JOURNAL OF VIROLOGY, June 2006, p. 5976–5983
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Vol. 80, No. 12
Properties and Dissemination of H5N1 Viruses Isolated during an
Influenza Outbreak in Migratory Waterfowl in Western China†
Hualan Chen,1* Yanbing Li,1Zejun Li,1Jianzhong Shi,1Kyoko Shinya,2,3Guohua Deng,1Qiaoling Qi,1
Guobin Tian,1Shufang Fan,1Haidan Zhao,1Yingxiang Sun,4and Yoshihiro Kawaoka2,5,6
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; Institute of Medical Sciences, University of Tokyo, Tokyo 108-8639, Japan2;
Avian Zoonosis Research Centre, Tottori University, Faculty of Agriculture, 4-101 Minami, Koyama-cho,
Tottori 680-8550, Japan3; Division of Animal Production and Veterinary Medicine Bureau of
Agri-Animal Production of Qinghai Province, 2 Jiaotong Road, Xining 810008,
People’s Republic of China4; Department of Pathobiological Sciences, School of
Veterinary Medicine, University of Wisconsin—Madison, 2015 Linden Drive,
Madison, Wisconsin 537065; and CREST, Japan Science and
Technology Agency, Saitama 332-0012, Japan6
Received 16 January 2006/Accepted 13 March 2006
H5N1 influenza A viruses are widely distributed among poultry in Asia, but until recently, only a limited
number of wild birds were affected. During late April through June 2005, an outbreak of H5N1 virus infection
occurred among wild birds at Qinghai Lake in China. Here, we describe the features of this outbreak. First
identified in bar-headed geese, the disease soon spread to other avian species populating the lake. Sequence
analysis of 15 viruses representing six avian species and collected at different times during the outbreak
revealed four different H5N1 genotypes. Most of the isolates possessed lysine at position 627 in the PB2 protein,
a residue known to be associated with virulence in mice and adaptation to humans. However, neither of the two
index viruses possessed this residue. All of the viruses tested were pathogenic in mice, with the exception of one
index virus. We also tested the replication of two viruses isolated during the Qinghai Lake outbreak and one
unrelated duck H5N1 virus in rhesus macaques. The Qinghai Lake viruses did not replicate efficiently in these
animals, producing no evidence of disease other than transient fever, while the duck virus replicated in
multiple organs and caused symptoms of respiratory illness. Importantly, H5N1 viruses isolated in Mongolia,
Russia, Inner Mongolia, and the Liaoning Province of China after August 2005 were genetically closely related
to one of the genotypes isolated during the Qinghai outbreak, suggesting the dominant nature of this genotype
and underscoring the need for worldwide intensive surveillance to minimize its devastating consequences.
Highly pathogenic H5N1 influenza viruses have repeatedly
caused serious outbreaks of disease at poultry farms since 1997
and pose a significant threat to human health due to their
ability to infect humans, resulting in high mortality (3, 16, 20).
Since late 2003, H5N1 viruses have spread in an unprece-
dented manner across Asia, resulting in more than 60 human
fatalities in Thailand, Vietnam, Cambodia, and Indonesia and
in the slaughter or infectious deaths of more than 150 million
birds. Despite extensive efforts to contain these outbreaks,
H5N1 viruses continue to circulate among poultry in Asia
(Office International des Epizooties [http://www.oie.int]) and
remain a threat to both veterinary and human public health. In
fact, the viruses have now spread to Europe, increasing the
likelihood of an H5N1 pandemic.
Wild aquatic birds harbor all 16 hemagglutinin (HA) and
all nine neuraminidase (NA) subtypes of influenza A virus
and therefore serve as the natural reservoir for this patho-
gen. Although influenza viruses in wild aquatic birds are
occasionally transmitted to avian (e.g., chickens and tur-
keys) and mammalian (e.g., humans, pigs, horses, minks,
whales, and seals) species, where they may produce out-
breaks of severe disease, they persist in evolutionary equi-
librium (stasis) in their natural reservoir and do not gener-
ally cause disease in wild waterfowl (22).
Highly pathogenic H5N1 viruses do not appear to have en-
tered the wild-bird populations to any appreciable extent until
late April to June 2005, when a large outbreak of H5N1 infec-
tion occurred in Qinghai Lake in western China (2, 12), a
major breeding site for migratory birds whose flyways extend to
Southeast Asia, India, Siberia, Australia, and New Zealand (4).
