Archives of Virology

, Volume 159, Issue 7, pp 1651–1661 | Cite as

Full-genome analysis of avian influenza virus H9N2 from Bangladesh reveals internal gene reassortments with two distinct highly pathogenic avian influenza viruses

  • Rokshana Parvin
  • Kristin Heenemann
  • Mohammad Y. Halami
  • Emdadul H. Chowdhury
  • M. R. Islam
  • Thomas W. Vahlenkamp
Original Article

Abstract

Low-pathogenic avian influenza viruses (LPAIVs) of subtype H9N2 have become widespread in poultry in many Asian countries with relevance to respiratory diseases of multifactorial origin. In Bangladesh, LPAIVs of subtype H9N2 co-circulate simultaneously with highly pathogenic avian influenza viruses (HPAIVs) of subtype H5N1 in commercial and backyard poultry. The aim of this study was to characterize LPAIVs of subtype H9N2 currently circulating in Bangladesh. The selected isolate A/Chicken/Bangladesh/VP01/2006 (H9N2) was propagated in chicken embryos. All eight gene segments were amplified by RT-PCR, cloned, and subjected to full-length sequencing. The sequence data obtained were compared with reference strains available in GenBank. Phylogenetic analysis of LPAIV H9N2 from Bangladesh revealed a close relationship to Indian, Pakistani and Middle Eastern isolates and identified an ancestor relationship to LPAIV H9N2 Quail/HK/G1/1997. The internal genes M and NP belong to lineage G1, whereas NS, PA, PB1 and PB2 belong to the prototype virus A/Chicken/Korea/38349-p96323/96. The internal genes showed high sequence homology to an HPAIV of subtype H7N3 from Pakistan, whereas the PB1 gene showed similarly high nucleotide homologies to recently circulating HPAIV H5N1 from Bangladesh, revealing two independent reassortment events. Examination of the hemagglutinin cleavage site of LPAIV H9N2 confirmed its low pathogenicity. The receptor-binding sites indicated a binding preference for human-type receptors. Several mutations in internal proteins are associated with increased virulence and altered host range, while other amino acids were found to be highly conserved among LPAIV H9N2 isolates.

Introduction

Avian influenza viruses (AIV) are enveloped viruses containing eight genome segments of single-stranded RNA with negative polarity. AIV H9N2 infections display only mild or even asymptomatic clinical signs and are described as low-pathogenic avian influenza virus (LPAIVs). LPAIV H9N2 was first detected in the United States in 1966 [1]. In Asia, LPAIV H9N2 was detected until 1985 only in domestic ducks in Hong Kong. During the 1990s, however, infections became more prevalent in domestic poultry [2], causing disease outbreaks in chickens in many parts of the world including Asia, the Middle East, Africa and Europe. Currently, LPAIV H9N2 is panzootic in multiple avian species [3]. The first zoonotic transmission to humans was reported in Hong Kong and China, in 1997 [4, 5, 6]. Human infections were associated with mild flu-like symptoms [7, 8] with no evidence of human-to-human transmission. Some evolved LPAIV H9N2 strains acquired human-like α2,6 sialic acid receptor specificity [9]. LPAIV H9N2 was also isolated from pigs [10] in Hong Kong, China, and Indonesia, demonstrating its broad host range and suggesting that it might play a role in the generation of new influenza strains of subtype H9 [11]. Three genetically distinct sublineages of H9N2 have become established in Asia [12, 13]. They are represented by the prototype viruses A/Quail/Hong Kong/G1/97 (HK-G1), A/Duck/Hong Kong/Y280/97 (Y-280), and A/Chicken/Korea/38349-p96323/96 (Kr-p96323) in the Eurasian lineage. Recently it has been proposed to distinguish four clades (H9.1 to H9.4) among the H9 HA genes [14]. The prevalence of H9N2 viruses in poultry around the world provides ample opportunity for the acquisition of mutations and reassortment events [15, 16]. Experimentally, LPAIV H9N2 strains also replicate in mice without adaptation [17].

