JOURNAL OF VIROLOGY, Sept. 2010, p. 8389–8397
Copyright © 2010, American Society for Microbiology. All Rights Reserved.
Vol. 84, No. 17
Continued Evolution of H5N1 Influenza Viruses in Wild Birds,
Domestic Poultry, and Humans in China from 2004 to 2009?†
Yanbing Li,‡ Jianzhong Shi,‡ Gongxun Zhong,‡ Guohua Deng,‡ Guobin Tian, Jinying Ge,
Xianying Zeng, Jiasheng Song, Dongming Zhao, Liling Liu, Yongping Jiang,
Yuntao Guan, Zhigao Bu, and Hualan Chen*
Animal Influenza Laboratory of the Ministry of Agriculture and National Key Laboratory of Veterinary Biotechnology,
Harbin Veterinary Research Institute, CAAS, 427 Maduan Street, Harbin 150001, People’s Republic of China
Received 24 February 2010/Accepted 28 May 2010
Despite substantial efforts to control H5N1 avian influenza viruses (AIVs), the viruses have continued to
evolve and cause disease outbreaks in poultry and infections in humans. In this report, we analyzed 51
representative H5N1 AIVs isolated from domestic poultry, wild birds, and humans in China during 2004 to
2009, and 21 genotypes were detected based on whole-genome sequences. Twelve genotypes of AIVs in southern
China bear similar H5 hemagglutinin (HA) genes (clade 2.3). These AIVs did not display antigenic drift and
could be completely protected against by the A/goose/Guangdong/1/96 (GS/GD/1/96)-based oil-adjuvanted
killed vaccine and recombinant Newcastle disease virus vaccine, which have been used in China. In addition,
antigenically drifted H5N1 viruses, represented by A/chicken/Shanxi/2/06 (CK/SX/2/06), were detected in
chickens from several provinces in northern China. The CK/SX/2/06-like viruses are reassortants with newly
emerged HA, NA, and PB1 genes that could not be protected against by the GS/GD/1/96-based vaccines. These
viruses also reacted poorly with antisera generated from clade 2.2 and 2.3 viruses. The majority of the viruses
isolated from southern China were lethal in mice and ducks, while the CK/SX/2/06-like viruses caused mild
disease in mice and could not replicate in ducks. Our results demonstrate that the H5N1 AIVs circulating in
nature have complex biological characteristics and pose a continued challenge for disease control and pan-
The highly pathogenic H5N1 influenza viruses that emerged
over a decade ago in southern China have evolved into over 10
distinct phylogenetic clades based on their hemagglutinin
(HA) genes. The viruses have spread to over 63 countries and
to multiple mammalian species, including humans, resulting in
498 cases of infection and 294 deaths by 6 May 2010 according
to the World Health Organization (WHO) (http://www.who
.int). To date, none of the different H5N1 clades has acquired
the ability to consistently transmit among mammalian species.
The currently circulating H5N1 viruses are unique in that they
continue to circulate in avian species. All previous highly
pathogenic H5 and H7 viruses have naturally “burned out” or
were stamped out because of their high pathogenicity in do-
mestic poultry. While there is growing complacency about the
potential of H5N1 “bird flu” to attain consistent transmissibil-
ity in humans and develop pandemicity, it is worth remember-
ing that we have no knowledge of the time that it took the 1918
Spanish, the 1957 Asian, the 1968 Hong Kong, and the 2009
North American pandemics to develop their pandemic poten-
tials. We may therefore currently be witnessing in real time the
evolution of an H5N1 pandemic influenza virus.
H5N1 avian influenza viruses (AIVs) were first detected in
sick geese in Guangdong province in 1996, and both nonpatho-
genic and highly pathogenic (HP) H5N1 viruses were de-
scribed (18). In 1997, H5N1 reassortant viruses that derived
the HA gene from A/goose/Guangdong/1/96 (GS/GD/1/96)-
like viruses and the other genes from H6N1 and/or H9N2
viruses caused lethal outbreaks in poultry and humans in Hong
Kong (6, 7). Since then, long-term active surveillance of influ-
enza viruses in domestic poultry has been performed, and
multiple subtypes of influenza viruses have been detected in
chickens and ducks in China (16, 19, 37). H5N1 influenza
viruses have been repeatedly detected in apparently healthy
ducks in southern China since 1999 (4, 13) and were also
detected in pigs in Fujian province in 2001 and 2003 (39).
