Vertical transmission of Zika virus (ZIKV) is associated with high fetal demise rates and fetal malformations, such as microcephaly, and the mechanisms behind these are not well-understood. Viral burden in the placenta and placental pathology is frequently observed following maternal infection in humans and rhesus macaques, and compromised placental function likely contributes to adverse pregnancy outcomes, hence a deeper understanding of the molecular events that occur in response to placental ZIKV exposure is needed. Currently, there are no minimally-invasive techniques to detect when ZIKV has crossed the maternal-fetal interface. The analysis of EVs and their cargo in biological fluids is currently being used in the diagnosis of cancers and infections and can be done with minimally-invasive methods. Based on the utilization of EVs and their cargo as a diagnostic tool in oncology, this proposal will define a method for isolating placental extracellular vesicles (PEVs) to detect placental ZIKV infection as well as assess subsequent alterations in PEV cargo to understand viral impact on placental function. Studies with several viruses, such as human immunodeficiency virus and hepatitis viruses have shown that EVs released by infected cells are involved in mediating viral transmission and can directly transmit infectious virus. We have routinely observed prolonged viremia in pregnant but not nonpregnant macaques, however the reservoir of ZIKV remains unknown. Therefore, we hypothesize PEVs are a readout of placental function and infection and harbor infectious virions that contribute to prolonged maternal viremia. To test this hypothesis, we propose 2 Specific Aims. Specific Aim 1. To isolate PEVs and analyze their cargo. In Aim 1A we will develop an immunoprecipitation protocol to isolate PEVs from macaque placental cell conditioned medium. In Aim 1B we will conduct in vitro infection experiments to compare PEVs from trophoblast cells exposed to ZIKV to uninfected cells by analyzing mRNA, miRNA and lncRNA cargo with RNAseq, and the proteome with mass spectrometry. Specific Aim 2. To analyze and compare peripheral blood EV and PEV cargo across gestation in healthy and ZIKV-exposed pregnant macaques. We will analyze the EV cargo by RNAseq and mass spectrometry, and determine if EVs contain Zika virus genomes and harbor virions. These studies will develop a minimally-invasive means of detecting ZIKV infection of the placenta and expand our molecular knowledge of placental function during a ZIKV infection in real time. The development of this animal model and minimally-invasive tool will revolutionize the field by enabling us to test therapies and verify therapeutic safety by monitoring placental health throughout gestation.