Local Innate Immune Responses and Influenza Virus Transmission and Virulence in Ferrets

M A J O R A R T I C L E

Local Innate Immune Responses and InfluenzaVirus Transmission and Virulence in Ferrets

Taronna R. Maines,1 Jessica A. Belser,1 Kortney M. Gustin,1 Neal van Hoeven,1 Hui Zeng,1 Nicholas Svitek,2

Veronika von Messling,2 Jacqueline M. Katz,1 and Terrence M. Tumpey1

1Influenza Division, National Center for Immunization and Respiratory Disease, Centers for Disease Control and Prevention, Atlanta, Georgia; and2INRS-Institut Armand-Frappier, University of Quebec, Canada

Host innate immunity is the first line of defense against invading pathogens, including influenza viruses.

Ferrets are well recognized as the best model of influenza virus pathogenesis and transmission, but little is

known about the innate immune response of ferrets after infection with this virus. The goal of this study was

to investigate the contribution of localized host responses to influenza virus pathogenicity and transmissibility

in this model by measuring the level of messenger RNA expression of 12 cytokines and chemokines in the upper

and lower respiratory tracts of ferrets infected with H5N1, H1N1, or H3N2 influenza viruses that exhibit diverse

virulence and transmissibility in ferrets. We found a strong temporal correlation between inflammatory

mediators and the kinetics and frequency of transmission, clinical signs associated with transmission, peak

virus shedding, and virulence. Our findings point to a link between localized innate immunity and influenza virus

transmission and disease progression.

Influenza A viruses are a persistent global public health

problem causing seasonal epidemics, occasional pan-

demics, and sporadic zoonotic infections. The disease

burden of influenza viruses depends predominately on

transmission efficiency and virulence in the human

population. Seasonal influenza viruses cause a highly

contagious disease and can be associated with.300 000

deaths annually worldwide, mostly among those aged

$65 years [1]. The last 4 influenza pandemics have

displayed a wide range of morbidity, mortality, and

transmissibility [2–5]. Human influenza virus infections

are typically characterized by respiratory tract symp-

toms, including rhinorrhea, congestion, sore throat,

cough, and sneezing, in addition to systemic symptoms,

including fever, chills, and muscle aches [6–8]. More

severe disease involving the lower respiratory tract

is often associated with age and underlying health

conditions [1, 9]. Highly pathogenic avian influenza

(HPAI) H5N1 viruses have caused hundreds of human

infections in 15 countries with a case fatality rate of

�60%. Human infections are primarily a result of ex-

posure to infected poultry, with no sustained trans-

mission among humans [10]. Individuals infected with

H5N1 viruses generally present with fever, headache,

sore throat, dyspnea, and cough, with severe infections

progressing to pneumonia, acute respiratory distress

syndrome, and death [11].

The ability of influenza viruses to cause disease and

spread easily among people depends on host and viral

factors. With influenza virus infection, the host innate

immune response is triggered through pathogen rec-

ognition receptors, such as RIG-I and TLR-7, which

initiate the release of proinflammatory mediators

[12, 13]. This early production of cytokines and

chemokines at the site of infection has been shown to

be the basis for many of the clinical and pathological

manifestations of disease [14, 15]. During severe

HPAI virus infections, a dysregulation of these

proinflammatory cytokines, often called a cytokine

storm, has been associated with pulmonary damage

[15–17]. Although the host innate immune response’s

Received 21 July 2011; accepted 23 September 2011; electronically published 9December 2011.Correspondence: Terrence M. Tumpey, Influenza Division, National Center for

Immunization and Respiratory Disease, Centers for Disease Control and Prevention,1600 Clifton Rd NE, MS-G16, Atlanta, GA 30333 ([email protected]).

The Journal of Infectious Diseases 2012;205:474–85Published by Oxford University Press on behalf of the Infectious Diseases Society ofAmerica 2011.DOI: 10.1093/infdis/jir768

474 d JID 2012:205 (1 February) d Maines et al

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role in influenza disease progression has been studied to some

extent, its role in influenza virus transmission remains poorly

understood.

