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Journal of Virology, July 2007, p. 7529-7539, Vol. 81, No. 14
0022-538X/07/$08.00+0     doi:10.1128/JVI.00327-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Characterization of Low-Pathogenic H5 Subtype Influenza Viruses from Eurasia: Implications for the Origin of Highly Pathogenic H5N1 Viruses

L. Duan,^1,2 L. Campitelli,³ X. H. Fan,¹ Y. H. C. Leung,¹ D. Vijaykrishna,^1,2 J. X. Zhang,^1,2 I. Donatelli,³ M. Delogu,⁴ K. S. Li,² E. Foni,⁵ C. Chiapponi,⁵ W. L. Wu,^1,2 H. Kai,^1,2 R. G. Webster,^1,6 K. F. Shortridge,^1,2 J. S. M. Peiris,^1,2 Gavin J. D. Smith,^1,2 H. Chen,^1,2* and Y. Guan^1,2*

State Key Laboratory of Emerging Infectious Diseases, Department of Microbiology, The University of Hong Kong, Faculty of Medicine Building, 21 Sassoon Road, Pokfulam, Hong Kong, SAR, China,¹ International Institute of Infection and Immunity, Shantou University, Shantou, Guangdong 515031, China,² Department of Infectious, Parasitic and Immune-Mediated Diseases, Istituto Superiore di Sanità, Viale Regina Elena 299, Rome, Italy,³ Department of Public Health and Animal Pathology, Faculty of Veterinary Medicine, University of Bologna, Ozzano Emilia (BO), Italy,⁴ Istituto Zooprofilattico Sperimentale of Lombardia and Emilia, Parma, Italy,⁵ Virology Division, Department of Infectious Diseases, St. Jude Children's Research Hospital, Memphis, Tennessee 38015⁶

Received 14 February 2007/ Accepted 3 May 2007

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Highly pathogenic avian influenza (HPAI) H5N1 viruses are now endemic in many Asian countries, resulting in repeated outbreaks in poultry and increased cases of human infection. The immediate precursor of these HPAI viruses is believed to be A/goose/Guangdong/1/96 (Gs/GD)-like H5N1 HPAI viruses first detected in Guangdong, China, in 1996. From 2000 onwards, many novel reassortant H5N1 influenza viruses or genotypes have emerged in southern China. However, precursors of the Gs/GD-like viruses and their subsequent reassortants have not been fully determined. Here we characterize low-pathogenic avian influenza (LPAI) H5 subtype viruses isolated from poultry and migratory birds in southern China and Europe from the 1970s to the 2000s. Phylogenetic analyses revealed that Gs/GD-like virus was likely derived from an LPAI H5 virus in migratory birds. However, its variants arose from multiple reassortments between Gs/GD-like virus and viruses from migratory birds or with those Eurasian viruses isolated in the 1970s. It is of note that unlike HPAI H5N1 viruses, those recent LPAI H5 viruses have not become established in aquatic or terrestrial poultry. Phylogenetic analyses revealed the dynamic nature of the influenza virus gene pool in Eurasia with repeated transmissions between the eastern and western extremities of the continent. The data also show reassortment between influenza viruses from domestic and migratory birds in this region that has contributed to the expanded diversity of the influenza virus gene pool among poultry in Eurasia.

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Aquatic birds are considered the natural reservoirs of influenza A virus, as all known subtypes (H1 to H16 and N1 to N9) have been isolated from aquatic birds (1, 16, 21, 25, 38, 45, 48). The results of genetic analyses have also suggested that all influenza viruses found in other hosts were derived from those viruses resident in aquatic birds (45). Influenza viruses from aquatic birds have occasionally been transmitted to terrestrial poultry, leading to disease outbreaks; however, only some H5 and H7 subtypes of influenza viruses have evolved into highly pathogenic strains that have caused significant mortality in poultry (2, 4, 13, 14, 23).

The H5N1 "bird flu" incident in Hong Kong, China, in 1997 demonstrated that a "pure" avian influenza virus could be directly introduced into humans and cause infection (9, 10, 41), suggesting a possible pathway for the generation of a new pandemic influenza (31). Genetic analyses suggested that the H5N1 virus causing human infection in Hong Kong, represented by A/Hong Kong/156/97 (H5N1/97), might have been generated by multiple reassortment between A/goose/Guangdong/1/96 (Gs/GD)-like and H9N2 or H6N1 viruses found in terrestrial poultry (19, 22, 50). Following the 1997 outbreak, highly pathogenic avian influenza (HPAI) H5N1 variants with Gs/GD-like hemagglutinin (HA) and neuraminidase (NA) genes became endemic in domestic poultry in southern China (6, 17, 18, 31, 42, 46). Those variants arose through a series of reassortment events to incorporate different gene segments with different origins (17, 18, 31). The endemicity of HPAI H5N1 viruses in poultry in China and its subsequent spread throughout Eurasia and to Africa continue to result in outbreaks in poultry and occasional human infections that has escalated into a new pandemic threat (5, 31, 42). However, the fundamental question of how the Gs/GD-like virus and its variants emerged in southern China has not been completely determined.

Previous studies have demonstrated that, in the case of H5 and H7 subtypes, low-pathogenic avian influenza (LPAI) virus precursors may evolve into HPAI viruses once transmitted to domestic poultry (2, 23). Since 1997, several LPAI H5 and H7 subtype outbreaks have been observed in Europe (2, 4, 13, 26, 35). Influenza surveillance in migratory birds and domestic poultry in Italy revealed that the precursor virus responsible for LPAI outbreaks in domestic poultry originated from migratory birds (4, 11-13). Moreover, a study in northern Europe also suggested that LPAI subtypes of H5 and H7 viruses resident in migratory duck were introduced to poultry and caused HPAI H5N2 and H7N7 outbreaks (35). Since 2003, outbreaks of LPAI H5N2 virus have also occurred in chickens in Japan and Taiwan. However, phylogenetic analyses revealed that the virus from Taiwan was most closely related to an LPAI H5N2 virus that caused multiple outbreaks in Mexico in the mid-1990s and not to Eurasian lineage viruses (8). This virus does not match the natural evolutionary pathway of influenza A virus, thereby implying another mechanism of virus emergence.

