Sequence and phylogenetic analysis of neuraminidase genes of H9N2 avian influenza viruses isolated from commercial broiler chicken in Iran (2008 and 2009)

Abstract

A total of 512 tissue samples collected from 30 farms located in various states of Iran during 2008–2009 as part of a program to monitor avian influenza viruses (AIVs) infection in Iran’s poultry population. To determine the genetic relationship of Iranian viruses, neuraminidase (NA) genes from ten isolates of H9N2 viruses isolated from commercial chickens in Iran during 2008–2009 were amplified and sequenced. The viruses’ neuraminidase gene was >90% similar to those of A/Quail/Hong Kong/G1/97 (H9N2) sublineage. The neuraminidase stalk regions in these Viruses had no deletion as compared to that of chicken/Beijing/1/94 sublineage (Beijing-like viruses) and the two human isolates A/HK/1073/99, A/HK/1074/99. Phylogenetic analysis of neuraminidase (NA) gene showed that it shares a common ancestor A/Quail/Hong Kong/G1/97 isolate which had contributed the internal genes of the H5N1 virus. The results of this study indicated that No (Beijing-like) virus and (Korean-like) virus were found in chickens in Iran, and the NA genes of H9N2 influenza viruses circulating in Iran during the past years were well conserved and the earlier Iranian isolates may be considered to represent such a progenitor.

Introduction

Influenza viruses belong to the orthomyxoviridae family of RNA viruses and are divided into five genera: Influenza A, B, C, Thogtovirus and Isavirus (Bouvier and Plese 2008; Lee and Saif 2009). Genetically and antigenically, avian influenza viruses (AIVs) exist as multiple subtypes based on the two glycoproteins (HA and NA) on the virion surface. To date, 16 HA (HA1–16) and nine NA (NA1–9) subtypes have been identified in aquatic birds (Fouchier et al. 2005; Cardona et al. 2009). The influenza A virion is studded with glycoprotein spikes of HA and NA, in a ratio of approximately 4:1. During replication, influenza A viruses require the activities of two viral surface glycoproteins: hemagglutinin (HA), which is responsible for binding to terminal sialic acid on cell surface glycoconjugates, and neuraminidase (NA), with an associated enzymatic activity that removes sialic acid from host cell glycoconjugates as well as newly synthesized viral proteins to facilitate the budding of progeny virions from cells (Kobasa et al. 2001).

Phylogenetic analysis of the NA N2 genes of the influenza viruses revealed that there were three distinct lineages: North American avian, human and swine, and Eurasian lineages. The Eurasian lineage consists of three sublineages. These included viruses represented by A/chicken/Beijing/1/94 or A/duck/HongKong/Y280/97 (Beijing-like or Y280-like) A/Quail/HongKong/G1/97 (G1-like), A/chicken/Korea/38349-p96323/96 (Korean-like) (Fig. 1). Sublineage I (G1-like) consists of human H9N2, Middle East, Indian and Pakistan H9N2 viruses. Sublineage II (Beijing- like) consists of Hong Kong H9N2 viruses isolated in 1997, which were characterized by no amino acid deletion, and all the mainland China H9N2 strains which had three amino acid deletions at positions 63, 64 and 65 except for the primary chicken H9N2 viruses identified in Beijing 1994. Sublineage III (Korean-like) included the Korean strains and viruses isolated from domestic ducks in Hong Kong in 1997 (Xu et al. 2007; Tosh et al. 2008; Wu et al. 2008).

Fig. 1

Phylogenetic relationships of neuraminidase genes from H9N2 viruses isolated in Iran during 2008–2009. All Iranian isolates fall into a special group, related to the G1 sublineage, and distributed among two NA gene subgroups associated with the time of their isolation

Some avian H9 viruses have acquired receptor binding characteristics typical of human strains, increasing the potential for reassortment in both human and pig respiratory tracts. In1999 and 2003, human cases of H9N2 virus were recorded in Hong Kong although no death was reported (Butt et al. 2005; Lupiani and Reddy 2009). Crossing the species barrier to mammals highlights the pandemic potential of H9N2 virus. With the emergence of non-human H9N2 isolates with avian characteristics, it is important to study the H9N2 isolates from avian hosts in addition to those obtained from humans for pandemic influenza understanding and preparedness.