The ferret is widely recognized as an excellent model of in-

fluenza virus transmission and pathogenesis, but little is known

about innate immune responses of ferrets, primarily due to the

lack of ferret-specific immunological reagents. Recently, ferret

genomic sequences have been reported, facilitating assessment

of messenger RNA (mRNA) expression levels of a number of

cytokines and chemokines [18, 19]. Although these studies have

identified a role of select immune mediators in influenza virus

pathogenesis [20–22], a comprehensive analysis of the local in-

nate immune responses in upper respiratory tract (URT) and

lower respiratory tract (LRT) tissues has not been performed.

Additionally, direct comparison of viruses of multiple sub-

types with differing transmission and virulence properties has

not been undertaken. Therefore, we have selected an H3N2

human influenza virus (A/Panama/2007/99 [PN99]), a 2009

pandemic H1N1 virus (A/Mexico/4482/09 [MX82]) and

2 H5N1 HPAI viruses (A/Hong Kong/486/97 [HK97] and

A/Thailand/16/04 [TH16]) and assessed for each the mRNA

expression levels of a panel of cytokines and chemokines in

ferret URT and LRT tissues after infection to characterize

temporal relationships with disease progression and trans-

mission. Our results establish a strong correlation between the

frequency of URT clinical signs, peak virus shedding, the

Figure 1. Transmission and clinical signs observed in ferrets infected with 4 influenza viruses: an H3N2 human influenza virus (A/Panama/2007/99[PN99]), a 2009 pandemic H1N1 virus (A/Mexico/4482/09 [MX82]) or an H5N1 HPAI virus (A/Hong Kong/486/97 [HK97] or A/Thailand/16/04 [TH16]).Kinetics and frequency of respiratory droplet transmission (A ), sneezing (B ), nasal discharge (C ), lethargy (D ), fever (E ), and dyspnea (F ) in ferrets(n 5 2–9 per time point) are presented for the days shown after inoculation. Respiratory droplet transmission is defined here as detection of virus innasal wash samples from contact animals housed in cages with perforated side walls adjacent to infected ferrets, so that virus is transmitted through theair in the absence of direct or indirect contact. All transmission data were published elsewhere [23, 29, 30]; clinical signs observed in the current study (at0.5, 2, and 6 days after inoculation) are presented in the context of data from those studies. The relative inactivity index for each virus is listedparenthetically in panel D. Fever is defined as $1.5�C higher than baseline.

Influenza Innate Immunity in Ferrets d JID 2012:205 (1 February) d 475



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Figure 2. Detection of influenza virus in the respiratory tracts of ferrets infected with 4 influenza viruses: an H3N2 human influenza virus (A/Panama/2007/99 [PN99]), a 2009 pandemic H1N1 virus (A/Mexico/4482/09 [MX82]) or an H5N1 HPAI virus (A/Hong Kong/486/97 [HK97] or A/Thailand/16/04




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kinetics of transmission in ferrets, and the local expression of

cytokine mRNAs that have been implicated in causing similar

clinical signs in humans infected by highly transmissible vi-

ruses. Furthermore, increased expression of proinflammatory

mediators and reduced expression of anti-inflammatory me-

diators in the LRT of ferrets correlates with severe disease and

lethal outcome.

MATERIALS AND METHODS

VirusesVirus stocks for the H5N1 and H3N2 viruses were propagated

in the allantoic cavity of 10-day-old embryonated hens’ eggs, as

described elsewhere [23]. The H1N1 virus stock was propagated

in Madin-Darby canine kidney cells. All research with HPAI

viruses was conducted under BSL3 containment, including en-

hancements required by the US Department of Agriculture and

the Select Agent Program [24–26].

Ferret ExperimentsMale Fitch ferrets aged 7–10 months old (Triple F Farms) and

serologically negative for currently circulating influenza viruses

were housed in cages within a Duo-Flo Bioclean mobile clean

room (Lab Products) throughout each experiment. Baseline

measurements and ketamine sedation of ferrets was performed

as described elsewhere [27] before intranasal inoculation with

106 50% egg infectious dose (EID50) or plaque-forming units of

virus (6 ferrets per virus) in 1 mL of phosphate-buffered saline

or phosphate-buffered saline alone for the mock-infected con-

trol animals. To target the timing of innate immune responses

and transmission, 2 ferrets each were humanely killed at 0.5,

2 and 6 days after inoculation. Nasal wash and lung tissue

specimens (harvested from each lobe and pooled) were collected

from each ferret for virus titration, as described elsewhere [27].

