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J Virol. Nov 2011; 85(22): 11626–11637.
PMCID: PMC3209293

2009 Pandemic H1N1 Influenza Virus Causes Disease and Upregulation of Genes Related to Inflammatory and Immune Responses, Cell Death, and Lipid Metabolism in Pigs[down-pointing small open triangle]


There exists limited information about whether adaptation is needed for cross-species transmission of the 2009 pandemic H1N1 influenza virus (pH1N1). Here, we compare the pathogenesis of two pH1N1 viruses, one derived from a human patient (A/CA/04/09 [CA09]) and the other from swine (A/swine/Alberta/25/2009 [Alb09]), with that of the 1918-like classical swine influenza virus (A/swine/Iowa/1930 [IA30]) in the pig model. Both pH1N1 isolates induced clinical symptoms such as coughing, sneezing, decreased activity, fever, and labored breathing in challenged pigs, but IA30 virus did not cause any clinical symptoms except fever. Although both the pH1N1 viruses and the IA30 virus caused lung lesions, the pH1N1 viruses were shed from the nasal cavities of challenged pigs whereas the IA30 virus was not. Global gene expression analysis indicated that transcriptional responses of the viruses were distinct. pH1N1-infected pigs had an upregulation of genes related to inflammatory and immune responses at day 3 postinfection that was not seen in the IA30 infection, and expression levels of genes related to cell death and lipid metabolism at day 5 postinfection were markedly different from those of IA30 infection. These results indicate that both pH1N1 isolates are more virulent due in part to differences in the host transcriptional response during acute infection. Our study also indicates that pH1N1 does not need prior adaptation to infect pigs, has a high potential to be maintained in naïve swine populations, and might reassort with currently circulating swine influenza viruses.


The 2009 pandemic H1N1 virus (pH1N1) spread to more than 200 countries and caused more than 18,000 human deaths worldwide by August 2010, when the World Health Organization announced the official end of the pandemic (60). Two unusual features of pH1N1 are its efficient human-to-human transmission and its ability to cross the species barrier. The virus has likely been transmitted from humans to other species such as turkeys, dogs, cats, ferrets, and pigs; pH1N1 infection in pigs has been reported worldwide (41, 43, 44, 50), including in the United States (53). The pH1N1 virus, a reassortant virus derived from North American triple reassortant and Eurasian swine influenza viruses (SIVs), contains the neuraminidase (NA) and matrix (M) genes from Eurasian SIVs and six other genes (PB1, PB2, PA, hemagglutinin [HA], NP, and NS) from North American triple reassortant SIVs (11, 49). The HA gene of pH1N1 is in the classical swine lineage, which is derived from the 1918 Spanish pandemic virus (2). Recent studies showed that the 2009 pH1N1 vaccine protects against 1918 Spanish influenza virus and vice versa in mice (33, 35), indicating antigenic similarities among these viruses. The A/swine/IA/15/30 (IA30) H1N1 virus is the first isolate from pigs that is antigenically similar to the 1918 virus (57) and is pathogenic in pigs (56).

Although pH1N1 is thought to have been generated in swine and circulated in pig populations before cross-species transmission to humans (37), this has not been confirmed despite significant research. The pathogenesis and transmission capacity of pH1N1 have been investigated in mice (2, 19, 32), ferrets (32, 37), miniature pigs (19), guinea pigs (51), monkeys (19), and pigs (25, 55, 59). The 1918 Spanish H1N1 virus caused the most devastating pandemic in human history, with approximately 50 million deaths, and is able to infect and cause a respiratory disease in swine (58). In this study, we compared the pathogenesis of two pH1N1 viruses, one derived from a human patient (A/CA/04/09 [CA09]) and the other from swine (A/swine/Alberta/25/2009 [Alb09]), with the 1918-like classical SIV IA30 in a pig model and analyzed host gene expression after inoculation with these viruses.


Viruses and cells.

The 2009 pH1N1 influenza virus human isolate CA09, the swine isolate Alb09, and the classical H1N1 SIV IA30 were propagated in embryonated chicken eggs. Madin-Darby canine kidney (MDCK) cells were maintained in minimum essential medium (MEM) with 5% fetal bovine serum (FBS; HyClone, Logan, UT) and 1% antibiotics (Invitrogen, Carlsbad, CA). They were then infected with bronchoalveolar lavage fluid (BALF) and nasal swabs obtained from experimentally infected pigs and incubated with infecting MEM containing 0.3% bovine serum albumin (BSA), l-glutamine (Invitrogen, Carlsbad, CA), MEM vitamins (Invitrogen, Carlsbad, CA), and 1% antibiotics.

