Genetic Characteristics and Pathogenicity of Recent G4 Eurasian Avian-like H1N1 Swine Influenza Viruses in China

G4 Eurasian avian-like H1N1 (G4 EA-H1N1) swine influenza viruses continue to be detected in pigs in China and remain a concern because of their reassortant genomes and zoonotic potential. Here, six H1N1 viruses isolated from pigs with respiratory disease in six Chinese provinces during 2023–2025 were characterized. Phylogenetic analysis showed that all six isolates belonged to the G4 genotype and retained the characteristic genomic constellation, with EA-lineage HA and NA genes, pdm09-derived PB2, PB1, PA, NP, and M segments, and a triple-reassortant NS segment. Sequence analysis identified amino acid substitutions in HA antigenic and receptor-binding-associated regions and differences in predicted HA and NA glycosylation patterns. Molecular docking indicated that the HAs could accommodate both α2,3- and α2,6-linked sialic acid receptor analogs. Hemagglutination inhibition testing of 96 field swine sera showed that 62 samples (64.6%) had titers ≥ 1:40 against the representative isolate JS0602, with seroreactivity to JS0602 detected among sera from all sampled regions. JS0602 replicated efficiently in MDCK cells and caused respiratory signs, pulmonary lesions, viral antigen deposition, and viral shedding in experimentally infected pigs. These findings provide updated genetic and biological information on contemporary G4 EA-H1N1 viruses and support continued surveillance in swine populations.