Initial reports (2, 12) of this outbreak identified a single intro-
duction of an H5N1 virus into four species of waterfowl, in-
cluding bar-headed geese (Anser indicus), brown-headed gulls
(Larus brunnicephalus), great black-headed gulls (Larus ich-
thyaetus), and great cormorants (Phalacrocorax carbo). The
virus was shown to be pathogenic in chickens and mice and was
shown to possess a Lys-to-Glu substitution at position 627 of
PB2, an alteration previously associated with high virulence in
mice and found only in H1N1 and H3N2 human isolates as well
as some H5N1 isolates from humans and tigers (7, 9, 10, 19, 23)
but not from wild birds.
In this report, we provide information on the magnitude of
* Corresponding author. Mailing address: Harbin Veterinary Research
Institute, 427 Maduan Street, Harbin 150001, People’s Republic of China.
Phone: 86-451-82761925. Fax: 86-451-82733132. E-mail: hlchen1@yahoo
† Supplemental material for this article may be found at http://jvi
the Qinghai Lake outbreak of H5N1 viral disease, the evolu-
tion of the viruses during the course of the outbreak, subse-
quent transmission to birds in other remote locations, and the
pathological properties of the viruses in animal models, includ-
ing nonhuman primates.
MATERIALS AND METHODS
Virus isolation and identification. Organ samples and cloacal swabs were
collected within 24 h from birds that had died and were inoculated into 10-day-
old embryonated specific-pathogen-free eggs for virus isolation, as described
previously (1). The HA and NA subtypes were determined by conventional
hemagglutination inhibition and neuraminidase inhibition tests as described pre-
viously (15). All experiments with the H5N1 isolates were performed in a bio-
safety level 3 laboratory, and animal experiments were performed in high-efficiency
particulate air-filtered isolators. Abbreviations of the isolates are given in Table 1.
Genetic and phylogenetic analyses. Viral RNA was extracted with the RNeasy
Mini kit (QIAGEN, Valencia, CA) and was reverse transcribed. PCR amplifi-
cation was performed by using segment-specific primers (primer sequences are
available upon request). The PCR products were purified with the QIAquick
PCR purification kit (QIAGEN) and sequenced by using the CEQ DTCS-Quick
Start kit on a CEQ 8000 DNA sequencer (Beckman Coulter). Sequence data
were compiled with the SEQMAN program (DNASTAR, Madison, WI), and
the phylogenetic analysis was carried out with the PHYLIP program of the
CLUSTALX software package (version 1.81), implementing a neighbor-joining
Animal experiments. The intravenous pathogenicity index (IVPI) of the iso-
lates in chickens was determined according to the recommendations of the Office
International des Epizooties (14). Briefly, groups of 10 specific-pathogen-free
6-week-old White Leghorn chickens housed in isolator cages were inoculated
intravenously with 0.2 ml of a 1:10 dilution of bacterium-free virus-containing
allantoic fluid (see Table 2 for titers of the isolates).
Groups of eight 7-week-old female BALB/c mice (Beijing Experimental An-
imal Center, Beijing, People’s Republic of China) were lightly anesthetized with
CO2and inoculated intranasally with 10650% egg infectious doses (EID50) of
the virus in a volume of 50 ?l. On day 3, three of the eight mice were euthanized
for virus titration. The remaining five mice were monitored daily for weight loss
and mortality. To determine the 50% mouse lethal doses (MLD50) of the viruses,
we inoculated six groups of mice (n ? 5 mice each) intranasally with 10-fold
serial dilutions of the virus containing 101to 106EID50in a 50-?l volume (1).
The MLD50was calculated by the method of Reed and Muench (17).
Three-year-old, colony-bred, female rhesus macaques (Macaca mulatta) were
individually placed in a negative-pressure isolator. The macaques were infected
intranasally with 107EID50of the viruses in 2 ml of phosphate-buffered saline
(four animals infected with A/bar-headed goose/Qinghai/1/05 [BHGs/QH/1/05],
four animals infected with A/great cormorant/Qinghai/3/05 [GC/QH/3/05], and
three animals infected with A/duck/Guangxi/35/2001 [H5N1] [DK/GX/35/01]).
