Generation and Characterization of Drug-Resistant Influenza B Viruses Selected In Vitro with Baloxavir Acid

Abstract

Baloxavir marboxil (BXM) is an antiviral drug that targets the endonuclease of the influenza polymerase acidic (PA) protein. Antiviral resistance, mainly mediated by the I38T PA substitution, readily occurs in both A(H1N1) and A(H3N2) viruses following a single dose of BXM. Influenza B resistance to BXM remains poorly documented. We aimed to generate baloxavir-resistant contemporary influenza B/Yamagata/16/1988- and B/Victoria/2/1987-like viruses by in vitro passages under baloxavir acid (BXA) pressure to identify resistance mutations and to characterize the fitness of drug-resistant variants. Influenza B/Phuket/3073/2013 recombinant virus (rg-PKT13, a B/Yamagata/16/1988-like virus) and B/Quebec/MCV-11/2019 (MCV19, a B/Victoria/2/1987-like isolate) were passaged in ST6GalI-MDCK cells in the presence of increasing concentrations of BXA. At defined passages, viral RNA was extracted for sequencing the PA gene. The I38T PA substitution was selected in MCV19 after six passages in presence of BXA whereas no PA change was detected in rg-PKT13. The I38T substitution increased the BXA IC₅₀ value by 13.7-fold in the MCV19 background and resulted in reduced viral titers compared to the wild type (WT) at early time points in ST6GalI-MDCK and at all time-points in human epithelial cells. By contrast, the I38T substitution had no impact on MCV19 polymerase activity, and this mutation was genetically stable over four passages. In conclusion, our results show a similar pathway of resistance to BXA in influenza B viruses highlighting the major role of the I38T PA substitution and suggest that I38T may differently impact the fitness of influenza variants depending on the viral type, subtype, or lineage.

Keywords: I38T substitution; Influenza B; PA; antiviral resistance; baloxavir.

Figure 1

Replication kinetics of MCV19 WT and its I38T variant in ST6GalI-MDCK cells. Confluent ST6GalI-MDCK cells were infected with an MOI of 0.0001. Supernatants were collected at the given time points and titrated by TCID₅₀. Mean viral titers of triplicates ± standard error of the mean were determined as TCID₅₀/mL. The mean of three independent experiments was calculated. * p < 0.05.

Figure 2

Replication kinetics of MCV19 and rg-PKT13 WT and I38T variants on human nasal airway epithelium. Infections were performed at the apical pole of HAE at an MOI of 0.002 for each virus. (A) MCV19 WT and its I38T variant generated by passaging (B) rg-PKT13 and its I38T variant generated by reverse genetics. Two experimental conditions were performed and viral loads were determined by qRT-PCR in triplicates for each of the two conditions and analyzed with t-test; # p < 0.001.

Figure 3

Polymerase activity of MCV19 WT and its I38T variant measured by minigenome assay. The pBZ plasmids: NP, PA, PB1, PB2, and the reporter luciferase plasmid were co-transfected in HEK293T cells. Luciferase activity was measured in supernatant after 18 h of incubation at 37 °C.