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Influenza A Viruses Grow in Human Pancreatic Cells and Cause
Pancreatitis and Diabetes in an Animal Model
Ilaria Capua,
a
Alessia Mercalli,
b
Matteo S. Pizzuto,
a
Aurora Romero-Tejeda,
a
Samantha Kasloff,
a
Cristian De Battisti,
a
Francesco Bonfante,
a
Livia V. Patrono,
a
Elisa Vicenzi,
c
Valentina Zappulli,
d
Vito Lampasona,
b
Annalisa Stefani,
e
Claudio Doglioni,
f,g
Calogero Terregino,
a
Giovanni Cattoli,
a
Lorenzo Piemonti
b
Department of Comparative Biomedical Sciences, Istituto Zooprofilattico Sperimentale delle Venezie, Legnaro, Padova, Italy
a
; Diabetes Research Institute-DRI, San Raffaele
Scientific Institute, Milan, Italy
b
; Viral Pathogens and Biosafety Unit, Division of Immunology, Transplantation and Infectious Diseases, San Raffaele Scientific Institute,
Milan, Italy
c
; Department of Public Health, Comparative Pathology and Veterinary Hygiene, Faculty of Veterinary Medicine, Agripolis, Legnaro, Padova, Italy
d
; Animal
Health and Welfare Department, Istituto Zooprofilattico Sperimentale delle Venezie, Legnaro, Padova, Italy
e
; Unit of Pathology, San Raffaele Scientific Institute, Milan, Italy
f
;
Università Vita-Salute, Milan, Italy
g
Influenza A viruses commonly cause pancreatitis in naturally and experimentally infected animals. In this study, we report the
results of in vivo investigations carried out to establish whether influenza virus infection could cause metabolic disorders linked
to pancreatic infection. In addition, in vitro tests in human pancreatic islets and in human pancreatic cell lines were performed
to evaluate viral growth and cell damage. Infection of an avian model with two low-pathogenicity avian influenza isolates caused
pancreatic damage resulting in hyperlipasemia in over 50% of subjects, which evolved into hyperglycemia and subsequently dia-
betes. Histopathology of the pancreas showed signs of an acute infection resulting in severe fibrosis and disruption of the struc-
ture of the organ. Influenza virus nucleoprotein was detected by immunohistochemistry (IHC) in the acinar tissue. Human sea-
sonal H1N1 and H3N2 viruses and avian H7N1 and H7N3 influenza virus isolates were able to infect a selection of human
pancreatic cell lines. Human viruses were also shown to be able to infect human pancreatic islets. In situ hybridization assays
indicated that viral nucleoprotein could be detected in beta cells. The cytokine activation profile indicated a significant increase
of MIG/CXCL9, IP-10/CXCL10, RANTES/CCL5, MIP1b/CCL4, Groa/CXCL1, interleukin 8 (IL-8)/CXCL8, tumor necrosis factor
alpha (TNF-␣), and IL-6. Our findings indicate that influenza virus infection may play a role as a causative agent of pancreatitis
and diabetes in humans and other mammals.
I
nfluenza A viruses (IAVs) originate from the wild-bird reservoir
and infect a variety of hosts, including domestic birds. These
viruses are also able to infect a significant number of mammals, in
which they may become established. Among these are pigs,
equids, mustelids, sea mammals, canids, felids, and humans. IAVs
cause systemic or nonsystemic infection, depending on the strain
involved. The systemic disease occurs mostly in avian species and
is known as highly pathogenic avian influenza (HPAI). It is char-
acterized by extensive viral replication in vital organs and death
within a few days after the onset of clinical signs in the majority of
infected animals. The nonsystemic form, which is by far the most
common, occurs in birds and mammals and is characterized by
mild respiratory and enteric signs. To differentiate it from HPAI,
in birds it is known as low-pathogenicity avian influenza (LPAI).
The different clinical presentation results from the fact that non-
systemic influenza A viruses are able to replicate only in the pres-
ence of trypsin or trypsin-like enzymes, and thus, their replication
is believed to be restricted to the respiratory and enteric tracts.
Avian IAVs have a tropism for the pancreas (1–4). Necrotizing
pancreatitis is a common finding in wild and domestic birds in-
fected with HPAI virus (5–8), and the systemic nature of HPAI is
in line with these findings. In contrast, it is difficult to explain
pancreatic colonization by LPAI viruses, which is a common find-
ing in infected chickens and turkeys (9–14). Previous studies have
reported that certain IAVs can also cause pancreatitis in mammals
following natural or experimental infection (15–18). Recently,
there have been reports of pancreatic damage in human cases
associated with H1N1pdm influenza A virus infection, including
both acute pancreatitis and the onset of type 1 diabetes (T1D)
(19–23).
To date, there has been no attempt to establish whether influ-
enza viruses are able to grow in pancreatic cells in vitro, and no
data are available on the consequences of influenza virus replica-
tion in the pancreas in vivo. In this study, we explored the impli-
cations of influenza virus infection for pancreatic endocrine func-
tion in an animal model, and we performed in vitro experiments
aiming to establish the occurrence, extent, and implications of
influenza A virus infection in human cells of pancreatic origin. For
the in vivo studies, we selected the turkey as a model, due to the fact
that turkeys are highly susceptible to influenza virus infection and
pancreatic damage is often observed as a postmortem lesion. Ex-
perimental infections were performed with two LPAI viruses,
A/turkey/Italy/3675/1999 (H7N1) and A/turkey/Italy/2962/2003
(H7N3), as both viruses had been associated with pancreatic le-
sions in naturally infected birds. For the in vitro studies, in addi-
tion to the previously mentioned avian strains, we selected A/New
Caledonia/20/99 (H1N1) and A/Wisconsin/67/05 (H3N2), as
Received 27 March 2012 Accepted 17 October 2012
Published ahead of print 24 October 2012
Address correspondence to Ilaria Capua, icapua@izsvenezie.it.
Supplemental material for this article may be found at http://dx.doi.org
/10.1128/JVI.0714-12.
Copyright © 2013, American Society for Microbiology. All Rights Reserved.
doi:10.1128/JVI.00714-12
January 2013 Volume 87 Number 1 Journal of Virology p. 597– 610 jvi.asm.org 597
these viruses have circulated for extensive periods in humans, and
existing epidemiological data would be suitable for a retrospective
study. The strains were used to infect two established human pan-
creatic cell lines (including human insulinoma and pancreatic
duct cell lines) and primary cultures of human pancreatic islets.
MATERIALS AND METHODS
In vivo experiment. The aim of this study was to establish whether two
natural nonsystemic avian influenza viruses obtained from field out-
breaks, without prior adaptation, could cause endocrine or exocrine pan-
creatic damage following experimental infection of young turkeys. The
study was performed in strict accordance with the relevant national and
local animal welfare bodies [Convention of the European Council no. 123
and National Guidelines (Legislative Decree 116/92)]. The protocol was
approved by the Committee on the Ethics of Animal Experiments of the
Istituto Zooprofilattico Sperimentale delle Venezie (IZSVe) (permit
number CE.IZSVE.022012).
Animals. Sixty-eight female meat turkeys obtained at 1 day of age
from a commercial farm were used in this study. The birds were housed in
negative-pressure, HEPA-filtered isolation cabinets for the duration of
the experimental trial. Before carrying out the infection, the animals were
housed for 3 weeks to allow adaptation and growth, received feed and
water ad libitum, and were identified by means of wing tags.
Viruses. Two LPAI viruses isolated during epidemics in Italy were
used for the experimental infection: A/turkey/Italy/3675/1999 (H7N1)
and A/turkey/Italy/2962/2003 (H7N3). Stocks of AI viruses were pro-
duced by a single passage in 9-day-old embryonated specific-pathogen-
free (SPF) chicken eggs via the allantoic cavity, according to EU Council
Directive 2005/94/EC (24). The allantoic fluid was harvested 48 h postin-
oculation, aliquoted, and stored at ⫺80°C until use. For viral titration, 100
l of 10-fold-diluted viral suspension was inoculated in SPF embryonated
chicken eggs, and the median embryo infectious dose (EID
50
) was calcu
-
lated according to the Reed and Muench formula (54).
