Download The PB2 E627K mutation contributes to the high polymerase activity

Journal of General Virology (2014), 95, 779–786
DOI 10.1099/vir.0.061721-0
The PB2 E627K mutation contributes to the high
polymerase activity and enhanced replication of
H7N9 influenza virus
Hong Zhang,13 Xuyong Li,23 Jing Guo,2 Li Li,1 Chong Chang,1
Yuanyuan Li,2 Chao Bian,3 Ke Xu,1 Hualan Chen2 and Bing Sun1,3
1
Correspondence
Key Laboratory of Molecular Virology & Immunology, Institut Pasteur of Shanghai,
Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences,
Shanghai 200031, PR China
Bing Sun
[email protected]
Hualan Chen
2
State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute,
Chinese Academy of Agricultural Sciences, Harbin 150001, PR China
[email protected]
3
State Key Laboratory of Cell Biology, Institute of Biochemistry and Cell Biology,
Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences,
Shanghai 200031, PR China
Received 14 November 2013
Accepted 4 January 2014
Human infection by H7N9 influenza virus was first identified in China in March 2013. As of 12
August 2013, a total of 135 documented cases with 44 fatalities had been reported. Genetic and
laboratory analyses of the novel H7N9 viruses isolated from patients indicate that these viruses
possess several polymerase gene mutations previously associated with human adaptation and
potential pandemic capabilities. However, the function of these mutations in the emergence and
pathogenicity of the viruses is not well known. In this study, we demonstrate that the PB2 E627K
mutation, which occurs in over 70 % of the H7N9 patient isolates, promotes the replication of
H7N9 virus by enhancing PB2 polymerase activity and enhances virulence in mice. Our results
show the PB2 E627K mutation has played an important role in this H7N9 influenza outbreak and
in the pathogenicity of the H7N9 virus.
INTRODUCTION
On 29 March 2013 the Chinese Center for Disease Control
and Prevention confirmed the first case of human infection
with H7N9 influenza A virus (Gao et al., 2013b). As of 12
August 2013, 135 cases with 44 fatalities had been confirmed, causing worldwide concern (Zhang et al., 2013).
Patients infected with H7N9 viruses have a rapidly progressive pneumonia, leading to respiratory failure and
acute respiratory distress syndrome (Gao et al., 2013a).
Phylogenetic analysis suggests that the novel H7N9 virus
is a triple reassortant and that its viral genes are of avian
origin. The surface glycoprotein haemagglutinin (HA)
was derived from the H7N3 virus from domestic ducks
in Zhejiang, whereas the neuraminidase (NA) was derived
from the wild bird H7N9 virus in South Korea. All six
internal genes show high similarity to the poultry H9N2
virus (Gao et al., 2013b; Kageyama et al., 2013). Human
3These authors contributed equally to this work.
The GenBank/EMBL/DDBJ accession numbers for the H7N9 PB2
influenza virus sequences are AGK84850, AGK84856, AGK84859,
AGO51410, AGO51398, AGO51442, AGJ73498, AGL44433,
AGI60293, AGN69469, AGN69457, AGJ51961, and AGM16245.
061721 G 2014 SGM
infections with H7N9 virus have not been reported
previously, and animal infections with H7N9 viruses had
not been detected in China before this outbreak.
The influenza virus is a continuous threat to human health.
In addition to the annual seasonal epidemic, the influenza virus occasionally causes pandemics. During the past
century, several pandemics have occurred, including 1918
(H1N1), 1957 (H2N2), 1968 (H3N2) and 2009 (pH1N1);
all the pandemic viruses bear HA and NA genes of avian or
pig origin (Liu et al., 2013). Owing to the low fidelity of
the viral RNA-dependent polymerase, it is easy for viruses
to acquire adaptive mutations that allow transmission to
hosts of different species. In modern times, direct human
infections with avian influenza viruses occur only sporadically, including with the H7N2 (Ostrowsky et al., 2012),
H7N3 (Skowronski et al., 2006; Tweed et al., 2004), H7N7
(Fouchier et al., 2004), H9N2 (Blair et al., 2013; Peiris et al.,
1999) and H5N1 (de Jong et al., 1997) subtypes. Most
human infections result in mild illness and conjunctivitis,
except for the H5N1 subtype, which is associated with
greater than 50 % mortality. Patients infected with the
H7N9 subtype have been detected in over 10 provinces of
China, and most experienced a severe clinical syndrome.
