﻿Phenotypic Differences in Virulence and Immune
Response in Closely Related Clinical Isolates of Influenza
A 2009 H1N1 Pandemic Viruses in Mice
Jeremy V. Camp1
, Yong-Kyu Chu2
, Dong-Hoon Chung1,2
, Ryan C. McAllister1
, Robert S. Adcock1
,
Rachael L. Gerlach1
, Timothy L. Wiemken3
, Paula Peyrani3
, Julio A. Ramirez3
, James T. Summersgill3
,
Colleen B. Jonsson1,2
*
1 Department of Microbiology and Immunology, University of Louisville, Kentucky, United States of America, 2 Center for Predictive Medicine for Biodefense and
Emerging Infectious Diseases, University of Louisville, Kentucky, United States of America, 3 Division of Infectious Diseases, Department of Medicine, University of
Louisville, Louisville, Kentucky, United States of America
Abstract
To capture the possible genotypic and phenotypic differences of the 2009 influenza A virus H1N1 pandemic (H1N1pdm)
strains circulating in adult hospitalized patients, we isolated and sequenced nine H1N1pdm viruses from patients
hospitalized during 2009­2010 with severe influenza pneumonia in Kentucky. Each viral isolate was characterized in mice
along with two additional H1N1 pandemic strains and one seasonal strain to assess replication and virulence. All isolates
showed similar levels of replication in nasal turbinates and lung, but varied in their ability to cause morbidity. Further
differences were identified in cytokine and chemokine responses. IL-6 and KC were expressed early in mice infected with
strains associated with higher virulence. Strains that showed lower pathogenicity in mice had greater IFNc, MIG, and IL-10
responses. A principal component analysis (PCA) of the cytokine and chemokine profiles revealed 4 immune response
phenotypes that correlated with the severity of disease. A/KY/180/10, which showed the greatest virulence with a rapid
onset of disease progression, was compared in additional studies with A/KY/136/09, which showed low virulence in mice.
Analyses comparing a low (KY/136) versus a high (KY/180) virulent isolate showed a significant difference in the kinetics of
infection within the lower respiratory tract and immune responses. Notably by 4 DPI, virus titers within the lung,
bronchoalveolar lavage fluid (BALf), and cells within the BAL (BALc) revealed that the KY/136 replicated in BALc, while KY/
180 replication persisted in lungs and BALc. In summary, our studies suggest four phenotypic groups based on immune
responses that result in different virulence outcomes in H1N1pdm isolates with a high degree of genetic similarity. In vitro
studies with two of these isolates suggested that the more virulent isolate, KY/180, replicates productively in macrophages
and this may be a key determinant in tipping the response toward a more severe disease progression.
Citation: Camp JV, Chu Y-K, Chung D-H, McAllister RC, Adcock RS, et al. (2013) Phenotypic Differences in Virulence and Immune Response in Closely Related
Clinical Isolates of Influenza A 2009 H1N1 Pandemic Viruses in Mice. PLoS ONE 8(2): e56602. doi:10.1371/journal.pone.0056602
Editor: Earl G. Brown, University of Ottawa, Canada
Received October 24, 2012; Accepted January 14, 2013; Published February 18, 2013
Copyright: ß 2013 Camp et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: Support was provided in part by the Commonwealth of Kentucky as a Clinical and Translational Science Pilot Project Program at the University of
Louisville to CBJ. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: cbjons01@louisville.edu
Introduction
The 2009 pandemic H1N1 influenza virus (H1N1pdm) arose
through reassortment of two preexisting swine influenza viruses,
a Eurasian avian-like virus and a North American triple
reassortant virus [1­3]. The risk factors associated with human
cases of H1N1pdm mirrored those of seasonal influenza [4]. As
observed with seasonal influenza, the most common underlying
chronic conditions among hospitalized patients were respiratory
disease, asthma, cardiac disease, and diabetes [4­9]. However, in
contrast to seasonal influenza, a greater proportion of severe and
fatal cases had a pre-existing chronic illness. A second notable
difference was the age distribution of hospitalized and severe cases.
Children less than 17 years old had the greatest rates of
hospitalization per capita and adults over 64 had the greatest
rates of mortality per capita. Retrospective clinical studies focused
on surveillance of H1N1pdm sequences present in ICU admissions
suggest that pre-existing medical conditions may be a more
important factor in severity rather than particular viral variants
[10]. However, this does not explain why a greater proportion of
persons with pre-existing medical conditions had more severe
disease than typically observed for seasonal influenza. Further,
previously unreported comorbidities such as morbid obesity have
been widely suggested for H1N1pdm for increased risk for
admission to the ICU and death [11]. At this time, it is difficult
to rule out the contribution of viral variants to the resulting illness
observed with the various comorbidities. Nonetheless, the course
of illness and the progression to more severe disease are most likely
due to the combined interplay of the individual's health, the
intrinsic phenotype of the infecting viral variant and the treatment
regime.
In contrast to seasonal influenza viruses, the H1N1pdm viruses
replicate well and show greater pathogenicity with viral antigen in
the bronchiolar epithelia and the alveolus by day 3 post-infection
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(DPI) [2,3,12]. In the BALB/c mouse, A/California/04/09 and
other H1N1pdm viruses show lethality, but only at the highest
dose of 106.5
plaque forming units (PFU) [2,12]. Studies of
infection of BALB/c with several different 2009 H1N1pdm virus
isolates show high virus titers in nasal turbinates (NT) and lung
tissues by 3 DPI. Virus titers show a slight decrease by 6 DPI in
lung and NT although the decrease varies among strains, too.
Proinflammatory cytokines and chemokines were elevated for
most mice in whole lung specimens at 3DPI for KC, IL-6, IL-
12(p40), G-CSF, M-CSF, MCP-1 MIG, MIP-1b, and LIF. By 6
DPI, IL-10 was present, albeit low, whereas some of the cytokines
were reduced (e.g., MIP-2, IL-6), but most remained elevated
[2,12]. The levels of cytokines and chemokines in humans or mice
do not suggest hypercytokinemia common to H5N1 and 1918
viruses [13­15].
The overall genetic distance among H1N1pdm isolates
remained low with 7 distinct clades in the first wave of the
pandemic, while in the second wave a single viral lineage
dominated [16]. Molecular surveillance, while important, will
not confirm potential phenotypic differences in vivo resulting from
amino acid changes associated with viral variants in newly
emerging strains or the potential for mixed infections and the
genetic diversity of the intrahost viral populations [17]. Animal
studies can complement molecular surveillance in monitoring the
potential pathogenicity and immunogenicity of circulating strains.
Further, such studies have potential to reveal the efficacy of
treatment regimes. Herein we report the isolation, sequence, and
characterization of nine H1N1pdm influenza A viruses from adult
patients hospitalized in Kentucky during the second pandemic
wave, September 2009 and April 2010. Four of the nine patients
died and all of the patients for whom data was available had an
underlying chronic condition. This group of clinical isolates, with
high genetic similarity, was characterized for virus load, virulence,
and host immune response in mice. Immune responses in the lungs
suggest four distinct immunological phenotypes that correlate with
the observed mortality in mice. This study underscores the
potential variability in the virulence of 2009 H1N1 influenza A
strains circulating in Kentucky during the pandemic. These data
suggest the hypothesis that the high severity of disease seen in
certain hospitalized patients may be related to infection with
H1N1pdm viral variants that, due to cell tropism and replication
levels, may exacerbate certain types of disease associated with
comorbidity.
Results
Isolation and Sequence Analyses of H1N1pdm Isolates
from Hospitalized Patients
H1N1pdm strains were isolated from nasopharyngeal swab
samples obtained from nine de-identified patients enrolled in the
Severe Influenza Pneumonia Surveillance (SIPS) project, a clinical
study of hospitalized patients with severe community-acquired
pneumonia in Kentucky from December 2008­ December 2011
(Table 1). Hospitals included in the SIPS project included two
rural areas of Kentucky, one in the east and one in the midwest,
and one in an urban area (Louisville). The midwest is active in
agriculture and one of the highest livestock producers of hogs and
pigs. The age of the patients ranged from 31 to 58 and included 5
females and 4 males. Many of those patients had underlying
comorbidities commonly associated with severe influenza disease
such as obesity, diabetes and chronic obstructive pulmonary
disorder (COPD) (Table S1). Four of the nine patients died.
