Zika Virus Infection at Different Pregnancy Stages: Anatomopathological Findings, Target Cells and Viral Persistence in Placental Tissues

Lucia de Noronha; Camila Zanluca; Marion Burger; Andreia Akemi Suzukawa; Marina Azevedo; Patricia Z. Rebutini; Iolanda Maria Novadzki; Laurina Setsuko Tanabe; Mayra Marinho Presibella; Claudia Nunes Duarte dos Santos

The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. . https://doi.org/10.1101/370528 doi: bioRxiv preprint chorionic villi, in agreement with the results shown in Table 2 (Martines et al., 2016b; Ritter et al., 2017) . Morphometric analysis showed enhancement of the number of HCs and syncytial sprouts (indicating a villous maturation disorder) in the placentas of women infected with ZIKV at late gestational periods compared to the numbers of these features in the control group placentas. Syncytial knots are specializations in the syncytiotrophoblast, and an increase in these structures in late gestation indicates placental pathology and can be used to evaluate villous maturity. Syncytial sprouts are markers of trophoblast proliferation; they are seen frequently during early pregnancy and are increased in many diseases (Loukeris et al., 2010) . These findings agree with the results of a study showing the damaging effect of type I interferon (INF I) on ex vivo human midgestation placental tissues treated with INF-β. The authors demonstrated that type I INFs trigger fetal death in a mouse model and that treatment of ex vivo human placentas with INF-β induces morphological changes in the villi, such as syncytial knots and sprouts (Yockey et al., 2018) . Immunohistochemical analysis revealed that HCs were the only ZIKV-positive fetal cells in the naturally infected human placental samples examined in this study, regardless of the gestational period at which infection occurred, and that HCs remain persistently infected until the time of delivery. It is noteworthy that even in the tissue samples from women infected in late pregnancy, i.e., with a short interval between the acute phase of infection and delivery time (LRV/16854, 16859, 16931, 16848, 16927 and 16284) , ZIKV was detected exclusively in HCs (Table 1) . HCs are placental villous macrophages of fetal origin, and alterations in their numbers (hyperplasia) and biological features are associated with complications in pregnancy. HCs play a role in diverse functions such as placental vasculogenesis, immune regulation and the secretion of enzymes and cytokines across the maternal-fetal barrier. In addition, the results from a double-labeling assay showed the association of Sprouty proteins (implicated in villus branch morphogenesis) with HCs, suggesting the involvement of HCs in the development of placental villi (Anteby et al., 2005) . HCs are categorized as placental M2 macrophages, and their location and migratory behavior confer an ability to move around the villous stroma and to make transient contacts with other macrophages and villous core cells (Khan et al., 2000; Tang et al., 2011) . Another fact worth mentioning is the apparent correlation between villous immaturity and congenital disorders caused by ZIKV infection since we observed the occurrence of fetal malformations in four out of six cases (66.7%) of villous immaturity (observed in H&E staining and morphometric analysis). Furthermore, villous immaturity may be related to an increase in HCs, the cells that sustain the presence of ZIKV in the placenta. These data could be valuable to pathologists and neonatologists following up on newborns exposed to ZIKV by vertical transmission. Of note, the case of the first trimester placenta with villitis (LRV/15 572) exhibited ZIKV-positive maternal inflammatory cells in the intervillous space. Although positivity in fetal endothelium and maternal l