*3.3. ZIKV Interaction Induces Degranulation*

The results from the infection of mast cells by ZIKV suggested that the response of HMC–1 could be contributing to the observations. We chose to explore the activation and degranulation of mast cells by β-hexosaminidase, a resident enzyme released in response to degranulation. Initially, flow cytometry was used to analyze the percentage of cells that display degranulation following incubations of HMC-1 cells with ZIKV at different MOIs for different times. After a 30 min incubation, all three MOIs showed similar percentages (Figure 3A). The percentage of cells decreased following a 6 h incubation and returned to the levels of mock infections after a 24 h incubation suggesting that, at the later time points, the granulosome recuperated or the released enzyme lost activity.

**Figure 3.** Kinetics of mast cell degranulation after interaction with ZIKV. (**A**) Percentage of degranulated cells after incubation with different MOIs of ZIKV in 30 min, 6 h and 24 h by flow cytometry. (**B**) Percentage of β-hexosaminidase release with different MOIs of ZIKV after 30 min. The synthetic compound 48/80 was used to elicite mast cell degranulation. \* Statistically significant differences between groups (same time of infection) assessed by a Mann–Whitney test (*p* < 0.05). Data represent the mean of duplicate values for each sample, in three independent experiments.

To evaluate the early kinetics of mast cell activation, the amount of β-hexosaminidase, normalized to the total cellular β-hexosaminidase, was measured at 30 min for each of the MOIs (Figure 3B). Despite the β-hexosaminidase levels not reaching the percentage of the cells stimulated with the synthetic compound 48/80, the release was gradually increased according to the amount of viral particles, suggesting that the activation of these cells actually occurs due to adsorption of the virus to cell receptors.

#### *3.4. ZIKV Led to Release of Cytokines and VEGF*

To analyze the release of the cytokines TNF- α, IL-6, and IL-10, along with VEGF, during infection with ZIKV, we performed ELISAs on the supernatant of mast cells activated with 30 min of contact with the virus as well as the extracellular levels produced by 1 h of virus presence and an incubation of 6 or 24 h. After the shortest interaction time, the levels of TNF-<sup>α</sup>, IL-6 and IL-10 increased greater in response to exposure to the control than with ZIKV (Figure 4). The levels of VEGF were nearly equal. The levels of the cytokines and VEGF in the supernatant were significantly greater after a hour incubation with ZIKV stocks with an additional 6 h incubation than the mock stocks. This difference grew with the increase in the secondary incubation time to 24 h although the absolute levels of these cytokines and VEGF were lower compared to 6 h. The observed release of TNF-<sup>α</sup>, IL-6, IL-10 and VEGF at 30 min suggested that they responded to a range of external stimuli. Meanwhile, the elevated levels of these mediators 6 h or 24 h after the infection with ZIKV infection suggests a stimulation in expression and release of these mediators after infection.

**Figure 4.** Cytokine and VEGF release by HMC-1 cells in response to ZIKV interactions. The supernatants of HCM-1 cells were collected after incubation with ZIKV or mock viral stocks for 30 min or following a 1 h incubation with an additional 6 or 24 h incubation. Commercial ELISAs were used to measure the level of released (**A**) TNF-<sup>α</sup>, (**B**) IL-6, (**C**) IL-10 levels and (**D**) VEGF. Data represent the mean of triplicate values for each sample obtained from three independet experiments. \* Statistically significant differences assessed by a Mann–Whitney test (*p* < 0.05).

## *3.5. Ultrastructural Changes Caused by ZIKV Infection*

To explore changes to aspects of the ultrastrutucture of mast cells in response to ZIKV infections, an infection with an MOI of 1 was used for the best conditions of infection as well as activation and degranulation of HMC-1. As a control for the analysis, the ultrastructure of cells incubated with the mock viral stock for 30 min was evaluted. Representative cells presented normal aspects for a mast cell in terms of the formation of the nucleus, and the volume of the mitochondria and normal endoplasmic reticulum with a high density of granules (Figure 5A–C). While cells incubated with the ZIKV for 30 min have a lower rate of infection, our previous data show they are at the optimal moment of adsorption and trigger degranulation. The ultrastructure of representative cells shows a decrease in cellular granules (Figure 5D–F), with no other major alterations. After 24 h incubation with the mock viral stock, we observed that the mastocytes continued to have a high density of granules, endoplasmic reticulum with closed cisterns, and mitochondria with some structural alterations, such as swollen and ruptured (Figure 5G–I). After the same period of incubation, the infected mast cells presented various organelle alterations observed as the formation of numerous vesicles, dilated endoplasmic reticulum cisterns, swollen mitochondria, ruptures in cellular membranes and, in some cells, the absence of a nucleus suggesting that a subset of cells may no longer be viable (Figure 5J–K). In several instances, the presence of viral-like particles were detected that match with the size of a ZIKV particle (Figure 5L).

**Figure 5.** Ultrastructural changes in HMC-1 mast cells infected with ZIKV. HCM-1 cells were exposed to ZIKV or mock for 30 min or 1 h with a post 24 h incubation before processing and imaging of ultrathin sections by electron microscopy. (**A**–**C**) Control HMC-1s incubated with mock for 30 min. (**D**–**F**) An HMC-1 cell incubated with ZIKV for 30 min with decreased granules. (**G**–**I**) An HMC-1 cell incubated with mock for 24 h with a high density of granules. (**J**–**L**) HMC-1 cell infected with ZIKV for 24 h. Panel L shows a virus-like particle (VLP) with a diameter of approximately 30 nm, consistent with ZIKV. Granules (**G**), nucleus (**N**), mitochondria (**M**) and endoplasmic reticulum (ER).
