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Molecular phylogeny and evolutionary dynamics of matrix gene of avian influenza viruses in China
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Molecular phylogeny and evolutionary dynamics of matrix gene of avian influenza viruses in China Peng Xiaorong, Wu Haibo, Peng Xiuming, Jin Changzhong, Lu Xiangyun, Xie Tiansheng, Cheng Linfang, Liu Fumin, Wu Nanping ⇑ State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, School of Medicine, Zhejiang University, 310003 Hangzhou, China
a r t i c l e
i n f o
Article history: Received 11 February 2015 Received in revised form 19 May 2015 Accepted 30 May 2015 Available online xxxx Keywords: Matrix gene Avian influenza virus Phylogenetic analysis Evolution China
a b s t r a c t In China, several subtype avian influenza viruses consistently circulate in poultry. Numerous studies have focused on the evolution of the hemagglutinin gene; however, studies on the evolution of the matrix (M) gene are limited. In this study, a large-scale phylogenetic analysis of M gene sequences of avian influenza viruses isolated in China revealed that the M gene has evolved into six different lineages denoted as I–VI. The majority of lineages I and IV were isolated in terrestrial birds, while the majority of lineages II, III, V and VI were isolated in aquatic birds. Lineage I included 148 H9N2 subtype viruses (72.2%), lineage II comprised of 63 H6 subtype viruses (100%), and lineage IV included 157 H5 subtype viruses (97.5%). The mean substitution rates of different lineages ranged from 1.32 10 3 (lineage III) to 3.64 10 3 (lineage IV) substitutions per site per year. According to the most recent common ancestor of all lineages, lineage III was the oldest lineage, formed in 1981 or even earlier. And lineage V was the most recent, established around the year 2000. Selective pressure on M2 was stronger than that on M1. The strongest selection pressure was observed in lineage IV. In addition, site-by-site analyses identified 8 positive selection sites, all in M2. Most of the sites (5 out of 8) were located in the extracellular domain, which is an antigen for vaccine development. The positive selection sites (amino acid positions 66, 82 and 97) are likely associated with virus budding. This study enhanced our knowledge of M gene evolution of avian influenza viruses, and is expected to improve the early detection of new viruses and lead to vaccine development. Ó 2015 Published by Elsevier B.V.
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1. Introduction
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In China, several subtype avian influenza viruses (AIVs) consistently circulate in poultry, including highly pathogenic avian influenza (HPAI, i.e. H5) (Ma et al., 2015), and low pathogenic avian influenza (i.e. H9 and H6) (Huang et al., 2012). Through gene reassortment and antigenic drift, many novel subtype AIVs have emerged (i.e. H7N9, H10N8 and H5N8) (Chen et al., 2014; Chen et al., 2013; Wu et al., 2014). These AIVs not only kill thousands of poultry, causing huge economic losses, but also represent a threat to humans. Thus, it is necessary to understand the evolutionary processes of AIV to gain a better knowledge of this pandemic virus. Numerous studies have focused on the evolution of the hemagglutinin antigen (HA) gene (Huang et al., 2012; Ma et al., 2015; Wu et al., 2010); however, only a few studies have
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⇑ Corresponding author. E-mail addresses: [email protected] (P. Xiaorong), fl[email protected] (W. Nanping).
addressed the evolution of the matrix (M) gene (Chander et al., 2013). The M gene encodes two partly overlapping proteins, a highly conserved 252-amino-acid M1 protein and a 97-amino-acid M2 protein (Scholtissek et al., 2002; Sun et al., 2010). The M1 protein binds to the cytoplasmic tails of HA and neuraminidase (NA), and bridges interactions between the viral lipid membrane and the ribonucleoprotein (RNP) core (Schmitt and Lamb, 2005). M1 and M2 play a vital role in viral assembly and budding. During viral assembly, M1 recruits several viral components (HA, NA, M2 and RNP) to the site of assembly (Gomez-Puertas et al., 2000). M2 initially stabilizes the site of budding, and subsequently alters membrane curvature, causing membrane scission and the release of the progeny virion (McCown and Pekosz, 2006; Rossman and Lamb, 2011). The M2 protein is a transmembrane protein: position 1–24 is the extracellular domain, position 25–43 is the transmembrane domain and position 44–97 is the cytoplasmic domain (Scholtissek et al., 2002). The transmembrane domain of M2 has
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Please cite this article in press as: Xiaorong, P., et al. Molecular phylogeny and evolutionary dynamics of matrix gene of avian influenza viruses in China. Infect. Genet. Evol. (2015), http://dx.doi.org/10.1016/j.meegid.2015.05.033
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Fig. 1. Phylogenetic trees for the M gene. The phylogenetic trees were constructed using the maximum likelihood method. Scale bar shows the evolutionary distance. The subtype, host and year of each M gene are indicated by different colored bars (see legend). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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ion channel activity, which is the target of adamantanes, the first class of antiviral drugs approved for treatment of human influenza (Salter et al., 2011). The widespread use of adamantanes against influenza has led to the emergence of resistant virus strains (Bright et al., 2005). Adamantine resistance is characterized by a mutation in one of five sites (positions 26, 27, 28, 31 or 34) in the M2 gene, although the most commonly observed mutation is S31N (Wang et al., 2013). The extracellular domain of the M2 protein (M2e), which contains 24 residues at the N-terminus, is highly conserved in all human epidemic strains, independent of subtype (Liu et al., 2005). Therefore, M2e serves as an attractive target for the development of universal influenza subunit vaccines (Fiers et al., 2004). Nevertheless, an avian-type M2e consensus amino acid sequence is up to 5 positions different from a human-type consensus sequence (Liu et al., 2005). Whether the M2e of AIVs in China is conserved or is under positive selection is unknown. Therefore, understanding of evolution of the M gene is very important and is practically relevant. In this study, a large-scale phylogenetic analysis of M genes of AIVs isolated in China was conducted to infer their evolutionary relationship. The substitution rate and the most recent common ancestor (TMRCA) were estimated using a Bayesian Markov Chain Monte Carlo (MCMC) method. We also estimated non-synonymous to synonymous substitution rate ratios (dN/dS ratio), and investigated the positive selection sites for each lineage. These analyses increase our knowledge of M gene evolution in AIVs, and are expected to improve AIVs surveillance and vaccine development.
2. Materials and methods
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2.1. Sequence data
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The nucleotide sequences of M gene were obtained from GenBank, hosted by the National Center for Biotechnology Information, on December 31, 2014 (Bao et al., 2008). All sequence data for the strains with a full-length M gene of any subtypes of AIVs isolated in China were included. Identical sequences in a dataset were represented by the oldest sequence in the group. Sequencing data were obtained together with information about the host, subtype, isolation year and isolation place. The sequences were highly similar; therefore, we picked at least one sequence per year, per subtype, per place for further study. A total of 644 sequences were obtained (the accession numbers are listed in additional file 1). All segments were aligned using the default settings in MUSCLE v3.5 (Edgar, 2004).
