?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.
We isolated a large number of low pathogenicity avian influenza A(H9N2) viruses and an increasing number of highly pathogenic avian influenza A(H5N1) viruses from apparently healthy birds in Bangladesh. During 2008–2011, subtype H9N2 viruses were isolated from multiple poultry species (predominantly chickens and quail) throughout the year, without seasonal prevalence; subtype H5N1 viruses were predominantly isolated from quail during the winter of 2011. Phylogenetic analyses revealed that the subtype H9N2 viruses are of the G1 clade; antigenically, the G1 clade consists of 2 branches distinguished by host species, but the Bangladesh viruses were divergent from the prototype G1 virus. Our comparison of the deduced amino acid sequence showed that the subtype H9N2 viruses have acquired mammalian host–specific mutations in their surface glycoproteins and internal genes.
We isolated subtype H9N2 and H5N1 viruses individually and in viral mixtures; the latter may result from co-infection of the same host species with multiple influenza subtypes. In our surveillance, we isolated influenza subtypes other these than mostly during 2008–2009, with very few other subtypes isolated after 2009. This finding suggests that subtypes H9N2 and H5N1 predominate in live-bird markets.
Phylogenetic analyses showed that, with 1 exception, the isolates we analyzed were homogenous and shared the common ancestor A/Qa/HK/G1/97 (H9N2). G1 was the only lineage identified. The viruses we analyzed shared close nucleotide homology with those of G1 lineage isolated from birds in Pakistan or India, which suggests that the Bangladesh viruses may have their origin in those countries (or vice versa). We speculate that, during the early 2000s, subtype H9N2 viruses circulating in Pakistan were introduced into Bangladesh through regional poultry or pet trade and then established themselves in domestic poultry. Although subtype H9N2 and H5N1 viruses were co-isolated, phylogenetic analysis did not identify any reassortment between the subtypes. However, the subtype H9N2 viruses still maintained the internal genes that were part of the subtype H5N1 reassortant previously implicated in human infection (7).
Results from our analyses support our hypothesis that the subtype H9N2 isolates from Bangladesh are related to those from Pakistan and have evolved into 2 distinct subpopulations based on host species. The chicken viruses we analyzed were antigenically homogenous and are related to human viruses but distinct from quail viruses. Furthermore, small changes in the quail viruses should be monitored closely because quail were the host species implicated during the emergence of the subtype H9N2 virus that jumped to humans in 1999 (7). In addition, quail were suggested to be the contributing host species of the H9N2/H5N1 reassortant of the subtype H5N1 virus that infected humans in 1997 (4)
Our analyses also shows that subtype H9N2 viruses circulating in Bangladesh show a trend toward accumulating molecular markers that favor interspecies transmission. Distinct mutations throughout the viral genomes have been implicated in the adaptation of viruses to a mammalian host (Table 4, Appendix; and Table 5, Appendix). In almost all of the Bangladesh viruses, the RBS of HA has the Q226L substitution, which has been implicated in human virus–like receptor specificity and is critical for replication and direct transmission of these viruses in ferrets (33,34).
Although the viruses we isolated have low pathogenicity and do not possess a polybasic cleavage site, they carry a motif similar to cleavage sites seen in the highly pathogenic H5N1 and H7N3 viruses. This result suggests that subtype H9N2 viruses have the potential to acquire a polybasic site and become highly pathogenic. In the HA of nearly all the isolates that we analyzed, 2 potential glycosylation sites were lost; this process was also seen in the adaptation of duck viruses to poultry (32,35). Because the duck isolate A/Env/BD/1041/2009 retained its glycosylation site, we hypothesize that the introduction of the Y439 lineage via ducks was unsuccessful because the G1 lineage was already dominant in the local poultry species.
In most of the viruses we analyzed, we observed residues in the avian–human signature positions becoming fixed (HA 226L and 183H, M1 V15I, M2 L55F, NS E227K, NP R214K, E372D, PB1-L13P); in certain isolates, the mutations were fixed by unique substitutions whose functions are unknown (HA Q227I, M2 E16V, I28F, PB1-F2 R79L). In addition, these viruses have acquired substitutions similar to those seen in highly pathogenic subtype H5N1 and H7N3 and pandemic subtype H1N1 and H3N2 viruses (36). Noticeably, in the NS gene’s PDZ motif, most subtype H9N2 isolates carried the KSEV motif seen in the 1918 pandemic subtype H1N1 virus (11,37). In the PA genes of 2 subtype H9N2 viruses, we identified R57Q and S409N substitutions, both of which were in the 1968 pandemic subtype H3N2 virus. These observations confirm that the Bangladesh subtype H9N2 viruses have accumulated molecular markers that influence host specificity and pathogenesis.
In conclusion, our results show that the Bangladesh subtype H9N2 viruses are genetically similar to, but distinct from, the A/Qa/HK/G1/97 isolate, which has been previously implicated in human infection (7). However, the Bangladesh viruses have accumulated molecular characteristics required for infecting humans and are antigenically similar to the human subtype H9N2 virus isolated from a patient in Bangladesh. These viruses have a tendency to reassort with highly pathogenic subtype H5N1 (7) and H7 viruses (38,39), which is particularly concerning in light of the emergence of avian influenza A(H7N9) virus in China that contains 6 gene segments from H9N2 influenza viruses and causes lethal infection in humans (40). The emergence of this reassortant virus emphasizes the potential for, and the danger of, transmission of subtype H9N2 viruses to humans.
No comments:
Post a Comment