Characterization of H9 subtype influenza viruses from the ducks of southern China: a candidate for the next influenza pandemic in humans?

Abstract

A current view of the emergence of pandemic influenza viruses envisages a gene flow from the aquatic avian reservoir to humans via reassortment in pigs, the hypothetical "mixing vessel." Understanding arising from recent H5N1 influenza outbreaks in Hong Kong since 1997 and the isolation of avian H9N2 virus from humans raises alternative options for the emergence of a new pandemic virus. Here we report that H9N2 influenza viruses established in terrestrial poultry in southern China are transmitted back to domestic ducks, in which the viruses generate multiple reassortants. These novel H9N2 viruses are double or even triple reassortants that have amino acid signatures in their hemagglutinin, indicating their potential to directly infect humans. Some of them contain gene segments that are closely related to those of A/Hong Kong/156/97 (H5N1/97, H5N1) or A/Quail/Hong Kong/G1/97 (G1-like, H9N2). More importantly, some of their internal genes are closely related to those of novel H5N1 viruses isolated during the outbreak in Hong Kong in 2001. This study reveals a two-way transmission of influenza virus between terrestrial and aquatic birds that facilitates the generation of novel reassortant H9N2 influenza viruses. Such reassortants may directly or indirectly play a role in the emergence of the next pandemic virus.

FIG. 1.

Phylogenetic trees for the H9 HA1 (a), NP (b), and PB2 (c) genes of influenza A viruses. The nucleotide sequences were analyzed by PAUP using a maximum-parsimony algorithm. The H9 HA1 phylogenetic tree is rooted to Duck/Alberta/60/76 (H12N5). The NP and PB2 gene trees were rooted to A/Equine/Prague/1/57 (H7N7). The lengths of the horizontal lines are proportional to the minimum number of nucleotide differences required to join the nodes. Vertical lines are used for spacing branches and labels. All viruses sequenced in the present studies are underlined, and the remaining sequences can be found in GenBank. Dk, duck; Gs, goose; Ty, turkey; Qa, quail; Ck, chicken; Pg, pigeon; SCk, silky chicken; WDk, wild duck; Sw, swine; Eq, equine; AqB, aquatic bird; HK, Hong Kong; ST, Shantou; Bei, Beijing; Kor, Korea; Ger, Germany; Gd, Guangdong.

FIG. 2.

Phylogenetic trees for the PB1 (a), PA (b), and NS (c) genes of influenza A viruses. Evolutionary analyses of nucleotide sequences of PB1, PA, and NS genes were performed as described in the legend to Fig. 1. The sequence ranges were listed in Materials and Methods. The PB1 and PA trees were rooted to B/Lee/40 and B/Singapore/222/79, respectively. The NS gene tree was rooted to A/Equine/Prague/1/56 (H7N7). Virus names and abbreviations can be found in the legend of Fig. 1.

FIG. 3.

Current understanding of the ecology of influenza virus in southern China that favors the emergence of H9N2 as a possible new pandemic influenza virus. The concept of the classic gene pool and the interplay between aquatic birds and influenza viruses may be explained by frequent interspecies transmission between terrestrial and aquatic poultry in the southern region of China.