Establishment of influenza A virus (H6N1) in minor poultry species in southern China

Abstract

An H6N1 virus, A/teal/Hong Kong/W312/97 (W312), was isolated during the "bird flu" incident in Hong Kong in 1997. Genetic analysis suggested that this virus might be the progenitor of the A/Hong Kong/156/97 (HK/97) H5N1 virus, as seven of eight gene segments of those viruses had a common source. Continuing surveillance in Hong Kong showed that a W312-like virus was prevalent in quail and pheasants in 1999; however, the further development of H6N1 viruses has not been investigated since 2001. Here we report influenza virus surveillance data collected in southern China from 2000 to 2005 that show that H6N1 viruses have become established and endemic in minor poultry species and replicate mainly in the respiratory tract. Phylogenetic analysis indicated that all H6N1 isolates had W312-like hemagglutinin and neuraminidase genes. However, reassortment of internal genes between different subtype virus lineages, including H5N1, H9N2, and other avian viruses, generated multiple novel H6N1 genotypes in different types of poultry. These novel H6N1/N2 viruses are double, triple, or even quadruple reassortants. Reassortment between a W312-like H6N1 virus and an A/quail/Hong Kong/G1/97 (HK/97)-like H9N2 virus simultaneously generated novel H6N2 subtype viruses that were persistent in poultry. Molecular analyses suggest that W312-like viruses may not be the precursors of HK/97 virus but reassortants from an HK/97-like virus and another unidentified H6 subtype virus. These results provide further evidence of the pivotal role of the live poultry market system of southern China in generating increased genetic diversity in influenza viruses in this region.

FIG. 1.

Rates of isolation of H6N1/N2 influenza viruses from terrestrial minor poultry species in southern China, July 2000 to December 2005. Surveillance was conducted in live poultry markets in Shantou, China.

FIG. 2.

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

FIG. 3.

Phylogenetic relationships of the H6 HA (A), N1 NA (B), and N2 NA (C) genes of representative influenza A viruses. Trees were generated by the NJ method in the PAUP* program. Numbers above and below branches indicate NJ bootstrap values and Bayesian posterior probabilities, respectively. Analysis was based on nucleotides 49 to 1032, 1 to 1353, and 1 to 1229 of the H6 HA, N1 NA, and N2 NA gene segments, respectively. The H6 HA and N2 NA trees are rooted to turkey/Canada/63 (H6N2), and the N1 NA tree is rooted to Wisconsin/1/33 (H1N1). Virus subtypes are indicated in parentheses, and viruses with no subtype designation are of the H6N1 subtype. Viruses in blue are viruses characterized in this study, and those in red are prototype viruses. Genotypes for viruses characterized in this study are indicated in parentheses along with their clade designations. Abbreviations: Aq, aquatic; AB, aquatic bird; AS, African starling; BS, Bewick's swan; Bei, Beijing; Ck, chicken; Ck/Bei, chicken/Beijing/1/94-like; Cu, chukar; Dk, duck; EU, Europe; FJ, Fujian; FPV, fowl plague virus; G1, Qa/HK/G1/97-like; GD, Guangdong; Gf, guinea fowl; Gs, goose; Gs/GD, goose/Guangdong/1/96-like; GX, Guangxi; GY, Guiyang; HK, Hong Kong; HLJ, Heilongjiang; HN, Hunan; JX, Jiangxi; Mall, mallard; MDk, migratory duck; Pa, partridge; Ph, pheasant; Qa, quail; SA, South Africa; SCk, silky chicken; ST, Shantou; Sw, swine; Tw, Taiwan; Ty, turkey; VNM, Vietnam; WDk, wild duck; W312, teal/HK/W312-like. Bar, 0.01 substitution per site.

FIG. 4.

Phylogenetic relationships of the PB2 (A), PB1 (B), and PA (C) polymerase genes of representative influenza A viruses isolated in Asia. Trees were generated by the NJ method in PAUP*. Numbers above and below branches indicate NJ bootstrap values and Bayesian posterior probabilities, respectively. Analysis was based on the following nucleotides: PB2, 1156 to 2109; PB1, 43 to 1215; and PA, 1411 to 2111. The PB2, PB1, and PA trees are rooted to equine/Prague/1/56 (H7N7), pintail duck/Alberta/628/79, and Ann Arbor/6/60, respectively. Viruses characterized in this study are highlighted in blue. Viruses in red represent prototype viruses. Genotypes for viruses characterized in this study are indicated in parentheses along with their clade designations. “Mix” indicates cocirculating H5N1, H6N1/N2, and H9N2 subtype viruses. Abbreviations: AST, Ashtrakhan; BHG, bar-headed goose; FJ, Fujian; HN, Hunan; QH, Qinghai; YN, Yunnan. Other abbreviations are listed in the legend to Fig. 3. Bar, 0.01 substitution per site.

FIG. 5.

Phylogenetic relationships of nucleoprotein (NP) (A), matrix (M) (B), and nonstructural (NS) protein (C) genes of representative influenza A viruses isolated in Asia. Trees were generated by the NJ method in PAUP*. Numbers above and below branches indicate NJ bootstrap values and Bayesian posterior probabilities, respectively. Analysis was based on the following nucleotides: NP, 22 to 915; M, 70 to 835; and NS, 1 to 815. The NP, M, and NS trees are rooted to equine/Prague/1/56 (H7N7). Viruses characterized in this study are highlighted in blue. Viruses in red represent prototype viruses. Genotypes for viruses characterized in this study are indicated in parentheses along with their clade designations. “Mix” indicates cocirculating H5N1, H6N1/N2, and H9N2 subtype viruses. Abbreviations are listed in the legends to Fig. 3 and 4. Bar, 0.01 substitution per site.

FIG. 6.

Genotypes of H6N1/N2 influenza viruses of chickens and other minor poultry species in southern China. The figure shows progenitors of H6 influenza viruses and internal gene donors (A) and H6N1 genotypes A (B) and B (C) in southern China. Dashed lines represent transient and short-lived genotypes. Details of transient genotypes are given in Table 3. The eight gene segments (horizontal bars starting at the top downward) are PB2, PB1, PA, HA, NP, NA, M, and NS. Each color represents a virus lineage. Genotype definitions are described in Results. Abbreviations are listed in the legend to Fig. 3.