Arch Virol. 2014 Jul;159(7):1651-61.

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

Parvin R, Heenemann K, Halami MY, Chowdhury EH, Islam MR, Vahlenkamp TW.

Faculty of Veterinary Medicine, Institute of Virology, Center for Infectious Diseases, University of Leipzig, An den Tierkliniken 29, 04103, Leipzig, Germany.



Low pathogenic avian influenza viruses (LPAIV) of subtype H9N2 have become widespread in poultry in many Asian countries with relevance to respiratory diseases of multifactorial origin. In Bangladesh LPAIV of subtype H9N2 co-circulate simultaneously with highly pathogenic avian influenza viruses (HPAIV) of subtype H5N1 in commercial and backyard poultry.The project aimed to characterize LPAIV of subtypes H9N2 currently circulating in Bangladesh. The selected isolate A/Chicken/Bangladesh/VP01/2006 (H9N2) was propagated in chicken embryo. All eight gene segment were amplified by RT-PCR, cloned and subjected to full length sequencing. The obtained sequence data were compared with the references strains available in GenBank. The phylogenetic analysis of LPAIV H9N2 from Bangladesh revealed close relationship with Indian, Pakistani and Middle Eastern isolates and maintained ancestor relation with LPAIV H9N2 Quail/HK/G1/1997.The internal genes M and NP belong to lineage G1 whereas NS, PA, PB1 and PB2 encoding genes belong to the prototype virus A/Chicken/Korea/38349-p96323/96.The internal genes showed high sequence homology with a HPAIV of subtype H7N3 from Pakistan, whereas the PB1 gene showed similarly high nucleotide homologies with recently circulating HPAIV H5N1 from Bangladesh revealing two independent reassortment events. The hemagglutinin cleavage site of LPAIV H9N2 confirmed its low pathogenicity. The receptor binding sites indicates a binding preference to 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.

PMID: 24420161



The phylogenetic analysis of the surface glycoprotein (HA and NA) suggested that the Bangladeshi AIV H9N2 belonging to the G1 lineage is closely related to other South Asian and the Middle Eastern isolates. The all six internal genes have shown 96 to 98% nucleotide homology with the HPAIV of subtype H7N3 from Pakistan (NARC-100/H7N3). Furthermore, the PB1 gene of BD-VP01 showed maximum identity (98%) with a HPAIV H5N1 isolate from Bangladesh (Ck-BD/H5N1) and clustered together in a same branch of the phylogenetic tree. Briefly, the results suggested that HPAIV subtype H7N3 had all six internal genes like H9N2 (like BD-VP01, other South-Asian and Middle-Eastern H9N2) and at the same time the HPAIV subtype H5N1 (CK-BD/H5N1) contained a BD-VP01-like PB1gene. That means AIV H9N2 act as a donor for those internal genes to the respective HPAI virus subtypes. For a better understanding we illustrated the possible reassortment events as shown in figure 1.

AIV H9N2 have contributed in several cases to the genetic reassortment either by receiving genes from or donating genes to other subtype of AIV (Gao et al. 2013; Kwon et al. 2006; Zhang et al. 2009; Munir et al. 2009; Guan et al., 1999;). In the current study, reassortment events occurred by indirect donation of six internal genes to a HPAIV of subtype H7N3 and by the direct donation of the PB1 gene to a HPAIV of subtype H5N1. AIV BD-VP01 was isolated in 2006 while HPAIV NARC-100/H7N3 was isolated in 2004. The current study suggested that all six internal proteins of HPAIV H7N3 showed genetic similarities with the previously mentioned circulating AIV H9N2 viruses in South-East Asia from 2000-2006. Therefore, those AIV H9N2 strains (Middle East and South-East Asia’ 2000-2004) might have served as a direct donor for those internal genes to the HPAIV of subtype H7N3. It remains open in which direction the gene donation occurred. The current study, however, strongly recommended that the AIV H9N2 has to be the donor of those genes because during the NCBI blast search all mentioned internal genes of HPAIV of subtype H7N3 always branched into the H9N2 strains– never the other way around. In case of the secondly described reassortment of the PB1 gene, it was surely a direct donation as AIV Ck-BD/H5N1 has a BD-VP01/H9N2 like PB1 gene. The isolation period and the country of origin of BD-VP01 and Ck-BD/H5N1 also supported this notion.


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Fig 1. Avian influenza virus (AIV) reassortment events between AIV BD-VP01/H9N2, high pathogenic AIV NARC-100/H7N3 and high pathogenic AIV Ck-BD/H5N1.


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