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Increased prevalence of a rare mutant of pandemic H1N1 influenza virus in a Eurasian region
Infection, Genetics and Evolution 11 (2011) 227–231
Contents lists available at ScienceDirect
Infection, Genetics and Evolution journal homepage: www.elsevier.com/locate/meegid
Short communication
Increased prevalence of a rare mutant of pandemic H1N1 influenza virus in a Eurasian region Ting-Ting Yang a, Zhao-Guo Wang a,*, Shan-Peng Li a, Xiao-Lin Liu a, Ying Yi a, Yu Yang a, Ping Yu a, Ji-Ming Chen b,** a b
Qingdao Center for Disease Control and Prevention, Qingdao 266032, PR China China Animal Health and Epidemiology Center, Qingdao 266032, PR China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 6 August 2010 Received in revised form 22 September 2010 Accepted 27 September 2010 Available online 8 October 2010
In 2009, a novel swine-origin H1N1 influenza virus sparked an influenza pandemic. The emergence of mutations in the viral genome is therefore of ongoing concern. In this study, the hemagglutinin (HA) gene sequences of 3444 pandemic H1N1 influenza viruses reported to the GenBank database and the sequences of 48 pandemic H1N1 influenza viruses detected in the Chinese city of Qingdao were analyzed. Among the 3492 viruses, 101 carried a serine to proline substitution at position 128 (S128P) in the viral HA gene. All the 101 S128P mutants belonged to Clade 7 which has become dominant worldwide since the summer of 2009. Among the 3492 viruses, 1646 were collected before July 25, 2009, and none of these viruses carried the S128P mutation. Furthermore, after July 25, 2009, the prevalence of the S128P mutant was 33.56% (99/295) in a region of Eurasia including Russia, Mongolia, mainland China and South Korea, but only 0.11% (2/1846) in the rest of the world. The data suggested that the originally rare S128P mutant has become prevalent in the Eurasia region, indicating that the S128P mutant likely transmitted more efficiently than other strains of the virus. Therefore, it is of significance to observe whether the S128P mutant will be more dominant worldwide in the coming future and investigate the exact effects of the S128P mutation. ß 2010 Elsevier B.V. All rights reserved.
Keywords: Pandemic influenza Influenza virus Mutation Epidemiology
1. Introduction On April 24, 2009, human infections with a novel swine-origin H1N1 subtype influenza virus in the USA and Mexico were reported by the World Health Organization (Smith et al., 2009). Within a few months the virus spread to many other countries, mainly through international air travel, and sparked the first human influenza pandemic of the 21st century (Garten et al., 2009; Smith et al., 2009). The emergence of mutations in the viral genome is of ongoing public and scientific concern, because such changes may affect the infectivity, pathogenicity, antigenicity, host tropism and drug sensitivity of the virus (Garten et al., 2009; Rambaut and Holmes, 2009; Glinsky, 2010; Janies et al., 2010; Maurer-Stroh et al., 2010). For example, it has been found that a glutamine to lysine substitution at position 391 (E391K) in the viral hemagglutinin (HA) is globally on the rise (Maurer-Stroh et al., 2010). This mutation may alter viral antigenicity and the HA oligomerization
* Corresponding author. Tel.: +86 532 85652104; fax: +86 532 85652104. ** Corresponding author. Tel.: +86 532 85623396; fax: +86 532 85623396. E-mail addresses: [email protected] (Z.-G. Wang), [email protected] (J.-M. Chen). 1567-1348/$ – see front matter ß 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.meegid.2010.09.014
interface which is important for membrane fusion. Additionally, a histidine to tyrosine substitution at position 274 (H274Y) in the viral neuraminidase protein has also been under intensive surveillance because it can render the virus resistant to the neuraminidase inhibitor oseltamivir (Dharan et al., 2009; Hauge et al., 2009). In this study, the HA gene sequences of 3444 pandemic H1N1 influenza viruses available in the GenBank database and the sequences of 48 pandemic H1N1 influenza viruses detected in Qingdao, a beach city in China, were analyzed to investigate whether a new mutation, a serine to proline substitution at position 128 (S128P), in the viral HA gene is of any epidemiological significance. 2. Materials and methods The 48 pandemic H1N1 influenza viruses detected in Qingdao, China, were from 15 randomly selected imported cases (individuals who just arrived from other countries or regions via air travel prior to sample collection) and 33 randomly selected domestic cases (those with no recent travel history to other countries or regions prior to sample collection). Case confirmation and gene sequencing were performed by the methods reported previously (Wang et al., in press). Briefly, the pandemic H1N1 influenza cases