Experimental design. Birds were divided into three experimental
groups (A [H7N1], B [H7N3], and K [control]). Groups A and B, each
consisting of 24 animals, were infected via the oral-nasal route with 0.1 ml
of allantoic fluid containing 10
6.83
EID
50
of the A/turkey/Italy/3675/1999
(H7N1) virus and 10
6.48
EID
50
of the A/turkey/Italy/2962/2003 (H7N3)
virus. Group K, consisting of 20 animals, received 0.1 ml of negative al-
lantoic fluid via the oral-nasal route and served as a negative control. All
birds were observed twice daily for clinical signs. On days 0, 3, 6, 9, 13, 15,
20, 23, 27, 31, 34, 41, and 45 postinfection (p.i.), blood was collected from
the brachial veins of all animals using heparinized syringes in order to
determine glucose and lipase levels in plasma. On days 2 and 3 p.i., tra-
cheal swabs were collected to evaluate viral replication. On day 3 p.i.,
blood was also collected to determine the presence of viral RNA in the
blood. On days 4 and 7 p.i., two birds from each infected group were
humanely sacrificed, and the pancreas and lungs were processed for the
detection of viral RNA and for histopathology and immunohistochemis-
try (IHC). Similarly, on days 8 and 17 p.i., one subject from each experi-
mental group was euthanized, and the pancreas was collected for histo-
logical and immunohistochemical studies. For this purpose, we selected
hyperglycemic subjects that had also shown an increase in lipase levels.
Biochemical analyses. Blood samples were collected in Gas Lyte 23 G
pediatric syringes containing lyophilized lithium heparin as an anticoag-
ulant. At each sampling, 0.3 ml of blood was collected and refrigerated at
4°C until it was processed. To obtain plasma, samples were immediately
centrifuged at 1,500 ⫻ g for 15 min at 4°C. To determine the levels of
glucose and lipase in plasma, commercially available kits (Glucose HK
and LIPC; Roche Diagnostics GmbH, Mannheim, Germany) were applied
to the computerized system Cobas c501 (F. Hoffmann-La Roche Std.,
Basel, Switzerland). The Glucose HK test is based on a hexokinase enzy-
matic reaction. The linearity of the reaction is 0.11 to 41.6 mmol/liter (2 to
750 mg/dl), and its analytic sensitivity is 0.11 mmol/liter (2 mg/dl). The
LIPC test is based on a colorimetric enzymatic reaction with a linearity of
3 to 300 U/liter and an analytic sensitivity of 3 U/liter.
Molecular tests. Tracheal swabs, blood samples, and organs (pancreas
and lungs) were tested for viral RNA by means of real-time reverse trans-
criptase PCR (RRT-PCR) for the identification of the influenza virus ma-
trix (M) gene.
RNA extraction. Viral RNA was extracted from 100 l of blood using
the commercial NucleoSpin RNA II kit (Macherey-Nagel) and from 50 l
of phosphate-buffered saline (PBS) containing a tracheal-swab suspen-
sion using the Ambion MagMax-96 AI-ND Viral RNA Isolation Kit for
the automatic extractor. One hundred fifty milligrams of homogenized
lung and pancreas tissues was centrifuged, and viral RNA was extracted
from 100 l of clarified suspension using the NucleoSpin RNA II kit
(Macherey-Nagel).
One-step RRT-PCR. The isolated RNA was amplified using the pub-
lished primers and probes from Spackman et al. (25), targeting the con-
served matrix (M) gene of type A influenza virus. Five microliters of RNA
was added to the reaction mixture, composed of 300 nM forward and
reverse primers (M25F and M124-R, respectively) and 100 nM fluores-
cent-label probe (M⫹64). The amplification reaction was performed in a
final volume of 25 l using the commercial QuantiTect Multiplex RT-
PCR kit (Qiagen, Hilden, Germany). The PCR was performed using the
following protocol: 20 min at 50°C and 15 min at 95°C, followed by 40
cycles at 94°C for 45 s and 60°C for 45 s. In vitro-transcribed target RNA
was obtained using Mega Short Script 7 (high-yield transcription kit; Am-
bion) according to the manufacturer’s instructions, quantified by Nano-
Drop 2000 (Thermo Scientific), and used to create a standard calibration
curve for viral-RNA quantification. To check the integrity of the isolated
RNA, the -actin gene was also amplified using a set of primers designed
in house (primer sequences are available upon request). The reaction
mixture was composed of 300 nM forward and reverse primers and 1⫻
EvaGreen (Explera, Jesi, Italy). The amplification reaction was performed
in a final volume of 25 l using the commercial Superscript III kit (Invit-
rogen, Carlsbad, CA). The PCR was performed using the following pro-
tocol: 30 min at 55°C and 2 min at 94°C, followed by 45 cycles at 94°C for
30 s and 60°C for 1 min.
Histology and immunohistochemistry. Formalin-fixed, paraffin-
embedded pancreas sections were cut (3 m thick). Slides were stained
with hematoxylin and eosin (H&E) (Histoserv, Inc., Germantown, MD).
Representative photographs were taken with SPOT Advanced software
(version 4.0.8; Diagnostic Instruments, Inc., Sterling Heights, MI). The
reagents and methodology for influenza virus IHC were as follows: poly-
clonal antibody anti-type A influenza virus nucleoprotein (NP) and
mouse anti-influenza virus A (NP subtype A, clone EVS 238; European
Veterinary Laboratory), 1:100 in PBS-2.5% bovine serum albumin (BSA)
for1hatroom temperature; secondary antibody, goat anti-mouse IgG2a
horseradish peroxidase (HRP) (Southern Biotech), 1/200 in PBS-2.5%
BSAfor1hatroom temperature. Antigen retrieval was performed by
incubating the slides for 10 min at 37°C in trypsin (Digest-all Kit; Invit-
rogen). Endogenous peroxidase was blocked with 3% H
2
O
2
for 10 min at
room temperature. Before incubation with primary antibody, a blocking
step was performed with PBS-5% BSA for 20 min at room temperature.
Diaminobenzidine (DAB) was applied as a chromogen (DakoCytoma-
tion; code K3468). IHC for insulin and glucagon was performed with
polyclonal guinea pig anti-swine insulin, 1:50 (A0564; Dako, Carpinteria,
CA), and polyclonal rabbit anti-glucagon, 1:200 (NCL-GLUC; Novocas-
tra, Newcastle, United Kingdom), using as a detection system the En Vi-
sion Ap (K1396; Dako, Carpinteria, CA) and nuclear fast red (K139;6
Dako) for influenza virus A staining and En Vision⫹System HRP-labeled
polymer anti-rabbit (K4002; Dako, Carpinteria, CA) and DAB (K3468;
Dako, Carpinteria, CA) for insulin and glucagon staining.
In vitro experiment. The aim of these experiments was to establish
whether human and avian influenza viruses could grow on cell lines de-
rived from the human pancreas and to investigate the effect of human
influenza virus replication in human pancreatic islets.
Capua et al.
598 jvi.asm.org Journal of Virology
Cell lines. Madin-Darby canine kidney (MDCK) cells were main-
tained in Alpha’s modified Eagle medium (AMEM) (Sigma) supple-
mented with 10% fetal bovine serum (FBS), 1% 200 mM
L-glutamine, and
a 1% penicillin-streptomycin-nystatin (pen-strep-nys) solution. The hu-
man insulinoma cell line hCM (26) and immortalized human ductal ep-
ithelial cell line HPDE6 (27) were maintained in RPMI (Gibco) supple-
mented with 1%
L-glutamine, 1% antibiotics, and FBS (5% and 10%,
respectively). MDCK and HPDE6 cells were passaged twice weekly at a
subcultivation ratio of 1:10 and 1:4, while hCM cells were split three times
per week at a ratio of 1:4. All cells were maintained in a humidified incu-
bator at 37°C with 5% CO
2
.
