Bioinformatic analysis reveals the expression of unique transcriptomic signatures in Zika virus infected human neural stem cells
© The Author(s) 2016
Received: 13 May 2016
Accepted: 3 June 2016
Published: 10 June 2016
The single-stranded RNA Flavivirus, Zika virus (ZIKV), has recently re-emerged and spread rapidly across the western hemisphere’s equatorial countries, primarily through Aedes mosquito transmission. While symptoms in adult infections appear to be self-limiting and mild, severe birth defects, such as microcephaly, have been linked to infection during early pregnancy. Recently, Tang et al. (Cell Stem Cell 2016, doi: 10.1016/j.stem.2016.02.016) demonstrated that ZIKV efficiently infects induced pluripotent stem cell (iPSC) derived human neural progenitor cells (hNPCs), resulting in cell cycle abnormalities and apoptosis. Consequently, hNPCs are a suggested ZIKV target.
We analyzed the transcriptomic sequencing (RNA-seq) data (GEO: GSE78711) of ZIKV (Strain: MR766) infected hNPCs. For comparison to the ZIKV-infected hNPCs, the expression data from hNPCs infected with human cytomegalovirus (CMV) (Strain: AD169) was used (GEO: GSE35295). Utilizing a combination of Gene Ontology, database of human diseases, and pathway analysis, we generated a putative systemic model of infection supported by known molecular pathways of other highly related viruses.
We analyzed RNA-sequencing data for transcript expression alterations in ZIKV-infected hNPCs, and then compared them to expression patterns of iPSC-derived hNPCs infected with CMV, a virus that can also induce severe congenital neurological defects in developing fetuses. We demonstrate for the first time that many of cellular pathways correlate with clinical pathologies following ZIKV infection such as microcephaly, congenital nervous system disorders and epilepsy. Furthermore, ZIKV activates several inflammatory signals within infected hNPCs that are implicated in innate and acquired immune responses, while CMV-infected hNPCs showed limited representation of these pathways. Moreover, several genes related to pathogen responses are significantly upregulated upon ZIKV infection, but not perturbed in CMV-infected hNPCs.
The presented study is the first to report enrichment of numerous pro-inflammatory pathways in ZIKV-infected hNPCs, indicating that hNPCs are capable of signaling through canonical pro-inflammatory pathways following viral infection. By defining gene expression profiles, new factors in the pathogenesis of ZIKV were identified which could help develop new therapeutic strategies.
Zika Virus (ZIKV) is a single-stranded positive sense RNA Flavivirus  that is primarily transmitted though the Aedes mosquito . Until recently this virus had remained relatively obscure, with the exception of a few scattered outbreaks [1–3]. Currently, 26 countries across South and Central America have reported widespread ZIKV infections, with cases also emerging in Europe and the United States . Typically, adult ZIKV infections are mild or asymptomatic, with exanthema, fever, conjunctivitis, and joint pain being the most common symptoms [2, 5]. However, ZIKV infection is also believed to be related to the concurrent increase in adult Guillain–Barré syndrome (GBS) cases . This is supported by a case controlled prospective study of the French Polynesian outbreak, where ZIKV was linked with increased incidence of GBS . Aside from its effects in adults, ZIKV infection of pregnant women can result in birth defects ranging from low birth weight to craniofacial and eye abnormalities , as well as microcephaly [7, 8]. The detection of ZIKV in fetal brain tissues indicates that ZIKV can also cross the placental barrier . Consequently, curbing the current ZIKV epidemic has become a top priority for a number of international health initiatives.
