Combined interferential contrast and fluorescent photomicrographs of positive and negative controls (A, B) and Curionopolis- (C, D) or Itacaiunas (E,F)-infected neurons after TUNEL immunolabeling.

<p>Apoptotic nuclei (arrows) are observed in control cultures exposed to UV light (A) and after Curionopolis (D) and Itacaiunas (F) virus inoculation at 4 and 5 days post-inoculation, respectively. Negative control cultures (B) and infected cultures at 1 day post-inoculation (C, E) present few stained nuclei.</p

Bright-field (A, G) and interferential contrast (B–F, H) photomicrographs of infected mouse brain sections illustrating viral antigen-immunolabeled cells 2 (A–D) and 4 (E–G) days after inoculation with Curionopolis virus and TUNEL immunolabeling at 6 days (G, H).

<p>Low (square) (A), medium (B) and high (C) power photomicrographs of labeled olfactory bulb neurons. High power images illustrating isolated neurons of the olfactory bulb with immunolabeled soma (arrow) and other neuronal appendages (arrowheads) (C); immunolabeled meningeal cells are also indicated (arrow) (D); cortical (E) and thalamic (F) neurons immunostained with viral antigens distributed in the cell appendages. TUNEL-positive neurons in infected brain sections (TUNEL POD procedure) 6 days after inoculation with Curionopolis virus into the ventral olfactory bulb (G, H). The arrows indicate immunostained neuronal nuclei.</p

Transmission electron photomicrographs of ultrathin sections obtained from control (A) and mouse brain infected intracerebrally with Curionopolis for 36 (B,C), 60 (D) and 96 h (E), and with Itacaiunas for 24 (F), 60 (G), 72 (H), 96 (I) and 108 h (J).

<p>Normal tissue with intact neuronal soma and appendages (A); viral particles (arrow), interstitial edema (stars) and cellular rarefaction (lozenge) are seen 36 h post-inoculation (p.i.) (B, C); necrotic cells were observed at 60 h p.i. (D); intense perivascular edema (stars), hyperplastic endotheliocytes and reduced vessel luminal area (E); well-preserved brain parenchyma and vessels at 24 h p.i. (F); viral particles, endotheliocyte hyperplasia, and mild interstitial edema (stars) at 60 h p.i. (G); membrane viral budding in rich polyribosomes oligodendrocyte-like cell at 72 h p.i. (H); brain parenchyma at 96 h p.i. presenting a large number of viral particles (I); apoptotic features were more marked at 108 h p.i. (J). AC = apoptotic cell, M = mitochondria, OL = oligodendrocyte, EC = endothelial cells, VL = vascular lumen, N = cell nucleus, NC = necrotic cells.</p

Transmission electron photomicrographs of ultrathin sections obtained from primary neuronal cultures.

<p>Control cultures of normal cells (A) and after Itacaiunas (B) and Curionopolis (C, D) infection at 4 and 5 days post-inoculation, respectively. Apoptotic cells (C) and virus budding (rectangle and arrows) (C, D) after Curionopolis infection. Itacaiunas virus particles (arrows) in culture at 5 days post-inoculation (B). N: nucleus; AN: apoptotic nucleus.</p

Photomicrographs of hematoxylin-eosin-stained sections from control (A) and infected brain sections at 96 h post-inoculation with Itacaiunas virus (B–D) and 60 h post-inoculation with Curionopolis virus (E–H).

<p>Multiple foci of congestion with sparse distribution of leukocytes characterized by the margination phenomenon (leukocytes becoming flat and the plasma membrane sticking to the capillary endothelium) (B); hypertrophic endotheliocytes (ellipse) and perivascular edema (stars) (C). Apoptotic like-cells with nuclear pyknosis and cytoplasmic condensation were detected among tissue vacuolation (arrows) (D). Meningeal congestion and edema with lymphomononuclear cells (arrows) (E). Groups of pyknotic cells presenting cytoplasmic condensation (circles), mixed with normal cells and vacuolated parenchyma (stars) (F). Recent hemorrhagic points (presence of red blood cells) presenting parenchymatous edema (lozenge), mainly in the areas of worst cell damage (84 h post-inoculation) (G). Nuclear fragmentation or karyorrhexis (arrows) and edema (lozenge) (96 h post-inoculation) (H).</p

Bright-field (A, D, F) and interferential contrast (B, C, E, G) photomicrographs of Itacaiunas virus-infected mouse brain at 4 (A–C), 6 (D, E) and 8 (F, G) days post-inoculation.

<p>Low (A) and medium (B) power photomicrographs of the olfactory neuronal group (smaller rectangle). Details of immunolabeled neurons of the frontal cortex (C) (arrows and arrowheads). Low (D) (rectangle) and medium (E) power photomicrographs of a group (arrows) of hippocampal neurons showing low viral antigen condensation (arrowhead). TUNEL-positive midbrain neurons of infected brain sections (TUNEL POD procedure) 12 days after inoculation with Itacaiunas virus (F, G). The arrows indicate immunostained neuronal nuclei.</p

Phase-contrast (A, C, E) and fluorescence photomicrographs (B, D, F) of primary neuronal cultures to illustrate normal noninfected cell morphology (A, B) and cytopathic effects 3 days after inoculation of Curionopolis virus (C, D) and 4 days after inoculation of Itacaiunas virus (E, F).

<p>Arrows point to neurite fragmentation and circles indicate refringent points probably corresponding to apoptotic nuclei (A, C). Arrows and arrowheads indicate infected soma and dendrites, respectively (D, F). Neurons were stained by indirect immunofluorescence for anti-neurofilament antibodies. Secondary antibodies were conjugated with fluorescein isothiocyanate.</p

Epidemiological Data: Numbers of suspected ZIKV cases and suspected microcephaly cases per state and per epidemiological week.

Contains 1) CSV file with number suspected ZIKV cases from January 2015 to the end of December 2015; 2) CSV file with number of suspected microcephaly cases from January 2015 to the first week of January 2016. Numbers correspond to suspected microcephaly cases at week 20 of pregnancy; 3) CSV file with codes of state of residence and municipality of residence in Brazil; and 4) R scripts for correlation analysis described in SI Section 1.5

Sequence data details and alignments for dataset A and B.

Contains (1) table with accession numbers, isolate names, cell passage history, publication details, country/location of sampling, sampling dates and (2) Fasta format sequence alignments of datasets A and B

BEAST XML input file used for genetic analysis.

BEAST XML input file used to generate Figure 3 under a strict clock model, a Bayesian skyline coalescent prior and a CTMC prior on the clock rate