Characterization of the Localized Immune Response in the Respiratory Tract of Ferrets following Infection with Influenza A and B Viruses

Louise A Carolan; Steve Rockman; Kathryn Borg; Teagan Guarnaccia; Patrick Reading; Jennifer Mosse; Anne Kelso; Ian Barr; Karen L Laurie

doi:10.1128/JVI.02797-15

. 2016 Feb 26;90(6):2838–2848. doi: 10.1128/JVI.02797-15

Characterization of the Localized Immune Response in the Respiratory Tract of Ferrets following Infection with Influenza A and B Viruses

Louise A Carolan ^a, Steve Rockman ^b,^c, Kathryn Borg ^a,^d, Teagan Guarnaccia ^a,^d, Patrick Reading ^a, Jennifer Mosse ^d, Anne Kelso ^a, Ian Barr ^a, Karen L Laurie ^a,^✉

Editor: A García-Sastre

PMCID: PMC4810641 PMID: 26719259

ABSTRACT

The burden of infection with seasonal influenza viruses is significant. Each year is typically characterized by the dominance of one (sub)type or lineage of influenza A or B virus, respectively. The incidence of disease varies annually, and while this may be attributed to a particular virus strain or subtype, the impacts of prior immunity, population differences, and variations in clinical assessment are also important. To improve our understanding of the impacts of seasonal influenza viruses, we directly compared clinical symptoms, virus shedding, and expression of cytokines, chemokines, and immune mediators in the upper respiratory tract (URT) of ferrets infected with contemporary A(H1N1)pdm09, A(H3N2), or influenza B virus. Gene expression in the lower respiratory tract (LRT) was also assessed. Clinical symptoms were minimal. Overall cytokine/chemokine profiles in the URT were consistent in pattern and magnitude between animals infected with influenza A and B viruses, and peak expression levels of interleukin-1α (IL-1α), IL-1β, IL-6, IL-12p40, alpha interferon (IFN-α), IFN-β, and tumor necrosis factor alpha (TNF-α) mRNAs correlated with peak levels of viral shedding. MCP1 and IFN-γ were expressed after the virus peak. Granzymes A and B and IL-10 reached peak expression as the virus was cleared and seroconversion was detected. Cytokine/chemokine gene expression in the LRT following A(H1N1)pdm09 virus infection reflected the observations seen for the URT but was delayed 2 or 3 days, as was virus replication. These data indicate that disease severities and localized immune responses following infection with seasonal influenza A and B viruses are similar, suggesting that other factors are likely to modulate the incidence and impact of seasonal influenza.

IMPORTANCE Both influenza A and B viruses cocirculate in the human population, and annual influenza seasons are typically dominated by an influenza A virus subtype or an influenza B virus lineage. Surveillance data indicate that the burden of disease is higher in some seasons, yet it is unclear whether this is due to specific virus strains or to other factors, such as cross-reactive immunity or clinical definitions of influenza. We directly compared disease severities and localized inflammatory responses to different seasonal influenza virus strains, including the 2009 pandemic strain, in healthy naive ferrets. We found that the disease severities and the cytokine and chemokine responses were similar irrespective of the seasonal strain or the location of the infection in the respiratory tract. This suggests that factors other than the immune response to a particular virus (sub)type contribute to the variable impact of influenza virus infection in a population.

INTRODUCTION

Morbidity and mortality associated with human influenza virus infection are significant worldwide. Influenza is caused by three virus types (A, B, and C), among which types A and B are responsible for most of the diagnosed cases of seasonal influenza. Influenza A viruses attract the greatest attention due to infrequent infections of humans with zoonotic viruses, the potential to give rise to pandemic viruses through genetic reassortment and antigenic shift, sustained antigenic drift, extensive spread, and varied severities of disease (1). In contrast, influenza B viruses are confined largely to humans, antigenic drift of these viruses occurs at a lower rate, and these viruses do not undergo antigenic shift (1, 2). The severity of disease caused by influenza virus is considered unpredictable (3), yet, until recently, infections with influenza B viruses were considered to be mild compared to infections with influenza A viruses (4) and to account for a reduced burden of disease. However, analysis of autopsy tissues following fatal infections indicated that influenza B viruses accounted for 30 to 40% of the burden of severe disease in children (5). These disparate findings may reflect the complexity of prior immunity and/or the impacts of preexisting medical conditions, highlighting the difficulties associated with understanding the pathogenesis and predicting the impact of disease induced by infection with seasonal influenza A and B viruses in humans.

Characterization of the immune response to influenza virus infection can contribute to our understanding of disease severity and may explain the virulence, pathogenicity, and tropism of particular virus strains. Respiratory epithelial cells and cells of the innate immune system produce different cytokines and chemokines following exposure to influenza viruses, and distinct patterns of inflammatory mediators can provide insights about infection severity with different virus strains in animal models of infection (6). In general terms, high levels of proinflammatory cytokines have been associated with severe disease following infection with highly pathogenic influenza viruses (reviewed in reference 7). Moreover, specific cytokines have been correlated with disease severity in both human (8 –13) and animal (14 –16) studies.

