Rare recovery of reassortants between contemporary clade 2.3.4.4b H5N1 viruses and human influenza A viruses in vitro

Influenza A virus (IAV) pandemics can arise when animal-derived and human-adapted IAVs exchange genome segments; however, whether contemporary clade 2.3.4.4b H5N1 viruses can generate viable reassortants with human IAVs is unclear. Here, we tested the reassortment potential of two clade 2.3.4.4b H5N1 viruses with human-adapted H1N1 and H3N2 IAVs using a biosafe reassortment system. Across multiple coinfection conditions, we genotyped 2,698 plaque-purified viral clones and recovered 20 reassortants (0.74%). Polymerase assays identified human IAV subunits, particularly H1N1 PA or H3N2 PB2, that increased H5N1 polymerase activity. Thus, contemporary clade 2.3.4.4b H5N1 viruses can reassort with human IAVs, but factors beyond polymerase compatibility may limit their recovery.