The economy of Bangladesh heavily depends on its agricultural resources. Livestock, especially poultry farming, is a promising sector for poverty reduction. This sector is becoming strongly under pressure due to continuous AIV outbreaks ever since the first detection in 2007. Both HPAI H5N1 and LPAI H9N2 co-circulate among poultry and village chickens, causing great economic losses. Bangladesh has also reported human infection with both subtypes [18]. Genetic information about those circulating AIVs is very limited. Although some epidemiological and ecological data [19, 20, 21, 22] as well as a very few molecular analyses of HPAI H5N1 from Bangladesh have been reported [23, 24], most of the information regarding influenza in Bangladesh has been obtained from passive surveillance. An active surveillance focusing on live-bird markets showed a high seroprevalence (94 %) for AIV H9N2 [25]. To understand the genetic background better and to elucidate potential reassortments, molecular analysis of AIV H9N2 from Bangladesh is necessary. We here report the first full-genome analysis of the first confirmed LPAIV H9N2 virus derived from a parent stock poultry farm at 2006 in Bangladesh.

Materials and methods

Sample

The H9N2 virus isolate A/Chicken/BD/VP01/2006 (BD-VP01) was obtained from a survey of a Bangladeshi parent stock poultry farm in September 2006. Primary virus isolation was done at the Department of Pathology, Bangladesh Agricultural University, Bangladesh. Further isolation and molecular characterization were carried out at the Institute of Virology, University of Leipzig, Germany.

Virus propagation and isolation

Virus isolation was carried out in 10-day-old specific-pathogen-free (SPF) embryonated chicken eggs (VALO BioMedia GmbH, Germany) using a standard procedure [26]. Briefly, viral samples were filtered through a 0.2-nm filter to exclude bacterial contamination during transport. The filtered suspension (200 μl) was inoculated via the allantoic cavity route into the SPF eggs. The eggs were incubated for 72 hours (h) at 37 °C. The harvested allantoic fluid (AF) was subjected to hemagglutination tests for virus titration using 1 % chicken red blood cells.

Virus characterization

Viral RNA was extracted from the AF using a QIAamp Viral RNA Mini Kit (QIAGEN, Hilden, Germany) and reverse transcribed under standard conditions with RevertAid Reverse Transcriptase (Thermo Scientific, Germany) using the Uni 12 primer [27]. The cDNA was tested for influenza A virus by real-time RT-PCR with IAV-M1.2-Mix-FAM targeting the matrix (M) gene as described previously [28]. Subsequently, a standard RT-PCR was performed for subtyping H9 and N2 using gene-specific primers [29]. A positive control (kindly provided by T.C. Harder, FLI, Germany) and a negative control (AF from uninfected egg) were used to check the specificity of the RT-PCR results and to avoid any unexpected amplifications.

Full-length cloning and sequencing

RT-PCR was performed for the full-length amplification of all eight genome segments of BD-VP01 using a slightly modified protocol based on that of Hoffmann et al. [27]. Briefly, 3 μl of cDNA was added to each 50-μl reaction mixture containing 10 μl of 5x Phusion buffer (Thermo Scientific, Germany), 1 μl of 10 mM dNTPs, 1 μl of each primer (10 pmol), 1.5 μl of DMSO, 0.5 μl of Phusion DNA polymerase (2 units/μl) and 32 μl of DEPC H2O. The first cycle of amplification was at 98 °C for 30 seconds (s) followed by 35 cycles of 98 °C for 10 s, 58 °C for 30 s and 72 °C for 1 min. The final extension was at 72 °C for 10 min. PCR products were purified after agarose gel electrophoresis (GeneJET Gel Extraction Kit, Thermo Scientific, Germany). The products were cloned (CloneJET PCR Cloning Kit, Thermo Scientific, Germany), and plasmid isolation was done from six selected colnes for each gene segment. Individual clone analyses excluded mixed infection by other subtypes. The purified plasmids were used for direct nucleotide sequencing using a Rhodamine Dye-Terminator Cycle Sequencing Ready Reaction Kit (Big Dye® Terminator v1.1; Applied Biosystems), followed by analysis in an ABIPRISM 310 Genetic Analyzer (Applied Biosystems).

