The genesis and evolution of H9N2 influenza viruses in poultry from southern China, 2000 to 2005

Abstract

H9N2 influenza viruses have become established in terrestrial poultry in different Asian countries over the last 2 decades. Our previous study demonstrated that quail harbor increasingly diverse novel H9N2 reassortants, including both Chicken/Beijing/1/94 (Ck/Bei-like) and Quail/Hong Kong/G1/97 (G1-like) viruses. However, since 1999, the genesis and evolution of H9N2 viruses in different types of poultry have not been investigated systematically. In the present study, H9N2 viruses isolated from chickens, ducks, and other minor poultry species were characterized genetically and antigenically. Our findings demonstrate that Ck/Bei-like H9N2 viruses have been introduced into many different types of poultry in southern China, including quail, partridges, chukar, pheasant, guinea fowl, and domestic ducks, while G1-like viruses were commonly detected in quail, less frequently detected in other minor poultry species, and not detected in chickens and ducks. Genetic analysis revealed 35 genotypes of H9N2 viruses, including 14 novel genotypes that have not been recognized before. Our results also suggested that two-way interspecies transmission exists between different types of poultry. Our study demonstrates that the long-term cocirculation of multiple virus lineages (e.g., H5N1 and H9N2 viruses) in different types of poultry has facilitated the frequent reassortment events that are mostly responsible for the current great genetic diversity in H9N2 and H5N1 influenza viruses in this region. This situation favors the emergence of influenza viruses with pandemic potential.

FIG. 1.

Comparison of H9N2 influenza virus isolation rates from chickens (a), other minor poultry species (b), and ducks (c) from southern China, July 2000 to December 2005. Surveillance was conducted in live poultry markets in Fujian, Guangdong, Guangxi, Guiyang, Hunan, and Yunnan Provinces. *, positive sampling occasions with low isolation rates of <0.3%.

FIG. 2.

Numerical analysis of HI titers (Table 3) by nonmetric multidimensional ordination in two dimensions (a) and by using hierarchical agglomerative clustering (b).

FIG. 3.

Phylogenetic relationships of HA (a) and NA (b) genes of representative influenza A viruses isolated in Asia. Trees were generated by the neighbor-joining method in the PAUP* program. Numbers above and below branches indicate neighbor-joining bootstrap values and Bayesian posterior probabilities, respectively. Analysis was based on nucleotides 129 to 1042 of the HA gene and 231 to 1297 of the NA gene. The HA and NA trees were rooted to Qa/Arkansas/29209-1/93 (H9N2) and Ck/Pennsylvania/8125/83 (H5N2), respectively. Viruses characterized in this study are highlighted in green. Genotypes characterized in this study are shown in parentheses and defined in Table 4. Abbreviations: BJ and Bei, Beijing; Ck, chicken; Dk, duck; FJ, Fujian; GD, Guangdong; Gf, guinea fowl; GX, Guangxi; HK, Hong Kong; HLJ, Heilongjiang; HN, Hunan; NC, Nanchang; Pg, pigeon; Ph, pheasant; Qa, quail; SCk, silky chicken; SD, Shandong; SH, Shanghai; ST, Shantou; Ty, turkey; WDk, wild duck; YN, Yunnan. Bar, 0.01 substitution per site.

FIG. 4.

Phylogenetic relationships of the PB2 (a), PB1 (b), PA (c), NP (d), M (e), and NS (f) genes of representative influenza A viruses isolated in Asia. Trees were generated by the neighbor-joining method in PAUP*. Numbers above and below branches indicate neighbor-joining bootstrap values and Bayesian posterior probabilities, respectively. Analysis was based on the following nucleotides: PB2, 1079 to 2138; PB1, 42 to 1217; PA, 1429 to 2127; NP, 31 to 917; M, 49 to 864; and NS, 88 to 815. The PB2, PA, NP, and M trees were rooted to A/equine/Prague/1/56 (H7N7), the PB1 tree was rooted to Qa/Arkansas/29209-1/93 (H9N2), and the NS tree was rooted to A/swine/Hong Kong/168/93 (H1N1). Viruses characterized in this study are highlighted in green. Aq, aquatic bird. Other virus names and abbreviations can be found in the legend to Fig. 3. Bar, 0.01 substitution per site.

FIG. 5.

Genotypes of H9N2 influenza viruses of chickens and other minor poultry species in southern China. The figure shows progenitors of H9N2 influenza virus genotype A and B series and internal gene donors (a) and genotypes from other minor poultry species (b) and chickens (c) in southern China. Dashed lines represent transient and short-lived genotypes. Details of transient genotypes are given in Table 6. The eight gene segments (horizontal bars starting from the top) are PB2, PB1, PA, HA, NP, NA, M, and NS. Each color represents a virus lineage. Genotype definitions are described in Materials and Methods. Abbreviations are listed in Table 4.

FIG. 6.

Ecology of H9N2 influenza viruses in southern China. “A” and “B” shown in black boxes indicate Qa/HK/G1/97-like and Ck/Bei/1/94-like virus lineages, respectively. Solid lines represent confirmed gene flow directions. Dashed lines indicate indirect evidence of gene flow.