﻿Amino Acids Transitioning of 2009 H1N1pdm in Taiwan
from 2009 to 2011
Guang-Wu Chen1,2.
, Kuo-Chien Tsao2,3,6
*.
, Chung-Guei Huang2,3,6
, Yu-Nong Gong5
, Shih-
Cheng Chang2,3
, Yi-Chun Liu6
, Hsiao-Han Wu6
, Shu-Li Yang6
, Tzou-Yien Lin2,4,7
, Yhu-Chering Huang4,7
,
Shin-Ru Shih2,3,6
1 Department of Computer Science and Information Engineering, Chang Gung University, Taoyuan, Taiwan, Republic of China, 2 Research Center for Emerging Viral
Infections, Chang Gung University, Taoyuan, Taiwan, Republic of China, 3 Department of Medical Biotechnology and Laboratory Science, Chang Gung University, Taoyuan,
Taiwan, Republic of China, 4 College of Medicine, Chang Gung University, Taoyuan, Taiwan, Republic of China, 5 Graduate Institute of Electrical Engineering, Chang Gung
University, Taoyuan, Taiwan, Republic of China, 6 Department of Laboratory Medicine, Chang Gung Memorial Hospital, Linkou, Taoyuan, Taiwan, Republic of China,
7 Department of Pediatrics, Chang Gung Memorial Hospital, Linkou, Taoyuan, Taiwan, Republic of China
Abstract
A swine-origin influenza A was detected in April 2009 and soon became the 2009 H1N1 pandemic strain (H1N1pdm). The
current study revealed the genetic diversity of H1N1pdm, based on 77 and 70 isolates which we collected, respectively,
during the 2009/2010 and 2010/2011 influenza seasons in Taiwan. We focused on tracking the amino acid transitioning of
hemagglutinin (HA) and neuraminidase (NA) genes in the early diversification of the virus and compared them with
H1N1pdm strains reported worldwide. We identified newly emerged mutation markers based on A/California/04/2009,
described how these markers shifted from the first H1N1pdm season to the one that immediately followed, and discussed
how these observations may relate to antigenicity, receptor-binding, and drug susceptibility. It was found that the amino
acid mutation rates of H1N1pdm were elevated, from 9.2961023
substitutions per site in the first season to 1.4661022
in
the second season in HA, and from 5.2361023
to 1.1061022
in NA. Many mutation markers were newly detected in the
second season, including 11 in HA and 8 in NA, and some were found having statistical correlation to disease severity. There
were five noticeable HA mutations made to antigenic sites. No significant titer changes, however, were detected based on
hemagglutination inhibition tests. Only one isolate with H275Y mutation known to reduce susceptibility to NA inhibitors
was detected. As limited Taiwanese H1N1pdm viruses were isolated after our sampling period, we gathered 8,876 HA and
6,017 NA H1N1pdm sequences up to April 2012 from NCBI to follow up the dynamics of mentioned HA mutations. While
some mutations described in this study seemed to either settle in or die out in the 2011­2012 season, a number of them still
showed signs of transitioning, prompting the importance of continuous monitoring of this virus for more seasons to come.
Citation: Chen G-W, Tsao K-C, Huang C-G, Gong Y-N, Chang S-C, et al. (2012) Amino Acids Transitioning of 2009 H1N1pdm in Taiwan from 2009 to 2011. PLoS
ONE 7(9): e45946. doi:10.1371/journal.pone.0045946
Editor: Patrick C. Y. Woo, The University of Hong Kong, China
Received June 20, 2012; Accepted August 23, 2012; Published September 24, 2012
Copyright: ß 2012 Chen et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: Grants from Chang Gung Memorial Hospital: CMRPG390601, CMRPG390602, CMRPG320018, CMRPD180213. http://www.cgmh.org.tw. Grants from
National Science Council, Taiwan: NSC97-2221-E-182-034-MY3. http://www.nsc.gov.tw. The funders had no role in study design, data collection and analysis,
decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: kctsao@adm.cgmh.org.tw
. These authors contributed equally to this work.
Introduction
A swine-origin influenza A virus (S-OIV) was first found in
North America in April 2009 [1] and soon became the 2009
H1N1 pandemic strain (H1N1pdm). This novel virus has been
identified as a re-assortment of previously known human, avian,
and swine influenza A viruses, following a complete deciphering of
its 8 segmented RNA fragments [2]. In August 2010 the World
Health Organization (WHO) announced that H1N1pdm infection
had moved into the post-pandemic period, and predicted that
localized outbreaks of various magnitudes were likely to occur for
a few years, which would resemble the behavior of a seasonal
influenza virus (WHO Media centre ­ H1N1 in post-pandemic
period. 10 August 2010). While H1N1pdm was still globally seen
in 2010/2011 season, the number of isolates declined considerably
from its debut in 2009 season. Recent WHO reports indicated
influenza A(H3N2) as the most detected virus in the northern
hemisphere in 2011/2012 season. The number of global
H1N1pdm cases continued to go down from its previous two
seasons (less than 10% of all positive specimens for influenza), and
was only found dominating in Mexico and central America (WHO
FluNet, 27 April 2012).
Influenza hemagglutinin (HA) is a major antigenic glycoprotein
responsible for binding the virus to the cell that is being infected.
Influenza neuraminidase (NA) is another viral glycoprotein which
cleaves the glycosidic linkages of neuraminic acids to free the
newly formed virions away from the host cell receptors. NA is also
an important drug target for the prevention of influenza infection.
This is especially true because the other influenza matrix protein,
M2, has evolved to significantly lose its susceptibility to
adamantanes (including amantadine and rimantadine) that has
been used to treat the disease for more than 30 years [3,4].
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Neuraminidase inhibitors (NAIs), including oseltamivir (Tamiflu)
and zanamivir (Relenza), are the other class of antivirals used to
control influenza infection. Recent reports, however, have shown
that oseltamivir-resistant seasonal H1N1 viruses became wide-
spread since the 2007/2008 season in the northern hemisphere
[5].
