JOURNAL OF VIROLOGY, June 2010, p. 5715–5718
Copyright © 2010, American Society for Microbiology. All Rights Reserved.
Vol. 84, No. 11
Unseasonal Transmission of H3N2 Influenza A Virus
During the Swine-Origin H1N1 Pandemic?†
Elodie Ghedin,1David E. Wentworth,2,3Rebecca A. Halpin,4Xudong Lin,2Jayati Bera,4Jay DePasse,1
Adam Fitch,1Sara Griesemer,2Erin Hine,4Daniel A. Katzel,4Larry Overton,4Kathleen Proudfoot,4
Jeffrey Sitz,4Bridget Szczypinski,4Kirsten St. George,2David J. Spiro,4and Edward C. Holmes5,6*
Center for Vaccine Research, Department of Computational Biology, University of Pittsburgh School of Medicine, Pittsburgh,
Pennsylvania 152611; Wadsworth Center, NYSDH, Albany, New York 122012; School of Public Health, State University of
New York, Albany, New York 122013; J. Craig Venter Institute, 9704 Medical Center Drive, Rockville, Maryland 208504;
Center for Infectious Disease Dynamics, Department of Biology, The Pennsylvania State University, University Park,
Pennsylvania 168025; and Fogarty International Center, National Institutes of Health, Bethesda, Maryland 208926
Received 5 January 2010/Accepted 11 March 2010
The initial wave of swine-origin influenza A virus (pandemic H1N1/09) in the United States during the spring
and summer of 2009 also resulted in an increased vigilance and sampling of seasonal influenza viruses (H1N1
and H3N2), even though they are normally characterized by very low incidence outside of the winter months.
To explore the nature of virus evolution during this influenza “off-season,” we conducted a phylogenetic
analysis of H1N1 and H3N2 sequences sampled during April to June 2009 in New York State. Our analysis
revealed that multiple lineages of both viruses were introduced and cocirculated during this time, as is typical
of influenza virus during the winter. Strikingly, however, we also found strong evidence for the presence of a
large transmission chain of H3N2 viruses centered on the south-east of New York State and which continued
until at least 1 June 2009. These results suggest that the unseasonal transmission of influenza A viruses may
be more widespread than is usually supposed.
The recent emergence of swine-origin H1N1 influenza A
virus (pandemic H1N1/09) in humans has heightened aware-
ness of how the burden of morbidity and mortality due to
influenza is associated with the appearance of new genetic
variants (5) and of the genetic and epidemiological determi-
nants of viral transmission (8). The emergence of pandemic
H1N1/09 is also unprecedented in recorded history as it means
that three antigenically distinct lineages of influenza A virus—
pandemic H1N1/09 and the seasonal H1N1 and H3N2 viruses—
currently cocirculate within human populations.
Although the presence of multiple subtypes of influenza A
virus may place an additional burden on public health re-
sources, it also provides a unique opportunity to compare
the patterns and dynamics of evolution in these viruses on a
similar time scale. Indeed, one of the most interesting sec-
ondary effects of the current H1N1/09 pandemic has been an
increased vigilance for cases of influenza-like illness and
hence an intensified sampling of seasonal H1N1 and H3N2
viruses during the typical influenza “off-season” (i.e., spring-
summer) in the northern hemisphere. Because the influenza
season in the northern hemisphere generally runs from No-
vember through March, with a usual peak in January or
February, influenza viruses sampled outside of this period
are of special interest.
The current model for the global spatiotemporal dynamics
of influenza A virus is that the northern and southern hemi-
spheres represent ecological “sinks” for this virus, with little
ongoing viral transmission during the summer months (9). In
contrast, more continual viral transmission occurs within the
tropical “source” population (13) that is most likely centered
on an intense transmission network in east and southeast Asia
(10). However, the precise epidemiological and evolutionary
reasons for this major geographic division, and for the season-
ality of influenza A virus in general, remain uncertain (1, 4).