Primary cells. Pancreatic islets were isolated and purified at San Raf-
faele Scientific Institute from pancreases of multiorgan donors according
to Ricordi’s method. Briefly, after cannulation of the pancreatic duct,
collagenase solution (2,000 U; Serva, Germany) at 4°C was injected
through the duct (perfusion). Subsequently, the pancreas was cut into
small pieces and loaded into a digestion chamber, named Ricordi’s cham-
ber, for an enzymatic and mechanical digestion at 37°C. Final purification
of digested pancreas was performed using a continuous gradient (Ficoll;
Biochrom, Berlin) in a computerized centrifuge system (COBE 2991 cell
processor). Islet preparations with purities of ⬎80% ⫾ 8% (mean ⫾
standard deviation [SD]; n ⫽ 6) not suitable for transplantation were used
after approval by the local ethical committee. Cells were seeded in 24-well
plates and 25-cm
2
flasks at 150 islets/ml and maintained in final wash
culture medium (Mediatech, Inc., Manassas, VA) at 37°C with 5% CO
2
.
Sialic acid receptor characterization on hCM and HPDE6 cells. The
presence of alpha-2,3- and alpha-2,6-linked sialic acid residues was deter-
mined by flow cytometry. Following trypsinization, 1 ⫻ 10
6
cells were
washed twice with PBS-10 mM HEPES (PBS-HEPES) for 5 min at 1,200
rpm and then treated with an avidin/biotin-blocking kit (Vector Labora-
tories) according to the manufacturer’s instructions, with cells incubated
for 15 min with 100 l of each solution. Alpha-2,3 and alpha-2,6 sialic acid
linkages were detected by incubating cells for 30 min with 100 l of bio-
tinylated Maackia amurensis (MAA) lectin II (Vector Laboratories; 5 g/
ml), followed by 100 l of phycoerythrin (PE)-streptavidin (BD Biosci-
ences; 10 g/ml) for 30 min at 4°C in the dark or with 100 lof
fluorescein-conjugated Sambucus nigra (SNA) lectin (Vector Laborato-
ries; 5 g/ml). Cells were washed twice with PBS-HEPES between all
blocking and staining steps and resuspended in PBS with 1% formalin
prior to analysis. To confirm the specificity of lectins, cells were pretreated
with 1 U per ml of neuraminidase (NA) from Clostridium perfringens
(Sigma) for 1 h prior to the avidin/biotin block. Samples were analyzed on
a BD Facscalibur or BD LSR II (BD Biosciences), and a minimum of 5,000
events were recorded.
Viruses and viral titration. Stocks of A/New Caledonia/20/99 (H1N1)
and A/Wisconsin/67/05 (H3N2) viruses were kindly provided to San Raf-
faele Hospital (HSR) by Nadia Naffakh, Pasteur Institute, CNR Virus
Influenza (Paris, France), and were serially expanded 2 times in MDCK
cells prior to use.
To propagate IAV, monolayer-cultured MDCK cells were washed
twice with PBS and infected with A/NewCaledonia/20/99 (H1N1) or
A/Wisconsin/67/05 (H3N2) at a multiplicity of infection (MOI) of 0.001.
After virus adsorption for1hat35°C, the cells were washed twice and
incubated at 35°C with Dulbecco’s modified Eagle’s medium (DMEM)
without serum supplemented with tosylsulfonyl phenylalanyl chlorom-
ethyl ketone (TPCK)-treated trypsin (1 g/ml; Worthington Biomedical
Corporation, Lakewood, NJ). Supernatants were recovered 48 h postin-
fection.
LPAI H7N1 A/turkey/Italy/3675/1999 and H7N3 A/turkey/Italy/
2962/2003 viruses isolated during epidemics in Italy were grown in 9-day-
old embryonated SPF chicken eggs as described above. The sequences for
A/New Caledonia/20/99 (H1N1), A/Wisconsin/67/05 (H3N2), and
A/turkey/Italy/3675/1999 (H7N1) isolates were previously published.
For viral titration, plaque assays were performed as previously de-
scribed (28). Briefly, MDCK monolayer cells, plated in 24-well plates at
2.5 ⫻ 10
5
cells/well, were washed twice with DMEM without serum, and
serial dilutions of virus were adsorbed onto cells for 1 h. The cells were
covered with 2⫻ minimal essential medium (MEM)-Avicel (FMC Biopo-
lymer, Philadelphia, PA) mixture supplemented with TPCK-treated tryp-
sin (1 g/ml). Crystal violet staining was performed 48 h postinfection,
and visible plaques were counted.
Virus replication kinetics in pancreatic-cell lines. Semiconfluent
monolayers of HPDE6 and hCM cells seeded on 24-well plates were
washed twice with PBS and then infected with human viruses (H1N1 and
H3N2) at an MOI of 0.001 and with avian viruses (H7N1 and H7N3) at an
M.O.I of 0.01 using 200 l of inoculum per well. The inoculum was
removed after1hofabsorption and replaced with 1 ml of serum-free
medium containing 0.05 g/ml TPCK-trypsin (Sigma). At 1, 24, 48, and
72 h postinfection, supernatants from three infected wells and one control
well were harvested, and viral titers were determined by virus isolation
using the 50% tissue culture infectious dose (TCID
50
) assay, as well as by
real-time RT-PCR detection of the matrix gene. All replication kinetics
experiments were repeated three times.
TCID
50
. Confluent monolayers of MDCK cells seeded onto 96-well
plates were washed twice in serum-free medium and inoculated with 50 l
of 10-fold serially diluted samples in serum-free MEM. After1hofad-
sorption, an additional 50 l of serum-free medium containing 2 g/ml
TPCK-trypsin was added to each well. Cytopathic-effect (CPE) scores
were determined after 3 days of incubation at 37°C by visual examination
of infected wells using a light microscope. The TCID
50
value was deter
-
mined using the method of Reed and Muench.
Growth assay in pancreatic islets. Islets were infected with H1N1 and
H3N2 influenza viruses by adding 4.8 ⫻ 10
2
or 4.8 ⫻ 10
3
PFU/well, re
-
spectively. Viral growth was performed with and without the addition of
TPCK-trypsin (Sigma) (1 g/ml). Uninfected islets were left as a negative
control. Samples were collected every 48 h from the day of infection (time
zero [t
0
]) until day 10 (t
5
, the fifth time point that occurred at 10 days
postinfection). Each sample was centrifuged at 150 ⫻ g for 5 min. The
supernatant was collected and stored at ⫺80°C for quantitative real-time
PCR, virus titration, and cytokine expression profiling. The pellet was
washed twice with PBS, stored at ⫺80°C, and subsequently processed for
real-time PCR. All pellets and supernatants were tested by real-time PCR
in triplicate.
Detection of viral RNA from pancreatic tissue. The total RNAs from
pancreatic islet pellets and supernatants were isolated using the commer-
cial NucleoSpin RNA II kit (Macherey-Nagel) according to the manufac-
turer’s instructions. The RNAs were eluted in 60 l of elution buffer and
tested using one-step RRT-PCR for the influenza virus matrix gene to
evaluate viral growth.
A quadratic regression model (C
T
[threshold cycle] ⫽
0
⫹
1
TPCK-
trypsin ⫹
2
time ⫹
3
time
2
⫹
4
time ⫻ TPCK-trypsin ⫹
5
time
2
⫻
TPCK-trypsin) for each virus and specimen was used to analyze the trend
of C
T
values over time. The influence of TPCK-trypsin presence and the
interaction between its presence and the time point were evaluated. The
regression model took into account the influence of the intragroup cor-
relation among repeated measurements for each observed time in the
confidence interval (CI) calculation. A residual postestimation analysis
was performed to verify the validity of the model.
One-step RRT-PCR. Quantitative real-time PCR targeting the con-
served M gene of type A influenza virus was applied according to the
protocol described above. To check the integrity of the isolated RNA, the
-actin gene was also amplified using the primers and probe previously
described (29). The reaction mixture consisted of 400 nM forward and
reverse primers (Primer-beta act intronic and Primer-beta act reverse,
respectively) and 200 nM fluorescent-label probe (5= Cy5 or 3= black hole
quencher 1 [BHQ1]). The amplification reaction was performed in a final
volume of 25 l using the commercial QuantiTect Multiplex RT-PCR kit
(Qiagen, Hilden, Germany). The PCR protocol was 20 min at 50°C and 15
min at 95°C, followed by 45 cycles at 94°C for 45 s and 55°C for 45 s.
