FIG 4.

Molecular target of IY7640. (A) Escape mutations (L49I, M403T, and E447K) identified in the HA protein of rK09 after IY7640 treatment are adjacently located with the CR6261 epitopes in the HA structure. The colors represent the following: slate, HA1; light pink, HA2; red, escape mutations against IY7640; and orange, CR6261 epitopes. (B) Replication kinetics of escape mutant viruses were evaluated in MDCK cells. (C and D) Viral infection and replication efficiency were measured by the number of the newly generated virus particles via plaque assays (C), and the amounts of newly expressed HA were compared by Western blotting (D). (E) A hemagglutination assay using the K09 virus and 0.5% (vol/vol) tRBCs was performed in the presence of PBS (negative control), 2-fold serially diluted anti-K09 guinea pig sera (α-K09 #1 and #2; positive controls), or IY7640 (starting at 90 μM).