On-Demand Patient-Specific Phenotype-to-Genotype Ebola Virus Characterization
Abstract
:1. Introduction
2. Materials and Methods
2.1. Cell Culture
2.2. Viruses
2.3. Recombinant EBOV Templates
2.4. Synthesis of the EBOV Genome-Encoding Plasmids and Helper Plasmids
2.4.1. Genome Plasmids
2.4.2. Helper Plasmids
2.4.3. Plasmid Sequencing
2.5. EBOV/Mak-G3864.1 Minigenome
2.6. Recombinant EBOV Rescue
2.7. Small Molecules
2.8. EBOV Assay in HeLa Cells
2.9. Immunostaining
2.10. Image and Data Analysis
2.11. Sequencing Data Analysis
2.12. Laboratory Mouse Experiments
3. Results
3.1. Recombinant EBOV Rescue
3.1.1. Selection of EBOV Isolate Sequences for De Novo Genome Synthesis and Rescue
3.1.2. Plasmid Establishment and Sequence Verification
3.2. EBOV Rescue
3.2.1. Cell Culture Adaptations Can Alter Susceptibility to MCMs
3.2.2. Recombinant EBOV/Mak-G3864.1 Mutants Cause Weight Loss in Interferon-Alpha/Beta Receptor Knockout (INFAR−/−) Laboratory Mice
4. Discussion
5. Conclusions
Disclaimer
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Designation for This Study | Genotype Compared to EBOV/ Mak-G3864.1 Sequence 412 | Characteristics | GenBank Accession Number |
---|---|---|---|
rEBOV/Mak-G3864.1 mutant r412 (parental) | C3579A (VP35 gene, silent) | MN975530 | |
rEBOV/Mak-G3864.1 mutant r414 | C1498T (NP gene, silent) | Detected during a flare-up of seven EVD cases that occurred in June and July of 2015 in Liberia [41]. The genome sequence of this virus indicated a markedly reduced rate of genome evolution compared to previously circulating viruses. | MN975531 |
G9355A (nonsense, truncation of last 7 amino acids of VP30) | |||
A10130G (VP24 gene UTR) | |||
rEBOV/Mak-G3864.1 mutant r424 | T3008C (NP gene UTR) | The double NP mutation was first identified in viruses from September 2014 and affects expression of NP in an EBOV minigenome assay [42]. | MN975532 |
T3011C (NP gene UTR) | |||
C3579A (VP35 gene, silent) | |||
rEBOV/Mak-G3864.1 mutant r440 | C6283T (GP1,2, V82A) | EBOV/Mak genome sequences obtained at the very beginning of the 2013–2016 EVD were all of the 82A genotype, but the 82V genotype quickly emerged and >90% of all of the sequenced EBOV genomes from the outbreak are of the 82V genotype [39]. The A82V change enhances EBOV infection in nonhuman primate cells [26,27,28,43,44,45,46], but it does not appear to alter virulence in an interferon alpha receptor knock-out (IFNAR−/−) laboratory mouse model and a rhesus monkey (Macaca mulatta) model of EVD [47]. | MN975533 |
C3579A (VP35 gene, silent) | |||
rEBOV/Itu mutant r865 | N/A | MN975534 | |
rEBOV/Tum mutant r866 | N/A | MN975535 |
Sample | Variants >5% | % 8U |
---|---|---|
HeLa 1 | G7692A (31.5%): GP1,2 D552N | 0.8 |
HeLa 2 | T331C (26%) in NP UTR | 1.8 |
ins11514A (38.9%) in VP24/L UTR | ||
Huh7D-12/T7 | none | 1.1 |
Vero E6 (1) | none | 0.9 |
Vero E6 (2) | A670G (25.8%): NP Q67R | 9.1 |
C16881T (7.9%): L T1767I | ||
Huh7 plus T7 * | none | 1.2 |