Pancreatic Damage by Influenza A Virus
January 2013 Volume 87 Number 1 jvi.asm.org 599
Western blot analysis. Cellular pellets were resuspended in lysis buf-
fer (50 mM Tris-HCl, pH 8, 1.0% SDS, 350 mM NaCl, 0.25% Triton-X,
proteases inhibitor cocktail) and then mixed and incubated on ice for 30
min. The suspension was sonicated three times for 5 min each time and
then centrifuged at maximum speed for 10 min. A Bradford test was
performed in order to calculate the total protein concentration for each
sample. Based on this calculation, the same amount of protein per sample
was loaded and electrophoresed in 12% polyacrylamide gels. Following
SDS-PAGE, the proteins were transferred from the gel onto immunoblot
polyvinylidene difluoride (PVD) membranes (Bio-Rad) by electro-
blotting. The membranes were saturated overnight at 4°C in 5% grade
blocker nonfat dried milk (Bio-Rad) in PBS and then incubated for1hat
room temperature under constant shaking in PBS containing 0.05%
Tween 20 (Sigma), 5% dried milk, and mouse monoclonal influenza A
virus nucleoprotein antibody (Abcam). -Actin antibody (Abcam) was
used as a loading control. After incubation with the primary antibody, the
membranes were exposed for1htoHRP-rabbit polyclonal secondary
antibody to mouse IgG (Abcam), followed by visualization of positive
bands by enhanced chemiluminescence (ECL) using Hyperfilm ECL
(Amersham Biosciences).
Visualization of viral growth in pancreatic cell lines. HPDE6 and
hCM cells were grown on slides to 80% confluence and infected with
either H1N1or H3N2 virus at an MOI of 0.1 with 0.05 mg/ml of TPCK-
trypsin. The cells were fixed and permeabilized at 0, 24, 48, and 72 h p.i.
with chilled acetone (80%). After blocking with PBS containing 1% BSA,
the cells were incubated for1hat37°C in a humidified chamber with
mouse monoclonal antibody to fluorescein isothiocyanate (FITC)-conju-
gated influenza A virus nucleoprotein (Abcam) in PBS containing 1%
BSA and 0.2% Evan’s Blue. The staining solution was decanted, and the
cells were washed three times. Nuclei of negative-control cells were
stained with DAPI (4=,6-diamidino-2-phenylindole) (Sigma) and then
washed with PBS and observed under UV light.
In situ visualization of viral RNA in pancreatic islets. To visualize
viral RNA localized within cells, purified human pancreatic islets were
harvested at 2, 5, and 7 days postinfection with human influenza viruses.
The islets were then incubated for 24 h in methanol-free 10% formalin,
deposited at the bottom of flat-bottom tubes, embedded in agar to immo-
bilize them, dehydrated, and finally embedded in paraffin. Islet samples
were sectioned at 4 m. For colocalization experiments, islets were enzy-
matically digested into single cells with a trypsin-like enzyme (12605-01;
TrypLE Express; Invitrogen, Carlsbad, CA) and cytocentrifuged onto
glass slides. In situ hybridization was performed using the Quantigene
ViewRNA technique, based on multiple oligonucleotide probes and
branched-DNA signal amplification technology, according to the manu-
facturer’s instructions (Affymetrix, Santa Clara, CA). The probe set used
was designed to hybridize the H1N1/A/New Caledonia/20/99 virus (Gen-
Bank sequence DQ508858.1). Due to sequence homology in the region
covered by the probes, the same set also recognized the H3N2 virus RNA,
as confirmed in pilot experiments. To identify cell types within islets, the
following Quantigene probes were used: insulin for beta cells (INS gene;
NCBI reference sequence NM_000207), alpha-amylase 1 for exocrine
cells (AMY1A gene; NCBI reference sequence NM_004038), and cytoker-
atin 19 for duct cells (KRT19 gene; NCBI reference sequence NM_002276).
Quantification of cells positive for each probe was performed within 8 ran-
domly chosen fields using the IN Cell Investigator software (GE Healthcare
United Kingdom Ltd.).
Determination of insulin secretion in infected islets. Aliquots of 100
islet equivalents (uninfected or infected with H1N1/A/New Caledonia/
20/99 and H3N2/A/Wisconsin/67/05) per column were loaded onto Sep-
hadex G-10 columns with medium at low glucose concentration (2 mM)
and preincubated at 37°C for 1 h. After preincubation, the islets were
exposed to sequential 1-h incubations at low (2 mM) and high (20 mM)
glucose concentrations. Supernatants were collected with protease inhib-
itor cocktail (Roche Biochemicals, Indianapolis, IN) and stored at ⫺80°C
at the end of each incubation. The insulin content was determined with an
insulin enzyme-linked immunoassay kit (Mercodia AB, Uppsala, Swe-
den) following manufacturer’s instructions. Insulin secretion indices
were calculated as the ratio between the insulin concentration at the end of
high-glucose incubation and the insulin concentration at the end of low-
glucose incubation.
Cytokine expression profile. The capability of H1N1 and H3N2 vi-
ruses to induce cytokine expression in human pancreatic islets was mea-
sured using multiplex bead-based assays based on xMAP technology (Bio-
Plex; Bio-Rad Laboratories, Hercules, CA). The parallel wells of
pancreatic islets were infected with viruses or were mock infected. The
culture medium supernatant was collected before infection and 2, 4, 6, 8,
and 10 days postinfection and assayed for 48 cytokines. Selected cytokines,
the limits of detection, and the coefficients of variability (intra-assay per-
cent coefficient of variation [%CV] and interassay %CV) of the cytokine/
chemokine are reported in Table S5 in the supplemental material.
Evaluation of cell death following infection (LIVE/DEAD assay).
The viability of islet cells after infection with human viruses was measured
using the LIVE/DEAD cell assay kit (L-3224; Molecular Probes, Inc., Le-
iden, The Netherlands). The assay is based on the simultaneous determi-
nation of live and dead cells with two fluorescent probes. Live cells are
stained green by calcein due to their esterase activity, and nuclei of dead
cells are stained red by ethidium homodimer 1. Islets harvested after 5
days of culture were further enzymatically digested into single cells with
trypsin-like enzyme (12605-01; TrypLE Express; Invitrogen, Carlsbad,
CA). According to the manufacturer’s instructions, single cells were incu-
bated with the labeling solution for 30 min at room temperature in the
dark, cytocentrifuged onto glass slides, and assessed with a Carl Zeiss
Axiovert 135TV fluorescence microscope. Analysis of dead cells was per-
formed on cytospin preparations using IN Cell Investigator software (GE
Healthcare United Kingdom Ltd.). Positive cells in each category were
quantified with 10 systematically random fields.
Statistical analysis. Data were generally expressed as mean ⫾ stan-
dard deviation or median (minimum-maximum). Differences between
parameters were evaluated using Student’s t test when parameters were
normally distributed and a Mann-Whitney U test when parameters were
not normally distributed. Kaplan-Meier and/or Cox regression analysis
was used to analyze the incidence of events during the time. A P value of
less than 0.05 was considered an indicator of statistical significance. Anal-
ysis of data was done using the SPSS statistical package for Windows (SPSS
Inc., Chicago, IL).
Nucleotide sequence accession numbers. The sequences of all eight
influenza virus genome segments belonging to H7N3 A/turkey/Italy/
2962/2003 were submitted to GenBank and assigned accession numbers
JX515660, JX515661, JX515662 , JX515663, JX515664, JX515665,
JX515666, and DQ090062.
RESULTS
In vivo experiment. (i) Clinical disease. Turkeys from both
H7N1-challenged (A) and H7N3-challenged (B) groups showed
clinical signs typical of LPAI infection, such as conjunctivitis, si-
nusitis, diarrhea, ruffled feathers, and depression on day 2 p.i.
Mild symptoms regressed by day 20 p.i. Only two subjects from
group A showed sinusitis until day 30 p.i. Mortality rates were low
in both groups: one subject in group A died on day 8 p.i., and one
subject in group B died on day 19 p.i.