Viruses were isolated by passage through both Madin-Darby
canine kidney (MDCK) cells and eggs and virus isolates were
designated according to a tracking number that contains no
patient identifier information. Herein, isolates will be referred to
by the tracking number and virus isolation method (e.g., A/
Kentucky/180/2010 egg-passaged isolate will be referred to as
``KY/180E''). Viruses were amplified and the virus titers were
determined by 50% tissue culture infectious dose (TCID50), plaque
forming units (PFU) and hemagglutination assays in MDCK cells
(data not shown). Viral titers measured in TCID50 and PFU assays
were similar for MDCK-adapted viruses and egg-adapted isolates
(e.g., median titer ,107
). Some viruses (A/KY/96/09, A/KY/
99/09, A/KY/108/09, A/KY/180/10) showed a 10­100 fold
higher titers in MDCK cell culture than in egg, but some (A/KY/
80/09, KY/110/09, KY/136/09) did not. Egg-adapted viruses
that were not recovered included A/KY/104/09 and A/KY/
190/10. Hemagglutination titers were generally lower from egg-
adapted viruses and showed variability among isolates (data not
shown).
Universal and gene-specific primers were designed and used to
amplify and sequence full-length cDNAs of all viral segments from
each isolate. The amino acid sequences encoded by each gene
were deduced and compared. All of the isolates have stop codons
in PB1-F2 at positions 12, 58, and 88 (data not shown) [18].
Amino acids that showed polymorphisms are listed for each viral
protein for the majority of genes (Table S2). The most amino acid
changes were observed in HA1 and acidic polymerase (PA) with
18 and 9 mutations collectively across the 7­8 the KY isolates (not
all isolates were completely sequenced). Neuraminidase and the
two basic polymerase subunits (NA, PB1 and PB2) each had 4­5
mutations while the nonstructural protein 1 (NS1) and HA2
showed 3 mutations collectively. In contrast, matrix (M1),
nucleoprotein (NP) had only 1 mutation and there were none in
M2, NS2 or PA-X. Of the mutations only a few have been
previously studied functionally to our knowledge. For example, in
the HA1/HA2 of KY/180E we noted changes in D222G, S83P,
S183P, and E374K. E374K was identified as a vaccine escape
mutation [19]. The S183P and the Q293H mutations in HA1
(found in KY/180E and KY/96E, respectively) have been
reported by others [20­23], and have been associated with
alterations in receptor binding and increases in disease severity in
humans [24,25].
Differential Progression of Virus Replication and Disease
in Mice Infected with H1N1pdm Strains
To make an initial assessment of the levels of replication and
virulence potential of each of the clinical H1N1pdm isolates,
groups of six DBA2 mice were intranasally-infected and monitored
daily for clinical signs and body weight. In addition to the nine
clinical isolates, we included two pandemic strains (A/CA/07/09
and A/NY/18/09) and a seasonal H1N1 strain (A/BN/59/07).
On days three and six, three mice each were euthanized and
analyzed for viral load and antibody titers (Table 2). All mice
infected with influenza strains showed weight loss (Table 2). Mice
infected with KY/96M, KY/80M, KY/180E, KY/80E, and NY/
18E showed the greatest weight loss in this short study (Table 2).
Mice infected with the seasonal influenza strain BN/59E showed
the least weight loss (about 9%). All other mice infected with
H1N1pdm influenza isolates (KY/136M, KY/99M, KY/136E,
KY/110E, CA/07E, KY/108M, KY/110M, KY/104M, and
KY/108E) showed weight loss averages ranging from 13% to
26%, respectively.
The mortality rates for mock- and virus-infected groups were
expressed as a percentage of lethal infections (Table 2). BN/59,
CA/07, KY/110 and KY/136 showed no lethality and low
morbidity; whereas KY/190 and KY/180 showed the greatest
Phenotypic Differences of H1N1pdm Isolates
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lethality and substantial weight loss. Both KY/80 and NY/18
showed lethality and weight loss. On 3 and 6 days post-infection
(DPI), three animals were sacrificed and the lung tissues were
collected for virus titration analysis (TCID50 assay of lung tissue
homogenate using MDCK cells). Virus titers of lungs from each
mouse-infected group showed relatively little difference between
the various isolates on 3 and 6 DPI (p.0.05 using pairwise
Wilcoxon Rank-Sum test without correction for multiple compar-
isons, Table 2). Mice infected with egg-adapted viruses showed
a higher mortality than several of the corresponding MDCK
isolates (e.g., see KY/96, KY/99, KY/108). However, both egg
and MDCK-adapted KY/80 and KY/180 showed lethality in
mice.
The influenza-specific serum IgG antibody responses in mice
were measured at 6 DPI by ELISA using antigen prepared from
inactivated NY/18 virus (Table 2) and reciprocal endpoints were
determined. Strains KY/80E, KY/180, and KY/190 had no
detectable IgG antibody by 6 DPI. The seasonal strain and all
other pandemic strains produced detectable IgG antibody; the
highest endpoint titers were collected from BN/59, KY/108E,
NY/18E, KY99M, KY/136, and KY/110M. In some cases, these
numbers reflect a small numbers of animals remaining at 6 DPI.
Cytokine and Chemokine Responses in H1N1pdm
Isolates Show Four Distinct Phenotypic Profiles
Mouse cytokine and chemokine panels were employed to gain
a broad overview of the immune response at 3 and 6 DPI in the
lungs and sera of mice infected with each of the viral isolates
(Figures 1 and 2). No significant difference was seen between egg-
and MDCK-passaged isolates; therefore, the combined data are
presented in these two figures. There was an overall increase in
proinflammatory cytokines (TNFa, IL-1b, IL-6 and KC) albeit
different levels were noted. The levels of TNFa and IL-1b were
greater in the lungs on day 3 in viral isolates with greater lethality
(e.g., KY/180, KY/190) as compared to those with no lethality
(e.g., KY/136, p,0.05). These same cytokines were higher in the
mice with no lethality on day 6 as compared to those showing
lethality. A similar temporal pattern was noted with some
chemokines (e.g., RANTES, Figure 2). Interferon-gamma (IFNc)
and IL-10, an anti-inflammatory cytokine, were present on day 6
in mice infected with all but the most lethal strains, KY/180, KY/
190 (Figure 1, p,0.05).
With the exception of isolate KY/110 and KY/104, the
chemokines CCL2 (MCP-1), CCL3 (MIP1a), CXCL9 (MIG), and
CXCL10 (IP-10) displayed similar levels in all isolates on 3 DPI
followed by a relative increase in animals infected with nonlethal
versus a decrease in animals infected with lethal isolates at 6 DPI
(Figure 2 and Table 2). IFNc stimulates both IP-10 and MIG, and
as expected the IFNc levels were low in lethal isolates as compared
to nonlethal isolates at 6 DPI. Cytokines and chemokines that
showed limited response in mice with any viral infection included
Eotaxin, GM-CSF, IL-1a, M-CSF, IL-2, IL-3, IL-4, IL-5, IL-7,
IL-9, IL-12p40, IL-13, IL-15, IL-17, MIP2, LIX, MIP1b,
RANTES, IL-12p70, and VEGF (data not shown).
A principal components analysis (PCA) was performed using 11
cytokine/chemokine measurements from the lungs of mice
infected with each virus (Figure S1). The cytokines and
chemokines included in the analysis were selected because they
were determined to be the most significantly different between
isolates on 3 and 6 DPI by generalized linear model fitness testing
(data not shown). The first two components explained 72% of the
variance (Figure S1) and the third component explained an
additional 10% (data not shown). CCL3, TNFaa, IL-10, IL1b,
and IFNc were all highly correlated with the first component
dimension (.80%), meaning these cytokines were principally
important in differentiating the isolates. CXCL10, KC and G-CSF
were similarly highly correlated with the second component
(.70%), and CXCL9 was highly correlated with the third
component dimension (83%, data not shown). The remaining
variables, IL-6 and CCL2, were not highly correlated with the
components used in this model, although they were significantly
different between isolates on 3 and 6 DPI. The mean for each
isolate on 3 and 6 DPI are plotted according to the coordinates of
the first two components in Figure 3 (and Figure S1).
The method used for computing this PCA is similar to k-means
cluster analysis, and therefore we expected to observe the isolates
to group according to their cytokine/chemokine signatures. To
better visualize these clusters, we connected each isolates' 3 DPI
coordinate to its 6 DPI coordinate with an arrow (Figure 3).
According to this method of using a ``time-resolved'' PCA, the
isolates were observed to cluster clearly into four groups
representing various trajectories of disease.
The first three groups of isolates could be differentiated by their
immunogenicity and low to moderate lethality. The first group
Table 1. General Patient Data for Nasal Swabs Used for Virus Isolation.
