Molecular Immunology 46 (2009) 17441749

Contents lists available at ScienceDirect

Molecular Immunology
journal homepage: www.elsevier.com/locate/molimm

Immune-related gene expression in response to H11N9 low pathogenic avian

inuenza virus infection in chicken and Pekin duck peripheral blood
mononuclear cells
Sean C. Adams a , Zheng Xing b,c , Jinling Li a , Carol J. Cardona a,b,
a

Department of Population Health and Reproduction, University of California, Davis, Davis, CA 95616, USA
Veterinary Medicine Extension, University of California, Davis, Davis, CA 95616, USA
c
Department of Pathology, Microbiology and Immunology, University of California, Davis, Davis, CA 95616, USA
b

a r t i c l e

i n f o

Article history:
Received 20 December 2008
Accepted 29 January 2009
Available online 27 February 2009
Keywords:
Comparative immunology
Pekin duck
Chicken
Avian inuenza virus
Cytokines
Interferon alpha
Interferon beta
Interferon gamma
Interleukin 1 beta
Interleukin 6
Quantitative real-time PCR
Peripheral blood mononuclear cells
Toll-like receptor 7
MHC

a b s t r a c t
The duck and chicken are important hosts of avian inuenza virus (AIV) with distinctive responses to
infection. Frequently, AIV infections in ducks are asymptomatic and long-lasting in contrast to the clinically apparent and transient infections observed in chickens. These differences may be due in part to
the host response to AIV infection. Using real-time quantitative PCR, we examined the expression of
immune-related genes in response to low pathogenic AIV H11N9 infection in peripheral blood mononuclear cells (PBMC) isolated from the blood of chickens and Pekin ducks. While chicken PBMC expressed
IL-1 and IL-6 at high levels similar to mammalian species, duck PBMC expression levels were minimal or unchanged. Similarly, duck IFN- expression was nearly unaffected, whereas chicken expression
was highly upregulated. Chicken IFN- was expressed to higher levels than duck IFN-, while IFN- was
expressed similarly by both species. IL-2 was elevated early in infection in duck PBMC, but returned to
baseline levels by the end of the experiment; in contrast, IL-2 was weakly induced in chicken PBMC at late
time points. TLR-7 and MHC class I molecule expressions were conserved between species, whereas duck
MHC class II expression was downregulated and chicken expression was unchanged. These results show
distinct PBMC expression patterns of pro-inammatory cytokines and IFNs between species. The differences in pro-inammatory cytokine and IFN expression reect the asymptomatic and lasting infection
observed in ducks and the tendency towards clinical signs and rapid clearance seen in chickens. These
results highlight important differences in the host response to AIV of two species thought to be critical
in the genesis and maintenance of epidemic strains of AIV.
2009 Elsevier Ltd. All rights reserved.

1. Introduction
Avian inuenza viruses (AIV) belong to the Orthomyxoviridae,
are of the Type A genus, and have negative-sense, single-stranded,
segmented genomes. Avian inuenza viruses are encapsulated by
envelopes containing the surface proteins hemagglutinin (HA) and
neuraminidase (NA) and are classied by 16 identied HA and 9 NA
subtypes (Fouchier et al., 2005), all of which occur in their reservoir hosts, free-ying waterfowl and shorebirds (Spackman, 2008;
Webster et al., 1992).
The roles of birds as reservoir hosts and hosts in which viruses
with pandemic potential can be amplied and transmitted to
humans have become a focus of interest with the emergence

Corresponding author at: Room 1383, Surge III, One Shields Avenue, University
of California, Davis, Davis, CA 95616, USA. Tel.: +1 530 754 5041;
fax: +1 530 752 7563.
E-mail address: cjcardona@ucdavis.edu (C.J. Cardona).
0161-5890/$ see front matter 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.molimm.2009.01.025