Evidence for this “sink-source” ecological model is that viruses
sampled from successive seasons in localities such as New York
State do not usually form linked clusters on phylogenetic trees,
indicating that they are not connected by direct transmission
through the summer months (7). Similar conclusions can be
drawn for the United States as a whole and point to multiple
introductions of phylogenetically distinct lineages during the
winter (6), followed by complex patterns of spatial diffusion
(14). However, despite the growing epidemiological and phy-
logenetic data supporting this model, it is also evident that
there is relatively little sequence data from seasonal influenza
viruses that are sampled from April to October in the northern
hemisphere. Hence, it is uncertain whether extended chains of
transmission can occur during this time period, even though
this may have an important bearing on our understanding of
To address these issues, we examined the evolutionary
behavior of seasonal H1N1 and H3N2 viruses as they cocir-
culated during a single time period—(late) April to June
2009—within a single locality (New York State). Not only
are levels of influenza virus transmission in the northern
hemisphere usually very low during this time period, but in
this particular season the human host population was also
experiencing the emerging epidemic of pandemic H1N1/09.
* Corresponding author. Mailing address: Department of Biology,
The Pennsylvania State University, University Park, PA 16802. Phone:
(814) 863-4689. Fax: (814) 865-9131. E-mail: firstname.lastname@example.org.
† Supplemental material for this article may be found at http://
?Published ahead of print on 17 March 2010.
MATERIALS AND METHODS
Sequence data. The New York State sequence data for the seasonal specimens
were identified by the influenza virus surveillance program and the genome
amplified directly from the swab specimen (16) at the Wadsworth Center, New
York State Department of Health. Genome sequence data was generated at the
J. Craig Venter Institute for the Influenza Genome Sequencing Project and are
freely available on GenBank from where they were downloaded. A number of
procedures were used to minimize the possibility of contamination. Portions of
primary clinical specimens for molecular analysis, including those for whole-
genome sequencing, were divided into aliquots and lysed in an accessioning room
located far from the culture facility, and forwarded directly to the extraction
laboratory. Once there, they were handled in a separate biosafety cabinet from
cultured isolates and extracted on automated nucleic acid extractors dedicated to
primary samples that are never used to extract cultured isolates. In addition,
negative extraction controls were included on every run to check for carryover of
contaminating material, and the negative extraction controls contained carrier
nucleic acid to ensure that even extremely low levels of contamination would be
carried through and be detectable in the subsequent assay. The entire workflow
is unidirectional and staff were not permitted to return to the accessioning or
other ‘clean’ areas. For the multisegment reverse transcription-PCR amplifica-
tion, the RNA was isolated in clean rooms and using equipment dedicated to
preamplification procedures. No template controls were included in both the
RNA extraction and genomic amplification procedures to ensure that amplifi-
cation was specific to the respective specimens.
For H1N1 we obtained seven new sequences sampled between 27 April and 26
May 2009 from five different counties in New York State. For H3N2, a larger
sample of 41 viruses was obtained between 27 April and 1 June 2009 and
representing 20 different counties, although predominantly from the southeast of
the state (Duchess, Nassau, Orange, Putnam, Ulster, and Westchester counties).
These sequences were compared to all those global H1N1 and H3N2 sequences
available on GenBank and covering the years 2008 and 2009 (although most of
the “background” viruses are also sampled from the United States) and for which
the exact day of sampling was available. Because of the very large number of
background HA1 and NA gene sequences available for these viruses, which are
therefore more representative, our analyses focused on these gene segments.
This resulted in data sets of the following size: seasonal H1N1 HA1 ? 652
sequences, 1,050 nucleotides (nt); NA ? 365 sequences, 1,410 nt; H3N2 HA1 ?
1,258 sequences, 1,040 nt; and NA ? 375 sequences, 1,407 nt. Because of the lack
of insertions and deletions all sequence alignments were undertaken by hand
using the SE-AL program (kindly distributed by Andrew Rambaut).
