The dengue viruses (DENV) constitute a single species within the genus Flavivirus of the Flaviviridae family and are transmitted to humans through infected mosquitoes. DENV causes dengue fever, an often severe flu-like illness, and the more serious dengue hemorrhagic fever. DENV contains a single-stranded, positive-sense RNA genome of ~10
kb in length, which encodes three structural proteins involved in particle formation (capsid [C], membrane [prM], and envelope [E]) and seven nonstructural proteins (NS) involved in viral replication (NS1, NS2a, NS2b, NS3, NS4a, NS4b, and NS5).1
The myriad strains of DENV can differ from each other by as much as 35% at the nucleotide level, but all strains belong to one of four serotypes (DENV-1–4),2
based on antigenic criteria. DENV enters cells by receptor-mediated endocytosis and replicates in the cytoplasm.
Over the past 30 years, infection with DENV has emerged as one of the most significant health risks in tropical and subtropical regions worldwide, with upward of 50 million people infected yearly.3
Further, according to the World Health Organization, two-fifths of the world's population is at risk for infection with DENV.4
Presently, no vaccine or specific antiviral agent is approved for use and the need to develop novel interventional strategies is pressing. Several recent reports have identified small-molecule compounds with anti-DENV activity, including ribavirin,5
mycophenolic acid,6 N
and an adenosine analog NITD008.11
However, only few of these compounds appear to have the pharmacologic properties appropriate for a commercial drug and no drug against DENV has yet shown efficacy in a clinical trial.
To date, most antiviral drug candidates target viral gene products and their use typically results in the generation of mutant drug-resistant virus. In contrast, the targeting of host factors may reduce or avoid the generation of resistance and allow the development of more durable therapies. Recently, considerable investigation has helped elucidate host–virus interactions, which occur during DENV infection and pathogenesis,12–14
although understanding of the cellular and molecular mechanisms involved remains far from complete.
Traditionally, antiviral drug development has employed cytopathic and plaque reduction assays to screen and evaluate compound efficacy.15
Plaque reduction assays are labor intensive and not amenable to screen large numbers of compounds, because of their low-throughput nature. Various methods to evaluate antiflaviviral efficacy and cytotoxicity have been developed, and these methods have been adapted to 96-well and, in some cases, 384-well microtiter plate formats from low- to high-throughput applications.16–19
To rapidly identify new antiviral drugs against DENV, screening strategies must be robust and able to evaluate large libraries of compounds for both antiviral efficacy and cytotoxicity. One of the most powerful tools available for drug discovery is high-content screening, which combines high-throughput screening (HTS) with the ability to collect cellular images of biological processes. Automated microscope technology has now advanced to the point where images from microtiter plates can be rapidly visualized and recorded with clarity.20–22
Changes in virus quantity or cell viability can be extracted from the acquired images and quantified using sophisticated analysis tools. When performed in parallel at a primary screening level, compounds can be rapidly evaluated for antiviral activity and cytotoxicity in living cells. Once successfully integrated, high-content technology can minimize both lead times for candidate generation and the probability of failures in eventual clinical trials.
In this article, we describe adaptation, validation, and screening of a high-content assay against a chemical library of known drugs and bioactives. Our assay utilizes a multiparametric readout to directly monitor viral replication in host cells, simultaneously evaluating cell viability to rapidly score compounds for activity. In the screening of a well-characterized collection of bioactives with our high-content assay, we identified hits that target several prominent classes of cellular factors including transporters, receptors, and enzymes, potentially allowing for repurposing of use as a new indication for antiviral therapies against DENV infections.