Sample ID/Locality
Code* Patient Age Sex
Hospital Admission
Date Nasal Swab Date LOS Y
(Days) Comorbidity Mortality
80/3 31/F 9/30/2009 9/30/2009 10 COPD, MRSA Died
96/2 51/M 10/24/2009 10/26/2009 4 BMI = 123.6 Survived
99/3 35/M 10/28/2009 10/29/2009 3 ND Survived
104/2 58/M 10/30/2009 11/4/2009 2 ND Died
108/6 57/F 11/2/2009 11/3/2009 19 ND Survived
110/6 54/F 11/3/2009 11/4/2009 3 COPD, diabetes, renal
disease, BMI = 79.7
Survived
136/3 46/M 12/10/2009 12/11/2009 8 Diabetes, MRSA Survived
180/2 53/M 3/24/2010 4/1/2010 19 COPD, renal disease Died
190/2 55/F 4/10/2010 4/15/2010 12 ND Died
*locality, 2 = western Kentucky (KY); 3 = eastern KY; 6 = Louisville metro;
Y
LOS, Length of stay in hospital; MRSA, methicillin-Resistant Staphylococcus aureus; COPD, chronic obstructive pulmonary disease; BMI- body mass index; ND, none
determined.
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Phenotypic Differences of H1N1pdm Isolates
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includes isolates with a low lethality in mice, and was exemplified
by infection with the seasonal strain BN/59, and 2009 pandemic
strains CA/07, KY/80 and KY/136. The second group (KY/96,
KY/99, and KY/104) included those strains with moderate
lethality in the mouse model, and was similar to the first group in
course of disease, but was characterized by increased inflamma-
tory cytokines (e.g., IL-6, KC-like, and G-CSF) and chemokines
(e.g., CXCL9). In contrast to the first group, these had a higher
level of the anti-inflammatory cytokine IL-10 later in disease, as
well as increased IFNc at 6 DPI. The third group of influenza
strains (KY/108 and KY/110) was the most immunogenic
compared to the other isolates, and differed from the two groups
above in that they showed the highest levels of inflammatory
cytokines late in infection (e.g., IL-6, TNF-a, IL-1b) but were
similar to Group 2 in terms of lethality. They showed the highest
level of chemoattractant chemokines, particularly CCL2, CCL3,
and CXCL10, and produced the highest levels of IL-10 and IFNc
detected in this study.
Finally, the fourth group of isolates, consisting only of KY/180,
KY/190, and NY/18, were the most lethal of the viruses screened
in mice. Their proinflammatory cytokines were elevated through-
out the course, but not different from 3 DPI in Group 1 isolates.
Most notably, these isolates failed to produce any IFNc or IL-10
by 6 DPI. In Figure 3, the trajectory of these isolates point in an
opposite direction from all other isolates, perhaps indicating
a different course of disease. In summary (see Table S5), the
immune responses of the virus isolates clustered similar to other
phenotypic markers of virulence and lethality (Table 2).
In-depth Comparison of the Temporal Progression of
Survival, Viral Load and Immune Responses of Two
Clinical Isolates, KY/136E and KY/180E, with Low and
High Virulence, Respectively
KY/136E and KY/180E were selected from the nine
H1N1pdm clinical isolates for further characterization as repre-
sentatives of lower and higher virulence based on the apparent
disease in mice. These isolates had similar concentrations of lung
cytokines and chemokines on 3 and 6 DPI compared to other
isolates and clustered together in the PCA. However, the time-
resolved PCA revealed that the isolates differed in the progression
of immune responses in the course of infection. To gain additional
insight, we first assessed dose response to infection in DBA/2 mice.
Mice were infected with 100
, 102
or 105
TCID50 of KY/180E or
KY/136E and examined daily for clinical signs. Each day, mice
were weighed and data from each dose group are presented in
Figure 4A (KY/136E) and Figure 4B (KY/180E). The Kaplan-
Meier survival curves of mice infected with KY/180E or KY/
136E (Figure 4C and 4D, respectively) confirmed the observations
reported above in terms of the general lethality of each virus,
although KY/136E did show lethality in 1­2 mice on 10 DPI
(Figure 4C). Mice infected with KY/180E succumbed to infection
starting at 3 DPI at the high dose and on 6 DPI for the middle
dose (Figure 4D). Animals were humanely euthanized upon
showing a moribund state or upon a 25% loss in body weight as
described in the materials and methods.
The levels of infectious virus present in the upper and lower
respiratory tracts, nasal turbinates (NT) and lung, respectively,
were measured at 1, 3 and 5 DPI at the three different doses (100
,
102
or 105
PFU per mouse) of KY/180E or KY/136E (Table S3).
As expected, levels of virus were greatest in mice with higher doses
of infection. Mice infected with KY/180E persisted at higher levels
of virus over the 5 day period as compared to KY/136E which
had approximately 2­3 logs lower virus in the lungs and had virus
levels that decreased from 3 to 5 DPI. The infectious dose (ID50)
and lethal dose (LD50) were ,100
TCID50/mouse and 101.2
TCID50 per mouse, respectively, for KY/180E (data not shown).
To further dissect the immune responses of these two viruses,
multiplex cytokine/chemokine bead arrays were employed to
provide insight into the temporal patterns for the key cytokine
and chemokine profiles noted in the broad survey of all the
Table 2. Summary of viral titers, lethality of H1N1pdm
isolates in DBA2 mice.
Inoculum DPI Virus titer (Lung) Lethality
Mock 3 ,1.5 * 0.0
6 ,1.5 * 0.0
KY/80M 3 7.260.5 0.0
6 6.460.9 33.3
KY/80E 3 7.660.1 0.0
6 7.060.3 33.3
KY/96M 3 7.060.5 0.0
6 7.260.5 0.0
KY/96E 3 7.360.4 0.0
6 7.460.4 100.0
KY/99M 3 6.460.2 0.0
6 5.860.7 0.0
KY/99E 3 7.460.9 0.0
6 7.360.3 33.3
KY/104M 3 6.660.9 0.0
6 6.960.5 33.3
KY/108M 3 7.560.6 0.0
6 6.660.4 0.0
KY/108E 3 6.560.5 0.0
6 6.460.8 33.3
KY/110M 3 5.960.4 0.0
6 6.260.9 0.0
KY/110E 3 5.960.2 0.0
6 6.860.7 0.0
KY/136M 3 5.360.3 0.0
6 5.860.9 0.0
KY/136E 3 5.860.4 0.0
6 5.260.5 0.0
KY/180M 3 6.860.3 0.0
6 5.760.7 100.0
KY/180E 3 6.960.4 33.3
6 6.460.1 66.7
KY/190M 3 6.860.5 0.0
6 6.360.3 100.0
NY/18E 3 6.960.6 0.0
6 6.861.0 33.3
CA/07E 3 7.260.6 0.0
6 6.960.8 0.0
BN/59E 3 6.760.0 0.0
6 5.960.1 0.0
Legend: Virus titer (log10 TCID50/mL at a limit of detection = 101.5
TCID50/mL).
doi:10.1371/journal.pone.0056602.t002
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isolate. Mice (5 per group/per day) were infected with each of
the isolates and humanely euthanized on 1, 3, and 5 DPI
(Figure 5 and 6). There was an overall increase in proin-
flammatory cytokines (TNFa, IL-1b, IL-6 and KC) in the lungs
of all mice, however, there were much higher levels of all
cytokines in lungs of mice infected with KY/180E. Further the
responses were much earlier and correlated with the high levels
of infection noted in the lung. In contrast with KY/180E, KY/
136E-infected mice showed a gradual progression in immune
responses, however, the overall responses on 6 DPI of KY/
136E remained low. Interferon-alpha (IFNa) and IL-12p70
levels were higher in the lungs of KY/180E-infected mice as
compared to KY/136E-infected mice (Figure S2). Virus-infected
mice that recovered from KY/136E infection showed a high IL-
10 response by 6 DPI whereas KY/180E-infected mice showed
no response (Figures 1 and 5, p,0.05). As expected, and also
shown in Figure 1, the IFNc levels were low in the lethal KY/
180E isolate as compared to the robust response observed in the
Figure 1. Cytokine levels in mice infected with pandemic and seasonal H1N1 influenza viruses. Six to eight week old DBA/2 mice were
infected intranasally with 105
TCID50 with a seasonal virus isolate (BN/59), Kentucky (KY/80, KY/136, KY/96, KY/99, KY/104, KY/108, KY/108, KY/110, KY/
180, KY/190) or other H1N1 pandemic isolates (CA/07, NY/18) from 2009. Cytokine levels were measured at 3 and 6 DPI as described in the materials
and methods and presented as mean +/2 SEM (n = 6 per group, although fewer animals were available for lethal isolates).
doi:10.1371/journal.pone.0056602.g001
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nonlethal KY/136E isolates at 6 DPI (no statistically significant
difference).
