# Sequence and Phylogenetic Analysis of Three Isolates of Avian Influenza H9N2 from Chickens in Southern China

Authors: Zhixun Xie; Jianbao Dong; Xiaofei Tang; Jiabo Liu; Mazhar I. Khan; Yaoshan Pang; Xianwen Deng; Zhiqin Xie
Journal: Scholarly Research Exchange
 (2008)

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## Abstract

Three isolates of H9N2 subtype of influenza virus have been isolated from chickens in Guangxi province. In this study, eight full-length genes of each of the H9N2 isolates, A/Chicken/Guangxi/1/00, A/Chicken/Guangxi/14/00, and A/Chicken/Guangxi/17/00, were obtained. Sequence analysis and phylogenetic studies were conducted by comparing eight genes of each isolate with those of the available H9N2 strains at GenBank. Results showed a high degree of homology between the Guangxi isolates and isolates from Guangdong and Jiangsu provinces, suggesting that Guangxi isolates originated from the same source. However, the eight genes of these three isolates from Guangxi were not in the same sublineages in the phylogenic trees, which suggest that they were products of natural reassortment between H9N2 avian influenza viruses from different sublineages. The 9 nucleotides ACAGAGATA encoding amino acids T, E, I were absent between nucleotide 205 and 214 in the open reading frame of NA genes in the Guangxi isolates. AIV strains that infect human have in their HA proteins, Leucine at position 226. The analysis of deduced amino acid sequence of HA proteins of C/GX/1/00, C/GX/14/00, and C/GX/17/00 showed that the position 226 of these isolates was glycine instead of leucine, suggesting that these three isolates differ from H9N2 AIV strains isolated from human infections.


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## Body

## 1. Introduction

Avian influenza virus (AIV) belongs
to theOrthomyxoviridae family of RNA viruses. It is an enveloped virus with a
helical nucleocapsid and eight segments of single-stranded negative-sense RNA.
The envelope contains the haemagglutinin (HA) and neuraminidase (NA) proteins,
of which there are currently 16 HA (H1–H16) and 9 NA (N1–N9) subtypes
[1, 2]. The H9N2 subtype that
is worldwide spread in poultry could infect humans and is prevalent in China
[3–8]. The first outbreak of disease attributed to H9N2
in China happened in Guangdong province in 1994. Other strains of this subtype were subsequently
isolated [6, 7, 9, 10].Studies to characterize these strains were focused on the HA genes for one of the surface viral proteins that plays a crucial role in the pathogenicity [10]. Recent studies
characterized the nonstructural [11] and neuraminidase genes
[12] of H9N2 viruses in China. Others showed that some
novel reassortant H9N2 influenza viruses may possess certain genes from H5
subtype viruses [7, 13, 14]. In this
study, we have characterized three strains of H9N2 influenza viruses that were
isolated from chicken farms in Guangxi province. All eight full-length genes of
these isolates were obtained individually by means of RT-PCR. Sequence analysis
and phylogenetic study were conducted by comparing the eight genes of each
isolate with sequences available in Genbank (H9N2 isolates from 1966–2005).


## 2. Materials and Methods

### 2.1. Viruses

Three isolates of H9N2 subtype influenza viruses, A/Chicken/Guangxi/1/00 (C/GX/1/00),
A/Chicken/Guangxi/14/00 (C/GX/14/00), A/Chicken/Guangxi/17/00 (C/GX/17/00)
were recovered from chickens in different areas of Guangxi province
during 2000 and 2001. Initial isolation was performed in 10-day-old
specific-pathogen-free (SPF) embryonated chicken eggs. Identification of the
viruses was determined by standard hemagglutination-inhibition and
neuraminidase-inhibition tests [15]. Allantoic fluids were harvested
from SPF egg-passaged viruses and used as stock viruses for further analysis.


