Eurasian avian-like swine influenza A viruses escape human MxA restriction by distinct mutations in their nucleoprotein.

To cross the human species barrier, influenza A viruses (IAV) of avian origin have to overcome the interferon-induced host restriction factor MxA by acquiring distinct mutations in their nucleoprotein (NP). We recently demonstrated that North American classical swine IAV are able to escape MxA restriction partially. Here, we investigated whether the Eurasian avian-like swine IAV lineage currently circulating in European swine would likewise evade restriction by human MxA. We found that the NP of the isolate A/swine/Belzig/2/2001 (Belzig) exerts increased MxA escape similar in extent to human IAV NPs. Mutational analysis revealed that the MxA escape mutations in Belzig-NP differ from the known MxA resistance cluster of the North American classical swine lineage and human-derived IAV NPs. A mouse-adapted avian IAV of the H7N7 subtype encoding Belzig-NP showed significantly enhanced viral growth in both MxA-expressing cells and MxA-transgenic mice compared to control viruses lacking the MxA escape mutations. Similarly, growth of recombinant Belzig virus was only marginally affected in MxA-expressing cells and MxA-transgenic mice compared to Belzig mutant viruses lacking MxA escape mutations in NP. Phylogenetic analysis of the Eurasian avian-like swine IAV revealed that the NP amino acids required for MxA escape were acquired successively and were maintained after their introduction. Our results suggest that circulation of IAV in the swine population can result in the selection of NP variants with a high degree of MxA resistance, thereby increasing the zoonotic potential of these viruses. SIGNIFICANCE The human MxA protein efficiently blocks replication of IAV from non-human species. In rare cases, however, these IAV overcome the species barrier and become pandemic. All known pandemic viruses have acquired and maintained MxA escape mutations in the viral NP and are thus not efficiently controlled by MxA. Intriguingly, partial MxA resistance can also be acquired in other hosts that express antivirally active Mx proteins such as swine. To perform a risk assessment of IAV circulating in the European swine population, we analyzed the degree of MxA resistance of Eurasian avian-like swine IAV. Our data demonstrate that these viruses carry yet undescribed Mx resistance mutations in NP that mediate efficient escape from human MxA. We conclude that Eurasian avian-like swine IAV possess a substantial zoonotic potential.