JOURNAL OF VIROLOGY, Dec. 2005, p. 15460–15466
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Vol. 79, No. 24
New Genotype of Avian Influenza H5N1 Viruses Isolated from
Tree Sparrows in China
Z. Kou,1F. M. Lei,2* J. Yu,3Z. J. Fan,1Z. H. Yin,2C. X. Jia,2K. J. Xiong,1Y. H. Sun ,2
X. W. Zhang,3X. M. Wu,4X. B. Gao,4and T. X. Li1*
State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, 430071 China1;
Institute of Zoology, Chinese Academy of Sciences, Beijing, 100080 China2; Beijing Genomics Institute,
Chinese Academy of Sciences, Beijing, 101300 China3; and
Shaanxi Institute of Zoology, Xi’an, 710032 China4
Received 28 June 2005/Accepted 27 September 2005
The 2004 outbreaks of highly pathogenic avian influenza H5N1 disease in China led to a great poultry loss
and society attention. A survey of avian influenza viruses was conducted on tree sparrows (Passer montanus)
collected in China in 2004. Four viruses were isolated from free-living tree sparrows. The results of the
whole-genome analysis indicated that an H5N1 virus with a new genotype is circulating among tree sparrows.
The hemagglutinin and neuraminidase genes of the new genotype were derived from Gs/Gd/96-like viruses and
the nuclear protein gene descended from the 2001 genotype A H5N1 viruses, while the other inner genes
originated from an unknown influenza virus. In experimental infection, all four viruses were highly pathogenic
to chickens but not pathogenic to ducks or mice. The four tree sparrow viruses were different from the 2003 tree
sparrow strain (genotype Z) in Hong Kong. The results suggested that H5N1 viruses might be distributed
widely in tree sparrows.
Highly pathogenic H5N1 influenza virus has caused serious
poultry loss, and since 1997 it has been reported to cause
human deaths. It has been determined that H5N1 influenza
viruses have undergone reassortment in recent years (12, 17).
The Hong Kong influenza H5N1 virus that infected humans in
1997 was confirmed to be a reassortant virus that had acquired
the hemagglutinin (HA) gene from A/goose/GuanGdong/1/96
(Gs/Gd/96; H5N1)-like viruses, the neuraminidase (NA) gene
from A/teal/HongKong/W312/97 (Teal/HK/W312/97; H6N1)-
like viruses, and the internal genes from A/quail/HongKong/
G1/97(Qa/HK/G1/97; H9N2)-like or Teal/HK/W312/97 viruses
(1, 5). Multiple genotypes of H5N1 were detected from 2001 to
2004, which were designated A, B, C, D, E, V, W, X, Y, Z, and
Z? (6, 12). Since 2002, genotype Z has been the dominant
H5N1 virus in southern China and was responsible for the
2003-2004 outbreaks in Asia (12).
Although the aquatic bird has been considered a natural
reservoir for avian influenza viruses (20), in recent years H5N1
viruses have also been isolated from terrestrial birds. From
2002 to 2004, genotype Z viruses were isolated from a feral
pigeon (Columba livia), a tree sparrow (Passer montanus), and
a peregrine falcon (Falco peregrinus) (12), and two genotype V
viruses were isolated from crows in Japan (13). The systematic
surveillance of avian H5N1 influenza viruses in resident birds
has not been well established. As the tree sparrow is a very
common terrestrial bird in China and has frequent contact with
humans, we conducted a survey of epidemic avian influenza
viruses in tree sparrows in the Henan province of China in
2004. Four H5N1 avian influenza viruses were isolated. All the
viruses were sequenced, and their pathogenicity to chickens,
ducks, and mice were tested. The results indicated that they
were new-genotype H5N1 viruses and highly pathogenic to
MATERIALS AND METHODS
Virus isolation. A survey of epidemic avian influenza virus in tree sparrows was
conducted in Pingyu country in the Henan province of China in May 2004.
Cloacal swabs were collected from 38 captured free-living tree sparrows and were
eluted with 0.5 ml of phosphate-buffered saline (PBS). After centrifugation at
6,000 ? g/min for 5 min at 4°C, the supernatant was identified by sandwich
enzyme-linked immunosorbent assay with an avian influenza virus antigen de-
tection kit (Keqian Ltd.). The antigen-positive supernatant of cloacal swabs was
mixed with an equal volume of PBS containing antibiotics (penicillin G, 4,000
U/ml; streptomycin sulfate, 800 U/ml) for 4 h at 4°C and was inoculated into the
allantoic cavities of 10-day-old, specific-pathogen-free, embryonated eggs (Bei-
jing MERIAL Ltd.). After incubation at 37°C for 48 to 72 h, the allantoic fluid
of the inoculated eggs was collected. Fifty percent egg infectious dose (EID50)
titers were calculated by the method of Reed-Muench. Allantoic fluid containing
virus stocks had been stored at ?70°C before being used.