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2.2. Phylogenetic tree analysis
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To enhance the phylogenetic analysis of M gene of AIVs in China, we also included several M genes of AIVs from several geographical origins (North-American and other Eurasian out of China) and different host origins (human, swine, canine and equine). The sequence data for the coding regions only were used; i.e. from nucleotide position 26 to 1007. Phylogenetic analysis was conducted using the maximum likelihood (ML) method in RAxML (Stamatakis, 2014). Analyses of 1000 bootstrap replicates were performed using GTR-GAMMA, the GTR model of nucleotide
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Please cite this article in press as: Xiaorong, P., et al. Molecular phylogeny and evolutionary dynamics of matrix gene of avian influenza viruses in China. Infect. Genet. Evol. (2015), http://dx.doi.org/10.1016/j.meegid.2015.05.033
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P. Xiaorong et al. / Infection, Genetics and Evolution xxx (2015) xxx–xxx Table 1 Lineages and annotations of AIV M genes. Representative sequence
Total number
HA subtype
Host
Year
Lineage I
A/Chicken/Beijing/1/1994 (Ck/BJ/1/94)
205
H9 H7 H6 H5
Duck (12.1%) Chicken (64.4%) Other terrestrial birds (18.3%)
2011– (28.3%) 2006–2010 (27.8%) 2001–2005 (28.3%) –2000 (13.6%)
Lineage II
A/wild duck/shantou/2853/2003 (WDK/ST/2853/03)
63
H6 (100%)
Duck (74.6%), Wild duck (11.1%), Chicken (7.9%), Other aquatic birds (6.3%)
2011– (6.3%), 2006–2010 (69.8%), 2001–2005 (19.0%), –2000 (3.1%)
Lineage III
A/duck/Nanchang/1749/1992 (DK/NC/1749/92)
37
H6 (35.14%) H3 24.32%) H7 (10.81%)
Duck (62.16%), Other aquatic birds (29.73%), Terrestrial birds (8.11%)
2011– (10.81%) 2006–2010 (45.95%) 2001–2005 (29.73%) –2000 (13.51%)
Lineage IV
A/goose/Guangdong/1/1996 (Gs/GD/1/96)
161
H5 (97.5%)
Duck (29.8%) Wild duck (4.3%), Chicken (34.1%) Others (31.8%)
2011– (12.4%) 2006–2010 (39.1%) 2001–2005 (47.8%) –2000 (0.6%)
Lineage V
A/duck/Shanghai/28–1/2009 (DK/SH/29–1/09)
H11 (15.38%) H7 (19.23%) H3 (26.92%)
Duck (80.77%) Other aquatic birds (7.69%) Chicken (7.69%) Other terrestrial birds (3.85%)
2011– (80.77%) 2006–2010 (11.54%) 2001–2005 (7.69%)
Lineage VI
A/duck/Nanchang/1681/1992 (DK/NC/1681/92-like)
H6 H4 H7 H3 H5
Duck (59%), Other aquatic birds (25%) Wild aquatic birds (14%) Chicken (1%)
2011– (21%) 2006–2010 (34%) 2001–2005 (36%) –2000 (9%)
26
100
(73.2%) (7.8%) (12.6%) (4.8%)
(31%) (14%) (11%) (11%) (9%)
Bold values are the most common characteristics of each lineage.
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substitution with the Gamma model of rate heterogeneity. The tree was color-coded by using FigTree (ver.1.4.2) (http://www.tree.bio. ed.ac.uk/software/figtree/).
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2.3. Evolutionary rate
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The substitution rate and TMRCA were estimated using a MCMC method as implemented in BEAST (Drummond et al., 2012). The SRD06 nucleotide substitution model (Shapiro et al., 2006) and coalescent Bayesian skyline model (Minin et al., 2008) were incorporated in the MCMC method. A relaxed molecular clock model with uncorrelated lognormal distribution was used to inferred the time-scaled maximum clade credibility phylogenies (Drummond et al., 2006). Multiple independent MCMC runs were performed and assessed for consistency. They were combined to give a total chain length of 0.5–4 108 steps, with sampling at every 100 steps. Convergence of relevant parameters was assessed by effective sample sizes over 200 in Tracer v1.5 (http://tree.bio.ed. ac.uk/software/tracer/).
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2.4. Selection pressure analysis To examine the selection pressure experienced by each lineage, we estimated the ratio of non-synonymous (dN) to synonymous (dS) substitutions for each lineage, using the HyPhy package (Pond et al., 2005). Selection pressure analysis acting on the codons of M1 and M2 of each lineage was carried out using HyPhy open-source software package available under the datamonkey web-server (http://www.datamonkey.org/) (Delport et al., 2010). The ratio of dN to dS substitutions per site was estimated using three different approaches: single likelihood ancestor counting (SLAC), fixed effects likelihood (FEL) (Kosakovsky Pond and Frost, 2005), and internal fixed effects likelihood (IFEL) (Pond et al., 2006). Best nucleotide substitutions models adopted in the analyses for different data sets were determined through the available tool in the datamonkey server.
Table 2 Substitution rates of viral M lineages. Lineage
I II III IV V VI
Substitution rate (10 site/year)
3
subs/
tMRCA(calendar year)
Mean
95% HPD lower
95% HPD upper
Mean
95% HPD lower
95% HPD upper
2.27 2.09 1.32 3.64 3.15 1.59
1.92 1.64 0.84 3.04 2.11 1.23
2.60 2.56 1.83 4.21 4.31 2.00
1990 1994 1981 1989 2000 1991
1994 1999 1990 1994 2001 1992
1982 1989 1970 1983 1997 1988
2.5. Protein structural analyses
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To map positive selection sites onto the protein structures and to better understand how the positive selection sites might interact with other proteins, we carried out structural analyses of M2 proteins using the Molecular Modeling Database (MMDB) (Madej et al., 2012). The three-dimensional structure of the extracellular domain of M2 was downloaded from the database (MMDB ID: 124024). Site 10 of M2e was highlighted in yellow using the Cn3D 3.4 software (Porter et al., 2007).