and perpetuation of H5N1 highly pathogenic avian inuenza virus

(HPAIV). Despite this, few studies have been done to elucidate basic
viral pathogenesis and host response questions in avian species
resulting in incomplete and confusing data on AIV in avian hosts.
For example, chickens and ducks generally respond to AIV infections differently and there are many instances demonstrating that
infection with a specic AIV isolate may cause lesions and even
death in a chicken host, while infection of a duck with the same
virus would be asymptomatic, rarely resulting in death (Homme
and Easterday, 1970; Kida et al., 1980; Narayan et al., 1969; Otsuki
et al., 1982; Slemons and Easterday, 1972; Slemons et al., 1990). AIV
shedding in chickens is transient with a rapid clearance by the host
(Kwon et al., 2008; Lee et al., 2004; Otsuki et al., 1982; Smith et al.,
1980); in contrast, prolonged, intermittent shedding is observed
in infected ducks (Higgins et al., 1987). While chickens can mount
a strong humoral immune response to AIV infection (Suarez and
Schultz-Cherry, 2000), it has been reported that ducks do not (Kida
et al., 1980; Philpott et al., 1989). Further, though AIV replication
has been reported in both the respiratory system and the intestinal

S.C. Adams et al. / Molecular Immunology 46 (2009) 17441749

tract for both species (Wood et al., 1995), AIV are usually limited in
distribution to the intestinal tract in the duck (Scholtissek, 1995),
while replication in the upper respiratory tract of chickens with
some migration to the intestinal tract is more common (Lee et al.,
2007; Swayne, 1997). To add to the complexity, inuenza viruses
adapted to efcient growth in ducks, do not always grow in chickens and vice versa and thus, very few studies have compared viral
pathogenesis of or host responses to the same AIV.
AIV replicates primarily in the epithelial cells of the lung and
intestines, however, spread to resident macrophages and recruited
monocytes are also a feature of infection (Herold et al., 2006).
Macrophages, among other cells that make up PBMC, possess a
battery of receptors responsible for the recognition of conserved
molecular patterns expressed by pathogens and the subsequent
induction of a signaling cascade that culminates in the activation of an immune response. One such receptor, toll-like receptor
(TLR) 7 is found in the endosomal compartment and recognizes
single-stranded viral RNA released during the uncoating of internalized virus (Barton, 2007). The recognition of viral RNA results
in the secretion of pro-inammatory cytokines such as IL-1 and
IL-6 as well as anti-viral cytokines such as the interferons (IFNs)
(MacDonald et al., 2008). Expression of IFNs and pro-inammatory
cytokines inuences both viral clearance and clinical disease
presentation. MHC class I and II antigen presentation, though selectively utilized on the basis of pathogen uptake, both serve to activate
cellular members of the adaptive immune response such as B cells
and T cells (CD4+ and CD8+) (Gromme and Neefjes, 2002; Williams
et al., 2002). Endogenously processed antigen is presented by MHC
class I molecules and activates CD8+ cytotoxic lymphocytes (CTL),
while antigen processed exogenously is presented by MHC class II
molecules activating antibody secreting B cells and helper CD4+
T cells (Germain, 1994; Lennon-Dumenil et al., 2002). The differential expression of the MHC molecules and the expression of
cytokines such as IL-2 and IL-6 may provide clues as to whether a
cell-mediated (Th1) or antibody (Th2) response are being initiated
by the cells responding to inuenza virus infection.
The in vitro infection of chicken and duck PBMC with AIV serves
as a starting point for observing host responses. By comparing the
expression of cytokines involved in pathogen responses including
the pro-inammatory, anti-viral, and cell-mediated and adap-

1745

tive responses in PBMC, we can better understand how immune

responses and thus, pathogenesis might differ between two highly
relevant agricultural species. Here we present the results of quantitative real-time RT-PCR analysis of several cytokines, the TLR-7, and
the MHC class I and II molecules expressed in response to infection
with the same low pathogenic avian inuenza virus (LPAIV) H11N9
in chicken and duck PBMC.
2. Materials and methods
Birds: Two 1-year-old Pekin ducks serologically negative for
AIV antibodies were obtained from a commercial breeder (Metzer
Farms, Gonzales, CA). Two Hyline W-36 egg-laying hens serologically negative for AIV antibodies were obtained from the University
of California, Davis avian research facility (Hopkins Avian Research
Facility, UC Davis). Blood was collected from all birds by wing
venipuncture into heparinized RPMI medium (Invitrogen Corp.,
Carlsbad, CA). Ten millilitres of blood was collected from each bird
and subsequently pooled by species.
2.1. PBMC cell culture
Peripheral blood mononuclear cells (PBMC) were puried by
Ficoll gradient (Lymphocyte Separation Media, Mediatech, Inc.,
Herndon, VA) separation. The PBMC were grown overnight in RPMI
supplemented with 10% fetal bovine serum, 5% chicken serum,
100 U/mL penicillin, and 100 g/mL streptomycin at 37 C, 5% CO2
with a cell density of approximately 5 107 cells/60 mm tissue culture dish. After overnight growth, non-adherent cells were removed
by washing the monolayers with sterile PBS to enrich the cultures
for adherent macrophages, monocytes, and dendritic cells.
2.2. Virus and cell culture infection
The AIV strain used in this study was A/duck/WA/663/97
(H11N9), a duck-adapted virus that has been well-characterized in
our laboratory (Li et al., 2008). The virus stocks were propagated
in SPF chicken eggs (Charles River, CA) using standard methods
(Woolcock, 2008) to a titer of 107.6 TCID50 /mL as determined in
Madin-Darby Canine Kidney (MDCK) cells.