Since the beginning of 2004, there have been significant
outbreaks of H5N1 avian influenza virus infection involving
multiple poultry farm flocks in more than 20 provinces in
China (2). H5N1 viruses resulted in the deaths of millions of
domestic poultry, including chickens, ducks, and geese, as the
result of infection or of culling and the deaths of thousands of
wild birds (5, 20). Thirty-eight human cases of HP H5N1 in-
fection with 25 fatalities have been associated with direct ex-
posure to infected poultry (WHO; http://www.who.int). Since
2004, the vaccination of domestic poultry has been used for the
control of HP H5N1 influenza virus in China. While this strat-
egy has been effective at reducing the incidence of HP H5N1 in
poultry and at markedly reducing the number of human cases,
it is impossible to vaccinate every single bird due to the enor-
mous poultry population. Outbreaks of H5N1 influenza virus
still continue to occur in poultry although at a reduced fre-
* Corresponding author. Mailing address: Harbin Veterinary Re-
search Institute, 427 Maduan Street, Harbin 150001, People’s Republic
of China. Phone: 86-451-82761925. Fax: 86-451-82733132. E-mail:
† Supplemental material for this article may be found at http://jvi
‡ Y.L., J.S., G.Z., and G.D. equally contributed to this paper.
?Published ahead of print on 10 June 2010.
A previous study by Smith et al. reported that a “Fujian-like”
H5N1 influenza virus emerged in late 2005 and predominated
in poultry in southern China (26). Those authors suggested
that vaccination may have facilitated the selection of the “Fu-
jian-like” sublineage. Here, we analyzed 51 representative
H5N1 viruses that were isolated from wild birds, domestic
poultry, and humans from 2004 to 2009 in China and described
their genetic evolution and antigenicity profiles. Our results
indicate that H5N1 influenza viruses in southern China, includ-
ing the “Fujian-like” viruses, are complicated reassortants,
which could be well protected against by GS/GD/1/96 virus-
based vaccines. We documented the emergence of the latest
variant of H5N1 (A/chicken/Shanxi/2/06 [CK/SX/2/06]) that
broke through existing poultry vaccines. We show that this
variant is less pathogenic in mice and ducks than the earlier
strains and propose that the variant was not selected by the use
MATERIALS AND METHODS
Viruses. The 51 H5N1 viruses used in this study were isolated from the
samples that were sent to the National Avian Influenza Reference Laboratory for
the diagnosis of suspected cases of avian influenza virus infection during 2004 to
2009 in China (see Table S1 in the supplemental material). All experiments with
the H5N1 isolates were performed in a biosafety level 3 laboratory facility, and
animal experiments were performed in HEPA-filtered isolators.
Genetic and phylogenetic analyses. Viral RNA was extracted with the RNeasy
minikit (Qiagen, Valencia, CA) and was reverse transcribed. PCR amplification
was performed by using segment-specific primers (primer sequences are avail-
able upon request). The PCR products were purified with the QIAquick PCR
purification kit (Qiagen) and sequenced by using the CEQ DTCS-Quick Start kit
with a CEQ 8000 DNA sequencer (Beckman Coulter). Sequence data were
compiled with the SEQMAN program (DNASTAR, Madison, WI), and phylo-
genetic analyses were carried out with the PHYLIP program of the CLUSTALX
software package (version 1.81) using a neighbor-joining algorithm. Bootstrap
values of 1,000 were used.
Antigenic analyses. Antigenic analyses were performed by hemagglutinin in-
hibition (HI) tests using chicken antisera generated against the tested viruses. To
generate the antisera, 6-week-old specific-pathogen-free (SPF) chickens were
injected with 0.5 ml of oil emulsion-inactivated vaccine derived from the selected
viruses, and sera were collected at 3 weeks after injection. We used 0.5% chicken
erythrocytes in the HI assay.
Animal experiments. All animal studies were approved according to the na-
tional guidelines by the Review Board of Harbin Veterinary Research Institute,
Chinese Academy of Agricultural Sciences.
Groups of eight 6-week-old female BALB/c mice (Beijing Experimental An-
imal Center, Beijing, China) were lightly anesthetized with CO2and inoculated
intranasally (i.n.) with 106.050% egg infective doses (EID50) of H5N1 influenza
virus in a volume of 50 ?l. Three of the eight mice were killed on day 3
postinoculation (p.i.) for virus titration in the lungs, kidneys, spleen, and brain.
The remaining five mice were monitored daily for weight loss and mortality. The
50% mouse lethal dose (MLD50) was determined for viruses that caused lethal
infection of mice by the i.n. inoculation of groups of five mice. The MLD50was
calculated by the method of Reed and Muench (23).
Groups of eight 4-week-old SPF ducks were intranasally inoculated with 106
EID50of H5N1 influenza virus in a volume of 0.1 ml. Three of eight ducks were
killed on day 3 p.i., and organs were collected for virus titration. The remaining
five ducks were observed for 2 weeks for disease, death, and serum conversion.
Swabs from all of the ducks were collected on days 3, 5, and 7 for the detection
of virus shedding.