Systematic influenza surveillance of domestic poultry in Hong Kong and southern China was conducted from 1976 to 1980 and 2000 onwards, while surveillance of migratory bird populations in southern China has been conducted since 2002 (6, 15, 31, 39-42). These surveillance studies accumulated comprehensive data on the influenza virus gene pool in this region and provide a unique opportunity to explore the possible evolutionary pathways of HPAI Gs/GD-like virus and its variants, i.e., different genotypes. In the present study, LPAI H5 subtype viruses isolated from surveillance of both domestic poultry and migratory birds in southern China, together with some H5 subtype viruses from Italy, were genetically and antigenically characterized. Our findings revealed the dynamic nature of the influenza virus gene pool in Eurasia with repeated transmissions between the eastern and western extremities of the continent. Furthermore, our findings suggest that current H5N2 and H5N3 influenza viruses in domestic birds of southern China were directly derived from those viruses resident in migratory birds, particularly from migratory ducks. Epidemiological and genetic findings also suggest that those LPAI H5 subtype viruses detected in our surveillance have not become established in poultry in southern China. Accumulated evidence suggests that Gs/GD-like virus might be derived from viruses resident in migratory birds along the western Pacific flyway. Some Gs/GD-like variants from poultry appeared to have resulted from further reassortment from viruses in migratory birds. However, there are some H5N1 variants that did not match natural evolutionary pathways, suggesting other origins for those viruses. The current study highlights the necessity of continued systematic influenza surveillance in both domestic and migratory birds in Eurasia to track the emergence and reemergence of novel influenza viruses with pandemic potential.

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Surveillance and virus studied. Influenza surveillance was conducted from 1976 to 1980 in live-poultry markets in Hong Kong, China, and from 2000 onwards in six different provinces in southern China, as previously described (6, 17, 31, 39-42). Migratory birds were sampled at aggregation sites of Poyang Lake, Jiangxi Province, China, and Mai Po Marshes, Hong Kong, China, as previously described (6). Further sampling of sentry domestic ducks from farms at Poyang Lake adjacent to these migratory bird aggregation sites was conducted year round from October 2002 onwards. In addition, influenza surveillance in wild waterfowl, both migratory and resident, was conducted from 1992 to 2002 at Orbetello wetland in central Italy (4, 11-13). Isolation of H5 influenza viruses from domestic poultry in Italy in 1997 to 1998 has been previously reported (14). Influenza isolates were subtyped by the standard hemagglutination inhibition (HI) test using a panel of the World Health Organization reference antisera (32, 49).

Three different representative groups of LPAI H5 subtype viruses were characterized and analyzed (Tables 1 and 2). These included 12 viruses isolated from domestic poultry in Hong Kong between 1975 and 1980, 19 viruses isolated in southern China since 2000, and eight viruses isolated from poultry and mallards in Italy. To provide further information on the influenza virus gene pool in both migratory birds and domestic ducks of this region, 11 viruses from seven different subtypes isolated from our surveillance were also characterized and analyzed (Table 2).

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TABLE 1. H5N3/H5N2 viruses sampled in southern China from 1976 to 1980 and 2000 to 2005

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TABLE 2. Additional viruses characterized in this study

Antigenic analysis. Viruses characterized in this study were passaged in 9- to 11-day-old chicken embryos as previously described (32). The antigenic characterization of H5 influenza viruses were conducted by HI assay performed with panels of monoclonal antibodies (MAbs) against different hemagglutinin (HA) proteins. MAbs against Ck/PA/1370/83, Ck/PA/8125/83, and VNM/1203/04 were produced by the Department of Infectious Diseases at St. Jude Children's Research Hospital, while MAbs against Ck/HK/YU22/02 were produced in our laboratory. The HI assay was started at a 1:100 dilution.

To visualize similarity between the antigenic reaction patterns of different viruses, numerical analysis of HI titers was conducted using PRIMER version 5.2.9 (PRIMER-E, Plymouth, United Kingdom). Titers of <100 and >12,800 in the HI assay were converted to 0 and 25,600 in this analysis, respectively. The data were square root transformed, and the Bray-Curtis coefficient (3) was used to construct a similarity matrix. Hierarchical agglomerative clustering with group-average linking (43) was conducted, and a dendrogram was produced. Nonmetric multidimensional scaling (28) was also used to produce two- and three-dimensional ordinations over 100 iterations. The two-dimensional configuration with lowest overall stress was presented.

Phylogenetic and molecular analyses. To better understand the origins of the Gs/GD lineage of H5 HPAI, 18 LPAI H5N2/N3/N8 viruses obtained from 2002 to 2005 and 12 LPAI H5N2/N3 viruses isolated during 1976 to 1980 from southern China, together with eight H5N2/N3 viruses from Italian poultry or mallards, were sequenced for each of the eight gene segments (Tables 1 and 2). To provide the background genetic information from a further 10 different subtypes, viruses isolated from migratory ducks in the last several years were also sequenced and phylogenetically analyzed with all available sequence data in public databases (Tables 1 and 2).

RNA extraction, cDNA synthesis and PCR were carried out as previously described (6). Sequence assembly, editing, alignment, and residue analysis were performed as previously described (6, 20). Phylogenetic analysis using MRMODELTEST 2.2 (36), PAUP* 4.0 (44), and MRBAYES 3.1 (24) also was carried out as previously described (6).

Nucleotide sequence accession numbers. The nucleotide sequences obtained in this study are available from GenBank under accession numbers EF597247 to EF597498.

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Prevalence of low-pathogenic H5 influenza viruses. Between July 2000 and December 2005, a total of 167,858 samples from domestic ducks, geese, and migratory ducks were collected in southern China and Hong Kong, China. Seventy-two LPAI H5 viruses were isolated on 10 sampling occasions during winter (isolation rate of 0.043%) (Table 1). The main body of those isolates were from sentry farm ducks or directly from migratory birds. Only two isolates, Dk/YN/435/02 and Gs/GY/3799/05 were detected from retail market birds. In the surveillance in the 1970s, among 11,798 market samples, 23 LPAI H5 viruses were isolated on 10 sampling occasions (isolation rate of 0.2%) (39, 40). Most of the viruses were from domestic ducks, with only one isolated from a goose (Table 1). It must be noted that no LPAI H5 viruses were recognized in terrestrial poultry in our surveillance. Thus, even though HPAI H5N1 viruses were persistently detected in different types of poultry, from 2000 onwards, LPAI H5 viruses were isolated predominantly from migratory ducks or sentry duck and barely detected in market waterfowl in southern China.

