Identification of the progenitors of Indonesian and Vietnamese avian influenza A (H5N1) viruses from southern China

Abstract

The transmission of highly pathogenic avian influenza H5N1 virus to Southeast Asian countries triggered the first major outbreak and transmission wave in late 2003, accelerating the pandemic threat to the world. Due to the lack of influenza surveillance prior to these outbreaks, the genetic diversity and the transmission pathways of H5N1 viruses from this period remain undefined. To determine the possible source of the wave 1 H5N1 viruses, we recently conducted further sequencing and analysis of samples collected in live-poultry markets from Guangdong, Hunan, and Yunnan in southern China from 2001 to 2004. Phylogenetic analysis of the hemagglutinin and neuraminidase genes of 73 H5N1 isolates from this period revealed a greater genetic diversity in southern China than previously reported. Moreover, results show that eight viruses isolated from Yunnan in 2002 and 2003 were most closely related to the clade 1 virus sublineage from Vietnam, Thailand, and Malaysia, while two viruses from Hunan in 2002 and 2003 were most closely related to viruses from Indonesia (clade 2.1). Further phylogenetic analyses of the six internal genes showed that all 10 of those viruses maintained similar phylogenetic relationships as the surface genes. The 10 progenitor viruses were genotype Z and shared high similarity (>/=99%) with their corresponding descendant viruses in most gene segments. These results suggest a direct transmission link for H5N1 viruses between Yunnan and Vietnam and also between Hunan and Indonesia during 2002 and 2003. Poultry trade may be responsible for virus introduction to Vietnam, while the transmission route from Hunan to Indonesia remains unclear.

FIG. 1.

Numerical analysis of HI titers (see Table 2) by nonmetric multidimensional ordination in two dimensions (A) and using hierarchical agglomerative clustering (B). Each antigenic group (circled) has been labeled on the cluster (see also Table 2).

FIG. 2.

Phylogenetic relationships of the HA (A) and NA (B) genes of representative influenza A viruses. The numbers above and below the branch nodes indicate neighbor-joining bootstrap values of ≥50% and Bayesian posterior probabilities of >95, respectively. Analyses were based on nucleotides 22 to 1,032 of the HA gene and nucleotides 1 to 1,090 of the NA gene. The HA and NA gene trees were rooted to duck/Hokkaido/51/96 and chicken/Scotland/59, respectively. Colors indicate viruses isolated from southern China (blue), Vietnam (red), and Indonesia (red). Scale bar, 0.01 nucleotide substitutions per site. Abbreviations: BHGs, bar-headed goose; BHgull, brown-headed gull; Ck, chicken; Cu, chukkar; Dk, duck; FJ, Fujian; Gf, Guinea fowl; Gs, goose; GD, Guangdong; GX, Guangxi; GY, Guiyang; HK, Hong Kong; HN, Hunan; IDN, Indonesia; JX, Jiangxi; Mall, mallard; MDk, migratory duck; MYS, Malaysia; NGA, Nigeria; Pa, partridge; Ph, pheasant; Qa, quail; QH, Qinghai; SCk, silky chicken; ST, Shantou; THA, Thailand; VNM, Vietnam; WDk, wild duck; YN, Yunnan.

FIG. 3.

Phylogenetic relationships of the PB2 (A), PB1 (B), PA (C), NP (D), M (E), and NS (F) genes of representative influenza A viruses. The numbers above and below the branch nodes indicate neighbor-joining bootstrap values of ≥50% and Bayesian posterior probabilities of >95, respectively. Analyses were based on nucleotides 986 to 2288, 1 to 1480, 1404 to 2151, 1 to 990, 1 to 998, and 1 to 842, respectively. All gene trees were rooted to A/equine/Prague/1/56 except PB1 and PA, which were midpoint rooted. Colors indicate viruses isolated from southern China (blue), Vietnam (red), and Indonesia (red). Scale bar, 0.01 nucleotide substitutions per site. Abbreviations: BHGs, bar-headed goose; BHgull, brown-headed gull; Ck, chicken; Cu, chukkar; Dk, duck; FJ, Fujian; Gf, Guinea fowl; Gs, goose; GD, Guangdong; GX, Guangxi; GY, Guiyang; HK, Hong Kong; HN, Hunan; IDN, Indonesia; JX, Jiangxi; Mall, mallard; MDk, migratory duck; MYS, Malaysia; NGA, Nigeria; Pa, partridge; Ph, pheasant; Qa, quail; QH, Qinghai; SCk, silky chicken; ST, Shantou; THA, Thailand; VNM, Vietnam; WDk, wild duck; YN, Yunnan.

FIG. 4.

Map of southern China showing the provinces under influenza surveillance and wave 1 transmission pathway of H5N1 viruses from southern China to other Southeast Asian regions.