Prompt laboratory-based efforts by Tang et al. provided RNA-seq data (GEO: GSE78711) from ZIKV-infected human neural progenitor cells (hNPCs). They found that when compared to neurons and embryonic stem cells, ZIKV infects hNPCs with high efficiency, and that infection results in abnormal cell cycle dynamics and apoptosis . For the current study, we analyzed this dataset using bioinformatic methods to uncover links between neuro-inflammatory pathways and ZIKV infection of hNPCs, as well as its correlation with clinical neurological symptoms. Since congenital CMV infection is also associated with higher incidence of neurological birth defects , a CMV-infected hNPC dataset (GEO: GSE19345) was used for comparison . We demonstrate for the first time that ZIKV activates several inflammatory signals within infected hNPCs that are implicated in both innate and acquired immune responses. Moreover, several genes related to pathogen responses are significantly up-regulated in response to ZIKV infection, but not perturbed in CMV-infected hNPCs. Our approach offers novel insight into the potential mechanisms underlying ZIKV infection and provides useful directions for future clinical research.
Comprehensive gene set gene ontology analysis
Top 30 genes up-regulated in ZIKV infected hNPCs compared to controls
Interferon-induced protein with tetratricopeptide repeats 2
Tumor necrosis factor receptor superfamily, member 14
Basic helix-loop-helix family, member e41
Small nucleolar RNA, C/D box 116-4
CCAAT/enhancer binding protein (C/EBP), beta
SEC24 family, member D (S. cerevisiae)
ChaC, cation transport regulator homolog 1 (E. coli)
cAMP responsive element binding protein 3-like 1
DNA-damage-inducible transcript 3
Interleukin 20 receptor beta
Discoidin domain receptor tyrosine kinase 2
UL16 binding protein 1
Solute carrier family 7 (anionic amino acid transporter light chain, xc-system), member 11
Gamma-aminobutyric acid (GABA) A receptor, rho 2
FOS-like antigen 1
v-rel reticuloendotheliosis viral oncogene homolog B
Basic helix-loop-helix family, member e40
Inhibin, beta E
X-box binding protein 1
Selectin P ligand
Solute carrier family 7 (amino acid transporter light chain, L system), member 5
Rho GTPase activating protein 9
Homocysteine-inducible, endoplasmic reticulum stress-inducible, ubiquitin-like domain member 1
Kruppel-like factor 15
CDC42 effector protein (Rho GTPase binding) 1
CCAAT/enhancer binding protein (C/EBP), delta
Family with sequence similarity 129, member A
Top 30 genes down-regulated in ZIKV infected hNPCs compared to controls
Sialic acid binding Ig-like lectin 10
Cell division cycle 20 homolog B (S. cerevisiae)
Olfactory receptor, family 51, subfamily E, member 2
Solute carrier organic anion transporter family, member 4A1
Sphingosine-1-phosphate phosphatase 2
Ribonucleotide reductase M2
Collectin sub-family member 12
Cholinergic receptor, nicotinic, alpha 1 (muscle)
Proline rich 22
Antigen p97 (melanoma associated) identified by monoclonal antibodies 133.2 and 96.5
Mitogen-activated protein kinase 15
Chromosome 21 open reading frame 58
Sperm flagellar 1
Autophagy related 16-like 2 (S. cerevisiae)
Chemokine (C-X-C motif) ligand 14
Sushi domain containing 2
Chromosome 6 open reading frame 118
DLX6 antisense RNA 1
ADAM metallopeptidase domain 28
Aquaporin 7 pseudogene 1
EF-hand calcium binding domain 1
Wingless-type MMTV integration site family, member 8B
Peptidase inhibitor 15
Chromosome 1 open reading frame 189
By also assessing cellular compartment sub-ontology, we elucidated the cellular location of the infection induced gene expression alterations (Fig. 1b). Genes down-regulated by ZIKV were highly enriched in centromeric and nuclear sites, the extracellular matrix, and mitochondria. Furthermore, down-regulated genes were associated with exosomes, suggesting that the infection can influence exosome biology. Up-regulated genes were enriched specifically in the Golgi complex, the cytoplasm, and the monocytic leukemia zinc-finger protein/related factor (MOZ/MORF) histone acetyltransferase complex, which serves as a regulator of neural and hematopoietic stem-cell identity .
