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Background: The human filarial parasites cause diseases that are among the most important causes of morbidity in the developing world. The elimination programs targeting these infections rely on a limited number of drugs, making the identification of new chemotherapeutic agents a high priority. The study of these parasites has lagged due to the lack of reverse genetic methods. Methodology/Principal findings: We report a novel co-culture method that results in developmentally competent infective larvae of one of the human filarial parasites (Brugia malayi) and describe a method to efficiently transfect the larval stages of this parasite. We describe the production of constructs that result in integrative transfection using the piggyBac transposon system, and a selectable marker that can be used to identify transgenic parasites. We describe the production and use of dual reporter plasmids containing both a secreted luciferase selectable marker and fluorescent protein reporters that will be useful to study temporal and spatial patterns of gene expression. Conclusions/Significance: The methods and constructs reported here will permit the efficient production of integrated transgenic filarial parasite lines, allowing reverse genetic technologies to be applied to all life cycle stages of the parasite. [ABSTRACT FROM AUTHOR]

Background: A homologue of the ecdysone receptor has previously been identified in human filarial parasites. As the ecdysone receptor is not found in vertebrates, it and the regulatory pathways it controls represent attractive potential chemotherapeutic targets. Methodology/ Principal Findings: Administration of 20-hydroxyecdysone to gerbils infected with B. malayi infective larvae disrupted their development to adult stage parasites. A stable mammalian cell line was created incorporating the B. malayi ecdysone receptor ligand-binding domain, its heterodimer partner and a secreted luciferase reporter in HEK293 cells. This was employed to screen a series of ecdysone agonist, identifying seven agonists active at sub-micromolar concentrations. A B. malayi ecdysone receptor ligand-binding domain was developed and used to study the ligand-receptor interactions of these agonists. An excellent correlation between the virtual screening results and the screening assay was observed. Based on both of these approaches, steroidal ecdysone agonists and the diacylhydrazine family of compounds were identified as a fruitful source of potential receptor agonists. In further confirmation of the modeling and screening results, Ponasterone A and Muristerone A, two compounds predicted to be strong ecdysone agonists stimulated expulsion of microfilaria and immature stages from adult parasites. Conclusions: The studies validate the potential of the B. malayi ecdysone receptor as a drug target and provide a means to rapidly evaluate compounds for development of a new class of drugs against the human filarial parasites. [ABSTRACT FROM AUTHOR]