﻿RESEARCH Open Access
Novel genetic reassortants in H9N2 influenza A
viruses and their diverse pathogenicity to mice
Yuhai Bi1
, Lu Lu1,2
, Jing Li1
, Yanbo Yin3
, Yi Zhang5
, Huijie Gao5
, Zhuoming Qin4
, Basit Zeshan1
, Jinhua Liu5
,
Lei Sun1*
and Wenjun Liu1*
Abstract
Background: H9N2 influenza A viruses have undergone extensive reassortments in different host species, and
could lead to the epidemics or pandemics with the potential emergence of novel viruses.
Methods: To understand the genetic and pathogenic features of early and current circulating H9N2 viruses, 15
representative H9N2 viruses isolated from diseased chickens in northern China between 1998 and 2010 were
characterized and compared with all Chinese H9N2 viruses available in the NCBI database. Then, the representative
viruses of different genotypes were selected to study the pathogenicity in mice with the aim to investigate the
adaptation and the potential pathogenicity of the novel H9N2 reassortants to mammals.
Results: Our results demonstrated that most of the 15 isolates were reassortants and generated four novel
genotypes (B62-B65), which incorporated the gene segments from Eurasian H9N2 lineage, North American H9N2
branch, and H5N1 viruses. It was noteworthy that the newly identified genotype B65 has been prevalent in China
since 2007, and more importantly, different H9N2 influenza viruses displayed a diverse pathogenicity to mice. The
isolates of the 2008-2010 epidemic (genotypes B55 and B65) were lowly infectious, while two representative viruses
of genotypes B0 and G2 isolated from the late 1990s were highly pathogenic to mice. In addition, Ck/SD/LY-1/08
(genotype 63, containing H5N1-like NP and PA genes) was able to replicate well in mouse lungs with high virus
titers but caused mild clinical signs.
Conclusion: Several lines of evidence indicated that the H9N2 influenza viruses constantly change their genetics
and pathogenicity. Thus, the genetic evolution of H9N2 viruses and their pathogenicity to mammals should be
closely monitored to prevent the emergence of novel pandemic viruses.
Keywords: avian influenza virus, H9N2, reassortant, genotype, pathogenicity
Background
H9N2 influenza viruses are panzootic in birds worldwide.
Statistical analysis of the host range and location of
H9N2 subtype influenza A viruses in the NCBI database
according to HA gene revealed that approximately 60%
of all the H9N2 influenza viruses were isolated from
chickens, with the remainder from wild birds (16.8%),
ducks (8.9%), turkeys (6.7%), and other domestic avian
populations (3.7%) (data not shown). In addition, the
overwhelming majority (94.2%) of H9N2 influenza
viruses were isolated in Asia, with > 65% coming from
mainland and Hong Kong of China (data not shown).
Since in China the H9N2 virus was first time isolated in
1994 while approximately 74 different genotypes have
been observed till now and new lineages and genotypes
continuously identified throughout China [1]. Two main
distinct lineages of H9N2 influenza viruses represented
by A/Chicken/Beijing/1/94 (Ck/Bei-like) and A/Quail/
Hong Kong/G1/97 (G1-like) have become endemic in
China since the mid-1990s [2-5]. The G1-like viruses
were mainly detected in quail of southern China [3,4].
While, the Ck/Bei-like viruses were first prevalent among
chickens, ducks, and other minor poultry species in both
southern and northern China [4,5] and then were gradu-
ally replaced by the F98-like (represented by A/Chicken/
* Correspondence: sunlei362@im.ac.cn; liuwj@im.ac.cn
 Contributed equally
1
Center for Molecular Virology, Key Laboratory of Pathogenic Microbiology
and Immunology, Institute of Microbiology, Chinese Academy of Sciences,
Beijing 100101, China
Full list of author information is available at the end of the article
Bi et al. Virology Journal 2011, 8:505
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© 2011 Bi et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.
Shanghai/F/98) viruses since 2000 onward [5,6]. These
viruses evolved from the Ck/Bei-like lineage but repli-
cated and transmitted more effectively than the antece-
dent viruses in chickens under experimental conditions
[5]. Recent studies revealed that most of the viruses origi-
nated from the Ck/Bei-like viruses and formed multiple
genotypes through complicated reassortments with
G1-like, G9-like (represented by A/Chicken/Hong Kong/
G9/97), Y439-like (represented by A/duck/Hong Kong/
Y439/97), and F98-like viruses [2].
In order to systematically analyze the evolution of H9N2
influenza viruses, the genealogy of H9N2 viruses was
further studied. In early reports, the methods of genealogy
classification were mainly determined on the basis of
genetic relationship of all the eight gene segments with
representative strains, such as Ck/BJ/1/94, Ck/SH/F/98,
Qa/HK/G1/97 [2,7]. Later, the H9N2 influenza viruses iso-
lated in southern China were classified into A (viruses
with a G1-like HA) and B (viruses with a Ck/Bei-like HA)
genotype series with different sources and gene constella-
tions [3,4]. On these basis, the H9N2 viruses were further
divided into seven different series (A ~ G) according to
their HA lineages. And in each series, different genotypes
were designated sequentially by additional 0, 1, 2 and
so on, according to their systematic nomenclature [1,3,4].
For example, in genotype B series, non-reassortment Ck/
Bei-like viruses were designated as B0, while reassortment
Ck/Bei-like viruses were designated sequentially as B1, B2,
and so on, according to when the novel genotype was first
identified [1]. The nomenclature system systematically
unified the lineages and genotypes of H9N2 influenza
viruses, which revealed the phylogenetic diversity and gen-
otypic complexity of H9N2 influenza viruses worldwide
[1].