One animal from each group was euthanized on days 4 and 7 postinfection by
exsanguination under ketamine anesthesia. Nasal swabs, bronchoalveolar lavage
specimens, and organs from the euthanized animals were collected for virus
titration and for histological and immunohistochemical examinations. The re-
maining animals were observed for 2 weeks. Sera collected from all animals prior
to infection and 2 weeks postinfection were used to detect anti-influenza virus
Histopathological studies. Samples of the lung, liver, spleen, kidney, pancreas,
heart, muscle, trachea, proventriculus, and large intestine from naturally infected
bar-headed geese and samples of tonsil, lung, liver, spleen, and heart from
nonhuman primates experimentally infected with the H5N1 viruses were fixed in
10% neutral buffered formalin solution at necropsy. They were dehydrated,
embedded in paraffin, and cut into 5-?m sections, which were then stained with
routine hematoxylin and eosin (HE). For viral antigen detection, sections were
processed for immunohistochemistry by a two-step dextran polymer method
(DAKO Japan Inc., Kyoto, Japan) using polyclonal rabbit antibody to an H5
virus. Nonhuman primate tissues infected with DK/GX/35/01 were subjected to
immunostaining to detect viral antigens; in these studies, polyclonal anti-H5 goat
serum was used as a primary antibody, and peroxidase-labeled anti-goat rabbit
serum served as a secondary antibody. Immunoreactions were visualized with
Nucleotide sequence accession numbers. The nucleotide sequences analyzed
in this study are available at the Influenza Sequence Database under accession
numbers ISDN137990 to ISDN138151.
Course of the avian influenza outbreak at Qinghai Lake.
The islets and wetlands of Qinghai Lake, located in western
China, are part of a protected natural reserve for wild birds.
TABLE 1. H5N1 viruses isolated from wild birds in Qinghai Lake
in the present study
A/great black-headed gull/
A/great black-headed gull/
A/great black-headed gull/
aIsolation date is shown as month/day/year.
TABLE 2. Lethality of the H5N1 avian influenza isolates in chickens and mice
Titer of virus stock
Virus titers in organs of mice (log10EID50/ml ? SD)a
Lung SpleenBrain Kidney
5.7 ? 0.6
4.3 ? 0.1
6.3 ? 0.9
5.6 ? 0.7
6.1 ? 0.2
5.9 ? 0.5
6.5 ? 0.7
6.7 ? 1.0
1.3 ? 0.1
2.0 ? 0.3
1.6 ? 0.3
1.6 ? 0.3
1.3 ? 0.0
1.5 ? 0.1
3.3 ? 0.3
2.2 ? 0.3
2.9 ? 0.7
1.6 ? 0.1
1.6 ? 0.1
1.6 ? 0.2
1.5 ? 0.1
2.9 ? 0.6
1.8 ? 0.2
2.5 ? 0.3
1.6 ? 0.1
1.8 ? 0.2
1.6 ? 0.2
1.8 ? 0.4
3.3 ? 0.3
aSix-week-old BALB/c female mice were infected intranasally with 106EID50of virus in a 50-?l volume. Three mice from each group were euthanized on day 3
postinoculation, and virus in the organs was titrated in eggs. SD, standard deviation; ?, virus was detected only in undiluted samples; ?, virus was not detected.
bThe MLD50dose was determined by inoculating groups of five 6-week-old female mice intranasally with 10-fold serial dilutions of each virus containing doses
ranging from 101to 106EID50in 50 ?l.
cThe IVPI was determined according to recommendations of the Office International des Epizooties (14).
VOL. 80, 2006ANALYSIS OF WILD BIRD H5N1 VIRUSES5977
More than 100,000 wild birds, representing 189 species, spend
the spring and summer at this reserve every year. Since the end
of April 2005, bar-headed geese arriving at Qinghai Lake from
southern Asia have shown signs of disease, including tremor
On 4 May 2005, two bar-headed geese (Anser indicus) were
found dead in the wetlands of Qinghai Lake, with 105 dead
geese reported on the following day (Fig. 1 and 2). On 13 May,
a total of 437 dead birds were collected. The species identified
extended to great black-headed gulls (Larus ichthyaetus) and
brown-headed gulls (Larus brunnicephalus), whose habitats on
the lake overlap closely with those of bar-headed geese. Dis-
ease signs and deaths were observed among ruddy shelducks
(Tadorna ferruginea) beginning on 13 May, with 90 and 12 dead
shelducks collected on 24 and 25 May, respectively. A limited
number of dead great cormorants (Phalacrocorax carbo), gath-
ered on two islets located 2 miles away from concentrations of
bar-headed geese and gulls, were first observed on 16 May, and
a large number of these birds were found dead on 24 to 26 May
and 1 June (Fig. 1 and 2). Altogether, 6,184 dead gulls, geese,
great cormorants, and ruddy shelducks were found from 4 May
to 29 June; bar-headed geese accounted for more than half of
this total. A limited number of whooper swans (Cygnus cygnus),
black-headed cranes (Grus nigricollis), and pochards (Aythya
ferina) also died during this outbreak.
