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. Author manuscript; available in PMC: 2012 Dec 1.
Published in final edited form as: Infect Genet Evol. 2011 Jul 7;11(8):2125–2132. doi: 10.1016/j.meegid.2011.07.002

Evolutionary dynamics of influenza A nucleoprotein (NP) lineages revealed by large-scale sequence analyses

Jianpeng Xu 1, Mary C Christman 1, Ruben O Donis 2, Guoqing Lu 1,*
PMCID: PMC3204331  NIHMSID: NIHMS310125  PMID: 21763464

Abstract

Influenza A viral nucleoprotein (NP) plays a critical role in virus replication and host adaptation, however, the underlying molecular evolutionary dynamics of NP lineages are less well-understood. In this study, large-scale analyses of 7,517 NP nucleotide sequences revealed eight distinct evolutionary lineages, including three host-specific lineages (human, classic swine and equine), two cross-host lineages (Eurasian avian-like swine and swine-origin human pandemic H1N1 2009) and three geographically isolated avian lineages (Eurasian, North American and Oceanian). The average nucleotide substitution rate of the NP lineages was estimated to be 2.4 × 10−3 substitutions per site per year, with the highest value observed in pandemic H1N1 2009 (3.4 × 10−3) and the lowest in equine (0.9 × 10−3). The estimated time of most recent common ancestor (TMRCA) for each lineage demonstrated that the earliest human lineage was derived around 1906, and the latest pandemic H1N1 2009 lineage dated back to Dec 17, 2008. A marked time gap was found between the times when the viruses emerged and were firstly sampled, suggesting the crucial role for long-term surveillance of newly emerging viruses. The selection analyses showed that human lineage had six positive selection sites, whereas pandemic H1N1 2009, classical swine, Eurasian avian and Eurasian swine had only one or two sites. Protein structure analyses revealed several positive selection sites located in epitope regions or host adaptation regions, indicating strong adaptation to host immune system pressures in influenza viruses. Along with previous studies, this study provides new insights into the evolutionary dynamics of influenza A NP lineages. Further lineage analyses of other gene segments will allow better understanding of influenza A virus evolution and assist in the improvement of global influenza surveillance.

Keywords: influenza, nucleoprotein (NP), lineage, substitution rate, TMRCA, selection

1. Introduction

Influenza A virus, which can infect a wide range of avian and mammalian species, has been considered an extremely important pathogen of respiratory infections (Webster et al., 1992). The virus can cause moderate to severe epidemics annually and catastrophic pandemics sporadically (Guan et al., 2010). The occurrence of pandemic H1N1 2009 attracted great public attention concerning the global burden of morbidity and mortality caused by influenza viruses (Garten et al., 2009). The influenza A virus is a negative strand RNA virus with eight gene segments, each of which encodes either one or two proteins. The subtype of influenza A virus is determined by the antigenicity of two surface glycoproteins, namely, hemaglutinin (HA) and neuraminidase (NA). Currently, 16 HA subtypes (H1-H16) and 9 NA subtypes (N1-N9) have been classified within influenza A viruses (Fouchier et al., 2005). The subtypes currently circulating in the human population are H1N1 and H3N2. More than 100 of the possible 144 influenza A virus HA - NA combinations are maintained in wild bird populations, with many variants found in each subtype (Munster et al., 2007).

Influenza A virus exhibits rapid evolution and complicated molecular dynamics due to diverse hosts, high mutation rates, and rapid replication (Holmes, 2010). The virus has also shown the propensity to escape immunity because of continuous antigenic drift, during which the viral protein epitopes are altered by non-synonymous mutations, as a result of interactions with the host immune system (Bush et al., 1999; Ferguson et al., 2003; Smith et al., 2004). In addition, the influenza virus can cross species barriers due to rapid mutation, genetic drift, genome reassortment, or simply transmitted from one host to another (Holmes, 2010; Kuiken et al., 2006; Pensaert et al., 1981). Each of the influenza viral genes plays a significant role in viral interactions with hosts and subsequent infection; therefore, understanding the evolution of each viral gene can provide new insights into the epidemiological dynamics of influenza viruses (Fourment et al., 2010; Furuse et al., 2009). This will be useful in predicting the genetic basis and periodicity of future influenza epidemics and pandemics (Pybus and Rambaut, 2009).

