Fig. 1: Virus rescue experiments of natural mutations.

a The amino acid sequences of HK68 NA and Vic11 NA differed by 70 amino acid substitutions. These 70 mutations were individually introduced into HK68 NA. Their replication fitness was examined by a virus rescue experiment. Virus titer was measured by 50% cell culture infectious dose (TCID₅₀). Each bar represents the mean of two independent biological replicates. Each datapoint represents one biological replicate. b The locations of the eight mutations that showed at least two-log decrease in virus titer compared to the wild type (WT) are shown as spheres on one protomer of NA that is in white cartoon representation, while the other three protomers are shown as semitransparent black surface (PDB 3TIA)²¹. Sialic acid in the active site is shown as yellow sticks. Locations of the two mutations that did not yield any detectable titer, namely N336H and N387K, are in orange. The other six mutations are in blue. c, d Fitness effects of c N387K and d N/Y336H on different strains were examined by a virus rescue experiment. While the WTs of HK68 and Bil69 had an Asn at residue 336, the WTs of Bil71, Alb76, Bk79, Bei89, and SD93 had a Tyr at residue 336. Virus titer was measured by TCID₅₀. Each bar represents the mean of three independent biological replicates. Each datapoint represents one biological replicate. The green dashed line represents the lower detection limit. Source data are provided as a Source data file.