Pathological examination. We observed severe hyperemia
and edema in the brain, hemorrhage and necrosis in the pan-
creas, and severe cloudy swelling of the kidneys in the bar-
headed geese, great black-headed gulls, brown-headed gulls,
and great cormorants that were examined immediately after
death. Histologic analysis of organ samples from two moribund
bar-headed geese revealed typical nonsuppurative encephalitis
FIG. 1. Course of migratory waterfowl deaths due to H5N1 viruses at Qinghai Lake. A total of 6,184 dead birds were collected from 4 May to
29 June 2005: 3,282 bar-headed geese, 929 great black-headed gulls, 570 brown-headed gulls, 1,302 great cormorants, and 145 ruddy shelducks.
FIG. 2. Pattern of spread of H5N1 viruses of different genotypes
among wild birds at Qinghai Lake. The disease began in bar-headed
geese, spread to brown-headed gulls and great black-headed gulls, and
then spread to great cormorants and ruddy shelducks. The viruses from
these species represent four genotypes, two of which appear to have
spread from bar-headed geese to the other three avian species, although
it is uncertain whether the genotype D virus originated in the bar-headed
5978 CHEN ET AL. J. VIROL.
characterized by perivascular cuffing of mononuclear cells, mi-
crogliosis, degeneration of nerve cells, and edema (Fig. 3a), all
indicative of viral infection. Influenza virus infection was con-
firmed by immunohistochemical studies with antibodies to an
H5 virus (Fig. 3b). There was also evidence of coagulative
necrosis with nonsuppurative inflammation in the pancreas
(Fig. 3c) as well as degenerative foci of cardiomyocytes with
nonsuppurative inflammation (Fig. 3d).
Virus isolation and identification. To isolate the virus, we
inoculated cloacal swab samples and homogenates of organs
from sick and dead birds into 10-day-old embryonated spe-
cific-pathogen-free chicken eggs. Fifteen H5N1 viruses were
isolated from multiple organs (brain, lung, spleen, kidney,
and intestine) and cloacal swabs of bar-headed geese, a
brown-headed gull, a great cormorant, great black-headed
gulls, and a whooper swan and from the feces of a ruddy
shelduck (Table 1).
Sequence analysis. To understand the genetic relationship
between the Qinghai Lake isolates and other H5N1 viruses, we
sequenced the entire genomes of the 15 isolates. Previously
published sequences of H5N1 viruses isolated from the same
outbreak by Chen et al. (2) and Liu et al. (12) were included in
the analysis for comparison. The HA, NA, and nucleoprotein
genes of all viruses isolated during the Qinghai Lake outbreak
were similar to each other and closely resembled those of the
A/chicken/Jiangxi/25/04 virus isolated during the 2004 out-
break in China (Fig. 4; also see Fig. S1 and S2a and b in the
supplemental material). As reported previously (2, 12), all of
these viruses had a series of basic amino acids at the HA
cleavage site (RRRKKR) characteristic of other influenza vi-
ruses that are highly pathogenic in chickens; they also had a
20-amino-acid deletion in the NA stalk (residues 49 to 68)
compared with the NA of the Goose/Guangdong/1/96 virus.
Unlike previous reports (2, 12), the PB2 genes of viruses
isolated during the Qinghai Lake outbreak differed from each
other (Fig. 4b). The PB2 genes of BHGs/QH/1/05 and A/bar-
headed goose/Qinghai/2/05 (BHGs/QH/2/05) viruses shared
99.8% identity but were less than 97% identical to those of the
other isolates. Phylogenetically, the PB2 genes of the Qinghai
isolates were divided into three clades. The A/ruddy shelduck/
Qinghai/1/05 (RS/QH/1/05) virus formed a clade by itself;
BHGs/QH/1/05 and BHGs/QH/2/05, both isolated early in the
FIG. 3. Histopathologic analysis of a moribund bar-headed goose from the H5N1 virus outbreak at Qinghai Lake. (a) Brain showing scattered
nonsuppurative inflammatory foci characterized by perivascular cuffing of mononuclear cells, microgliosis, degeneration of nerve cells, and edema
(HE stain). (b) Brain with numerous nerve and glial cells positive for viral antigen (brown pigments) by immunohistological staining with an
anti-H5 polyclonal antibody. (c) Pancreas showing scattered coagulative necrotic foci in parenchyma with nonsuppurative inflammation (HE stain).
(d) Heart showing small nonsuppurative inflammatory foci with degenerating cardiomyocytes (HE stain).
