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Phylogenetic Analysis Reveals the Global Migration of Seasonal Influenza A Viruses
http://pathogens.plosjournals.org/perlserv/?request=get-document&doi=10.1371/journal.ppat.0030131
Martha I. Nelson1,2, Lone Simonsen 3, Cecile Viboud 4, Mark A. Miller 4, Edward C. Holmes 1,2,4*
1 Center for Infectious Disease Dynamics, The Pennsylvania State University, University Park, Pennsylvania, United States of America, 2 Department of Biology, The Pennsylvania State University, University Park, Pennsylvania, United States of America, 3 National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America, 4 Fogarty International Center, National Institutes of Health, Bethesda, Maryland, United States of America
The winter seasonality of influenza A virus in temperate climates is one of the most widely recognized, yet least understood, epidemiological patterns in infectious disease. Central to understanding what drives the seasonal emergence of this important human pathogen is determining what becomes of the virus during the non-epidemic summer months. Herein, we take a step towards elucidating the seasonal emergence of influenza virus by determining the evolutionary relationship between populations of influenza A virus sampled from opposite hemispheres. We conducted a phylogenetic analysis of 487 complete genomes of human influenza A/H3N2 viruses collected between 1999 and 2005 from Australia and New Zealand in the southern hemisphere, and a representative sub-sample of viral genome sequences from 413 isolates collected in New York state, United States, representing the northern hemisphere. We show that even in areas as relatively geographically isolated as New Zealand's South Island and Western Australia, global viral migration contributes significantly to the seasonal emergence of influenza A epidemics, and that this migration has no clear directional pattern. These observations run counter to suggestions that local epidemics are triggered by the climate-driven reactivation of influenza viruses that remain latent within hosts between seasons or transmit at low efficiency between seasons. However, a complete understanding of the seasonal movements of influenza A virus will require greatly expanded global surveillance, particularly of tropical regions where the virus circulates year-round, and during non-epidemic periods in temperate climate areas.
Author Summary
The winter seasonality of influenza A virus in temperate climates is one of the most puzzling epidemiological patterns in infectious disease. To help resolve the issue of influenza seasonality, we studied, using viral genome sequence data, the patterns of global migration of influenza A virus, particularly between the northern and southern hemispheres. A phylogenetic analysis of approximately 900 complete genomes of the H3N2 subtype of human influenza A virus sampled from New Zealand and Australia (southern hemisphere), and New York state, United States (northern hemisphere), revealed that cross-hemisphere migration frequently occurs in both directions and involves multiple viral strains. Such global viral traffic therefore contributes significantly to the introduction of new influenza epidemics in both northern and southern hemispheres. These results also show that influenza A virus migrates afar during non-epidemic periods, rather than persisting locally at low levels during the influenza “off-season”. However, although this represents the largest and first bihemisphere study of its kind to our knowledge, the results highlight the need for sampling from tropical regions and during non-epidemic periods in temperate areas. Studies of this kind are critical to fully understand the geographical dispersal of influenza A virus and the role of climate in triggering seasonal epidemics.
Introduction
Influenza A virus is able to persistently re-infect human populations by continually evading host immunity through the rapid evolution of surface antigens (“antigenic drift”) [1]. Influenza virus epidemics strike temperate latitudes of the world each winter, from November to March in the northern hemisphere and from May to September in the southern hemisphere [2]. In the United States alone, these influenza epidemics are associated with an annual average of 36,000 human deaths [3] and 226,000 hospitalizations [4]; globally, the virus is associated with as many as half a million annual deaths [5]. While rapid antigenic change is a hallmark of influenza evolution, recent studies have failed to detect antigenic drift over an epidemic season, suggesting that important evolutionary processes may occur during non-epidemic periods, either locally or perhaps elsewhere [6–8]. However, surveillance during non-epidemic periods is not conducted routinely by the network of World Health Organization influenza reference centers [9] and, consequently, little is known about how and where the virus persists in the human population in between winter epidemics at low levels. A key question is therefore whether the virus remains locally within its host population in between epidemics, perhaps persisting within hosts in a latent state [10], or whether the virus migrates afar to other reservoirs, such as the tropics, and is later reintroduced.
