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1 Systems Biology Laboratory, Department of Microbiology, Miami University, Oxford, OH 45056, USA
2 School of Biology, Georgia Institute of Technology, Atlanta, GA 30332, USA
3 State Key Laboratory of Emerging Infectious Diseases, The University of Hong Kong, Hong Kong SAR
4 Department of Avian Medicine, College of Veterinary Medicine, University of Georgia, Athens, GA 30602, USA
Correspondence
Xiu-Feng Wan
wanhenry{at}yahoo.com
or
xwan{at}cdc.gov
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Present address: Influenza Division, Centers for Disease Control and Prevention, Atlanta, GA 30333, USA. 
The GenBank/EMBL/DDBJ accession numbers for the sequences reported in this paper are EU627685 and EU636682–EU636696.
Supplementary material is available with the online version of this paper.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Wan, 1998). About 1 year later, a related H5N1 genotype caused human deaths in Hong Kong having been isolated first from chicken farms and later from the live poultry markets (Claas et al., 1998). This Hong Kong 1997 (HK97) H5N1 highly pathogenic avian influenza outbreak was the first documented incident of a purely avian influenza virus (AIV) causing human respiratory disease and death. After slaughtering 1.5 million chickens the avian influenza outbreak stopped and was followed by banning trade of live poultry for 7 weeks (Shortridge et al., 1998; Sims et al., 2003). There have been no known indigenous human H5N1 cases in Hong Kong since the 1997 incident. In early 2003, a new genetic variant was isolated from two Hong Kong residents one of whom subsequently died soon after returning from a visit to the Fujian Province, China to the north of Hong Kong (http://www.who.int/csr/disease/avian_influenza/ai_timeline/en/index.html). This new H5N1 genetic variant led to avian influenza outbreaks in South-east Asia at the end of the year. In May 2005, another H5N1 genetic variant was identified in Qinghai Lake, western China that spread to central Asia and beyond (Chen et al., 2005; Liu et al., 2005). Up to April 17, 2008, 381 human cases had been confirmed and 235 were fatal (www.who.int). Since 2003, at least 209 million birds have been slaughtered or have died because of this disease (www.fao.org).
Southern China has an abundance and diversity of AIV in domestic ducks, which are raised in close proximity to humans (Shortridge, 1992) in a region designated a hypothetical epicentre for the emergence of pandemic influenza viruses (Shortridge & Stuart-Harris, 1982). However, with such an abundance of AIVs, why doesn't influenza pandemics occur more frequently? There must be something very complex that is needed to cause one or more AIVs to give rise to a pandemic virus. Therefore, by reconstructing genetic events that may have led to the appearance of HK97 and the current H5N1 HPAIVs in humans, the information generated here might provide a higher order of preparedness for a pandemic in the future.
The isolation of H5N1 viruses from chickens and humans in Hong Kong in 1997 possibly pre-empted a pandemic. The availability of viral sequences provides us with an opportunity to track down the genetic origins of these viruses. It has been reported that the haemagglutinin (HA) of A/Goose/Guangdong/1/96(H5N1) most likely provided the HA of HK97 H5N1 HPAIV (Xu et al., 1999). During the past decade, a range of H5N1 reassortants has been reported (Chen et al., 2004, 2006; Guan et al., 2002, 2004; Li et al., 2004). Recently, segments from AIVs isolated 30 years ago were identified in these H5N1 AIVs (Duan et al., 2007; Wan et al., 2007b). However, the genetic genesis of these H5N1 AIVs has not been well characterized yet due to (i) lack of systematic surveillance in avian hosts in this region from 1980 to 2000 and (ii) limitation of current phylogenetic analysis approaches. Recently, a new quantitative genotype method called Genotype In Network (GIN) was developed (Wan et al., 2007b). Different from conventional phylogenetic tree construction approach, GIN does not perform multiple sequence alignment or tree construction and is able to analyse a large number of viruses. By combining with phylogenetic tree construction, our new method provides an opportunity towards a more systematic genetic analysis of a wider range of viruses pre- and post-1996 (Wan et al., 2007b).
