7: Potential mechanisms of contemporary strain Zika virus replication in human placental trophoblasts


      Zika virus (ZIKV) is an emerging mosquito-borne flavivirus. A causal link explaining the delay between maternal symptoms and fetal infection is missing, and why prior African outbreaks were not associated with fetal malformations is unknown. In this study, we sought to answer these questions using state-of-the-science approaches with a single passage contemporary ZIKV strain and infection of high purity trophoblasts from uninfected donors.

      Study Design

      ZIKV-FLR was isolated by inoculating A. albopictus C6/36 mosquito cells with serum from a non-pregnant subject infected in Barranquilla, Colombia. Subsequent deep sequencing on the Illumina platform was performed, and alignments to all 77 sequenced genomic strains was made. We isolated n=20 uninfected human primary trophoblasts, and examined expression of putative ZIKV cell entry receptors. We documented intracellular infection in trophoblasts using single molecule RNA FISH to both negative and positive viral strands. Finally, we examined the potential role of miRNAs in modulating trophoblast infection.


      Phylogenetic trees were generated from all currently available complete ZIKV sequences (n of 77; Fig 1A). The resultant phylogenetic tree demonstrates phylogenetic delineation noted between African strains (non known to cause microcephaly) and the current Americas and recent Asian strains. Moreover, primary human trophoblasts express putative cell entry receptors for ZIKV (Fig 1B) prior to infection with ZIKV. Upon co-culturing with a current strain of ZIKV, we observe intracellular localization of ZIKV as evidenced by single molecule FISH (Fig 1C). Finally, ZIKV infection is associated with a significant diminution of the ssRNA-ligand sensing miRNA, mir21 (0.7 fold lower, p<0.01) but not the C19 cluster miRNA species.


      Contemporary ZIKV strains are genomically distinct from historic epidemic strains, mirroring their association with microcephaly and fetal malformations. Placental trophoblasts express putative ZIKV entry receptors, and cytoplasmic replication can visualized by single molecular RNA FISH. Select placental miRNAs (mir-21) are significantly and specifically down-modulated following ZIKV infection. We speculate that these findings are of potential mechanistic interest to ZIKV perinatal pathogenesis.
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