While the 2009 influenza pandemic caused a significant social and economic burden, the A(H1N1)pdm09 virus was not as virulent as other influenza viruses, such as A(H5N1) or A(H7N9) (14, 17, 18). Compared to infections with circulating seasonal influenza A and B virus strains, infection with A(H1N1)pdm09 virus was associated with more severe disease in some human studies (19) but with comparable disease in others (20, 21). Comparisons of disease severities and immune responses induced by infection with A(H1N1)pdm09 virus and seasonal influenza A virus strains are limited in animal models (14, 22, 23), with no studies focusing on coexistent influenza A and B virus strains. We recently developed TaqMan real-time reverse transcription-PCR (RT-PCR) assays to detect 15 cytokines and chemokines associated with infection in the ferret (24). In the present study, these assays were used to characterize the immune responses elicited in ferrets following infection with two influenza A viruses [A(H1N1)(pdm09) and A(H3N2)] and an influenza B virus that cocirculated in the Australian population in 2009 and 2010.

MATERIALS AND METHODS

Ferrets.

Adult male and female ferrets (weight, 500 to 1,500 g) were purchased from independent breeders and housed at bioCSL (Parkville, Australia), using services provided under a support services agreement with the Victorian Infectious Diseases Reference Laboratory. Serum samples from ferrets were tested by hemagglutination inhibition (HI) assay to ensure seronegativity (titer of <10) for currently circulating influenza virus strains, as previously described (25). Experiments using ferrets were conducted with approval from the bioCSL/Pfizer Animal Ethics Committee, in accordance with the Australian Government National Health and Medical Research Council's Australian code of practice for the care and use of animals for scientific purposes.

Viruses.

A/Tasmania/2004/2009 [A(H1N1)pdm09], A/Perth/16/2009 [A(H3N2)], and B/Brisbane/1/2007 (B/Florida/4/2006-like virus; B/Yamagata lineage) viruses were passaged in the allantoic cavity of embryonated hen's eggs and stored in aliquots at −80°C.

TCID₅₀ assay.

Fifty percent tissue culture infective dose (TCID₅₀) assays were performed as described previously (26) on MDCK-SIAT1 cells, using hemagglutination of turkey red blood cells (RBC) as the readout for the presence of influenza virus.

Virus infection of ferrets.

Naive ferrets were anesthetized (with ketamine at 12.5 mg/kg of body weight and Xylazil-20 at 2.5 mg/kg in a 1:1 [vol/vol] mixture; Troy Laboratories) and then infected artificially by intranasal inoculation with 10^3.5 TCID₅₀ of influenza virus in 0.5 ml. Animals were visually inspected daily. Body weight was measured on the day of challenge and then daily thereafter. Temperature was measured daily after challenge by using implanted temperature transponders fitted to identification chips (LifeChip Bio-Thermo; Digivet).

Generation and collection of ferret URT and LRT samples.

Nasal wash (upper respiratory tract [URT]) and lung tissue (lower respiratory tract [LRT]) samples were collected from naive and influenza virus-infected adult ferrets. Nasal washes were collected daily under light sedation (5 mg/kg Xylazil-20; Troy Laboratories) by delivery of 1 ml phosphate-buffered saline (PBS) supplemented with 50 U/ml penicillin, 50 μg/ml streptomycin (SAFC Biosciences), and 1% (wt/vol) bovine serum albumin (BSA; Sigma-Aldrich) into the nostril. Expelled liquid was collected, and 200 μl of nasal wash was aliquoted immediately into 1 ml RNAprotect saliva reagent (Qiagen, Germany) and stored at −80°C until RNA extraction; the remainder was stored at −80°C for TCID₅₀ assays. For collection of tissues, ferrets were anesthetized and then euthanized by intravenous, cardiac, or peritoneal injection of pentobarbitone. Respiratory tissues (individual lung lobes) were divided in half, cut into sections, and incubated in RNAlater tissue collection/RNA stabilization solution (Ambion, Life Technologies, Australia) at 4°C overnight; the remainder was stored at −80°C for TCID₅₀ assays. Following incubation, tissues were removed from solution and stored at −80°C until RNA extraction.

RNA extraction.

Cells in ferret URT (nasal wash) samples treated with RNAprotect saliva reagent were pelleted and resuspended in 350 μl buffer RLT. RNA was extracted by use of an RNeasy microkit (Qiagen) according to the manufacturer's instructions and eluted with 14 μl RNase-free water. Total RNA was extracted from ferret LRT (lung tissue) samples by use of an RNeasy maxikit (Qiagen) according to the manufacturer's instructions. Briefly, 3 ml RLT buffer containing 143 mM β-mercaptoethanol was added directly to the frozen tissue, along with three 1/4-in. ceramic spheres (MP Biomedicals, New South Wales, Australia) and 2 g silicone-carbide sharp particles (1 mm; Daintree Scientific, Tasmania, Australia). The sample was homogenized using a Fastprep-24 instrument with a CoolTeenPrep adapter, and the lysate was clarified twice by centrifugation at 300 × g for 10 min. RNA was extracted using the animal tissue protocol and eluted with an 800-μl volume. RNA purity was assessed for all samples by spectrophotometry, using the A₂₆₀/A₂₈₀ value. The RNA yield was 449 ± 576 ng/200 μl for URT samples and 112 ± 90 μg/g for LRT samples.

