﻿JOURNAL OF VIROLOGY, Feb. 2008, p. 1146­1154 Vol. 82, No. 3
0022-538X/08/$08.000 doi:10.1128/JVI.01698-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
A Single-Amino-Acid Substitution in the NS1 Protein Changes the
Pathogenicity of H5N1 Avian Influenza Viruses in Mice
Peirong Jiao,1
Guobin Tian,1
Yanbing Li,1
Guohua Deng,1
Yongping Jiang,1
Chang Liu,1
Weilong Liu,1
Zhigao Bu,1
Yoshihiro Kawaoka,2,3,4
and Hualan Chen1
*
Animal Influenza Laboratory of the Ministry of Agriculture and National Key Laboratory of Veterinary Biotechnology,
Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, 427 Maduan Street, Harbin 150001,
People's Republic of China1
; Division of Virology, Department of Microbiology and Immunology,2
and International Research Center for Infectious Diseases, Institute of Medical Science,3
University of Tokyo, Tokyo 108-8639, Japan; and Department of Pathobiological Sciences,
School of Veterinary Medicine, University of Wisconsin, Madison, Wisconsin 537064
Received 6 August 2007/Accepted 6 November 2007
In this study, we explored the molecular basis determining the virulence of H5N1 avian influenza viruses in
mammalian hosts by comparing two viruses, A/Duck/Guangxi/12/03 (DK/12) and A/Duck/Guangxi/27/03 (DK/
27), which are genetically similar but differ in their pathogenicities in mice. To assess the genetic basis for this
difference in virulence, we used reverse genetics to generate a series of reassortants and mutants of these two
viruses. We found that a single-amino-acid substitution of serine for proline at position 42 (P42S) in the NS1
protein dramatically increased the virulence of the DK/12 virus in mice, whereas the substitution of proline for
serine at the same position (S42P) completely attenuated the DK/27 virus. We further demonstrated that the
amino acid S42 of NS1 is critical for the H5N1 influenza virus to antagonize host cell interferon induction and
for the NS1 protein to prevent the double-stranded RNA-mediated activation of the NF-B pathway and the
IRF-3 pathway. Our results indicate that the NS1 protein is critical for the pathogenicity of H5N1 influenza
viruses in mammalian hosts and that the amino acid S42 of NS1 plays a key role in undermining the antiviral
immune response of the host cell.
H5N1 highly pathogenic avian influenza virus (HPAIV) is
not only a catastrophic pathogen for poultry, but it poses a
severe threat to the public health and may cause a future
influenza pandemic. In 1997, highly pathogenic H5N1 avian
influenza virus caused outbreaks in chickens in Hong Kong and
was transmitted to humans, causing the deaths of 6 of 18
people infected (4, 31). The H5N1 outbreaks in poultry, which
became widespread in late 2003, affected at least 10 Asian
countries initially, but since then, H5N1 viruses have been
isolated from wild birds (3) and poultry in multiple countries in
Asia, Europe, and Africa (http://www.oie.int). H5N1 influenza
virus infections have occurred in several mammalian species,
such as pigs, domestic cats, tigers, and leopards (http://www
.oie.int). More importantly, human cases of H5N1 infections
have been reported in many countries (http://www.who.int),
with greater than 50% mortality caused by H5N1 viruses
among infected humans. Such findings have sparked great in-
terest in pandemic preparedness as well as in understanding
the genetic determinants of influenza virus pathogenicity and
the ability of the virus to cross species barriers to mammalian
hosts.
The pathogenicity of influenza viruses is determined by many
factors, including virus-specific determinants encoded within the
virus genome. In the H5 and H7 subtypes of influenza viruses,
the multiple basic amino acids adjacent to the cleavage site of the
hemagglutinin (HA) glycoprotein are a prerequisite for lethality
in chickens and mice (12, 13, 30). For H5N1 influenza viruses, a
reverse genetics study demonstrated that a single-amino-acid sub-
stitution at position 627 of the PB2 protein from glutamic acid to
lysine is responsible for virulence in mammalian species (12).
Moreover, the amino acid at position 701 in PB2 plays a crucial
role in the ability of H5N1 viruses of duck origin to replicate and
be lethal in mice (16). This same PB2 amino acid residue con-
tributes to the increased lethality of an H7N1 avian influenza
virus in a mouse model (9).
Several studies have reported that the NS1 protein is also
associated with the virulence and host range of influenza vi-
ruses in different animal models (17, 23, 27, 28). Influenza
viruses in which the NS1 gene was deleted exhibited an atten-
uated phenotype in mice and pigs (23, 28). The glutamic acid
at position 92 of the NS1 protein of the H5N1 influenza virus
that transmitted to humans in 1997 was shown to be critical in
conferring virulence and resistance to antiviral cytokines in
pigs (27). However, H5N1 virus with this amino acid residue is
no longer circulating in nature and glutamic acid is not found
in the NS1 proteins of other influenza viruses. Another amino
acid substitution at position 149 of the NS1 protein from valine
to alanine was shown to be responsible for the replication of a
goose H5N1 influenza virus in chickens (17); however, this
mutation did not affect virus virulence in mammals (H. Chen,
unpublished data). Thus, the specific amino acid residues in
avian NS1 that are responsible for conferring high virulence in
mammals remain unclear.
Host factors, such as the immune responses, also play a role
in determining influenza virus pathogenicity (14). The inter-
* Corresponding author. Mailing address: Harbin Veterinary Research
Institute, CAAS, 427 Maduan Street, Harbin 150001, People's Republic
of China. Phone: 86-451-85935079. Fax: 86-451-82733132. E-mail:
hlchen1@yahoo.com.

Published ahead of print on 21 November 2007.
