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Citation:
Emerging Microbes & Infections (2013) 2, e50;
doi:10.1038/emi.2013.50
Published online 7 August 2013
Infection of inbred BALB/c and C57BL/6 and outbred
Institute of Cancer Research mice with the emerging H7N9 avian influenza virus
Open
Zhaoqin Zhu.nature.com/emi/journal/v2/n8/full/emi201350a.html#aff1″ title=”affiliated with 1″>1,.nature.com/emi/journal/v2/n8/full/emi201350a.html#note1″ title=”author note”>*, Yuqin Yang.nature.com/emi/journal/v2/n8/full/emi201350a.html#aff1″ title=”affiliated with 1″>1,.nature.com/emi/journal/v2/n8/full/emi201350a.html#note1″ title=”author note”>*, Yanling Feng.nature.com/emi/journal/v2/n8/full/emi201350a.html#aff1″ title=”affiliated with 1″>1,.nature.com/emi/journal/v2/n8/full/emi201350a.html#note1″ title=”author note”>*, Bisheng Shi.nature.com/emi/journal/v2/n8/full/emi201350a.html#aff1″ title=”affiliated with 1″>1, Lixiang Chen.nature.com/emi/journal/v2/n8/full/emi201350a.html#aff1″ title=”affiliated with 1″>1, Ye Zheng.nature.com/emi/journal/v2/n8/full/emi201350a.html#aff1″ title=”affiliated with 1″>1, Di Tian.nature.com/emi/journal/v2/n8/full/emi201350a.html#aff1″ title=”affiliated with 1″>1, Zhigang Song.nature.com/emi/journal/v2/n8/full/emi201350a.html#aff1″ title=”affiliated with 1″>1, Chunhua Xu.nature.com/emi/journal/v2/n8/full/emi201350a.html#aff1″ title=”affiliated with 1″>1, Boyin Qin.nature.com/emi/journal/v2/n8/full/emi201350a.html#aff1″ title=”affiliated with 1″>1, Xiaonan Zhang.nature.com/emi/journal/v2/n8/full/emi201350a.html#aff1″ title=”affiliated with 1″>1, Wencai Guan.nature.com/emi/journal/v2/n8/full/emi201350a.html#aff1″ title=”affiliated with 1″>1, Fang Liu.nature.com/emi/journal/v2/n8/full/emi201350a.html#aff1″ title=”affiliated with 1″>1, Tao Yang.nature.com/emi/journal/v2/n8/full/emi201350a.html#aff1″ title=”affiliated with 1″>1, Hua Yang.nature.com/emi/journal/v2/n8/full/emi201350a.html#aff1″ title=”affiliated with 1″>1, Dong Zeng.nature.com/emi/journal/v2/n8/full/emi201350a.html#aff1″ title=”affiliated with 1″>1, Wenjiang Zhou.nature.com/emi/journal/v2/n8/full/emi201350a.html#aff1″ title=”affiliated with 1″>1, Yunwen Hu.nature.com/emi/journal/v2/n8/full/emi201350a.html#aff1″ title=”affiliated with 1″>1,.nature.com/emi/journal/v2/n8/full/emi201350a.html#aff2″ title=”affiliated with 2″>2 and Xiaohui Zhou.nature.com/emi/journal/v2/n8/full/emi201350a.html#aff1″ title=”affiliated with 1″>1,.nature.com/emi/journal/v2/n8/full/emi201350a.html#aff2″ title=”affiliated with 2″>2
1.
1Shanghai Public Health Clinical Center, Fudan University,
Shanghai 201508, China
2.
2Key laboratory of Medical Molecular Virology of the
Ministries of Education, School of Basic Medical Science, Fudan Univeristy,
Shanghai 200032, China
Correspondence:
XH Zhou, E-mail:.org”>zhouxiaohui@shaphc.org; YW Hu, E-mail:.com”>ywhu0117@126.com
*These authors contributed equally to this work.