In this study, we analysed the genes of H5N1 HPAIVs from 1996 onward and attempted to identify possible progenitors for them. The results indicate that at least 21 reassortants have emerged from combinations of these genes since then. The information generated may be useful for pandemic preparedness as well as avian influenza prevention and control.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Datasets.
Besides the two archive avian influenza isolates we sequenced, our dataset contains 44 398 influenza gene sequences from the Influenza Virus Resource database (Bao et al., 2004), which were updated in August of 2007. This dataset includes 554 H5N1 AIVs, which have complete or almost-complete genes for all eight gene segments.
Progenitor gene identification.
To identify the potential progenitor genes for each isolate, we applied our newly developed GIN method (Wan et al., 2007b). Briefly, GIN first measures the genetic distances between viral genes using Complete Composition Vector (CCV) (Wan et al., 2007a) and then identifies influenza modules, a cluster of viral genes with small evolutionary distances, using a local optimization program based on thresholds derived from Bayesian analysis (Wan et al., 2007b). The genes among and within the modules were further analysed by phylogenetic methods.
Phylogenetic analyses.
Multiple sequence analysis of influenza genes was made by the MUSCLE program (Edgar, 2004) and checked against amino acid sequences wherever possible. The amino acid divergence for specific lineage in the phylogenetic tree was also identified. To reconstruct the tree topology, maximum-parsimony and neighbour-joining methods implemented in PAUP* 4.0β (Swofford, 1998) were used as described previously (Wan et al., 2005). The maximum-likelihood (ML) tree estimation was evaluated using GARLI version 0.951 (Zwickl, 2006). Bayesian inference of phylogeny was performed with BEAST version 1.4.6 (Drummond & Rambaut, 2007) with the General Time Reversible (GTR) model estimated by MODELTEST 3.7 (Posada & Crandall, 1998). The GTR model was run assuming a gamma-distribution of substitution and invariable rates across sites for 1 million iterations (mcmc ngen=1 000 000). The Tracer version 1.4 was used to estimate the confidence of Markov chain Monte Carlo (MCMC) analyses from BEAST, and the effective sample size (ESS) must have a minimum of 100 as suggested by the manual. The TreeAnnotator version 1.4.6 was applied to extract the tree with the highest clade credibility. The tree topologies were confirmed for the four methods used. The influenza gene trees (except NP) shown in Fig. 1 were the Bayesian inference trees from BEAST. The ML tree from GARLI was used for the NP gene as shown in Fig. 1 since ESS from BEAST analysis did not meet our minimum requirement even with 50 million iterations (mcmc ngen=50 000 000). Control and log files for all stand-alone programs run here and other methodological materials are available on request.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). The results show that the HA genes of these H5N1 viruses are phylogenetically close to Tk/England/50-92/91(H5N1) (Tk/E91-like viruses) (Fig. 1a
). However, the neuraminidase (NA) and six internal gene segments originated from three to seven different lineages: NA has three lineages; polymerase PB2 has five; polymerase PB1 has five; polymerase PA has four; the nucleoprotein gene (NP) has six; matrix (M) has seven and non-structural (NS) has two (Table 1, Fig. 1 and Supplementary Fig. S1 available in JGV Online). Most of these pre-1996 AIVs were isolated from southeastern or eastern Asia, especially from southern China. Besides these Asian strains, some H5N1 segments are closely related to strains isolated earlier in Europe, such as African starling/England.Q/983/79(H7N1) and Tk/England/N28/73(H5N2) (Fig. 1 and Table 1).
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). The reassortant nomenclature for the 554 H5N1 AIVs is listed in Supplementary Table S1 (available in JGV Online).
