Mouse adaptation results in increased virulence and lung pathology.
BALB/c mice were infected with 106
PFU of either CA/04 or MA-CA/04 virus; weight loss, survival, and viral titers were determined at different days p.i. While mice infected with CA/04 lost minimal weight and recovered by day 8 p.i., infection with MA-CA/04 resulted in continuous weight loss and 100% lethality by day 7 p.i. ( and ). This increase in virulence was accompanied by increased replication of MA-CA/04 virus in lungs, as quantified by plaque assay (). Since extrapulmonary replication was previously reported for MA-CA/04 (16
) but not for other 2009 H1N1 mouse-adapted viruses, we verified that under our experimental conditions, MA-CA/04 disseminated to the brain and spleen on days 3 and 5 p.i. but CA/04 did not.
Fig 1 Characterization of mortality, weight loss, and viral titers in MA-CA/04- and CA/04-infected mice. Mice were inoculated with 106 PFU of each virus or PBS as a mock control. (A) Mortality data from mice inoculated with influenza virus or mock inoculated (more ...)
Microscopic lesions were examined by hematoxylin-and-eosin staining of lung tissue sections from infected mice, and viral antigen was detected by immunohistochemistry. Infection with either CA/04 or MA-CA/04 virus produced lesions typical of influenza A infections: bronchiolitis and bronchitis with accompanying necrosis of respiratory epithelium and associated histiocytic alveolitis (; see also Table S1 in the supplemental material). In general, the lesions produced by MA-CA/04 were more severe than the lesions observed with CA/04. At 1 day p.i., both viruses produced mild to moderate bronchiolitis and bronchitis with degenerative changes, including vacuolation, of the respiratory epithelium. Viral staining was commonly present in bronchioles and bronchial respiratory epithelium (). The MA-CA/04 virus also produced mild histiocytic alveolitis at this time point, and infrequent viral staining was present in alveolar epithelium and macrophages. At 3 days p.i., mild to moderate bronchiolitis and bronchitis with necrosis and loss of the respiratory epithelium, accompanied by mild to moderate peribronchiolar histiocytic to lymphocytic alveolitis, was observed with both viruses ( and ). Alveolar necrosis with congestion and edema was more evident in mice infected with the MA-CA/04 virus. Infrequent viral staining in bronchioles and bronchial respiratory epithelium was seen with both viruses, but viral staining in alveolar epithelium and macrophages was more common in tissues from mice infected with MA-CA/04 ( and , inserts). At 5 days p.i., mice infected with CA/04 had mild lymphocytic to histiocytic peribronchiolar to diffuse alveolitis, as well as bronchiolitis and bronchitis with necrosis and loss of the respiratory epithelium. In some cases neutrophils were also present (). Rare to common viral staining was present in alveolar epithelium, in macrophages, and in bronchioles and bronchial respiratory epithelium. At this same time point, mice infected with MA-CA/04 had moderate to severe peribronchiolar to diffuse alveolitis, with histiocytes, lymphocytes and uncommon neutrophils present (). Moderate to severe bronchiolitis and bronchitis with necrosis and loss of the respiratory epithelium were also observed. Viral staining was infrequent in bronchioles and bronchial respiratory epithelium but common in alveolar epithelium and macrophages.
Fig 2 Histopathologic changes and immunostaining of tissues from H1N1 virus-inoculated mice. Photomicrographs of lung tissue sections stained with hematoxylin and eosin and for the presence of viral antigen (×400). (A) Lung tissue from mock-infected (more ...)
In summary, increased virulence of MA-CA/04 was associated with more severe lung lesions, more evident infection of alveolar epithelium and macrophages, and indications of a greater number of histiocytes, lymphocytes, and neutrophils infiltrating the lung than with wild-type CA/04 virus.
MA-CA/04 induces a strong host transcriptional response after infection.