RNA extraction and nucleotide sequencing. Viral RNA was extracted from
virus-infected allantoic fluid with Trizol reagent (Invitrogen). The cDNAs were
amplified by the TaKaRa rTAQ enzyme (TaKaRa Bio) with avian influenza virus
primers (10). After being purified with Montage PCR cleanup filter plates (Mil-
lipore Corporation), the PCR products were used for sequencing with an Am-
ersham ET dye terminator kit (Amersham Pharmacia Bio) and ABI PRISM 370
DNA sequencer (PE Applied Biosystems). The sequencing primers were avail-
able at the website http://www.genomics.org.cn/AI/index.jsp. All sequence data
were edited by BioEdit version 5.0.9 and aligned by Clustal X (version 1.8).
Phylogenetic trees were generated with MEGA version 2.0.
Pathogenicity tests. To determine the pathogenicity of the virus isolated, the
viruses were inoculated into chickens, ducks, and mice. Groups of eight specific-
pathogen-free, 6-week-old chickens (Beijing MERIAL Ltd.) were tested accord-
ing to the recommendation of the Office International des E´pizooties (OIE).
Each chicken was intravenously injected with 0.2 ml of a 1:10 dilution of allantoic
fluid containing virus, and mortality was observed over a 10-day period. Groups
of eight 3-week-old ducks were inoculated intranasally with 0.1 ml of allantoic
fluid, and mortality was also observed over a 10-day period. Groups of 10 6- to
* Corresponding author. Mailing address for T. X. Li: State Key
Laboratory of Virology, Wuhan Institute of Virology, Chinese Acad-
emy of Sciences, Wuhan, 430071 China. Phone: 86-27-87198465. Fax:
86-27-87198465. E-mail: firstname.lastname@example.org. Mailing address for F. M. Lei:
Institute of Zoology, Chinese Academy of Sciences, Beijing, 100080
China. Phone: 86-10-62562713. Fax: 86-10-62565689. E-mail: leifm
8-week-old female BALB/c mice (Experimental Animal Center of Hubei Control
Disease Center, Wuhan, China) were inoculated intranasally with the virus-
allantoic fluid in a volume of 50 ?l and were observed daily for 14 days for signs
of disease. On the third day, three mice of each group were killed purposely, and
the EID50s of the viruses in the lungs and brains were determined. The remain-
ing mice were monitored daily for mortality. All the animal experiments were
performed in a biosafety level 3 laboratory, and the inoculated viral doses of the
allantoic fluid were 105.5to 106.5EID50s. Control groups of chickens, ducks, and
mice were inoculated with PBS.
Nucleotide sequence accession numbers. All sequences have been deposited in
GenBank. The accession numbers are AY741215 to AY741222 and DQ073399
Virus isolation and molecular characterization. Among the
38 cloacal swabs from captured free-living tree sparrows, 25
tested avian influenza virus antigen positive by enzyme-linked
immunosorbent assay. These 25 samples were inoculated into
embryonated eggs, and four avian influenza viruses were iso-
lated. They were named A/Tree sparrow/Henan/1/04 (H5N1)
(Ts/HN/1/04) to A/Tree sparrow/Henan/4/04 (H5N1) (Ts/HN/
4/04). Each of eight gene segments of the four viruses was
sequenced. The four isolates shared a high homology with each
other (96% to 99%), except in their NA and PB2 genes. For
the NA gene, only Ts/HN/4/04 did not contain a 20-amino-acid
(aa) deletion in the stalk of the NA molecule, so the homology
between Ts/HN/4/04 and other sparrow viruses was low (91%
to 92%). The homology between the PB2 genes of Ts/HN/2/04
and Ts/HN/3/04 was 99%, but the homology between Ts/HN/
1/04 and Ts/HN/2/04 (or Ts/HN/3/04) was 91%, while that
between Ts/HN/4/04 and Ts/HN/2/04 (or Ts/HN/3/04) was
95%. Nucleotide sequence analysis revealed that they origi-
nated from different sources (Table 1.).