Antigenic analysis. Antigenic analysis with MAbs against representative H5 influenza viruses demonstrated a diversity of reaction patterns (Table 3). LPAI H5 viruses isolated from both domestic and wild waterfowl in southern China since 2003 onwards were antigenically conserved and did not differ significantly (greater than fourfold) from each other in the HI assay. Those viruses obtained from both poultry and mallards in Italy also had very similar reactivity patterns. Only Ck/Italy/9097/97 had a significantly lower titer to MAb 15A3 against VNM/1203/04. It is interesting to note that most LPAI H5 viruses isolated from Italy and southern China from the 1970s to the mid-1990s also showed similar reaction patterns (Table 3). In contrast, HPAI H5N1 representative strains showed high antigenic diversity with different reaction patterns, as has been previously observed (6, 42). It was noted that the reaction pattern of Ck/PA/1370/83 was different from those of all Eurasian viruses tested (Table 3).

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TABLE 3. Antigenic analysis of H5 subtype influenza viruses by hemagglutinin inhibition test

In the numerical analysis of HI titers, all LPAI H5 viruses from southern China and Italy isolated from the 1970s to the mid-1990s clustered together, with the exception of Dk/JX/7820/03 that had lower titers against most MAbs tested (Fig. 1). This highly conserved antigenicity likely reflects the evolutionary stasis of those viruses in their natural reservoir. It was noted that an early Gs/GD-like virus, Gs/HK/437.4/99, and three HPAI H5N2 viruses (Ck/Italy/312/97, Ty/Italy/392/97, and Ck/Italy/8/98) isolated from outbreaks in terrestrial poultry in Italy, also grouped with those LPAI H5 viruses from Eurasia. This is likely due to those viruses having been introduced to poultry from this gene pool in the recent past. This analysis also demonstrated the high antigenic diversity of HPAI H5N1 variants currently prevailing in different regions (Fig. 1).

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FIG. 1. Numerical analysis of HI titers (see Table 2) by nonmetric multidimensional ordination in two dimensions (A) and using hierarchical agglomerative clustering (B). Colors indicate viruses isolated from Italy (blue), southern China (green), and A/goose/Guangdong/1/96-like (red). Abbreviations: IDN, Indonesia; Mall, mallard; MDk, migratory duck; BHG, bar-hooded goose; QH, Qinghai.

Phylogenetic analysis of the surface genes. Phylogenetic analysis of the H5 HA gene showed that all viruses separated into the American and Eurasian lineages (Fig. 2A). Within the Eurasian lineage, two major sublineages of "early" and "contemporary" viruses are apparent. The first sublineage consists predominantly of older viruses from across the continent, including those viruses isolated in Hong Kong from 1976 to 1980, along with viruses from Germany, Japan, and the United Kingdom. There is only one LPAI H5 virus from Italy (Ck/Italy/9097/97) that falls into this group which was introduced recently to chicken from this gene pool. Therefore, this sublineage of H5 subtype viruses probably represents the early domestic Eurasian gene pool of H5 viruses (Fig. 2A).

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FIG. 2. Phylogenetic relationships of the HA (A) and M (B) genes of representative influenza A viruses isolated in Asia, America, and Europe. 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 1032 of the HA gene and 26 to 947 of the M gene. The HA and M gene trees were rooted to A/Japan/305/57 and A/equine/Prague/1/56, respectively. Colors indicate viruses isolated from Italy (blue), southern China (green), and A/goose/Guangdong/1/96 (red). Red branches indicate A/goose/Guangdong/1/96-like highly pathogenic influenza virus and its variants. Bar, 0.1 nucleotide substitution per site. Abbreviations: Am, America; Mall, mallard; MDk, migratory duck; Gf, guinea fowl; SCk, silky chicken; BH goose, bar-hooded goose; QH, Qinghai; Qa, quail; WDk, wild duck.

The second major Eurasian sublineage is composed of three distinct groups (Fig. 2A). Group 1 contains all but one of the recent viruses from China and Italy characterized in the present study, along with other Eurasian isolates, from both domestic and migratory birds. However, four Italian viruses isolated during the outbreaks from 1997 to 1998 and one obtained from a mallard clustered together within this group, suggesting that after introduction into terrestrial poultry, those viruses had undergone significant evolution (Fig. 2A). The phylogeny of this group, together with that of the "early" sublineage, indicates the continued genetic exchange within this influenza virus gene pool between the eastern and western extremities of the Eurasian continent.

Group 2 consists exclusively of viruses from Western Europe, with the exception of the virus Dk/Chany Lake/9/03 that was isolated in Novosibirsk, Russia in Central Asia (Fig. 2A). In contrast, group 3 contained viruses from the western Pacific flyway isolated from domestic ducks and swans, along with HPAI Gs/GD-like H5N1 viruses that have subsequently spread throughout Eurasia (6, 31, 42). This group therefore seems to match one of the major migratory flyways in this region (47).

In the American lineage, viruses from two major outbreaks and those from migratory birds clustered together (Fig. 2A). It has been noted that a virus from Japan, Dk/Hokkaido/84/02 also joins this lineage, providing an example of gene exchange between the American and Eurasian influenza virus gene pools. Interestingly, a virus that recently caused outbreaks in poultry in Japan in 2005 (Ck/Ibaraki/1/2005) also fell in the American lineage and was most closely related to Ck/Hidalgo/28159-232/94 and Ck/Taiwan/1209/03 that caused outbreaks in Mexico and Taiwan, respectively (Fig. 2A) (8).

