**2. Materials and Methods**

#### *2.1. Placentae Collection, Patient Clinical History and Ethical Approval*

At delivery, samples from the placentae were collected and fixed in 10% formaldehyde. Samples were collected at the Hospital Plantadores de Cana, Campos dos Goytacazes, RJ, Brazil. As a control, a sample of a full-term placenta from a healthy donor was included.

Case 1: A 23-year-old patient. Symptoms: fever, arthralgia, exanthema and pruritus in the third trimester of gestation. At 38 weeks of gestation, her baby girl was born by cesarean delivery, with 37 cm of cephalic circumference. The mother's IgM serology was positive for Zika. The test for dengue NS1 was negative.

Case 2: A 34-year-old patient. Symptoms: exanthema and pruritus in the third trimester of gestation. Her baby girl was born at term, by cesarean delivery, with 38 weeks of gestation. She presented with a normal 34 cm of cephalic circumference. The mother's IgM serology was positive for Zika.

Patient recruitment and the procedures performed were pre-approved by the Ethics Committee of the Oswaldo Cruz Foundation/FIOCRUZ (CAEE: 65924217.4.0000.5248) and by the Ethics Committee of Faculty of Campos Medicine/Benedito Pereira Nunes Foundation (CAEE: 65924217.4.3001.5244). The patients were fully informed of the research plans and provided written consent to participate, which included permission to publish all data without identifying information.

#### *2.2. Histopathology and Histological Detection of Mast Cells in ZIKV Infected Placentae*

All histological processing of the sample was performed as described previously by our group [25]. The histopathological analysis was performed on the images observed and captured by hematoxylin and eosin (H&E) staining. The staining used to highlight the mast cells was Toluidine Blue 1%. Stained specimens were visualized by light microscopy (Olympus, Tokyo, Japan), and digital images were obtained using Image-Pro Plus software version 7.0.