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3. Results
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3.1. Phylogenetic analysis
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The phylogenetic tree of all M genes was constructed (Fig. 1), using the ML approach with bootstrapping analyses to assess clade robustness. We defined ‘‘lineage’’ as an aggregate of large branches. These lineages were denoted as I–VI. Several reference sequences were chosen to represent main differences in each lineage. Lineage I (82% bootstrap support) contained 205 sequences, including 148 H9N2 subtype viruses (72.2%), 13 H7N9 subtype viruses (6.3%) and many other different subtypes. The H7N9
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Fig. 2. Bayesian inferences of different lineages. A: (lineage I), B: (lineage II), C: (lineage III), D: (lineage IV), E: (lineage V), F: (lineage VI). Branch coloring indicates inferred rates of nucleotide substitution from blue (0 10 3 subs/site/year) to red (0.16 10 3 subs/site/year) (see legend). The scale bar indicates the number of years before the present. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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subtype AIVs isolated in 2013 were caused major outbreaks of human infection (Chen et al., 2014; Liu et al., 2013). One hundred and sixty-eight sequences (82.7%) were isolated in terrestrial birds, 133 (64.8%) of them in chickens (Table 1). A/Chicken/Beijing/1/1994 (Ck/BJ/1/94) was chosen to represent lineage I, which was the first H9N2 subtype AIV isolated in China. Lineage II (100% bootstrap support) comprised of 63 H6 subtype viruses (100%). Fifty-eight sequences (92%) were isolated in aquatic birds, 47 (74.6%) in ducks, 7 (11.1%) in wild ducks. A/wild duck/shantou/2853/2003 (WKD/ST/2853/03) was chosen to represent lineage II. Lineage III (58% bootstrap support) contained 37 sequences. Thirty-four sequences (91.9%) were isolated in aquatic birds, 21
(62.2%) in ducks. A/duck/Nanchang/1749/1992 (DK/NC/1749/92) was chosen to represent lineage III. Lineage IV (70% bootstrap support) comprised of 161 sequences, including 157 H5 subtype viruses (97.5%). These viruses were detected in multiple species, mostly in chicken (34.1%). These AIVs were responsible for several HPAI outbreaks in China (Li et al., 2004). A/goose/Guangdong/1/1996 (Gs/GD/1/96) was chosen to represent lineage IV, which was the first H5N1 subtype AIV isolated in China. Lineage V (51% bootstrap support) contained 26 sequences. Twenty-three sequences (88.4%) were isolated in aquatic birds, 21 (80.77%) in ducks. Twenty-one sequences (80.77%) were isolated after 2011, which suggested that this lineage was a newly
Please cite this article in press as: Xiaorong, P., et al. Molecular phylogeny and evolutionary dynamics of matrix gene of avian influenza viruses in China. Infect. Genet. Evol. (2015), http://dx.doi.org/10.1016/j.meegid.2015.05.033
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Fig. 3. Selective pressure among different lineages. Selective pressures for the entire sequence (dN/dS) were calculated separately for M1 and M2. The error bar shows the 95% confidence interval.
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formed one. A/duck/Shanghai/28-1/2009 (DK/SH/29-1/2009) was chosen to represent lineage V. Lineage VI (12% bootstrap support) comprised of 100 sequences, including 31 H6 subtype viruses (31%), 14 H4 subtype viruses (14%), 11 H7 subtype viruses (11%) and 11 H3 subtype viruses (11%). Ninety-eight sequences (98%) were isolated from aquatic birds, 14 (14%) in wild ducks. A/duck/Nanchang/1681/1992 (DK/NC/1681/92) was chosen to represent lineage VI.
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3.2. Evolutionary rate
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We calculated the evolutionary rate and TMRCA of all lineages, which are shown in Table 2. The mean substitution rates of the M gene ranged from 1.3 10 3 (lineage III) to 3.6 10 3 (lineage IV) substitutions per site per year. The special evolutionary rates in each branch are shown in Fig. 2. The mean TMRCA of lineage III was dated back to 1981, with the 95% highest posterior density (HPD) interval between 1970 and 1990. And lineage V was the newly formed one, established in about 2000, with the 95% HPD interval between 1997 and 2001.
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3.3. Selective pressures
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We defined the magnitude of the pressure as the estimate of the dN/dS ratio. A higher selective pressure indicated that the gene (or the site) was under stronger selection (positive selection) for amino acid substitution ( Pond et al., 2007). Selective pressure was statistically stronger in M2 than that in M1 for all lineages (Fig. 3).
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The highest dN/dS ratio was observed in lineage IV: the dN/dS ratio for M1 genes was 0.1 and was 0.6 for M2 genes, while the lineage III appeared to be under the strongest negative selection (the dN/dS ratios for M1 genes was 0.04; and for M2 genes was 0.23).
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3.4. Site-by-site analyses
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Site-by-site (by each codon) analyses for all lineages were conducted by SLAC, FEL and iFEL. Positive selection sites by at least one test were identified in different M lineages (Table 3). No codons in M1 of all lineages were under significant positive selection (P < 0.05). Six sites were positively selected in M2 of lineage I: four sites in the extracellular domain (positions 10, 13, 16 and 21), and two sites in the cytoplasmic domain (positions 82 and 97). There were three positive selection sites in lineage IV (positions 14, 66 and 82). The rest of lineages had no positive selection sites. The 3D structure of M2e is shown in Fig. 4. According to the structural analysis, positions 10 located at the middle of M2e is important for the binding of M2e to antibodies. The most common amino acids at the M2e 10–20 site of AIVs in our study remained PTRNGWECKCS (Table 4). However, we noted that sites 10, 13, 14 and 16 were under positive selection in at least one lineage.
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4. Discussion
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In this study, we compiled a comprehensive dataset and carried out a large-scale sequence analysis of the M genes in AIVs isolated in China. Since only a small portion of M gene in AIVs gets sequenced, this data set in our study is just a small sample of all the viruses that circulated in Chinese poultry. This issue is a potential weakness of our study. Active surveillance of AIVs could enhance our knowledge about the evolution of M gene. In previous reports, the M genes of AIVs were divided into two different lineages, Eurasian avian and North American avian (Furuse et al., 2009; Webster et al., 1992). The phylogenetic tree showed that the M genes in our study all belonged to the Eurasian lineage and evolved independently in six lineages, denoted as I–VI. The majority of lineage I was the H9N2 subtype AIV. The H9N2 influenza virus is now the most prevalent in chickens in china (Sun and Liu, 2015). Lineage I also included several different subtype AIVs (H5N1, H6, H7N9 and H10N8), which suggested the H9N2 influenza virus reassorted with multiple other subtype viruses (Xu et al., 2007). The H9N2 influenza did not induce obvious clinical signs or deaths in chickens, which suggested that it can persistently evolve to adapt to this special host (Webster, 2004).
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Table 3 Positive selection sites for M1 and M2 detected using SLAC, FEL and IFEL methods. Lineage
Protein
I
M1 M2
IV
M1 M2
Domaina
Codon
Normalized dN-dS
p-Value
Normalized dN-dS
p-Value
Normalized dN-dS
p-Value
ex ex ex ex cy cy
None 10 13 16 21 82 97
2.88 1.673 2.471 2.428 5.093 5.597
0.042 0.199 0.083 0.081 0.0064 0.021
1.771 0.811 1.387 1.372 2.628 3.025
0.017 0.049 0.046 0.043 0.0063 0.061
4.643 0.946 1.75 2.33 3.567 2.223
0.0007 0.063 0.044 0.018 0.0039 0.207
ex cy cy
None 14 66 82
4.52 8.57 7.95
0.163 0.057 0.04
1.86 3.92 3.05
0.047 0.02 0.1
3.23 2.52 2.78
0.024 0.17 0.143
SLAC
FEL
iFEL
Bold values are those deemed to indicate significantly positive or negative selection (P < 0.05). a Ex indicates extracellular domain; Tr, transmembrane domain; and Cy, cytoplasmic domain.