Phylogenetic analysis showed that three N2 genes from recent southern China H5N2 isolates (Gs/GY/3799/05, Dk/JX/1286/05, and Dk/JX/3345/05) were closely related to the N2 genes of viruses obtained from mallards in Italy and ducks in Hokkaido, Japan (data not shown). Analysis also showed that the N3 genes from the recent H5N3 viruses isolated in southern China are closely related to H7N3 subtype viruses from domestic and migratory birds in Italy from 2001 to 2003 (data not shown). Therefore, both the HA and NA genes of those H5N2 and H5N3 viruses recently isolated from domestic ducks or migratory birds in southern China were from the Eurasian lineage and appear to be derived from viruses resident in migratory birds. Phylogenetic analysis of N1 NA also revealed that the NA gene of Gs/GD-like virus was most closely related to Dk/Hokkaido/55/95 (H1N1) (data not shown). Another H5N1 variant (Ck/Hebei/718/01) had an N1 that was almost identical to African starling/England-Q/983/79 (H7N1) (unpublished data), which was investigated as a vaccine candidate in China (7, 33, 37).

Phylogenetic analysis of the internal genes. In general, all internal gene trees were divided into the American and Eurasian lineages, and the "early" and "contemporary" sublineages were observed within the Eurasian lineage (Fig. 2B and 3 and data not shown). The internal gene phylogenies further highlight the dynamic nature of the influenza virus gene pool in Eurasia with viruses from the east and west frequently clustering together. For the LPAI H5N2 virus Ck/Ibaraki/1/05, all available gene segments clustered with the Mexico-like HPAI from 1994. It is noteworthy that several Gs/GD-like variants isolated from northern China from 1997 to 2004 consistently clustered within the "early" Eurasian sublineage (Fig. 2B and 3 and data not shown). Most of the LPAI H5 viruses from southern China clustered or grouped together with some viruses from European viruses, including those Italian HPAI and LPAI viruses (Fig. 2B and 3 and data not shown).

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FIG. 3. Phylogenetic relationships of the NP (A), PA (B), and PB2 (C) genes of representative influenza A viruses isolated in Asia, America (Am), and Europe. Numbers above and below the branch nodes indicate neighbor-joining bootstrap values of 50% and Bayesian posterior probabilities of >95, respectively. Analyses of NP, PA, and PB2 genes were based on nucleotides 41 to 984, 1425 to 2135, and 1065 to 2085, respectively. The gene trees were rooted to A/equine/Prague/1/56 (NP, PB2) and B/Lee/40 (PA), respectively. Colors indicate viruses isolated from Italy (blue), southern China (green), and A/goose/Guangdong/1/96 (red). Red branches indicate A/goose/Guangdong/1/96-like highly pathogenic influenza virus and its variants. Bar, 0.01 nucleotide substitution per site. Abbreviations: Am, America; Qa, quail; Mall, mallard; Mdk, migratory duck; Gf, guinea fowl; Sck, silky chicken; BH goose, bar-hooded goose; QH, Qinghai; Ph, pheasant.

In the M-gene tree, the Eurasian lineage separated into four different sublineages, except for those H9N2 influenza viruses from terrestrial poultry in China that formed an outgroup sublineage to the rest (Fig. 2B). All recent H5 viruses from both southern China and Italy, along with those viruses recently isolated from migratory birds, fell into two distinguishable groups (groups 1 and 2) that appeared to represent a natural evolutionary pathway. It is noteworthy that the Gs/GD-like viruses and its variants, which formed an independent sublineage, were most closely related to Dk/Hokkaido/120/01 (H6N2), a virus that consistently has a close phylogenetic relationship to those viruses from migratory ducks, suggesting it may have originated in migratory birds (Fig. 2B and 3 and data not shown). Furthermore, while almost all 1970s H5 viruses from Hong Kong clustered together with the "early" sublineage, there was a single virus (Dk/HK/23/76) that fell at the base of those group 2 viruses (Fig. 2B).

In the NP gene tree, all recent LPAI H5 viruses from sentry ducks and retail markets in southern China clustered together with viruses isolated directly from migratory ducks at Mai Po Marshes in Hong Kong or Poyang Lake in China (Fig. 3B). All 1970s H5 viruses joined the "early" sublineage, while the Italian viruses were most closely related to other viruses from Europe. Interestingly, Dk/Hokkaido/120/01 solely clustered with three viruses all isolated from migratory or sentry ducks at Poyang Lake (Fig. 3B). These relationships were also observed in the PB2 and PA trees, suggesting that this virus might also have originated in migratory ducks (Fig. 3). The PA gene tree had a phylogenetic topology similar to that of the NP gene tree (Fig. 3B). It is noteworthy that those Gs/GD-like variants clustered together with other viruses from migratory birds and formed different clades within the contemporary Eurasian sublineage.

In the PB2 phylogeny, the Eurasian lineage formed three groups. The first group consisted of some early LPAI H5 viruses from domestic ducks in southern China and Japan, along with recent H5N1 viruses from northern China (Fig. 3C). The other two groups consist of both early lineage viruses and recent isolates, in contrast to the other gene trees where the early viruses almost exclusively formed a single group. Interestingly, the PB2 gene of Gs/GD/1/96 is most closely related to that of an H11N3 virus (WDk/ST/1411/00) that was isolated from mallard at a market in Shantou, China. Whether this bird was farmed or truly migratory is not known. Also these two viruses were then most closely related to Ck/Italy/9097/97, a virus that appeared to have been recently introduced to poultry from migratory birds. Moreover, many Gs/GD-like variants, including Qinghai-like virus, clustered with an LPAI H5 virus (Dk/YN/435/02) that incorporates several gene segments of migratory bird origin. These findings provide further evidence that Gs/GD-like virus and some of its variants may have been introduced to poultry from migratory birds.

To identify the possible source of Gs/GD-like HPAI H5N1 viruses and its variants, we summarized their most phylogenetically closely related viruses for each of the gene segments (Table 4). For Gs/GD-like viruses, three of the gene segments were likely derived from migratory birds, and four of the genes are most closely related to those viruses isolated from ducks in Hokkaido, Japan. Furthermore, some of the variants isolated from our surveillance during 2001 to 2005 in southern China contain gene segments that are most closely related to gene segments from viruses in the migratory bird gene pool (Table 4). It is noteworthy that these analyses confirm that several H5N1 variants isolated from northern China from 1997 to 2004 contained many gene segments belonging to the early Eurasian gene pool identified from viruses isolated in the 1970s and 1980s (27). Moreover, it is surprising that one H5N1 virus (Ck/Hebei/718/01) contained an NA N1 gene with very high homology (98.3%) to that of African starling/England-Q/983/79 (H7N1).