The 2009 H1N1pdm virus acquired its HA gene directly from
the classic swine influenza A virus of North American lineage,
which can be further traced back to the 1918 virus [6]. The virus
took its NA and M genes from Eurasian swine, which equipped it
with a completely different set of NA and M genes from those of
seasonal H1N1 or H3N2­which are reportedly resistant to the
above-mentioned antivirals at different levels. Thus far all
H1N1pdm viruses, unfortunately, have been found to be resistant
to amantadine and remantadine (WHO fourth NIC Meeting
Report in the Western pacific Region, May, 2010). Although most
of the tested H1N1pdm viruses at the end of 2009/2010 season
were still susceptible to zanamivir and oseltamir, rare cases were
shown to share a single amino acid substitution H275Y in their
NA gene, which costs drug susceptibility [7]. More than one study
also indicated that the excessive use of NAI drugs is likely to
increase the chance of NAI-resistant viruses evolving [8,9].
The current study elucidated the evolutionary dynamics of
H1N1pdm, based on 77 and 70 isolates which we collected,
respectively, during the 2009/2010 and 2010/2011 influenza
seasons in Taiwan. It was found that the amino acid mutation
rates for both HA and NA nearly doubled in the second season
than they were in the first season. In particular that some of the
newly found mutation markers in the second season showed
statistical correlation to disease severity. Although there were five
noticeable HA mutations made to antigenic sites, no visible titer
changes were detected based on hemagglutination inhibition tests.
All Taiwanese isolates maintained susceptibility to NAIs, except
one isolate observed with drug resistance marker H275Y in early
2011.
Materials and Methods
Ethics Statement
This study has been approved by the Institutional Review Board
(IRB) of Chang Gung Medical Foundation, Linkou Medical
Center, Taoyuan, Taiwan. The IRB approved number is ``98-
2707B'' and the topic is titled ``Antiviral Susceptibility Surveillance
of Novel Swine-Origin Influenza A H1N1''. We used residual
virus isolates grown from the specimens of potential influenza A
H1N1 patients during their routine checkup. Since no extra
clinical specimens were collected from patients and human
specimens were not directly used in this research, the IRB agreed
that no written or verbal informed consent was necessary.
Sample Collection
We collected 1,590 H1N1pdm isolates at Chang Gung
Memorial Hospital (CGMH), Taoyuan, Taiwan, from June
2009 to February 2011. Two to four of these isolates per week
were recruited and their HA and NA sequences were produced
and analyzed. A total of 147 samples were obtained, including 77
from June 2009 to May 2010 in the first pandemic season, and
another 70 from August 2010 to February 2011 in the second
season.
Statistical Methods
Student's t-test was used to assess the overall difference in terms
of mutation frequencies between severe (pneumonia, acute
respiratory distress syndrome, or expired) and non-severe (other
upper respiratory tract infections) cases of H1N1pdm infections.
The correlation of site-specific mutations between severe and non-
severe cases was evaluated using two-tailed Fisher's exact test.
Figure 1. Chronological surveillance of influenza viruses in Chang Gung Memorial Hospital from 2007 to 2012.
doi:10.1371/journal.pone.0045946.g001
Amino Acids Transitioning of 2009 H1N1pdm
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Nucleotide Sequencing
Nucleotide sequences of HA and NA were obtained using RT-
PCR and the Sanger dideoxy sequencing method. A total of six
primer pairs were used for sequencing HA and NA genes, and are
listed in Table S1. The obtained amplicons were assembled into
a full-length 1,701-bp span for HA and 1,410-bp for NA, using
DNASTAR Lasergene (DNAStar, Madison, WI).
Sequence Analysis
Nucleotide sequences were aligned and translated into protein
sequences using BioEdit (Tom Hall, Ibis Biosciences, Carlsbad,
CA). The prototype strain A/California/04/2009-the first
H1N1pdm isolate and candidate vaccine virus used by the
Centers for Disease Control and Prevention (CDC/USA), served
as a reference strain to display the amino acid changes in the
investigated Taiwanese strains. GenBank accession numbers, for
HA and NA of A/California/04/2009, are FJ966082 and
FJ966084, respectively.
Nucleotide Sequence Accession Numbers
All newly-reported sequences in this study have been deposited
at GenBank database under the accession numbers CY045226,
CY045234, CY045242, CY047744, CY053474, CY053482,
CY053490, CY053498, CY053506, and JN381203-JN381340
for the HA genes; and CY045228, CY045236, CY045244,
Table 1. Monthly HA gene mutation frequencies (in percentage) for H1N1pdm.
Mutation Type 2009/10 2010/11 Total (%)
Jun Jul Aug Sep Oct Nov Dec Jan Feb May Aug Sep Oct Nov Dec Jan Feb
L8M II.b 14 17 30 5.4
T14I II.c 17 30 6.1
P100S I.a 100 100 100 100 100 100 100 100 100 100 83 100 100 100 100 100 95 98.6
D114N II.c 28 45 9.5
N142D II.a 50 50 60 43 83 25 11 12.9
S160G II.b 29 25 50 40 14.3
S202T II.b 43 17 63 72 65 23.8
T214A I.b 92 100 100 100 100 100 100 100 100 100 100 100 57 100 50 50 55 82.3
S220T I.a 92 100 86 100 89 89 100 100 100 50 100 100 100 100 100 100 90 95.2
R222K I.c 14 17 30 6.8
I233V II.c 17 35 6.8
V266L I.c 7 11 28 35 9.5
K300E I.c 8 11 30 6.1
I338V I.a 100 100 100 93 100 100 100 100 100 100 100 100 100 100 75 100 95 97.3
E391K I.b 8 29 43 78 75 100 75 100 50 67 60 86 100 75 33 80 59.2
S468N I.b 8 13 43 17 63 72 65 25.2
Samples/month 13 5 7 14 9 8 9 8 2 2 6 5 7 6 8 18 20 147
doi:10.1371/journal.pone.0045946.t001
Table 2. Monthly NA gene mutation frequencies (in percentage) for H1N1pdm.
