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

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.

Figure 1. Phylogenetic relationships of HA genes of H9N2 avian influenza viruses isolated from Pakistan between 2005 and 2008.

The Phylogenetic tree was generated using minimum evolution analysis with maximum composite likelihood using the Tamura-Nei model with MEGA (version 4.1 beta). Numbers below branches indicate bootstrap value percentages from 1000 replicates, bootstrap values >70% are shown. Analysis was based on complete open reading frames of all gene segments. The scale bar represents the distance unit between sequence pairs. Bhgs, bar headed goose, GF, guinea fowl; Ph, pheasant, Ck, chicken; Dk, duck; Qa, quail; Ty, turkey; Par, parakeet, Sw, swine; Pak, Pakistan; RP, Rawalpindi; Afg, Afghanistan; HK, Hong Kong; ST, Shantou; VN, Vietnam; Wis, Wisconsin; Cal, California; Kor, Korea; GZ, Guangzhou; Rus, Russia; UP, Uttar Pradesh. The viruses characterised in this report are indicated as red. The sequences of H9N2 and H7N3 viruses previously isolated from Pakistan are indicated blue and pink respectively.

Figure 2. Phylogenetic relationships of the NA genes.

The phylogenetic methods and abbreviations were as described for figure 1.

Figure 3. Phylogenetic relationships of the M genes.

The phylogenetic methods and abbreviations were as described for figure 1.

Figure 4. Phylogenetic relationships of the NP genes.

The phylogenetic methods and abbreviations were as described for figure 1.

Figure 5. Phylogenetic relationships of the PB2 genes.

The phylogenetic methods and abbreviations were as described for figure 1.

Figure 6. Phylogenetic relationships of the PB1 genes.

The phylogenetic methods and abbreviations were as described for figure 1.

Figure 7. Phylogenetic relationships of the PA genes.

The phylogenetic methods and abbreviations were as described for figure 1.

Figure 8. Phylogenetic relationships of the NS genes of H9N2 avian influenza viruses isolated from poultry in Pakistan.

Analysis was based on nucleotides 27–870 of segment eight of all viruses included in this analysis. NS gene sequences were compared with closely related H5, H7 and H9 viruses (the viruses highlighted in red were sequenced in this study and H9N2, H7N3 and H5N1 viruses isolated in Pakistan previously are indicated in blue, pink and brown respectively). The phylogenetic methods and abbreviations were as described for figure 1.

Figure 9. Representation of the newly identified genotypes of H9N2 viruses in poultry in Pakistan.

The possible genetic progenitors of these new genotypes are (A) H9N2 (LPAI) viruses represented by A/Ck/Pak/2/99, belonging to G1-lineage, (B) H9N2 (LPAI) viruses represented as Indian/Middle East lineage containing distinct polymerase complex genes, (C) H7N3 (HPAI) viruses represented by the prototype A/Ck/Pak/NARC-100/2004 virus, (D) H7N3 (HPAI) viruses represented by the prototype A/Ck/Pak/34668/95 virus containing a 13 amino acid deletion in their NS1 polypeptide and (E) H5N1 (HPAI) viruses represented by A/Ck/Afghanistan/1207/2006 belonging to genotype Z. The three newly identified H9N2 genotypes (F, G and H) contained PB2, PB1, and PA genes similar to an Indian/Middle East lineage of H9N2 virus, along with the NS genes derived from two distinct H7N3 parental viruses and an H5N1 parental virus and the HA, NA, NP and M genes from H9N2 viruses of the G1 lineage.