Although influenza virus has long been regarded a “cold-weather” pathogen due to its marked winter epidemics in temperate zones, recent studies show that tropical regions experience significant year-round influenza virus activity [11]. In theory, such a “tropical belt” could serve as a year-round reservoir that harbors endemic populations of influenza virus that seasonally reintroduce viral isolates into temperate zones to trigger new epidemics [12,13]. Whereas population crashes at the end of seasonal epidemics create severe evolutionary bottlenecks that limit genetic diversity, tropical zones may function as permanent mixing pools for viruses from around the world. Historically, Southeast Asia has been considered a potential epicenter for emergence of pandemic viruses due to the proximity with which humans live with their domestic animals [14]. However, abundant data from these regions is currently unavailable, so the origins of influenza pandemics and epidemics remain unclear.
Given the ease and speed with which the influenza virus is thought to spread between humans, it is generally accepted that global chains of direct person-to-person transmission are sufficient to maintain the influenza virus in the human population [15]. However, a complete understanding of how the influenza virus transmits between humans is lacking [16], and whether human-to-human spread alone accounts for the seasonal emergence of epidemics has been questioned [17]. The simultaneous appearance of influenza outbreaks separated by large longitudinal distances, as well as sporadic influenza cases during summer months, suggests that the virus may instead already be “seeded” and somehow reactivated by environmental stimuli. Thus, the alternating pattern of northern and southern hemisphere bi-epidemics could, in principle, also result from opposite climatic forces independently reactivating viral activity in these two hemispheres at alternating six-month intervals. Hence, instead of continually migrating across the equator, separate viral populations could persist locally in an asymptomatic latent state over the summer months until climatic stimuli sufficiently increase host susceptibility and/or viral transmissibility to induce another epidemic. However, hypotheses of how climatic change may directly or indirectly influence viral activity and/or host susceptibility remain largely untested [18,19].
Crucially, some theories for influenza seasonality produce testable phylogenetic hypotheses. On one hand, if influenza A virus persists locally over the summer in a latent state, then isolates sampled over multiple seasons from a single locality would cluster together on a phylogenetic tree, separate from isolates from other geographic regions (Figure 1). Alternatively, if the virus did not evolve in situ between epidemic seasons, but rather traveled globally between epidemics, then the resulting phylogeny would show extensive intermixing of isolates from different localities. To determine whether influenza virus migrates away between the northern and southern hemispheres during non-epidemic summer months, or remains there latently, we conducted an extensive phylogenetic analysis of 399 whole-genome A/H3N2 influenza A viruses sampled from New Zealand (most commonly Canterbury, South Island) from 2000 to 2005 (six seasons), 88 viral genome sequences from Australia (most commonly Western Australia) from 1999 to 2005 (seven seasons), along with a carefully selected sub-sample of 52 isolates that are representative of the clades present in a larger sample of 413 viruses from New York state, United States, collected between 1998 and 2005 and analyzed previously [7]. Given the relative geographic isolation and low population densities of New Zealand's South Island and Western Australia, as well as sampling limitations, this analysis provides a conservative estimate of the extent of cross-hemisphere migration occurring during this time period.
Figure 1. Models for the Global Evolution of Influenza Virus
Two phylogenetic hypotheses depicting the evolution of A/H3N2 influenza virus by (A) global migration, in which isolates from adjacent seasons in the northern and southern hemispheres are interspersed topologically, or by (B) reactivation of latent virus, in which isolates from the northern hemisphere give rise, in situ, to isolates that circulate in the same locality the next season, and thus are topologically linked (likewise for southern hemisphere viruses).
Results
Extensive Viral Genetic Diversity in Australia and New Zealand
Populations of A/H3N2 influenza virus in Australia and New Zealand from 1999 to 2005 exhibit extensive genetic diversity across the entire genome (Figures 2–4), comparable to the diversity observed previously in New York state [7] (in all phylogenies, clades from Australia are shaded blue, those from New Zealand in green, those from New York state in orange, and global isolates in pink). In particular, multiple viral clades co-circulate during each influenza season in New Zealand and Australia, defined as clusters of isolates with high bootstrap support (>70%), or which are separated by exceptionally long branches (Table 1). As with the New York state data, these viruses were collected in the context of seasonal surveillance efforts in Australia and New Zealand and therefore likely provide a representative sample of the overall genetic composition of the viral populations.