Phylogenetic and molecular genetic analysis

The sequencing data were initially checked by NCBI BLAST search, assembled, and edited using EditSeq (DNASTAR Inc., Madison, Wisconsin). Additional influenza virus sequences data available in GenBank were downloaded for comparative phylogenetic analysis of all eight gene segments. The selection was based on reference group representatives described in previous studies on H9 lineages and nucleotide homology found in the NCBI BLAST search. Selected sequences were subjected to Clustal W multiple sequence alignment. Residue analyses were done using the BioEdit 7.1.5 program. A nucleotide sequence identity matrix was calculated to determine the homology between the BD VP01 isolate and other selected reference isolates. Phylogenetic trees were generated by the distance-based neighbor-joining (NJ) method using MEGA version 5.10. Bootstrap values were calculated based on 1,000 NJ replicates of the alignment. The nucleotide (nt) sequences obtained from this study are available in the GenBank database under the accession numbers KC986287 to KC986294. Individual amino acid (aa) changes in defined conserved regions of the eleven proteins of BD-VP01 were analyzed and compared with those of LPAI H9N2 and other subtypes.

Results

Homology and phylogenetic analysis

RT-PCR for all genome segments – hemagglutinin (HA), neuraminidase (NA), polymerase (PA, PB1 and PB2), nucleoprotein (NP), matrix (M) and nonstructural genes (NS) – of AIV H9N2 from Bangladesh (BD-VP01) yielded amplification products of the expected size for each segment. After sequence determination, phylogenetic analyses were carried out to determine the evolutionary relationships between the Bangladeshi AIV H9N2 isolate and the selected isolates from the GenBank database.

BLAST search and primary sequence homology analysis from pairwise alignment of all eight full-length genes of BD-VP01 revealed that the genes for the two surface glycoproteins, HA and NA, had the highest nt sequence homology of 98.8% to those of A/chicken/Bangladesh/627/2007 (BD-627), followed by A/Chicken/Chandigarh/2048/2003 (Ck-CD) and A/Chicken/Tripura/105131/2008 (Ck-TP). With respect to the internal gene segments, BD-VP01 had homology of 98.8% to Indian H9N2 and 96.0% nt sequence homology to Pakistani and Middle Eastern H9N2 isolates. The M and NS genes had >95% nt homology to those of subtype H5N1 from humans in Hong Kong and subtypes H5N2 and H7N7 from aquatic birds in Europe. Most interestingly, all internal genes of BD-VP01 except PB2 (89.6%) showed more than 95% nt sequence homology to a HPAIV A/Chicken/Karachi/NARC-100/04 H7N3 (NARC-100) from Pakistan (Table 1). Again, the PB1 gene of BD-VP01 also shared the highest nt sequence homology (97.7%) with two recent HPAIV isolates of subtype H5N1 from Bangladesh, A/chicken/Bangladesh/12VIR-7140-7/2012 and A/chicken/Bangladesh/12VIR-7140-16/2012 (Ck-BD/H5N1). Accordingly, the aa homologies also showed genotypic diversification within the internal proteins (data not shown).
Table 1

Nucleotide sequence comparisons between the Bangladeshi BD-VP01/H9N2 and representatives of subtype H9N2

BD-VP01

Represented H9N2 group

NARC-100/H7N3

Ck-BD/H5N1

Maximum identity with

HK-G1

HK-Y280

Kr- p96323

Wis-66

HA

91.8

87.6

79.6

79.1

-

-

BD-627 (98.8%)

NA

92.6

90.7

85.0

85.3

-

-

Ck-CD (97.1%)

M

95.4

92.9

90.0

90.6

97.3

89.2

Ck-TP (98.8%)

NS

88.2

88.6

91.6

90.1

96.1

87.2

Ck-TP (97.8%)

NP

96.2

87.6

-

86.0

97.5

88.3

Ck-CD (98.5%)

PA

88.7

86.2

91.9

85.1

96.0

90.2

Ck-TP (98.5%)

PB1

90.0

88.8

91.5

88.9

96.6

97.7

Ck-TP (98.7%)

PB2

86.4

85.1

87.9

82.6

89.6

84.7

Ck-TP (97.4%)

HK-G1, A/Quail/Hong Kong/G1/1997; HK-Y280, A/Chicken/Hong Kong/Y280/1997; Kr-p96323, A/Chicken/Korea/38349-p96323/96; Wis-66, A/Turkey/Wisconsin/66; NARC-100, A/Chicken/Karachi/NARC-100/2004; BD-627, A/chicken/Bangladesh/627/2007; Ck-CD, A/Chicken/Chandigarh/2048/2003; Ck-TP, A/Chicken/Tripura/105131/2008; Ck-BD/H5N1, A/chicken/Bangladesh/12VIR-7140-7/2012 and A/chicken/Bangladesh/12VIR-7140-16/2012