(ii) Detection of viral RNA. Viral RNA was detected from the
tracheal swabs collected from 17/20 birds infected with H7N1 vi-
rus and 19/20 birds infected with H7N3 virus on day 2 p.i. and in
all animals on day 3 p.i. Viral RNA was also detected from the
blood of two birds of group A H7N1 and four birds of group B
H7N3 on day 3 p.i. and from the pancreases and lungs collected on
days 4 and 7 p.i. (see Table S4 in the supplemental material). No
viral RNA was detected from the uninfected controls.
(iii) Biochemical analyses. In blood samples collected intra
Capua et al.
600 jvi.asm.org Journal of Virology
vitam to reveal metabolic alterations, a significant increase in
plasma lipase levels (10 to 100 times the values of the control
birds) was evident in H7N1-challenged (12/20) and H7N3-chal-
lenged (10/20) turkeys between days 3 and 9 p.i. (Fig. 1A), while
no uninfected controls showed modification of lipase levels (20/
20; P ⬍ 0.001; Pearson chi-squared test). A clear trend between the
presence of viral RNA in blood at day 3 and the increase in lipase
was evident in infected animals (hazard ratio, 2.51; 95% confi-
dence interval, 0.92 to 6.81; P ⫽ 0.07). Lipase levels within the
normal range were rapidly reestablished in all cases, and it was
decided to no longer evaluate this parameter on day 23 (see Tables
S1 and S2 in the supplemental material). After day 9 p.i., 5 birds in
group A and 5 birds in group B developed hyperglycemia
(Fig. 1B). Of these, two birds maintained the hyperglycemic status
throughout the experiment, while in all the other birds, the levels
of blood glucose returned to levels similar to those of controls (see
Table S3 in the supplemental material). A clear association be-
tween the increase in lipase between days 3 and 9 p.i. and the
development of hyperglycemia after day 9 p.i. was evident. In fact,
hyperglycemia was present only in the birds that developed high
lipase values postinfection and never appeared in birds with nor-
mal lipase levels (10/22 and 0/18, respectively; P ⫽ 0.001), with a
median time between development of hyperlipasemia and hyper-
glycemia of 4.5 days (minimum-maximum, 3 and 7).
(iv) Histopathology and immunohistochemistry. None of
the control turkeys showed significant histological changes or pos-
itive immunohistological reactions to avian influenza virus (AIV)
(Fig. 2A). In all infected birds, histopathologic lesions were evi-
dent, although markedly different in samples collected at different
times postinfection. At early stages (days 4 to 8 p.i.), acute pancre-
atitis with necrotic acinar cells and massive inflammatory infiltra-
tion composed of macrophages, heterophils, lymphocytes, and
plasma cells dominated over areas of healthy/uninvolved/spared
tissue (Fig. 2B). From day 8 p.i., these necrotic inflammatory le-
sions/necrotic inflammatory areas were gradually replaced by
ductule hyperplasia and lymphocytic infiltration with a mild de-
gree of fibroplasia. At later stages (day 17 p.i), extensive fibrosis
with lymphoid nodules replaced pancreatic parenchyma, and dis-
ruption of the normal architecture of the organ was evident (Fig.
2C). Variable numbers of necrotic acinar cells were observed dur-
ing the entire experimental period. Obstructive ductal lesions
were not seen in any case or stage.
By immunohistochemistry, degenerating and necrotic acinar
cells showed specific reaction to virus nucleoprotein antigen anti-
body during the experimental period (Fig. 2D). Some of the vas-
cular endothelial cells also showed a positive reaction, as well as
occasional ductal epithelial cells. In uninfected controls, the insu-
lin-positive tissues of the pancreas were scattered singly or in small
groups of islets of various shapes and sizes in the interstitium of
the exocrine part (Fig. 3A). At day 8 p.i., the normal structure of
FIG 1 Biochemical analysis. Kaplan-Meier analysis for the appearance of hyperlipasemia (A) and hyperglycemia (B) (plasma glucose, ⬎27.78 mmol/liter)
among mock-, H7N1-, and H7N3-infected turkeys. Differences were tested using the log rank statistic. The bar graphs show the frequency of events in relation
to hyperlipasemia, hyperglycemia, and presence of viremia.
Pancreatic Damage by Influenza A Virus
January 2013 Volume 87 Number 1 jvi.asm.org 601
islets was partially destroyed and the number of islet cells was
reduced. The remaining islets were smaller and distorted, with
irregular outlines or dilated intraislet capillaries; the number of
cells staining for insulin was also reduced, and these cells pre-
sented enlarged cytoplasm and sometimes appeared to have gran-
ular degeneration and even necrosis. Fibrous bands appeared in-
side the islet, with islet fragmentation and dislocation of small and
large clusters of endocrine cells (Fig. 3B). At day 17 p.i., separate
large clusters of endocrine insulin-positive cells were evident em-
bedded in or close to the extensive fibrosis that replaced the acinar
component (Fig. 3C). Beyond day 17 p.i., groups of very large
(⬎200 m in diameter), usually irregular islet-like areas of mainly
insulin immunoreactivity were clearly present scattered in exten-
sive acinar fibrosis (Fig. 3D and E).
In vitro experiment. (i) Susceptibility of human pancreatic
cell lines to human influenza A viruses. The susceptibility of en-
docrine (hCM insulinoma) and ductal (HPDE6) cell lines to
H1N1/A/New Caledonia/20/99 and H3N2/A/Wisconsin/67/05
infections was investigated.
(ii) Receptor distribution. Our investigation using lectin
staining for receptor detection on both the hCM and HPDE6 cell
lines revealed high levels of alpha-2,6 sialic acid-linked sialic acid
molecules (required by human-tropic viruses), as well as alpha-
2,3-linked residues (used by avian-tropic viruses). The mean peak
intensities of hCM incubated with MAA lectin II (alpha-2,3 spe-
cific) and SNA lectin (alpha-2,6 specific) were nearly identical at
approximately 2.6 ⫻ 10
4
for both receptors. HPDE6 also had
high-level expression of both receptor types, with 3.7 ⫻ 10
4
for
SNA and 1.6 ⫻ 10
4
for MAA. MDCK cells were also included as a
positive-control line for both receptor types, as these cells are
widely used for the isolation of viruses of human and avian origin.
Fluorescence-activated cell sorter (FACS) analysis showed that
MDCK cells expressed levels of alpha-2,3 receptors similar to
those of HPDE6 cells, with the mean peak intensity near 1.8 ⫻ 10
4
,
while alpha-2,6 expression was equal to that of hCM cells, with
mean fluorescence at 2.5 ⫻ 10
4
. Therefore, both pancreatic cell
lines can be said to express sialic acid receptors at levels compara-
ble to those of MDCK cells, and in the case of hCM cells, expres-
sion of the human-virus receptors was even higher (see Fig. S1 in
the supplemental material). Pretreatment of all cells with 1 U/ml
of NA from Clostridium perfringens resulted in decreased fluores-
cence for both lectin types, confirming specificity (data not
shown).
(iii) Virus replication kinetics in pancreatic cell lines. hCM
and HPDE6 cells were infected with human influenza H1N1 and
H3N2 viruses at an MOI of 0.001. Visual examination of the in-
FIG 2 Histopathology and immunohistochemistry. (A) Turkey pancreas. Normal tissue is shown. Acinar cells containing zymogen granules in their cytoplasm
are evident, associated with two nests of normal islet cells and a ductal structure (H&E). (B) Turkey pancreas. Shown is diffuse and severe necrosis of acinar cells
(arrows) with severe inflammatory infiltrate (asterisks) (H&E). (C) Turkey pancreas. Most of the pancreas has been replaced by foci of fibrous connective tissue
and lymphoid nodules, with some ductular proliferation. (D) Turkey pancreas. Shown is immunohistochemistry for avian influenza virus NP, with an areaof
necrosis, and positive nuclei and cytoplasm in both acinar and ductal cells.
Capua et al.
602 jvi.asm.org Journal of Virology
fected cells by light microscopy revealed no cytopathic effect at
any time point postinfection on hCM or HPDE6 cells. TCID
50
results revealed a continued increase in viral titers in HPDE6 cells
over the 72-h course, though the H1N1 viral titers were only
slightly higher at 72 h than at 48 h postinfection. In contrast, viral
titers reached in hCM cells remained quite similar from 48 to 72 h
postinfection for both H1N1 and H3N2 isolates (Fig. 4A and B).
