Review

Veterinary Pathology
2014, Vol 51(2) 410-426
Influenza A Virus Infections in Swine: ª The Author(s) 2013
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Pathogenesis and Diagnosis DOI: 10.1177/0300985813513043
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B. H. Janke1

Abstract
Influenza has been recognized as a respiratory disease in swine since its first appearance concurrent with the 1918 ‘‘Spanish flu’’
human pandemic. All influenza viruses of significance in swine are type A, subtype H1N1, H1N2, or H3N2 viruses. Influenza
viruses infect epithelial cells lining the surface of the respiratory tract, inducing prominent necrotizing bronchitis and
bronchiolitis and variable interstitial pneumonia. Cell death is due to direct virus infection and to insult directed by leukocytes
and cytokines of the innate immune system. The most virulent viruses consistently express the following characteristics of
infection: (1) higher or more prolonged virus replication, (2) excessive cytokine induction, and (3) replication in the lower
respiratory tract. Nearly all the viral proteins contribute to virulence. Pigs are susceptible to infection with both human and
avian viruses, which often results in gene reassortment between these viruses and endemic swine viruses. The receptors on the
epithelial cells lining the respiratory tract are major determinants of infection by influenza viruses from other hosts. The
polymerases, especially PB2, also influence cross-species infection. Methods of diagnosis and characterization of influenza
viruses that infect swine have improved over the years, driven both by the availability of new technologies and by the necessity
of keeping up with changes in the virus. Testing of oral fluids from pigs for virus and antibody is a recent development that
allows efficient sampling of large numbers of animals.

Keywords
influenza virus, swine, pathogenesis, cytokines, apoptosis, virulence, cross-species infection, hemagglutinin, receptors, poly-
merases, diagnosis

The evolution and epidemiology of influenza A virus (IAV) Pathogenesis of Infection

infections in swine have been integrally tied to the segmented
nature of the genome of the virus, which facilitates gene
reassortment, and to the adaptability of these genes through
mutation, which sometimes facilitates replication in a new
host.8,19,77,85,89 Influenza was first recognized as a respiratory
disease entity in swine during the 1918 human Spanish flu pan-
demic.67 The virus was not isolated until 1931, when nasal dis-
charge from pigs was inoculated into ferrets and subsequently
into embryonated chicken eggs.129 In 1933, a similar virus was
recovered from humans with this technique.131 The viruses
were essentially identical and served as the prototype for type
A subtype H1N1 influenza viruses.
All influenza viruses of significance that infect swine are
type A viruses, defined by the highly conserved internal
nucleoproteins and matrix proteins. Influenza A virus subtypes
are based on the external hemagglutinin (HA) and neuramini-
dase (NA) proteins. The predominant viruses in swine popula-
tions in different parts of the world may have genes of different
origin, even though the subtypes may be similar.10,73,171 The
original classic H1N1 swine influenza virus, which remained
the predominant influenza virus in North American swine
populations for nearly 70 years, has been almost completely
displaced by subsequent reassortant viruses.157
Although the genetic makeup of influenza viruses in swine var-
ies, the clinical disease and lesions induced by the classic H1N1
swine influenza virus,52,53,101,139,158,166 avian-like H1N1 viruses
adapted to swine,9,24,29 and various H1N1,21,84,134,155,158,159
H1N2,29,56,155,158 H3N2,76,118 and H2N387 reassortants (includ-
ing the recent 2009 human pandemic H1N178,156 and subsequent
reassortants15,63) are essentially the same. The gross and micro-
scopic lesions, clinical signs, virus distribution in tissues, and
virus shedding patterns have been described with variable
emphasis in the above studies and in other reports on isolates
of interest, in comparison studies, and in vaccine trials. Two
of the earliest studies, one employing histopathology and immu-
nofluorescence,101 the other histopathology and ultrastructural
1
Veterinary Diagnostic Laboratory, Department of Veterinary Diagnostic and
Production Animal Medicine, College of Veterinary Medicine, Iowa State
University, Ames, IA, USA
Corresponding Author:
B. H. Janke, DVM, PhD, Veterinary Diagnostic Laboratory, Department of
Veterinary Diagnostic and Production Animal Medicine, College of Veterinary
Medicine, Iowa State University, Ames, IA 50011, USA.
Email: bhjanke@iastate.edu
Janke
characterization,166 still provide some of the most detailed histo-
pathologic descriptions.
In experimentally infected pigs inoculated with a sufficient
dose of challenge virus (2 ml of 106 egg infectious dose 50
[EID50] or tissue culture infectious dose 50 [TCID50] intratra-
cheally) and in the individual pig with natural infection, the
course of infection is very short (5–7 days) with minimal virus
detected after this time. In the immunofluorescent study,101 the
first evidence of virus infection was a pale fluorescence in the
nucleus of bronchial epithelial cells by 2 hours postinoculation
(PI). By 4 hours PI, virus antigen was abundant in both nucleus
and cytoplasm of infected cells. In the ultrastructural study,166
virus was observed budding from the surface of type II pneu-
mocytes as early as 5 hours PI. In a more recent ultrastructural
study on a differentiated human airway epithelial cell line, vir-
ions in high numbers were budding from cell surfaces at 24
hours PI, predominantly from the tips of microvilli of both
ciliated and secretory cell types.69 Although virus has been
detected in mediastinal lymph nodes, blood, and other tissues
such as brain in a few studies in swine,9,24,77 productive influ-
enza virus replication with consequent tissue damage is limited
in this species to the respiratory tract.
Because the virus enters the lung through the airways, the
most consistent macroscopic manifestation of influenza virus
infection is cranioventral bronchopneumonia that affects a
variable percentage of the lung. In milder infections, small
clusters of medium to dark red lobules are located in the cranial
and middle lung lobes and occasionally in cranioventral por-
tions of the caudal lobes and in accessory lobes (Fig. 1). In
some pigs, these foci of consolidation are somewhat linear and
confluent and situated in the hilar region at the junction of the
lung lobes. In more severe or extensive infections, most or all
of the cranial and middle lung lobes and cranioventral portions
of the caudal lobes may be consolidated. Less frequently,
severe acute infection presents as a diffusely congested lung
with prominent interlobular edema and extensive foam in the
bronchi and trachea. Such cases likely represent an exaggerated
cytokine response, and in such lungs, the characteristic cranio-
ventral lobular consolidation is obscured by the more diffuse
interstitial pneumonia and edema.
In swine, influenza viruses primarily infect the epithelial cells
lining the surface of the respiratory tract, from nasal mucosa to
alveoli, but virus also has been detected in the glandular epithe-
lial cells associated with the larger airways. The hallmark micro-
scopic lesion is necrotizing bronchitis and bronchiolitis. During
the first 24 hours PI, neutrophils begin to accumulate in the vas-
culature adjacent to bronchioles, and light loose lymphocytic
cuffs appear around the airways, sometimes before epithelial
damage is evident. Necrosis and sloughing of airway epithelial
cells and neutrophil transmigration into airway lumens occur
at 24 to 48 hours PI (Fig. 2). By 48 hours PI, affected airways
are lined by flattened epithelial layers, and neutrophils in the
luminal debris have been replaced or are accompanied by macro-
phages. Lymphocyte accumulation around airways and adjacent
vessels becomes more prominent. Continuing in repair, the
epithelial cells lining the airways proliferate and mature into
411
tightly packed tall columnar cells, resulting in an irregular hyper-
plastic appearance, often by 72 hours PI (Fig. 3). In severely
damaged airways, proliferation of fibroblasts from exposed
subepithelial lamina propria may result in formation of endo-
bronchial polyps or incomplete splitting of repairing airways
(bronchiolitis obliterans) (Fig. 4). Such damage is most likely
to occur in the smaller airways.