ZIKV DEGs represented in canonical human neurological diseases
Neurological clinical phenotypes associated with ZIKV DEGs
Dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 1A
Calcium/calmodulin-dependent serine protein kinase (MAGUK family)
ADP-ribosylation factor guanine nucleotide-exchange factor 2
Asp (abnormal spindle) homolog, microcephaly associated
Centromere protein J
Centrosomal protein 152 kDa
Solute carrier family 25 (mitochondrial thiamine pyrophosphate carrier), member 19
WD repeat domain 62
CDK5 regulatory subunit associated protein 2
Cancer susceptibility candidate 5
Elongation factor Tu GTP binding domain containing 2
Immediate early response 3 interacting protein 1
SCL/TAL1 interrupting locus
Centrosomal protein 135 kDa
Zinc finger protein 335
nudE nuclear distribution E homolog 1
Diaphanous homolog 1
Kinesin family member 11
Congenital nervous system disorders
ADP-ribosylation factor guanine nucleotide-exchange factor 2 (brefeldin A-inhibited)
TGFB-induced factor homeobox 1
Glutamate receptor, ionotropic, N-methyl d-aspartate 2B
Solute carrier family 12 (potassium/chloride transporter), member 5
Filamin A, alpha
SIX homeobox 3
CDK5 regulatory subunit associated protein 2
nudE nuclear distribution E homolog 1
WD repeat domain 62
Asp (abnormal spindle) homolog, microcephaly associated
Centromere protein J
SCL/TAL1 interrupting locus
Protein O−linked mannose beta1,2−N−acetylglucosaminyltransferase
Centrosomal protein 152 kDa
GLI family zinc finger 2
Zinc finger protein 335
Cyclin−dependent kinase−like 5
Cystatin B (stefin B)
Tuberous sclerosis 1
Calcium channel, voltage−dependent, beta 4 subunit
Chloride channel, voltage−sensitive 2
Potassium voltage−gated channel, KQT−like subfamily, member 2
Ceroid−lipofuscinosis, neuronal 5
ATPase, Na +/K + transporting, alpha 2 polypeptide
Leucine−rich, glioma inactivated 1
Sodium channel, voltage−gated, type I, beta subunit
Aldehyde dehydrogenase 5 family, member A1
EF−hand domain (C−terminal) containing 1
DEAD (Asp−Glu−Ala−Asp) box polypeptide 58
Ribosomal protein SA
Three prime repair exonuclease 1
Proteolipid protein 1
Fas (TNF receptor superfamily, member 6)
GO network mapping reveals connections between hNPC ZIKV infection and the immune system
Gene expression in ZIKV-infected hNPCs differs from the expression profile of CMV-infected hNPCs
A number of common human viruses are known to infect fetal NPCs, such as Herpes simplex virus 1 (HSV-1), CMV, human immunodeficiency virus (HIV), and Varicella-zoster virus (VZV), and are linked to the development of severe birth defects [6, 9, 11, 13, 22–24]. To explore if the alterations in immune related gene expression seen in ZIKV-infected hNPCs was consistent with other viral infections, we compared the ZIKV expression profile to that of CMV-infected human induced pluripotent stem (iPS) derived NPCs. This gene set provided well matched data for comparison, since the hNPCs in both studies were derived from iPS cells and implement Illumina transcript analysis [9, 23]. Additionally, hNPCs have demonstrated permissiveness to both viruses and infection in early fetal development with either virus has been linked to similar congenital neurological defects [9, 11, 23, 24].
Immune and inflammatory responses in ZIKV-infected hNPCs
Cell and tissue type analysis of the whole ZIKV DEG gene set revealed representation of tissue types associated with immune and hematological function (Fig. 5c). Tissue and cell types exclusively associated with ZIKV up-regulated genes included the bone marrow, erythrocytes, and platelets. B-cells and dendritic cells were the only cell types exclusive to the down-regulated gene set. Both up- and down-regulated genes were enriched for monocytes, T-cells, leukocytes, and the thymus. The immune specific enrichment of the genes differentially expressed in ZIKV-infected hNPCs indicate involvement of both the innate and adaptive immune system. Dendritic cells are important antigen-presenting cells (APC) while T- and B-cells are primarily responsible for adaptive response. This enrichment points to a role for ZIKV inducing NPCs to take on a yet unclear immune-modulating phenotype.
