1 Division of Virology, Department of Infectious Diseases, St Jude Children's Research Hospital, 332 North Lauderdale Street, Memphis, TN 38105-2794, USA
2 Department of Pathology, University of Tennessee Health Science Center, Memphis, TN, USA
3 Canadian Wildlife Service, Environment Canada, Edmonton, Alberta, Canada
Correspondence
Robert G. Webster
Robert.Webster{at}stjude.org
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ABSTRACT |
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INTRODUCTION |
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Genes from this pool have crossed over the species barrier into human and other domestic avian and swine species, either as complete avian viruses or as genetically reassorted viruses following interspecies transmission. Phylogenetic studies have shown that the human pandemic strains of 1957 and 1968 were derived from reassortant viruses with genes of human and avian origin (Kawaoka et al., 1989; Schafer et al., 1993
; Scholtissek et al., 1978
).
Swine have been proposed as the mixing vessel in which these pandemic influenza viruses arise (Scholtissek & Naylor, 1988). Recently, H3N2 viruses have established a stable lineage in USA swine. In these animals, two reassortants were identified, a double reassortant, between the classical swine H1N1 and human H3N2 viruses and a triple reassortant, which included two avian internal protein genes (PA and PB2) in addition to those seen in the double reassortant (Zhou et al., 1999a
). The triple reassortant has become the dominant circulating strain in USA swine and continues to evolve. Subsequent reassortant events with the co-circulating classical H1N1 swine viruses have generated novel H1N2 viruses. Two of these H1N2 isolates have a third avian internal protein gene (PB1), which is closely related to an avian H5N2 virus (Choi et al., 2002
; Karasin et al., 2002
). In Europe, avian-like H1N1 viruses have replaced the classical H1N1 in European swine and multiple reassortant events similar to those seen in the USA have occurred in pigs in Great Britain (Brown et al., 1997
, 1998
; Scholtissek et al., 1983
). These findings suggest that although influenza viruses continue to evolve in swine, the conservation of these avian internal protein genes confer a selective advantage.
Although avian influenza viruses can infect other mammals, it was thought that direct transmission to humans in nature was unlikely (reviewed by Horimoto & Kawaoka, 2001). However in 1997, a highly pathogenic H5N1 avian influenza virus was directly transmitted to humans from poultry in Hong Kong, suggesting that adaptation in an intermediate mammalian host was not necessary (Claas et al., 1998
; De Jong et al., 1997
; Subbarao et al., 1998
).
Despite the depopulation of the bird markets and institution of preventative measures, the H5N1 subtype re-emerged in 2000, 2001, 2002 and 2003 with multiple genotypes (Brammer et al., 2003; Guan et al., 2002a
, b
; Webster et al., 2002
). Although a number of recent studies have examined phylogenetic data from virus samples collected in the bird markets of Hong Kong and southern China, little data are available regarding the gene pool in feral waterfowl (Guan et al., 2000
, 2002a
, b
; Hoffmann et al., 2000
; Lin et al., 1994
; Liu et al., 2003
; Webster et al., 2002
; Zhou et al., 1999b
).
In the current study, we provide the first genotypic analysis of this vast gene pool by detailing phylogenetic data from 35 viruses collected from Canadian ducks sampled in the Alberta wilderness during a 17-year period. From this data, we sought to enhance the current database and answer the following questions: do these duck isolates support the current dogma that influenza in waterfowl has achieved evolutionary stasis' and that genes can be grouped into separate North American and Eurasian avian clades? Do these gene segments reassort within this pool? Are there specific haemagglutinin (HA) subtypes that have a greater propensity for reassortment? Does this feral waterfowl gene pool contribute to infection in domestic species and, if so, which genotypes have a propensity to crossover into other species?
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METHODS |
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RNA isolation, RT-PCR and sequencing.
Viral RNA was isolated from allantoic fluid using the Qiagen RNeasy kit (Qiagen) according to the manufacturer's protocol. Reverse transcription of viral RNA and subsequent PCR was performed using primers specific for each viral gene, as described previously (Hoffmann et al., 2001). PCR products were gel purified and cleaned using the QIAquick PCR purification kit (Qiagen) according to the manufacturer's protocol. PCR amplicons were sequenced by the Hartwell Center for Bioinformatics and Biotechnology at St Jude Children's Research Hospital using rhodamine or dRhodamine dye-terminator cycle sequence-ready reaction kits with AmpliTaq DNA polymerase FS (Perkin-Elmer Applied Biosystems; PE/ABI) and synthetic oligonucleotides. Electrophoresis and analysis of the samples were done using the PE/ABI model 373 or model 377 DNA sequencers.
