A PB1 T296R substitution enhance polymerase activity and confer a virulent phenotype to a 2009 pandemic H1N1 influenza virus in mice

- PB1-T296R significantly enhance the virulence of 2009 pH1N1 in mammals.
- PB1-T296R enhance 2009 pH1N1 replication in vivo and in vitro.
- PB1-T296R enhance 2009 pH1N1 polymerase activity.

While the 2009 pandemic H1N1 virus has become established in the human population as a seasonal influenza virus, continued adaptation may alter viral virulence. Here, we passaged a 2009 pandemic H1N1 virus (A/Changchun/01/2009) in mice. Serial passage in mice generated viral variants with increased virulence. Adapted variants displayed enhanced replication kinetics in vitro and vivo. Analysis of the variants genomes revealed 6 amino acid changes in the PB1 (T296R), PA (I94V), HA (H3 numbering; N159D, D225G, and R226Q), and NP (D375N). Using reverse genetics, we found that a PB1-T296R substitution found in all adapted viral variants enhanced viral replication kinetics in vitro and vivo, increased viral polymerase activity in human cells, and was sufficient for enhanced virulence of the 2009 pandemic H1N1 virus in mice. Therefore, we defined a novel influenza pathogenic determinant, providing further insights into the pathogenesis of influenza viruses in mammals.