Figure 2. Phylogenetic Relationships of the NA Gene of A/H3N2 Influenza Viruses Sampled from New York State (n = 52), New Zealand (n = 51), Australia (n = 45), and Globally (n = 22) from 1998 to 2005, Estimated Using ML
Larger image
Viral isolates from New York state are highlighted in orange, isolates from New Zealand in green, isolates from Australia in blue, and global isolates in pink. Light yellow rectangles identify viral clades, with numbers in white boxes giving bootstrap values for key nodes (>70%). To clarify reassortment events, capital letters in blue refer to clades that appear in section I of the tree (denoted in pink) on the phylogeny of the concatenated six non-surface glycoproteins; lowercase letters in red refer to clades contained within section II (denoted in light green); and lowercase roman numerals in dark green refer to clades outside sections I and II. Bootstrap values highlighted in yellow identify nodes that define a cross-hemisphere migration event. The tree is rooted by isolate A/New York/327/1999 from the 1998–1999 season (i.e., the earliest sampled isolate), and all horizontal branch lengths are drawn to a scale of nucleotide substitutions per site.
Figure 3. Phylogenetic Relationships of the HA Gene of A/H3N2 Influenza Viruses Sampled from New York State (n = 52), New Zealand (n = 51), Australia (n = 45), and Globally (n = 13) from 1998 to 2005, Estimated Using ML
larger image
Color scheme, rooting, scale, and symbols are the same as those used in Figure 2.
Figure 4. Phylogenetic Relationships of the Concatenated Six Non-Surface Glycoprotein Segments of A/H3N2 Influenza Viruses Sampled from New York State (n = 52), New Zealand (n = 51), and Australia (n = 45) from 1998 to 2005, Estimated Using ML
Larger image
Color scheme, rooting, scale, and symbols are the same as those used in Figure 2.
Table 1.
Clades in the HA, NA, and Concatenated Six Non-Surface Glycoprotein Segment Phylogenies Comprised of Viruses from New York State, New Zealand, Australia, and Globally
On the neuraminidase (NA) tree (Figure 2), viruses from New Zealand and Australia fall into at least 15 distinct clades, some of which appear in multiple seasons: three clades circulated in the 1999 season (clades v, vi, and ix; when data was only available from Australia), five in 2000 (clades i, ii, iii, iv, viii), four in 2001 (clades i, iv, v, g), one in 2002 (clade a), one in 2003 (clade b; when a new reassortant virus predominated), two in 2004 (clades A and c), and three in 2005 (clades A, B, E). Clade lettering reflects the three main sections of the phylogeny, based on topology and time: clades A–E contain viral isolates from 2003 to 2005 that fall within section I of the tree (large pink rectangle in upper portion of tree); clades a–e contain isolates from 2001–2003 that fall within section II (large light green rectangle in middle portion of tree); clades i–xiii contain isolates from 1998 to 2000 that fall outside of sections I and II. The hemagglutinin (HA) tree (Figure 3) contains at least 15 clades from New Zealand and Australia: two circulated in 1999 (clades v and x), four in 2000 (clades i, ii, iii, iv), four in 2001 (clades g, i, iv, xiii), two in 2002 (clades h and j), one in 2003 (clade b), two in 2004 (clades A and c), and three in 2005 (clades A, B, E) (Figure 3). Finally, the phylogeny of the concatenated six non-surface glycoprotein segments (PB2, PB1, PA, NP, M1, NS1) contains the largest number of clades, presumably because this larger data set (9,636 bp) provides the greatest resolution. Southern hemisphere isolates form at least 18 clades on this phylogeny: four were present in 1999 (clades v, vi, x, xii), three in 2000 (clades i/ii, iii, iv), four in 2001 (clades g, i/ii, iv, xiii), three in 2002 (clades a, k, l), one in 2003 (clade b), three in 2004 (clades A, F, c), and three in 2005 (clades A, B, E) (Figure 4).
Differences in the number of clades among segments may also be indicative of reassortment, especially involving the HA gene, as previously demonstrated in New York state [7]. Indeed, several major reassortment events are immediately evident from topological incongruities among these three phylogenies. On the HA tree, clades b, c, and e fall in section I along with isolates from 2004 and 2005, while these clades fall in section II amidst 2002 and 2003 isolates in the NA and concatenated six non-surface glycoprotein phylogenies (Figures 2–4). Clade c also falls in section I on the PB2 phylogeny, showing a similar reassortment pattern as HA. However, aside from this lone reassortment event, the phylogenies of the six non-surface glycoprotein segments are very similar, enabling us to study them as a single concatenated entity (phylogenetic trees for individual segments are provided as Figures S1–S6).