### 2.2. RNA Extraction and RT-PCR

Viral RNA was obtained from allantoic fluid by TRIzol extraction
according to the method described by Xie et al. [16]. The concentrations of
RNA were determined by spectrophotometry using the UV2501PC (Shimadzu
Cooperation, Tokyo, Japan) and then stored at −20°C.
Reverse transcription was done by using random primers with
RNA PCR (AMV) V3.1 kit (TaKaRa Biotechnology, Dalian, China). Amplification of the eight full-length genes was carried out by PCR as described
previously by Xie et al. [16] using pairs of specific primers as
described in Table 1. The reaction was
carried out at 30°C for 5 minutes followed by 60 minutes incubation at 37°C. PCR was
performed in a reaction mixture of 50 μL containing master mix buffer with
dNTPs, 50-time Advantage 2 polymerase (Clontech Mountain View, California, USA), 10 μM
of each specific primer, and 200 ng cDNA template. The PCR condition for the
amplification of Pb2, Pb1, and HA was
95°C for 2 minutes denaturation, and 35 cycles of 95°C for 30 seconds, 62°C for 45 seconds
(annealing), and 68°C for 3 minutes, followed by 70°C for 10 minutes final extension. The PCR condition for the
amplification of NP, NA, M, and NS genes was the same as above, except that the
annealing temperature was reduced to 60°C for NP and NA genes and 58°C for M
and NS genes.Table 1
The nucleotide sequences of the eight pairs of specific primers for H9N2 subtypes.
GeneSequence of primersHAF :5′-AGCAAAAGCAGGGGAATTTCAC-3′R :5′-AGTAGAAAACAAGGGTGTTTTTGC-3′:NAF :5′-AAAAGCAGGCAAACCATTTGA-3′R :5′-TTTTCATGAAGGACAAGCTAA-3′NPF :5′-GCAGGTAGATAATCACTCACTG-3′R :5′-AGTAGAAACAAGGGTATTTTT-3′MF :5′-AGCAAAAGCAGGTAGATGTTTAAAG-3′R :5′-AGTAGAAACAAGGTAGTTTTTTAC-3′NSF :5′-AAAGCAAGGGTGACAAAGACAT-3′R :5′-TAGAAACAAGGGTGTTTTTTATCA-3′PAF :5′-AGCAAAAGCAGGTACTGAT-3′R :5′-AGTAGAAACAAGGTACTTTT-3′Pb1F :5′-AAAAGCAGGCAAACCATTTGA-3′R :5′-TTTTCATGAAGGACAAGCTAA-3′Pb2F :5′-AAAAGCAGGTCAATTATATTC-3′R :5′-AAGGTCGTTTTTAAACTATTCA-3′


### 2.3. Sequence Analysis

All PCR products were subjected to electrophoresis in a
1% (w/v) agarose gel. DNA fragments of the expected length were extracted and
purified with DNA Glass-milk Rapid Purification Kit (MK001-2, BioDev, Beijing, China). The purified DNA fragments were
cloned into pMD18-T easy vector according to the manufacturer protocol (TaKaRa
Biotechnology Co. Ltd., Dalian, China). Three clones of each of the 8 fragments were sequenced by the Takara Biotechnology Co.
Ltd., Dalian, China. DNA sequences of eight genes were compared with the GenBank
database. DNA sequences of each cloned gene were repeated twice to confirm the
similarity of sequence data. The nucleotide sequences obtained in this study
are available in the GenBank database under accession numbers: DQ485205 to
DQ485228.


### 2.4. Phylogenetic Analysis

The DNA sequences of the eight genes of three Guangxi H9N2
subtypes were compared initially with the Megalign program of the package with
the Clustal alignment algorithm (DNASTAR Inc., Madison, Wis, USA) against the twelve H9N2 virus
sequences as listed in Table3. Pairwise sequence alignments were also performed
with the Clustal alignment algorithm in the Megalign program to determine
sequence similarity and phylogenetic relationship of different H9N2 subtype
viruses. The ancestral relationships within eight genes among the fifteen H9N2
subtypes are presented in a phylogenetic tree created by the Megalign program.