VOL. 80, 2006 ANALYSIS OF WILD BIRD H5N1 VIRUSES5979
Qinghai Lake outbreak, formed the second clade with A/per-
egrine falcon/Hong Kong/04 and A/chicken/Yamaguchi/7/04
(CK/Yamaguchi/7/04) viruses; and the remaining isolates, in-
cluding those reported by previously by Chen et al. (2) and Liu
et al. (12), formed the third clade (Fig. 4b). The PB2 protein of
all the Qinghai Lake viruses in the last clade had a lysine at
position 627, which is conserved in authentic human viruses
and is associated with the high virulence of H5N1 viruses in
mice (8). Interestingly, however, two of the index isolates in the
second clade, together with other viruses in this clade and in
clade 1, contain glutamic acid at position 627.
The phylogenetic trees of the PA (Fig. 4c), PB1, M, and NS
genes (see Fig. S2c to e in the supplemental material) of these
H5N1 viruses were similar to each other. BHGs/QH/2/05 and
CK/Yamaguchi/7/04 were closely related to each other and
formed a single clade, while the remaining viruses formed
another clade. The PA gene of the BHGs/QH/2/05 virus shared
less than 93% identity with other wild-bird viruses isolated at
Qinghai Lake but was 98.2% identical to the corresponding
gene of the CK/Yamaguchi/7/04 virus. The PB1 gene of the
BHGs/QH/2/05 virus shared less than 93% identity with the other
wild-bird viruses but was 98.7% identical to that of the CK/
Yamaguchi/7/04 virus. Thus, the Qinghai Lake isolates repre-
sented four genotypes, genotypes A to D (Fig. 2). The BHGs/
QH/2/05 (genotype B) isolate shares the PB2 gene with genotype
A viruses, but four of its other genes appear to be unique among
the wild-bird viruses detected in this study.
Experimental infection of chickens, mice, and nonhuman
primates. Using chickens, we next tested the pathogenicity of
eight H5N1 viruses, including at least one virus of each geno-
type, according to recommendations of the Office Interna-
tional des Epizooties (14). All viruses killed chickens within
24 h and had an intravenous pathogenicity index of 3.0, the
highest value possible (Table 2). Similarly, in mice, all isolates,
with the exception of BHGs/QH/2/05, were highly lethal when
administered intranasally with an MLD50of less than 0.5 log
EID50(Table 2). This group of lethal viruses was readily re-
covered from each of the organs tested, indicating its ability to
cause systemic infection.
To test the pathogenic potential of these isolates in pri-
mates, we intranasally infected rhesus macaques with 107
EID50s of BHGs/QH/1/05 or GC/QH/3/05. Half of the animals
infected with either virus showed an increased body tempera-
ture for 1 to 3 days (see Fig. S3a and b in the supplemental
material) and anorexia on day 1 or 2 postinfection. Increased
respiratory rates were observed on days 5 to 6 postinfection in
all animals, but none died or showed severe symptoms during
the 2-week observation period. Surprisingly, virus was not re-
covered by nasal swabs, lung lavages, or organ samples col-
lected on day 4 or 7 postinfection from animals infected with
either virus (Table 3), even though sera collected at 2 weeks
FIG. 4. Phylogenetic analyses of the H5N1 viruses isolated during the Qinghai Lake outbreak. The phylogenetic trees were generated with the
PHYLIP program of the CLUSTALX software package (version 1.81) by using the neighbor-joining algorithm and bootstrap values of 1,000. (a)
HA (nucleotides 105 to 1659); (b) PB2 (nucleotides 82 to 2264); (c) PA (nucleotides 67 to 2151). The phylogenetic tree of HA was rooted to
A/mallard/Denmark/64650/03 (H5N7), and the PB2 and PA phylogenetic trees were rooted to A/Memphis/1/90 (H3N2). The sequences of the
wild-bird viruses obtained in this study are shown in red, and the viruses isolated after the Qinghai Lake outbreak are shown in green, while those
of the wild-bird viruses reported previously by Liu et al. and Chen et al. are shown in blue. Dates of virus isolation during Qinghai Lake outbreak
are also shown.
TABLE 3. Titers of viruses isolated from rhesus macaques
infected with H5N1 avian influenza virusesa
Virus titers in organs of nonhuman primates
Tonsil Lung Spleen Liver Heart
aThree-year-old, colony-bred, female rhesus macaques (Macaca mulatta)
were infected intranasally with 107EID50of the viruses in 2 ml of phosphate-
buffered saline. One animal from each group was euthanized on days 4 and 7
postinfection by exsanguination under ketamine anesthesia. Nasal swabs, bron-
choalveolar lavage specimens, and organs from the euthanized animals were
collected for virus titration. ?, virus was not detected; ND, not determined; ?,
virus was detected only in the original samples.
5980 CHEN ET AL.J. VIROL.
postinfection had antibody titers of more than 1:2,000 as de-
termined by an enzyme-linked immunosorbent assay.