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Table 1 Background of the 48 pandemic H1N1 influenza viruses detected in Qingdao, China. GenBank accession number
Strains
Case sources
Sample collection date
Residue at position 128
CY050262 GU168018 GU144805 CY050263 CY050264 CY050265 CY050266 CY050267 CY050268 CY050269 CY050270 CY050271 CY050272 CY050273 CY050274 CY050140 CY050100 CY050098 CY050112 CY050113 CY050117 CY050119 CY050121 CY050130 CY067613 CY067614 CY067615 CY067616 CY067617 CY067618 CY067619 CY067620 CY067621 CY067622 CY067623 CY067624 CY067625 CY067626 CY067627 CY067628 CY067629 CY060631 CY060632 CY060633 CY060634 CY067611 CY067612 CY060630
A/Qingdao/148/2009 A/Qingdao/222/2009 A/Qingdao/231/2009 A/Qingdao/283/2009 A/Qingdao/292/2009 A/Qingdao/320/2009 A/Qingdao/322/2009 A/Qingdao/333/2009 A/Qingdao/359/2009 A/Qingdao/362/2009 A/Qingdao/375/2009 A/Qingdao/383/2009 A/Qingdao/399/2009 A/Qingdao/477/2009 A/Qingdao/492/2009 A/Qingdao/1008/2009 A/Qingdao/1006/2009 A/Qingdao/1020/2009 A/Qingdao/1170/2009 A/Qingdao/1215/2009 A/Qingdao/1261/2009 A/Qingdao/1268/2009 A/Qingdao/1269/2009 A/Qingdao/1364/2009 A/Qingdao/1508/2009 A/Qingdao/1517/2009 A/Qingdao/1530/2009 A/Qingdao/1568/2009 A/Qingdao/1577/2009 A/Qingdao/1609/2009 A/Qingdao/1610/2009 A/Qingdao/1626/2009 A/Qingdao/1629/2009 A/Qingdao/1631/2009 A/Qingdao/1642/2009 A/Qingdao/1669/2009 A/Qingdao/1773/2009 A/Qingdao/1775/2009 A/Qingdao/1776/2009 A/Qingdao/1779/2009 A/Qingdao/1780/2009 A/Qingdao/63/2010 A/Qingdao/66/2010 A/Qingdao/70/2010 A/Qingdao/94/2010 A/Qingdao/101/2010 A/Qingdao/123/2010 A/Qingdao/220/2010
Flying from Flying from Flying from Flying from Flying from Flying from Flying from Flying from Flying from Flying from Flying from Flying from Flying from Flying from Flying from Domestic Domestic Domestic Domestic Domestic Domestic Domestic Domestic Domestic Domestic Domestic Domestic Domestic Domestic Domestic Domestic Domestic Domestic Domestic Domestic Domestic Domestic Domestic Domestic Domestic Domestic Domestic Domestic Domestic Domestic Domestic Domestic Domestic
2009-06-13 2009-06-24 2009-06-27 2009-07-05 2009-07-06 2009-07-10 2009-07-10 2009-07-12 2009-07-16 2009-07-18 2009-07-19 2009-07-21 2009-07-23 2009-08-12 2009-08-17 2009-09-01 2009-09-04 2009-09-04 2009-09-08 2009-09-11 2009-09-15 2009-09-15 2009-09-15 2009-09-18 2009-10 2009-10 2009-10 2009-11 2009-11 2009-11 2009-11 2009-11 2009-11 2009-11 2009-11 2009-11 2009-12 2009-12 2009-12 2009-12 2009-12 2010-01 2010-01 2010-01 2010-01 2010-01 2010-02 2010-03
S S S S S S S S S S S S S S S S P P P S P P P P S P S P P S S S S P S L S S P P P P P P P S P P
were confirmed by a commercial real-time RT-PCR kit, and the fulllength HA gene was amplified by RT-PCR with a pair of universal primers for influenza A virus (Hoffmann et al., 2001), and sequenced thereafter. More information about the 48 cases and the relevant GenBank accession numbers are given in Table 1. The viral HA gene sequences were searched and aligned using the web server of the Influenza Virus Resource in NCBI (Bao et al., 2008). The geographical distribution and collection dates of the relevant viruses were also obtained from this web server. Prevalences were compared statistically by x2 test. The S128P mutants and some other pandemic H1N1 influenza viruses (reference viruses) were phylogenetically analyzed using the software MEGA 4.1 with the neighbor-joining (NJ) method and the model of maximum composition likelihood (Tamura et al., 2007). Substitution rates were set as different among sites and among lineages. Gaps were treated by pairwise-deletion. Bootstrap values were calculated out of 1000 replicates. At least one representative of each of the 7 clades identified previously (Nelson et al., 2010), and at most one of the viruses collected in the same city in the same month with known collection dates were selected
the USA Australia Chile Australia Australia South Korea France France Hong Kong Philippines South Korea India South Korea Indonesia Hong Kong