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TABLE 4. Possible sources of gene segments of highly pathogenic H5N1 influenza viruses isolated in China, 1996 to 2005

Molecular characterization. Seventeen of 21 H5 subtype viruses characterized in this study, including those isolated in Hong Kong from 1976 to 1980, had a Q-R-E-T-R motif in the connecting peptide that is typical for LPAI H5 viruses (Table 5). Three H5N2 viruses, Ck/Italy/312/97, Ty/Italy/392/97, and Ck/Italy/8/98, isolated from 1997/98 outbreaks in northern Italy, had multiple basic amino acids in the connecting peptide (Q-R-R-R-K-K-R) that is typical of HPAI H5 subtype influenza viruses. It was also noted that Ck/Italy/9097/97 had a unique Q-K-E-T-R motif at its connecting peptide that has not been recognized before in other H5 subtype influenza viruses.

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TABLE 5. Connecting peptide of H5 subtype viruses characterized in this study

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The findings of the present study revealed that LPAI H5 viruses were isolated predominantly from migratory or sentry ducks during the winter, barely detected in market waterfowl, and not found in terrestrial poultry in southern China from 2000 to 2005. This suggests that interspecies transmission of LPAI H5 virus from migratory birds to domestic waterfowl did occur but that those viruses did not subsequently become prevalent in aquatic or terrestrial domestic birds. In contrast, influenza surveillance in the 1970s revealed that LPAI H5 viruses were found year-round in domestic waterfowl, indicating that there has been a change in the ecology of influenza in southern China. This likely resulted from the long-term endemicity of HPAI H5N1 viruses and extensive vaccination in poultry, leading to increased host selection pressure.

Phylogenetic and antigenic analyses demonstrated that LPAI H5 viruses obtained from southern China in recent years were closely related to those H5 viruses isolated from Italy in the mid-1990s (2, 4, 11-14), while different subtypes of influenza viruses isolated from migratory birds in southern China clustered together with either viruses from Hokkaido (Japan), Mongolia, and Siberia, or with viruses from Europe. These findings provide further evidence of the dynamic influenza virus gene pool in this region. Along the western Pacific migratory flyway, the influenza virus gene pool in the domestic waterfowl of southern China has "mixed" longitudinally with viruses isolated from Japan, Mongolia, and Siberia. However, it appears that there has also been "mixing" latitudinally through overlapping migratory flyways, thereby facilitating interaction between the influenza virus gene pool in domestic waterfowl in the eastern and western extremities of the Eurasian continent. This helps to explain the latitudinal spread of the Qinghai-like (clade 2.2) H5N1 virus in the last 2 years, while H5N1 outbreaks in Korea and Japan may represent the longitudinally transmitting pathway (5).

In the present study, the phylogenies of Gs/GD-like H5N1 virus genes either clustered with viruses from migratory birds or with viruses isolated from ducks in Hokkaido, Japan (Table 4). As no systematic surveillance was conducted from 1980 to 1997 in southern China, we failed to identify the direct precursor of the Gs/GD-like virus which emerged in 1996 (50). Even though it is not clear whether those Hokkaido viruses were from domestic or migratory ducks, many of them clustered directly with the viruses isolated from migratory ducks at Poyang Lake, Jiangxi Province, China, and Mai Po Marshes, Hong Kong, since 2002 (Fig. 2 and 3 and data not shown). We therefore speculate that the duck viruses from Hokkaido may also be of migratory bird derivation. Thus, the available findings suggest that Gs/GD-like H5N1 virus may be derived from an LPAI H5N1 virus in migratory waterfowl along the western Pacific migratory flyway. A precedent for such an introduction and subsequent change from LPAI to HPAI has also been observed in Europe (2, 4, 35).

Phylogenetic analysis also showed that those H5N1 variants isolated in southern China from 2001 to 2005 were mainly reassortants between Gs/GD-like virus and virus from migratory birds. Even though there are still some gene segments for which we could not identify the source, current results suggest that these reassortment events very likely occurred in the field. However, those H5N1 variants from northern China in 1997 and afterwards contained almost all internal genes that were obviously from early gene pool viruses (Table 5). The remaining question is to how those old gene segments had been incorporated into Gs/GD-like virus to generate those variants.

Two possibilities could explain this situation, either these data were a result of laboratory contamination or the viruses were isolated in the field through the use of incompletely inactivated poultry vaccines that utilized older strains of LPAI H5 virus. For instance, one virus (Ck/Hebei/718/01) contained an N1 gene that was almost identical to that from African starling/England-Q/983/79 (H7N1). This H7N1 virus, along with Ty/Wisconsin/1/66 (H9N2) and Ty/England/N28/73 (H5N2), were investigated as vaccine candidates in China (33, 37). An H9N2 virus (Ck/Heilongjiang/35/00), isolated in a field in northern China, had almost identical HA and NP genes with Ty/Wisconsin/1/66 (30, 34). Furthermore, genetic analyses of viruses recently isolated from LPAI H5N2 outbreaks in Taiwan (8) and Japan confirmed that those H5N2 viruses were derived from a Mexico-like LPAI H5N2 virus, suggesting that those viruses may have been directly derived from incompletely inactivated vaccine (29). Therefore, it seems likely that those H5N1 variants containing early gene segments were not generated under natural conditions.

Currently, HPAI H5N1 viruses are endemic in poultry in many Eurasian countries (6, 31, 42), and as this article was being prepared, a new outbreak wave was occurring across eastern and southeastern Asia. Genetic analysis of the recent outbreak suggested that the outbreaks in Korea and Japan were caused by BH goose/QH/65/05-like virus. If this is the case, it is not impossible that those Gs/GD-like variants may have successfully become established in the natural reservoirs of influenza viruses, i.e., migratory waterfowls. If so, control of HPAI H5N1 in the short term has become an impossible mission.

