Abstract
Highly pathogenic H5Nx viruses of clade 2.3.4.4b have spread worldwide, causing major economic losses and increased human exposure. Since 2020, multiple mammalian infections have been reported, raising concerns about further adaptation to mammalian hosts. We analyzed influenza A virus sequences from the Influenza Virus Database at the National Center for Biotechnology Information to identify new mammalian adaptation markers in the polymerase complex and nucleoprotein, using recursive partitioning. These markers were grouped into "proteotypes" to assess their co-occurrence and association with host origin. This analysis revealed distinct groups of proteotypes linked to mammalian adaptation, including those seen in historical and pandemic human strains. Identified mutations were introduced alone or in combination into a 2.3.4.4b H5N8 virus to evaluate their impact on polymerase activity in mammalian cells using a minigenome assay. PB1 V336I and PB2 K702R increased polymerase activity in human cells, particularly with PB2 E627K, supporting enhanced surveillance of 2.3.4.4b H5Nx viruses. These findings highlight mutation combinations relevant for enhanced surveillance of 2.3.4.4b H5Nx viruses.