Zika virus co-opts miRNA networks to persist in placental microenvironments detected by spatial transcriptomics [visium]

We and others have demonstrated Zika virus (ZIKV) congenital infection evades double-stranded RNA detection, and may persist in the placenta for the duration of pregnancy without accompanying overt histopathologic inflammation. We used orthogonal approaches to test the hypothesis that ZIKV disrupts placental miRNAs to enable viral persistence and fetal pathogenesis. In primary human trophoblasts, high-throughput sequencing crosslinking and immunoprecipitation (AGO-HITS-CLIP) demonstrated an unexpected disruption of placental miRNA-regulated TGF-ß networks. In gnotobiotic mice, absence of microbes rendered normally resistant mice susceptible to congenital ZIKV infection, and placental spatial transcriptomics revealed distinct microenvironments defined by significant upregulation of complement cascade components. Finally, treatment of ZIKV-infected mice with the RNAi-enhancer enoxacin led to loss of ZIKV placental persistence and rescue of fetal growth restriction. These results collectively suggest that ZIKV co-opts miRNA inflammatory regulatory networks to persist in the placenta reservoir, a conduit of vertical transmission and fetal pathogenesis. Overall design: Gnotobiotic congenital ZIKV mouse model. Following timed mattings of four-to-six-week-old Swiss/webster mice from the Baylor College of Medicine Gnotobiotics Core, plugged dams were weighed and transferred to separate sterile insulators. Daily intraperitoneal injections of enoxacin (Sigma-Aldrich, Cat. 557304) at 10 mg/kg were previously shown to be safe and effective in a model of obesity32 and were done from embryonic days E1.5-E18.5. Dams were either received mock United States Pharmacopeia (USP)-grade phosphate-buffered saline (PBS) (VWR, Cat. 76081-514) subdermal injections or ZIKV infection with HN16 strain (first passage in Vero-E6 cells) at an MOI of 1x105 PFU on embryonic days E7.5, E8.5, and E9.5. On day 18.5, dams were humanely sacrificed, and uterine horns were dissected. Images of the uterine horns were taken to record resorption events. Individual fetuses were weighed. Placental tissues were stored at -80C in Trizol. All mouse experiments were conducted under the supervision of Baylor College of Medicine Institutional Animal Care and Use Committee approved protocols.