Antigenic and Molecular Characterization of Avian Influenza A(H9N2) Viruses, Bangladesh

Karthik Shanmuganatham, Mohammed M. Feeroz, Lisa Jones-Engel, Gavin J.D. Smith, Mathieu Fourment, David Walker, Laura McClenaghan, S.M. Rabiul Alam, M. Kamrul Hasan, Patrick Seiler, John Franks, Angie Danner, Subrata Barman, Pamela McKenzie, Scott Krauss, Richard J. Webby, and Robert G. Webster

Author affiliations: St. Jude Children’s Research Hospital, Memphis, Tennessee, USA (K. Shanmuganatham, D. Walker, L. McClenaghan, P. Seiler, J. Franks, A. Danner, S. Barman, P. McKenzie, S. Krauss, R.J. Webby, R.G. Webster); Jahangirnagar University, Dhaka, Bangladesh (M.M. Feeroz, S.M.R. Alam, M.K. Hasan); University of Washington, Seattle, Washington, USA (L. Jones-Engel); Duke-NUS Graduate Medical School, Singapore (G.J.D. Smith, M. Fourment); Duke Global Health Institute, Durham, North Carolina, USA (G.J.D. Smith)

Abstract

Human infection with avian influenza A(H9N2) virus was identified in Bangladesh in 2011. Surveillance for influenza viruses in apparently healthy poultry in live-bird markets in Bangladesh during 2008–2011 showed that subtype H9N2 viruses are isolated year-round, whereas highly pathogenic subtype H5N1 viruses are co-isolated with subtype H9N2 primarily during the winter months. Phylogenetic analysis of the subtype H9N2 viruses showed that they are reassortants possessing 3 gene segments related to subtype H7N3; the remaining gene segments were from the subtype H9N2 G1 clade. We detected no reassortment with subtype H5N1 viruses. Serologic analyses of subtype H9N2 viruses from chickens revealed antigenic conservation, whereas analyses of viruses from quail showed antigenic drift. Molecular analysis showed that multiple mammalian-specific mutations have become fixed in the subtype H9N2 viruses, including changes in the hemagglutinin, matrix, and polymerase proteins. Our results indicate that these viruses could mutate to be transmissible from birds to mammals, including humans.

Initially infecting poultry, avian influenza A(H9N2) viruses have been sporadically identified in pigs and humans, which suggests that some of these viruses have adapted to bind mammalian host receptors or have acquired mutations that increase mammalian receptor specificity (1–3). Human infection with avian influenza A(H9N2) virus was initially identified in Hong Kong and China in 1999 (4); in 2011, infection with this subtype was reported for a patient in Bangladesh (5). Detection of these viruses in humans outside of China highlights the necessity and urgency for comprehensive surveillance because of the viruses’ expanding host range.
Phylogenetically, avian influenza A(H9N2) viruses can be grouped into 3 distinct sublineages represented by their prototype strains: A/Qa/HK/G1/97 (G1-like), A/Dk/HK/Y280/97 (Y280-like), and A/Ck/Korea/38349-p96323/96 (Korean-like) (1,6,7). Genetic and antigenic analyses of subtype H9N2 isolates from the past 2 decades have shown that these viruses are gradually evolving from the Eurasian lineage into several distinct sublineages and are becoming established in domestic poultry (7–18). Phylogenetic analyses of subtype H9N2 viruses isolated in China and the Middle East have shown that these viruses have undergone reassortment with other subtypes to generate multiple novel genotypes consisting of gene segments from different lineages (7,11,13,19,20).
Worldwide, Bangladesh is among countries with the highest numbers of reported outbreaks of highly pathogenic avian influenza (HPAI) (H5N1) (21). Since an initial outbreak in February 2007, Bangladesh has reported 550 outbreaks of infection with HPAI (H5N1) virus (493 at commercial farms and 57 among backyard poultry) (22–24). In Bangladesh, live-bird markets are the most common outlets for purchase of poultry and poultry meat; an estimated 95% of poultry meat and eggs sold in the country are sold at these markets (25). Previous surveillance conducted at live-bird markets in Bangladesh found that avian influenza virus (AIV) is prevalent (23%); the low pathogenicity H9N2 subtype predominated, but other subtypes were isolated, including H5N1, H1N2, H1N3, H3N6, H4N2, and H10N7 (26). However, this surveillance report did not include information about the molecular properties of circulating subtype H9N2 viruses.
We reviewed surveillance data and conducted molecular and genetic analyses of influenza A(H9N2) viruses circulating among poultry in Bangladesh. Our study had 3 primary goals: 1) characterize the antigenic and molecular properties of subtype H9N2 isolates; 2) define genetic and phylogenetic relationships between the genes identified in these viruses and those of other AIVs; and 3) determine whether these viruses have acquired genomic changes that could facilitate transmission from avian to mammalian hosts.