Please cite this article in press as: Xiaorong, P., et al. Molecular phylogeny and evolutionary dynamics of matrix gene of avian influenza viruses in China. Infect. Genet. Evol. (2015), http://dx.doi.org/10.1016/j.meegid.2015.05.033
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interface/EN_GIP_20150106CumulativeNumberH5N1cases.pdf). Those two lineages represent a serious threat as potential pandemic virus for humans. Therefore, active surveillance of the molecular evolution of these viruses is the best option to detect and interrupt their transmission. Aquatic birds are accepted as the natural reservoirs of influenza A viruses, and these viruses have been introduced to other animals, shaping the current ecology of influenza viruses (Webster et al., 1992). Introduction of influenza viruses from migratory aquatic birds through domestic ducks to other poultry was frequently observed in the recent decades (Chen et al., 2013; Ma et al., 2015). The influenza virus pool in aquatic birds is a never-ending source of genetic and antigenic material (Macken et al., 2006). In the present study, the genepool in aquatic birds is represented by the sequences in lineages III and VI. The evolutionary rates and selective pressure of these two lineages were lower than others. TMRCA of lineage III indicated that it is the oldest one. M genes from the H6N2 subtype AIVs were established as a monophyletic group (lineage II), just as in a previous study (Huang et al., 2012). Lineage V was a new group never reported before. Interestingly, the evolutionary rates of these two lineages were significantly higher than those from lineage III and VI. One possible explanation is that strains in a lineage that appeared more recently have had to evolve more rapidly to adapt better to the host or viral replication (Furuse et al., 2009). These findings suggest that domestic ducks can facilitate significant genetic and antigenic changes of viruses. The selective pressure is stronger on M2 than that on M1; more sites under positive selection were identified in M2 than in M1. Among them, most of the sites (5 out of 8) under positive selection in M2 are located in the extracellular domain. Although the most common amino acids at the M2e 10–20 site remained PTRNGWECKCS (Liu et al., 2005), the positive selection would change this fact in the near future. Since M2e is an antigen for vaccine development, especially the position 10 located at the middle of M2e which was important with the binding to antibodies, surveillance of these positive selection positions is an important part in development of the universal influenza A vaccine. The cytoplasmic domain of M2 is important for interaction with M1, genome packaging, and formation of virus particles. One site was under positive selection in the cytoplasmic domain of M2 (position 82). Positions 82–89 were important for infectious virus production (McCown and Pekosz, 2006). Another study showed that vRNP packaging is mediated by amino acids at positions 70–89 of the M2 gene (McCown and Pekosz, 2005). The M2 gene must, therefore, have evolved with several functions. In summary, we analyzed the M genes of AIVs isolated in China and studied their evolutionary dynamics. The M gene has evolved into different lineages with varied evolutionary rates and selection
Fig. 4. 3D crystal structure of the interaction between the extracellular domain of M2 and a monoclonal antibody. The pink protein represents the heavy chain of the monoclonal antibody from mouse (Mus musculus) and the brown protein is the light chain of monoclonal antibody. Site 10 under positive selection for Lineage H9 and DK is highlighted in yellow. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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Moreover, H9N2 influenza viruses from poultry could occasionally be transmitted from poultry to mammalian species, including humans (Butt et al., 2005). A serological surveillance study in Guangzhou in 2008 showed that the anti-H9 antibody positive rate among poultry retailers was up to 15.5% (Wang et al., 2009), which highlighted that these viruses can adapt to humans. H7N9 and H10N8 are novel human avian influenza viruses, which have emerged in humans in China from 2013, acquiring their internal gene by reassortment with H9N2 viruses (Chen et al., 2014; Liu et al., 2013). Therefore, poultry carrying H9N2 could be incubators for novel AIVs capable of infecting humans. The majority of lineage IV viruses were from the H5 subtype AIV. The H5N1 HPAI virus has been a known pathogen of chickens for many years (Li et al., 2004), responsible for several outbreaks in recent years (Kang et al., 2014; Kim et al., 2008). The H5N8 subtype virus, isolated in Zhejiang, was most closely related to isolates from poultry in countries in eastern Asia (Wu et al., 2014). H5N1 has spread to cause infection and death in many species including domestic birds and mammals (i.e. tigers, dogs, cats and humans) (Ma et al., 2009). From 2003 to 2014 the cumulative number of humans infected with the H5N1 AIVs cases in China reached 47 with 30 fatalities (http://www.who.int/influenza/human_animal_
Table 4 Amino acid sequence of all M2 protein on 10–20. Position
10
11
12
13
14
15
16
17
18
19
20
Conserved amino acid Amino acida
P P (73.5%) L (20.2%) H (6.1%)
T T (99.0%) I (0.8%) S (0.2%)
R R (96.3%) K (3.7%)
N N (81.5%) T (14.6%) S (1.6%) A (0.3%) K (0.3%)
G G (77.2%) E (22.8%)
W W (100%)
E E (90.4%) G (8.5%) V (1.0%) A (0.2%)
C C (99.5%) Y (0.3%) G (0.2%)
K K (43.1%) R (36.4%) N (19.7%) S (0.6%) I (0.2%)
C C (99.8%) Y (0.2%)
S S (95.2%) N (4.3%) G (0.3%) I (0.2%)
Bold values are those positions highly conserved (>99%). a Highly conserved amino acid sequence of M2e in avian influenza viruses is PTRNGWECKCS (Liu et al., 2005).
Please cite this article in press as: Xiaorong, P., et al. Molecular phylogeny and evolutionary dynamics of matrix gene of avian influenza viruses in China. Infect. Genet. Evol. (2015), http://dx.doi.org/10.1016/j.meegid.2015.05.033
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pressures. In addition, mutations of amino acids at the positive selection sites are likely associated with virus budding and antigen recognition. This study enhances our knowledge of gene evolution of AIVs, and is expected to improve the early detection of new viruses and lead to M2e vaccine development.
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This study was supported by Grants from the National Key Technologies R&D Programme for the 12th Five-Year Plan of China (2012ZX1000-004-005) and the Independent Task of State Key Laboratory for Diagnosis and Treatment of Infectious Diseases (Nos. 2010ZZ04 and 2014ZZ12). We gratefully acknowledge the support of the IBM high performance computing cluster of Bio-macromolecules Analysis Lab, Analysis Center of Agrobiology and Environmental Sciences, Zhejiang University.
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Appendix A. Supplementary data
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Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.meegid.2015.05. 033.
