Molecular Immunology 55 (2013) 200–211

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

Review: Influenza virus in pigs

Elisa Crisci a,1 , Tufária Mussá a,1 , Lorenzo Fraile b , Maria Montoya a,c,∗
a
Centre de Recerca en Sanitat Animal (CReSA), UAB-IRTA, Campus de la Universitat Autònoma de Barcelona, 08193 Bellaterra (Cerdanyola del Vallès), Spain
b
Universitat de Lleida, Lleida, Spain
c
Institut de Recerca i Tecnologia Agroalimentàries (IRTA), Barcelona, Spain

a r t i c l e i n f o a b s t r a c t

Article history: Influenza virus disease still remains one of the major threats to human health, involving a wide range
Received 8 December 2012 of animal species and pigs play an important role in influenza ecology. Pigs were labeled as “mixing
Received in revised form 23 February 2013 vessels” since they are susceptible to infection with avian, human and swine influenza viruses and genetic
Accepted 25 February 2013
reassortment between these viruses can occur. After the H1N1 influenza pandemic of 2009 with a swine
Available online 21 March 2013
origin virus, the most recent research in “influenzology” is directed at improving knowledge of porcine
influenza virus infection. This tendency is probably due to the fact that domestic pigs are closely related to
Keywords:
humans and represent an excellent animal model to study various microbial infectious diseases. In spite
Pig
Influenza virus
of the role of the pig in influenza virus ecology, swine immune responses against influenza viruses are
Immune responses not fully understood. Considering these premises, the aim of this review is to focus on the in vitro studies
performed with porcine cells and influenza virus and on the immune responses of pigs against human,
avian and swine influenza viruses in vivo. The increased acceptance of pigs as suitable and valuable models
in the scientific community may stimulate the development of new tools to assess porcine immune
responses, paving the way for their consideration as the future “gold standard” large-animal model in
immunology.
© 2013 Elsevier Ltd. All rights reserved.

1. Introduction
1.1. Influenza virus and pigs
Since the “obscuri coeli influentia”, defined by the ancient as the
“astral influence” during influenza virus (IV) illness, to today, much
progress has been made with regard to the knowledge of this dis-
ease. Nevertheless, IV disease still remains one of the major threats
to human health, and involves a wide range of animal species.
Influenza viruses (IVs) are enveloped, single stranded RNA
viruses of the family Orthomyxoviridae. This family comprises dif-
ferent genera, in particular influenza A, B and C viruses. Influenza A
viruses are further classified into subtypes based on the antigenic-
ity of their hemagglutinin (HA) and neuraminidase (NA) molecules.
Currently, 17 HA (H1–H17) (H17 recently described in Tong et al.
(2012)) and 9 NA subtypes (N1–N9) are known.
Influenza A and B viruses possess segmented genomes of
eight single-stranded negative-sense RNA molecules that typically
encode 11 or 12 viral proteins (Medina and Garcia-Sastre, 2011).
∗ Corresponding author at: Centre de Recerca en Sanitat Animal (CReSA),
UAB-IRTA, Campus de la Universitat Autònoma de Barcelona, 08193 Bellaterra (Cer-
danyola del Vallès), Spain. Tel.: +34 935814562; fax: +34 935814490.
E-mail address: maria.montoya@cresa.uab.es (M. Montoya).
1
These authors contributed equally to this paper.
The viral envelope, derived from the host plasma membrane, con-
sists of a lipid bilayer containing transmembrane proteins (HA and
NA) and matrix proteins (M1 and M2). HA, is the major enve-
lope protein forming projection like spikes on the virion surface
(Webster et al., 1992), it provides the receptor-binding site and
elicits neutralizing antibodies. HA binds to host cell receptors that
contain terminal ␣-2,6-linked or ␣-2,3-linked sialic acid (␣-2,6-SA
or ␣-2,3-SA) moieties. Cleavage of HA is essential for fusion and
virus infectivity. NA removes the cell surface receptor (sialic acid);
it is critical for release of virus particles from the cell surface and
spread of virus. M2, an ion channel, is crucial during uncoating
for dissociating the virus ribonucleocapsids (vRNP) from M1 in the
early phase of the infectious cycle. The viral core consists of helical
vRNP containing vRNA (negative stranded) and nucleoprotein (NP)
along with minor amounts of the nuclear export protein (NEP) (also
called non-structural protein NS2) and three polymerase proteins
(PB1, PB2, and PA) which form the viral RNA polymerase complex
(Arranz et al., 2012; Nayak et al., 2004, 2009; Palese and Shaw,
2007).
IVs are thought to infect different animal species and pigs
(Sus scrofa) are one of the natural hosts of these viruses. Swine
influenza is a highly important respiratory swine disease and the
main causative viruses are H1N1, H1N2 and H3N2 subtypes (Brown,
2012), which are antigenically related to human IVs. Pigs are sus-
ceptible to infection with avian and human IVs, and are supposed
to play an important role in human influenza ecology. In fact,

0161-5890/$ – see front matter © 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.molimm.2013.02.008
 E. Crisci et al. / Molecular Immunology 55 (2013) 200–211
genetic reassortment between human and/or avian and/or swine
IVs can occur. The potential to generate novel IVs has resulted in
swine being labeled “mixing vessels”. There are three facts sup-
porting the mixing vessel hypothesis: (1) swine are susceptible to
avian and human viruses; (2) reassortment of swine/avian/human
viruses occurs in pigs and (3) pigs can transmit reassortant IVs to
humans (Ma et al., 2009). However, infection of pigs could result in
viruses gaining the adaptations necessary to either maintain infec-
tion within pigs, thereby providing a further reservoir for human
infection, or provide the mutations required to allow transmission
in humans, leading to establishment of this virus in the human
population (Londt et al., 2012).
Other animal models, such as ferrets and guinea pigs, have been
successfully used for influenza virus studies (Belser et al., 2011;
Sun et al., 2010), but after the H1N1 influenza pandemic of 2009 (A
(H1N1)pdm/2009) with a swine origin virus (Garten et al., 2009),
much effort has been made to improve the knowledge of IV infec-
tion in pigs. Various studies have demonstrated the pathogenesis
and transmission of A (H1N1)pdm/2009 IV in swine (Brookes et al.,
2010; Busquets et al., 2010; Lange et al., 2009) and further studies
have been performed on pathogenesis and epidemiology of other
IVs in pigs (De Vleeschauwer and Van Reeth, 2010; Khatri et al.,
2010; Kitikoon et al., 2012; Zhu et al., 2011). In this way, the recent
studies in “influenzology” have been directed at swine IV infec-
tion. This tendency is probably due to the fact that domestic pigs
are closely related to humans anatomically, genetically and physio-
logically, and represent an excellent animal model to study various
microbial infectious diseases. Indeed, experiments in pigs are much
more likely to be predictive of therapeutic treatments in humans
than experiments in rodents. A recent review (Meurens et al., 2012)
highlighted the numerous advantages of the pig model for infec-
tious disease research and vaccine development. Based on the same
subtypes that infect pigs and human and the similarities between
clinical diseases, pathogenesis and tissue tropism of IV infections
in pigs and humans, pig are ideal experimental animals to study
different aspects of influenza infections (Meurens et al., 2012).
In spite of the role of the pig in IV ecology, swine immune
responses against IVs are not fully understood. Taking into account
these premises, the aim of this review is to focus on the immune
responses of pigs against swine (swIV), human (huIV) and avian
(aIV) influenza viruses.
2. In vitro interaction between influenza viruses and the
porcine immune system
IVs are thought to infect mainly the respiratory-tract epithelial
cells of animals and humans in vivo. IVs are able to infect a variety of
cells in vitro, including the Madin-Darby canine kidney (MDCK) cell
line (Tobita et al., 1975), the African Green Monkey Kidney (VERO)
cell line (Genzel et al., 2010), human monocytes (Bussfeld et al.,
1998) and human monocyte-derived macrophages (Hofmann et al.,
1997).
Several in vitro studies have investigated the interaction
between IV and different systems of mammalian cells. These
reports mainly focused attention on the binding ability of the virus
and on the pathogenesis, but few data have been published on the
immune responses elicited after the IV-cell interaction. Here, we
review the in vitro studies performed with different IVs and porcine
cell systems. Other studies have been performed using the porcine
system considering the co-infection of IV and other pathogens.
These data are out of the scope of this review.
2.1. Epithelial cells
Epithelial cells are the first host defence barrier and the primary
target for IVs; for this reason different studies have investigated the
201
interaction between IV and the respiratory epithelium. In vitro stud-
ies with progenitors or differentiated respiratory epithelial cells are
possible, as are air–liquid interface culture or explant culture or ex
vivo organ culture. Nowadays, the latest tendency is to use differ-
ent porcine cell systems for studying IV infection, probably because
swine-origin cells are considered a more suitable tool to investigate
the host cell contributions to pathogenesis in pigs.
In the case of differentiated airway epithelial cells from pigs,
infection studies with IVs have been reported with explant cul-
tures from different parts of the respiratory tract (Van Poucke et al.,
2010). These explants are viable for four days and different IVs were
used in this system (Table 1). The infectivity of aIVs was shown
to be lower in the upper respiratory tract, while the pattern of
huIVs more closely resembled that of swIVs. Compared to swIVs
and huIVs, replication of the aIVs was limited in all cultures but
most strikingly in nasal and tracheal explants (Van Poucke et al.,
2010).
Nunes et al. also described an ex vivo organ culture (EVOC) sys-
tem using porcine trachea (Nunes et al., 2010). In this study the
EVOC system was maintained for 7 days and the ciliary move-
ment exhibited a gradual decrease after 4 days. They challenged
the EVOC with swIV H1N1 (Table 1) and the system showed his-
tological changes similar to in vivo IV infection and supported
productive viral replication over multiple cycles of infection with
dose-dependent kinetics (Nunes et al., 2010). Recently, Londt et al.
(2012) used tracheal, bronchi and lung explants and tested dif-
ferent IV subtypes (Table 1). Pandemic viruses and HPAI H5N1
subtype were able to infect pig ex vivo organ culture, and the
detection of virus was higher in the tissues of the lower respira-
tory tract (i.e. lungs). Endemic swIV was able to replicate to higher
levels in trachea when compared to avian and pandemic viruses.
A(H1N1)pdm/2009 and aIVs appeared to replicate preferentially
in the bronchi and lung tissue explants. This may be due to the
increase in ␣2–3 receptor density or the number of susceptible
cells in the lower respiratory tract. Thus, also this report supports
the use of ex vivo explants for screening viruses for their ability to
infect pigs prior to in vivo studies (Londt et al., 2012).
An interesting new culture system for porcine differentiated
respiratory epithelial cells, precision-cut lung slices (PCLS) was also
set up. Punyadarsaniya et al. (2011) used this culture system to
compare the infection of respiratory epithelial cells by swine IV and
two LPAI aIVs (Table 1). Interestingly, this system is much more sus-
ceptible to aIV H9N2 than to the H7N7 subtype and swIV growth
was faster than aIVs. The ciliary activity was maintained for up to
48 h post infection (h pi) with all the three viruses and ciliostasis
appeared to be dependent on the amount of the virus generated
during the infection. The results have shown that all the viruses
were able to infect ciliated epithelial cells and swIV also infected
mucus-producing cells (Punyadarsaniya et al., 2011).
Olsen’s group also described two different types of porcine
airway epithelial culture for studying IV infection. Recently, dif-
ferentiated swine airway epithelial (SAE) cell cultures were grown
at an air–liquid interface (ALI) varying amounts of retinoic acid and
epidermal growth factor and infected with reverse genetics(rg)-
created viruses from triple reassortant rH3N2 and human-lineage
swine IV (Table 1). The expression of ␣2–3 and ␣2–6-linked sialic
acid on SAE cultures is similar to that in the swine lower trachea. IVs
replicated successfully in these multi-layered cultures, even in the
absence of exogenous trypsin (Bateman et al., 2013). Infection and
replication characteristic of the IVs used were similar to results pre-
viously seen in vivo and in monolayer of primary swine respiratory
epithelial cells (SRECs) in vitro (Busch et al., 2008).
Another fascinating recent report has shown the possibility of
isolating and using stem/progenitor epithelial cells from the lungs
of pigs for in vitro studies (Khatri et al., 2012). These Oct4+ SSEA-
1+ cells have extensive self-renewal potential and the capacity to
202 E. Crisci et al. / Molecular Immunology 55 (2013) 200–211

Table 1
In vitro studies on influenza viruses and porcine cell systems.

