PA from a Recent H9N2 (G1-Like) Avian Influenza A Virus (AIV) Strain Carrying Lysine 367 Confers Altered Replication Efficiency and Pathogenicity to Contemporaneous H5N1 in Mammalian Systems
Abstract
:1. Introduction
2. Materials and Methods
2.1. Cells, Viruses and Plasmids
2.2. Reverse Genetics (Rg) Systems for H5N1EGY and H9N2EGY and Generation of Reassortant, Mutant and Wild-Type Strains
2.3. In Vitro Replication Efficiency of Reassortant and Wild-Type Strains
2.4. Flow Cytometry Analysis of Viral Polymerase Activity
2.5. Temperature-Dependent Replication Kinetics of Reassortant and Wild-Type Strains
2.6. Pathogenicity of Reassortant Strains versus Wild-Type H5N1EGY in BALB/c Mice
2.7. Luciferase Reporter Assay
2.8. Pathogenicity of Mutated Strains versus Wild-Type H5N1EGY in BALB/c Mice
2.9. Ethical Statement and Biosafety
2.10. Statistical Analysis
3. Results
3.1. Gene segments of H9N2EGY Show High Genetic Compatibility in the Genetic Background of H5N1EGY
3.2. Improved Replication Efficiency of H5N1EGY Reassortants Expressing PB2 and PA of H9N2EGY in MDCK-II Cells
3.3. Impact of the Polymerase Subunits of H9N2EGY on the Polymerase Activity of Reassortant H5N1EGY
3.4. Replication Efficiency of Reassortant and Wild-Type H5N1EGY in Human Lung Cells Increases at Elevated Temperature Resembling Lung Ambient Temperature in Humans
3.5. Reassortants H5N1PB2-H9N2EGY and H5N1PA-H9N2EGY Showed Similar to Lower Virulence When Compared to the Wild-Type H5N1EGY in Mice
3.6. The Genetic Analysis of the Polymerase-Encoding Segments from H9N2EGY Revealed that the PAH9N2EGY and PB2H9N2EGY Encode for Distinct Mammalian-Like Variations
3.7. The H5N1EGY Mutant Expressing PAR367K Replicates Efficiently in Primary Human Bronchial Epithelial Cells and Continuous Human Cell Culture Models in a Temperature-Dependent Manner
3.8. Replication Efficiency of H5N1PA_R367K Is Not Associated with an Enhanced Polymerase Activity
3.9. The PA_R367K Contributes to the Pathogenicity of H5N1EGY in Mice
4. Discussion
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Mutant Virus | Mutation | Primer Name | Mutagenesis Primer Sequence |
---|---|---|---|
H5N1PA_R367K | R367K | PA-367F | 5’-GAAAAAAACGAGCCAGTTAAAGTGGGCACTCGGTGAGAACATG-3’ |
PA-367R | 5’-CATGTTCTCACCGAGTGCCCACTTTAACTGGCTCGTTTTTTTC-3’ | ||
H5N1PA-H9N2EGY_K367R | K367R | PA-K367R-H9EGY-F | 5’-GAAGAAAACAAGCCAATTAAGATGGGCACTCGGTGAGAATATG-3’ |
PA-K367R-H9EGY-R | 5’-CATATTCTCACCGAGTGCCCATCTTAATTGGCTTGTTTTCTTC-3’ |
Amino Acid (aa) Residue | PB2 | Amino Acid (aa) Residue | PA | ||
---|---|---|---|---|---|
H5N1EGY | H9N2EGY | H5N1EGY | H9N2EGY | ||
6 | E | G | 38 | I | V |
64 | I | M | 58 | S | G |
66 | I | M | 94 | V | I |
80 | R | K | 101 | E | D |
106 | A | T | 129 | T | I |
129 | N | T | 184 | A | V |