Table 1
Sequence of the oligonucleotide primers used in quantitative RT-PCR.
RNA target

Size of PCR product (bp)

Target accession number

5 -CATCTGCCCATTTGATGTTG-3
5 -TGTCGATGTCCCGCATGA-3
5 - CGGTCGACTTATTTTTGCAGATATCT-3
5 -TTCCAGGTAGGTCTGAAAGGCGAA-3
5- AGGCGCTGTAATCGTTGTCT-3
5 - TGGATTCTCAAGTCGCTCATCG-3
5 -AAGCAGTGCAGGCAAAGAAT-3
5 -TGTAAACGTCTCCCCTTTGG-3
5 -ATTATCTTTGGGCCCCAGTC-3

200
80
80
171
387
248
222
312
218

V00407
NM204524
AY510091
EU170468
EU367971
DQ906156
NM001031338
DQ008588
DQ780342

Duck and chicken

Interferon
5 -CCTCAACCAGATCCAGCATT-3

5 - GGATGAGGCTGTGAGAGGAG-3

259

AY831397

Duck
GAPDH
IL-1
IL-2
IL-6
Interferon
Interferon
MHC class I
MHC class II
TLR-7

5 -CTGTCTTCGTGTGTGGCTGT-3
5 -GAGCTTGTAGCCCTTGATGC-3
5 -ATCGCCCACACTAAGAGCAT-3
5 -CCTTATCGTCGTTGCCAGAT-3
5 -GGGCTGTAGGTGTGGTTCTG-3
5 - GGATTTTCAAGCCAGTCAGC-3
5 -CTCTCCTCTCCAGTACGTCCTTCC-3
5 -CCGTTCTTCATCCAGGTGAT-3
5 -TCAAGAAATATCAAGATAATCACATCA-3

176
185
137
150
232
247
196
229
154

AY436595
DQ393268
AF294323
AB191038
EF053034
AJ012254
AB115246
AY905539
AY940195

Chicken
GAPDH
IL-1
IL-2
IL-6
Interferon
Interferon
MHC class I
MHC class II
TLR-7

Primer sequences
Forward

Reverse

5 -CCTCTCTGGCAAAGTCCAAG-3
5 -GCTCTACATGTCGTGTGTGATGAG-3
5 -CGGGATCCATGATGTGCAAAGTACTG-3
5 -ATGTGCAAGAAGTTCACCGTG-3
5 -ATGCCACCTTCTCTCACGAC-3
5 -GCTGACGGTGGACCTATTATT-3
5 -AAGAAGGGGAAGGGCTACAA-3
5 -CTCGAGGTCATGATCAGCAA-3
5 -TGTGATGTGGAAGCCTTTGA-3

5 -ATGTTCGTGATGGGTGTGAA-3
5 -TCGACATCAACCAGAAGTGC-3
5 -GCCAAGAGCTGACCAACTTC-3
5 -TTCGACGAGGAGAAATGCTT-3
5 -TCCTCCAACACCTCTTCGAC-3
5 -GCTGATGGCAATCCTGTTTT-3
5 -GAAGGAAGAGACTTCATTGCCTTGG-3
5 -CCACCTTTACCAGCTTCGAG-3
5 -CCTTTCCCAGAGAGCATTCA-3

PCR primers and accession numbers of gene targets.