To characterize the impact of mouse adaptation on host responses, pulmonary gene expression was compared between time matched mock-infected mice and mice infected with either CA/04 or MA-CA/04 virus at 1, 3, and 5 days p.i. We observed that MA-CA/04 infection resulted in more differentially expressed (DE) genes at each time point than did infection with CA/04 (). The number of DE genes was greatest on day 3 p.i. for both viruses; however, importantly, transcriptional responses to CA/04 infection decreased dramatically on day 5, whereas for MA-CA/04, the number of DE genes decreased only marginally. Functional analysis of upregulated genes showed that MA-CA/04 infection resulted in a sustained increase in the expression of genes associated with the inflammatory response, immune cell trafficking, cell death, and cellular growth and proliferation (see Fig. S1 in the supplemental material). After CA/04 infection, only 195 transcripts were significantly upregulated on day 1 p.i., and they were not strongly associated with any biological function (see Fig. S1). In contrast, on day 3 p.i., 1,374 genes were upregulated after CA/04 infection, and their functional enrichment profile was very similar to that of genes induced by MA-CA/04 at the same time. At day 5 p.i., lipid and amino acid metabolism processes were upregulated in CA/04-infected mice but not in mice infected with MA-CA/04. Genes downregulated following MA-CA/04 and CA/04 virus infection were enriched to a limited extent in different general biological functions, such as tissue development, genetic disorder, cellular function, and maintenance (see Fig. S1). On day 3 p.i., 60% of the functions associated with downregulated genes after CA/04 or MA-CA/04 infection were in common (see Fig. S1).
Fig 3 Number of genes differentially expressed (DE) after infection for each virus compared to results for time-matched mocks (A) or overlap between genes differentially regulated between MA-CA/04 and CA/04 at each time point (B). (A) Numbers of upregulated (more ...)
The enhanced virulence of MA-CA/04 is therefore characterized by an increased host response, with upregulated genes strongly associated with immune/inflammatory processes across all time points. In contrast, whereas CA/04 infection also induced a strong immune response, the response was limited at day 3, with metabolism functions being induced later in infection.
MA-CA/04 and CA/04 elicit different transcriptional programs.
To directly examine differences in the host response to CA/04 and MA-CA/04, we determined the genes that were DE between CA/04 and MA-CA/04 at each day p.i. (Welch t test, absolute log2 ratio > 1 and q value < 0.05). A total of 4,202 unique DE transcripts were identified, with an overlap between time points of 36%. At day 3, only 25% of the transcripts discriminating CA/04 and MA-CA/04 were not DE at the other time points, whereas at days 1 and 5 p.i., there were 41% and 53% unique genes, respectively ().
Genes that were DE between MA-CA/04 and CA/04 were categorized into three sets based on their general expression patterns (; see also Data Set S1 in the supplemental material). Genes in set 1 (upregulated lethal signature; 2,107 genes) were more highly expressed during MA-CA/04 infection at at least one time point, and many of these genes remained highly expressed throughout infection. These genes were only transiently upregulated, and to a lesser extent, on day 3 p.i. in CA/04-infected animals. Genes in set 2 (survival/recovery signature; 968 genes) were upregulated in CA/04-infected animals at at least one time point while unchanged or downregulated during MA-CA/04 infection. The largest differential within this group was found on day 5 p.i. Finally, genes in set 3 (downregulated lethal signature; 1,126 genes) were more downregulated in MA-CA/04-infected mice at at least one time point and showed a trend opposite from that of genes in set 1. Many of the genes in set 3 were highly downregulated early after MA-CA/04 infection and remained so throughout infection, while they were only transiently downregulated, and to a lesser extent, on day 3 p.i. in CA/04-infected animals.
Fig 4 Genes DE between MA-CA/04 and CA/04 infection formed 3 main transcriptional programs. The heat map represents expression levels (in log2 ratios) of 4,202 transcripts that were DE between MA-CA/04 and CA/04 infection at at least one time point. Genes were (more ...) Genes highly induced after MA-CA/04 infection are associated with the immune response and enriched with interferon regulatory factor (IRF) binding motifs.
