Genomic reassortment of influenza A virus in
North American swine, 1998–2011
Martha I. Nelson,1Susan E. Detmer,2David E. Wentworth,3Yi Tan,1
Aaron Schwartzbard,1Rebecca A. Halpin,3Timothy B. Stockwell,3
Xudong Lin,3Amy L. Vincent,4Marie R. Gramer5and Edward C. Holmes1,6
Martha I. Nelson
Received 9 July 2012
Accepted 17 September 2012
1Fogarty International Center, National Institutes of Health, Bethesda, MD 20892, USA
2Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, SK, Canada
3J. Craig Venter Institute, Rockville, MD 20850, USA
4Virus and Prion Research Unit, National Animal Center, USDA-ARS, Ames, IA 50010, USA
5University of Minnesota Veterinary Diagnostic Laboratory, St Paul, MN 55108, USA
6Center for Infectious Disease Dynamics, Department of Biology, The Pennsylvania State
University, University Park, PA 16802, USA
Revealing the frequency and determinants of reassortment among RNA genome segments is
fundamental to understanding basic aspects of the biology and evolution of the influenza virus. To
estimate the extent of genomic reassortment in influenza viruses circulating in North American
swine, we performed a phylogenetic analysis of 139 whole-genome viral sequences sampled
during 1998–2011 and representing seven antigenically distinct viral lineages. The highest
amounts of reassortment were detected between the H3 and the internal gene segments (PB2,
PB1, PA, NP, M and NS), while the lowest reassortment frequencies were observed among the
H1c, H1pdm and neuraminidase segments, particularly N1. Less reassortment was observed
among specific haemagglutinin–neuraminidase combinations that were more prevalent in swine,
suggesting that some genome constellations may be evolutionarily more stable.
Swine express forms of the sialic acid receptor that can be
preferentially bound by both avian (N-acetylneuraminic
acid-a2,3-galactose – NeuAc-a2,3Gal) and human influenza
viruses (NeuAc-a2,6Gal), facilitating inter-species reassort-
ment and the emergence of novel virus variants (Imai &
Kawaoka, 2012). The recent emergence of the pandemic
H1N1/09 (H1N1pdm09) influenza virus, with segments that
have their ultimate origins in avian, human and multiple
swine influenza viruses, exemplifies the capacity of swine to
serve as a ‘mixing vessel’ (Garten et al., 2009; Smith et al.,
2009). Since July 2011 novel reassortant swine-origin
H3N2v influenza viruses containing M segments of
H1N1pdm09 origin were identified in .100 cases of human
infection in the USA, and continue to be monitored closely
for pandemic potential (Centers for Disease Control and
Prevention, 2012; Lindstrom et al., 2012).
Certain constellations of genome segments show evidence
of conservation in swine, such as the combination of six
triple reassortant internal genes (TRIG) that has remained
prevalent in North American swine since 1998. The low fre-
quency of H3N1 subtype viruses in swine similarly suggests
evolutionary constraints on other genomic combinations.
To assess pandemic potential, experimental studies have
used reverse genetics to determine the viability of various
reassortant virusesin animal models, including avian H5N1/
human H3N2 (Chen et al., 2008; Li et al., 2010), avian
H9N2/human H1N1pdm09 (Kimble et al., 2011) and avian
H9N2/human H3N2 reassortants (Sorrell et al., 2009). To
date, however, the patterns of genomic reassortment in
nature have not been estimated using a phylogenetic approach
forthe various influenzaA viruslineagesthat havecirculated
in North American swine since the late 1990s.
To determine the patterns of reassortment in North
American swine, we performed an analysis of 139 whole-
genome sequences collected from North American swine
during 1998–2011. In particular, we compared reassortment
between the neuraminidase (NA) segment, six internal gene
segments and haemagglutinin (HA) segments, which are
associated with seven antigenically distinct swine influenza
virus lineages that have been identified previously in North
American swine: namely, H1a, H1b, H1d, H1c, H1pdm09,
H3-I and H3-IV (Vincent et al., 2008; Ducatez et al., 2011).