1146
feron (IFN) response represents an early host defense mech-
anism against viral infections and is an important component
of innate immunity (33). The presence of double-stranded
RNA (dsRNA) is a signal to the host cell that virus infection
and replication are occurring and triggers a plethora of anti-
viral host defense mechanisms (5, 29). The presence of dsRNA
induces the synthesis of alpha/beta IFN (IFN-/) proteins
through the activation of several transcription factors, includ-
ing IRF-3, IRF-7, NF-B, and c-Jun/ATF2. Influenza viruses
have dsRNA species of replication intermediates that elicit the
host IFN response. The secreted IFN-/ induces an antiviral
state in influenza virus-infected and uninfected neighboring
cells by stimulating the transcription of IFN-stimulated re-
sponse element promoter-containing genes via the JAK/STAT
pathway (29). However, influenza and other viruses have de-
veloped strategies to counteract host IFN-/ production,
through inhibiting the activation of transcription factors in-
volved in IFN activation (10, 18) and by attenuating host gene
expression (20). Antagonism of the innate response by influ-
enza virus is a property of the NS1 protein (7, 11, 20). Al-
though data in this area of research are growing, there remain
facets of host range and virulence determination that need
further examination.
In this study, we characterized two H5N1 avian influenza
viruses that were isolated from ducks, A/Duck/Guangxi/12/
2003 (DK/12) and A/Duck/Guangxi/27/2003 (DK/27), in the
Guangxi province of China in 2003. These two viruses are
highly pathogenic for chickens but differ in their virulences in
mice. We used reverse genetics to determine the molecular
basis for the difference in virulence in mice and to explore the
possible underlying mechanisms. We found a specific amino
acid in NS1 that confers lethality in mice to an avian H5N1
virus.
MATERIALS AND METHODS
Cells and viruses. Human embryonic kidney cells (293T) and Vero cells were
grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal
bovine serum plus antibiotics. Human lung epithelial cells (A549) were grown in
nutrient mixture F-12 Ham Kaighn's modified medium with 10% fetal bovine
serum. The cells were incubated at 37°C in 5% CO2. Recombinant vesicular
stomatitis virus (VSV) expressing green fluorescent protein (VSV-GFP) was
generated by inserting the G protein gene of VSV into the VSVG*GFP vector
by use of reverse genetics as described previously (15, 17).
Construction of plasmids. The construction of plasmids for virus rescue was
performed as described previously (16). Mutations were introduced into the NS1
gene by site-directed mutagenesis (Invitrogen) with the set of primers shown in
Table 1. We also generated plasmids expressing two wild-type and two mutant
NS1 protein sequences by use of the pCAGGS plasmid vector for the reporter
gene assay. These plasmids were designated as p12NS1 and p27NS1 for the
wild-type NS1 proteins and p12NS1P42S and p27NS1S42P for the mutant NS1
proteins. To investigate the IRF-3-dependent promoter activation, we synthe-
sized the mouse ISG-54 promoter fragment based on the available sequence
information (GenBank accession number X77259) and inserted it between the
NheI and BglII sites of the pLuc 3-enhancer plasmid (Promega) in front of the
firefly luciferase open reading frame and designate the construct as pISG54-Luc.
All of the constructs were completely sequenced to ensure the absence of un-
wanted mutations.
Generation of reverse genetic reassortant viruses. Reassortant viruses were
generated by reverse genetics as described previously (12, 16). The rescued
viruses were detected by hemagglutination assay, and RNA was extracted and
analyzed by reverse transcription-PCR (RT-PCR). Each viral segment was se-
quenced to confirm the identity of the reassortant viruses.
Animal experiments. Determination of the intravenous pathogenicity index
value (IVPI) with chickens was performed according to the recommendations of
the Office International Des Epizooties (21). For the mouse study, groups of 11
6-week-old female BALB/c mice (Beijing Experimental Animal Center) were
lightly anesthetized with CO2 and inoculated intranasally with 106.0
50% egg
infectious doses (EID50) of H5N1 influenza virus in a volume of 50 l. Three
mice in each group were euthanized on days 4 and 6 postinoculation (p.i.).
Organs were collected and titrated for virus infectivity in eggs as described
previously (2). The remaining mice were monitored for 14 days for weight loss
and mortality. The 50% mouse lethal dose (MLD50) was determined by inocu-
lating groups of five mice with 10-fold serial dilutions containing 101
to 106
EID50
TABLE 1. Primers used for pBD cDNA construction to amplify the full-length cDNAs of the viruses and for introducing the mutations in
the NS1 gene
Purpose
Primer (5­3)a
Forward Reverse
For PB2 amplification CCAGCAAAAGCAGGTCAAATATATTCA 5TTAGTAGAAACAAGGTCGTT
For PB1 amplification CCAGCAAAAGCAGGCAAACCA 5TTAGTAGAAACAAGGCATTTTTTC
For PA amplification CCAGCAAAAGCAGGTACTGATC 5TTAGTAGAAACAAGGTACTTTTTTGGAC
For HA amplification CCAGCAAAAGCAGGGGTCCAATC 5TTAGTAGAAACAAGGGTGTTTTTAACTAC
For NP amplification CCAGCAAAAGCAGGGTAGATAATC 5TTAGTAGAAACAAGGGTATTTTTC
For NA amplification CCAGCAAAAGCAGGAGTTCAAAATGAAT 5TTAGTAGAAACAAGGAGTTTTTTGAACAA
For M amplification CCAGCAAAAGCAGGTAGATGTTGAAAGATG 5TTAGTAGAAACAAGGTAGTTTTTTACTC
For NS amplification CCAGCAAAAGCAGGGTGACAA 5TTAGTAGAAACAAGGGTGTTTTTTATCAT
For DK/12NS1P42S NS
mutation
TCAGAAGTCCCTAAGAGGAAGAGGC 5GCCTCTTACTCTTAGGGACTTCTGA
For DK/12NS1N48S NS
mutation
TAAGAGGAAGAGGCAGCACCCTTGG 5CCAAGGGTCCTGCCTCTTCCTCTTA
For DK/27NS1S42P NS
mutation
TCAGAAGCCCCTAAGAGGAAGAGGC 5GCCTCTTCCTCTTAGGGGCTTCTGA
For DK/27NS1S48N NS
mutation
TAAGAGGAAGAGGCAACACCCTTGG 5CCAAGGGTTCTGCCTCTTCCTCTTA
For GX27NS1R38A NS
mutation
5ACCGGCTTCGCGCAGATCAGAAGTC 5TAGGGACTTCTGATCTGCGCGAAGC
For GX27NS1K41A NS
mutation
5CGCCGAGATCAGGCGTCCCTAAGAG 5CTCTTAGGGACGCCTGATCTCGGCG
For GX27NS1R38AK41A
NS mutation
5TTCGCGCAGATCAGGCGTCCCTAAG 5CTTAGGGACGCCTGATCTGCGCGAA
a
The nucleotides that have been changed are underlined and in boldface.