Reverse transcription and TaqMan real-time PCR.

Reverse transcription was performed as previously described (24). Primers and probes were previously described for all of the cytokine, chemokine, and housekeeping genes (24). Ferret granzyme B was amplified using previously described methods (24). Briefly, cDNA was prepared from ferret leukocytes stimulated in vitro for 72 h with concanavalin A (5 μg/ml; Sigma) and recombinant human interleukin-2 (IL-2) (0.1 μg/ml; Roche Diagnostics GmbH). The ferret granzyme B sequence was amplified using forward (5′-GCAGGGGAGATCATCGGG-3′) and reverse (5′-TCCATAGGAGACAATGCCCTG-3′) primers designed to match the sequences of human, mouse, and bovine granzyme B. The product was cloned into the pGEM-T Easy vector (Promega). The granzyme B sequence was detected in the cDNA by a real-time PCR assay using the forward primer 5′-GCAGGAGGTGAGGCTGGAA-3′, the reverse primer 5′-GGGATAGCGGGATTTGCA-3′, and the probe 6-carboxyfluorescein (FAM)-TGCAGGACGATGGCA-MGBNFQ. Influenza A virus was detected using influenza A virus matrix primers and probes as previously described (24), and influenza B virus was detected using CDC universal influenza B virus primers and probes targeting the influenza B virus NS gene from the CDC influenza virus real-time RT-PCR influenza/B typing panel, obtained through the Influenza Reagent Resource, Influenza Division, WHO Collaborating Centre for Surveillance, Epidemiology and Control of Influenza, Centers for Disease Control and Prevention, Atlanta, GA. The reverse transcription reaction mix volume resulted in 1 ng initial RNA/μl final cDNA preparation for URT samples and 10 ng initial RNA/μl final cDNA preparation for LRT culture samples. cDNA standards were generated in parallel with cDNA test samples for each experiment, using RNAs pooled from a range of test samples within each experiment. At least 80 μl at 10 ng initial RNA/μl final cDNA was prepared for URT experiments, and at least 80 μl at 100 ng initial RNA/μl final cDNA was prepared for LRT experiments. Tenfold and 2-fold serial dilutions (5 dilutions of each) were prepared and used for standard curve generation for efficiency calculations. cDNAs were stored at −20°C. TaqMan real-time PCR assays were performed using cDNA samples, standards, and plasmid controls as previously described (24). All calculations were performed as previously described (24). The time zero control sample for all in vivo infection time courses was a pool of nasal washes from 50 uninfected ferrets (URT measures) or lung tissues from uninfected ferrets (LRT measures).

Statistical analysis.

Data were analyzed using R-2.15.3 software. mRNA expression levels in nasal wash samples from ferrets infected with A(H1N1)pdm09 virus were assessed over time by using a random-effects model with Tukey's post hoc analysis. mRNA expression levels in lung samples from ferrets infected with A(H1N1)pdm09 virus were compared over time by using one-way analysis of variance (ANOVA) with Tukey's post hoc test. Temperatures, or gene expression levels for nasal wash samples, were compared between ferrets infected with A(H1N1)pdm09, A(H3N2), or influenza B virus for each day, using one-way ANOVA with Tukey's post hoc test. The correlations between virus shedding and mRNA expression (fold change) were estimated by determining the Pearson correlation coefficients.

Nucleotide sequence accession number.

The sequence encoding ferret granzyme B is available in GenBank under accession number KT324658 .

RESULTS

Patterns of cytokine and chemokine expression are similar in the upper and lower respiratory tracts following A(H1N1)pdm09 influenza virus infection.

We assessed clinical signs of disease and expression levels of cytokine and chemokine mRNAs following intranasal infection of ferrets with A(H1N1)pdm09 virus. Nasal wash samples were collected daily to measure upper respiratory tract (URT) infection, while lung tissues from ferrets sacrificed daily were harvested to evaluate lower respiratory tract (LRT) infection.

Ferrets lost weight over the course of the infection but recovered over the period of analysis (Fig. 1A). A modest temperature peak was detected on day 2 after infection (Fig. 1B); minimal other clinical signs were observed, consistent with previous studies with the A(H1N1)pdm09 virus (27, 28). In the URT, viral RNA levels peaked at day 2 postinfection and declined thereafter, with viral clearance occurring 8 or 9 days after infection (Fig. 1C). The kinetics of virus replication were different in the LRT, with viral RNA levels remaining below detection for the first 2 days after infection before peaking at days 4 to 6 postinfection and declining thereafter (Fig. 1C). The real-time PCR fold changes and infectious virus titers (TCID₅₀) were determined for a subset of samples to indicate the cutoff for infectious virus for all real-time PCR data (Fig. 1D). Seroconversion (HI titer of 160 to 640) was detected on day 7 after infection (data not shown).

