Virus Genes

, Volume 48, Issue 3, pp 479–485

Sequence and phylogenetic analysis of surface protein genes of emerging H9N2 influenza viruses isolated from poultry in two geographical regions of China

Authors

  • Yu Xue
    • College of Animal ScienceSouth China Agricultural University
    • Guangdong Wen’s Foodstuff Group Co. LtdGuangdong Enterprise Key Laboratory for Animal Health and Environmental Control
  • Jing-Lan Wang
    • College of Animal ScienceSouth China Agricultural University
  • Zhuan-Qiang Yan
    • College of Animal ScienceSouth China Agricultural University
  • Guang-Wei Li
    • College of Animal ScienceSouth China Agricultural University
  • Shun-Yan Chen
    • College of Animal ScienceSouth China Agricultural University
  • Xiang-Bin Zhang
    • College of Animal ScienceSouth China Agricultural University
  • Jian-Ping Qin
    • Guangdong Wen’s Foodstuff Group Co. LtdGuangdong Enterprise Key Laboratory for Animal Health and Environmental Control
  • Hai-Yan Li
    • Guangdong Wen’s Foodstuff Group Co. LtdGuangdong Enterprise Key Laboratory for Animal Health and Environmental Control
  • Shuang Chang
    • Guangdong Wen’s Foodstuff Group Co. LtdGuangdong Enterprise Key Laboratory for Animal Health and Environmental Control
  • Feng Chen
    • College of Animal ScienceSouth China Agricultural University
  • Ying-Zuo Bee
    • College of Animal ScienceSouth China Agricultural University
    • College of Animal ScienceSouth China Agricultural University
Article

DOI: 10.1007/s11262-014-1060-1

Cite this article as:
Xue, Y., Wang, J., Yan, Z. et al. Virus Genes (2014) 48: 479. doi:10.1007/s11262-014-1060-1

Abstract

Subtype H9N2 avian influenza viruses (AIVs) circulating in China have aroused increasing concerns for their impact on poultry and risk to public health. The present study was an attempt to elucidate the phylogenetic relationship of H9N2 AIVs in two geographically distinct regions of China where vaccination is routinely practiced. A total of 18 emerging H9N2 isolates were identified and genetically characterized. Phylogenetic analysis of hemagglutinin (HA) and neuraminidase (NA) genes confirmed that the isolates belonged to the Y280 lineage. Based on the HA genes, the isolates were subdivided into two subgroups. The viruses from Zhejiang Province were clustered together in Group I, while the isolates from Guangdong Province were clustered together in Group II. Antigenic characterization showed that the tested viruses were antigenically different when compared to the current used vaccine strain. It was notable that 14 out of total 18 isolates had an amino acid exchange (Q→L) at position 216 (226 by H3 Numbering) in the receptor-binding site, which indicated that the virus had potential affinity of binding to human like receptor. These results suggest that the emerging viruses have potential risk to public health than previously thought. Therefore, continuous surveillance studies of H9N2 influenza virus are very important to the prognosis and control of future influenza pandemics.

Keywords

Phylogenetic analysisH9N2 subtypeAvian influenza virusSurface protein

Introduction

Avian influenza viruses (AIVs) belong to the orthomyxoviridae family of RNA viruses and have eight single stranded RNA segments encoding at least ten proteins, including two major surface glycoproteins (HA and NA), nucleoprotein (NP), three polymerase proteins (PB2, PB1 and PA), matrix protein (M1), ion channel protein (M2), and two non-structural (NS1 and NS2) proteins. Of these ten proteins, HA and NA, both highly variable and diverse, are primarily responsible for facilitating influenza virus infection [1]. Influenza viruses exist as multiple subtypes based on the glycoproteins (HA and NA) on the virion surface. Currently, 16 HA (HA1–16) and 9 NA (NA1–9) subtypes have been identified in aquatic birds [2].