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Infection, Genetics and Evolution journal homepage: www.elsevier.com/locate/meegid 5 6 3 4 7 8 9 10 11 12 1 2 4 7 15 16 17 18 19 20 21 22 23 24 25 26
Molecular phylogeny and evolutionary dynamics of matrix gene of avian influenza viruses in China Peng Xiaorong, Wu Haibo, Peng Xiuming, Jin Changzhong, Lu Xiangyun, Xie Tiansheng, Cheng Linfang, Liu Fumin, Wu Nanping ⇑ State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, School of Medicine, Zhejiang University, 310003 Hangzhou, China
a r t i c l e
i n f o
Article history: Received 11 February 2015 Received in revised form 19 May 2015 Accepted 30 May 2015 Available online xxxx Keywords: Matrix gene Avian influenza virus Phylogenetic analysis Evolution China
a b s t r a c t In China, several subtype avian influenza viruses consistently circulate in poultry. Numerous studies have focused on the evolution of the hemagglutinin gene; however, studies on the evolution of the matrix (M) gene are limited. In this study, a large-scale phylogenetic analysis of M gene sequences of avian influenza viruses isolated in China revealed that the M gene has evolved into six different lineages denoted as I–VI. The majority of lineages I and IV were isolated in terrestrial birds, while the majority of lineages II, III, V and VI were isolated in aquatic birds. Lineage I included 148 H9N2 subtype viruses (72.2%), lineage II comprised of 63 H6 subtype viruses (100%), and lineage IV included 157 H5 subtype viruses (97.5%). The mean substitution rates of different lineages ranged from 1.32 10 3 (lineage III) to 3.64 10 3 (lineage IV) substitutions per site per year. According to the most recent common ancestor of all lineages, lineage III was the oldest lineage, formed in 1981 or even earlier. And lineage V was the most recent, established around the year 2000. Selective pressure on M2 was stronger than that on M1. The strongest selection pressure was observed in lineage IV. In addition, site-by-site analyses identified 8 positive selection sites, all in M2. Most of the sites (5 out of 8) were located in the extracellular domain, which is an antigen for vaccine development. The positive selection sites (amino acid positions 66, 82 and 97) are likely associated with virus budding. This study enhanced our knowledge of M gene evolution of avian influenza viruses, and is expected to improve the early detection of new viruses and lead to vaccine development. Ó 2015 Published by Elsevier B.V.
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1. Introduction
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In China, several subtype avian influenza viruses (AIVs) consistently circulate in poultry, including highly pathogenic avian influenza (HPAI, i.e. H5) (Ma et al., 2015), and low pathogenic avian influenza (i.e. H9 and H6) (Huang et al., 2012). Through gene reassortment and antigenic drift, many novel subtype AIVs have emerged (i.e. H7N9, H10N8 and H5N8) (Chen et al., 2014; Chen et al., 2013; Wu et al., 2014). These AIVs not only kill thousands of poultry, causing huge economic losses, but also represent a threat to humans. Thus, it is necessary to understand the evolutionary processes of AIV to gain a better knowledge of this pandemic virus. Numerous studies have focused on the evolution of the hemagglutinin antigen (HA) gene (Huang et al., 2012; Ma et al., 2015; Wu et al., 2010); however, only a few studies have
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⇑ Corresponding author. E-mail addresses: [email protected] (P. Xiaorong), fl[email protected] (W. Nanping).
addressed the evolution of the matrix (M) gene (Chander et al., 2013). The M gene encodes two partly overlapping proteins, a highly conserved 252-amino-acid M1 protein and a 97-amino-acid M2 protein (Scholtissek et al., 2002; Sun et al., 2010). The M1 protein binds to the cytoplasmic tails of HA and neuraminidase (NA), and bridges interactions between the viral lipid membrane and the ribonucleoprotein (RNP) core (Schmitt and Lamb, 2005). M1 and M2 play a vital role in viral assembly and budding. During viral assembly, M1 recruits several viral components (HA, NA, M2 and RNP) to the site of assembly (Gomez-Puertas et al., 2000). M2 initially stabilizes the site of budding, and subsequently alters membrane curvature, causing membrane scission and the release of the progeny virion (McCown and Pekosz, 2006; Rossman and Lamb, 2011). The M2 protein is a transmembrane protein: position 1–24 is the extracellular domain, position 25–43 is the transmembrane domain and position 44–97 is the cytoplasmic domain (Scholtissek et al., 2002). The transmembrane domain of M2 has
http://dx.doi.org/10.1016/j.meegid.2015.05.033 1567-1348/Ó 2015 Published by Elsevier B.V.
Please cite this article in press as: Xiaorong, P., et al. Molecular phylogeny and evolutionary dynamics of matrix gene of avian influenza viruses in China. Infect. Genet. Evol. (2015), http://dx.doi.org/10.1016/j.meegid.2015.05.033
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Fig. 1. Phylogenetic trees for the M gene. The phylogenetic trees were constructed using the maximum likelihood method. Scale bar shows the evolutionary distance. The subtype, host and year of each M gene are indicated by different colored bars (see legend). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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ion channel activity, which is the target of adamantanes, the first class of antiviral drugs approved for treatment of human influenza (Salter et al., 2011). The widespread use of adamantanes against influenza has led to the emergence of resistant virus strains (Bright et al., 2005). Adamantine resistance is characterized by a mutation in one of five sites (positions 26, 27, 28, 31 or 34) in the M2 gene, although the most commonly observed mutation is S31N (Wang et al., 2013). The extracellular domain of the M2 protein (M2e), which contains 24 residues at the N-terminus, is highly conserved in all human epidemic strains, independent of subtype (Liu et al., 2005). Therefore, M2e serves as an attractive target for the development of universal influenza subunit vaccines (Fiers et al., 2004). Nevertheless, an avian-type M2e consensus amino acid sequence is up to 5 positions different from a human-type consensus sequence (Liu et al., 2005). Whether the M2e of AIVs in China is conserved or is under positive selection is unknown. Therefore, understanding of evolution of the M gene is very important and is practically relevant. In this study, a large-scale phylogenetic analysis of M genes of AIVs isolated in China was conducted to infer their evolutionary relationship. The substitution rate and the most recent common ancestor (TMRCA) were estimated using a Bayesian Markov Chain Monte Carlo (MCMC) method. We also estimated non-synonymous to synonymous substitution rate ratios (dN/dS ratio), and investigated the positive selection sites for each lineage. These analyses increase our knowledge of M gene evolution in AIVs, and are expected to improve AIVs surveillance and vaccine development.
2. Materials and methods
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2.1. Sequence data
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The nucleotide sequences of M gene were obtained from GenBank, hosted by the National Center for Biotechnology Information, on December 31, 2014 (Bao et al., 2008). All sequence data for the strains with a full-length M gene of any subtypes of AIVs isolated in China were included. Identical sequences in a dataset were represented by the oldest sequence in the group. Sequencing data were obtained together with information about the host, subtype, isolation year and isolation place. The sequences were highly similar; therefore, we picked at least one sequence per year, per subtype, per place for further study. A total of 644 sequences were obtained (the accession numbers are listed in additional file 1). All segments were aligned using the default settings in MUSCLE v3.5 (Edgar, 2004).