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H. Zhang and others
At this time, no investigations have revealed evidence of
sustained spread of this virus in humans. However, the
possibility of limited human-to-human spread cannot be
excluded in a few small clusters of human H7N9 virus
infections (Qi et al., 2013).
Further analysis of the H7N9 gene segments has shown the
presence of signature amino acids associated with adaptation to the human host and with virulence, such as the
Q226L mutation in HA, which is associated with increased
binding to mammalian-like receptors in the human upper
airway, and the E627K and D701N mutations in PB2, which
are associated with enhanced replication and virulence
(Kageyama et al., 2013; Li et al., 2005; Liu et al., 2013).
During the past 2 months, many studies have shown that
H7N9 viruses have a mixed a-2,3/a-2,6 receptor preference,
which is regulated by the single Q226L mutation (Belser
et al., 2013). The H7N9 viruses were transmitted through
close contact between ferrets, but they were not well transmitted by droplets. However, one isolate of H7N9 from a
patient in Anhui was demonstrated to be highly transmissible between ferrets by respiratory droplets (Zhang et al.,
2013). The PB2 E627K mutation has been recognized as
one of the most important mammalian adaptive markers.
Since the beginning of the outbreak of highly pathogenic
avian H5N1, the percentage of H5N1 isolates containing
PB2 627K has increased. In this current outbreak of H7N9,
a large percentage of the viruses isolated from patients
contain 627K in PB2, whereas all the viruses isolated from
poultry and from the environment contain 627E (Li et al.,
2013; Shi et al., 2013). Thus, it is reasonable to suppose
that the PB2 E627K substitution plays an important role in
the transmission and virulence of the novel H7N9 virus.
However, the role of PB2 627K in the emergence of H7N9
needs to be experimentally demonstrated.
In this study, we tested the polymerase activity and replication kinetics of the H7N9 virus harbouring 627K/E in
cell cultures. In addition, the pathogenicity of H7N9 was
tested in mice by measuring body mass for two weeks postinfection and determining organ viral load in selected
organs. We demonstrate that the PB2 E627K substitution
contributes to the replication and pathogenicity of the
H7N9 virus by regulating polymerase activity.
RESULTS
The PB2 E627K mutation was prevalent in human
isolates of H7N9 virus
Gene analysis of the H7N9 virus has shown that the new
viruses contain some mutations associated with adaptation
to human hosts, such as PB2 E627K. To evaluate the role
of PB2 E627K in the emergence of H7N9, we collected all
the sequences of H7N9 isolated from humans and avian
hosts. The percentage of viruses containing the PB2 E627K
mutation was calculated. The percentage of H7N9 patient
isolates containing PB2 E627K is high, at 71.4 % [PB2 627K
780
was found in 10 of 14 human H7N9 isolates and only
four isolates downloaded from the National Center for
Biotechnology Information (NCBI) were PB2 627E], and
the fatality rate was approximately 30 % (http://www.who.
int/influenza/human_animal_interface/influenza_h7n9/Data_
Reports/en/). All of the 37 H7N9 viruses isolated from
avian or environmental sources contain PB2 627E (Zhang
et al., 2013). Together, these data suggest that the PB2
E627K mutation plays an important role in the occurrence of H7N9 from avian sources in human hosts.
The PB2 E627K mutation contributes to
high polymerase activity and enhanced
replication of H7N9
To test the contribution of PB2 E627K to H7N9 polymerase activity, we performed a luciferase reporter assay.