Cells Culture cell system/tissue Viruses References

Epithelial cells Explant culture/ swIV Van Poucke et al. (2010)

Nasal respiratory mucosa Sw/Belgium/1/98 (H1N1)
Trachea Sw/Flanders/1/98(H3N2)
Bronchi Sw/Gent/7625/99 (H1N2)
Lung huIV
A/New Caledonia/20/99 (H1N1)
A/Panama/2007/99 (H3N2)
LP aIV
Duck/Italy/1447/05 (H1N1)
Mallard/Alberta/279/98 (H3N8)
Duck/Belgium/06936/05 (H4N6)
Chicken/Belgium/150/99 (H5N2)
Chicken/Italy/1067/V99 (H7N1)
Mallard/Italy/3401/2005 (H5N1)
Explant culture/ HP aIV Londt et al. (2012)
Trachea A/chicken/Netherlands/3219-3/03 (H7N7)
Bronchi A/turkey/Turkey/1/2005 (H5N1)
Lung LPAI aIV
A/chicken/England/4054/06 (H7N3)
huIV
A/England/195/09 (H1N1)
A/California/7/09 (H1N1)
swIV
A/swine/England/195852/92 (H1N1)
Ex vivo organ culture (EVOC) system/Trachea swIV Nunes et al. (2010)
A/swine/England/453/06 (H1N1)
Differentiated respiratory epithelial cells, swIV Punyadarsaniya et al. (2011)
precision-cut lung slices (PCLS)/ A/sw/Bissendorf/IDT1864/2003 (H3N2)
Lung LP aIV
A/duck/Potsdam/15/80 (H7N7)
A/chicken/Saudi Arabia/CP7/98 (H9N2)
Progenitor epithelial cells swIV Sw/OH/07, H1N1 Khatri and Saif (2011)
Type II pneumocytes/ huIV Hu/OH/06, H1N1
Bone Marrow aIV Ch/NY/95, H7N2
Stem/progenitor epithelial cells swIV Sw/OH/07 H1N1 Khatri et al. (2012)
Type I and II pneumocytes/ huIV Hu/OH/06, H1N1
Lung aIV Ch/NY/95, H7N2
Differentiated swine airway epithelial (SAE) swIV H3N2 Bateman et al. (2013)
cells, multi-layered culture/Distal trachea rg (A/Swine/Minnesota/593/99)
rg (A/swine/Ontario/00130/97)
Monolayer of primary swine respiratory swIV H3N2 Busch et al. (2008)
epithelial cells (SRECs)/ rg (A/Swine/Minnesota/593/99)
Distal tracheal-proximal bronchi rg (A/swine/Ontario/00130/97)
A/Swine/Iowa/533/99
A/Swine/Iowa/569/99
A/Swine/Nebraska/209/98
A/Swine/CO/1/77
Intestinal epithelial cell line (SD-PJEC) 25 influenza virus isolates of human, swine or Sun et al. (2012)
subclone of IPEC-J2/ avian origin
Jejunal epithelia of a neonatal unsuckled piglet
Newborn swine kidney (NSK, BS CL 177)/ LP aIV Lombardo et al. (2012)
Kidney A/Mallard/Italy/3401/05 (H5N1)
A/Turkey/Italy/2962/V03 (H7N3)
A/Turkey/Wisconsin/66 (H9N2)
HP aIV
A/Chicken/Italy/8/A98 (H5N2)
swIV
A/Swine/Italy/25674/09 (H1N1)
A/Swine/Italy/290271/09 (H1N1)
A/Swine/BS2156 (H1N2)
A/Swine/Oedenrode/1/96 (H3N2)

Stromal cells Mesenchymal stromal cells (MSCs) from bone A/Sw/OH/24366 (H1N1) Khatri and Saif (2011)
marrow A/Hu/OH/K1130/06 (H1N1)

Macrophages M/BAL A/Hu/Sydney/5/97 (H3N2) Seo et al. (2004)

A/New Caledonia/1/99 (H1N1)
B/Shichuan/99
M/BAL swIV H1N2 (not specified) Kim et al. (2009)
3D/4 spontaneously transformed line/ A/Hu/Nanjing/108/2009 (H1N1) Gao et al. (2012)
Myelomonocytic cell line alveolar M
 E. Crisci et al. / Molecular Immunology 55 (2013) 200–211
Table 1 (Continued )
Cells Culture cell system/tissue
Dendritic cells cDCs (BMDCs)/
Bone Marrow
cDCs/
Bone Marrow
pDCs/
Blood PBMCs
undergo differentiation to alveolar type I- and type II-like pneu-
mocytes. These cells express sialic acid receptors and supported
the active replication of IVs (Table 1), which was accompanied by
cell lysis. The data provided new insights into the pathogenesis of
IV, suggesting that the infection and lysis of lung progenitor cells by
IV is responsible for the loss of lung functions, delayed lung repair
and delayed epithelial regeneration after IV infection (Khatri et al.,
2012). The authors also studied the permissiveness of mesenchi-
mal stromal cells and their cytokine production after swine and
human H1N1 infection (Table 1). The findings demonstrated that
mesenchimal stromal cells are susceptible to IVs and the infection
resulted in cell lysis and in the production of TNF-␣ and IL-6 (Khatri
and Saif, 2011).
Lombardo et al. (2012) compared different mammalian and
avian cell lines in their ability to support different IV subtypes
(Table 1). The most interesting finding was that newborn swine kid-
ney (NSK, BS CL 177) permitted viral isolation of aIVs, either LPAI
or HPAI strains, from target tissues of infected chicken. In addition,
NSK cells appeared more susceptible than MDCK cells for primary
isolation from clinical specimens. These cells also allowed serial
virus cultivation of both aIVs and swIVs, yielding high quantities of
IV mainly for HPAI (Lombardo et al., 2012).
Finally, the porcine intestinal epithelial cell line (SD-PJEC) was
characterized for IV production (Sun et al., 2012). The SD-PJEC cell
line is a subclone of the IPEC-J2, which is a non-transformed, non-
tumorigenic small intestinal epithelial cell line originally derived
from jejunal epithelia of a neonatal, unsuckled piglet (Rhoads et al.,
1994). Sun et al. (2012) demonstrated that SD-PJEC preferentially
expresses ␣2–6 receptors and 25 IV isolates were tested (Table 1).
This cell line was permissive to infection with human, swine and
some avian IVs, but poorly supported the growth of human-origin
influenza B viruses. The authors suggested this system as an appro-
priate cellular model for swine-origin IV, since then to date, findings
concerning the pathogenic mechanism of IV within the digestive
system have been limited due to a lack of a good in vitro cellular
system (Sun et al., 2012).
2.2. Innate immune system
The innate immune system forms the first line of defence
against IV infection (Fig. 1). It consists of a plethora of components,
such as mucus, collectins and acute phase proteins, which aim
203
Viruses References
swIV A/Swine/Spain/SF32071/2007 (H3N2) Mussá et al. (2011, accepted for
huIV A/Catalonia/63/2009 (H1N1) publication)
LP aIV A/Anas
plathyrhynchos/Spain/1877/2009 (H7N2)
HP aIV A/Chicken/Italy/5093/1999 (H7N1)
aIV A/chicken/Yamaguchi/7/04 (H5N1) Ocana-Macchi et al. (2012)
swIV A/swine/Belzig/2/01 (H1N1) Bel et al. (2011)
HP aIV H5N1
A/turkey/Turkey/05
A/Cygnus/Italy/742/06
A/mallard/Italy/835/06
HP aIV H7N1
A/turkey/Italy/4580/98
A/ostrich/Italy/2332/00
LP aIV H7N1
A/turkey/Italy/3675/99
huIV
A/New Caledonia/20/99 (H1N1)
A/Wisconsin/67/05 (H3N2)
A/Hong Kong/8/68 (H3N2)
swIV
A/swine/Iowa/1976/31 (H1N1)
A/swine/Belgium/1/98 (H1N1)
A/swine/Flanders/1/98 (H3N2)
to prevent the infection of respiratory epithelial cells. Therefore,
innate immune cells like macrophages, neutrophils, dendritic
cells and natural killer cells are recruited with the objective of
controlling virus replication and dissemination and to orchestrate
the immune response. These cells secrete different types of chem-
ical mediators such as cytokines that will activate the T cells and
induce their differentiation into memory cells or elicit an adaptive
response by humoral (virus specific antibodies) and cellular immu-
nity (T cells) (Fig. 1). In the following section we will consider each
component of the porcine innate immunity separately.
2.2.1. Collectins
Surfactant protein D (SP-D) belongs to the family of C-type
lectins (collectins) which are important effectors of innate immune
system. Hillaire et al. (2011) evaluated the potential of recombi-
nant porcine SP-D as an antiviral agent against influenza A viruses
in vitro. Thirty IVs (Table 1) from bird, pig and human origins were
tested for their sensitivity to recombinant SP-D. They showed that
porcine SP-D bind HA, inhibited NA activity and was more potent
than recombinant human SP-D. Porcine SP-D was active against a
broad range of influenza A virus strains and neutralized a variety of
H1N1 and H3N2 viruses, including pandemic H1N1 viruses. Finally,
SP-D prevented attachment of human seasonal H1N1 and H3N2 to
receptor on epithelial cells of the upper respiratory tract (Hillaire
et al., 2011).
2.2.2. Macrophages
Upon infection of the alveoli, alveolar macrophages (M)
become activated and phagocytose (apoptotic) IV infected cells
limiting viral spread in pigs (Kim et al., 2008). Seo et al. (2004) iso-
lated alveolar M in bronco-alveolar lavage (BAL) of post-mortem
pigs. Thus, M that constitutively reside in the respiratory tract of
pigs, were infected with huIVs (Table 1) and the infectivity titers
of the viruses were similar. The inflammatory cytokines IL-1␤, IL-6
and TNF-␣ were induced by alveolar M and of these, TNF-␣ was
the most highly induced. H3N2 huIV induced significantly greater
expression of TNF-␣ than did H1N1 or influenza B virus. TNF-␣
protein increased as the duration of infection increased and alve-
olar M did not undergo apoptosis when infected with IVs. Taken
together, these findings suggest that alveolar M are involved in
the pathogenesis of IV infection and in the associated immune
response (Seo et al., 2004).
204 E. Crisci et al. / Molecular Immunology 55 (2013) 200–211

Fig. 1. Immunity during influenza virus infection. The innate immune system forms the first line of defence against influenza infection. It consists of plethora of components,
such as mucus, collectins and acute phase proteins, which aim to prevent infection of respiratory epithelial cells. Therefore, rapid innate immune cells like macrophages,
dendritic cells, natural killer and  T cells are recruited with the objective of controlling and blocking virus replication and dissemination and to orchestrate the immune
response. These cells secrete different types of chemical mediators such as cytokines that will activate the T cells and induces their differentiation or elicit an adaptive
response with the production of specific IV-antibodies responses. This figure is mainly based on human and murine studies.