The nucleoprotein (NP) gene encodes a protein with around 500 amino acids; it plays an important role in assembly and budding of influenza virus and has a putative role in host range (Ruigrok et al., 2010; Snyder et al., 1987). The primary function of NP is to bind with the viral RNA segments to form the nucleocapsid of a virus particle. RNA hybridization techniques were used to investigate the genetic diversity and grouping of NP sequences, which revealed five RNA hybridization groups (Bean, 1984). Sequence analyses of the NP gene using the maximum-parsimony algorithm supports the hypothesis that NP is involved in the maintenance of a host-specific gene pool (Gorman et al., 1990). In addition, phylogenetic analysis of 89 NP nucleotide sequences demonstrated that H1N1 human and classical swine virus share a common ancestor (Gorman et al., 1991). The phylogenetic analyses of 436 NP sequences provides a view of phylogenetic diversity and distribution of lineages; however, some lineages were assigned based upon the information of as few as two sequences (Chen et al., 2009). On the other hand, the phylodynamics parameters of the NP gene (i.e., evolutionary rates and TMRCAs) were analyzed in genomic studies but limited to certain specific hosts (Chen and Holmes, 2006, 2010). Our large-scale sequence analyses of the influenza A viral NP gene were conducted to fill the above gaps.

2. Materials and Methods

2.1 Sequence data

A complete set of nucleoprotein (NP) nucleotide sequences, together with the information of host, subtype, isolation time, and isolation place, were downloaded from GenBank (Bao et al., 2008). After excluding the sequences from laboratory strains or duplicate strains, a total of 7,517 sequences longer than 1,440 nucleotides were obtained. The identical sequences were removed using a Perl script, which resulted in 5,094 sequences for further analysis.

2.2 Phylogenetic analysis

Sequences were aligned with MUSCLE (Edgar, 2004). The alignments were adjusted manually using TranslatorX (Abascal et al., 2010) based upon corresponding protein sequences. Sequences were tested for recombination using RDP3 (Martin et al., 2010); no definitive recombinant was detected. Phylogenetic analysis was conducted using the maximum-likelihood (ML) method in RAxML (Stamatakis et al., 2005). RAxML uses rapid algorithms for bootstrap and maximum likelihood searches and is considered one of the fastest and most accurate phylogeny programs for large datasets. Analyses of1000 bootstrap replicates were performed using GTRGAMMA, i.e., GTR model of nucleotide substitution with Gamma model of rate heterogeneity. All the analyses were conducted on a Supercomputer cluster at the University of Nebraska Holland Computing Center (http://hcc.unl.edu). A set of Perl scripts were written by us to facilitate their computational analyses.

2.3 Classification and nomenclature of lineages

Lineages and major clusters were determined based upon the groupings with strong bootstrap support in the ML tree. Additional information such as hosts, geographic regions and circulation years were also considered in the classification. The trees were visualized and color-coded with FigTree (http://tree.bio.ed.ac.uk/software/figtree/). We used the same lineage nomenclature as we described previously: Roman alphabetic letters such as A, B and C to represent different lineages (Lu et al., 2007).

2.4 Measurement of selection pressures

To determine selection pressure acting on NP lineages, we estimated the ratio of non-synonymous (dN) to synonymous (dS) substitutions per site (ratio dN/dS) for each lineage, using all the sequences included in this study. Positively selected codons were detected using the single likelihood ancestor counting (SLAC), fixed effects likelihood (FEL) and internal fixed effects likelihood (IFEL) methods with a significance level of 0.05. In the SLAC method, the nucleotide and codon model parameter estimates are used to reconstruct the ancestral codon sequences at the internal nodes of the tree. The single most likely ancestral sequences are then fixed as known variables, and applied to infer the expected number of non-synonymous or synonymous substitutions that have occurred along each branch, for each codon position. The FEL method is based on maximum-likelihood estimates. The FEL method estimates the ratio of non-synonymous to synonymous substitutions on a site-by-site basis for the entire tree or only the interior branches (IFEL). These methods are implemented in the HYPHY package (Pond et al., 2005). Positive selection sites were mapped on the NP protein structure using Molecular Operating Environment (MOE) (Ye et al., 2006).

2.5 Substitution rate and time of most recent common ancestor (TMRCA) analyses

The substitution rate and the time of most recent common ancestor (TMRCA) of each lineage were estimated using a Bayesian Markov Chain Monte Carlo (MCMC) method as implemented in the BEAST version 1.5.4 package (Drummond and Rambaut, 2007). For computational tractability, samples from the complete datasets of size 120 - 200 sequences were carefully selected by removing sequences collected from the same location and at the same point in time (Felsenstein, 2006). In all cases, the GTR + Γ4 nucleotide substitution model was employed as this was the best-fit model supported by Modeltest (Posada and Crandall, 1998). For each analysis the Bayesian skyline coalescent model was used, as it describes the fluctuating population dynamics characteristics of influenza virus (Rambaut et al., 2008).