Bold indicates the highest nucleotide identity of gene segments with respective group

–, not included in the analysis or sequence not available in GenBank

Phylogenetic analysis revealed that the BD-VP01 broadly clustered with AIVs from India, Pakistan, and the Middle East (2001 to 2008). The HA phylogeny incorporated it into the Eurasian lineage, as represented by the HK-G1 group (clade H9.4.1), sharing a common ancestor (Fig. 1a). The NA phylogeny revealed a somewhat similar clustering to that of HA (Fig. 1b). The internal gene segments showed more diversified relationships compared to the surface glycoproteins. The M and NP genes formed clades similar to those of the surface genes and were associated with the prototype HK-G1 group (Fig. 1c and e). These genes also had a close relationship to the subtypes H5N1 and H6N1, which are associated with the group HK-G1. Otherwise, the NS (Fig. 1d), PA, PB1 and PB2 (Fig. 1f-h) segments clustered with those of isolates from India, Pakistan and the Middle East but maintained a direct out-group relationship to representatives of group Kr-p96323 (clade H9.3). Interestingly, apart from the above-mentioned relationships, all six internal gene segments maintained a direct group relationship to HPAIV NARC-100/H7N3 (Fig. 1C-H). Again, in the PB1 gene tree, Ck-BD/H5N1 grouped into the same cluster, and even in the same branch, along with BD-VP01 as represented by the prototype Kr-p96323 (Fig. 1g). Taken together, the results strongly suggest that reassortment events had occurred between the gene segments of BD-VP01/H9N2 and HPAIV of subtypes H7N3 and H5N1.
Fig. 1

Phylogenetic tree of the gene segments HA, NA, M, NS, NP, PA, PB1 and PB2 (a-h) of LPAIV H9N2 from Bangladesh. The tree was generated by the distanced-based neighbor-joining method using MEGA 5.10. The reliability of the tree was assessed by bootstrap analysis with 1,000 replications. Bootstrap values ≥50% are shown. The scale bar of each tree indicates substitutions per site. Abbreviations are listed in Table 1

Molecular genetic analysis

To analyze specific conserved regions or motifs related to molecular determinants of pathogenicity and virulence, a detailed aa sequence comparison of BD-VP01 with other representative isolates was performed.

The surface glycoproteins HA and NA

The HA aa sequences of some Bangladeshi isolates (BD-VP01 and BD-627) exhibited a 325KSSR*GLF motif, which is, in contrast to the RSSR*GLF motif determined in another Bangladeshi isolate (A/Chicken/Bangladesh/FDIL(M)112/07), at the peptide site connecting HA1 and HA2. Although most of the H9N2 isolates included in this study contained RSSR cleavage motifs, the KSSR motif confers the cleavage feature of an LPAIV. The Bangladeshi HA contained specific conserved residues at the receptor-binding site, but they carried a Q234L substitution, like G1. At the left and right edge of the binding pocket motif was NGLIGR and GTSKS, respectively. Analysis of the N-X-T/S motif (X can be any aa except proline) revealed that the Bangladeshi isolates had five potential glycosylation sites, at positions 29, 105, 141, 298, 305 respectively, within the HA1 molecule (Table 2).
Table 2

Analysis of the amino acid sequences of the HA and NA proteins of the BD-VP01/H9N2 isolate

Selected strain

HA*

NA

Cleavage site

Left edge of RBP

Right edge of RBP

Total glyco site

Neuraminidase active site (HB)

Stalk deletion

335-341

232-237

146-150

(N-X-T/S motif)

