The influenza A (H1N1) pdm09 virus emerged in 2009 and has been continuously circulating in humans for over ten years. Here, we analyzed a clinical influenza A (H1N1) pdm09-infected patient case hospitalized for two months in Guangdong (from December 14, 2019 to February 15, 2020). This isolate, named A/Guangdong/LCF/2019 (LCF/19), was genetically sequenced, rescued by reverse genetics, and phylogenetically analyzed in the context of other relevant pdm09 isolates. Compared with earlier isolates, this pdm09 virus's genetic sequence contains four substitutions, S186P, T188I, D190A, and Q192E, of the hemagglutinin (HA) segment at position 186–192 (H3 numbering) in the epitope Sb, and two of which are located at the 190-helix. Phylogenetic analysis indicated that the epitope Sb started undergoing a rapid antigenic change in 2018. To characterize the pathogenicity of this novel substitution motif, a panel of reassortant viruses containing the LCF/2019 HA segment or the chimeric HA segment with the four substitutions were rescued. Kinetic growth data revealed that the reassortant viruses, including the LCF/2019 with the PTIAAQE substitution, propagated faster than those rescued ones having the STTADQQ motif in the epitope Sb in Madin-Darby Canine Kidney (MDCK) cells. The HI test showed that the binding activity of escape mutant to 2018 pdm09 sera was weaker than GLW/2018, suggesting that old vaccines might not effectively protect people from infection. Due to the difference in the selection of vaccine strains, people vaccinated in the southern hemisphere could still suffer a severe infection if infected with this antigenic drift pdm09 virus.

Influenza A virus (IAV) is a major human pathogen associated with significant morbidity and mortality worldwide. Through serial passage in mice, we generated a recombinant pdmH1N1 2009 IAV, A/Guangdong/GLW/2018 (GLW/18-MA), which encodes an mCherry gene fused to the C-terminal of a polymerase acidic (PA) segment and demonstrated comparable growth kinetics to the wild-type. Nine mutations were identified in the GLW/18-MA genome: PA (I61M, E351G, and G631S), NP (E292G), HA1 (T164I), HA2 (N117S and P160S), NA (W61R), and NEP (K44R). The recombinant IAV reporter expresses mCherry, a red fluorescent protein, at a high level and maintains its genetic integrity after five generations of serial passages in Madin-Darby Canine Kidney cells (MDCK) cells. Moreover, the imaging is noninvasive and permits the monitoring of infection in living mice. Treatment with oseltamivir or baicalin followed by infection with the reporter IAV led to a decrease in fluorescent protein signal in living mice. This result demonstrates that the IAV reporter virus is a powerful tool to study viral pathogenicity and transmission and to develop and evaluate novel anti-viral drugs, inhibitors, and vaccines in the future.