document: human metapneumovirus ( hmpv ) is a negative single - stranded rna virus of the family paramyxoviridae and closely related to the avian metapneumovirus ( ampv ) subgroup c [ 1 , 2 ] . 
   hmpv is an important aetiological agent of respiratory tract infection ( rti ) in infants , or senior and immunocompromised individuals . 
   this infection caused different symptoms ranging from influenza like syndromes ( i.e. , fever , cough , and rhinorrhea ) to severe lower respiratory tract infection . 
   previous studies have shown that many children exposed to this virus and also easily to be reinfected as common [ 35 ] . 
   therefore , hmpv is becoming as a major concern in child respiratory tract viral infection . 
   genome sequencing and comparative analysis provides us a useful approach to analyze the pathogenicity of organisms . 
   moreover , this analysis might also provide us an approach to understand its evolution history and cell - host interaction . as previously reported hmpv genome is approximately 13  kb in length , and the gene composition from 3 terminal to 5 terminal 
   is n - p - m - f - m2 - 1/m2 - 2-sh - g - l [ 6 , 7 ] . 
   comparative analysis suggests that its genomic organization is similar to human respiratory syncytial virus ( hrsv ) , which just lacks 2 nonstructural genes , ns1 and ns2 . 
   moreover , hmpv has been demonstrated the existence with two main genetic lineages termed as subtype a and b , which also containing within them the subgroups a1/a2 and b1/b2 , respectively . 
   the genetic diversity analysis shows the a2 sublineage exhibits the greatest diversity among all the sublineages of hmpv . 
   as we all know , there are differences in the frequency of occurrence on synonymous codons in coding dna , which termed as synonymous codon usage bias . 
   briefly , there are 64 different codons ( 61 codons encoding for amino acids plus 3 stop codons ) in each organism , but only 20 different translated amino acids . 
   these alternative codons for the same amino acids are termed as synonymous codons . in general 
   , codon usage variation may be the product of natural selection and/or mutation pressure for accurate and efficient translation in various organisms [ 810 ] . 
   synonymous codon usage bias on virus can provide us with a better understanding on the evolution profile , gene expression , and virus - host interaction [ 1114 ] . however , there is still lacking about codon usage pattern of hmpv genome and its major influence factors . 
   herein , we firstly performed the comparative analysis of synonymous codon usage in hmpv genomes and analyzed their influencing factors . 
   this study will provide a new insight to understand the pathogenicity and its evolution history of hmpv . 
   in this study , a total 17 complete hmpv genomes which representing two genotypes were retrieved from ncbi ( http://www.ncbi.nlm.nih.gov/ ) until december , 2011 . the serial number ( sn ) , genbank number , genotype , and other information 
   moreover , 10 ampv genomes sequences were retrieved from ncbi database as reference frame ( in supplemental table  1 supplementary material available online at doi:10.1155/2012/460837 ) . to investigate the characteristics of synonymous codon usage , relative synonymous codon usage ( rscu ) values of each complete coding region in 17 hmpv genomes and 
   the rscu value of each codon for their amino acid was calculated as previously described . 
   a codon with an rscu value of more than 1.0 has a positive codon usage bias , while a value of less than 1.0 has a negative codon usage bias . 
   when the codon with rscu values close to 1.0 , it means that this codon is chosen equally and randomly . 
   the codon usage data of human cell and bird cell were obtained from the codon usage database online ( http://www.kazusa.or.jp/codon/ ) . 
   the effective number of codons ( enc ) is used to measure deviation from expected random codon usage of hmpv and is independent of hypotheses involving natural selection . 
   if only one codon is used for each amino acid , this value would be 20 , while all of codons are used equally , it will be 61 . 
   moreover , the index of gc3s was used to calculate the fraction of the nucleotides g + c content at the synonymous third codon position ( excluding aug [ met ] , ugg [ trp ] , and the termination codons ) . 
   multivariate statistical analysis can be used to explore the relationships between variables and samples . in this study , 
   correspondence analysis was used to investigate the major trend in codon usage variation among genomes . in this study , 
   the complete coding region of all 17 hmpv genomes was represented as a 59 dimensional vector , and each dimension corresponds to the rscu value of one sense codon ( excluding met , trp , and the termination codons ) . 
   correlation analysis was used to identify the relationship between nucleotide composition and synonymous codon usage pattern . 
   in order to investigate the synonymous codon usage of hmpv , we calculated various rscu values of various codons from 17 different strains , including different genotypes . 
   as shown in table 3 , the preferred codons in hmpv are gca , aga , aau , gau , ugu , caa , gaa , gga , cau , cau , auu , uua , aaa , uuu , cca , uca , agu , aca , uau , guu . interestingly , all preferred codons in hmpv genomes are ended with a / u , while none of them is ended with g / c . 
   this result suggests that hmpv genome has a great synonymous codon usage bias , and this phenomenon might highly associate with the nucleotide composition in its genomes . 
   as shown in table 2 , the g + c content of hmpv genome is 36.91% , which shares similar extent with another rna virus . 
   there are over 68.57% codons are ended with a / u among hmpv genomes , and 40.87% codons are a3 end , and 27.7% codons are u3 end . 
   this high abundance of a / u nucleotides is consistent with all preferred codons , which are ended with a / u . 
   this phenomenon reflects that nucleotide composition is the main force to affect the codon usage bias in hmpv genome . 
   moreover , as shown in table 2 , the enc values among hmpv genomes show a range from 45.127 to 48.28 , and its average value of 45.785 and sd value of 0.8458 . 
   the stable enc values suggest that their genomic compositions are much conserved among hmpv genomes . 
