携带增强绿色荧光蛋白的重组甲型流感病毒构建及体外应用

Development and in vitro application of a recombinant influenza A virus expressing enhanced green fluorescent protein

摘要:

目的 构建表达增强绿色荧光蛋白(enhanced green fluorescent protein, EGFP)报告基因的重组甲型流感病毒，并研究其体外生物学特性、抗病毒药物筛选及中和抗体检测的应用。方法 运用流感病毒反向遗传学技术在H1N1-PR8病毒神经氨酸酶(NA)基因的C端插入EGFP基因，成功构建并拯救出重组甲型流感病毒，命名为PR8NAEGFP/WSN。采用逆转录聚合酶链式反应(reverse transcription-polymerase chain reaction, RT-PCR)与测序分析验证EGFP插入位置的准确性及遗传稳定性；通过EGFP报告基因表达、复制动力学分析和空斑形态观察，对重组病毒(PR8NAEGFP/WSN)和对照病毒(PR8NA/WSN，无EGFP插入)进行鉴定；采用病灶形成试验(focus-forming assay, FFA)、定量聚合酶链式反应(quantitative polymerase chain reaction, qPCR)和EGFP荧光检测，评估流感病毒阳性药物法匹拉韦的体外抗病毒药效；同时采用病灶减少中和试验(focus reduction neutralization test, FRNT)和报告基因活性检测，开展甲型流感病毒(influenza A virus, IAV)中和抗体检测。结果 重组病毒(PR8NAEGFP/WSN)在鸡胚中经过5代盲传后仍能保持遗传稳定。与PR8NA/WSN相比，该重组病毒在48 h测得的病毒滴度略低，结晶紫噬斑形态与对照病毒相似。采用重组病毒测得的法匹拉韦半数有效浓度(half maximal effective concentration, EC₅₀)与对照病毒一致，且通过FFA、qPCR和EGFP荧光等多种检测方法所得的EC??值之间具有良好的相关性。FRNT和EGFP荧光测定的中和抗体滴度相关系数r为0.930 4，结果具有良好的一致性。结论 携带报告基因的重组流感病毒为IAV的基础研究、抗病毒药物和疫苗评价提供了实时可视化的工具。

Abstract:

Objective To develop a recombinant influenza A virus (IAV) expressing a reporter (enhanced green fluorescent protein, EGFP), study its biological characteristics in vitro, and explore its application in antiviral drug screening and neutralizing antibody (nAb) detection.Methods The EGFP gene was inserted into the C-terminus of NA (derived from H1N1-PR8) by reverse genetics of influenza virus, and the recombinant IAV was successfully constructed, rescued, and named PR8NAEGFP/WSN. The EGFP insertion position and genetic stability were analyzed by RT-PCR and sequencing. PR8NAEGFP/WSN and the control virus (PR8NA/WSN, without EGFP insertion) were identified based on EGFP reporter gene expression, replication kinetics, and plaque morphology. The in vitro antiviral efficacy of favipiravir, a positive drug for influenza virus, was evaluated by focus-forming assay (FFA), qPCR, and EGFP fluorescence detection. The focus reduction neutralization test (FRNT) and reporter gene activity detection were conducted for IAV nAb detection.Results PR8NAEGFP/WSN remained genetically stable after five passages in chicken embryos. Compared with PR8NA/WSN, PR8NAEGFP/WSN showed a slightly declined titer at the time point of 48 h and similar crystal violet plaque morphology. The EC₅₀ of favipiravir measured with the recombinant virus is consistent with that with the control virus, and the EC₅₀ values obtained through various detection methods, including FFA, qPCR, and EGFP fluorescence, show good correlations. The correlation coefficient r of the nAb titer determined by FRNT and EGFP fluorescence was 0.930 4, which indicated good consistency.Conclusion PR8NAEGFP/WSN, a recombinant IAV carrying the reporter gene, might be used as a real-time visualization tool for the basic research on IAV and the evaluation of antivirals and vaccines for IAV.