Phylogenetic studies.
DNA sequences were aligned by the Lasergene sequence analysis software package (DNASTAR). Multiple sequences were aligned using CLUSTAL X (v 1.81; Thompson et al., 1997), and phylogenetic trees were generated using the neighbour-joining and parsimony algorithms and bootstrap analysis in PAUP (v 4.0b10; D. Swofford, Sinauer Associates). Phylogenetic trees were generated based on the alignment of the complete or partial nucleotide sequences of each viral gene. The regions of each internal gene used in analysis included: PB2, nt position 65603; PB1, 354565; PA, 47525; NP, 41631; M, 40668 and NS, 101621. The regions used in the analysis of the HA and NA genes and trees not illustrated in this paper can be viewed at http://www.stjuderesearch.org/data/feralduck.
Generation of phylogenetic groupings.
Related viruses were assigned phylogenetic groupings on the basis of their position in the trees, which were generated by neighbour-joining and maximum-parsimony methods and the following criteria. (i) Phylogenetic distinctions generated by the trees must be supported by high bootstrap scores (>95 %) at the nodal branches (Nei & Kumar, 2000). (ii) When the bootstrap scores at the nodal branches are less than this arbitrary cut-off value, the sequence similarities of the gene segments for each virus in the proposed genotype group must have a 95 % or greater sequence similarity with other members of the group and less than 94·9 % identity with the gene segments of viruses belonging to other phylogenetic groups. (iii) In cases where the trees predict groupings in which some viral gene segments within a group have less than 95 % similarities (e.g. 94·5 %), those segments may be included in that group, as long as the sequence identity is higher with members of the proposed grouping than it is with members from other groupings. (iv) If the position of the virus isolate is in the same phylogenetic grouping in trees generated by two different methods (neighbour-joining and maximum-parsimony), but there is discrepancy between groupings based on sequence similarity, the genotype group of the virus isolate will be dictated by where it is placed in the calculated phylogenetic trees. (v) If the virus isolate is not in the same position in both trees generated by the two different methods and there is discrepancy between sequence identities, then a definitive grouping cannot be assigned.
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RESULTS |
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Identification of matrix genes that form a distinct early branch in the phylogenetic tree
Although matrix genes are clustered into three main groups within the North American avian clade, two genetically identical viruses isolated from two different species of ducks during the same year, A/mallard/Alberta/98/85 (H6N2) and A/pintail/Alberta/113/85 (H6N2) form a distinct branch early in the phylogenetic tree. To establish better their phylogenetic relationship, these two outlier matrix genes were completely sequenced and compared with the complete sequences from 219 matrix genes compiled from viruses sequenced in this study and from the influenza database (Macken et al., 2001). The resulting tree suggests that these isolates form their own distinct branch with sequence similarities less than 92·6 %, compared with that of the other 219 sequences. Bootstrap analysis using the neighbour-joining method rooted to A/equine/Prague/56 (H7N7), places these two outliers at the base of the North American avian clade (Fig. 2
); bootstrap analysis using the maximum-parsimony method places the outliers just off the root of the tree (see website for tree). However, the numeric value was low (i.e. 34 of 100 trees) with either method.
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Although most of the predicted proteins of the Canadian feral duck matrix genes clustered closely together in the North American avian clade, with >99 % amino acid identity, the phylogenetic analysis of the M1 and M2 sequences and their predicted proteins of A/mallard/Alberta/98/85 (H6N2) and A/pintail/Alberta/113/85 (H6N2) confirm the unique placement of these outlier genes. The similarity between the predicted M1 protein and an avian consensus sequence generated using sequence data from 73 North American duck and shorebird viruses obtained from the database (Macken et al., 2001), and sequences provided by L. Widjaja (personal communication), was 97·6 %; that of the predicted M2 protein was 93·5 %. The M1 protein of these outliers had 5 amino acid changes throughout the length of the protein (I15V, S116A, S142V, N224S and N232D). Similarly, the predicted sequence of the M2 protein of the outliers differed from the consensus sequence by 6 amino acids distributed throughout the cytoplasmic, transmembrane and surface portions of the protein (S13N, I27V, F28I, Y50C, K70E and Q77R).