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2.2. Phylogenetic tree analysis
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To enhance the phylogenetic analysis of M gene of AIVs in China, we also included several M genes of AIVs from several geographical origins (North-American and other Eurasian out of China) and different host origins (human, swine, canine and equine). The sequence data for the coding regions only were used; i.e. from nucleotide position 26 to 1007. Phylogenetic analysis was conducted using the maximum likelihood (ML) method in RAxML (Stamatakis, 2014). Analyses of 1000 bootstrap replicates were performed using GTR-GAMMA, the GTR model of nucleotide
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P. Xiaorong et al. / Infection, Genetics and Evolution xxx (2015) xxx–xxx Table 1 Lineages and annotations of AIV M genes. Representative sequence
Total number
HA subtype
Host
Year
Lineage I
A/Chicken/Beijing/1/1994 (Ck/BJ/1/94)
205
H9 H7 H6 H5
Duck (12.1%) Chicken (64.4%) Other terrestrial birds (18.3%)
2011– (28.3%) 2006–2010 (27.8%) 2001–2005 (28.3%) –2000 (13.6%)
Lineage II
A/wild duck/shantou/2853/2003 (WDK/ST/2853/03)
63
H6 (100%)
Duck (74.6%), Wild duck (11.1%), Chicken (7.9%), Other aquatic birds (6.3%)
2011– (6.3%), 2006–2010 (69.8%), 2001–2005 (19.0%), –2000 (3.1%)
Lineage III
A/duck/Nanchang/1749/1992 (DK/NC/1749/92)
37
H6 (35.14%) H3 24.32%) H7 (10.81%)
Duck (62.16%), Other aquatic birds (29.73%), Terrestrial birds (8.11%)
2011– (10.81%) 2006–2010 (45.95%) 2001–2005 (29.73%) –2000 (13.51%)
Lineage IV
A/goose/Guangdong/1/1996 (Gs/GD/1/96)
161
H5 (97.5%)
Duck (29.8%) Wild duck (4.3%), Chicken (34.1%) Others (31.8%)
2011– (12.4%) 2006–2010 (39.1%) 2001–2005 (47.8%) –2000 (0.6%)
Lineage V
A/duck/Shanghai/28–1/2009 (DK/SH/29–1/09)
H11 (15.38%) H7 (19.23%) H3 (26.92%)
Duck (80.77%) Other aquatic birds (7.69%) Chicken (7.69%) Other terrestrial birds (3.85%)
2011– (80.77%) 2006–2010 (11.54%) 2001–2005 (7.69%)
Lineage VI
A/duck/Nanchang/1681/1992 (DK/NC/1681/92-like)
H6 H4 H7 H3 H5
Duck (59%), Other aquatic birds (25%) Wild aquatic birds (14%) Chicken (1%)
2011– (21%) 2006–2010 (34%) 2001–2005 (36%) –2000 (9%)
26
100
(73.2%) (7.8%) (12.6%) (4.8%)
(31%) (14%) (11%) (11%) (9%)
Bold values are the most common characteristics of each lineage.
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substitution with the Gamma model of rate heterogeneity. The tree was color-coded by using FigTree (ver.1.4.2) (http://www.tree.bio. ed.ac.uk/software/figtree/).
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2.3. Evolutionary rate
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The substitution rate and TMRCA were estimated using a MCMC method as implemented in BEAST (Drummond et al., 2012). The SRD06 nucleotide substitution model (Shapiro et al., 2006) and coalescent Bayesian skyline model (Minin et al., 2008) were incorporated in the MCMC method. A relaxed molecular clock model with uncorrelated lognormal distribution was used to inferred the time-scaled maximum clade credibility phylogenies (Drummond et al., 2006). Multiple independent MCMC runs were performed and assessed for consistency. They were combined to give a total chain length of 0.5–4 108 steps, with sampling at every 100 steps. Convergence of relevant parameters was assessed by effective sample sizes over 200 in Tracer v1.5 (http://tree.bio.ed. ac.uk/software/tracer/).
136 137
141 142 143 144 145 146 147 148 149 150 151 152
153 154 155 156 157 158 159 160 161 162 163 164 165 166 167
2.4. Selection pressure analysis To examine the selection pressure experienced by each lineage, we estimated the ratio of non-synonymous (dN) to synonymous (dS) substitutions for each lineage, using the HyPhy package (Pond et al., 2005). Selection pressure analysis acting on the codons of M1 and M2 of each lineage was carried out using HyPhy open-source software package available under the datamonkey web-server (http://www.datamonkey.org/) (Delport et al., 2010). The ratio of dN to dS substitutions per site was estimated using three different approaches: single likelihood ancestor counting (SLAC), fixed effects likelihood (FEL) (Kosakovsky Pond and Frost, 2005), and internal fixed effects likelihood (IFEL) (Pond et al., 2006). Best nucleotide substitutions models adopted in the analyses for different data sets were determined through the available tool in the datamonkey server.
Table 2 Substitution rates of viral M lineages. Lineage
I II III IV V VI
Substitution rate (10 site/year)
3
subs/
tMRCA(calendar year)
Mean
95% HPD lower
95% HPD upper
Mean
95% HPD lower
95% HPD upper
2.27 2.09 1.32 3.64 3.15 1.59
1.92 1.64 0.84 3.04 2.11 1.23
2.60 2.56 1.83 4.21 4.31 2.00
1990 1994 1981 1989 2000 1991
1994 1999 1990 1994 2001 1992
1982 1989 1970 1983 1997 1988
2.5. Protein structural analyses
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To map positive selection sites onto the protein structures and to better understand how the positive selection sites might interact with other proteins, we carried out structural analyses of M2 proteins using the Molecular Modeling Database (MMDB) (Madej et al., 2012). The three-dimensional structure of the extracellular domain of M2 was downloaded from the database (MMDB ID: 124024). Site 10 of M2e was highlighted in yellow using the Cn3D 3.4 software (Porter et al., 2007).