## 2.1. Viruses

Three isolates of H9N2 subtype influenza viruses, A/Chicken/Guangxi/1/00 (C/GX/1/00),
A/Chicken/Guangxi/14/00 (C/GX/14/00), A/Chicken/Guangxi/17/00 (C/GX/17/00)
were recovered from chickens in different areas of Guangxi province
during 2000 and 2001. Initial isolation was performed in 10-day-old
specific-pathogen-free (SPF) embryonated chicken eggs. Identification of the
viruses was determined by standard hemagglutination-inhibition and
neuraminidase-inhibition tests [15]. Allantoic fluids were harvested
from SPF egg-passaged viruses and used as stock viruses for further analysis.


## 2.2. RNA Extraction and RT-PCR

Viral RNA was obtained from allantoic fluid by TRIzol extraction
according to the method described by Xie et al. [16]. The concentrations of
RNA were determined by spectrophotometry using the UV2501PC (Shimadzu
Cooperation, Tokyo, Japan) and then stored at −20°C.
Reverse transcription was done by using random primers with
RNA PCR (AMV) V3.1 kit (TaKaRa Biotechnology, Dalian, China). Amplification of the eight full-length genes was carried out by PCR as described
previously by Xie et al. [16] using pairs of specific primers as
described in Table 1. The reaction was
carried out at 30°C for 5 minutes followed by 60 minutes incubation at 37°C. PCR was
performed in a reaction mixture of 50 μL containing master mix buffer with
dNTPs, 50-time Advantage 2 polymerase (Clontech Mountain View, California, USA), 10 μM
of each specific primer, and 200 ng cDNA template. The PCR condition for the
amplification of Pb2, Pb1, and HA was
95°C for 2 minutes denaturation, and 35 cycles of 95°C for 30 seconds, 62°C for 45 seconds
(annealing), and 68°C for 3 minutes, followed by 70°C for 10 minutes final extension. The PCR condition for the
amplification of NP, NA, M, and NS genes was the same as above, except that the
annealing temperature was reduced to 60°C for NP and NA genes and 58°C for M
and NS genes.Table 1
The nucleotide sequences of the eight pairs of specific primers for H9N2 subtypes.
GeneSequence of primersHAF :5′-AGCAAAAGCAGGGGAATTTCAC-3′R :5′-AGTAGAAAACAAGGGTGTTTTTGC-3′:NAF :5′-AAAAGCAGGCAAACCATTTGA-3′R :5′-TTTTCATGAAGGACAAGCTAA-3′NPF :5′-GCAGGTAGATAATCACTCACTG-3′R :5′-AGTAGAAACAAGGGTATTTTT-3′MF :5′-AGCAAAAGCAGGTAGATGTTTAAAG-3′R :5′-AGTAGAAACAAGGTAGTTTTTTAC-3′NSF :5′-AAAGCAAGGGTGACAAAGACAT-3′R :5′-TAGAAACAAGGGTGTTTTTTATCA-3′PAF :5′-AGCAAAAGCAGGTACTGAT-3′R :5′-AGTAGAAACAAGGTACTTTT-3′Pb1F :5′-AAAAGCAGGCAAACCATTTGA-3′R :5′-TTTTCATGAAGGACAAGCTAA-3′Pb2F :5′-AAAAGCAGGTCAATTATATTC-3′R :5′-AAGGTCGTTTTTAAACTATTCA-3′


## 2.3. Sequence Analysis

All PCR products were subjected to electrophoresis in a
1% (w/v) agarose gel. DNA fragments of the expected length were extracted and
purified with DNA Glass-milk Rapid Purification Kit (MK001-2, BioDev, Beijing, China). The purified DNA fragments were
cloned into pMD18-T easy vector according to the manufacturer protocol (TaKaRa
Biotechnology Co. Ltd., Dalian, China). Three clones of each of the 8 fragments were sequenced by the Takara Biotechnology Co.
Ltd., Dalian, China. DNA sequences of eight genes were compared with the GenBank
database. DNA sequences of each cloned gene were repeated twice to confirm the
similarity of sequence data. The nucleotide sequences obtained in this study
are available in the GenBank database under accession numbers: DQ485205 to
DQ485228.