VOL. 82, 2008 PATHOGENICITY OF H5N1 AVIAN INFLUENZA VIRUSES 1147
of the virus in a 50-l volume and calculated by using the method of Reed and
Muench (24). The chicken and mouse studies have been approved by the Review
Board of Harbin Veterinary Research Institute, Chinese Academy of Agricul-
tural Sciences.
Sequence analysis. Reassortant viruses and the plasmids used for virus rescue
were fully sequenced to confirm the absence of unwanted mutations. Viral RNA
was extracted from allantoic fluid and was reverse transcribed. A set of fragment-
specific primers (primer sequences available on request) were used for the PCR
amplification and sequence analysis. The sequence data for the two viruses used
in these studies are available in GenBank (accession no. pending).
Detection of IFN secretion. Monolayers of 80% confluent A549 cells were
infected at a multiplicity of infection (MOI) of 2. Following infection, cells were
incubated with Opti-MEM (Gibco/BRL) and the supernatants were harvested
24 h p.i. Viruses present in the supernatants were UV inactivated by placing
samples on ice 70 cm below a 30-W UV lamp for 20 min, and inactivation of the
virus was confirmed by egg propagation. The UV-inactivated supernatants were
then added to A549 cells and incubated for 24 h. The cells were then infected
with 0.001 MOI of VSV-GFP. At 14 h p.i., cells expressing GFP were visualized
by fluorescence microscopy.
Analysis of IFN-/ mRNA by RT-PCR. A549 cells were infected with influ-
enza viruses at an MOI of 2, and at 20 h p.i., total RNA was extracted and
digested with DNase I (Roche). RT-PCR was performed by using primer pairs
specific for human IFN- and human IFN- mRNA (GenBank accession num-
bers M54886, BC112302, and NM002176). A 550-bp fragment of human -actin
was amplified as a control. The products were sequenced and confirmed to be
derived from the expected mRNAs.
Quantification of IFN-/ production by ELISA. For quantification of se-
creted IFN-/, A549 cells were either mock infected or infected with different
H5N1 viruses at an MOI of 2. The supernatants were harvested 24 h p.i. Viruses
present in the supernatants were UV inactivated by placing samples on ice 70 cm
below a 30-W UV lamp for 20 min. Production of IFN-/ in culture superna-
tants was measured using a human IFN- or IFN- enzyme-linked immunosor-
bent assay (ELISA) kit (human IFN- and human IFN- ELISA kit; Adlitteram
Diagnostic laboratories Inc.) according to the manufacturer's instructions. For
each treatment, three sets of sample were collected, and each sample was tested
in duplicate by ELISA.
Western blot analysis of the viral protein levels in the virus-infected A549
cells. Six-well plates of 90% confluent A549 cells were mock infected or infected
with viruses at an MOI of 2. Cells were lysed 12 h p.i. and the lysates subjected
to Western blot analysis using mouse anti-truncated A/Goose/Guangdong/1/96
NS1 and chicken anti-A/Goose/Guangdong/1/96 NP antibodies and monoclonal
anti- actin antibody (Sigma) as the control.
Reporter gene assay. 293T cells were used for investigating NF-B promoter
activation, and Vero cells were used for the IRF-3-dependent promoter, ISG54,
activation assay. Cells were transfected with an NF-B-responsive promoter-
driven firefly luciferase reporter plasmid, pNF-B-Luc (Stratagene, La Jolla,
CA) or pISG54-Luc. In addition, an internal control plasmid to normalize trans-
fection efficiency, pTK-RL (Promega), encoding the Renilla luciferase protein,
was transfected into the cells. The reporter gene plasmids pNF-B-Luc or pISG-
54K-Luc and the pTK-RL plasmids were cotransfected at 0.5 g along with 4.0
g of the NS1 (wild-type or mutant) plasmid into 80% confluent cells by using
Lipofectamine 2000 (Invitrogen). At 24 h posttransfection, the cells were mock
treated or transfected with 40 g poly(I:C) (Amersham Pharmacia) by using
Lipofectamine 2000. At 24 h posttreatment, the cells were lysed and luciferase
activities were determined with a dual-luciferase reporter assay system (Pro-
mega) and normalized on the basis of the Renilla luciferase activities.
Nucleotide sequence accession numbers. The sequence data for the two vi-
ruses used in these studies have been deposited in GenBank under accession
numbers EV263342 to EV263357.
RESULTS
Biological properties of the two H5N1 avian influenza vi-
ruses isolated from ducks. We isolated two H5N1 viruses,
DK/12 and DK/27, from apparently healthy ducks in the
Guangxi province of China in 2003 during routine surveillance.
The pathogenicity analysis of these two viruses in chickens
(following the recommendation by the Office International
Des Epizooties [21]) revealed that the DK/27 virus killed all 10
chickens within 24 h and yielded an IVPI of 3 (with 3.0 being
the most pathogenic and 0 being the least pathogenic). Al-
though the DK/12 virus was milder than the DK/27 virus, it
killed 7 of 10 chickens within 10 days, and its IVPI value was
1.4. Therefore, both of the viruses were highly pathogenic for
chickens.
We then tested the virulence of these two viruses in a mam-
malian mouse model described previously (2). Three mice
from each group were killed on day 4 and day 6 after intranasal
inoculation of 106
EID50 of virus, and their organs were col-
lected for virus titration in eggs. The DK/12 virus replicated in
the mouse lungs without adaptation, and the mean titer
reached 3.9 log EID50 on day 4 p.i.; however, the virus was not
detected in any other organ tested (Fig. 1A). DK/12 virus
caused 5% body weight loss by day 9 p.i. at which point the
mice started to regain the weight over the course of the re-
maining observation period (data not shown). In contrast,
DK/27 caused a systemic infection, replicated to the high titer
of 7.3 log EID50 in the lungs and to the titers of 3.6, 2.9, and 2.2
log EID50 in the spleen, kidney, and brain, respectively (Fig.