Of the 139 genomes, 36 were collected in the USA during
2002–2010 as part of routine diagnostic investigations from
Supplementary material is available with the online version of this paper.
Journal of General Virology (2012), 93, 2584–2589
2584 045930Printed in Great Britain
samples submitted by veterinarians and were selected largely
at random from the influenza virus archive at the University
of Minnesota Veterinary Diagnostic Laboratory. These were
sequenced as part of this study by our colleagues at the
J. Craig Venter Institute using multiplex real-time PCR
(Djikeng et al., 2008; Zhou et al., 2009) (Table S1, available
in JGV Online). The remaining 133 genome sequences were
downloaded from the Influenza Virus Resource available at
NCBI’s GenBank (Bao et al., 2008), 63 of which were
collected from the USA, 36 from Canada and four from
Cuba. Finally, as considerably more sequence data were
available for the HA and NA than for the internal gene
segments, an additional analysis of the large N1 (n5319)
and N2 (n5285) datasets was conducted (Table S2).
Sequence alignments were manually constructed for each
genome segment using Se-Al (Rambaut, 2002). Phylogenetic
trees were inferred separately for each segment using the
BayesianMarkovchain MonteCarlo methodavailableinthe
MRBAYES package (version v.3.1.2; Huelsenbeck & Ronquist,
2001). This analysis incorporated a GTR model of nucleo-
tide substitution with gamma-distributed rate heterogeneity
and a proportion of invariable sites, as determined by
MODELTEST (Posada & Crandall, 1998; parameter values
available from the authors upon request). The analysis was
run for 10 million generations, with two hot chains and one
cold chain, until convergence was achieved, with a 25%
burn-in (for the PA segment convergence was reached
after 25 million generations). For comparison, phylogenies
were also inferred using the maximum-likelihood method
available in PAUP* (Swofford, 2003), again utilizing the
GTR+C+I model substitution model (data available upon
request). Dueto thedeepdivergence betweentheN1andN2
and the H1 and H3 antigens, separate trees were inferred for
these groups of sequences. Genetically distinct HA lineages
were identified on the H1 (five clusters) and H3 phylogenies
(two clusters) (Figs S1 and S2, respectively). The H1a, H1b,
H1c and H1pdm clusters fall within the lineage of ‘classical’
H1 influenza viruses that have circulated in North American
swine since 1918. The H1d viruses comprise two closely
related lineages of human-origin seasonal H1 viruses that
were both introduced into North American swine during
2002–2003 (Karasin et al., 2006; Nelson et al., 2011). The
H3-I and H3-IV clusters are both in the triple reassortant
lineage that was identified in North American swine in 1998
(Zhou et al., 1999). Of the 139 isolates, 14 were of the H1a
lineage, 17 were H1b, 21 were H1d, 17 were H1c, 21 were
H1pdm, 11 were H3-I and 35 were H3-IV (Table S1). In
addition, three isolates exhibited mixed infections, com-
prising H1a/H3-IV, H1b/H3-IV and H1c/H3-IV combina-
tions. Although the greater genetic similarity of the H3
viruses (92–95% sequence similarity between the H3-I and
H3-IV clusters, versus 73–93% similarity among the H1
clusters) meant that the H3-I and H3-IV clusters could have
been analysed as a single H3 cluster, we conservatively
considered the H3-I and H3-IV clusters separately, as
combining them could have inflated the estimate of H3
reassortment. To account for potential sample bias and for
uncertainties in segment assignments in three cases of mixed
infection, we randomly subsampled our data and ran
additional analyses (Text S1).