In both field cases and experimental challenge studies, the
sizes of airways that are affected may vary. In the author’s
experience, the largest noncartilaginous airways (primary
bronchioles) are most consistently and most severely affected,
likely due to the dynamics of airflow that affect droplet suspen-
sion and deposition or possibly due to receptor distribution.
There is some variation between cases, and the larger lobar or
segmental bronchi may be spared or only minimally affected,
or conversely, only these larger airways may be damaged. Like-
wise, the smallest terminal or respiratory bronchioles also are
frequently spared but in severe extensive infections may exhibit
the characteristic epithelial necrosis. An early immunofluores-
cent study on sequentially sampled pigs described infection as
progressive, beginning in the small bronchi followed by distal
spread to terminal bronchioles and alveoli as well as proximally
to bronchi and the trachea,101 which may suggest that some of
the variation in lesion distribution is a function of time after
infection or of the position of the lobule in question along the
infected branch of the respiratory tree.
Simultaneous to the onset and progression of bronchial and
bronchiolar damage and inflammation, degenerative and inflam-
matory lesions develop in the alveoli.101,166 During the first
24 hours PI, alveolar walls, primarily peribronchiolar in location,
are widened by vascular congestion and interstitial edema.
Endothelial cells lining the capillaries are swollen, and neutro-
phils accumulate in capillary lumens and marginate against
vessel walls. Type II pneumocytes become swollen. Neutrophils,
macrophages, and lymphocytes lightly infiltrate the alveolar
interstitium and migrate into alveolar lumens. As in the airways,
neutrophils predominate early in infection, but subsequently
(by 48 hours PI), the alveolar luminal exudate becomes pre-
dominantly mononuclear. These cells appear to be a mixture
of sloughed swollen pneumocytes and macrophages. By close
examination, necrosis of constituent cells may be identified
in alveolar walls, but the septa usually remain intact. In severe
infections, necrotic cellular debris accumulates in alveolar
lumens, or rarely, the necrotic debris and exudative proteins
form hyaline membranes. In such cases, alveolar and interlobu-
lar edema is usually evident. Swollen alveolar epithelial cells
lining the lumen add to the thickness of the alveolar septa. In
some infections, in contrast to the alveolar lesions described
above, alveoli may be affected only minimally.
Consistent with the multifocal macroscopic lesions in the
lung, lobules within the same histopathologic section of lung
may differ in the degree of involvement, and unaffected lobules
often sit adjacent to severely affected lobules. This likely
reflects irregular distribution of virus through the branches
of the respiratory tract via inhalation of aerosolized virus.
Experimentally, the virus titer in the inoculum and the route
412 Veterinary Pathology 51(2)

Figure 1. Lung in situ, 4-week-old pig, 5 days after experimental intratracheal inoculation with triple reassortant H3N2 swine influenza virus.
Multifocal lobular consolidation in cranial and middle lung lobes is evident. Figure 2. Lung, pig, field case. The acute phase of influenza A virus
infection is characterized by necrosis and sloughing of bronchiolar epithelial cells and flattening of the remaining cells lining the airway. The lumen
contains clusters of neutrophils and sloughed epithelial cells. Light peribronchiolar lymphocyte infiltration is evident. Hematoxylin and eosin
(HE). Figure 3. Lung, pig, field case. The subacute phase of influenza A virus infection is characterized by an irregular hyperplastic epithelial
layer lining the airway. The lumen contains mucus and a few neutrophils and macrophages. Transmigration of the epithelial layer by lymphocytes
and a few neutrophil clusters is evident. HE. Figure 4. Lung, pig, field case. A small bronchiole is disrupted by an endobronchiolar polyp (bronch-
iolitis obliterans), a sequel sometimes observed following severe infection with influenza A virus. HE.

of inoculation can affect lesion development.24,76 Inducing
clinical disease and significant lung lesions can be difficult
with intranasal inoculation as pigs may swallow much of the
inoculum, reducing the volume of inoculum that reaches the
lung in contrast to intratracheal inoculation. If the virus dose is
too light, lesions may be minimal but multifocally asynchronous
in development. The latter issue can interfere with studies that
evaluate lesion progression in sequential necropsies.
Subsequently, the epithelial hyperplasia resolves and all that
remain are peribronchiolar lymphocytic cuffing and partially
atelectatic alveoli with variable leukocyte populations. Although
somewhat dependent on the extent of damage, lungs return
to normal by 2 weeks PI. However, in pigs that survive severe
infection, airways disrupted by bronchiolitis obliterans may
provide evidence of earlier infection.
Mechanisms of Cell Injury
Research into the molecular mechanisms of influenza-induced
cell injury has been conducted primarily in vitro in cell lines of
nonswine origin with viruses not isolated from swine. Although
in vitro studies have their limitations because of the artificial
nature of the medium, control of variables is more readily
accomplished than in the in vivo studies. The segmented gen-
ome of influenza viruses and the ability to isolate the effect
of specific proteins through generation of virus with the desired
Janke
genes by reverse genetics facilitate these studies. Commonal-
ities in mechanisms of infection likely exist regardless of the
source of virus or host involved. Thus, in attempting to under-
stand pathogenesis of influenza virus infection at the cellular
level and the resulting disease in swine (or in other species), the
results of such studies warrant consideration. Findings suggest
necrosis, apoptosis, and cytokines of the innate immune
response all contribute to the destruction of influenza virus–
infected cells.
Necrosis
Much of the damage to the respiratory tract by influenza virus
can be attributed to direct viral destruction of infected cells.55,126
Influenza virus infection induces cytolysis of infected cells
through suppression of host cell gene expression and protein
synthesis. Viral polymerases bind to host cell polymerase tran-
scripts and cleave host messenger RNAs (mRNAs) (50 cap
removal), which then degrade. Viral NS1 protein inhibits export
of host mRNAs from the nucleus, which greatly reduces transla-
tion in the cytoplasm. Blockage of host cell protein synthesis, in
concert with initiation of viral protein synthesis and virus parti-
cle budding from cytoplasmic membranes, results in cytolytic
death within 20 to 40 hours of infection. Necrosis is character-
ized by disruption of cell membranes and swelling of cytoplasm
and mitochondria, followed by disintegration of organelles and
total cell lysis. Cell death by necrosis stimulates a strong inflam-
matory response through cytokine induction.
Apoptosis
Cell death induced by influenza virus infection also occurs
through apoptosis by both extrinsic and intrinsic pathways.46
Apoptosis has been most thoroughly studied in established cell
lines of both respiratory and nonrespiratory origin74,75,126 but
has also been detected in respiratory tract cells collected fol-
lowing in vivo infection.98,145 Viral components that have been
most strongly implicated in the induction and regulation of
apoptosis include nonstructural proteins NS1 and PB1-F2 and
the ion channel structural protein M2.46,74,75 NS1 appears capa-
ble of exerting either proapoptotic or antiapoptotic influences,
depending on intracellular conditions.46,74,75 Some studies sug-
gest viral neuraminidase99,124 and M1 proteins176 and single- or
double-stranded RNA intermediates3 also influence apoptosis.
Inconsistent results between different studies with regard to the
induction of necrosis vs apoptosis appear to be related to the
cell types studied and the viruses used.