Expression of cytokines and chemokines was also measured in the URT and LRT at various times after infection with A(H1N1)pdm09 virus. Because the amounts of total RNA in URT and LRT samples varied over time (Fig. 2A and B, respectively), a consistent amount of total RNA was assessed for each sample. Expression of mRNAs for cytokines and chemokines was standardized to the mRNA expression of housekeeping (HKP) genes for each tissue. The HKP genes encoding hypoxanthine phosphoribosyltransferase (HPRT), ATF4, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were consistently expressed in URT samples and thus were used for standardization of URT data (Fig. 3A and C), while the HKP genes encoding L32, GAPDH, HPRT, and ATF4 were consistently expressed in LRT samples and thus were used for standardization of LRT data (Fig. 3B and D). The efficiencies of real-time PCRs for all cytokine and chemokine genes were consistent and overlapped those for the HKP genes (Fig. 2C and D).

FIG 2 — Quantitation of total RNAs isolated from URT and LRT samples and assessment of efficiency of TaqMan assays of ferret cytokines by using cDNA. (A) Each point indicates the amount of RNA isolated from a nasal wash (URT) sample collected from an individual ferret at a particular time point after A(H1N1)pdm09, A(H3N2), or influenza B virus infection. There was no difference in the amounts of RNA at each time point between different viruses. (B) Each point indicates the amount of RNA isolated from a single lung lobe (LRT) sample from a ferret infected with A(H1N1)pdm09 virus. (C and D) Efficiencies of reaction for all genes for a cDNA pool generated from URT (C) and LRT (D) samples as determined by 2-fold and 10-fold dilutions. Housekeeping genes (ATF4, HPRT, GAPDH, and L32) are indicated by white circles, and outliers are indicated by white squares. The mean for each group is indicated by a horizontal line. The percent reaction efficiency and corresponding PCR efficiency (E) are indicated by dotted lines.

FIG 3 — Assessment of housekeeping gene expression in URT and LRT samples from ferrets. Expression of housekeeping genes was determined for URT (A) and LRT (B) samples collected daily after A(H1N1)pdm09 virus infection. The mean for each group is indicated by a horizontal line, and P values indicate overall differences between groups. (B) Each point indicates the amount of RNA isolated from a single lung lobe (LRT) sample from a ferret. (C and D) After stepwise exclusion of the more variable genes, the average expression stability values (M) of the remaining housekeeping genes were calculated using geNorm (54), for both the URT (C) and the LRT (D). The least stable gene is shown on the left, and the most stable gene is shown on the right. Note the consistency of expression of HPRT, ATF4, and GAPDH for URT samples and the consistency of expression of L32, HPRT, ATF4, and GAPDH for LRT samples.

In general terms, we noted that induction of a number of inflammatory mediators tended to correlate with levels of viral replication, and for the purposes of this study, these were classified as having an “early” induction profile (Fig. 4). In the URT, expression of IL-1β, IL-6, tumor necrosis factor alpha (TNF-α), alpha interferon (IFN-α), and IFN-β peaked at day 2 postinfection (P values of <0.001 to 0.05) (Fig. 4A). Expression of IL-1α and IL-12p40 also appeared to peak at day 2 postinfection, though this was not significant (Fig. 4A). There were strong correlations between the expression patterns of IL-1α, IL-1β, IL-6, IL-12p40, TNF-α, IFN-α, and IFN-β and expression of influenza virus in the URT (Table 1). In the LRT, expression of IL-1α, IL-6, IL-12p40, and IFN-β peaked at days 3 to 7, which again correlated with the kinetics of viral replication (Fig. 4B; Table 1).

FIG 4 — Early peak gene expression in the URT and LRT following A(H1N1)pdm09 virus infection. Ferrets were infected intranasally with A(H1N1)pdm09 virus. URT (A) and LRT (B) samples were assayed, and the data are presented as described in the legend to Fig. 1C. The IFN-β gene was not detected in the day 0 sample from the URT, so the *C_T* was given a value of 40; all other genes had detectable *C_T* values at day 0. Dotted lines indicate the baseline values for samples from uninfected ferrets. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

TABLE 1.