Analysis of cytokine/chemokine associated intracellular signaling pathways
Our results show that collectively, ZIKV induced transcriptomic alterations are associated with established clinical pathologies. Following viral infection there is a reallocation of host nucleic acids and other metabolites towards viral specific replication processes , thus the enrichment of biological processes related to nucleic acid metabolism is an expected finding, as well as the enrichment of cell cycle regulation. The cell cycle perturbations of infected cells suggest the initiation of a G1/S checkpoint and impaired progression through G1 . A possible explanation for these cell cycle abnormalities could be inhibition of S-phase progression due to limited nucleic acid reserves as the virus reallocates them for its own replication. Down-regulation of critical epigenetic markers represents a possible driver for abnormal gene expression in infected cells. If NPCs in the developing human brain are diverted from normal development, it can result in conditions like microcephaly. Moreover, this raises questions about the possible long-term neurological effects in adult following ZIKV infection of adult neural progenitors. Although, the ZIKV associated clinical phenotypes were highly enriched for congenital CNS disorders, comparatively few genes were associated with adult CNS inflammation and demyelination diseases. This may be the due to the fact that the current study implemented the MR766 Ugandan isolate which has not been associated with an increased incidence of GBS and myelitis. As a result, future studies that employ more neoteric strains will likely observe increased representation of related genes.
The significant enrichment of canonical immune system pathways in ZIKV-infected hNPCs proved to be another unexpected finding. Our study provides evidence at the transcriptional level that two major mediators of inflammation, NF-κB and the STAT family of proteins, regulate inflammatory response in hNPCs. NF-κB is one of the main transcriptional factors activated in response to pro-inflammatory cytokines, such as TNF-α and IL-1. It is also triggered by TLRs via pathogen-associated molecular patterns (PAMPs). The Janus Kinase (JAK)-Signal transducer and activator of transcription (STAT) pathway is also a major signaling pathway that regulates inflammation and mediates the responses of target cells to inflammatory cytokines. Activation of these pathways has a central role in inflammation through the regulation of genes encoding pro-inflammatory cytokines, chemokines, and inducible enzymes such as cyclooxygenase-2 (COX2) and inducible nitric oxide synthase (iNOS) . Moreover, it has recently been reported that ZIKV-infected fetal brains display a number of inflammatory markers, including diffuse astrogliosis, and activations of macrophages and microglia . It is conceivable that ZIKV-infected NPCs may exert immunomodulatory and inflammatory effects on CNS cells such as neurons as well as astrocytes in an autocrine, paracrine, and juxtacrine manner, thus amplifying brain inflammation. Interestingly, although congenital CMV infection can also result in microcephaly and general impaired brain development , the inflammatory signals were not observed in CMV-infected hNPCs. The significance of this is still under investigation.
With the currently limited molecular data regarding transcriptomic alterations in infected host cells, large scale analysis is difficult. In spite of this, we present analysis of the currently available data in an effort to guide future studies. The RNA-seq was performed on only one infected cell type, hNPCs, thus we implemented stringent Benjamini Hochberg (BH)  false discovery rate (FDR) q-value cut-offs to limit false positives in enriched pathways. The use of the older MR766 isolate for the experiments likewise limits extrapolation of the results, even though it shares 88.9 % nucleotide and 96.5 % amino acid identity with the current strain . Considering that reported pathogenicity has been increasing over time, the presented data likely represents a milder host response should any host transcript differences exist.