Vaccination and challenge study of chickens. An oil-adjuvanted inactivated
vaccine produced with the seed virus Re-1, which is a reassortant virus, generated
by reverse genetics, containing the modified HA and neuraminidase (NA) genes
of GS/GD/1/96 virus and the six internal genes of A/Puerto Rico/8/34 (PR8) virus
(31), had been used in poultry since 2004 (3). Recombinant Newcastle disease
virus (NDV) strain LaSota, expressing the HA gene of the GS/GD/1/96 virus, was
constructed as previously described (11) and used as a bivalent vaccine to protect
chickens from Newcastle disease virus and H5N1 avian influenza virus infection
(3). To evaluate the protective efficacy of these two vaccines, 3-week-old SPF
chickens were vaccinated with 0.3 ml of the H5N1 inactivated vaccine containing
2.8 ?g of the HA protein (31) via intramuscular injection or vaccinated i.n. with
106EID50of a recombinant NDV vaccine. Three weeks after vaccination, chick-
ens were challenged with 105EID50of different H5N1 influenza viruses. Oro-
pharyngeal and cloacal swabs of the chickens were collected for virus titration on
days 3 and 5 postchallenge (p.c.), and chickens were observed for signs of disease
or death for 2 weeks p.c.
Nucleotide sequence accession numbers. The nucleotide sequences analyzed
in this study are available at the GenBank database under accession numbers
HM172069 to HM172484 (see Table S1 in the supplemental material).
Molecular and phylogenetic analysis. Between 2004 and
2009, H5N1 viruses caused over 100 outbreaks of HP influenza
virus infection in poultry and wild birds in China. To under-
stand the genetic relationships, 51 representative viruses iso-
lated from chickens, ducks, geese, wild birds, and humans were
sequenced and compared with 10 representative viruses that
were previously reported (4, 5, 35, 36).
The HA genes of the 61 isolates were divided phylogeneti-
cally into five groups (Fig. 1). Group 1 contained six viruses,
with five of them being representative viruses isolated during
the 2004 outbreak. All of them shared over 98% homology and
grouped with the previously reported A/duck/Guangxi/50/01
(DK/GX/50/01) virus (4). We included the previously reported
clade 8 and 9 H5N1 viruses (1, 33) in this group, as their
intergroup homology is over 98%. The HA gene of the group
2 viruses (clade 2.3) included 25 viruses isolated mainly from
southern China (Fig. 1). This group was isolated mainly from
ducks but also includes chicken, goose, and human isolates.
Eight viruses formed group 3 (clade 2.2), with one virus being
the representative virus isolated from chickens in Liaoning in
2005, one virus being isolated from poultry outbreaks in 2004,
and six viruses being isolated from wild birds in Qinghai, Tibet,
and Liaoning provinces during 2005 to 2006 (Fig. 1). Group 4
(clade 7.1) contained nine poultry viruses and one human
isolate. The nine poultry viruses were isolated from chickens in
Xinjiang province during 2005 to 2006, and the HA genes of
these viruses shared 97.4% to 98.5% homology with the HA
gene of A/Beijing/1/03 (BJ/1/03) (H5N1) virus, a human rep-
resentative strain of the clade 7 viruses (1, 33). Group 5 in-
cluded 11 viruses: one virus was isolated from Vietnam (22),
and 10 viruses were isolated from chickens in six provinces in
northern China and one province in southern China during
2006 to 2009. These viruses, represented by A/chicken/Shanxi/
2/2006 (CK/SX/2/06), represent a novel group of H5N1 viruses
not previously characterized; their HA genes share less than
95% homology with the group 1, group 2, and group 3 viruses
and less than 97% homology with the group 4 viruses. The
closest sequence to that of the CK/SX/2/06-like viruses from
previously available databases is the BJ/1/03 virus, which has
only 96.5% nucleotide sequence identity. A previous report (1)
designated the BJ/1/03 and CK/SX/2/06 viruses as the repre-
sentative strains of clade 7; however, in our analysis, these two
viruses were mapped into two different groups, and we there-
fore designated the group 4 and group 5 viruses as clade 7.1
and clade 7.2, respectively.
The neuraminidase (NA) genes of all of the viruses tested
had a 20-amino-acid deletion in the NA stalk (residues 49 to
68). The NA genes of these viruses formed seven groups (Fig.
1, and see Fig. S1 in the supplemental material). H5N1 viruses
8390LI ET AL.J. VIROL.
isolated in 2004 formed groups 1, 2, and 3 and contained the
viruses isolated in poultry in southern China from 2004 to
2007. Groups 4, 5, and 6 contained the viruses isolated from
chickens in Xinjiang (XJ chicken-like), from chickens in other
northern provinces (SX chicken-like), and from wild birds (QH
wild bird-like), respectively. Group 7 contained three viruses
that were isolated in 2008 and 2009. The homology of the
genes within groups is over 98%, and the homology of the
genes is less than 97% between the groups.
