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.
Highlights
The mutational basis for the control of host switching and virulence in influenza A viruses (IAV) or their interrelationship is poorly understood [1,2,3,4] and the identification of genetic markers of host adaptation is the subject of much debate [5]
The existing knowledge of the evolution of virulence and host switching in IAV is incomplete as recently demonstrated by the introduction of a novel HK/1/68-MA (H3N2) and A/FM/1/ 47-MA (H1N1) IAV from swine into humans without the genetic markers associated with virulence and interspecies transmission [6,7,8]
Novel mutations were selected at passage 20, including mutations in PB2, NP, M1, M2, non-structural protein 1 (NS1), and nuclear export protein (NEP) (Table 2)
Summary
The mutational basis for the control of host switching (hostspecific infection) and virulence (disease severity) in influenza A viruses (IAV) or their interrelationship is poorly understood [1,2,3,4] and the identification of genetic markers of host adaptation is the subject of much debate [5]. Parallel evolution is characteristic of drug resistance and is increasingly being observed among organisms that have evolved common traits The links between IAV evolution, adaptation and virulence have yet to be elucidated
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