Mice infected with the middle dose of KY/180E that survived
after 6 DPI, and produced high concentrations of IFNc, eventually
became moribund with all but one euthanized by Day 10. These
data suggest that the immune response differences between the
two isolates are due to the high levels of infection in the lung by
KY/180E. The levels of cytokines and chemokines in the lung
support this observation (Figures S3­S6), showing a dose response
for both isolates on 1, 3, and 5 DPI in mouse lung homogenate.
Notably, a dose of 102
pfu/mouse of KY/180E showed a pro-
gressive increase in chemotactic chemokines to 5DPI whereas
a higher dose (105
PFU/mouse) began declining in concentration
of these analytes after 3DPI (Figure S3). Similar patterns were seen
for proinflammatory cytokines in the lung (Figure S5) and
cytokines involved in adaptive immunity initiation, IFNc and
IL12p70 (Figure S6). The endpoint titer IgG responses of the mice
for the low and middle doses were similar for both viruses
(Figure 7; all significantly different from control using Student's t-
test, p,0.05). Further, the HI titers were also very similar except
Figure 2. Chemokine levels in mice infected with pandemic and seasonal H1N1 influenza viruses. Six to eight week old DBA/2 mice were
infected intranasally with 105
TCID50 with a seasonal virus isolate (BN/59), Kentucky (KY/80, KY/136, KY/96, KY/99, KY/104, KY/108, KY/108, KY/110, KY/
180, KY/190) or other H1N1 pandemic isolates (CA/07, NY/18) from 2009. Chemokine levels were measured at 3 and 6 DPI as described in the
materials and methods and presented as mean +/2 SEM (n = 6 per group when possible).
doi:10.1371/journal.pone.0056602.g002
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for the lower dose, which was about 2-fold higher in mice infected
with KY/180E (Figure 7).
The leukocyte chemoattractant chemokines CCL2, CXCL10,
KC (CXCL8-like), and G-CSF were greater and earlier in the
lungs of mice infected with high doses of KY/180E than with
a similar dose of KY/136E (Figure 6, p,0.05). As expected,
cytospins of BAL revealed that KY/180E contained more
neutrophils and monocytes on Day 4 compared to KY/136E
(Figure 8A,B, p,0.05). Additionally, we observed a higher pro-
portion of macrophages in the lungs in the low dose group of KY/
136E compared to the higher dose group (p,0.05, Figure 8C).
Despite there being a greater number of infiltrating leukocytes in
the lungs of KY/180E-infected mice, these mice had a delayed
clearance of virus in the lung.
Given the apparent clearance of virus from the lungs of KY/
136E (Table S4), we were interested in whether the apparent
differences in lethality of the two viruses could be due to the site of
virus replication. We tested virus titers from bronchoalveolar
lavage fluid (BALf), from the cellular pellet of the BALc (BALc) of
the mice, and from the homogenized lung after lavage using
TCID50 assay. Samples were collected on 3 and 4 DPI because we
began to see decreases in viral titer in the lungs of KY/136E-
infected mice after 3DPI. In our earlier studies (Table S4), the
virus titer in lung reflects lung, BALf, and BAL cells combined and
we observed a nearly 3 log difference between KY/136E and KY/
180E. When BAL is collected before isolation of lung tissue, we
found the majority of infectious KY/136E virus present in lungs of
mice was localized in the cellular fraction of the BAL (Figure 9). In
contrast to KY/180E, no infectious KY/136E virus was present in
the BALf by 4 DPI. Flow cytometric analysis revealed that the
influenza NP-positive cells in the BAL from both isolates were also
Gr1-positive (clone RB6-8C5), a marker of mouse phagocytic
leukocytes, i.e., Ly6C/Ly6G positive (Figure 8D).
Infection and Replication of H1N1pdm Viruses in
Macrophage Cell Lines
Given the differences noted within the levels of virus in BALf
and BALc, we used a BALB/c mouse macrophage cell line
(RAW264.7) and a C57BL/6 mouse macrophage cell line (NR-
9465) to test for the ability of KY/136E and KY/180E to infect
and replicate in macrophage cells. Confocal microscopy at 24
hours post-infection showed that both viruses were able to infect
macrophage cell lines (Figure 10). However, KY/180E was more
efficient at replication, reaching 10-fold higher virus titers that
persisted longer in both cell culture systems (Figure 11 and Figure
S7). Although the cell culture supernatant was positive at 24 hours
post-infection for both viruses in vitro, KY/180E was the only
isolate detected from the cellular component in BAL (Figure 8;
p,0.05 at 24 and 48 hours post-infection). These observations
agree with our findings in the DBA/2 mouse model and suggest
that KY/180 is more successful in vivo in replication and
production of infectious virus in macrophages (Figure 8).
Discussion
Herein we present data characterizing several isolates of
H1N1pdm influenza virus taken from human patients with severe
pneumonia in Kentucky, USA. Clinical surveillance from foci
around the world showed that the majority of human cases of
H1N1pdm were mild [26,27]. Severe disease from H1N1pdm
infection was more likely to be seen in patients with pre-existing
chronic illness [28]. It was initially observed that antigenic and
genetic variation in circulating pdmH1N1 2009 influenza viruses
was less than what is seen during seasonal human influenza A
(H1N1) [29]. Similarly, the isolates listed here show relatively low
variation, differing at only 43 amino acid positions. Each isolate
contained from 2 to 12 unique non-synonymous mutations from
the consensus sequence.
Three of the four lethal cases occurred in western Kentucky,
and 1 of these lethal cases (KY/180) contained a virus isolate
with the avian-like HA RBS (G222). The D222 is most
commonly associated with human H1 strains while the G222 is
common in avian strains. The HA 222 position, D222G/N/S/
E/Y, was a common polymorphism in severe cases of influenza
in human patients, which is within the HA receptor binding
site, RBS [10,30­32] [33]. Additionally, the D222G has been
associated with increased pathogenesis in some animal models
possibly due to increased replication of the virus [30,31,34­38].
The aspartic acid functions in binding of the receptor and as
a calcium antigenic site. Each of these amino acids will give rise
to a specific binding affinity to the a2-6-sialic acid receptor with
the D222G increasing a2-3-sialic acid receptor binding speci-
ficity [35,37,39]. However, it is clear that other amino acids
constellations at positions 183, 186, 187, 216 and 224 will also
influence these interactions [3], and may result in different
overall outcomes. Additional amino acid signatures in closely
related H1N1pdm isolates have also been implicated in
differences in virulence in animal models. For example, in
Figure 3. Principal components analysis of immune responses
in lungs of mice to infection with pandemic and seasonal
influenza viruses. A principal components analysis was performed
using the 14 cytokines/chemokines analytes shown to be the most
significantly different across all isolates from days 3 and 6 (determined
by generalized linear model fitness testing, data not shown). The data
were normalized and scaled (zero mean-centered) cytokine responses
after influenza infection at 3 and 6 DPI for all viruses. The ordinate and
abscissa represent the first and second components from the PCA,
which explain approximately 72% of the variance. Each arrow
represents the mean of a virus isolate tested in mice. Arrow tails
represent day 3 components and arrow heads represent day 6
components. Using this tool to visualize the immune response, the
arrows depict the trajectory of disease of the various influenza isolates
as tested in DBA/2 mice. Additionally, the 12 virus isolates clustered into
four distinct patterns: Group 1, BN/59, CA/07, KY/80, KY/136; Group 2,
KY/96, KY/99, KY/104, KY/108; Group 3, KY/108, KY/110; Group 4, KY/
180, KY/190, NY/18 (See Table S5).
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a study in a cynomolgus macaque model of two highly similar
strains of H1N1pdm, A/Mexico/4487 and A/Mexico/4108,
showed notable heterogeneity in virulence [40]. The amino acid
variations responsible for the differences noted in these studies
have not yet been reported. The HA1 of KY/180E also has
a P183 (noted as 186 in [20]) rather than a S183 at the Sb
antigenic site, which also has been noted to affect receptor
specificity ([21­23]). This site, independently or together with
the D222G mutation, is thought to allow binding to a-2,3
linked sialic acid residues. This substitution may allow the virus
to more efficiently enter and replicate in the lower airway
epithelium [23,41­43].