Functional analysis was performed using IPA to determine the canonical pathways and biological functions () that were most significantly associated with differences in gene expression between the two viruses (). Set 1 was strongly enriched in pathways related to innate immunity and relationships between innate and adaptive immunity (). Approximately 14% of the transcripts (291 probes) in set 1 encoded factors involved in immune cell trafficking (; see also Data Set S1 in the supplemental material), including a large number of CCL and CXCL cytokines and their receptors. We also noted an increase in the expression of genes coding for inflammatory molecules, such as the classical proinflammatory cytokines IL-1b, IL-6, and TNF-α, and molecules involved in tissue destruction and cell death, including matrix metallopeptidases, those encoded by FAS, TNF, and the TNF receptor, TNFRSF1B, and several receptors of Fc fragments. We also compared the list of genes in set 1 with the list of genes that were DE following intranasal treatment of mice with alpha/beta interferon (IFN-α/β) (see Data Set S2) (9
). Among the 2,107 transcripts in set 1, 39% (825 probes) were thus defined as IFN-regulated genes (Fisher's exact P
value < 2.2e−16), suggesting that a large part of the set 1 transcriptional program was driven by an upregulation of the innate immune response.
IPA canonical pathways and biological functions enriched in each transcriptional program discriminating MA-CA/04 and CA/04a
To identify potential transcriptional regulators of the genes within set 1, we used PSCAN to analyze the promoter regions of these genes for binding motifs of 130 transcription factors (TFs) (; see also Data Set S3 in the supplemental material). We found a strong enrichment for IRF1 and -2 binding sites, as well as NF-κB and STAT3. IRF3 and NF-κB have been previously described as being highly activated during pathogenic influenza virus infection of cell lines (36
), and STAT TFs are extensively involved in the antiviral response in vivo
). It may be interesting to note that among ISGF3 components (STAT1-STAT2-IRF9), only STAT1 is present in PSCAN. STAT1 binding motif was enriched for set 1 (P
value = 3.6e−4) (see Data Set S3) but not for the other sets. The sequence of the ISGF3 binding site partially overlaps the IRF binding element, and we found significant enrichment for IRF binding element in set 1. This may suggest that ISGF3 also plays a role in regulating the transcriptional program associated with lethality. Besides, since IRF1 and -2 were the only IRFs in the JASPAR database (see Data Set S3), and since IRFs bind similar DNA motifs (though with some specificities [38
]), it is likely that other IRFs could also control the expression of genes that were upregulated after MA-CA/04 infection. Other novel TFs potentially regulating the set 1 transcriptional response based on binding motif enrichment (; see also Data Set S3) were myeloid zinc finger protein 1 (MZF-1) and SPI1 (also known as PU-1), TFs that are both involved in myeloid cell development.
TF binding motifs enriched in each transcriptional program discriminating MA-CA/04 and CA/04a
Together, these results suggest that MA-CA/04 may trigger a greater activation of pattern recognition receptors (PRRs) resulting in IRF and NF-κB activation, leading subsequently to a high IFN and proinflammatory response, mediated by STAT TFs. This cytokine environment could affect the functions of infiltrating innate (dendritic cells and macrophages) and adaptive immune cells and also be responsible for lung lesions. In contrast, in CA/04 virus-infected animals, this activation was more limited and restrained to day 3 p.i.
Survival is associated with increased expression of lipid metabolism genes and genes enriched with nuclear receptor binding sites.