The high evolution rates with complicated reassort-
ments prompted that H9N2 virus may threat to human
health by genetic contribution to the generation of novel
pandemic influenza virus. A recent study highlighted the
importance and risk of H9 reassortants between avian
H9N2 virus and the pandemic H1N1/2009 influenza virus
exhibited higher pathogenicity to mice than both the par-
ental viruses [8]. In fact, H9N2 viruses have sporadically
crossed the barrier from birds to mammals and several
recently emerging F98-like viruses were suspected to have
high pathogenicity to mice [9]. Noticeably, H9N2 virus has
caused several human infections with mild respiratory dis-
ease in Hong Kong and mainland of China since 1997
[10]. Moreover, some H9N2 viruses isolated from live bird
markets in Hong Kong, with Leu (L) residue at amino acid
position 226 (numbered by H3) in hemagglutinin (HA)
receptor binding site (RBS), displayed human virus-like
receptor specificity [11]. Further studies revealed that the
Leu226 (numbered by H3) in HA gene was found to be
important for the transmission of the H9N2 viruses in
ferrets, and mixing the surface glycoproteins of an H9N2
virus with the internal genes of an human H3N2 virus
resulted in enhanced replication and efficient direct trans-
mission in ferrets [12]. These results raised great concerns
about viral evolution and suggested that the H9N2 avian
virus could be of pandemic importance.
Results
Phylogenetic analysis
Eight gene segments of the 15 isolates were phylogeneti-
cally analyzed together against the full length sequences of
379 representative viruses in China. The phylogenetic tree
of HA gene segments demonstrated that the Ck/Bei-like
was the predominate lineage and could be separated into
four sub-lineages in China (Additional file 1, Figure.S1A).
The G9-like sub-lineage and the F98-like sub-lineage were
prevailing around the year 2000. Whereas the JS98-like
viruses (represented by A/ckichen/Jiangsu/1/98) circulated
from 2000 to 2005, and the Y280-like sub-lineage (repre-
sented by A/duck/Hong Kong/Y280/97), which composes
the majority of H9N2 influenza viruses, has circulated in
the recent 5 years (Additional file 1, Figure.S1A). In the
present study, the 13 viruses isolated from 2007 to 2010
fell into the Y280-like sub-lineage, and Ck/SD/WF/98
belonged to the G9-like cluster (Table 1 and Additional
file 1, Figure.S1A). Interestingly, the Ck/HLJ/u/98 isolate
displayed high identity (99.4%) with Ck/HLJ/35/00 [Gen-
bank: DQ064366], which belonged to the Ty/WI/1/66-like
North American lineage (Additional file 1, Figure.S1A). A
similar phylogenetic structure was seen in NA gene, but in
contrast to HA, the NA gene of the Ck/Bei-like lineage
could be divided into three major subgroups: F98-, G9-,
and Y280-like (Additional file 1, Figure.S1B). Most of the
isolates were in the Y280-like sub-lineage, while Ck/SD/
lx929/07, Ck/SD/lx1023/07 and Ck/HLJ/u/98 grouped
with G9-like viruses (Table 1 and Additional file 1, Figure.
S1B), which did not contain a stalk deletion (Additional
file 2, Table S1). It was also noteworthy that a human
H9N2 isolate A/Guangzhou/333/99 [Genbank: AY043024]
shared high identity (98.3%) and formed a sister cluster
with Ck/HLJ/u/98 (Additional file 1, Figure.S1B).
The M genes of Ck/Bei-like and G1-like lineages were
predominant in H9N2 viruses (Additional file 1, Figure.
S1C). And the G1-like M gene was more prevalent than
that of other gene segments and was clearly separated
into three sub-lineages among different periods of time
(Additional file 1, Figure.S1C). Ten of the eleven 2008-
2010 isolates fell into the same G1-like lineage, while the
other five isolates grouped into the Ck/Bei-like branch
(Table 1 and Additional file 1, Figure.S1C). The phyloge-
netic tree of NS gene displayed that all the 15 isolates
grouped into the Ck/Bei-like lineage, which predomi-
nated in China and generated three sub-lineages (Addi-
tional file 1, Figure.S1D). Most of the 2008-2010 isolates
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belonged to the F98-like group, while except Ck/SD/WF/
98 was in the Ck/Bei-like cluster, the other four viruses
clustered into the SH96-like group, which represented by
A/Quail/Shanghai/8/96 (Table 1 and Additional file 1,
Figure.S1D).
Concerning the PB2 gene, Chinese H9N2 viruses mainly
fell into three different lineages: Ck/Bei-, F98-, and G1-like
(Additional file 1, Figure.S1E). Interestingly, seven isolates
together with other six 2007-2010 viruses from Genbank,
formed a novel independent lineage that originated from
an unknown source and were genetically adjacent to the
G1-like cluster (named as H-like lineage, represented by
A/chicken/Shandong/H/09) (Table 1 and Additional file 1,
Figure.S1E). A/swine/Yangzhou/1/2008(H9N2) [Genbank:
HM998919] also located into this new cluster (Additional
file 1, Figure.S1E). Analysis of the PB1 gene revealed that
China H9N2 viruses could also be separated into three
major lineages. The viruses isolated since 2008 in this
study mainly belonged to the F98-like lineage, while other
early isolates clustered into the Ck/Bei-like lineage, and
the Ck/HLJ/u/98 belonged to the G1-like lineage (Table 1
and Additional file 1, Figure.S1F).