Two types of AIV have been described based on their pathogenicity, namely highly pathogenic type AIVs (HPAIV), which cause severe diseases that can lead to large-scale devastating outbreaks with flock mortality as great as 100 %, and low pathogenic type AIVs (LPAIV), which only induce asymptomatic or mild infections [3]. H9N2 AIVs usually cause mild clinical signs in birds. However, they might also elicit severe outbreaks in poultry as a result of co-infection with other pathogens [4, 5]. Notably, previous studies have defined two distinct lineages of H9N2 influenza viruses: the North American lineage and the Eurasian lineage. The Eurasian lineage can be further divided into three major sublineages represented by their prototype strains: A/quail/Hong Kong/G1/97 (G1), A/duck/Hong Kong/Y280/97 (Y280), and A/Hong Kong/Y439/97 (Y439) [6].

H9N2 AIVs were first isolated from turkeys in the USA in 1966 [7]. Since then, avian influenza outbreaks caused by H9N2 have been reported in various regions including Hong Kong, mainland China, South Korea, Japan, India, Pakistan, the Middle East, Europe, South Africa, and South America [814]. In 1994, H9N2 viruses were firstly isolated from diseased chickens in Guangdong Province, China and have since spread to domestic poultry in other provinces [15, 16]. H9N2 virus infections have greatly affected not only the poultry industry but also public health [17]. Some H9N2 viruses have acquired receptor-binding characteristics typical of human strains (2,6-NeuAcGal) [18]. In addition, H9N2 viruses have shown striking changes in the hemadsorbing (HB) sites similar to pandemic human viruses [19]. These findings have demonstrated the possibility of interspecies transmission of H9N2 viruses and its ability to be a new endemic strain.

In 2011, H9N2 influenza viruses seriously affected chickens on many farms in Eastern China and Southern China. The purpose of this study was to characterize the phylogenetic relationships of surface protein genes of H9N2 AIV field strains from these two regions. The study would provide useful information for understanding of the evolution of pandemic H9N2 strains in China.

Materials and methods

Viruses

A total of 18 H9N2 viruses were isolated from chickens in vaccinated poultry flocks in Zhejiang Province and Guangdong Province, in Eastern China and Southern China, respectively, from October to December 2011 (Table 1). All viruses were propagated in specific pathogen free (SPF) embryonated chicken eggs via allantoic route and were incubated for 72 h at 37 °C. The allantoic fluids of the embryos were collected and subjected to hemagglutination inhibition (HI) and neuraminidase inhibition (NI) tests to identify the virus subtypes using antisera specific to the reference strains of influenza viruses.
Table 1

