﻿Published Ahead of Print 7 September 2011.
2011, 85(22):11626. DOI: 10.1128/JVI.05705-11.
J. Virol.
Richard Webby, Michael G. Katze and Juergen A. Richt
Stigger-Rosser, Qinfang Liu, Chuanling Qiao, Jake Elder,
Wenjun Ma, Sarah E. Belisle, Derek Mosier, Xi Li, Evelyn
Metabolism in Pigs
Responses, Cell Death, and Lipid
Related to Inflammatory and Immune
Genes
Causes Disease and Upregulation of
2009 Pandemic H1N1 Influenza Virus
http://jvi.asm.org/content/85/22/11626
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JOURNAL OF VIROLOGY, Nov. 2011, p. 11626­11637 Vol. 85, No. 22
0022-538X/11/$12.00 doi:10.1128/JVI.05705-11
Copyright © 2011, American Society for Microbiology. All Rights Reserved.
2009 Pandemic H1N1 Influenza Virus Causes Disease and Upregulation
of Genes Related to Inflammatory and Immune Responses,
Cell Death, and Lipid Metabolism in Pigs
Wenjun Ma,1
 Sarah E. Belisle,2
 Derek Mosier,1
Xi Li,1
Evelyn Stigger-Rosser,3
Qinfang Liu,1
Chuanling Qiao,1
 Jake Elder,1
Richard Webby,3
Michael G. Katze,2
and Juergen A. Richt1
*
Department of Diagnostic Medicine/Pathobiology, College of Veterinary Medicine, Kansas State University,
Manhattan, Kansas1
; Department of Microbiology, University of Washington, Seattle, Washington2
; and
St. Jude Children's Research Hospital, Memphis, Tennessee3
Received 15 July 2011/Accepted 29 August 2011
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 Organiza-
tion 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), indi-
cating 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 trans-
mission to humans (37), this has not been confirmed despite
significant research. The pathogenesis and transmission capac-
ity 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 approxi-
mately 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.
MATERIALS AND METHODS
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
* Corresponding author. Mailing address: Department of Diagnos-
tic Medicine/Pathobiology, College of Veterinary Medicine, Kansas
State University, K224B Mosier Hall, Manhattan, KS 66506. Phone:
(785) 532-2793. Fax: (785) 532-4039. E-mail: jricht@vet.k-state.edu.
 W. Ma and S. Belisle contributed equally to the work.
 Present address: Harbin Veterinary Research Institute, Harbin,
China.

Published ahead of print on 7 September 2011.
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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 per-
formed 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 vari-
ant), 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 clas-
sical H1N1 SIV experiment, 10 4-week-old crossbred pigs were inoculated in-
tratracheally 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, Ma-
nassas, 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 en-
tered 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 deter-
mined 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 path-
ways. 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 Path-
ways 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.
Nucleotide sequence accession numbers. All sequences have been deposited
in the GenBank database under accession numbers JF915184 to JF915191,
CY097778 to CY097785, and JN617971 to JN617978.
RESULTS
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).
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 signifi-
cantly higher than that in IA30-inoculated pigs, but there were
no significant differences between the CA09- and Alb09-inoc-
TABLE 1. Comparison of sequences between IA30 and BM1918,
CA09, and Alb09 virusesa
IA30
gene
BM1918 CA09 Alb09
% Nucleotide
identity
% Amino
acid
identity
% Nucleotide
identity
% Amino
acid
identity
% Nucleotide
identity
% Amino
acid
identity
PB2 98.7 97.4 86.1 95.1 86.0 95.1
PB1 95.3 97.9 81.1 95.6 80.9 95.5
PA 97.6 96.8 84.4 95.4 84.3 95.5
HA 94.2 94.3 82.5 85.5 82.3 85.3
NP 97.8 97.8 88.6 94.4 88.6 94.4
NA 95.2 93.8 84.2 87.8 84.3 87.8
M 98.0 89.0 89.0
M1 98.4 94.8 94.8
M2 96.9 90.7 90.7
NS 96.1 90.7 90.7
NS1 91.3 84.0 83.6
NS2 94.2 92.6 92.6
a
IA30, A/swine/Iowa/1930; BM1918, A/Brevig Mission/1/1918; CA09, A/CA/
04/09; Alb09, A/swine/Alberta/25/2009.