## 2.4. Phylogenetic Analysis

The DNA sequences of the eight genes of three Guangxi H9N2
subtypes were compared initially with the Megalign program of the package with
the Clustal alignment algorithm (DNASTAR Inc., Madison, Wis, USA) against the twelve H9N2 virus
sequences as listed in Table3. Pairwise sequence alignments were also performed
with the Clustal alignment algorithm in the Megalign program to determine
sequence similarity and phylogenetic relationship of different H9N2 subtype
viruses. The ancestral relationships within eight genes among the fifteen H9N2
subtypes are presented in a phylogenetic tree created by the Megalign program.


## 3. Results

### 3.1. Result of Sequencing

The lengths of all genes were obtained
for the three isolates (C/GX/1/00, C/GX/14/00,             and
C/GX/17/00): HA 1.7 kb, NA 1.4 kb, NP 1.5 kb, M 1.0 kb, NS 890 bp, PA 2.2 kb, Pb1 2.3 kb, Pb2 2.3 kb.
BLAST software and Megalign programs were used to determine the sequence similarity
of the eight genes from the three isolates, as shown in Table2.Table 2
Length of each gene of AIV C/GX/1/00, C/GX/14/00, C/GX/17/00, and virus with the greatest homology to each gene.
Gene segmentStrainOpen reading frameLength of peptidesVirus with greatest homologyHomology (%)HAC/GX/1/0034∼1716560A/Chicken/Guangdong/4/0099.3C/GX/14/0034∼1716560A/Chicken/Guangdong/4/0098.5C/GX/17/0034∼1716560A/Chicken/Henan/26/0099.2NAC/GX/1/0020∼1429469A/Chicken/Guangdong/4/0097.0C/GX/14/0020∼1420466A/Chicken/Jiangsu/1/0097.7C/GX/17/0020∼1429469A/Chicken/Jiangsu/1/0097.7NPC/GX/1/0039∼1435498A/Chicken/Jiangsu/1/0098.7C/GX/14/0039∼1435498A/quail/Hongkong/G1/9798.0C/GX/17/0039∼1435498A/quail/Hongkong/G1/9798.0MC/GX/1/0026∼784252A/Chicken/Guangdong/4/0099.6C/GX/14/0026∼784252A/Chicken/Guangdong/4/0098.4C/GX/17/0026∼784252A/Chicken/Guangdong/4/0099.6NSC/GX/1/0025∼720231A/Chicken/Guangdong/47/0198.9C/GX/14/0066∼719217A/Chicken/Guangdong/4/0098.7C/GX/17/0027∼680217A/Chicken/Guangdong/4/0098.5PAC/GX/1/0025∼2175716A/Chicken/Guangdong/47/0194.0C/GX/14/0025∼2175716A/Duck/Hongkong/Y439/9793.8C/GX/17/0025∼2175716A/Chicken/Guangdong/4/0093.0Pb1C/GX/1/0022∼2298758A/Chicken/Guangdong/4/0096.2C/GX/14/0022∼2298758A/Chicken/Jiangsu/1/0095.9C/GX/17/0022∼2298758A/Chicken/Guangdong/4/0094.8Pb2C/GX/1/0025∼2304759A/Chicken/Jilin/53/0195.5C/GX/14/0025∼2304759A/Hongkong/1047/9992.4C/GX/17/0025∼2304759A/quail/Hongkong/G1/9797.9Table 3
Comparison of ORF nucleotide
sequences of NA gene; nt = nucleotide.
H9N2 subtypesSite in the ORF↓190 nt     ↓205     ↓214     ↓230 ntA-chicken-Guangxi-1-00AATAGAAAGGAACATA--------GTGCACTTGAATAGCA--------A-chicken-Guangxi-14-00AATGGAAAGGAACATA--------GTGTATCTGAATAGCAA-chicken-Guangxi-17-00AATGGAAAGGAACATA--------GTGTATCTGAATAGCAA-chicken-Pakistan-2-99AATAGAAAGGAACATAACAGAGATAGTGCATTTGAATAGCAA-chicken-Shanghai-F-98AATAGAAAGGAACATA--------GTGCATTTGAATAGTAA-duck-Hong Kong-Y280-97AATAGAAAGGAACATA--------GTGCATTTGAATAGTAA-duck-Hong Kong-Y439-97AATAGAAAGGAACATAACAGAGATAGTGCATTTGAATAATAA-Quail-Hong Kong-G1-97AATAGAAAGGAACATAACAGAGATAGTGCATTTGAATAATAA-chicken-Guangdong-47-01AATAGAAAGGAACATAACAGAGATAGTGCATTTGAATAGCAA-chicken-Korea-S1-2003AATAGAAAAGAACATAACAAAAATAGTGTATTTGAATAATAA-chicken-Shenzhen-9-97AATAGAAAGGAACATAACAGAGATAGTGCATTTGAATAATAA-duck-Nanjing-2-97AATAGAAAGGAGCATA--------GTGCATTTGAATAGTAA-chicken-Guangdong-4-00AATAGAAAAGAACATAACAGAGATAGTGCATTTGAATAGTAA-chicken-Fujian-25-00AATAGAAAGGAACATAACAGAGATAGTGCATTTGAATAA--------A-chicken-Jiangsu-1-00AATAGAAAGGAACATAACAGAGATAGTGCATTTGAATAGCA