HI assays.

To confirm that pigs were SIV free, sera from all experimental pigs were tested by a hemagglutination inhibition (HI) assay before infection (40). For HI assays, serum was heat inactivated at 56°C, treated with a 20% suspension of kaolin (Sigma-Aldrich, St. Louis, MO) to eliminate nonspecific inhibitors, and adsorbed with 0.5% chicken or turkey red blood cells. The HI assay was performed to test antibodies against the following panel of reference SIV strains: A/swine/IA/1973 (H1N1), A/swine/Texas/98 (H3N2), A/sw/NC/2001 (H1N1 variant), and 2009 pandemic A/swine/Alberta/25/2009 (H1N1).

Pathogenesis studies in pigs.

Pigs were obtained from a healthy herd free of SIV and porcine reproductive and respiratory syndrome virus. These studies included two experiments: the classical H1N1 SIV (IA30) study was completed at Kansas State University's biosafety level 2 (BSL-2) facility in compliance with the Institutional Animal Care and Use Committee at Kansas State University, and the pH1N1 virus study was completed at the Central States Research Center (CSRC), Inc., BSL-3 facility (Oakland, NE), in compliance with the Institutional Animal Care and Use Committee at CSRC. The inoculation protocol used for both studies was described previously (45). In each experiment, 10 pigs were inoculated with noninfectious cell culture supernatant as controls. For the classical H1N1 SIV experiment, 10 4-week-old crossbred pigs were inoculated intratracheally with 106 50% tissue culture infective doses (TCID50)/pig of egg-derived IA30 virus. For the pH1N1 virus experiment, 10 4-week-old crossbred pigs were inoculated intratracheally with 106 TCID50/pig of either egg-derived CA/09 or Alb/09 virus. Five animals per group were euthanized at 3 and 5 days postinfection (dpi), respectively. Nasal swabs were taken at 0, 3, and 5 dpi, placed in 2 ml of MEM, and stored at −80°C. Blood was collected from all inoculated and control pigs at 0, 3, and 5 dpi. Each lung was lavaged with 50 ml of MEM to obtain BALF. The viral load in BALF and nasal swabs was determined in a 96-well plate, as previously described (31, 45).

Examination of lungs and testing of samples from experimental pigs.

During necropsy, an experienced veterinarian recorded the percentage of gross lesions (defined as purple-red consolidation typical of an SIV infection) of each lung lobe. A mean value was calculated for the seven pulmonary lobes of each pig (45). Tissue samples from the trachea, the right cardiac pulmonary lobe, and other affected lobes were fixed in 10% buffered formalin, routinely processed, and stained with hematoxylin and eosin for microscopic examination. Lung sections were assigned a score of 0 to 3 on the basis of the extent of bronchial epithelial and parenchymal injury, using previously described criteria (45). A single board-certified veterinary pathologist scored all slides and was blind to the treatment groups. The avidin-biotin-peroxidase complex method was used to detect influenza A virus nucleoprotein using immunohistochemistry (IHC). Briefly, slides were pretreated with protease-1 and incubated with a mouse anti-influenza A virus nucleoprotein monoclonal antibody (HB-65; ATCC, Manassas, VA) for 32 min at 37°C, with biotinylated goat anti-mouse secondary antibody (Ventana Medical Systems, Carol Stream, IL) for 8 min, and with avidin-biotin-peroxidase conjugate substrate (Ventana Medical Systems, Carol Stream, IL) for 4 min.

Expression microarray analysis and bioinformatics.

Total RNA isolation and mRNA amplification were performed on equal masses of total RNA isolated from lungs of influenza virus- and mock-infected pigs. Reverse transcription-PCR (RT-PCR) was performed on RNA isolated for array analysis, using probes designed against the HA gene of each isolate to confirm the presence of influenza virus in the lung samples analyzed for gene expression.