Received
27 May 2013; Revised 3 July 2013; Accepted
7 July 2013
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ABSTRACT
A new
avian-origin influenza virus A (H7N9) recently crossed the species barrier and
infected humans; therefore, there is an urgent need to establish mammalian
animal models for studying the pathogenic mechanism of this strain and the
immunological response. In this study, we attempted to develop mouse models of
H7N9 infection because mice are traditionally the most convenient models for
studying influenza viruses. We showed that the novel A (H7N9) virus isolated
from a patient could infect inbred BALB/c and C57BL/6 mice as well as outbred
Institute of Cancer Research (ICR) mice. The amount of bodyweight lost showed
differences at 7 days post infection (d.p.i.) (BALB/c mice 30%, C57BL/6 and ICR mice approximately 20%), and the lung indexes were increased both at 3 d.p.i.
and at 7 d.p.i.. Immunohistochemistry demonstrated the existence of the H7N9
viruses in the lungs of the infected mice, and these findings were verified by
quantitative real-time polymerase chain reaction (RT-PCR) and 50% tissue culture infectious dose (TCID50)
detection at 3 d.p.i. and 7 d.p.i.. Histopathological changes occurred in the
infected lungs, including pulmonary interstitial inflammatory lesions,
pulmonary oedema and haemorrhages. Furthermore, because the most clinically
severe cases were in elderly patients, we analysed the H7N9 infections in both
young and old ICR mice. The old ICR mice showed more severe infections with
more bodyweight lost and a higher lung index than the young ICR mice. Compared
with the young ICR mice, the old mice showed a delayed clearance of the H7N9
virus and higher inflammation in the lungs. Thus, old ICR mice could partially
mimic the more severe illness in elderly patients.
Keywords:
avian
influenza virus; H7N9; mice
.nature.com/emi/journal/v2/n8/full/emi201350a.html#top”>Top of page
INTRODUCTION
Avian influenza viruses are a persistent threat to global
public health. Recently, a novel avian-origin influenza A (H7N9) virus that
originated from three existing avian influenza A viruses (with hemagglutinin
from H7N3, neuraminidase from H7N9 and six internal genes from H9N2) has caused
over 127 cases in China, 26 of which resulted in deaths..nature.com/emi/journal/v2/n8/full/emi201350a.html#bib1″>1,.nature.com/emi/journal/v2/n8/full/emi201350a.html#bib2″>2 As
previous pandemic viruses have been derived from avian host origins,.nature.com/emi/journal/v2/n8/full/emi201350a.html#bib3″>3,.nature.com/emi/journal/v2/n8/full/emi201350a.html#bib4″>4
further adaptation of the novel H7N9 to mammals in the future could make H7N9 a
pandemic virus..nature.com/emi/journal/v2/n8/full/emi201350a.html#bib5″>5,.nature.com/emi/journal/v2/n8/full/emi201350a.html#bib6″>6,.nature.com/emi/journal/v2/n8/full/emi201350a.html#bib7″>7
To predict whether this virus can become pandemic, it is
important to understand the high pathogenicity and potential risk of
transmission of the novel H7N9 avian influenza viruses among humans by
utilizing mammalian models. Clinical and epidemiological data collected from
human cases could partially reveal features of infection with the novel H7N9
avian influenza virus, but such data are insufficient to dissect the biological
or molecular mechanisms of virus pathogenicity in the host. Mammalian models
are urgently needed to study the pathogenic and immunological mechanisms of
H7N9 infection and to establish efficient vaccines and therapeutic drugs.
Several mammalian species can support influenza
replication and are employed as infectious models, including mice, ferrets,
guinea pigs, cats, pigs and non-human primates..nature.com/emi/journal/v2/n8/full/emi201350a.html#bib8″>8 Very
recently, Zhu et al..nature.com/emi/journal/v2/n8/full/emi201350a.html#bib7″>7 reported that the human H7N9 virus
infected ferrets and pigs, and those models were employed to study the
infectivity and transmission of this novel virus, as well as the pathology that
ensues. Although infections in ferrets and pigs can mimic the typical signs of
human symptoms, a major limitation of those models is the lack of specific
reagents to study the immune responses; additionally, these species lack the
abundant resources of transgenic, gene-knockout or knockin strains to analyse
the function of individual genes, which are characteristics present in mice.
Mice are one of the most common and convenient mammalian
models for studying the pathogenesis and immunology of many influenza viruses.nature.com/emi/journal/v2/n8/full/emi201350a.html#bib9″>9,.nature.com/emi/journal/v2/n8/full/emi201350a.html#bib10″>10,.nature.com/emi/journal/v2/n8/full/emi201350a.html#bib11″>11,.nature.com/emi/journal/v2/n8/full/emi201350a.html#bib12″>12
because of their well-characterized genome, advanced techniques for genomic
manipulation, the convenient availability of mouse-specific immunological
reagents, easy husbandry and low cost. Those features have allowed mice to
become one of the best tools for drug evaluation and vaccine studies for the
influenza virus. However, not every influenza virus can infect mice easily without
adaptation by the virus. With respect to this new avian-origin virus that
recently crossed the species barrier and infected humans, it is not yet known
whether it can infect mice.