### 3.2. Comparison of Nucleotide and Deduced Amino Acid Sequences of Eight Genes

In this study, the nucleotide sequences obtained from these three H9N2 isolates were compared
with those of all the available full-length genomes of H9N2 strains isolated
during 1966–2005 deposited in the GenBank database. Results indicate
that nucleotide homology among these isolates with HA genes is between 82.6% to
99.3%; for NA genes is between 91.8% to 97.7%; for NP genes is between 88.0% to
98.7%; for M genes is between 92.7% to 99.6%: for NS genes is between 92.1% to 98.9%;
for PA genes is between 86.1% to 94.1%; for Pb1 genes is between 86.8% to 96.2%;
and for Pb2 genes is between 84.3% to 97.9%.This sequence comparison also shows that 9 nucleotides between nucleotide positions 205 and
214, lying between position 187 to 195 open reading (ORF) in the
NA genes, are absent in these three  Guangxi isolates (see Table3) analyzed here. These 9 nucleotides
encode for amino acids T, E, I. The deduced amino acid sequences of HA genes of
C/GX/1/00, C/GX/14/00, and C/GX/17/00 at the cleavage site of HA contain a
PARSSR/GL motif, which denotes the sequence found in low pathogenicity avian
influenza (LPAI) viruses, as described previously [4, 10, 17].


