华东地区新分离的 H9N2 AIV 的分子进化特征及波形蛋白在 MDCK 细胞中对病毒复制的功能。
The molecular evolutionary characteristics of new isolated H9N2 AIV from East China and the function of vimentin on virus replication in MDCK cells.
机构信息
Key Laboratory of Animal Microbiology of China's Ministry of Agriculture, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, 210095, China.
Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences, Nanjing, 210014, China.
出版信息
Virol J. 2020 Jun 17;17(1):78. doi: 10.1186/s12985-020-01351-9.
BACKGROUND
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.
METHODS
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.
RESULTS
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.
CONCLUSIONS
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.
背景
低致病性 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的 H9N2 病毒的分子进化特征，并研究 H9N2 AIV 在敏感细胞中的复制的细胞内靶蛋白。
方法
从山东健康鸡的泄殖腔中分离出 AIV A/鸡/山东/LY1/2017（H9N2），并通过 RT-PCR 扩增和分析该分离的 H9N2 AIV 的全长 8 个基因片段。MDCK 细胞用作靶细胞模型，通过 VOPBA 测定和 LC-MS/MS 鉴定 H9N2 AIV 的病毒结合蛋白。用特异性抗体和 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 Vimentin-pcDNA3.0，并转染至 MDCK 细胞中，然后用 RT-PCR 检测 H9N2 AIV mRNA。
结果
系统进化分析表明，分离的 H9N2 AIV 的 HA、NA、PB2、PB1、PA、NP 和 M 七个基因来源于 A/鸡/上海/F/98，而 NS 基因来源于 A/鸭/香港/Y439/97。分离的 H9N2 AIV 的 HA 基因的裂解位点序列为 PARSSR G 模式，受体结合位点的左侧序列（224~229）为 NGQQGR 模式，与 A/鸡/上海/F/98 相似。经 VOPBA 测定，我们发现一种约 50KDa 的蛋白质与 H9N2 AIV 结合，LC-MS/MS 分析结果证实，波形蛋白是与 H9N2 AIV 结合的重要蛋白。用 H9N2 AIV 处理前孵育特异性抗体和 siRNA 可降低 MDCK 细胞中的病毒 RNA 水平。此外，我们发现过表达波形蛋白可增加 MDCK 细胞中 H9N2 AIV 的复制。
结论
这些发现表明，分离的 H9N2 AIV 可能是最近临床常见的 H9N2 株，波形蛋白可能是 H9N2 AIV 在 MDCK 细胞中复制的重要因素之一，可能是设计和开发抗病毒药物的新靶点。
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