H9N2 viruses circulated widely in the Middle East (Iran, United Arab Emirates Israel) and were associated with serious disease in poultry (Aamir et al. 2007; Alexander 2007; Mosleh et al. 2009; Perk et al. 2009). In this study, we characterized NA genes and proteins of ten Iranian isolates which have been isolated from commercial broiler chicken in Iran in 2008 and 2009. These isolates resulted in rapid mortality due to tracheitis and respiratory congestion. We delineated the genomes and hemadsorbing (HB) sites of NA genes of these field isolates and we also established their phylogenetic relationship to the other Asian H9N2 viruses.

Materials and methods

Sampling and virus isolation

Samples collected from 30 farms were received from various parts of the country between April 2008 and February 2009. The tissue samples usually included trachea and lung. The samples were collected from flocks with the history of acute respiratory illness.

Virus isolation was carried out in 9- to 11-day-old SPF (specific pathogen free) embryonated chicken eggs following the standard protocol (Swayne et al. 1998). Briefly, triturated tissue samples treated with two antibiotic and antimycotic solutions (penicillin 10,000 U/ml, streptomycin 10,000 U/ml, nystatin 20,000 U/ml) for 30 min at 37°C, and clarified by centrifugation at 1,500 rpm for 10 min were inoculated through allantoic routes. The allantoic fluid was harvested after incubation at 37°C for 72–96 h and clarified by centrifugation at 1,500×g for 15 min at 4°C. HA test negative samples were given two more passages and tested again before being declared negative for AIV isolation.

The identification of viruses subtype was determined by a standard hemagglutination inhibition (HI) and neuraminidase inhibition (NI) tests (Alexander and Spackman 1981).

Ten virus isolates obtained in this study were named as follows: (A/chicken/Iran/RZ28/2008, A/chicken/Iran/RZ36/2008, A/chicken/Iran/RZ37/2008, A/chicken/Iran/RZ42/2008, A/chicken/Iran/RZ53/2008, A/chicken/Iran/RZ69/2008, A/chicken/Iran/RZ70/2008, A/chicken/Iran/RZ71/2009, A/chicken/Iran/RZ75/2009, A/chicken/Iran/RZ77/2009).

RT-PCR and sequence analysis

The viral RNA was extracted directly from the allantoic fluid by means of the High pure viral Nucleic Acid Kit (Roche, Germany). Purified genomic RNA was used to generate cDNA clones by reverse-transcription polymerase chain reaction (RT-PCR) according to the standard procedure. RT-PCR was performed by using specific primers for 940-bp (nucleotides 1–940) and 560-bp (nucleotides 920–1,470) fragments of NA.

Primers used for NA amplication were:

• Forward primer (980 bp): 5′- GCAAAAGCAGGAGTGAAAATG-3′

• Reverse primer (980 bp): 5′-AGTCCTGAGCACAAATAACTGG-3′

• Forward primer (560 bp): 5′-TTAGATGTGTTTGCAGGGAC-3′

• Reverse primer (560 bp): 5′- GGTTCTAAAATTGCGAAAGC-3′

The PCR products were purified by using High pure product purification kit (Roche Germany). PCR products were applied to low melting point (LMP) agarose and the distinct bands were purified from gel for sequencing (MWG Co., Germany)

Nucleotide and deduced amino acid sequences of the NA gene were edited with the Editseq (DNASTER Software package, Version 5.2). Nucleotide and deduced amino acid sequences were aligned by ClustalW, Version 1.4.

Nucleotide sequences of the NA gene were used for phylogenetic tree construction. The phylogenetic analysis was performed with the MegAlign program2.8.

Nucleotide sequence accession numbers

The sequences determined in this study are available at GenBank under accession numbers GU071969-73 and HM855266-70.

