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

<p>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 <i>wt</i> HA sequence; (B), two HA mutations relative to <i>wt;</i> and (C) 3–5 mutations relative to <i>wt</i> 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 <i>wt</i> 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 <i>wt</i> A/SLOV15 NA sequence.</p>