.nature.com/emi/journal/v2/n8/full/emi201350a.html#top”>Top of page
MATERIALS
AND METHODS
Viral isolation
The prototype virus,
A/Shanghai/4664T/2013(H7N9) (GenBank No. KC853225-KC853232), was isolated from
throat swab specimens obtained from an H7N9 influenza patient whose status was
confirmed by the Chinese Center for Disease Control and Prevention. This case
was a 27-year-old male who was admitted to the Fifth People’s Hospital of
Shanghai with symptoms of dizziness and chills on 2nd March 2013. The specimen
was collected before the patient received oseltamivir. Although he received
supplemental oxygen and symptomatic and supportive treatment, his symptoms were
not relieved and his chest computed tomography indicated ‘white lung’. He died
of pneumonia and respiratory failure on 9th March 2013.
Madin–Darby canine kidney (MDCK) cells were maintained in
complete minimum essential medium (HyClone, Utahi, USA) that was supplemented
with 100 U/mL penicillin, 100 µg/mL streptomycin and foetal bovine
serum to a final concentration of 10% (Hyclone) for 24 h before viral inoculation.
Subsequently, 400 µL of the swab sample was incubated with the MDCK cells
for one hour, washed with phosphate-buffered saline (PBS), and further
cultivated in the MDCK cells for 96 h. The supernatant from the cell
culture was collected and was subjected to titration before animal infection.
All experiments related to the H7N9 virus, including the
virus isolation, cultivation, amplification, titration and animal infection,
were conducted in the Animal Biosafety Level 3 Laboratory following the
standard operating protocols approved by the Institutional Biosafety Committee
at the Shanghai Public Health Clinical Center, Fudan University.
Mice
Six- to eight-week-old
female BALB/c, C57BL/6 and ICR mice as well as old female ICR mice (60 weeks
old) were purchased from the B&K Universal Group Limited (Shanghai, China)
and housed under specific pathogen-free conditions at the animal facilities of
the Shanghai Public Health Clinical Center, Fudan University (Shanghai, China).
The mice were transferred to the Animal Biosafety Level 3 Laboratory before
infection. All mouse-related procedures were performed in accordance with the
Institutional Animal Care and Use Committee-approved protocols.
Infection and sample collection
Female mice of the BALB/c,
C57BL/6 and ICR strains (B&K Universal Group Limited, Shanghai, China) were
used in this study. The young mice were 6–8 weeks old, and the old ICR mice
were 60 weeks old. Twelve or fourteen mice of each strain were divided into
experimental and control groups. The mice were inoculated with 5×104
TCID50 of the virus or with PBS after sevoflurane (Hearem, Osaka,
Japan) inhalation anaesthesia. The mice were monitored for clinical signs and
survival, and bodyweight was measured daily during the 7-day observation
period. Bodyweight loss was calculated using the formula: (weightinfected
day − weight0 day)/weight0 day×100%.
Three or four mice in each of the experimental groups of BALB/c, C57BL/6, ICR
and old ICR mice were euthanized by cervical dislocation at 3 and 7 days post
inoculation (d.p.i.); three mice from each control group were also euthanized
at the same time. The lungs, livers, hearts and brains were removed under
aseptic conditions and washed three times in sterile PBS. A portion of the
lungs was removed for pathology, and the remaining organs were mechanically and
ultrasonically homogenized (Jxfstprp-24, Shanghai, China) in sterile PBS
(500 µL PBS for 1 g of lung tissue, and 800 µL PBS for 1 g
of other organs). The centrifuged supernatant was aliquoted and stored at
−80 °C until use. The whole lung was weighed to calculate the lung index
(LI), using the following formula: LI=weight of lung ×100/bodyweight. The lung
index increase rate was also calculated, using the following formula: (LIinfected−LImock)/LImock×100%.