We used WSN (Influenza A/WSN/1933 (H1N1)) virus as a
reference strain. The polymerase activity of H7N9 627K
was approximately 20-fold higher than that of H7N9 627E
in 293T cells (Fig. 1a). However, there was not as big a
difference between the polymerase activity of H7N9 627K
and 627E in the chicken embryo fibroblast (CEF) cell line
DF-1 as that in mammalian cells (Fig. 1b). The polymerase
activity pattern of PB2 627K/E in H7N9 was similar to the
activity observed in H5N1 cases (Mänz et al., 2012). To
further confirm the contribution of PB2 627K to H7N9
replication, we compared the replication kinetics of H7N9
viruses containing PB2 627K and 627E in the A549 human
alveolar epithelial cell line and primary CEFs. The virus
titre of H7N9 was approximately 100-fold higher than that
of H7N9 PB2 K627E from 12 h onwards after infection in
A549 cells. The viral titre of H7N9 PB2 K627E did not
increase during the infection, suggesting that the virus is
not well adapted to the A549 cell line (Fig. 1c). There was
no apparent difference in viral titre between H7N9 and
H7N9 PB2 627E in CEFs (Fig. 1d). Taken together, these
results indicate that PB2 627K enhances polymerase
activity and viral replication in mammalian cells.
Polymerase activity of H7N9 with PB2 627K is
higher than with PB2 627E and is capable of
promoting virus replication at 33 ‡C
Although the mechanism of how PB2 E627K exerts its
effects is not yet clear, one possible function of PB2 627K is
to facilitate replication of avian viruses in the human upper
respiratory tract, which generally has a temperature of
33 uC. In contrast, the temperature of the avian intestinal
tract is closer to 41 uC, a temperature at which PB2 627E
facilitates efficient viral replication (Hatta et al., 2007; Steel
et al., 2009). To address whether the PB2 E627K mutation
influences polymerase activity at a lower temperature, we
tested the polymerase activity of WSN and H7N9 viruses
with PB2 627K or 627E at 33 uC in 293T cells. The
polymerase activity of H7N9 was still relatively high and
about 1.5-fold higher than that of WSN at 33 uC. It was
obvious that the polymerase activity of H7N9 viruses with
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Journal of General Virology 95
(a) 300
(b) 800
Relative polymerase activity
Relative polymerase activity
PB2 E627K promotes high polymerase activity of H7N9
50
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H7N9-PB2 K627E
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Viral titres (log10 EID50 ml–1)
Viral titres (log10 EID50 ml–1)
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Fig. 1. Polymerase activity and replication kinetics of H7N9 in cell cultures. The polymerase activity of WSN, WSN 627E, H7N9
and H7N9 627E was tested in 293T cells (a) and DF-1 cells (b). Fifty nanograms each of PB1, PB2, PA and NP in pCAGGS;
100 ng of human pPolI-NP-luc or avian pPolI-NS-luc; and 10 ng of pRLSV40 (Promega) were co-transfected into 293T cells
or DF-1 cells, which were harvested 24 h after transfection. All data were normalized to the activity of the WSN sample. Growth
curves of H7N9 and H7N9 PB2 K627E were studied in A549 cells (c) and CEFs (d). Cells were infected with viruses at
an m.o.i. of 0.01. At 12, 24, 48 and 72 h post-inoculation, the supernatants were harvested, and virus titres were determined
in eggs.
PB2 627K was about 60-fold higher than that of H7N9
viruses with PB2 627E (Fig. 2a). These data suggest that the
H7N9 viruses may have the ability to replicate well in
the human upper respiratory tract at 33 uC. To test the
contribution of PB2 627K to viral replication at 33 uC, the
replication kinetics of H7N9 were tested in A549 cells. As
shown in Fig. 2b, compared with H7N9 PB2 K627E, H7N9
grew well in A549 cells at 33 uC. These data show that
H7N9 is more adapted to mammalian cells than H7N9 PB2
K627E at 33 uC.