Clustering of PB2 and PA genes from swine and quail viruses within the same phylogenetic grouping
One of the most interesting results from the phylogenetic analysis is the clustering of the PB2 and PA genes from quail and swine isolates with feral Canadian duck viruses in the same phylogenetic grouping (Fig. 1). To illustrate this, the phylogenetic analysis of the PA gene is presented below.
The PA genes clustered into two main groups, with isolates having 88·5 % nucleotide similarities between the two groups. The two groups were further subdivided as follows: the first group, which included 10 isolates related to influenza A viruses from shorebirds and gulls, divided into two distinct phylogenetic clusters (1a and b). The second group comprised the remaining 25 viruses, which clustered within the North American avian clade. The second group was divided into four phylogenetic subgroupings (2ad). Key features of this tree include: (i) the clustering of one shorebird virus [A/ruddy turnstone/NewJersey/47/85 (H4N6)] and two Eurasian viruses from the influenza database [i.e. A/duck/Hokkiado/8/80 (H3N8) and A/swine/HongKong/81/78 (H3N2)] with the feral duck genes in the North American avian clade; (ii) the placement of a number of swine and quail viruses [i.e. A/swine/Texas/4199-2/98 (H3N2) (sw/TX/98), A/swine/HongKong/81/78 (H3N2) (sw/HK/78) and A/quail/Arkansas/29209-1/93 (H9N2) (qu/AR/93)] within the same phylogenetic subgrouping (2c), which share high nucleotide sequence similarity with the PA gene from one particular duck virus A/mallard/Alberta/743/83 (H9N1) (96·7, 98·3 and 98·5 % sequence similarity, respectively). The other North American swine virus with an avian PA gene, A/swine/Ontario/01911-1/99 (H4N6) (sw/ONT/99) belongs to phylogenetic group 2d and is most closely related to A/mallard/Alberta/202/96 (H2N5) and A/pintail/Alberta/22/97 (H2N9) (95·4 %) but also shares a 95 % sequence identity with A/mallard/Alberta/743/83.
Placement of three H6 and four H9 genes in the Eurasian lineage
The phylogenetic analyses of the genes encoding the surface glycoproteins circulating in the feral Canadian ducks have similar features, such as high degree of nucleotide and amino acid identities between genes from viruses isolated many years apart. Interestingly, three of eight H6 and all four of the H9 genes in these North American duck viruses clustered with Eurasian isolates, whereas the remaining NA and HA genes were all placed in one or two groupings within the North American avian clade. The phylogenetic analysis of the H6 gene is presented (Fig. 3) in greater detail to illustrate this unique feature. The phylogenetic tree for H9 and other HA and NA genes can be viewed at www.stjuderesearch.org/data/feralduck.
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A high degree of similarity of genes from influenza A isolated from domestic species and viruses isolated from feral ducks
Feral aquatic birds in North America have been the reservoir of some or all of the genes in influenza viruses that have crossed over into domestic species in the USA, including sw/TX/98 (H3N2), sw/ONT/99 (H4N6) and ck/CA/01 (H6N2) (Karasin et al., 2000; Webby et al., 2002
; Zhou et al., 1999a
). The nucleotide sequences of the internal genes of these viruses have a higher degree of similarity with genes in feral Canadian duck viruses than they have with previously published sequences (Table 3
). Although A/swine/Korea/cy02/02 (H1N2) also has PB2 and PA genes that clustered with North American duck isolates, all of the genes in this virus share close similarity with currently circulating H1N2 swine viruses in North America and is the result of a swine virus having been imported to Korea from North America (Y. K. Choi, personal communication).
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DISCUSSION |
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Consistent with accepted dogma, genes from influenza viruses isolated from Canadian feral ducks are in evolutionary stasis and the majority of the genes clustered with other avian species in the North American clade. However in contrast to this, the H9 and some of the H6 HA genes in this study clustered with viruses in the Eurasian avian clade.