TCID50 detection
The MDCK cells were seeded
in 96-well plates at 5000 cells/well in 100 µL of Dulbecco’s modification
of Eagle’s medium with 10% foetal bovine serum and antibiotics (penicillin and
streptomycin) for approximately 24 h. The viruses or samples were diluted
1:10 to a final concentration of 1:1 000 000 in a separate V-bottom 96-well
plate with serum-free Dulbecco’s modification of Eagle’s medium medium. Ten
microlitres of each dilution was transferred to the MDCK cells mixed with
90 µL Dulbecco’s modification of Eagle’s medium containing bovine serum
albumin Fraction V. The plates were placed in an incubator at 37 °C with 5% CO2
for approximately 5 days, and the cytopathic effect was observed daily in each
well using a microscope (Olympus, Shinjuku, Japan). The supernatants were
collected and subjected to viral load quantitation. The TCID50
values were calculated using the Reed–Muench method.
Extraction of total RNA and quantitative
RT-PCR for viral load
Total RNA was extracted
from whole homogenized tissue using an extraction kit (Qiagen, Stow Kark,
German) according to the manufacturer’s instructions. The quality of the RNA
samples was measured by detecting the optical density (280/260); the absorption
ratios ranged from 1.9 to 2.25. The total RNA samples were stored at
−80 °C until use. To remove any residual DNA contamination, the RNA
samples were incubated with RNase-free DNase I at 37 °C for 20 min,
followed by stopping the reaction at 65 °C for 10 min.
The TaqMan real-time quantitative PCR amplification
reactions were carried out in an Eppendorf Mastercycler ep realplex RT-PCR
system. The RT-PCR was carried out with the One-Step RT-PCR kit (TaKaRa, Tokyo,
Japan). A 25 µL reaction mixture consisting of a 12.5 µL TaqMan
Universal PCR Master Mix, 400 nM primers and a 300 nM TaqMan probe
was used for each of the target genes. A master mix (22.5 µL) was prepared
into the wells of a 0.2 mL optical-grade 96-well PCR plate. Then,
2.5 µL of an RNA template was added to a final volume of 25 µL.
Hemagglutinin gene fragments, amplified with primers designed according to the
published sequence of the H7 subtype (GenBank No. KC853228), were inserted into
the recombinant T plasmids (TaKaRa) and were used as a quantitative standard.
We used the housekeeping gene glyceraldehyde phosphate dehydrogenase as a reference
gene (sense primer: 5′-CAA TGT GTC CGT CGT GGA TCT-3′; antisense primer: 5′-GTC
CTC AGT GTA GCC CAA GAT G-3′; probe primer: 5′-(6-FAM)-CGT GCC GCC TGG AGA AAC
CTG CC-(TAMRA)-3′). All of the reactions were carried out in triplicate without
a template control as well as with the influenza A virus
(A/Shanghai/4664T/2013(H7N9)) as a positive control. The thermal cycle
conditions were as follows: 42 °C for 10 min (AmpErase activation)
followed by 95 °C for 1 min and 45 cycles of 95 °C for 15 s
and 60 °C for 45 s. All of the data were analysed using the
REALPLE×2.2 software (Mastercycler reprealplex real-time PCR system, Eppendorf,
German). The quantification of the H7N9 virus was shown as gene copies
per µg total RNA of tissue, which was further normalized by the expression
level of the housekeeping gene glyceraldehyde phosphate dehydrogenase.
Pathology and immunohistochemistry
The mouse lungs were fixed
in 4%
paraformaldehyde, dehydrated, embedded in paraffin and cut into 5-mm-thick
sections, which were stained with haematoxylin and eosin (H&E). For the
immunohistochemistry (IHC) assay, the slides were incubated with serum
collected from a male H7N9 patient on day 15 of hospitalisation, which was used
as the primary antibody (1:100, titred by immunofluorescent assay). Serum from
a healthy male donor was used as the control. Rabbit anti-human immunoglobulin
G (Dako Corp., Carpinteria, CA, USA) was used as the secondary antibody for the
IHC analysis in accordance with the manufacturer’s instructions. The slides were
viewed using an Olympus BX51 microscope, and the images were captured and
analysed by the corresponding acquisition software (DP controller; Olympus).
Only the cells with red-stained nuclei were considered positive in the IHC
assay.
Data analysis
All of the statistical
analyses were performed using GraphPad Prism Software (Version 5.0; GraphPad,
La Jolla, CA, USA). Normality tests were conducted, after which parametric
(unpaired t-test) or nonparametric (Mann–Whitney U test) tests
were used to analyse the differences between the control and experimental
groups. The statistical significance was set at P≤0.05.