Three clock models were compared statistically for each dataset using a Bayes factor test in the Tracer program (Suchard et al., 2001): a strict clock, an uncorrelated lognormal relaxed clock (UCLD) and an uncorrelated exponential relaxed clock (UCED) (Drummond et al., 2006). The UCED model was found to provide the best fit for all lineages (Bayes factor > 30). We thus used this model for the estimation of evolutionary dynamic parameters. In each case, MCMC chains were run for sufficient time to achieve convergence, with uncertainty in parameter estimates reflected in the 95% highest probability density (HPD). The Maximum Clade Credibility (MCC) tree across all plausible trees was then computed from the BEAST trees using the TreeAnnotator program, with the first 10% of trees removed as burn-in.

3. Results

3.1 Phylogeny and major lineages of influenza A viral NP genes

Eight distinct lineages, denoted as A to H, were identified based upon the phylogeny of 7,517 NP sequences sampled between 1918 and 2010 (Figure 1). All the lineages are characterized by groupings with strong bootstrap support values. The seasonal human influenza lineage (A), the pandemic H1N1 2009 human lineage (B), and the classical swine lineage (C) are grouped together and appear to share a common ancestor, while the remaining five lineages are grouped together and are likely to have the same evolutionary origin (Figure 1). The annotations, representative sequences and main subtypes for each lineage and isolation periods of viruses within lineage are detailed in Table 1. Multiple subtypes were found in avian lineages, whereas only a few subtypes were found in non-avian lineages (Table 1).

Figure 1.

Figure 1

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The maximum-likelihood phylogenetic tree of influenza A viral NP genes. Eight lineages are denoted as A through H. The bootstrap support values are shown on the major nodes. The scale bars indicate the numbers of nucleotide substitutions per site.

Table 1.

Information of influenza A viral NP lineages and annotation

Lineage Annotation Isolation period Representative sequence Subtypes
A Human 1918-2009 A/Brevig Mission/1/1918(H1N1) H1N1, H2N2, H3N2
B Pandemic H1N1 2009 2009-2010 A/Texas/05/2009(H1N1) H1N1
C Classical swine 1930-2009 A/swine/Iowa/15/1930(H1N1) H1N1, H3N2, H1N2
D Eurasian avian 1927-2009 A/fowl/Dobson/1927(H7N7) H7N1, H2N3, H3N8, H3N3, H5N2, H5N1, H6N2, H9N2,
H4N6, H7N3, H10N7
E Eurasian swine 1979-2009 A/swine/Belgium/WVL1/1979(H1N1) H9N2, H3N2, H1N2, H1N1, H5N1
F North Amecian avian 1953-2008 A/duck/Manitoba/1/1953(H10N7) H5N2, H9N2, H6N2, H7N3, H3N8, H7N3, H2N2, H5N2,
H1N1, H4N6, H12N5
G Oceanian avian 1975-1984 A/shearwater/Australia/751/1975(H5N3) H6N5, H3N8, H7N7, H11N3, H4N6, H4N4,H4N8, H6N2,
H15N9, H1N2, H9N1, H6N1
H Equine 1963-2008 A/equine/Miami/1/1963(H3N8) H3N8
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Lineage A is composed of mostly human influenza viruses (n = 2,647) , which includes H1N1 between 1918 and 1957, H2N2 between 1957 and 1968, H3N2 after 1968, and H1N1 after 1977, indicating that the NP genes of the viruses circulating in human populations were originally derived from the 1918 Spanish flu viruses (Figure 1). Lineage A also includes 38 viral strains from pigs and 1 from birds. Lineage B corresponds to pandemic H1N1 2009, which is grouped with lineage C, indicating the classical swine origin of the pandemic H1N1 2009 NP gene. Lineage C mainly consists of classical swine viruses (n = 235), but it also has 15 human viruses and 18 avian viruses. In addition, the triple-reassortant swine viruses, which contains genes from classic swine (NP, NS and M), human (HA, NA, PB1) and avian (PB2 and PA) influenza viruses, were also found in this lineage.