Nasal turbinates and lung specimens (�30 mg excision in

proximity to a bronchus) were collected and stabilized in

RNAlater (Ambion). Serum samples were analyzed using

VetScan Classic and Comprehensive Diagnostic Profile rotors

(Abaxis). Ferrets were monitored for clinical signs of infection,

and relative inactivity indexes were determined, as described

elsewhere [27]. Animal research was conducted under the

guidance of the Centers for Disease Control and Prevention

Institutional Animal Care and Use Committee in an Association

for Assessment and Accreditation of Laboratory Animal Care

International–accredited animal facility.

Quantification of mRNARNA isolations were performed on ferret nasal turbinates and

lung tissue samples with an RNeasy Mini Kit using the RNA

stabilization and on-column DNase digestion protocols (Qiagen).

Semiquantitative reverse-transcription polymerase chain reaction

was performed as described elsewhere [18], using 0.5 lg of RNAand a Mx3005P (Stratagene). Expression levels were normal-

ized to Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH)

and are reported as fold change compared with mock-infected

animals. Primer sequences for interferon (IFN) a, IFN-c, tu-mor necrosis factor (TNF) a, interleukin (IL) 6, IL-10, IL-

12p40, GAPDH [18], and FluM1 [28] were published elsewhere.

Primer sequences for the remaining genes are as follows: IFN-b,Forward 5#-GGTGTATCCTCCAAACTGCTCTCC-3#, Reverse

5#-CACTCCACACTGCTGCTGCTTAG-3#; IL-4, Forward 5#-

TCACCGGCACTTTCATCCACGGACATAACTT-3#, Reverse

5#-GAGCTGCTGAAGCACAGTTGCAGCTCTGC-3#; IL-8,

Forward 5#-AACCCACTCCACGCCTTTCCATC-3#, Reverse 5#-

GGCACACCTCTTTTCCATTGAC-3#; CXCL9, Forward 5#-

GGTGGTGTTCCTCTTTTGTTGA-3#, Reverse 5#-GTTCCTTG

GGTGGTGGTGAT-3#; CXCL10, Forward 5#-CCTGGCTTCAC

CGAGTTCT-3#, Reverse 5#-AGTAGCAGCCCATGGAGTAAAA-

3#; CXCL11, Forward 5#-CCTTCTTTCACATTCAGCGTTTCT-

3#, Reverse 5#-CTATAGCCATGCCCTTCACACTCA-3#.

StatisticsStatistical significance was determined using area under the

curve and 1-way analysis of variance with Bonferroni’s multiple-

comparison posttest. The correlations between observational

data (area under the curve) and peak mRNA expression (fold

rise compared with values in mock-infected ferrets) were esti-

mated by Pearson correlation coefficients.

RESULTS

Transmission and Disease Observed in Influenza Virus–InfectedFerretsWork from this laboratory published elsewhere describes the

respiratory droplet transmissibility of each of the viruses used in

this study [23, 29, 30]. Typical of seasonal H3N2 viruses, PN99

transmitted efficiently (3 of 3 ferrets) as early as 2 days after

contact (Figure1A). Although the 2009 pandemic H1N1 virus,

MX82, also transmitted by respiratory droplets, transmission

was somewhat less efficient than for the seasonal virus, with only

2 of 3 contact ferrets shedding virus by 3 days after contact. No

Figure 2 continued. [TH16]. Virus titers were measured in nasal wash samples (A ) and lungs (B ) collected from ferrets (n 5 2–8 per time point) on thedays shown after inoculation and are expressed as the log10 mean6 standard deviation, with a limit of detection of 101.5 50% egg infectious dose/mL or g.Nasal wash samples may contain secretions from the upper or lower respiratory tracts, because sneezing occurs during collection. Data generated in thecurrent study (striped columns) are presented in the context of work published elsewhere [23, 29, 30]. Viral messenger RNA (mRNA) levels were measuredin nasal turbinates (C ) and lungs (D ) of ferrets (n5 2 per time point) by real-time reverse-transcription polymerase chain reaction and are presented asfold increases relative to levels in mock-infected animals for the days shown after inoculation.