.nature.com/emi/journal/v2/n8/full/emi201350a.html#top”>Top of page
RESULTS
General appearance, body weight and lung index
The three mouse strains
were intranasally inoculated with influenza virus A (H7N9). No change in the
general appearance of the mice was observed during the first 2 days after
inoculation. However, after the third day, all three strains of mice showed
reduced activity, ruffled fur and difficulty breathing (tachypnea and laboured
respiration) accompanied by reduced food and water intake and obvious weight
loss.
From 3 to 7 d.p.i., bodyweight loss and a lung-index
increase were observed. A similar trend of bodyweight loss occurred in all
three strains of mice (.nature.com/emi/journal/v2/n8/full/emi201350a.html#fig1″>Figure 1A).
However, the extent of the bodyweight loss in the BALB/c mice was greater than
that in the C57BL/6 and ICR mice. The percentage of bodyweight loss reached 30% in
the BALB/c mice and 20% in the C57BL/6 and ICR mice at 7 d.p.i. (.nature.com/emi/journal/v2/n8/full/emi201350a.html#fig1″>Figure 1A). The
rate of increase in the lung index of the BALB/c mice (70%±17.76%) was higher than those of
the C57BL/6 (40%±1.34%) and
ICR mice (5%±1.04%) at 3
d.p.i. However, the lung index of the C57BL/6 mice increased more than that of
the ICR and BALB/c mice at 7 d.p.i.; the lung index increase rates were
approximately 160%±10.07%, 100%±45.47% and
70%±10.01%,
respectively (.nature.com/emi/journal/v2/n8/full/emi201350a.html#fig1″>Figure 1B).
.nature.com/emi/journal/v2/n8/fig_tab/emi201350f1.html#figure-title”>Figure 1.
.nature.com/emi/journal/v2/n8/fig_tab/emi201350f1.html#figure-title”>.0/msohtmlclip1/01/clip_image001.jpg” alt=”Description: figure 1 – unfortunately we are unable to provide accessible alternative text for this. if you require assistance to access this image, please contact help@nature.com or the author”>
Characterisation of bodyweight lost and lung index post
influenza virus A (H7N9) infection in mice. Inbred BALB/c, C57BL/6 and outbred
ICR mice were intranasally infected (i.n.) with 4×105 TCID50
influenza A virus (A/Shanghai/4664T/2013(H7N9)). The bodyweight loss was monitored.
Lungs were collected at day 3 d.p.i or 7 d.p.i.. The results of lung index are
expressed as the mean±s.d.,*P<0.05, **P<0.01, n=3. (A) Bodyweight change. (B) Lung index. .nature.com/emi/journal/v2/n8/fig_tab/emi201350f1.html#figure-title">Full figure and legend (38K)

Viral infection in the lungs of the three
mouse strains
Lung samples from the
infected mouse groups were tested by viral-specific RT-PCR, viral titration on
the MDCK cells and IHC to confirm whether the H7N9 virus could successfully
establish an infection in mice. Evidence of a viral infection could be found in
the lungs of all three strains in the form of a viral-specific gene that was
detected in the supernatant of the homogenized lung tissues by RT-PCR (.nature.com/emi/journal/v2/n8/full/emi201350a.html#fig2″>Figure 2A);
moreover, the virus could be recovered when inoculating the supernatant onto
MDCK cells (.nature.com/emi/journal/v2/n8/full/emi201350a.html#fig2″>Figure 2B). The
IHC of the lung tissue also showed viral antigen in the pulmonary tissues of
the infected mice (.nature.com/emi/journal/v2/n8/full/emi201350a.html#fig2″>Figure 2C), and
virus inclusion bodies could be found in the cytoplasm of macrophages and
alveolar epithelial cells (data not shown). The peak of the viral load in the
lungs was at 3 d.p.i. in all three strains of mice, but the BALB/c mice had a
higher viral load than the other two strains (log virus copies were
5.839±0.2367/ µg total RNA in the BALB/c mice, 5.427±0.3576/ µg total
RNA in the C57BL/6 mice and 4.809±0.5772/ µg total RNA in the ICR mice).