Three avian lineages (D, F and G) were identified corresponding to geographic distinctions (Figure 1). Lineage D is mainly composed of Eurasian avian viruses (n = 1,805), but with a few exceptions: 167 human, 116 swine, 8 environmental samples, 5 tiger, 2 mink, 3 feline, 1 stone marten and 3 canine viruses. Highly pathogenic H5N1 avian influenza viruses were found to be in lineage D as well. Lineage E is a Eurasian swine lineage derived from lineage D (Eurasian avian), possibly through direct transmission. Lineage F, although mostly from North American avian (n = 1,090), contain some strains from environmental samples (99), swine (4) and humans (2). Lineage G consists of Oceanian avian influenza viruses (35). Lineage H corresponds mainly to equine influenza viruses (n = 92), but a few are from canine (4) and swine (2). From the viewpoint of tree topology, a typical ladder-like tree structure was observed in equine viruses over time (Figure 1).

3.2 Substitution rates and times of most recent common ancestor (TMRCA) of influenza A NP lineages

The nucleotide substitution rates and TMRCAs are summarized in Table 2. The estimated substitution rates of NP lineages ranged from 0.9 × 10−3 to 3.43 × 10−3 substitutions per site per year (nts/st/yr). Lineage B (pandemic H1N1 2009) was found to be the fastest evolving lineage, with a mean substitution rate of 3.43 × 10−3 and a 95% highest posterior density interval (HPD) of 2.7 × 10−3 - 4.12 × 10−3. Lineage H (equine) is the slowest evolving lineage, with a mean estimate of 0.9 × 10−3 (95% HPD: 0.68 × 10−3 - 1.13 × 10−3), which is significantly slower than those of other lineages.

Table 2.

Substitution rates and times of most recent common ancestor (TMRCAs) of influenza A viral NP lineages*

Lineage Substitution rate (× 10−3)
subs/site/year)
 TMRCA (calendar year)
Mean 95% HPD
lower		 95% HPD
upper		 Mean 95% HPD
lower		 95% HPD
upper
A: Human 1.98 1.69 2.27 1906 1884 1918
B: Pandemic (H1N1) 2009 3.43 2.7 4.12 17-Dec-08 14-Jul-08 30-Mar-09
C: Classical Swine 2.29 1.99 2.58 1911 1888 1927
D: Eurasian avian 3.41 2.83 4.01 1920 1908 1927
E: Eurasian swine 2.8 2.4 3.21 1972 1963 1978
F: North American avian 2.25 1.95 2.53 1946 1932 1953
H: Equine 0.9 0.68 1.13 1947 1926 1959
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*

Oceanian avian lineage (G): without enough sequences for reliable estimation of the parameters

The TMRCA of lineage A (human) was dated to 1906, and the 95% HPD interval was between 1884 and 1918 (Figure 2), which is 12 years before the outbreak of pandemic H1N1 1918. The mean TMRCA of lineage B (pandemic H1N1 2009) was estimated to be at Dec 17, 2008 (95% HPD: July 14, 2008 –Mar 30, 2009) (Supplementary Figure S1). The TMRCA of lineage C (classical swine) was estimated at 1911 (95% HPD: 1888 – 1927) (Supplementary Figure S2). The TMRCA of lineage D (Eurasian avian) was estimated to be 1920 (95% HPD: 1908 – 1927) (Supplementary Figure S3). The time of most recent common ancestor of lineage E (Eurasian swine) was estimated at 1972 (95% HPD: 1963 – 1978), seven years earlier than the first detection of Eurasian swine lineage in Belgium in 1979 (Supplementary Figure S4). The emergence of the North American avian lineage was dated to 1946 (95% HPD: 1932 – 1953) (Supplementary Figure S5). Finally, the most recent common ancestor of NP genes of equine influenza viruses can be traced back to 1947 (95% HPD: 1926 – 1959) (Supplementary Figure S6).

Figure 2.

Figure 2

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Maximum clade credibility (MCC) tree of the influenza A NP human lineage. The tree is scaled to the time (horizontal axis), with blue horizontal bars at nodes representing the 95% HPDs of TMRCAs. The scale bar indicates the number of years before the present.

3.3 Selection pressure of NP lineages

The estimation of dN/dS ratios revealed different selection pressures occurred in different lineages (Table 3). The highest dN/dS ratio was observed in lineage H (equine: dN/dS =0.13). This is slightly higher than that of lineage A (human), which has a dN/dS ratio of 0.12. Interestingly, lineage G (Oceanian avian) and F (North American avian) appear to be under the strongest negative selection (dN/dS ratios: 0.04 and 0.05, respectively). The two swine lineages experienced similar selection pressures (0.09 for classical swine and 0.08 for Eurasian swine).