PB2 627K contributes to the replication and
transcription processes in mammalian cells
As we have demonstrated that PB2 627K contributes to
the high polymerase activity and enhanced replication of
H7N9 viruses in mammalian cells, we further investigated
whether the higher activity associated with PB2 627K was
http://vir.sgmjournals.org
related to an increase in transcription (mRNA) and/or
replication (cRNA and vRNA synthesis) by using a quantitative PCR-based assay. H7N9 viruses harbouring PB2
627K had a higher transcription activity at 33 uC, as demonstrated by a .10-fold higher level of PB2 627K than PB2
627E mRNA. The cRNA of H7N9 viruses harbouring PB2
627K was about threefold higher than that of H7N9 viruses
harbouring PB2 627E, although the vRNA level of H7N9
with PB2 627K was slightly higher than that of H7N9 with
PB2 627E (Fig. 3b). Conversely, the H7N9 viruses
containing PB2 627K had a higher replication
and transcription activity at 37 uC, as demonstrated by
an approximately twofold increase in the level of all three
kinds of RNA of PB2 627K compared with that of PB2
627E (Fig. 3a). These data indicate that PB2 627K enhances
polymerase activity by regulating the viral replication and
transcription process in mammalian cells.
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H. Zhang and others
(b)
Viral titre (log10 EID50 ml–1)
200
150
100
50
8
H7N9
H7N9-PB2 K627E
6
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Time post infection (h)
12
K6
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H
W
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SN
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K6
27
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Relative polymerase activity
(a)
Fig. 2. Polymerase activity and replication kinetics of H7N9 harbouring PB2 627K or 627E at 33 6C. (a) Polymerase activity of
H7N9 and H7N9 627E in 293T cells at 33 6C. Fifty nanograms each of PB1, PB2, PA and NP in pCAGGS; 100 ng of human
pPolI-NP-luc; and 10 ng of pRLSV40 (Promega) were co-transfected into 293T cells, which were harvested 24 h after
transfection. All data were normalized to the activity of the WSN sample. (b) Replication kinetics of H7N9 harbouring PB2 627K
or 627E at 33 6C. A549 cells were infected with viruses at an m.o.i. of 0.01. At 12, 24, 48 and 72 h post-inoculation, the
supernatants were harvested and virus titres were determined in eggs.
To further determine the pathogenicity and replication of
H7N9 harbouring 627K or 627E in mammals, mice were
infected with 106 median 50 % egg-infectious dose (EID50)
of both viruses, and organs were harvested at 3 and 5 days
post-infection. The body mass of mice infected with H7N9
decreased by approximately 20 %, while the body mass of
mice infected with H7N9 PB2 K627E did not decrease (Fig.
4a). The replication of H7N9 and H7N9 PB2 K627E was
100
80
60
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20
0
vRNA
cRNA
mRNA
33 °C
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Relative vRNA level
vRNA
cRNA
mRNA
37 °C
(a) 120
detected in nasal turbinates and lungs but not in other
organs (brain, spleen and kidneys). The viral titres of H7N9
in nasal turbinates and lungs was significantly higher than
the titres of H7N9 PB2 K627E (Fig. 4b, c). The replication
of H7N9 was similar in nasal turbinates and the lung,
whereas the viral titre of H7N9 PB2 K627E in nasal
turbinates was lower than that in the lung. Our data
suggest that PB2 627K promotes viral replication in
mice, and H7N9 virus containing PB2 627K is better
adapted to the upper and lower respiratory tracts than
virus containing PB2 627E.
Relative vRNA level
PB2 627K enhances the replication of H7N9
in mice
Fig. 3. Quantification of viral RNA levels of H7N9RNP complexes containing PB2 627K or 627E at 37 6C and 33 6C. 293T
cells were co-transfected with expression plasmids encoding NP, PA, PB1 and PB2 together with pPolI-NA plasmid and
incubated at (a) 37 6C or (b) 33 6C. Total cellular RNA was isolated 24 h post-transfection and was subjected to quantitative
RT-PCR for segment 6 (NA gene) transcripts. All data were normalized to viral RNA, cRNA and mRNA of the H7N9 sample.