Other mutations observed in the HA gene of the Kentucky
isolates have also been reported by others. Belser, et al. (2010)
published data that showed A/CA/04/09 also has the S83P
mutation in HA (as in KY/180E), although this mutation is not
a known pathogenicity determinant [12]. Ilyushina, et al. (2010)
found the S183P (as seen in KY/180E) mutation arose from
serial passage of A/CA/04/09 in mice and resulted in increased
pathogenicity [21]. Xu, et al. (2011) report isolates from China
show similar variability and share some of our mutations in HA
(S83P, T203S, and V321I; seen in KY/180E, KY/96E, and
KY/80E, respectively), as well as mutations in NS1 (V123I,
seen in KY/99E) and PA (V14I, seen in KY/180E) [44].
Melidou et al. have data that suggest V321I mutation in HA
may be associated with increased disease severity [24]. Finally,
HA1 amino acid mutation Q293H, identified in KY/96E, has
been associated with increased severity of disease observed in
human cases during the pandemic [24,25]. We include
a summary of those mutations that correlate with virulence in
humans and mice (Table S6). An understanding of these and
other signatures associated with more virulent phenotypes will
benefit insight into the biology of the virus, patient management
and public health responses.
In total, KY/180E contains mutations that differ from KY/
136E in seven amino acid sites in the HA protein, four sites in PA,
two sites in NA, PB1 and PB2, and one site in each of M1, NS1,
and NP. Comparison of the CA/07/09, NY/18, NL/602 isolates
with the KY isolates showed additional variation among all isolates
(Table S2). Specifically, CA/07/09, NY/18, NL/602 were similar
to the KY consensus in M, HA2, PA, PB1 and PB2, In HA1,
a Q223R occurred in NY/18 only. In NA, a D248N changed
occurred in CA/07 and NL/602. In NS1, a D247N was noted for
KY/110, CA/07, NY/18, and NL/602. Most additional changes
were conserved hydrophobic changes such as Val to Iso or Met to
Leu. No differences were observed among the KY, CA/07/09,
NY/18, NL/602 isolates in M2 or NS2 (Table S2). Despite the
small apparent genomic variation between H1N1pdm isolates, we
and others have shown that there exists substantial variation in the
course of disease, pathogenicity, and immune response in human
patients, in animal models, and in primary cell culture [25,40,44­
46]. As virulence determinants of influenza viruses typically
involve the genes encoding the HA, NA, and polymerase proteins,
we would expect changes in these genes to be of future interest for
Figure 4. Weight loss and Kaplan-Meier curve of mice infected with KY/136 and KY/180. Mean weight change (+/2 SEM) after six to eight
week old DBA/2 mice were infected intranasally with 100
, 102
and 105
TCID50 with KY/136E (A) or KY/180E (B). Mice were examined daily for clinical
signs and weighed (n = 10 mice per virus group). Kaplan-Meier Survival curves for these mice show KY/136E to have low lethality (C) compared to KY/
180E (D).
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biological function [47]. Addition genome diversity that might give
rise to unique phenotypes during infection may be revealed be
deep sequencing or isolation of clones that sample the population
of the virus [17].
To gain insight into the potential phenotypic variability inferred
from the genotypic variability each virus was screened in DBA2
mice. All IAV tested so far, H1N1pdm, 1918 H1N1, and seasonal
influenza A H1N1 virus, infect DBA2 with varying levels of
Figure 5. Cytokine levels in mice infected with KY/136 or KY/180. The levels of notable cytokine responses are shown for 1, 3 and 5 DPI in six
to eight week old DBA/2 mice that were infected intranasally with 105
TCID50 of KY/180E or KY/136E (n = 10 mice per virus group per time point).
Statistical significance was determined by day using Kruskal-Wallis test followed by pairwise Wilcoxon Rank Sum post hoc test with Holm's
adjustment for multiple comparisons. P-values ,0.05 are indicated by the following method: ``a'' = KY/180 is significantly different from mock;
``b'' = KY/136 is significantly different from mock; ``c'' = KY/180 is significantly different from KY/136.
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Figure 6. Chemokine levels in mice infected with KY/136 or KY/180. The levels of notable chemokine responses are shown for 1, 3 and 5 DPI
in six to eight week old DBA/2 mice that were infected intranasally with 105
TCID50 of KY/180E or KY/136E (n = 10 mice per virus group per time
point). Statistical significance was determined by day using Kruskal-Wallis test followed by pairwise Wilcoxon Rank Sum post hoc test with Holm's
adjustment for multiple comparisons. P-values ,0.05 are indicated by the following method: ``a'' = KY/180 is significantly different from mock;
``b'' = KY/136 is significantly different from mock; ``c'' = KY/180 is significantly different from KY/136.
doi:10.1371/journal.pone.0056602.g006
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lethality without adaptation [48], and express comparatively high
levels of proinflammatory cytokines and chemokines [49,50].
Herein all DBA2 mice showed high levels of infection with all
isolates similar to infection of BALB/c mouse with H1N1pdm
viruses [2,12] with relatively little difference between the various
isolates on 3 and 6 DPI (p.0.05 using pairwise Wilcoxon Rank-
Sum test without correction for multiple comparisons, Table 2).
However, the isolates did show three groupings in lethality (lethal,
moderately lethal and not lethal) in DBA2 mice with KY/180,
KY190 and KY/96 showing the highest lethality. Using a statistical
method, PCA, we asked if based on these differences in lethality
we might be able to cluster cytokine and chemokine responses
across the H1N1pdm isolates. To our knowledge, this is the first
time this method (a ``time-resolved'' PCA) has been used to
discriminate phenotypic characteristics of viruses. Classically PCA
is used as a method to reduce multivariate data so that they are
better suited for a predictive statistical model. The most common
contemporary usage of PCA in microbiology is during the analysis
of gene expression arrays to check reproducibility of replicates. We
chose to use the PCA method because of its similarity to common
clustering algorithms (specifically, k-means clustering), and there-
fore its potential to elucidate clusters of the immune response of
mice after infection with genetically similar influenza isolates. Our
approach relied on two defined time points to detect differences
between virus isolates using expression of multiple immune
markers. Although some of the markers used in this analysis
may be redundant in a virus model (e.g., pro-inflammatory IL-6
and CXCL10), subtle differences that may go unnoticed are seen
using this multivariate approach, particularly in the timing of these
responses (and not necessarily the magnitude). Therefore, using
indicators of adaptive immune priming (IFNc) and reduction of
inflammation (IL-10) were critical to the successful implementa-
tion of this strategy. More data of this kind from other influenza
isolates and other respiratory virus infections may assist in the
development of this method, and may reduce the number of
soluble immune markers needed for analysis in a clinical setting.
Analyses of our data suggest that there are at least two main
trajectories of H1N1 influenza infection in these mice: one that
successfully resolves infection, and one that does not. The majority
of the isolates followed a trajectory that led to resolution and
clearance of the virus from the lungs. These isolates could be
further clustered into three subgroups, and these subgroups differ
according to the overall immunogenicity of the isolates (i.e., the
levels of cytokines/chemokines in the lung). In general, the isolates
showing the least mortality in DBA2 mice share in common
a trajectory of disease that is characterized by a gradual increase,
then decrease, in inflammatory cytokines and leukocyte chemoat-
tractant chemokines, and concomitant increase in anti-inflamma-
tory cytokines and IFNc. The isolates causing the highest lethality
in the DBA2 mouse (KY/180E, KY/180M, and KY/190M)
showed an early rise in the proinflammatory cytokines and
chemokines, and a delayed or absent rise in IL-10 and IFNc. The
lethal isolates were clearly differentiated using the time-resolved
PCA in that they showed a trajectory that was opposite of all other
isolates. The small sample size of human patient data and their
confounding comorbidities limit the inferences that can be made
connecting locale, age, sex, and length of hospital stay with
genotype and pathogenicity of the isolates in the human patients.
Recent data suggest that human polymorphisms in genes that
restrict virus infection (e.g., IFITM3 gene) may also play an
important role in outcome [51]. A final challenge is there are no
relevant scoring systems for influenza pneumonia. A pneumonia
severity index (PSI) is commonly used in hospitals by clinicians for
community acquired pneumonia, but the comorbidities (e.g. high
BMI, diabetes) complicate interpretation.