Although apoptosis has been proposed as a host defense
mechanism that reduces virus spread through rapid death of
virus-infected cells and dampens inflammation through phago-
cytosis of apoptotic cell debris, recent studies suggest viruses
can use the process to their advantage. Viruses may enhance
their infectivity by inhibiting caspase activity until sufficient
replication of viral proteins has been completed.46,79,94,168
Contrarily, in a case of Highly Pathogenic Avian Influenza
(HPAI) zoonosis, an H5N1 virus isolated from a human fatality
413
induced more cellular apoptosis in vitro than less virulent
viruses of other subtypes.75
Immunopathology
Numerous studies have described the cellular and cytokine
response to influenza virus infection as measured in vivo in var-
ious animal models and in vitro in cell cultures.11,14,54,55,59,126,148
Contradictory results from various studies on the interactions
of components of the inflammatory response suggest that
the severity of tissue damage is determined by the balance
between proinflammatory and anti-inflammatory cytokine
activity. An in-depth discussion of the complex multitude of
molecules involved in the innate immune response to influ-
enza virus infection is beyond the scope of this review, but
an excellent review article on immunopathology has recently
been published.23 A brief summary of the mechanisms of
destruction of influenza virus–infected cells by the innate
immune response is given below.
Type 1 interferon (IFN), predominantly INF-a, is generated
within a few hours by infected respiratory epithelial cells to
inhibit viral replication and infection of adjacent cells31 and
to initiate proinflammatory cytokine/chemokine responses.107
Surface proteins expressed by the infected cells serve as signals
for their destruction by cells of the innate immune system
(killer cells, neutrophils, macrophages) resident in nearby tis-
sues. Neutrophils destroy virus-infected cells through genera-
tion of oxidases and engulf debris from cells killed by virus
but also may damage tissue by release of other enzymes (eg,
elastase, myeloperoxidase).51,61
The leukocytic cells also effect destruction of virus-infected
cells during the early innate immune response through genera-
tion of a variety of cytokines,* which also recruit additional
leukocytes to the site of injury. The primary proinflammatory
cytokines are tumor necrosis factor (TNF)–a, interleukin
(IL)–1, and IL-6. TNF-a and IL-1 are strong upregulators of
leukocyte adhesion molecules on endothelial cells. TNF-a also
increases microvascular permeability in the lung. IFN-a, TNF-
a, and IL-1, IL-6, and IL-8 can be detected in bronchoalveolar
lavage fluid (BALF) of influenza virus-infected pigs, reaching
peak levels within 18 to 24 hours PI, followed by a drop to
insignificant levels by 48 to 72 hours PI. This wave corre-
sponds to a similar rise and fall in neutrophil numbers in BALF,
virus titers in lung, lesions of acute necrotizing bronchiolitis,
and clinical illness. By 48 hours, macrophages have replaced
neutrophils as the predominant inflammatory cell in the
lung.149
Cytokines released by innate immune cells also have effects
that reach beyond the localized cellular environment. TNF-a
has other well-described actions in the lung, including
increased bronchoconstriction and bronchial hyperreactivity,
as well as changes in mucus and surfactant production. IFN-
*References 36, 51, 61, 62, 109, 116, 144.
414
a and IL-6 also have systemic effects such as induction of fever
and production of acute phase proteins by the liver.4,116
Although endothelial cells have not been considered targets of
influenza virus infection, recent studies suggest that endothelial
cells in the lung are significant modulators of both intracellular
and cytokine aspects of the acute response.34,107,138 These studies
have been conducted primarily in mouse tissues and human
endothelial cell lines using human viruses considered highly viru-
lent but not necessarily pandemic viruses. Various cytokines and
cell surface signals play significant roles in the endothelial
damage. Endothelial cells variably exhibit necrosis or apoptosis,
but the predominant change is loss of intercellular junctions,
which results in vascular leakage.2 When prominent, this leakage
corresponds to extremely high cytokine levels in the tissues
(cytokine storm).162 Although some studies have described rec-
eptors for influenza virus on the surface of human endothelial
cells170 and virus infection of avian28,65 and human endothelial
cells,175 these studies suggest the endothelial cell damage is due
to cytokines. Influenza virus infection of endothelial cells in
swine lungs has not been documented.
In summary, injury and destruction of the cells lining the
respiratory tract by influenza virus is effected by both direct virus
infection and by the cytokines of the innate immune response. The
most severe infections are characterized by high cytokine levels
in the tissues. In humans, therapy directed toward important com-
ponents of the innate immune response may be as effective as
antiviral products in alleviating severe lung damage.107
Immune-Mediated Enhancement of Disease
In an experimental trial evaluating the effect of an inactivated
H1N1 vaccine on subsequent challenge with a heterologous
virus, several pigs developed lung lesions dramatically more
severe than that induced in nonvaccinated control pigs. The
enhanced lesions were produced in a second trial with the same
vaccine and challenge virus and in additional trials with differ-
ent vaccines and challenge viruses.32,33,64,157
The enhanced lesions were characterized macroscopically
by involvement of a greater percentage of lung and microsco-
pically by more severe necrotizing and proliferative bronchio-
litis, more prominent peribronchiolar lymphocytic cuffing, and
severe lymphohistiocytic interstitial pneumonia. There was
more intense lymphocyte infiltration into the epithelial layer
and subepithelial lamina propria of the large- to medium-
sized airways and suppurative bronchitis and bronchiolitis of
greater severity than that observed in the nonvaccinated, chal-
lenged pigs. The epithelial lining of the trachea was also more
frequently disrupted and disorganized, and the subepithelial
inflammation was more intense in the vaccinated pigs.
The antigenic relationship between vaccine virus and chal-
lenge virus or other factors that might contribute to this
enhanced inflammatory response have not been determined.
Such overzealous reactions likely occur in field situations, but
the frequency of concurrent multipathogen infections that
result in more severe pneumonia makes identification of such
cases of immune-augmented disease difficult. The researchers
Veterinary Pathology 51(2)
speculate that the potential may exist for similar vaccine-
associated disease enhancement in humans.33
Virulence
In discussions on the pathogenesis of disease induced by influ-
enza virus infections, the question soon arises whether viruses
vary in inherent virulence capabilities. The clinical signs asso-
ciated with natural influenza virus infections in both swine and
humans can vary considerably, from mild respiratory illness to
severe dyspnea followed shortly by death. Secondary bacterial
infection can contribute to more severe pneumonia and higher
mortality rates in both species. Rapidly spreading epidemics,
sometimes accompanied by higher mortality rates, usually sig-
nal introduction of a new virus strain against which the popu-
lation has little immune protection. However, some influenza
viral infections without bacterial augmentation and some epi-
demics with excessively high morbidity and/or mortality rates
suggest the capacity for increased virulence may reside within
some viruses. In swine, only very occasional influenza field
outbreaks present with high mortality that appears to be
effected by virus alone.86 In humans, viruses responsible for
pandemics usually elicit higher morbidity rates (likely reflec-
tive of greater transmissibility) and/or greater severity of clin-
ical disease (likely reflective of higher virus replication rates)
than that observed with most seasonal influenzas, but overall
mortality rates may or may not be higher. Depending on the
pandemic, the incidence of infection may be higher in certain
age segments of the population.