### 3.3. Phylogenetic Analysis of Eight Genes

The phylogenetic relationship between
each of the eight genes from three isolates, C/GX/1/00, C/GX/14/00, and
C/GX/17/00, was analyzed based on their nucleotide sequence
(Figures1–8). The analysis indicates
that there are three sublineages in the Eurasian lineage as described
previously [4, 10, 18]. The NP and Pb1 genes of C/GX/14/00 and NP, Pb1,
Pb2 genes of C/GX/17/00 were incorporated into the sublineage represented by
A/Quail/Hongkong/G1/97 (see Figures 4, 7,
and 8). The HA, M, NP, Pb1, Pb2 genes of C/GX/1/00, the HA and M genes of C/GX/14/00, and HA and M genes of C/GX/17/00 were incorporated
into the sublineage represented by A/duck/Hongkong/Y280/97 (see Figures 1,
2, and 3).
No gene of the three isolates was incorporated into the sublineage
represented by A/duck/Hongkong/Y439/97. The PA, NA, NS genes of C/GX/1/00, NA, PA, NS, Pb2 genes of C/GX/14/00, and the NA, NS, PA genes of C/GX/17/00 were not incorporated into any of sublineage
of these three isolates (see Figures 4, 5,
6, and 8).Figure 1
Phylogenetic tree of HA gene of AIV.Figure 2
Phylogenetic tree of NA gene of AIV.Figure 3
Phylogenetic tree of M gene of AIV.Figure 4
Phylogenetic tree of NP gene of AIV.Figure 5
Phylogenetic tree of NS gene of AIV.Figure 6
Phylogenetic tree of PA gene of AIV.Figure 7
Phylogenetic tree of Pb1 gene of AIV.Figure 8
Phylogenetic tree of Pb2 gene of AIV.


## 3.1. Result of Sequencing

The lengths of all genes were obtained
for the three isolates (C/GX/1/00, C/GX/14/00,             and
C/GX/17/00): HA 1.7 kb, NA 1.4 kb, NP 1.5 kb, M 1.0 kb, NS 890 bp, PA 2.2 kb, Pb1 2.3 kb, Pb2 2.3 kb.
BLAST software and Megalign programs were used to determine the sequence similarity
of the eight genes from the three isolates, as shown in Table2.Table 2
Length of each gene of AIV C/GX/1/00, C/GX/14/00, C/GX/17/00, and virus with the greatest homology to each gene.
Gene segmentStrainOpen reading frameLength of peptidesVirus with greatest homologyHomology (%)HAC/GX/1/0034∼1716560A/Chicken/Guangdong/4/0099.3C/GX/14/0034∼1716560A/Chicken/Guangdong/4/0098.5C/GX/17/0034∼1716560A/Chicken/Henan/26/0099.2NAC/GX/1/0020∼1429469A/Chicken/Guangdong/4/0097.0C/GX/14/0020∼1420466A/Chicken/Jiangsu/1/0097.7C/GX/17/0020∼1429469A/Chicken/Jiangsu/1/0097.7NPC/GX/1/0039∼1435498A/Chicken/Jiangsu/1/0098.7C/GX/14/0039∼1435498A/quail/Hongkong/G1/9798.0C/GX/17/0039∼1435498A/quail/Hongkong/G1/9798.0MC/GX/1/0026∼784252A/Chicken/Guangdong/4/0099.6C/GX/14/0026∼784252A/Chicken/Guangdong/4/0098.4C/GX/17/0026∼784252A/Chicken/Guangdong/4/0099.6NSC/GX/1/0025∼720231A/Chicken/Guangdong/47/0198.9C/GX/14/0066∼719217A/Chicken/Guangdong/4/0098.7C/GX/17/0027∼680217A/Chicken/Guangdong/4/0098.5PAC/GX/1/0025∼2175716A/Chicken/Guangdong/47/0194.0C/GX/14/0025∼2175716A/Duck/Hongkong/Y439/9793.8C/GX/17/0025∼2175716A/Chicken/Guangdong/4/0093.0Pb1C/GX/1/0022∼2298758A/Chicken/Guangdong/4/0096.2C/GX/14/0022∼2298758A/Chicken/Jiangsu/1/0095.9C/GX/17/0022∼2298758A/Chicken/Guangdong/4/0094.8Pb2C/GX/1/0025∼2304759A/Chicken/Jilin/53/0195.5C/GX/14/0025∼2304759A/Hongkong/1047/9992.4C/GX/17/0025∼2304759A/quail/Hongkong/G1/9797.9Table 3
Comparison of ORF nucleotide
sequences of NA gene; nt = nucleotide.
H9N2 subtypesSite in the ORF↓190 nt     ↓205     ↓214     ↓230 ntA-chicken-Guangxi-1-00AATAGAAAGGAACATA--------GTGCACTTGAATAGCA--------A-chicken-Guangxi-14-00AATGGAAAGGAACATA--------GTGTATCTGAATAGCAA-chicken-Guangxi-17-00AATGGAAAGGAACATA--------GTGTATCTGAATAGCAA-chicken-Pakistan-2-99AATAGAAAGGAACATAACAGAGATAGTGCATTTGAATAGCAA-chicken-Shanghai-F-98AATAGAAAGGAACATA--------GTGCATTTGAATAGTAA-duck-Hong Kong-Y280-97AATAGAAAGGAACATA--------GTGCATTTGAATAGTAA-duck-Hong Kong-Y439-97AATAGAAAGGAACATAACAGAGATAGTGCATTTGAATAATAA-Quail-Hong Kong-G1-97AATAGAAAGGAACATAACAGAGATAGTGCATTTGAATAATAA-chicken-Guangdong-47-01AATAGAAAGGAACATAACAGAGATAGTGCATTTGAATAGCAA-chicken-Korea-S1-2003AATAGAAAAGAACATAACAAAAATAGTGTATTTGAATAATAA-chicken-Shenzhen-9-97AATAGAAAGGAACATAACAGAGATAGTGCATTTGAATAATAA-duck-Nanjing-2-97AATAGAAAGGAGCATA--------GTGCATTTGAATAGTAA-chicken-Guangdong-4-00AATAGAAAAGAACATAACAGAGATAGTGCATTTGAATAGTAA-chicken-Fujian-25-00AATAGAAAGGAACATAACAGAGATAGTGCATTTGAATAA--------A-chicken-Jiangsu-1-00AATAGAAAGGAACATAACAGAGATAGTGCATTTGAATAGCA