The PB2 genes of these viruses formed four groups (Fig. 1,
and see Fig. S1 in the supplemental material). Group 1 con-
tained most viruses that were isolated from domestic poultry in
2004 and the viruses isolated from chickens in northern China
from 2006 to 2009, including the XJ chicken-like viruses and
SX chicken-like viruses. Groups 2 and 3 contained viruses
causing outbreaks in domestic poultry in southern China dur-
ing 2005 to 2007. Group 4 contained viruses causing outbreaks
in wild birds in Qinghai and Tibet during 2005 to 2006 and two
viruses isolated from chickens in southern China in 2008. The
PB2 genes of the viruses in group 3 shared less than 92%
homology with those in the other three groups, and the ho-
mology among the other three groups was between 95% and
97%. All of the PB2 genes of the group 4 viruses have a lysine
at position 627 that is conserved among influenza viruses that
have adapted to mammals (30) and is associated with the high
virulence of H5N1 viruses in mice (12).
The PB1 genes of these H5N1 viruses were classified into
four groups (Fig. 1, and see Fig. S1 in the supplemental ma-
terial). Group 1 contained most of the viruses that were iso-
lated from domestic poultry in southern China between 2004
and 2009 and the XJ chicken-like viruses. Groups 2 and 3
contained the QH wild bird-like viruses and the SX chicken-
like viruses, respectively. The PB1 gene of A/chicken/Guizhou/
7/08 (CK/GZ/7/08) formed group 4 by itself.
The PA genes of these viruses were divided into three
groups. Group 1 contained most of the viruses isolated in 2004
and QH wild bird-like viruses. Group 2 contained viruses iso-
lated mainly from domestic poultry in southern China from
2004 to 2009. The XJ chicken- and SX chicken-like viruses
formed group 3 (Fig. 1, and see Fig. S1 in the supplemental
The NP genes of all of these viruses shared over 97% ho-
mology and were considered one lineage, although they
formed different forks in the phylogenetic tree (see Fig. S1 in
FIG. 1. Phylogenetic analyses of the H5N1 viruses isolated from 2004 to 2009 in China. The phylogenetic trees were generated with the
PHYLIP program of the CLUSTALX software package (version 1.81). The five trees were generated based on the following sequences: HA
nucleotides (nt) 29 to 1732, NA nt 21 to 1730, PB2 nt 28 to 2307, PB1 nt 25 to 2298, and PA nt 25 to 2175. The phylogenetic tree of HA was rooted
to A/mallard/Denmark/64650/03 (H5N7), the NA phylogenetic tree was rooted to A/green-winged teal/Ohio/72/99 (H1N1), and the PB2, PB1, and
PA phylogenetic trees were rooted to A/Memphis/1/90 (H3N2). The colors of the viruses in the NA, PB2, PB1, and PA trees match with those used
in the HA tree. Abbreviations: BHG, bar-headed goose; CK, chicken; DK, duck; GS, goose; GC, great cormorant; SK, shrike; AH, Anhui; FJ,
Fujian; GD, Guangdong; GX, Guangxi; HB, Hubei; HN, Hunan; HeB, Hebei; HeN, Henan; JX, Jiangxi; LN, Liaoning; NX, Ningxia; QH, Qinghai;
SD, Shandong; SX, Shanxi; TB, Tibet; XJ, Xinjiang. Sequences labeled with a red “#” in the HA tree were downloaded from the available
databases. Viruses labeled with a red arrow were selected for antiserum generation.
VOL. 84, 2010EVOLUTION OF H5N1 INFLUENZA VIRUSES IN CHINA8391
the supplemental material). The M genes of the viruses formed
three groups (Fig. S1). Group 1 contained three viruses iso-
lated in 2004 and the QH wild bird-like viruses. Group 2
contained viruses from chickens in northern China from 2005
to 2009, and the remaining viruses formed group 3. The NS
genes of these viruses could be divided into two groups (Fig.
S1). Group 1 contained the QH wild bird-like viruses and a few
viruses isolated in 2004, and the remaining viruses formed
On the basis of genomic diversity, the viruses investigated in
this study were divided into 21 genotypes (Fig. 2, and see Table
S2 in the supplemental material). Genotype 1 represents the
majority of H5N1 viruses that were isolated from influenza
virus outbreaks in 2004, while genotypes 2 through 7 and ge-
notype 17 contain viruses from individual outbreaks in 2004.