Survival of infection with CA/04 was associated with the induction of 968 genes that were significantly more upregulated in CA/04-infected mice than in MA-CA/04 infected mice (). Functional analysis of these genes found that 268 transcripts (28%) encoded molecules involved in lipid metabolism (see Data Set S1 in the supplemental material), including several ATP-binding cassette (ABC) transporters, apolipoprotein, diverse enzymes involved in fatty acid metabolism, vitamin D, arachidonic acid, lipid synthesis and uptake, several solute carrier family members (SLC), cytochrome P450 members, and surfactant proteins (SP-D and A1). Induction of these genes in CA/04-infected mice could be involved in viral clearance, as with SP-D (25
), and in limiting the inflammatory response, as with CYP2C and -2J synthesizing anti-inflammatory metabolites (44
), and enzymes involved in arachidonic acid metabolism. In addition, functional analysis showed a significant enrichment in genes related to amino acid metabolism. The upregulation of amino acid and lipid metabolism genes may be required to build cellular components essential for recovery from lung tissue damage.
To find potential regulators of the survival/recovery signature, we looked for TF binding motifs in promoters of genes from set 2. The top-ranked TFs were nuclear receptors acting in a heterodimer with RXR (NR1H2::RXRA and PPARG::RXRA) or as a homodimer (NR2F1 and several hepatocyte nuclear factors). These results were consistent with canonical pathway analysis, because three out of the five most enriched pathways involved RXR and one pathway (FXR/RXR activation) involved hepatocyte nuclear factor TFs (). Overall, these data indicate that nuclear receptors may be involved in lipid and amino acid metabolism induction potentially required for recovery from influenza infection.
Lethal infection is associated with suppression of coagulation genes.
The last set of genes DE between MA-CA/04 and CA/04 included genes that were more downregulated in MA-CA/04 mice at at least one time point (). Most of these genes were consistently more downregulated throughout infection with MA-CA/04 while being unchanged in CA/04-infected animals, but a small fraction were upregulated in these animals on days 1 and 5 p.i. and therefore also belonged to set 2. Overall enrichment scores were less significant for this set than for the other two sets (). However, we found a significant representation of lipid metabolism and coagulation-related genes in this set (; see also Data Set S1 in the supplemental material). Lipid enrichment was only partially explained by overlap with set 2, since several genes belonging only to set 3 were also associated with lipid metabolism. Procoagulant (F2, fibrinogen) and anticoagulant (Serpin C1 = Antithrombin III) factors, as well as fibrinolytic (Plasminogen) and antifibrinolytic (Serpin F2) factors, were more downregulated in MA-CA/04 mice on day 3 p.i. On day 5 p.i., several anticoagulants (encoded by Protein C, Serpin D1, C1, and A1) and profibrinolytic factors (encoded by Plasminogen, F12 and Kallikrein B) were more upregulated in CA/04-infected than in MA-CA/04-infected mice (see Data Set S1) and could limit fibrin deposition involved in acute lung injury.
Transcription factor analysis using PSCAN found significant enrichment for PPARG::RXRA, which could therefore also be implicated in downregulation of set 3 genes, as well as several transcriptional repressors (ZEB1, INSM1, SP1, and PLAG1). In conclusion, MA-CA/04 infection induced a consistent downregulation of lipid metabolism and coagulation-associated genes, which could be due to regulation of PPARG and/or transcriptional repressors.
Prediction of TF activation state supports IRF and nuclear receptor regulation of transcription.
Because we found several TF binding motifs that were highly enriched in each set of genes discriminating CA/04 and MA-CA/04, we were interested in determining their activation state following infection. For this purpose, we used the IPA TF analytic tool, which can predict the activation of TFs based on the expression levels of their known targets. We performed this analysis using log2 ratio expression values for the 4,203 genes discriminating CA/04- and MA-CA/04-infected animals. represents z scores for 16 TFs that were found to be very likely (|z| > 4) to be involved in the transcriptional response to infection at at least one time point and in one group. Based on the annotation of their target genes, we found that these TFs were involved either in lipid metabolism control or in the inflammatory response.
Fig 5 TF activation state prediction in MA-CA/04- and CA/04-infected mice. (A) z scores predicting activation state of TFs (|z| > 4) based on expression values of genes discriminating CA/04 and MA-CA/04. Dashed lines depict |z| = 2, which is considered (more ...)