Mutation Type 2009/10 2010/11 Total (%)
Jun Jul Aug Sep Oct Nov Dec Jan Feb May Aug Sep Oct Nov Dec Jan Feb
M15I II.a 50 17 40 29 67 11 8.2
N44S II.b 43 17 75 50 45 19.0
V106I I 100 100 100 100 100 100 100 100 100 50 100 100 100 100 100 100 100 99.3
N189S II.a 17 60 29 67 11 8.2
V241I II.b 43 17 88 72 60 24.5
N248D I 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100
S299A II.c 17 35 6.8
I365T II.a 33 20 29 50 5.4
N369K II.b 43 17 88 72 65 25.2
I374V II.c 17 35 6.8
Samples/month 13 5 7 14 9 8 9 8 2 2 6 5 7 6 8 18 20 147
doi:10.1371/journal.pone.0045946.t002
Amino Acids Transitioning of 2009 H1N1pdm
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CY047746, CY053476, CY053484, CY053492, CY053500,
CY053508, and JN381343-JN381480 for the NA genes.
Results
Influenza Epidemiology in Taiwan
Figure 1 shows the chronological surveillance of influenza
viruses at Chang Gung Memorial Hospital (CGMH) in Taiwan
from 2007 to 2012. Although influenza B appeared to dominate in
the winter of 2006/2007, positive cases of seasonal H1 viruses rose
during the following two winters. During the 2009 H1N1pdm
outbreak, large number of H1N1pdm cases were detected between
approximately June 2009 and January 2010, similar to what was
observed elsewhere in the world. H1N1 pdm activity seemed to die
out after its historical debut, and influenza B viruses took over in
the first half of 2010, followed by predominantly influenza H3
viruses in the summer months. Sporadic H1N1pdm cases were still
observed in the summer months of 2010, until a surge of
H1N1pdm cases emerged from mid- to late 2010 until early 2011.
Influenza B viruses were mostly detected since April 2011, and
peaked in the winter months of 2011/2012. Some influenza A
H3N2 cases were also observed, with very limited H1N1pdm
viruses detected in the 2011/2012 season. In the discussion that
follows, for the sake of convenience we define the first H1N1pdm
season as having spanned from June 2009 to May 2010, and the
second season from August 2010 to February 2011. These time
periods were ascertained from the distribution of isolation counts,
shown in Figure 1.
Amino Acid Transitioning of Taiwanese H1N1pdm
Viruses
All sequences were compared to A/California/04/2009 to
highlight their amino acid changes. Among the 566 amino acid
positions of the full-length HA gene in 147 Taiwanese H1N1pdm
strains (spanning the two influenza seasons from June 2009 to
February 2011), 100 positions (17.7%) were found with amino acid
substitutions. Although many of these changes were merely
transient, 16 positions showed a mutation frequency of more than
5% among the 147 HA samples. Table 1 lists their monthly
mutation frequencies; March, April, June, and July of 2010 were
excluded as no H1N1pdm viruses were isolated in CGMH during
those months. It is clear that P100S (98.6%), S220T (95.2%), and
I338V (97.3%) dominated since June 2009, and still maintained
over 90% frequencies in season-ending months in 2011. These
three mutations quickly settled and dominated in the virus
population were assigned to type I.a in Table 1. T214A was also
frequently seen throughout the entire sampling time, yet it
appeared in only approximately half the viruses isolated from
December 2010 to February 2011. E391K was another sub-
stitution that was less seen at the beginning of pandemic but
largely emerged only after September 2009. It resembled the
temporal distribution of T214A which appeared less frequently in
season-ending months, in contrast to the total dominance observed
in type I.a mutations. Both T214A and E391K were grouped as
type I.b mutations, together with S468 which was sporadically
seen in the first season and commonly appeared since October
2010 in the second season.
The remaining 10 signatures appeared at various stages in these
two seasons. For example, three group I.c mutations were
sporadically observed relatively early in 2009, including R222K
in August, V266L in September/October, and K300E in June.
They remained completely silent, however, for the entire 2010 and
appeared again only in early 2011. Seven other group II mutations
appeared only in the second season, including one type II.a
mutation N142D since the summer of 2010; three type II.b
mutations L8M, S160G and S202T since October 2010; and three
type II.c mutations T14I, D114N and I233V since January 2011.
Interestingly, T14I, D144N, R222K, I233V, V266L, and K300E
seemed to have a synchronized appearance in January and
February of 2011, either for the first time or as a re-emergence
after being absent for the entire 2010. All 16 HA mutations still
showed at least 30% frequency (6 out of 20 cases) in February
2011, except for N142D which was last seen in only two out of 18
viruses in January 2011 but none in February. The complete
amino acid mutation statistics of 147 Taiwanese HA sequences
can be seen in Table S2.
NA protein sequences of 469-aa for the same 147 Taiwanese
H1N1pdm strains were also analyzed. We found 69 positions
(14.7%) that had different amino acid residues from A/California/
04/2009. Many of these NA mutations were rarely seen, similar to
what was observed in HA. Table 2 shows ten major NA sites with
more than 5% mutation frequency, among which V106I and
N248D of type I were present at all times. Other eight mutations
entered no earlier than May 2010, with three type II.a mutations
M15I, N189S, and I365T ceasing to appear in the final months of
the 2010/11 season. In other words, only five new primary NA
mutations survived at the end of February 2011; of these, three
type II.b mutations N44S, V241I, and N369K had not been seen
until October 2010, and two type II.c mutations S299A and
Table 3. Monthly accumulated amino acid mutation counts
and frequencies (mutations per sequence) for 147 H1N1pdm
HA and NA genes isolated in Chang Gung Memorial Hospital,
Taiwan.
HA NA
Seq cnt Accu. mut. Mut. freq. Accu. mut. Mut. freq.
Jun/200913 59 4.54 29 2.23
Jul 5 21 4.20 11 2.20
Aug 7 35 5.00 19 2.71
Sep 14 73 5.21 33 2.36
Oct 9 50 5.56 21 2.33
Nov 8 44 5.50 20 2.50
Dec 9 50 5.56 22 2.44
Jan/2010 8 47 5.88 22 2.75
Feb 2 14 7.00 5 2.50
May 2 12 6.00 7 3.50
Aug 6 45 7.50 21 3.50
Sep 5 39 7.80 24 4.80
Oct 7 57 8.14 35 5.00
Nov 6 54 9.00 35 5.83
Dec 8 65 8.13 48 6.00
Jan/2011 18 138 7.67 97 5.39
Feb 20 180 9.00 101 5.05
Season 1 77 405 5.24a
189 2.45b
Season 2 70 578 8.26c
361 5.16d
Total 147 983 6.69 550 3.74
a
9.2961023
,
b
5.2361023
,
c
1.4661022
,
d
1.1061022
substitutions per amino acid site.
doi:10.1371/journal.pone.0045946.t003
Amino Acids Transitioning of 2009 H1N1pdm
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I374V were only observed after January 2011. Interestingly, the
appearance of NA mutations of type II.a, II.b and II.c completely
synchronized with type II.a, II.b and II.c HA mutations listed in
Table 1, respectively. The complete amino acid mutation statistics
of the 147 Taiwanese NA sequences can be seen in Table S3.