Results

Disease signs in the field

Avian influenza virus was isolated from 20 out of 512 tissue samples processed for virus isolation between 2008 and 2009. The symptoms included anorexia, reduced water consumption, gasping, and conjunctivitis with facial edema. The major post-mortem findings were tracheitis, respiratory congestion and hemorrhages on proventriculus. Mortality rose sharply (1% to 10%), resulting in considerable economic losses.

Neuraminidase gene of the Iranian viruses

In this study, 1,447 bp of the NA genes were sequenced and amino acid sequences 469 of the NA genes, ten isolates were deduced from the nucleotide sequence. Nucleotide sequence analysis of the NA gene showed that the Iranian isolates are very closely related (90.6–100%) and shared a homology of (92.0–96%) with H9N2 isolates of A/Quail/Hong Kong/G1/97 sublineage and the two human isolates A/HK/1073/99 and A/HK/1074/99. These viruses differed from other viruses by ten amino acid substitutions at the following positions: 9 (V to I) ،40 (T to A) ،270 (M to L)، 271 (K to R) 280 ، (N to S) ، 285 (D to N) ، 301) R to k)، 335 (H to L) 405) K to R) ، 416 (C to S).

Analysis of NA protein sequences showed that these H9N2 viruses have seven glycosylation sites with the N-X-T/S motif, in which X may be any amino acid except proline (positions 61, 69, 86, 146, 200, 234, 402).

Amino acids of HB site at position 366–373 were IRKDSRAG except for A/chicken/Iran/RZ53/2008 isolate, which was IRKDSREG. Amino acids at position 399–404 were of two types: DSDNWS or DSDNLS (Table 1).

Table 1 Amino acid substitutions in the hemadsorbing sites of N2 NAs compared to established H9N2

The neuraminidase stalk regions in these viruses had no deletion as compared to that of chicken/Beijing/1/94 sublineage (Beijing-like viruses) and the two human isolates (A/HK/1073/99 and A/HK/1074/99). All NA genes of the Iranian H9N2 viruses fall into a single group within a G1-like sublineage (Fig. 1). In turn, this group subdivides into two main subgroups: the first of these includes viruses isolated from 1998 to 2004 and surprisingly the isolate A/chicken/Iran/RZ53/2008, the second subgroup includes strains isolated after 2004 (Fig. 1).

Discussion

H9N2 AIVs have been circulating in domestic poultry in Iran for 12 years and were still isolated from chickens, even from some vaccinated flocks (Nili and Asasi 2003; Karimi and Bozorogmehri 2004). Although H9N2 viruses are characterized as low pathogenic avian influenza (LPAI) viruses, they may cause high morbidity and mortality (Nili and Asasi 2002; Kim et al. 2006; Aamir et al. 2007). In this study, overt clinical signs in chickens observed at affected farms included acute respiratory signs and 1–10% mortality rates during the outbreak. Bano et al. (2003) and Nili and Asasi (2002) reported that H9N2 viruses with an IVP index of 0.00/3.00 could cause high mortality (20–65%) in the field due to environmental stress and secondary bacterial (Escherichia coli) and Mycoplasma gallisepticum infections. Kishida et al. (2004) reported that co-infection of Staphylococcus aureus or Haemophilus paragallinarum exacerbates H9N2 influenza A virus infection in chicken. Haghighat-Jahromi et al. (2008) have reported that these viruses produce significant disease problems in poultry, especially when poultry are co-infected with other respiratory pathogens such as infectious bronchitis virus. These results indicated that clinical signs and mortality observed under field conditions could be due to the concurrent infections or environmental factors.

The sequence and length of stalk region is known to vary among and within NA subtypes. Shortening of NA stalk by deletion of amino acid is characterization of highly pathogenic H5 and H7 influenza viruses (Aimer et al. 2009; Pfeiffer et al. 2009) but it is not known whether deletion in the NA of H9N2 is correlated with pathogenicity in chicken. No deletion was observed in N2 NA of Iranian viruses. Some viruses in the chicken/Beijing/1/94 sublineage (Beijing-like viruses) and the two human isolates (A/HK/1073/99 and A/HK/1074/99) had deletion at residues 63–65 and 38–39, respectively (Aamir et al. 2007; Xu et al. 2007; Wu et al. 2008). This finding indicated that the NAs of the Iranian H9N2 viruses were different from those of chicken/Beijing/1/94 sublineage and suggested that the NAs of H9N2 viruses in Iran evolved independently of these N2 NAs.

