不同H9N2亚型鸭流感病毒NS1基因克隆和功能进化分析

摘要

[Objective] The study aimed to lay a foundation for the further studies on function mechanism of NS1 protein in the interspecies transmission of waterfowl influenza virus. [Method] Using the serologic assay and the specific RT-PCR method, some strains of H9 subtype waterfowl influenza virus were isolated from the 12 to 20 day-old muscovy duck flocks without any clinical symptoms in different areas of Guangdong Province. Four of these strains, including A/duck/ZQ/303/2007(H9N2) (A3 for short), A/Duck/FJ/301/2007(H9N2) (C1 for short), A/Duck/NH/306/2007(H9N2) (D6 for short), A/duck/SS/402/2007(H9N2) (E2 for short), and a strain named A/duck/ZC/2007(H9N2) (L1 for short) from a muscovy duck died of avian influenza virus (AIV), were used for NS1 gene cloning and sequencing. Subsequently, the obtained NS1 gene sequences were compared with other NS1 sequences registered in GenBank, and the phylogenetic analysis was also conducted. [Result] When compared with the H9N2 AIV NS1 sequences in GenBank, the NS1 genes of the four AIV strains A3, C1, D6 and E2 displayed homologies ranging from 99% to 100% at nucleotide level, and 95% to 100% at amino acid level; while the NS1 gene of L1 strain displayed homology ranging from 94% to 97% at nucleotide level, and 93% to 98% at amino acid level. The phylogenetic tree demonstrated that A3, C1, D6 and E2 were highly resemblant, and L1 was closest to AY66473 (chicken, 2003). By comparison with the NS1 gene sequences of L1, AF523514 (duck), AY664743 (chicken) and EF155262.1 (quail) using DNAstar, A3, C1, D6 and E2 presented nucleotide variations at site 21(R→Q), 70, 71(KE→EG), 86 (A→S), 124 (V→M) and 225 (S→N), and amino acid variations at site 21, 70, 71 and 86 in dsRNA-dependent protein kinase (PKR) binding domain of NS1 gene, which induced the evident variations of antigenic determinant and surface probability plot of NS1 protein. [Conclusion] This study suggested that the amino acid sequence variation in PKR binding domain of NS1 protein had something to do with the virus pathogenicity.