An examination of viral-RNA replication by qRRT-PCR showed a
continued increase in viral replication up to 72 h postinfection in
both cell lines and for both human viruses tested (Fig. 4C and D).
Despite the higher MOI used to perform the infections (MOI ⫽
0.01), both H7N1 and H7N3 avian influenza viruses showed lower
levels of replication in both pancreatic cell lines than the human
viruses (see Fig. S2 in the supplemental material), with a trend
characterized by steady levels of virus RNA up to 48 h p.i. and a
decrease for both cell lines at 72 h p.i. Based on the RRT-PCR
results, hCM cells appeared to be more sensitive to avian viruses,
since the total amount of “M gene” RNA on average was 2 log units
higher than in HPDE6 cells (see Fig. S2A and B in the supplemen-
tal material). This was also confirmed by TCID
50
results (see Fig.
S2C and D in the supplemental material), which for both viruses
reached higher titers in hCM. In the latter, however, the H7N1
strain exhibited a higher replication efficacy in hCM than in
H7N3. This result is not reflected in the RRT-PCR results, in
which comparable amounts of viral RNA were detected for both
viruses.
No significant differences in viral replication between the two
avian viruses were observed in HPDE6 cells.
(iv) Western blot analysis for detection of virus nucleopro-
tein. Results of H1N1 and H3N2 influenza virus nucleoprotein
expression in the hCM and HPDE6 cell lines are reported in Fig.
5a, b, e, and f. No differences relating to the viral strain or the cell
type were shown in the trend of NP expression. As expected, in-
fluenza virus nucleoprotein was not observed at t
0
(before infec
-
tion), while it was detected at 24 (t
24
), 48 (t
48
), and 72 (t
72
) hours
p.i. for both viruses in hCM and HPDE6 cells. Comparing the
bands obtained from samples at t
24
to those obtained at t
48
and t
72
,
an increase in the NP expression was observable. On the other
hand, the amount of -actin, used as loading control, was at the
same level in all the samples tested (Fig. 5c, d, g, and h).
(v) Immunofluorescence targeting the NP protein. Human
influenza virus replication was also detected by a fluorescent sig-
nal derived from FITC conjugate in hCM cells at 24 h p.i. (see Fig.
S3A and B in the supplemental material) for both viruses tested,
which increased at 48 and 72 h p.i. No differences were observed
between the viral strains tested. The fluorescent signal for both
viruses was also observed at 24 h postinfection in HPDE6 cells (see
Fig. S3C and D in the supplemental material). In this case also, the
number of cells marked continued to increase at 48 and 72 h p.i.,
demonstrating the enhancement of the nucleoprotein expression
over time (data not shown).
(vi) Susceptibility of human pancreatic islets to human influ-
enza A viruses. The regression model indicated that the C
T
values
for both viruses, in the presence or absence of TPCK-trypsin,
tested both in pellets and in supernatant specimens, decreased
significantly over time (P ⬍ 0.05) (Fig. 6). The statistical analysis
showed that the virus titer increased over time independently of
the virus subtype and type of sample (pellet or supernatant). In-
terestingly, C
T
values for the viruses grown with TPCK-trypsin
decreased significantly more than those obtained without the ex-
ogenous proteases (P ⬍ 0.01) only for H1N1 pellets and superna-
tant samples (Fig. 6A and C). TPCK-trypsin seemed to enhance
H3N2 virus growth, but the difference did not reach statistical
significance (P ⬎ 0.10) (Fig. 6B and D). The residual postestima-
tion analysis indicated that the model used was appropriate (data
not shown). Analysis of data was done using STATA 12.0 statisti-
cal software.
In situ hybridization was performed to visualize viral RNA lo-
FIG 3 Immunohistochemistry for insulin. Shown is turkey pancreas with representative islet structures before and after H7N3 infection at different time points.
Pancreatic Damage by Influenza A Virus
January 2013 Volume 87 Number 1 jvi.asm.org 603
calized within islet cells. The results clearly demonstrate the pres-
ence of viral RNA after both H1N1 and H3N2 virus infection (Fig.
7A). Since human islet primary cultures contain both endocrine
and exocrine cells, a fluorescence-based multiplex in situ hybrid-
ization strategy was applied to determine which and how many
cells were infected in the islets. For this purpose, distinctly labeled
probes were combined to analyze viral RNA and insulin, amylase,
or cytokeratin in 19 transcripts simultaneously, and after hybrid-
ization, human islets were disaggregated and cell positivity was
quantified. Five days after infection, 0%, 10.8%, and 4.3% of total
cells were positive for viral RNA in mock-, H1N1-, and H3N2-
infected islets, respectively (P ⬍ 0.001) (Fig. 7B). Of the H1N1-
positive cells, 49% ⫾ 27% stained positive for insulin, 26% ⫾ 16%
for amylase, and 1.6 ⫾ 2.4% for CK19, and 25% ⫾ 21% were
negative for the tested transcripts. Of the H3N2-positive cells,
40% ⫾ 23% stained positive for insulin, 20% ⫾ 20% for amylase,
and 2.3 ⫹ 5% for CK19, and 41% ⫾ 45% were negative for the
tested transcripts (Fig. 7C). On the other hand, of the insulin-
positive cells, 14% ⫾ 10% and 8% ⫾ 8% were positive for viral
RNA 5 days after H1N1 and H3N2 infection, respectively (P ⫽
0.023). Of the amylase-positive cells, 27% ⫾ 9% and 9% ⫾ 6%
were positive for viral RNA after H1N1 and H3N2 infection, re-
FIG 5 Western blot analysis for the detection of viral nucleoprotein in pan-
creatic cell lines. Western blot analysis of H1N1 (a and b) and H3N2 (e and f)
influenza virus NP (56 kDa) in hCM and HPDE6 cells. (c, d, g, and h) Samples
were collected before infection (t
0
) and 24 (t
24
), 48 (t
48
), and 72 (t
72
) hours
postinfection. -Actin (42 kDa) was used as a loading control in order to
ensure that the same amount of protein was tested for each sample.
FIG 4 Human influenza virus replication kinetics in pancreatic cell lines. Shown is the replication kinetics of A/New Caledonia/20/99 (H1N1) and A/Wisconsin/
67/2005 (H3N2) in hCM and HPDE6 cells. hCM and HPDE6 cells were infected with each virus at an MOI of 0.001, and at 24, 48, and 72 h postinfection,
supernatants from three infected and one mock-infected control well were harvested for virus isolation and qRRT-PCR. (A) Virus isolation results of H1N1 in
hCM and HPDE6 cells. (B) Virus isolation results for H3N2 in hCM and HPDE6 cells. (C) qRRT-PCR results for H1N1 in hCM and HPDE6 cells. (D) qRRT-PCR
results for H3N2 in hCM and HPDE6 cells. All results represent means plus standard deviations for three independent experiments.
Capua et al.
604 jvi.asm.org Journal of Virology
spectively (P ⬍ 0.001). Of the CK19-positive cells, 3% ⫾ 4% and
1.3% ⫾ 3% were positive for viral RNA after H1N1 and H3N2
infection, respectively (P ⫽ 0.36) (Fig. 8).
(vii) Modulation of survival, insulin secretion, and innate
immunity in human pancreatic islets infected with human in-
fluenza A viruses in vitro. Visual examination of the infected
islets by light microscopy and LIVE/DEAD assay revealed no sig-
nificant cytopathic effect at any time point postinfection (days 0 to
7). Five days after infection, uninfected cells showed an overall
mortality of 3.26%, H3N2-infected cells 5.21%, and H1N1-in-
fected cells 7.38% (P, nonsignificant versus mock-infected cells)
(see Fig. S4A in the supplemental material). Moreover, exposure
of islets to either H1N1 or H3N2 virus did not affect their ability to
respond to high glucose, as tested in a static perfusion system (see
Fig. S4B in the supplemental material).