Genotypes 8 through 15, containing the HA gene of clade 2.3,
represent the viruses isolated from domestic poultry from 2005
to 2009 in southern China, and genotype 16 is the only virus
detected from northern China that bears the HA gene of clade
2.3. It is noteworthy that 17 genotypes (genotypes 1 to 17) were
originally detected in domestic poultry, mainly from waterfowl,
in southern China. The wild bird viruses included in this study
are genetically similar to our previously reported genotype C
virus that was detected in Qinghai Lake in 2005 (5), and they
were designated genotype 18 (Fig. 2). Genotype 19 represents
viruses isolated in chickens in Xinjiang, and genotypes 20 and
21 represent viruses isolated in other provinces in northern
China. The viruses in genotypes 10, 11, 13, 18, and 19 are
associated with human infections in China and other countries
Studies with mice. In our previous study of the viruses iso-
lated from ducks in Southern China during 1999 to 2002, we
observed an increasing level of pathogenicity in mice with the
progression of time (4). Maines et al. also reported previously
that H5N1 viruses exhibited increased lethality over time in
ferrets (21). To investigate the virulences of the viruses iso-
lated in recent years in China, we selected and tested 22 viruses
from eight different genotypes (genotypes 6, 7, 10, 11, 12, 18,
19, and 20) in mice. We found that 9 of 13 viruses isolated from
domestic poultry in southern China, including the viruses of
genotypes 6, 7, 10, 11, and 12, replicated systemically and killed
mice at a very low dosage (50% minimal lethal dose [MLD50]
of ?3 log10EID50) (Fig. 3). The DK/GD/23/04 (genotype 7),
DK/HN/70/04 (genotype 6), CK/SC/81/05 (genotype 10), and
DK/HN/11/07 (genotype 12) virus replicated well in the lungs
of mice but were not detected in the kidneys or brains of the
animals. These four viruses killed mice only at a high dosage
(MLD50of ?5.2 log10EID50) (Fig. 3). All three wild bird
viruses (genotype 18 viruses) were highly lethal in mice
(MLD50of ?1 log10EID50). However, all six other viruses
isolated from chickens in northern China (genotypes 19 and
FIG. 2. Genotypic evolution of the H5N1 viruses isolated in China
from 2004 to 2009. The eight gene segments are indicated at the top of
each bar. The number in each bar shows the group of genes indicated
in Fig. 1 and Fig. S1 in the supplemental material.
FIG. 3. Replication and virulence of H5N1 influenza viruses in
mice. Virus replication was tested as described in Materials and Meth-
ods. The data shown are the mean titers for three mice. A value of 0.5
was assigned if the virus was not detected from the undiluted sample.
The MLD50is shown as the log10EID50. Genotypes were determined
on the basis of the diversity of the gene nucleotide sequences, as
described in the legends of Fig. 1 and 2. The red dashed line indicates
the lower limit of detection.
8392LI ET AL.J. VIROL.
20) replicated only in the lungs and did not kill any mice, even
at the highest inoculation dosage (MLD50of ?6.5 log10EID50)
(Fig. 3, and see Table S2 in the supplemental material).
Studies with ducks. The H5N1 viruses that were isolated
from apparently healthy ducks did not kill any ducks in the
laboratory setting even though they were shed from the trachea
or the cloaca (4). Since late 2003, however, H5N1 viruses have
caused disease and deaths in ducks in multiple countries (27,
29). To understand the virulence in ducks of the viruses that
were isolated in China in recent years, specific-pathogen-free
(SPF) ducks were intranasally inoculated (106EID50in a
0.1-ml volume) with eight representative viruses isolated from
different species, geographic locations, and time periods.
As shown in Fig. 4, three viruses, two viruses from ducks and
one virus from bar-headed goose, could replicate to high titers
in the lungs and brains of ducks. These viruses were shed
through tracheae and/or cloacae after being intranasally inoc-
ulated (Fig. 4A) and killed all of the inoculated ducks within 5
days postinoculation (p.i.) (Fig. 4B). The GS/HB/65/05 virus
replicated in the lungs of ducks as well as the duck and wild
bird viruses, but the virus titer in the brain was significantly
lower than that observed for the inoculated duck and wild bird
viruses. Low titers of this virus were detected on day 3 p.i. (Fig.
4A). The CK/XJ/27/06 virus replicated in the lung at lower
titers and was shed through the trachea, but virus replication
and shedding were not detected for any ducks that were inoc-
ulated with three SX/CK/2/06-like viruses isolated from chick-
ens in northern China (Fig. 4A). All of the ducks inoculated
with GS/HB/65/05, CK/XJ/27/06, CK/SX/2/06, CK/HeN/A7/06,
and CK/LN/A1/06 stayed healthy and survived during the
2-week observation period (Fig. 4B).
