The authors have declared that no competing interests exist.
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.
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 9.29×10−3 substitutions per site in the first season to 1.46×10−2 in the second season in HA, and from 5.23×10−3 to 1.10×10−2 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 2011–2012 season, a number of them still showed signs of transitioning, prompting the importance of continuous monitoring of this virus for more seasons to come.
A swine-origin influenza A virus (S-OIV) was first found in North America in April 2009
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
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
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.
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.
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.
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 |
Student’s
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
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 |
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.
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, CY047746, CY053476, CY053484, CY053492, CY053500, CY053508, and JN381343-JN381480 for the NA genes.
HA | NA | ||||
Seq cnt | Accu. mut. | Mut. freq. | Accu. mut. | Mut. freq. | |
Jun/2009 | 13 | 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 |
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189 |
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Season 2 | 70 | 578 |
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361 |
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Total | 147 | 983 | 6.69 | 550 | 3.74 |
9.29×10−3,
5.23×10−3,
1.46×10−2,
1.10×10−2 substitutions per amino acid site.
HA | NA | |||
Mutation frequency | Mutation frequency | |||
Non-severe cases |
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 |
ARDS |
8.5 | <0.001 | 4.6 | <0.05 |
Upper respiratory tract infection.
Acute respiratory distress syndrome.
Student’s
HA (%) | NA (%) | |||||||
S160G | S202T | V266L | S468N | N44S | V241I | S299A | N369K | |
Non-severe cases |
8(9.7) | 14(17) | 2(2.4) | 16(19.5) | 0 (0) | 13 (16.0) | 0 (0) | 13 (16.0) |
Severe cases |
12(23.5) | 21(41.2) | 9(17.6) | 21(41.2) | 15 (29.4) | 21 (41.2) | 4 (7.8) | 21 (41.2) |
0.04 | 0.003 | 0.003 | 0.009 | <0.0001 | 0.002 | 0.02 | 0.002 |
Upper respiratory tract infection.
Pneumonia, Acute respiratory distress syndrome, Expired.
Fisher’s exact test.
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.
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
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.
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) |
Time | Cnt | K2E | I4T | F12L | A13V | A15T | K39R | V47A | D52N | P100S | S101N | |||||||||
TW | 6∼9/2009 | 39 | 1 | 1 | 3 | 1 | 3 |
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IND | 5∼9/2009 | 13 | 2 | 2 | 1 |
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RUS | ∼1/2010 | 23 |
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CAN | 5∼12/2009 | 210 |
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TW | 6∼9/2009 | 39 | 1 | 1 |
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IND | 5∼9/2009 | 13 | 1 |
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RUS | ∼1/2010 | 23 | 1 | 7 | 1 | 1 | 1 |
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CAN | 5∼12/2009 | 210 |
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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 |
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TW | 6∼9/2009 | 39 | 1 | 1 |
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IND | 5∼9/2009 | 13 |
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RUS | ∼1/2010 | 23 | 1 |
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CAN | 5∼12/2009 | 210 | 8 |
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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.
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
Igarashi et al.
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
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
Following the observation that a number of HA amino acid changes were located on the predicted antigenic sites, hemagglutination 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 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.
NA antigenicity was less studied in the past than was HA antigenicity. Maurer-Stroh et al.
Several amino acid substitutions of influenza A virus are known to confer resistance to NAI
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.
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.
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
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 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.
A nationwide molecular surveillance of H1N1pdm genomes in Canada
Earlier we discussed the difference in HA mutating sites between Taiwanese and Canadian studies over the same sampling period (
Furuse et al.
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
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 (
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