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Among the 21 reassortants, H5N1-PR1 contained Gs/Gd/1/96; only H5N1-PR2 and H5N1-PR7 have been identified in the confirmed human cases. H5N1-PR1 was a reassortment between Tk/E91-like (HA, NA and NS), Dk/NC/1681/92(H3N8)-like (PB2, PB1, PA and NP) and an unknown donor for MP (lineage VII) (Supplementary Fig. S1c). The only lineage difference between H5N1-PR7 and H5N1-PR1 is located in the NS gene (Supplementary Fig. S1d and Table 2). NS in H5N1-PR1 was likely to have been derived from Tk/E91-like viruses, while the NS in H5N1-PR7 has a close phylogenetic distance to Dk/HK/542/79(H10N9)-like and Dk/HK/147/77(H9N6)-like viruses (Fig. 1 and Supplementary Fig. S1).
Frequent reassortments involved some strains isolated 20–30 years ago. The NA and NP genes of Ck/Hebei/718/2001(H5N1) (H5N1-PR10) were very close to African starling/England/983/79(H7N1)-like viruses (Fig. 1), while the other four internal segments had diverse evolutionary origins: its PB2 and PA is close to AIVs isolated in China as well as swan/Hokkaido/51/96(H5N3). It is worth mentioning that the NP gene of Ck/Hebei/1/02(H7N2) is virtually identical to that of Tk/England/N-28/73(H5N2) in both nucleotide and amino acid compositions (Fig. 1f).
Relationships of H5N1-PRs with reported reassortants
During the past decade there has been a number of H5N1 genotypes reported in mainland China and Hong Kong, which generally identify a reassortant by combining lineages/sublineages defined by tree topologies (instead of progenitor genes used in this study) from NA and internal segments (Guan et al., 2002; Chen et al., 2004, 2006; Li et al., 2004). Since multiple lineages may be derived from the same progenitor genes, an H5N1-PR may include multiple reassortants reported earlier. For instance, H5N1-PR7 includes genotypes A, B, C, E, G, V, W, Y, Z and Z+ (Guan et al., 2002; Li et al., 2004; Smith et al., 2006). H5N1-PR7 also includes the recently reported Qinghai-like and Fujian-like lineages (Chen et al., 2005; Smith et al. 2006). As shown in Table 2, 13 H5N1-PRs are first reported in this study.
Emergence of low pathogenic AIVs through progenitor gene combinations
The combinations of the progenitor genes have also resulted in at least four reassortants in eastern Asia: O-PR1, Swan/HK/51/96(H5N3)-like viruses; O-PR2, Dk/HK/55/96(H1N1)-like viruses; O-PR3, Dk/Mongolia/54/01(H5N2)-like viruses; O-PR4, Ck/Hebei/1/02(H7N2)-like viruses (Table 2). Swan/HK/51/96(H5N3) and Dk/HK/55/96(H1N1) were identified in Japan in 1996 the same year as Gs/Gd/1/96(H5N1) (Okazaki et al., 2000). The phylogenetic analyses suggest both HA and NA of Gs/Gd/1/96(H5N1) are likely to be derived from Tk/E91-like viruses (Fig. 1 and Table 2). It is interesting that the HA of Swan/HK/51/96(H5N3)-like viruses and the NA of Dk/Hokkaido/55/96(H1N1)-like viruses are likely to have been derived from Tk/E91-like viruses. However, Swan/Hokkaido/51/96(H5N3)-like viruses have internal segments possibly derived from Dk/NC/1904/92(H7N1)-like (PB2, PB1 and PA), Tk/England/N28/73(H5N2) (NP)-like and Tk/E91-like (MP and NS) viruses.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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and Supplementary Table S1). The newly developed GIN drew upon publicly available H5N1 virus sequences facilitating a more definitive characterization of the gene pool and a revised nomenclature system, H5N1-PR. Compared with the earlier genotyping system (Guan et al., 2002; Chen et al. 2004), H5N1-PR may provide a more definitive, mutually inclusive research based tool since it focuses on genetic origins instead of reassortment events.