Antigenic analysis. Antisera, generated in SPF chickens or
ferrets, to the selected H5N1 viruses and two monoclonal an-
tibodies (MAbs) derived from GS/GD/1/96 and CK/SX/2/06
viruses, respectively, were used for antigenic analyses by hem-
agglutination inhibition (HI) assays with 0.5% chicken eryth-
rocytes. As indicated in Table 1, the antisera generated in
chickens against GS/GD/1/96, DK/AH/1/06, and BHG/QH/
3/05 cross-reacted well with most viruses that were isolated
from domestic poultry in southern China, the viruses from wild
birds (genotype 18), and the viruses from chickens in Xinjiang
(genotype 19). The HI titers of these viruses, however, were 2-
to 8-fold lower than the homologous titers. It is worth noting
that the antisera of these three viruses reacted poorly with the
viruses isolated from chickens in northern China (genotype
20). The antisera derived from CK/SX/2/06 reacted well with
the genotype 20 viruses but poorly with all other viruses. Their
heterologous HI titers were ?8- to 16-fold lower than the
homologous HI titers (Table 1). Unlike the chicken antisera
generated against the DK/AH/1/06 virus, which cross-reacted
with most of the viruses, ferret antisera against AH/1/05 re-
acted poorly with the CK/SX/2/06-like viruses and with the
BHG/QH/3/05 virus, suggesting that the oil adjuvant antigen
may have induced greater cross-reactivity. MAb DD7, derived
from GS/GD/1/96, did not react with the chicken viruses from
northern China, including the genotype 20 and 21 viruses, but
reacted with other viruses with various titers. MAb SCiC1,
derived from CK/SX/2/06 virus, reacted with the majority of
the tested viruses but not with the wild bird viruses (genotype
18) and the CK/FJ/1/07 virus (Table 1). These results suggest
that the H5N1 influenza viruses circulating in China are anti-
genically different. The viruses isolated from chickens in sev-
eral provinces in northern China (genotypes 20 and 21) espe-
cially exhibited severe antigenic drift.
Protective efficacy of the H5N1 vaccines used in China.
China is one of the countries that use vaccines to control H5N1
influenza virus in poultry. An inactivated vaccine containing
the HA and NA genes of the GS/GD/1/96 virus and internal
genes from the A/Puerto Rico/8/34 (PR8) virus was developed
by reverse genetics and has been used in the field since 2004
(31). Recombinant NDVs containing the HA genes of H5N1
influenza virus were also generated as bivalent live, attenuated
vaccines against both NDV and avian influenza virus infection
in poultry (11). The bivalent vaccine that contains the HA gene
of GS/GD/1/96 has been used in the field since 2006. The
GS/GD/1/96-based vaccines induced cross-reactive antisera
(Table 1) and completely protected chickens from challenges
against the viruses from southern China (clade 2.3) and the
viruses from wild birds (clade 2.2) (Table 2). However, the
GS/GD/1/96-based antisera reacted poorly with the CK/SX/2/
06-like virus. To evaluate whether the GS/GD/1/96-based vac-
FIG. 4. Replication and virulence of H5N1 influenza viruses in
ducks. (A) Viral titers and shedding in ducks on day 3 p.c. Data shown
are means ? standard deviation. For statistical purposes, a value of 0.9
was assigned if virus was not detected from the undiluted sample.*,
P ? 0.01 compared with the titers in the corresponding samples in the
DK/GD/23/04, DK/HB/43/05, and BHG/QH/1/06 virus-inoculated
ducks. (B) Death pattern for ducks inoculated with different H5N1
influenza viruses. The dashed line indicates the lower limit of
VOL. 84, 2010EVOLUTION OF H5N1 INFLUENZA VIRUSES IN CHINA8393
cine could provide protection against the CK/SX/2/06-like vi-
ruses, we performed challenge studies with chickens. As shown
in Table 2, virus shedding was detected on day 5 postchallenge
(p.c.) in 4 of 20 chickens that were inoculated with the inacti-
vated vaccine and in 12 of 20 chickens that received the re-
combinant NDV vaccine. Only 80% and 40% of chickens vac-
cinated with inactivated vaccine and recombinant NDV,
respectively, survived during the 2-week observation period
after challenge. All of the chickens in the control group died
before day 4 p.c. (Table 2).
Despite substantial efforts to control infection of poultry,
H5N1 viruses have continued to evolve and spread. The viruses
have caused outbreaks in poultry in more than 60 countries
and have caused human infections in 15 countries, which have
resulted in 294 fatalities by 6 May 2010 according to the WHO
(http://www.who.int). Here, we analyzed H5N1 influenza vi-
ruses isolated from domestic poultry, wild birds, and humans
from 2004 to 2009 in China. Our results demonstrate that these
H5N1 influenza viruses formed 21 different genotypes and
exhibited distinct virulence in ducks and in a mammalian
mouse model. We also determined that the H5N1 influenza
viruses that emerged in chickens in northern China in 2006 had
antigenically drifted and could not be protected efficiently by
the GS/GD/1/96 virus-derived vaccines used in China.