Table 3 shows the statistics of amino acid substitutions for the
two H1N1pdm seasons investigated. The mutation counts shown
here represent all amino acid substitutions based on A/California/
04/2009 within the given month. A mutation frequency was
computed by dividing the accumulated mutations by the sample
count per month, per season, and so on. The mutation frequencies
were higher for HA than for NA protein in all months. We also
found that the mutation frequencies in the second season were
8.26 sites per 566-aa HA sequence (or 1.4661022
per amino acid
site) and 5.16 sites per 469-aa NA sequence (or 1.1061022
per
amino acid site). These rates were elevated compared with the
5.24 and 2.45 sites per sequence for HA and NA (or 9.2961023
and 5.2361023
per amino acid site), respectively, found for the
previous season­that is, when the pandemic first took place in
2009.
We further traced 133 samples whose clinical records are
available and compared their disease severity with the observed
HA/NA diversity. Among these, 82 are non-severe cases of upper
respiratory tract infection and 51 are severe cases of lower
respiratory tract infection, including 37 with pneumonia and 14
with acute respiratory distress syndrome (ARDS) or expired. As
shown in Table 4, viruses from patients that were very sick did
seem to mutate more. Table 5 lists a number of amino acid sites
that exhibit significant difference in mutation frequencies between
the non-severe and severe cases. Interestingly, all these eight
signatures emerged only in second season (no earlier than October
Table 4. Correlation of mutation frequencies on HA and NA genes between severe and non-severe cases of H1N1pdm infection.
HA NA
Mutation frequency p-value*
Mutation frequency p-value*
Non-severe casesa
(N = 82) 6.1 ­ 3.3 ­
Severe cases (N = 51) 7.1 0.001 4.4 ,0.001
Pneumonia (N = 37) 6.7 0.09 4.3 ,0.001
ARDSb
or Expired (N = 14) 8.5 ,0.001 4.6 ,0.05
a
Upper respiratory tract infection.
b
Acute respiratory distress syndrome.
*Student's t-test.
doi:10.1371/journal.pone.0045946.t004
Table 5. Correlation of significant mutation sites on HA and NA genes between severe and non-severe cases of H1N1pdm
infection.
HA (%) NA (%)
S160G S202T V266L S468N N44S V241I S299A N369K
Non-severe casesa
(N = 82)
8(9.7) 14(17) 2(2.4) 16(19.5) 0 (0) 13 (16.0) 0 (0) 13 (16.0)
Severe casesb
(N = 51)
12(23.5) 21(41.2) 9(17.6) 21(41.2) 15 (29.4) 21 (41.2) 4 (7.8) 21 (41.2)
p-value*
0.04 0.003 0.003 0.009 ,0.0001 0.002 0.02 0.002
a
Upper respiratory tract infection.
b
Pneumonia, Acute respiratory distress syndrome, Expired.
*Fisher's exact test.
doi:10.1371/journal.pone.0045946.t005
Table 6. NA amino acid substitutions and their frequencies
(mutation count divided by total count) in drug-associated
sites of Taiwanese H1N1pdm viruses.
Month/Residue/
Pos I223 S247 H275 N295
Jun/2009
Jul
Aug
Sept
Oct
Nov
Dec
Jan/2010 T(1/8)
Feb
May
Aug
Sept
Oct
Nov
Dec N(1/8)
Jan/2011 K(2/18)
Feb Y(1/20)
doi:10.1371/journal.pone.0045946.t006
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of 2010), suggesting their roles in complicating the disease as the
virus evolved.
HA Antigenicity and Receptor Binding
Igarashi et al. [10] predicted a number of antigenic sites of
H1N1pdm hemagglutinin by homology modeling of the earlier
H1N1 viruses. Approximately half of these antigenic residues (7 of
13 Sa sites, 6 of 12 Sb sites, and 8 of 19 Ca sites) showed amino
acid substitutions in the investigated Taiwanese HA sequences; an
exception was the Cb site, for which all six antigenic residues
remained unchanged. Many of these substitutions, however,
occurred in rather limited cases. For example, some substitutions
occurred only once among 77 Taiwanese viruses collected in the
first pdm season, and were no longer observed at all during the
second season. These substitutions included P141S and K177T in
Sa, A203T and A212V in Sb, and H155Y and I183F in Ca.
Others emerged only in the second season, typically only once but
at most twice; these substitutions included G172E, N173T, P176S,
and K180T in Sa; T201N, S207I, and L208I in Sb; and G187R in
Ca. Some transient substitutions were found in Ca sites for both
seasons. For example, A158S and D239E were found in the first
season, whereas in the second season they were changed into
different residues of E and G, respectively. Aside from these rare
substitutions, S220T in Ca was found to dominate in all months of
both seasons, as shown in Table 1. Antigenic mutations seen
mainly in the second season included N142D in Sa and S202T in
Sb, although the former seemed to die out after December 2010
and had completely disappeared by February 2011 (Table 1). In
contrast, S202T has recently become dominant in Taiwan only in
end-of-season months. The last two noticeable antigenic mutations
included A156T and R222K in Ca. Mutation A156T occurred
only in four of 13 viruses collected from October to November
2010, and went completely silent for the remainder of the season.
Mutation R222K was first seen only in August 2009, and re-
emerged in nine out of 38 viruses collected in January and
February 2011. Details of the described antigenic mutations can
be seen in panels 1 to 4 of Table S4.
Following the observation that a number of HA amino acid
changes were located on the predicted antigenic sites, hemagglu-
tination inhibition (HI) tests were additionally performed to assess
if the mutated viruses have changed their antigenic profile. Ferret
anti-serum against A/California/07/2009 (kindly provided by Dr.