The HA genes from the four tree sparrows all have the same
multiple basic amino acids (RRKKR) at the connecting pep-
tide between HA1 and HA2, which was considered a charac-
teristic of influenza viruses that are highly pathogenic for
chickens (11). All amino acids relevant to receptor binding (aa
91, 130 to 134, 149, 151, 179, 186, 190 to 191, and 220 to 225)
were identical to those of Gs/Gd/1/96 (3). Therefore, it is likely
that these viruses bind to cellular receptors with 2,3-NeuAcGal
linkages, as has been shown for Gs/Gd/1/96 (7).
The HA protein of Ts/HN/1/04, Ts/HN/2/04, and Ts/HN/
3/04 had an additional glycosylation site (aa 170 to 172) at the
head of the HA molecule and contained a 20-aa deletion in the
stalk of the NA molecule, while Ts/HN/4/04 did not. The ad-
ditional glycosylation site in the HA protein and the deletion of
20 aa in the NA protein were also dominant in other 2004
viruses isolated from poultry and human, which was suggested
to be connected with the adaptation of viruses for spreading
more efficiently in terrestrial domestic poultry (15).
Four tree sparrow viruses had a 5-aa deletion (aa 80 to 84)
in the middle of the NS molecule but did not have a mutation
of Glu92 in the NS1 protein or Lys627 in the PB2 protein,
which had been suggested to be associated with the increased
virulence of H5N1 viruses in mice (4, 8). Moreover, the four
viruses did not have mutations at the amino acids Ser31,
Leu26, Val27, and Ala30 in the transmembrane region of the
M2 protein, which occur in many genotype Z viruses and which
have been proved to be associated with amantadine resistance
Phylogenetic analysis. Phylogenetic analysis revealed that
the HA genes of tree sparrows formed a branch in the phylo-
genetic tree with 2000-2004 isolates from Asia (genotypes Z,
Z?, Y, A, B, C, D, E, and X). The HK/97 isolates formed
another branch. Both branches originated from Gs/Gd/96-like
viruses (Fig. 1).
The NA gene tree showed that tree sparrows branched with
genotypes A, B, C, D, E, X, Y, Z, and Z? and with Gs/Gd/96
and formed a branch of avian influenza viruses. Isolates of
HK/97 formed a separate branch. Unlike the other three tree
sparrow isolates, Ts/HN/02 clustered into the branch of geno-
type Y, Z, and Z? viruses of 2002 to 2004 (Fig. 1).
Analysis of the internal protein genes shows that four H5N1
sparrow viruses originated from multiple avian influenza vi-
ruses. The PB2 genes of Ts/HN/2/04 and Ts/HN/3/04 were
almost identical, but they were different from Ts/HN/1/04 and
Ts/HN/4/04. Ts/HN/1/04 and Ts/HN/4/04 formed a branch to-
gether with genotypes A, B, C, D, E, Y, Z, Z?, and X and with
Gs/Gd/96. This branch was distinctly related to the branch of
Ts/HN/2/04 and Ts/HN/3/04 and the branch of H5N1/97 (Fig.
1). Interestingly, the PB2 gene of DK/Yolohama/aq10/03,
which was a genotypically unique H5N1 influenza virus iso-
lated from duck meat in 2003 (14), was found to be most
closely related to that of the tree sparrows (Fig. 1).
In the phylogenetic tree of the PB1 gene, the four sparrow
viruses formed a separate fork and clustered with the branch of
genotypes A, B, C, D, E, Y, Z, and Z? and the branches of
Gs/Gd/96 and HK/97. According to the phylogenetic tree, the
PB1 gene of tree sparrows might be the ancestor of genotypes
A, B, C, D, E, Y, Z, and Z? and of Gs/Gd/96 and HK/97. The
PB1 gene of genotype X is distinctly related to those of all the
above-named genotypes (Fig. 1).
In the phylogenetic tree of the PA gene, the four sparrow
viruses formed a separate branch. The branch was distinctly
related to the branch of genotypes A, B, C, D, E, Y, Z, Z?,
and X and to the branch of Gs/Gd/96, the branch of H5N1/97,
and that of Gf/HK/38/02 (Fig. 1).
In the phylogenetic tree of the NP gene, the four sparrow
viruses formed a separate fork and clustered with the branch of
genotypes B, C, E, Y, Z, Z?, and X, the branch of HK/97, and
that of DK/Yolohama/aq10/03 (14). The NP genes of the
above-named genotypes were similar to those of genotype A
and Gs/Gd/96 but distinctly related to that of genotype D
TABLE 1. Percentages of sequence similarity between the tree
sparrow virus isolates and other influenza viruses
Segment Virus with the highest similaritya
A/swine/Hong Kong/126/82 (H3N2)
A/swine/Hong Kong/81/78 (H3N2)
A/Duck/Hong Kong/289/78 (H9N2)
A/Duck/Hong Kong/698/79 (H5N3)
A/Duck/Hong Kong/610/79 (H9N2)
aPercentages of sequence similarity were calculated based on the nucleotide
sequences of the complete open reading frames of eight genes, with Ts/HN/2/04
as the representative strain. The nucleotide sequences of Ts/HN/2/04 were com-
pared to those in GenBank.