1st loop 367-372

2nd loop 400-403

3rd loop 431-433

Ck/BD/VP01/06

KSSR~GLF

NGLIGR

GTSKS

5

KKDSRA

SNNW

PQE

No

Ck/BD/627/07

KSSR~GLF

NGLIGR

GTSKS

5

-

-

-

-

Ck/BD/FDIL(M)112/07

RSSR~GLF

NGLIGR

GTSKS

5

-

-

-

-

Ql/HK/G1/97

RSSR~GLF

NDLQGR

GISRA

6

KKDSRS

SDIR

PQE

Yes

Dk/HK/Y280/97

RSSR~GLF

NGLQGR

GTSKA

5

KEDSRS

SDNW

PQE

Yes

Ck/Kr/38349-p96323/96

ASYR~GLF

NGQQGR

GTSKA

5

SKDSRS

NNNW

PQE

No

Ck/Emirates/R66/02

RSSR~GLF

NGQLGR

GTSKA

6

KEDLRA

SDNW

PQE

No

Av/SA/910135/06

RSSR~GLF

NGLIGR

GTSKS

5

KKDSRV

SDNW

PQE

No

Ck/Tripura/105131/08

KSSR~GLF

NGLIGR

GTSKS

5

NKDSRA

SNNW

PRE

No

Ck/Pak/UDL-01/05

RSSR~GLF

NGLIGR

GTSKS

5

KKDSRA

SDNW

PQE

No

Ck/Dubai/338/01

RSSR~GLF

NGLMGR

GTSKA

6

KKDLRA

SDNW

PQE

No

Ty/Wis/66

VSSR~GLF

NGQQGR

GTSRA

5

SKDSRS

SNNW

PQE

No

HK/1073/99

RSSR~GLF

NGLQGR

GTSRA

6

KKDSRS

SDNW

PQE

Yes

Ck/Kar/NARC-100/04 (H7N3)

337KR-RKR~GLF

233NGQSGR228

142GATSS146

2

366SNSGRS371

399NNDW402

NKN

Yes

HK/156/97(H5N1)

340RERRRKKR ~ GLF

238NGQSGR243

148GVSSA152

5

345STSSRS350

377ITDW380

412PKE414

No

Ck/HK/220/97 (H5N1)

340RERRRKKR ~ GLF

238NGQSGR243

148GVSSA152

5

345STSSRS350

377ITDW380

412PKE414

No

BD/207095/08(H5N1)

340RERRRKKR ~ GLF

238NGQSGR243

148GVSSA152

5

345STNSRS

377ITDR380

412PKE414

Yes

BD/3233/11(H5N1)

340RERRRKKR ~ GLF

238NGQSGR243

148GVSSA152

5

345STNSRS

377ITDW380

412PKE414

Yes

Ck/BD/12VIR-7140-7/12(H5N1)

340QRERRRK-R~GLF

238NGQSGR243

148GVSAA152

6

345STNSRS

377ITDW380

412PKE414

Yes

Ck/BD/12VIR-7140-16/12(H5N1)

340QRERRRK-R~GLF

238NGQSGR243

148GVSAA152

6

345STNSRS

377ITDW380

412PKE414

Yes

Analysis was done by pairwise multiple alignments, using the Cluster W method in BioEdit7.1

Bold indicate the Q234L mutation which is impotant substitution for receptor binding site and defining the title

RBP, receptor-binding pocket; Ql, quail; Dk, duck; Ck, chicken; Av, avian; Wc, watercoot, Ty, Turkey; HK, Hong Kong; Kr, Korea; SA, Saudi Arabia; Pak, Pakistan; Wis, Wisconsin; Kar, Karachi; BD, Bangladesh

–, not included or not available in GenBank

* Numbering according to H9 for H9N2, H7 for H7N3, H5 for H5N1

The NA of BD-VP01/H9N2 contained aa mutations on three loops of the hemadsorbing (HB) site, which interacts directly with sialic acids and is associated with neuraminidase inhibitor drug resistance. The first loop containing three serine (S) motifs contained substitutions by lysine (K) and alanine (A) at positions 367 and 372, respectively. A K430Q substitution in the third loop was observed, as in other H9N2 strains. A neuraminidase stalk deletion was absent in BD-VP01 but was present in the two representative groups (HK-G1 and Y-280) of clade H9.4, as summarized in Table 2.