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virus shedding was detected in any of the contacts of animals

infected by H5N1 viruses (TH16 or HK97), indicative of the

inability of these viruses to transmit through respiratory drop-

lets. In addition to the ferrets in these published studies, 6 ferrets

each were intranasally inoculated with 106 EID50 or plaque-

forming units of TH16, HK97, MX82, or PN99 virus and were

observed for up to 6 days for clinical signs of disease. Sneezing

was most prominent in PN99 virus–infected ferrets; frequency

was 3–9 times greater for these ferrets compared with animals

infected by any other virus (P, .01) (Figure 1B). Nasal discharge

was observed more frequently among PN99 virus–infected

animals up to 4 days after inoculation, although at later time

points, TH16 and, to a lesser degree, HK97 virus, exhibited

increased nasal discharge (Figure 1C). Although nasal discharge

was observed in a majority of H5N1 virus–infected ferrets later

in the course of infection, the exudates were opaque, viscous,

and often associated with acute nasal obstruction, whereas

rhinorrhea in PN99 virus–infected ferrets was characterized

by clear, flowing discharge at early time points. Nasal discharge,

congestion, and sneezing are often reported in humans ex-

perimentally infected with seasonal influenza viruses and peak

2–4 days after inoculation [8, 12, 13, 31–33]. Similar kinetics

Figure 3. Innate immunity in upper respiratory tract tissues of ferrets infected with 4 influenza viruses: an H3N2 human influenza virus (A/Panama/2007/99 [PN99]), a 2009 pandemic H1N1 virus (A/Mexico/4482/09 [MX82]) or an H5N1 HPAI virus (A/Hong Kong/486/97 [HK97] or A/Thailand/16/04[TH16]. Cytokine and chemokine messenger RNA expression in nasal turbinates of ferrets (n 5 2 per time point) was measured by real-time reverse-transcription polymerase chain reaction and is presented as the mean fold change (6 standard deviation) compared with values in mock-infected animalson the days shown after inoculation. Abbreviations: IFN, interferon; IL, interleukin; TNF, tumor necrosis factor.




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of nasal symptoms were observed in ferrets infected with the

seasonal influenza PN99 virus, which peaked 3–4 days after

inoculation (Figure 1B and 1C). In contrast, we observed

significantly reduced sneezing and less rhinorrhea early in

infection in ferrets inoculated with H5N1 influenza viruses

that do not transmit efficiently via respiratory droplets (P, .01)

(Figure 1A–C).

Lethargy was observed in all H5N1 virus–infected ferrets by

3 days after inoculation and in 27% of PN99 virus–infected

ferrets and 91% of MX82 virus–infected ferrets at 2 days after

inoculation and was typically associated with fever ($1.5�Cabove baseline), which peaked in all groups by 2 days after in-

oculation (Figure 1D and 1E). Severe lethargy was most evident

in H5N1-infected ferrets (relative inactivity index, .2.0),

whereas human influenza viruses induced more modest lethargy

(Figure 1D). We observed an inverse correlation between

transmission and both the frequency and severity of lethargy in

ferrets (r , 20.9; P , .05) (Figure 1A and 1D). Dyspnea was

pronounced in TH16 virus infection and ultimately required

euthanasia, resulting in a 100% lethality rate by 7 days after

inoculation for this virus (Figure 1F). Euthanasia was required

for 35% of HK97 virus–infected ferrets because of the onset of

neurological dysfunction by 7–13 days after inoculation, whereas

MX82 virus had a 50% lethality rate due to excessive weight loss

(mean maximum, 17.5%) requiring euthanasia by 8 days after

inoculation; no ferrets died after PN99 virus infection (data not

shown; [27, 29]). Serum chemistry analysis of ferrets infected

by TH16 virus reflected significantly altered albumin, alkaline

phosphatase, alanine aminotransferase, amylase, total bili-

rubin, calcium, sodium, and total protein levels by 6 days

Figure 3. Continued.




