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Novel genotypes of H9N2 influenza A viruses isolated from poultry in Pakistan containing NS genes similar to highly pathogenic H7N3 and H5N1 viruses.

Iqbal M, Yaqub T, Reddy K, McCauley JW - PLoS ONE (2009)

Bottom Line: The NS gene segment showed greater genetic diversity and shared a high level of similarity with NS genes from either H5 or H7 subtypes rather than with established H9N2 Eurasian lineages.These results indicate that during recent years the H9N2 viruses have undergone extensive genetic reassortment which has led to the generation of H9N2 viruses of novel genotypes in the Indian sub-continent.The novel genotypes of H9N2 viruses may play a role in the increased problems observed by H9N2 to poultry and reinforce the continued need to monitor H9N2 infections for their zoonotic potential.

View Article: PubMed Central - PubMed

Affiliation: Division of Microbiology, Institute for Animal Health, Compton Laboratory, Compton, Newbury, Berkshire, UK. munir.iqbal@bbsrc.ac.uk

ABSTRACT
The impact of avian influenza caused by H9N2 viruses in Pakistan is now significantly more severe than in previous years. Since all gene segments contribute towards the virulence of avian influenza virus, it was imperative to investigate the molecular features and genetic relationships of H9N2 viruses prevalent in this region. Analysis of the gene sequences of all eight RNA segments from 12 viruses isolated between 2005 and 2008 was undertaken. The hemagglutinin (HA) sequences of all isolates were closely related to H9N2 viruses isolated from Iran between 2004 and 2007 and contained leucine instead of glutamine at position 226 in the receptor binding pocket, a recognised marker for the recognition of sialic acids linked alpha2-6 to galactose. The neuraminidase (NA) of two isolates contained a unique five residue deletion in the stalk (from residues 80 to 84), a possible indication of greater adaptation of these viruses to the chicken host. The HA, NA, nucleoprotein (NP), and matrix (M) genes showed close identity with H9N2 viruses isolated during 1999 in Pakistan and clustered in the A/Quail/Hong Kong/G1/97 virus lineage. In contrast, the polymerase genes clustered with H9N2 viruses from India, Iran and Dubai. The NS gene segment showed greater genetic diversity and shared a high level of similarity with NS genes from either H5 or H7 subtypes rather than with established H9N2 Eurasian lineages. These results indicate that during recent years the H9N2 viruses have undergone extensive genetic reassortment which has led to the generation of H9N2 viruses of novel genotypes in the Indian sub-continent. The novel genotypes of H9N2 viruses may play a role in the increased problems observed by H9N2 to poultry and reinforce the continued need to monitor H9N2 infections for their zoonotic potential.

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Phylogenetic relationships of the NP genes.The phylogenetic methods and abbreviations were as described for figure 1.
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pone-0005788-g004: Phylogenetic relationships of the NP genes.The phylogenetic methods and abbreviations were as described for figure 1.