Expression oligonucleotide arrays were performed on RNA isolated from lung tissue from four to five individual animals per group at 3 and 5 dpi. Probe labeling and microarray slide hybridization were performed with Porcine Gene Expression Microarray V1 (G2519F; Agilent Technologies). All data were entered into a custom-designed Oracle 9i-backed relational database and then uploaded into Rosetta Resolver System, version 5.0 (Rosetta Biosoftware), and Spotfire Decision Site, version 8.1 (Spotfire). All primary expression microarray data, in accordance with the proposed minimum information about a microarray experiment (MIAME) standards (4), are available at http://viromics.washington.edu.

To determine gene expression in response to infection, the ratios of data from individual samples to data from time- and experiment-matched mock samples were determined. A Bonferoni-adjusted one-way analysis of variance (ANOVA) was used to determine differences in gene sequence expression between strains on each day postinfection (false discovery rate [FDR]-adjusted P of <0.05); gene sequences that specifically distinguished the strains from each other were determined by applying a Student's posthoc test (P < 0.1) to ANOVA results. To select for genes that were most relevant to infection, transcripts that were differentially expressed between strains on a given day were filtered to include only transcripts that were 1.5-fold different from values of mock infections (P < 0.05) in at least one of the strains being compared on that day postinfection.

Functional analysis of statistically significant gene expression changes was performed with Ingenuity Pathways Analysis (IPA; Ingenuity Systems). This software analyzes RNA expression data in the context of known biological response and regulatory networks as well as other higher-order response pathways. Ingenuity functional analysis identified biological functions and/or diseases that were most significant among differentially regulated genes at each time point. For all analyses, a Benjamini-Hochberg test correction was applied to the IPA-generated P value to determine the probability that each biological function assigned to that data set was due to chance alone. In the functional networks, genes are represented as nodes, and the biological relationship between two nodes is represented as an edge (line). All edges are supported by at least one published reference or from canonical information stored in the Ingenuity Pathways Knowledge Base.

Statistical analyses.

Virological and clinical data are shown as mean (or geometric mean) and standard error of the mean (SEM) within the text and in graphical formats. Macroscopic pneumonia scores, microscopic pneumonia scores, log10 transformed BALF and nasal swab virus titers, and temperatures were analyzed using ANOVA, with a P value of ≤0.05 considered statistically significant (GraphPad Prism, GraphPad Software, La Jolla, CA). Virological measures shown to be significantly different by treatment group were compared pairwise by using a Tukey-Kramer test.


Sequence comparison of viruses.

To compare pathogenicity of the 2009 pH1N1 virus with the 1918-like H1N1 virus in pigs, the IA30 virus was used in this study because it is the first mammalian influenza virus isolate and a derivative of the 1918 Spanish H1N1 virus. The IA30 virus showed 94.2% to 98.7% homology with the genome of the 1918 (A/Brevig Mission/1/1918) H1N1 virus at nucleotide level and 91.3% to 98.4% homology at the amino acid level (Table 1). Less homology (80.9% to 90.7%) was observed between the pH1N1 (CA09 and Alb09) and IA30 viruses at the nucleotide level. At the amino acid level, the polymerase subunits (PB1, PB2, and PA), M1, and NP of the IA30 virus showed high homology (94.4% to 95.6%) with the proteins of the pH1N1 (CA09 and Alb09) viruses (Table 1).

Table 1.
Comparison of sequences between IA30 and BM1918, CA09, and Alb09 virusesa

Clinical symptoms.

All animals were monitored for clinical symptoms through the entire course of the study (0 to 5 dpi). Rectal temperature measurements showed that pigs inoculated with either the classical IA30 or pH1N1 (CA09 or Alb09) virus had a fever starting at 1 dpi, but control animals did not (Fig. 1). Rectal temperature in Alb09-inoculated pigs was significantly higher than that in IA30-inoculated pigs, but there were no significant differences between the CA09- and Alb09-inoculated pigs or between CA09- and IA30-inoculated pigs. All pH1N1-inoculated pigs had pronounced clinical infection compared with IA30-infected animals. From 2 dpi, both CA09- and Alb09-inoculated pigs showed clinical symptoms of infection, such as coughing, sneezing, lethargy, inappetence, and labored breathing. Starting on 4 dpi, more than 50% of pigs from both pH1N1-inoculated groups showed labored breathing and depression, including lethargy and inappetence, but there was no significant difference in clinical signs between the groups. IA30-inoculated pigs did not show any clinical symptoms from 1 to 5 dpi.