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after inoculation, indicative of acute dehydration and liver

dysfunction (data not shown). Abnormal analyte levels of

ferrets infected by any of the other viruses were sporadic and

not significant. Together, these data highlight the diversity in

transmissibility and virulence among the influenza viruses

included in this study and provide a platform for investigating

differences in innate immunity that are associated with

transmission and disease severity.

Influenza Virus Replication in the Ferret Respiratory TractVirus titers were measured in ferret nasal wash and lung samples

collected at 0.5, 2, and 6 days after inoculation with human

or avian influenza virus. Nasal wash titers are presented in the

context of virus shedding kinetics reported elsewhere from

studies conducted in this laboratory (Figure 2A) [23, 27, 29].

In the current study, H5N1 virus infection resulted inmean peak

virus titers in nasal wash samples of 105.4 or 105.5 EID50/mL at

2 or 6 days after inoculation, respectively, as virus titers remain

elevated throughout TH16 virus infection. In comparison, nasal

wash virus titers were $2.5 times higher for the 2009 pandemic

H1N1 virus, MX82 (105.9 EID50/mL), and 25 times higher for

the seasonal influenza virus, PN99 (106.9 EID50/mL), at 2 days

after inoculation when respiratory droplet transmission is first

observed with the latter virus (Figure 1A). Conversely, in the

LRT, virus titers in TH16 virus–infected ferrets were greatest,

significantly higher than either of the human influenza viruses

(P , .01), and they remained elevated throughout infection

(Figure 2B). Viral mRNA was measured in nasal turbinate

and lung tissues of ferrets at the same time points after in-

oculation (Figure 2C and 2D). For the human influenza viruses,

.200 times more viral mRNA was detected in the URT at

0.5 days after inoculation compared with the H5N1 viruses,

and viral mRNA levels remained significantly greater at 2 days

after inoculation (P , .01), but by 6 days after inoculation

viral mRNA levels were more comparable among all groups

(Figure 2C). At 0.5 days after inoculation, viral mRNA levels in

lungs were greatest in TH16-infected animals (�200-fold in-

crease compared with mock-infected animals) (Figure 2D), and

at 2 days after inoculation, H5N1 virus mRNA levels were sig-

nificantly higher compared with the human influenza viruses

(.75-fold; P , .01). By 6 days after inoculation, levels were

reduced to ,90-fold for all viruses except TH16 virus, which

remained elevated at levels nearly 800 times those in mock-

infected animals. These comparative differences in virus rep-

lication in the URT versus the LRT for human and avian

influenza viruses prompted us to examine the localized innate

immune responses in the URT and LRT and to investigate

their relationship with disease progression and transmission.

URT Expression of Cytokine and Chemokine mRNA in FerretsExpression of a panel of proinflammatory cytokine and che-

mokine mRNAs was measured in ferret nasal turbinates collected

at 0.5, 2, and 6 days after inoculation with human influenza or

HPAI viruses (Figure 3). Twelve hours after inoculation with

PN99 virus, mean peak levels of TNF-a and IL-6 mRNA in

ferrets were significantly higher in nasal turbinates compared

with any other virus group (P , .01) (Figure 3A and 3B). By

2 days after inoculation, PN99 and MX82 virus–infected ferrets

expressed these cytokines at levels significantly greater than in

either H5N1 virus–infected group; levels of mRNA in the PN99

virus group were at least twice that of MX82 virus (P , .01).

In particular, levels of IL-6 mRNA at 2 days after inoculation

were highest in ferrets infected by human influenza viruses,

whereas H5N1 virus–infected ferrets exhibited levels that were

Table 1. Correlation of Transmission and Associated Clinical Signs With Cytokine Expression in Nasal Turbinates of TH16, HK97, MX82,or PN99 Virus–Infected Ferrets

Pearson r (P )a

Cytokine or Clinical Sign Transmission Sneezing Rhinorrheab Nasal Wash Titersc

TNF-a 0.977 (.011) 1.000 (.00006) 0.998 (.001) 0.984 (.008)

IL-6 0.918 (.041) 0.989 (.005) 0.986 (.007) 0.999 (.0003)