Intermixing among Viral Populations from Australia, New Zealand, and New York State
Isolates from the 52 representative New York state genomes are clearly interspersed with southern hemisphere isolates throughout our phylogenetic trees (Figures 2–4; Table 1), indicating that these populations regularly intermix as a result of cross-hemisphere migration. However, patterns of viral intermixing are both variable and complex, as clades from all three phylogenies contain an array of different combinations of viral populations from New Zealand, Australia, and/or New York state. For example, the phylogeny of the concatenated six non-surface glycoproteins contains a relatively even mix of mono-hemisphere and bi-hemisphere clades (Table 1), with five New York state–only clades, seven southern hemisphere–only clades, six clades that contain New York state isolates and isolates from only one of the southern hemisphere countries, and three clades that contain isolates from all three countries. Thus, on an annual basis some, but not all, viral populations mix with other populations from the same and/or opposite hemisphere, and this number is likely to increase with additional sampling. Indeed, a majority (nine) of the 16 clades containing southern hemisphere isolates also contained northern hemisphere isolates, suggesting widespread viral traffic across the equator (Table 1). Migration between Australia and New Zealand is also extensive, as almost all clades containing isolates from New Zealand also contained Australian isolates, and vice versa, except for the 1999 season, for which no New Zealand isolates were available. Further, these figures are likely to be underestimates, as mono-national and mono-hemisphere clades may be at frequencies too low to be detected in the genome collections currently available. Alternatively, clades could also have originated in areas not sampled in our study, such as tropical regions, where influenza viruses typically circulate year-round [12].
Strikingly, even for those viral clades that do not exhibit cross-hemisphere migration, there is very little evidence for in situ evolution within specific localities. For example, in no case on any phylogeny are clades of A/H3N2 from New York state directly linked over multiple seasons. Rather, New Zealand and Australia viruses are always interspersed among New York state clades from different seasons, indicating that they are not evolving in geographic isolation across seasons. Most clades from New Zealand and Australia show equivalent patterns of discontinuous evolution, with very few clusters of southern hemisphere clades that are not separated by New York state viruses (although in situ evolution cannot be ruled out between a few 2004 and 2005 clades without additional sampling). Thus, even in relatively isolated areas of New Zealand and Australia, viruses do not regularly evolve in geographic isolation. Rather, evolution appears to be shaped by frequent cross-hemisphere migration and recurrent reintroduction.
Bi-Directional Cross-Hemisphere Migration Occurs between Influenza Seasons
Importantly, our phylogenetic analysis suggests that seasonal migration occurs from the northern hemisphere to the southern hemisphere, as well as south-to-north. Although inferring the direction of viral migration is in part dependent on sample composition, definitive evidence of a migration event are clades containing a single population of northern hemisphere viruses and a single population of southern hemisphere viruses supported by a high level of bootstrap support (>70%).
Because winter influenza seasons alternate by six-month intervals between the northern and southern hemispheres, it was also possible to determine, within the confines of sampling, the timescale and hence direction of cross-hemisphere migration. The inferred directionality of 11 cases of definitive cross-hemisphere migration evident on the HA, NA, and non-surface glycoprotein phylogenies are summarized in Table 2. Of these, all but two involve the concatenated non-surface glycoproteins, which provide a more reliable phylogeny as previously described. Eight of these migration events occur in a north-to-south direction, versus three in a south-to-north direction, suggesting that viruses may migrate more frequently from New York state to the southern hemisphere than in the opposite direction, although this will need to be confirmed with larger sample sizes. For example, on the concatenated gene tree, viral isolates from the 2003–2004 season in New York state form a single well-supported phylogenetic clade (clade b, 100% bootstrap support) with viruses from 2003 from New Zealand and Australia (Figure 4). Since the 2003 New Zealand and Australia viruses predate the 2003–2004 New York state viruses (i.e., the northern hemisphere winter), we infer that the lineage that gave rise to these southern hemisphere viruses migrated northward to infect New York state between the 2003 southern hemisphere winter (May to October) and the 2003–2004 winter in New York state.