List of H9N2 isolates sequenced in this study

Virus

Abbreviation

Accession number

HA

NA

A/chicken/Zhejiang/WXQ12/2011

WXQ12

JQ770124

JQ770156

A/chicken/Zhejiang/KHZ12/2011

KHZ12

JQ770125

JQ770144

A/chicken/Zhejiang/HGH12/2011

HGH12

JQ770126

JQ770160

A/chicken/Zhejiang/FYQ12/2011

FYQ12

JQ770127

JQ770159

A/chicken/Zhejiang/FXK12/2011

FXK12

JQ770128

JQ770155

A/chicken/Zhejiang/SYP12/2011

SYP12

JQ770138

JQ770149

A/chicken/Zhejiang/DJR12/2011

DJR12

JQ770139

JQ770147

A/chicken/Zhejiang/ZGPL12/2011

ZGPL12

JQ770142

JQ770148

A/chicken/Zhejiang/XBF12/2011

XBF12

JQ770143

JQ770157

A/chicken/Guangdong/ZW12/2011

ZW12

JQ770131

JQ770146

A/chicken/Guangdong/SYX12/2011

SYX12

JQ770132

JQ770152

A/chicken/Guangdong/LJT12/2011

LJT12

JQ770133

JQ770151

A/chicken/Guangdong/LWZ11/2011

LWZ11

JQ770134

JQ770150

A/chicken/Guangdong/SJS12/2011

SJS12

JQ770135

JQ770162

A/chicken/Guangdong/LFQ12/2011

LFQ12

JQ770136

JQ770161

A/chicken/Guangdong/CYH12/2011

CYH12

JQ770137

JQ770158

A/chicken/Guangdong/LYH12/2011

LYH12

JQ770140

JQ770145

A/chicken/Guangdong/SQL10/2011

SQL10

JQ770141

JQ770154

RT-PCR and sequencing

The viral RNA was extracted directly from the allantoic fluid using TRIzol reagent (Invitrogen, USA) following the manufacture’s protocol. One-step RT-PCR was performed using the PrimeScript One-Step RT-PCR Kit (TaKaRa Biotechnology, Dalian, China) according to the standard procedure. Primers used for the surface protein genes were: HA-F: 5′-CAAGATGGAAGTAGTATCACT-3′. HA-R: 5′-TTGCCAATTATATACAAATGT-3′. NA-F: 5′-AGCA/GAAGCAGGAGTG/AAAATGAAC/TC-3′. NA-R: 5′-AGTAGAAACAAGGAGTTTTTTTCTAAAATTGCG-3′.

The RT-PCR amplification conditions were as follows: 50 °C for 30 min, 94 °C for 5 min, 32 cycles of 94 °C for 40 s, 53 °C (HA) and 56 °C (NA) for 40 s, 72 °C for 2 min, respectively, and finally, extension at 72 °C for 10 min. The PCR products were purified using an AxyPrep DNA Gel Extraction Kit (Axygen Inc., USA) according to the manufacturer’s instructions and then ligated with pMD19-T vector for later sequencing (TaKaRa Biotechnology, Dalian, China).

Phylogenetic analysis

We performed multiple-sequence alignment with the ClustalW program using MEGA software, version 5.05 [20]. The nucleotide sequence homologies were further accessed through the Clustal W method of DNAStar software (DNAStar Inc.). Phylogenetic trees were generated by the neighbor-joining method with the use of MEGA software, version 5.05, and bootstrap values were calculated on 1,000 replications of the alignment.

Antigenic analysis

Antigenic analyses were performed by HI tests using chicken antisera generated against the H9N2 viruses. Six week-old SPF chickens were injected with 1 ml of oil emulsion-inactivated vaccine derived from eight tested viruses and the commercial vaccine strain A/chicken/Guangdong/SS/94(SS/94), and sera were collected at 3 weeks after injection. Institutional and national guidelines for the care and use of animals were followed, and all animal procedures were conducted under the protocol (SCAU-AEC-2010-0416) approved by the Animal Ethics Committee of South China Agricultural University. The cross HI test was performed as previously described with a starting sera dilution of 1:10 [21]. The dataset consisted of a table of nine chicken sera by 12 H9N2 viruses resulting in 108 individual HI measurements (Table 2).
Table 2