1A; Table 2). Infection with DK/27 virus caused a more than
30% reduction in body weight, and all of the mice died before
day 8 p.i. The two H5N1 viruses markedly differed in the dose
FIG. 1. Replication and lethality of the DK/12 and DK/27 viruses
in mice. (A) Six-week-old SPF BALB/c mice (three/group) were inoc-
ulated intranasally with 106
EID50 of each virus in a 50-l volume and
killed on day 3 p.i., and organs were collected for virus titration in eggs.
Data shown are the mean virus titers  standard deviation. (B to E)
Death patterns of the mice infected with different H5N1 viruses,
DK/12 (B), DK/27(C), R-DK/12 (D), and R-DK/27 (E), with the doses
of 101
to 106
EID50 (101
EID50, F; 102
EID50, E; 103
EID50, ,; 104
EID50, OE; 105
EID50, ; 106
EID50, }).
1148 JIAO ET AL. J. VIROL.
required to kill 50% of infected mice (MLD50): 6.4 log EID50
for DK/12 and 0.6 log EID50 for DK/27 (Fig. 1B and C).
To determine the genetic relationship between the two viruses,
we sequenced their genomes and compared them with the avail-
able sequences of H5N1 viruses. We found that the two viruses
are closely related, with all of their eight segments sharing over
99% homology with the previously reported A/Duck/Fujian/01/02
virus (2). DK/12 and DK/27 share the same PB1, NP, M1, M2,
and NS2 genes at the amino acid levels. At the amino acid level,
we mapped a total of eight differences between the two viruses in
their PB2, PA, HA, NP, and NS1 genes (Table 3). These data
suggest that single- or multiple-amino-acid combinations among
these eight different amino acids contribute to the difference in
virulence in mice of the two viruses.
Rescued DK/12 and DK/27 viruses maintained the biologi-
cal properties of the wild-type viruses. To investigate the ge-
netic basis of the virulence of the DK/12 and DK/27 viruses, we
established a reverse genetics system for the two viruses. We
inserted cDNAs of each full-length RNA segment of DK/12
and DK/27 into the viral RNA-mRNA bidirectional expression
plasmid pBD, as described in Materials and Methods. Using
these plasmids, we generated the DK/12 and DK/27 viruses from
cloned cDNA, designated R-DK/12 and R-DK/27, respectively.
After confirmation by sequence analysis, we prepared virus stocks
by use of 10-day-old specific-pathogen-free (SPF) eggs and tested
the replications and lethalities of these viruses in mice. R-DK/12
and R-DK/27 exhibited properties similar to those of their re-
spective original viruses in terms of virus titers in organs and with
respect to MLD50s (Fig. 1D and E; Table 2).
The NS gene plays a major role in the difference in patho-
genicities in mice between the DK/12 and DK/27 viruses. To
identify the genes responsible for the difference in pathogenic-
ities between the DK/12 and DK/27 viruses, we generated five
single-gene recombinant viruses, each bearing the PB2, PA,
HA, NP, or NS gene from DK/27 and the other seven genes
from DK/12. The recombinant viruses that contained the PB2,
PA, HA, or NP gene of DK/27 (designated DK/12-27PB2,
DK/12-27PA, DK/12-27HA, or DK/12-27NP, respectively) dis-
played the same low pathogenicity in mice as the wild-type
DK/12 virus (MLD50, 6.4 log EID50) and replicated only in the
lungs (Table 2), although the virus titers in the lungs of mice
infected with DK/12-27PA or DK/12-27NP virus were signifi-
cantly higher than that of the DK/12 virus-infected mice. DK/
12-27PA also caused about 20% weight loss in inoculated mice,
whereas DK/12-27PB2, DK/12-27HA, and DK/12-27NP vi-
ruses caused only a transient reduction in body weight (Fig.
TABLE 2. Replication of transfectant viruses in micea
Virus
Mean virus titer (log10 EID50/ml  SD) at indicated day p.i. in:
MLD50
(log EID50)
Lung Spleen Kidney Brain
Day 4 p.i. Day 6 p.i. Day 4 p.i. Day 6 p.i. Day 4 p.i. Day 6 p.i. Day 4 p.i. Day 6 p.i.
DK/12 3.9  0.5 3.6  0.7       6.4
DK/27 7.3  0.1 6.9  0.4 3.6  0.3 2.0  0.9 2.9  0.9 3.0  0.1 2.2  0.8 3.1  0.1 0.6
R-DK/12 3.5  0.1 3.5  0.6       6.4
R-DK/27 7.2  0.3 6.9  0.4 3.8  0.3 1.8  0.6 2.7  0.7 2.9  0.1 2.0  0.5 2.8  0.3 0.6
DK/12-27PB2 4.5  0.5 4.5  0.7       6.4
DK/12-27PA 5.9  0.4c
4.1  1.0       6.4
DK/12-27HA 3.8  0.3 4.5  0.4       6.4
DK/12-27NP 5.1  0.3c
5.5  0.5b
      6.4
DK/12-27NS 5.8  0.6c
6.9  0.4c
2.3  0.1 2.6  0.3 1.6  0.3 1.6  0.5  3.1  0.3 2.0
DK/27-12PB2 5.6  0.5e
5.9  0.5 2.2  0.8e
2.8  0.4 2.0  0.5 2.9  0.5 1.5  0.1 2.9  0.5 0.6
DK/27-12PA 5.7  0.4e
6.4  0.1 2.1  0.6d
2.3  0.8 1.3  0.1d
1.9  0.4d
1.9  0.7 2.5  0.4 1.5
DK/27-12HA 6.8  0.3 5.8  0.5d
3.5  0.7 3.0  0.5d
2.4  0.1 2.5  0.4 1.4  0.1 2.4  0.9 1.5
DK/27-12NP 6.5  0.4 6.3  0.6 3.1  0.6 2.5  0.4 2.4  0.1 2.2  0.1 1.4  0.1 2.9  0.4 0.6
DK/27-12NS 3.9  0.5e
4.9  0.5e
      6.4
DK/12NS1P42S 6.5  0.4c
6.8  0.3c
2.0  0.7 2.0  0.7 1.3  0.1 1.3  0.1  3.3  0.8 2.2
DK/12NS1N48S 3.4  0.1 4.5  0.4       6.4
DK/27NS1S42P 4.5  0.4e
5.8  0.3d
      6.4
DK/27NS1S48N 7.3  0.1 7.5  0.1 2.5  1.1 2.8  0.8 1.7  0.7 3.1  0.8 2.2  0.9 3.1  0.3 0.8
DK/27NS1R38A 7.6  0.3 7.3  0.8 2.1  0.8 1.4  0.1 2.1  0.6 2.1  0.6 1.4  0.1 2.4  0.1 0.8
DK/27NS1K41A 6.6  0.3 6.5  0.4 1.7  0.4 1.4  0.1 1.6  0.3 2.2  0.3 1.6  0.5 2.9  0.4 0.6
DK/27NS1R38AK41A 5.0  0.5d
4.6  0.7e
      6.4
a
Six-week-old SPF BALB/c mice were inoculated intranasally with 106
EID50 of each virus in a 50-l volume. Three mice from each group were killed on days 4
and 6 p.i., and virus titers were determined in samples of lung, spleen, kidney, and brain in eggs. , no virus was isolated from the sample.
