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Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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RiboSeq of African green monkey (Vero) cells and human malignant glioblastoma multiforme (U251) cells infected at a multiplicity of infection (MOI) of 3 with the Asian/American Zika virus (ZIKV), PE243; and the African ZIKV strain, Dak84

ABSTRACT: Vero and U251 cells were infected with the Asian/American ZIKV (PE243) and the African ZIKV (Dak84) at MOI:3 to assess the viral transcriptome in two cell lines. Samples were harvested at 24 hours post-infection (h p.i.) by flash-freezing, without cycloheximide pre-treatment. RNase I treatment was carried out, following which ribosomes and enclosed RNA were isolated by centrifugation through a sucrose cushion. RNA was extracted, ribosomal RNA was removed using Illumina's RiboZero kit, and remaining RNA was gel purified to select fragments 25-35nt long. Fragments were cloned into adapters using the TruSeq small RNA adapter kit and sequenced on Illumina NextSeq.

INSTRUMENT(S): NextSeq 500

ORGANISM(S): Homo sapiens

SUBMITTER: Nerea Irigoyen

PROVIDER: E-MTAB-12970 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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RiboSeq of African green monkey (Vero) cells infected at a multiplicity of infection (MOI) of 3 with the Asian/American Zika virus (ZIKV) strain, PE243

Project description:Vero cells were infected with the Asian/American Zika virus (ZIKV) strain, PE243, at MOI:3 to assess the viral transcriptome. Samples were harvested at 24 hours post-infection (h p.i.) by flash-freezing, without cycloheximide pre-treatment. RNase I treatment was carried out, following which ribosomes and enclosed RNA were isolated by centrifugation through a sucrose cushion. RNA was extracted, ribosomal RNA was removed using Illumina's RiboZero kit, and remaining RNA was gel purified to select fragments 25-35nt long. Fragments were cloned into adapters using the TruSeq small RNA adapter kit and sequenced on Illumina NextSeq.

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Project description:Vero and U251 cells were infected with the Asian/American ZIKV (PE243) and the African ZIKV (Dak84) at MOI:3 to assess the viral transcriptome in two cell lines. Samples were harvested at 24 hours post-infection (h p.i.) by flash-freezing, without cycloheximide pre-treatment. Ribosomal RNA was removed using Illumina's RiboZero kit, and RNA was hydrolysed and then gel purified to select fragments 25-35nt long. Fragments were cloned into adapters using the TruSeq small RNA adapter kit and sequenced on Illumina NextSeq.

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RiboSeq of African green monkey (Vero) cells infected at a multiplicity of infection (MOI) of 3 with different Zika virus (ZIKV) mutants (American WT, African-like, uORF1-KO and uORF2-PTC1) generated by reverse genetics

Project description:Vero cells were infected with the different Zika virus (ZIKV) mutants (American WT, African-like, uORF1-KO and uORF2-PTC1) generated by reverse genetics at MOI:3 to assess their viral transcriptome. Samples were harvested at 24 hours post-infection (h p.i.) by flash-freezing, without cycloheximide pre-treatment. RNase I treatment was carried out, following which ribosomes and enclosed RNA were isolated by centrifugation through a sucrose cushion. RNA was extracted, ribosomal RNA was removed using Illumina's RiboZero kit, and remaining RNA was gel purified to select fragments 25-35nt long. Fragments were cloned into adapters using the TruSeq small RNA adapter kit and sequenced on Illumina NextSeq.

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RNASeq of African green monkey (Vero) cells infected at a multiplicity of infection (MOI) of 3 with different Zika virus (ZIKV) mutants (American WT, African-like, uORF1-KO and uORF2-PTC1) generated by reverse genetics

Project description:Vero cells were infected with the different Zika virus (ZIKV) mutants (American WT, African-like, uORF1-KO and uORF2-PTC1) generated by reverse genetics at MOI:3 to assess their viral transcriptome. Samples were harvested at 24 hours post-infection (h p.i.) by flash-freezing, without cycloheximide pre-treatment. Ribosomal RNA was removed using Illumina's RiboZero kit, and RNA was hydrolysed and then gel purified to select fragments 25-35nt long. Fragments were cloned into adapters using the TruSeq small RNA adapter kit and sequenced on Illumina NextSeq.