Similar TCID50 values were observed among the three strains in the in
vitro assay ((3.83±0.22)×104 TCID50/g lung weight in
the BALB/c mice, (2.62±1.45)×104 TCID50/g lung weight in
the C57BL/6 mice and (1.22±1.19)×104 TCID50/g lung weight
in the ICR mice). The virus in the lungs of the BALB/c and ICR mice were
cleared at 7 d.p.i. (log virus copies were 5.074±0.2850/ µg total RNA in
the BALB/c mice, 5.477±0.08649/ µg total RNA in the write C57BL/6 in one
line mice and 3.798±0.04081/ µg total RNA in the ICR mice), whereas the
C57BL/6 mice maintained a level similar to the 3 d.p.i. level.
.nature.com/emi/journal/v2/n8/fig_tab/emi201350f2.html#figure-title”>Figure 2.
.nature.com/emi/journal/v2/n8/fig_tab/emi201350f2.html#figure-title”>.0/msohtmlclip1/01/clip_image002.jpg” alt=”Description: figure 2 – unfortunately we are unable to provide accessible alternative text for this. if you require assistance to access this image, please contact help@nature.com or the author”>
Virus loads in the lungs. After intranasally infected
(i.n.) with 4×105 TCID50 influenza A virus
(A/Shanghai/4664T/2013(H7N9)), lungs were collected and homogenized for virus
detection at 3 d.p.i or 7 d.p.i.. Total RNAs in homogenates were extracted and
reverse transcribe to cDNA. (A) The virus loads in each mouse lung was
determined quantitative PCR to detect the copy number of H7N9 gene. (B)
TCID50 assay in MDCK cells were performed to check the titre of H7N9
in infected lung by inoculation of serial diluted supernatant of lung homogenates.
The results of RT-PCR are expressed as the average±s.d. (n=3) of
the log 10
virus copies per µg total RNA. The results of TICD50 are expressed
as the average±s.d. (n=3).*P<0.05, **P<0.01. (C) The lung from infected mice (C-a, C-b) or uninfected mice (C-c, C-d) were fixed and prepared for immunochemistry staining of the H7N9 virus using H7N9 patient’s serum (C-a, C-c) and control serum from healthy donor (C-b, C-d) as primary antibody. Images were obtained at magnification is ×400. (C-a) Slide of infected lung detected by H7N9 patient’s serum. Alveolar epithelial cells and macrophages with positive signals of H7N9 antigen in slurry and nuclei (arrows). (C-b) Slide of infected lung incubated with control serum from healthy donor. The control lung of non-infected mice incubated with H7N9 patient’s serum (C-c) and control serum from healthy donor (C-d). .nature.com/emi/journal/v2/n8/fig_tab/emi201350f2.html#figure-title">Full figure and legend (96K)

Histopathological changes in the infected
lungs
The majority of the
pathological changes in the lungs among all three strains of infected mice
consisted of focal inflammation at 3 d.p.i. Compared with the normal lungs in
mock-infected mice (.nature.com/emi/journal/v2/n8/full/emi201350a.html#fig3″>Figures
3A, 3B, 3G, 3H, 3M and 3N), most of the lungs from the infected mice showed the
infiltration of inflammatory cells, including monocytes/macrophages and
lymphocytes, in the pulmonary parenchyma and in the alveolar septa (.nature.com/emi/journal/v2/n8/full/emi201350a.html#fig3″>Figures
3C, 3D, 3I, 3J, 3O and 3P). However, the pathological changes were more severe at
7 d.p.i. and were different among the three strains. These changes presented as
a focal pulmonary consolidation in the BALB/c mice (.nature.com/emi/journal/v2/n8/full/emi201350a.html#fig3″>Figures 3E and 3F), pulmonary oedema and haemorrhage in the C57BL/6 mice (.nature.com/emi/journal/v2/n8/full/emi201350a.html#fig3″>Figures 3K and 3L), and the typical pulmonary interstitial inflammatory
lesions in the ICR mice (.nature.com/emi/journal/v2/n8/full/emi201350a.html#fig3″>Figures 3Q and 3R) at 7 d.p.i.
.nature.com/emi/journal/v2/n8/fig_tab/emi201350f3.html#figure-title”>Figure 3.