Correlations between virus shedding and cytokine, chemokine, or immune mediator gene expression for URT and LRT samples from ferrets infected with A(H1N1)pdm09 virus

Cytokine, chemokine, or immune mediator	Correlation coefficient (P value)^a
Cytokine, chemokine, or immune mediator	URT	LRT
IL-1α	0.2284 (0.0117)	0.7886 (<0.0001)
IL-1β	0.4221 (<0.0001)	0.1569 (0.2118)
IL-6	0.6139 (<0.0001)	0.7855 (<0.0001)
IL-12p40	0.2539 (0.001)	0.4303 (0.0003)
TNF-α	0.2898 (0.0001)	0.3332 (0.00667)
IFN-α	0.61 (<0.0001)	0.6292 (<0.0001)
IFN-β	0.9136 (<0.0001)	0.6759 (<0.0001)
MCP1	−0.06217 (0.4192)	0.8161 (<0.0001)
IFN-γ	−0.01088 (0.888)	0.6291 (<0.0001)
Granzyme A	−0.1866 (0.0413)	−0.06643 (0.5990)
IL-10	−0.1539 (0.0445)	0.2984 (0.0157)
IL-17	−0.09944 (0.2799)	0.4319 (0.0003)
IL-8	−0.09259 (0.2284)	0.7886 (<0.0001)
IL-2	Not detected	0.1376 (0.2742)

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Pearson correlation coefficients were estimated for the relationship between virus shedding (fold change) and gene expression (fold change). P values shown in bold represent significant correlations.

Additional genes showed patterns of expression that did not correlate as strongly with the kinetics of viral replication, and in some cases no clear peak in expression could be detected (Fig. 5). In the URT, (i) MCP1 expression remained high from days 3 to 7 postinfection, (ii) IFN-γ was expressed at high levels on days 3 to 6 (P < 0.05 on days 4 and 5), (iii) granzyme A expression increased from days 3 to 5 before reaching a clear peak at day 6 (P < 0.001) and declining thereafter, and (iv) IL-10 expression peaked at days 8 and 9 postinfection (Fig. 5A). Note that expression of granzyme A and IL-10 was negatively correlated with the virus peak (Table 1). No clear peak was detected for the expression of IL-17 or IL-8, whereas IL-2 and IL-4 were not detected in the URT at any time point (Fig. 5A and data not shown). In the LRT, (i) MCP1 and IFN-γ were expressed at high levels on days 5 to 7, (ii) granzyme A expression peaked on days 7 and 8, and (iii) IL-10 expression peaked at days 7 to 9. There was no clear peak in expression of IL-17, IL-8, or IL-2 in the LRT (Fig. 5B). IL-4 was not detected at any time point (data not shown).

FIG 5 — Later peak gene expression in URT and LRT following A(H1N1)pdm09 virus infection. Ferrets were infected intranasally with A(H1N1)pdm09 virus, and data for the URT (A) and LRT (B) are presented as described in the legends to Fig. 1C and 2. The granzyme A gene was not detected in the day 0 sample from the URT, so the *C_T* was given a value of 40; all other genes had detectable *C_T* values at day 0. IL-2 was not detected in URT samples. Note that the expression of granzyme B mRNA was not assessed for these samples. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Seasonal influenza A(H3N2) virus and influenza B virus induce cytokine and chemokine patterns similar to those with A(H1N1)pdm09 virus following infection of the URT.

Naive ferrets were infected with A(H1N1)pdm09, A(H3N2), and influenza B viruses to determine whether there were differences in cytokine/chemokine responses to different influenza virus (sub)types. URT samples were collected after viral infection for analysis. No virus was detected in the LRT (data not shown). Animals lost weight but recovered to baseline over the period of examination (Fig. 6A). There was a small temperature peak in ferrets infected with A(H1N1)pdm09 and influenza B viruses on day 2 after infection (Fig. 6B). No other clinical symptoms were observed.

FIG 6 — Gene expression in the URT following A(H1N1)pdm09, A(H3N2), or influenza B virus infection. Ferrets were infected intranasally with influenza A(H1N1)pdm09 (black circles and bars), B (white triangles and striped bars), or A(H3N2) (white circles and bars) virus (n = 3 or 4 per group). Weights (A) and temperatures (B) were measured daily. The percent weight change was calculated from the starting measurement on the day of infection. Data shown are means ± SD. (C) URT samples were collected daily for analysis. Infectious virus was measured by the TCID₅₀ assay, and fold changes in expression of all genes are presented as described in the legend to Fig. 1C. The IFN-β gene was not detected in the day 0 samples from the URT, so the *C_T* was given a value of 40; all other genes had detectable *C_T* values at day 0. *, P < 0.05; **, P < 0.01.

Following infection with each of the three strains, virus titers in the URT peaked at 2 to 3 days postinfection and were cleared by day 8 postinfection (Fig. 6C). Moreover, expression patterns of each cytokine, chemokine, or immune mediator were similar following infection of ferrets with A(H1N1)pdm09, A(H3N2), or influenza B virus. Based on studies of A(H1N1)pdm09-infected ferrets (Fig. 4 and 5), cytokines and chemokines with clear peaks in expression (IL-6, IFN-α, IFN-β, MCP1, IFN-γ, granzyme A, and granzyme B) were compared between ferrets infected with different viruses (Fig. 6C). Data are not shown for the other genes, which did not exhibit clear peaks in expression. Expression levels of IL-6, MCP1, and granzyme A mRNAs did not differ between ferrets infected with the three viruses (Fig. 6C). Expression levels of IFN-α, IFN-β, IFN-γ, and granzyme B were generally similar, although different expression levels were detected on particular days (Fig. 6C). No difference in cytokine profile was observed when ferrets were infected with the same dose (TCID₅₀) of cell-grown A(H3N2) virus or the same virus isolated in eggs (data not shown).

