Tunneling Nanotubes Contribute to Zika Virus Pathogenesis at the Maternal-Fetal Interface in Vivo.

Zika virus (ZIKV) infection during pregnancy can result in severe fetal outcomes, yet the mechanisms of transplacental dissemination remain incompletely defined. We previously showed that ZIKV induces tunneling nanotubes (TNTs), actin-rich intercellular conduits that enable direct cell-to-cell transfer of viral components. Here, we investigated the in vivo role of TNTs in ZIKV maternal-fetal transmission using complementary pregnancy models. A TNT-deficient ZIKV mutant (ZIKVΔTNT), harboring a change between residues 40 and 52 of the nonstructural protein 1 (NS1), showed markedly reduced viral dissemination to maternal and fetal tissues across all models tested, whereas the TNT-competent ZIKV established a robust infection. ZIKVΔTNT infection was associated with reduced placental pathology, altered junctional-to-labyrinth architecture, improved placental efficiency, and protection from fetal growth restriction. Loss of TNT-forming capacity also limited viral persistence despite maternal type III interferon (IFN-λ) responses, suggesting a role for TNTs in immune evasion. Together, these findings provide in vivo evidence that TNTs are a key mechanism by which ZIKV enhances dissemination, promotes placental dysfunction, and drives fetal pathogenesis.IMPORTANCEZika virus infection during pregnancy can cause severe fetal abnormalities, yet how the virus overcomes the placenta remains incompletely understood. Here, we show that ZIKV exploits direct intercellular connections called tunneling nanotubes (TNTs) to facilitate cell-to-cell transmission and impact placental infection and fetal outcomes. Using multiple pregnancy models, we demonstrate that viruses capable of forming these structures disseminate more efficiently, damage the placenta, and lead to fetal growth restriction, whereas TNT-deficient viruses show reduced infection and milder disease. Importantly, TNT-mediated dissemination is associated with viral persistence despite maternal interferon responses, suggesting a role in immune evasion. These findings identify TNTs as a previously underappreciated pathway of viral transmission during pregnancy and suggest new therapeutic targets. More broadly, TNTs may contribute to the pathogenesis of other vertically transmitted or emerging viral infections.