Evolution and molecular epidemiology of H9N2 influenza A viruses from quail in southern China, 2000 to 2005

Abstract

H9N2 influenza viruses have become established and maintain long-term endemicity in terrestrial poultry in Asian countries. Occasionally these viruses transmit to other mammals, including humans. Increasing epidemiological and laboratory findings suggest that quail may be an important host, as they are susceptible to different subtypes of influenza viruses. To better understand the role of quail in influenza virus ecology and evolution, H9N2 viruses isolated from quail during 2000 to 2005 were antigenically and genetically characterized. Our results showed that H9N2 viruses are prevalent year-round in southern China and replicate mainly asymptomatically in the respiratory tract of quail. Genetic analysis revealed that both the G1-like and Ck/Bei-like H9N2 lineages were cocirculating in quail since 2000. Phylogenetic analyses demonstrated that most of the isolates tested were double- or multiple-reassortant variants, with four G1-like and 16 Ck/Bei-like genotypes recognized. A novel genotype of G1-like virus became predominant in quail since 2003, while multiple Ck/Bei-like genotypes were introduced into quail, wherein they incorporated G1-like gene segments, but none of them became established in this host. Those Ck/Bei-like reassortants generated in quail have then been introduced into other poultry. These complex interactions form a two-way transmission system between quail and other types of poultry. The present study provides evidence that H9N2 and H5N1 subtype viruses have also exchanged gene segments to generate currently circulating reassortants of both subtypes that have pandemic potential. Continuing influenza virus surveillance in poultry is critical to understanding the genesis and emergence of potentially pandemic strains in this region.

FIG. 1.

H9N2 influenza virus isolation rate in quail from southern China, July 2000 to December 2005.

FIG. 2.

Numerical analysis of HI titers (see Table 2) by using nonmetric multidimensional scaling (A) and hierarchical agglomerative clustering (B).

FIG. 3.

Phylogenetic relationships of the HA (a) and NA (b) genes of representative influenza A viruses isolated in Asia. Trees were generated by the neighbor-joining method with the PAUP* program (Bayesian analysis revealed similar relationships.) Numbers above and below branches indicate neighbor-joining bootstrap values and Bayesian posterior probabilities, respectively. Not all supports are shown because of space constraints. 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 was shown in brackets. Bars, 0.01 substitution per site. BJ and Bei, Beijing; Ck, chicken; Dk, duck; GD, Guangdong; Gf, guinea fowl; GX, Guangxi; HLJ, Heilongjiang; HN, Henan; HK, Hong Kong; NC, Nanchang; Pg, pigeon; Ph, pheasant; Qa, quail; SCk, silky chicken; SD, Shandong; SH, Shanghai; ST, Shantou; Ty, turkey; WDk, wild duck.

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 with the PAUP* program (Bayesian analysis revealed similar relationships.) Numbers above and below branches indicate neighbor-joining bootstrap values and Bayesian posterior probabilities, respectively. Not all supports are shown because of space constraints. 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 to Qa/Arkansas/29209-1/93 (H9N2), and the NS tree to A/swine/Hong Kong/168/93 (H1N1). Viruses characterized in this study are highlighted in green. Bars, 0.01 substitution per site. Virus names and abbreviations are in the legend to Fig. 3.

FIG. 5.

Genotypes of H9N2 influenza viruses from quail in southern China. The eight gene segments (horizontal bars, starting at the top) are PB2, PB1, PA, HA, NP, NA, M, and NS. Each color represents a virus lineage. Genotypes were defined by gene phylogeny (Fig. 3 and 4); a distinct phylogenetic lineage with bootstrap support of ≥80% (≥60% for PB2 genes) indicated a common origin. Question marks indicate that the corresponding genotype was not detected in that year, and red crosses indicate the genotype was not detected in two consecutive years.