as the reference viruses. The reported HA gene sequences of the reference viruses should be no shorter than 1.2 kb. Some S128P mutants collected in same city in the same month were also excluded for the phylogenetic analysis if they were genetically similar to each other (genetic distances <1%, which were calculated by the same method used for the phylogenetic analysis). To evaluate the results obtained with the NJ method, the phylogenetic relationships among the sequences were also calculated using RAxML version 7.0.4 software with the General Time Reversible (GTR) substitution matrix and the maximum likelihood (ML) method (Stamatakis et al., 2008). 3. Results and discussion 3.1. Spatial distribution of the S128P mutant On July 26, 2010, the HA gene sequences of 3444 pandemic H1N1 influenza viruses isolated from humans were available in the GenBank database with known amino acid residues at position 128 and known collection months. Among the 3444 viruses and the 48
[()TD$FIG]
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Fig. 1. Distribution of the cities in Russia, Mongolia, mainland China and South Korea with the HA sequences of pandemic H1N1 influenza viruses available in GenBank on July 26, 2010. The cities with and without the S128P mutant in GenBank are marked with triangles and circles, respectively, in a human population distribution map downloaded from the Wikipedia website (http://en.wikipedia.org/wiki/World_population).
viruses we detected in Qingdao, 366 were from a region of Eurasia including 123 from Russia, 217 from mainland China, 9 from Mongolia and 17 from South Korea (Fig. 1). This region is referred to below as the RCMK region. The other 3126 viruses were from other parts of Eurasia, North and South America, Africa or Oceania. Among the 366 viruses from the RCMK region, 99 (85 from mainland China, six from Russia, five from South Korea, three from Mongolia) carried the S128P mutation in the HA gene. However, only two of the other 3126 viruses from the rest of the world carried the S128P mutation. Detailed information about the 101 S128P mutants is given in Supplementary File 1. In addition, four of the 3391 (3444 + 48–101) pandemic viruses lacking the S128P mutation showed other changes at this position (two S128T and two S128L mutations). According to the information provided in the GenBank database, the 366 viruses from the RCMK region and the 99 S128P mutants among these viruses were reported by 35 and 18 entities, respectively. The relevant samples were collected from 75 and 25 cities, respectively. Distribution of these cities is largely consistent with the host population distribution in this region (Fig. 1), indicating that the 366 viruses and the 99 mutants were geographically representative.
After July 26, 2009, the prevalence of the S128P mutation in the RCMK region was 33.56% [99/(366–71)], significantly higher (P < 0.01) than its counterpart in the rest of the world, 0.11% [2/ (3492–1646)]. Moreover, as shown in Table 2, the prevalence of the S128P mutant was rising in the RCMK region in 2009 from the percentage of the cities reporting the S128P mutant. However, because few viruses were collected in 2010 in the RCMK region, it remains unknown how prevalent the S128P mutant was in this region in 2010. 3.3. Distribution of the S128P mutation among the viruses detected in Qingdao Among the 48 viruses detected in Qingdao, 20 from the 33 domestic cases and none from the 15 imported cases carried the S128P mutation. Twelve of the 15 imported cases flied from out of the RCMK region. These data are consistent with the global distribution of the S128P mutant mentioned above. Because the viruses from the 33 domestic cases and the 15 imported cases were collected before and after August 18, 2009, respectively, the observed discrepancy between the domestic and the imported cases could be explained by the spatial distribution or the temporal distribution of the S128P mutant.
3.2. Temporal distribution of the S128P mutant 3.4. Variation at position 128 in other H1 subtype influenza viruses According to the collection dates (Supplementary File 1), the S128P mutant was found firstly in the city of Chengdu in Sichuan, China on July 26, 2009. In addition, among the 3492 (3444 + 48) viruses analyzed, 1646 were collected before July 25, 2009, including 71 from the RCMK region. None of these 1646 viruses carried the S128P mutation, indicating that this mutation was rare worldwide during the first three months of the pandemic. However, it remains unknown when and where the mutant came out.
On July 26, 2010, sequences of the viral HA gene of 554 avian, 669 swine and 3607 human seasonal H1 subtype influenza viruses were available in the GenBank database with known amino acid residues at position 128. All the 554 avian and 81.17% (543) of the 669 swine H1 subtype influenza viruses harbored threonine at position 128, while 93.15% (3360) of the 3607 human seasonal H1 subtype influenza viruses harbored another amino acid, valine, at the position.