Similar results in M culture supernatant from M infected
with swIV H1N2 were shown by Kim et al. (2009). Significant
differences in TNF-␣ concentration between swIV-infected and
uninfected alveolar M were detected at different h pi, with a peak
at 36 h pi. These results suggested that TNF-␣ may be an impor-
tant mediator in the pathophysiology of swIV infection (Kim et al.,
2009).
Another in vitro study used 3D/4 cells, a spontaneoulsy-
transformed line of swine M (ATCC), infected with a pandemic
H1N1 virus (Table 1) (Gao et al., 2012). This report demonstrated
that A (H1N1)pdm/2009 retains the ability to infect and replicate
in swine M, inducing a typical cytopathic effect (16 h pi) and
destroying the cell monolayer (32 h pi). This study also examined
the pattern of cytokine responses in pH1N1-infected swine M by
real time RT-PCR. IL-6 and IL-8 levels were up-regulated at 16 h
and level of IL-8 continued to rise up at 36 h pi. Robust induction of
antiviral IFN-␤ and TNF family members, which may be attributable
to cell death, was observed. FasL and TNF-␣ remained undetectable,
while the TNF-related apoptosis-inducing ligand (TRAIL) seemed
to be the most abundant one before infection. FasL and TNF-␣
were induced most robustly, but TRAIL was only mildly induced
in response to infection. The level of IL-1␤ remained unchanged
throughout the infection (different from Barbe et al., 2011; Van
Reeth, 2000), indicating that IL-6 and IL-8, as well as TNF-␣ were
the main pro-inflammatory cytokines up-regulated. The authors
also observed the induction of RIG-1 and MDA-5, which appeared
to be suppressed completely by inhibitors of ERK1/2 or JNK1/2. This
indicated that the induction of RIG-1 or MDA-5 depends on the
activation of ERK1/2 and JNK1/2 in pig M (Gao et al., 2012).
2.2.3. Dendritic cells
Conventional dendritic cells (cDCs), situated underneath the air-
way epithelium barrier and above the basal membrane, monitor
the airway lumen via their dendrites that are extended through the
tight junctions between the airway epithelial cells (Kreijtz et al.,
2011). cDCs can detect and opsonise (neutralize) virions and apop-
totic bodies from infected cells and can also be infected themselves.
Upon entry of the virion into the cells, the cDCs migrate via the
afferent lymphatic system to the draining lymph node. Here they
present the IV derived antigen through the MHC to T cells and
activate them (GeurtsvanKessel and Lambrecht, 2008). Among the
various other activities of DCs in IV infection, they can also exert
cytolytic activity and contribute to the formation of bronchus asso-
ciated lymphoid tissue (BALT) (GeurtsvanKessel and Lambrecht,
2008; Kreijtz et al., 2011). Although DCs have an important role
during IV infection, few studies have investigated the interaction
between IV and porcine DCs.
Our group described for the first time the interaction between
porcine bone marrow-derived DCs (poBMDCs) (cDCs) and swIV
H3N2 (Table 1) in vitro. Infection of poBMDCs resulted in struc-
ture resembling IV inside vesicles and also free in the cytoplasm
of the cells. Viral progeny was undetectable in the supernatant but
limited replication was detected in the first 8 h pi. However, viral
particles from infected-poBMDCs were able to induce cytopathic
effect in susceptible cells only when cell-to-cell interaction was
favored (Mussá et al., 2011). Additionally Mussá et al. (accepted for
publication), observed that similarly to the swIV H3N2, porcine DCs
also supported a limited replication of other IVs (Table 1) during
the first 8 h pi, without release of infectious progeny. These viruses
also similarly modulated the expression of NF-␬B, TGF-␤ and IL-
10 genes. However, they induced different kinetics and levels of
inflammatory cytokines. Infection of poBMDCs with swIV induced
a peak of IFN-␣ secretion at 24 h pi, whereas with the others the
production of IFN-␣ was not detected. SwIV and HPAI induced more
TNF-␣ compared to huIV and LPAI. SwIV, LPAI and HPAI induced an
increase of IL-12 from 16 to 24 h pi and all viruses used induced
IL-18 secretion in a time-dependent manner.
Summerfield’s group also used GM-CSF derived DCs and other
avian and porcine IVs (Table 1). They also generated recombinant
reassortants by reverse genetics to elucidate the role of the sin-
gle gene segments in the activation of cDCs. The highest IFN type I
responses were achieved by the porcine virus reassortant contain-
ing the avian polymerase gene PB2. This finding was not due to
the differential tropism since all viruses infected DCs equally (and
also porcine PK-15 epithelial cells) and infectivity was independent
of the HA expressed by the virus. All viruses induced MHC-II, but
porcine H1N1 expressing the avian viral PB2 induced more promi-
nent nuclear NF-kB translocation compared to its parent virus.
 E. Crisci et al. / Molecular Immunology 55 (2013) 200–211
Therefore, for porcine DCs, PB2 was defined as an important viral
element controlling IFN type I. While all viruses had a comparable
ability to infect DCs, to initiate replication and to activate the cells
in terms of MHC-II induction, only those expressing PB2 derived
from H5N1 were unable to prevent IFN type I induction; however
no viral progeny was detected (Ocana-Macchi et al., 2012).
Plasmacytoid DCs (pDCs), a subset of the DC family, are highly
specialized in sensing viruses and readily secreting IFN-␣ after
exposure to swIV (Calzada-Nova et al., 2009). Therefore, consid-
ering the distinct features of pDCs and the crucial role of IFN in
fighting virus infections, Bel et al. (2011) analyzed the interac-
tions of different influenza A viruses (Table 1) isolated from avian,
human and swine with pDCs obtained from pigs. Their results
demonstrated that porcine pDCs can produce high levels of IFN-␣ in
response to all tested strains, with subtype-specific differences in a
virus-dose dependent manner. High levels of IFN-␣ were detected
upon live, chemically inactivated or UV-inactivated virus stimula-
tion. In contrast, heat-inactivated virus failed to induce a response.
The observation that chemical and UV-inactivation did not abol-
ish IFN-␣ release indicated that non-infectious particles are also
stimulatory for pDCs. Additionally, these treatments did not abol-
ish hemagglutination, suggesting that the integrity of HA and its
binding function are necessary to induce IFN-␣ responses in pDCs.
At low viral doses, H5N1 and H7N1 avian viruses, are more efficient
in infecting pDCs when compared to human H1N1, and at inducing
the secretion of IFN-␣ (Bel et al., 2011).
2.2.4. Natural killer cells
NK cells are important effector cells of the innate immune
response. They can recognize antibody-bound IV infected cells and
lyse these cells, a process called antibody dependent cell cyto-
toxicity (ADCC). These cells can recognize IV-infected cells with
their cytotoxicity receptors NKp44 and NKp46. Upon binding to
the influenza virus HA the receptors trigger the NK cells to lyse
the infected cell (Mandelboim et al., 2001). It has been suggested
that invariant NKT (iNKT) cells stimulate the induction of cellular
immunity and regulate infection induced pathology (Paget et al.,
2011). An NK specific monoclonal antibody from pigs was recently
reported (Mair et al., 2012) and iNKT cells were identified in pig
lungs (Renukaradhya et al., 2011). With the identification of NK cell
markers in pigs, many studies on the role of these cells in influenza
infected pigs will probably be addressed in the future.
3. In vivo immune responses during influenza infection in
pigs
The three swIV subtypes (H1N1, H1N2 and H3N2) have been
associated with disease in swine. Subclinical infections are also
very common and many, if not most pigs become infected with one
or more IV subtypes without ever showing clinical signs (Charley
et al., 2006; Van Reeth, 2007). Serological profiles after consecutive
experimental infections of pigs with European H1N1, H3N2, and
H1N2 swIV have been described by Van Reeth et al. (2006). The
disease caused by IVs in pigs is essentially similar to that recorded
in human, with low mortality but high morbidity (Brown, 2000;
Fouchier et al., 2003).
Pigs are susceptible to infection with IV from both avian and
human origin with occasional transmission of virus to human.
Examples are the pandemic of 1957, 1968 and the 2009 pandemic
which were caused by a reassortant influenza (Fouchier et al., 2003)
that had been generated in pigs (Garten et al., 2009).
The purpose of this section is to summarize the immune
responses during an IV infection in swine considering pigs exper-
imentally or naturally infected with human, avian or porcine
subtypes of IV (swIVs summarized in Fig. 2).
205
Lung viral load Cytokines APPs
IFN- T cells Lymphocytes IV-specific Abs
Magnitude of infection/responses
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
Days after infection
Fig. 2. Swine influenza virus infection in pigs. General kinetics of lung viral load,
acute phase proteins (APPs), cytokines (e.g. TNF-␣, IL-6, and IL-8) and different
immune system components (lymphocyte, IFN-␥ producing cells and influenza
virus (IV)-specific antibodies) during infection with different swine influenza viruses
(H1N1 and H3N2) in pigs. The magnitude of responses is related to the days after
infection (0–14).
3.1. Acute phase proteins
The acute phase response is a nonspecific early response of
the organism caused by various stimuli. This reaction is mediated
by pro-inflammatory cytokines and involves local and systemic
reactions, including changes in the concentration of acute phase
proteins (APPs) (Heegaard et al., 1998; Pomorska-Mol et al.,
2012a,b). APPs in swine are C-reactive protein (CRP), haptoglobin
(Hp), serum amyloid A (SAA) and pig major acute phase protein
(Pig-MAP) and only a few studies have investigated the role of APPs
in pigs during IV infection.
An increase of CRP and Hp concentrations in pigs was reported
by Brookes et al. (2010) during acute IV infection caused by pan-
demic H1N1 2009 virus (Table 2). The CRP levels peaked at 4 days
post infection (dpi)/days post contact (dpc), while Hp responses
were more protracted, peaking at 9–11 dpi/dpc in both infected and
in-contact pigs. CRP responses were higher in infected pigs than in
pigs at first-fourth transmission cycles (TC1-4), and were more ele-
vated in the infected and TC1 animals than the later transmission
cycles (Brookes et al., 2010).
Barbe et al. (2011) also described in intratracheal swH1N1
(A/Swine/Belgium/1/98) infected pigs that CRP, Hp, lipopolysaccha-
ride (LPS)-binding protein (LBP) peaked in broncho-alveolar lavage
fluid (BALF) at 2 dpi (CRP, Hp) or 30 dpi (LBP) and then decreased.
While the concentration of CRP and Hp peaked at 48 h in serum, the
level of LBP remained relatively constant (LBP increased in BALF and
not in serum). APPs reached higher levels in serum than in BALF and
did not directly correlate with viral load. Given the small number of
pigs, APPs concentration post infection did not differ significantly
from control values (Barbe et al., 2011).
A recent study of APP responses after subclinical intranasal
infection with swine H1N1 (A/Sw/Poland/KPR9/2004) in pigs
showed that in this model Hp and SAA were significantly induced
from 1 to 2 dpi, before specific Abs in serum were present
(Pomorska-Mol et al., 2012b) (Fig. 2). Although the concentration
of CRP and Pig-MAP remained generally unchanged to the end of
the study, in half of the infected pigs, the concentration of CRP
tended to increase at 1 dpi (Pomorska-Mol et al., 2012b). A similar
study was also performed by the same group using H1N2 subtype
(Pomorska-Mol et al., 2012a) (Table 2). In infected pigs concentra-
tion of CRP, Hp, SAA increased significantly at the time when the
most amount of virus was shed (from 1 to 3 dpi) (Fig. 2) and the
206 E. Crisci et al. / Molecular Immunology 55 (2013) 200–211