b
P value was 0.05 compared with the titers in the corresponding organs of the DK/12- or R-DK/12-inoculated mice.
c
P value was 0.01 compared with the titers in the corresponding organs of the DK/12- or R-DK/12-inoculated mice.
d
P value was 0.05 compared with the titers in the corresponding organs of the DK/27- or R-DK/27-inoculated mice.
e
P value was 0.01 compared with the titers in the corresponding organs of the DK/27- or R-DK/27-inoculated mice.
TABLE 3. Amino acid differences between the DK/12 and
DK/27 viruses
Gene
segment
Position of
amino acid
Amino acid in:
DK/12 virus DK/27 virus
PB2 497 Ser (S) Asn (N)
607 Leu (L) Val (V)
612 Ala (A) Thr (T)
PA 44 Glu (G) Val (V)
HA 216 Val (V) Ile (I)
NP 105 Met (M) Val (V)
NS1 42 Pro (P) Ser (S)
48 Asn (N) Ser (S)
VOL. 82, 2008 PATHOGENICITY OF H5N1 AVIAN INFLUENZA VIRUSES 1149
2A). In contrast to the other single-gene reassortants, the sin-
gle-gene recombinant containing the NS gene of DK/27 (DK/
12-27NS) caused systemic infection (Table 2). The MLD50 of
the DK/12-27NS virus was over 104
-fold higher than that of the
DK/12 virus (MLD50, 2.0 versus 6.4 log EID50).
The effect of individual genes derived from the DK/12 virus
on the virulence of DK/27 virus was also examined by gener-
ating five single-gene recombinant viruses, each containing the
PB2, PA, HA, NP, or NS gene from DK/12 virus and the
remaining segments from DK/27 virus. The viruses that carried
the PB2, PA, HA, and NP gene of DK/12 (DK/27-12PB2,
DK/27-12PA, DK/27-12HA, and DK/27-12NP, respectively)
replicated in all four organs tested and caused rapid weight
loss. The mice died within 9 days of inoculation (Fig. 2B; Table
2), although the virus titers in the lungs of DK/27-12PB2- and
DK/27-12PA-inoculated mice were significantly lower than
that of the DK/27 virus-inoculated animals. The MLD50s of
these four recombinant viruses were similar to that of the
DK/27 virus (ranging from 0.6 to 1.5 log EID50). The recom-
binant virus containing the NS gene of DK/12 in a background
of genes from the DK/27 virus (DK/27-12NS), however, repli-
cated only in the lungs, and the virus was dramatically atten-
uated in mice (MLD50, 0.6 versus 6.4 log EID50). In addition,
DK/27-12NS caused a reduction in the body weight of the mice
over the first 10 days after infection, but the mice regained
weight over the remaining observation period (Fig. 2B). These
results indicated that the NS gene plays a major role in the
difference in pathogenicities in mice between the DK/12 and
DK/27 viruses.
Amino acid substitution at position 42 in the NS1 protein changes
the pathogenicity of the DK/12 and DK/27 viruses in mice. There
are only two amino acid differences in the NS gene between
DK/12 and DK/27, and both of the changes are located in the
NS1 protein; that is, at positions 42 and 48 (Table 3). To
elucidate the molecular basis of the virulence and replication
discrepancy between the DK/12 and DK/27 viruses, we gener-
ated four mutant viruses each containing a substitution of the
NS1 amino acid residues at position 42 or 48 and tested their
pathogenicities in mice. A mutant DK/12 virus, designated
DK/12NS1P42S and containing a substitution at amino acid
position 42 that reflected the sequence found in the DK/27
NS1 protein (a Pro-to-Ser substitution), was highly pathogenic
in mice, in contrast to the DK/12 virus (MLD50, 2.2 versus 6.4
log EID50). This mutant caused systemic infection in animals
(Fig. 3; Table 2). A mutant DK/12 virus encoding an Asn-to-
Ser mutation at position 48 of NS1 (DK/12NS1N48S) was not
lethal and replicated only in the lung of mice (MLD50, 6.4 log
EID50) (Fig. 3; Table 2). Similarly, a Ser-to-Pro substitution at
position 42 in the NS1 protein of DK/27 that reflected the
sequence found in the DK/12 virus resulted in marked atten-
uation of the DK/27 virus (MLD50, 0.6 versus 6.4 log EID50).
This mutant, DK/27NS1S42P, replicated only in the lung and
caused a transient reduction in the body weight of the mice
(Table 2; Fig. 3). However, the mutant virus DK/27NS1S48N,
containing a Ser-to-Asn mutation introduced at position 48 of
the NS1 protein, was not attenuated with respect to the DK/27
virus. These results suggest that the amino acid at position 42
of NS1 protein is critical for the difference in virulence be-
tween the DK/12 and DK/27 viruses in mice.