Pathologic studies on tissues from natural infections reveal
some differences. In lungs of pigs submitted from high mortal-
ity field cases, diffuse noncollapse and prominent interlobular
edema may mask the characteristic cranioventral consolida-
tion. Microscopically, the extent of airway involvement (the
number of affected airways, both larger and smaller, within
each lobule) and the severity of alveolar inflammation (wide-
spread prominence of pneumocyte swelling, increased numbers
of infiltrating cells in alveolar walls and lumens, and abun-
dance of proteinaceous fluid in alveolar lumens) are the most
remarkable differences from routine infections. In humans,
microscopic lesions induced by the most virulent influenza
viruses, the 1918 H1N1 and HPAI H5N1 viruses, have been
described in some studies as not differing markedly from non-
pandemic infections but with the most significant differences
involving the degree of inflammatory changes at the alveolar
level.137 The alveolar inflammation described in these studies
resembles that observed in severe swine cases.86 Others have
described the lesions induced by the 1918 and 2009 pandemic
H1N1 viruses and the HPAI H5N1 virus as involving both the
upper and lower portions of the respiratory tract, whereas a sea-
sonal H3N2 virus infection induced lesions primarily limited to
the upper tract.42 Thus, results of pathologic examination of
lungs of both naturally infected swine and humans suggest
viruses that induce inflammation in the lower respiratory tract
cause the most severe disease.
Janke
In vivo challenge studies also have been used in the search for
more virulent strains. With swine-origin viruses, comparative
challenge studies conducted in pigs have not provided definitive
results. Attempts to experimentally reproduce severe disease
with isolates from high mortality field cases have not been suc-
cessful.86 Pigs challenged with viruses adapted to or endemic in
swine usually develop only mild clinical disease and similar
lesions of mild to moderate severity. Although some studies
describe more extensive lung involvement, comparison of
results from different researchers must consider the impact of
factors that may vary between studies, such as virus titer in
inoculum, method of restraint and route of inoculation, age of
pigs, and so on.24,76,118 In most comparative studies with ende-
mic viruses run concurrently under the same protocols, lesion
differences have not tended to be dramatic. However, measure-
ment of quantifiable parameters such as fever, cytokine levels in
lung or BALF, and virus titers in nasal secretions, lung tissue, or
BALF can supply additional data that often correlate well with
gross and microscopic lung scores. Such studies provide further
evidence of possible virulence differences between viruses.
Some studies suggest that the triple reassortant viruses may be
more virulent than the classic H1N1 viruses,158 especially those
reassortants that have been recovered from circumstances in
which both pigs and humans were infected.159
Pigs also appear to be relatively resistant to infection with
viruses that cause disease in other host species. Infection of pigs
with low-pathogenicity avian influenza (LPAI) viruses or seaso-
nal human influenza viruses usually results in limited viral repli-
cation with mild clinical disease and lesions, unless the virus
adapts through genetic mutation.77 Even inoculation with viruses
such as the 1918 human pandemic H1N1 virus and HPAI H5N1
that induce severe disease in other mammals (humans, primates,
ferrets, and mice) only results in disease of similar or less sever-
ity than that induced by endemic swine viruses.82,164
With viruses associated with human disease, challenge stud-
ies seeking to identify or characterize virulent strains have of
necessity been conducted in animal models, primarily mice,
ferrets, and macaques, and in the in vitro studies in influenza-
susceptible cell lines. Mice have been used extensively to study
influenza viruses but are not a natural host for these viruses;
human viruses do not cause disease in mice unless first adapted
through serial passage.6 Ferrets are a better model for human
influenza infections.5,7,153 Pigs probably would be an even closer
model,59 but the logistics and cost of maintaining swine for
experimental studies precludes their use in many research insti-
tutions. In mouse and ferret models, clear differences in lesion
severity between the HPAI H5N1 or 1918 human pandemic
H1N1viruses and the seasonal viruses are evident. The severity
of disease induced in ferrets by the 2009 human pandemic H1N1
virus is intermediate.42,146
In summary from these studies, influenza viruses that are
more virulent in humans consistently express 2 characteristics
of infection: (1) higher or more prolonged levels of virus repli-
cation and (2) induction of a strong proinflammatory response
(cytokine storm), a significant result of which is increased per-
meability of alveolar vasculature.70,81,113,162 Dysregulation of
415
the early innate immune response appears to be responsible for
both of these characteristics.70
Numerous studies, using both in vivo animal models and in
vitro cell culture systems, have investigated the viral proteins
as the basis for these differences in virulence. Parameters eval-
uated in animal studies include severity of clinical disease or
lesions and virus titers or cytokine levels resulting from infec-
tion. Although evaluation of virulence in the in vitro systems
is not technically plausible, increased production of virus or
cytokines (if the cell type is capable of such production) is
interpreted in these studies to be evidence for possible contri-
bution to virulence. These studies often use viruses generated
by reverse genetics to evaluate the effect of gene replacement
or gene mutation from a more or less virulent strain on an
otherwise identical virus.
Many such studies report findings that run counter to earlier
work or reach opposite or different conclusions when initial
studies are extended to other viruses, hosts, or experimental
protocols. Nearly all viral proteins have been implicated as
virulence factors, either individually or in concert with other
proteins.17,88,123 Such studies emphasize the polygenic nature
of virulence attributes in influenza viruses, the complexity of
the interactions between viral proteins, and the importance of
differences between hosts (and tissues within hosts) in the dis-
ease process. Several recent articles have summarized current
knowledge of how viral proteins affect virulence.6,70,143 The
reported roles for various specific viral proteins in virulence are
described below.
Hemagglutinin
The hemagglutinin (HA) is a protein protruding from the sur-
face of the virus that is responsible for attachment of virus to
host cells, internalization of virus, and intracellular fusion of
viral capsid with endosomal membranes. In avian species, the
cleavage site in the HA plays an important role in virulence,
which is manifested as systemic infection. The single basic
amino acid adjacent to the cleavage site in LPAI viruses is sus-
ceptible to proteases that are limited to the gastrointestinal and
respiratory tracts. The H5 (and H7) in HPAI viruses has a
sequence of multiple basic amino acids that allows cleavage
by furin-like proteases that are present in many tissues, thus
permitting lethal systemic infection.135
Influenza viruses that naturally infect mammalian hosts do
not have a multibasic cleavage site, and systemic infection is
not a normal characteristic of influenza virus infection in mam-
mals. Artificially inserting a multiple basic amino acid
sequence by reverse genetics into the HA of mildly virulent
viruses does not result in increased virulence or systemic infec-
tion in ferrets.122 However, the HPAI H5N1 virus, which does
have a multibasic cleavage site, can be lethal for mice and fer-
rets in which it induces increased cytokine levels and virus
titers and results in systemic infection.136 This does not occur
in pigs,82 which may reflect a difference in tissue proteases
in this species. The HA from the 1918 human pandemic
H1N1virus, which does not have a multibasic cleavage site but
416
still appears to be highly cleavable, also confers increased
virulence to reassortants as reflected in increased virus titers
and inflammation in mice and human airway cells.66,111 Thus,
no consistent association has been established between a
sequence of multiple basic amino acids in the HA and virulence
of viruses possessing such a sequence for mammalian species.
Neuraminidase
Neuraminidase (NA) is an external protein that prevents aggre-
gation of viral progeny upon release from infected cells.