In conclusion, we report that ZIKV infection induces alterations to NPC immune response gene expression. While the primary infection typically occurs through the bite of an infected mosquito, the mechanism for entry into the restricted CNS is currently unclear. Presumably the specific physiology of the fetal blood brain barrier is permissible to ZIKV as it is to other viruses . Regardless, it has been shown that the virus can infect NPCs with high efficiency leading to increased apoptosis . In addition, the presented study is the first to report enrichment of numerous pro-inflammatory pathways in ZIKV-infected hNPCs, indicating that hNPCs are capable of activating pro-inflammatory immune system pathways following viral infections. This represents, in addition to direct activation of apoptotic pathways, a mechanism by which the virus could induce cell death via the propagation a cytotoxic pro-inflammatory environment in the CNS. Although it is possible that ZIKV can infect immune cells in a similar manner as the related Dengue virus [30–33], the ability of ZIKV to infect hNPCs may exacerbate CNS inflammation. Consequently, a better understanding of the cellular and molecular mechanisms regulation of the cross-talk between NPCs, CNS resident cells, and immune cells may be crucial for the development of effective strategies to treat ZIKV infection.
Gene sets and expression analysis
We analyzed the transcriptomic sequencing (RNA-seq) data (GEO: GSE78711) of ZIKV (MR766) infected human neural progenitor cells (hNPCs). Briefly, Tang et al. infected hNPCs derived from human induced pluripotent stem (iPS) cells with ZIKV. The infected hNPCs were incubated for 56 h, a time frame already proven sufficient for a greater than 60 % infection rate. RNA extracted from ZIKV-infected samples and mock infected samples was used for library generation and sequencing . For comparison to the ZIKV-infected hNPCs, the expression data from iPCS derived NPCs infected with human CMV (Ad169) was implemented (GEO: GSE35295). The cells were infected with CMV, and the RNA was extracted 24 after infection for analysis on an Illumina HumanHT-12 V4.0 expression beadchip. Three biological replicates for control and infected cells were used, and a list of significantly altered genes were generated with a BH FDR q-value cut-off of < 0.05 .
Availability of data and materials
The datasets supporting the conclusions of this article are available in the gene expression omnibus (GEO) repository, http://www.ncbi.nlm.nih.gov/geo/.
Biological process and cellular compartment analysis
Using FunRich , up- and down-regulated genes with associated Log2 FCs were analyzed for enrichment against the human annotated genome. Biological process, cellular compartment, and biological pathway significance was defined as a FDR q-value less than 0.05. All significant biological processes were presented. All 11 significant cellular compartments for the ZIKV up-regulated genes were presented, along with the top 11 most significant pathways enriched for ZIKV down-regulated genes.
Subsetting for immune specific biological pathway enrichment
The significant biological pathways associated with both ZIKV up- and down-regulated genes were filtered such that non-immune specific biological pathways were excluded. Significance was defined as a FDR q-value less than 0.05.
ZIKV genes associated with clinical phenotypes
Clinically relevant diseases associated with ZIKV DEGs were queried using the MalaCards  integrated database of human diseases. The absolute percentage of ZIKV DEGs present in the reference list derived from MalaCards was reported, as well as the total number of genes in the reference list.
Gene ontology network analysis
DEGs with an absolute fold change greater than 0.5 were imported to Cytoscape , where Gene Ontology clustering was performed using the ClueGO plugin . The human genome was used as the background, and the GO biological process and GO immune system terms were queried for enrichment. Terms were generated such that GO Term Fusion was implemented for pathways with a less than 0.01 FDR q-value. Groups with less than three connections were excluded from the final network.
induced pluripotent stem cell
human neural progenitor cell
monocytic leukemia zinc-finger protein/related factor
differentially expressed genes
central nervous system
herpes simplex virus 1
human immunodeficiency virus
granulocyte–macrophage colony stimulating factor
myeloid differentiation primary response gene 88
tumor necrosis factor
C-X-C motif chemokine
signal transducer and activator of transcription
nuclear factor kappa-light-chain-enhancer of activated B cells
pathogen-associated molecular patterns
inducible nitric oxide synthase
AR and YR conceived and designed the study. AR analyzed the data. AR, DB, JW, RN, JF and YR interpreted the results and discussed the study. AR, DB and YR wrote the paper. All authors read and approved the final manuscript.
Thanks to Stephanie Hurwitz for review of the manuscript. This work was supported by the National Institutes of Health (R01GM100474 and R01GM072611).
The authors declare that they have no competing interests.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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