The topologies of the PA and NP gene segments were
similar with the PB1 gene, and the majority of the 15
H9N2 isolates located in the F98-like lineage. For the PA
gene, most of the isolates from 2008 to 2010 clustered in
the F98-like group, while two early isolates (Ck/SD/WF/98
and Ck/HLJ/u/98) fell into the Ck/Bei-like and G1-like
lineages, respectively. Two isolates, Ck/SD/lx929/07 and
Ck/SD/lx1023/07, belonged to the Y439-like lineage,
which was an established H9N2 lineage originating from
H5N1-like virus (Additional file 1, Figure.S1G). Impor-
tantly, Ck/SD/LY-1/08 contained H5N1-origined PA and
NP genes. The NP genes of the H9N2 isolates mainly
grouped with the F98-like viruses. However, the NP genes
of Ck/HLJ/u/98 and Ck/SD/WF/98 belonged to the
Ty/WI/1/66-like North America lineage and the Ck/Bei-
like lineage, respectively (Table 1 and Additional file 1,
Figure.S1H).
Genotype analysis
In the present study, the 15 H9N2 isolates were further
classified into seven genotypes according to the systematic
and effective methods of genotype classification defined in
previous studies [1]. Six genotypes were Ck/Bei-like (geno-
type B0, B55, B62, B63, B64 and B65), and one genotype
was Ty/WI/1/66-like (genotype G2) (Figure 1, Table 1).
Ck/SD/WF/98 was a typical Ck/Bei-like virus with non-
reassortment and therefore classified into the genotype B0
group. Two isolates (Ck/SD/BD/08 and Ck/SD/KD/09)
originated from the F98-like lineage and the M genes
came from the G1-like lineage, belonged to genotype B55
which contains viruses frequently isolated in recent years
[9,13].
Four novel H9N2 genotypes identified in this study
were designated as B62, B63, B64 and B65. Genotype B62
viruses (Ck/SD/lx929/07 and Ck/SD/lx1023/07) were
quintuple reassortments with Ck/Bei-, F98-, G9-, Y439-
like and SH96-like viruses, which originated from Ck/
Bei-like and had F98-like PB2 gene, G9-like NA gene,
Table 1 Gene constellations of different genotypes of the H9N2 AIVs
Virus Genotype Genetic source*
PB2 PB1 PA HA NP NA M NS
Ck/SD/WF/98 B0 BJ94 BJ94 BJ94 G9 BJ94 Y280 BJ94 BJ94
Ck/HLJ/u/98 G2 G1 G1 G1 Nor A Nor A G9 BJ94 SH96
Ck/SD/lx929/07 B62 F98 BJ94 Y439 Y280 F98 G9 BJ94 SH96
Ck/SD/lx1023/07 B62 F98 BJ94 Y439 Y280 F98 G9 BJ94 SH96
Ck/SD/LY-1/08 B63 F98 BJ94 H5N1 Y280 H5N1 Y280 BJ94 SH96
Ck/SD/BD/08 B55 F98 F98 F98 Y280 F98 Y280 G1 F98
Ck/SD/KD/09 B55 F98 F98 F98 Y280 F98 Y280 G1 F98
Ck/SD/02/08 B64 F98 BJ94 F98 Y280 F98 Y280 G1 F98
Ck/SD/01/09 B65 H F98 F98 Y280 F98 Y280 G1 F98
Ck/SD/02/09 B65 H F98 F98 Y280 F98 Y280 G1 F98
Ck/SD/H/09 B65 H F98 F98 Y280 F98 Y280 G1 F98
Ck/SD/BD/10 B65 H F98 F98 Y280 F98 Y280 G1 F98
Ck/SD/01/10 B65 H F98 F98 Y280 F98 Y280 G1 F98
Ck/SD/02/10 B65 H F98 F98 Y280 F98 Y280 G1 F98
Ck/SD/03/10 B65 H F98 F98 Y280 F98 Y280 G1 F98
* PB, polymerase basic protein; PA, polymerase acidic protein; HA, hemagglutinin; NP, nucleocapsid protein; NA, neuraminidase; M, matrix; NS, nonstructural;
BJ94, Ck/Bei-like (represented by A/Chicken/Beijing/1/94); Y280, Y280-like (represented by A/duck/Hong Kong/Y280/97); F98, F98-like (represented by A/Chicken/
Shanghai/F/98); G1, G1-like (represented by A/Quail/Hong Kong/G1/97); G9, G9-like (represented by A/Chicken/Hong Kong/G9/97); Y439, Y439-like (represented
by A/duck/Hong Kong/Y439/97); SH96, SH96-like (represented by A/Quail/Shanghai/8/96); H, unknown source and named as H-like (represented by A/chicken/
Shandong/H/09); H5N1, H5N1-like.
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SH96-like NS gene, and PA gene from the Y439-like line-
age; The Ck/SD/LY-1/08 isolate was classified into geno-
type B63, which came from Ck/Bei-like virus and
contained H5N1-like PA and NP gene segments; The
Ck/SD/02/08 isolate had the same genetic source with
genotype B55 except the PB1 gene came from Ck/Bei-
like lineage, was classified into genotype B64. Seven gene
segments of genotype B65 viruses (Ck/SD/01/09, Ck/SD/
02/09, Ck/SD/H/09, Ck/SD/BD/10, Ck/SD/01/10, Ck/
SD/02/10, Ck/SD/03/10) were identical to genotype B55,
but their PB2 gene was derived from an unknown origin.