Phylogenetic analysis. Phylogenetic trees were first inferred using the maxi-
mum-likelihood (ML) method available in the PHYML package (2) and using
SPR branch-swapping. In all cases, the GTR??4model of nucleotide substitu-
tion was utilized. We also performed PHYML analyses using the simpler
HKY85??4substitution model and recovered tree topologies that contained the
same major groupings with respect to the New York State viruses (which are the
only phylogenetic patterns of interest in the present study). To further assess
the robustness of our analyses, we undertook an additional phylogenetic analysis
utilizing the ML approach available in the GARLI package (15), adjusting the
run length parameter (to 100,000 steps) to improve the search quality. This
analysis again made use of the GTR??4substitution model. Because of the
similarity of the resulting phylogenies, only those generated by PHYML are
shown here. Finally, for all data sets, a bootstrap resampling procedure was
undertaken using 1,000 replicate neighbor-joining trees and the parameter set-
tings for the GTR??4model of nucleotide substitution determined in PHYML.
This analysis was undertaken using the PAUP* package (12). All relevant pa-
rameter values for the analyses described above are available from the authors
RESULTS AND DISCUSSION
Seasonal H1N1. Because of the very small size of the sea-
sonal H1N1 data set sampled from New York State (n ? 7)
from April to May 2009, few strong conclusions could be drawn
on the overall molecular epidemiology of this virus in this
population. However, it is clear that all but two of the HA1
sequences from New York State fall into disparate locations on
the phylogenetic tree, suggesting that they represent indepen-
dent entries into this population and for which there has likely
been no significant onward transmission (Fig. 1). A similar
pattern is observed in the NA phylogeny (see Fig. S1 in the
supplemental material). The interesting exception are two vi-
ruses (A/New York/3768/2009 and A/New York/3467/2009)
sampled on 13 and 26 May from the adjacent Steuben and
Ontario counties, respectively, and which form a monophyletic
group in the NA phylogeny and which are closely related in the
HA1 tree (forming a clade with A/Massachusetts/4/2009 sam-
pled on 27 January 2009; Fig. 1, asterisk). Such close clustering
is compatible with the hypothesis that these viruses are linked
by a direct chain of transmission. However, it is also notewor-
thy that these viruses are characterized by relatively long
branch lengths (in marked contrast to the H3N2 transmission
cluster described below), which may be indicative of multiple
unsampled links within the New York State transmission chain,
independent entries from other locations, or greater antigenic
diversity which may in turn increase the possibility of transmis-
sion. Similarly, it is notable that all seven newly sampled New
York State viruses are closely related to viruses sampled in the
early months (January to March) of 2009 in the United States,
most notably the isolate from Massachusetts. It is therefore
possible that despite their diverse phylogenetic locations, these
viruses have also been transmitted within the greater U.S.
population to at least the end of April. Clearly, a greater
sampling of viruses from multiple locations is needed to con-
firm this hypothesis.
Seasonal H3N2. As with H1N1, our analysis of the H3N2
virus population from April to June 2009 reveals that there
have again been multiple entries of the virus into New York
State, reflected in the presence of distinct clusters of viruses on
both the HA1 (Fig. 2) and NA (see Fig. S2 in the supplemental
material) phylogenies. The most striking result, however, is the
presence of a cluster of 21 very closely related viruses that was
sampled over a time period of 5 weeks from 27 April to 1 June
FIG. 1. ML tree (unrooted) of 652 HA1 sequences of seasonal
H1N1 virus sampled globally during the period from 2008 to 2009. The
seven viruses sampled from New York State during the period 27 April
to 26 May are shaded red, with each likely introduction event marked
by a square. A possible transmission cluster is denoted with an asterisk;
the bootstrap value for this node is also shown.
5716 GHEDIN ET AL.J. VIROL.
2009 and characterized by very short branch lengths (including
predominantly identical sequences). That all but one of these
viruses was sampled from six adjacent counties in the most
populous south-east region of New York State strongly sug-
gests that they have been sampled from a direct chain of
transmission (Fig. 2b and c). Importantly, this putative trans-
mission cluster was observed in both the HA and the NA
phylogenies. In addition, the strict quality controls in place at
the Wadsworth Center and the fact that members of this clus-
ter were extracted at different times makes cross-contamina-
tion highly unlikely. That a single member of this cluster was
sampled from Tompkins county in central New York State
suggests that this transmission chain may extend even further
in time and space. Indeed, that an additional 13 viruses fall
very close to the transmission cluster in all phylogenies, being
separated from the main cluster by other U.S. viruses (Fig. 2),
supports the notion that this transmission chain is widespread.