Table 2 Temporal distribution of the S128P mutant based on 366 sequences reported from the RCMK region with known collection dates. Collection dates (day/month/year)
Number of S128P mutants
Number of total viruses
Number of cities reporting the S128P mutant
Number of cities reporting the pandemic viruses
Before 01/07/2009 01/07/2009–31/08/2009 01/09/2009–31/10/2009 01/11/2009–31/12/2010 After 31/12/2009
0 13 26 52 8
44 68 69 171 14
0 3 7 28 3
11 21 18 60 7
a b
(0.00%) (19.12%)a (37.68%)b (30.41%) (57.14%)
12 of the 13 mutants were collected in the same cluster in a city on July 26, 2009. Seven of the 26 mutants were collected in the Chinese city of Xian in September 2009.
(0.00%) (14.28%) (38.89%) (46.67%) (42.86%)
[()TD$FIG]
230
T.-T. Yang et al. / Infection, Genetics and Evolution 11 (2011) 227–231
Fig. 2. Phylogenetic tree of 84 S128P mutants and other 122 pandemic H1N1 influenza viruses based on the viral HA gene sequences. The S128P mutants and the other viruses are marked with triangles and circles, respectively. The tree is rooted by two earliest isolates of the pandemic virus, A/California/04/2009 and A/California/07/2009. Bootstrap values (>50) are given at the relevant nodes. All the viral designations are available in the Supplementary File 2, and they were elongated with the collection date plus ‘‘P’’ for the S128P mutants or ‘‘Cx’’ for the representatives of Clade x (x = 1, 2, . . ., 7).
3.5. Phylogenetic analysis of the S128P mutant Phylogenetic analysis of the HA gene sequences of 206 pandemic H1N1 influenza viruses, as shown in Fig. 2, suggested that all the S128P mutants were located in the terminal branches of the phylogenetic tree rooted with two earliest pandemic H1N1 viruses, A/California/04/2009 and A/California/07/2009 (Clade 1). All the S128P mutants belong to Clade 7 which has become dominant worldwide since the summer of 2009 (Valli et al., 2010).
It should be mentioned that the clades were classified according to the viral genomic sequences rather than the viral HA gene sequences, and therefore some clades were located together in the tree of Fig. 2. Most of the bootstrap values of the phylogenetic tree are rather low, which should be caused by that some intermediate sequences were involved in the analysis (Chen et al., 2007). The phylogenetic tree calculated using the ML method (data not shown) was of no obvious differences from that calculated using the NJ method (Fig. 2).
T.-T. Yang et al. / Infection, Genetics and Evolution 11 (2011) 227–231
3.6. Epidemiological inference of the distribution of the S128P mutant The aforementioned distribution data of the S128P mutation revealed an interesting scenario about the pandemic virus, namely, that the originally rare S128P mutant has become prevalent in the RCMK region. This indicated that the S128P mutant spread significantly more efficiently than other strains of this virus, resulting in its high prevalence there. Therefore, the S128P mutant likely has higher transmissibility than other strains of this virus in humans. This further indicates that this mutant may become more dominant worldwide in the coming future. It may be argued that the emerging dominance of the S128P mutant resulted not from its higher transmissibility, but from geographical isolation. However, geographical isolation can be excluded in this case due to the large-scale domestic and international travel that occurs worldwide (Suzuki and Nei, 2002; Chen et al., 2009; Garten et al., 2009). Previous reports indicated position 128 in the viral HA gene is near to one motif of the viral receptor-binding site (RBS) covering positions 131–135, and one of the viral antigenic epitopes covering positions 124, 125, 153, 157, 159 and 164 (Garten et al., 2009; Igarashi et al., 2010; Yang et al., 2010). Therefore, the S128P mutation may change the receptor-binding property and the antigenicity of the virus. The aforementioned variation of position 128 in the HA gene in avian, swine and human seasonal H1 subtype influenza viruses also suggested that this position is of some hostspecificity. However, the exact effects of the S128P mutation remain unknown. It is also unknown whether the S128P mutation changed the viral virulence in humans. Further studies are therefore needed to investigate the exact biomedical significance of the S128P mutation. Acknowledgments We thank Dr. Jiwang Chen in Northwest University in the USA for his critical comments on data analysis. This work was supported by the Pandemic Influenza Research Funds of Qingdao City (No. 09-1-1-49-nsh, 2008-wszd119 and 10-3-3-8-1-nsh) and Chinese Ministry of Agriculture. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.meegid.2010.09.014. References Bao, Y., Bolotov, P., Dernovoy, D., Kiryutin, B., Zaslavsky, L., Tatusova, T., Ostell, J., Lipman, D., 2008. The influenza virus resource at the National Center for Biotechnology Information. J. Virol. 82, 596–601.