Binding motifs for several TFs likely to be involved in inflammatory response control were also found in our previous PSCAN analysis to be present in promoters of genes from the upregulated lethal signature (; see also Data Set S3 in the supplemental material). These TFs were predicted to be activated throughout infection with MA-CA/04 virus, while in CA/04-infected mice they would be activated only by day 3 p.i., with a possible decrease in their activation on day 5 p.i. (). Consistently, most of their target genes belonged to the upregulated lethal signature (set 1) and were more expressed in MA-CA/04-infected mice than in CA/04-infected mice (). Two additional IRFs (IRF7 and -8) were also predicted to be more activated in MA-CA/04-infected mice, which supports the general implication of IRFs in controlling the transcriptional program associated with lethality. Additionally, two negative regulators, GFI1 and TRIM24, were found to be constantly inactivated in MA-CA/04 samples, and we verified that their targets were also more upregulated in MA-CA/04-infected mice ().
Among the four TFs that could control lipid metabolism during infection, three were previously found in PSCAN (HNF1A, HNF4A, and PPARG). The hepatocyte nuclear factors (HNFs) were predicted to be activated in CA/04-infected lungs on days 1 and 5 p.i., while they would be repressed or unchanged at the same time points in MA-CA/04-infected animals (). Consistently, most of their target genes were more expressed in CA/04 animals than in MA-CA/04 animals on days 1 and 5 p.i. (). PPARG was another nuclear receptor found by the two complementary TF analyses. It was predicted to be inhibited throughout infection with MA-CA/04 virus, while not with infection with CA/04, on days 1 and 5 p.i. (). Most of its targets were more downregulated in MA-CA/04 samples and were associated with lipid metabolism. Notably, the small subset of PPARG targets that were found to be more upregulated in MA-CA/04-infected mice are genes that are known to be repressed by PPARG, including those encoding several proinflammatory cytokines (IL-1, IL-6, IFNG, and TNF) and several chemokines (CXCL-1, CXCL-3, CXCL-6, and CXCL-14 and CCL-13, CCL-17, and CCL-22). This was consistent with an inhibition of PPARG activity in MA-CA/04-infected animals and indicates that this regulation could be involved in both the absence of lipid metabolism upregulation and increased inflammatory response observed in MA-CA/04-infected animals.
These results overall support the implication of several nuclear receptors (HNF1A, HNF4A, and PPARG), IRFs, and the NF-κB complex in the differential host response to MA-CA/04 or CA/04 infection. We also found novel transcription regulators not present in the JASPAR database, notably TRIM24, whose repression could play a role in increasing the inflammation in MA-CA/04 virus-infected animals.
Genes associated with inflammatory macrophages and granulocytes are highly induced during MA-CA/04 infection.
Since several pathways differentially induced between MA-CA/04 and CA/04 were related to specific immune cells or included chemoattractants, we hypothesized that observed transcriptional differences elicited by MA-CA/04 and CA/04 infection could be partially due to differences in immune cell populations and/or their activation state in the lungs. To further explore this possibility, we compared our gene expression profiles to microarray data for each immune cell population present in the GNF Mouse Gene Atlas v3 database (42
). We defined, for each immune cell population present in the database, a list of genes that were expressed 20-fold more in a given cell subset than in whole lung. We then analyzed whether there was significant enrichment in genes that were more upregulated in response to infection with either MA-CA/04 (, orange lines) or CA/04 (, blue lines). We could therefore evaluate whether differences in gene expression profiles between MA-CA/04 and CA/04 infection were associated with immune cell infiltration.
Fig 6 Enrichment in immune cell highly expressed genes for genes that were differentially changed between MA-CA/04 and CA/04 infection. Genes specific to each immune cell population were determined using the Gene Atlas v3 data set to be genes that were 20-fold (more ...)