Antigenic analysis of H9N2 avian influenza viruses tested in this study

Virus

Subgroup

Antiseraa

WXQ12

KHZ12

HGH12

FYQ12

ZW12

SYX12

LJT12

LWZ11

SS/94

WXQ12

I

1280b

1280

160

320

1280

1280

1280

160

160

KHZ12

I

2560

2560

320

2560

1280

1280

1280

320

320

HGH12

I

160

80

320

80

80

640

320

80

40

FYQ12

I

1280

160

160

320

80

640

640

40

160

FXK12

I

640

640

320

640

1280

1280

1280

160

320

SYP12

I

2560

2560

320

1280

160

2560

640

80

640

ZW12

II

20

20

20

20

80

160

160

20

40

SYX12

II

2560

2560

160

1280

640

2560

1280

320

640

LJT12

II

160

<c

40

80

80

80

160

40

40

LWZ11

II

1280

320

160

320

1280

1280

640

320

320

SJS12

II

1280

320

160

20

40

1280

80

640

80

SS/94

Vaccine strain

80

40

<

80

40

40

160

<

320

aAntisera were tenfold diluted

bHomologous titers are shown in bold

c< titer <20

Results

Homology analysis

The coding sequences of the HA and NA genes of the 18 tested H9N2 viruses were 1,683 and 1,401 nucleotides long, respectively. Homology analysis was performed to compare the nucleotide sequences of surface protein genes of the tested viruses with other sequences available in GenBank. Analysis of the HA and NA genes showed that the homology among the tested viruses were ranged from 88.2 to 99.8 % and 93.9 to 100 %, respectively. The HA and NA genes of the tested H9N2 isolates shared 91.9–93.2 % and 92.6–94.1 % nucleotide homology, respectively, with the representative virus Y280, Which indicated that they belong phylogenetically to the Y280-like lineage. As compared to the vaccine strains SS/94, A/chicken/Shandong/6/96(6/96), and A/chicken/Shanghai/F/98(F/98), the isolated HA and NA genes showed a low homology ranged from 90.0 to 92.5 % and 92.1 to 94.6 %, respectively. The results of homology analysis indicated that most of the tested strains have genetic distances with the vaccine strain.

Phylogenetic analysis

To determine the evolutionary relationships between tested viruses and the other representative H9N2 viruses, phylogenetic analysis was carried out for the two surface protein gene segments. Phylogenetic analyses based on the HA genes revealed that the 18 tested strains belonged to the Y280-like lineage (Fig. 1). Subsequently, this group was subdivided into two subgroups. The viruses isolated from Zhejiang Province were clustered together in Group I, while the isolates from Guangdong Province were clustered together in Group II. None of the present sequences were clustered within the G1-like and Y439-like lineage. The results indicated that the phylogenetic distributions on HA vary were associated with the geography.
https://static-content.springer.com/image/art%3A10.1007%2Fs11262-014-1060-1/MediaObjects/11262_2014_1060_Fig1_HTML.gif
Fig. 1

Phylogenetic tree of H9N2 avian influenza viruses (AIVs) isolated in China in 2011 based on the HA gene sequences. The tested viruses were marked with triangles, vaccine strains were marked with squares, and the isolates marked with stars stand for representative strains of sublineage grouping

The NA genes of these viruses were also clustered in the Y280 lineage (Fig. 2). However, instead of all ten sequences from Guangdong Province clustered together in one group, three of them (SYX12, LJT12, and LYH12) formed a single group named Group NAII, while the remaining seven Guangdong isolates and all viruses from Zhejiang Province fell into another predominant group named Group NAI.
https://static-content.springer.com/image/art%3A10.1007%2Fs11262-014-1060-1/MediaObjects/11262_2014_1060_Fig2_HTML.gif
Fig. 2

Phylogenetic tree of H9N2 viruses isolated in China in 2011 based on the NA gene sequences. The tested viruses were marked with triangles, vaccine strains were marked with squares, and the isolates marked with stars stand for representative strains of sublineage grouping

Antigenic analysis of H9N2 isolates

To understand the antigenic evolution of the tested H9N2 influenza viruses, six viruses from Zhejiang Province (Group I), five viruses from Guangdong Province (Group II), and the vaccine strain SS/94 were investigated with sera raised against H9N2 viruses. SS/94 was one of the earliest isolates from Guangdong Province and was widely used as a vaccine strain; however, the HI test showed that 6 of 11 new emerging viruses were antigenic heterologous with this virus (the HI titers were two-fold lower than the homologous HI titer). The antisera to the Group I virus WXQ12 and KHZ12 could cross-react well with both of Group I and Group II viruses, except the ZW12 virus, but not with the vaccine strain SS/94. The antisera derived from Group II virus SYX12 reacted well with the all of the 11 new emerging viruses but poorly with the vaccine strain SS/94. These results suggested that the H9N2 influenza viruses circulating in these two provinces of China were quite different antigenically with the vaccine strain which illustrated the necessity to update the vaccine formulation for H9N2.

