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1 Supplementary Online Content Melo AS, Aguiar RS, Amorim MMR, et al. Congenital Zika virus infection: beyond neonatal microcephaly. JAMA Neurol. Published online October 3, doi: /jamaneurol eappendix 1. Phylogenetic analysis eappendix 2. Zika serology done with Chembio DPP Zika IgM/IgG efigure 1. Serological results of the prototype rapid test from Chembio DPP Zika IgM/IgG efigure 2. Additional brain tissue pathology for case 7 efigure 3. Additional brain tissue pathology for case 1 efigure 4. Additional brain tissue pathology for case 1 efigure 5. Additional brain tissue pathology for case 1 efigure 6. Additional brain tissue pathology for case 1 efigure 7. Phylogenetic analysis of ZIKV sequences from microcephaly cases isolated from amniotic fluid (AF), placenta (PL), brain (BR), lung (LG) etable 1. Images modalities etable 2. Summary of image findings This supplementary material has been provided by the authors to give readers additional information about their work.

2 eappendix 1. Phylogenetic analysis All (106) ZIKV sequences for env gene available on Virus Pathogen Database Analysis resource (ViPR - accessed in 11/03/16) were downloaded for the phylogenetic analysis. Phylogenetic reconstruction was performed using maximum likelihood inference methods on alignments of the ENV region of the polyprotein coding sequence. Maximum likelihood analysis was performed using PhyML 3.0 phylogeny online server, with the approximate likelihood-ratio (alrt) test to assign statistical significance to internal branches. The best-fit substitution model used in the analysis was determined with the automatic model selection by SMS (Smart Model Selection) on Phyml 3.0, using the AIC (Akaike Information Criterion). The GTR+G6+I was the chosen model. The low support values are probably due to the highly sequence conservation and to the short fragment sequenced, consequently few substitutions could be observed in the alignment. Therefore, although the tree indicates that several American strains are clustered with Asian-Pacific lineages, this issue still needs to be analyzed using longer sequences, since the support values are low. The phylogenetic analyses also showed a genetic variation between ZIKV sequences of different tissues of post-mortem cases 1 and 7 that does not grouped in the same branch (efigure 7A green and blue sequences). Some Brazilian sequences here analyzed presented a V23I substitution (valine to isoleucine) at amino acid position 23 of the ZIKV envelope protein (efigure 7B highlighted red box). This variation poses a difference from all African and Asian isolates herein analyzed and represented in efigure 7B by the prototypic isolates NC_ from Uganda (hashed in green), and KJ from French Polynesia (hashed in red). All sequences that presented this polymorphism clustered together on the phylogeny in a highly supported (0.92) clade that is contrasted with red branches (efigure 7A). Of note, case 1 (highlighted sequence in bold green in efigure 7A) showed the V23I polymorphism in both viruses isolated from the brain tissue (BR_Case1) and the amniotic fluid (AF_Case1), however, virus isolated from the amniotic fluid of case1 (AF_Case1(prenatal)) collected at 22 weeks of gestational age presented a valine amino acid at the position 23. As mentioned, the all sequences are very conserved and few substitutions could be observed. For instance, there are only 2 substitutions between prenatal and the other case 1 sequences that have the V23I polymorphism. Moreover, the viruses isolated from case 7 (highlighted in blue) only presented V23I when present in the amniotic fluid (AF_Case7) or lungs (LG_Case7), but in the brain (BR_Case7; 23V), and this is the only polymorphism between these sequences. Whether this V23I polymorphism in the virus envelope protein results in a different behavior of virus tropism or other virological effects should be further investigated.

3 eappendix 2. Zika serology done with Chembio DPP Zika IgM/IgG In order to test the presence of IgG and IgM in affected babies we utilized a rapid test from Chembio, DPP Zika IgM/IgG. In order to validate this test we used a seroconversion panel sera from a Zika acute infection adult case using sera from day 0 (during acute symptoms, fever, rash and conjunctivitis), 33 and 72 days after symptoms were we could see the appearance of IgM and IgM during this time course (See efigure 1). There was the appearance of IgM after 33 days of infection and fade away after 77 days. On the other hand IgG showed up 33 days after infection and remained after 77 days. When we analyzed the IgM and IgG reaction of the two babies (cases 1 and 2) showing evidence of ZIKV infection with cases 11 and 12, there was a similar band intensity. This fact suggests that cases 11 and 12 are also seroreactive to ZIKV.