Table 2
In vivo studies on influenza viruses and pigs.

Type Virus subtype identification Route of inoculation Age of pigs References

SwIV H1N1 A/Sw/Belgium/1/83 Intratracheal 3-week-old Van Reeth et al. (1998)

Cesarian
Clostrum-deprived
H1N1 A/Sw/Belgium/1/98 Intratracheal 10- to 14-week-old Van Reeth et al. (2002a,b)
H3N2 A/Sw/Flanders/1/98
H1N2 A/Sw/Gent/7625/99
H1N1 A/Sw/Indiana/1726/88 Intranasal 8-week-old Larsen et al. (2000)
H1N1 A/Sw/Best/96 Culture supernatant 10-week-old Heinen et al. (2001)
H3N2 A/Sw/Oedenrode/96 aereosola
H3N2 A/Sw/Neth/St.Oedenrode/96 Culture supernatant SPF Heinen et al. (2000)
aereosola 10-week-old

H1N1 A/Sw/Neth/Best/96 Culture supernatant 7- to 15-week-old Loeffen et al. (2003)

aereosola
H1N1 A/Swine/IA/40776/92 Lung homogenate 5-week-old Kim et al. (2006)
H3N2 A/Swine/IA/41305/98 nebulization
H1N2 A/Swine/Korea/S5/05 Intranasal 6-week-old Jo et al. (2007)
H1N1 A/Swine/IA/40776/92 Intratracheal 5-week-old Kitikoon et al. (2008)
H1N2 A/Swine/Wisconsin/R33f/01
H3N2 A/Swine/Texas/4199-2/98
H1N1 A/Swine/IA/35233/99
H1N2 A/Swine/Indiana/9K035/99
H3N2 A/Swine/Wisconsin/R7c/01
H1N1 A/Sw/Finistere Intranasal 8-week-old Ferrari et al. (2008)
Intrapharingeal
H1N1 A/Sw/OH/24366/07 Intranasal 5-week-old Khatri et al. (2010)
Intratracheal
H1N2 A/Sw/Grandstedt/IDT3475/2004 Intranasal 5-week-old Pomorska-Mol et al. (2012a,b)
SwIV (rH3N2p) A/Swine/Illinois/A01201606/2011 Intranasal 3-week-old Kitikoon et al. (2012)
HuIV (H3N2-TRIG) A/Swine/Pennsylvania/62170-1/2010 Intratracheal
HuIV H3N2 A/Indiana/08/2011
HuIV H3N2 A/Sidney/5/97 Intranasal 4-week-old Seo et al. (2004)
H1N1 A/New Caledonia/1/99
Influenza B/Shichuan/99
H1N1 A/New Caledonia/20/99 Intranasal 6-week-old Kim et al. (2008)
H1N1 1918/rec Intranasal 4-week-old Weingartl et al. (2009)
Intratracheal
H1N1 A/Regensburg/D6/09 Intranasal 10-week-old Lange et al. (2009)
H1N1 A/California/04/09 Intranasal 8-week-old SPF miniature Itoh et al. (2009)
pigs Brookes et al. (2010)
H1N1 A/California/07/09 4- to 5-week-old
aIV 42 strains of IV (38 aIV) Intranasal 50- to 80-day-old Kida et al. (1994)
aIV subtypes:
H2N2, H2N8, H2N9,H3N2,H3N6,
H3N8,H4N5,H4N6,H4N8,H4N9,H5N2,H5N3,H6N1,
H6N2,H7N1, H7N2,H7N3,H8N4,H9N2,H9N5,
H10N1,H10N3,H10N8,H10N9,H11N9,H12N5,H13N6
aIV H5N1 A/Ck/Vt/C-58/04 Intranasal 4-week-old Choi et al. (2005)
HPAI H5N1 A/Dk/Th/D4AT/04
H5N1 A/Gs/Th/G7CS/04
H5N1 A/Chicken/Yamaguchi/7/04 Intranasal 8-week-old Isoda et al. (2006)
H5N1 A/Duck/Yokohama/aq-10/03 Miniature pigs
H5N1 R
(A/Duck/Mongolia/54/01-A/Duck/Mongolia/47/01)
H5N1 A/Chicken/Indonesia/7/03 Intranasal 2- to 3-week-old Lipatov et al. (2008)
H5N1 A/Whooper swan/Mongolia/244/05
H5N1 A/Muscovy duck/Vietnam/209/05
H5N1 A/goose/Hubei/ZFE/2004 Intranasal 4-week-old Li et al. (2008)

Intranasal route mimics natural infection, whereas intratracheal challenge is a better way to investigate the pathogenesis.
a
Aereosol produced by nebulization of 2 ml culture supernatant using airbrush device (Badger).