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15462 KOU ET AL.J. VIROL.
FIG. 1. Phylogenetic trees of virus genes isolated from tree sparrows in China. Trees were generated by using neighbor-joining analysis with the Tamura-Nei-gamma model in the MEGA
program (version 2.0). Numbers below branches indicate bootstrap value percentages from 1,000 replicates. Analysis was based on nucleotides 1 to 1,012 (1,012 bp) of the HA gene, 1 to 1,055
(1,055 bp) of the NA gene, 1,058 to 2,227 (1,170 bp) of the PB2 gene, 23 to 1,316 (1,294 bp) of the PB1 gene, 1,450 to 2,148 (699 bp) of the PA gene, 22 to 976 (955 bp) of the NP gene, 25
to 759 (725 bp) of the M gene, and 46 to 685 (640 bp) of the NS gene. The scale bar represents the distance unit between sequence pairs. Th, Thailand; VN, Vietnam; In, Indonesia; HK, Hong
Kong; Gd, Guangdong; YN, Yunnan; Sh, Shanghai; Sd, Shandong; ST, Shantou; Ck, chicken; Dk, duck; Qa, quail; Gs, Goose; SCk, silky chicken. The sequences are named in concordance
with their GenBank nomenclature. The tree sparrow genes are indicated in red.
VOL. 79, 2005 H5N1 VIRUSES ISOLATED FROM TREE SPARROWS IN CHINA15463
FIG. 1. Continued.
15464 KOU ET AL.J. VIROL.
In the phylogenetic tree of the M gene, the four sparrow
viruses formed a separate branch with H3 and H6 subtype
viruses. The branch was distinctly related to the branch of
genotypes A, B, C, D, E, X, Y, Z, and Z?, that of Gs/Gd/96,
and that of HK/97 (Fig. 1). The NS genes of four tree sparrows
were distinctly related to that of genotypes A, B, C, D, E, X, Y,
Z, and Z?, that of H5N1/97, and that of the H3, H4, H6, H9,
and H11 subtypes (Fig. 1).
According to the above-described analysis of phylogenetic
relationships, the four tree sparrow isolates were different from
other reported H5N1 genotypes. They were reassortants of
Gs/Gd/96-like viruses; the outer genes are derived form Gs/
Gd/96-like viruses, and the inner genes are derived from un-
known influenza viruses resident in wild birds (Fig. 1).
Pathogenicity tests. Four isolates from tree sparrows were
inoculated into chickens, ducks, and mice. As shown in Table
2, all the isolates killed seven or eight out of eight infected
chickens within 10 days. According to the OIE criteria, all the
isolates were highly pathogenic to chickens. It was reported
that pathogenicity to duck varied according to different H5N1
isolates (16). The result of animal experiments indicated that
the tree sparrow isolates were not pathogenic to ducks (Table
2) and so were different from the highly pathogenic viruses
isolated from wild aquatic birds in 2002 (16). In the mice
experiments, although the viruses were unable to kill mice,
they could be detected in all the lungs of mice examined, as
well as in the brains of mice infected with Ts/HN/2/04 (Ta-
A survey of epidemic avian influenza viruses in tree sparrows
was conducted in Pingyu country, Henan province, China, in
May 2004. Four viruses were isolated from 38 free-living tree
sparrows and were identified to be the highly pathogenic H5N1
subtype avian influenza virus. By molecular characterization
and an analysis of phylogenetic relationships, their genetic
characterizations were different from those of the dominant
genotype Z viruses and other H5N1 genotype viruses. Accord-
ing to phylogenetic analysis results, only the HA, NA, and NP
genes were found connected with other reported genotypes
viruses. The HA and NA genes are descended from Gs/Gd/
96-like virus, while the NP gene might be from genotype A
viruses (Fig. 1 and 2). But other inner genes, such as PB2, PB1,
PA, M, and NS, were novel and might have originated from an
unknown virus (Fig. 1 and 2). The PB2 gene of DK/Yolohama/
aq10/03 might also have originated from this unknown virus.
The possible derivation of the tree sparrow viruses and their
relationship to other H5N1 genotypes are summarized in Fig.
2. Reassortants were found to be very popular in the origin of
influenza viruses (12).