Reassortment events involving the HA and another
genome segment were identified visually on each phylo-
genetic tree by colour-coding each isolate according to the
HA lineage associated with that viral isolate, as identified
on the H1 and H3 phylogenies: H1a, H1b, H1d, H1c,
H1pdm, H3-I and H3-IV (for illustration, the PB2 phylo-
geny is presented in Fig. 1; phylogenies for the PB1, PA,
NP, N1, N2, M and NS segments are presented in Figs S3–
S9). To identify major reassortment events, isolates were
characterized on each phylogeny by broad classifications of
evolutionary origins: TRIG (avian) for PB2 and PA, TRIG
(human) for PB1, TRIG (classical) for NP, M and NS;
classical swine; human; pandemic; and N2-2002 and N2-
1998 for NA (Table S3). This broad classification scheme is
useful for identifying major reassortment events, particu-
larly those involving segments of pandemic H1N1pdm09
origin. However, it greatly underestimates the amount of
reassortment between more genetically similar viruses
within these broad categories, for example the extensive
intermixing of viruses of the HA types among the multi-
ple clades positioned within the TRIG section of the PB2
phylogeny (Fig. 1). Therefore, additional reassortment
events were visually identified by well-supported nodes (i.e.
posterior probability ¢90%) defining clades containing
more than one HA type. Using these criteria, 22 reassort-
ment events were identified on the PB2 phylogeny, in-
cluding several cases where multiple reassortment events
were defined by the same node (Fig. 1). For example, the
node defining the pandemic H1N1pdm09 clade is asso-
ciated with two separate reassortment events with H1b
and H1d viruses. Similarly, 22 reassortment events were
identified on the PA, M and NS phylogenies; 23 on the NP
tree; and 18 on the PB1 and N2 trees (Table S4, Figs S3–
S9). In contrast, only six reassortment events were evident
on the N1 tree (Fig. S6). The difference in the frequency of
reassortment between the N1 and N2 segments cannot be
explained by differences in sampling, as more N1 sequences
(n573) were available than N2 sequences (n569). In fact,
the addition of 246 N1 sequences, for which whole-genome
sequences were not available for this study, did not result in
the detection of further reassortment events on the larger N1
phylogeny (Fig. S10).
Inferring the directionality of reassortment by the HA type
is complicated when reassortment is so frequent that the
HA type of internal branches is frequently ambiguous. In a
few cases, the direction of reassortment is clear, such as
reassortment events involving the large, well-defined clade
of pandemic H1N1pdm09 viruses. But estimating the
extent of reassortment of other HA types is considerably
more difficult. Rather, it was feasible to estimate the extent
of phylogenetic clustering of each HA type. This provided
another measure of reassortment between a given segment
and the HA, as phylogenies for internal genes would
remain strongly clustered by the HA type in the absence of
Reassortment of influenza virus in swine
reassortment (Fig. 1). As an extreme example, the most
highly clustered H1pdm segment was associated with only
one reassortment event across all eight phylogenetic trees
(with the NP segment, Table S4, Fig. S5). In contrast, the
H3-IV segment exhibited the least evidence of phylogenetic
clustering, with no monophyletic H3-IV clusters con-
taining more than two isolates on the PB2 tree, for example
Fig. 1. This clustering analysis was performed using the
Bayesian Tip-Significance (BaTS) method (Parker et al.,
2008), which determines whether a phenotypic trait, in this
case the HA type, is more associated with the underlying
phylogeny than expected by chance alone. To account for
possible phylogenetic error, BaTS estimates the association
between the HA type and phylogeny over a posterior
distribution of trees generated by MRBAYES.