The capability of H1N1 and H3N2 viruses to induce cytokine/
chemokine expression in human pancreatic islets was shown by
detectable expression of all but three (interleukin 1b [IL-1b], IL-5,
and IL-7) of the cytokines tested. In mock-infected cells, the high-
est concentrations were detected for CCL2/MCP1 (maximum,
25,558 pg/ml, day 4), ICAM-1 (maximum, 14,063, day 10),
CXCL8/IL-8 (maximum, 11,635 pg/ml, day 10); IL-6 (8,452 pg/
ml, day 4), CXCL1/GRO-␣ (maximum, 8,581 pg/ml, day 4),
VCAM-1 (maximum, 5,566 pg/ml, day 6), VEGF (maximum,
3,225 pg/ml, day 10), SCGF-b (maximum, 1,427 pg/ml, day 6),
hepatocyte growth factor (HGF) (maximum, 1,195 pg/ml, day 6).
MIF (maximum, 806 pg/ml, day 6), granulocyte colony-stimulat-
ing factor (G-CSF) (maximum, 794 pg/ml day 6), CXCL9/MIG
(maximum, 448 pg/ml, day 6), granulocyte-macrophage colony-
stimulating factor (GM-CSF) (maximum, 280 pg/ml, day 4), IL-
2Ra (maximum, 230 pg/ml, day 6), IL-12p40 (maximum, 215
pg/ml, day 6), macrophage colony-stimulating factor (M-CSF)
(maximum, 212 pg/ml, day 10), LIF (maximum, 185 pg/ml, day
6), and CXCL4/SDF1 (maximum, 121 pg/ml, day 6) showed lower
but consistent expression. CXCL10/IP-10, PDGF-BB, IL-1Ra, IL-
12p70, CCL11/eotaxin, FGFb, CCL5/RANTES, CCL4/MIP-1,
CCL7/MCP-3, IL-3, IL-16, SCF, TRAIL, alpha 2 interferon (IFN-
␣2), IFN-␥, and CCL27/CTAK showed low but consistent expres-
sion (maximum, between 10 and 100 pg/ml). Very low (maxi-
mum, ⬍10 pg/ml) but detectable expression was present for IL-2,
IL-4, IL-9, IL-10, IL-13, IL-15, CCL3/MIP-1␣, tumor necrosis fac-
tor alpha (TNF-␣), IL-17, IL-18, IL-1a, -NGF, and TNF-. Two
inflammatory cytokines (IL-6 and TNF-␣) and six inflammatory
chemokines (CXCL8/IL-8, CXCL1/GRO-␣, CXCL9/MIG,
CXCL10/IP-10, CCL5/RANTES, and CCL4/MIP-1) showed
over 5-fold increase in influenza virus-infected cell supernatants
compared to mock-infected controls (Fig. 9A). Among these, the
IFN-␥-inducible chemokines CXCL9/MIG and CXCL10/IP-10
FIG 6 Viral-RNA detection by RRT-PCR of the M gene in human pancreatic islets. Shown are two-way quadratic prediction plots with CIs for RRT-real-time
C
T
values (ct) obtained from H1N1 (A and C) and H3N2 (B and D) (4.8 ⫻ 10
3
PFU/well) pancreatic islet infection. For each virus, the C
T
trends in pancreatic
islet pellets and supernatants from the day of infection (t
0
) until day 10 (t
5
) in the presence (left) or absence (middle) of TPCK-trypsin and as an average of the
previous two conditions (right) are represented.
Pancreatic Damage by Influenza A Virus
January 2013 Volume 87 Number 1 jvi.asm.org 605
showed the strongest response to H1N1 or H3N2 infection (over
100-fold increase). Both peaked 6 to 8 days p.i. and showed a
stronger response to higher doses of the viruses (Fig. 9B).
DISCUSSION
The objective of our work was to assess IAV replication in pancre-
atic cells and to evaluate its consequences both at the cellular level
in vitro and at the tissue level in vivo. In fact, despite previous
reports on a potential role of IAV in pancreatic damage in both
animals and humans (2–4, 19–23), to date, there has been no
attempt to establish whether IAVs are able to grow in pancreatic
cells, and no data are available on the consequences of IAV repli-
cation in the pancreas. Our studies have generated novel in vitro
data indicating that human influenza A viruses are able to grow in
human pancreatic primary cells and cell lines. The addition of
exogenous trypsin appears to enhance but not to be essential for
viral replication in human pancreatic islets. In vitro studies are
corroborated and become of greater significance if combined with
animal studies, in which two nonsystemic strains of IAVs were
able to colonize the pancreases of experimentally infected young
turkeys and to cause metabolic consequences reflecting endocrine
and exocrine damage.
Colonization of the pancreas by IAV has been reported follow-
ing a number of natural and experimental infections of animals,
primarily in birds undergoing both systemic and nonsystemic in-
fection. To date, there is no direct evidence of influenza virus
infection of the pancreas in humans; however, several reports
yield indirect evidence of its occurrence. Here, we have for the first
time demonstrated that two nonsystemic avian influenza viruses
cause severe pancreatitis resulting in a dysmetabolic condition
comparable with diabetes as it occurs in birds. Literature is avail-
able on the clinical implications of endocrine and exocrine dys-
functions of the pancreas in birds, including poultry. With refer-
ence to endocrine function, several studies indicate that with a
total pancreatectomy, birds suffer severe hypoglycemic crisis lead-
ing to death (30). If a residual portion of the pancreas as small as
1% of the pancreatic mass is left in situ, a transient (or reversible)
hyperglycemic condition is observed in granivorous birds, in
which normal glycemia is reestablished within a couple of weeks
(31, 32). This indicates that the pancreatic tissue of birds has sig-
nificant compensatory potential and is also influenced by the fact
that there is evidence of the presence of some endocrine tissue able
to secrete insulin outside the pancreas (33). Insulin is the domi-
nant hormone in well-fed birds, while glucagon is the dominant
hormone in fasting birds. In our experiment, which was carried
FIG 7 Viral-RNA detection by in situ hybridization in human pancreatic islets. Islets were infected with H1N1 and H3N2 by adding 100 l of viral suspension
containing a viral dilution of 4.8 ⫻ 10
3
PFU/well. Mock-infected islets were left as a negative control. (A) Two days after infection, the presence of the viral-RNA
molecules was detected on cytoembedded pancreatic islets upon addition of the Fast Red alkaline phosphatase substrate due to the formation of a colored
precipitate. Bound viral mRNA was then visualized using either a standard bright-field or a fluorescence microscope (magnification, ⫻40). Arrows, viral-mRNA-
positive cells. (B and C) Five days after infection, multiplex fluorescence-based in situ hybridization was performed, and after disaggregation, islet cells were
cytocentrifuged onto glass slides. Viral-RNA-, insulin-, amylase-, and CK19-positive cells were assessed with a Carl Zeiss Axiovert 135TV fluorescence micro-
scope. Quantification was performed using IN Cell Investigator software. Each dot represents the percentage of positive cells quantified on one systematically
random field. Results from two experiments are shown. A Mann-Whitney U test was used for statistical analysis.
Capua et al.
606 jvi.asm.org Journal of Virology
out with food ad libitum, damage to the endocrine component of
the pancreas would most likely manifest itself as hyperglycemia.
With reference to the exocrine function, pancreatitis in birds is
characterized by malaise, reluctance to feed, enteritis, and depres-
sion. Intra vitam investigations have been based on increased he-
matic lipase concentration (32). In our study, pancreatitis was
evaluated both in vivo, by measuring the lipase concentration in
the blood, and postmortem, by histopathologic examination of
pancreases collected at different time points. As it occurs in mam-
mals, pancreatic damage results in a rapid increase of the hematic
lipase levels that is transient, and values returned to normal by day
15 p.i. Interestingly, the birds that had shown increased lipase
levels in the blood and thus supposedly the most severe pancreatic
damage exhibited high blood glucose levels in subsequent days,
which in only a few cases persisted until the termination of the
experiment. This is in keeping with the clinical and metabolic
presentation of diabetes in birds. The histological investigations
clearly demonstrated viral replication in the exocrine portion of
the pancreas, resulting in fibrosis and disruption of the architec-
ture of the organ. While it is clear that both isolates under study
replicated extensively in the acinar component of the pancreas, we
were unable to determine whether viral replication also occurred
in the islets. In any case, it is reasonable to believe that viral infec-
tion resulted in severe acute pancreatitis, which impaired both the
endocrine and exocrine functions.