Ming-Tsan Liu, Taiwanese CDC) was used in these tests. It was
mentioned earlier that there are five HA locations with noticeable
Table 7. Various HA mutations in the first H1N1pdm season from four different geographical locations.
Time Cnt K2E I4T F12L A13V A15T K39R V47A D52N P100S S101N
TW 6,9/2009 39 1 1 3 1 3 39 1
IND 5,9/2009 13 2 2 1 13
RUS ,1/2010 23 23
CAN 5,12/2009 210 55
Time Cnt D103N P141S S145P H155Y K171E G172E S179N A212G T214A S220T
TW 6,9/2009 39 1 1 38 37
IND 5,9/2009 13 1 13 10
RUS ,1/2010 23 1 7 1 1 1 23 21
CAN 5,12/2009 210 72
Time Cnt R222K K226E K228E D239G/E Q240R V251I V266L N277D K300N/E Q310H
TW 6,9/2009 39 1 1E 1 1E
IND 5,9/2009 13 1G 1 1 2
RUS ,1/2010 23 1 1 6G, 1E 2 1 3 1N 1
CAN 5,12/2009 210 6 2G, 3E 55
Time Cnt P314S P321S I338V S343Y G356R D381G N387K E391K K419T V428I
TW 6,9/2009 39 1 1 38 1 2 9 1
IND 5,9/2009 13 13 1 1
RUS ,1/2010 23 1 21
CAN 5,12/2009 210 8 37
Time Cnt N458D N461K S468N I477T K521N R526K I527T I564T
TW 6,9/2009 39 1 1 1 1
IND 5,9/2009 13 3 3
RUS ,1/2010 23 1 1
CAN 5,12/2009 210 6
TW: Taiwan; IND: India; RUS: Russia; CAN: Canada.
doi:10.1371/journal.pone.0045946.t007
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amino acid changes in the predicted antigenic sites, including
A156T, S220T and R222K in Ca, D142D in Sa and S202T in Sb.
We therefore selected nine viruses (all in winter season of 2010/
2011) each contains at least two of these five transitions and found
their HI titers ranging from 1:1280 to 1:5120, suggesting no
antigenic change for the investigated Taiwanese H1N1pdm
viruses regardless that many HA mutations were observed (data
not shown).
Yang et al. [11] listed HA 148,152 (the 130-loop), 201,208
(the 190-helix), and 235,242 (the 220-loop) as the receptor-
binding sites (RBS) of the 2009 H1N1pdm viruses. Seven RBS
positions displayed amino acid changes in the investigated
Taiwanese strains. Six of them were also found in antigenic sites
described in the previous paragraphs, including T201N, S202T,
A203T, S207I, and L208I in Sb, and D239G/E in Ca. The only
non-antigenic RBS mutation was A151T, with only one out of five
cases reported in September and one of eight cases in December
2010 during the second Taiwanese H1N1pdm season. Details of
the RBS mutations can be seen in panel 5 of Table S4.
NA Antigenicity
NA antigenicity was less studied in the past than was HA
antigenicity. Maurer-Stroh et al. [12] identified a number of NA
antigenic regions of the new H1N1pdm via a homology-based 3D
structure modeling and epitopes mapping. These regions included
positions 83,99, 103,144, 156,190, 252,303, 330, 332,
340,345, 368, 370, 386,395, 400, 431,435, and 448,468.
Among these 193 predicted NA antigenic sites, only 31 (16.1%)
showed amino acid changes in our collected samples, compared
with 21 out of 50 (42.0%) of the HA antigenic sites that showed
variations. Similar to what has been observed in mutating HA
antigenic sites, most of the NA antigenic mutations identified were
found to occur only rarely. Details of the described antigenic
mutations can be seen in panel 6 of Table S4.
NA Genetic Diversity and Susceptibility to NAI
Several amino acid substitutions of influenza A virus are known
to confer resistance to NAI [13­18]. These include E119V,
D151N, S247N, H275Y and N295S, among which H275Y has
been used as a primary marker for diagnosing resistant viruses.
Recent homology modeling for the NA gene of the new
H1N1pdm also identified additional 13 NA residues (118, 152,
156, 179, 180, 223, 225, 228, 277, 278, 293, 368, and 402) that
were important for contacting sialyloligosaccharide substrates
directly, participating in catalysis, or providing a structural
framework to the bound drug [12]. Table 6 summarizes the
above-mentioned drug-associated sites that showed mutations in
our sequenced Taiwanese NA gene. Only four mutations were
found in a limited count of viruses. In particular, only one (A/
Taiwan/90252/2011) of 20 isolates was found with H275Y in
Figure 2. HA mutation dynamics of Taiwanese H1N1pdm
viruses versus publicly available H1N1pdm viruses. Horizontal
axis represents the year/month the sampling took place, and the
vertical axis represents the frequency (in percentage) that one particular
mutation occurred in the month. Taiwanese data range from June 2009
to February 2011, and are graphed by various markers (circles, squares,
triangles, and diamonds). A total of 8,876 H1N1pdm HA sequences are
collected from NCBI which cover a 3-year span from April 2009 to March
2012 (no case in April 2012), and are graphed in thick lines. Mutations
are grouped according to the transitioning types described in Table 1,
and are displayed from top to bottom as type I.a, I.b, I.c, II.a, II.b and II.c.
Note that some lines are broken due to that no samples are found in
these months. Various lines may have different breakpoints since some
NCBI sequences are partial, making total positional counts vary. For
example, there is no amino acid 468 (shown in a gray line in the second
subpanel) available in September 2011, as well as in February and
March of 2012.
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Amino Acids Transitioning of 2009 H1N1pdm
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February 2011. Other than H275Y, two more mutations occurred
in late months of the second season, including one S247N in
December 2010 and two isolates with N295K in January 2011.
Note that the two mutations in NA 295 in Taiwan were converted
into K rather than into S; the latter has been reported to affect
susceptibility to NAIs in seasonal viruses [13,17]. The last drug-
associated mutation I223T appeared earlier in January 2010 at the
wake of the first H1N1pdm season which structural modeling has
predicted as a drug binding site [12].