The list of genes that exhibited no change in expression after CA/04 infection but which were strongly induced by MA-CA/04 at days 1 and 5 p.i. was highly enriched in genes associated with lipopolysaccharide (LPS)-stimulated macrophages (from both bone marrow-derived macrophages [BMM] and thioglycolate-elicited peritoneal macrophages [TEPM]) (). LPS was used as an in vitro
stimulus in the Mouse Gene Atlas study to trigger proinflammatory pathways activating macrophages (24
). Higher infiltration of inflammatory macrophages in MA-CA/04-infected lungs may therefore contribute to the transcriptional program associated with lethality. Similarly, analysis of the other immune cell-associated genes () suggested a higher infiltration of NK cells at day 1 p.i. and of granulocytes and mast cells for all time points after MA-CA/04 infection. In contrast, on day 3 p.i., there were fewer genes more highly expressed in MA-CA/04-infected mice, and there were fewer associated with macrophages, suggesting a similar proportion of macrophages in CA/04- and MA-CA/04-infected lungs on day 3 p.i. Macrophage infiltration after CA/04 infection may therefore be transient, while such infiltration is sustained in MA-CA/04-infected animals.
Closer examination of enrichment scores at day 3 p.i. revealed that genes highly induced by CA/04 included genes that were highly expressed in unstimulated bone marrow-derived macrophages (, “BMM_0h,” enrichment score of 11.1,), B cells, and FoxP3+ T cells (). However, most of the genes from the transcriptional program associated with survival/recovery (set 2) that were highly expressed on day 5 p.i. with CA/04 infection were not associated with immune cells ().
Overall, these results suggest that macrophages present in lungs after infection with MA-CA/04 or CA/04 could be functionally different, with noninflammatory macrophages contributing to the gene expression response to CA/04, while inflammatory macrophage, granulocyte, and mast cell infiltration contributes to the transcriptional response measured in lung samples from MA-CA/04-infected animals.
Transcriptional programs associated with lethality and recovery in the context of other H1N1 influenza virus infections.
We next examined whether the transcriptional signatures associated with lethality and recovery that were derived from the comparison of CA/04- and MA-CA/04-infected mice corresponded to the expression patterns elicited by other lethal and nonlethal H1N1 influenza viruses. Several nonlethal H1N1 viruses were selected for comparison. These included Mex/4482 and NJ/8/76, both of which were isolated from patients with severe respiratory illness, and Brisbane/59/07, a seasonal H1N1 virus. Intranasal infection by these viruses in BALB/c mice resulted in a range of virulence, in agreement with a previous report of H1N1 murine virulence (5
). Brisbane/59/07 and NJ/8/76 caused minimal weight loss (see Fig. S2A in the supplemental material) and were classified as low pathogenicity. Mex/4482 caused significant weight loss, which was intermediate between those of CA/04 and MA-CA/04 infection (Student's t
< 0.05 from days 4 and 2 p.i., respectively) (see Fig. S2A). Viral titers in the lungs were much lower in Brisbane/59/07-infected animals than in all other groups (P
< 0.05), while titers were similar in mice infected with Mex/4482, NJ/8/76, or CA/04 (see Fig. S2B). Stored RNA isolated from the lungs of mice infected with the reconstructed 1918 pandemic influenza virus (r1918) was used as an additional lethal H1N1 comparator (see Materials and Methods). As described previously, mice infected with r1918 had 75% weight loss at day 4 p.i., and there was 100% lethality by day 6 p.i. (21
). Viral titers in the spleen and brain of animals infected with these lethal and nonlethal viruses indicated that none of these viruses disseminated systemically in this mouse model.
