Modelling West Nile Virus and Usutu Virus Pathogenicity in Human Neural Stem Cells
Abstract
:1. Introduction
2. Materials and Methods
2.1. Cells and Culture Protocols
2.1.1. Reprogramming of Erythroblasts into hiPSCs
2.1.2. Differentiation of hiPSCs into NSCs
2.2. Viral Strains
2.3. Infections with WNV, USUV and ZIKV
2.4. Analysis of Virus Replication Kinetics
2.4.1. Quantitative Real-Time RT-PCR Analysis of Virus RNA Load
2.4.2. Virus Titration by 50% Tissue Culture Infective Dose (TCID50) Assay
2.5. End-Point RT-PCR Analysis of Differentiation Marker Expression
2.6. qRT-PCR Analysis of mRNA Levels of Genes Involved in Innate Antiviral Response
2.7. Immunofluorescence Assays
2.8. Apoptosis Assay
2.9. Cell Viability Assay
2.10. Statistical Analysis
3. Results
3.1. Human iPSC-Derived NSCs Are Permissive to WNV and USUV Infection
3.2. Kinetics of WNV, USUV, and ZIKV Production in hNSCs
3.3. Effects of WNV, USUV and ZIKV Infection on hNSC Viability and Caspase-3 Activity in Infected Cells
3.4. Expression of Innate Antiviral Immune Genes in hNSCs in Response to WNV, USUV and ZIKV Infection
4. Discussion
Author Contributions
Funding
Conflicts of Interest
References
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Target Gene | Forward/Reverse Primers (5′–3′) | References | |
---|---|---|---|
Neural stem cells markers | NESTIN | GAAGGTGAAGGGCAAATCTG CCTCTTCTTCCCATATTTCCTG | [40] |
PAX6 | TCTAATCGAAGGGCCAAATG TGTGAGGGCTGTGTCTGTTC | [41] | |
SOX1 | GCGGAAAGCGTTTTCTTTG TAATCTGACTTCTCCTCCC | [42] | |
SOX2 | TTGTCGGAGACGGAGAAGCG TGACCACCGAACCCATGGAG | [42] | |
Pluripotency marker | OCT4 | GTGGAGGAAGCTGACAACAA CAGGTTTTCTTTCCCTAGCT | [43] |
Housekeeping gene | ACTIN | GGACTTCGAGCAAGAGATGG AGCACTGTGTTGGCGTACAG | [44] |
Target Gene | Forward/Reverse Primers/TaqMan Probe (5′–3′) | Reference |
---|---|---|
RIG1 | ACCAGAGCACTTGTGGACGCT TGCCGGGAGGGTCATTCCTGT | [45] |
MDA5 | CAGAAGGAAGTGTCAGCTGCTTAG TGCTGCCACATTCTCTTCATCT | [46] |
CGAS | CCTGCTGTAACACTTCTTAT TTAGTCGTAGTTGCTTCCT | [47] |
TLR3 | GAAAGGCTAGCAGTCATCCA CATCGGGTACCTGAGTCAAC | [48] |
TLR7 | CTTGGCACCTCTCATGCTCT GTCTGTGCAGTCCACGATCA | [49] |
TLR8 | AGTTTCTCTTCTCGGCCACC GGAACATGTTTTCCATGTTTCTGT | [49] |
TLR2 | GGCCAGCAAATTACCTGTGTG AGGCGGACATCCTGAACCT FAM-CCATCCCATGTGCGTGG-MGB | [50] |
MAVS | AGCAAGAGACCAGGATCGACTG CGCAATGAAGTACTCCACCCA | [46] |
NFKB | GCCAACAGATGGCCCATACC TGCTGGTCCCACATAGTTGC | [51] |
IRF3 | AGCAGAGGACCGGAGCAA AGAGGTGTCTGGCTGGGAAA FAM-ACCCTCACGACCCACATAAAATCTACGAGTTTG-TAMRA | [52] |
IRF7 | TACCATCTACCTGGGCTTCG AGGGTTCCAGCTTCACCA | [46] |
TNFA | CCAGACCAAGGTCAACCTCC CCCTCCCAGATAGATGGGCT | [53] |
IFNA1 | AGAATCTCTCCTTTCTCCTG TCTGACAACCTCCCAGGCAC | [54] |
IFNB1 | GAGCTACAACTTGCTTGGATTCC CAAGCCTCCCATTCAATTGC FAM-ACAAAGAAGCAGCAATTTTCAGTGTCAGAAGCT-TAMRA | [52] |
IFNL1 | GAGGCCCCCAAAAAGGAGTC AGGTTCCCATCGGCCACATA | [49] |
IFNL2 | AATTGTGTTGCCAGTGGGGA GCGACTGGGTGGCAATAAAT | [49] |
IFNL3 | AGGGCCAAAGATGCCTTAG CAGCTCAGCCTCCAAAGC | [49] |
IFIT1 | TCTCAGAGGAGCCTGGCTAA TGACATCTCAATTGCTCCAG | [49] |
IFIT2 | AAGAGTGCAGCTGCCTGAA GGCATTTTAGTTGCCGTAGG | [49] |
RSAD2 | CTTTGTGCTGCCCCTTGAG TCCATACCAGCTTCCTTAAGCAA | [55] |
CASP1 | AAGACCCGAGCTTTGATTGACTC AAATCTCTGCCGACTTTTGTTTCC | [56] |
CASP3 | TGCATACTCCACAGCACCTG TTCTGTTGCCACCTTTCGGT | [57] |
IL1B | GAGCAACAAGTGGTGTTCTCC AACACGCAGGACAGGTACAG | [58] |
GAPDH | GAAGGTGAAGGTCGGAGTC GAAGATGGTGATGGGATTTC FAM-CAAGCTTCCCGTTCTCAGCC-TAMRA | [59] |
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Riccetti, S.; Sinigaglia, A.; Desole, G.; Nowotny, N.; Trevisan, M.; Barzon, L. Modelling West Nile Virus and Usutu Virus Pathogenicity in Human Neural Stem Cells. Viruses 2020, 12, 882. https://doi.org/10.3390/v12080882
Riccetti S, Sinigaglia A, Desole G, Nowotny N, Trevisan M, Barzon L. Modelling West Nile Virus and Usutu Virus Pathogenicity in Human Neural Stem Cells. Viruses. 2020; 12(8):882. https://doi.org/10.3390/v12080882
Chicago/Turabian StyleRiccetti, Silvia, Alessandro Sinigaglia, Giovanna Desole, Norbert Nowotny, Marta Trevisan, and Luisa Barzon. 2020. "Modelling West Nile Virus and Usutu Virus Pathogenicity in Human Neural Stem Cells" Viruses 12, no. 8: 882. https://doi.org/10.3390/v12080882