Balanced function of HA and NA results in the most efficient
virus replication, and alterations in the NA protein can interfere
with this balance, resulting in decreased virulence. In studies of
HPAI H5N1 in mice, changes in the length and glycosylation of
NA influenced virulence.93 Some NA proteins inherently
appear to contribute to greater virulence. In a study with reas-
sortant 1918 human pandemic virus and contemporary H1N1
viruses, NA from the 1918 human pandemic H1N1 virus con-
tributed to high virus replication rates in both mouse lungs and
human airway cells.111 In studies of reassortants between pan-
demic 2009 H1N1 and seasonal human viruses inoculated into
ferrets, reassortants containing the H3N2 NA were more viru-
lent, whereas other reassortants were attenuated.123 Some stud-
ies suggest the NA protein also contributes to apoptosis.99,124
Polymerases
The polymerases direct cell processes toward virus replication
over cell maintenance functions. Within the virus, a trimeric
polymerase gene complex is bound with the nucleoprotein
attached to each genome segment. After virus entry into the cell
and release from the endosome, the viral ribonucleoproteins
and template RNA are transported to the nucleus, where viral
mRNA is transcribed by the polymerases (RNA-dependent
RNA polymerase). PB1 is the catalyst, and PB2 removes 50
caps from host pre-mRNA molecules (cap-snatching), assisted
by the endonuclease PA, for use as primers for viral mRNA
production. These viral mRNAs are moved to the cytoplasm for
translation of viral proteins and for additional rounds of tran-
scription.6,12 It is to the advantage of the virus to maintain the
function of cellular mRNA synthesis. The polymerases control
the steps of production of viral components, and increased rates
of activity could result in increased viral replication.6
Reverse genetics techniques have been used extensively to
study the individual polymerases and to compare the virulence
of different gene reassortmentsy and point mutations within spe-
cific polymerases.12,83,177,178 All 3 polymerases have some effect
on virulence in various studies, including PA47,133,178 and PB1,133
but PB2 protein seems to most significantly alter pathogeni-
city.88,133,177 The amino acid at position 627 of the PB2 protein
influences virulence and host preference. A glutamic acid at this
site (627E) favors replication in avian hosts, possibly through
y
References 6, 17, 45, 47, 84, 88, 123, 133, 143.
Veterinary Pathology 51(2)
temperature sensitivity (ie, increased replication rates at the war-
mer body temperatures of birds). Lysine at this site (627K) favors
adaptation to and increased virulence in mammalian hosts, which
have cooler temperatures in the upper respiratory tract.90 Another
mutation in PB2 that favors mammalian host infection is D701N
(aspartic acid to asparagine at position 701), as manifested by
increased virus replication.30 In larger studies using single and
multiple gene reassortants, it became obvious that interaction
between the polymerases was as important as the individual poly-
merases. Compatibility of the polymerases with each other as well
as with the other viral proteins appeared to be critical.17,88,133
Surprisingly, the pandemic 2009 H1N1 virus, which was
more virulent than most seasonal influenza viruses for humans
and in experimental mammalian hosts, did not have the reputed
virulence marker mutations in its PB2 protein.45 When the
molecular mutations in PB2 suggested to be virulence markers
for avian viruses were introduced individually into the avian-
origin PB2 of the pandemic 2009 H1N1 virus, virulence was
not altered.45 In summary, the polymerases appear to influence
virulence through control of protein synthesis processes that
favor virus production over cell maintenance functions, but
sequence variations between polymerases will likely preclude
identification of definitive virulence markers for all viruses.
PB1-F2
The PB1-F2 protein is a nonstructural protein that is expressed
during infection as an alternative open reading frame of the
PB1 gene.72,130,151 Within host cells, the protein associates most
extensively with mitochondria but has also been detected in both
cytoplasm and nucleus.18,94 The results of multiple in vitro and
in vivo studies in various cell lines and experimental animals
suggest the protein contributes to virulence through several func-
tions: (1) apoptosis induction, primarily in cells of the innate
immune system; (2) suppression of early interferon response
by infected cells; (3) increased viral replication rates or delayed
viral clearance; and (4) increased inflammation in host tissues.
Nearly all of these functions require cooperative interaction of
the PB1-F2 with other cellular or virus-induced proteins.41,80,169
Some effects of the protein involve dampening of the host
response to infection. Localization of the protein to mitochondria
in macrophages results in mitochondrial membrane depolariza-
tion and permeabilization, initiating death of these cells via the
intrinsic apoptosis pathway.18,172 Studies also have demonstrated
suppression of interferon induction by PB1-F2 in both epithelial
and immune cells in the in vitro and in vivo models.22,151
Other studies have focused on the effect of the protein on
virus multiplication and host tissue response. Co-localization
of the protein with PB1 in the nucleus leads to increased poly-
merase activity and likely consequent increased viral replica-
tion.94 In a mouse model, insertion of PB1-F2 from the 1918
H1N1 pandemic virus into a laboratory-adapted virus resulted
in a higher viral production rate per cell and a higher infected
cell rate.130 Contrarily, in another mouse model study, virus
replication rates were not increased, but virus clearance was
delayed.173 Studies in mice have associated PB1-F2 with
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increased cytokine concentrations in BALF, infiltration of
alveolar walls and lumens with neutrophils and macrophages,
and deposition of fibrin and necrotic cell debris in alveoli.95
Although the PB1-F2 protein appears to contribute to viru-
lence in many studies, the length (full vs truncated) of the pro-
tein and association with virulence are not consistent for all
influenza viruses.44,96,121 The protein is expressed at full length
(87–90 aa) by most avian influenza viruses, including H5N1
HPAI viruses, and by the 1918 (H1N1), 1957 (H2N2), and
1968 (H3N2) human pandemic viruses, but in truncated form
in many viruses from mammalian hosts, including the pan-
demic 2009 H1N1 virus. Exceptions to these generalizations
abound.72,143,174 Insertion of genes for the full-length protein
into viruses with truncated forms does not necessarily enhance
virulence, calling into question the protein’s role as a virulence
factor. Similarly in swine, most but not all swine triple reassor-
tant H3N2 viruses express full-length PB1-F2 proteins, but in
vitro and in vivo studies have not consistently associated the
presence of these proteins with increased virulence.114,115
NS1
The NS1 protein is not a structural part of the virion and is pres-
ent only within the cytosol of infected cells. The protein contri-
butes to virulence by interference with the host antiviral
response, including IFN-a/b production and expression of other
cytokines such as TNF-a and IL-6.6,132 Both proapoptotic and
antiapoptotic activities have been documented for NS1.46,74,75
Inhibiting apoptosis of cells that produce viral progeny and pro-
moting apoptosis of cells of the innate immune system that
attack infected cells would be beneficial for the virus.152
In conclusion, influenza viruses evaluated under a variety of
experimental protocols do appear to express a range of virulence
or pathogenic capabilities. Identification of specific virulence
determinants has proven to be difficult because of polygenic
contributions to pathogenicity. The polymerase proteins effect
alterations in virulence through misdirection of host cell protein
synthetic processes to viral protein production. Viral proteins
NS1 and PB1-F2 contribute to virulence through inhibition in
innate immune responses of the host. The roles of HA and NA
proteins as virulence attributes in the infection of mammalian
species are less clear, but balanced function of these 2 proteins
is necessary for efficient virus multiplication. Thus, not all
HA-NA pairings that emerge from reassortment will likely result
in viruses of equivalent virulence.
Cross-Species Infections
Although only tangentially related to pathogenesis of infection
or virulence, infection of swine with viruses originating from
other species has been a major factor in the evolution of ende-
mic strains within swine populations. In any discussion of
influenza in swine, the question of what to expect from
cross-species infection receives consideration.