The genomes of the viruses recovered on days 4 and 6 p.i.
from the lungs of mice infected with the NS reassortants or
mutant viruses were sequenced. No amino acid residue change
was found in any of the gene segments except for PB2, for
which we found that a mutation corresponding to a change
FIG. 2. Comparison of weight changes in mice infected with differ-
ent H5N1 avian influenza viruses. Mice (five mice/group) were intra-
nasally infected with 106
EID50 of virus. (A) Mice infected with R-
DK/12 and the reassortants in the DK/12 background. (B) Mice
infected with R-DK/27 and the reassortants in the DK/27 background.
FIG. 3. NS1 mutant viruses and their virulences in mice. The color
of the bar indicates the origin of the gene as follows: blue, DK/12; red,
DK/27. The corresponding amino acids are shown as single-letter ab-
breviations with the positions numbered at the top. The red dots in the
mouse figures indicate tissue tropism (upper left, brain; lower left,
lung; upper right, kidney; lower right, spleen). The mutated amino
acids are shown in red or blue and italics. Amino acid abbreviations: R,
Arg; K, Lys; P, Pro; S, Ser; A, Ala.
1150 JIAO ET AL. J. VIROL.
from Glu to Lys at position 627 occurred in about 30% of the
DK/12-27NS and DK/12NS1P42S viruses isolated from inocu-
lated mice on day 6 p.i. but not from any samples recovered
from other virus-inoculated mice (Table 4).
The amino acid at position 42 of the NS1 protein affects
DK/12 and DK/27 virus antagonization of IFN-/ action in
human lung epithelial A549 cells. The amino-terminal 73
amino acids of the NS1 protein of influenza virus are respon-
sible for binding to RNAs, in particular dsRNA, conferring
upon the virus the ability to escape the IFN-/ response by
inhibiting the activation of transcription factors involved in
IFN activation (10, 18). To determine whether the difference in
the replication and virulence of DK/12 and DK/27 was directly
correlated with the abilities of these viruses to inhibit the
IFN-/ system, the IFN-inducing properties of influenza vi-
ruses expressing wild-type or mutant NS1 proteins were inves-
tigated. Human epithelial lung A549 cells were infected at an
MOI of 2 with wild-type DK/27, wild-type DK/12, mutant DK/
27NS1S42P virus, or mutant DK/12NS1P42S virus. At 24 h
postinfection, supernatants from infected A549 cells were used
to determine the levels of secreted IFN-/ in a bioassay based
on the inhibition of VSV-GFP viral replication. The results are
shown in Fig. 4. Supernatants from mock-infected cells caused
no inhibition of GFP expression by VSV-GFP in A549 cells.
FIG. 4. Induction of IFN-/ synthesis in A549 cells infected with influenza A viruses expressing wild-type or mutant NS1 proteins. (A) IFN-/
bioassay. A549 cells were pretreated for 24 h with UV-inactivated supernatants from A549 cells infected with the indicated influenza viruses. The
pretreated A549 cells were then infected with VSV-GFP, and, 14 h p.i., the cells expressing GFP were monitored by fluorescence microscopy.
(B) RT-PCR analysis of IFN-/ mRNA levels in virus-infected A549 cells. Cells were infected at an MOI of 2, and, 20 h p.i., total RNA was
extracted and RT-PCR was done using primer pairs specific for human IFN-/ and -actin mRNA. The fragments of IFN-, IFN-, and -actin
are shown on the left. (C) Quantification of IFN-/ production. A549 cells were either mock infected or infected at an MOI of 2. Twenty-four
hours postinfection, the amounts of IFN- and IFN- released into the culture supernatant were measured by ELISA. (D) Levels of virus protein
expression in infected cells. A549 cells were infected with virus at an MOI of 2 and lysed 12 h postinfection. Lysates of mock-infected cells or of
cells infected with the indicated influenza viruses were incubated with mouse anti-truncated NS1 antiserum (I) or with chicken antiserum that was
generated by inoculating SPF chickens with inactivated GS/GD/1/96 virus (II). Expression of -actin protein was also examined as a control (III).
Binding was visualized with DAB (3,3-diaminobenzidine) reagent after incubation with peroxidase-conjugated secondary antibodies. The NS1,
NP, and -actin proteins are indicated on the right. Numbers 1 to 5 in panels B, C, and D indicate the type of infection as follows: 1, mock; 2,
R-DK/27; 3, R-DK/12; 4, DK/27NS1S42P; 5, DK/12NS1P42S.
TABLE 4. Amino acids at the key positions of the PB2 and NS1 genes of the viruses recovered from mice
Virus
Amino acid at indicated key position of the viruses recovered from the mice on:
Day 4 p.i Day 6 p.i.
PB2 NS1 PB2 NS1
627 701 42 48 627 701 42 48
R-DK/12 E D P N E D P N
R-DK/27 E D S S E D S S
DK/12-27NS E D S S E (67%)  K (33%) D S S
DK/27-12NS E D P N E D P N
DK/12-NS1P42S E D S N E (67%)  K (33%) D S N
DK/12-NS1N48S E D P S E D P S
DK/27-NS1S42P E D P S E D P S
DK/27-NS1S48N E D S N E D S N
VOL. 82, 2008 PATHOGENICITY OF H5N1 AVIAN INFLUENZA VIRUSES 1151
However, VSV-GFP replication was completely abolished in
cells pretreated with the supernatant of wild-type DK/12- and
mutant DK/27NS1S42P-infected cells. By contrast, the super-
natant of wild-type DK/27- or mutant DK/12NS1P42S-infected
cells did not inhibit VSV-GFP replication (Fig. 4A).
To determine whether the induction of the antiviral state in
the treated cells correlated with the level of IFN-/ induction
in A549 cells infected with different viruses, the relative levels
of IFN-/ mRNA in the infected A549 cells were examined
by RT-PCR. A549 cells were infected with various influenza
viruses, and the cells were harvested for RNA extraction 20 h
after infection. Total RNA was digested with DNase I to re-
move DNA from RNA samples. RT-PCR analysis confirmed
that infection with wild-type DK/12 or mutant DK/27NS1S42P
virus induces higher levels of IFN-/ mRNA than infection
with wild-type DK/27 or mutant DK/12NS1P42S virus (Fig.