Fig. 1.
Rectal temperature of pigs infected with IA30, CA09, and Alb09 H1N1 viruses or mock infected from day 0 to 5 dpi. Control 1 is a control for the pH1N1 study (CA09 and Alb09) and control 2 is for the IA30 study.


Postmortem evaluation revealed severe macroscopic lung lesions (plum-colored, consolidated areas) in pigs inoculated with either pH1N1 virus (CA09 or Alb09 virus) or with the classical IA30 virus and few to no lesions in control pigs (Fig. 2A and B). There were significant differences between the overall mean number of lung lesions in the three groups of virus-inoculated pigs and control pigs on 3 and 5 dpi (P < 0.001). However, there were no significant differences in lung lesions between pigs inoculated with both pH1N1 viruses and the classical IA30-inoculated pigs or between CA09- and Alb09-inoculated pigs on 3 and 5 dpi.

Fig. 2.
Macroscopic lung lesions in pigs inoculated with pH1N1 and classical IA30 viruses or mock infected at 3 dpi (A) and 5 dpi (B). The values for the average macroscopic lung lesions of each group on each day are means ± SEM.

The microscopic score (0 to 3), indicative of the extent of damage to lung architecture, was between 1.40 to 2.30 at 3 and 5 dpi in all three inoculated groups compared with a score of 0.00 to 0.20 in control pigs (Table 2). Although microscopic scores in Alb09- and CA09-inoculated pigs were higher than those in IA30-inoculated pigs, there were no significant differences in the histopathologic lung damage among the three groups. All infected pigs had variable degrees of damage, ranging from mild to moderate bronchointerstitial pneumonia, atelectasis, acute to subacute bronchiolitis with epithelial necrosis, and variable lymphocytic cuffing of bronchioles at 3 and 5 dpi (Fig. 3). Some control pigs had incidental noninfluenza-associated lung lesions (Table 2) but were negative for SIV infection. All three viruses replicated well in the lungs, with virus titers reaching approximately 104.29 to 105.21 TCID50/ml in the BALF (Table 2). In contrast, shedding in the nasal cavity differed by viral strain: virus was isolated from 90% of nasal swabs at 3 dpi and in 100% of nasal swabs at 5 dpi in pH1N1-inoculated pigs (Table 2), but no virus was detected in nasal swabs of IA30-inoculated pigs. Interestingly, a large amount of viral antigens were detected in the bronchiole epithelium by IHC staining from pigs infected with pH1N1 viruses at 3 dpi, but only limited viral antigens were found from those infected with the classical IA30 (Fig. 3), as previously reported (58). IHC reactivity on the IA30 virus was inconsistent and when present was mild and not substantially different from that on the controls.

Fig. 3.
Microscopic sections of bronchioles in the lungs from control and infected pigs at 3 dpi. (A) Inoculation with noninfectious cell culture supernatant. Note terminal bronchiole with normal ciliated and nonciliated epithelium. (B) Inoculation with the IA30 ...
Table 2.
Virus titers in BALF and nasal swabs and percentage of nasal shedding and microscopic lung lesions in pigs inoculated with pH1N1 and classical IA30 viruses or mock infected

Sequence analysis.

To investigate whether molecular adaptation occurs during replication of the pH1N1 virus in pigs, whole genomes of CA09 and Alb09 were sequenced and compared before and after infection in pigs. The human and swine isolates showed 96% to 100% nucleotide sequence identity, depending on the gene segment. Notably, there were few amino acid differences between the genomes of both viruses before inoculation (Table 3). In particular, there were five amino acid differences (P100S, N104K, T214A, S220T, and T338V) in the surface HA proteins of CA09 and Alb09 (Table 3). Two BALF samples from infected pigs at 3 and 5 dpi from each pH1N1 challenge group were used to amplify and sequence the viral genomes postinfection. After the human isolate CA09 was passaged in pigs, only the HA protein showed an amino acid change from S to P at position 200. There were no mutations in the other viral proteins and genes, except for the presence of several mutations in the PB2 gene at the nucleotide level. For the swine isolate Alb09, there was an amino acid substitution from D to E at position 144 in the HA protein; all other viral proteins were conserved. At the nucleotide level, there were several mutations in PB2 and one mutation in PB1 from Alb09 virus isolated at 5 dpi. The PA, NP, NA, M, and NS genes showed 100% nucleotide homology to the Alb09 virus before infection.