To assess whether virus replication was required for localized cytokine and chemokine mRNA expression, we assessed LRT samples from ferrets infected with influenza B and A(H3N2) viruses. Although expression of mRNAs for several immune mediators was detected in URT samples, none was detected in LRT samples from the same animals (data not shown).

Overall, these data demonstrate that the recent human influenza viruses used in our study do not induce major differences in the kinetics of virus replication or the induction of chemokines and cytokines in the URT following infection in ferrets and that inflammation and cell recruitment rely on the presence of replicating virus.

DISCUSSION

Clinical and epidemiological reports provide a complex picture of the impacts of different seasonal influenza virus (sub)types on infection risk and disease severity, with both influenza A and B viruses contributing to morbidity and mortality (21, 29 –34). However, the understanding of disease due to seasonal influenza viruses in “normal” cases (nonhospitalized patients with nonfatal cases) is limited. Ferrets can be infected directly with human influenza viruses, without the need for adaptation, and display clinical disease symptoms similar to those of humans, providing an ideal model for human influenza (35 –37). We characterized the immune responses following seasonal influenza A and B virus infections in normal healthy ferrets. There was consistency in the pattern and overall magnitude of cytokine expression following infection with seasonal influenza A or B viruses in the URT. Furthermore, a consistent pattern of cytokine and chemokine expression, although of a lower magnitude, was also detected in the LRT following A(H1N1)pdm09 virus infection. These data provide a baseline reading for responses to seasonal influenza virus infection and suggest that disease levels associated with seasonal and pandemic influenza virus infections in healthy individuals are similar.

Patterns of gene expression were broadly similar in the URT and LRT following A(H1N1)pdm09 virus infection (Fig. 7). The same genes were expressed in unison with peak viral shedding in the URT and LRT, indicating that induction of the early innate immune response is related to the presence of localized virus irrespective of the site. Markers of the adaptive immune response were expressed on the same days after infection in both the URT and LRT, which is suggestive of systemic activation of virus-specific T and B lymphocytes in draining lymph nodes, most likely by antigen-presenting cells migrating from the URT, and subsequent recruitment of activated lymphocytes to the infected respiratory tract. Importantly, activated leukocytes were recruited only to sites of infection and inflammation, as no cytokines or chemokines were detected in the LRT of ferrets infected with A(H3N2) or influenza B virus, where there was no detectable virus replication at any time after infection. A number of cytokines and chemokines had lower fold changes of mRNA expression in the LRT than in the URT. This most likely reflects the different compositions of tissues collected from lungs and nasal washes; similar results have been reported by others (14).

The in vivo cytokine profiles following infection with A(H1N1)pdm09 virus presented in this study are consistent with those in other studies using the SYBR green real-time PCR assay and microarrays in ferrets (14, 15, 22, 23, 38 –40). Similar cytokine and chemokine profiles have also been detected in plasmas from children infected with A(H1N1)pdm09 virus (41), autopsy tissues from fatal cases of A(H1N1)pdm09 virus infection (42), and the lungs of pigs (43), mice, and macaques (44) infected with A(H1N1)pdm09 virus.

This study demonstrated a remarkable consistency in the cytokine and chemokine patterns in the URT following infection with seasonal influenza A and B viruses. Furthermore, there was no clear hierarchy for the magnitudes of expression of all cytokines after infection with the three viruses assessed. Because the A(H1N1)pdm09 virus replicates in both the URT and the LRT, it has been suggested that the immune response is greater for this virus than for seasonal influenza A viruses. Our data demonstrate that, provided the viral load is equivalent, there is no overall change in magnitude or kinetics of localized cytokine and chemokine production. Comparisons of cytokine and chemokine production levels following seasonal A(H1N1) (pre-2009) and A(H1N1)pdm09 infections, A(H3N2) and A(H1N1)pdm09 infections, or A(H1N1) (pre-2009) and influenza B virus infections have also been performed by other groups, using SYBR green real-time PCR on various respiratory tissue samples (14, 22, 45). A hierarchy was observed for gene expression levels, which mirrored that for virus loads, suggesting that the magnitude of the response is directly related to the amount of virus present (14, 22, 45). We titrated inoculum doses of other viruses in the ferret model and observed delayed virus shedding when animals were inoculated with lower virus doses and more rapid kinetics when animals were inoculated with higher virus doses (data not shown). Infection with lower inoculum doses or infection of animals naturally by transmission may delay the kinetics of cytokine and chemokine production. Others have also shown that the route of virus administration can affect the clinical disease (46, 47) and that the volume of virus inoculum can influence virus spread (48). Although a larger inoculum volume (1 ml) is associated with more consistent LRT replication than that with the small volume used in our study (0.5 ml), all lung lobes were collected and analyzed in our study, minimizing any sampling bias associated with dosing, as previously reported (48). Furthermore, while we cannot completely rule out that the inability to detect growth of A(H3N2) or influenza B virus in the LRT was due to intranasal inoculation, others have reported only limited replication of a 2008 A(H3N2) virus in the LRT when dosed intratracheally in a ferret model of pneumonia (49). Assessments of cytokine and chemokine profiles following infections with other seasonal influenza virus strains are also of interest.