Wild waterfowl serve as the reservoir of nearly all known
subtypes of influenza A viruses (16 HA, 9 NA), and the few
417
subtypes that have successfully adapted to mammalian host
species originated from this virus gene pool.163 Recently, an
influenza virus expressing a novel 17thHA was detected in
bats.141 The other genes in this virus also were widely diver-
gent from other type A viruses. Until this discovery, all influ-
enza viruses endemic in mammalian host species, if traced to
their source, appeared to be the result of crossover by avian
viruses. The genes in influenza viruses that currently circulate
in swine populations originated from avian viruses that directly
infected pigs or that adapted first to humans before contributing
genes to swine viruses.39,77,87 Domestic avian species are
infected by a larger subset of the subtypes that infect wild birds,
but routine infection of humans and swine is limited to only a
few subtypes. Subtypes H1N1, H3N2, and H1N2 routinely
infect pigs. Humans are infected with these same subtypes but
historically also were infected with an H2 subtype, the 1957
H2N2 pandemic virus (Asian flu). No infection of swine with
an H2 subtype virus had been documented until the report of
the one localized H2N3 outbreak in the Midwestern United
States in 2006.87
A point of frequent discussion is whether swine must serve
as the intermediate host for infection of humans with avian
viruses, particularly the pandemic viruses, a question that
goes back to the origin of the classic swine influenza virus and
the 1918 Spanish flu pandemic. Early studies that reported
experimental infection of pigs with nearly all avian HA sub-
types60 and identified receptors for both avian and human
viruses in the trachea of swine led to speculation that pigs
likely serve as the ‘‘mixing vessel’’ from which pandemic
human influenza viruses arise.50 However, there is no evi-
dence that pigs were previously infected with the subtypes
responsible for the 1918, 1957, and 1968 pandemics but rather
that, with the exception of the 1918 virus, the pandemic
viruses were reassortants of avian viruses and the preexisting
viruses circulating in human populations. Recent receptor
studies on the respiratory tracts of humans and swine have
revealed very similar distribution patterns, suggesting that
avian viruses could just as readily infect the human respira-
tory tract as the swine respiratory tract, negating the need of
the pig as an intermediate host.142,147
Avian and human viruses do not readily infect swine,77 but
infection of pigs with intact avian and human viruses has been
reported.77,85 The presence of avian- and human-origin genes
in the triple reassortants that comprise most of the influenza
viruses currently circulating in swine also provides evidence
for such infections.73,157,171 These triple reassortant viruses
with the cassette of internal protein genes from human-,
avian-, and swine-origin viruses appear to have an increased
capability of adding genes from viruses currently circulating
in human populations. Since 1998, when the triple reassortants
were first identified, the incidence of new reassortant viruses that
contain genes from human viruses appearing in swine populations
has increased. These new reassortant viruses cause significant
problems for the swine industry by becoming endemic, adding
to the antigenic diversity of influenza viruses against which pigs
need protection.103
418
Infection of humans with swine viruses also has been doc-
umented,z including more than 70 cases since 1958, with 41
cases in the United States alone from 1990 to 2009. Viruses that
apparently reassorted in pigs and infected humans have been
documented.13 The pandemic 2009 H1N1 virus, the initial
source of which is still unknown, is the most recent well-
known example.37 Interestingly, in different locations in
the world, this virus first caused widespread clinical disease in
the human population and only subsequently was detected in
swine populations (in which it promptly reassorted with endemic
swine viruses154). This pandemic virus highlights the possibility
of ‘‘reverse zoonosis’’ that exists where humans and swine
closely interact.125 Serologic surveys have been conducted that
suggest people who work with swine are more frequently
infected with swine viruses than people who do not.40,108 Results
of these surveys should be interpreted with caution because of
possible partial cross-reactivity in the serologic assays between
human and swine viruses of the same subtype. Thus, the inci-
dence of human infection with swine viruses is unknown.
Studies have been conducted to determine the factors that
favor movement of intact virus across species and reassort-
ment.77,87 Two factors influencing cross-species infection have
received the most focus in swine studies: (1) the relationship
between viral HA and the host cell receptors to which the
HA attaches and (2) the polymerase gene complex, the viral
proteins responsible for usurping cellular processes for virus
replication.
Hemagglutinin and Host Cell Receptors
The presence or absence of specific cell receptors dictates which
hosts can be successfully infected by a given virus, and the dis-
tribution of those receptors in the respiratory tract may influence
extent of infection and thus secondarily the severity of disease.
Gene reassortment that results in emergence of a virus with a
new combination of genes can occur only in a host species with
receptors for the viruses that contributed genes to the new virus.
The molecular structure of the HA selects the type of cells
within the host that are infected and thus strongly influences the
host range. The HA occurs as a trimer molecule projecting
from the surface of the influenza virion with a receptor pocket
at the distal end that binds to sialic acids linked in either a2,3 or
a2,6 conformation to the terminal galactose of oligosaccharide
chains on glycoproteins or glycolipids that project from the
host cell surface.112 Avian viruses attach preferentially to
a2,3 sialic acids, whereas mammalian-adapted viruses prefer
to attach to a2,6 sialic acids.
The relative distribution of these receptors in avian and
mammalian tissues and the binding of viruses recovered from
different hosts to these receptors have been studied by a variety
of techniques: lectin immunohistochemistry on frozen or
formalin-fixed tissue sections, ex vivo biopsy tissues, live
cells derived from respiratory tracts or respiratory cell
z
References 1, 63, 71, 77, 100, 127, 159, 167.
Veterinary Pathology 51(2)
lines,48,50,92,105,128,140 and fluorescein isothiocyanate–labeled
inactivated viruses on sections of formalin-fixed, paraffin-
embedded tissues.26,150 Information derived through use of the
various methods has tended to be in agreement, but conflicting
or confusing results are sometimes reported, and initial ‘‘facts’’
may actually be more complicated.91,106 Early studies tried to
define the association of receptors with specific cell types
(ciliated, nonciliated, goblet, type I pneumocyte, type II pneu-
mocyte), with inconsistent results. Later studies revealed that
viruses infect more than one type of cell, that some cells
express more than one type of receptor,48,68,140 and that after
virus entry, cells may cease to express receptors.142 In the in
vitro studies, removal of sialic acid residues with neuramini-
dase did not prevent infection,38,43,140 and virus infection of
cells with few or no recognized receptors has been reported.106
In 2 studies, virus attachment did not always correlate with effi-
cient virus replication.20,119 These studies may suggest a core-
ceptor is necessary for virus entry, or at least caution is
warranted against overinterpretation of binding patterns to sia-
lic acids.35 Studies that include discussion of problems with the
use of lectins48 and some of the discrepancies in these stud-
ies106 have been published.
Results of more recent studies suggest that other factors
beyond the type of sialic acid linkage also influence attach-
ment: conformation of the glycan, the structure and length of
the oligosaccharide chain underlying the sialic acid, the density
of receptors on the surface of the cell, the avidity of the binding
between HA and receptor, the balance between HA binding
avidity and neuraminidase activity, and temperature sensitiv-
ity.49,161 Mutations at HA amino acids 226, 228, and 190 influ-
ence host receptor specificity.104
Several recent studies have provided excellent detail on re-
ceptor distribution in the pig respiratory tract.102,147,150 Although
early studies identified both a2,3 and a2,6 receptors on tracheal
epithelial cells, setting the stage for establishing the pig’s reputa-
tion as ‘‘a mixing vessel,’’ the more recent studies indicate that
very few swine tracheal cells express a2,3 receptors. Receptor
distribution in human and swine respiratory tracts is very similar,
suggesting that swine are no more likely than humans to be suc-
cessfully infected by avian viruses and thus are not a necessary
intermediate host for avian-to-human infections. The ferret
respiratory tract is also very similar to that of human and swine,
supporting the use of this species as a laboratory model. Contra-
rily, receptor distribution in the mouse, which is not a natural host
for influenza virus, is quite dissimilar. Very few a2,6 receptors
are present at any level of the mouse respiratory tract.