http://pathogens.plosjournals.org/perlserv/?request=get-document&doi=10.1371/journal.ppat.0030131
Martha I. Nelson1,2, Lone Simonsen 3, Cecile Viboud 4, Mark A. Miller 4, Edward C. Holmes 1,2,4*
1 Center for Infectious Disease Dynamics, The Pennsylvania State University, University Park, Pennsylvania, United States of America, 2 Department of Biology, The Pennsylvania State University, University Park, Pennsylvania, United States of America, 3 National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America, 4 Fogarty International Center, National Institutes of Health, Bethesda, Maryland, United States of America
The winter seasonality of influenza A virus in temperate climates is one of the most widely recognized, yet least understood, epidemiological patterns in infectious disease. Central to understanding what drives the seasonal emergence of this important human pathogen is determining what becomes of the virus during the non-epidemic summer months. Herein, we take a step towards elucidating the seasonal emergence of influenza virus by determining the evolutionary relationship between populations of influenza A virus sampled from opposite hemispheres. We conducted a phylogenetic analysis of 487 complete genomes of human influenza A/H3N2 viruses collected between 1999 and 2005 from Australia and New Zealand in the southern hemisphere, and a representative sub-sample of viral genome sequences from 413 isolates collected in New York state, United States, representing the northern hemisphere. We show that even in areas as relatively geographically isolated as New Zealand's South Island and Western Australia, global viral migration contributes significantly to the seasonal emergence of influenza A epidemics, and that this migration has no clear directional pattern. These observations run counter to suggestions that local epidemics are triggered by the climate-driven reactivation of influenza viruses that remain latent within hosts between seasons or transmit at low efficiency between seasons. However, a complete understanding of the seasonal movements of influenza A virus will require greatly expanded global surveillance, particularly of tropical regions where the virus circulates year-round, and during non-epidemic periods in temperate climate areas.
Author Summary
The winter seasonality of influenza A virus in temperate climates is one of the most puzzling epidemiological patterns in infectious disease. To help resolve the issue of influenza seasonality, we studied, using viral genome sequence data, the patterns of global migration of influenza A virus, particularly between the northern and southern hemispheres. A phylogenetic analysis of approximately 900 complete genomes of the H3N2 subtype of human influenza A virus sampled from New Zealand and Australia (southern hemisphere), and New York state, United States (northern hemisphere), revealed that cross-hemisphere migration frequently occurs in both directions and involves multiple viral strains. Such global viral traffic therefore contributes significantly to the introduction of new influenza epidemics in both northern and southern hemispheres. These results also show that influenza A virus migrates afar during non-epidemic periods, rather than persisting locally at low levels during the influenza “off-season”. However, although this represents the largest and first bihemisphere study of its kind to our knowledge, the results highlight the need for sampling from tropical regions and during non-epidemic periods in temperate areas. Studies of this kind are critical to fully understand the geographical dispersal of influenza A virus and the role of climate in triggering seasonal epidemics.
Introduction
Influenza A virus is able to persistently re-infect human populations by continually evading host immunity through the rapid evolution of surface antigens (“antigenic drift”) [1]. Influenza virus epidemics strike temperate latitudes of the world each winter, from November to March in the northern hemisphere and from May to September in the southern hemisphere [2]. In the United States alone, these influenza epidemics are associated with an annual average of 36,000 human deaths [3] and 226,000 hospitalizations [4]; globally, the virus is associated with as many as half a million annual deaths [5]. While rapid antigenic change is a hallmark of influenza evolution, recent studies have failed to detect antigenic drift over an epidemic season, suggesting that important evolutionary processes may occur during non-epidemic periods, either locally or perhaps elsewhere [6–8]. However, surveillance during non-epidemic periods is not conducted routinely by the network of World Health Organization influenza reference centers [9] and, consequently, little is known about how and where the virus persists in the human population in between winter epidemics at low levels. A key question is therefore whether the virus remains locally within its host population in between epidemics, perhaps persisting within hosts in a latent state [10], or whether the virus migrates afar to other reservoirs, such as the tropics, and is later reintroduced.
