ZIKA virus effects on neuroprogenitors are exacerbated by the main pyriproxyfen metabolite via thyroid hormone signaling disruption

North-Eastern Brazil saw intensive application of the insecticide pyriproxyfen (PPF) during the microcephaly outbreak caused by Zika virus (ZIKV). ZIKV requires the neural RNA-binding protein Musashi-1 to replicate. TH represses MSI1. Being a suspected TH disruptor, we hypothesized that co-exposure to the main metabolite of PPF, 49-OH-PPF, would exacerbate ZIKV effects through increased MSI1 expression. This was tested using in vitro mouse neurospheres and an in vivo TH signaling reporter model, Xenopus laevis. TH signaling was decreased by 49-OH-PPF in both models. In mouse-derived neurospheres the metabolite reduced neuroprogenitor proliferation as well as markers of neuronal differentiation. The results demonstrated that 49-OH-PPF significantly induced MSI1 at both the mRNA and protein level, as well as Fasn mRNA. Other TH target genes were also significantly modified. Importantly, several key genes implicated in neuroprogenitor fate and commitment were not dysregulated by 49-OH-PPF alone, but were in combination with ZIKV infection. These included the neuroprogenitor markers Nestin, Egfr, Gfap, Dlx2 and Dcx. Unexpectedly, 49-OH-PPF decreased ZIKV replication, although only at the fourth and last day of incubation, and RNA copy numbers stayed within the same order of magnitude. However, intracellular RNA content of neuroprogenitors was significantly decreased in the combined presence of the PPF metabolite and ZIKV. We conclude that 49-OH-PPF interferes with TH action in vivo and in vitro, inhibiting neuroprogenitor proliferation. In the presence of ZIKV, TH signaling pathways crucial for cortical development are significantly impacted. This provides another example of viral effects that are exacerbated by drug or pesticide use.