Selecting two isolates, KY/180E and KY/136E we sought to
better define two of these trajectories in DBA2 mice. We initially
observed KY/136E had a low level of immunogenicity in mice,
which included an increase in IL-10 and IFNc by day 6. Although
mice infected with KY/136E are similar to KY/180E in
concentration of IFNa, G-CSF, CCL2, KC, IL-6, CXCL10,
and other inflammatory cytokines and chemokines in the lung, the
isolates differ in the timing of these responses. Increases in these
cytokines and chemokines are typically seen during infections with
H1N1pdm influenza isolates [2,52,53]. For example, some studies
show that IL-6 is released in response to influenza infection and
that levels of IL-6 in the upper respiratory tract and in blood
correlate with symptoms [54­56].
Type-I interferons are known to initiate the anti-viral response
to influenza virus infection. One hypothesis to explain a more
severe course of illness is that the virus is capable inhibiting the
effect of type-I interferon signals via NS1 protein [57,58]. The
NS1 protein from KY/136E differs from KY/180E in a single
amino acid at site 112 (112M versus 1121I, respectively). By day 1
post-infection, mice infected with KY/180E had a higher level of
IFNa in the lung. Despite an increased IFNa and IL12-p70
response, mice infected with KY/180 failed to produce IFNc, IL-
10, and serum antibodies by 6 DPI. This most closely resembles
the type of aberrant immune response seen during infections with
the pandemic 1918 Influenza A (H1N1) [59,60] and with severe
seasonal influenza isolates [61]. Others have shown similar
patterns of inflammation with isolates of pandemic 2009 H1N1
influenza [32,46,62].
Cytokines and chemokines such as CCL2 (MCP-1), CCL3
(MIP1a), CCL5 (RANTES), and CXCL10 (IP10) are responsible
for activating leukocytes and attracting them to the lung
compartment to clear infection [63]. Indeed, there were a higher
number of leukocytes, including elevated numbers of neutrophils
and monocytes, seen in the lungs of KY/180E-infected mice on
days 3 and 4 post-infection. It is known that certain pathogenic
H1N1 strains, such as the 1918 H1N1 strain, causes the increased
neutrophil recruitment to the lung due to increased chemokine
Figure 7. IgG responses of mice infected with different doses of
KY/136 or KY/180. Serum from day 20 post-infection was taken from
six to eight week old DBA/2 mice that were infected intranasally with
100
, 102
and 105
TCID50 of KY/180E or KY/136E (n = 10 mice per virus
group per time point). Influenza-specific (A/NY/18/09 BPL-inactivated
whole viral antigen) IgG titers were measured by ELISA and presented
as average log2 endpoint titers (+/2SEM). No mice survived to day 20 at
the high dose of KY/180. All endpoint titers for animals infected with
influenza virus isolates were significantly different from mock (p,0.05).
Endpoint titers for both IgG and HI from animals infected with KY/180
were significantly different from the same dosage amount of KY/136
(p,0.05).
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responses [64,65], which was seen with KY/180E. Neutrophils are
important for assisting in clearing influenza-infected cells from the
lungs directly and indirectly [66,67], but the precise mechanisms
remain to be discovered [64,68,69]. Although important to the
resolution of influenza virus infection, neutrophils contain
cytotoxic granules that may cause severe pathology, and may
contribute to morbidity and mortality [70]. An increase in
neutrophils late in disease confers the pathogenicity seen in highly
pathogenic strains of influenza viruses [69].
Natural Killer (NK) cells are known to be potent innate immune
cells that recognize influenza hemagglutinin [71]. Classically, NK
cell effector functions include release of IFNc and direct
cytotoxicity to influenza-infected cells by granule exocytosis (e.g.,
granzyme B and perforin). Mice infected with KY/180E have
higher numbers of peripheral NK cells at 3 DPI compared to KY/
136E using flow cytometry (data not shown). Consistent with this
finding, CCL5 (RANTES) was increased in the lungs and serum of
mice infected with KY/180E compared to KY/136E (Figure 2).
NK cell deficiency in mouse models of influenza infection leads to
increased pathogenicity [72,73]. Despite the increase in NK cells
in KY/180E-infected mice, it has been shown that some influenza
isolates may counteract this effector response, causing lysosomal
degradation of the f chain, which is a critical component of the
NK-activating receptor [74]. Additionally, DBA2 mice lack the
NKG2A activating receptor that is responsible for recognizing the
down-regulation of MHC-I that typically occurs with a viral
infection [75]. Therefore, KY/180E may be able to exacerbate
this deficiency by decreasing NK cell effector function. It has been
shown that NK cells also contribute to immune pathology in
response to influenza and other respiratory virus infections [76].
Finally, this study and others have shown that some strains of
influenza virus are capable of infecting macrophages [77­80], and
may alter their functionality in response to infection [81,82].
During influenza infection, alveolar macrophages are critical in
clearing virus from the lungs and are key producers of IFNc to
stimulate the adaptive immunity [64,65,76,83­88]. Upon in-
fection, influenza viruses may interfere with their normal function
[86,89,90]. Of the two isolates tested here, mice infected with KY/
136E showed clearance of virus from the lung by day 4.
[86,89,90]. Our in vitro data using mouse macrophage cell lines
indicates that KY/180E was able to produce infectious virus better
than KY/136E. Additionally, mice infected with KY/180E had
a higher number of Gr-1 positive infiltrating cells into the lungs by
day 4, a delayed IL-10 production, and very low levels of
Figure 8. Cells within bronchial lavage fluid of mice infected with KY/136 or KY/180. The cells within the bronchoalveolar lavage fluid
(BALf) at 4 DPI from DBA/2 mice infected with 105
TCID50 of KY/136E (A) or KY/180E (B) (n = 5 mice per virus group per time point) were affixed to
slides by cytospin centrifuge and stained (Kwik-Diff). Microscopic images from three fields per slide were counted by three blinded, independent
observers. (C) The average number of macrophages, non-specific monocytes, and neutrophils are presented as a percent of the total cell count (+/2
SEM). There were significantly more neutrophils in the BALf of mice infected with KY/180E virus (p,0.05, indicated by asterisks). (D) Cells in the BALf
were fixed, permeabilized, and stained with anti-influenza nucleoprotein (NP)-FITC antibody conjugate and analyzed by flow cytometry. There were
significantly more NP-positive cells in mice infected with KY/180 at a dose of 102
pfu, and in mice infected with KY/136 compared to mock-infected
controls (p,0.05, indicated by asterisks). 80% of NP-positive cells were Gr-1 positive (macrophage or neutrophil).
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IFNcresulting in a deficient virus clearance. Both our in vivo and
in vitro data show that KY/180E can infect macrophages, which
may affect the cell's ability to release IFNc and explain the
increase pathogenicity observed with this virus compared to KY/
136E. Future studies will focus on understanding the mechanism(s)
involved in the virus-induced deregulation of macrophage function
in vitro and in vivo. Understanding the genetic basis for the
differences in the interaction between the virus and macrophages
as well as other pulmonary and immune cells will contribute to
understanding the course of influenza infection in patients, and
assist in predicting potential virulence during future outbreaks.
Materials and Methods
Detection of Influenza in Clinical Nasopharyngeal Swab
Specimens
Nasal swab samples were provided by the SIPS project. A
nasopharyngeal swab was taken from all patients meeting the case
definition, which was defined as a patient admitted to an intensive
care unit with the physician diagnosis of community-acquired
pneumonia. The University of Louisville Institutional Review
Board Human Subject Protection Program Office (HSPPO)
approved this study prior to any data collection (#08.0399).
Informed consent was waived because it was a retrospective chart
review and the data were analyzed anonymously. The nasopha-
ryngeal swabs were taken as part of `standard of care' diagnosis.
Detection of influenza virus was accomplished using the Re-
spiratory Viral Panel (RVP) detection kit (Luminex Corp., Austin,
TX), recently approved for in vitro diagnostics by the FDA. The
RVP is a reverse-transcriptase, real-time PCR assay which is
multiplexed to detect 12 viral targets in a single reaction well.
Those targets include Influenza A, Influenza A-subtype H1,
Influenza A-subtype H3, Influenza B, RSV-A, RSV-B, Parain-
flunza 1, Parainfluenza 2, Parainfluenza 3, human metapneumo-
virus, Rhinovirus and Adenovirus. Nasal swabs were collected
from each patient and placed in Universal Transport Media
(Copan Diagnostics, Inc, Murietta, CA). Nucleic acid was
extracted using the QIAmp Mini-Elute Viral Spin KitH (Qiagen,
Valencia, CA) into a final volume of 50.0 ml. A 5.0 ml aliquot was
combined with 20 ml of RVP mastermix and processed through
several rounds of amplification, including a single-tube multiplex
reverse-transcriptase PCR, followed by a multiplex target-specific
primer extension protocol, a bead-hybridization step, and finally
a data acquisition step in the Luminex reader. Results are
qualitative and read-outs are Positive, Negative or a No Call
(equivalent).