Discussion
Evolution of H1N1pdm HA Gene in Taiwan
In this work we analyzed 147 Taiwanese H1N1pdm viruses to
portray the evolutionary dynamics of its historical debut. We
collected 77 samples for the 2009/2010 season and 70 samples for
the 2010/2011 season, and we divided the two seasons by
including the two cases in May of 2010 into the first season.
Because no data were collected for March and April 2010 and for
June and July 2010, the two May cases might just as well have
been assigned to the beginning of the second season instead. The
overall statistics discussed here, however, would most likely not
have been affected either way. We detected 51 HA sites showing
changes in the first pdm season in Taiwan. Moving into the second
pdm season, only 22 of the 51 HA mutations had maintained such
changes. The other 29 HA sites were found to have recovered
their amino acids to what was originally observed in A/
California/04/2009. Nevertheless, 49 new HA sites showed
amino acid substitutions that had not been observed in the first
pdm season, bringing the number of HA sites showing amino acid
changes in the second season (relative to the original 2009
California strain) to 71.
Pan et al. [19] analyzed H1N1pdm strains deposited to
GenBank between April 1 and December 31, 2009, reported
only one dominant HA mutation S220T and described its location
near the receptor-binding domain (RBD). They did not observe
the other four major HA mutations that we have reported in
Table 1. Potdar et al. [20] reported an Indian study involving 13
H1N1pdm isolates from May to September 2009. They found the
HA sequences dominated by four mutations, namely P100S,
T214A, S220T, and I338V. These results are consistent with our
findings, except that Potdar et al. did not notice any E391K
mutation. Despite approximately the same time-frame between
the two studies (May-September 2009 for India, and June-
September 2009 for Taiwan), only six mutations were found in
common between 16 mutations detected from 13 Indian viruses
and 26 mutations from 39 Taiwanese viruses (shown in Table 7).
Ilyicheva et al. [21] performed a sequence analysis of 23 Russian
H1N1pdm strains and reported 20 HA mutation sites. Graham et
al. [22] performed HA sequence analysis of 235 Canadian
H1N1pdm strains, of which 210 were collected from May to
December 2009. Taking together the Indian and Taiwanese cases
spanning approximately the same period of time in the first
H1N1pdm season, these mutations were detected from viruses
isolated in four distinct geographical locations, representing an
H1N1pdm HA mutation spectrum around the globe. Most of the
sporadic mutations did not occur at sites that are common across
these four locations, suggesting that a geographical variation did
exist in the early diversification of the viral genome.
All primary HA mutations observed in the first Taiwanese
H1N1pdm season remained abundant in the second season. These
include P100S, S220T and I338V. Although T214A substitution
was also found dominated throughout the two seasons, its
appearance was less frequent (,50%) in the end-of-season months
December 2010 to February 2011. Table 1 also notes other
dynamic patterns of HA mutations, including the early emerging
and re-emerging mutations of types I.b and I.c, and second-season
emerging types II.a, IIb and II.c. The possible association between
these dynamic patterns and HA evolution, particularly at a stage
after the so-called early diversification of this new virus, is an
interesting question to address. Many of these mutations were still
present with over 50% occurrence at the season-ending month.
The late-breaking mutations (T14I, D114N and I233V of type
II.c, and R222K, V266L and K300E of type I.c) were seen at
approximately the same frequencies of 11­28% in January 2011,
which had nearly doubled to 30­45% in February 2011. Although
their prevalence was not as obvious as that of other mutations, our
data suggest they may re-appear in the subsequent seasons.
As limited Taiwanese H1N1pdm viruses were isolated and
investigated after our sampling period, we gathered 8,876
H1N1pdm HA sequences up to April 2012 from National Center
for Biotechnology Information (NCBI) and analyzed the dynamics
of mentioned HA mutations. As shown in Figure 2, the temporal
appearance of T214A was in general declining yet in an oscillatory
manner after March 2011. For example, T214A was only seen in 2
out of 15 HA sequences in February 2012, and none among 5
cases in March 2012. Figure 2 also supports our finding for the two
late emerging/re-emerging type II.c mutations D114N and
I233V, and two type I.c mutations R222K and V266L. Their
dynamic patterns after March 2011 seem to resemble what was
observed in T214A. Interestingly, the remaining type I.c mutation
K300E and type II.c mutation T14I are hardly seen in these NCBI
HA sequences. In contrast to the generally declining T214A, the
other two type I.b mutations E391K and S468N and two type II.b
mutations S160G and S202T after the second season appear to be
stabilizing in the virus population. Continuous monitoring these
mutations in more seasons to come is important to better
understand the HA evolutionary spectrum of this virus.
Evolution of H1N1pdm NA Gene in Taiwan
We mentioned that only 22 out of 51 HA mutations (43.1%)
detected in the first season showed up again at least once in the
second pdm season in Taiwan. Of all 71 HA mutations observed
in the second season, 49 mutations (69.0%) had not appeared at all
in the first season. Such diversification for giving up old and
acquiring new mutations across seasons was even more noticeable
for NA, in which only 25% of the mutations (7 out of 28) from the
first season survived in the second season, and 85.4% (41 out of 48)
of the mutations found in the second season were newly emerged.
A number of the newly emerged second-season mutations were
short lived and had disappeared completely in the final months of
the season, including the three type II.a mutations M15I, N189S,
and I365T. The three type II.b mutations N44S, V241I, and
Figure 3. NA mutation dynamics of Taiwanese H1N1pdm viruses versus publicly available H1N1pdm viruses. Horizontal axis
represents the year/month the sampling took place, and the vertical axis represents the frequency (in percentage) that one particular mutation
occurred in the month. Taiwanese data range from June 2009 to February 2011, and are graphed by various markers (circles, squares, triangles, and
diamonds). A total of 6,017 H1N1pdm NA sequences are collected from NCBI which cover a 3-year span from April 2009 to April 2012, and are
graphed in thick lines. Mutations are grouped according to the transitioning types described in Table 2, and are displayed from top to bottom as type
I, II.a, II.b and II.c.
doi:10.1371/journal.pone.0045946.g003
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N369K began in October 2010 and appeared to persist until the
season's end, although they never reached 100% peak as did
V106I and N248D. Recall that the previously mentioned HA
mutations of L8M, S160G, S202T, and S468N were also found to
emerge or re-emerge in October 2010. Nevertheless, similar to
a number of type I.c and type II.c HA mutations observed in the
final two months of the second season (T14I, D114N, R222K,
I233V, V266L and K300E), we also found that the two type II.c
NA mutations S299A and I374V emerged only in January and
February 2011. Such co-incidence in the evolutionary dynamics of
HA and NA suggests that a fitness or co-evolution occurs between
the two genes, which may play an important role in shaping the
viral genome for many seasons to come.
