Cell-based antiviral assays for screening and profiling inhibitors against dengue virus.

Dengue, a mosquito-borne virus of the Flaviviridae family, is reemerging as one of the most important human pathogens in tropical and subtropical regions of the world. It is estimated that 2.5 billion people live in areas at risk for transmission of dengue virus (DENV). Furthermore, it causes significant morbidity and mortality with 50-100 million infections per year. Currently, there are no vaccines commercially available and no effective antiviral drugs for treatment of DENV infections. In this chapter, we describe a plaque reduction assay and a cell-based high-throughput antiviral assay for identifying inhibitors against DENV. The latter is a homogeneous high-throughput assay that has been fully validated according to the Food and Drug Administration (FDA) guidelines for assay validation and can be used for screening compound libraries.

A duplex real-time RT-PCR assay for profiling inhibitors of four dengue serotypes.

We have developed a duplex real-time RT-PCR assay for profiling antiviral inhibitors of four dengue virus (DENV) serotypes. In this assay, the primers and the probe for amplifying DENV were designed in the conserved regions of the genome after aligned more than 300 nucleotide sequences of four dengue serotypes deposited in the GeneBank. To discriminate the antiviral activity from the cytotoxicity of compounds, a housekeeping gene of the Vero cells, ß-actin, was used to design the primers and the probe for the second set of PCR as an internal control, which is used to normalize the RNA levels of dengue-specific PCR due to the cellular toxicity of test compounds. For compound profiling, the duplex PCR is performed using LightCycler(®) in a single tube to simultaneously amplify both the dengue target gene and the Vero cell housekeeping gene from the compound-treated Vero cell lysates. This assay was validated against a panel of reference compounds. The results show that the universal primers and probe in this duplex RT-PCR assay can efficiently amplify all four dengue serotypes and that the PCR efficiency for both the dengue target gene and the Vero cells ß-actin gene is 100%.

Expression and purification of dengue virus NS5 polymerase and development of a high-throughput enzymatic assay for screening inhibitors of dengue polymerase.

The nonstructural protein 5 (NS5) of dengue virus (DENV) plays a central role in the virus replication. It functions as a methyltransferase and an RNA-dependent RNA polymerase. As such, it is a promising target for antiviral drug development. To develop a high-throughput biochemical assay for screening compound libraries, we expressed and purified the polymerase domain of the dengue NS5 protein in bacterial cells. The polymerase activity is measured using a scintillation proximity assay. This homogeneous and high--throughput assay enables screening of compound libraries for identifying polymerase inhibitors against DENV. In this chapter we describe the methods to express and purify the dengue NS5 polymerase from E. coli and a validated high-throughput enzymatic assay for screening inhibitors of NS5 polymerase.

Methods for screening and profiling inhibitors of herpes simplex viruses.

Herpes simplex viruses (HSV) establish lifelong latent infections in infected hosts that reactivate -periodically and result in virus shedding and recurrent diseases, such as genital herpes. Sexually transmitted infections (STIs) caused by HSV are a major public health problem worldwide. At present, the only effective antiviral drugs for treatment of HSV are nucleoside analogues, which are incorporated into the DNA chain and terminate the chain elongation during virus replication. With increasing emergence of drug resistance, novel drugs for new viral targets are warranted. In this chapter, several screening and profiling assays including plaque reduction assays, cytopathic effect inhibition assay, and in vitro cytotoxicity assay for identifying and evaluating inhibitors of HSV are described. Assays for mode of action studies, such as virus adsorption and penetration, are also presented.

A fluorescence-based high-throughput screening assay for identifying human cytomegalovirus inhibitors.

Human cytomegalovirus (HCMV) is a common opportunistic pathogen that can cause devastating -morbidity and mortality amongst neonates and immune-compromised patients. The current standard of care for HCMV infection is limited to four antiviral compounds that have major limitations in terms of long--term use, toxicity, and use during pregnancy. To provide patients with alternative treatment options to decrease HCMV-related morbidity and mortality, new drugs with novel modes of action are warranted. Here, we describe a validated high-throughput fluorescence antiviral screening assay based on infection of fibroblast cells with a fluorescently tagged reference strain of HCMV (AD169-GFP) to screen and profile HCMV inhibitors.

Development of a high-throughput antiviral assay for screening inhibitors of chikungunya virus and generation of drug-resistant mutations in cultured cells.

Chikungunya virus (CHIKV) is a mosquito-borne Alphavirus that has already infected millions of people in recent large-scale epidemics in Africa, the islands of the Indian Ocean, South and Southeast Asia, and northern Italy. The infection is still ongoing in many countries, such as India. Although the fatal rate is approximately 0.1% in the La Réunion outbreak, it causes painful arthritis-like symptoms that can last for months or even years. Currently, neither vaccine nor approved antiviral therapy exists to protect humans from chikungunya infection. Therefore, there is an urgent unmet medical need for the development of antiviral drugs for pre-exposure prophylaxis and/or treatment of chikungunya infections. In this chapter, we describe a fully validated ATP/luminescence assay that is effective for high-throughput screening of CHIKV inhibitors. Protocols for growing CHIKV stocks and generating drug-resistant viral variants for modes of action studies of compounds are also described.