Table 3.

The interim PA sequence-based analysis to assess baloxavir susceptibility of influenza A viruses.^a.

PA CEN substitution		Common characteristics	Interpretation

Primary^b	I38T	• Primary treatment-emergent; associated with diminished virologic response in clinical settings • Rarely as natural polymorphism • Confers elevated baloxavir EC₅₀ in cell culture (11–614-fold)	• Reduced susceptibility • Phenotypic testing is not required, but helpful
Primary^b	I38 F, M, N, S	• Treatment-emergent • Rarely as natural polymorphisms • Confer elevated baloxavir EC₅₀ (4–112-fold)
Secondary	E18G E23 G, K, R Y24C K34R A36V A37T I38L L106R E119D V122A E198K E199 D, G, K	• Most are natural polymorphisms • Some emerged post baloxavir exposure or introduced for assessment • Confer variably elevated baloxavir EC₅₀ (<3–19 fold)	• Suspected reduced susceptibility • Phenotypic testing is required
Tertiary (this study only)^c	A20 T, V Y24F L28M R84K L106I I120 L, V V122 I, L	• Natural polymorphism • No or little change in EC₅₀ (<3-fold)	• Normal susceptibility • Phenotypic testing is not required, but helpful

CEN, cap-dependent endonuclease; EC₅₀, 50 % effective concentration; PA, polymerase acidic subunit.

PA CEN domain for type A viruses spans from amino acid residue 1 to 198 (Omoto et al., 2018); residue 199 is adjacent to CEN and belongs to the linker region. The consensus amino acid at residue 28 is proline (P) and leucine (L) for A (H1N1)pdm09 and A (H3N2) backgrounds, respectively. See Table S1 for additional information on the residues and substitutions shown.

In type B viruses, PA-I38T confers 5- to 15-fold elevated baloxavir EC₅₀. PA-I38F and PA-I38M were shown to confer variably elevated EC₅₀ (<3- to 8-fold) and phenotypic testing would be required to report reduced susceptibility in type B viruses.

Previously reported tertiary category PA amino acid substitutions for type A and B viruses are listed in Table S7.