The development, optimization and validation of an assay for high throughput antiviral drug screening against Dengue virus

International Journal of Clinical and Experimental Medicine

Lihua Wang¹  Pulin Che¹  Qianjun Li¹ 

关键词: Dengue virus;    high throughput screening;    HTS;    cytopathic effect;    CPE;    assay development;    assay optimization;    assay validation;    antiviral;   

DOI  :  

学科分类：医学（综合）

来源: e-Century Publishing Corporation

【 摘 要 】

Dengue virus (DENV) is listed as one of the NIAID Category A priority pathogens. Dengue disease is endemic in most tropical countries, with an estimated 2.5 billion people living in areas at risk of DENV infection. Due to the lack of vaccines and antiviral drugs, it is now a huge public health burden around the world. In order to screen large compound libraries for the identification of novel antivirals targeting DENV, it is essential to develop a high throughput screening (HTS) amenable assay. Here, we present the development, optimization and validation of a cytopathic effect-based assay against Dengue virus serotype-2 (DENV-2). The assay conditions, including cell culturing conditions, DMSO tolerance and the multiplicity of infection, were optimized in both 96- and 384-well plates. Assay robustness and reproducibility were determined under the optimized conditions in 96-well plate, including Z'-value of 0.71, signal-to-background ratio of 6.88, coefficient of variation of 6.3% in mock-infected cells and 12.3% in DENV-2 infected cells. This assay was further miniaturized into a 384-well plate format with similar assay robustness and reproducibility comparing with these in the 96-well plate format. This assay was then validated using the LOPAC¹²⁸⁰ compound library, demonstrating its repeatability with comparable assay robustness and reproducibility. This fully developed and validated HTS amenable assay could be used in future studies to screen large compound libraries for the identification of novel antivirals against dengue disease.

【 授权许可】

Unknown