1 Adaptive mutations in influenza A / California / 07 / 2009 2 enhance polymerase activity and infectious virion 3 production 4

Mice are not natural hosts for influenza A viruses (IAVs), but they are useful models for 17 studying antiviral immune responses and pathogenesis. Serial passage of IAV in mice invariably 18 causes the emergence of adaptive mutations and increased virulence. Typically, mouse-adaptation 19 studies are conducted in inbred laboratory strains BALB/c and C57BL/6, which have defects in the 20 antiviral Mx1 gene that results in increased susceptibility to infection and disease severity. Here, we 21 report the adaptation of IAV reference strain A/California/07/2009(H1N1) (a.k.a. CA/07) in outbred 22 Swiss Webster mice. Serial passage led to increased virulence and lung titers, and dissemination of 23 the virus to brains. We adapted a deep-sequencing protocol to identify and enumerate adaptive 24 mutations across all genome segments. Among mutations that emerged during mouse-adaptation, 25 we focused on amino acid substitutions in polymerase subunits: polymerase basic-1 (PB1) T156A 26 and F740L, and polymerase acidic (PA) E349G. These mutations were evaluated singly and in 27 combination in minigenome replicon assays, which revealed that PA E349G increased polymerase 28 activity. By selectively engineering these three adaptive PB1 and PA mutations into the parental 29 CA/07 strain, we demonstrated that adaptive mutations in polymerase subunits decreased the 30 production of defective viral genome segments with internal deletions, and dramatically increased 31 the release of infectious virions from mouse cells. Together, these findings increase our 32 understanding of the contribution of polymerase subunits to successful host adaptation. 33