Influenza A virus NS1 gene mutations F103L and M106I increase replication and virulence

Abstract

BackgroundTo understand the evolutionary steps required for a virus to become virulent in a new host, a human influenza A virus (IAV), A/Hong Kong/1/68(H3N2) (HK-wt), was adapted to increased virulence in the mouse. Among eleven mutations selected in the NS1 gene, two mutations F103L and M106I had been previously detected in the highly virulent human H5N1 isolate, A/HK/156/97, suggesting a role for these mutations in virulence in mice and humans.ResultsTo determine the selective advantage of these mutations, reverse genetics was used to rescue viruses containing each of the NS1 mouse adapted mutations into viruses possessing the HK-wt NS1 gene on the A/PR/8/34 genetic backbone. Both F103L and M106I NS1 mutations significantly enhanced growth in vitro (mouse and canine cells) and in vivo (BALB/c mouse lungs) as well as enhanced virulence in the mouse. Only the M106I NS1 mutation enhanced growth in human cells. Furthermore, these NS1 mutations enhanced early viral protein synthesis in MDCK cells and showed an increased ability to replicate in mouse interferon β (IFN-β) pre-treated mouse cells relative to rPR8-HK-NS-wt NS1. The double mutant, rPR8-HK-NS-F103L + M106I, demonstrated growth attenuation late in infection due to increased IFN-β induction in mouse cells. We then generated a rPR8 virus possessing the A/HK/156/97 NS gene that possesses 103L + 106I, and then rescued the L103F + I106M mutant. The 103L + 106I mutations increased virulence by >10 fold in BALB/c mice. We also inserted the avian A/Ck/Beijing/1/95 NS1 gene (the source lineage of the A/HK/156/97 NS1 gene) that possesses 103L + 106I, onto the A/WSN/33 backbone and then generated the L103F + I106M mutant. None of the H5N1 and H9N2 NS containing viruses resulted in increased IFN-β induction. The rWSN-A/Ck/Beijing/1/95-NS1 gene possessing 103L and 106I demonstrated 100 fold enhanced growth and >10 fold enhanced virulence that was associated with increased tropism for lung alveolar and bronchiolar tissues relative to the corresponding L103F and I106M mutant.ConclusionsThe F103L and M106I NS1 mutations were adaptive genetic determinants of growth and virulence in both human and avian NS1 genes in the mouse model.

Avian influenza virus H5N1 induces rapid interferon-beta production but shows more potent inhibition to retinoic acid-inducible gene I expression than H1N1 in vitro

“…Previous studies have demonstrated that highly pathogenic avian influenza virus H5N1 replicated more efficiently than human influenza viruses due to its PA, PB1, and PB2 [33-35]. Our results also proved that A/tree sparrow/Henan/1/04(H5N1) was consistent with other H5N1 strains and that it rapidly and efficiently amplified in mammalian cells, which resulted in the rapid activation of IFN-β signaling in early infection.…”

Genetic and antigenic evolution of H9N2 avian influenza viruses circulating in Egypt between 2011 and 2013

“…In addition, Egyptian strains exhibited no substitutions at position 92, which is related to virulence of H5N1 and cytokine resistance when changed to E [45]. F103L and M106I amino acid substitutions, which are known to be genetic determinants of pathogenicity and virulence in both human and avian hosts, were not observed in NS1 [12]. Phylogenetic analysis of NS genes showed that the Egyptian H9N2 isolates are highly homogenous and cluster together with Israeli isolates in group B, which are closely related to an H7N3 virus isolated from Pakistan in 2004 (Fig.…”

Identification of Host Genes Linked with the Survivability of Chickens Infected with Recombinant Viruses Possessing H5N1 Surface Antigens from a Highly Pathogenic Avian Influenza Virus

“…Two viral proteins, NS1 and NS2, are translated from the NS gene. Leucine at aa 103 and isoleucine at aa 106 of the NS1 amino acid sequence enhance the replication ability of viruses in vitro and in vivo and mortality in vivo (7). Their substitution expanded viral tropism in the lungs and increased the extent of infection.…”