Genomic and Protein Structural Maps of Adaptive Evolution of Human Influenza A Virus to Increased Virulence in the Mouse

Abstract

Adaptive evolution is characterized by positive and parallel, or repeated selection of mutations. Mouse adaptation of influenza A virus (IAV) produces virulent mutants that demonstrate positive and parallel evolution of mutations in the hemagglutinin (HA) receptor and non-structural protein 1 (NS1) interferon antagonist genes. We now present a genomic analysis of all 11 genes of 39 mouse adapted IAV variants from 10 replicate adaptation experiments. Mutations were mapped on the primary and structural maps of each protein and specific mutations were validated with respect to virulence, replication, and RNA polymerase activity. Mouse adapted (MA) variants obtained after 12 or 20–21 serial infections acquired on average 5.8 and 7.9 nonsynonymous mutations per genome of 11 genes, respectively. Among a total of 115 nonsynonymous mutations, 51 demonstrated properties of natural selection including 27 parallel mutations. The greatest degree of parallel evolution occurred in the HA receptor and ribonucleocapsid components, polymerase subunits (PB1, PB2, PA) and NP. Mutations occurred in host nuclear trafficking factor binding sites as well as sites of virus-virus protein subunit interaction for NP, NS1, HA and NA proteins. Adaptive regions included cap binding and endonuclease domains in the PB2 and PA polymerase subunits. Four mutations in NS1 resulted in loss of binding to the host cleavage and polyadenylation specificity factor (CPSF30) suggesting that a reduction in inhibition of host gene expression was being selected. The most prevalent mutations in PB2 and NP were shown to increase virulence but differed in their ability to enhance replication and demonstrated epistatic effects. Several positively selected RNA polymerase mutations demonstrated increased virulence associated with >300% enhanced polymerase activity. Adaptive mutations that control host range and virulence were identified by their repeated selection to comprise a defined model for studying IAV evolution to increased virulence in the mouse.

Identification of PB2 Mutations Responsible for the Efficient Replication of H5N1 Influenza Viruses in Human Lung Epithelial Cells

“…Human influenza A virus acquired the PB2 amino acid mutation E249G upon mouse adaptation (25), suggesting that the amino acid at position 249 probably is involved in some mechanism for adaptation to mammals. Sequence analysis showed that the prevalence of PB2 amino acid 249G in human seasonal H3N2 viruses was almost 50%.…”

Naturally Occurring Mutations in the PA Gene Are Key Contributors to Increased Virulence of Pandemic H1N1/09 Influenza Virus in Mice

“…The PB2-588 position is located within the 627 domain and has been identified as a host-range-specific amino acid position (20,21); it is characteristic of human influenza viruses, contributes to virus replication in mammalian cells, and partially compensates for the lack of 627K (20,21). The PA-P224S mutation is located in the nuclear localization signal domain and may influence the transport and accumulation of PA in the nuclei of virus-infected cells (22). The PA-A70V mutation was identified in a mouse-adapted H1N1/09 virus and on its own in CA04 background did not cause lethality or a more than 10% weight loss (23), but we showed here that PA-A70V combined with PA-P224S significantly boosted the lethality and lung pathology of PA-P224S alone.…”

Alternative Reassortment Events Leading to Transmissible H9N1 Influenza Viruses in the Ferret Model

“…induction, weight loss, pathology, and mortality in mice in an H3N2 background (29,30). We found D2N in 60% of sequencing reads in RCP5 and in 0% of reads in P1.…”