A Mutated PB1 Residue 319 Synergizes with the PB2 N265S Mutation of the Live Attenuated Influenza Vaccine to Convey Temperature Sensitivity
Abstract
:1. Introduction
2. Materials and Methods
2.1. Cells and Media
2.2. Plaque Purification
2.3. Tissue Culture Infectious Dose for 50% Infection (TCID50)
2.4. Single Reaction Genomic Amplification for IAV Gene Sequencing
2.5. Minigenome Assay
2.6. Site-Directed Mutagenesis
2.7. Viral Rescue
2.8. Growth Curves
2.9. Viral Attenuation
2.10. Ethics Statement
2.11. Semi-Infectious Particles
3. Results
3.1. Identification of a Single-Gene Replacement Virus That is More Temperature Sensitive than LAIV
3.2. A Single Residue Conveyed the Majority of Phenotypic Reversion
3.3. PB1 319Q and PB2 265S Impart Temperature Sensitivity to the Viral Polymerase of Two Additional IAV Strains
3.4. PB1 319Q and PB2 265S Impart Temperature Sensitivity to PR8
3.5. PB1 319Q and PB2 N265S Confer Synergistic Attenuation on PR8 In Vivo
3.6. The Combination of PB1 319Q and PB2 265S Increases the Formation of Semi-Infectious Particles
4. Discussion
5. Conclusions
6. Patents
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Virus | PB2 | PB1 | PA | NP | M | NS | HA | NA |
---|---|---|---|---|---|---|---|---|
LAIV | A/AA-LAIV | A/AA-LAIV | A/AA-LAIV | A/AA-LAIV | A/AA-LAIV | A/AA-LAIV | A/Korea | A/Korea |
SGR | A/AA-LAIV | A/Korea | A/Korea | A/Korea | A/Korea | A/Korea | A/Korea | A/Korea |
SGR-rev | A/AA-LAIV | A/Korea | A/Korea | A/Korea | A/Korea | A/Korea | A/Korea | A/Korea |
Amino Acid | Incidence among Human Isolates | |||
---|---|---|---|---|
SGR | SGR-Rev | |||
PB2 | 73 | Q | K | 32,794 D 12 K |
PB1 | 145 | S | N | 145 S 23,711 N |
319 | Q | L | 0 Q 23,671 L | |
PA | 347 | D | N | 33,464 D 21 N 14,589 S |
409 | S | N | 18,919 N | |
632 | S | P | 33,445 S 18 P |
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Cox, A.; Schmierer, J.; D’Angelo, J.; Smith, A.; Levenson, D.; Treanor, J.; Kim, B.; Dewhurst, S. A Mutated PB1 Residue 319 Synergizes with the PB2 N265S Mutation of the Live Attenuated Influenza Vaccine to Convey Temperature Sensitivity. Viruses 2020, 12, 1246. https://doi.org/10.3390/v12111246
Cox A, Schmierer J, D’Angelo J, Smith A, Levenson D, Treanor J, Kim B, Dewhurst S. A Mutated PB1 Residue 319 Synergizes with the PB2 N265S Mutation of the Live Attenuated Influenza Vaccine to Convey Temperature Sensitivity. Viruses. 2020; 12(11):1246. https://doi.org/10.3390/v12111246
Chicago/Turabian StyleCox, Andrew, Jordana Schmierer, Josephine D’Angelo, Andrew Smith, Dustyn Levenson, John Treanor, Baek Kim, and Stephen Dewhurst. 2020. "A Mutated PB1 Residue 319 Synergizes with the PB2 N265S Mutation of the Live Attenuated Influenza Vaccine to Convey Temperature Sensitivity" Viruses 12, no. 11: 1246. https://doi.org/10.3390/v12111246