Fig. 2: HA-K130N and HA-K130E mutations confer oseltamivir resistance.

a, b Plaque-reduction assay of the A/California/07/2009 wild-type (Cal09-WT) virus and HA-K130N and HA-K130E mutants. This assay was conducted in the absence (0 μM) or presence of 0.01, 0.1, 1, 10, or 100 μM oseltamivir. Recombinant viruses generated from two independent rescue experiments were subjected to plaque-reduction experiments. Each dot represents the diameter (mm) of a single plaque (n = 20). c Time course of virus replication of the Cal09-WT virus, HA-K130N, and HA-K130E mutant viruses in MDCK cells infected at an MOI of 0.001 in the absence of oseltamivir. At the indicated time points, the virus titers in the culture supernatant were determined by a plaque assay. Data are presented as mean values ± SEM from n = 2 independent biological replicate experiments. d Virus replication of the Cal09-WT virus, HA-K130N, and HA-K130E mutant viruses in MDCK cells infected at an MOI of 0.001 in the presence of oseltamivir. The virus titers in the culture supernatant at 72 h postinfection were determined by a plaque assay. Data are presented as mean values ± SEM from n = 3 independent biological replicate experiments. e–g Competitive co-infection experiments between Cal09-WT (HA-130K) and HA-K130N mutant viruses (e), Cal09-WT (HA-130K) and HA-K130E mutant viruses (f), and HA-K130N and HA-K130E mutant viruses (g). Viruses were infected in MDCK cells with an MOI of 0.01 and serially passaged in the absence and presence of 0.1 and 160 μM oseltamivir. Virion RNA was isolated, amplified by RT-PCR, and sequenced. The sequence traces of residue 130 of HA from P1 and P2 viruses are shown. The illustrations of viruses were adapted from a royalty-free vector graphic sourced from pixabay.com. Source data are provided as a Source Data file.