Internal proteins

The results of the M1 and M2 protein analysis associated with host range, increased virulence in mammalian cells, and drug resistance against M2 blockers are summarized in Table 3. The BD-VP01 had the aa substitution I15 V in M1, which is quite common in AIV H9N2 isolates but differs from regular AIV signatures. E16 and I28 in the M2 protein are characteristics of AIVs, but BD-VP01 had rare V and F mutations at those positions, which differs from the usual substitution pattern seen before in AIV H9N2. There were no reported substitutions at positions L26, V27, A30, S31 or G34, and the virus therefore remained sensitive to antiviral M2 blockers. The NS1 protein was 230 aa long and contained the ‘KSEV’ PDZ ligand (PL) motif at the C-terminal end (Table 3). Most AIVs possess ESEV PL motifs at the end of NS1 protein. However for many H9N2 isolates, frequent mutations and changes in the PDZ ligand were observed.
Table 3

Analysis of the amino acid sequences of the M, NS, NP, PA, PB1 and PB2 proteins of BD-VP01/H9N2

Selected strain

M

NS1

NP

PA

PB1

PB2

M1

M2

PL motif

Deletion 80-84

136

372

305

337

367

PB1

PB1-F2

627

15

16

28

27

13

20

158

66

Ck/BD/VP01/06

I

V

F

V

KSEV

No

I

D

C

T

R

P

I

S

N

E

Ql/HK/G1/97

I

G

V

V

EPEV

No

M

E

Y

A

M

P

T

N

N

E

Dk/HK/Y280/97

I

G

V

V

EPEV

No

L

E

Y

A

K

P

T

N

N

E

Ck/Kr/38349-p96323/96

I

E

I

V

ESEV

No

-

-

Y

A

K

P

T

N

N

E

Ck/Emirates/R66/02

I

G

V

V

KQKR

No

M

E

Y

A

K

P

T

N

N

E

Av/SA/910135/06

I

D

V

V

GSEV

No

M

D

Y

A

K

P

I

S

N

E

Ck/Tripura/105131/08

I

V

F

V

KSEV

No

I

D

C

T

K

P

I

S

N

E

Ck/Pak/UDL-01/05

I

G

V

V

ESKV

Yes

I

D

Y

A

K

P

I

S

N

E

Ck/Dubai/338/01

I

G

V

A

KQKR

No

M

E

Y

A

K

P

T

N

N

E

Tk/Wis/66

V

E

I

V

ESEV

No

L

E

Y

A

K

P

T

N

D

E

HK/1073/99

I

E

V

V

EPEV

 

M

E

Y

A

M

P

T

N

N

E

Ck/Kar/NARC-100/04

I

D

V

V

KSEV

No

I

D

Y

A

K

P

I

S

N

E

HK/156/97

I

G

V

V

EPEV

No

M

E

Y

A

M

P

T

N

S

E

Ck/HK/220/97

I

G

V

V

EPEV

No

M

E

Y

A

M

P

T

N

N

E

BD/207095/2008

I

E

I

V

-

-

L

E

Y

T

R

P

T

N

N

K

BD/3233/2011

I

E

I

V

ESKV

Yes

L

E

Y

T

K

P

T

N

N

K

Ck/BD/12VIR-7140-7/2012

I

E

V

V

ESEV

Yes

L

E

Y

A

K

P

I

S

N

E

Ck/BD/12VIR-7140-16/2012

I

E

V

I

ESEV

Yes

L

E

Y

A

K

P

I

S

N

E

The ribonucleoprotein complex (RNP), which includes NP, PA, PB1 and PB2, was analyzed and compared with those of selected reference isolates. The NP and PA of BD-VP01/H9N2 had mostly an avian-like signature, although there were some substitutions, such as L136I, E372D in NP as well as Y305C, A337T and K367R in PA gene. Interestingly, the same aa substitutions at position 337 and 367 in PA was observed in HPAIV Bangladeshi isolates of subtype H5N1 from humans but absent in other H5N1 isolates related to HK-G1 (Table 3). PB1 of selected isolates exhibited proline (P) at position 13, along with all other strains included in this study. This aa serves as a marker of virulence in mice, whereas the avian residue at this position is L. The existence of full-length PB1-F2 suggested increased pathogenicity of AIVs. Analysis showed that BD-VP01 displayed a full-length (90 aa) PB1-F2 protein. The important mutation N66S in the PB1-F2 protein, however, which is thought to cause increased virulence, was not present in the Bangladeshi isolates (Table 3). The most important aa code in PB2 is at position 627. The Bangladeshi isolates of subtype H9N2 and chicken BD-H5N1 lacked this kind of substitution, which is present in human BD-H5N1 isolates (Table 3).

