Cloning and sequence analysis of HA gene and NA gene of swine influenza virus H9N2 subtype Jinan strain

Two pairs of primers were designed based on HA genes and NA genes of the strains of the H9N2 subtype of influenza virus according to GenBank.Swine influenza virus H9N2 Jinan isolate (SW/SD/JT/07) as template,the HA gene and NA gene were amplified with RT-PCR,which were cloned into pMD18-T vector,sequenced and analyzed.As a result,the ORF of the HA gene of SW/SD/JT/07 was 1 701 bp and encoded 566 amino acids.The amino acid sequence at HA0 cleavage site in SW/SD/JT/07 was R-S-S-R-G,which is non-highly pathogenic.There were the same eight glycosylation sites in the HA proteins of SW/SD/JT/07 and the reference strains.SW/SD/JT/07 HA has five receptor binding sites,but the 190th amino acid residue showed a difference between SW/SD/JT/07 and the reference strains.The ORF of the NA gene of SW/SD/JT/07 was 1 413 bp and encoded 470 amino acids.The amino acid sequence in the NA protein matrix was very conservative among SW/SD/JT/07 and the reference strains.There were five antigenic sites in the NA protein,the 325th amino acid residue was D in SW/SD/JT/07 and was N in the reference strains,the 398th amino acid residue was E in SW/SD/JT/07,DC/HB/W1/04 and CK/GS/2/99 and was D or N in the other strains.The 63th,64th and 65th amino acid residues were deleted in the NA protein of SW/SD/JT/07,CK/SH/F/98,DC/HB/W1/04,CK/GS/2/99,SW/GD/WXL/04,SW/SD/W4/03 and SW/YN/Simao2/07.The nucleotide homologies of the HA genes between SW/SD/JT/07 and the reference strains were from 82.5% to 98.6%,the identity of the NA genes from 82.2% to 99.1%.On the basis of HA gene and NA gene phylogenetic analysis,it implied that SW/SD/JT/07 and the avain influenza virus H9N2 subtype had a nearer inherit distance.