Replicative Fitness of Seasonal Influenza A Viruses With Decreased Susceptibility to Baloxavir

Abstract

Susceptibility of influenza A viruses to baloxavir can be affected by changes at amino acid residue 38 in the polymerase acidic (PA) protein. Information on replicative fitness of PA-I38-substituted viruses remains sparse. We demonstrated that substitutions I38L/M/S/T not only had a differential effect on baloxavir susceptibility (9- to 116-fold) but also on in vitro replicative fitness. Although I38L conferred undiminished growth, other substitutions led to mild attenuation. In a ferret model, control viruses outcompeted those carrying I38M or I38T substitutions, although their advantage was limited. These findings offer insights into the attributes of baloxavir-resistant viruses needed for informed risk assessment.

Keywords: baloxavir acid; drug resistance; ferret; influenza; polymerase acidic protein.

Figure 1.

(A and B) Effect of polymerase acidic (PA) substitutions I38M and I38T on A(H3N2) virus replication in ferrets. Animals (4 per group) were intranasally inoculated with 10³ 50% tissue culture infectious dose (TCID₅₀) of virus, (A) control I38 and I38M viruses, and (B) control I38 and I38T viruses. Nasal washes were collected at indicated days postinfection (dpi) and virus titers were determined. Dotted line indicates the lower limit of virus detection, 1.75 log₁₀ TCID₅₀/mL. Data shown are mean ± standard deviation, and the unpaired t test with Welch’s correction was used for statistical comparisons (*, P < .05). (C–H) Proportions of virus subpopulations in the nasal washes of ferrets. Ferrets (4 per group) were inoculated with 10³ TCID₅₀ containing control and PA-I38-substituted A(H3N2) virus mixed at ratios 10:90, 30:70, or 70:30; (C–E) control I38 and I38M viruses and (F–H) control I38 and I38T viruses. Nasal washes were collected at indicated dpi, and proportion of the respective PA subpopulations were determined by pyrosequencing using single-nucleotide polymorphism analysis.