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| IJCEM Copyright © 2008-All rights reserved. Published by e-Century Publishing Corporation, Madison, WI 53711 |
Int J Clin Exp Med 2(4):363-373;2009
Original Article
The development, optimization and validation of an assay for high throughput
antiviral drug screening against Dengue virus
Pulin Che, Lihua Wang, Qianjun Li
Department of Microbiology, Division of Infectious Diseases, Department of Medicine, University of Alabama at
Birmingham, AL 35294, USA
Received November 5, 2009, accepted November 30, 2009, available online: December 8, 2009
Abstract: 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 (IJCEM911001).
Key words: Dengue virus, high throughput screening, HTS, cytopathic effect, CPE, assay development, assay optimization,
assay validation, antiviral
Full Text PDF
Address all correspondence to:
Qianjun Li, PhD,
Division of Infectious Diseases,
Department of Medicine, University of Alabama at
Birmingham, Birmingham, AL 35294,
Tel: 205-975-7601, Fax: 205-934-5600
E-mail: liq@uab.edu
Original Article
The development, optimization and validation of an assay for high throughput
antiviral drug screening against Dengue virus
Pulin Che, Lihua Wang, Qianjun Li
Department of Microbiology, Division of Infectious Diseases, Department of Medicine, University of Alabama at
Birmingham, AL 35294, USA
Received November 5, 2009, accepted November 30, 2009, available online: December 8, 2009
Abstract: 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 (IJCEM911001).
Key words: Dengue virus, high throughput screening, HTS, cytopathic effect, CPE, assay development, assay optimization,
assay validation, antiviral
Full Text PDF
Address all correspondence to:
Qianjun Li, PhD,
Division of Infectious Diseases,
Department of Medicine, University of Alabama at
Birmingham, Birmingham, AL 35294,
Tel: 205-975-7601, Fax: 205-934-5600
E-mail: liq@uab.edu