1746

S.C. Adams et al. / Molecular Immunology 46 (2009) 17441749

Fig. 1. Cytokine expression in duck and chicken PBMC in response to H11N9 infection. (A) Fold change expression of pro-inammatory cytokines IL-1 and IL-6. (B) Fold
change expression of type I and II interferons. (C) Fold change expression of Th1-related cytokine IL-2. Cytokine expression was evaluated by qRT-PCR and normalized to the
expression of the reference gene glyceraldehyde-3-phosphate-dehydrogenase. Fold change was calculated by the Ct method. All results were obtained in triplicate. (* )
indicates p < 0.05 for the comparison of duck and chicken transcripts as determined by the Students T-test. Error bars represent standard error.

Cultures of PBMC were made in triplicate for both birds. One

PBMC culture was trypsinized and the cells counted. This representative count was used to calculate the volume of virus stock
necessary to infect the other three PBMC cultures at an m.o.i. of 4
for duck. Chicken PBMC were infected at an m.o.i. of 0.5 because an
m.o.i. of 4 resulted in the death of all cells in the culture by 8 hpi
and the failure to recover RNA (data not shown). To infect cells,
growth media was replaced with infection media containing RPMI
and supplemented as before, but without serum, and with the addition of viral allantoic uid (VAF). Negative control PBMC cultures
were set up identically but without the addition of VAF. Culture
plates were gently rocked every 15 min for 1 h after which the
media was replaced with RPMI supplemented with 0.2% fetal calf
serum (Invitrogen Corp., Carlsbad, CA) and 1 g/mL TPCK trypsin
(SigmaAldrich, St. Louis, MO). Cultures were incubated and RNA
extracted from the cell monolayer at 8, 24, and 36 h post-infection
(hpi).
2.3. RNA and cDNA preparation
Total RNA was isolated from infected and control PBMC at each
time point using QIAshredder columns and the RNeasy mini RNA
Purication kit following manufacturers instructions (Qiagen Inc.,

Valencia, CA). RNA in each sample was quantied using Ultrospec

2000 mass spectrophotometer (Pharmacia Biotech, Uppsala, Sweden). Three hundred nanograms of RNA from each culture was
treated with DNase to remove genomic DNA and then used to produce cDNA using a Quantitect Reverse Transcription kit (Qiagen
Inc., Valencia, CA).
2.4. Quantitative real-time RT-PCR
qRT-PCR was performed using primers designed by Primer3
software (http://frodo.wi.mit.edu/) based on published target
sequences. Primers were developed for IL-1, IL-2, IL-6, interferon
alpha (IFN-), interferon beta (IFN-), interferon gamma (IFN-),
TLR-7, and MHC class I and class II molecules based on published
sequences and the predicted product sizes are shown in Table 1.
Primers pairs were selected based on specicity as determined
by dissociation curves. qRT-PCR was performed using 7500 RealTime PCR System (Applied Biosystems) and Smartcycler (Cepheid)
thermocyclers. PCR conditions were the same for each targeted
gene and are as follows: 1 min at 50 C, 95 C for 5 min, followed
by 45 cycles of 95 C for 15 s and 58 C for 32 s. Cycling was terminated after 45 cycles with 95 C for 15 s, 60 C for 1 min, and
95 C for 15 s. Dissociation curves of the products were generated

S.C. Adams et al. / Molecular Immunology 46 (2009) 17441749

Fig. 2. Fold change expression of TLR-7 by duck and chicken PBMC in response
to H11N9 infection. (* ) indicates p < 0.05 for the comparison of duck and chicken
transcripts as determined by the Students T-test. Error bars represent standard error.