Molecular analysis
The deduced HA, NA, and PB2 amino acid sequences of
the 15 viruses were respectively compared with those of
representative viruses. Interestingly, the HA gene of Ck/
SD/lx929/07 had a 21-nucleotide deletion from 1267 to
1287 encoding 7 amino acids, while another virus (Ck/SD/
lx1023/07) isolated from the same poultry farm at the
same time did not contain a deletion in its HA protein.
This deletion located in the HA2 domain and did not
affect any potential glycosylation site of HA (Additional
file 2, Table S1). There were seven conserved potential gly-
cosylation sites in the HA proteins of most H9N2 viruses
(amino acid positions 29-31, 141-143, 218-220, 298-300,
305-307, 492-494, and 551-553, H9 numbering). The addi-
tion and deletion of the potential glycosylation sites in the
present isolates were shown in Additional file 2, Table S1,
but whether the change in the glycosylation sites might
affect the viral characteristics needed to be explored
further. Moreover, most of the H9N2 viruses tested in this
study had the same P-A-R-S-S-R  G amino acid motif at
the cleave site (arrow) (Additional file 2, Table S1), which
was characteristic of H9N2 virus from land-based poultry
[7]. However, Ck/HLJ/u/98 had a P-A-V-S-S-R  G motif,
i.e., contained a Val instead of an Arg at position 335 (H9
numbering) (Additional file 2, Table S1).
The RBS in HA protein associates with the receptor
binding specificity, and determines the infection spectrum
of influenza virus. Ten of the fifteen characterized viruses
had the human-like motif Leu226 (H3 numbering), and
four isolates contained the avian-like motif Gln226 in the
RBS (Additional file 2, Table S1). Interestingly, almost all
of the Ck/BJ/1/94-like viruses had a RBS-related Ala-to-
Val/Thr mutation at position 190 (H3 numbering) (Addi-
tional file 2, Table S1).
It was also well know that the amino acids at position
627 and 701 of the PB2 gene (H3 numbering) were
recognized as the critical mammalian host determinant.
All the present H9N2 isolates contained the Glu (E) and
Asp (D) in the two sites, respectively (Additional file 2,
Table S1), which represented the weak virulence in mam-
malian host. Furthermore, the length of NA stalk may
affect the pathogenicity of influenza virus, and most of
the H9N2 isolates had the same "marking" deletion of
three amino acids (positions 63-65) at the NA stalk
region, except for three isolates (Ck/SD/WF/98, Ck/SD/
lx929/07, and Ck/SD/lx1023/07). The analysis showed
Figure 1 Genotypes of H9N2 influenza viruses isolated from chickens in northern China. The seven different genotypes are designated B0,
G2, B62, B63, B55, B64 and B65. The eight horizontal bars in ovals represent eight gene segments of AIV (from top to bottom): PB2, PB1, PA, HA,
NP, NA, M, and NS. Each color represents a virus lineage. "?", displays an unknown source lineage.
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that the diverse molecular characters existed in H9N2
viruses, such as RBS, amino acid motif at the cleave site,
and deletion in HA or NA protein, but the significance of
the diversity should be further studied.
Pathogenicity to mice
To investigate the adaptation and potential pathogenicity
of different H9N2 genotypes to mammals, we selected
eight representative isolates from different genotypes with
good reproductive capacity in SPF eggs (Table 2) and then
these viruses were inoculated intranasally (i.n.) into the
exposure groups of 6-week-old female BALB/c mice. The
onset and the duration of clinical symptoms induced by
representative H9N2 isolates at the dose of 107
EID50 were
as follows: five isolates from 2008 to 2010 (genotype B55
and B65) caused no gross lesions in lungs of the inocu-
lated mice at 3 days post-infection (d.p.i.), as the mice
remained healthy and gained body weight during the
infection (Figure 2, Table 3). In contrast, the mice inocu-
lated with Ck/SD/WF/98 (genotype B0) or Ck/HLJ/u/98
(genotype G2) started showing inactivity, ruffled fur, and
obvious body weight loss at 2 and 3 d.p.i. (Figure 2 and
Table 3). From 4 to 6 d.p.i., the mice of the two infectious
groups exhibited severe inappetence, emaciation, and the
most significant weight loss (14.77 and 14.72% for the Ck/
SD/WF/98- and Ck/HLJ/u/98-inoculated groups, respec-
tively) (Figure 2 and Table 3). The inappetence and inac-
tivity were correlated with a gradual loss of body weight,
and death was observed after inoculation of mice with Ck/
SD/WF/98 or Ck/HLJ/u/98 viruses (Figure 3). Another
strain, Ck/SD/LY-1/08 (genotype 63) displayed milder
clinical signs and less weight loss (the maximum weight
loss was 6.09%) compared to the Ck/SD/WF/98 and Ck/
HLJ/u/98 strains (Figure 2 and Table 3). The survived
mice clinically recovered from infection at 7 d.p.i., and
their body weight reached the same level as the mock-
infected control groups at 14 d.p.i. (Figure 2).
The replication ability of the six H9N2 isolates in mice
was evaluated by the virus titer in the lungs of each
infected mice group at 3 d.p.i. (Table 3). Four strains repli-
cated well in mice lungs without prior adaptation, among
which Ck/SD/WF/98 (genotype B0), Ck/HLJ/u/98
(genotype G2), and Ck/SD/02/09 (genotype 65) were able
to replicate well in mice with different virus titers at 3 d.p.i.