4 efigure 1. Serological results of the prototype rapid test from Chembio DPP Zika IgM/IgG A Zika Acute Infection Day 0 Day 33 Day 73 B Microcephaly cases Case 1 Case 2 Case 11 0 Case 12 1 C 350 Chembio micoreader unit #1 #2 #10 #11 Acute infection Microcephaly case Serological results of the prototype rapid test from Chembio, DPP Zika IgM/IgG. (A) photographs of rapid test strips showing the results of IgM band (left) and IgG band (right) from serially collected samples of an acute infection case by Zika virus: days 0 (during symptoms), 33 and 72 days after symptoms. The upper band on the strips is the assay positive control band. The lower band is the test band (IgM marked with red arrow or IgG). (B) photographs of rapid test strips showing the results of IgM band (left) and IgG band (right) of four cases of microcephaly. Both cases 1 and 2, are positive for PCR on amniotic fluid and brain tissue, but PCR data is missing for cases 10 and 11. (C) graph showing the band intensity (in arbitrary units) of IgM (blue bar) and IgG (red bar) for each strip shown on (A) and (B), according to the Chembio microreader. The microreader is a small gadget that scans the test strip and provides quantification for the IgM and IgG band intensity, in arbitrary units.

5 efigure 2. Additional brain tissue pathology for case 7 A B A Coronal section of the upper part of one hemisphere showing shallow sulci, greyish translucid white matter and enlarged lateral ventricle; B- Ventricular surface with erosion of the ependyma (arrow). (H&E x 100).

6 efigure 3. Additional brain tissue pathology for case 1 Enlarged lateral ventricle with very thin cortex and white matter

7 efigure 4. Additional brain tissue pathology for case 1 Occasional perivascular lymphocytes and calcification in the brainstem (arrow) H&E x 400

8 efigure 5. Additional brain tissue pathology for case 1 Leptomeningeal glioneuronal heterotopia in the brainstem (middle and left side of the picture) H&E x 100

9 efigure 6. Additional brain tissue pathology for case 1 Immunopositivity for CD3 (T lymphocytes) around a vessel (x400).

10 efigure 7. Phylogenetic analysis of ZIKV sequences from microcephaly cases isolated from amniotic fluid (AF), placenta (PL), brain (BR), lung (LG)

11 Phylogenetic analysis of ZIKV sequences from microcephaly cases isolated from amniotic fluid (AF), placenta (PL), brain (BR), lung (LG). (A) Maximum likelihood phylogeny of envelope region from ZIKV. Envelope sequences generated from cases 1, 3, 4, 5, 6, 7 and 8 (in bold letters) were compared with available sequence data from other countries. Approximate likelihood-ratio test support values greater than 0.7 are shown. References sequences were marked as suggested by the world map colors: American sequences as with grey boxes; Asian and Pacific with pink boxes, whereas African sequences are highlighted with green boxes. Four (4) monophyletic African clades were recovered and are shown collapsed. All Red branches grouped the sequences that share the V23I polymorphism (substitution of a valine for isoleucine at envelope position 23). Sequences obtained from different tissues in the post-mortem cases 1 and 7 were separate in internal branches (blue and green sequences). This diversity is highlighted with green (case 1) and blue (case 7) in the phylogeny. Country and year of isolation are shown when available. NA= not available. (B) Partial alignment of the ZIKV envelope protein showing the region of the V23I polymorphism at amino acid position 23 (highlighted with surrounding red box). All American sequences are hashed in grey, African lineages are represented by the prototypic isolates NC_ from Uganda (hashed in green), and KJ from French Polynesia (hashed in red).

12 etable 1. Images modalities Imaging Case 1 Case Case 3 Case 4 Case Case 6 Case 7 Case Case 9 Case Case Modalities Fetal US yes yes yes yes yes yes yes yes yes yes yes Fetal MRI yes yes yes yes no yes yes no no no no Post-Natal US Post-Natal TC Post-Natal RMI Post- Mortem CT Post- Mortem RMI Status yes yes no no no no no no no no no no yes no no yes no no yes no yes yes no no no no yes no no no no no no yes no no no no no yes no no no no yes no no no no no yes no no no no Perinatal Death Alive Gestation in course Gestation in course Alive Gestation in course Perinatal Death Alive Perinatal Death Alive Alive

13 etable 2. Summary of image findings Imaging Findings Case 1 Case Case 3 Case 4 Case 5 Case 6 Case 7 2 Cerebral Volume yes yes yes yes yes yes yes Reduction Abnormal Cortical yes yes yes yes yes yes yes Development (Lissencephaly) (Lissencephaly) Ventriculomegaly yes yes yes yes yes yes yes Callosal Hypoplasia yes yes yes yes yes yes yes Abnormalities of Basal Ganglia and/or Thalami Cerebellar Hypoplasia Brainstem Hypoplasia Subcortical Calcification Periventricular Calcification Basal ganglia, Brainstem and/ or Thalamic Calcification yes yes yes yes yes yes yes yes yes yes yes Yes yes yes yes yes yes yes yes yes yes yes yes yes yes yes yes yes Suggestive no no no no no no yes yes no yes suggestive suggestive yes