by increasing the temperature of samples incrementally from 55

to 100 C as the nal step of the real-time PCR. Amplied products
were run on a gel and extracted using a PCR and Gel Purication
kit (Qiagen Inc., Valencia, CA). For the purpose of assay validation, puried products were cloned into pCR-TOPO2.1 subcloning
vectors (Invitrogen, Carlsbad, CA) and sequenced to verify proper
target amplication using M13 forward and reverse primers as provided by Davis Sequencing (Davis, CA).
2.5. Calculations and statistics
Expression fold change was determined by the Ct method
using glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) as the
endogenous reference gene to normalize the level of target gene
expression. Logarithmic transformation was performed on fold
change values before being analyzed by the Students T-test
(Microsoft Excel 2007). The Students T-test was used to determine signicant difference between fold change values of chicken
and duck transcripts. Standard error was calculated using the fold
change values of three replicates for each gene measured.
3. Results
3.1. Differential expression of pro-inammatory, anti-viral, and
Th1-associated cytokines in duck and chicken PBMC
The expression of pro-inammatory cytokines IL-1 and IL-6
were statistically different (at p < 0.01) at each time point for duck
and chicken PBMC infected with H11N9. In chicken, both IL-1 and
IL-6 messages were induced by 8 hpi (9- and 1006-fold respectively)
and peaked at 24 hpi (16- and 1322-fold). While also upregulated,
IL-6 expression in duck cells was induced at a far lower level (peak
expression was 12-fold at 36 hpi). In contrast, IL-1 expression was
suppressed for the duration of the experiment 1.4- to 2-fold in
duck cells (see Fig. 1A). Taken together, these results indicate weak

1747

pro-inammatory signaling in duck PBMC, which is different from

the robust expression of pro-inammatory cytokines observed in
chicken PBMC.
We compared type I and II IFN expression in chicken and
duck cells infected with H11N9 and observed a similar speciesdependent response. The pattern of IFN- responses in chicken
and duck were similar with peak expression occurring at 8 hpi followed by a decrease to baseline expression between 24 and 36 hpi.
The differences in expression levels for IFN- were not statistically different (p > .05) between duck and chicken. The comparative
expression of IFN- represents the most striking nding in this
study: chicken IFN- is highly expressed and peaks at 238-fold
expression while the duck response is never more than 1.6-fold elevated (24 hpi). IFN- is induced, although insignicantly different
(p = .32) to 406-fold for duck and 757-fold for chicken cells by 8 hpi,
but a decline in expression in duck is observed by 24 hpi (39-fold
induction), whereas chicken IFN- continues to rise and peaks at
1698-fold expression at 24 hpi (see Fig. 1B).
The expression of the Th1-involved cytokine, IL-2, was signicantly higher in duck cells compared to chicken cells at 8 hpi, while
at the other time points IL-2 was not signicantly different between
the species. Despite the lack of statistically signicant differences
at the 24 and 36 hpi points, the trend of IL-2 expression is clearly
different: duck PBMC express elevated levels (4-fold induction) of
IL-2 early during the infection, followed by a gradual decrease to
baseline expression by 36 hpi. In contrast, chicken PBMC expression of IL-2 was gradually induced from baseline levels at 8 hpi to a
3-fold induction by 36 hpi.
3.2. Conserved downregulation of TLR-7 expression in chicken
and duck PBMC
We compared the expression of TLR-7 in response to infection
with H11N9 in chicken and duck PBMC. The results showed that
both duck and chicken expressed TLR-7 only transiently at the early
stage of infection followed by a decline (see Fig. 2.). Peak expression occurred at 8 hpi for both species PBMC, and never surpassed
baseline expression levels. At 24 and 36 hpi, the infection process
appeared to result in approximately the same level of downregulation of TLR-7, between 1.5- and 2-fold decrease in expression in
duck and chicken cells (see Fig. 2).
3.3. MHC class I and II molecule expression preference by species
MHC class I molecule expression was upregulated in duck PBMC
throughout the duration of the experiment. By 8 hpi, MHC class
I was upregulated 2-fold whereas MHC class II molecules were
downregulated by 1.5-fold. The trend of MHC class II suppression
continued through the experimental period, reaching its lowest
level at 36 hpi with a 3-fold downregulation. MHC class I, on the

Fig. 3. Fold change expression of major histocompatibility complexes by duck and chicken PBMC in response to H11N9 infection. (A) MHC class I molecule. (B) MHC class II
molecule. (* ) indicates p < 0.05 for the comparison of duck and chicken transcripts as determined by the Students T-test. Error bars represent standard error.

1748

S.C. Adams et al. / Molecular Immunology 46 (2009) 17441749

other hand, was upregulated to 5-fold by 24 hpi, followed by a

decrease to a 3-fold induction at 36 hpi. In chicken and duck PBMC,
MHC class I molecules were expressed at nearly identical levels,
whereas MHC class II molecules were maintained at baseline levels with no signicant changes. The difference in MHC class I and
class II expression between duck and chicken PBMC was not significant for the 8 and 24 hpi time points (p = 0.19, p = 0.13), however,
by 36 hpi, the MHC class II expression levels were signicantly
different (p = .01) with duck suppression by 3-fold and chicken
upregulation at 1.4-fold induction (see Fig. 3).