(Table 3). And mice inoculated with Ck/SD/LY-1/08 had a
much higher virus titer (5.0, 5.5 and 5.25 log10EID50/0.1
ml) than those inoculated with other strains (1.5 to 2.75
log10 EID50/0.1 ml) at 3 d.p.i. (Table 3).
The damage to respiratory tissues of the infected mice
was consistent with the clinical symptoms and lung virus
titers. Ck/SD/WF/98 (genotype B0)- and Ck/HLJ/u/98
(genotype G2)-infected mice displayed severe bronchop-
neumonia and interstitial pneumonia (Figure 4). Ck/SD/
LY-1/08 (genotype 63)-infected mice also suffered heavy
bronchopneumonia and a higher virus titer in their
lungs, although it caused less severe clinical syndromes,
with 6.09% of the maximum weight loss and little ruffled
fur (Table 3 and Figure 2). Additionally, there were no
obviously tissue lesions in the respiratory system among
the other five isolates in genotype 55 or 65 (i.e., Ck/SD/
02/09, Ck/SD/BD/10, Ck/SD/03/10, Ck/SD/KD/09, and
Ck/SD/BD/08). The pathogenicity test of the representa-
tive H9N2 viruses was repeated twice in mice and the
similar results were obtained, which indicated that the
different H9N2 viruses in our study possessed diverse
replication ability and virulence in mice.
Discussion
H9N2 viruses have undergone extensive reassortments to
generate multiple reassortants and genotypes
[1-7,9,13,14]. In the present study, extensive sequence
data was used to characterize the evolutionary pattern by
genetic and pathogenic diversity of the H9N2 influenza
viruses in China. Phylogenetic analysis indicated that
both Ck/Bei-like and F98-like viruses co-circulated in
China from 1998 to 2010 (Additional file 1, Figure.S1).
The internal gene segments of most viruses isolated after
2008 were derived from the F98-like group, indicating
that F98-like has been the dominant lineage in recent
years. Phylogenetic analysis also indicated multiple sub-
lineages in each gene segments were well-separated and
evolved independently, which indicated that each gene
segment of the H9N2 viral genome displayed a high evo-
lutionary rate.
Table 2 Reproductive capacity of the representative H9N2 AIVs in embryonated eggs
Virus HA EID50(log10/0.1 ml) ELD50(log10/0.1 ml) Genotype
Ck/SD/BD/08 27
7.75 5.0 B55
Ck/SD/KD/09 28
7.75 5.25 B55
Ck/SD/BD/10 211-12
8.75 < 7.5 B65
Ck/SD/03/10 29
8.75 8.5 B65
Ck/SD/02/09 29~10
8.25 6.75 B65
Ck/SD/LY-1/08 211~12
8.5 8.0 B63
Ck/HLJ/u/98 29~10
8.0 < 6.5 G2
Ck/SD/WF/98 29~10
7.75 7.5 B0
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Multiple reassortants and an increasing number of
H9N2 influenza virus genotypes have been continuously
identified throughout China in recent years, although
most of the viruses were transients and could not be
established in avian hosts [1,4,5]. In the present study, all
of the representative strains isolated from diseased
chicken in northern China were reassortants exception
Ck/SD/WF/98 (which was a pure Ck/Bei-like strain)
(Table 1 and Additional file 1, Figure.S1). One novel
stable and predominant lineage (genotype B65) was iden-
tified in our study. This triple-reassortant lineage pos-
sessed a G1-like M gene segment, a PB2 gene with an
unknown origin, and other gene segments from F98-like
viruses (Table 1 and Additional file 1, Figure.S1).
Together with our 7 isolates, 13 H9N2 viruses have so far
been detected in this genotype which were mainly iso-
lated from chickens in northern and southeastern China
from 2007 to 2010 (Additional file 1, Figure.S1E). Inter-
estingly, A/swine/Yangzhou/1/2008 (isolated from swine)
also belonged to this new genotype (Additional file 1, Fig-
ure. S1E), indicating that genotype B65 virus has been
established in chickens in different regions of China and
has spread to mammals. Other new identified genotypes
were composed of one or two strains (Table 1, Figure 1).
Figure 2 Mean changes in body weight of mice infected with H9N2 viruses. BALB/c mice were inoculated i.n. with each of eight H9N2
viruses at a dose of 107
EID50. The body weights were monitored daily for a 14-day observation period and expressed as a percentage of the
initial value. The data represents the mean of four mice in each group.
Table 3 Differences in pathogenicity among the H9N2 AIVs to BALB/c mice
Virus Virus replication in mouse lung at 3 d.p.i. (log10EID50/0.1ml) Clinical syndromes Seroconversion
(HI mean titer)
Body weight (%)c
Fur
Ck/SD/BD/08 1/3 (<a
) gain (16.27) unruffled < 10
Ck/SD/KD/09 1/3 (<a
) gain (12.86) unruffled 20-160
Ck/SD/BD/10 1/3 (<a
) gain (13.97) unruffled 40-320
Ck/SD/03/10 -b
gain (13.82) unruffled 10-20
Ck/SD/02/09 2/3 (1.5, 2.75)* gain (10.55) unruffled 160-320
Ck/HLJ/u/98 3/3 (2.5, 3.75, 2.5)** lose (14.72) ruffled < 10
Ck/SD/LY-1/08 3/3 (5.0, 5.5, 5.25)** lose (6.09) Little ruffled 20-40
Ck/SD/WF/98 3/3 (1.5, 2.75, 2.0)** lose (14.77) ruffled 40-80
Mock-infected control - gain (13.04) unruffled -
a
<, Virus titer < 1 log10EID50.
b
-, No virus or haemagglutination inhibition (HI) titer was detected.
c
The percentage of the mean maximum weight loss or gain among the inoculated mice over the course of infection.