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ulated pigs or between CA09- and IA30-inoculated pigs. All
pH1N1-inoculated pigs had pronounced clinical infection com-
pared 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 de-
pression, 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.
Pathogenicity. Postmortem evaluation revealed severe mac-
roscopic 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 be-
tween 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.
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 differ-
ences in the histopathologic lung damage among the three
groups. All infected pigs had variable degrees of damage, rang-
ing from mild to moderate bronchointerstitial pneumonia, at-
electasis, acute to subacute bronchiolitis with epithelial necro-
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.
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.
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sis, 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.
Sequence analysis. To investigate whether molecular adap-
tation occurs during replication of the pH1N1 virus in pigs,
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
Detection time
and virus
or group
Virus titer in BALF
(TCID50/ml)a
Microscopic lung
lesion scoreb
% Positive nasal
swabs (virus
titer)c
3 dpi
CA09 4.68  0.67 1.60  0.24 90 (3.67  0.32)
Alb09 5.21  0.24 2.10  0.33 90 (4.60  0.42)
IA30 4.29  0.44 1.40  0.33 0 (1)d
pH1N1 control 1 0.00 0 (1)
IA30 control 1 0.00 0 (1)
5 dpi
CA09 5.14  0.73 2.20  0.25 100 (4.46  0.66)
Alb09 4.77  0.57 2.30  0.30 100 (3.30  0.53)
IA30 4.40  0.10 1.50  0.35 0 (1)
pH1N1 control 1 0.00 0 (1)
IA30 control 1 0.20  0.12 0 (1)
a
Values are log10 geometric mean TCID50/ml  SEM.
b
Lung sections were assigned a score of 0 to 3. Values are mean  SEM.
c
Percentages are based on the total number of swabs. Values in parentheses
are the log10 geometric mean nasal swab TCID50/ml  SEM.
d
Lower than the detection limit of 10 TCID50/ml.
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 virus. Note
acute bronchiolitis and bronchointerstitial pneumonia. The mucosa and submucosa are slightly thickened and disorganized, and there are
submucosal neutrophils and lymphocytes. Intralumenal cellular debris and pyknotic neutrophils are present. There are few peribronchiolar
and interstitial lymphocytes and localized parenchymal hemorrhages. (C) Inoculation with the CA09 virus. Note acute bronchiolitis and
bronchointerstitial pneumonia. There is mucosal thickening and disorganization with epithelial degeneration and hyperplasia. Intraepithelial
and intralumenal neutrophils are present along with degenerate sloughed epithelial cells within the lumen. There are a few peribronchiolar
and interstitial lymphocytes. (D) Inoculation with the Alb09 virus. Note acute bronchiolitis and alveolar atelectasis. There is attenuation of
the mucosa with degeneration and necrosis of airway epithelium with sloughed epithelial cells within the airway lumen. Associated
parenchyma is atelectatic. IHC staining in panels E, F, and G shows viral antigens within bronchiolar epithelium in lungs of a pig infected
with IA30, CA09, and Alb09 virus at 3 dpi.
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whole genomes of CA09 and Alb09 were sequenced and com-
pared 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 se-
quence the viral genomes postinfection. After the human iso-
late 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 nucle-
otide level, there were several mutations in PB2 and one mu-
tation 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.
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 transcrip-
tomic analysis on lung tissues from infected and control pigs
during acute infection (3 and 5 dpi), using a commercial mi-
croarray. There were 2,840 transcripts whose expression dis-
tinguished 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). Interest-
ingly, the differences in host transcriptional responses to infec-
tion mirrored the clinical manifestations of the disease, with a
clear response profile distinguishing pH1N1 from IA30 on
both days (Fig. 4).