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Project description:Zika virus (ZIKV) infection causes microcephaly and has been linked to other brain abnormalities. How ZIKV impairs brain development and function remains elusive. Here we systematically profiled transcriptomes of human neural progenitor cells (hNPCs) and astrocytes exposed to Asian ZIKVC, African ZIKVM, and Dengue virus (DENV). DENV causes distinct gene expression changes; and ZIKV has a broader impact on the expression of gene involved in DNA replication and repair. While overall expression profiles are similar, ZIKVC, but not ZIKVM, induces upregulation of viral response genes. Upon ZIKV infection, astrocytes exhibit more prominent transcriptome changes than hNPCs, particularly enriching genes related to inflammatory response and cytokine production pathways. Our analyses reveal cell-type- and strain-specific molecular signatures associated with ZIKV infection, and identify astrocytes as a major direct target of ZIKV. Further investigation of ZIKV-host interactions based our transcriptomic datasets may help to illuminate neural virulence determinants of ZIKV in patients. Gene Expression Analyses of the cells infected with different flaviviruses

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2018-09-03 | PXD009551 | Pride

Extended ZIKV-NS4B interactome (AP-MS/MS analysis of ZIKV interacting proteins)

Project description:To identify the cellular interactors of ZIKV-NS4B-interacting proteins and their differential composition upon ZIKV infection, individual cDNAs expressing FLAG-tagged variants of the following ZIKV-NS4B-interacting proteins were generated (GeneBank and UniProt Accession numbers are given in parenthesis): CEND1 (BC034732.1; Q8N111), CHP1 (BC031293; Q99653), CLN6 (BC010849.1; Q9NWW5), TMEM41b (BC035034.1; Q5BJD5), RBFOX2 (NM_001082579.1; O43251-6), BSG (BC009040.2;P35613). Lentiviruses expressing each of these proteins were used to transduce SK-N-BE2 cells, which were either mock-infected or infected with ZIKV (H/PF/2013, MOI=3), and 72 hours later subjected to AP-MS/MS to identify the respective cellular interactors.

2018-09-03 | PXD009561 | Pride

Effect of ZIKV on pre-implantation embryos

Project description:To determine the effect of Zika virus infection on pre-implantation embryonic development, we performed single blastocyst RNA-Seq on MOCK and ZIKV infected embryos. ZIKV infection results in an increased risk of spontaneous abortion and poor intrauterine growth although the mechanisms underlying fetal loss remain undetermined. Little is known about the impact of ZIKV infection during the earliest stages of pregnancy, or pre- and peri-implantation, because most current studies of ZIKV infection in pregnancy models focus on post-implantation stages. Here, we demonstrate that trophectoderm cells of pre-implantation human and mouse embryos can be efficiently infected with ZIKV, and that trophectoderm can propagate virus causing cell death of neural progenitors. These findings were corroborated by our demonstration that hESC-derived trophectoderm cells are infected by ZIKV in a dose dependent manner. RNAseq of single blastocysts revealed key transcriptional changes in cellular and physiologic functions upon ZIKV infection, including nervous system development and function, prior to commitment to the neural cell lineage. Finally, the pregnancy rate of mice infected pre-implantation was > 50% lower than females infected at E4.5. These results demonstrate that pre-implantation ZIKV infection of trophectoderm leads to miscarriage or spontaneous abortion. Moreover, pre- and peri-implantation ZIKV infects trophectoderm cells that propagate virus over time causing cell death in neural progenitors. Cumulatively, these data demonstrate that vertical pre- and peri-implantation ZIKV infection of trophectoderm impairs fetal development and causes neural progenitor cell death, elucidating a previously unappreciated association of pre- and peri-implantation ZIKV infection and microcephaly.

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RiboSeq of murine 17Cl-1 cells infected at MOI 10 with MHV-A59, compared to mock-infected controls, harvested at 5 and 8 hpi, plus a control tunicamycin-treated sample.

Project description:Murine 17Cl-1 cells were infected with the model Betacoronavirus mouse hepatitis virus (MHV) strain A59 and subjected to ribosome profiling to observe changes in the host translatome in infected cells compared to mock-infected cells. Samples were harvested at 5 and 8 hpi by flash-freezing, without cycloheximide pre-treatment. RNase I treatment was carried out, following which ribosomes and enclosed RNA were isolated by centrifugation through a sucrose cushion. RNA was extracted, ribosomal RNA was removed using Illumina's RiboZero kit, and remaining RNA was gel purified to select fragments 25-35nt long. Fragments were cloned into adapters using the TruSeq small RNA adapter kit and sequenced on Illumina NextSeq, then differential translation efficiency analysis was carried out on the data deposited data. One uninfected sample was treated with 2ug/mL tunicamycin for 6 hours before harvesting as a positive control for unfolded protein response activation.

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Human neurospheres ZIKV infection

Project description:Zika virus (ZIKV) has been associated with microcephaly and other brain abnormalities; however, the molecular consequences of ZIKV to human brain development are still not fully understood. Here we describe alterations in human neurospheres derived from induced pluripotent stem (iPS) cells infected with the strain of Zika virus that is circulating in Brazil. Combining proteomics and mRNA transcriptional profiling, over 500 proteins and genes associated with the Brazilian ZIKV infection were found to be differentially expressed. These genes and proteins provide an interactome map, which indicates that ZIKV controls the expression of RNA processing bodies, miRNA biogenesis and splicing factors required for self-replication. It also suggests that impairments in the molecular pathways underpinning cell cycle and neuronal differentiation are caused by ZIKV. These results point to biological mechanisms implicated in brain malformations, which are important to further the understanding of ZIKV infection and can be exploited as therapeutic potential targets to mitigate it.

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