In this comparison, we infected and sampled ferrets with the three viruses concurrently, generated cDNAs, and then performed real-time PCR on all samples in the same assays to enable a direct comparison. Measuring mRNA levels enables many genes to be detected in limited URT samples. Our assays require 1 ng RNA and can be multiplexed (24), enabling expression of over 20 genes to be tested in a single nasal wash sample, typically with limited RNA. Confirmation of leukocyte cell recruitment to the URT and LRT by flow cytometry assays would complement the real-time PCR data shown here. Antibodies have been described for some leukocytes of the ferret (50 –53), yet these are limited and can give variable results in different laboratories. Studies to assess cytokine and chemokine production by ferret leukocytes by using flow cytometry analyses are ongoing.

This study shows a high level of consistency in patterns of cytokines, chemokines, and immune mediators induced following influenza virus infection of the ferret, regardless of the type or subtype of virus. These findings contribute to our understanding of seasonal influenza virus infections and suggest that any seasonal influenza virus may have a significant impact on immunocompromised individuals.

ACKNOWLEDGMENTS

The Melbourne WHO Collaborating Centre for Reference and Research on Influenza is supported by the Australian Government Department of Health and Ageing.

We are grateful for advice provided by Steve Vander Hoorn. We are also grateful for technical assistance provided by the bioCSL animal house staff.

Funding Statement

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

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[B2] 2.Vijaykrishna D, Holmes EC, Joseph U, Fourment M, Su YCF, Halpin R, Lee RTC, Deng YM, Gunalan V, Lin X, Stockwell TB, Fedorova NB, Zhou B, Spirason N, Kühnert D, Bošková V, Stadler T, Costa AM, Dwyer DE, Huang QS, Jennings LC, Rawlinson W, Sullivan SG, Hurt AC, Maurer-Stroh S, Wentworth DE, Smith GJ, Barr IG. 2015. The contrasting phylodynamics of human influenza B viruses. eLife 4:e05055. doi: 10.7554/eLife.05055. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B3] 3.CDC. 19 August 2015, posting date Flu symptoms and severity. http://www.cdc.gov/flu/about/disease/symptoms.htm.

[B4] 4.McCullers JA, Hayden FG. 2012. Fatal influenza B infections: time to reexamine influenza research priorities. J Infect Dis 205:870–872. doi: 10.1093/infdis/jir865. [DOI] [PubMed] [Google Scholar]

[B5] 5.Paddock CD, Liu L, Denison AM, Bartlett JH, Holman RC, Deleon-Carnes M, Emery SL, Drew CP, Shieh WJ, Uyeki TM, Zaki SR. 2012. Myocardial injury and bacterial pneumonia contribute to the pathogenesis of fatal influenza B virus infection. J Infect Dis 205:895–905. doi: 10.1093/infdis/jir861. [DOI] [PubMed] [Google Scholar]

[B6] 6.Tate MD, Schilter HC, Brooks AG, Reading PC. 2011. Responses of mouse airway epithelial cells and alveolar macrophages to virulent and avirulent strains of influenza A virus. Virus Immunol 24:77–88. doi: 10.1089/vim.2010.0118. [DOI] [PubMed] [Google Scholar]

[B7] 7.La Gruta NL, Kedzierska K, Stambas J, Doherty PC. 2007. A question of self-preservation: immunopathology in influenza virus infection. Immunol Cell Biol 85:85–92. doi: 10.1038/sj.icb.7100026. [DOI] [PubMed] [Google Scholar]

[B8] 8.Oshansky CM, Gartland AJ, Wong SS, Jeevan T, Wang D, Roddam PL, Caniza MA, Hertz T, Devincenzo JP, Webby RJ, Thomas PG. 2014. Mucosal immune responses predict clinical outcomes during influenza infection independently of age and viral load. Am J Respir Crit Care Med 189:449–462. doi: 10.1164/rccm.201309-1616OC. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B9] 9.de Jong MD, Simmons CP, Thanh TT, Hien VM, Smith GJ, Chau TN, Hoang DM, Chau NV, Khanh TH, Dong VC, Qui PT, Cam BV, Ha do Q, Guan Y, Peiris JS, Chinh NT, Hien TT, Farrar J. 2006. Fatal outcome of human influenza A (H5N1) is associated with high viral load and hypercytokinemia. Nat Med 12:1203–1207. doi: 10.1038/nm1477. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B10] 10.Chiaretti A, Pulitanò S, Conti G, Barone G, Buonsenso D, Manni L, Capozzi D, Ria F, Riccardi R. 2013. Interleukin and neurotrophin up-regulation correlates with severity of H1N1 infection in children: a case-control study. Int J Infect Dis 17:e1186–93. doi: 10.1016/j.ijid.2013.07.006. [DOI] [PubMed] [Google Scholar]