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Contents lists available at ScienceDirect
Infection, Genetics and Evolution journal homepage: www.elsevier.com/locate/meegid
Short communication
Increased prevalence of a rare mutant of pandemic H1N1 influenza virus in a Eurasian region Ting-Ting Yang a, Zhao-Guo Wang a,*, Shan-Peng Li a, Xiao-Lin Liu a, Ying Yi a, Yu Yang a, Ping Yu a, Ji-Ming Chen b,** a b
Qingdao Center for Disease Control and Prevention, Qingdao 266032, PR China China Animal Health and Epidemiology Center, Qingdao 266032, PR China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 6 August 2010 Received in revised form 22 September 2010 Accepted 27 September 2010 Available online 8 October 2010
In 2009, a novel swine-origin H1N1 influenza virus sparked an influenza pandemic. The emergence of mutations in the viral genome is therefore of ongoing concern. In this study, the hemagglutinin (HA) gene sequences of 3444 pandemic H1N1 influenza viruses reported to the GenBank database and the sequences of 48 pandemic H1N1 influenza viruses detected in the Chinese city of Qingdao were analyzed. Among the 3492 viruses, 101 carried a serine to proline substitution at position 128 (S128P) in the viral HA gene. All the 101 S128P mutants belonged to Clade 7 which has become dominant worldwide since the summer of 2009. Among the 3492 viruses, 1646 were collected before July 25, 2009, and none of these viruses carried the S128P mutation. Furthermore, after July 25, 2009, the prevalence of the S128P mutant was 33.56% (99/295) in a region of Eurasia including Russia, Mongolia, mainland China and South Korea, but only 0.11% (2/1846) in the rest of the world. The data suggested that the originally rare S128P mutant has become prevalent in the Eurasia region, indicating that the S128P mutant likely transmitted more efficiently than other strains of the virus. Therefore, it is of significance to observe whether the S128P mutant will be more dominant worldwide in the coming future and investigate the exact effects of the S128P mutation. ß 2010 Elsevier B.V. All rights reserved.
Keywords: Pandemic influenza Influenza virus Mutation Epidemiology
1. Introduction On April 24, 2009, human infections with a novel swine-origin H1N1 subtype influenza virus in the USA and Mexico were reported by the World Health Organization (Smith et al., 2009). Within a few months the virus spread to many other countries, mainly through international air travel, and sparked the first human influenza pandemic of the 21st century (Garten et al., 2009; Smith et al., 2009). The emergence of mutations in the viral genome is of ongoing public and scientific concern, because such changes may affect the infectivity, pathogenicity, antigenicity, host tropism and drug sensitivity of the virus (Garten et al., 2009; Rambaut and Holmes, 2009; Glinsky, 2010; Janies et al., 2010; Maurer-Stroh et al., 2010). For example, it has been found that a glutamine to lysine substitution at position 391 (E391K) in the viral hemagglutinin (HA) is globally on the rise (Maurer-Stroh et al., 2010). This mutation may alter viral antigenicity and the HA oligomerization
* Corresponding author. Tel.: +86 532 85652104; fax: +86 532 85652104. ** Corresponding author. Tel.: +86 532 85623396; fax: +86 532 85623396. E-mail addresses: [email protected] (Z.-G. Wang), [email protected] (J.-M. Chen). 1567-1348/$ – see front matter ß 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.meegid.2010.09.014
interface which is important for membrane fusion. Additionally, a histidine to tyrosine substitution at position 274 (H274Y) in the viral neuraminidase protein has also been under intensive surveillance because it can render the virus resistant to the neuraminidase inhibitor oseltamivir (Dharan et al., 2009; Hauge et al., 2009). In this study, the HA gene sequences of 3444 pandemic H1N1 influenza viruses available in the GenBank database and the sequences of 48 pandemic H1N1 influenza viruses detected in Qingdao, a beach city in China, were analyzed to investigate whether a new mutation, a serine to proline substitution at position 128 (S128P), in the viral HA gene is of any epidemiological significance. 2. Materials and methods The 48 pandemic H1N1 influenza viruses detected in Qingdao, China, were from 15 randomly selected imported cases (individuals who just arrived from other countries or regions via air travel prior to sample collection) and 33 randomly selected domestic cases (those with no recent travel history to other countries or regions prior to sample collection). Case confirmation and gene sequencing were performed by the methods reported previously (Wang et al., in press). Briefly, the pandemic H1N1 influenza cases