Although influenza virus has long been regarded a “cold-weather” pathogen due to its marked winter epidemics in temperate zones, recent studies show that tropical regions experience significant year-round influenza virus activity [11]. In theory, such a “tropical belt” could serve as a year-round reservoir that harbors endemic populations of influenza virus that seasonally reintroduce viral isolates into temperate zones to trigger new epidemics [12,13]. Whereas population crashes at the end of seasonal epidemics create severe evolutionary bottlenecks that limit genetic diversity, tropical zones may function as permanent mixing pools for viruses from around the world. Historically, Southeast Asia has been considered a potential epicenter for emergence of pandemic viruses due to the proximity with which humans live with their domestic animals [14]. However, abundant data from these regions is currently unavailable, so the origins of influenza pandemics and epidemics remain unclear.
Given the ease and speed with which the influenza virus is thought to spread between humans, it is generally accepted that global chains of direct person-to-person transmission are sufficient to maintain the influenza virus in the human population [15]. However, a complete understanding of how the influenza virus transmits between humans is lacking [16], and whether human-to-human spread alone accounts for the seasonal emergence of epidemics has been questioned [17]. The simultaneous appearance of influenza outbreaks separated by large longitudinal distances, as well as sporadic influenza cases during summer months, suggests that the virus may instead already be “seeded” and somehow reactivated by environmental stimuli. Thus, the alternating pattern of northern and southern hemisphere bi-epidemics could, in principle, also result from opposite climatic forces independently reactivating viral activity in these two hemispheres at alternating six-month intervals. Hence, instead of continually migrating across the equator, separate viral populations could persist locally in an asymptomatic latent state over the summer months until climatic stimuli sufficiently increase host susceptibility and/or viral transmissibility to induce another epidemic. However, hypotheses of how climatic change may directly or indirectly influence viral activity and/or host susceptibility remain largely untested [18,19].
Crucially, some theories for influenza seasonality produce testable phylogenetic hypotheses. On one hand, if influenza A virus persists locally over the summer in a latent state, then isolates sampled over multiple seasons from a single locality would cluster together on a phylogenetic tree, separate from isolates from other geographic regions (Figure 1). Alternatively, if the virus did not evolve in situ between epidemic seasons, but rather traveled globally between epidemics, then the resulting phylogeny would show extensive intermixing of isolates from different localities. To determine whether influenza virus migrates away between the northern and southern hemispheres during non-epidemic summer months, or remains there latently, we conducted an extensive phylogenetic analysis of 399 whole-genome A/H3N2 influenza A viruses sampled from New Zealand (most commonly Canterbury, South Island) from 2000 to 2005 (six seasons), 88 viral genome sequences from Australia (most commonly Western Australia) from 1999 to 2005 (seven seasons), along with a carefully selected sub-sample of 52 isolates that are representative of the clades present in a larger sample of 413 viruses from New York state, United States, collected between 1998 and 2005 and analyzed previously [7]. Given the relative geographic isolation and low population densities of New Zealand's South Island and Western Australia, as well as sampling limitations, this analysis provides a conservative estimate of the extent of cross-hemisphere migration occurring during this time period.
Figure 1. Models for the Global Evolution of Influenza Virus
Two phylogenetic hypotheses depicting the evolution of A/H3N2 influenza virus by (A) global migration, in which isolates from adjacent seasons in the northern and southern hemispheres are interspersed topologically, or by (B) reactivation of latent virus, in which isolates from the northern hemisphere give rise, in situ, to isolates that circulate in the same locality the next season, and thus are topologically linked (likewise for southern hemisphere viruses).
Results
Extensive Viral Genetic Diversity in Australia and New Zealand
Populations of A/H3N2 influenza virus in Australia and New Zealand from 1999 to 2005 exhibit extensive genetic diversity across the entire genome (Figures 2–4), comparable to the diversity observed previously in New York state [7] (in all phylogenies, clades from Australia are shaded blue, those from New Zealand in green, those from New York state in orange, and global isolates in pink). In particular, multiple viral clades co-circulate during each influenza season in New Zealand and Australia, defined as clusters of isolates with high bootstrap support (>70%), or which are separated by exceptionally long branches (Table 1). As with the New York state data, these viruses were collected in the context of seasonal surveillance efforts in Australia and New Zealand and therefore likely provide a representative sample of the overall genetic composition of the viral populations.
