Naturally occurring mutations in the PA gene are key contributors to increased virulence of pandemic H1N1/09 influenza virus in mice

Abstract

We examined the molecular basis of virulence of pandemic H1N1/09 influenza viruses by reverse genetics based on two H1N1/09 virus isolates (A/California/04/2009 [CA04] and A/swine/Shandong/731/2009 [SD731]) with contrasting pathogenicities in mice. We found that four amino acid mutations (P224S in the PA protein [PA-P224S], PB2-T588I, NA-V106I, and NS1-I123V) contributed to the lethal phenotype of SD731. In particular, the PA-P224S mutation when combined with PA-A70V in CA04 drastically reduced the virus's 50% mouse lethal dose (LD50), by almost 1,000-fold.

FIG 1

Relative pathogenicities of pandemic H1N1/09 influenza viruses with different amino acid mutations in mice. (A) Amino acid differences between the avirulent CA04 and pathogenic SD731 viruses. (B) Survival percentages of mice infected with 10⁵ PFU of each virus. (C) Photomicrographs of hematoxylin and eosin (H&E)-stained lungs from mice infected with 10⁴ PFU of virus at 6 dpi. Solid arrows, erosion of epithelial lining in the bronchioles; open arrow, extensive epithelial damage and inflammatory cell infiltration in the bronchioles; triangle, alveolar interstitial consolidation and extensive inflammatory cell infiltration. Magnification, ×100.

FIG 2

Polymerase activities of pandemic H1N1/09 influenza mutant viruses as determined by minigenome replication assays. Values shown are the means ± SD of data from three independent experiments and are normalized to those of SD731 (100%). #, the value for CA04 was significantly lower than those for the other mutants (P < 0.05 or P < 0.01; analysis of variance [ANOVA]); **, the values of corresponding RNPs were significantly different (P < 0.01, ANOVA).