Results are means±SD from three independent assays.
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Journal of General Virology 95
PB2 E627K promotes high polymerase activity of H7N9
H7N9
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ey
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Lu
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H7N9 PB2 K627E
Viral titres
(log10 EID50 ml–1)
Body mass change (%)
(a)
Organs
Fig. 4. Replication of H7N9 and H7N9 PB2 K627E in mice. (a) Body mass of mice infected with H7N9 and H7N9 PB2 K627E.
Mice were inoculated with 106 EID50 of virus H7N9 or H7N9-PB2 K627E in a volume of 50 ml. Body mass was recorded for
14 days after infection. Virus titres of H7N9 (b) and H7N9 PB2 K627E (c) in mouse organs were determined. Nasal turbinates,
lungs, spleen, kidneys and brain were harvested at 3 days and 5 days post-infection, and virus titres were determined in eggs.
Dashed lines indicate the lower limit of detection.
DISCUSSION
The emergence of a new H7N9 avian influenza is a global
health concern due to the severity of infection and associated
mortality in humans. During the past several decades,
human infections with other H7-subtype avian viruses have
been reported, but most of them resulted in only mild illness
and conjunctivitis, except for one death caused by H7N7 in
The Netherlands (Fouchier et al., 2004). However, this is the
first time that the N9 subtype has been reported in human
infections. Genetic analysis has predicted that the viruses
contain some mutations related to human adaptation,
such as PB2 E627K, a hallmark of influenza virulence and
transmission to humans. Therefore, in this study we
experimentally clarified the contribution of PB2 627K to
the replication and pathogenicity of H7N9.
To evaluate the role of PB2 627K in the outbreak of H7N9,
we collected all the PB2 sequences of human-isolated
and avian or environment-isolated H7N9 from NCBI.
The percentage of PB2 627K was higher in H7N9 isolated
from patients, but there was no PB2 627K in the avian or
environment-isolated H7N9. The evidence suggests that PB2
627K may play an important role in human H7N9 infection.
However, the PB2 627K is not the only factor that
determines polymerase activity and adaptation to humans.
There may be other mutations in polymerase genes that
promote the viral replication in H7N9, such as PB2 591K/R,
PB2 701N (Gabriel et al., 2005; Mehle & Doudna, 2009;
Yamada et al., 2010) and PB1 (473V and 598P) (Xu et al.,
2012). Four H7N9 isolates in our collections contained PB2
627E: A/Nanjing/1/2013, A/Jiangsu/1/2013, A/Jiangsu/2/
2013 and A/Zhejiang/DTID-ZJU01/2013. The isolate from
Zhejiang harbours PB2 701N, which is predicted to complement the loss of PB2 627K. In contrast, the isolates from
Nanjing and Jiangsu have PB2 591K and PB1 473V. In
future, we should monitor the evolution of H7N9 in case
these viruses acquire more adaptive mutations.
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PB2 627K contributes to high polymerase activity in mammalian cells, but not in avian cells. Using a luciferase
reporter assay, we demonstrated that PB2 627K promotes
the polymerase activity of H7N9 in mammalian cells, but
not in avian cells. In addition, viral titre studies in mice
demonstrate that PB2 627K contributes to the enhanced
replication of H7N9 in vivo. Many potential mechanisms of
the high polymerase activity associated with PB2 627K have
been proposed. Amino acid 627 lies on the surface of a
polymerase PB2 subunit and is involved in the interaction
with the host factor importin (Tarendeau et al., 2008). The
differential use of importin-a isoforms governs cell tropism
and host adaptation of influenza virus based on studies
investigating the transmission of avian influenza H5N1
(Gabriel et al., 2008, 2011). Some studies have shown that
the enhanced interaction between NP and PB2 mediated by
PB2 627K contributes to the high polymerase activity in
mammalian cells but not in avian cells (Labadie et al., 2007;
Rameix-Welti et al., 2009). Some studies suggested that
the adaptive mutation PB2 E627K was mediated by an
inhibitory or stimulatory factor in host cells (Mehle &
Doudna, 2008; Moncorgé et al., 2010). The crystallography
indicated that the amino acid PB2 627 was in the Cterminal RNA binding domain and the PB2 627K had
higher RNA binding activity than the PB2 627E (Kuzuhara
et al., 2009). More experiments should be performed to
test this mechanism in the H7N9 virus. The stability of
influenza virus is temperature dependent. To cause a pandemic, a virus needs to maintain a stable state and replicate
well at a lower temperature (33 uC), which is the approximate temperature of the human upper respiratory tract.