* The data were compared with the Ck/SD/BD/08 group by 2-way ANOVA, respectively, *P < 0.01, **P < 0.001.
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However, further sequence data collection is needed to
confirm if they are transient reassortants or have been
established in avian.
H9N2 influenza viruses can also generate novel reassort-
ment or genotype viruses that carry gene segments with
wide evolutionary distances. It has been reported that
H9N2 strain A/swine/Korea/S190/2004 was a reassortant
between a Eurasian lineage and a North American lineage
viruses [1]. In China, Ck/HLJ/35/00 has only been
reported as H9N2 virus that possesses HA [Genbank:
DQ064366] and NP [Genbank: DQ064447] genes from an
early North American lineage (represented by Ty/WI/1/
66) [1,2]. Interestingly however, the Ck/HLJ/u/98 isolate
analyzed in our study possessed each of eight gene seg-
ments with the highest homology to those of Ck/HLJ/35/
00 (Additional file 1, Figure.S1). These findings suggested
that the two strains might have the same origin, and this
genotype was not transient rather circulated in China for
several years.
The novel H9N2 reassortants or genotypes with wide
evolutionary distances not only generated by hybridization
among different lineages of H9N2 viruses, but also by
reassortment between H9N2 and H5N1 viruses. The first
reported evidence of hybridization between H9N2 and
H5N1 was the case of human infected by highly patho-
genic avian influenza (HPAI) H5N1 virus in 1997, and the
Qa/HK/G1/97(H9N2)-like viruses were hypothesized to
have been involved in the generation of the H5N1 virus
[15]. Previously isolated H9N2 influenza viruses that reas-
sorted with H5N1 viruses could also be found elsewhere:
those possessing the H5N1-like PB1 gene segments were
from southeastern China [14], and H9N2 viruses with NS
genes originating from the HPAI H5N1 lineage were iso-
lated from Pakistan [16]. However, the pathogenicity of
these reassortants was not clear [14,16]. Noticeably, in our
study, both the PA and NP genes of Ck/SD/LY-1/08 (gen-
otype 63) shared high homology (98%-99%) with the
H5N1 viruses circulating around the year 2005, such as A/
chicken/China/1/02 (H5N1) [GenBank: DQ023146], A/
swine/Shandong/2/03 (H5N1) [GenBank: AY646426,
AY700213], and A/environment/Qinghai/1/2008 (H5N1)
[GenBank: FJ455823, FJ455825]. Interestingly, one H5N1
reassortment virus, A/plateau pika/Qinghai/04/2007
(H5N1), was closely related to Ck/SD/LY-1/08 in its PB2
(98.4% homology) [GenBank: FJ390058], PA (98.5%
homology) [GenBank: FJ390060], and NP (99.6% homol-
ogy) [GenBank: FJ390062] gene segments (Additional file
1, Figure.S1E, G and H). It also contained the Ck/Bei-like
M and NS genes, as well as a Y439-like PB1 gene. It was
demonstrated that multiple reassortments have occurred
between H9N2 and H5N1 subtypes since the early out-
breaks, and they continued exchanging internal gene seg-
ments and generating novel viruses.
H9N2 viruses of different genotypes and reassortment
patterns could have huge differences in their pathogenicity
and transmission in mammals (BALB/c mice and swine)
under experimental conditions [2,7,9]. In the present study,
the majority of the recently circulated genotype B65 and
B55 viruses, which include the PB2 gene segment from an
unknown origin and/or the M gene segment from the G1-
like lineage, displayed low infective ability in mice (Table 3
and Figure 2). In addition, Ck/SD/LY-1/08 (genotype B63)
was able to replicate well in mice lungs with high virus
titer but caused mild clinical symptoms (Table 3 and
Figure 3 Survival percentage of mice infected with H9N2 viruses. Groups of mice (n = 4) were inoculated i.n. with each of eight H9N2
viruses at a dose of 107
EID50. The percent survival was observed daily for 14 days.
