1008 Autophagy and ZIKV viral replication co-occur in human and primate placentae

Abstract

Evidence suggests that ZIKV induces autophagy via LC3-dependent mechanisms in trophoblasts, and inhibition of autophagy restricts placental infection in mice and cytotrophoblasts in vitro. Thus, anti-autophagal drugs (i.e, hydroxychloroquine) could inhibit vertical transmission. To generate preclinical data, we assessed primary cell culture and placentae from human congenital infections, and an experimental non-human primate (Callithrix jacchus) model for associated autophagal activity. Primary trophoblast cells were isolated from placenta (n=10) and infected in vitro with ZIKV. Autophagy-associated gene expression (ULK-1, BECN1, ATG5, ATG7, ATG12, ATG16L1, MAP1LC3A, MAP1LC3B, p62) was determined by TaqMan qPCR for changes with ZIKV. In in vivo validation experiments, autophagy genes LC3B and p62 were probed using in situ hybridization (ISH) in the placentae of Congenital Zika Syndrome (n=3) and ZIKV-infected primate placenta and fetal tissue (n=1). Infected and uninfected villi were compared for mean density and colocalization of autophagal markers. Studies of primary human trophoblasts revealed key autophagy genes ATG5 and p62 were decreased ∼2 fold in ZIKV-infected trophoblasts (p=0.04 and p=0.03). Histologic examination by ISH confirmed ZIKV replication in primate congenital infection cases (Fig 1A). Immunohistological identification of autophagal proteins LC3B and p62 demonstrates co-localization (Fig 1B-C). Protein expression was assessed via densitometry and revealed ∼0.8 fold decrease in p62 in villi with active ZIKV replication (p=0.007), confirming autophagal flux (Fig 2). We demonstrate for the first time that the association of autophagy at the maternal fetal barrier is observed following ZIKV in vivo using three methodologies. Downregulation of key autophagy genes is consistent with findings in Dengue virus, where autophagy is initially induced then downregulated in late stage infection. These findings collectively suggest antiautophagal drugs may be effective in mediating vertical transmission of ZIKV and potentially other novel RNA viruses.View Large Image Figure ViewerDownload Hi-res image Download (PPT)