level of Pig-MAP remained unchanged during the study. They found
a significant positive correlation between maximum concentration
of SAA in serum and lung scores, suggesting SAA as a useful indi-
cator in experimental infection or as a marker for disease severity
(Pomorska-Mol et al., 2012a).
3.2. Swine influenza virus
3.2.1. Role of maternal antibodies
Few studies have been performed to determine the role of
maternally derived antibodies (MDA) against IV in the clini-
cal protection of piglets and particularly their effect on the
development of the active immunity after an infection with a
homologous or heterologous virus. Loeffen et al. (2003) used
homologous infection with H1N1 SwIV (Table 2) and showed
that MDA protected piglets against the clinical consequences
of a primary IV, but that this protection was not complete.
Piglets with and without MDA were protected against clinical
consequences of a secondary infection; but pigs with MDA devel-
oped weaker immunity than other animals without MDA. This
study opened the way to consider the role of MDA in IV infec-
tion and overall growth performance from weaning to slaughter
showed a trend in favor of pigs without MDA (Loeffen et al.,
2003).
 E. Crisci et al. / Molecular Immunology 55 (2013) 200–211
Concerning the presence of MDA and the use of vaccination,
Kitikoon et al. (2006) have shown that MDA suppressed serum Ab
responses and the induction of SwIV-specific memory T cells fol-
lowing the administration of a bivalent inactivated vaccine in pigs.
Enhancement of lung pneumonia was observed in immunized pigs
in the presence of MDA when challenged with a heterologous H1N1
virus (Kitikoon et al., 2006). These authors also tested levels of M2e-
specific Abs in pigs that were positive for SwIV-specific MDA. These
Abs were low and did not appear to be correlated with the higher
HI Ab titers. Variation in the SwIV exposure profile of the sow may
account for the difference in M2e-specific Ab levels of the MDA+
pigs (Kitikoon et al., 2008). Finally, when compared to the previ-
ous study with H1N1, similar results have also been observed with
H3N2 based vaccines using homologous and heterologous infection
(Vincent et al., 2012).
3.2.2. H1N1 and H3N2 influenza viruses
The first article regarding pigs that demonstrated the produc-
tion of TNF-␣ and IL-1 in the BAL of pigs was published in the late
90s by Van Reeth et al. (1998) and later reviewed in (Van Reeth,
2000; Van Reeth et al., 2002b). Biological active IFN-␣, TNF-␣ and
IL-1 were detected in the BALF of pigs inoculated with H1N1 swIV
(Table 2). Cytokine titers and lung virus titers were significantly
higher 18–24 h pi than at 48–72 h pi. All the cytokines were posi-
tively correlated with a 3- to 4-fold increase in BAL cell numbers and
with a drastic neutrophil infiltration. In addition, cytokine produc-
tion coincided with the onset of general and respiratory symptoms
of influenza and with the development of a necrotizing bronchop-
neumonia (Van Reeth et al., 1998). This study was complemented
by a follow up article from the same group reporting conventional
pigs inoculated with all the circulating subtypes of swIV (Table 2)
(Van Reeth et al., 2002a). The major finding in this last study was
that as previously seen, IFN-␣, TNF-␣ and also IL-6 correlated with
virus titer in the lungs (Fig. 2) and symptom severity, while there
was not a clear correlation for IL-1. They suggested that the discrep-
ancy in IL-1 between the two studies may be due to differences
in the sanitary status of the pigs. They also assessed for the first
time IL-8 in swIV-infected pigs. IL-8 correlated with virus titers
and appeared to be induced by swIV infection. These two studies
argued for the role of IFN-␣, IL-6 and TNF-␣ in swIV pathogenesis
(Van Reeth et al., 1998, 2002a). Another study highlighted the role
of IFN-␣ in IL-6 and IL-12 induction in swIV infection using IFN-␣
neutralizing Abs (Barbe et al., 2009).
In 2000, a more detailed investigation on systemic and mucosal
responses was performed during H1N1 swIV infection in pigs by
Olsen’s group (Larsen et al., 2000) (Table 2). Virus-specific serum
IgG, IgA and HI titers all peaked 2–3 weeks after the primary
infection and did not substantially increase after re-challenge. As
expected, the predominant virus-specific isotype in serum was IgG
whereas IgA was the predominant isotype in upper and lower respi-
ratory tract. Nevertheless, pigs also responded with virus-specific
IgG in both upper (nasal washes) and lower (BAL) airways. Results
of Ab-secreting cell ELISPOT assays demonstrated that the numbers
of both IgG and IgA secreting cells in the nasal mucosa were higher
than in any other tissue examined. IFN-␥ secreting cells (Fig. 2), as
a measure of T-cell responses, were predominantly localized to the
spleen and tracheo-bronchial lymph nodes and peaked on day 21
after primary infection (Larsen et al., 2000). Similar results were
described by Ferrari et al. (2008) with swIV H1N1 (Table 2). Virus
was isolated from 1 to 5 dpi only after the first inoculation and
cell-mediated response showed an increase of T lymphocyte pro-
liferation from 14 to 20 dpi after the first infection (Fig. 2). Further
enhancement, was observed even earlier (7 days) during the sec-
ond infection and a plateau phase at high values until the end of the
experiment (47 dpi). IFN-␥ producing cells increased in spleen and
in peripheral blood from 14 to 40 dpi (Fig. 2) (Ferrari et al., 2008).
207
These studies were complemented by Kim et al. (2006))who
described Ab responses to specific viral proteins in pigs infected
with H1N1 and H3N2 swIV (Table 2). Both IgM and IgG against HA,
NP, NS1 and NS2 were detected by 7 dpi. IgG against these pro-
teins were still detectable at 28 dpi. In contrast, IgG against NA and
M1 were not detected until 14 dpi and no IgM against these pro-
teins was detected. This study was useful in determining that the
NP protein was a suitable antigen for a subtype-unrestricted sero-
logic assay, whereas NS1 was for serologic assays that differentiates
between infected and vaccinated pigs (Kim et al., 2006). Taking
into account humoral responses, the responses to the extracellu-
lar region of M2 (M2e) and M1 were also investigated (Kitikoon
et al., 2008). In this study, pigs were tested with different swIV
(Table 2) and the primary objective was to characterize the M2-
specific antibody response. Low M2 antibody responses occurred
following primary infection and the antibody titers did not increase
following homologous challenge, but did increase following het-
erologous challenge. When seroconversion occurred, the infection
dose had a major effect on HA, M2e but not the M1 antibody level
(Kitikoon et al., 2008).
Another experimental design used swIV H1N1 (Table 2) in con-
ventional pigs (Khatri et al., 2010). In this study, they focused on
the host mucosal immune responses and they were the first to
demonstrate the phenotype of different immune cells associated
with innate, adaptive, and regulatory immune responses during
the acute phase of the disease, in particular at the site of viral
replication. Infected pigs showed: (1) higher frequency of CD8+
cells in BAL, tonsil, lung and tracheobronchial lymph nodes (TBLN),
indicating the proliferation of IV-specific CD8+ cells; (2) increased
frequency of activated CD4+ cells associated with enhanced gen-
eration of swIV-specific mucosal IgA and an increased frequency
of activated CD8+ cells in lungs; (3) higher frequencies of CD4+
CD8+ cells in BAL, TBLN and tonsils; (4) NK cells in lungs, BAL and
PBMCs decreased; (5) enrichment of the DC fraction and larger
amounts of IL-12 in lungs; (6) increased amount of IFN-␥ in lungs
and TBLN; (7) increase of ␥␦ T cells in BAL and lung; 8) increase
of Treg, IL-10 and TGF-␤ in lungs. In conclusion they detected
innate, proinflammatory, Th1, Th2 and Th3 cytokines, as well as
swIV-specific IgA in lungs of infected pigs. Production of IFN-␥ by
lymphocytes of the TBLN was also detected. Higher frequencies
of CTLs ␥␦ T cells, DCs, activated T cells, CD4+ and CD8+ T cells
were detected in the infected-pig lungs. Concomitantly, higher fre-
quencies of the immunosuppressive T regulatory cells were also
detected in the infected pigs. This study highlighted the striking
similarities between the pathogenesis of swIV and the pandemic
H1N1 virus in humans, suggesting the pig as a useful model for
pathogenesis studies (Khatri et al., 2010).
In the same way, Heinen et al. (2001) previously described the
primary infection of pigs with swIV H1N1 and H3N2 (Table 2),
focusing on cellular immunity. An acute inflammatory response
characterized by a massive increase in neutrophils from 6 to 40% (2
dpi) of all leukocytes in the lungs was observed. From 8 dpi the % of
lymphocytes in the lungs was higher than controls and remained
elevated until 42 dpi for both subtypes. Additionally after H3N2
infection different events occurred: (a) an increase in the percent-
age of Th cells starting at 2 dpi, (b) peak in the percentage of Th
cells between 4 and 11 dpi, (c) a massive infiltration of CTLs on 8
dpi, leading to a decrease in the percentage of NKs, (d) the tem-
porary disappearance of the CTLs from lung around 11 dpi and (e)
a long-lived increase in the percentage of CTLs in the lungs and
the decrease in NK to the pre-infection level. The same sequence
was also observed after H1N1 infection, but it developed faster
than after H3N2 infection. Finally, they demonstrated the existence
of a clear heterosubtypic immunity (Het-I) in pigs, that partially
protected H1N1-infected pigs from H3N2 secondary infection and
they suggested that CD8+ T cells have a role in Het-I (Heinen et al.,
208 E. Crisci et al. / Molecular Immunology 55 (2013) 200–211
2001). The same authors (Heinen et al., 2000), in a previous study,
described the systemic and mucosal Ab responses in serum, BAL and
nasal lavage fluid (NLF). The Ab response to infection was character-
ized by high IgA titers in BAL and NLF. Moreover, substantial titers
of IgG1 were detected in BAL and less in NLF. The specific activity
of IgA was much higher in NLF and BAL than in serum. The specific
activity of IgG1 was higher in BALF than in serum, suggesting a local
production of this isotype in the lung also. IgA was the predominant
Ig in NLF and IgG in BAL. The appearance of influenza-specific IgM
between 4 and 7 dpi coincided with the clearance of virus from the
oropharyngeal tract. The authors suggested that IgM are respon-
sible for viral clearance after a primary infection, but in contrast
to IgA, IgM probably has a minor role in long lasting immunity to
secondary infection (Heinen et al., 2000).
With regard to H3N2 infection, one of the most recent articles
describes infection in pigs with the novel porcine H3N2 reassor-
tant viruses (H3N2-TRIG and rH3N2p) and a human H3N2 virus
(Table 2) (Kitikoon et al., 2012). This study did not detect an increase
in virulence in human H3N2 or rH3N2p viruses compared to the
endemic H3N2-TRIG virus and the Abs specific to the H3N2-TRIG
virus cross-reacted to both reassortant H3N2 viruses. No further
immunological studies were performed during these infections.