4. Discussion
In mammals, signs of inuenza stem from the hosts inammatory response to infection (Durbin et al., 2000). It has been shown
that infected cells and the immune cells that respond to infection
express early cytokines in response to the presence of the pathogen.
The cytokines, IL-1, IL-6, TNF-, and IFN-, are expressed by primary and continuous macrophage cultures from mice, swine, and
humans immediately after inuenza virus infection (Peschke et al.,
1993; Van Reeth, 2000). The results from our study indicate that,
with the exception of IFN-, the expression of the pro-inammatory
cytokines IL-1 and IL-6 in chicken and duck PBMC are distinct.
The increased expression of IL-1 and IL-6 in chicken and the
skew towards a Th2 response are similar to the responses of mammals. In contrast, the downregulation of IL-1 and IL-6 and the
skew towards a weak Th1 response in duck PBMC is very different both from the responses of chickens and mammals infected
with inuenza A viruses. In addition, it has been demonstrated in
mice, swine, and humans that disease outcome is correlated with
the in vitro expression of IL-1, IL-6, TNF-, and IFN- (Hayden et
al., 1998; Kaiser et al., 2001; Skoner et al., 1999; Van Reeth, 2000).
Though our results were derived in vitro, they are consistent with
what is observed in experiments carried out in vivo, i.e., an absence
of signs of disease in ducks correlates with low pro-inammatory
cytokine levels and the presence of lesions in chickens correlated
with high pro-inammatory cytokine levels.
Differential expression of the type I IFNs in chicken and duck
PBMC evidenced by the weak induction of IFN- in duck PBMC and
the approximately 100-fold induction in chicken PBMC may suggest
differing susceptibilities of the two species to the suppressive effect
of the NS1 gene on IFN expression. Reduction in IFN- expression
after infection with inuenza virus was likely due to NS1A expression, a virally encoded immunomodulator. The interaction between
NS1A and the cellular protein cleavage and polyadenylation specicity factor, which is required for the processing of the 3 -end of
mRNA, results in suppression of IFN- expression in human airway
epithelial cells (Noah et al., 2003). In comparing the expression levels of both type I IFNs, chicken PBMC express higher levels of both
IFN- and IFN- than duck PBMC. A weak induction of IFN has been
correlated in chickens with higher virus titers and a longer shedding period, whereas strong IFN expression results in lower virus
titers and a shorter shedding period (Cauthen et al., 2007). Based
on these ndings in chickens, we conclude that the lower overall
expression of IFNs by duck PBMC in response to AIV infection reect
what happens at the organismal level, longer shedding and weaker
viral clearance observed in duck, and more rapid clearance and thus
a relatively shorter shedding period in chicken.
Ducks and chickens are among the most important hosts in
the maintenance and transmission cycles of inuenza A viruses.
Some of the reasons that the duck and chicken agricultural systems have been so critical in the emergence of epidemic AIV are
that AIV can replicate at low levels in duck for at least 32 days and
be shed intermittently (Higgins et al., 1987) and infected ducks are
frequently asymptomatic (Webby and Webster, 2001; Webster et

al., 1978) and thus may contact many new individuals while shedding virus. However, it may also be that different species may exert
variable immunological pressures on AIV and their combined presence results in greater changes in the virus than would happen
if a single species were infected. It has been suggested that since
ducks and chickens are short-lived, they do not put immunological pressure on pathogens such as AIV and therefore there is little
antigenic drift (Austin and Webster, 1986; Kida et al., 1987). Despite
this argument, our ndings would suggest that antigenic drift probably does occur in chickens, as others have reported (Garcia et al.,
1997; Suarez et al., 1999) and it is driven by a robust host response
to infection. In contrast, our ndings of MHC II downregulation
would suggest that there is a much less robust antibody response
in ducks and that antigenic drift may not occur in ducks as has
been suggested by others (Kida et al., 1987). However, ducks do
immunologically respond to AIV infection and their responses may
exert a different type of selective pressure whose impact is yet
to be understood. The real-life complexities of understanding the
emergence of novel disease agents in agricultural systems surely
requires more study, including more attention to host immune
responses.

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