Although phylogenetic studies have demonstrated that avian influenza viruses have evolved into two separate lineages (i.e. the North American and Eurasian clades) (Ito et al., 1991, 1995
; Kawaoka et al., 1988
; Lin et al., 1994
; Okazaki et al., 2000
; Suarez & Perdue, 1998
; Webster et al., 1992
), geographical intermixing has been described in the H2 subtype in shorebirds in North America. This may be because of the intermixing of birds from different continents in the arctic and subarctic regions and rare trans-Atlantic migration of some of these birds, particularly ruddy turnstones, whose migratory patterns are not fully elucidated (Gorman et al., 1990
; Del Hoyo et al., 1992
; Makarova et al., 1999
). This hypothesis is further supported by the results of this study, which place the PB2 gene of A/ruddy turnstone/New Jersey/47/85 (H4N6) with Eurasian isolates. Despite evidence that there may be a distinct gene pool in shorebirds (Kawaoka et al., 1988
), the clustering of duck PA and PB2 genes with those of shorebirds and gulls in this study suggests an intermixing of genes between the two pools. Alternatively, the possible existence of two major phylogenetic subgroups that do not correlate with the geographical origin of the virus isolates, and therefore do not fit the current classification system, could explain these data. Although convergent evolution has also been suggested as an alternative hypothesis (Webby et al., 2002
), this is less likely given that the virus has achieved evolutionary stasis in these avian hosts.
Genes within the feral Canadian duck gene pool reassort independently. Although dominant genotypes may be present in any given year, there was no clear evolution of these dominant genotypes from year to year and the majority of the HA subtypes examined were equally diverse. However, some subtypes had conserved a phylogenetic grouping for a particular gene such as the NS gene in H2, the M gene in H3 and the PB2 gene in H4 viruses. The significance of this finding is not clear and may be related to the limited number of virus isolates examined over a short-time frame. However, the remaining genes in these viruses had multiple phylogenetic groupings, a finding that suggests multiple genotypes that reflect the diverse nature of the gene pool. This contrasts previous findings that suggest although co-infection with two or more influenza A viruses can occur in ducks, it does not occur randomly; thus, the number and types of possible reassortants are limited (Sharp et al., 1997).
The H9 subtype was the only example of restricted diversity in a particular HA subtype in this study; the genotypic pattern of three out of four H9 viruses maintained a high degree of similarity from 1983 to 1991. These data differ from previous studies, which showed that the H9N2 subtypes circulating in domestic species within the avian markets of southeast China had a greater diversity and showed more evidence of reassortment than did other virus subtypes in the gene pool, suggesting that the H9 subtype has a greater propensity for reassortment (Liu et al., 2003). Alternatively, because there is greater host variation in these domestic species, the increased selection pressures may be driving the increase in reassortment seen within the domestic population.
Why the H9 subtypes in the current study did not reassort is unclear but may be related to the infrequency with which this subtype is isolated in the North American duck gene pool. Sharp et al. (1993) found that the predominant subtypes that were isolated consistently in feral Alberta ducks included H3, H4 and H6; H9 subtypes were isolated in small, infrequent clusters and would disappear from the gene pool for periods as long as 6 years. In contrast, the H9 subtype is more common in shorebirds and gulls, and this gene pool may spillover into the duck gene pool as these birds migrate north in the spring (Kawaoka et al., 1988
). However, if there is limited circulation of this subtype, the lack of diversity over an 8-year period suggests that the source of periodic reintroduction of H9 is either reassorting at a slow rate or in a state of frozen evolution, similar to what has been suggested for equine strains isolated from South America in 1987 and from India in 1988 (Endo et al., 1992
; Lindstrom et al., 1998
). These speculations are based on a limited sample size and need to be interpreted with caution. More genomic data of H9 viruses circulating in Canadian ducks is needed to explore these observations further.
Viruses sequenced in this study were clearly related to viruses that have been isolated from domestic species. By examining each of the eight genes in the feral duck viruses, we can determine better whether there are common genotypes or phylogenetic groupings that crossover the species barrier more frequently. We have shown that the PA genes of swine and quail species and the PB2 genes of swine species that have North American avian related genes clustered together in the same phylogenetic grouping.
