Genetic evolution of H9 subtype influenza viruses from live poultry markets in Shanghai, China

Abstract

H9N2 influenza viruses have become established and maintain long-term endemicity in poultry. The complete genomes of seven avian H9N2 influenza viruses were characterized. These seven influenza virus isolates were obtained from live poultry markets in Shanghai, China, in 2002 and from 2006 to 2008. Genetic analysis revealed that all seven isolates had an RSSR motif at the cleavage site of hemagglutinin (HA), indicating low pathogenicity in chickens. Phylogenetic analyses indicated that the seven avian H9N2 viruses belonged to the lineage represented by Duck/Hong Kong/Y280/97 (H9N2), a virus belonging to the Chicken/Beijing/1/94-like (H9N2) lineage, and that they are all quadruple reassortants consisting of genes from different lineages. The six internal genes of the isolates possessed H5N1-like sequences, indicating that they were reassortants of H9 and H5 viruses. All of the viruses had nonstructural (as well as HA and neuraminidase) genes derived from the Duck/Hong Kong/Y280/97-like virus lineage but also had other genes of mixed avian virus origin, including genes similar to those of H5N1 viruses (Gs/GD-like). The infected chickens showed no signs of disease. These results show the genetic and biological diversity of H9N2 viruses in Shanghai and support their potential role as pandemic influenza agents.

FIG. 1.

Phylogenetic tree of the HA (positions 115 to 999) and NA (positions 59 to 1235) genes of the H9N2 avian influenza viruses in Shanghai, China, in 2002 and from 2006 to 2008. The unrooted phylogenetic trees were generated by the distance-based neighbor-joining method using MEGA, version 3.1. The reliability of the trees was assessed by bootstrap analysis with 1,000 replications; only bootstrap values of >90% are shown. Abbreviations: BJ and Bei, Beijing; CA, California; Ck, chicken; Dk, duck; GD, Guangdong; Gf, guinea fowl; Gs, goose; GX, Guangxi; GZ, Guangzhou; HK, Hong Kong; HLJ, Heilongjiang; JS, Jiangshu; NC, Nanchang; NJ, Nanjing; Pg, pigeon; Ph, pheasant; Qa, quail; SD, Shandong; SH, Shanghai; ST, Shantou; Ty, turkey; VNM, Vietnam; WDk, wild duck; WI, Wisconsin; YN, Yunnan.

FIG. 2.

Phylogenetic trees of the PB2 (positions 32 to 2228), PB1 (positions 182 to 1080), PA (positions 23 to 1257), M (positions 270 to 765), NP (positions 2 to 1445), and NS (positions 34 to 693) genes of the H9N2 avian influenza viruses in Shanghai, China, in 2002 and from 2006 to 2008. The method used is described in the legend to Fig. 1. Virus abbreviations are defined in the legend to Fig. 1.

FIG. 2.

Phylogenetic trees of the PB2 (positions 32 to 2228), PB1 (positions 182 to 1080), PA (positions 23 to 1257), M (positions 270 to 765), NP (positions 2 to 1445), and NS (positions 34 to 693) genes of the H9N2 avian influenza viruses in Shanghai, China, in 2002 and from 2006 to 2008. The method used is described in the legend to Fig. 1. Virus abbreviations are defined in the legend to Fig. 1.

FIG. 2.

Phylogenetic trees of the PB2 (positions 32 to 2228), PB1 (positions 182 to 1080), PA (positions 23 to 1257), M (positions 270 to 765), NP (positions 2 to 1445), and NS (positions 34 to 693) genes of the H9N2 avian influenza viruses in Shanghai, China, in 2002 and from 2006 to 2008. The method used is described in the legend to Fig. 1. Virus abbreviations are defined in the legend to Fig. 1.