The development and genetic diversity of H5N1 influenza virus in China, 1996-2006

Abstract

Since it was first detected in 1996, the Goose/Guangdong/1/1996 (Gs/GD) H5N1 influenza virus and its reassortants have spread to over 60 countries, with over 20 distinct genetic reassortants previously recognized. However, systematic analysis of their interrelationship and the development of genetic diversity have not been explored. As each of those reassortants was first detected in China, here 318 full-length H5N1 virus genomes isolated from 1996 to 2006 in this region were phylogenetically analyzed. Our findings revealed two major group reassortment events in 2001 and 2002 that were responsible for the generation of the majority of the 44 distinct Gs/GD genotypes identified, excepting those 1997 variants. Genotype replacement and emergence occurred continually, with 34 transient genotypes detected while only 10 variants were persistent. Two major replacements of predominant genotypes were also observed: genotype B replaced by Z in 2002 and then genotype Z replaced by the now predominant genotype V in 2005.

Fig. 1

Phylogenetic relationships of the HA (A) and NA (B) genes of representative influenza A viruses. Analyses were based on nucleotides 1–1,696 of the HA gene and nucleotides 1–1,355 of the NA gene. The HA and NA gene trees were rooted to duck/Hokkaido/51/96 and chicken/Scotland/59, respectively. The numbers above and below the branch nodes indicate Bayesian posterior probabilities of ≥95 and neighbor-joining bootstrap values of ≥50%, respectively. H5N1 prototype virus names are in red. Virus genotypes are given in parenthesis following the virus names. Major genotypes are indicated with bold text. Numbers labeled on the HA tree indicate WHO H5N1 clade designations (http://www.who.int/csr/disease/avian_influenza/guidelines/nomenclature/en). In the NA tree, 19-aa and 20-aa indicate the presence of amino acid deletions in the stalk region. *Indicates viruses without the 20-aa NA deletion. Scale bar, 0.01 nucleotide substitutions per site. Abbreviations: AS, African starling; BHG, bar-headed goose; Ck, chicken; Dk, duck; Gf, Guinea fowl; Gs, goose; HK, Hong Kong; MDk, migratory duck; Pa, partridge; Ph, pheasant; SCk, silky chicken; ST, Shantou; TS, tree sparrow; Ty, Turkey; WDk, wild duck.

Fig. 2

Phylogenetic relationships of the polymerase genes PB2 (A) and PB1 (B) of representative influenza A viruses isolated in Eurasia. Analysis was based on nucleotides 1–2,277 of the PB2 gene and 1–2,271 of the PB1 gene. The PB2 and PB1 trees are rooted to equine/Prague/1/56 (H7N7), pintail duck/Alberta/628/79, respectively. The numbers above and below the branch nodes indicate Bayesian posterior probabilities of ≥95 and neighbor-joining bootstrap values of ≥50%, respectively. Red branches indicate H5N1 viruses with H5N1 prototype virus names in red. Virus genotypes are given in parenthesis following the virus names. Major genotypes are indicated with bold text. Lineage names are indicated in normal text: Aquatic, contemporary Eurasia aquatic virus; Ck/Bei, Ck/Beijing/1/94-like (H9N2) virus; Early, 1970s Eurasia aquatic virus; G1, Qa/HK/G1/97-like (H9N2) virus; H9N2, contemporary reassortant H9N2 virus. Scale bar, 0.01 substitutions per site. Abbreviation: AS, African starling; BHG, bar-headed goose; Ck, chicken; Dk, duck; Gf, Guinea fowl; Gs, goose; HK, Hong Kong; MDk, migratory duck; Pa, partridge; Ph, pheasant; Qa, Quail; SCk, silky chicken; ST, Shantou; TS, tree sparrow; Ty, Turkey; WDk, wild duck.

Fig. 3

Phylogenetic relationships of polymerase acidic (PA) (A) and nucleoprotein (NP) (B) genes of representative influenza A viruses isolated in Eurasia. Analysis was based on nucleotides 1–2,148 of the PA gene and 1–1,482 of the NP gene. The PA and NP trees are rooted to equine/Prague/1/56 (H7N7). The numbers above and below the branch nodes indicate Bayesian posterior probabilities of ≥95 and neighbor-joining bootstrap values of ≥50%, respectively. Red branches indicate H5N1 viruses with H5N1 prototype virus names in red. Virus genotypes are given in parenthesis following the virus names. Major genotypes are indicated with bold text. Lineage names are provided in the Figure 2 legend. Scale bar, 0.01 substitutions per site. Abbreviation: AS, African starling; BHG, bar-headed goose; Ck, chicken; Dk, duck; Gf, Guinea fowl; Gs, goose; HK, Hong Kong; MDk, migratory duck; Pa, partridge; Ph, pheasant; Qa, Quail; SCk, silky chicken; ST, Shantou; TS, tree sparrow; Ty, Turkey; WDk, wild duck.

Fig. 4

Phylogenetic relationships of matrix (M) (A) and non-structural (NS) (B) genes of representative influenza A viruses isolated in Eurasia. Analysis was based on nucleotides 1–1,002 of the M gene and 1–863 of the NS gene. The M and NS trees are rooted to equine/Prague/1/56 (H7N7). The numbers above and below the branch nodes indicate Bayesian posterior probabilities of ≥95 and neighbor-joining bootstrap values of ≥50%, respectively. Red branches indicate H5N1 viruses with H5N1 prototype virus names in red. Virus genotypes are given in parenthesis following the virus names. Major genotypes are indicated with bold text. Lineage names are provided in the Figure 2 legend. Scale bar, 0.01 substitutions per site. Abbreviation: AS, African starling; BHG, bar-headed goose; Ck, chicken; Dk, duck; Gf, Guinea fowl; Gs, goose; HK, Hong Kong; MDk, migratory duck; Pa, partridge; Ph, pheasant; Qa, Quail; SCk, silky chicken; ST, Shantou; TS, tree sparrow; Ty, Turkey; WDk, wild duck.

Fig. 5

Genotypes of H5N1 influenza viruses in southern China, 1996–2006. The eight gene segments, represented by horizontal bars are, from top to bottom, PB2, PB1, PA, HA, NP, NA, M and NS. Each different color represents a distinct phylogenetic lineage. Genotype definitions are described in the results section. Only persistent genotypes that were detected for more than two years are represented. Numbers of transient genotypes detected in only one year is given in boxed text. All detected genotypes are given in Figure S1 and a representative virus for each genotype in each year is listed in Table S1.

Fig. 6

Simplified cladograms representing major lineages of the PB2 (A), PB1 (B), PA (C), NP (D), M (E) and NS (F). The cladograms are based on the phylogenetic trees shown in Figure 1–Figure 4. Major H5N1 genotypes are shown in blue text and prototype virus lineages are shown in red text. Lineage names are provided in the Figure 2 legend.