.nature.com/emi/journal/v2/n8/fig_tab/emi201350f3.html#figure-title”>.0/msohtmlclip1/01/clip_image003.jpg” alt=”Description: figure 3 – unfortunately we are unable to provide accessible alternative text for this. if you require assistance to access this image, please contact help@nature.com or the author”>
Pathological analysis of the lungs. Lungs were collected
from mice infected with influenza virus A/Shanghai/4664T/2013(H7N9) at 3 d.p.i
or 7 d.p.i., or from uninfected mice and prepared or histology studies as
described in the section on ‘MATERIALS AND METHODS’. Images of H&E-stained slides
were observed under microscopy at magnitudes of ×100 and ×400. (A)
BALB/c mice PBS negative control (×100). (B) BALB/c mice PBS negative
control (×400). (C) BALB/c mice at 3 d.p.i (×100). (D) BALB/c
mice at 3 d.p.i (×400). (E) BALB/c mice at 7 d.p.i (×100). (F)
BALB/c mice at 7 d.p.i (×400). (G) C57BL/6 mice PBS negative control
(×100). (H) C57BL/6 mice PBS negative control (×400). (I) C57BL/6
mice at 3 d.p.i (×100). (J) C57BL/6 mice at 3 d.p.i (×400). (K)
C57BL/6 mice at 7 d.p.i (×100). (L) C57BL/6 mice at 7 d.p.i (×400). (M)
ICR mice PBS negative control (×100). (N) ICR mice PBS negative control
(×400). (O) ICR mice at 3 d.p.i (×100). (P) ICR mice at 3 d.p.i
(×400). (Q) ICR mice at 7 d.p.i (×100). (R) ICR mice at 7 d.p.i
(×400).
.nature.com/emi/journal/v2/n8/fig_tab/emi201350f3.html#figure-title”>Full figure and legend (123K)

Old ICR mice showed more severe inflammation
and delayed viral clearance than their young counterparts
When comparing the young
and old ICR mice, the bodyweight loss of the old mice was only slightly greater
than that of the young mice (22% vs. 15%) at 7 d.p.i. (.nature.com/emi/journal/v2/n8/full/emi201350a.html#fig4″>Figure 4A).
However, the lung index increase rates of the old ICR mice were approximately
27.4%±7.31% and
204%±29.03% at 3
and 7 d.p.i., respectively, which were much higher than those of their younger
counterparts (3.8%±1.03% and
87.7%±45.47%,
respectively) at the corresponding time points (.nature.com/emi/journal/v2/n8/full/emi201350a.html#fig4″>Figure 4B).
.nature.com/emi/journal/v2/n8/fig_tab/emi201350f4.html#figure-title”>Figure 4.
.nature.com/emi/journal/v2/n8/fig_tab/emi201350f4.html#figure-title”>.0/msohtmlclip1/01/clip_image004.jpg” alt=”Description: figure 4 – unfortunately we are unable to provide accessible alternative text for this. if you require assistance to access this image, please contact help@nature.com or the author”>
Comparison of influenza virus A (H7N9) infections between
young mice and old ICR mice. Outbred mice ICR young (6–8 weeks) or old (60
weeks) were infected by 4×105 TCID50 influenza virus A
(H7N9). Lungs were collected at day 3 d.p.i or 7 d.p.i, and LI was calculated
using the formula: LI=weight of lung×100/bodyweight. Lung index percentage=(LIinfected−LImock)/LImock×100%. The
virus loads of lung homogenates were determined by RT-RCR and TCID50
detection (R). H7N9 titres in lungs were collected at 3 d.p.i or 7 d.p.i and
fixed in 4%
paraformaldehyde and stained with H&E. Magnification was ×400.
Cytokines/chemokines in lungs were examined. (A) Bodyweight lost curve.
(B) Lung index. (C) Virus loads of lungs detected by RT-PCR
(left) and TCID50 (right). The results are expressed as the average±s.d.
(n=3).*P<0.05,**P<0.01. (D) Slides of lungs with H&E staining (×400). (D-a) Young ICR mice with PBS; (D-b) old ICR mice with PBS; (D-c) young ICR mice at 3 d.p.i; (D-d) old ICR mice at 3 d.p.i.; (D-e) young ICR mice at 7 d.p.i.; (D-f) old ICR mice at 7 d.p.i.. .nature.com/emi/journal/v2/n8/fig_tab/emi201350f4.html#figure-title">Full figure and legend (143K)

Interestingly, the duration of the viral shedding was
different between the old ICR mice and the young ICR mice. The kinetics of the
viral loads in the young ICR mice peaked at 3 d.p.i. and decreased at 7 d.p.i.