Key site analyses

The deduced amino acid sequences of surface protein genes of the tested viruses were respectively determined and compared with those of representative H9N2 viruses (Table 3). In this study, the isolates had two types of cleavage site motifs on the HA protein. The Group I viruses had the cleavage site motifs PSRSSR↓GLF. While In Group II, except one strain LYH12 harbored PAKSSR↓GLF at the cleavage sites, the others had the motifs PARSSR↓GLF, which were identical to the isolates such as representative strains HK-G1, HK-Y280, and vaccine strains. All isolates lacked multiple basic amino acids at the cleavage site. Among the amino acids residues in the receptor-binding site (RBS) of HA protein, most of them were conserved; however, a remarkable substitution mutation A180 V (or T) was observed. It was notable that 14 out of total 18 isolates had an amino acid exchange (Q→L) at position 216 (226 by H3 Numbering) in the RBS, which indicated that the virus had high affinity of binding to human like receptor [22].
Table 3

Comparison of amino acid sequences of HA and NA genes between field isolates and other published strains of H9N2

Viruses

Residue at RBSa position

Cleavage site of HA

NA Stalk deletions

Hemadsorbing site

173

180

216

218

366–373

399–404

431–433

LWZ11

N

T

Q

G

PARSSR↓GLF

63–65

IKSDSRRG

DSDSWS

PLE

LJT12

L

IKNGSRRG

DSDDWS

PQE

SYX12

V

L

IKNGSRRG

DSDDWS

PQE

ZW12

A

IKGDSRRG

PQE

SQL10

L

PQE

LYH12

PKRSSR↓GLF

IKNGSRRG

DSDDWS

PQE

CYH12

A

L

PQE

LFQ12

L

PQE

SJS12

A

IKSGSRRG

PQE

WXQ12

L

PSRSSR↓GLF

PQE

KHZ12

A

L

PSRSSR↓GLF

PQE

HGH12

L

PSRSSR↓GLF

IKNDSRRG

PQE

FYQ12

L

PSRSSR↓GLF

PQE

FXK12

L

PSRSSR↓GLF

IKSGSRRG

PQE

DJR12

L

PSRSSR↓GLF

IKSGSRRG

PQE

SYP12

A

L

PSRSSR↓GLF

IKSDLRRG

PKE

ZGPL12

L

PSRSSR↓GLF

IESDSRRG

PQE

XBF12

L

PSRSSR↓GLF

IKSGSRRG

PQE

SS/94

A

PAGSSR↓GLF

IKEDLRRG

DSDNWS

PQE

6/96

A

IKKDLRRG

DSDNWS

PQE

F/98

A

IKKDSRRG

DSDNWS

PQE

Y280

L

IKEDSRRG

DSDNWS

PQE

G1

H

E

L

38–39

IKKDSRRG

DSDIWS

PQE

Y439

H

E

PAASNR↓GLF

None

DNNNWS

PQE

aRBS, receptor-binding site

“En dashes” indicated sequences identical to those of the LWZ11 strain

The sequences in bold stand for amino acid substitutions

Mutations of HB sites on NA protein of the tested strains were analyzed. The results revealed that amino acids of HB sites of the Group NAI strains at position 366–373 had great genetic diversity (Table 3), while the Group NAII strains only exhibited IKNGSRSG at the same positions. The amino acid sequences at position 399–404 had two types: DSDSWS and DSDDWS. Interestingly, the mold of DSDSWS only existed in Group NAI strains and DSDDWS belonged to Group NAII strains. Most of these amino acid residues at position 431–433 were PQE among the test strains. Globally, these results were similar to those detected in the strain HK-Y280 and in H9N2 human isolates from Hong Kong in 1999. In addition, the tested strains all showed 3-residue deletion (position 63–65) in the NA stalk regions, which was in agreement with the data on most of Chinese H9N2 viruses.