According to the phylogenetic analysis, all Iranian isolates fall into a special group, related to the G1 sublineage — a finding that may indicate a common origin of all tIranian isolates from a single progenitor. Previous studies indicated that isolates from Pakistan, Iran, Germany, and Saudi Arabia all showed very close relationships, and they might have originated from a common source (Toroghi and Momayez 2006; Homayounimehr et al. 2010).

No Beijing-like virus and Korean-like virus was found in the local chicken population since the outbreak of H9N2 influenza in Iran in 1997. This evidence indicates that the NA genes of H9N2 influenza viruses circulating in Iran during the past years were well conserved and the earlier Iranian isolates or those from neighbouring countries may represent such a progenitor. Nevertheless, according to the phylogenetic analysis, the Iranian group was not homogeneous and these viruses may be distributed among two (NA gene) subgroups, associated with the time of their isolation. It seems that these differences are the result of accumulation of mutations among the viruses circulating within the poultry reservoir. One of the factors driving the evolution of low-pathogenic influenza viruses is the immunological pressure, which increases in the case of the ongoing vaccination. The postvaccinal specific immunity of the population could be decisive as a selective factor in ensuring the prevalence of a viral strain which differed from the vaccinal strain and earliest Iranian isolates; however, there are numerous reports indicating that the group viruses in Eurasian sublineage confer an immunity to each other (Aamir et al. 2007; Xu et al. 2007).

The HB site of NA is highly conserved in equine and aquatic bird influenza viruses. However, avian viruses co-circulating in mammals such as pigs and humans accumulate various substitutions in the HB site, thus decreasing their HB capacity (Baum and Paulson 1991). The NA of these new isolates carried substitutions in the HB site similar to those of other avian H9N2 viruses from Asia and human H9N2 viruses (Table 1). Mutations in the HB site were previously seen in viruses that bind to α-2,6-linked receptors (Gambarian et al. 2002). Matrosovich et al. (2001) have shown the HB site in the NA of Asian H9N2 viruses to be under positive selection pressure for mutations, which results in compatible combinations of HA and NA.

As Aamir et al. (2007) reported, our result also showed a close relationship between Iranian and human isolates with a maximum identity of 96%. Like the Pakistan isolates the NAs of the Iranian Isolates, human and quail G1 viruses shared seven amino acids that distinguish them from the N2s of other H9N2 viruses (Table 2), and they possessed a similar pattern of glycosylation sites (Cameron et al. 2000). Previous studies of H9N2 isolates from Iran have shown that Iranian isolates possessed amino acid leucine (L) at position 226 instead of glutamine (Q) at the receptor binding site of hemagglutinins (HA) which is similar to A/Quail/HongKong/G1/97 and the two human isolates (A/HK/1073/99 and A/HK/1074/99 (Karimi and Bozorogmehri 2004; Homayounimehr et al. 2010). Amino acid differences in the receptor binding sites of HAs have been shown to be associated with differences in receptor binding specificity, (Wan and Perez 2007). So Iranian H9N2 isolates can bind to α (2, 6) receptors, and considering the homology of these isolates with human H9N2 strains, it seems that the potential of these avian influenza isolates to infect human should not be overlooked.

Table 2 Amino acids characteristic of the proteins of the H9N2 viruses, A/Hong kong/1074/99, A/Quail/Hong kong/G1/97, A/chicken/Pakistan/2/99, and Iranian viruses

In summary, our study has demonstrated that the Iranian H9N2 viruses belong to G1-like sublineage, and that the NA genes of H9N2 influenza viruses circulating in Iran during the past years were well conserved. With the present threat of H5N1 virus in Iran (Shoushtari et al. 2007), continuous surveillance would help to improve the understanding of the evolution and the emergence of pandemic strains in this region.