T.-T. Yang et al. / Infection, Genetics and Evolution 11 (2011) 227–231
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Table 1 Background of the 48 pandemic H1N1 influenza viruses detected in Qingdao, China. GenBank accession number
Strains
Case sources
Sample collection date
Residue at position 128
CY050262 GU168018 GU144805 CY050263 CY050264 CY050265 CY050266 CY050267 CY050268 CY050269 CY050270 CY050271 CY050272 CY050273 CY050274 CY050140 CY050100 CY050098 CY050112 CY050113 CY050117 CY050119 CY050121 CY050130 CY067613 CY067614 CY067615 CY067616 CY067617 CY067618 CY067619 CY067620 CY067621 CY067622 CY067623 CY067624 CY067625 CY067626 CY067627 CY067628 CY067629 CY060631 CY060632 CY060633 CY060634 CY067611 CY067612 CY060630
A/Qingdao/148/2009 A/Qingdao/222/2009 A/Qingdao/231/2009 A/Qingdao/283/2009 A/Qingdao/292/2009 A/Qingdao/320/2009 A/Qingdao/322/2009 A/Qingdao/333/2009 A/Qingdao/359/2009 A/Qingdao/362/2009 A/Qingdao/375/2009 A/Qingdao/383/2009 A/Qingdao/399/2009 A/Qingdao/477/2009 A/Qingdao/492/2009 A/Qingdao/1008/2009 A/Qingdao/1006/2009 A/Qingdao/1020/2009 A/Qingdao/1170/2009 A/Qingdao/1215/2009 A/Qingdao/1261/2009 A/Qingdao/1268/2009 A/Qingdao/1269/2009 A/Qingdao/1364/2009 A/Qingdao/1508/2009 A/Qingdao/1517/2009 A/Qingdao/1530/2009 A/Qingdao/1568/2009 A/Qingdao/1577/2009 A/Qingdao/1609/2009 A/Qingdao/1610/2009 A/Qingdao/1626/2009 A/Qingdao/1629/2009 A/Qingdao/1631/2009 A/Qingdao/1642/2009 A/Qingdao/1669/2009 A/Qingdao/1773/2009 A/Qingdao/1775/2009 A/Qingdao/1776/2009 A/Qingdao/1779/2009 A/Qingdao/1780/2009 A/Qingdao/63/2010 A/Qingdao/66/2010 A/Qingdao/70/2010 A/Qingdao/94/2010 A/Qingdao/101/2010 A/Qingdao/123/2010 A/Qingdao/220/2010
Flying from Flying from Flying from Flying from Flying from Flying from Flying from Flying from Flying from Flying from Flying from Flying from Flying from Flying from Flying from Domestic Domestic Domestic Domestic Domestic Domestic Domestic Domestic Domestic Domestic Domestic Domestic Domestic Domestic Domestic Domestic Domestic Domestic Domestic Domestic Domestic Domestic Domestic Domestic Domestic Domestic Domestic Domestic Domestic Domestic Domestic Domestic Domestic
2009-06-13 2009-06-24 2009-06-27 2009-07-05 2009-07-06 2009-07-10 2009-07-10 2009-07-12 2009-07-16 2009-07-18 2009-07-19 2009-07-21 2009-07-23 2009-08-12 2009-08-17 2009-09-01 2009-09-04 2009-09-04 2009-09-08 2009-09-11 2009-09-15 2009-09-15 2009-09-15 2009-09-18 2009-10 2009-10 2009-10 2009-11 2009-11 2009-11 2009-11 2009-11 2009-11 2009-11 2009-11 2009-11 2009-12 2009-12 2009-12 2009-12 2009-12 2010-01 2010-01 2010-01 2010-01 2010-01 2010-02 2010-03
S S S S S S S S S S S S S S S S P P P S P P P P S P S P P S S S S P S L S S P P P P P P P S P P
were confirmed by a commercial real-time RT-PCR kit, and the fulllength HA gene was amplified by RT-PCR with a pair of universal primers for influenza A virus (Hoffmann et al., 2001), and sequenced thereafter. More information about the 48 cases and the relevant GenBank accession numbers are given in Table 1. The viral HA gene sequences were searched and aligned using the web server of the Influenza Virus Resource in NCBI (Bao et al., 2008). The geographical distribution and collection dates of the relevant viruses were also obtained from this web server. Prevalences were compared statistically by x2 test. The S128P mutants and some other pandemic H1N1 influenza viruses (reference viruses) were phylogenetically analyzed using the software MEGA 4.1 with the neighbor-joining (NJ) method and the model of maximum composition likelihood (Tamura et al., 2007). Substitution rates were set as different among sites and among lineages. Gaps were treated by pairwise-deletion. Bootstrap values were calculated out of 1000 replicates. At least one representative of each of the 7 clades identified previously (Nelson et al., 2010), and at most one of the viruses collected in the same city in the same month with known collection dates were selected
the USA Australia Chile Australia Australia South Korea France France Hong Kong Philippines South Korea India South Korea Indonesia Hong Kong