In the pig, epithelial cells lining the nasal passages, trachea,
and bronchi almost exclusively express a2,6 receptors; only a
very few scattered cells with a2,3 receptors were identified
in the upper airways in these studies. At the level of the termi-
nal bronchioles, the number of cells expressing a2,3 receptors
begins to increase. At the level of respiratory bronchioles and
alveoli, both a2,6 and a2,3 receptors can be identified,
although the percentages of each and the types of cells expres-
sing these receptors vary with the study. In summary, a2,6
receptors are present in abundance throughout the porcine
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respiratory tract, but a2,3 receptors are limited to the small
bronchioles and alveoli and are of similar or lesser abundance
than a2,6 receptors in this portion of the tract. Interestingly,
many cells other than the airway epithelial cells also appear
to express sialic acid receptors, including peribronchial muco-
sal gland epithelial cells, subepithelial connective tissue cells,
and endothelial cells.170
The placement of these receptors in various portions of the
respiratory tract (and other tissues) dictates to a large degree the
distribution of infection and severity of disease in the individ-
ual animal of a given host as well as the likelihood of transmis-
sion between individuals of the same species and between
species. Thus, a preponderance of receptors for a virus in the
smaller bronchioles and alveoli will predispose a host to devel-
oping severe disease (pneumonia) rather than bronchitis, but
transmission of virus between individuals may be limited. Con-
versely, if most receptors for a particular virus are in the upper
respiratory tract and larger airways, virus shedding and thus
transmission between individuals may be facilitated, but the
disease induced will be relatively milder (bronchitis).
Polymerases and Other Viral Proteins
After hemagglutinins and receptors, the polymerases have
received the most study directed toward defining determinants
of host range. These studies have been conducted in the in vitro
systems and in various experimental hosts (mice, primates, fer-
rets) but infrequently in pigs. As with much of the basic
research on virulence factors, inconsistent results in studies
on host range conducted in different experimental hosts pre-
cludes definitive extrapolation of observations to other host
species until supported by additional evidence.
The techniques that employ viruses generated by reverse
genetics to investigate virulence also have been used to provide
information about host range, and results should be interpreted
with caution. A gene substitution or mutation in a given virus
that results in reduced replication in the same host or in vitro sys-
tem could be interpreted to demonstrate attenuation of the viru-
lence capability of that virus. However, a similar gene change (in
the same virus or in another virus) that results in reduced repli-
cation when inoculated into a different host could be due to
attenuation of the virulence of the virus or be a reflection of host
range, that is, a lesser ability to replicate in the second host,
regardless of the gene change. Particularly in the investigation
of the effect of a specific gene change in additional viruses or
in other hosts, sufficient control groups must be included to sup-
port interpretation of results and conclusions. Herein lies the
problem with studies seeking to clarify viral virulence factors
that affect severity of disease in humans since inoculation of
humans with potentially virulent viruses is not morally or ethi-
cally justifiable. The use of cell culture systems derived from
humans is as close to the actual host as such studies can get even
though results may not accurately reflect human infection. Like-
wise, results of studies on swine viruses that are conducted in
mice or other laboratory animals or in nonswine cell culture sys-
tems may not reflect what happens in the pig.
419
In investigation of host range, reduced virus replication
in a second host may not necessarily be the result of dif-
ferences in cell tropism but could involve differing ability
to efficiently use the protein synthetic machinery or an
incompatibility with other viral proteins. As an example,
in a study that compared cell tropism, replication, and
cytokine induction by HPAI H5N1, pandemic 2009 H1N1,
human seasonal H1N1 and H3N2, LPAI H12N5, and classic
swine H1N1 viruses in human lung explants, all viruses
replicated only in type II pneumocytes, but only the HPAI
H5N1 and seasonal viruses replicated to high titer. The
avian viruses (HPAI and LPAI) stimulated high cytokine
responses, whereas the seasonal viruses and the pandemic
2009 H1N1 stimulated only weak responses. The classic
swine virus replicated poorly and stimulated only a weak
cytokine response.165
Evidence is strongest for a major role of the PB2 polymerase
protein in host range.129 One of the most consistent results of
studies indicates a glutamic acid (E) at amino acid 627 favors
infectivity of avian species, whereas lysine at this position
(627K) favors infectivity of mammalian species.129 Passage
of avian viruses in mammalian hosts will result in selection
of E627K mutants. Another potential determinant of host range
occurs at amino acid 701 of PB2, with aspartic acid favoring
avian hosts and asparagine favoring mammalian hosts. In other
studies, most of the viral proteins have been shown to have
some influence on host range.16,58,77,97,104
Diagnosis of Influenza Virus Infections in
Swine
Methods of diagnosis of influenza virus infections have
improved over the years with change being driven by both the
availability of new technologies and the necessity of keeping
up with changes in the virus. A recent well-referenced review
article describes the various tests used in the diagnosis of influ-
enza virus infection in swine and addresses attributes and ratio-
nale behind influenza surveillance systems.25
Pig Selection
Diagnosis of influenza virus infection in swine is most effec-
tively accomplished by sampling multiple pigs rather than
individual animals. This approach may be critical because,
within a group of affected pigs at any given point in time, not
all pigs will be at the same point in the course of infection.
Sampling multiple animals increases the chances of catching
some pigs at the peak of virus shedding or with maximal viral
titers in lung tissues. Given the short course of infection by
this virus, selection of the right pig for individual sampling
cannot be emphasized enough. Such pigs have high fevers
(105 F) and clear nasal discharge and exhibit lethargy, reluc-
tance to move, and dyspnea or hyperpnea. Although cough is
a clinical hallmark of influenza virus infection, pigs at this
stage of disease will already have passed the point of highest
virus load in the lungs.
420
Samples of Choice
Although samples to be used for diagnostic efforts may involve
collection from individual animals, these samples often are
analyzed in pools. Antemortem sampling involves collection
of nasal secretions on synthetic swabs and suspension of the
fluid in saline or similar physiologic solution. Postmortem sam-
ples include collection of bronchial swabs or bronchoalveolar
lavage fluid flushed from the intact respiratory tract and fresh
and formalin-fixed portions of lung with characteristic lesions.
Histopathology
Although a variety of assays are available, histopathologic
examination of lung tissue remains a valuable tool in the diagno-
sis of influenza infection in swine: (1) the acute lesions are
nearly pathognomonic for this virus, and (2) lesions induced
by the virus will remain after the virus is gone, although resol-
ving lesions become less definitive. No other commonly encoun-
tered respiratory pathogen in swine induces widespread
necrotizing bronchitis and bronchiolitis. Even severe purulent
bronchopneumonia will not cause similar damage to the airways.
Severe porcine circovirus type 2 (PCV2) infections may
result in necrosis and fibrosis of medium to small airways that
can cause confusion with influenza infection; however, other
features of that virus infection differ. In PCV2-infected lungs,
the damage to airways may be ‘‘one-sided’’ and not involve the
entire circumference of the cross section, and the fibrosis may
extend irregularly into the surrounding interstitium. Peri-
bronchiolar inflammation in PCV2 infection also tends to be
more histiocytic than lymphocytic.
Resolving influenza infections have lesions that resemble
Mycoplasma hyopneumoniae infection, and additional testing
may be required to differentiate these possibilities. Airways are
lined by tightly packed, somewhat irregular, hyperplastic
columnar epithelial cells and surrounded by prominent lym-
phocytic cuffs. A subtle difference sometimes evident is early
nodular hyperplasia of bronchial-associated lymphoid tissue
associated with the medium to larger airways (in addition to the
lymphocytic cuffing) in the M. hyopneumoniae–infected lungs,
whereas the lymphocytic infiltration is relatively evenly dis-
tributed around airways in resolving influenza infections.
