As shown in Fig.
4A, at 72 h postinoculation, the majority of genes regulated more than 1.5-fold during 1918 HA/NA:WSN virus infection (70%) were common to all infections, with 20% in common with WSN and 4% in common with New Caledonia HA/NA:WSN infections. A closer inspection of the genes preferentially regulated greater than 1.5-fold (
P < 0.05;
n = 4) in the 1918 HA/NA:WSN infections showed elevated expression of the protease cathepsin H (Ctsh); several transcription factors, such as Sp1 and Satb1; and the transforming growth factor β family member Gdf3 (data not shown; see full data at
http://expression.microslu.washington.edu ). However, this analysis indicated that the genes preferentially regulated in the 1918 HA/NA:WSN infections were only modestly changed, with respect to magnitude but not direction, compared to changes observed for the New Caledonia HA/NA:WSN and parental WSN recombinant viruses. These data indicate that few alterations in the expression of genes present on the microarrays employed in these studies were uniquely attributable to infections with the 1918 HA/NA:WSN virus (see Materials and Methods). However, when we examined the population of genes that showed preferential regulation of greater than 1.5-fold (
P < 0.05;
n = 4) in the 1918 HA/NA:WSN and WSN infections, clear differences could be observed in comparison with the New Caledonia HA/NA:WSN infections. By using set analysis, 384 genes that were regulated more than 1.5-fold only in the lungs of mice infected with 1918 HA/NA and WSN viruses were identified, with approximately 190 genes that had available functional annotation. A closer inspection of these data showed 19 annotated genes whose expression was anticorrelated between the two high-pathogenicity infections and New Caledonia HA/NA:WSN (Fig.
4B). The genes that showed increased expression in the 1918 HA/NA:WSN and WSN infections but were down-regulated in the New Caledonia infection included several cytokine-regulated genes, including those for TNF-α-induced protein 2 (Tnfaip2), the cold shock RNA binding protein Rbm3, and the IL-10-induced metalloprotease ADAMTS4 (EST_H3022E09). Also included in this group of genes that were up-regulated during highly pathogenic influenza infections were the oxidative stress response glutathione peroxidase 3 (Gpx3) gene and the DNA damage-induced growth arrest Gadd45g gene. These data suggested that infections with the 1918 HA/NA:WSN and WSN viruses resulted in a more significant inflammatory response and reactive oxygen species-induced stress, as was similarly observed at 24 h p.i. When we examined the genes that showed preferential regulation in the New Caledonia HA/NA:WSN infections, we identified 200 genes, of which 97 had at least inferred functional annotation. This analysis further showed that the expression of 11 genes was anticorrelated with either the 1918 HA/NA:WSN or parental WSN infections. However, as shown in Fig.
4C only a macropain protease subunit (Psma6) showed anticorrelation with both of the highly virulent recombinant influenza viruses. Given the minimal correlation of these genes with the previously reported virulence of these viruses, a direct relationship between virus-induced regulation of these genes could not be inferred (
69). We next examined the genes whose expression was regulated more than 1.5-fold in all of the recombinant influenza virus infections. To identify the dominant viral response genes, we focused on the expression of genes with significant regulation (>4-fold with
P < 0.05 [
n = 4]) at 72 h that were common to all infection groups (Fig.
4D). Many of the genes commonly regulated by all three infection groups were those involved in immune responses. These include the genes for the IL-2 receptor (EST_H3024G06), which is involved in T-cell activation and proliferation; cytochrome
b245 (EST_H3060F11), a critical component of membrane-bound oxidase activity of phagocytes (macrophages and neutrophils); the transcription factor Stat1, which is a critical component of the IFN-α/β and IFN-γ response; the Gro1 oncogene (EST_H3051F10), which is the mouse orthologue of human CXCL1, a potent neutrophil chemokine; the C-type lectin Clecsf9, a downstream target of NF-IL-6 that plays roles in macrophage function; and the IFN-regulated transcription factor Irf1, which activates expression of IFN-α/β and IFN-inducible MHC class I genes. Other commonly up-regulated genes include those for cyclin D2 (Ccnd2), which is essential for control of the cell cycle at the G
1-to-S transition and is an indicator of cell proliferation. Taken together, the pattern of these gene expression data showed that clear differences were observed between the highly virulent 1918 HA/NA:WSN and parental WSN recombinant viruses and the attenuated New Caledonia HA/NA:WSN recombinant virus, which mirrored the histopathology data, and that many critical events determining the severity of viral pathogenicity occurred relatively early in infection (by 24 h p.i. in our studies).