A second possible transmission cluster concerns two viruses
(A/New York/3104/2009 and A/New York/3280/2009) sampled
on 29 April and 3 May in Wyoming and Orange counties,
respectively. Although this cluster contains only two isolates,
and from counties that do not share a border, the sequences in
question are identical, which again suggests that they form part
of a larger transmission chain.
The intense interest in pandemic H1N1/09 has ignited an
extensive program of surveillance for influenza-like illness and
genome sequencing. This has enabled us to detect, for the first
time, the extended transmission of seasonal influenza A virus
during the influenza “off-season” in the northern hemisphere.
Although most lineages of influenza virus undoubtedly die-off
FIG. 2. (a) ML tree (unrooted) of 1,258 HA1 sequences of seasonal H3N2 sampled globally during the period from 2008 to 2009. The 41 viruses
sampled from New York State during the period 27 April to 1 June are shaded red, with each likely introduction event marked by a square. Possible
transmission clusters are noted by asterisks, and their associated bootstrap values are also shown. Although a bootstrap value ?70% is observed
for the main transmission cluster, this group of sequences is strongly supported (0.84) under the approximate likelihood ratio test available in
PHYML. (b) Counties of New York State from where the putative H3N2 transmission cluster was sampled (shaded circles). (c) Magnification of
the 21-sequence cluster that provides strong evidence for the unseasonal transmission of H3N2.
VOL. 84, 2010TRANSMISSION OF H3N2 VIRUS DURING THE H1N1 PANDEMIC 5717
during the summer months, so that the influenza epidemics
that occur during any specific season are most likely caused by
the seeding, each winter, of viruses ultimately derived from a
global reservoir population, our study clearly shows that the
onset of spring in the northern hemisphere does not necessar-
ily spell the immediate end of virus transmission: that at least
21 viruses (and likely many more) sampled across 5 weeks in
seven counties form a tight monophyletic group in the H3N2
phylogenies represents strong evidence that influenza A virus
has been transmitted in the population for at least this period.
In addition, our phylogenetic analysis reveals that multiple
lineages of H1N1 and H3N2 also circulate during the influenza
off-season, again illustrating how influenza A virus is easily
able to exploit human contact networks. The remaining ques-
tion is whether such unseasonal transmission will produce a
chain long enough that it enables the virus to persist from
epidemic to epidemic; that is, to survive the entire summer
period? From the large-scale phylogenetic data presented to
date there is no strong evidence for such extended transmis-
sion, as lineages of viruses sampled from successive influenza
seasons tend not to cluster together (7).
More generally, the observation of the unseasonal transmis-
sion of H3N2 (and perhaps H1N1) may have implications for
our understanding of the causes of seasonality in influenza
virus. In particular, that the virus is able to establish transmis-
sion networks in the warmer and more humid months of late
April to June in New York State indicates that the virus is able
to spread in what may be suboptimal climatic conditions (3, 11)
if a sufficient number and density of susceptible hosts is avail-
able. Although the spring and summer of 2009 was highly
unusual in that H1N1/09 as well as seasonal influenza viruses
cocirculated in the U.S. population, summer waves of influenza
virus transmission are often observed during pandemics. We
therefore suggest that the more intensive sampling of influenza
virus genetic diversity during the summer months in the north-
ern hemisphere may represent a useful additional way to ex-
plore the issues that underpin the distinctive seasonality of
influenza A virus.
This study was in part funded by NIH grant GM080533 and NIH
contract HHSN272200900007C. D.E.W. is funded in part by NIH/
NIAID P01 AI059576-05 and the New York State Department of
Health. K.S.G. is supported in part by the New York State Department
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