Table 3.
Comparison of sequences between the CA09 and Alb09 genomes before passage in pigs

Host gene expression analysis. (i) pH1N1 elicits an early inflammatory response that is absent in IA30 infection.

To study the mechanisms underlying the differences in response among Alb09, CA09, and IA30, we performed global transcriptomic analysis on lung tissues from infected and control pigs during acute infection (3 and 5 dpi), using a commercial microarray. There were 2,840 transcripts whose expression distinguished the viruses at 3 dpi and 2,044 transcripts whose expression distinguished the viruses at 5 dpi, as calculated by one-way ANOVA (FDR-adjusted P of <0.05; 1.5-fold change compared with mock infection in at least one group). Interestingly, the differences in host transcriptional responses to infection mirrored the clinical manifestations of the disease, with a clear response profile distinguishing pH1N1 from IA30 on both days (Fig. 4).

Fig. 4.
Differential gene expression patterns in the lungs of CA09-, Alb09-, and IA30-infected pigs. Pigs were infected with a 106 TCID50/pig of IA30, CA09, or Alb09 (see Materials and Methods). Animals from each infection group and a mock group were euthanized ...

At 3 dpi, these differences corresponded to increased early transcriptional activation of inflammatory and immune pathways in pH1N1-infected but not IA30-infected pigs (Table 4 and Fig. 5). Pathway analysis suggested that the increase in immune response included pattern recognition receptor (PRR) signaling and antiviral responses in pH1N1-infected pigs. This response was generally absent in IA30-infected pigs (Fig. 6, blue boxes indicate PRR and antiviral signaling molecules). PRR signaling is a component of the innate immune response that is initiated through the recognition of conserved pathogen-associated molecular patterns. Therefore, it is notable that these PPR signaling molecules are induced in pH1N1-infected pigs while expression is largely unchanged in IA30-infected pigs despite equal influenza virus titers across all three infections. This suggests that the ability of the host to detect and respond to IA30 infection through PRR-related signaling is decreased or absent, which correlates to fewer clinical symptoms in these animals. Furthermore, there were very few differences among inflammatory and immune genes by 5 dpi, indicating that the timing of this host transcriptional response may be a critical factor in response to the pH1N1 and classical viruses.

Fig. 5.
Alb09 and CA09 infection results in an upregulation of immune response genes at day 3 postinfection that is absent or repressed in IA30-infected pigs. Pigs were infected with a 106 TCID50/pig of IA30, CA09, or Alb09 (see Materials and Methods). Animals ...
Fig. 6.
IA30-infected pigs fail to induce immune response genes associated with pattern recognition of viruses and cytokine and immune cell responses in the lung. Pigs were infected with a 106 TCID50/pig of IA30, CA09, or Alb09 (see Materials and Methods). Animals ...
Table 4.
Top 10 select biological functions that distinguish host transcriptional response in the lungs at 3 and 5 dpi between CA09-, Alb09-, and IA30-infected pigsa

(ii) pH1N1 virus infection results in higher expression of cell death and lipid metabolism genes than IA30 infection.

Among the host genes with differential transcriptional responses to pH1N1 viruses and IA30 at 5 dpi, we found significant differences in those related to cell death and lipid metabolism (Table 4 and Fig. 7A). The majority of these genes were induced with pH1N1 infection but were unchanged or repressed with IA30 infection. Closer examination of the top biological functions that distinguished the groups showed a functional link between the cell death and lipid metabolism genes, with approximately two-thirds of the lipid metabolism genes playing roles in cell death. Network analysis revealed that the top-scoring network among the differentially expressed genes at 5 dpi was statistically enriched for genes related to cellular differentiation and lipid metabolism (Fig. 7B). Expression of molecules within the network indicated a concurrent suppression of lipid metabolism-related molecules in IA30-infected animals only. Interestingly, this included downregulation of peroxisome proliferator-activated receptor gamma (PPARG) (Fig. 7B, blue box), which has been reported to be expressed in immune cells of weaned pigs and plays a role in mediating the inflammatory response and the function of immune cells, including dendritic cells (28, 29). In contrast, many molecules within this network, including PPARG, were unchanged or upregulated in pH1N1-infected pigs. The differential regulation of these genes may be influenced in part by distinct immune and inflammatory responses of classical virus- and pH1N1 virus-infected pigs at 3 dpi.