Discussion

H9N2 subtype Influenza viruses continue to circulate widely in domestic poultry in Asia [13, 14, 23]. As low pathogenicity viruses, the H9N2 viruses infect a number of species, including chickens, quail, turkeys, ducks, geese, and pigs [24]. More importantly, they may be significant donors of genetic material to emerging human pathogens. Recent studies have demonstrated that the H9N2 viruses contributed the internal genes to the human H7N9 viruses that caused panic in Eastern China [25]. Previous studies have reported that H9N2 AIVs were the mainly prevalent virus subtype in chickens in Eastern China and South China [16, 26, 27]. In the present study, we characterized genetically 18 H9N2 viruses isolated from chickens in our surveillance in Zhejiang and Guangdong provinces, China, in 2011. Phylogenetic analysis demonstrated that the current isolates from these two geographical distinct regions were derived from Y280-like lineage, which indicated that they have a common ancestor. However, the viruses from these two regions strictly divided into two groups, according to the phylogenetic relationship of HA genes. Furthermore, it is remarkable that the Group I viruses from Zhejiang Province had the A316S substitution at cleavage site, which was reported to increase HA cleavage efficiency and strengthen the virulence of H9N2 virus in chickens and mice [28].

It has been well documented that the RBS motif of HA is critical for cellular receptor specificity and also determining virus host range [29]. Particularly, the residue at position 180 has been reported to influence the affinity of virus binding to SA receptor, high affinity to the human like receptor being with V at position 180, intermediate with T and low with A [30]. In this study, 5 out of the 18 tested strains had A180, one of the viruses had V180, and the others had threonine residue at this position. These results were in accordance with findings for other H9N2 viruses isolated in China [31]. Furthermore, all isolates from Group I and five isolates in Group II had leucine at position 216, instead of glutamine at this position of the vaccine strains. The switch from glutamine to leucine at position 216 indicates a preference for NeuAca2, 6-Gal linkage, which possesses human influenza virus-like receptor specificity [22].

The HB site of NA is highly conserved in equine and aquatic bird influenza viruses. However, avian viruses co-circulating in mammals accumulate various substitutions in the HB site, thus decreasing their HB capacity [19]. The NA sequences of the tested strains carried substitutions in the HB site were typical of human pandemic H2N2 and H3N2 viruses [30]. The most striking mutation was at position 402, from asparagine to serine, in Group NAI strains, and from asparagine to aspartic acid in Group NAII strains. Furthermore, the NA stalk is crucial for balancing the complementary activity of HA and NA and has been linked with efficiency of virus replication and pathogenesis [32]. Amino acid alignment of the NA gene with deletion of three amino acids residues at positions 63–65 was found in the tested strains. This deletion was reported typical of viruses isolated from China.

As it is well known, vaccination is an effective method to prevent AIVs epidemic. But, in order for the vaccine to be effective, it needs to target current local strains, and selected based on its pathogenic, antigenic, and genetic properties [33]. In the present study, the homology analysis of HA and NA genes showed that the current isolates shared less homology with the vaccine strains. Furthermore, antigenic characterization showed that the tested viruses were antigenically different when compared to the current used vaccine strain. Our findings potently indicated that the vaccine strains need to be updated and the candidate vaccine strains should be selected from the predominant clade.

In summary, the present study has demonstrated that the circulating H9N2 AIVs belong to Y280-like lineage, and also indicated that different geographical factors played a significant role in the evolution of H9N2 virus. In spite of the low virulence, these strains had potential affinity of binding to human like receptor and exhibits genetic disparity with vaccine strains. This situation could give rise to great uncertainty for the future evolution and ecosystem of H9N2 viruses in China. Therefore, it is imperative that particular attention be paid to the ecology and evolution of avian-originated H9N2 viruses in order to avoid future influenza pandemics in humans.

Acknowledgments

This research was supported by a grant from the Science and Technology Planning Project of Guangdong Province China (No. 2010B090301019, 2009B020201008).

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© Springer Science+Business Media New York 2014