This study demonstrates the dynamic nature of the influenza virus gene pool in Eurasia with continuing gene exchanges between the eastern and western parts of the continent. Our findings suggest that the HPAI Gs/GD-like virus were likely derived directly from viruses resident in migratory birds. The failure to identify the source for all gene segments of Gs/GD-like viruses and its variant highlights the need for continued and extensive surveillance in both migratory and domestic populations in larger regions. Such surveillance is crucial for effective pandemic influenza preparedness.

 
This study was supported by the Research Fund for Control of Infectious Diseases and Research Grants Council (HKU 7512/06 M and HKU1/05C) of the Hong Kong SAR Government, the Li Ka-Shing Foundation, and the National Institutes of Health (NIAID contract HHSN266200700005C).

We gratefully acknowledge the excellent technical assistance of Li-Juan Zhang and Justin Bahl and the continual and invaluable collaboration of Marco Carsughi, Antonio Canu, and Luigi Calchetti (WWF Italian Delegation).

 
* Corresponding author. Mailing address: State Key Laboratory of Emerging Infectious Disease, Department of Microbiology, The University of Hong Kong, Li Ka Shing Faculty of Medicine Building, 21 Sassoon Road, Pokfulam, Hong Kong, SAR, China. Phone: (852) 28199830. Fax: (852) 28171958. E-mail for H. Chen: hlchen{at}hkucc.hku.hk. E-mail for Y. Guan: yguan{at}hkucc.hku.hk

Published ahead of print on 16 May 2007.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