Discussion

AIV, once introduced in to land-based domestic poultry or mammalian species, can evolve rapidly [30]. In Bangladesh, AIV H9N2 infections in domestic poultry, especially in commercial and backyard chickens has become geographically widespread across the country, causing serious economic losses. Besides AIV H9N2, Bangladesh has experienced HPAIV H5N1 infections, both in chickens and humans, which has raised the possibility of reassortments between the gene segments of LPAIV and HPAIV or the occurrence of mixed infections. This study aimed to understand the genetic relationship and evolution of AIV H9N2 strains from Bangladesh.

Phylogenetic analysis suggested that the HA and NA of Bangladeshi H9N2 isolates are closely related to those of South Asian and Middle Eastern isolates, forming a distinct cluster. This defined cluster maintained a direct out-group relationship to the prototype HK-G1, confirming that BD-VP01 belongs to the G1 lineage or clade H9.4.1. The internal genes of the Bangladeshi isolate, which showed more genotypic differentiation, are directed to G1 (M and NP) and Kr-p96323 (NS, PA, PB1 and PB2) prototypes within the H9 lineage. More interestingly, the internal genes were more similar to those of HPAIV NARC-100/H7N3 and in their phylogeny always maintained a direct group relationship with BD-VP01, along with other South Asian and Middle Eastern H9N2 isolates. The PB1 and PB1-F2 proteins of BD-VP01 showed maximum identity to those of HPAIV Ck-BD/H5N1, and in PB1 phylogeny, they clustered together in a same branch. The results suggest that all of the internal genes of HPAIV subtype H7N3 were like those of BD-VP01, and at the same time, CK-BD/H5N1 also had a BD-VP01-like PB1gene. H9N2 was described previously as a donor for internal genes of subtype H5N1 [31]. A reassortant genotype of AIV H9N2 containing the NS and NP genes similar to AIV of subtypes H7N3, H5N1, H7N1, H6N1 was described recently [32, 33, 34], where H9N2 received genes from HPAIV, but in the current study, reassortment events in the opposite direction were found. A very recent human infection with H7N9 in China was recorded where H7N9 contained all six internal genes from AIV H9N2 [35, 36], which supports the current study. Therefore, the H9N2 strains isolated from the Middle East and South East Asia (2000-2004), which have similar genetic relationships to those we found for BD-VP01, might have served as the donor of those internal genes to HPAIV subtype H7N3. Therefore it remains open whether H7N3 received those internal genes directly from BD-VP01. BD-VP01 was isolated in 2006, while NARC-100/H7N3 was isolated in 2004, but as they showed homology in all internal genes to BD-VP01, alongside other H9N2 viruses, we suggest that HPAIV H7N3 received its internal genes from H9N2 circulating in South East Asia. H9N2 has to be the donor of those genes, as all of these internal genes of H7N3 always grouped with H9N2 strains in NCBI BLAST searches. In the case of the second described reassortment of the PB1 gene, it is surely a direct donation, as Ck-BD/H5N1 has a BD-VP01/H9N2-like PB1 gene.

HA is the critical determinant of pathogenicity, with a clear structure of the cleavage site [37, 38]. The analyzed Bangladeshi isolates contain dibasic KSSR and RSSR motifs, suggesting low pathogenicity of these viruses. The receptor-binding specificity of HA is responsible for the host-range restriction, and aa changes cause alteration of binding specificity. BD-VP01 contained substitutions such as G1, which is typical for human influenza virus association with preferential binding to an α2,6 linkage [39, 40] and has also been found to be important for transmission in ferrets [41]. The HA1 proteins of Bangladeshi isolates contain five potential glycosylation site motifs and therefore lack one site compared to HK-G1. It has been described that an alteration in the glycosylation pattern influences the adaptation of viruses to poultry [42, 43].