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3. Results
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3.1. Phylogenetic analysis
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The phylogenetic tree of all M genes was constructed (Fig. 1), using the ML approach with bootstrapping analyses to assess clade robustness. We defined ‘‘lineage’’ as an aggregate of large branches. These lineages were denoted as I–VI. Several reference sequences were chosen to represent main differences in each lineage. Lineage I (82% bootstrap support) contained 205 sequences, including 148 H9N2 subtype viruses (72.2%), 13 H7N9 subtype viruses (6.3%) and many other different subtypes. The H7N9
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Fig. 2. Bayesian inferences of different lineages. A: (lineage I), B: (lineage II), C: (lineage III), D: (lineage IV), E: (lineage V), F: (lineage VI). Branch coloring indicates inferred rates of nucleotide substitution from blue (0 10 3 subs/site/year) to red (0.16 10 3 subs/site/year) (see legend). The scale bar indicates the number of years before the present. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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subtype AIVs isolated in 2013 were caused major outbreaks of human infection (Chen et al., 2014; Liu et al., 2013). One hundred and sixty-eight sequences (82.7%) were isolated in terrestrial birds, 133 (64.8%) of them in chickens (Table 1). A/Chicken/Beijing/1/1994 (Ck/BJ/1/94) was chosen to represent lineage I, which was the first H9N2 subtype AIV isolated in China. Lineage II (100% bootstrap support) comprised of 63 H6 subtype viruses (100%). Fifty-eight sequences (92%) were isolated in aquatic birds, 47 (74.6%) in ducks, 7 (11.1%) in wild ducks. A/wild duck/shantou/2853/2003 (WKD/ST/2853/03) was chosen to represent lineage II. Lineage III (58% bootstrap support) contained 37 sequences. Thirty-four sequences (91.9%) were isolated in aquatic birds, 21
(62.2%) in ducks. A/duck/Nanchang/1749/1992 (DK/NC/1749/92) was chosen to represent lineage III. Lineage IV (70% bootstrap support) comprised of 161 sequences, including 157 H5 subtype viruses (97.5%). These viruses were detected in multiple species, mostly in chicken (34.1%). These AIVs were responsible for several HPAI outbreaks in China (Li et al., 2004). A/goose/Guangdong/1/1996 (Gs/GD/1/96) was chosen to represent lineage IV, which was the first H5N1 subtype AIV isolated in China. Lineage V (51% bootstrap support) contained 26 sequences. Twenty-three sequences (88.4%) were isolated in aquatic birds, 21 (80.77%) in ducks. Twenty-one sequences (80.77%) were isolated after 2011, which suggested that this lineage was a newly
Please cite this article in press as: Xiaorong, P., et al. Molecular phylogeny and evolutionary dynamics of matrix gene of avian influenza viruses in China. Infect. Genet. Evol. (2015), http://dx.doi.org/10.1016/j.meegid.2015.05.033
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Fig. 3. Selective pressure among different lineages. Selective pressures for the entire sequence (dN/dS) were calculated separately for M1 and M2. The error bar shows the 95% confidence interval.
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formed one. A/duck/Shanghai/28-1/2009 (DK/SH/29-1/2009) was chosen to represent lineage V. Lineage VI (12% bootstrap support) comprised of 100 sequences, including 31 H6 subtype viruses (31%), 14 H4 subtype viruses (14%), 11 H7 subtype viruses (11%) and 11 H3 subtype viruses (11%). Ninety-eight sequences (98%) were isolated from aquatic birds, 14 (14%) in wild ducks. A/duck/Nanchang/1681/1992 (DK/NC/1681/92) was chosen to represent lineage VI.
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3.2. Evolutionary rate
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We calculated the evolutionary rate and TMRCA of all lineages, which are shown in Table 2. The mean substitution rates of the M gene ranged from 1.3 10 3 (lineage III) to 3.6 10 3 (lineage IV) substitutions per site per year. The special evolutionary rates in each branch are shown in Fig. 2. The mean TMRCA of lineage III was dated back to 1981, with the 95% highest posterior density (HPD) interval between 1970 and 1990. And lineage V was the newly formed one, established in about 2000, with the 95% HPD interval between 1997 and 2001.
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3.3. Selective pressures
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We defined the magnitude of the pressure as the estimate of the dN/dS ratio. A higher selective pressure indicated that the gene (or the site) was under stronger selection (positive selection) for amino acid substitution ( Pond et al., 2007). Selective pressure was statistically stronger in M2 than that in M1 for all lineages (Fig. 3).
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The highest dN/dS ratio was observed in lineage IV: the dN/dS ratio for M1 genes was 0.1 and was 0.6 for M2 genes, while the lineage III appeared to be under the strongest negative selection (the dN/dS ratios for M1 genes was 0.04; and for M2 genes was 0.23).
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3.4. Site-by-site analyses
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Site-by-site (by each codon) analyses for all lineages were conducted by SLAC, FEL and iFEL. Positive selection sites by at least one test were identified in different M lineages (Table 3). No codons in M1 of all lineages were under significant positive selection (P < 0.05). Six sites were positively selected in M2 of lineage I: four sites in the extracellular domain (positions 10, 13, 16 and 21), and two sites in the cytoplasmic domain (positions 82 and 97). There were three positive selection sites in lineage IV (positions 14, 66 and 82). The rest of lineages had no positive selection sites. The 3D structure of M2e is shown in Fig. 4. According to the structural analysis, positions 10 located at the middle of M2e is important for the binding of M2e to antibodies. The most common amino acids at the M2e 10–20 site of AIVs in our study remained PTRNGWECKCS (Table 4). However, we noted that sites 10, 13, 14 and 16 were under positive selection in at least one lineage.
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4. Discussion
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In this study, we compiled a comprehensive dataset and carried out a large-scale sequence analysis of the M genes in AIVs isolated in China. Since only a small portion of M gene in AIVs gets sequenced, this data set in our study is just a small sample of all the viruses that circulated in Chinese poultry. This issue is a potential weakness of our study. Active surveillance of AIVs could enhance our knowledge about the evolution of M gene. In previous reports, the M genes of AIVs were divided into two different lineages, Eurasian avian and North American avian (Furuse et al., 2009; Webster et al., 1992). The phylogenetic tree showed that the M genes in our study all belonged to the Eurasian lineage and evolved independently in six lineages, denoted as I–VI. The majority of lineage I was the H9N2 subtype AIV. The H9N2 influenza virus is now the most prevalent in chickens in china (Sun and Liu, 2015). Lineage I also included several different subtype AIVs (H5N1, H6, H7N9 and H10N8), which suggested the H9N2 influenza virus reassorted with multiple other subtype viruses (Xu et al., 2007). The H9N2 influenza did not induce obvious clinical signs or deaths in chickens, which suggested that it can persistently evolve to adapt to this special host (Webster, 2004).
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Table 3 Positive selection sites for M1 and M2 detected using SLAC, FEL and IFEL methods. Lineage
Protein
I
M1 M2
IV
M1 M2
Domaina
Codon
Normalized dN-dS
p-Value
Normalized dN-dS
p-Value
Normalized dN-dS
p-Value
ex ex ex ex cy cy
None 10 13 16 21 82 97
2.88 1.673 2.471 2.428 5.093 5.597
0.042 0.199 0.083 0.081 0.0064 0.021
1.771 0.811 1.387 1.372 2.628 3.025
0.017 0.049 0.046 0.043 0.0063 0.061
4.643 0.946 1.75 2.33 3.567 2.223
0.0007 0.063 0.044 0.018 0.0039 0.207
ex cy cy
None 14 66 82
4.52 8.57 7.95
0.163 0.057 0.04
1.86 3.92 3.05
0.047 0.02 0.1
3.23 2.52 2.78
0.024 0.17 0.143
SLAC
FEL
iFEL
Bold values are those deemed to indicate significantly positive or negative selection (P < 0.05). a Ex indicates extracellular domain; Tr, transmembrane domain; and Cy, cytoplasmic domain.