In 2003, an avian influenza H5N1 virus was isolated once
from a tree sparrow in Hong Kong, and the genotype of this
strain was Z. The tree sparrow viruses that we isolated were
quite different from the genotype Z virus. The fact that a
diversity of H5N1 viruses was isolated from tree sparrows sug-
gested that H5N1 viruses might be distributed widely in tree
sparrows and that infections might not be occasional cases. The
aquatic birds of the world were considered the natural reser-
voirs of influenza A viruses. Recently, genotype V and Z H5N1
viruses were isolated from different terrestrial birds (12). Our
FIG. 2. Possible derivation of the tree sparrow viruses and their
relationship to other H5N1 genotypes. The eight gene segments in
each schematic virus particle are (from top to bottom) the PB2, PB1,
PA, HA, NP, NA, M, and NS genes. Genes of the same lineage are
shown in the same color. The capital letters indicate the genotypes and
Sparrow 1 to Sparrow 4 indicate the four sparrow isolates.
TABLE 2. Results of animal infection experiments
(no. dead/no. inoculated)
(no. of days to death)
Virus in lung
Virus in brain
VOL. 79, 2005 H5N1 VIRUSES ISOLATED FROM TREE SPARROWS IN CHINA15465
results indicated that a new genotype H5N1 virus was found in Download full-text
tree sparrows. Multiple genotypes of viruses isolated from ter-
restrial birds indicated that terrestrial birds may play an im-
portant role in the natural reservoir and transmission of influ-
enza virus. The tree sparrows from which the virus was isolated
appeared normal when captured, indicating that they were
carriers of the viruses instead of dead end hosts.
The origin of the tree sparrow viruses is unclear. The simi-
larity of the HA and NA genes with those of Gs/Gd/96 indi-
cated that they had some connection with aquatic birds. When
migrating birds fly to their habitants, viruses that they have can
be transmitted to local aquatic and terrestrial birds. In con-
trast, migrating birds can be infected by the viruses reserved in
resident birds. So, viral genes are transmitted between the
migrating gene pool and resident gene pool, and new-genotype
viruses are created. Tree sparrows are in close contact with
aquatic birds and domestic poultry; therefore, the chance of
viral infection from other birds is high.
The result of animal experiments indicated that the tree
sparrow isolates were highly pathogenic to chickens but not
pathogenic to ducks. In mice experiments, the viruses did not
kill mice but were detected in the lungs as well as some of the
brains. Nowadays, the mechanism of the transmissibility of
avian influenza viruses to mammals is not resolved and was
proposed to involve multiple viral genes. It is firmly believed
that avian gene constellations promote transmission to mam-
mals (19, 21). The possible transmission and adaptation to
mammals need to be further studied.
Highly pathogenic H5N1 avian influenza viruses resulted in
a windstorm of disease outbreaks in China and other Asian
countries in 2004. As there were cases in which viruses were
fatal to humans (2, 9), the viruses have been a serious threat to
the public health and the poultry industry. Wild birds were
considered to be significantly related to the increasing spread
of the virus in Asia, and H5N1 viruses with pandemic potential
become endemic in regions and were not easily eradicable
(12). Tree sparrows are residents and widespread in the whole
of China and other Eurasian countries. They are more or less
attached to human habitations and are found to have close
contact with poultry, wild birds, domestic animals, and human
beings. As H5N1 viruses were isolated from these birds, pre-
caution is needed to prevent potential threats to the poultry
industry and human health. In the region of China where the
H5N1 viruses in this study were isolated, pigs and ducks are
housed closely in farming villages. By means of possible trans-
mission between swine and birds, the existence of the H5N1
virus in tree sparrows may serve as a new reservoir for the
reemergence of a highly pathogenic avian influenza virus
H5N1 outbreak. Additional surveys, especially long-term sur-
veillance of tree sparrows and other wild birds, are necessary.
This research was supported by the Ministry of Science and Tech-
nology of China program (grant no. 2004BA519A11), the 973 Project
(grant no. 2005CB523000), the Innovation Program (grant no.
INF105-SDB-3-A2) of the Chinese Academy of Sciences (CAS), the
863 project (grant no. 2005 AA219070), and the Innovation Program
of the Institute of Zoology, CAS.
We appreciate the staff of the State Forestry Administration of
China and local Forestry Departments for their help in collecting
wild-bird samples. We acknowledge S. L. Chen, Z. Zhang, Q. S. Yan,
H. J. Hu, H. F. Zhao, F. S. Zou, H. P. Liu, and many others for field
sample collection and assistance in the laboratory. We also acknowl-
edge J. Wang for sequencing efforts and Z. H. Hu for critical review of
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