PB2 627K improves polymerase activity at lower temperatures and is thought to confer stability on viruses, which
allows the virus to replicate better at the lower temperature
(Steel et al., 2009). We tested the polymerase activity of
PB2 627K at 33 uC. H7N9 harbouring PB2 627K had
relatively high polymerase activity at 33 uC. The viral titre
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H. Zhang and others
in nasal turbinates also indicated that H7N9 is well adapted
to the upper respiratory tract. In addition, PB2 627K may
improve polymerase activity by regulating the transcription
and replication processes at different temperatures.
37 uC, respectively. Culture supernatant was collected at the indicated
time points post-infection and titrated in eggs. The growth data shown
are the average results of three independent experiments.
Our study characterized the H7N9 virus (harbouring
PB2 627K) and found that it possessed higher polymerase
activity and improved replication in mammalian cells
when compared to H7N9 harbouring PB2 K627E. More
importantly, PB2 627K promoted the replication of H7N9
in nasal turbinates and lungs in mice and can cause illness.
Our data indicate that the PB2 627K plays an important
role in the outbreak and pathogenicity of H7N9.
PA and NP; 100 ng of pPolI-NP-luc; and 10 ng of pRLSV40
(Promega) were co-transfected into the cells using Lipofectamine
2000 (Invitrogen) according to the manufacturer’s instructions.
Cells were harvested 24 h after transfection at 37 uC and 33 uC.
Luciferase assays were performed using the Dual-Luciferase reporter
assay system (Promega) according to the manufacturer’s protocol.
The firefly and Renilla luciferase activities were measured using a
microplate luminometer (Veritas). The ratio of firefly luciferase
activity to Renilla luciferase activity was calculated to represent the
efficiency of the transcription/replication of the viral-like reporter
RNA. All experiments were performed in triplicate. Results are
presented as the mean±SD.
METHODS
Facility. All experiments with live H7N9 viruses were conducted
within the enhanced animal biosafety level 3 (ABSL3+) facility in
the Harbin Veterinary Research Institute of the Chinese Academy
of Agricultural Sciences, approved for such use by the Ministry of
Agriculture of China and the China National Accreditation Service for
Conformity Assessment.
Plasmids. PB1, PB2, PA and NP from A/Anhui/1/2013 (H7N9)
were commercially synthesized and cloned into the vector pCAGGS
with restriction enzyme EcoRI (Takara). Viral cDNAs from the
A/WSN/33 (H1N1) virus were kindly provided by Professor Hans
Klenk (Marburg University, Germany) and were cloned into the vector
pCAGGS (kindly provided by Dr Jun-ichi Miyazaki, Osaka University,
Japan). The pPolI-NP-luc and pPolI-NA were also provided by
Professor Hans Klenk. The avian pPolI-NP-luc was constructed by
replacing the human pPolI-NP-luc promoter by the avian promoter.
Cells. Human embryonic kidney 293T cells and immortalized CEFs
(DF-1) were purchased from the ATCC and maintained in Dulbecco’s
modified Eagle’s medium (HyClone) supplemented with 10 % FBS
(Gibco) plus penicillin and streptomycin. Alveolar basal epithelial
cells (A549) were maintained in F-12K Nutrient Mixture (Gibco)
containing 10 % FBS plus antibiotics. Madin–Darby canine kidney
cells were grown in minimum essential medium with Eagle’s salts
containing 4 % FBS, 4 mM L-glutamine, and antibiotics.