Viruses and Virus Isolation
The viruses CA/07, NY/18, and BN/59 were kindly provided
by the Centers for Disease Control and Prevention, Virus
Surveillance and Diagnosis Branch, Influenza Division. Viruses
from clinical cases from KY present in nasopharyngeal swab
specimens were isolated and propagated in the chorioallantoic
cavity of 10-day-old embryonated chicken eggs (Charles River)
and MDCK cell line purchased from ATCC (CCL-34). The
allantoic fluid containing infectious particles or MDCK supernate
was harvested 72 h after inoculation. MDCK cells were infected in
DMEM virus culture medium (DMEM containing 0.2% BSA, 1%
PEN/STREP, and 2 mg/mL of Trypsin-TPCK). The infectious
virus titer of the resulting seed stock was determined by TCID50
and the titer calculated by Reed and Muench [91], and confirmed
by plaque assay on MDCK cells. Egg passage E2 was used for the
studies involving KY/180E and KY/136E reported herein.
RNA Isolation and Sequencing
To sequence each of the gene segments of each H1N1pdm
isolate, we followed the method described by Inoue et al [92]
with minor modifications. In brief, total RNA was isolated with
MagMAX AI/ND Viral RNA isolation kit (Ambion) from each
virus seed stock solutions. Five mL of RNA was used to
synthesize cDNA with SuperScriptase III (Invitrogen) using the
FWuni12 and RVuni13 primers based on those reported by
Inoue et al [92] and the cDNAs were PCR-amplified based
with Accuprime (Invitrogen) The amplified products were
separated by agarose gel electrophoresis and the DNA bands
corresponding to the size of each segment were purified using
the Wizard SV gel Clean-Up System (Promega). The purified
DNAs were used as templates for automated dideoxy sequenc-
ing with BigDye 3.1 cycle sequencing kit (Applied Biosystems).
To sequence the entire gene we used approximately 500 bp
overlapping gene-specific primers in both directions. For the
sequencing of HA gene, we used FWuni12, RVuni13, HA462_f
(59-GACTCGAACAAAGGTGTAACGG-39) and HA1202_r
(59-GTCAATGGCATTCTGTGTGCTC-39) as sequencing pri-
mers. For the sequencing of NA gene, we used FWuni12,
RVuni13, NA_521r (59-TGACCAAGCGACTGACTCAA-39),
NA376_f (59-CCCTTGGAATGCAGAACCTT-39) and
NA905_f (59-CGTGGGTGTCTTTCAACCAGAA-39). The se-
quences were assembled with the SeqScapeTM
program
(Applied Biosystems). Alignments of nucleic and amino acid
sequences were completed to identify polymorphisms with other
H1N1pdm isolates and sequences for all isolates were submitted
through the Influenza Research Database and uploaded into the
GenBank (accession numbers provided in Table S4).
Ethics Statement for Animal Studies
Mice studies were approved by the University of Louisville
Institutional Animal Care and Use Committee with Veterinary
Medicine tasked to monitor and support all animal experiments.
Research was conducted in compliance with the Animal
Figure 9. Virus titer the in lung compartments of mice infected
with KY/136 or KY/180. Bronchoalveolar lavage was taken from six
to nine week old DBA/2 mice that were infected with 105
TCID50 KY/
136E or KY/180E on days 3 and 4 post-infection. Cells were separated
from the lavage fluid by centrifugation and the fluid (BALf), cellular (BAL
cell), and whole lung homogenate were tested separately for virus by
TCID50 assay on MDCK cells. Lung compartments taken from mice
infected with KY/180 had statistically higher virus titers at 3 DPI than
from mice infected with KY/136 (p,0.05, indicated by asterisks on the
figure). It is not possible to compute statistical differences from 4 DPI, as
there were only two mice infected with KY/180 in that experiment that
survived to 4 DPI.
doi:10.1371/journal.pone.0056602.g009
Phenotypic Differences of H1N1pdm Isolates
PLOS ONE | www.plosone.org 13 February 2013 | Volume 8 | Issue 2 | e56602
Welfare Act and other federal statutes and regulations relating
to animals and experiments involving animals and adheres to
principles stated in the Guide for the Care and Use of Laboratory
Animals, National Research Council, 1996. The facilities where
this research was conducted in a fully accredited by the
Association for Assessment and Accreditation of Laboratory
Animal Care International.
Mice Studies
Six to nine-week-old female DBA/2 mice were purchased from
Jackson Laboratories (Bar Harbor, ME) and housed in the
vivarium managed by UofL at the Research Resource Center or
at the Regional Biocontainment Laboratory. The mice received
food and water ad libitum, and all experiments were conducted in
accordance with rules of the Institutional Animal Control and Use
Committee of UofL. Mice were anaesthetized by isofluorane
inhalation and infected intranasally with in a total volume of 30 ml
at the dose as outlined in the figure legends and text. Viruses were
adjusted to the dose required in PBS (pH 7.2). Animals were
observed daily for morbidity and measured for weight loss. All
mice showing more than 25% body weight loss were considered to
have reached the experimental end point and were euthanized
humanely.
Mice were euthanized on the days noted in the results and on
figure legends and blood, nasal turbinates and lung tissues were
collected and stored appropriately until analyses. For collection of
BAL after euthanasia, the neck and thoracic area were disinfected
by soaking with 70% ethanol, and the trachea exposed by
dissection. An 18G catheter needle was inserted into upper
trachea, the needle removed, and the outer catheter sheath moved
into the lower trachea. A 3 mL syringe filled with 1 mL of DPBS
was used to carefully inject DPBS into the lungs, and aspirate BAL
fluid. Collected fluids were transferred to an ice-cold, sterile 15 mL
conical tube on ice and repeated once more as above. BAL fluids
were immediately centrifuged at 500g for 10 minutes to separate
fluids and cells. After centrifugation, supernatants are separated
and kept separately at 280uC freezer until used for TCID50 assay.
Cells pellets were washed once by adding 2 mL ice-cold DPBS
and centrifuged again at 500g for 10 min. Cells were resuspended
in 1 mL DPBS and kept at 280uC until used.
Figure 10. Detection of Influenza in mouse macrophage cell lines. Mouse macrophage cell line, RAW264.7, was infected in vitro with MOI = 1
of each influenza isolate (high pathogenic isolate, KY/180, and low pathogenic isolate, KY/136) on chambered microscopy slides. At 24 hours post-
infection cells were fixed, permeabilized, and stained using a FITC antibody conjugate specific for influenza A (H1N1) nucleoprotein (green). Cells
were counterstained with a nuclear dye (TO-PRO3, Molecular Probes, red), and visualized on a Zeiss LSM710 confocal microscope. Both isolates tested
were observed to infect macrophages. Scale bars indicate 100 microns.
doi:10.1371/journal.pone.0056602.g010
Phenotypic Differences of H1N1pdm Isolates
PLOS ONE | www.plosone.org 14 February 2013 | Volume 8 | Issue 2 | e56602
TCID50 Assay for Viral Load in Tissues and Bronchial
Lavage Fluid
Tissues were homogenized in ice-cold virus culture medium
(DMEM containing 0.2% BSA, 1% PEN/STREP) within a bio-
safety cabinet. Tissue homogenates were clarified by centrifuga-
tion (4000g for 20 min at 4uC) prior to storage at 280uC, and later
analyzed by TCID50 or Luminex cytometric bead array (next
section). For measurement of the level of infectious virus present in
tissue samples, the titers of the virus were determined by TCID50
assay by titration of the clarified tissue homogenates or BALf on
MDCK cells. The limit of virus detection was typically 101.5
TCID50/ml or as indicated in each figure. Virus titers were
calculated by the method of Reed and Muench [91], and are
expressed as the mean log10 TCID50 per milliliter. Tissues in
which no virus was detected were given a value of 101.0
TCID50/
ml for calculation of the mean titer.
Quantification of Cytokine and Chemokine Levels in
Lungs
Sera and lungs from mice euthanized on days as noted in figure
legends and text were analyzed by using LuminexH xMAPH
technology-based assay kit (Millipore) according to the manufac-
turer's protocol. The final reaction plate was read with a Luminex
100 or FlexMAP 3D machine and specific concentrations were
calculated from a standard using Luminex xPONENT software.
