Luciferase-encoded influenza viruses for antiviral screening

描述（由申请人提供）：目前正在努力设计新型疫苗和抗病毒药，以应对流感大流行的威胁。此外，已经报道了耐药突变体的出现，包括对现有药物M2阻断剂和神经氨酸酶（NA）抑制剂双重耐药的那些。高通量筛选试验是鉴定具有新的和尚未鉴定的作用机制的抗病毒药物的重要工具。在这里，我们建议开发这样一种检测方法，使用编码荧光素酶而不是NA（delNA/luc-mutant）的基因工程流感病毒和MDCK细胞系，这允许NA-非依赖性病毒生长。将基于delNA/luc-mutant-感染的细胞中发光的减少来评价抗病毒效果。之前，我们产生了编码eGFP的流感病毒，并使用它来监测病毒在活细胞中的传播。该突变体不需要NA活性的细胞培养物中的感染产量的生产，但在体内高度减毒。随着反向遗传技术的使用，我们最近产生了携带编码萤火虫或海肾萤光素酶的修饰的delNA基因的病毒。这两种酶都是单体和小的（61 kDa和36 kDa），并且都不需要翻译后加工。为了测量酶活性，我们利用了Dual-Glo荧光素酶测定试剂盒（Promega），其被设计为允许对在96孔或384孔板中生长的含有萤火虫和海肾荧光素酶基因的细胞进行机器人高通量分析。将试剂直接加入到生长培养基中的delNA/luc-mutant-感染的细胞中，无需洗涤或预处理。使用Victor 3读板器（PerkinElmer）测量发光，该仪器可以配备有板堆叠器、振荡器、分配器模块、增强安全模式和其他选项，以便于集成到机器人系统中。在展示了原理证明之后，我们现在计划1）鉴定荧光素酶的最佳编码策略; 2）评估嵌合基因的稳定性;以及3）评估各种参数（例如，细胞培养接种密度，M.O.I.，时间P.I.，中等体积等）解读的结果该测定是通用的，并且可以进行修改以产生对现有药物（包括NA抑制剂）敏感的编码腺苷三磷酸酶的突变体。因为所述酶使用不同的底物，所以所述测定还允许同时评价针对两种病毒的抗病毒活性（即，H1和H5）。

DESCRIPTION (provided by applicant): Significant efforts are being directed toward design of novel vaccines and antivirals to confront an influenza pandemic threat. Moreover, emergence of drug resistant mutants, including those dually resistant to existing drugs M2-blockers and neuraminidase (NA) inhibitors has been reported. High-throughput screening assays are essential tools for identification of antivirals with novel and as-yet-unidentified mechanisms of action. Here we propose to develop such an assay with the use of the genetically engineered influenza virus encoding the luciferase instead of NA (delNA/luc-mutant) and MDCK cell line, which allows the NA-independent virus growth. The antiviral effect will be evaluated based on reduction of luminescence in the delNA/luc-mutant-infected cells. Previously, we generated the influenza virus encoding eGFP and used it to monitor the virus spread in live cells. The mutant did not require NA activity for production of infectious yields in cell culture and yet was highly attenuated in vivo. With the use of the reverse genetic technique, we have recently generated viruses carrying the modified the delNA-gene that encodes either Firefly or Renilla luciferase. Both enzymes are monomeric and small (61kDa and 36kDa), and neither requires post-translational processing. To measure the enzyme activity, we took advantage of the Dual-Glo Luciferase Assay kit (Promega), which was designed to allow robotic high-throughput analysis of cells containing genes of Firefly and Renilla luciferases grown in 96- or 384-well plates. The reagent was added directly to the delNA/luc-mutant-infected cells in growth medium without washing or preconditioning. The luminescence was measured with the use of Victor3 plate reader (PerkinElmer), the instrument that could be equipped with a plate stacker, a shaker, dispenser modules, an Enhanced Security mode, and other options for easy integration into a robotic system. Having shown the proof-of-principal, we now plan 1) to identify the optimal coding strategy for luciferase; 2) to evaluate the chimeric gene stability; and 3) to assess the effects of various parameters (e.g., cell culture seeding density, M.O.I., time p.i., medium volume etc.) on the outcome of the readings. The assay is versatile, and modifications can be made to generate the luciferase-encoding mutants susceptible to the existing drugs, including NA inhibitors. Because the luciferases use different substrates, the assay also permits evaluation of antiviral activity against two viruses at once (i.e., H1 and H5).