Abstract
We previously demonstrated ZIKV replicates in trophoblasts and other placental cells using human and primate models. Recent evidence also suggests that ZIKV induces autophagy via LC3-II-dependent mechanisms. While inhibition of autophagy restricts ZIKV placental infection in mice and cytotrophoblasts (CT) in vitro, leading to suggestion that anti-autophagal drugs could inhibit transmission, primary human and primate data is missing. To generate preclinical data, we directly assessed placental and trophoblast-specific autophagal activity following ZIKV infection in human cases and primate models. Placentas of clinical cases (n=4) and primates (n=2) with confirmed ZIKV infection and CZS, alongside negative controls (n=4), were assessed for co-localization of LC3-II and the ZIKV virus by histology. Trophoblast cells were isolated (n=10), and infected with ZIKV or mock. Viral replication was confirmed by single molecule FISH, strand-specific PCR, and immunofluorescence. The relative expression of autophagy-associated genes (ULK-1, BECN1, ATG5, ATG7, ATG12, MAP1LC3A, MAP1LC3B, ATG16L1, SQSTM1) were quantitated by TaqMan qPCR. Significance was determined by p<0.05. Blinded histologic exam demonstrated ZIKV replication in case but not control human and primate placentas in syncytiotrophoblasts (ST), CT and Hofbauer cells (Fig 1A). Co-localization of autophagy marker LC3-II in human and primate placentas however is limited to decidua and villous parenchyma and sparse in ST (Fig 1B). In validation studies, 4G2 antibody labeling confirmed ZIKV replication in ex vivo trophoblasts (Fig 2A). Of 9 autophagy related genes, ATG5 and SQSTM1 were decreased ∼2 fold in these ZIKV-infected trophoblasts (p=0.04 and p=0.03) (Fig 2B). Two alternate transcripts of LC3-II (MAP1LC3A and MAP1LC3B) were unchanged. We have demonstrated for the first time that while ZIKV replicates in CT, ST, and Hofbauer cells, autophagy appears limited to cells in the decidual and villous parenchyma and not ST. These findings explain observations that the placenta serves as a conduit and a reservoir for infection, and why the ST still function during ongoing low level ZIKV replication. Second, they provide preclinical evidence supporting use of an anti-autophagal drug as potential adjunctive therapy to prevent vertical ZIKV transmission.View Large Image Figure ViewerDownload Hi-res image Download (PPT)
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