## 3.2. Comparison of Nucleotide and Deduced Amino Acid Sequences of Eight Genes

In this study, the nucleotide sequences obtained from these three H9N2 isolates were compared
with those of all the available full-length genomes of H9N2 strains isolated
during 1966–2005 deposited in the GenBank database. Results indicate
that nucleotide homology among these isolates with HA genes is between 82.6% to
99.3%; for NA genes is between 91.8% to 97.7%; for NP genes is between 88.0% to
98.7%; for M genes is between 92.7% to 99.6%: for NS genes is between 92.1% to 98.9%;
for PA genes is between 86.1% to 94.1%; for Pb1 genes is between 86.8% to 96.2%;
and for Pb2 genes is between 84.3% to 97.9%.This sequence comparison also shows that 9 nucleotides between nucleotide positions 205 and
214, lying between position 187 to 195 open reading (ORF) in the
NA genes, are absent in these three  Guangxi isolates (see Table3) analyzed here. These 9 nucleotides
encode for amino acids T, E, I. The deduced amino acid sequences of HA genes of
C/GX/1/00, C/GX/14/00, and C/GX/17/00 at the cleavage site of HA contain a
PARSSR/GL motif, which denotes the sequence found in low pathogenicity avian
influenza (LPAI) viruses, as described previously [4, 10, 17].