In conclusion swIV H1N1 and H3N2 infections showed similar
patterns in various studies: (1) a rapid increase of cytokines corre-
lated with viral lung titers and APPs in the early stage; (2) the peak
of humoral responses after 14 dpi; and (3) the beginning of cellu-
lar responses at the site of infection after 7 dpi. Some discrepancy
between the different systems may be related to the animals and
the experimental design used.
3.2.3. H1N2 influenza viruses
This subtype kindled little interest in swine research and the
first work on porcine H1N2 pathogenesis was performed by Jo
et al. (2007) (Table 2). In this work high levels of IL-1, IL-8 and
TNF-␣ were detected in lungs of infected pigs, while the induced
amount of IL-4, IL-10 and IFN-␥ was similar in infected and control
pigs. Among the cytokines tested TNF-␣ was the highest induced
cytokine and peaked at 5 dpi, after the beginning of virus shedding
by nasal discharge (3 dpi) (Jo et al., 2007).
Subsequently, Kim et al. (2009) described the same cytokines
in BAL of 3-week-old pigs intranasally inoculated with swIV H1N2.
In BAL the TNF-␣ concentration was maximal at 1 dpi and declined
markedly by 3 dpi. The body temperatures were correlated with the
levels of TNF-␣ in BAL and the pulmonary lesion scores correlated
with TNF-␣ positive cells by IHQ (Kim et al., 2009).
The most recent study was performed by Pomorska-Mol et al.
(2012a) (Table 2), focusing attention on the APPs and the immune
responses. In this study Abs against swIV H1N2 in the serum of
infected pigs were detected from 7 dpi and a specific T cell response
was observed from 5 dpi. Starting from 7 dpi a significant antigen-
specific response was seen in all infected animals. A significant
drop in lymphocyte numbers and an increase in medium-sized
cell counts with no accompanying leucopenia were observed from
3 to 7 dpi in all infected animals. The mean number of lympho-
cytes did not return to the mean pre-inoculated concentration and
the mean number of medium-sized cells returned to the normal
concentration at 10 dpi (Pomorska-Mol et al., 2012a).
In conclusion, immune responses to swIV H1N2 infection seem
to be slightly delayed compared to the other H1N1 and H3N2 sub-
types.
3.3. Human influenza virus
The first study in vivo that demonstrated that huIV can infect
alveolar porcine M that constitutively reside in the respiratory
tract, without causing apoptosis in pigs, was published in 2004
(Seo et al., 2004). Alveolar M infected with human H3N2 (Table 2)
showed greater expression of TNF-␣ than did alveolar M infected
with human H1N1 or influenza B virus (Table 2) and they did not
undergo apoptosis when infected. IL-8 and IL-1 were not induced
in alveolar M in vivo and TNF-␣ peaked at 5 dpi without a strong
correlation with viral titers (peak 3 dpi) (Seo et al., 2004). Succes-
sively, Kim et al. demonstrated that alveolar M are essential for
controlling human H1N1 (Table 2) in pigs. In fact, in vivo deple-
tion of alveolar M results in 40% mortality when pigs are infected
with currently circulating human H1N1, while none of the infected
control pigs dies. Additionally, more severe respiratory signs were
found in depleted pigs and TNF-␣ was significantly lower and IL-10
higher in depleted animals than controls. Lower Ab titers and CD8+
T cells expressing IFN-␥ were detectable in depleted pigs (Kim et al.,
2008).
Human 1918 pandemic IV was tested in pigs; the virus was not
lethal, in contrast to other species (mice, ferrets and macaques), and
pigs did not develop severe respiratory distress (Table 2) (Weingartl
et al., 2009). In this study virus was isolated at 3 and 5 dpi, and
piglets developed antibodies at 7 dpi, but a profound investigation
on immune responses was not performed (Weingartl et al., 2009).
The first work with the recent A(H1N1)pdm/2009 was per-
formed by Lange et al. (2009) (Table 2). NP- and H1-specific Abs
could be detected 7 dpi and CD4+ T cells became activated imme-
diately after the infection. Both CD4+ and CD8+ T lymphocytes
expanded from 3 to 7 dpi coinciding with clinical signs (Lange
et al., 2009). Subsequently, Brookes et al. (2010) used a similar
virus (Table 2) that in infected pigs produced clinical signs, viral
pathogenesis restricted to the respiratory tract, infection dynamics
consistent with endemic pig strains, virus transmissibility between
pigs and virus-host adaptation events (Brookes et al., 2010). In
miniature pigs, the pandemic virus did not cause any signs and
titers were detected in lungs on 3 dpi (Itoh et al., 2009) (Table 2).
In summary, pigs are susceptible to different types of huIV and
alveolar M have been shown to have an important role during this
infection. Further studies have to be performed to full understand
the immune responses during huIV infection in pigs.
3.4. Avian influenza virus
Pigs are clearly susceptible to infection with both LPAI and HPAI
viruses and LPAI viruses have occasionally been isolated from pigs
in the field (Table IV (Van Reeth, 2007)). Most of these viruses have
an H1 or H3, HA subtype that are usually restricted to birds and
can also cross the species barrier to pigs. Serological studies in Asia
have shown evidence for infections of pigs with avian H4, H5 and
H9 viruses (Van Reeth, 2007). Few studies have been performed on
the avian IV infection in pigs and most of them have been focused on
the pathogenesis and transmission of HPAI H5N1, since this virus
infected humans causing high mortality (Choi et al., 2005; Isoda
et al., 2006; Lipatov et al., 2008). In this section we summarize the
most important studies performed with aIV in pigs.
One of the first experimental studies in pigs with different aIV
was performed by Kida et al. (1994) in 1994. In this work pigs were
inoculated with 38 LPAI aIVs (Table 2), mainly of duck origin, and
27 with non-human-type HA. At least one strain of each HA sub-
type replicated in the porcine respiratory tract for 5–7 days and
most of them were detected in nasal swabs and induced a sero-
logical response. Sera of infected pigs showed high Ab titers in
ELISA and neutralization tests, but did not inhibit haemagglutina-
tion of homologous viruses. Co-infection of pigs with a swIV and
with an aIV able to replicate in these animals generating reassor-
tant viruses. Thus, these results indicated that aIV with or without
non-human-type HA can be transmitted to pigs (Kida et al., 1994).
Most recent studies have focused attention on H5N1 aIV to
determine whether this virus associated with human infections had
 E. Crisci et al. / Molecular Immunology 55 (2013) 200–211
been transmitted to pigs. Choi et al. (2005) studied different 2004
Asian H5N1 viruses (Table 2) in pigs, revealing that all viruses tested
replicated in the swine respiratory tract but none were transmit-
ted to contact pigs. Virus titers from nasal swabs peaked on day
2 pi and low titers were detected in the liver of two of the four
pigs tested. Their findings indicated that pigs can be infected with
highly lethal Asian H5N1 viruses but that these viruses are not
readily transmitted between pigs under experimental conditions
(Choi et al., 2005). In the same way, a follow up paper, assessed HPAI
H5N1 viruses (Table 2) in different species of birds and mammals
(Isoda et al., 2006). In this study the authors showed that miniature
pigs were resistant to infection with the viruses tested (Isoda et al.,
2006). Finally, Lipatov et al. (2008) tested other HPAI H5N1 viruses
(Table 2) in pigs. Strains were different from the other studies,
but also in this case pigs demonstrated low susceptibility to infec-
tion with HPAI H5N1 viruses. Inoculation of pigs with these viruses
resulted in asymptomatic to mild symptomatic infection restricted
to the respiratory tract, mainly to the lungs, and tonsils (Lipatov
et al., 2008). This was also confirmed by recent surveillance stud-
ies that observed that H5N1 aIV was circulating in pigs in Indonesia
without causing disease. In this study the authors reported that one
isolate recognized human-type receptors, meaning that the H5N1
can replicate for long periods undetected and facilitate the adapt-
ion of aIV to mammalian host (Nidom et al., 2010). Li et al. (2008)
describe H5N1 infection (Table 2) in different species, and in this
experimental infection pigs showed “flu-like” clinical symptoms.
Animals developed severe respiratory symptoms within 5 dpi and
a continuous fever of over 40 ◦ C for 5–7 days. Specific Abs against
the virus were detected at 28 and 45 dpi (Li et al., 2008).
In recent years porcine research with aIVs were directed mainly
toward the role of the cross-protection between different IV. Two
studies performed by Van Reeth’s group showed how prior infec-
tion with swIV may be a total or partial barrier to infection with aIVs
(De Vleeschauwer and Van Reeth, 2010; Van Reeth et al., 2009).
In conclusion, pigs are susceptible to different aIVs but H5
subtype infections are in general abortive and not able to be trans-
mitted between pigs. Thus, the risk of aIV infection is mainly
related to the possibility of reassortment and not to the morbid-
ity/transmissibility. Further studies have to be performed to clarify
the transmission between pigs using different avian subtypes.
4. Conclusions
Influenza virus represents one of the human pathogen for
which the porcine model has been successfully used, improving
the knowledge in this infectious human disease. Pigs are well
accepted as “mixing vessels” for IVs and their role in the emer-
gence and epidemiology of influenza pandemic is well documented.
For this reason, new strategies are gaining attention in porcine
vaccination to develop future IV vaccine such as live-attenuated,
subunit, vectored and DNA vaccines. This topic was extensively
discussed in a recent review by Van Reeth and Ma (2012). There-
fore, immunological features associated with influenza infection
are now of great interest in influenza research for the similari-
ties with human immunity. An exhaustive recent review (Meurens
et al., 2012) highlighted the importance of the pig model for
human infectious disease research and vaccine development, and
humanized piglets have been described as “stand-ins” for micro-
biome studies (Hvistendahl, 2012). Additionally, pig-to-primate
organ xenotransplantation have been described (Ibrahim et al.,
2006) and transgenic pigs have been proposed as a model for
translational biomedical research (Aigner et al., 2010) or develop-
mental immunology (Rothkotter et al., 2002). Thus, the increased
acceptance of pigs as a suitable and valuable model in the scien-
tific community may stimulate the development of new tools to
209
assess porcine immune responses, which will facilitate the future
development of pigs as the “gold standard” large-animal model in
immunology, and more precisely for IV immunology.
Funding
Our work in this field was funded by the project AGL2010-
22200-C02-01 of Spanish Ministry of Science and Innovation.
Conflict of interests
The authors declare no financial conflict of interests.
Acknowledgement
We want to thank Davide Boscolo for Fig. 1 (www.
davideboscolo.org).
References