The NA stalk length correlates with efficiency of viral replication. BD-VP01 did not show a stalk deletion when compared to the reference group G1, Y280, and recent Bangladeshi HPAIV H5N1 isolates. It is known that longer stalk length results in better replication [44], and a deletion in the stalk region may be required during adaptation processes [45]. Analysis of the neuraminidase active site or hemadsorbing site (HB) of NA revealed the acquisition of mutations in three loops associated with resistance to neuraminidase drug inhibitors. Almost all analyzed isolates in this study had the same features, indicating that H9N2 viruses are commonly resistant to neuraminidase inhibitors.

The influenza virus polymerase is responsible for pathogenicity and host adaptation [46, 47]. At position 627 of the PB2 protein, BD-VP01 contained glutamic acid (E), which is commonly seen in AIV, while human influenza virus possesses lysine (K). A single aa substitution (K627E) can dramatically increase pathogenicity and enhance replication in mammalian hosts [48, 49]. Proline (P) at position 13 of the PB1 protein, which can increase polymerase activity in new hosts, was also observed in BD-VP01. The PB1-F2 protein is important for promoting viral pathogenicity, and the existence of the full-length protein contributes to increased virulence [50]. Genetic analysis of BD-VP01 showed a full-length PB1-F2 protein but did not show the N66S mutation, which confers increased disease severity and cytokine production in mice [51]. Based on PA protein analysis, BD-VP01 contains mutations at positions 305, 337 and 365, which were not usual in other representative H9N2 isolates, but their specific function and relationship to molecular determinants is currently unknown. Bangladeshi H9N2 also exhibited the recently identified PA-X protein [52], which is 252 aa in length. The main function of the NP is encapsulation of the viral genome to form an RNP complex, which is associated with major determinants of the genetic signature of the host [53]. BD-VP01 had aa substitutions at positions 136 and 372, differing from the H9N2 prototypes but similar to some Pakistani H9N2 strains and to NARC-100/H7N3.

Molecular analysis of the NS1 protein showed that it is 230 aa in length, which is typical for H9N2 viruses. It was previously shown that the NS1 protein possesses a strain-specific aa length in birds and mammalian hosts [54] and a deletion of five amino acids (80-84) along with the D29E mutation caused increase virulence in chickens and mice [55]. BD-VP01 exhibited a KSEV C-terminal motif, which is also found in some Indian and Pakistani isolates and NARC-100. However, the role of the C-terminal aa of NS1 with regard to the pathogenesis of H9N2 viruses in poultry remains unknown. Bangladeshi H9N2 contained an NEP of 121 aa in length that did not contain an important mutation. The conserved aa residues in M are associated with host range, increase virulence, or replication in mammalian hosts [53]. BD-VP01/H9N2 contained an isoleucine (I) at position 15 of M1, which is conserved among H9N2 viruses.

In conclusion, we have investigated and characterized an LPAIV H9N2 strain isolated from a retrospective analysis of a poultry farm in Bangladesh from 2006 and compared the virus with selected reference viruses from GenBank. It is clear that LPAIV H9N2 was circulating in Bangladesh before the first declaration of an HPAIV H5N1 event in the country. The Bangladeshi isolates exhibited particular genetic features, and the most important concern is genetic reassortment of internal genes with two distinct HPAIVs of subtypes H7N3 and H5N1. Considering the above-mentioned mutations and gene segment reassortments, further studies will be designed to investigate growth kinetics, the molecular basis of cell tropism, the host response, and the mode of transmission. As influenza outbreaks are still ongoing events in Bangladesh, it is highly recommended to continue monitoring, surveillance, and molecular genetic analysis of circulating H9N2 throughout the country.

Notes

Acknowledgements

This study was supported by the German Academic Exchange Service (DAAD), reference number A/10/98746. Initial funding for studies in Bangladesh was supported by the Third World Academy of Science (TWAS).

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Copyright information

© Springer-Verlag Wien 2014

Authors and Affiliations

  • Rokshana Parvin
    • 1
    • 2
  • Kristin Heenemann
    • 1
  • Mohammad Y. Halami
    • 1
  • Emdadul H. Chowdhury
    • 2
  • M. R. Islam
    • 2
  • Thomas W. Vahlenkamp
    • 1
  1. 1.Faculty of Veterinary Medicine, Institute of Virology, Center for Infectious DiseasesUniversity of LeipzigLeipzigGermany
  2. 2.Department of Pathology, Faculty of Veterinary ScienceBangladesh Agricultural UniversityMymensinghBangladesh

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