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. 2020 Jan 1;221(1):63-70.
doi: 10.1093/infdis/jiz418.

Impact of the Baloxavir-Resistant Polymerase Acid I38T Substitution on the Fitness of Contemporary Influenza A(H1N1)pdm09 and A(H3N2) Strains

Liva Checkmahomed  1 , Zeineb M'hamdi  1 , Julie Carbonneau  1 , Marie-Christine Venable  1 , Mariana Baz  1 , Yacine Abed  1 , Guy Boivin  1
Affiliations

Impact of the Baloxavir-Resistant Polymerase Acid I38T Substitution on the Fitness of Contemporary Influenza A(H1N1)pdm09 and A(H3N2) Strains

Liva Checkmahomed et al. J Infect Dis. .

Abstract

Background: Baloxavir is a cap-dependent inhibitor of the polymerase acid (PA) protein of influenza viruses. While appearing virologically superior to oseltamivir, baloxavir exhibits a low barrier of resistance. We sought to assess the impact of the common baloxavir-resistant I38T PA substitution on in vitro properties and virulence.

Methods: Influenza A/Quebec/144147/2009 (H1N1)pdm09 and A/Switzerland/9715293/2013 (H3N2) recombinant viruses and their I38T PA mutants were compared in single and competitive infection experiments in ST6GalI-MDCK cells and C57/BL6 mice. Virus titers in cell culture supernatants and lung homogenates were determined by virus yield assays. Ratios of wild-type (WT) and I38T mutant were assessed by digital RT-PCR.

Results: I38T substitution did not alter the replication kinetics of A(H1N1)pdm09 and A(H3N2) viruses. In competition experiments, a 50%:50% mixture evolved to 70%:30% (WT/mutant) for A(H1N1) and 88%:12% for A(H3N2) viruses after a single cell passage. The I38T substitution remained stable after 4 passages in vitro. In mice, the WT and its I38T mutant induced similar weight loss with comparable lung titers in both viral subtypes. The mutant virus tended to predominate over the WT in mouse competition experiments.

Conclusion: The fitness of baloxavir-resistant I38T PA mutants appears relatively unaltered in seasonal subtypes warranting surveillance for its dissemination.

Keywords: A(H1N1)pdm09; A(H3N2); I38T; PA; baloxavir; influenza; resistance.

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Figures

Figure 1.
Figure 1.
Polymerase activity of contemporary wild-type (WT) and I38T A(H1N1)pdm09 and A(H3N2) viruses by minigenome assays. 293T cells were cotransfected with NP, PA, PB1, PB2, and the reporter luciferase plasmids. Mean luciferase values of 3 independent experiments were measured. The data were analyzed with 1-way ANOVA Dunnett multiple comparisons test. **, P < .01.
Figure 2.
Figure 2.
In vitro replicative capacity of recombinant (A) A(H1N1)pdm09 and (B) A(H3N2) viruses. Confluent ST6GalI-MDCK cells were infected with recombinant viruses at a multiplicity of infection of 0.0001. Supernatants were harvested at the indicated time points and viral titers (TCID50/mL) were determined in ST6GalI-MDCK cells. Data were analyzed using the Mann-Whitney test. Abbreviation: WT, wild type.
Figure 3.
Figure 3.
In vitro quantification of wild-type (WT) and I38T populations in (A) A(H1N1)pdm09 and (B) A(H3N2) viruses. Proportion of WT viruses and their respective I38T mutants are indicated in competition experiments performed in ST6GalI-MDCK cells. Cells were infected using a multiplicity of infection of 0.001. Proportions of each viral population were determined by digital droplet reverse transcription polymerase chain reaction and are expressed as a percentage of the total viral population. Mean proportion values from quadruplicate experiments are shown.
Figure 4.
Figure 4.
Impact of the I38T PA mutation in mice experimentally infected with A(H1N1)pdm09 viruses. A, Mean body weight loss ± standard deviation of mice infected with recombinant A(H1N1)pdm09 wild-type (WT) and mutant viruses. Groups of 6 mice were infected with 5 × 104 plaque-forming units of the recombinant A(H1N1)pdm09 and its I38T variant. Percent body weight losses as compared to initial weights were recorded daily until day 14 postinoculation. *P < .05. B, Mean viral titers in lungs ± standard deviation were determined as TCID50/mL in ST6GalI-MDCK cells for groups of 4 mice euthanized on days 3 and 6 postinoculation. C, Proportions of WT and I38T viral populations in lungs of mice as determined by digital droplet reverse transcription polymerase chain reaction.
Figure 5.
Figure 5.
Impact of the I38T PA mutation in mice experimentally infected with A(H3N2) viruses. A, Mean body weight losses ± standard deviation of mice infected with recombinant A(H3N2) wild-type (WT) and I38T viruses. Groups of 6 mice were infected with 104 plaque-forming units of each virus. Percent body weight losses as compared to initial weights were recorded daily until day 14 postinoculation. B, Mean viral titers in lungs ± standard deviation were determined as TCID50/mL in ST6GalI-MDCK cells for groups of 4 mice euthanized on days 3 and 6 postinoculation. C, Proportions of WT and I38T viral populations in lungs of mice as determined by digital droplet reverse transcription polymerase chain reaction.
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