[B11] 11.Paquette SG, Banner D, Zhao Z, Fang Y, Huang SS, Leün AJ, Ng DC, Almansa R, Martin-Loeches I, Ramirez P, Socias L, Loza A, Blanco J, Sansonetti P, Rello J, Andaluz D, Shum B, Rubino S, de Lejarazu RO, Tran D, Delogu G, Fadda G, Krajden S, Rubin BB, Bermejo-Martin JF, Kelvin AA, Kelvin DJ. 2012. Interleukin-6 is a potential biomarker for severe pandemic H1N1 influenza A infection. PLoS One 7:e38214. doi: 10.1371/journal.pone.0038214. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B12] 12.Yu X, Zhang X, Zhao B, Wang J, Zhu Z, Teng Z, Shao J, Shen J, Gao Y, Yuan Z, Wu F. 2011. Intensive cytokine induction in pandemic H1N1 influenza virus infection accompanied by robust production of IL-10 and IL-6. PLoS One 6:e28680. doi: 10.1371/journal.pone.0028680. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B13] 13.Hayden FG, Treanor JJ, Fritz RS, Lobo M, Betts RF, Miller M, Kinnersley N, Mills RG, Ward P, Straus SE. 1999. Use of the oral neuraminidase inhibitor oseltamivir in experimental human influenza: randomized controlled trials for prevention and treatment. JAMA 282:1240–1246. doi: 10.1001/jama.282.13.1240. [DOI] [PubMed] [Google Scholar]

[B14] 14.Maines T, Belser J, Gustin K, van Hoeven N, Zeng H, Svitek N, von Messling V, Katz J, Tumpey T. 2012. Local innate immune responses and influenza virus transmission and virulence in ferrets. J Infect Dis 205:474–485. doi: 10.1093/infdis/jir768. [DOI] [PubMed] [Google Scholar]

[B15] 15.Meunier I, Embury-Hyatt C, Stebner S, Gray M, Bastien N, Li Y, Plummer F, Kobinger G, von Messling V. 2012. Virulence differences of closely related pandemic 2009 H1N1 isolates correlate with increased inflammatory responses in ferrets. Virology 422:125–131. doi: 10.1016/j.virol.2011.10.018. [DOI] [PubMed] [Google Scholar]

[B16] 16.Svitek N, Rudd P, Obojes K, Pillet S, von Messling V. 2008. Severe seasonal influenza in ferrets correlates with reduced interferon and increased IL-6 induction. Virology 376:53–59. doi: 10.1016/j.virol.2008.02.035. [DOI] [PubMed] [Google Scholar]

[B17] 17.Korteweg C, Gu J. 2010. Pandemic influenza A (H1N1) virus infection and avian influenza A (H5N1) virus infection: a comparative analysis. Biochem Cell Biol 88:575–587. doi: 10.1139/O10-017. [DOI] [PubMed] [Google Scholar]

[B18] 18.Wang C, Yu H, Horby PW, Cao B, Wu P, Yang S, Gao H, Li H, Tsang TK, Liao Q, Gao Z, Ip DKM, Jia H, Jiang H, Liu B, Ni MY, Dai X, Liu F, Van Kinh N, Liem NT, Hien TT, Li Y, Yang J, Wu JT, Zheng Y, Leung GM, Farrar JJ, Cowling BJ, Uyeki TM, Li L. 2014. Comparison of patients hospitalized with influenza A subtypes H7N9, H5N1, and 2009 pandemic H1N1. Clin Infect Dis 58:1095–1103. doi: 10.1093/cid/ciu053. [DOI] [PMC free article] [PubMed] [Google Scholar]

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PERMALINK

Characterization of the Localized Immune Response in the Respiratory Tract of Ferrets following Infection with Influenza A and B Viruses

Louise A Carolan

Steve Rockman

Kathryn Borg

Teagan Guarnaccia

Patrick Reading

Jennifer Mosse

Anne Kelso

Ian Barr

Karen L Laurie

Roles

ABSTRACT

INTRODUCTION

MATERIALS AND METHODS

Ferrets.

Viruses.

TCID50 assay.

Virus infection of ferrets.

Generation and collection of ferret URT and LRT samples.

RNA extraction.

Reverse transcription and TaqMan real-time PCR.

Statistical analysis.

Nucleotide sequence accession number.

RESULTS

Patterns of cytokine and chemokine expression are similar in the upper and lower respiratory tracts following A(H1N1)pdm09 influenza virus infection.

FIG 1.

FIG 2.

FIG 3.

FIG 4.

TABLE 1.

FIG 5.

Seasonal influenza A(H3N2) virus and influenza B virus induce cytokine and chemokine patterns similar to those with A(H1N1)pdm09 virus following infection of the URT.

FIG 6.

DISCUSSION

FIG 7.

ACKNOWLEDGMENTS

Funding Statement

REFERENCES

ACTIONS

PERMALINK

RESOURCES

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Links to NCBI Databases

TCID₅₀ assay.