Bi et al. Virology Journal 2011, 8:505
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Figure 2). Natural selection suggests that a less virulent
strain is more likely to co-exist with the host population
because mobile, living hosts will transmit the strains most
effectively [17]. Thus, whether the changes in the host
adaptation and replication ability of Ck/SD/LY-1/08 are
associated with the H5N1 donor of the PA and NP gene
segments must be explored further. Importantly, two early
strains in our study (Ck/HLJ/u/98 in genotype G2 and Ck/
SD/WF/98 in genotype B0) caused the death of infected
mice without prior adaptation (Figure 3), though a previous
study of several H9N2 viruses belonging to the same geno-
types reported non-lethality in mice [2]. The similar discor-
dant infectivity in mice with same genotype viruses was
also observed in this study, Ck/SD/BD/10, Ck/SD/03/10
and Ck/SD/02/09 were all in the genotype B65, and the
former two viruses nearly couldn't replicate in mice lungs,
but the third strain were able to replicate well in mice
lungs without prior adaptation. It was reported that the
mutations of E627K and D701N in PB2 gene were the key
factors for a virus to acquire the ability to adapt to increase
Figure 4 Representative histopathological changes in Hematoxylin and Eosin (H&E)-stained respiratory system tissues (nose, trachea,
and lung). Ck/SD/WF/98 virus-infected mice displayed severe bronchopneumonia and interstitial pneumonia in lung tissues (A and D), which
showed interstitial edema and thickening of the alveolar walls, alveolar lumen flooded with dropout of alveolar cells, erythrocytes, and
inflammatory cells (black square), bronchial epithelial cell desquamation (thick solid arrow) and extensive lymphocyte, neutrophil, and plasma cell
infiltrates around the bronchiolitis and blood vessels (thick white arrow); congestion in the blood vessels (black triangle); Light (B, H) and intense
(E) congestion in the blood vessels of the nasal submucosa caused by the Ck/SD/WF/98 and Ck/SD/03/10 viruses, respectively (black triangle);
Congestion in the blood vessels of the tracheal submucosa (black triangle) and dropout of the mucous epithelium in the trachea (thick solid
arrow) caused by the Ck/SD/WF/98 virus (C and F). There were no obvious histopathological changes in the respiratory system tissues of the Ck/
SD/03/10-infected group and no significant difference compared to the PBS mock-infection group.
Bi et al. Virology Journal 2011, 8:505
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virulence in a new mammalian host [18]. Molecular analy-
sis demonstrated that all of the viruses in our study pos-
sessed the conservative residues Glu627 and Asp701 in
PB2 gene. Therefore, it is probable that the virulence of
H9N2 influenza viruses is not fully relevant to whole-gene
homology, and some amino-acid mutations or deletions
may dramatically alter the virulence of influenza viruses.
In light of the persistent evolution of H9N2 viruses
with high evolution rate, it is highly necessary to moni-
tor the evolution and evaluate the virulence of novel
avian H9N2 viruses to mammal animals, which may dis-
cover and prohibit the potential threat to human by
novel viruses, and provide the key clues for preventing
new pandemic influenza.
Conclusions
Here, we described the genetic and pathogenic characteris-
tics of the representative H9N2 viruses isolated from
chicken farms in northern China over a 12-year span. Phy-
logenetic analyses revealed that H9N2 influenza viruses
evolved quickly in China, and new genotypes were fre-
quently generated in chicken flocks. The present study
identified two stable H9N2 lineages (genotype B55 and
B65) that have been circulating in China in recent years.
In addition, different H9N2 viruses possessed diverse
replication capacity and pathogenicity in mice.
In summary, our study provides evidences to further
understand the genetic evolution and inter-species trans-
mission of H9N2 influenza A viruses. Their different
pathogenicitiy behavior and host adaptations to mice may
likely be associated with both the gene reassortments and
key amino acid mutations. Thus, surveillance followed by
large-scale sequencing efforts and animal experiments are
critically important to detect the virulent variants and pro-
vide insight into the key features for emergence of pan-
demic viruses.
Materials and methods
Virus isolation and identification
Surveillance was conducted in northern China from 1998
to 2010. Tracheal, cloacal and fecal swabs, and lung tissues
were collected from sick or dead chickens from chicken
farms. The tissue homogenates of clinical specimens were
centrifuged for 10 minutes at 8, 000 × g, and after remov-
ing bacterial contaminants by micropore membrane filters
(0.22 m) the supernatant was incubated in the allantoic
cavities of 10-day-old specific pathogen free (SPF) chicken
eggs (Beijing Merial Vital Laboratory Animal Technology
Company) at 37°C for 72 h for viral isolation, finally the
allantoic fluids was harvested and stored at -80°C. The
purification of all the strains was done by the end-point
dilution assay [19], and subtyping was performed as pre-
viously described [20]. Fifteen avian H9N2 viruses were
chosen for detailed analysis (Additional file 3, Table S2).
Viral gene sequencing
Viral RNA was extracted from infected allantoic fluids
using Trizol reagent (Invitrogen). Reverse transcription
(RT) was performed by using the Uni12 primer
(AGCAAAAGCAGG), and specific primers were
designed amplifying the eight full-length gene segments
of the virus. The PCR products were purified and
sequenced from the Beijing Genomics Institute using
synthetic oligonucleotides.
Genetic and phylogenetic analyses
To determine the molecular evolutionary characteristics of
H9N2 viruses isolated in China, eight gene segments from
each of 15 isolates were phylogenetically analyzed together
with all the full length sequences of the representative
viruses of China available in GenBank. The nucleotide and
amino acid sequences of each gene segment were analyzed
by DNAMAN (Version 5.2.2, Lynnon Biosoft, USA) and
DNASTAR (DNASTAR, Inc., Madison, MI, USA) soft-
wares. The nucleotide sequence-based phylogenetic trees
were generated with MEGA 4.1 http://www.megasoftware.
net via Neighbor-Joining algorithm and the reliability of
the trees was assessed by bootstrap analysis with 1, 000
replications.
Genotype analysis
Genotype analysis was performed systematically for each
of the eight gene segments based on the distribution of
lineages in phylogenetic trees, and the genes sharing over
95% homology in the same lineage were considered as one
genotypic group [1]. H9N2 viruses according to their HA
lineages were divided into seven series, designated as
A~G. Viruses with G1-like, Ck/Bei-like, Ck/Korea/38349-
p96323/96-like, Y439-like, Dk/HK/289/78-like, Qa/HK/
AF157/92-like, Ty/WI/1/66-like HA genes were classified
into genotype A, B, C, D, E, F, and G series, respectively
[1]. In each of the series, different genotypes were desig-
nated sequentially by additional 0, 1, 2 and so on, accord-
ing to different gene constellations with the systematic
nomenclature [1,3,4].