Fig. 7.
Significant differences in the expression of genes related to cell death and lipid metabolism distinguish pH1N1- and IA30-infected pigs at 5 dpi. Pigs were infected with a 106 TCID50/pig of IA30, CA09, or Alb09 (see Materials and Methods). Animals from ...


The 2009 pH1N1 viruses not only infected and caused death in humans but also were able to transmit virus to and infect other species. Our study confirms that pigs are susceptible to either the swine or human 2009 pH1N1 isolates, as reported previously (5, 25, 55). In addition, we found that virus replication in pH1N1-infected and classical IA30 virus-infected pigs was similar, but pH1N1 induced clear symptoms in pigs while IA30 did not. A previous study showed that IA30 virus caused mild respiratory symptoms in two pigs out of six infected animals (58); however, in our current and previous studies no obvious clinical symptoms except fever were observed in infected pigs (30). This divergence might be due to differences in the genetics, ages of pigs, and housing conditions. Both CA09 and Alb09 were shed more efficiently from the upper nasal cavity than IA30, indicating that pH1N1 might have a high potential to maintain itself in swine populations.

The H1N1 virus caused the 1918 pandemic, leading to the death of 50 to 100 million people, mostly healthy young adults. It is striking that those who died in the 2009 pandemic were predominantly young and middle-aged adults although the overall pH1N1-associated mortality rate was very low (26). The classical H1N1 SIVs are derivatives of the 1918 Spanish influenza virus. The IA30 H1N1 virus was originally isolated by R. Shope (57) from Iowa pigs with respiratory disease and was the first mammalian influenza A virus ever isolated. IA30 is pathogenic to pigs and mice (30, 58) and was chosen as a representative 1918 pandemic H1N1-derived virus. The HA genes from both the IA30 and the 2009 pH1N1 viruses are derived from the classical SIVs, but they have a different internal genetic make-up. Interestingly, the polymerase subunit (PB1, PB2, and PA) and NP of the IA30 virus showed a high homology with those of both the 1918 and 2009 pandemic viruses at the amino acid level although they had a lower homology with those of the 2009 pandemic virus at the nucleotide level. For the polymerase subunit and NP, which forms the ribonucleoprotein (RNP) with influenza viral RNA, there was a high degree of homology (94.4% to 96.7%) at the amino acid level between the 1918 and 2009 pandemic H1N1 viruses. Further research is necessary to determine if these similarities in RNP are important contributors to pandemic potential in both the 1918 and 2009 viruses. Although both the pH1N1 and classical H1N1 viruses reached similar titers in pig lungs and caused similar severe lung lesions in our study, pH1N1 was shed efficiently via the upper respiratory tract, but the IA30 virus was not. It is not clear why shedding of IA30 in the nasal cavity was limited.

Although there was a difference in 11 amino acids (aa) between Alb09 and CA09, the isolates replicated similarly in pigs. A change of only a single amino acid in the HA of the human and swine pH1N1 isolates after they were passaged in pigs suggests that these pH1N1 genomes were conserved in a single passage in vivo, indicating that molecular adaptation of a human-derived pH1N1 virus to the pig may not be necessary.

Microarray analysis revealed that pH1N1-infected pigs mounted a more potent early immune response (3 dpi) and had higher expression levels of genes related to cell death and lipid metabolism (5 dpi) than IA30-infected pigs. The absence of a clinical response to IA30 may be associated with a decrease in PRR and antiviral signaling in response to the virus during early acute infection, followed by downregulation of lipid- and cell death-related genes at 5 dpi. These differences in host transcriptional response may act independently or synergistically to account for the increase in clinical disease in pH1N1-infected animals. Previous studies of influenza viruses, including seasonal human H1N1, lethal avian H5N1, and the reconstructed 1918 virus (r1918), in macaques and mice suggest that an early and sustained inflammatory response is associated with increased influenza virus pathogenesis (1, 21, 23). These studies report augmented immune responses associated with cytokine, chemokine, and interferon release and a cell death response mediated by the more pathogenic influenza viruses. Similarly, pH1N1 infection of nonhuman primates resulted in higher proinflammatory gene expression and cytokine production than infection with seasonal influenza virus, which was resolved as animals recovered from infection (46). Our results in pigs somewhat mirror these findings in macaques as we showed an early increase of proinflammatory gene expression in infected pigs that was resolved as all of the animals in the study recovered. However, in contrast to previous studies with lethal influenza viruses in other animal models, differences in immune responses observed in the present study were not maintained at later time points. This may reflect the resolution of infection in both the pH1N1- and IA30-infected pigs.

