携带荧光素酶的新型可示踪流感病毒及其体内外生物学特性研究

Influenza virus has major impact on human and animal health. Mouse models are used to study viral replication, define viral and host determinants of pathogenesis and screen antiviral drugs. Conventional studies of influenza infection in mice model rely upon euthanizing and dissecting cohorts of animals at multiple time points to identify sites of infection and quantify viral titers in different types of organs. These experimental paradigms are expensive and time consuming because of the need of sufficient animals at each time point to generate statistically significant data. These limitations of conventional experimental approaches to study viral infection highlight the need for new, alternative strategies to investigate viral infection in vivo. Bioluminescence imaging has becoming a new powerful method to study viral infection in small animal models. This method offers monitoring of spatial and temporal progression of infection in the same living animal. Compared to traditional techniques, bioluminescence imaging could provide more intuitive and sensitive real-time data with low costs. In this study, we aim to generate replicate-competent recombinant influenza virus carrying luciferase reporter gene in viral genome. By modulating the packaging signal sequences and the insertion of autocleave 2A peptide sequence, the recombinant virus has the genetic stability of luminescence reporter in viral genome and at the same time maintain the virulence of wild type virus. After intravenous injection of substrate of luciferase, the dynamics of viral replication could be directly detected by In Vivo Imaging System in infected mice. This replication-competent influenza virus carrying luciferase reporter gene could open additional avenues of research on developing antiviral therapeutics and vaccines and on in vivo studies of viral dissemination and pathogenicity.

流感病毒给人类和动物健康带来巨大威胁，小鼠模型可用来研究流感病毒感染、病毒与宿主间相互作用以及药物抗病毒活性等。传统的研究方法需要在多个时间点分批处死动物，进行组织器官分离、病毒滴定等实验，存在耗时、耗力、对动物和试剂需求量较大等缺点。相比之下，生物发光活体成像则可以对同一实验动物体内病毒复制进行连续、实时的活体动态监测，具有操作简单、结果直观、灵敏度高、成本低等特点。本研究拟对流感病毒基因组进行改造，制备可自主复制的、携带生物发光荧光素酶报告基因的重组流感病毒。通过基因两端包装信号的调节与口蹄疫病毒2A蛋白自剪切序列的插入，使重组病毒既稳定表达荧光素酶报告基因，同时又保持病毒毒力，能够真实地反映流感病毒体内外复制特性。尾静脉注射荧光素酶底物后，利用活体动物体内可见光成像系统，直接监控活体小鼠体内病毒的复制动态，为流感病毒致病机制研究、新型抗流感药物与疫苗的研发提供新的策略和有效手段。

流感病毒能引起季节性流感及流感大流行，如H1N1猪流感、H5N1禽流感及最近新出现的H7N9禽流感感染人并伴有高病死率。由于流感病毒与宿主间相互作用机制尚未明确、尚缺乏有效的治疗手段以及广谱流感疫苗，使得流感病毒的防控与治疗仍然是对人类的一大挑战。小鼠是流感研究中常用的动物模型，传统的研究方法都是通过监测小鼠体重变化、生存率及脏器中病毒滴定来评判病毒致病力、宿主反应性及评估疫苗与抗病毒疗效的指标。这些方法都不能实时动态的直接反应病毒在活体宿主中的复制特点。为此，本项目利用流感病毒反向遗传操作技术对流感病毒基因组进行改造，制备可自主复制的、携带生物发光荧光素酶报告基因的重组流感病毒IAV-luc。IAV-luc与PR8剂量的病毒通过滴鼻分别感染小鼠，感染后48h静脉注射底物腔肠素后，将小鼠置于活体成像仪中观察。IAV-luc感染鼠鼻腔与胸腔位置有明显发光，而PR8感染对照小鼠未见发光。将小鼠安乐死后，解剖去除小鼠肺脏，体外灌注腔肠素底物，可见IAV-luc小鼠感染肺脏明显发光，而PR8感染对照鼠肺脏未见发光。取不同剂量的IAV-luc滴鼻感染小鼠，感染小鼠鼻腔与胸腔的发光强度同病毒感染剂量呈正相关。IAV-luc滴鼻感染小鼠，在感染后1-6天，检测感染小鼠的发光，可见IAV-luc感染小鼠在感染后的体内发光与发光部位光子值呈现动态变化。感染后1天时基本未见发光，感染后2天发光最强，随后开始逐渐回落。取出小鼠肺脏，用传统方法对肺脏病毒滴定，结果显示，IAV-luc感染小鼠肺脏光子值同pfu滴度呈现很好的相关性。. IAV-luc感染细胞与感染小鼠模型，能够快速简便的反应抗血清对流感病毒的抑制效果及小鼠体内抗病毒疗效。可复制型IAV-luc荧光报告流感病毒将为流感病毒致病机制研究、新型抗流感药物与疫苗的研发提供新的策略和有效手段。