Reverse genetics. An eight-plasmid reverse genetics system was
used to generate H7N9 and H7N9-PB2 K627E viruses. As described
previously, cDNA from the human-infecting H7N9 influenza virus
A/Anhui/1/2013 (AH1) was inserted into the bidirectional transcription vector pBD. We introduced the mutation PB2 K627E into the
AH1-PB2 plasmid by site-directed mutagenesis with the QuikChange
Site-Directed Mutagenesis kit (Stratagene) according to the manufacturer’s protocol. The plasmids used for virus rescue and the genes
from the rescued viruses were fully sequenced to confirm the absence
of unwanted mutations. Virus rescue was performed as previously
described (Li et al., 2005). Briefly, 293T cells were co-transfected with
0.5 mg of each of the eight plasmids mixed with 10 ml Lipofectamine
LTX (Invitrogen) according to the manufacturer’s instructions.
Eight hours later, the DNA-transfection mixture was replaced
by Opti-MEM (Gibco). The supernatant was harvested and injected
into 10-day-old specific pathogen-free embryonated eggs for virus
propagation after 48 h. The rescued virus was detected by haemagglutination assay.
Luciferase reporter assay. Fifty nanograms each of PB1, PB2,
Strand-specific real-time RT-PCR assay. Fifty nanograms each of
PB1, PB2, PA and NP in pCAGGS and 100 ng of pPolI-NA (provided
by Professor Hans Klenk) were co-transfected into the cells using
Lipofectamine 2000 (Invitrogen) according to the manufacturer’s
instructions. Cells were harvested 24 h after transfection at 37 uC
and 33 uC, and the total cellular RNA was extracted with TRIzol
RNA isolation reagents (Invitrogen). RNA (0.5 mg of each sample)
was reverse-transcribed by using the strand-specific tagged primers
(Kawakami et al., 2011) for the NA gene using a ReverTra Ace qPCR
RT kit (Toyobo) according to the manufacturer’s instructions. Realtime quantitative PCR (qPCR) was performed with SYBR Green Realtime PCR Master Mix (Toyobo) according to the manufacturer’s
instructions on an ABI PRISM 7900HT. The qPCR cycle conditions
were 95 uC for 10 min followed by 40 cycles of 95 uC for 15 s and
60 uC for 1 min. The vRNA, cRNA and mRNA levels were expressed
relative to GAPDH mRNA as ratios.
Mouse infection. To evaluate the virulence of H7N9 influenza
virus in a mammalian host, two groups (n511) of 6–7-week-old
female BALB/c mice (Vital River) were inoculated with 106 EID50
of the H7N9 virus or the H7N9-PB2 K627E virus in a volume of
50 ml. Three mice were euthanized at 3 and 5 days post-inoculation,
respectively, and their nasal turbinates, lungs, spleen, kidneys and
brain were suspended in 1 ml of cold sterile PBS and subsequently
homogenized for viral titration. The other mice were weighed and
observed for signs of disease for 2 weeks.
ACKNOWLEDGEMENTS
This work was supported by grants from the National 973 key project
(2013CB530504), and the National 863 project (2012AA02A404,
2012AA020103), the National Science and Technology Major Project (2012ZX10002-007-003, 2013ZX10004-101-005, 2013ZX10004003-003), grants from the National Natural Science Foundation
of China (31030029, 31230024, 81201280), a grant from CAS Key
Project (KSZD-EW-Z-002-3) and a grant from the Science and Technology Commission of Shanghai Municipality (12ZR1435000).
Finally this work is the following study of European Project SP5BCT-2006-044161 (to B. S.). This work was also supported by grants
from the Ministry of Science and Technology (2012ZX10004214)
(to H. C.).
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