Autopsy and Neuropathological Findings

Figure 2. Figure 2. Microscopic Analysis of Brain Tissue. Panel A shows thickened leptomeninges (black arrowhead) and irregular cortical and subcortical calcifications (asterisks) associated with cortical displacement (arrows), with preserved germinative matrix (white arrowhead); gyration is absent. Panel B shows higher magnification of calcifications with filamentous structures (arrow), possibly representing encrusted, damaged axons and dendrites, and oval and polygonal structures (arrowheads), possibly representing encrusted, damaged neuronal-cell bodies (hematoxylin and eosin staining in Panels A and B). Panel C shows immunohistochemical labeling of proliferated reactive astrocytes that extend into the subarachnoid space (asterisk) (glial fibrillary acid protein, clone 6F2 [Dako]). Panel D shows immunohistochemical labeling of numerous activated microglial cells and macrophages in the cortex (full thickness marked with a line) and subcortical white matter (lower part of the figure). Nonspecific staining of the calcifications is present (arrow). Focal leptomeningeal infiltrates of macrophages are seen (arrowhead) (HLA-DR, clone TAL 1B5 [Dako]). Panel E shows neurofilament immunohistochemical staining of axons in a cross-section of the lumbar spinal cord with severe Wallerian degeneration of the lateral corticospinal tracts (black asterisks), moderate involvement of other descending tracts (arrows), and well-preserved ascending tracts in the dorsal columns (white asterisk) (neurofilament, clone 2F11 [Dako]). Panel F shows indirect immunofluorescence of fetal brain tissue, revealing a green granular intracytoplasmic reaction (see also inset). The yellow signals adjacent to the green granules indicate autofluorescence of lipofuscin, suggesting that viral particles are located in the cytoplasm of neurons.

The fetal body weight was 1470 g (5th percentile), the length 42 cm (10th percentile), and the head circumference 26 cm (1st percentile). The only external anomaly that was noted was microcephaly. The placenta weighed 200 g, resulting in a placental–fetal weight ratio of 0.136 (<3rd percentile). Macroscopic examination of the CNS revealed micrencephaly with a whole-brain weight of 84 g (4 SD below average), widely open sylvian fissures, and a small cerebellum and brain stem. Almost complete agyria and internal hydrocephalus of the lateral ventricles were observed. There were numerous variable-sized calcifications in the cortex and subcortical white matter in the frontal, parietal, and occipital lobes. The subcortical nuclei were quite well developed (Figure 1C and 1D). In spite of some autolysis, microscopic examination revealed appropriate cytoarchitecture of the fetal brain. The most prominent histopathological features were multifocal collections of filamentous, granular, and neuron-shaped calcifications in the cortex and subcortical white matter with focal involvement of the whole cortical ribbon, occasionally associated with cortical displacement (Figure 2A and 2B). Diffuse astrogliosis was present with focal astrocytic outburst into the subarachnoid space, mostly on the convexity of the cerebral hemispheres (Figure 2C). Activated microglial cells and some macrophages expressing HLA-DR were present throughout most of the cerebral gray and white matter (Figure 2D). Scattered mild perivascular infiltrates composed of T cells and some B cells were present in the subcortical white matter (Fig. S1 in the Supplementary Appendix). The cerebellum, brain stem, and spinal cord showed neither inflammation nor dystrophic calcifications. The brain stem and spinal cord showed Wallerian degeneration of the long descending tracts, especially the lateral corticospinal tract, whereas ascending dorsal columns were well preserved (Figure 2E). Indirect immunofluorescence revealed granular intracytoplasmic reaction in destroyed neuronal structures, which pointed to a possible location of the virus in neurons (Figure 2F, and Fig. S1 in the Supplementary Appendix). Histologic examination of the placenta confirmed focal calcifications in villi and decidua, but no inflammation was found. There were no relevant pathological changes in other fetal organs or in the umbilical cord or fetal membranes. Fetal karyotyping with the use of microarray technology showed a normal 46XY (male) profile.

Electron Microscopy

Figure 3. Figure 3. Electron Microscopy of Ultrathin Sections of Fetal Brain and Staining of a Flavivirus-like Particle. Panel A shows a damaged brain cell with a cluster of dense virions located in the disrupted endoplasmic reticulum. Remains of membranes derived from different cellular compartments and filamentous structures are also seen. A magnified view of the boxed area with virions clearly visible (arrows) is shown in Panel B. Panel C shows a group of enveloped structures with a bright interior, presumably indicating viral replication (arrow). Panel D shows a negatively stained viral particle with morphologic characteristics consistent with those of Flaviviridae viruses (arrow).

Although analysis of the ultrathin sections of the brain showed poorly preserved brain tissue with ruptured and lysed cells, clusters of dense virus-like particles of approximately 50 nm in size were found in damaged cytoplasmic vesicles. Groups of enveloped structures with a bright interior were also detected. At the periphery of such groups, the remains of membranes could be seen. Negative staining of homogenized brain revealed spherical virus particles measuring 42 to 54 nm with morphologic characteristics consistent with viruses of the Flaviviridae family (Figure 3).

Microbiologic Investigation

Positive results for ZIKV were obtained on RT-PCR assay only in the fetal brain sample, where 6.5×107 viral RNA copies per milligram of tissue were detected. In addition, all autopsy samples were tested on PCR assay and were found to be negative for other flaviviruses (dengue virus, yellow fever virus, West Nile virus, and tick-borne encephalitis virus), along with chikungunya virus, lymphocytic choriomeningitis, cytomegalovirus, rubella virus, varicella–zoster virus, herpes simplex virus, parvovirus B19, enteroviruses, and Toxoplasma gondii (Table S2 in the Supplementary Appendix).

Figure 4. Figure 4. Phylogenetic Analysis of the Complete Genome of Zika Virus. The evolutionary history was inferred by means of the neighbor-joining method under a GTR+G+I substitution model. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (2000 replicates) is shown next to the branches. The GenBank accession number, year of isolation, and country of origin are indicated on the ZIKV branches for all strains except for those identified in 2015 and 2016. ZIKV strain Bahia, Brazil (KU527068), was obtained in this study. The complete genome sequence was recovered from fetal brain tissue. The 0.01 scale bar denotes the genetic distance in nucleotide substitutions per site.

A complete ZIKV genome sequence (10,808 nucelotides) was recovered from brain tissue. Phylogenetic analysis showed the highest identity (99.7%) with the ZIKV strain isolated from a patient from French Polynesia in 2013 (KJ776791) and ZIKV detected in Sao Paolo, Brazil, in 2015 (KU321639), followed by a strain isolated in Cambodia in 2010 (JN860885, with 98.3% identity) and with a strain from the outbreak in Micronesia in 2007 (EU545988, with 98% identity) (Figure 4). In the ZIKV polyprotein, 23 polymorphisms were detected in comparison with the strain from Micronesia and 5 polymorphisms in comparison with the isolate from French Polynesia; three amino acid changes were found in the NS1 region (K940E, T1027A, and M1143V), one in the NS4B region (T2509I), and one in the FtsJ-like methyltransferase region (M2634V).