## 3.3. Phylogenetic Analysis of Eight Genes

The phylogenetic relationship between
each of the eight genes from three isolates, C/GX/1/00, C/GX/14/00, and
C/GX/17/00, was analyzed based on their nucleotide sequence
(Figures1–8). The analysis indicates
that there are three sublineages in the Eurasian lineage as described
previously [4, 10, 18]. The NP and Pb1 genes of C/GX/14/00 and NP, Pb1,
Pb2 genes of C/GX/17/00 were incorporated into the sublineage represented by
A/Quail/Hongkong/G1/97 (see Figures 4, 7,
and 8). The HA, M, NP, Pb1, Pb2 genes of C/GX/1/00, the HA and M genes of C/GX/14/00, and HA and M genes of C/GX/17/00 were incorporated
into the sublineage represented by A/duck/Hongkong/Y280/97 (see Figures 1,
2, and 3).
No gene of the three isolates was incorporated into the sublineage
represented by A/duck/Hongkong/Y439/97. The PA, NA, NS genes of C/GX/1/00, NA, PA, NS, Pb2 genes of C/GX/14/00, and the NA, NS, PA genes of C/GX/17/00 were not incorporated into any of sublineage
of these three isolates (see Figures 4, 5,
6, and 8).Figure 1
Phylogenetic tree of HA gene of AIV.Figure 2
Phylogenetic tree of NA gene of AIV.Figure 3
Phylogenetic tree of M gene of AIV.Figure 4
Phylogenetic tree of NP gene of AIV.Figure 5
Phylogenetic tree of NS gene of AIV.Figure 6
Phylogenetic tree of PA gene of AIV.Figure 7
Phylogenetic tree of Pb1 gene of AIV.Figure 8
Phylogenetic tree of Pb2 gene of AIV.


## 4. Discussion

AIV has a segmented negative-strand
genome that includes HA, NA, M, NS, NP,
PA, Pb1, Pb2 genes. The
results of sequencing and comparisons with the other H9N2 virus sequences showed
that we have obtained each of eight genes from three Chinese isolates,
C/GX/1/00, C/GX/14/00, and C/GX/17/00 successfully.Previous studies have defined two
distinct lineages of H9N2 influenza viruses: North American and Eurasian
lineages. The Eurasian lineage consists of at least three sublineages [7, 13, 14]. Phylogenetic
analysis in our study showed similar patterns (Figures 1–8). Viral genomes
of H9N2 viruses analyzed in previous studies had shown a common source of
origin from southern China  [6, 9, 10, 18]. Sequence
comparison and phylogenetic analysis illustrated that NP and Pb1 genes of C/GX/14/00, and NP, Pb1,
and Pb2 genes of C/GX/17/00 were incorporated into the sublineage represented by A/Quail/Hongkong/G1/97. HA,
M, NP, Pb1, and Pb2 genes of C/GX/1/00, HA and M genes of
C/GX/14/00, and HA and M genes of C/GX/17/00 were incorporated
into the sublineage represented by A/duck/Hongkong/Y280/97. None of the genes from these three isolates
was incorporated into the sublineage represented by A/duck/Hongkong/Y439/97. PA,
NA, and NS genes of C/GX/1/00, NA, PA, NS, and Pb2 genes of C/GX/14/00, and the NA, NS, and PA genes of C/GX/17/00 were not incorporated into any of the sublineages. So, it is possible that AIV H9N2 subtype strains C/GX/1/00, C/GX/14/00,
and C/GX/17/00 were products of natural reassortment of avian influenza viruses,
suggesting that there may be a specific gene pool in China, as described by Lu
et al. [18].Interestingly, gene sequence comparison indicates that there is a 9-nucleotide deletion
between nucleotides 205 and 214 (187 position to 195 position of ORF) in the NA genes of
the three isolates C/GX/1/00, C/GX/14/00, and C/GX/17/00. These 9 nucleotides
encode for amino acids T, E, and I. This deletion did not change the ORF of the
NA gene. Noteworthy, about 80% of China mainland isolates carry a
similar deletions. Further studies are needed to determine if the lack of 9 nucleotides
in NA affects the function of the NA protein.HA protein determines the infectious host range of AIV strains. AIV strains infecting
humans carry leucine at position 226 of the HA gene [7, 13, 14]. The analysis of deduced amino acid sequence of
HA proteins of C/GX/1/00, C/GX/14/00, and C/GX/17/00 isolates showed a glycine
at position 226, therefore these three isolates do not belong to H9N2 AIV
strains isolated from human infections.



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*Source: 802317-2008-04-14.xml*