Mentions: To determine the genetic relationship of H9N2 avian influenza viruses currently prevalent in poultry in Pakistan, we selected twelve H9N2 viruses isolated between May 2005 and March 2008 from different districts of the Punjab and the NWFP of Pakistan, Supplementary Table 1. These districts have a large concentration of commercial poultry farms and a number of H5N1 and H7N3 outbreaks have also been reported in this region over the last few years (18). The H9N2 isolates were all defined as LPAI viruses by an intravenous pathogenicity test of diluted infectious allantoic fluid (Table 1). Complete full length sequencing of all 8 segments was performed on at least 5 cDNA clones for each segment and a consensus sequence for each virus was produced; there was only very limited polymorphism in the analysed sequences. Phylogenetic relationships were examined between these viruses and representative H9N2 viruses from Asia and the Middle East, along with the established Eurasian H9N2 lineages: namely the G1-lineage, the Y280-lineage and the Korean-lineage represented by prototype viruses A/Quail/Hong Kong/G1/97, A/Duck/Hong Kong/Y280/97 and A/Chicken/Korea/38349-p96323/96 respectively, and with viruses identified in the Far East as emerging H9N2 lineages [6], [25]. Phylogenetic analysis of the HA revealed that all twelve isolates cluster together with G1-lineage viruses and show a very close relationship (93.0 to 96.4% nucleotide identity) with recent poultry-isolated H9N2 viruses from Iran (Figure 1 and Table 2), which shares a border with Pakistan. A slightly higher divergence (6.3 to 9.7%) was seen between HA genes of H9N2 viruses isolated from Pakistan in 1999 (Ck/Pakistan/2/99, Ck/Pakistan/4/99, and Ck/Pakistan/5/99); another set of viruses from Middle East isolated between 2000 and 2003 [14] also cluster with this group (Figure 1). The phylogenetic relationship of NA and M genes of the Pakistan viruses also fell within the G1-lineage and showed close identity (93.0–97.0% in the NA gene) with Ck/Pakistan/2/99, Ck/Pakistan/4/99, and Ck/Pakistan/5/99 (Figure 2, 3 and Table 2) and for the M gene 96.5 to 97.6% nucleotide identity with A/Ck/Pakistan/2/99. Like the HA, NA and M genes, the NP genes also grouped together with G1-lineage viruses (Figure 4): the closest nucleotide sequence identity (95.8–97.3%, Table 2) was found with viruses isolated from Pakistan during 1999 and A/Parakeet/Chiba/1/97, an introduction thought to be imported from Pakistan [26]. There was relatively little nucleotide divergence (2.4–7.0%) of NA, M and NP gene compared with H9N2 viruses isolated from Pakistan in 1999 (Ck/Pakistan/2/99, Ck/Pakistan/4/99 and Ck/Pakistan/5/99), which were of the G1-lineage but within this lineage some sub-clustering is evident (Figure 1, 2, 3 and 4). The phylogenetic analyses of each of the three polymerase complex genes (PB2, PB1 and PA), in contrast to the HA, NA, M and the NP genes, did not branch with exemplars of the G1-lineage or any of the other established Eurasian lineages (Figure 5, 6 and 7). All three polymerase complex genes were most closely related to those of H9N2 viruses isolated from the Persian Gulf and India between 2000 and 2004 [14], [27]. Nucleotide identities were from 94.1 to 96.4% for the PA gene, 91.2–94.4% for the PB1 gene and 94.5–96.6% identity in PB2; these similarities may indicate a separate Indian sub-continental lineage of H9N2 virus [6], [25]. This notion is supported by conclusions based on analyses of H9N2 viruses from the Middle East [14] where it was observed that reassortment of the PB2 gene had taken place and it was postulated that a UAE lineage of H9N2 viruses may have emerged.


Novel genotypes of H9N2 influenza A viruses isolated from poultry in Pakistan containing NS genes similar to highly pathogenic H7N3 and H5N1 viruses.

Iqbal M, Yaqub T, Reddy K, McCauley JW - PLoS ONE (2009)

Phylogenetic relationships of the NP genes.The phylogenetic methods and abbreviations were as described for figure 1.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2690689&req=5