1
1. Alexander, D. J. 2000. A review of avian influenza in different bird species. Vet. Microbiol. 74:3-13.[CrossRef][Medline]
2
2. Banks, J., E. S. Speidel, E. Moore, L. Plowright, A. Piccirillo, I. Capua, P. Cordioli, A. Fioretti, and D. J. Alexander. 2001. Changes in the haemagglutinin and the neuraminidase genes prior to the emergence of highly pathogenic H7N1 avian influenza viruses in Italy. Arch. Virol. 146:963-973.[CrossRef][Medline]
3
3. Bray, R. J., and J. T. Curtis. 1957. An ordination of the upland forest communities of southern Wisconsin. Ecol. Monogr. 27:325-349.[CrossRef]
4
4. Campitelli, L., E. Mogavero, M. A. De Marco, M. Delogu, S. Puzelli, F. Frezza, M. Facchini, C. Chiapponi, E. Foni, P. Cordioli, R. Webby, G. Barigazzi, R. G. Webster, and I. Donatelli. 2004. Interspecies transmission of an H7N3 influenza virus from wild birds to intensively reared domestic poultry in Italy. Virology 323:24-36.[CrossRef][Medline]
5
5. Chen, H., G. J. D. Smith, S. Y. Zhang, K. Qin, J. Wang, K. S. Li, R. G. Webster, J. S. M. Peiris, and Y. Guan. 2005. H5N1 virus outbreak in migratory waterfowl. Nature 436:191-192.[CrossRef][Medline]
6
6. Chen, H., G. J. D. Smith, K. S. Li, J. Wang, X. H. Fan, J. M. Rayner, D. Vijaykrishna, J. X. Zhang, L. J. Zhang, C. T. Guo, C. L. Cheung, K. M. Xu, L. Duan, K. Huang, K. Qin, Y. H. Leung, W. L. Wu, H. R. Lu, Y. Chen, N. S. Xia, T. S. Naipospos, K. Y. Yuen, S. S. Hassan, S. Bahri, T. D. Nguyen, R. G. Webster, J. S. Peiris, and Y. Guan. 2006. Establishment of multiple sublineages of H5N1 influenza virus in Asia: implications for pandemic control. Proc. Natl. Acad. Sci. USA 103:2845-2850.[Abstract/Free Full Text]
7
7. Chen, J. M., H. C. Ma, J. W. Chen, Y. X. Sun, J. M. Li, and Z. L. Wang. 2007. A preliminary panorama of the diversity of N1 subtype influenza viruses. Virus Genes 35:33-40.[CrossRef][Medline]
8
8. Cheng, M. C., M. S. Lee, and C. H. Wang. 2005. Molecular characterization of H5N2 low pathogenic avian influenza viruses from Taiwan poultry in 2004, p. 30. In Proceedings of the APEC Conference on Avian Influenza. National Chung Hsing University, Taichung, Taiwan.
9
9. Claas, E. C., A. D. Osterhaus, R. Vanbeek, J. C. de Jong, G. F. Rimmelzwaan, D. A. Senne, S. Krauss, K. F. Shortridge, and R. G. Webster. 1998. Human influenza A H5N1 virus related to a highly pathogenic avian influenza virus. Lancet 351:472-477.[CrossRef][Medline]
10
10. de Jong, J. C., E. C. J. Claas, A. D. M. E. Osterhaus, R. G. Webster, and W. L. Lim. 1997. A pandemic warning? Nature 389:554.[Medline]
11
11. de Marco, M. A., L. Campitelli, E. Foni, E. Raffini, G. Barigazzi, M. Delogu, V. Guberti, L. di Trani, M. Tollis, and I. Donatelli. 2004. Influenza surveillance in birds in Italian wetlands (1992-1998): is there a host restricted circulation of influenza viruses in sympatric ducks and coots? Vet. Microbiol. 98:197-208.[CrossRef][Medline]
12
12. de Marco, M. A., G. E. Foni, L. Campitelli, E. Raffini, L. di Trani, M. Delogu, V. Guberti, G. Barigazzi, and I. Donatelli. 2003. Circulation of influenza viruses in wild waterfowl wintering in Italy during the 1993-99 period: evidence of virus shedding and seroconversion in wild ducks. Avian Dis. 47(Suppl. 3):861-866.[Medline]
13
13. de Marco, M. A., E. Foni, L. Campitelli, M. Delogu, E. Raffini, C. Chiapponi, G. Barigazzi, P. Cordioli, L. di Trani, and I. Donatelli. 2005. Influenza virus circulation in wild aquatic birds in Italy during H5N2 and H7N1 poultry epidemic periods (1998 to 2000) Avian Pathol. 34:480-485.[CrossRef][Medline]
14
14. Donatelli, I., L. Campitelli, L. di Trani, S. Puzelli, L. Selli, A. Fioretti, D. J. Alexander, M. Tollis, S. Krauss, and R. G. Webster. 2001. Characterization of H5N2 influenza viruses from Italian poultry. J. Gen. Virol. 82:623-630.[Abstract/Free Full Text]
15
15. Ellis, T. M., R. B. Bousfield, L. A. Bissett, K. C. Dyrting, G. S. M. Luk, S. T. Tsim, K. Strum-Ramirez, R. G. Webster, Y. Guan, and J. S. M. Peiris. 2004. Investigation of outbreaks of highly pathogenic H5N1 avian influenza in waterfowl and wild birds in Hong Kong in late 2002. Avian Pathol. 33:492-505.[CrossRef][Medline]
16
16. Fouchier, R. A. M., V. Munster, A. Wallensten, T. M. Bestebroer, S. Herfst, D. Smith, G. F. Rimmelzwaan, B. Olsen, and A. D. M. E. Osterhaus. 2005. Characterization of a novel influenza A virus hemagglutinin subtype (H16) obtained from black-headed gulls. J. Virol. 79:2814-2822.[Abstract/Free Full Text]
17
17. Guan, Y., M. Peiris, K. F. Kong, K. C. Dyrting, T. M. Ellis, T. Sit, L. J. Zhang, and K. F. Shortridge. 2002. H5N1 influenza viruses isolated from geese in southeastern China: evidence for genetic reassortment and interspecies transmission to ducks. Virology 292:16-23.[CrossRef][Medline]
18
18. Guan, Y., J. S. M. Peiris, A. S. Lipatov, T. M. Ellis, K. C. Dyrting, S. Krauss, L. J. Zhang, R. G. Webster, and K. F. Shortridge. 2002. Emergence of multiple genotypes of H5N1 avian influenza viruses in Hong Kong SAR. Proc. Natl. Acad. Sci. USA 99:8950-8955.[Abstract/Free Full Text]
19
19. Guan, Y., K. F. Shortridge, S. Krauss, and R. G. Webster. 1999. Molecular characterization of H9N2 influenza viruses: were they the donors of the "internal" genes of H5N1 viruses in Hong Kong? Proc. Natl. Acad. Sci. USA 16:9363-9367.
20
20. Hall, T. A. 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp. Ser. 41:95-98.
21
21. Hinshaw, V. S., G. M. Air, V. J. Gibbs, L. Graves, B. Prescott, and D. Karunakaran. 1982. Antigenic and genetic characterization of a novel hemagglutinin subtype of influenza A viruses from gulls. J. Virol. 42:865-872.[Abstract/Free Full Text]
22
22. Hoffmann, E., J. Stech, I. Leneva, S. Krauss, C. Scholtissek, P. S. Chin, M. Peiris, K. F. Shortridge, and R. G. Webster. 2000. Characterization of the influenza A virus gene pool in avian species in southern China: was H6N1 a derivative or a precursor of H5N1? J. Virol. 74:6309-6315.[Abstract/Free Full Text]
23
23. Horimoto, T., E. Rivera, J. Pearson, D. Senne, S. Krauss, Y. Kawaoka, and R. G. Webster. 1995. Origin and molecular changes associated with emergence of a highly pathogenic H5N2 influenza virus in Mexico. Virology 213:223-230.[CrossRef][Medline]
24
24. Huelsenbeck, J. P., and F. R. Ronquist. 2001. MRBAYES: Bayesian inference of phylogenetic trees. Bioinformatics 17:754-755.[Abstract/Free Full Text]
25