To determine whether genes discriminating the transcriptional response to CA/04 and MA-CA/04 on each day p.i. were also able to discriminate low-pathogenicity H1N1 (Brisbane/59/07 and NJ/8/76) from the intermediate-pathogenicity Mex/4482 and from the high-pathogenicity r1918, we used nonparametric multidimensional scaling (MDS) to visualize global gene expression concordance between all samples and for each signature (). Kruskal's stress was used to measure the information loss during dimensionality reduction. We had very small stress values (<5%), indicating a good fit of the MDS representations. On day 1 p.i., and using the genes that were DE between mice infected with CA/04 and those infected with MA-CA/04 on that time point (1,868 genes), MA-CA/04- and r1918-infected samples were located in the same 2D space, indicating that these genes were expressed at similar levels for both lethal viruses. Low and medium pathogens were clustered closer to the mock inoculation. This confirmed that, as with CA/04, less pathogenic viruses induced limited changes in the expression of these genes on day 1 p.i., compared to results for the highly pathogenic viruses MA-CA/04 and r1918.
Fig 7 Comparison of MA-CA/04 and CA/04 transcriptional profiles with those of r1918, Mex/4482, Brisbane/59/07, and NJ/8/76. (A) Nonparametric multidimensional scaling (MDS) representing the Euclidian distances between samples on each day p.i. The matrix distance (more ...)
Likewise, MDS analysis showed that genes DE between mice infected with CA/04 or MA-CA/04 on day 3 p.i. (1,041 genes) also differentiated highly pathogenic (r1918 and MA-CA/04) from less pathogenic viruses (CA/04, Brisbane/59/07, and NJ/8/76). Interestingly, Mex/4482, which induced significant weight loss at this time point, elicited a host response similar to that for the lethal viruses on day 3 p.i. Finally, on day 5 p.i., we observed three different clusters of samples based on expression of 3,123 genes DE between mice infected with MA-CA/04 or CA/04. Mice infected with MA-CA/04 or r1918 expressed these genes similarly, since Mex/4482 elicited a response intermediate between those for low- and high-pathogenicity viruses, and a third cluster was formed by the less pathogenic viruses, with results for Brisbane/59/07 and NJ/8/76 being more similar to those for the mock samples than those for CA/04. These results indicate that genes DE between mice infected with CA/04 and MA-CA/04 discriminate different levels of pathogenicity of other H1N1 viruses.
We next compared these DE gene signatures across viruses by examining the distribution and correlation of gene expression across groups (). We observed that the upregulated lethal signature was expressed to similar levels between MA-CA/04 and r1918 (Pearson correlation, r = 0.81), with a higher median of expression than that for the other viruses on each day p.i. In addition, these genes were overall slightly more expressed in response to Mex/4482 than to CA/04. Downregulated genes from the lethal transcriptional signatures were also more downregulated by the lethal viruses on each day p.i. (Pearson correlation between MA-CA/04 and r1918, r = 0.51). Finally, the recovery signature on day 5 p.i. was upregulated only by less pathogenic viruses, with the highest upregulation for CA/04-infected mice but good correlation with results for Brisbane/59/07 (r = 0.89) and NJ/8/76 (r = 0.9). The intermediate-pathogenicity virus, Mex/4482, which induced weight loss continuously until day 8 p.i., did not upregulate the recovery-associated genes on day 5 p.i., confirming that these genes were associated with recovery and that their expression might be delayed during Mex/4482 virus infection. This analysis confirmed the general relevance of the transcriptional programs associated with lethality and survival/recovery for H1N1 pathogenicity.
Overall, using global gene expression profiling, we found that the increased virulence of the mouse-adapted CA/04 virus was associated with higher and earlier expression of inflammatory and immune cell-related genes following activation of IRF, NB-κB, and STAT TFs. Comparison of our data to metadata collected in other studies from multiple immune cell populations suggested that these differences may be due in part to increases in inflammatory macrophage infiltration in the lungs of mice infected with MA-CA/04. Lethality was also associated with the lack of upregulation of lipid and amino acid metabolism genes, which were upregulated in CA/04-infected mice. Transcription factor analysis suggested that this absence of signaling during lethal infection may be mediated in part by nuclear receptors. Finally, comparison of the gene expression patterns associated with lethality and recovery suggests similar patterns in the expression of these genes between CA/04 and other nonlethal H1N1 influenza viruses and between the lethal viruses MA-CA/04 and r1918.