pone-0005788-g004: Phylogenetic relationships of the NP genes.The phylogenetic methods and abbreviations were as described for figure 1.
Mentions: To determine the genetic relationship of H9N2 avian influenza viruses currently prevalent in poultry in Pakistan, we selected twelve H9N2 viruses isolated between May 2005 and March 2008 from different districts of the Punjab and the NWFP of Pakistan, Supplementary Table 1. These districts have a large concentration of commercial poultry farms and a number of H5N1 and H7N3 outbreaks have also been reported in this region over the last few years (18). The H9N2 isolates were all defined as LPAI viruses by an intravenous pathogenicity test of diluted infectious allantoic fluid (Table 1). Complete full length sequencing of all 8 segments was performed on at least 5 cDNA clones for each segment and a consensus sequence for each virus was produced; there was only very limited polymorphism in the analysed sequences. Phylogenetic relationships were examined between these viruses and representative H9N2 viruses from Asia and the Middle East, along with the established Eurasian H9N2 lineages: namely the G1-lineage, the Y280-lineage and the Korean-lineage represented by prototype viruses A/Quail/Hong Kong/G1/97, A/Duck/Hong Kong/Y280/97 and A/Chicken/Korea/38349-p96323/96 respectively, and with viruses identified in the Far East as emerging H9N2 lineages [6], [25]. Phylogenetic analysis of the HA revealed that all twelve isolates cluster together with G1-lineage viruses and show a very close relationship (93.0 to 96.4% nucleotide identity) with recent poultry-isolated H9N2 viruses from Iran (Figure 1 and Table 2), which shares a border with Pakistan. A slightly higher divergence (6.3 to 9.7%) was seen between HA genes of H9N2 viruses isolated from Pakistan in 1999 (Ck/Pakistan/2/99, Ck/Pakistan/4/99, and Ck/Pakistan/5/99); another set of viruses from Middle East isolated between 2000 and 2003 [14] also cluster with this group (Figure 1). The phylogenetic relationship of NA and M genes of the Pakistan viruses also fell within the G1-lineage and showed close identity (93.0–97.0% in the NA gene) with Ck/Pakistan/2/99, Ck/Pakistan/4/99, and Ck/Pakistan/5/99 (Figure 2, 3 and Table 2) and for the M gene 96.5 to 97.6% nucleotide identity with A/Ck/Pakistan/2/99. Like the HA, NA and M genes, the NP genes also grouped together with G1-lineage viruses (Figure 4): the closest nucleotide sequence identity (95.8–97.3%, Table 2) was found with viruses isolated from Pakistan during 1999 and A/Parakeet/Chiba/1/97, an introduction thought to be imported from Pakistan [26]. There was relatively little nucleotide divergence (2.4–7.0%) of NA, M and NP gene compared with H9N2 viruses isolated from Pakistan in 1999 (Ck/Pakistan/2/99, Ck/Pakistan/4/99 and Ck/Pakistan/5/99), which were of the G1-lineage but within this lineage some sub-clustering is evident (Figure 1, 2, 3 and 4). The phylogenetic analyses of each of the three polymerase complex genes (PB2, PB1 and PA), in contrast to the HA, NA, M and the NP genes, did not branch with exemplars of the G1-lineage or any of the other established Eurasian lineages (Figure 5, 6 and 7). All three polymerase complex genes were most closely related to those of H9N2 viruses isolated from the Persian Gulf and India between 2000 and 2004 [14], [27]. Nucleotide identities were from 94.1 to 96.4% for the PA gene, 91.2–94.4% for the PB1 gene and 94.5–96.6% identity in PB2; these similarities may indicate a separate Indian sub-continental lineage of H9N2 virus [6], [25]. This notion is supported by conclusions based on analyses of H9N2 viruses from the Middle East [14] where it was observed that reassortment of the PB2 gene had taken place and it was postulated that a UAE lineage of H9N2 viruses may have emerged.

Bottom Line: The NS gene segment showed greater genetic diversity and shared a high level of similarity with NS genes from either H5 or H7 subtypes rather than with established H9N2 Eurasian lineages.These results indicate that during recent years the H9N2 viruses have undergone extensive genetic reassortment which has led to the generation of H9N2 viruses of novel genotypes in the Indian sub-continent.The novel genotypes of H9N2 viruses may play a role in the increased problems observed by H9N2 to poultry and reinforce the continued need to monitor H9N2 infections for their zoonotic potential.

View Article: PubMed Central - PubMed

Affiliation: Division of Microbiology, Institute for Animal Health, Compton Laboratory, Compton, Newbury, Berkshire, UK. munir.iqbal@bbsrc.ac.uk

ABSTRACT
The impact of avian influenza caused by H9N2 viruses in Pakistan is now significantly more severe than in previous years. Since all gene segments contribute towards the virulence of avian influenza virus, it was imperative to investigate the molecular features and genetic relationships of H9N2 viruses prevalent in this region. Analysis of the gene sequences of all eight RNA segments from 12 viruses isolated between 2005 and 2008 was undertaken. The hemagglutinin (HA) sequences of all isolates were closely related to H9N2 viruses isolated from Iran between 2004 and 2007 and contained leucine instead of glutamine at position 226 in the receptor binding pocket, a recognised marker for the recognition of sialic acids linked alpha2-6 to galactose. The neuraminidase (NA) of two isolates contained a unique five residue deletion in the stalk (from residues 80 to 84), a possible indication of greater adaptation of these viruses to the chicken host. The HA, NA, nucleoprotein (NP), and matrix (M) genes showed close identity with H9N2 viruses isolated during 1999 in Pakistan and clustered in the A/Quail/Hong Kong/G1/97 virus lineage. In contrast, the polymerase genes clustered with H9N2 viruses from India, Iran and Dubai. The NS gene segment showed greater genetic diversity and shared a high level of similarity with NS genes from either H5 or H7 subtypes rather than with established H9N2 Eurasian lineages. These results indicate that during recent years the H9N2 viruses have undergone extensive genetic reassortment which has led to the generation of H9N2 viruses of novel genotypes in the Indian sub-continent. The novel genotypes of H9N2 viruses may play a role in the increased problems observed by H9N2 to poultry and reinforce the continued need to monitor H9N2 infections for their zoonotic potential.

Show MeSH
Related in: MedlinePlus