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

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: Primary human trophoblast isolation and ZIKV infection. Term placentas from 4 healthy patients were subject to cytotrophoblast purification as described previously 11. Cytotrophoblasts were seeded in 60 cm dishes and maintained with DMEM:F12 media (Gibco, Cat. 11320-033) supplemented with + 10% heat-inactivated fetal bovine serum (FBS; Gibco, Cat. 16140-071) + 1% penicillin-streptomycin (P/S; Gibco, Cat. 15140-122). Media was changed daily for 2-4 days until cells differentiated into syncytiotrophoblasts confirmed by peak ß-human chorionic gonadotropin expression. At 5 days post-isolation, primary human trophoblasts cells from each patient (105-90 million cells/replicate) were mock-infected with PBS or infected with a contemporary first-passage ZIKV strain HN16 (GenBank accession KY328289.1)10,14 at an MOI of 1 plaque-forming units (PFU) and harvested at 5 days post-infection.