Figure 2. Phylogenetic Relationships of the NA Gene of A/H3N2 Influenza Viruses Sampled from New York State (n = 52), New Zealand (n = 51), Australia (n = 45), and Globally (n = 22) from 1998 to 2005, Estimated Using ML
Larger image
Viral isolates from New York state are highlighted in orange, isolates from New Zealand in green, isolates from Australia in blue, and global isolates in pink. Light yellow rectangles identify viral clades, with numbers in white boxes giving bootstrap values for key nodes (>70%). To clarify reassortment events, capital letters in blue refer to clades that appear in section I of the tree (denoted in pink) on the phylogeny of the concatenated six non-surface glycoproteins; lowercase letters in red refer to clades contained within section II (denoted in light green); and lowercase roman numerals in dark green refer to clades outside sections I and II. Bootstrap values highlighted in yellow identify nodes that define a cross-hemisphere migration event. The tree is rooted by isolate A/New York/327/1999 from the 1998–1999 season (i.e., the earliest sampled isolate), and all horizontal branch lengths are drawn to a scale of nucleotide substitutions per site.
Figure 3. Phylogenetic Relationships of the HA Gene of A/H3N2 Influenza Viruses Sampled from New York State (n = 52), New Zealand (n = 51), Australia (n = 45), and Globally (n = 13) from 1998 to 2005, Estimated Using ML
larger image
Color scheme, rooting, scale, and symbols are the same as those used in Figure 2.
Figure 4. Phylogenetic Relationships of the Concatenated Six Non-Surface Glycoprotein Segments of A/H3N2 Influenza Viruses Sampled from New York State (n = 52), New Zealand (n = 51), and Australia (n = 45) from 1998 to 2005, Estimated Using ML
Larger image
Color scheme, rooting, scale, and symbols are the same as those used in Figure 2.
Table 1.
Clades in the HA, NA, and Concatenated Six Non-Surface Glycoprotein Segment Phylogenies Comprised of Viruses from New York State, New Zealand, Australia, and Globally
On the neuraminidase (NA) tree (Figure 2), viruses from New Zealand and Australia fall into at least 15 distinct clades, some of which appear in multiple seasons: three clades circulated in the 1999 season (clades v, vi, and ix; when data was only available from Australia), five in 2000 (clades i, ii, iii, iv, viii), four in 2001 (clades i, iv, v, g), one in 2002 (clade a), one in 2003 (clade b; when a new reassortant virus predominated), two in 2004 (clades A and c), and three in 2005 (clades A, B, E). Clade lettering reflects the three main sections of the phylogeny, based on topology and time: clades A–E contain viral isolates from 2003 to 2005 that fall within section I of the tree (large pink rectangle in upper portion of tree); clades a–e contain isolates from 2001–2003 that fall within section II (large light green rectangle in middle portion of tree); clades i–xiii contain isolates from 1998 to 2000 that fall outside of sections I and II. The hemagglutinin (HA) tree (Figure 3) contains at least 15 clades from New Zealand and Australia: two circulated in 1999 (clades v and x), four in 2000 (clades i, ii, iii, iv), four in 2001 (clades g, i, iv, xiii), two in 2002 (clades h and j), one in 2003 (clade b), two in 2004 (clades A and c), and three in 2005 (clades A, B, E) (Figure 3). Finally, the phylogeny of the concatenated six non-surface glycoprotein segments (PB2, PB1, PA, NP, M1, NS1) contains the largest number of clades, presumably because this larger data set (9,636 bp) provides the greatest resolution. Southern hemisphere isolates form at least 18 clades on this phylogeny: four were present in 1999 (clades v, vi, x, xii), three in 2000 (clades i/ii, iii, iv), four in 2001 (clades g, i/ii, iv, xiii), three in 2002 (clades a, k, l), one in 2003 (clade b), three in 2004 (clades A, F, c), and three in 2005 (clades A, B, E) (Figure 4).
Differences in the number of clades among segments may also be indicative of reassortment, especially involving the HA gene, as previously demonstrated in New York state [7]. Indeed, several major reassortment events are immediately evident from topological incongruities among these three phylogenies. On the HA tree, clades b, c, and e fall in section I along with isolates from 2004 and 2005, while these clades fall in section II amidst 2002 and 2003 isolates in the NA and concatenated six non-surface glycoprotein phylogenies (Figures 2–4). Clade c also falls in section I on the PB2 phylogeny, showing a similar reassortment pattern as HA. However, aside from this lone reassortment event, the phylogenies of the six non-surface glycoprotein segments are very similar, enabling us to study them as a single concatenated entity (phylogenetic trees for individual segments are provided as Figures S1–S6).