By contrast, when infected with the DK/GX/35/01 H5N1
virus isolated from a healthy duck in Guangxi Province in 2001
during routine surveillance (1, 11), all three macaques devel-
oped fever within the first 2 days postinfection (see Fig. S3c in
the supplemental material) and became anorexic on day 1.
Two of the animals recovered on day 3, while the third animal
remained ill until day 6. The DK/GX/35/01 virus was isolated
from nasal swabs, lung lavages, and samples of lung, spleen,
liver, and heart from animals euthanized on day 4 and from the
lungs of animals euthanized on day 7, but it was not recovered
from other organs tested (cerebellum, cerebrum, kidney, pan-
creas, intestines, inguinal lymph nodes, mesenteric lymph
gland, and blood). These findings confirm the susceptibility of
rhesus macaques to avian H5N1 virus infection and indicate
that the DK/CX/35/01 virus, but not BHGs/QH/1/05 and GC/
QH/3/05, can cause systemic disease in nonhuman primates.
Necropsy of macaques on day 7 postinfection revealed a
spectrum of macroscopic lesions in the lungs. A GC/QH/3/05
virus-infected animal had focal discoloration in the medial
lobe, and large foci of consolidation on the accessory and lower
lobes in a BHGs/QH/1/05 virus-infected animal were seen,
together with prominent swelling of the lymph nodes, while
extensive pulmonary consolidation was apparent on the medial
and lower lobes in DK/GX/35/01 virus-infected animals. His-
tologically, the consolidated area seen in the BHGs/QH/1/05-
infected macaque was consistent with prominent features of
bronchointerstitial pneumonia with massive recruitment of
lymphocytes (Fig. 5a). Prominent features within and on the
periphery of the lesions included proliferative and reactive
hyperplasia of alveolar cells (Fig. 5b), an accumulation of
foamy macrophages in alveolar spaces (Fig. 5c), severe alveo-
lar edema (Fig. 5d), and thickening of alveolar walls with
lymphocyte recruitment (Fig. 5d, arrows). In one of the ani-
mals infected with the DK/GX/35/01 virus, foci of peribronchi-
olitis detected at 4 days postinfection (Fig. 5e) had progressed
to a massive accumulation of foamy macrophages within alve-
olar spaces and severe alveolar edema by 7 days postinfection
(Fig. 5f), but lymphocyte recruitment into the alveolar wall and
regenerative changes of alveolar cells were rare compared with
findings in the BHGs/QH/1/05-infected macaque. In sharp
contrast to these observations, the lungs of animals infected
with the GC/QH/3/05 virus had only small lesions consisting of
FIG. 5. Histological findings from rhesus macaques infected with H5N1 viruses. (a) Section from a consolidated area from lungs shows
bronchointerstitial pneumonia with severe infiltration of inflammatory cells (BHGs/QH/1/05 virus, day 7 postinfection) (HE stain). The lung lesions
were distributed around the bronchioli. Asterisks indicate lumen of bronchioli. (b) Severe alveolar damage was observed within and along the
periphery of the consolidated area (BHGs/QH/1/05 virus, day 7 postinfection) (HE stain). Severe proliferative and reactive hyperplasia of alveolar
cells with massive recruitment of lymphocytes, fibrin exudates, and alveolar edema are shown. (c) A strong reaction with macrophages was one of
the prominent findings in the lungs (BHGs/QH/1/05 virus, day 7 postinfection) (HE stain). (d) Severe alveolar edema, thickening of alveolar wall
with lymphocyte recruitment (white arrow), and regeneration of alveolar cells (black arrow) were also observed (BHGs/QH/1/05 virus, day 7
postinfection) (HE stain). (e) The lung lesions were detected as peribronchiolitis in a macaque infected with DK/GX/35/01 virus at 4 days
postinfection (DK/GX/35/01 virus, day 4 postinfection) (HE stain). The asterisk indicates lumen of bronchioles. (f) Prominent alveolar edema and
strong reaction with foamy macrophages but scant regenerative change and scant lymphocytic recruitment in a macaque infected with DK/GX/
35/01 virus (DK/GX/35/01 virus, day 7 postinfection) (HE stain). (g) Viral antigens in tonsilar epithelium on day 4 postinfection (brown)
(BHGs/QH/1/05 virus, day 4 postinfection) (immunohistochemistry).