Please cite this article in press as: Xiaorong, P., et al. Molecular phylogeny and evolutionary dynamics of matrix gene of avian influenza viruses in China. Infect. Genet. Evol. (2015), http://dx.doi.org/10.1016/j.meegid.2015.05.033
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interface/EN_GIP_20150106CumulativeNumberH5N1cases.pdf). Those two lineages represent a serious threat as potential pandemic virus for humans. Therefore, active surveillance of the molecular evolution of these viruses is the best option to detect and interrupt their transmission. Aquatic birds are accepted as the natural reservoirs of influenza A viruses, and these viruses have been introduced to other animals, shaping the current ecology of influenza viruses (Webster et al., 1992). Introduction of influenza viruses from migratory aquatic birds through domestic ducks to other poultry was frequently observed in the recent decades (Chen et al., 2013; Ma et al., 2015). The influenza virus pool in aquatic birds is a never-ending source of genetic and antigenic material (Macken et al., 2006). In the present study, the genepool in aquatic birds is represented by the sequences in lineages III and VI. The evolutionary rates and selective pressure of these two lineages were lower than others. TMRCA of lineage III indicated that it is the oldest one. M genes from the H6N2 subtype AIVs were established as a monophyletic group (lineage II), just as in a previous study (Huang et al., 2012). Lineage V was a new group never reported before. Interestingly, the evolutionary rates of these two lineages were significantly higher than those from lineage III and VI. One possible explanation is that strains in a lineage that appeared more recently have had to evolve more rapidly to adapt better to the host or viral replication (Furuse et al., 2009). These findings suggest that domestic ducks can facilitate significant genetic and antigenic changes of viruses. The selective pressure is stronger on M2 than that on M1; more sites under positive selection were identified in M2 than in M1. Among them, most of the sites (5 out of 8) under positive selection in M2 are located in the extracellular domain. Although the most common amino acids at the M2e 10–20 site remained PTRNGWECKCS (Liu et al., 2005), the positive selection would change this fact in the near future. Since M2e is an antigen for vaccine development, especially the position 10 located at the middle of M2e which was important with the binding to antibodies, surveillance of these positive selection positions is an important part in development of the universal influenza A vaccine. The cytoplasmic domain of M2 is important for interaction with M1, genome packaging, and formation of virus particles. One site was under positive selection in the cytoplasmic domain of M2 (position 82). Positions 82–89 were important for infectious virus production (McCown and Pekosz, 2006). Another study showed that vRNP packaging is mediated by amino acids at positions 70–89 of the M2 gene (McCown and Pekosz, 2005). The M2 gene must, therefore, have evolved with several functions. In summary, we analyzed the M genes of AIVs isolated in China and studied their evolutionary dynamics. The M gene has evolved into different lineages with varied evolutionary rates and selection
Fig. 4. 3D crystal structure of the interaction between the extracellular domain of M2 and a monoclonal antibody. The pink protein represents the heavy chain of the monoclonal antibody from mouse (Mus musculus) and the brown protein is the light chain of monoclonal antibody. Site 10 under positive selection for Lineage H9 and DK is highlighted in yellow. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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Moreover, H9N2 influenza viruses from poultry could occasionally be transmitted from poultry to mammalian species, including humans (Butt et al., 2005). A serological surveillance study in Guangzhou in 2008 showed that the anti-H9 antibody positive rate among poultry retailers was up to 15.5% (Wang et al., 2009), which highlighted that these viruses can adapt to humans. H7N9 and H10N8 are novel human avian influenza viruses, which have emerged in humans in China from 2013, acquiring their internal gene by reassortment with H9N2 viruses (Chen et al., 2014; Liu et al., 2013). Therefore, poultry carrying H9N2 could be incubators for novel AIVs capable of infecting humans. The majority of lineage IV viruses were from the H5 subtype AIV. The H5N1 HPAI virus has been a known pathogen of chickens for many years (Li et al., 2004), responsible for several outbreaks in recent years (Kang et al., 2014; Kim et al., 2008). The H5N8 subtype virus, isolated in Zhejiang, was most closely related to isolates from poultry in countries in eastern Asia (Wu et al., 2014). H5N1 has spread to cause infection and death in many species including domestic birds and mammals (i.e. tigers, dogs, cats and humans) (Ma et al., 2009). From 2003 to 2014 the cumulative number of humans infected with the H5N1 AIVs cases in China reached 47 with 30 fatalities (http://www.who.int/influenza/human_animal_
Table 4 Amino acid sequence of all M2 protein on 10–20. Position
10
11
12
13
14
15
16
17
18
19
20
Conserved amino acid Amino acida
P P (73.5%) L (20.2%) H (6.1%)
T T (99.0%) I (0.8%) S (0.2%)
R R (96.3%) K (3.7%)
N N (81.5%) T (14.6%) S (1.6%) A (0.3%) K (0.3%)
G G (77.2%) E (22.8%)
W W (100%)
E E (90.4%) G (8.5%) V (1.0%) A (0.2%)
C C (99.5%) Y (0.3%) G (0.2%)
K K (43.1%) R (36.4%) N (19.7%) S (0.6%) I (0.2%)
C C (99.8%) Y (0.2%)
S S (95.2%) N (4.3%) G (0.3%) I (0.2%)
Bold values are those positions highly conserved (>99%). a Highly conserved amino acid sequence of M2e in avian influenza viruses is PTRNGWECKCS (Liu et al., 2005).
Please cite this article in press as: Xiaorong, P., et al. Molecular phylogeny and evolutionary dynamics of matrix gene of avian influenza viruses in China. Infect. Genet. Evol. (2015), http://dx.doi.org/10.1016/j.meegid.2015.05.033
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pressures. In addition, mutations of amino acids at the positive selection sites are likely associated with virus budding and antigen recognition. This study enhances our knowledge of gene evolution of AIVs, and is expected to improve the early detection of new viruses and lead to M2e vaccine development.
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Acknowledgements
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This study was supported by Grants from the National Key Technologies R&D Programme for the 12th Five-Year Plan of China (2012ZX1000-004-005) and the Independent Task of State Key Laboratory for Diagnosis and Treatment of Infectious Diseases (Nos. 2010ZZ04 and 2014ZZ12). We gratefully acknowledge the support of the IBM high performance computing cluster of Bio-macromolecules Analysis Lab, Analysis Center of Agrobiology and Environmental Sciences, Zhejiang University.
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Appendix A. Supplementary data
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Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.meegid.2015.05. 033.
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Please cite this article in press as: Xiaorong, P., et al. Molecular phylogeny and evolutionary dynamics of matrix gene of avian influenza viruses in China. Infect. Genet. Evol. (2015), http://dx.doi.org/10.1016/j.meegid.2015.05.033
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