Intermixing among Viral Populations from Australia, New Zealand, and New York State
Isolates from the 52 representative New York state genomes are clearly interspersed with southern hemisphere isolates throughout our phylogenetic trees (Figures 2–4; Table 1), indicating that these populations regularly intermix as a result of cross-hemisphere migration. However, patterns of viral intermixing are both variable and complex, as clades from all three phylogenies contain an array of different combinations of viral populations from New Zealand, Australia, and/or New York state. For example, the phylogeny of the concatenated six non-surface glycoproteins contains a relatively even mix of mono-hemisphere and bi-hemisphere clades (Table 1), with five New York state–only clades, seven southern hemisphere–only clades, six clades that contain New York state isolates and isolates from only one of the southern hemisphere countries, and three clades that contain isolates from all three countries. Thus, on an annual basis some, but not all, viral populations mix with other populations from the same and/or opposite hemisphere, and this number is likely to increase with additional sampling. Indeed, a majority (nine) of the 16 clades containing southern hemisphere isolates also contained northern hemisphere isolates, suggesting widespread viral traffic across the equator (Table 1). Migration between Australia and New Zealand is also extensive, as almost all clades containing isolates from New Zealand also contained Australian isolates, and vice versa, except for the 1999 season, for which no New Zealand isolates were available. Further, these figures are likely to be underestimates, as mono-national and mono-hemisphere clades may be at frequencies too low to be detected in the genome collections currently available. Alternatively, clades could also have originated in areas not sampled in our study, such as tropical regions, where influenza viruses typically circulate year-round [12].
Strikingly, even for those viral clades that do not exhibit cross-hemisphere migration, there is very little evidence for in situ evolution within specific localities. For example, in no case on any phylogeny are clades of A/H3N2 from New York state directly linked over multiple seasons. Rather, New Zealand and Australia viruses are always interspersed among New York state clades from different seasons, indicating that they are not evolving in geographic isolation across seasons. Most clades from New Zealand and Australia show equivalent patterns of discontinuous evolution, with very few clusters of southern hemisphere clades that are not separated by New York state viruses (although in situ evolution cannot be ruled out between a few 2004 and 2005 clades without additional sampling). Thus, even in relatively isolated areas of New Zealand and Australia, viruses do not regularly evolve in geographic isolation. Rather, evolution appears to be shaped by frequent cross-hemisphere migration and recurrent reintroduction.
Bi-Directional Cross-Hemisphere Migration Occurs between Influenza Seasons
Importantly, our phylogenetic analysis suggests that seasonal migration occurs from the northern hemisphere to the southern hemisphere, as well as south-to-north. Although inferring the direction of viral migration is in part dependent on sample composition, definitive evidence of a migration event are clades containing a single population of northern hemisphere viruses and a single population of southern hemisphere viruses supported by a high level of bootstrap support (>70%).
Because winter influenza seasons alternate by six-month intervals between the northern and southern hemispheres, it was also possible to determine, within the confines of sampling, the timescale and hence direction of cross-hemisphere migration. The inferred directionality of 11 cases of definitive cross-hemisphere migration evident on the HA, NA, and non-surface glycoprotein phylogenies are summarized in Table 2. Of these, all but two involve the concatenated non-surface glycoproteins, which provide a more reliable phylogeny as previously described. Eight of these migration events occur in a north-to-south direction, versus three in a south-to-north direction, suggesting that viruses may migrate more frequently from New York state to the southern hemisphere than in the opposite direction, although this will need to be confirmed with larger sample sizes. For example, on the concatenated gene tree, viral isolates from the 2003–2004 season in New York state form a single well-supported phylogenetic clade (clade b, 100% bootstrap support) with viruses from 2003 from New Zealand and Australia (Figure 4). Since the 2003 New Zealand and Australia viruses predate the 2003–2004 New York state viruses (i.e., the northern hemisphere winter), we infer that the lineage that gave rise to these southern hemisphere viruses migrated northward to infect New York state between the 2003 southern hemisphere winter (May to October) and the 2003–2004 winter in New York state.
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