Identification of mammalian-adapting mutations in the viral polymerase complex of avian influenza A viruses

Abstract

Infections of avian influenza A virus (AIAV) in humans have become a worldwide public health issue. Introduction of mammalian-adapting mutations in the viral polymerase complex is one of the main factors resulting in the direct transmission from avian species to humans. Although several mammalian-adapting mutations have been recently identified, it is still possible that there are hitherto unrecognized mutations in the viral polymerase complex that can also enhance the adaptability of AIAVs in mammalian hosts. In this study, novel mammalian-adapting mutants in the viral polymerase complex were identified by a high-throughput genetic approach and their pathogenic potential was evaluated.

I have established a reliable screening system by generating random mutations within the three subunits of the polymerase complex of H9N2, an avian influenza A virus that is a precursor of H5N1 viruses responsible for the 1997 outbreak in humans. More than twenty novel potential candidates were identified using the relative fitness index (RFI), which was calculated from the data obtained with next generation sequencing. Their viral polymerase activities and replication efficiencies were tested, with wild type H9N2 and the PB2-E627K mutant as controls. By mapping the novel candidates on the well-resolved structure of a viral polymerase complex of an AIAV, the “627 domain” of PB2 subunit and the N-terminal domain of PA subunit were determined as the essential regions in which mutations favor mammalian adaptation of AIAVs. Taken together, my study helps to gain further insight into the viral genetic determinants that contribute to pathogenicity of AIAVs in humans.