Both quail and swine are intermediate hosts, and swine, in particular, are thought to be the mixing vessel from which pandemic influenza arises (Perez et al., 2003; Scholtissek & Naylor, 1988
). Currently, the dominant influenza virus strains circulating in Eurasian and North American pigs have various genes encoding internal proteins of avian origin (Brown et al., 1997
; Zhou et al., 1999a
). This predominance suggests that the genes of the avian viruses impart a selective advantage. Lin et al. (1994)
suggested that influenza genes in the Eurasian avian gene pool may possess unique sequences' that permit transmission and infection of other hosts. The present study indicates that perhaps this constellation of polymerase genes is optimal for a polymerase complex that allows enough replicative error to optimize species adaptation. Although chickens have been implicated in the transmission of avian influenza viruses to humans, their role as an intermediate host has not been as defined as it has been for quail and swine species. Clustering of viruses isolated from chickens within these phylogenetic groupings would further support this, however, this was not the case (Fig. 1
).
Interestingly, although the 1957 and 1968 pandemics were associated with human avian reassortants that contained an avian PB1 gene (Kawaoka et al., 1989; Schafer et al., 1993
; Scholtissek et al., 1978
), genes from the two swine viruses that contain avian PB1 protein genes [swine/Kansas/13481-T/00 (H1N2) and sw/ONT/99 (H4N6)] did not cluster together in this analysis. However, qu/AR/93 (H9N2), ch/CA/01 (H6N2) and swine/Kansas/13481-T/00 (H1N2) did cluster in the same phylogenetic grouping.
Although there is no direct evidence and the dataset is small, one could speculate that North American avian viruses that possess polymerase protein genes which fall into these genotypic groupings might have a greater propensity to crossover the species barrier and adapt in an intermediate host (to view PB1 and PB2 trees see supplemental figures at www.stjuderesearch.org/data/feralduck).
Another unique finding in this study is the identification of a distinct lineage of matrix genes in feral ducks. These outlier genes are different from other matrix genes circulating in the Canadian duck gene pool, and although definitive placement in the phylogenetic tree is not possible, our analyses suggest that the viruses that contain these outlier genes diverge from other avian isolates at the base of the North American avian clade or earlier. The consequence of amino acid changes in the predicted proteins of the M1 and M2 genes of the outliers (compared with the avian consensus) is not clear. The five differing residues in the M1 protein do not lie within the nuclear targeting domain or within sites of the protein that influence the morphology of the virus (Bourmakina & Garcia-Sastre, 2003; Ye et al., 1995
). Although the M2 gene in the outlying duck viruses have an I27V substitution, this amino acid change is not known to confer amantadine resistance (Grambas et al., 1992
; Hay et al., 1985
).
It is not clear, why this unique lineage of matrix genes in other feral Canadian ducks was not identified. The virus isolate belonged to the H6 subtype that was frequently isolated and had multiple genotypes in different years, a finding that suggests it reassorted freely. Because this study included only 1 % of the viruses in our repository, other related viruses probably have yet to be identified. Alternatively, the matrix genes of the outlying viruses may have conferred a survival disadvantage and were subsequently subjected to negative selection. However, this hypothesis is unlikely to be correct; because of the comparatively low sequence identity between the outliers and other avian matrix genes suggests that the outliers' genes diverged from the other North American avian viral genes long ago and have been maintained.
This study is the first attempt at a systematic review of the influenza genes circulating in its natural host. The main criticism of genotype analysis as a method of analysing the patterns of gene reassortment is that it depends on the strictness of the criteria for grouping viruses. In this study, criteria were established for generation of phylogenetic groupings in an attempt to accurately reflect the relationship of one virus to another. Although arbitrary, this method and the time period chosen provide some indication of how diverse the gene pool is and how genes can reassort over a 17-year period. However, only a few of the genes were completely sequenced and the relatively small number of viruses chosen during this time period make it impossible to assess the year-to-year changes in the gene pool. In addition, sampling bias because of a small sample size cannot be excluded. A more comprehensive and complete genotypic analysis is needed to examine some of the concepts arising from this study, to gain a broader understanding of the depth of the influenza gene pool and to determine how influenza A viruses reassort in aquatic birds. Expansion of the database and alignment of the complete gene sequences or implementation of a set region of the gene segment for analysis would facilitate further genomic mining to help provide a more accurate reflection of the diversity of this gene pool.
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ACKNOWLEDGEMENTS |
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Received 4 December 2003;
accepted 19 April 2004.