Huh7D-12/T7 plus T7 * | none | 1.4 |
VeroE6 plus T7 * | A4943T (100%): VP40 Q155L | 2.4 |
A9124C (68.1%): VP30 E205D |
Virus | Phenotype | Compound ID | EC50 (μM) | SD | EC90 | CC50 (μM) | SI |
---|---|---|---|---|---|---|---|
EBOV/Mak-r440 | 82A 544T | 3.47 | 0.007 | 0.0004 | 0.02 | >10 | >1423 |
EBOV/Mak-r412 | 82V 544T | 0.01 | 0.001 | 0.08 | >10 | >1014 | |
EBOV/Mak-201403261 | 82V 544I | 0.04 | 0.008 | 1.67 | >10 | >248 | |
EBOV/May-tc | 82A 544I | 0.01 | 0.005 | 0.74 | >10 | >792 | |
EBOV/Mak-r440 | 82A 544T | 1412 | 0.15 | 0.12 | 5.65 | >20 | >131 |
EBOV/Mak-r412 | 82V 544T | 0.77 | 0.23 | 16 | >20 | >26 | |
EBOV/Mak-201403261 | 82V 544I | 2.93 | 1.21 | 79.7 | >20 | >7 | |
EBOV/May-tc | 82A 544I | 2.64 | 1.14 | 106 | >20 | >8 | |
EBOV/Mak-r440 | 82A 544T | 1413 | 0.08 | 0.02 | 0.68 | >20 | >266 |
EBOV/Mak-r412 | 82V 544T | 0.24 | 0.06 | 4.03 | >20 | >85 | |
EBOV/Mak-201403261 | 82V 544I | 0.85 | 0.33 | 27,8 | >20 | >24 | |
EBOV/May-tc | 82A 544I | 0.76 | 0.27 | 18.7 | >20 | >26 | |
EBOV/Mak-r440 | 82A 544T | 1466 | 0.04 | 0.007 | 0.14 | >20 | >497 |
EBOV/Mak-r412 | 82V 544T | 0.13 | 0.02 | 1.39 | >20 | >159 | |
EBOV/Mak-201403261 | 82V 544I | 0.39 | 0.17 | 24.4 | >20 | >52 | |
EBOV/May-tc | 82A 544I | 0.30 | 0.13 | 11.1 | >20 | >67 | |
EBOV/Mak-r440 | 82A 544T | 1476 | 0.001 | 0.0004 | 0.05 | >10 | >14,169 |
EBOV/Mak-r412 | 82V 544T | 0.002 | 0.001 | 0.17 | >10 | >5985 | |
EBOV/Mak-201403261 | 82V 544I | 0.005 | 0.002 | 0.4 | >10 | >2018 | |
EBOV/May-tc | 82A 544I | 0.002 | 0.002 | 0.37 | >10 | >4262 |
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Beitzel, B.F.; Radoshitzky, S.R.; Di Paola, N.; Brannan, J.M.; Kimmel, D.; Caviness, K.; Soloveva, V.; Yu, S.; Postnikova, E.N.; Finch, C.L.; et al. On-Demand Patient-Specific Phenotype-to-Genotype Ebola Virus Characterization. Viruses 2021, 13, 2010. https://doi.org/10.3390/v13102010
Beitzel BF, Radoshitzky SR, Di Paola N, Brannan JM, Kimmel D, Caviness K, Soloveva V, Yu S, Postnikova EN, Finch CL, et al. On-Demand Patient-Specific Phenotype-to-Genotype Ebola Virus Characterization. Viruses. 2021; 13(10):2010. https://doi.org/10.3390/v13102010
Chicago/Turabian StyleBeitzel, Brett F., Sheli R. Radoshitzky, Nicholas Di Paola, Jennifer M. Brannan, David Kimmel, Katie Caviness, Veronica Soloveva, Shuiqing Yu, Elena N. Postnikova, Courtney L. Finch, and et al. 2021. "On-Demand Patient-Specific Phenotype-to-Genotype Ebola Virus Characterization" Viruses 13, no. 10: 2010. https://doi.org/10.3390/v13102010
APA StyleBeitzel, B. F., Radoshitzky, S. R., Di Paola, N., Brannan, J. M., Kimmel, D., Caviness, K., Soloveva, V., Yu, S., Postnikova, E. N., Finch, C. L., Liu, H., Prugar, L., Bakken, R., Dye, J. M., Kugelman, J. R., Cunningham, J. M., Sanchez-Lockhart, M., Kuhn, J. H., & Palacios, G. (2021). On-Demand Patient-Specific Phenotype-to-Genotype Ebola Virus Characterization. Viruses, 13(10), 2010. https://doi.org/10.3390/v13102010