Computational protocol: Primary Human Placental Trophoblasts are Permissive for Zika Virus (ZIKV) Replication

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Protocol publication

[…] All currently available (n = 77; June, 2016 accession) ZIKV complete genome and complete polyprotein CDS assemblies together with Spondweni virus strain SM-6 V-1 were downloaded from the NCBI Nucleotide database (Accessions: KU955595.1, KU955594.1, KU955593.1, KU955592.1, KU955591.1, KU681082.3, KU681081.3, KX247646.1, KX185891.1, KU866423.1, KX056898.1, KJ776791.1, KX280026.1, KX197192.1, KU744693.1, KX117076.1, KU509998.3, KU963796.1, KU321639.1, KU991811.1, KU870645.1, KU926310.1, KU926309.1, KU922960.1, KU922923.1, KU820898.1, KU740184.2, KU853013.1, KU853012.1, KU729217.2, KU729218.1, KU761564.1, KU720415.1, KU497555.1, KU707826.1, KU527068.1, NC_012532.1, KU647676.1, KU501217.1, KU501216.1, KU501215.1, KU365780.1, KU365779.1, KU365778.1, KU365777.1, KU312312.1, KF268950.1, KF268949.1, KF268948.1, LC002520.1, KF383119.1, KF383118.1, KF383117.1, KF383116.1, KF383115.1, AY632535.2, EU545988.1, KU758877.1, KX247632.1, KX262887.1, KX253996.1, KU820897.2, KX087101.2, KX198135.1, KX198134.1, KX156776.1, KX156775.1, KX156774.1, KU937936.1, KX087102.1, KX051563.1, KU955590.1, KU955589.1, KU963574.1, KU963573.1, KU820899.2, DQ859059.1, DQ859064.1). Sequences were aligned using MAFFT v7.0 (Katoh) with the accurate (L-INS-i) setting. PAUP* v4.0 (Swofford) automated model selection was used to identify optimal likelihood parameters based on the best AICc (GTR + I + G; nst = 6 rclass = (abcdef) rmatrix = (1.0321439 6.5676849 1.547758 0.49119007 20.584045)basefreq = (0.27644007 0.23772344 0.28277434) rates = gamma shape = 1.8552617 pinv = 0.49424577). A Neighbor Joining tree was constructed and the tree was visualized using FigTree v1.4.2 (Rambaut; http://tree.bio.ed.ac.uk/software/figtree/). Leaf nodes were labelled as country_host_year of collection based on availability of this information at NCBI or in associated publications. [...] RT PCR. Trophoblasts were isolated from healthy donors as described above and cultured in vitro for 5 days with daily media changes of DMEM/F-12 (Gibco) with 10% FBS (Atlanta Biologicals). Cells were then washed with PBS and saved by scraping adherent cells in RNAlater (ThermoFisher) and subsequently stored at −80 °C for later extraction. For control, cryopreserved human hepatocytes or healthy human whole liver were used, alongside freshly isolated human buffy coat cells (1 × 106) or the cell line A549 cells. Isolated hepatocytes were purchased from CellzDirect (Life Technologies) and whole liver pieces were procured upon liver resections. Total RNA was then extracted from thawed cells using the Nucleospin RNA isolation kit (Macherey-Nagel, Duren, Germany) according to manufacturer’s instructions. The quality and yield of the RNA isolation was assessed by Nanodrop, and 1 μg cDNA libraries were prepared using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) according to instructions. Levels of putative receptors were then assessed by RT-PCR using GoTaq DNA Polymerase (Promega, Madison, WI) in 25 ng reactions at 57 °C for all primers. The primers for DC-SIGN, L-SIGN, TYRO3, Axl, and TIM-1 were designed against NCBI RefSeqs with the assistance of Primer3 software, and verified using UCSC Genome bowser in silico PCR (http://genome.ucsc.edu). To account for the many splice variants and sequence similarity of the highly homologous DC-SIGN and L-SIGN transcripts, primers were designed in regions common to the splice variants but unique to the respective transcripts with the assistance of SpliceCenter software (In Silico Solutions, Falls Church, VA). Major bands were found at 707 bp (DC-SIGN), and 229 bp (L-SIGN), and 166 bp (L-SIGN), corresponding to RefSeqs DC-SIGN (CD209) transcript variant 1, L-SIGN (CLEC4M) transcript variants 1, 8, 9 and 10, and L-SIGN (CLEC4M) transcript variants 7 and 11 respectively. These band sizes were therefore used to evaluate transcriptional changes of CD-SIGN and L-SIGN. For all primer sets, at least one primer was designed to span an exon junction to avoid genomic bands. Assessment of transcription was made by comparison against a previously validated GAPDH loading control. Primer sequences are listed in . […]

Pipeline specifications

Software tools Primer3, In-Silico PCR, SpliceCenter
Application qPCR
Organisms Homo sapiens, Zika virus
Diseases Abnormalities, Drug-Induced, Brain Diseases, Fetal Diseases, Infection, Microcephaly, HIV Infections