The low pathogenic H9N2 AIV caused the serious impact on the poultry industry and public safety. Our purpose was to investigate the molecular evolutionary characteristics of the new isolated H9N2 virus and investigate the intracellular target protein of H9N2 AIV replication in sensitive cells. AIV A/chicken/Shandong/LY1/2017 (H9N2) was isolated from the cloaca of the healthy chicken in Shandong, and the full-length eight gene segments of this isolated H9N2 AIV were amplified by RT-PCR and analyzed. MDCK cells were used as the target cell model, and VOPBA assay and LC-MS/MS were carried out to identify the virus-binding protein of H9N2 AIV. MDCK cells were pre-treated with the special antibody and siRNA, and treated with H9N2 AIV to detect the virus replication. Additionally, Vimentin-pcDNA3.0 was successfully constructed, and transinfected into MDCK cells, and then H9N2 AIV mRNA was detected with RT-PCR. Phylogenetic analysis revealed that HA, NA, PB2, PB1, PA, NP and M seven genes of the isolated H9N2 AIV were derived from A/Chicken/Shanghai/F/98, while NS gene was derived from A/Duck/Hong Kong/Y439/97. The cleavage site sequence of HA gene of the isolated H9N2 AIV was a PARSSR G pattern, and the left side sequence (224 ~ 229) of receptor binding site was NGQQGR pattern, which were similar to that of A/Chicken/Shanghai/F/98. Following VOPBA assay, we found one protein of about 50KDa binding to H9N2 AIV, and the results of LC-MS/MS analysis proved that vimentin was the vital protein binding to H9N2 AIV. The pre-incubation of the specific antibody and siRNA decreased the viral RNA level in MDCK cells treated with H9N2 AIV. Furthermore, we found that over-expressed vimentin increased H9N2 AIV replication in MDCK cells. These findings suggested that the isolated H9N2 AIV might be a recent clinical common H9N2 strain, and vimentin protein might be one vital factor for H9N2 AIV replication in MDCK cells, which might be a novel target for design and development of antiviral drug.

中文翻译：

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来自华东地区的新分离出的H9N2 AIV的分子进化特征以及波形蛋白对MDCK细胞中病毒复制的作用。

低致病性的H9N2 AIV对家禽业和公共安全造成了严重影响。我们的目的是研究新分离出的H9N2病毒的分子进化特征，并研究H9N2 AIV在敏感细胞中复制的胞内靶蛋白。从山东健康鸡的泄殖腔中分离出AIV A / chicken / Shandong / LY1 / 2017（H9N2），并通过RT-PCR扩增了该分离的H9N2 AIV的全长8个基因片段。将MDCK细胞用作靶细胞模型，并进行VOPBA测定和LC-MS / MS鉴定H9N2 AIV的病毒结合蛋白。用特殊抗体和siRNA预处理MDCK细胞，并用H9N2 AIV处理以检测病毒复制。此外，成功构建了波形蛋白pcDNA3.0，然后转染到MDCK细胞中，然后用RT-PCR检测H9N2 AIV mRNA。系统进化分析显示，分离出的H9N2 AIV的HA，NA，PB2，PB1，PA，NP和M七个基因分别来自A / Chicken / Shanghai / F / 98，NS基因来源于A / Duck / Hong Kong / Y439 / 97。分离出的H9N2 AIV HA基因的切割位点序列为PARSSR G模式，受体结合位点的左侧序列（224〜229）为NGQQGR模式，与A / Chicken / Shanghai / F /相似。 98。通过VOPBA分析，我们发现了一种与H9N2 AIV结合的约50KDa的蛋白质，LC-MS / MS分析的结果证明波形蛋白是与H9N2 AIV结合的重要蛋白质。特异性抗体和siRNA的预孵育降低了用H9N2 AIV处理的MDCK细胞中病毒RNA的水平。此外，我们发现过表达的波形蛋白增加了MDCK细胞中H9N2 AIV的复制。这些发现表明，分离的H9N2 AIV可能是最近临床上常见的H9N2菌株，波形蛋白可能是MDCK细胞中H9N2 AIV复制的重要因素，这可能是抗病毒药物设计和开发的新目标。