4B). The PCR products were sequenced and confirmed to be
derived from the expected mRNAs. We further investigated
the expression and secretion of the IFN-/ for A549 cells
infected by different viruses. The culture supernatants were
harvested 24 h after infection, and the amounts of IFN- and
IFN- were measured by ELISA. The results confirmed that
infection with wild-type DK/12 or mutant DK/27NS1S42P vi-
rus induces higher levels of IFN-/ expression and secretion
than infection with wild-type DK/27 or mutant DK/12NS1P42S
virus (Fig. 4C).
Since previous reports indicated that the ability of the influ-
enza virus to antagonize IFN-/ induction by the host was
related to NS1 protein expression levels in the infected cells
(17, 28), we compared the levels of NS protein expressed in
virus-infected cells by Western blotting (Fig. 4D). No signifi-
cant difference in the levels of viral proteins, including NS1 and
NP, was detected among the samples.
The amino acid at position 42 is critical for the NS1 protein
to inhibit dsRNA-mediated NF-B-responsive promoter acti-
vation. Activation of NF-B and IRF-3 is required for the
induction of the IFN- promoter (6, 25, 26, 32, 36). NF-B has
been shown to bind to the positive regulatory domain II of the
IFN- promoter and to play an essential role in regulating
IFN- transcription (1, 8). Expression of the NS1 protein of
influenza A virus is known to prevent virus- and/or dsRNA-
mediated activation of the NF-B pathway (35). To determine
whether the contribution of the NS1 protein to the pathoge-
nicities of the DK/12 and DK/27 viruses is associated with
blocking the activation of the NF-B pathway, we used an
NF-B reporter gene, pNF-B-Luc, which contains a lucifer-
ase reporter gene under the control of an NF-B-responsive
promoter. Cotransfection of pNF-B-Luc with expression plas-
mid p12NS1 or p27NS1S42P did not inhibit the expression of
dsRNA [poly(I:C)]-induced reporter gene activity in 293T
cells, whereas cotransfection of p27NS1 or p12NS1P42S sub-
stantially reduced it (Fig. 5A). These results demonstrate that
the NS1 proteins of DK/12 and DK/27 viruses differ in their
abilities to prevent the dsRNA-mediated activation of the
NF-B pathway, and the amino acid at position 42 in NS1 is
critical for this function. The difference in reporter gene in-
duction was not due to differences in NS1 protein levels, as
Western blotting demonstrated that wild-type and mutant NS1
proteins were expressed in 293T cells at similar levels (data not
shown).
The amino acid at position 42 is critical for the NS1 protein
to inhibit dsRNA-mediated IRF-3-dependent promoter activa-
tion. NS1 protein of influenza A virus has been reported to
inhibit the activation of IRF-3, which is a key regulator of IFN
gene expression (32). The ISG54 promoter is one of the pro-
moters that can be directly activated by the IRF-3, and it could
also be activated by IFN produced in response to dsRNA
treatment or to viral infection. To investigate if the amino acid
change in 42 affects the ability of NS1 protein to inhibit the
activation of the IRF-3-dependent promoter, we transfected
the Vero cells, which do not produce IFN-/, with a ISG54-
Luc reporter plasmid plus a constitutively expressed Renilla
luciferase plasmid and a mammalian expression plasmid. The
expression plasmid was empty vector pCAGGS or the
pCAGGS plasmid expressing the wild-type or mutant NS1
gene. Cotransfection of pISG54-Luc with the plasmid p12NS1
or p27NS1S42P did not inhibit the expression of dsRNA
[poly(I:C)]-induced reporter gene activity in Vero cells,
whereas cotransfection of p27NS1 or p12NS1P42S substan-
tially reduced it (Fig. 5B). These results demonstrate that the
NS1 proteins of DK/12 and DK/27 viruses differ in their abil-
ities to inhibit the dsRNA-mediated activation of the IRF-3-
dependent promoter, and the amino acid at position 42 in NS1
is critical for this function. Western blotting demonstrated that
FIG. 5. Prevention of poly(I:C)-induced activation of an NF-B
promoter and the IRF-3-depedent promoter by NS1 protein.
(A) NF-B promoter assay. 293T cells were cotransfected with pNF-
B-Luc and pTK-RL plasmids along with the specified NS1 plasmids,
with or without subsequent poly(I:C) transfection. (B) ISG-54 pro-
moter assay. Vero cells were cotransfected with pISG-54-Luc and
pTK-RL plasmids along with the specified NS1 plasmids, with or with-
out subsequent poly(I:C) transfection. Bars: 1, pCAGGS; 2, pCAGGS
with poly(I:C); 3, p12NS1 with poly(I:C); 4, p27NS1 with poly(I:C); 5,
p12NS1P42S with poly(I:C); 6, p27NS1S42P with poly(I:C).
1152 JIAO ET AL. J. VIROL.
wild-type and mutant NS1 proteins were expressed in Vero
cells at similar levels (data not shown).
At least one of the two basic amino acids at position R38 or
K41 in the NS1 protein is required for the virulence of the
DK/27 virus in mice. Amino acids R38 and K41 in the RNA-
binding domain of the NS1 protein have previously been
shown to be important in the inhibition of IFN production and
the virulence of the influenza viruses (6, 19). Both of our H5N1
avian influenza viruses contain R38 and K41 in the NS1 pro-
tein. To investigate whether these two basic amino acids con-
tribute to virulence, we generated three NS1 mutant viruses in
the DK/27 background, DK/27R38A, DK/27K41A, and DK/
27R38AK41A, and tested their replications and virulences in
mice. As shown in Table 2 and Fig. 3, the mutants with a
single-amino-acid substitution, DK/27R38A and DK/27K41A,
replicate systemically and were highly virulent in mice, with
MLD50s of 0.8 and 0.6 log EID50, respectively. However, the
double mutant DK/27R38AK41A replicated only in the lungs
and was highly attenuated in this animal (MLD50, 6.4 log
EID50) (Fig. 3; Table 2). DK/27R38AK41A still has Ser at
position 42 of NS1. These results demonstrate that the amino
acid S42 and at least one of the basic amino acids at position
R38 or K41 in NS1 are required for the virulence of DK/27 in
mice.