Aigner, B., Renner, S., Kessler, B., Klymiuk, N., Kurome, M., Wunsch, A., Wolf, E.,
2010. Transgenic pigs as models for translational biomedical research. Journal
of Molecular Medicine (Berlin) 88, 653–664.
Arranz, R., Coloma, R., Chichon, F.J., Conesa, J.J., Carrascosa, J.L., Valpuesta, J.M., Ortin,
J., Martin-Benito, J., 2012. The structure of native influenza virion ribonucleo-
proteins. Science 338 (December (6114)), 1634–1637.
Barbe, F., Atanasova, K., Van Reeth, K., 2011. Cytokines and acute phase proteins
associated with acute swine influenza infection in pigs. Veterinary Journal 187,
48–53.
Barbe, F., Saelens, X., Braeckmans, D., Lefevre, F., Reeth, K.V., 2009. Role of IFN-
alpha during the acute stage of a swine influenza virus infection. Research in
Veterinary Science 88, 172–178.
Bateman, A.C., Karasin, A.I., Olsen, C.W., 2013. Differentiated swine airway epithelial
cell cultures for the investigation of influenza A virus infection and replication.
Influenza and Other Respiratory Viruses 7 (March (2)), 139–150.
Bel, M., Ocana-Macchi, M., Liniger, M., McCullough, K.C., Matrosovich, M., Sum-
merfield, A., 2011. Efficient sensing of avian influenza viruses by porcine
plasmacytoid dendritic cells. Viruses 3, 312–330.
Belser, J.A., Katz, J.M., Tumpey, T.M., 2011. The ferret as a model organism to study
influenza A virus infection. Disease Models and Mechanisms 4, 575–579.
Brookes, S.M., Nunez, A., Choudhury, B., Matrosovich, M., Essen, S.C., Clifford, D.,
Slomka, M.J., Kuntz-Simon, G., Garcon, F., Nash, B., Hanna, A., Heegaard, P.M.,
Queguiner, S., Chiapponi, C., Bublot, M., Garcia, J.M., Gardner, R., Foni, E., Loeffen,
W., Larsen, L., Van Reeth, K., Banks, J., Irvine, R.M., Brown, I.H., 2010. Replication,
pathogenesis and transmission of pandemic (H1N1) 2009 virus in non-immune
pigs. PLoS ONE 5, e9068.
Brown, I.H., 2000. The epidemiology and evolution of influenza viruses in pigs. Vet-
erinary Microbiology 74, 29–46.
Brown, I.H., 2012. History and epidemiology of swine influenza in Europe. Current
Topics in Microbiology and Immunology January (Epub ahead of print).
Busch, M.G., Bateman, A.C., Landolt, G.A., Karasin, A.I., Brockman-Schneider, R.A.,
Gern, J.E., Suresh, M., Olsen, C.W., 2008. Identification of amino acids in the
HA of H3 influenza viruses that determine infectivity levels in primary swine
respiratory epithelial cells. Virus Research 133, 269–279.
Busquets, N., Segales, J., Cordoba, L., Mussá, T., Crisci, E., Martin-Valls, G.E., Simon-
Grife, M., Perez-Simo, M., Perez-Maillo, M., Nunez, J.I., Abad, F.X., Fraile, L., Pina,
S., Majo, N., Bensaid, A., Domingo, M., Montoya, M., 2010. Experimental infection
with H1N1 European swine influenza virus protects pigs from an infection with
the 2009 pandemic H1N1 human influenza virus. Veterinary Research 41, 74.
Bussfeld, D., Kaufmann, A., Meyer, R.G., Gemsa, D., Sprenger, H., 1998. Differential
mononuclear leukocyte attracting chemokine production after stimulation with
active and inactivated influenza A virus. Cellular Immunology 186, 1–7.
Calzada-Nova, G., Schnitzlein, W., Husmann, R., Zuckermann, F.A., 2009. Characteri-
zation of the cytokine and maturation responses of pure populations of porcine
plasmacytoid dendritic cells to porcine viruses and toll-like receptor agonists.
Veterinary Immunology and Immunopathology 135, 20–33.
Charley, B., Riffault, S., Van Reeth, K., 2006. Porcine innate and adaptative immune
responses to influenza and coronavirus infections. Annals of the New York
Academy of Sciences 1081, 130–136.
Choi, Y.K., Nguyen, T.D., Ozaki, H., Webby, R.J., Puthavathana, P., Buranathal, C.,
Chaisingh, A., Auewarakul, P., Hanh, N.T., Ma, S.K., Hui, P.Y., Guan, Y., Peiris,
J.S., Webster, R.G., 2005. Studies of H5N1 influenza virus infection of pigs by
using viruses isolated in Vietnam and Thailand in 2004. Journal of Virology 79,
10821–10825.
De Vleeschauwer, A., Van Reeth, K., 2010. Prior infection of pigs with swine influenza
viruses is a barrier to infection with avian influenza viruses. Veterinary Micro-
biology 15, 340–345.
Ferrari, M., Candotti, P., Lombardi, G., Amadori, M., Dotti, S., Guana, S., Petrini,
S., 2008. A comparison of the humoral and cell-mediated response of pigs
210 E. Crisci et al. / Molecular Immunology 55 (2013) 200–211
experimentally infected with either influenza or PRRS viruses. Veterinary
Research Communications 32 (Suppl. 1), S199–S201.
Fouchier, R.A., Osterhaus, A.D., Brown, I.H., 2003. Animal influenza virus surveillance.
Vaccine 21, 1754–1757.
Gao, W., Sun, W., Qu, B., Cardona, C.J., Powell, K., Wegner, M., Shi, Y., Xing, Z., 2012.
Distinct regulation of host responses by ERK and JNK MAP kinases in swine
macrophages infected with pandemic (H1N1) 2009 influenza virus. PLoS ONE 7,
e30328.
Garten, R.J., Davis, C.T., Russell, C.A., Shu, B., Lindstrom, S., Balish, A., Sessions, W.M.,
Xu, X., Skepner, E., Deyde, V., Okomo-Adhiambo, M., Gubareva, L., Barnes, J.,
Smith, C.B., Emery, S.L., Hillman, M.J., Rivailler, P., Smagala, J., de Graaf, M., Burke,
D.F., Fouchier, R.A., Pappas, C., Alpuche-Aranda, C.M., Lopez-Gatell, H., Olivera,
H., Lopez, I., Myers, C.A., Faix, D., Blair, P.J., Yu, C., Keene, K.M., Dotson Jr., P.D.,
Boxrud, D., Sambol, A.R., Abid, S.H., St George, K., Bannerman, T., Moore, A.L.,
Stringer, D.J., Blevins, P., Demmler-Harrison, G.J., Ginsberg, M., Kriner, P., Water-
man, S., Smole, S., Guevara, H.F., Belongia, E.A., Clark, P.A., Beatrice, S.T., Donis,
R., Katz, J., Finelli, L., Bridges, C.B., Shaw, M., Jernigan, D.B., Uyeki, T.M., Smith,
D.J., Klimov, A.I., Cox, N.J., 2009. Antigenic and genetic characteristics of swine-
origin 2009 A(H1N1) influenza viruses circulating in humans. Science 325,
197–201.
Genzel, Y., Dietzsch, C., Rapp, E., Schwarzer, J., Reichl, U., 2010. MDCK and Vero cells
for influenza virus vaccine production: a one-to-one comparison up to lab-scale
bioreactor cultivation. Applied Microbiology and Biotechnology 88, 461–475.
GeurtsvanKessel, C.H., Lambrecht, B.N., 2008. Division of labor between dendritic
cell subsets of the lung. Mucosal Immunology 1, 442–450.
Heegaard, P.M., Klausen, J., Nielsen, J.P., Gonzalez-Ramon, N., Pineiro, M., Lamp-
reave, F., Alava, M.A., 1998. The porcine acute phase response to infection with
Actinobacillus pleuropneumoniae. Haptoglobin, C-reactive protein, major acute
phase protein and serum amyloid A protein are sensitive indicators of infection.
Comparative Biochemistry and Physiology. Part B: Biochemistry and Molecular
Biology 119, 365–373.
Heinen, P.P., de Boer-Luijtze, E.A., Bianchi, A.T., 2001. Respiratory and systemic
humoral and cellular immune responses of pigs to a heterosubtypic influenza A
virus infection. Journal of General Virology 82, 2697–2707.
Heinen, P.P., van Nieuwstadt, A.P., Pol, J.M., de Boer-Luijtze, E.A., van Oirschot, J.T.,
Bianchi, A.T., 2000. Systemic and mucosal isotype-specific antibody responses
in pigs to experimental influenza virus infection. Viral Immunology 13,
237–247.
Hillaire, M.L., van Eijk, M., van Trierum, S.E., van Riel, D., Saelens, X., Romijn, R.A.,
Hemrika, W., Fouchier, R.A., Kuiken, T., Osterhaus, A.D., Haagsman, H.P., Rim-
melzwaan, G.F., 2011. Assessment of the antiviral properties of recombinant
porcine SP-D against various influenza A viruses in vitro. PLoS ONE 6, e25005.
Hofmann, P., Sprenger, H., Kaufmann, A., Bender, A., Hasse, C., Nain, M., Gemsa, D.,
1997. Susceptibility of mononuclear phagocytes to influenza A virus infection
and possible role in the antiviral response. Journal of Leukocyte Biology 61,
408–414.
Hvistendahl, M., 2012. Pigs as stand-ins for microbiome studies. Science 336, 1250.
Ibrahim, Z., Busch, J., Awwad, M., Wagner, R., Wells, K., Cooper, D.K., 2006. Selected
physiologic compatibilities and incompatibilities between human and porcine
organ systems. Xenotransplantation 13, 488–499.
Isoda, N., Sakoda, Y., Kishida, N., Bai, G.R., Matsuda, K., Umemura, T., Kida,
H., 2006. Pathogenicity of a highly pathogenic avian influenza virus,
A/chicken/Yamaguchi/7/04 (H5N1) in different species of birds and mammals.
Archives of Virology 151, 1267–1279.
Itoh, Y., Shinya, K., Kiso, M., Watanabe, T., Sakoda, Y., Hatta, M., Muramoto, Y.,
Tamura, D., Sakai-Tagawa, Y., Noda, T., Sakabe, S., Imai, M., Hatta, Y., Watan-
abe, S., Li, C., Yamada, S., Fujii, K., Murakami, S., Imai, H., Kakugawa, S., Ito, M.,
Takano, R., Iwatsuki-Horimoto, K., Shimojima, M., Horimoto, T., Goto, H., Taka-
hashi, K., Makino, A., Ishigaki, H., Nakayama, M., Okamatsu, M., Warshauer, D.,
Shult, P.A., Saito, R., Suzuki, H., Furuta, Y., Yamashita, M., Mitamura, K., Nakano,
K., Nakamura, M., Brockman-Schneider, R., Mitamura, H., Yamazaki, M., Sug-
aya, N., Suresh, M., Ozawa, M., Neumann, G., Gern, J., Kida, H., Ogasawara, K.,
Kawaoka, Y., 2009. In vitro and in vivo characterization of new swine-origin
H1N1 influenza viruses. Nature 460, 1021–1025.
Jo, S.K., Kim, H.S., Cho, S.W., Seo, S.H., 2007. Pathogenesis and inflammatory
responses of swine H1N2 influenza viruses in pigs. Virus Research 129, 64–70.
Khatri, M., Dwivedi, V., Krakowka, S., Manickam, C., Ali, A., Wang, L., Qin, Z.,
Renukaradhya, G.J., Lee, C.W., 2010. Swine influenza H1N1 virus induces acute
inflammatory immune responses in pig lungs: a potential animal model for
human H1N1 influenza virus. Journal of Virology 84, 11210–11218.
Khatri, M., Goyal, S.M., Saif, Y.M., 2012. Oct4+ stem/progenitor Swine lung epithe-
lial cells are targets for influenza virus replication. Journal of Virology 86,
6427–6433.
Khatri, M., Saif, Y.M., 2011. Epithelial cells derived from swine bone marrow express
stem cell markers and support influenza virus replication in vitro. PLoS One 6,
e29567, http://dx.doi.org/10.1371/journal.pone.0029567.
Kida, H., Ito, T., Yasuda, J., Shimizu, Y., Itakura, C., Shortridge, K.F., Kawaoka, Y., Web-
ster, R.G., 1994. Potential for transmission of avian influenza viruses to pigs.
Journal of General Virology 75 (Pt 9), 2183–2188.
Kim, B., Ahn, K.K., Ha, Y., Lee, Y.H., Kim, D., Lim, J.H., Kim, S.H., Kim, M.Y., Cho, K.D., Lee,
B.H., Chae, C., 2009. Association of tumor necrosis factor-alpha with fever and
pulmonary lesion score in pigs experimentally infected with swine influenza
virus subtype H1N2. Journal of Veterinary Medical Science 71, 611–616.
Kim, H.M., Lee, Y.W., Lee, K.J., Kim, H.S., Cho, S.W., van Rooijen, N., Guan, Y., Seo, S.H.,
2008. Alveolar macrophages are indispensable for controlling influenza viruses
in lungs of pigs. Journal of Virology 82, 4265–4274.
Kim, W.I., Wu, W.H., Janke, B., Yoon, K.J., 2006. Characterization of the humoral
immune response of experimentally infected and vaccinated pigs to swine
influenza viral proteins. Archives of Virology 151, 23–36.
Kitikoon, P., Nilubol, D., Erickson, B.J., Janke, B.H., Hoover, T.C., Sornsen, S.A., Thacker,
E.L., 2006. The immune response and maternal antibody interference to a
heterologous H1N1 swine influenza virus infection following vaccination. Vet-
erinary Immunology and Immunopathology 112, 117–128.
Kitikoon, P., Strait, E.L., Thacker, E.L., 2008. The antibody responses to swine influenza
virus (SIV) recombinant matrix 1 (rM1), matrix 2 (M2), and hemagglutinin
