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Zika Virus Alters DNA Methylation of Neural Genes in an Organoid Model of the Developing Human Brain


Zika Virus Alters DNA Methylation of Neural Genes in an Organoid Model of the Developing Human Brain

Sylvie Janssens et al. mSystems.


Zika virus (ZIKV) infection during early pregnancy can cause microcephaly and associated defects at birth, but whether it can induce neurologic sequelae that appear later in life remains unclear. Using a model of the developing brain based on embryonic stem cell-derived brain organoids, we studied the impact of ZIKV infection on the DNA methylation pattern across the entire genome in selected neural cell types. The virus unexpectedly alters the DNA methylome of neural progenitors, astrocytes, and differentiated neurons at genes that have been implicated in the pathogenesis of a number of brain disorders, most prominently mental retardation and schizophrenia. Our results suggest that ZIKV infection during fetal development could lead to a spectrum of delayed-onset neuropsychiatric complications. IMPORTANCE Scientific research on human neural stem cells and cerebral organoids has confirmed the congenital neurotropic and neurodestructive nature of the Zika virus. However, the extent to which prenatal ZIKV infection is associated with more subtle brain alterations, such as epigenetic changes, remains ill defined. Here, we address the question of whether ZIKV infection induces DNA methylation changes with the potential to cause brain disorders later in life.

Keywords: DNA methylation; Zika virus; astrocytes; brain disorders; microcephaly; neurons.


ZIKV infection induces global DNA methylation changes in multicellular human cerebral organoids. (A) (Top) Schematic of the samples used for whole-genome bisulfite sequencing (WGBS). (Bottom) ZIKV-positive cells (anti-flavivirus group E antigen, green) upon ZIKV infection (strain MR766) of cerebral organoid-derived 2D cultures. Bars, 100 µm. (B) Heat maps show methylation levels of all defined 1-kb tiles (left) and DMRs with an average methylation difference greater than 0.2 (right). (C) Enrichment [log2(hypergeometric P value)] of genomic features of differentially methylated 1-kb tiles (q value of <0.05 and methylation difference greater than 0.2). UTR, untranslated region; ncRNA, noncoding RNA; TTS, transcription termination site; SINE, short interspersed nuclear element; LINE, long interspersed nuclear element; LTR, long terminal repeat. (D) Examples of a hypomethylated (DDX3X) and hypermethylated (IRX3) gene locus. Gray, areas with nonsignificant sequencing signal; red dashed line, differentially methylated region.
ZIKV infection induces DNA methylation changes in human cerebral organoid-derived astrocytes, neurons, and neural progenitor cells. (A) Schematic of the samples used for reduced representation bisulfite sequencing (RRBS). (B) Immunofluorescence staining of ZIKV- and mock-infected multicellular 2D cerebral organoid cultures to detect ZIKV (strain MR766) infection (4G2, anti-flavivirus group E antigen, green) in neurons (DCX, red), astrocytes (GFAP, red), and neural progenitor cells (PAX6, red). Bar, 100 µm. DAPI, 4′,6-diamidino-2-phenylindole. (C) Principal-component analysis based on the mean methylation levels of 100-bp tiles. (D) Distribution of methylation levels in samples with or without ZIKV (strain MR766) infection, as indicated. (E) Heat maps of differentially methylated 100-bp tiles in each cell type (q value of <0.05 and methylation difference greater than 0.2). NPC, neural progenitor cells.
Disease ontology of genes associated with ZIKV-induced changes. (A) Numbers of hypomethylated and hypermethylated DMRs near gene loci (5,000 bp upstream to 500 bp downstream of transcription start sites [TSS]) per cell type. (B) Examples of hypomethylated (KDM6A, USP9X, and GSAP) and hypermethylated (GDI1, EBF3, and TWIST2) gene loci. Gray, areas with nonsignificant sequencing signal; red dashed line, differentially methylated region. (C) Venn diagram showing the number of affected gene loci in each cell type after ZIKV infection (strain MR766) and their overlap with genes defined by the human phenotype ontology for microcephaly (MCPH; total overlap of 53 out of 422 MCPH-related genes). (D) Dot plot of top DisGeNET disease categories that correlate with differentially methylated gene loci (5,000 bp upstream to 500 bp downstream of transcriptional start site). astro, astrocytes; neuro, neurons; npc, neural progenitor cells.
Correlation between neuropsychiatric disorders and genes affected by ZIKV-induced methylation changes. (A) Distribution of genes that associate with PsyGeNET disease categories according to cell type (SI, substance induced; UD, use disorders). (B) Analyses of gene networks and their association with psychiatric diseases. Included are all genes that were identified in at least one of the cerebral organoid-derived cell types. Yellow nodes represent diseases; gray nodes represent genes.

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