HA mutations provide a cumulative increase in A/SLOV15 replication in primary human nasal epithelial cells (hNEC).

Five day time-course infections in hNEC were conducted at an MOI of 0.01. Apical wash samples were collected every 24 hours and virus titre measured by TCID₅₀ assay. Mean virus titre per day was calculated from three independent experiments of three transwells each (total nine transwells per virus). Mean daily virus titre of A/SLOV15 V1 (blue) and A/SLOV15 variants carrying: (A) a single HA mutation relative to the wt HA sequence; (B), two HA mutations relative to wt; and (C) 3–5 mutations relative to wt are illustrated by the columns. Symbols depict mean daily virus titre for individual hNEC transwells while columns show group means and error bars indicate the standard deviation. A Kruskal-Wallis test with a post-hoc Dunn’s multi-comparison test was conducted to compare A/SLOV15 V1 to all other A/SLOV15 variants. P values indicating significant difference in hNEC replication relative to A/SLOV15 V1 are depicted in panels A-C as follows: **** P<0.0001, *** P<0.001, ** P<0.01 and * P<0.05. A/SLOV15 mutants S2-S13 were generated by introducing the following substitutions to wt A/SLOV15 HA (V1) sequence: S2: N125D; S3: D127E; S4: D222G; S5: R223Q; S6: N125D, D127E; S7: D127E, D222G; S8: D127E, R223Q; S9: D222G, R223Q; S10: N125D, D127E, R223Q; S11: D127E, D222G, R223Q; S12: N125D, D127E, D222G, R223Q; S13: N125D, D127E, D222G, R223Q, N380D. All variants contained wt A/SLOV15 NA sequence.