At 3 dpi, these differences corresponded to increased
early transcriptional activation of inflammatory and immune
pathways in pH1N1-infected but not IA30-infected pigs (Ta-
ble 4 and Fig. 5). Pathway analysis suggested that the in-
crease 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 recogni-
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 and
5 dpi, total RNA was isolated from lung tissue, and global gene ex-
pression was determined by microarray analysis. The figure shows a
one-dimensional clustering of transcripts with statistically significant
differences in expression in the lung on each day (2,840 transcripts at
3 dpi; 2,044 transcripts at 5 dpi) as determined by one-way ANOVA
(FDR-corrected P value of 0.05; selecting for transcripts with an
average 1.5-fold change [P  0.05] compared with mock infection in at
least 1 group) on each day. Gene expression for each animal is shown
as the log (ratio) of expression relative to that of experiment and
time-matched samples extracted from lungs of mock-infected pigs.
Red indicates that the gene expression is higher than mock infection
values; green indicates that gene expression is lower than mock values.
TABLE 3. Comparison of sequences between the CA09 and Alb09
genomes before passage in pigs
CA09 gene
Homology (%) with Alb09 gene
Amino acid (mutation s) Nucleotide (ORF)
PB2 100 99.8
PB1 100 99.9
PA 99.9 (P224S)a
99.8
HAb
99.1 (P100S, N104K, T214A,
S220T, T338V)
99.6
NP 99.6 (V88I, D362G) 99.7
NA 99.6 (V92I, N224D) 99.6
M1 100 99.7
M2 100 100
NS1 99.5 (I123V) 99.8
NS2 100 100
a
The amino acid P is an amino acid of the CA09 virus PA protein at position
224, and amino acid S belongs to the Alb09 virus PA protein.
b
Only one amino acid change was found in the HA of the CA09 virus (S200P)
and the Alb09 virus (D144E) after passage in pigs.
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tion 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 im-
mune 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.
(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 re-
sponses to pH1N1 viruses and IA30 at 5 dpi, we found signif-
icant differences in those related to cell death and lipid me-
tabolism (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 ex-
pressed 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 dif-
ferential 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.
DISCUSSION
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 replica-
tion 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 ani-
mals (58); however, in our current and previous studies no
obvious clinical symptoms except fever were observed in in-
fected 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 influ-
enza 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 patho-
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
dpi Biological function P valueb Total no. of molecules
in function
No. of molecules in function with
increased expression
IA30 Alb09 CA09
3 Immune response 2.06E-07 50 22 48 49
Developmental process of blood cells 2.91E-07 50 21 48 48
Differentiation of leukocytes 1.17E-06 33 18 32 32
Quantity of blood cells 1.17E-06 37 16 35 36
Hematological process 2.38E-06 47 19 44 44
Activation of blood cells 5.37E-06 34 15 32 32
Survival of leukocytes 5.50E-06 18 9 18 18
Inflammatory disorder of mammals 5.50E-06 29 12 28 29
Antiviral response of organism 5.50E-06 12 3 12 12
5 Accumulation of monounsaturated fatty acids 1.63E-02 3 1 2 2
Transport of lipoprotein 1.63E-02 4 0 3 3
Apoptosis 4.55E-02 56 21 46 44
Contraction of tissue 5.64E-02 11 3 8 7
Cell death of eukaryotic cells 5.64E-02 54 21 45 44
Polymerization of filaments 5.64E-02 6 4 4 3
Metabolism of lipid 5.64E-02 21 5 16 15
Developmental process of endothelial cells 5.64E-02 8 4 3 5
Depolarization of mitochondria 5.64E-02 4 2 3 3
a
Redundant biological functions and nonrelated, disease-specific functions have not been included in the table. Increased expression is relative to that of time
and experiment-matched mock samples.
b
Benjamini-Hochberg corrected.
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genic to pigs and mice (30, 58) and was chosen as a represen-
tative 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 poly-
merase 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 impor-
tant 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 de-
crease 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 syner-
gistically 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 sug-
gest that an early and sustained inflammatory response is as-
sociated 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 re-
sulted 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 expres-
sion 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
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 from each infection
group and a mock group were euthanized at 3 and 5 dpi, total RNA was isolated from lung tissue, and global gene expression was determined by
microarray analysis. The heat map depicts immune response-related genes (Table 4) that were differentially expressed between infection conditions
at 3 dpi. Gene expression for each animal is shown as the log (ratio) of expression relative to that of experiment and time-matched samples
extracted from lungs of mock-infected pigs. Red indicates that the gene expression is higher than mock infection values; green indicates that gene
expression is lower than mock values.