Table 3.

Selection sites detected using SLAC, FEL and IFEL methods (significant level = 0.05)

Positively selected sites
 No. of negatively selected sites
 Mean dN/dS
SLAC FEL IFEL SLAC FEL IFEL
A: Human 101, 131, 217, 451, 472 101, 131, 217, 451, 472 217, 450, 472, 473 328 352 322 0.12
B: pandemic H1N1 2009 None None 353 66 155 20 0.1
C: Classical Swine 217 217 217, 472 267 318 279 0.09
D: Eurasian avian 34, 456 34, 456 456 416 418 417 0.08
E: Eurasian swine None None 31 180 264 179 0.08
F: North American avian None None None 414 415 413 0.05
G: Oceanian avian None None None 39 156 136 0.04
H: Equine None None None 24 60 24 0.13
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Positive and negative selection sites were identified in different NP lineages (Table 3, Figure 3). Positive selection sites were found in sequences of lineages A, B, C, D and E. The human lineage A was found to have 6 positive selection sites, whereas lineages B, C, D and E were observed to have only one or two sites. No positively selected sites were found in the equine lineage (H), Oceanian avian (E) or North American avian (H). The amino acid position 217 of NP of human lineage A and position 472 of NP of classical swine lineage C were found to be under positive selection. The position 472 is significant, in that structure analysis showed that it is located in an antibody binding region (Figure 3). In addition, the positive selection codon at position 217 is located in an alpha helix, which runs parallel to an adjacent antibody binding region, indicating possible strong viral adaptation to host immune system pressures. Similarly, the position 34 is located in a proposed CTL epitope site, while position 353 is located in the flanking region of a proposed CTL epitope where mutations are known to disrupt cytosolic processing of viral CTL epitopes.

Figure 3.

Figure 3

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The structure of influenza A viral nucleoprotein (PDB: 2Q06), with positive selection sites denoted as yellow circles. Structural regions are denoted in different colors: pink for alpha-helices, red for beta sheets, and white for loops.

4. Discussion

Lineage classification is critical for long-term influenza surveillance, because it offers a database of lineage information for detecting evolutionary changes of new viral strains (Chen et al., 2009). In this study, we conducted large-scale sequence analyses of the NP segment (over 7,500 sequences) and identified eight distinct lineages. The results of our lineage classification and designation are largely in agreement with previous reports (see below); however, our study employs a much larger dataset and presents more detailed lineage annotations. The evolutionary relationship shown in our phylogenetic tree strongly suggests that human and classical swine lineages share the same evolutionary origin, which agrees with previous findings (Gorman et al., 1991). In addition, similar lineages (i.e., human, equine, classical swine, Eurasian avian, North American avian) were also designated based upon the evolutionary analyses of other gene segments (Chen et al., 2009; Dunham et al., 2009; Fourment et al., 2010; Gorman et al., 1990; Ito et al., 1991; Reid et al., 2004; Xu et al., 2011).

Lineage A includes human influenza viruses of three different subtypes isolated in different time periods. It is evident that the same NP gene remained in human influenza viruses, although two antigenic shifts occurred in 1957 and 1968, respectively. A similar situation was observed in the M gene (Furuse et al., 2009). Previous phylogenetic analyses revealed that the H2N2 virus, emerging in 1957, was a genetic reassortant between previously circulating human H1N1 virus dating from 1918 and avian viruses, with the novel H2, N2 and PB1 genes derived from Eurasian avian viruses (Kilbourne, 2006). In addition, the influenza A virus of the H3N2 subtype circulating in the human population since 1968 was also caused by reassortment between previously present human H2N2 virus and avian virus (H3, PB1) (Fang et al., 1981; Kawaoka et al., 1989).

With respect to evolutionary dynamics, the human lineage (A) of influenza NP has a substitution rate of 1.98 × 10−3 substitutions per site per year, a value close to previously reported ones (Jenkins et al., 2002). Although our mean TMRCA of the human lineage (A) is three years earlier than previously reported, the 95% HPD ranges are almost the same (1884–1918) (Smith et al., 2009a). A similar result was observed by Fourment et al., (2010) in the analysis of N1, with the most recent common ancestor dated back to 1906 (95% HPD: 1885-1905). Using the mutational bias towards an increased content of uracil in human influenza, Rabadan et al. (2006) estimated that the 20th century influenza viruses first diverged in 1910. Concerning the genetic origin of the 1918 pandemic influenza viruses, it appears that the NP gene, along with HA and NA, was first introduced into the human influenza viruses no more than two decades before that pandemic (Fourment et al., 2010).