(HA) proteins in pigs with different SIV exposure. Veterinary Microbiology 126,
51–62.
Kitikoon, P., Vincent, A.L., Gauger, P.C., Schlink, S.N., Bayles, D.O., Gramer, M.R., Dar-
nell, D., Webby, R.J., Lager, K.M., Swenson, S.L., Klimov, A., 2012. Pathogenicity
and transmission in pigs of the novel A(H3N2)v influenza virus isolated from
humans and characterization of swine H3N2 viruses isolated in 2010-2011.
Journal of Virology 86, 6804–6814.
Kreijtz, J.H., Fouchier, R.A., Rimmelzwaan, G.F., 2011. Immune responses to influenza
virus infection. Virus Research 162, 19–30.
Lange, E., Kalthoff, D., Blohm, U., Teifke, J.P., Breithaupt, A., Maresch, C., Starick, E.,
Fereidouni, S., Hoffmann, B., Mettenleiter, T.C., Beer, M., Vahlenkamp, T.W., 2009.
Pathogenesis and transmission of the novel swine-origin influenza virus A/H1N1
after experimental infection of pigs. Journal of General Virology 90, 2119–2123.
Larsen, D.L., Karasin, A., Zuckermann, F., Olsen, C.W., 2000. Systemic and mucosal
immune responses to H1N1 influenza virus infection in pigs. Veterinary Micro-
biology 74, 117–131.
Li, X., Jin, M., Yu, Z., Zhang, A., Chen, H., Qian, P., 2008. Pathogenicity of a goose isolate
of highly pathogenic H5N1 influenza A virus for chickens, mice, and pigs. Acta
Virologica 52, 41–46.
Lipatov, A.S., Kwon, Y.K., Sarmento, L.V., Lager, K.M., Spackman, E., Suarez, D.L.,
Swayne, D.E., 2008. Domestic pigs have low susceptibility to H5N1 highly
pathogenic avian influenza viruses. PLoS Pathogens 4, e1000102.
Loeffen, W.L., Heinen, P.P., Bianchi, A.T., Hunneman, W.A., Verheijden, J.H., 2003.
Effect of maternally derived antibodies on the clinical signs and immune
response in pigs after primary and secondary infection with an influenza H1N1
virus. Veterinary Immunology and Immunopathology 92, 23–35.
Lombardo, T., Dotti, S., Renzi, S., Ferrari, M., 2012. Susceptibility of different cell lines
to Avian and Swine Influenza viruses. Journal of Virological Methods 185, 82–88.
Londt, B.Z., Brookes, S.M., Nash, B.J., Nunez, A., Stagg, D.A., Brown, I.H., 2012. The
infectivity of pandemic 2009 H1N1 and avian influenza viruses for pigs: an
assessment by ex vivo respiratory tract organ culture. Influenza and Other Respi-
ratory Viruses June, http://dx.doi.org/10.1111/j. 1750-2659.2012.00397.x (Epub
ahead of print).
Ma, W., Lager, K.M., Vincent, A.L., Janke, B.H., Gramer, M.R., Richt, J.A., 2009. The role
of swine in the generation of novel influenza viruses. Zoonoses Public Health 56,
326–337.
Mair, K.H., Essler, S.E., Patzl, M., Storset, A.K., Saalmuller, A., Gerner, W., 2012. NKp46
expression discriminates porcine NK cells with different functional properties.
European Journal of Immunology 42, 1261–1271.
Mandelboim, O., Lieberman, N., Lev, M., Paul, L., Arnon, T.I., Bushkin, Y., Davis, D.M.,
Strominger, J.L., Yewdell, J.W., Porgador, A., 2001. Recognition of haemagglu-
tinins on virus-infected cells by NKp46 activates lysis by human NK cells. Nature
409, 1055–1060.
Medina, R.A., Garcia-Sastre, A., 2011. Influenza A viruses: new research develop-
ments. Nature Reviews Microbiology 9, 590–603.
Meurens, F., Summerfield, A., Nauwynck, H., Saif, L., Gerdts, V., 2012. The pig: a model
for human infectious diseases. Trends in Microbiology 20, 50–57.
Mussá, T., Ballester, M., Silva-Campa, E., Baratelli, M., Busquets, N., Lecours, M.P.,
Dominguez, J., Amadori, M., Fraile, L., Hernández, J., Montoya, M. Infection by
swine, human or avian influenza viruses differentially activates porcine den-
dritic cells cytokine profile. Veterinary Immunology and Immunopathology,
accepted for publication.
Mussá, T., Rodriguez-Carino, C., Pujol, M., Cordoba, L., Busquets, N., Crisci, E.,
Dominguez, J., Fraile, L., Montoya, M., 2011. Interaction of porcine conventional
dendritic cells with swine influenza virus. Virology 420, 125–134.
Nayak, D.P., Balogun, R.A., Yamada, H., Zhou, Z.H., Barman, S., 2009. Influenza virus
morphogenesis and budding. Virus Research 143, 147–161.
Nayak, D.P., Hui, E.K., Barman, S., 2004. Assembly and budding of influenza virus.
Virus Research 106, 147–165.
Nidom, C.A., Takano, R., Yamada, S., Sakai-Tagawa, Y., Daulay, S., Aswadi, D., Suzuki,
T., Suzuki, Y., Shinya, K., Iwatsuki-Horimoto, K., Muramoto, Y., Kawaoka, Y., 2010.
Influenza A (H5N1) viruses from pigs, Indonesia. Emerging Infectious Diseases
16, 1515–1523.
Nunes, S.F., Murcia, P.R., Tiley, L.S., Brown, I.H., Tucker, A.W., Maskell, D.J., Wood,
J.L., 2010. An ex vivo swine tracheal organ culture for the study of influenza
infection. Influenza and Other Respiratory Viruses 4, 7–15.
Ocana-Macchi, M., Ricklin, M.E., Python, S., Monika, G.A., Stech, J., Stech, O., Summer-
field, A., 2012. Avian influenza A virus PB2 promotes interferon type I inducing
properties of a swine strain in porcine dendritic cells. Virology 427, 1–9.
Paget, C., Ivanov, S., Fontaine, J., Blanc, F., Pichavant, M., Renneson, J., Bialecki, E.,
Pothlichet, J., Vendeville, C., Barba-Spaeth, G., Huerre, M.R., Faveeuw, C., Si-Tahar,
M., Trottein, F., 2011. Potential role of invariant NKT cells in the control of pul-
monary inflammation and CD8+ T cell response during acute influenza A virus
H3N2 pneumonia. Journal of Immunology 186, 5590–5602.
Palese, P., Shaw, M.L., 2007. Orthomyxoviridae: the viruses and their replication. In:
Knipe, D.M., Howley, P.M. (Eds.), Fields Virology. Lippincott Williams & Wilkins,
Philadelphia, pp. 1647–1689.
 E. Crisci et al. / Molecular Immunology 55 (2013) 200–211
Pomorska-Mol, M., Markowska-Daniel, I., Kwit, K., 2012a. Immune and acute phase
response in pigs experimentally infected with H1N2 swine influenza virus. FEMS
Immunology and Medical Microbiology 66, 334–342.
Pomorska-Mol, M., Markowska-Daniel, I., Pejsak, Z., 2012b. Acute phase protein
response during subclinical infection of pigs with H1N1 swine influenza virus.
Veterinary Microbiology 159, 499–503.
Punyadarsaniya, D., Liang, C.H., Winter, C., Petersen, H., Rautenschlein, S., Hennig-
Pauka, I., Schwegmann-Wessels, C., Wu, C.Y., Wong, C.H., Herrler, G., 2011.
Infection of differentiated porcine airway epithelial cells by influenza virus: dif-
ferential susceptibility to infection by porcine and avian viruses. PLoS ONE 6,
e28429.
Renukaradhya, G.J., Manickam, C., Khatri, M., Rauf, A., Li, X., Tsuji, M., Rajashekara, G.,
Dwivedi, V., 2011. Functional invariant NKT cells in pig lungs regulate the airway
hyperreactivity: a potential animal model. Journal of Clinical Immunology 31,
228–239.
Rhoads, J.M., Chen, W., Chu, P., Berschneider, H.M., Argenzio, R.A., Paradiso, A.M.,
1994. l-Glutamine and l-asparagine stimulate Na+ –H+ exchange in porcine jeju-
nal enterocytes. American Journal of Physiology 266, G828–G838.
Rothkotter, H.J., Sowa, E., Pabst, R., 2002. The pig as a model of developmental
immunology. Human and Experimental Toxicology 21, 533–536.
Seo, S.H., Webby, R., Webster, R.G., 2004. No apoptotic deaths and different levels
of inductions of inflammatory cytokines in alveolar macrophages infected with
influenza viruses. Virology 329, 270–279.
Sun, Y., Bi, Y., Pu, J., Hu, Y., Wang, J., Gao, H., Liu, L., Xu, Q., Tan, Y., Liu, M., Guo, X.,
Yang, H., Liu, J., 2010. Guinea pig model for evaluating the potential public health
risk of swine and avian influenza viruses. PLoS ONE 5, e15537.
Sun, Z., Huber, V.C., McCormick, K., Kaushik, R.S., Boon, A.C., Zhu, L., Hause, B., Webby,
R.J., Fang, Y., 2012. Characterization of a porcine intestinal epithelial cell line for
influenza virus production. Journal of General Virology 93, 2008–2016.
Tobita, K., Sugiura, A., Enomote, C., Furuyama, M., 1975. Plaque assay and primary
isolation of influenza A viruses in an established line of canine kidney cells
(MDCK) in the presence of trypsin. Medical Microbiology and Immunology 162,
9–14.
Tong, S., Li, Y., Rivailler, P., Conrardy, C., Castillo, D.A., Chen, L.M., Recuenco, S., Ellison,
J.A., Davis, C.T., York, I.A., Turmelle, A.S., Moran, D., Rogers, S., Shi, M., Tao, Y., Weil,
M.R., Tang, K., Rowe, L.A., Sammons, S., Xu, X., Frace, M., Lindblade, K.A., Cox, N.J.,
Anderson, L.J., Rupprecht, C.E., Donis, R.O., 2012. A distinct lineage of influenza A
virus from bats. Proceedings of the National Academy of Sciences of the United
States of America 109, 4269–4274.
Van Poucke, S.G., Nicholls, J.M., Nauwynck, H.J., Van Reeth, K., 2010. Replication of
avian, human and swine influenza viruses in porcine respiratory explants and
association with sialic acid distribution. Virology Journal 7, 38.
211
Van Reeth, K., 2000. Cytokines in the pathogenesis of influenza. Veterinary Micro-
biology 74, 109–116.
Van Reeth, K., 2007. Avian and swine influenza viruses: our current understanding
of the zoonotic risk. Veterinary Research 38, 243–260.
Van Reeth, K., Braeckmans, D., Cox, E., Van Borm, S., van den Berg, T., Goddeeris, B.,
De Vleeschauwer, A., 2009. Prior infection with an H1N1 swine influenza virus
partially protects pigs against a low pathogenic H5N1 avian influenza virus.
Vaccine 27, 6330–6339.
Van Reeth, K., Labarque, G., Pensaert, M., 2006. Serological profiles after consecutive
experimental infections of pigs with European H1N1, H3N2, and H1N2 swine
influenza viruses. Viral Immunology 19, 373–382.
Van Reeth, K., Ma, W., 2012. Swine influenza virus vaccines: to change or not to
change-that’s the question. Current Topics in Microbiology and Immunology
September (Epub ahead of print).
Van Reeth, K., Nauwynck, H., Pensaert, M., 1998. Bronchoalveolar interferon-alpha,
tumor necrosis factor-alpha, interleukin-1, and inflammation during acute
influenza in pigs: a possible model for humans? Journal of Infectious Diseases
177, 1076–1079.
Van Reeth, K., Van Gucht, S., Pensaert, M., 2002a. Correlations between lung
proinflammatory cytokine levels, virus replication, and disease after swine
influenza virus challenge of vaccination-immune pigs. Viral Immunology 15,
583–594.
Van Reeth, K., Van Gucht, S., Pensaert, M., 2002b. In vivo studies on cytokine
involvement during acute viral respiratory disease of swine: trouble-
some but rewarding. Veterinary Immunology and Immunopathology 87,
161–168.
Vincent, A.L., Ma, W., Lager, K.M., Richt, J.A., Janke, B.H., Sandbulte, M.R., Gauger,
P.C., Loving, C.L., Webby, R.J., Garcia-Sastre, A., 2012. Live attenuated influenza
vaccine provides superior protection from heterologous infection in pigs with
maternal antibodies without inducing vaccine-associated enhanced respiratory
disease. Journal of Virology 86, 10597–10605.
Webster, R.G., Bean, W.J., Gorman, O.T., Chambers, T.M., Kawaoka, Y., 1992.
Evolution and ecology of influenza A viruses. Microbiological Reviews 56,
152–179.
Weingartl, H.M., Albrecht, R.A., Lager, K.M., Babiuk, S., Marszal, P., Neufeld, J.,
Embury-Hyatt, C., Lekcharoensuk, P., Tumpey, T.M., Garcia-Sastre, A., Richt, J.A.,
2009. Experimental infection of pigs with the human 1918 pandemic influenza
virus. Journal of Virology 83, 4287–4296.
Zhu, H., Zhou, B., Fan, X., Lam, T.T., Wang, J., Chen, A., Chen, X., Chen, H., Webster,
R.G., Webby, R., Peiris, J.S., Smith, D.K., Guan, Y., 2011. Novel reassortment of
Eurasian avian-like and pandemic/2009 influenza viruses in swine: infectious
potential for humans. Journal of Virology 85, 10432–10439.