Statistics and PCA
R (version 2.13.0) base statistical package and GraphPad
software package were used in the analysis of data and generation
of figures. Generalized linear models were constructed using each
analyte included in the initial screen of Kentucky isolates in DBA/
2 mice as a response variable (cytokine/chemokine concentrations)
and isolate, DPI, and egg vs. MDCK stock, were used as
predictors. The stock designation of the virus was removed from
the model after it was seen that it did not contribute to explain
significant proportion of the variance. Log likelihood ratios were
used to compare the relative fit of each model, and the top 12
models showing the highest parameter estimates for the isolates
were chosen for inclusion into the PCA (i.e., the cytokines/
chemokines that were the most different between isolates,
controlling for DPI, selected from the best-fit models). The R
package ``FactoMineR'' (version 1.14) was used to perform the
PCA. Statistically significant differences between multiple groups
was assessed using Kruskal-Wallis tests followed by post hoc tests
using pairwise Wilcoxon Rank Sum tests unless otherwise noted.
Multiple comparisons were adjusted according to Holm's method
and a p-value ,0.05 is considered significant.
Cytospin and Flow Cytometry
BAL cells were centrifuged at 300g for 5 min and washed twice
in PBS. Cells were resuspended in 0.4 mL PBS and loaded into
Shandon CytospinTM
centrifuge funnels. The cytocentrifuge
chambers were spun at 1000 rpm for 5 min. Slides were air dried
in a BSC and then were fixed and stained with Eosin and
Methylene Blue (Kwik-Diff, Thermo Scientific). Slides were
inspected under a light microscope.
For flow cytometry analysis, BAL cells were washed in PBS as
above and stained with antibodies specific to mouse CD3 (500A2
Pacific Blue, BD Biosciences), Gr-1 (RB6-8C5 phycoerythrin,
eBioscience), and CD49b (DX5 phycoerythrin-Cy7, eBioscience).
Cells were then washed and fixed in 4% paraformaldehyde for 15
minutes at room temperature, followed by permeabilization with
0.2% saponin in 2% BSA for 20 minutes. Cells were then stained
intracellularly for influenza nucleoprotein using a fluorescein
isothiocyanate conjugated antibody (NP-FITC, AbCam
#ab20921) for 30 minutes. Cells were washed and analyzed on
a FACSAria II flow cytometer (BD).
Mouse Macrophages Experiments
RAW264.7 cells (ATCC #TIB-71) were seeded onto glass
chamber slides (LabTek) and allowed to rest overnight. The cells
were gently washed in PBS and virus was added at 1.0 MOI. The
cells were washed and fixed at 24 hours post-infection and
permeabilized with 0.2% saponin in 10% FBS. Cells were stained
for intracellular influenza nucleoprotein as above, and a nuclear
counter stain was included for the final 10 minutes (TO-PRO3,
Molecular Probes). Cells were washed, mounted under a coverslip
with ProLong Antifade media (Invitrogen), and visualized using
a Zeiss LSM/710 confocal microscope. Kinetics of infection were
determined by infecting mouse macrophage cell lines, RAW264.7
or BEI Resources #NR9456 (NIAID, NIH), in triplicate wells of
a 24-well plate in DMEM supplemented with 0.2% BSA, 1% Pen/
Strep, 2.5% L-glutamine, and 25 mM HEPES. Supernatants were
taken and centrifuged prior to performing a TCID50 assay using
MDCK cells. Virus titers were confirmed at 24 hours using
a plaque assay.
Supporting Information
Figure S1 Principal Components Analysis (PCA) of
mouse lung cytokine and chemokine expression after
challenge with clinical Influenza A (H1N1) virus isolates
from Kentucky, 2009. Standardized mean values for each
cytokine/chemokine are plotted from Day 3 and Day 6 post-
infection (n = 3, each) against the first two principal components,
accounting for 72% of the variation in the analysis.
(TIF)
Figure S2 Cytokine and chemokine profiles from mouse
lung homogenate. DBA/2 mice were infected with KY/180E,
KY/136E (105
pfu), or mock-infected with PBS. The mice were
sacrificed 1, 3, and 5 days post-infection (n = 5 mice per group-
Figure 11. Replication of KY/180E and KY/136E in mouse
macrophage cell lines. Mouse macrophage cell line, RAW264.7, was
infected in vitro with the influenza isolates on a 24-well plate at an
MOI = 1.0. After one hour of incubation, the cells were washed and
returned to the incubator. Cell culture supernatants were collected over
time and virus titer was determined by TCID50 assay. Both KY/180E and
KY/136E replicate in mouse macrophage cell lines. KY/180E had
significantly higher titers of virus detected at 24 and 48 hours post-
infection (p,0.05, indicated by asterisks on the figure). Pathogenic
isolate KY/180E was able to replicate better in mouse macrophages
compared to the low pathogenic isolate, KY/136E.
doi:10.1371/journal.pone.0056602.g011
Phenotypic Differences of H1N1pdm Isolates
PLOS ONE | www.plosone.org 15 February 2013 | Volume 8 | Issue 2 | e56602
day). Samples from moribund mice taken after Day 5 post-
infection were also analyzed when available. Bars indicate mean
concentration.
(TIF)
Figure S3.
(TIF)
Figure S4 Dose response of chemokines in the lungs of
mice infected with KY/136 or KY/180 influenza A
(H1N1) virus isolates. Mice were infected with 100
, 102
, or
105
pfu of virus and samples were collected upon euthanasia on
days 1, 3, or 5 post-challenge (D1, D3, and D5, respectively). n = 5
mice per dose-day for all groups except for mice infected with 105
pfu of KY/180 where only 2/5 mice survived to D5.
(TIF)
Figure S5 Dose response of cytokines in the lungs of
mice infected with KY/136 or KY/180 influenza A
(H1N1) virus isolates. Mice were infected with 100
, 102
, or
105
pfu of virus and samples were collected upon euthanasia on
days 1, 3, or 5 post-challenge (D1, D3, and D5, respectively). n = 5
mice per dose-day for all groups except for mice infected with 105
pfu of KY/180 where only 2/5 mice survived to D5.
(TIF)
Figure S6 Dose response of cytokines in the lungs of
mice infected with KY/136 or KY/180 influenza A
(H1N1) virus isolates. Mice were infected with 100
, 102
, or
105
pfu of virus and samples were collected upon euthanasia on
days 1, 3, or 5 post-challenge (D1, D3, and D5, respectively). n = 5
mice per dose-day for all groups except for mice infected with 105
pfu of KY/180 where only 2/5 mice survived to D5.
(TIF)
Figure S7 Replication kinetics of KY/180E and KY/
180E isolates in C57BL/6 mouse macrophages. The
macrophage cell line, BEIR #NR-9465, was infected at 1.0
MOI (two independent experiments at n = 3 per experiment) and
clarified supernatants were taken at 4, 24, 48, and 72 hour post-
infection. Virus titers were measured by TCID50 assay on MDCK
cells.
(TIF)
Table S1 Comorbidities associated with severe, hospi-
talized influenza pneumonia patients.
(DOCX)
Table S2 Variants noted in amino acid sequence
alignments of H1N1pdm clinical isolates.
(DOCX)
Table S3 GenBank accession numbers of H1N1pdm
isolates.
(DOCX)
Table S4 Virus titers (TCID50/ml*) on 1, 3, and 5 days
post-infection in lungs and nasal turbinates of DBA/2
mice infected with KY/180E and KY/136E.
(DOCX)
Table S5 Relative magnitude of immune responses of
H1N1pdm isolates in mice in groups revealed by
principal component analysis clustering.
(DOCX)
Table S6 Summary of references to mutations in
Influenza A (H1N1) isolates with observed virulence.
(DOCX)
Acknowledgments
We thank Jennifer Kraenzle for her technical support in some of the mouse
studies and Punya Mardhanan for her support of sequence analyses. We
are grateful to Dr. Haval Shirwan and Dr. Silvia Uriarte of the University
of Louisville for their helpful discussions and critical reviews of data
presented in this manuscript.
Author Contributions
Conceived and designed the experiments: YKC JVC CBJ. Performed the
experiments: YKC JVC DHC JTS RCM RSA. Analyzed the data: YKC
JVC CBJ PP TLW RLG RCM JAR. Wrote the paper: YKC CBJ JVC
DHC JTS RCM TLW.
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