We gathered 6,017 H1N1pdm NA sequences from NCBI to
follow up those mentioned NA mutations in Taiwan. Figure 3
describes their temporal dynamics for the entire three H1N1pdm
seasons from April 2009 to April 2012. Other than that V106I and
N248D are fixed in the population as expected, those three type
II.b mutations (N44S, V241I and N369K) that showed higher
prevalence in 2010­2011 season seem to appear in a oscillatory
manner. Note that a number of 2011­2012 months are missing in
Figure 3 because of no NA sequences are available in NCBI.
Nevertheless, the sample counts of the final four months in Figure 3
(August/October of 2011, and January/April of 2012) are only
three, two, three and one, respectively. More samples are needed
in order to better describe NA evolution in terms of amino acid
transitioning.
Mutation Rates of Taiwanese H1N1pdm Viruses in 2009­
2011
A nationwide molecular surveillance of H1N1pdm genomes in
Canada [22] revealed HA and NA genetic diversity at 1.9861023
and 2.3661023
amino acid substitution per protein site, re-
spectively. This study's sampling covered a period of only eight
months (from the emergence of H1N1pdm to December 5, 2009)
and the NA proteins seemed to display slightly more substitutions
than did the HA proteins. Our rates of amino acid substitution for
HA and NA in the first season (a 12-month period) were
9.2961023
and 5.2361023
, respectively. To make these compa-
rable with Canadian's study, a sub-sampling from June to
November 2009 was chosen and mutation rates were re-computed
as 8.9061023
for HA and 4.6861023
for NA. Not only did our
HA proteins display far more diversity than NA, our study also
found much higher mutation rates.
Earlier we discussed the difference in HA mutating sites
between Taiwanese and Canadian studies over the same sampling
period (Table 6). We noted that P100S, T214A, S220T, and
I338V occurred in almost all 39 Taiwanese H1N1pdm viruses
sampled. For the 210 Canadian viruses collected within the same
time period, however, only 72 (34.3%) were observed to contain
S220T; no P100S, T214A, or I338V changes were found. Two
other major mutations, K2E and Q310H, were detected in 55
(26.2%) of the Canadian HA proteins but not at all in Taiwanese
strains. However, the overall HA mutation rate for Taiwanese
viruses was apparently higher than that of the Canadian ones.
Such geographical disparity may explain the differences in
mutation rates described here.
Furuse et al. [23] compared the evolutionary rates among
seasonal H1N1 (1918­1957 and 1977­2009) virus, swine H1 virus,
and 2009 H1N1pdm virus; their results indicated that the rate of
H1N1pdm was much lower than that of the others. However, the
pdm data analyzed had been sampled over a period of less than
ten months, up to the end of the first pandemic season. This
sampling limitation may have led to an unreliable estimation of the
correlation coefficient for describing the evolutionary trend. The
Canadian study mentioned above was also based on an eight-
month period only, and many genetic variants were still evolving,
at least for the first pandemic season. Nevertheless, our second-
season evolutionary statistics indicated that the amino acid
mutation rates of H1N1pdm HA and NA are elevated than they
were in the first H1N1pdm season. For the mutation frequencies
shown in Table 3, the Taiwanese H1N1 viruses showed 5.24
substitutions per 566-aa HA segment in the first season, increasing
to 8.26 (a 57.6% boost) in the second season. NA displayed a lower
mutation frequency than HA in the first season, with 2.45
substitutions per 469-aa segment, but soared to 5.16 (a 111%
boost) in the second season. Ongoing surveillance data obtained
from various geographical locations and subsequent seasons will
enable more accurate descriptions of the evolutionary dynamics of
this novel H1N1pdm virus.
It is mentioned that the way these clinical samples were collected
did not take into consideration the demographic factors such as
gender, age or geographical location. Neither did we gather
vaccination history from the patients. As a result, these data are not
suitable for revealing correlations between these factors and amino
acid mutations. A large-scale, island-wide study by Taiwanese CDC
[24] revealed that the major affected groups were shifted to older
individualsofhigherage-specificcasefatalityratios(CFRs)fromMay
2009toApril2011inTaiwan.Theyalsodiscussedthepossibilitythat
the shift could be attributed to the vaccination program in which
adults aged 18­64 were the shortfall in influenza vaccination. How
such shifting of CFRs relates to underlying genome variations,
however, remains to be investigated.
In summary, we revealed amino acids transitioning of the two
surface glycoproteins of H1N1pdm viruses, particularly on how
these mutations shifted in 2010/2011 season after the H1N1pdm's
debut in 2009/2010. We found 17.7% of HA and 14.7% of NA
sites had their amino acids mutated based on A/California/4/
2009. Many of these mutations were transient, demonstrating how
the viral genome has been shaped dynamically. Among those
mutations that appeared more frequently (.5% incidence in all
147 viruses from June 2009 to February 2011), many were new
after August 2010 which were not seen throughout the first
pandemic season (Tables 1 and 2). Furthermore, some late-
breaking mutations are found to have statistical correlation to
disease severity. Although a number of mutations were made to
the antigenic sites, HI tests showed no titer changes for these
Taiwanese strains. There was only one recent isolate in February
2011 which contains the well-known resistance marker H275Y in
NA, suggesting an overall susceptibility for Taiwanese isolates to
NAIs.
Supporting Information
Table S1 Primer pairs used in sequencing HA and NA
genes.
(PDF)
Table S2 HA amino acid mutation statistics of 147
Taiwanese H1N1pdm viruses.
(PDF)
Table S3 NA amino acid mutation statistics of 147
Taiwanese H1N1pdm viruses.
(PDF)
Table S4 Monthly amino acid mutation statistics of 147
Taiwanese H1N1pdm viruses in HA antigenic sites,
receptor binding sites, and NA antigenic sites.