Our results demonstrated that the viruses from domestic
poultry, mainly from ducks, in southern China are complicated
reassortants. The HA (group 2, clade 2.3), NP, M (except the
DK/GD/23/04 virus), and NS (except the DK/HN/69/04 virus)
genes of the viruses isolated from 2004 to 2009 in southern
China (genotypes 5 to 16) are quite similar (Fig. 2), but their
NA, PB2, PB1, and PA genes show significant diversity. This
suggests that multiple subtypes of influenza viruses may have
actively cocirculated in waterfowl in this region and that reas-
sortment among different viruses frequently occurred. Smith et
al. previously reported that a “Fujian-like” H5N1 virus domi-
nated in southern China (26). However, our data indicate that
the viruses detected in China bearing the HA of clade 2.3.4
viruses are complicated reassortants. We cannot determine
which of these viruses dominates over the others. The HA
genes of the clade 2.3 viruses were detected as early as 2004,
and although these viruses exhibited some degree of antigenic
drift compared with the index virus GS/GD/1/96, they could
still be completely protected by the GS/GD/1/96-based vac-
cines. The continued circulation of these groups of viruses in
TABLE 1. Antigenic analysis of H5N1 avian influenza viruses isolated in China
Virus (HA gene group)HA cladeg
HI antibody titere
NDV (LaSota strain)f
aAntisera were generated by vaccinating SPF chickens with the oil-emulsified inactivated vaccine derived from the indicated viruses.
bAntisera were generated in ferrets.
cThe MAb was generated from GS/GD/1/96 virus.
dThe MAb was generated from CK/SX/2/06 virus.
e—, not done. Homologous titers are shown in boldface type.
fAn H9N2 virus and a Newcastle disease virus were used as negative antigen controls.
gNA, not applicable.
8394LI ET AL.J. VIROL.
southern China may have resulted from unqualified vaccina-
tion coverage in waterfowl, especially in ducks, and not as a
result of vaccination selection as was pointed out previously by
Smith et al. (26).
We reported previously that the viruses isolated from the
outbreak in wild birds in Qinghai Lake consisted of four ge-
notypes, and the dominant genotype, represented by the BHG/
QH/3/05 virus, derived the HA, NA, and NP genes from A/CK/
JX/25/04-like viruses (5). Our current results suggest that
BHG/QH/3/05-like viruses are likely a reassortant of A/CK/JX/
25/04-like viruses, a GS/GD/72/04-like virus, and an unknown
virus that contributed the PB2 gene containing a lysine at
position 627. This PB2 gene was detected in CK/TB/6/08 and
CK/GZ/7/08 viruses that were isolated from chickens in the
live-bird markets in Tibet and Guizhou in 2008. The lysine at
position 627 in PB2 is conserved in human viruses and is
associated with the high virulence of H5N1 viruses in mice (12)
and the transmission of influenza viruses in mammals (28). The
BHG/QH/3/05-like viruses were not detected in poultry in
China after 2005; however, they were repeatedly detected in
wild birds or domestic poultry in other countries in Europe,
Africa, and Asia (8, 15, 25, 32) and were reported to be de-
tected in pikas in the Qinghai Lake area in 2007 (38). These
widely distributed viruses bear two known genetic markers in
the PB2 and HA genes that have been proven to be critical for
the transmission of H5N1 influenza viruses in mammalian
hosts (10, 28) and represent a clear pandemic potential.
The CK/SX/2/06-like viruses were first detected in chickens
from the Shanxi province in northern China that were vacci-
nated with GS/GD/1/96-based inactivated avian influenza virus
vaccines. The viruses then spread to several other provinces in
northern China, including Ningxia, Henan, Shandong, and Lia-
oning provinces. The origin of these viruses is still unclear.
Their degree of antigenic drift may easily connect their emer-
gence as a result of vaccination selection; however, genomic
analyses confirmed that the CK/SX/2/06-like viruses are reas-
sortants with new HA, NA, and PB1 genes that were newly
introduced into poultry in China. Viruses with an HA gene
similar to that of CK/SX/2/06-like viruses were also detected in
chickens in Vietnam in 2008 (22). The sequences of the NA
gene and of the internal genes of these viruses from Vietnam
are not available; therefore, it is unknown if they have genomes
similar to those of the CK/SX/2/06-like viruses or if the viruses
acquired the HA gene from a similar ancestor. The limited
replication of the CK/SX/2/06-like viruses in mice and ducks
suggests that these viruses have not been transmitted into or
adapted to other species. However, it is concerning that the
CK/SX/2/06-like viruses reacted poorly with the antisera gen-
erated from other viruses, and therefore, it is important to
include viruses from this clade for vaccine development for the
possibility of an H5N1 influenza virus pandemic.