Our study also showed an absence of early PRR gene expression in the IA30-infected animals that could not be attributed to a difference in viral titers. Interestingly, previous studies of lethal avian H5N1 and r1918 viruses in wild-type mice noted that the expression of genes associated with viral sensing were unchanged or downregulated in the r1918 virus-infected mice and increased in the H5N1-infected mice despite equivalent viral titers in lungs at 1 dpi (9). This indicates that the virulence mechanisms utilized in the pH1N1 virus are different from those of the r1918 virus.

The PB1-F2, which is expressed from a +1 reading frame of the viral RNA polymerase subunit PB1 (8), is able to induce apoptosis and promote inflammation (24) and has been shown to be a virulence factor and promote secondary bacterial infections (34). However, the 2009 pH1N1 viruses do not express full-length PB1-F2 because they possess three stop codons at amino acid positions 12, 58, and 88 in the PB1-F2 reading frame. Interestingly, the 1918-like IA30 virus also expresses a truncated PB1-F2 (34 aa) since there are three stop codons (amino acid positions 35, 61, and 91) in the IA30 PB1-F2 reading frame, which is different from that previously reported in the 1918 Spanish influenza virus (34). Recent studies have demonstrated that the pH1N1 viruses containing full-length PB1-F2 do not show increased in vitro and in vivo growth kinetics and mouse pathogenicity (14, 39), questioning the role of PB1-F2 as a critical virulence gene for the pH1N1 viruses. It remains unknown whether higher expression of cell death-related genes in pH1N1-infected pigs at 5 dpi is related to the 11-aa truncated PB1-F2 found in these viruses; the function of the PB1-F2 in the cell death gene expression needs to be elucidated in future studies.

A limited number of studies have examined the relationship between lipid metabolism and influenza virus infection (3, 46). In the present study, pH1N1 was associated with an overall increase in lipid metabolism genes at 5 dpi, whereas the IA30 virus induced a significant concordant decrease in a network of related lipid metabolism and cell death genes which were related to the molecule PPARG. Lipids play multifaceted roles in the influenza virus life cycle, including virus entry, assembly, and budding within the cell, and are known mediators of virus-host interactions (7, 18). Although it is possible that the differences that we observed in lipid metabolism genes may reflect virus-specific shifts in virus entry or assembly favoring the pH1N1 virus, we did not observe any difference in virus titers between the groups that would support this theory. Instead, it is more likely that the differences that we observed are due to differential host regulation of lipid mediators of the immune response. The increase in lipid-related genes in the pH1N1-infected animals may reflect compensatory late reprogramming of cellular metabolism to restore membrane damage incurred during early immune and inflammatory responses. Differences in the early immune responses to the pH1N1 and IA30 viruses may also account for the relative downregulation of PPARG in IA30 infection as PPARG is expressed in the immune cells of pigs, and its activation in immune cells plays a role in the mediation of the host response to immunological stress (28, 29). As such, the observed decreases in PPARG in IA30-infected but not pH1N1-infected cells may be related to the relative absence of an early immune response in the IA30 infection and could indicate virus-specific differences in immune cell populations in the lungs of infected pigs.

To our knowledge, this is the first report that dysregulation of lipid metabolism occurs at the site of primary infection in pigs and that pigs without clinical symptoms exhibit a decreased activation of lipid-regulating genes. Recent studies of moderately pathogenic pH1N1 in nonhuman primates indicated an early downregulation of lipid metabolism gene expression in lungs which was not observed in the lungs of animals infected with a mildly pathogenic, seasonal variant of the H1N1 virus (46). Previous global gene expression studies of r1918 influenza virus infection in human cell lines indicated that the NS1 segment of the r1918 virus may contribute to virulence by repressing the expression of lipid metabolism genes including proinflammatory lipid mediators (3).

