**3. Results**

#### *3.1. Detection of Mast Cells, Histopathology and ZIKV Replication in Placental Infected Tissues*

First, we evaluated the presence of mast cells in the placentae of ZIKV infected women during pregnancy in comparison to a non-infected control sample. To detect mast cells, we performed immunohistochemistry with a Toluidine Blue stain and identified these cells in placental sections of these patients by the prominent purple coloration (Figure 1A–C, arrows). Next, fluorescence microscopy images (Figure 1D–F) were used to identify cells that displayed both the mast cell marker c-Kit (red) and ZIKV NS1 protein (green). As expected, no evidence of ZIKV NS1 protein was observed in control placenta (Figure 1D). In constrast, dually labeled cells were readily observed in placenta from both ZIKV seropostive patients (Figure 1E,F), which suggested that these cells were infected and supported virus replication (Figure 1E,F). To examine the histopathological aspects, H&E stainging was used to identify maternal portions (basal decidua) and fetal portions (chorionic villi), which were normal in the control placenta (Figure 1G). Within the placentae from the ZIKV infected patients, case 1 presented areas with immature chorionic villi, chronic villositis and chronic deciduitis with lymphocytes in chorionic villi and decidua (Figure 1H). The placenta from case 2 showed intervillitis with lymphocytes in the intevillous space and immature chorionic villi (Figure 1I). To extentd the search for cells supporting ZIKV replication, immunohistochemistry was used to provide broad staining of NS1 protein both in the maternal and fetal portions of the placentae. Again, the control, non-infected samples showed no reactivity against NS1. Within placentae from infected mothers, extensive reactivity was seen in not only immune cells, but also trophoblasts and decidual cells suggesting that they are also permissive to infection (Figure 1K,L).

**Figure 1.** Detection of ZIKV infected mast cells in placental tissue from seropositve mothers. Placentae were collected from mothers infected or not with ZIKV immediately after childbirth and preserved in formadehyde. (**A**–**C**) Brightfield images of sections stained with Toluidine Blue showing metacromatic granules (purple, arrows) in mast calls. (**D**–**F**) Immunofluorescent images of DAPI (nuclei; blue), c-Kit (mast cell marker; red) and NS1 (ZIKV marker; green) showing ZIKV infected mast cells with both red and green fluorscence. No NS1 antigen was observed in any sections from the control placenta. The histopathological analysis of the H&E stained placentae showed normal aspects in decidua and chorionic villi within the control placenta (**G**), whereas infected placentae showed areas with lymphocytic infiltrates and immature chorionic villi (**H**,**I**). Detection of ZIKV NS1 protein by immunohistochemisty did not identify any positive cells in control placentae (**J**). Numerous cells positive for NS1 were detected in placentae from infected mothers, in both maternal and fetal portions (**K**–**L**). CV, chorionic villi; Dec, decidua; Im, immature chorionic villi; Ly, lymphocytes.

#### *3.2. Infection Rate of ZIKV at Di*ff*erent MOIs*

After observing that placental mast cells were infected with ZIKV during a natural infection, the susceptibility to ZIKV entry and permissiveness to its replication was evaluated using the HMC-1 cell line under controlled conditions. Cells were exposed to three different MOIs (0.1, 0.2 and 1) of virus or an equal volume of mock as a control to determine conditions of infections. A mock viral stock was

generated from supernatants of Vero cells that were not exposed to ZIKV as a control. The percentage of cells infected by ZIKV was determined by counting the number of cells displaying the fluorescent detection of NS1, a protein that is present only after viral replication, by flow cytometry. Cells were either incubated with virus or mock for 30 min and processed for analysis, or for 1 h with a subsequent incubation for 6 h or 24 h. NS1 was detected under all conditions (Figure 2A), even after 30 min, which suggests that ZIKV can rapidly enter cells and begin replication. Considering that the percentage of cells was nearly equivalent across the three MOIs at 30 min, the results further sugges<sup>t</sup> that only a subset of cells were susceptible to rapid infection. By increasing the virus binding and entry time to 1 h, followed by a 6 h incubation, the percent of cells infected increased with a maximum percent observed with a MOI of 1. A slight increase in the percentage of cells was measured when the post-infection incubation increased to 24 h. Averaged histograms of the three conditions (Figure 2B–D) show a nearly equivalent low background from the mock and the highest levels of infection with a MOI of 1 in 6 h and 24 h, with a mean of 40.10 ± 4.81 and 45.30 ± 3.44% of infected cells in three independent experiments, respectively.

**Figure 2.** Percentage of HMC-1 cells infected with different MOIs of ZIKV. HMC-1 cells were incubated with ZIKV at MOIs of 0.1, 0.2 or 1 for 30 min and prepared for flow cytometry, or 1 h followed by 6 h or 24 h incubation before analysis. Cells were permeabilized, fixed and stained with the mouse monoclonal IgG antibody against ZIKV non-structural protein NS1 followed by incubation with the Alexa 488-conjugated anti-mouse. Panel (**A**) presents the individual percentages of HMC-1 cells expressing the NS1 protein under the different conditions from three independent experiments. Averaged histograms from the experiments with an MOI of 1 are shown in (**B**) 30 min, (**C**) 1 h with 6 h and (**D**) 1 h with 24 h infection. For negative control, cells were incubated with mock viral stocks. \* Statistically significant differences between groups (same time of infection) assessed by a Mann–Whitney test (*p* < 0.05).