To compare reassortment patterns among genomic seg-
ments, we determined the ratio of clustering by the HA
type on the internal gene and the NA trees that is expected
by chance alone, compared with the association that is
observed in the data. These expected/observed ratios are
summarized in a heat-map (Fig. 2), with the y-axis ordered
by the amount of reassortment observed among the seven
HA types and the x-axis ordered by the amount of
reassortment between the HA and the six internal genome
segments and N1 and N2. These expected/observed ratios
are largely consistent with the estimates of reassortment
based on well-supported phylogenetic nodes (above). Both
methods identified more reassortment between the HA and
the internal genome segments and relatively less between
the HA and NA segments, particularly N1 (Figs 1 and 2,
Fig. 1. Phylogenetic relationships of the (illustrative) PB2 segment of 139 influenza viruses collected in North American swine
during 1998–2011. Isolates are colour-coded and labelled by the HA lineage, as inferred from the H1 and H3 phylogenies: H1a
viruses are rust, H1b are green, H1c are blue, H1d are turquoise, H1pdm09 are pink, H3-I are yellow and H3-IV are purple.
Posterior probabilities are shown for key nodes, and nodes that define reassortment events are identified by small yellow circles
(single reassortment events) and larger yellow circles (multiple reassortment events). The tree is midpoint rooted for clarity.
Isolates are related to three major lineages of the PB2 segment in swine: classical swine influenza viruses, the TRIG
constellation (including pandemic H1N1pdm09) and human seasonal H1 viruses.
M. I. Nelson and others
2586 Journal of General Virology 93
Table S5). The relatively high rates of reassortment observed
among internal gene segments reflect how the conserved
TRIGconstellation hasremained dominantinNorthAmeri-
can swine by continually reassorting with different HA and
NA segments, displacing the internal gene segments in these
other viral lineages, as has also been observedexperimentally
in swine (Ma et al., 2010).
The BaTS analysis also provides a comparison of reassort-
ment frequencies among the HA types. Expected/observed
ratios were greatest among the H3-IV viruses, indicative of
higher levels of reassortment between the H3 and internal
gene segments: a ratio of 0.65 for PB2, 0.68 for PB1, 0.95
for PA, 0.52 for NP, 0.60 for M and 0.85 for NS (Table S5).
In contrast, the lowest ratios were observed for the H1c
antigen, with values of 0.19, 0.20, 0.17, 0.19, 0.39 and 0.19
for the PB2, PB1, PA, NP, M and NS phylogenies, res-
pectively. The relatively low reassortment frequency for the
H1pdm segment could also reflect limited sampling during
the recent emergence of these viruses in swine; indeed,
reassortment is likely to increase as H1pdm09 viruses
disseminate more widely in pigs and hence have more
opportunities to reassort with other swine viruses.
Overall, our data indicate that the HA reassorts more
frequently with internal gene segments than with NA,
particularly N1. This inference is based on the relatively
low numbers of reassortment events that were visually
identified on the N1 tree, and the relatively high levels of
phylogenetic clustering on the N1 phylogeny (Figs 2, S6
and S10, Table S5). Although only two H3N1 viruses were
identified here, both isolated in 2004 (Lekcharoensuk et al.,
2006), the lower reassortment frequencies of N1 and N2 are
not biased by the limited sampling of some low frequency
HA–NA pairings (e.g. H3N1, H1aN2 and H1pdmN2).
Rather, the lowest frequencies of reassortment are observed
between the most prevalent HA–NA pairings in North
American swine: the H1cN1, H1pdmN1, H1dN2 and H3-
IVN2 (Fig. 2, Table S5). Indeed, the HA–NA pairings
associated with low sample sizes (n,50 isolates with the
particular HA–NA pairing) are associated with higher fre-
quencies of reassortment (mean expected/observed ratio5
observed ratio50.08; P50.01, Wilcoxon non-parametric
t-test) (Table S6).
Understanding how the molecular compatibility between
various HA and NA segments relates to the frequency of
reassortment at a population level clearly requires further
data and analysis. Certain HA segments clearly have a
preference for N2, particularly H3 and H1d (~95% of H1d
viruses were H1N2), whereas 93% of H1c segments were
paired with N1. These observed preferences are consistent
with previous studies (Lorusso et al., 2011; Nelson et al.,
2012) (Tables S1 and S2). It remains unclear why the
human-origin H1d antigen might have a preference for N2
in swine when such pairings are very rare in humans.