(PDF)
Amino Acids Transitioning of 2009 H1N1pdm
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Author Contributions
Conceived and designed the experiments: GWC KCT SCC TYL YCH
SRS. Performed the experiments: CGH YCL HHW SLY. Analyzed the
data: GWC KCT YNG SRS. Contributed reagents/materials/analysis
tools: GWC CGH YNG TYL YCH. Wrote the paper: GWC KCT SRS.
References
1. Dawood FS, Jain S, Finelli L, Shaw MW, Lindstrom S, et al. (2009) Emergence
of a novel swine-origin influenza A (H1N1) virus in humans. N Engl J Med 360:
2605­2615.
2. Garten RJ, Davis CT, Russell CA, Shu B, Lindstrom S, et al. (2009) Antigenic
and genetic characteristics of swine-origin 2009 A(H1N1) influenza viruses
circulating in humans. Science 325: 197­201.
3. Bright RA, Medina MJ, Xu X, Perez-Oronoz G, Wallis TR, et al. (2005)
Incidence of adamantane resistance among influenza A (H3N2) viruses isolated
worldwide from 1994 to 2005: a cause for concern. Lancet 366: 1175­1181.
4. Deyde VM, Xu X, Bright RA, Shaw M, Smith CB, et al. (2007) Surveillance of
resistance to adamantanes among influenza A(H3N2) and A(H1N1) viruses
isolated worldwide. J Infect Dis 196: 249­257.
5. Renaud C, Kuypers J, Englund JA (2011) Emerging oseltamivir resistance in
seasonal and pandemic influenza A/H1N1. J Clin Virol 52: 70­78.
6. Smith GJ, Vijaykrishna D, Bahl J, Lycett SJ, Worobey M, et al. (2009) Origins
and evolutionary genomics of the 2009 swine-origin H1N1 influenza A
epidemic. Nature 459: 1122­1125.
7. Moscona A (2009) Global transmission of oseltamivir-resistant influenza.
N Engl J Med 360: 953­956.
8. Debarre F, Bonhoeffer S, Regoes RR (2007) The effect of population structure
on the emergence of drug resistance during influenza pandemics. J R Soc
Interface 4: 893­906.
9. Deyde VM, Okomo-Adhiambo M, Sheu TG, Wallis TR, Fry A, et al. (2009)
Pyrosequencing as a tool to detect molecular markers of resistance to
neuraminidase inhibitors in seasonal influenza A viruses. Antiviral Res 81: 16­
24.
10. Igarashi M, Ito K, Yoshida R, Tomabechi D, Kida H, et al. (2010) Predicting
the antigenic structure of the pandemic (H1N1) 2009 influenza virus
hemagglutinin. PLoS One 5: e8553.
11. Yang H, Carney P, Stevens J (2010) Structure and Receptor binding properties
of a pandemic H1N1 virus hemagglutinin. PLoS Curr 2: RRN1152.
12. Maurer-Stroh S, Ma J, Lee RT, Sirota FL, Eisenhaber F (2009) Mapping the
sequence mutations of the 2009 H1N1 influenza A virus neuraminidase relative
to drug and antibody binding sites. Biol Direct 4: 18; discussion 18.
13. Abed Y, Baz M, Boivin G (2006) Impact of neuraminidase mutations conferring
influenza resistance to neuraminidase inhibitors in the N1 and N2 genetic
backgrounds. Antivir Ther 11: 971­976.
14. de Jong MD, Tran TT, Truong HK, Vo MH, Smith GJ, et al. (2005)
Oseltamivir resistance during treatment of influenza A (H5N1) infection.
N Engl J Med 353: 2667­2672.
15. Gubareva LV, Kaiser L, Matrosovich MN, Soo-Hoo Y, Hayden FG (2001)
Selection of influenza virus mutants in experimentally infected volunteers treated
with oseltamivir. J Infect Dis 183: 523­531.
16. Hurt AC, Lee RT, Leang SK, Cui L, Deng YM, et al. (2011) Increased detection
in Australia and Singapore of a novel influenza A(H1N1)2009 variant with
reduced oseltamivir and zanamivir sensitivity due to a S247N neuraminidase
mutation. Euro Surveill 16.
17. Sheu TG, Deyde VM, Okomo-Adhiambo M, Garten RJ, Xu X, et al. (2008)
Surveillance for neuraminidase inhibitor resistance among human influenza A
and B viruses circulating worldwide from 2004 to 2008. Antimicrob Agents
Chemother 52: 3284­3292.
18. Ujike M, Ejima M, Anraku A, Shimabukuro K, Obuchi M, et al. (2011)
Monitoring and characterization of oseltamivir-resistant pandemic (H1N1) 2009
virus, Japan, 2009­2010. Emerg Infect Dis 17: 470­479.
19. Pan C, Cheung B, Tan S, Li C, Li L, et al. (2010) Genomic signature and
mutation trend analysis of pandemic (H1N1) 2009 influenza A virus. PLoS One
5: e9549.
20. Potdar VA, Chadha MS, Jadhav SM, Mullick J, Cherian SS, et al. (2010)
Genetic characterization of the influenza A pandemic (H1N1) 2009 virus isolates
from India. PLoS One 5: e9693.
21. Ilyicheva T, Susloparov I, Durymanov A, Romanovskaya A, Sharshov K, et al.
(2011) Influenza A/H1N1pdm virus in Russian Asia in 2009­2010. Infect Genet
Evol 11: 2107­2112.
22. Graham M, Liang B, Van Domselaar G, Bastien N, Beaudoin C, et al. (2011)
Nationwide molecular surveillance of pandemic H1N1 influenza A virus
genomes: Canada, 2009. PLoS One 6: e16087.
23. Furuse Y, Shimabukuro K, Odagiri T, Sawayama R, Okada T, et al. (2010)
Comparison of selection pressures on the HA gene of pandemic (2009) and
seasonal human and swine influenza A H1 subtype viruses. Virology 405: 314­
321.
24. Yang JR, Huang YP, Chang FY, Hsu LC, Lin YC, et al. (2011) New variants
and age shift to high fatality groups contribute to severe successive waves in the
2009 influenza pandemic in Taiwan. PLoS One 6: e28288.
Amino Acids Transitioning of 2009 H1N1pdm
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