Pathogenicity test in mice
H9N2 influenza viruses from each genotype were selected
to assess their potential pathogenicity to mammal. BALB/c
mice (6-week-old, female) were purchased from Vital
River Laboratories, Beijing. Mice in each group (n = 7)
were lightly anesthetized with Zoletil (tiletamine-zolaze-
pam; Virbac; 25 g/g) and inoculated i.n. with 107
EID50
of H9N2 virus in a volume of 50 l. Mock-infected control
mice were inoculated i.n. with 50 l phosphate-buffered
saline (PBS). Three mice were humanely euthanized at 3
d.p.i., and their lungs were collected, ground and homoge-
nized in cold PBS under sterile conditions. Then the solid
debris was pelleted by centrifugation at 5, 000 × g for
Bi et al. Virology Journal 2011, 8:505
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Page 9 of 11
10 min, and the homogenates were used for virus titra-
tions in 10-day-old embryonated chicken eggs. Virus titers
were reported in units of log10EID50 per 0.1 ml. Nose, tra-
chea, and part of left lung lobes were fixed in 10% buffered
formalin, embedded in paraffin, and the tissues were
stained with hematoxylin-eosin for histopathological eva-
luation. The rest of the mice were monitored daily for gen-
eral behavior and clinical signs, including food intake,
body weight, inactivity, and mortality, for 14 days.
Ethics Statement
The use of all laboratory animals and animal subjects in
this study was approved by the Beijing Association for
Science and Technology, with approval ID SYXK (Beij-
ing) 2007-0023, and followed by the Beijing Laboratory
Animal Welfare and Ethical Guidelines of the Beijing
Administration Committee of Laboratory Animals.
Nucleotide sequence accession numbers
The nucleotide sequences of 15 H9N2 influenza viruses
isolated in this study have been submitted to the Gen-
Bank database and have assigned the accession numbers
(JF795035-JF795148).
Additional material
Additional file 1: Figure.S1. Unrooted neighbor-joining phylogenetic
trees for the HA (34-1573 nt) (A), NA (20-1387 nt) (B), M (26-1007 nt) (C),
NS (27-849 nt) (D), PB2 (28-2289 nt) (E), PB1 (25-2242 nt) (F), PA (25-2175
nt) (G), and NP (46-1526 nt) (H) genes of H9N2 influenza A viruses. Trees
were generated by the Maximum Composite Likelihood model of
Neighbor-Joining algorithm with MEGA 4.1 http://www.megasoftware.
net. The reliability of the trees was assessed by bootstrap analysis with 1,
000 replicates and only bootstrap values  90% were shown. Different
lineages were indicated by different colors. The viruses obtained in the
present study were marked with red circles, and the representative
strains in each lineage were marked with yellow squares.
Additional file 2: Table S1. Comparison of amino acid sequences of HA,
NA, and PB2 genes of representative H9N2 viruses from northern China.
Additional file 3: Table S2. The clinical information of representative
avian H9N2 influenza viruses isolated from northern China.
Acknowledgements
This work was supported by the National Natural Science Foundation of
China (31101830), the Knowledge Innovation Program of the Chinese
Academy of Sciences (KSCX2-EW-Q-14), the funding of Animal Experimental
platform operation of the Chinese Academy of Sciences (CZBZX-1), and the
Ministry of Science and Technology of China program (2010BAD04B01).
Wenjun Liu is the principal investigator of the Innovative Research Group of
the National Natural Science Foundation of China (NSFC, Grant No.
81021003). We also thank Dr. Yanxin Hu, Dr. Honglei Sun for excellent
technical assistance during histopathological examination, thank Dr. Yipeng
Sun and Dr. Guoying Dong for suggestions of the manuscript.
Author details
1
Center for Molecular Virology, Key Laboratory of Pathogenic Microbiology
and Immunology, Institute of Microbiology, Chinese Academy of Sciences,
Beijing 100101, China. 2
Graduate University of Chinese Academy of Sciences,
Beijing 100101, China. 3
College of Animal Science and Veterinary Medicine,
Qingdao Agricultural University, Qingdao 266109, China. 4
Institute of Animal
Science and Veterinary Medicine, Shandong Academy of Agricultural
Sciences, Jinan, Shandong 250100, China. 5
Key Laboratory of Zoonosis of
Ministry of Agriculture, College of Veterinary Medicine, China Agricultural
University, Beijing 100193, China.
Authors' contributions
This study was elaborated and launched by YB, LS and WL, who leading the
research groups on virology and genetics. YB and LL accomplished the virus
sequencing, phylogenetic, molecular analyses and the animal experiments.
YB, ZQ, YY, JL, YZ, HG and JhL did much effort on virus collection and
isolation. YB, LL and BZ participated in the work of manuscript preparation
and revision. All authors have read and approved the final manuscript.
Competing interests
The authors declare that they have no competing interests.
Received: 24 June 2011 Accepted: 4 November 2011
Published: 4 November 2011
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doi:10.1186/1743-422X-8-505
Cite this article as: Bi et al.: Novel genetic reassortants in H9N2
influenza A viruses and their diverse pathogenicity to mice. Virology
Journal 2011 8:505.
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