Novel mutations in avian PA in combination with an adaptive mutation in PR8 NP exacerbate the virulence of PR8-derived recombinant influenza A viruses in mice

The polymerase complex of the low-pathogenic avian influenza virus [A/chicken/Korea/KBNP-0028/2000] (0028) has previously been characterized, and novel amino acid residues present in the polymerase acidic protein (PA) that likely contribute to pathogenicity toward mammals have been identified. In the present study, our aims were to generate A/Puerto Rico/8/34 (PR8)-derived recombinant viruses containing the 0028-PA gene with a single amino acid mutation and to test their pathogenicity and replication ability. We found that the recombinant viruses acquired additional single mutations in the nucleoprotein (NP). Because the additional mutations in NP did not affect viral pathogenicity, but rather attenuated viral replication and polymerase activity, the incompatibility of the avian PA gene within the PR8 backbone may have induced an adaptive mutation in NP. To minimize the differences due to NP mutations, we generated 0028-PA mutants with an E375G mutation, not affecting viral replication and pathogenicity, in the NP gene. The PR8-PA(0028)-E684G mutant showed significantly higher viral replication in mammalian cells as compared to PR8-PA(0028) and led to 100% mortality in mice, with significantly increased interferon β expression. Thus, the E684G mutation in the PA gene may play an important role in viral pathogenicity in mice by increasing viral replication and the host immune response.