Ouabain

Zika virus (ZIKV) is an arbovirus associated with neurological disorders accompanying congenital infections. With no vaccine or antiviral approved, there is an urgent need for the development of effective antiviral agents against ZIKV infection. We evaluated the anti-ZIKV and immunomodulatory activity of ouabain, a Na⁺/K⁺-ATPase inhibitor known to have immunomodulatory and antiviral activities, using human neural stem and progenitor cells (hNS/PCs) and a murine model of congenital Zika syndrome (CZS). Our data demonstrated that ouabain reduces ZIKV infection in hNS/PCs, mouse placenta, yolk sac, and the fetal head. Ouabain mitigated neurogenesis impairment triggered by ZIKV in hNS/PCs and prevented ZIKV-mediated reduction of fetus and head sizes. In addition, ouabain decreased tumor necrosis factor and interleukin-1β levels in the placenta, highlighting its immunomodulatory activity in the murine model. Our findings indicate that ouabain possesses anti-ZIKV and immunomodulatory activities, suggesting that it should be investigated further as a promising treatment for CZS.

Plants produce defensive toxins to deter herbivores. In response, some specialized herbivores evolved resistance and even the capacity to sequester toxins, affecting interactions at higher trophic levels. Here, we test the hypothesis that potential natural enemies of specialized herbivores are differentially affected by plant toxins depending on their level of adaptation to the plant-herbivore system. We focus on toxic cardiac glycosides (CGs) from milkweeds (Asclepias spp.), which inhibit animal Na+/K+-ATPases, and two CG-resistant insects, the large milkweed bug Oncopeltus fasciatus and a CRISPR-edited Drosophila melanogaster. Both have CG-resistant Na+/K+-ATPases through a set of key amino acid substitutions, which facilitate CG sequestration. We conducted infection experiments with entomopathogenic nematodes (Steinernema carpocapsae, S. feltiae, and S. hermaphroditum) as natural enemies on host insects containing mixtures of milkweed-derived CGs or purified CGs (ouabain, digoxin, and digitoxin) that vary in toxicity. The nematode S. carpocapsae is known to occur in soil near milkweed plants and naturally has several of the same Na+/K+-ATPase substitutions as the milkweed bug O. fasciatus and our Drosophila mutant. This nematode not only exhibited higher fecundity in hosts that carried CGs relative to the other nematode species (which have sensitive Na+/K+-ATPases), but also showed attraction to mixtures of CGs in milkweed root extracts and to purified ouabain when tested on agar plates. A coiling phenotype, which is a symptom of neurotoxicity, was observed more frequently in S. feltiae and S. hermaphroditum upon exposure to milkweed root extracts than in S. carpocapsae. Nematode behavior was further tested in sand, and while attraction to CGs was found for S. carpocapsae, nematodes of the other species tended to migrate away from milkweed root chemicals. Thus, S. carpocapsae can tolerate CGs and may use these as chemical cues to locate insect hosts that live on or around milkweed plants.