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immune responses observed in the present study were not main-
tained 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 ex-
pression in the IA30-infected animals that could not be attrib-
uted to a difference in viral titers. Interestingly, previous stud-
ies 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 equiv-
alent 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 in-
fections (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.
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 from each infection
group and a mock group were euthanized at 3 and 5 dpi, total RNA was isolated from lung tissue, and global gene expression was determined by
microarray analysis. Network diagram depicts the relationship between immune response genes that show differential expression in response to the
Alb09, CA09, and IA30 viruses. Molecules in the network were selected from differentially regulated immune response genes on the basis of their
direct interactions with each other, as determined by functional analysis by Ingenuity Pathway Analysis (Ingenuity Systems). Shading of molecules
reflects average expression compared with mock infection within each group at 3 dpi. Red indicates that the gene expression is higher than mock
values; green indicates that gene expression is lower than mock values. Molecules that changed less than 1.5-fold from mock infection values are
shaded in gray.
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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 re-
lated 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 dif-
ferences that we observed in lipid metabolism genes may re-
flect 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 reprogram-
ming 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 im-
mune 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 de-
creased activation of lipid-regulating genes. Recent studies of
moderately pathogenic pH1N1 in nonhuman primates indi-
cated an early downregulation of lipid metabolism gene ex-
pression in lungs which was not observed in the lungs of ani-
mals 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 patho-
genesis 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 iden-
tified 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 deter-
mine 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 differ-
ences in the NS1 proteins of the pH1N1 and IA30 viruses may
be influencing pH1N1 pathogenicity and transmission or ac-
count for the observed differences in host lipid and immune
responses.
This study demonstrates the importance of using high-through-
put 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 influ-
enza 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), circo-
virus (27), Actinobacillus pleuropneumoniae (13, 36, 48), and
Escherichia coli (6). Our data suggest that global transcription
profiling is a promising avenue for uncovering novel mecha-
nisms of response to influenza virus infection in swine. The
differential response signatures obtained in our study are in-
dicative of disparate host responses leading to differences in
clinical response, despite similar virologic findings.
pH1N1 viruses have been isolated from swine herds world-
wide, indicating that they are cocirculating in the pig popula-
tion with currently circulating SIVs. Furthermore, reassort-
ment 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
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 each infection group and
a mock group were euthanized at 3 and 5 dpi, total RNA was isolated from lung tissue, and global gene expression was determined by microarray
analysis. (A) Heat map depicts cell death and lipid metabolism genes (Table 4) that were differentially expressed between infection conditions at
5 dpi. Gene expression for each animal is shown as the log (ratio) of expression relative to that of experiment and time-matched samples extracted
from lungs of mock-infected pigs. Red indicates that the gene expression is higher than mock; green indicates that gene expression is lower than
mock. (B) Network diagram depicts the top-scoring network of genes as determined by Ingenuity Pathway Analysis (Ingenuity Systems) of the
differentially expressed genes at 5 dpi. Shading of the molecules reflects the average expression compared with mock values in each group. Red
indicates that the gene expression is higher than mock infection values; green indicates that gene expression is lower than mock values. Molecules
that changed less than 1.5-fold from mock infection values are shaded in gray. Groups or complexes of molecules are shaded in white.
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virus genes may potentially reassort with mammalian-adapted
viruses, which could lead to enhanced cross-species transmis-
sion. 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 addi-
tion to causing high mortality. This reassortant virus may be-
come 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.
ACKNOWLEDGMENTS
We thank Vani Shanker (Department of Scientific Editing at the St.
Jude Children's Research Hospital) for critical review of the manu-
script and Ruben Donis (CDC) for providing the 2009 human pan-
demic 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 ac-
knowledge the assistance of the Histopathology Section at the Depart-
ment 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 analy-
sis 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, Na-
tional Institutes of Health, Department of Health and Human Ser-
vices (P51 RR00166-45).
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