VOL. 80, 2006 ANALYSIS OF WILD BIRD H5N1 VIRUSES5981
focal alveolitis accompanied by hyperplasia of alveolar cells. In
extrapulmonary organs, suppurative tonsillitis and systemic ac-
tivation of lymph follicles were prominent by 4 days postinfec-
tion in the animal infected with BHGs/QH/1/05 virus. Viral
antigens could be detected only in tonsilar epithelium on day 4
postinfection in animals infected with BHGs/QH/1/05 or GC/
QH/3/05 (Fig. 5g); however, animals infected with DK/GX/
35/01 showed positive reactions to anti-H5 serum in several
tissues, including tonsil, lung, and spleen (data not shown).
These results demonstrate the varied pathogenic potential in
primates of the H5N1 viruses isolated from waterfowl.
Subsequent spread of the Qinghai Lake-like viruses. Subse-
quent to the outbreak in Qinghai Lake from April to June
2005, H5N1 viruses have continued to cause outbreaks in Asia
and have now even caused outbreaks in Europe (WHO report,
http://www.who.int). We sequenced the entire genome of some
H5N1 viruses isolated from wild birds in Mongolia in August
2005 and chickens during major outbreaks in the Liaoning
Province and Inner Mongolia (see Fig. S1 in the supplemental
material) in October and November 2005, respectively. Phylo-
genetic analysis of these viruses and a virus isolated from a wild
bird in Russia in August 2005 (GenBank access numbers
DQ230521, DQ230523, DQ234073, DQ230575, DQ230577,
DQ232605, DQ232607, and DQ232609) showed that these vi-
ruses belong to genotype C (Fig. 2; see Fig. S2 in the supplemen-
tal material); moreover, all of these viruses possessed Lys at
position 627 in PB2. Taken together, these findings suggest that
H5N1 viruses have spread to wild-bird populations and have been
Here, we report a detailed analysis of a large outbreak of
H5N1 avian influenza virus occurring in migratory waterfowl
from late April through June 2005 in the Qinghai Lake region
of western China. In contrast to previous reports of viruses
isolated during this outbreak (2, 12), our studies reveal a
marked heterogeneity among the causative viruses. For exam-
ple, sequence analyses of 15 viruses isolated from six different
avian species showed that at least four genotypes were respon-
sible for the outbreak. All of the viruses were highly lethal to
chickens, and seven of the eight test viruses replicated sys-
temically and were highly lethal in mice. We also found that
the viruses isolated early in the outbreak possessed a typical
avian virus signature amino acid at position 627 of PB2, Glu,
unlike later isolates, which had Lys at this position (2, 12).
Moreover, these index viruses possessed a phylogenetically
distinct PB2 gene compared with those of other Qinghai
isolates. This suggests that the virus introduced into the
Qinghai Lake waterfowl population may have possessed the
index virus-like PB2 gene but that during the outbreak, it
acquired a PB2 gene with Lys at position 627. Alternatively,
the newly introduced virus may have already possessed PB2
with Lys at this position, but viruses with the mutant type of
PB2 were not detected until 10 May.
A distinct temporal pattern of infection of different avian
species by the H5N1 viruses was also apparent (Fig. 1). Bar-
headed geese were the first species to be affected, followed by
brown headed gulls and great black-headed gulls about 10 days
later (13 May) and then by ruddy shelducks and great cormo-
rants after another 10 days (24 and 25 May). The time between
the detection of small numbers of deaths (13 and 16 May) and
the detection of considerably larger numbers of deaths (24 and
26 May) of ruddy shelducks and great cormorants was also
about 10 days. These findings could be interpreted to indicate
stepwise introduction of the virus into different avian species in
the lake. Our sequence analyses revealed that at least three
genotypes of H5N1 viruses were circulating among bar-headed
geese, while the viruses isolated from great black-headed gulls,
brown-headed gulls, great cormorants, and whooper swans
were similar to each other and belonged to only one of the
genotypes found in bar-headed geese (Fig. 4). Viruses repre-
senting genotypes A and B were isolated from the bar-headed
geese that died early during the outbreak and were likely not
spread to other species. We speculate that viruses of genotype
D may also have been present in bar-headed geese at the
beginning of the outbreak but were not identified because of
the limited number of dead birds analyzed.
The origin of the virus responsible for the Qinghai Lake
outbreak remains unclear. The disease was first recognized in
bar-headed geese (Fig. 1 and 2), suggesting at least two possi-
ble mechanisms for the introduction of the H5N1 virus into
wild-bird populations by this species using the lake as a habitat.