DISCUSSION
H5N1 avian influenza viruses have caused the deaths of
more than half of the humans they have infected since 1997
and clearly represent a threat to public health. Although dif-
ferent strains of H5N1 virus are known to have substantially
different pathotypes, the effects of specific amino acid changes
on the host ranges and virulences of H5N1 HPAIV remain
largely unexplored. Here, we characterized two H5N1 HPAIV,
DK/12 and DK/27, which have similar genomes but show
markedly different pathogenicities in mice. Using this pair of
viruses and single-gene reassortant viruses created from them,
we demonstrated that the NS1 protein contributes to the dif-
ferent pathogenicities of these two viruses in mice and that the
amino acid residue at position 42 in the NS1 protein of DK/27
is important for its ability to replicate and cause lethality in
mice and to antagonize IFN-/ production in A549 cells. We
further demonstrated that the amino acid at position 42 of NS1
is critical for the ability of these viruses to inhibit dsRNA-
mediated activation of the NF-B pathway and the IRF-3
pathway. This is the first demonstration that a single amino
acid at position 42 of the NS1 gene plays a major role in the
determination of virulence of H5N1 avian influenza virus in a
mammalian host.
Mutation or deletions within the effector domain of the NS1
gene have previously been shown to affect the ability of influ-
enza viruses to antagonize IFN-/ production, and the de-
creased ability of the virus to antagonize IFN-/ production is
linked with reduced expression levels of the NS1 protein in
infected cells (17, 28). In this study, the mutation of amino acid
42 within the RNA-binding domain of NS1 affected the ability
of the virus to antagonize IFN-/ production in A549 cells,
suggesting that the RNA-binding domain also contributes to
the ability of the NS1 protein to antagonize IFN-/ produc-
tion in host cells. However, NS1 protein levels were not sig-
nificantly different among the four viruses tested (Fig. 4D),
suggesting that the mechanisms by which the NS1 effector
domain and the NS1 RNA-binding domain antagonize host
IFN-/ production are different. In addition, the mutation of
amino acid 42 within the RNA-binding domain of NS1 blocked
the virus from preventing the dsRNA-mediated activation of
the NF-B pathway and the IRF-3-dependent promoter, dem-
onstrating the importance of the ability of NS1 to counteract
the host cell antiviral immune response.
The amino acid at position 42 of NS1 varies among avian
influenza viruses; however, the serine at this position is highly
conserved in the human, swine, and equine influenza viruses
(2,561 of 2,564 influenza viruses isolated from mammalian
hosts have S42 in their NS1 genes). Based on the available
sequence information, all 135 H5N1 influenza viruses that have
been isolated from humans and other mammals have serine at
position 42 in the NS1 protein. The amino acid at position 42
is located within the second -helix spanning amino acids 30 to
50 of the RNA-binding domain of NS1 protein (34). Two basic
amino acids, R38 and K41, in this same region have previously
been reported to be important for inhibiting host IFN induc-
tion and virulence of influenza A viruses (6). Using WSN virus,
Donelan et al. demonstrated that the basic amino acid arginine
at position 38 and lysine at position 41 of NS1 play a critical
role in the inhibition of IFN production and in the virulence of
the virus in mice, and an attenuated mutant containing NS1,
R38AK41A, acquired increased virulence in mice as a result of
the mutation of NS1 S42G (6). A recent study (19) reported
that a mutant virus designated A/Udorn/72 and containing
alanine instead of asparagine at position 38 was highly atten-
uated in MDCK cells. Our data indicate that DK/27 viruses
containing the single substitution of alanine for either of the
basic amino acids at positions 38 and 41 alone were not atten-
uated in mice, but substitution of alanine at both positions led
to complete attenuation. Together with our finding of the
importance of the amino acid at position 42 in virulence, these
data emphasize the critical role of the second -helix structure
of the RNA-binding domain of NS1 for IFN antagonism and
for virulence.
A recent study reported that the RNA helicase enzymes
retinoic acid-inducible gene I (RIG-I) acts as a single-stranded
RNA sensor and a potential target of viral immune evasion,
and NS1 protein of influenza A virus blocks the RIG-I activa-
tion mediated by viral genomic single-stranded RNA bearing
5 phosphates (22). It remains to be investigated if the contri-
bution of the NS1 protein to the pathogenicities of the DK/12
and DK/27 viruses is associated with blocking the activation of
RIG-I induced by the viral genomic 5-phosphorylated RNA.
In summary, we demonstrate here that the amino acid at
position 42 of NS1 plays an important role in the ability of
H5N1 influenza viruses to antagonize the host IFN response
and for the virulence of H5N1 avian influenza virus in a mam-
malian host. We also attest that the RNA-binding domain of
the NS1 protein plays an important role in preventing the
dsRNA-mediated activation of the NK-B pathway and the
IRF-3 pathway. Our results provide strong additional evidence
that the NS1 protein is a virulence factor for H5N1 avian
influenza viruses and that multiple domains within this protein
may be suitable targets for the development of antiviral drugs
and attenuated vaccines.
VOL. 82, 2008 PATHOGENICITY OF H5N1 AVIAN INFLUENZA VIRUSES 1153
ACKNOWLEDGMENTS
We thank Gloria Kelly and Susan Watson for editing the manu-
script, Michael Whitt for providing the reverse genetics system for
generating the recombinant VSV-GFP, and Nancy Cox and Kanta
Subbarao for providing the plasmid pBD.
This work was supported by Chinese National S&T Plan Grant
2004BA519A-57, the Chinese National Key Basic Research Program
(973) 2005CB523005 and 2005CB523200; by grants-in-aid and a con-
tract research fund from the Ministry of Education, Culture, Sports,
Science and Technology, Japan, Program of Founding Research
Centers for Emerging and Reemerging Infectious Diseases; and by
National Institute of Allergy and Infectious Diseases Public Health
Service research grants.
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