Background:Multiple studies have shown both induction and inhibition of autophagy during Zika virus (ZIKV) infection. While some have proposed mechanisms by which autophagic dysregulation might facilitate ZIKV vertical transmission, there is a lack ofin situdata in human and non-human primate models. This is an especially pertinent question as autophagy-inhibitors, such as hydroxychloroquine, have been proposed as potential therapeutic agents aimed at preventing vertical transmission of ZIKV and other RNA viruses.Objectives:Given the paucity of pre-clinical data in support of either autophagic enhancement or inhibition of placental ZIKV viral infection, we sought to assess cellular, spatial, and temporal associations between placental ZIKV infection and measures of autophagy in human primary cell culture and congenital infection cases, as well as an experimental non-human primate (marmoset,Callithrix jacchus) model.Study Design:Primary trophoblast cells were isolated from human placentae (n= 10) and infectedin vitrowith ZIKV. Autophagy-associated gene expression (ULK-1, BECN1, ATG5, ATG7, ATG12, ATG16L1, MAP1LC3A, MAP1LC3B, p62/SQSTM1) was then determined by TaqMan qPCR to determine fold-change with ZIKV-infection. Inin vivovalidation experiments, autophagy genesLC3Bandp62/SQSTM1were probed usingin situhybridization (ISH) in the placentae of human Congenital Zika Syndrome (CZS) cases (n= 3) and ZIKV-infected marmoset placenta (n= 1) and fetal tissue (n= 1). Infected and uninfected villi were compared for mean density and co-localization of autophagic protein markers.Results:Studies of primary cultured human trophoblasts revealeddecreasedexpression of autophagy genesATG5andp62/SQSTM1in ZIKV-infected trophoblasts [ATG5fold change (±SD) 0.734-fold (±0.722),p= 0.036; p62/SQSTM1 0.661-fold (±0.666),p= 0.029]. Histologic examination by ISH and immunohistochemistry confirmed spatial association of autophagy and ZIKV infection in human congenital infection cases, as well as marmoset placental and fetal tissue samples. When quantified by densitometric data, autophagic protein LC3B, and p62/SQSTM1 expression in marmoset placenta were significantlydecreasedinin situZIKV-infected villi compared to less-infected areas [LC3B mean 0.951 (95% CI, 0.930–0.971),p= 0.018; p62/SQSTM1 mean 0.863 (95% CI, 0.810–0.916),p= 0.024].Conclusion:In the current study, we observed that in the non-transformed human and non-human primate placenta, disruption (specifically down-regulation) of autophagy accompanies later ZIKV replicationin vitro, in vivo, andin situ. The findings collectively suggest that dysregulated autophagy spatially and temporally accompanies placental ZIKV replication, providing the firstin situevidence in relevant primate pre-clinical and clinical models for the importance of timing of human therapeutic strategies aimed at agonizing/antagonizing autophagy. These studies have likely further implications for other congenitally transmitted viruses, particularly the RNA viruses, given the ubiquitous nature of autophagic disruption and dysregulation in host responses to viral infection during pregnancy.