147 | T | I | 204 | K | R |
197 | R | K | 212 | L | R |
249 | K | E | 269 | K | R |
292 | M | I | 287 | S | A |
315 | I | M | 321 | G | N |
339 | T | K | 323 | V | A |
368 | Q | R | 337 | T | A |
369 | K | R | 342 | M | L |
377 | S | A | 351 | D | E |
390 | N | D | 367 | R | K |
393 | T | S | 382 | E | D |
451 | T | V | 388 | R | S |
498 | H | Q | 391 | K | R |
521 | T | A | 396 | D | G |
529 | V | I | 400 | T | S |
570 | I | M | 437 | H | Y |
591 | Q | K | 448 | E | A |
627 | K | E | 450 | A | V |
649 | I | V | 539 | K | R |
661 | T | A | 554 | V | I |
615 | R | K | |||
626 | R | K | |||
653 | S | P | |||
669 | V | I | |||
706 | L | F | |||
712 | A | I | |||
716 | N | K |
Distinct Amino Acid (aa) Residues | ||||||
---|---|---|---|---|---|---|
PA Segment | 321 | 342 | 351 | 367 | 705 | 707 |
PAH9N2EGY | N | L | E | K | S | F |
PAH5N1EGY | G | M | D | R | S | L |
PAH7N9Anhui (Human isolate) | N | L | E | K | S | F |
PAH5N1EGY (Human isolates 2006–2013) | S | L | E | R | S | F |
PAH5N1EGY (Avian isolates 2006–2013) | N | L | E | R | S | F |
PAH5N1EGY (Human isolates 2014–2017) | G | M | D | K | S | L |
PAH5N1EGY (Avian isolates 2014–2017) | N | L | E | R | Y | F |
PB2 Segment | 591 | 627 | 701 | |||
PB2H9N2EGY | K | E | D | |||
PB2H5N1EGY | Q | K | D | |||
PB2H7N9Anhui (Human isolate) | S | K | R | |||
PB2H5N1EGY (Human isolates 2006–2013) | Q | K | D | |||
PB2H5N1EGY (Avian isolates 2006–2013) | Q | K | D | |||
PB2H5N1EGY (Human isolates 2014–2017) | Q | K | D | |||
PB2H5N1EGY (Avian isolates 2014–2017) | Q | K | D |
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Mostafa, A.; Mahmoud, S.H.; Shehata, M.; Müller, C.; Kandeil, A.; El-Shesheny, R.; Nooh, H.Z.; Kayali, G.; Ali, M.A.; Pleschka, S. PA from a Recent H9N2 (G1-Like) Avian Influenza A Virus (AIV) Strain Carrying Lysine 367 Confers Altered Replication Efficiency and Pathogenicity to Contemporaneous H5N1 in Mammalian Systems. Viruses 2020, 12, 1046. https://doi.org/10.3390/v12091046
Mostafa A, Mahmoud SH, Shehata M, Müller C, Kandeil A, El-Shesheny R, Nooh HZ, Kayali G, Ali MA, Pleschka S. PA from a Recent H9N2 (G1-Like) Avian Influenza A Virus (AIV) Strain Carrying Lysine 367 Confers Altered Replication Efficiency and Pathogenicity to Contemporaneous H5N1 in Mammalian Systems. Viruses. 2020; 12(9):1046. https://doi.org/10.3390/v12091046
Chicago/Turabian StyleMostafa, Ahmed, Sara H. Mahmoud, Mahmoud Shehata, Christin Müller, Ahmed Kandeil, Rabeh El-Shesheny, Hanaa Z. Nooh, Ghazi Kayali, Mohamed A. Ali, and Stephan Pleschka. 2020. "PA from a Recent H9N2 (G1-Like) Avian Influenza A Virus (AIV) Strain Carrying Lysine 367 Confers Altered Replication Efficiency and Pathogenicity to Contemporaneous H5N1 in Mammalian Systems" Viruses 12, no. 9: 1046. https://doi.org/10.3390/v12091046