Variation in the HA–NA protein compatibility may be host-
dependent (e.g. differences in receptor specificity and/or
enzymic efficiency). Indeed, recent evidence that the func-
tional balance in the HA–NA activity is higher among
human H1N1pdm09 viruses than in swine influenza viruses
potentially explains the greater restrictions on reassortment
between the HA and NA in humans (Xu et al., 2012).
The relatively low frequencies of reassortment between HA
and NA may relate to constraints on maintaining a balance
between receptor binding (HA) and sialic acid cleavage
(NA) activities, which are required for successful viral entry
and release from host cells (Mitnaul et al., 2000). The lesser
N1 N2 PB1 NP PB2
M PA NS
Fig. 2. Relative frequencies of reassortment
for each genome segment among seven
antigenic lineages of influenza virus in North
American swine, coded by colour: lower
expected/observed ratios (,0.2) are yellow,
middle rates (0.2–0.6) are orange and higher
rates (.0.6) are red. Frequency estimates
represent the ratio of association between the
HA type and phylogeny as expected under the
null hypothesis of a random association
between the phylogeny and HA type versus
the association that is observed in the data;
hence, a higher number indicates higher levels
of reassortment. The x-axis and y-axis are
sorted in order of reassortment frequency,
such that the lowest frequencies appear in the
bottom left and the highest in the upper right.
Blank squares indicate a viral sample number
(n¡1) for which rates could not be estimated.
Reassortment of influenza virus in swine
extent of N1 reassortment could reflect differences in the
neuraminidase activity of the N1 protein compared with
N2. HA–NA combinations that are more prevalent in
North American swine (H3N2, H1cN1, H1pdmN1 and
H1dN2) are characterized by lower frequencies of reassort-
ment, which could signify a greater protein compatibility
and evolutionary stability of these pairings. In contrast, less
successful HA–NA combinations (H1bN2, H1dN1 and
H1cN2) do not appear to transmit as efficiently in swine
but are sporadically generated by reassortment, resulting in
phylogenies with less clustering by the HA type.
While experimental studies have advanced our under-
standing of RNA packaging (Gao & Palese, 2009) and the
viability of various avian–human influenza virus reassort-
ants with pandemic potential (e.g. Kimble et al., 2011),
fewer studies consider patterns of intra- and inter-subtype
reassortment at the epidemiological scale. Future studies
utilizing greater whole-genome influenza virus sequence
data may better link the frequency of reassortment with the
population dynamics of individual viral lineages and seg-
ment pairings. Such studies require systematic, population-
based surveillance of influenza in swine, which will facili-
tate comparisons in viral dynamics across space, time and
different virus lineages. Although we attempted here to
account for major biases in sampling, we cannot be certain
that biases in the past collection and sequencing of swine
viruses have notaffectedourfindings.Finally,itisimportant
to determine how the reassortment patterns observed here
among North American swine influenza viruses compare to
frequencies of reassortment among other swine populations,
particularly in regions with high genetic diversity of swine
influenza such as Asia (Vijaykrishna et al., 2011), as well as
with humans and avian species.
We are grateful to the pork producers and swine practitioners for
participating in the USDA Swine Influenza Surveillance System
through the National Animal Health Laboratory Network (NAHLN).
We thank Dan Weinberger and Ce ´cile Viboud of the Fogarty
International Center for technical input. This research was conducted
within the context of the Multinational Influenza Seasonal Mortality
Study (MISMS), an on-going international collaborative effort to
understand influenza epidemiology and evolution, led by the Fogarty
International Center, NIH, with funding from the Office of Global
Affairs at the Department of Health and Human Services (DHHS)
(A.S., E.C.H., M.I.N. and Y.T.). This work was supported in part by
funds from the National Institute of Allergy and Infectious Disease,
the National Institutes of Health and the Department of Health and
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Reassortment of influenza virus in swine