IFN-a 0.817 (.091) 0.993 (.003) 0.982 (.009) 0.972 (.014)

IFN-b 0.810 (.095) 0.985 (.007) 0.969 (.016) 0.966 (.017)

Transmission . 0.949 (.042) 0.991 (.004) 0.930 (.035)

Sneezing . . 0.913 (.043) 0.984 (.008)

Rhinorrhea . . . 0.969 (.016)

Abbreviations: IFN, interferon; IL, interleukin; TNF, tumor necrosis factor.a Pearson correlation coefficients are estimated for correlations between observational data and peak messenger RNA expression. P values are in parentheses;

values in bold represent significant correlations (P , .05). Ellipses indicate that no comparison was made or results are listed in another cell.b Rhinorrhea includes observations of clear, flowing nasal discharge up to 4 days after inoculation, not the opaque, obstructive discharge noted in H5N1-infected

ferrets later during infection.c Nasal wash virus titers measured 2 days after inoculation.




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elevated or sustained through 6 days after inoculation. The

URT peak expression levels of TNF-a and IL-6 correlated with

virus shedding, sneezing, rhinorrhea, and transmission among

the virus groups (r . 0.9; P , .05) (Figures 1–3, Table 1).

The induction of type I IFN (IFN-a and IFN-b) elicits one ofthe most effective defense mechanisms against influenza virus

infection [34]. The most striking difference in cytokine mRNA

levels in the URT among our ferret groups was with type I IFNs,

which peaked in all groups at 2 days after inoculation (Figure 3C

and 3D). In PN99 virus–infected ferrets, IFN-a was expressed

at levels 9 times higher than in H5N1-infected ferrets and 2

times higher than inMX82 virus–infected ferrets, whereas IFN-bwas expressed at levels 60 times higher than in H5N1-infected

ferrets and 10 times higher than in MX82 virus–infected ferrets.

The MX82 virus group also expressed significantly higher

levels of IFN-a and IFN-b mRNA (at least 4- and 6-fold, re-

spectively) than either H5N1 virus ferret group (P, .001). Thus,

viruses that induced the greatest clinical signs and peak virus

shedding in the URT also induced the highest levels of type I

IFN mRNA expression (r . 0.9; P , .05) (Figure 1, Figure 3C

and 3D; Table 1). Increased type II IFN (IFN-c) mRNA levels

were detected in nasal turbinates of all groups of infected ferrets,

peaking at 2 days after inoculation (Figure 3E). However, levels

of expression in TH16 virus–infected ferrets were 60-fold higher

than in mock-infected ferrets, whereas levels in HK97, MX82,

and PN99 virus groups were significantly higher, .120-fold

those for mock-infected animals (P , .001). A strong inducer

of IFN-c, IL-12 mRNA was expressed at increased levels in

all infected animals by 12 hours after inoculation, peaking

by 2 days after inoculation. PN99 virus-infected ferrets ex-

pressed 5 times more IL-12 mRNA than any other group

(P , .01) (Figure 3F).

IL-8 (CXCL8) is a neutrophil chemotactic cytokine that

peaked at 6 days after inoculation in ferrets infected with H5N1

viruses, with the highest levels attained by TH16 virus; PN99

and MX82 virus–infected ferrets exhibited peak levels of this

cytokine 2 days after inoculation (Figure 3G). Additional Th1

response chemokines in the CXC subfamily were elevated in

URT tissues of all ferret groups, peaking 2 days after inoculation

(Figure 3H–J). At this time point, CXCL9 (MIG) and CXCL11

(IP9) mRNAs were expressed in PN99 virus–infected ferret nasal

turbinates at levels almost 900-fold those in mock-infected fer-

rets and $3 times those than in ferrets infected by any other

virus (Figure 3H and 3J). CXCL10 (IP10) mRNA levels were

elevated in all groups of ferrets$400-fold compared with mock

infection (Figure 3I). These results demonstrate a gradient of

CXCL9 and CXCL11 mRNA expression in the URT, with the

highest levels achieved by PN99 virus–infected ferrets; there was

more comparable elevated expression of CXCL10 mRNAs

among all ferret groups.