as the reference viruses. The reported HA gene sequences of the reference viruses should be no shorter than 1.2 kb. Some S128P mutants collected in same city in the same month were also excluded for the phylogenetic analysis if they were genetically similar to each other (genetic distances <1%, which were calculated by the same method used for the phylogenetic analysis). To evaluate the results obtained with the NJ method, the phylogenetic relationships among the sequences were also calculated using RAxML version 7.0.4 software with the General Time Reversible (GTR) substitution matrix and the maximum likelihood (ML) method (Stamatakis et al., 2008). 3. Results and discussion 3.1. Spatial distribution of the S128P mutant On July 26, 2010, the HA gene sequences of 3444 pandemic H1N1 influenza viruses isolated from humans were available in the GenBank database with known amino acid residues at position 128 and known collection months. Among the 3444 viruses and the 48
[()TD$FIG]
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229
Fig. 1. Distribution of the cities in Russia, Mongolia, mainland China and South Korea with the HA sequences of pandemic H1N1 influenza viruses available in GenBank on July 26, 2010. The cities with and without the S128P mutant in GenBank are marked with triangles and circles, respectively, in a human population distribution map downloaded from the Wikipedia website (http://en.wikipedia.org/wiki/World_population).
viruses we detected in Qingdao, 366 were from a region of Eurasia including 123 from Russia, 217 from mainland China, 9 from Mongolia and 17 from South Korea (Fig. 1). This region is referred to below as the RCMK region. The other 3126 viruses were from other parts of Eurasia, North and South America, Africa or Oceania. Among the 366 viruses from the RCMK region, 99 (85 from mainland China, six from Russia, five from South Korea, three from Mongolia) carried the S128P mutation in the HA gene. However, only two of the other 3126 viruses from the rest of the world carried the S128P mutation. Detailed information about the 101 S128P mutants is given in Supplementary File 1. In addition, four of the 3391 (3444 + 48–101) pandemic viruses lacking the S128P mutation showed other changes at this position (two S128T and two S128L mutations). According to the information provided in the GenBank database, the 366 viruses from the RCMK region and the 99 S128P mutants among these viruses were reported by 35 and 18 entities, respectively. The relevant samples were collected from 75 and 25 cities, respectively. Distribution of these cities is largely consistent with the host population distribution in this region (Fig. 1), indicating that the 366 viruses and the 99 mutants were geographically representative.
After July 26, 2009, the prevalence of the S128P mutation in the RCMK region was 33.56% [99/(366–71)], significantly higher (P < 0.01) than its counterpart in the rest of the world, 0.11% [2/ (3492–1646)]. Moreover, as shown in Table 2, the prevalence of the S128P mutant was rising in the RCMK region in 2009 from the percentage of the cities reporting the S128P mutant. However, because few viruses were collected in 2010 in the RCMK region, it remains unknown how prevalent the S128P mutant was in this region in 2010. 3.3. Distribution of the S128P mutation among the viruses detected in Qingdao Among the 48 viruses detected in Qingdao, 20 from the 33 domestic cases and none from the 15 imported cases carried the S128P mutation. Twelve of the 15 imported cases flied from out of the RCMK region. These data are consistent with the global distribution of the S128P mutant mentioned above. Because the viruses from the 33 domestic cases and the 15 imported cases were collected before and after August 18, 2009, respectively, the observed discrepancy between the domestic and the imported cases could be explained by the spatial distribution or the temporal distribution of the S128P mutant.
3.2. Temporal distribution of the S128P mutant 3.4. Variation at position 128 in other H1 subtype influenza viruses According to the collection dates (Supplementary File 1), the S128P mutant was found firstly in the city of Chengdu in Sichuan, China on July 26, 2009. In addition, among the 3492 (3444 + 48) viruses analyzed, 1646 were collected before July 25, 2009, including 71 from the RCMK region. None of these 1646 viruses carried the S128P mutation, indicating that this mutation was rare worldwide during the first three months of the pandemic. However, it remains unknown when and where the mutant came out.
On July 26, 2010, sequences of the viral HA gene of 554 avian, 669 swine and 3607 human seasonal H1 subtype influenza viruses were available in the GenBank database with known amino acid residues at position 128. All the 554 avian and 81.17% (543) of the 669 swine H1 subtype influenza viruses harbored threonine at position 128, while 93.15% (3360) of the 3607 human seasonal H1 subtype influenza viruses harbored another amino acid, valine, at the position.
