In Vitro Characterization of Multidrug-Resistant Influenza A(H1N1)pdm09 Viruses Carrying a Dual Neuraminidase Mutation Isolated from Immunocompromised Patients
Abstract
:1. Introduction
2. Materials and Methods
2.1. Viruses
2.2. Antiviral Compounds
2.3. NA Inhibition Assay
2.4. Focus Reduction Assay
2.5. Cytopathic Effect Reduction Assay
2.6. Plaque Assay
2.7. Virus Replication Kinetics In Vitro
2.8. Competitive Virus Replication In Vitro
2.9. Deep Sequencing Analysis
2.10. Structural Analysis of the NA Protein
2.11. Statistical Analysis
3. Results
3.1. Immunocompromised Patients Infected with Dual H275Y Mutant Viruses
3.2. Antiviral Susceptibilities of the Dual H275Y Mutant Viruses
3.3. In Vitro Replication Kinetics of the Dual H275Y Mutant Viruses
3.4. Competitive Growth Capabilities of the Dual H275Y Mutant Viruses and the Wild-Type Viruses
3.5. Effects of Amino Acid Substitutions on the Stability of the NA Protein
4. Discussion
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Isolate Name | Date of Symptom Onset (Day/Month/Year) | Antiviral Administration (Day/Month/Year) | Date of Specimen Collection (Day/Month/Year) | NA Substitution |
---|---|---|---|---|
A/Hiroshima/57/2014 | 31-03-2014 | 31-03-2014: Peramivir 04-04-2014: Laninamivir | 04-04-2014 | H275Y/I223R |
A/Ibaraki/54/2016 | 09-02-2016 | 09-02-2016: Peramivir 24-02-2016 to 03-03-2016: Peramivir 26-02-2016 to 03-03-2016: Oseltamivir 05-03-2016: Laninamivir | 07-03-2016 | H275Y/I223K |
A/Hiroshima/13/2016 | 26-02-2016 | 23-02-2016: Laninamivir 26-02-2016 to 01-03-2016: Peramivir 07-03-2016 to 11-03-2016: Peramivir 28-03-2016 to 01-04-2016: Peramivir | 11-03-2016 | H275Y/G147R |
Isolate Name | GISAID Isolate ID | NA Substitution | IC50, nM (Fold-Change 1) | EC50, µM (Fold-Change 1) | ||||
---|---|---|---|---|---|---|---|---|
Oseltamivir 2 | Peramivir 2 | Zanamivir 2 | Laninamivir 2 | Baloxavir 3 | Favipiravir 4 | |||
A/Hiroshima/57/2014 | EPI ISL 160499 | H275Y/I223R | 6263.69 (20,000) | 944.20 (94,000) | 4.84 (48) | 4.48 (20) | 8.34 (2.1) | 7.99 (1.2) |
A/Osaka/8/2014 | EPI ISL 155839 | H275Y | 173.80 (560) | 28.25 (2,800) | 0.03 (0.3) | 0.14 (0.6) | 5.10 (1.3) | 4.76 (0.7) |
A/Sakai/23/2013 | EPI ISL 154461 | None (wild-type) | 0.31 | 0.01 | 0.10 | 0.22 | 4.05 | 6.64 |
A/Ibaraki/54/2016 | EPI ISL 221789 | H275Y/I223K | 10161.87 (16,000) | 501.62 (7,200) | 2.48 (12) | 1.94 (6.9) | 7.24 (1.2) | 10.20 (1.0) |
A/Yokohama/94/2016 | EPI ISL 218900 | H275Y | 466.79 (730) | 20.88 (300) | 0.33 (1.6) | 0.52 (1.9) | 3.49 (0.6) | 21.40 (2.1) |
A/Yokohama/40/2016 | EPI ISL 217919 | None (wild-type) | 0.64 | 0.07 | 0.21 | 0.28 | 6.16 | 10.01 |
A/Hiroshima/13/2016 | EPI ISL 220376 | H275Y/G147R | 1324.62 (1,500) | 114.14 (1,400) | 1.56 (4.5) | 0.37 (0.9) | 10.59 (1.0) | 15.13 (1.7) |
A/Aichi/83/2016 | EPI ISL 233222 | H275Y | 364.44 (420) | 31.93 (400) | 0.77 (2.2) | 0.77 (1.9) | 10.18 (0.9) | 14.83 (1.7) |
A/Yokohama/59/2016 | EPI ISL 217920 | None (wild-type) | 0.86 | 0.08 | 0.35 | 0.40 | 11.04 | 8.81 |
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Takashita, E.; Fujisaki, S.; Yokoyama, M.; Shirakura, M.; Morita, H.; Nakamura, K.; Kishida, N.; Kuwahara, T.; Sato, H.; Doi, I.; et al. In Vitro Characterization of Multidrug-Resistant Influenza A(H1N1)pdm09 Viruses Carrying a Dual Neuraminidase Mutation Isolated from Immunocompromised Patients. Pathogens 2020, 9, 725. https://doi.org/10.3390/pathogens9090725
Takashita E, Fujisaki S, Yokoyama M, Shirakura M, Morita H, Nakamura K, Kishida N, Kuwahara T, Sato H, Doi I, et al. In Vitro Characterization of Multidrug-Resistant Influenza A(H1N1)pdm09 Viruses Carrying a Dual Neuraminidase Mutation Isolated from Immunocompromised Patients. Pathogens. 2020; 9(9):725. https://doi.org/10.3390/pathogens9090725
Chicago/Turabian StyleTakashita, Emi, Seiichiro Fujisaki, Masaru Yokoyama, Masayuki Shirakura, Hiroko Morita, Kazuya Nakamura, Noriko Kishida, Tomoko Kuwahara, Hironori Sato, Ikuko Doi, and et al. 2020. "In Vitro Characterization of Multidrug-Resistant Influenza A(H1N1)pdm09 Viruses Carrying a Dual Neuraminidase Mutation Isolated from Immunocompromised Patients" Pathogens 9, no. 9: 725. https://doi.org/10.3390/pathogens9090725