It is possible that differences in immune and lipid responses between the pH1N1- and IA30-infected pigs could be due in part to the differences in the NS1 proteins of the viruses. NS1 is a multifunctional protein that plays a key role in the pathogenesis and virulence of influenza A viruses and is a well-known viral interferon antagonist (15). Although the pH1N1 NS1 is an effective interferon antagonist, it inefficiently blocks general host gene expression in human and swine cells (17). Compared to the NS1 of the IA30 virus, the pH1N1 NS1 is truncated at 220 aa and lacks the consensus C-terminal PDZ domain motif. Although this PDZ domain was previously identified as a virulence marker in other influenza virus strains (20, 38), recent studies indicate that this PDZ may not be critical for replication, pathogenicity, or transmission of the 2009 pH1N1 virus (16). Although the pH1N1 NS1 has only 84% identity with the IA30 NS1 at the amino acid level, both NS1 proteins possess similar amino acids at positions which have been previously postulated to be important for viral replication and virulence, such as position 92D, where 92E has been shown to be critical for escaping antiviral cytokine responses (47), and 103F and 106M, which have been shown to be critical for CPSF30 binding (10). Further study is necessary to determine the mechanisms by which the host and influenza virus alter lipid and immune responses during infection. These may include additional studies to determine if amino acid differences in the NS1 proteins of the pH1N1 and IA30 viruses may be influencing pH1N1 pathogenicity and transmission or account for the observed differences in host lipid and immune responses.

This study demonstrates the importance of using high-throughput technologies as a sensitive measure of host response to acute infection in pigs. Although the application of global expression profiles to infection in pigs is important, given that they are a reservoir for many zoonotic diseases such as influenza virus, its use has been rare compared with other model systems. Most previous studies using microarray in pigs have focused on response to classical swine fever virus (12), circovirus (27), Actinobacillus pleuropneumoniae (13, 36, 48), and Escherichia coli (6). Our data suggest that global transcription profiling is a promising avenue for uncovering novel mechanisms of response to influenza virus infection in swine. The differential response signatures obtained in our study are indicative of disparate host responses leading to differences in clinical response, despite similar virologic findings.

pH1N1 viruses have been isolated from swine herds worldwide, indicating that they are cocirculating in the pig population with currently circulating SIVs. Furthermore, reassortment between pH1N1 and circulating SIVs has been reported (22, 54). Importantly, it is possible to generate via reassortment in the pig a more virulent virus that may infect humans. In this context it should be noted that the human seasonal and avian influenza viruses have been occasionally isolated from pigs throughout the world (42, 54), indicating that human and avian virus genes may potentially reassort with mammalian-adapted viruses, which could lead to enhanced cross-species transmission. A recent study showed that eight reassortant avian H9N2 viruses containing PA and/or other genes from the 2009 pH1N1 exhibited higher virulence without prior adaptation than the parental virus in a mouse model (52). Therefore, if a novel virus is generated via reassortment between pH1N1 and other influenza A viruses in pigs or other animal species or humans, this virus might possess high transmissibility in addition to causing high mortality. This reassortant virus may become a potential causative agent for a future pandemic and may lead to much higher mortality rates than those reported for the 2009 pandemic. Therefore, influenza virus surveillance in susceptible hosts is crucial for the control and prevention of the next pandemic.


We thank Vani Shanker (Department of Scientific Editing at the St. Jude Children's Research Hospital) for critical review of the manuscript and Ruben Donis (CDC) for providing the 2009 human pandemic virus; we thank Deborah Clouser, Audree Gottlob, and Darlene Sheffer (Kansas State University) for animal studies and technical assistance and Sara Kelly and Sean Proll (University of Washington) for technical assistance with genomics data. We also gratefully acknowledge the assistance of the Histopathology Section at the Department of Diagnostic Medicine/Pathobiology, Kansas State University.

We acknowledge a grant from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services, to support this study (Contract No. HHSN266200700005C). Transcriptomics and bioinformatics analysis was supported by grants from the National Institute of Allergy and Infectious Diseases, National Institutes of Health (P01 AI058113-06), and National Center for Research Resources, National Institutes of Health, Department of Health and Human Services (P51 RR00166-45).


[down-pointing small open triangle]Published ahead of print on 7 September 2011.


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