.RT-PCR was employed to amplify the cDNA of 7 strains(including 3# and 18#) which are representative(cover different antigenicity and virulence),then linked to pGEM-T vector,transformed into E.coli JM-109,sequenced positive recombinant plasmid. NS gene sequence analysis shows that the nucleotide homology of 7 H9N2 strains is between 95.7% and 99.8%,deduced amino acid homology is between 93.2 % and 99.6%.It shows that the NS gene in china is high stable,belongs to the avian-swine branch of A allele. Like other isolates in china mainland ,there are 13 amino acide loss in the signal area of C'end of 7 H9N2 subtype strains .The nucleotide acide at 39 site of 10#,17#,24# mutate from C to T; The nucleotide acide at 64 site of 8#,10#,15#,18# mutate from A to G;The 139 and 225 site mutate from G to A,236 and 375 site mutate from T to C;339 site mutate from C to T. 8#, 10#, 15#, 17#, 18#, 24#'s 114 and 453 site mutate from A to G;267 site mutate from C to T;681 site mutate T to C. The effect of the nucleotide acide and amino acide's change still need further reseach. But 460 site of 18#'s NS1 coding area mutate from G to T,make the signal area of of NS1 protein's C'end lose 64 amino acid.l8# has more stroner virulence(ELD₅₀ is 10^-8.7,MDT is 69.9,ICPI is 0.44,IVPI is 0.51),and has loss of MoAb action site on HA epitope,the change of virulence and antigenicity may have certain relation to the loss of NS1 gene,but the mechanism and explaination still need further reseach. In this reseach,we study the variation of H9N2 subtype AIV on antigenicity and virulence,it will provide gist to the prevention and cure H9N2 AI ,provide guidance to the inoculation and the selection of H9N2 strain used to produce vaccine.