Methods to Detect Influenza Virus
The direct or indirect fluorescent antibody test using hyperim-
mune serum (polyclonal antibody) to detect virus antigen in fro-
zen lung sections was the primary assay employed in veterinary
diagnostic laboratories for many years. The staining techniques
could also be used on cell culture monolayers used for virus
isolation.
Immunohistochemistry applied to sections of formalin-fixed
tissue is now used more frequently to detect virus in tissue sec-
tions. The preferred antibody is a monoclonal antibody against
the conserved type A nucleoprotein because it will detect influ-
enza virus of all subtypes.160 Virus antigen is visualized in both
Veterinary Pathology 51(2)
cytoplasm and nucleus, and in many sections, the nuclear stain-
ing predominates. This probably reflects the fact that during viral
morphogenesis, nucleoprotein is formed within the nucleus.55
In situ hybridization methods applied to tissue sections also
have been described.57 This technique is not routinely used in
US veterinary diagnostic laboratories.
Commercially available antigen-detection enzyme-linked
immunosorbent assay (ELISA) kits developed for diagnosis
of human and avian influenza virus infections are used in some
laboratories to detect the virus in nasal or bronchial swabs or
bronchoalveolar lavage fluids. These tests can be used to detect
endemic swine influenza viruses because the antibodies in the
assays are directed against conserved type A antigens in matrix
proteins or nucleoproteins.
Virus isolation can be conducted in embryonated chicken
eggs or in a number of established cell lines. Before the develop-
ment of polymerase chain reaction (PCR) methodologies, virus
growth in allantoic fluids was monitored by HA tests. Currently,
PCR techniques are used to detect and characterize influenza
viruses isolated in eggs or cell cultures. The isolation procedures
are more frequently used for recovery of virus for autogenous
vaccine production or for amplification of virus titer to facilitate
subtype determination than for routine diagnosis.
PCR techniques are currently used routinely for diagnosis
because large numbers of samples can be handled efficiently
and because the method is least affected by sample quality.
Either in separate assays or as a multiplex PCR, genes for the
conserved nucleoproteins or matrix proteins are detected as a
screening test followed by assays for the HA and NA subtypes.
Serology
The hemagglutination inhibition (HI) test has served as the
standard serologic assay for the detection of antibody against
influenza viruses in swine. ELISAs have been developed, some
of which are available as commercial kits, to remove the sub-
jectivity of the HI test and to facilitate testing of large numbers
of samples. However, because of the variety of reassortant
influenza viruses circulating in swine populations and the anti-
genic variation within subtypes, and because of the widespread
use of commercial and autogenous vaccines, diagnosis of influ-
enza infection through the use of serologic assays has become
much more difficult. The ultimate result is a much reduced
ability to detect antibody against many reassortants.
To circumvent this problem due to antigenic variability, a
blocking ELISA has been developed that will detect antibody
against the nucleoprotein (NP), a highly conserved internal pro-
tein in all type A influenza viruses. If results of the blocking
ELISA suggest recent influenza infection, additional HI serologic
assays can be conducted with a panel of standard virus strains. An
ELISA also has been developed that will detect antibody against
the NS1 protein. This protein is not present in intact virions in
killed vaccines but is produced only in infected cells. Thus, anti-
body against NS1 will be stimulated in infected pigs but not in
vaccinated pigs, an example of a naturally occurring DIVA (Dif-
ferentiating Infected from Vaccinated Animals) vaccine.
Janke 421

More Than Detection—Virus Characterization large percentage of the pigs in a pen will take a turn at the rope
(S. T. Millman, personal communication, 2012). After approx-
Because of the propensity of influenza viruses for reassortment,
imately 10 to 20 minutes, the rope can be removed and the sal-
mere detection of influenza virus may not provide sufficient
iva wrung out into a container. Contaminating feed and fecal
information for full evaluation of the field situation and manage-
debris are removed by centrifugation and the supernatant pro-
ment decisions. Determination of H and N subtypes is routine in
cessed for virus detection.
many veterinary diagnostic laboratories, but a subtype descrip-
Virus is detected by PCR techniques, although determi-
tion by itself provides little information about the origin of the
nation of subtype may be less successful in some samples.
genes that make up an influenza virus or the possible source
Virus isolation has proven to be more difficult. Substances
of infection. Determining the full or partial nucleic acid
are present in oral fluids that may interfere with detection
sequence of the HA1 portion of the hemagglutinin protein allows
by PCR, neutralize live viruses, or are toxic to cell lines.
preparation of dendograms, which compare the current virus iso-
Studies are being conducted to remove or counteract these
late with well-characterized viruses endemic in swine popula-
substances and improve the efficiency of assays performed
tions, as well as prior isolates from the same operation. If this
on oral fluids.
historic information includes prior isolates from the same site
Oral fluids are not currently used for influenza serology, but
or production unit as well as different segments of the pig flow
testing will likely be available soon. Research studies indicate
in the same operation, this information may suggest whether the
that the commercial avian influenza multispecies ELISA
currently detected infection is due to reappearance of the same
(FlockChek; IDEXX Laboratories, Westbrook, ME) will detect
virus from that site or elsewhere in the operation, or the introduc-
antibody against a variety of influenza viruses infecting pigs,
tion of a new virus from the outside. The first possibilities may
including the 2009 pandemic H1N1.21,110 Antibody can be
suggest a need for changes in management and biosecurity mea-
detected in saliva as early as 5 days PI. Antibody induced by
sures within the operation. The latter possibility may be even
vaccination also can be detected. Because antibody titers in
more critical if pigs will now be exposed to an antigenically dif-
oral secretions are not as high as in serum, some alterations
ferent virus and additional reassortant viruses appear.
in methodology may be necessary for optimum results.
Although methodologies exist for characterization/compar-
Swine influenza has been a recognized respiratory disease in
ison of all genes, characterization efforts on routine cases usu-
pigs for more than 90 years, and over those years, the etiology
ally are limited to the HA gene. An exception would be the
and pathogenesis of infection have been determined and step-
development of PCR assays for the M gene that were used for
wise facilitated by advances in research technologies that allow
surveillance purposes to identify the potential entrance of the
us to probe further into the details of infection. The virus has
2009 H1N1 virus into swine populations.140 Research studies
continued to change, most significantly by the introduction of
in many veterinary diagnostic laboratories focus on character-
genes from influenza viruses from other hosts, to the extent that
ization of other genes in field isolates with the objective of
‘‘influenza A virus infection of swine’’ better describes this dis-
identifying new reassortants or reassortment trends.
ease than ‘‘swine influenza.’’ These changes in the virus, as
well as significant changes in swine production operations,
have compelled veterinary diagnostic laboratories to develop
Oral Fluids improved and more flexible diagnostic methods.
A relatively new approach to diagnosis involves collection of
oral fluids from groups or pens of pigs to detect virus shedding
Declaration of Conflicting Interests
in oral secretions, a method particularly valuable in detecting a
virus with such a short duration of infection in the individual The author(s) declared no potential conflicts of interest with respect to
the research, authorship, and/or publication of this article.
pig.27,110,117,120 More pigs can be sampled easily and with less
expense, without the need to identify individual pigs early in
infection. This method has a very high probability of detecting Funding
virus when only 10% to 20% of the pigs in the group are shed- The author(s) received no financial support for the research, author-
ding virus, and thus this approach can be used to suggest the ship, and/or publication of this article.
cause of current illness. Alternatively, through repeated sam-
pling of a single group of pigs as they move through various
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