One possibility is that the virus was carried to the lake by other
wild birds not susceptible to H5N1 infection and was then
transmitted to bar-headed geese. Another possibility is that
bar-headed geese infected elsewhere were the species that
brought the virus to Qinghai Lake, presumably via the East
Asian-Australian flyway or the Central Asian-Indian flyway. If
the first scenario is correct, the virus should have been trans-
mitted to all susceptible species at the same time, including
brown-headed gulls and great black headed gulls, which con-
gregate with bar-headed geese in the islet where H5N1 virus-
infected bar-headed geese were found. The fact that the dis-
ease was identified in these two species of gulls approximately
10 days after the discovery of fatal cases of H5N1 infection
among bar-headed geese supports the second scenario.
Importantly, a genotype C virus, which was found in multiple
species in Qinghai Lake, was responsible for the wild-bird
outbreak of H5N1 infection in Mongolia and Russia in August
2005 and also caused major outbreaks in chickens in the Liao-
ning Province and Inner Mongolia in October and November
2005 (Fig. 4; see Fig. S2 in the supplemental material), sug-
gesting that viruses of this genotype may be more pernicious
than those of other genotypes. These findings call for intensive
surveillance of wild migrating birds as biologic vectors that
possibly spread H5N1 viruses over a wide range of territories.
It is important that viruses of genotype C possess Lys at
position 627 in PB2, unlike any other avian viruses, and that
this residue is found in some human H5N1 (7, 9, 10) and H7N7
(5) isolates as well as in the virus responsible for the Spanish
influenza pandemic (21). Thus, it is worrisome that H5N1
viruses with a trait associated with human adaptation have
entered into migrating waterfowl populations.
In the present study, seven of eight test viruses replicated
systemically and killed mice. Among these seven lethal viruses,
five have a lysine at position 627 of the PB2 protein and one
has asparagine at position 701 of this molecule. Although both
of these changes are associated with high virulence in mice (6,
8, 11), our findings indicate the existence of additional muta-
5982 CHEN ET AL.J. VIROL.
tions that contribute to the virulence of avian H5N1 viruses in Download full-text
Several different animal models have been used to evaluate
the virulence of avian H5N1 influenza viruses in mammals.
Maines et al. demonstrated the general equivalence of mice
and ferrets for assessing the pathogenic potential of H5N1
viruses isolated from chickens and humans in Thailand and
Vietnam (13). Experimental infection of cynomolgous ma-
caques with the index H5N1 virus from the 1997 outbreak in
Hong Kong resulted in acute respiratory distress syndrome and
multiple-organ dysfunction, which was similar to findings in
humans (18, 24). By contrast, the clinical signs produced in
rhesus macaques by infection with two of the Qinghai Lake
isolates were quite mild. Although the A/duck/Guangxi/35/01
virus did cause systemic infection and symptoms of influenza-
like illness, the extent of the disease as judged by both its
clinical symptoms and histopathology was milder than that
reported previously by Rimmelzwaan et al. (18). This discrep-
ancy may reflect the experimental procedures used by Rim-
melzwaan et al. and our group. While we intranasally infected
macaques with 2 ml of virus in fluid, Rimmelzwaan and col-
leagues used 5 ml of viral fluid, applying 4 ml intratracheally,
0.5 ml to the tonsils, and 0.25 ml to each of the conjunctiva,
which would be expected to induce more severe disease than
that seen in our study.
In conclusion, the H5N1 viruses that caused a massive out-
break of lethal disease among wild birds at Qinghai Lake in
western China represent a phylogenetically and biologically
heterogeneous group reiterating the features of H5N1 viruses
now circulating in nature. The fact that viruses with a PB2
mutation associated with human adaptation of avian viruses
are circulating in migratory waterfowl and that an avian H5N1
virus was capable of causing systemic infection in primates is
worrisome. Moreover, migratory waterfowl may possibly
spread these viruses over a wide range of territories. If viruses
with the ability to replicate systemically in primates establish in
migratory waterfowl, there would be an even more critical
need for increased surveillance of poultry and the development
of control measures.
We thank Peirong Jiao, Gongxun Zhong, Krisna Wells, and Martha
McGregor for excellent technical assistance; John Gilbert for editing
the manuscript; and Kangzhen Yu for critical discussion. We appreci-
ate R. Sodnomdarjaa for providing the viruses isolated from wild birds
in Mongolia for comparison.
This work was supported by the Animal Infectious Disease Control
Program of the Ministry of Agriculture of China; by Chinese National
Natural Science Foundation 30440008; by the Chinese National Key
Basic Research Program (973) 2005CB523005 and 2005CB523200; by
the Chinese National S&T Plan; by Public Health Service research
grants from the National Institute of Allergy and Infectious Diseases;
by grants-in-aid for Scientific Research on Priority Areas from the
Ministries of Education, Culture, Sports, Science, and Technology,
Japan; and by CREST (Japan Science and Technology Agency).
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