25. Kawaoka, Y., S. Yamnikova, T. M. Chambers, D. K. Lvoy, and R. G. Webster. 1990. Molecular characterization of a new hemagglutinin, subtype H14, of influenza A virus. Virology 179:759-767.[CrossRef][Medline]
26
26. Koopmans, M., B. Wilbrink, M. Conyn, G. Natro, H. van der Nat, H. Vennema, A. Meijer, J. van Steenbergen, R. Fouchier, A. Osterhaus, and A. Bosman. 2004. Transmission of H7N7 avian influenza A virus to human beings during a large outbreak in commercial poultry farms in the Netherlands. Lancet 363:587-593.[CrossRef][Medline]
27
27. Kou, Z., F. M. Lie, J. Yu, Z. J. Fan, Z. H. Yin, C. X. Jia, K. J. Xiong, Y. H. Sun, X. W. Zhang, X. M. Wu, X. B. Gao, and T. X. Li. 2005. New genotype of avian influenza H5N1 viruses isolated from tree sparrows in China. J. Virol. 79:15460-15466.[Abstract/Free Full Text]
28
28. Kruskal, J. B. 1964. Nonmetric multidimensional scaling: a numerical method. Psychometrika 29:115-129.[CrossRef]
29
29. Lee, C. W., D. A. Senne, and D. L. Suarez. 2004. Effect of vaccine use in the evolution of Mexican lineage H5N2 avian influenza virus. J. Virol. 78:8372-8381.[Abstract/Free Full Text]
30
30. Li, C., K. Yu, G. Tian, D. Yu, L. Liu, B. Jing, J. Ping, and H. Chen. 2005. Evolution of H9N2 influenza viruses from domestic poultry in Mainland China. Virology 340:70-83.[CrossRef][Medline]
31
31. Li, K. S., Y. Guan, J. Wang, G. J. D. Smith, K. M. Xu, L. Duan, A. P. Rahardjo, P. Puthavathana, C. Buranathai, T. D. Nguyen, A. T. S. Estoepangestie, A. Chaisingh, P. Auewarakul, H. T. Long, N. T. H. Hanh, R. J. Webby, L. L. M. Poon, H. Chen, K. F. Shortridge, K. Y. Yuen, R. G. Webster, and J. S. M. Pieris. 2004. Genesis of highly pathogenic and potentially pandemic H5N1 influenza virus in eastern Asia. Nature 430:209-213.[CrossRef][Medline]
32
32. Li, K. S., K. M. Xu, J. S. M. Peiris, L. L. M. Poon, K. Z. Yu, K. Y. Yuen, K. F. Shortridge, R. G. Webster, and Y. Guan. 2003. Characterization of H9 subtype influenza viruses from the ducks of southern China: a candidate for the next influenza pandemic in humans? J. Virol. 77:6988-6994.[Abstract/Free Full Text]
33
33. Liu, L. L., X. R. Wang, H. L. Chen, G. B. Tian, K. Sun, and P. Wei. 2006. The growth characteristics of different subtypes of avian influenza virus in the MDCK cell line. Chin. J. Vet. Med. 42:18-20. (In Chinese.)
34
34. Lu, J. H., X. F. Liu, W. X. Shao, Y. L. Liu, D. P. Wei, and H. Q. Liu. 2005. Phylogenetic analysis of eight genes of H9N2 subtype influenza virus: a mainland China strain possessing early isolates' genes that have been circulating. Virus Genes 31:163-169.[CrossRef][Medline]
34
35. Mastrosovich, M., N. Zhou, Y. Kawaoka, and R. Webster. 1999. The surface glycoproteins of H5 influenza viruses isolated from humans, chickens, and wild aquatic birds have distinguishable properties. J. Virol. 73:1146-1155.[Abstract/Free Full Text]
35
36. Munster, V. J., A. Wallensten, C. Baas, G. F. Rimmelzwaan, M. Schutten, B. Olsen, A. D. M. E. Osterhaus, and R. A. M. Fouchier. 2005. Mallards and highly pathogenic avian influenza ancestral viruses, northern Europe. Emerg. Infect. Dis. 11:1545-1551.[Medline]
36
37. Nylander, J. A. A. 2004. MRMODELTEST 2. Evolutionary Biology Centre, Uppsala University, Uppsala, Sweden.
37
38. Qiao, C., G. Tian, Y. Jiang, Y. Li, J. Shi, K. Yu, and H. Chen. 2006. Vaccines developed for H5 highly pathogenic avian influenza in China. Ann. N. Y. Acad. Sci. 1081:182-192.[CrossRef][Medline]
38
39. Röhm, C., N. N. Zhou, J. Süss, J. Mackenzie, and R. G. Webster. 1996. Characterization of a novel influenza hemagglutinin, H15: criteria for determination of influenza A subtypes. Virology 217:508-516.[CrossRef][Medline]
39
40. Shortridge, K. F. 1982. Avian influenza A viruses of southern China and Hong Kong: ecological aspects and implications for man. Bull. W. H. O. 60:129-135.[Medline]
40
41. Shortridge, K. F. 1992. Pandemic influenza—a zoonosis? Semin. Respir. Infect. 7:11-25.[Medline]
41
42. Shortridge, K. F., N. N. Zhou, Y. Guan, P. Gao, T. Ito, Y. Kawaoka, S. Kodihallli, S. Krauss, D. Markwell, K. Gopal Murti, M. Norwood, D. Senne, L. Sims, A. Takada, and R. G. Webster. 1998. Characterization of avian H5N1 influenza viruses from poultry in Hong Kong. Virology 252:331-342.[CrossRef][Medline]
42
43. Smith, G. J. D., X. H. Fan, J. Wang, K. S. Li, K. Qin, J. X. Zhang, D. Vijaykrishna, C. L. Cheung, K. Huang, J. M. Rayner, J. S. M. Peiris, H. Chen, R. G. Webster, and Y. Guan. 2006. Emergence and predominance of an H5N1 influenza variant in China. Proc. Natl. Acad. Sci. USA 103:16936-16941.[Abstract/Free Full Text]
43
44. Sokal, R. R., and C. D. Michener. 1958. A statistical method for evaluating systematic relationships. Univ. Kans. Sci. Bull. 38:1409-1438.
44
45. Swofford, D. L. 2001. PAUP*: phylogenetic analysis using parsimony (and other methods) 4.0 Beta. Sinauer Associates, Sunderland, MA.
45
46. Webster, R. G., W. J. Bean, O. T. Gorman, T. M. Chambers, and Y. Kawaoka. 1992. Evolution and ecology of influenza A viruses. Microbiol. Rev. 56:152-179.[Abstract/Free Full Text]
46
47. Webster, R. G., Y. Guan, M. Peiris, D. Walker, S. Krauss, N. N. Zhou, E. A. Govorkova, T. M. Ellis, K. C. Dyrting, T. Sit, D. R. Perez, and K. F. Shortridge. 2002. Characterization of H5N1 influenza viruses that continue to circulate in geese in southeastern China. J. Virol. 76:118-126.[Abstract/Free Full Text]
47
48. Webster, R. G., J. S. M. Peiris, H. Chen, and Y. Guan. 2006. H5N1 outbreaks and enzootic influenza. Emerg. Infect. Dis. 12:3-8.[Medline]
48
49. Webster, R. G., M. Yakhno, V. S. Hinshaw, W. J. Bean, and K. G. Murti. 1978. Intestinal influenza: replication and characterization of influenza viruses in ducks. Virology 84:268-278.[CrossRef][Medline]
49
50. Xu, K. M., K. S. Li, G. J. D. Smith, J. W. Li, H. Tai, J. X. Zhang, R. G. Webster, J. S. M. Peiris, H. Chen, and Y. Guan. 2007. Evolution and molecular epidemiology of H9N2 influenza A viruses from quail in southern China, 2000 to 2005. J. Virol. 81:2635-2645.[Abstract/Free Full Text]
50
51. Xu, X., K. Subbarao, N. J. Cox, and Y. Guo. 1999. Genetic characterization of the pathogenic influenza A/goose/Guangdong/1/96 (H5N1) virus: similarity of its hemagglutinin gene to those of H5N1 viruses from the 1997 outbreaks in Hong Kong. Virology 261:15-19.[CrossRef][Medline]

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Journal of Virology, July 2007, p. 7529-7539, Vol. 81, No. 14
0022-538X/07/$08.00+0     doi:10.1128/JVI.00327-07
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