However, in the old ICR mice, the viral load grew slowly at the early stages of
infection, but remained higher at 7 d.p.i., which indicated delayed viral
clearance in the older ICR mice (.nature.com/emi/journal/v2/n8/full/emi201350a.html#fig4″>Figure 4C). The
lung viral loads of the young ICR mice were higher than those of the old ICR
mice at 3 d.p.i. (log virus copies were 4.089±0.2850/ µg total RNA in the
young ICR mice and 3.700±0.3131/ µg total RNA in the old ICR mice) while
less than that of the old ICR mice at 7 d.p.i. (log virus copies were
3.798±0.0408/ µg total RNA in the young ICR mice and 4.443±0.1144/ µg
total RNA in the old ICR mice, P=0.0003).
Compared with uninfected mock (.nature.com/emi/journal/v2/n8/full/emi201350a.html#fig4″>Figures 4D-a and 4D-b), both young ICR mice and old ICR mice have inflammatory
cells infiltration (.nature.com/emi/journal/v2/n8/full/emi201350a.html#fig4″>Figures 4D-c and 4D-d) at 3 d.p.i.. However, different pathological changes
observed between these two groups of mice at 7 d.p.i. Young ICR mice presented
the typical pulmonary interstitial inflammatory lesions, while old ICR mice
showed many inflammatory cell infiltrations and severe hemorrhages (.nature.com/emi/journal/v2/n8/full/emi201350a.html#fig4″>Figures 4D-e and 4D-f). Therefore, though old ICR mice showed severe
inflammation responses than the young mice, it did not seem to contribute to
the virus clearance.
.nature.com/emi/journal/v2/n8/full/emi201350a.html#top”>Top of page
DISCUSSION
Gene analysis data indicate that the novel H7N9 viruses
may be better adapted to infecting mammals than are other avian influenza
viruses..nature.com/emi/journal/v2/n8/full/emi201350a.html#bib1″>1,.nature.com/emi/journal/v2/n8/full/emi201350a.html#bib13″>13 In
contrast to the highly pathogenic avian influenza virus H5N1, these H7N9
viruses are thought to be weakly pathogenic..nature.com/emi/journal/v2/n8/full/emi201350a.html#bib5″>5
However, recent reports indicate that infection with H7N9 can cause severe
respiratory failure in humans..nature.com/emi/journal/v2/n8/full/emi201350a.html#bib1″>1,.nature.com/emi/journal/v2/n8/full/emi201350a.html#bib14″>14,.nature.com/emi/journal/v2/n8/full/emi201350a.html#bib15″>15 The
average age of the patients infected with H7N9 who display severe symptoms is
approximately 63 years;.nature.com/emi/journal/v2/n8/full/emi201350a.html#bib15″>15 by
contrast, most H5N1-infected patients are young..nature.com/emi/journal/v2/n8/full/emi201350a.html#bib16″>16,.nature.com/emi/journal/v2/n8/full/emi201350a.html#bib17″>17 The
mechanism of H7N9 pathogenicity is not yet understood, and no vaccine is
currently available. These gaps motivate us to develop appropriate mammalian
models for studying the pathogenesis of the H7N9 virus and evaluating candidate
vaccines and antiviral drugs.
Very recently, H7N9 infection models of ferrets and pig
have been established..nature.com/emi/journal/v2/n8/full/emi201350a.html#bib7″>7 The
ferrets supported H7N9 virus replication in their upper and lower respiratory
tracts and shed the virus for 6–7 days with relatively mild clinical signs. The
pigs infected by the H7N9 virus shed the virus for 6 days. However,
applications of the ferret model and the pig model were limited because of a
lack of immunological research reagents and genetically modified animal
resources, as well as high costs.
As a well-characterized mammalian laboratory animal, the
mouse is one of the most widely used mammalian models for the studies of
influenza virus pathogenesis and immunology, drug evaluations and vaccine
developments..nature.com/emi/journal/v2/n8/full/emi201350a.html#bib8″>8,.nature.com/emi/journal/v2/n8/full/emi201350a.html#bib9″>9,.nature.com/emi/journal/v2/n8/full/emi201350a.html#bib10″>10,.nature.com/emi/journal/v2/n8/full/emi201350a.html#bib11″>11,.nature.com/emi/journal/v2/n8/full/emi201350a.html#bib12″>12 In
this study, we aimed to construct mouse models for the H7N9 infection. We chose
inbred BALB/c and

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