The virulence of influenza virus is a polygenic trait. Several
amino acids in the PB2, NS1, and M1 genes have been proven
to be associated with the virulence of avian influenza viruses in
a mammalian host (9, 13, 17, 24, 34). All of the viruses of
genotypes 10 and 12 have the same amino acids in positions
that were reported to be important for the virulence of H5N1
influenza virus in mice (9, 13, 17, 24, 34); however, it is inter-
esting that the virulences of the CK/SC/81/05 virus of genotype
10 and of the DK/HN/11/07 virus of genotype 12 were over
1,000-fold lower in mice than those of other viruses of the same
genotypes (Fig. 3). These viruses could therefore be used as
model viruses to explore new genetic determinants for the
virulence of H5N1 viruses in mammals.
Vaccination is a major strategy used in China for the control
of H5N1 avian influenza virus. The H5 inactivated vaccine has
been proven to be efficacious in chickens, ducks, and geese (14,
31). The present study demonstrated that the GS/GD/1/96
virus-based vaccine could protect against different H5N1 vi-
ruses isolated in China, except the CK/SX/2/06-like viruses. An
inactivated vaccine containing the modified HA and NA genes
of the CK/SX/2/06 virus has been developed and used in sev-
eral provinces in northern China since 2006 (3). The surface
genes of a clade 2.3.4 virus, DK/AH/1/06, were used to con-
TABLE 2. Protective efficacies of Chinese vaccines against different H5N1 virusesa
(HA group ?clade?)
No. of swabs with virus shedding/total no. of swabs (mean log10EID50? SD)b
Day 3Day 5
Oropharyngeal CloacalOropharyngeal Cloacal
BHG/3/05 (3 ?2.2?) Inactivated
10/10 (3.0 ? 0.4)
10/10 (2.7 ? 1.0)
AH/1/05 (2 ?2.3.4?) Inactivated
10/10 (2.4 ? 0.6)
10/10 (1.7 ? 0.6)
3/3 (1.4 ? 0.1)
3/3 (1.8 ? 0.4)
DK/HN/21/05 (2 ?2.3.4?) Inactivated
10/10 (3.3 ? 0.4)
10/10 (3.4 ? 1.0)
CK/SX/2/06 (5 ?7.2?) Inactivated
20/20 (2.9 ? 0.6)
20/20 (1.9 ? 0.5)
3/20 (1.7 ? 0.3)c
11/20 (2.0 ? 0.4)c
4/20 (1.5 ? 0.2)c
12/20 (1.9 ? 0.2)c
aThree-week-old SPF chickens were used for this study.
b—, chickens died in that group.
cThe titers shown are the means ? standard deviations of the chickens that actually shed virus.
VOL. 84, 2010EVOLUTION OF H5N1 INFLUENZA VIRUSES IN CHINA8395
struct the PR8 reassortant and recombinant Newcastle disease
virus vaccine seed strains, and these seed viruses were used to
replace the GS/GD/1/96 virus-based strains for vaccine pro-
duction in the middle of 2008 (2). Although vaccine efficacy has
been confirmed in both laboratory and field tests (11, 31), and
the vaccine strain has been updated in a timely manner, the
control of H5N1 avian influenza virus by vaccination still faces
different challenges in different avian species. Older ducks are
more resistant to H5N1 viruses than are younger ducks (31).
Therefore, many adult ducks were not vaccinated and may still
serve as a reservoir for the virus. The continued circulation of
H5N1 viruses in southern China may then be a result of low
vaccination coverage rather than the viruses having undergone
a mutation that has allowed them to escape vaccine-induced
In summary, we fully analyzed 51 H5N1 influenza viruses
isolated from 2004 to 2009 in China. We characterized the
genetic and biological complexity of H5N1 AIVs and provided
important information for a more comprehensive understand-
ing of H5N1 AIV evolution. The multiple genotypes of viruses
detected in southern China imply that the environmental fac-
tors in that area may have favored the generation of reassor-
tants. It is therefore worrisome that these lethal H5N1 AIVs
may have more opportunities to acquire the ability to effi-
ciently transmit among humans. Moreover, the emergence of
antigenically drifted CK/SX/06-like viruses poses a challenge
for influenza virus pandemic preparedness. Clearly, there is a
critical need for the continued surveillance of poultry and for
regularly updated control measures.
We thank Gloria Kelly for editing the manuscript and Nancy Cox for
providing the ferret antisera against H5N1 influenza virus.
This work was supported by Chinese National Natural Science Foun-
dation grant 30825032; Chinese National Key Basic Research Program
(973) grants 2010CB534000, 2005CB523005, and 2005CB523200; and by
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