Table 2 Temporal distribution of the S128P mutant based on 366 sequences reported from the RCMK region with known collection dates. Collection dates (day/month/year)
Number of S128P mutants
Number of total viruses
Number of cities reporting the S128P mutant
Number of cities reporting the pandemic viruses
Before 01/07/2009 01/07/2009–31/08/2009 01/09/2009–31/10/2009 01/11/2009–31/12/2010 After 31/12/2009
0 13 26 52 8
44 68 69 171 14
0 3 7 28 3
11 21 18 60 7
a b
(0.00%) (19.12%)a (37.68%)b (30.41%) (57.14%)
12 of the 13 mutants were collected in the same cluster in a city on July 26, 2009. Seven of the 26 mutants were collected in the Chinese city of Xian in September 2009.
(0.00%) (14.28%) (38.89%) (46.67%) (42.86%)
[()TD$FIG]
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Fig. 2. Phylogenetic tree of 84 S128P mutants and other 122 pandemic H1N1 influenza viruses based on the viral HA gene sequences. The S128P mutants and the other viruses are marked with triangles and circles, respectively. The tree is rooted by two earliest isolates of the pandemic virus, A/California/04/2009 and A/California/07/2009. Bootstrap values (>50) are given at the relevant nodes. All the viral designations are available in the Supplementary File 2, and they were elongated with the collection date plus ‘‘P’’ for the S128P mutants or ‘‘Cx’’ for the representatives of Clade x (x = 1, 2, . . ., 7).
3.5. Phylogenetic analysis of the S128P mutant Phylogenetic analysis of the HA gene sequences of 206 pandemic H1N1 influenza viruses, as shown in Fig. 2, suggested that all the S128P mutants were located in the terminal branches of the phylogenetic tree rooted with two earliest pandemic H1N1 viruses, A/California/04/2009 and A/California/07/2009 (Clade 1). All the S128P mutants belong to Clade 7 which has become dominant worldwide since the summer of 2009 (Valli et al., 2010).
It should be mentioned that the clades were classified according to the viral genomic sequences rather than the viral HA gene sequences, and therefore some clades were located together in the tree of Fig. 2. Most of the bootstrap values of the phylogenetic tree are rather low, which should be caused by that some intermediate sequences were involved in the analysis (Chen et al., 2007). The phylogenetic tree calculated using the ML method (data not shown) was of no obvious differences from that calculated using the NJ method (Fig. 2).
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3.6. Epidemiological inference of the distribution of the S128P mutant The aforementioned distribution data of the S128P mutation revealed an interesting scenario about the pandemic virus, namely, that the originally rare S128P mutant has become prevalent in the RCMK region. This indicated that the S128P mutant spread significantly more efficiently than other strains of this virus, resulting in its high prevalence there. Therefore, the S128P mutant likely has higher transmissibility than other strains of this virus in humans. This further indicates that this mutant may become more dominant worldwide in the coming future. It may be argued that the emerging dominance of the S128P mutant resulted not from its higher transmissibility, but from geographical isolation. However, geographical isolation can be excluded in this case due to the large-scale domestic and international travel that occurs worldwide (Suzuki and Nei, 2002; Chen et al., 2009; Garten et al., 2009). Previous reports indicated position 128 in the viral HA gene is near to one motif of the viral receptor-binding site (RBS) covering positions 131–135, and one of the viral antigenic epitopes covering positions 124, 125, 153, 157, 159 and 164 (Garten et al., 2009; Igarashi et al., 2010; Yang et al., 2010). Therefore, the S128P mutation may change the receptor-binding property and the antigenicity of the virus. The aforementioned variation of position 128 in the HA gene in avian, swine and human seasonal H1 subtype influenza viruses also suggested that this position is of some hostspecificity. However, the exact effects of the S128P mutation remain unknown. It is also unknown whether the S128P mutation changed the viral virulence in humans. Further studies are therefore needed to investigate the exact biomedical significance of the S128P mutation. Acknowledgments We thank Dr. Jiwang Chen in Northwest University in the USA for his critical comments on data analysis. This work was supported by the Pandemic Influenza Research Funds of Qingdao City (No. 09-1-1-49-nsh, 2008-wszd119 and 10-3-3-8-1-nsh) and Chinese Ministry of Agriculture. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.meegid.2010.09.014. References Bao, Y., Bolotov, P., Dernovoy, D., Kiryutin, B., Zaslavsky, L., Tatusova, T., Ostell, J., Lipman, D., 2008. The influenza virus resource at the National Center for Biotechnology Information. J. Virol. 82, 596–601.
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