Expressionof anti-inflammatory cytokines such as IL-4 and IL-

10 is important for disease resolution, preventing inflammation

from going unchecked [35]. Similar to findings in human studies

[12], we observed no substantial changes in nasal turbinate levels

of IL-4 mRNAs, but a gradual increase in IL-10 mRNAs was

observed in all ferret groups (Figure 3K and 3L). However, the

mean peak IL-10 expression in PN99 virus–infected ferrets was

25-fold that in mock-infected ferrets, compared with increases of

14–15-fold for MX82 and HK97 and only 4-fold in TH16 group,

a significantly smaller difference (P , .01). We have demon-

strated a correlation between the expression of inflammatory

mediators in the URT of ferrets and the relative transmissibility of

these influenza viruses; however, innate immunity in the LRT is

probably a better indicator of severe disease.

LRT Expression of Cytokine and Chemokine mRNA in FerretsSevere disease and poor prognosis in H5N1 virus–infected

patients has been associated with hypercytokinemia and hy-

perchemokinemia [15, 36, 37]. In lung tissues of H5N1 virus–

infected ferrets, we detected significant increases in mRNA

levels of TNF-a and IL-6 cytokines at 2 days after inoculation

compared with animals infected by MX82 or PN99 viruses

(P , .05) (Figure 4A and 4B). TNF-a and IL-6 were $8 and

$54 times higher, respectively, than levels detected in ferrets

infected by human influenza viruses. IFN-a/b mRNA levels

were also elevated in H5N1 virus–infected ferrets compared with

those infected with human influenza viruses, with 3–161-fold

increases at 2 days after inoculation (P, .05) (Figure 4C and 4D).

IFN-c mRNA expression was significantly increased in lung

tissues of both H5N1 virus ferret groups compared with human

influenza viruses (P , .01) (Figure 4E), and IL-12 mRNA ex-

pression was highest at 0.5 days after inoculation in TH16 virus

lung tissue but at later time points was more similar between

ferret groups (Figure 4F).

Previous studies showed that increased expression of CXCL10

in lung tissues from H5N1 virus–infected ferrets was associ-

ated with disease severity [20]. We also found significantly

higher levels of CXCL10 mRNA in lung tissue at 2 days after

inoculation in H5N1 virus–infected ferrets compared with

those infected by human influenza viruses (P , .01), which

correlated with the presence of fever, dyspnea, and lethality

among the virus groups (r . 0.9; P , .05) (Figure 4I). At this

time point, levels of CXCL11 and CXCL9 mRNA were also

significantly higher in H5N1 virus–infected ferrets compared

with the human virus groups, whereas peak expression of

CXCL9 was not significantly greater at other time points

(Figure 4H and 4J). In humans, increased CXCL10 chemokine

also correlated with severe disease and was highest in fatal

cases, as was IL-8, which we found to be highest in H5N1-

infected ferrets at 2 days after inoculation.( Figure 4G) [15].

Overall, the more robust innate immune response in the lungs

of H5N1-infected ferrets was consistent with virus titers mea-

sured: 1–2 logs higher compared with human influenza viruses

(Figure 2B).




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Despite elevated levels of proinflammatory cytokine and

chemokine mRNAs in lung tissues of H5N1 virus–infected

ferrets, no significant up-regulation of anti-inflammatory

cytokines IL-4 or IL-10 was detected at any time point

(Figure 4K and 4L), even though increased IL-10 mRNA

levels were detected in nasal mucosa of HK97 but not TH16

virus (Figure 3L). The lack of anti-inflammatory regulation

of H5N1 virus infections may be site specific; without the

down-regulation of inflammation in the lungs, pulmonary

tissue damage is certain, potentially preventing disease res-

olution [35, 38].