The results suggest that the source of progenitor genes might have a critical impact on host adaptation and/or pathogenesis of these H5N1 viruses. For instance, H5N1-PR1 and H5N1-PR7 have only a single lineage difference in the NS gene (Supplementary Fig. S1d and Table 2). H5N1-PR1, containing Gs/Gd/1/96, was identified in goose, waterfowl and environmental samples, which generally referred at the time to any samples (faecal or otherwise) found on the poultry floor or cage (without clear host record). However, H5N1-PR7 viruses were identified directly in both waterfowl and land-based birds, such as chickens. Viruses from H5N1-PR7 caused most of the reported outbreaks in both domestic and wild birds and confirmed H5N1 human cases since 2003. In vitro experiments showed that the NS gene enhances virus replication in mammalian cells (Twu et al., 2007). The residue mutation from aspartic acid to glutamic acid at position 92 in the NS1 protein was reported to increase the virulence of avirulent A/Puerto Rico/8/34(H1N1) in pigs (Seo et al., 2002). However, viruses belonging to the NS lineage related to the 1997 Hong Kong outbreak have glutamic acid at residue 92 in NS1, and the other H5N1 viruses have aspartic acid at this position (data not shown). Since 2000, a similar 5 aa deletion at positions 80–84 in the NS1 gene of most H5N1 viruses, especially those isolates after the 2003/2004 H5N1 outbreak in eastern Asia (Wan et al., 2005), has been shown. Although viruses without deletions may still circulate in wild birds, e.g. A/mallard/Guangxi/wt/2004(H5N1) and A/slaty-backedgull/Shandong/38/04(H5N1), it was shown that this deletion can increase the pathogenesis of H5N1 viruses in chickens (Long et al., 2006).
These findings indicate that the H5N1-PR2 reassortant would have been involved with the 18 cases recorded in the H5N1 outbreak in Hong Kong in 1997, which included six fatalities (Sims et al., 2003). This reassortant disappeared after slaughtering 1.5 million chickens there and trade of live poultry stopped for 7 weeks (Sims et al., 2003). H5N1 97-like viruses have been recorded once since 1997 from egg shell washes taken from one goose and two duck eggs imported from Vietnam into China in 2005 (Li et al., 2006). The reason for this is unclear. It is not impossible that H5N1-PR2 reappearing in 2005 might have been detected as a consequence of HK97-like virus inactivated vaccine usage or laboratory contamination. Nevertheless it raises important issues on H5N1 ecology warranting intensive virus surveillance in the region. H5N1-PR2 has four different progenitor genes from H5N1-PR1, which includes Gs/Gd/1/96(H5N1). Thus, the 1997 Hong Kong outbreaks may have originated from a different reassortment event from that gave rise to Gs/Gd/1/96(H5N1).
Besides these reassortants, several other H5N1 HPAIVs, such as Ck/Hubei/wi/97, Ck/Jilin/hg/02, WildDk/GD/314/04 and Ck/Hubei/wk/97, have different combinations of the progenitor genes identified in this study (Supplementary Table S1). However, some genotypes did not form a statistically significant lineage or lacked complete genomic datasets and thus are not shown in Table 2. For instance, the MP gene of WildDk/GD/314/04 did not cluster with any other viruses to form a lineage with statistical significance. Therefore, the 21 H5N1 reassortants identified in this study are still incomplete; the emergence of new reassortants seemingly continues. By virtue of providing further genetic background to the origins of H5N1 HPAIVs, the findings of this study could be useful toward developing an influenza prevention and control strategy. Such strategy must be based on long-term systematic AIV surveillance, quick provision of gene sequences and isolates as appropriate and structured international coordination. It's conjectural, however, whether the genetic combination patterns recognized for HPAIV H5N1 viruses to date or of putative ones would be capable of endowing pandemicity upon other subtype AIVs. Genetic data available for pandemic viruses of the 20th century indicate that they are unlikely to have been highly pathogenic for chickens and other types of bird (www.oie.int). It remains to be seen whether there are other H5N1 viruses that have been stored in Asia or elsewhere, as yet ungenotyped, that could shed light on the functional range of progenitor genes.
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ACKNOWLEDGEMENTS |
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Received 6 March 2008;
accepted 7 May 2008.
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