Current knowledge of influenza virus replication indicates that
influenza viruses that do not exhibit a multibasic cleavage site on
the hemagglutinin (HA) protein do not become systemic. How-
ever, in our in vivo experiments, the virus reached the pancreas,
and we detected viral RNA on day 3 p.i. in the blood of 2/20 (group
A, H7N1) and 4/20 (group B, H7N3) infected turkeys. It is there-
fore possible that following replication in target organs, such as
the lung and the gut, in some individuals, a small amount of virus
reaches the bloodstream and thus the pancreas. Although the de-
tected C
T
values indicate low levels of viral RNA, this often re
-
sulted in the development of pancreatitis (detected in vivo by hy-
perlipasemia). This, in turn, in our experimental model, has
resulted in a hyperglycemic condition, in keeping with the presen-
tation of diabetes in granivorous birds.
The results of our in vitro experiments show that all IAVs
tested, both of avian (H7N1A/turkey/Italy/3675/1999 and H7N3
A/turkey/Italy/2962/2003) and of human (H1N1 Caledonia/
20/99 and H3N2 A/Wisconsin/67/2005) origin, are able to grow in
established pancreatic cell lines and that human viruses also grow
in pancreatic islets. Viral replication occurs in cells of both endo-
crine and exocrine origin. Our investigations also show that both
alpha-2,3 and alpha-2,6 receptors are present in pancreatic cells,
indicating that both human and avian influenza viruses could find
suitable receptors in the organ. The human viruses used in this
study did not induce significant mortality of islet cells, and insulin
secretion did not appear to be affected by infection in this system.
Whether other strains, such as H1N1pdm or viruses of high
FIG 8 Virus RNA and insulin/amylase/CK19 localization. Islets were infected with H1N1 and H3N2 by adding 100 l of viral suspension containing a viral
dilution of 4.8 ⫻ 10
3
PFU/well. Five days after infection, multiplex fluorescence-based in situ hybridization was performed using the Quantigene ViewRNA
technique, based on multiple oligonucleotide probes and branched-DNA signal amplification technology, according to the manufacturer’s instructions. (Left)
The red signal corresponds to the presence of influenza virus RNA, and the green signal corresponds to the presence of insulin, amylase, or CK18 transcripts
(magnification, ⫻63). White arrows, double-positive cells. (Right) Viral-RNA-, insulin-, amylase-, and CK19-positive cells were assessed with a Carl Zeiss
Axiovert 135TV fluorescence microscope. Quantification was performed using IN Cell Investigator software. Each dot represents the percentage of positive cells
quantified on one systematically random field. Results from two experiments are shown.
Pancreatic Damage by Influenza A Virus
January 2013 Volume 87 Number 1 jvi.asm.org 607
pathogenicity, may cause more significant damage remains to be
established. On the other hand, it was clear from the cytokine
expression profile that IAV infection is able to induce a strong
proinflammatory response in human pancreatic islets. The IFN-
␥-inducible chemokines MIG/CXCL9 and IP-10/CXCL10
showed the highest increase after infection. Large amounts of
RANTES/CCL5, MIP1b/CCL4, Gro␣/CXCL1, IL-8/CXCL8,
TNF-␣, and IL-6 were also released.
Of interest, many of these factors are described as key media-
tors in the pathogenesis of type 1 diabetes (34). Recently IP10/
CXCL10 was identified as the dominant chemokine expressed in
vivo in the islet environment of prediabetic animals and type 1
diabetic patients, whereas RANTES/CCL5 and MIG/CXCL9 pro-
teins were present at lower levels in the islets of both species (35).
The chemokine IP-10/CXCL10 attracts monocytes, T lympho-
cytes, and natural killer (NK) cells, and islet-specific expression of
CXCL10 in a mouse model of autoimmune diabetes caused by
viruses (rat insulin promoter [RIP]-lymphocytic choriomeningi-
tis virus [LCMV]) accelerates autoimmunity by enhancing the
migration of antigen-specific lymphocytes (36). This is in line
with other findings, in which neutralization of IP-10/CXCL10
(37) or its receptor (CXCR3) (38) prevents autoimmune disease
in the same mouse model (RIP-LCMV). Studies in nonobese di-
abetic (NOD) mice have demonstrated elevated expression of IP-
10/CXCL10 mRNA and/or protein in pancreatic islets during the
prediabetic stage (39). Increased levels of MIP1b/CCL4 and IP-10/
CXCL10 are present in the sera of patients who have recently been
diagnosed as having type 1 diabetes mellitus (T1D) (40–42).
We speculate that if influenza virus finds its way to the pan-
creas, either through viremia, as detected in human patients (43–
45), or through reflux from the gut through the pancreatic duct,
the virus would find a permissive environment. Here, the virus
would find appropriate cell receptors and susceptible cells belong-
ing to both the endocrine and exocrine components of the organ.
Viral replication would result in cell damage due to the activation
of a cytokine storm similar to the one associated with various
conditions linked to diabetes. Thus, we believe that influenza virus
infections should be investigated as potential agents of pancreatic
damage as reported previously (19, 20), resulting in acute pancre-
atitis and the onset of type 1 diabetes (21–23). The rapid world-
wide increase in the incidence of T1D suggests a major role for
environmental factors in the etiology. According to cross-sec-
tional and prospective studies on T1D patients and/or prediabetic
individuals, virus infections may be one of these. Possible viral
involvement in the etiology of T1D has been suggested by several
authors. Viruses associated with human T1D include enterovi-
ruses, such as coxsackievirus B (CVB) (46–48), but also measles
virus, congenital rubella virus, mumps virus, cytomegalovirus
(49–51), and influenza B virus (52). Rotavirus and reovirus have
additionally been shown to be diabetogenic in mice (49, 53).
In conclusion, our findings suggest that the pancreas could be
a potential target of IAV infection, and thus, we propose that IAV
could play a role in the etiopathogenesis of pancreatitis and dia-
betes in humans and perhaps in other mammals. Clearly, a thor-
ough analysis of existing clinical data and prospective collection of
human samples will be needed to fully address this issue. In the
FIG 9 Cytokine/chemokine expression profile modification induced by human influenza A virus infection. Islets were infected with H1N1 and H3N2 by adding
100 l of viral suspension containing two viral dilutions of 4.8 ⫻ 10
3
or 4.8 ⫻ 10
2
PFU/well. Mock-infected islets were left as a negative control. Samples were
collected every 48 h from the day of infection (t
0
) until day 10 (t
10
). The supernatant was collected and assayed for 50 cytokines. (A) Virus-induced modification
in the islet cytokine/chemokine profile. The data are expressed as the maximum fold increase for each factor detected during culture in relation to mock-infected
islets (n ⫽ 2). Dashed line, 5-fold-increase threshold. The error bars indicate standard deviations. Red items are factors with a ⬎5-fold increased threshold. (B)
IFN-␥-inducible chemokine CXCL9/MIG and CXCL10/IP-10 concentrations during 10 days of culture in the presence (⫹) or absence (⫺) of H1N1 and H3N2.
Capua et al.
608 jvi.asm.org Journal of Virology
meantime, a mammalian preclinical model should be tested to
evaluate the role of IAV infection directly or in the context of other
immune-mediated conditions in etiopathogenesis of islet damage
and beta cell autoimmunity leading to T1D. Similarly, IAV infec-
tion should be ruled out when clinical cases with symptoms of
endocrine or exocrine pancreatic damage of unknown etiology
occur.
ACKNOWLEDGMENTS
We acknowledge H. L. Shivaprasad (University of California, Davis) for
his support with histopathology and immunohistochemistry and Marzia
Mancin (IZSVe, Legnaro, Padova, Italy) for her support with statistical
analysis of data.
Preliminary studies were funded through the EU FP6 project “Train-
ing and Technology Transfer of Avian Influenza Diagnostics and Disease
Management Skills” (FLUTRAIN) (project no. 044212).
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