DISCUSSION

Host innate immunity plays a critical role during disease pro-

gression and resolution after influenza virus infection. We have

examined the localized innate immune responses in the URT

and LRT of ferrets after infection with 4 influenza viruses that

Figure 4. Innate immunity in lower respiratory tract tissues of ferrets infected with 4 influenza viruses: an H3N2 human influenza virus (A/Panama/2007/99[PN99]), a 2009 pandemic H1N1 virus (A/Mexico/4482/09 [MX82]) or an H5N1 HPAI virus (A/Hong Kong/486/97 [HK97] or A/Thailand/16/04 [TH16]. Cytokineand chemokine messenger RNA expression in lungs of ferrets (n 5 2 per time point) was measured by real-time reverse-transcription polymerase chainreaction and is presented as the mean fold change (6 standard deviation) compared with values in mock-infected animals on the days shown afterinoculation. Abbreviations: IFN, interferon; IL, interleukin; TNF, tumor necrosis factor.




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exhibit differential properties of virulence and transmission

in both humans and ferrets. Our findings demonstrate simi-

larities between the innate immune responses in the respiratory

tracts of ferrets and humans after influenza virus infection and

provide potential markers of innate immunity that may identify

properties of disease severity and transmission. In human

volunteers experimentally infected with seasonal influenza virus,

URT expression of TNF-a, IL-6, and IFN-a correlated with

virus shedding and nasal and systemic symptoms [12, 13, 31, 32].

We also observed a strong up-regulation of proinflammatory

mediators (TNF-a, IL-6, IFN-a/b, IL-12, CXCL9, CXCL11) inthe URT of ferrets at a point in infection when transmission,

clinical signs associated with transmission, and peak virus

shedding occur. Symptom onset in influenza virus–infected

individuals has been shown to be associated with the timing

of transmission to household contacts [39, 40]. Furthermore,

the relative level of expression of proinflammatory cytokine

mRNAs correlated with the relative ability of TH16, HK97,

MX82 and PN99 viruses to transmit in our ferret model.

Despite correlation with clinical signs associated with trans-

mission, concurrent peak expression of IFN-a/b, may also

reduce virus replication. Exogenous treatment of guinea pigs or

ferrets with type I IFN reduced virus shedding and transmission

of human influenza viruses [41–43]. Other immune mediators

(IFN-c, IL-8, CXCL10, IL-4, IL-10) were expressed in the URT

in patterns seemingly unrelated to transmission and may be as-

sociated more with virus clearance or disease severity. Our as-

sessment of LRT innate immunity revealed significant increases

Figure 4. Continued.




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in peak expression of nearly all of the proinflammatory me-

diators tested for one or both of the H5N1 viruses compared

with the human influenza viruses, and no significant increase

was observed in either anti-inflammatory cytokine, supporting

previous studies in ferrets, nonhuman primates, and humans

that link dysregulation of innate immunity with severe disease

after H5N1 virus infection [15, 20, 44, 45]. Amore detailed time-

course and, as more reagents become available, assessment of

cytokine and chemokine protein expression levels will provide

greater insight into the complexity of the host–virus interface

and the innate immune response of ferrets to influenza virus

infection.

Collectively, our findings demonstrate that the site of virus

replication serves a critical role in the local expression of in-

flammatory mediators and the onset of clinical signs that in-

fluence transmission and disease severity. Viral factors, such as

receptor specificity and infectivity, are known determinants

of influenza virus transmission, but here we report host innate

immunity profiles that correlate with clinical signs, such as

rhinorrhea and sneezing, at time points that overlap with peak

virus shedding and transmission of influenza virus in ferrets.

Therefore, we conclude that this combination of events orches-

trated during influenza virus infection may help facilitate

efficient transmission of seasonal influenza viruses and that

regulation of the host innate immune response in the LRT

influences disease severity.

Notes

Acknowledgments. The authors thank Paul Gargiullo and Vic Veguilla

for assistance with statistical analyses, Shannon Emery for expertise in

real-time RT-PCR analysis, and the CDC Animal Resources Branch for

exceptional animal care. The findings and conclusions in this report are

those of the authors and do not necessarily reflect the views of the

funding agency.

Financial support. This work was supported by the Oak Ridge Institute

for Science and Education (K. M. G.).

Potential conflicts of interest. All authors: No reported conflicts.

All authors have submitted the ICMJE Form for Disclosure of Potential

Conflicts of Interest. Conflicts that the editors consider relevant to the

content of the manuscript have been disclosed.

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Local Innate Immune Responses and Influenza Virus Transmission and Virulence in Ferrets

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