The evolution of human influenza viruses is probably influenced by positive selection, gene flow, immune selection and competition (Bush et al., 1999; Grenfell et al., 2004). In contrast, immunological memory may not be substantially involved in the evolution of swine and domestic poultry influenza viruses because of their short life spans, which means influenza viruses may infect these non-human hosts only once in a lifetime (Meier-Ewert and Dimmock, 1970). Interactions with human immunity, partial cross-immunity, and competition between influenza viruses result in stronger immune selection at some amino acid sites among the influenza viruses infecting people (Bush et al., 1999). This was not seen in the avian influenza viruses. We found at most two positively selected sites and relatively low dN/dS ratios in avian lineages, supporting the view that no strong selection occurs in avian NP genes.

In the human lineage (A), the codons encoding residues 101 and 131 were found to be under positive selection in this study, which are potentially involved in the host adaptation (Forsberg and Christiansen, 2003). In an analysis of 246 NP sequences using the parsimony method, Suzuki (2006) also identified position 131 as a positive selection site. The codons at positions 31 and 34, which appeared to be under positive selection for Eurasian swine and Eurasian avian, respectively, are located in a region proposed to be involved in nuclear exportation (Neumann et al., 1997).

A novel swine-origin virus was detected in April 2009 in Mexico and spread rapidly to the rest of the world (Fraser et al., 2009). This pandemic (H1N1) 2009 influenza is believed to arise from a new reassortment between North American and Eurasian swine lineages (Christman et al., 2011; Garten et al., 2009; Smith et al., 2009b). The substitution rate for NP genes of pandemic (H1N1) 2009 estimated using sequences from the entire pandemic period in this study (3.43 × 10−3) is higher than the rate estimated during the early outbreak period (2.59 × 10−3) (Smith et al., 2009b). In addition, the dN/dS ratio of the pandemic 2009 NP (0.1) is obviously higher than the ratio of the closely related swine NP (0.05) (Smith et al., 2009b). The increase of the dN/dS ratio, which occurred after the inter-species transmission from swine to human, could be attributed to the adaptation to the new host and/or intensive epidemic surveillance (Smith et al., 2009b).

The evolution of equine lineage (H) seems to be quite interesting. First, a typical ladder-like tree structure was observed for the equine lineage from the phylogenetic tree (Figure 1). This phylogenetic pattern shows that viruses isolated at earlier times are located toward the root of the tree, while viruses isolated at a later time are presented farther from the root. Such tree topology reflects a process of temporal replacement of new viral strains and continual turnover of the viruses within this lineage due mainly to natural selection as previously suggested (Ferguson et al., 2003; Grenfell et al., 2004; Plotkin et al., 2002; Smith et al., 2004). Secondly, among all the eight lineages, the equine lineage was found to have the highest dN/dS ratio. The same dN/dS ratio (0.13) was reported by Murcia et al. (2011). In addition, the number of negative selection sites (24 using the SLAC method) was found to be much less than that found in other lineages, which might explain why the equine lineage has a higher dN/dS ratio. In this study, we did not find any positive selection site, but one positive selection site was identified by Murcia et al. (2011). This discrepancy might be attributed to the differences of significance levels and data sets used in the two studies. Concerning the substitution rate of equine viruses, a mean rate of 0.9 × 10−3 estimated in this study is consistent with previous estimates (Jenkins et al., 2002; Murcia et al., 2011). Compared with other hosts, however, equine viruses evolve much slower, reflecting host differences in their immune systems, population sizes, motilities and life spans. Finally, lineage H also includes canine influenza viruses, which provide evidence that equine influenza viruses have broken the species barrier and become established as a highly contagious respiratory pathogen of dogs (Crawford et al., 2005).

Supplementary Material

01

Highlights.

  • Inline graphic We conducted large-scale phylogenetic analyses of influenza A NP genes

  • Inline graphic 8 lineages identified: 3 host specific, 2 cross-host, 3 geographic-specific

  • Inline graphic Substitution rates, TMRCAs, and selection pressures vary in different lineages

  • Inline graphic Evolution of influenza A viruses strongly associated with host adaptation

Acknowledgements

This publication was made possible by NIH grant numbers R01 LM009985-01A1. The authors also acknowledge the UCRCA, the University of Nebraska at Omaha, for continuous funding support to this research program. The authors are grateful to Andy Zhong and Todd Herpy for their help with structural analysis.

Footnotes

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