   natural selection and mutation pressure have been considered to be two key factors which have effect on codon usage patterns of organisms . in order to investigate whether mutation pressure or natural selection as a determinative factor for codon usage mutation in hmpv , we calculated correlation relation between a% , u% , g% , c% , gc% and a3% , u3% , g3% , c3% , gc3% . 
   as shown in table 4 , there exhibits a very complex correlation map observed in nucleotide compositions . in detail 
   , u3% has a significant positive correlation with u% ( r = 0.7821 , p < 0.01 ) , while shared negative correlation with c% ( r = 0.7153 , p < 0.01 ) and gc% ( r = 0.7474 , p < 0.01 ) . 
   c3% has positive correlation with c% ( r = 0.8331 , p < 0.01 ) and gc% ( r = 0.7880 , p < 0.01 ) , and negative correlation with a% ( r = 0.6199 , p < 0.01 ) and g% ( r = 0.5846 , p < 0.05 ) . 
   gc3% has significant positive correlation with c% ( r = 0.7178 , p < 0.01 ) and gc% ( r = 0.8052 , p < 0.01 ) , but has negative correlation with u% ( r = 0.7342 , p < 0.01 ) . 
   interestingly , the gc3% shows positive correlation with c% , while shows negative correlation with g% . 
   we calculated the gc3 skew by using formula as cg3  skew = ( c3  g3)/(c3 + g3 ) . 
   the gc3 skew of hmpv range s from 0.371 to 0.592 , which reveals that gc composition involved in the codon usage bias . 
   these data suggest that the nucleotide constraint might play an important role in influencing synonymous codon usage bias . 
   furthermore , the correlation analysis of first two principle axes ( f1 and f2 ) in hmpv and its nucleotide contents were performed ( table 5 ) . 
   apparently , the first principle axis ( f1 ) has a significantly negative correlation with u3% , and negative correlation with gc3% . 
   this result suggests that nucleotide u3% and gc3% are the major factor influencing the synonymous codon usage pattern in hmpv genome . 
   moreover , we observed the second principle axis ( f2 ) shared a significant positive correlation with g3% , and negative correlation with c3% and gc3% . 
   therefore , compositional constraint is a major factor which is involved in shaping the pattern of synonymous codon usage bias in hmpv genome . to investigate the effect of different hpmv genotypes on synonymous codon usage 
   , we analyzed the codon usage bias of different genotypes with correspondence analysis . from the correspondence analysis , the first dimension variable f1 and 
   the second dimension variable f2 can reflect 43.27% and 33.38% of total mutation , respectively . 
   as the plot of correspondence analysis shown ( figure 1 ) , each genotype is mainly separated and clustered into two clades . 
   however , the sublineage of each genotype did not exhibit any significant difference among them . 
   but this might be due to the limited number of hpmv genomes available in current study . 
   therefore , the phylogenetic distant might effect on the variation of synonymous codon usage in hmpv , and this difference might reflect on their biological effect , such as viral replication , virulence , and so forth . from the enc - gc3% plot analysis ( figure 2 ) , 
   the plots of each hmpv genomes are all under the expected curve , none of them shows above the curve . 
   moreover , there are still some other factors that can effect on the codon usage bias of hmpv . in the current study 
   as shown in table 3 , the pattern of synonymous codon usage in hmpv shows a complementary profile , which shows in human cell . in detail , hmpv and human host cell shared only 1 preferred codon ( aga ) , which encoded for arginine , while there are 17 different preferred codons between them . as a reference frame , 
   ampvs were enrolled in this study , and it also shows a complementary pattern with its host , bird cell . 
   the comparative analysis among hmpv and human host cell , hmpv and ampv host bird cell , and hmpv and ampv were analyzed . to compare the complementary ability of hmpv with bird cell , 
   there are more overlays ( 6 preferred codons overlay ) than hmpv with its human host cell ( only 1 preferred codons overlay ) . 
   this result shows human cell has much more complementary pattern with hmpv than bird cells . 
   this might be more suitable for hmpv survive and persist infect in human host environment . 
   this result also suggests that host factor plays an important role in codon usage bias in hmpv . 
   this complementary trend will benefit for virus replication instead of competitive with its host and it might help us to understand the mechanism of hmpv persistent infection . 
   interestingly , hmpvs are shares with more than 15 preferred codons to ampv , which might be due to a close phylogenetic distance . 
   synonymous codon usage analysis can reveal much about virus genome . to understand the extent and causes of codon usage bias is essential for studying the viral evolution , particularly the interaction between viruses and host immune response . in this study 
   as we know , the variation and evolution of virus generally happened in the changes of nucleotide composition . therefore , the nucleotide composition bias is the main force to influence the synonymous codon usage patterns . in this study 
   first of all , in hmpv , all preferred codons are ended in a / u nucleotide , which occupied the majority of nucleotide composition in hmpv genome . 
   this phenomenon confirmed that nucleotide composition was the main force in shaping the pattern of codon usage . 
   secondly , enc was used to quantify the codon usage bias , which is one of the best overall estimators of absolute synonymous codon usage bias . in this study , we observed enc of these genomes fluctuated from 45.13 to 48.28 with a mean 45.78  0.85 . this enc value of hmpv is consistent with other previously reported rna virus in the same family paramyxoviridae , ranging from 43.8 to 55.1 , that is , measles virus 55.1 , mumps virus 54.3 , parainfluenza-3 virus 43.8 , and respiratory syncytial virus 44.3 [ 8 , 24 , 25 ] . 
   moreover , the enc of hmpv is more close to respiratory infection agents , rsv and parainfluenza-3 , which reveals that the similar extent of codon usage bias among viruses might have similar infection syndrome . 
   this observation helps us to address an interesting assumption that synonymous codon usage bias of virus might associate with its pathogenicity . 
   mutation pressure and natural selection are generally treated as the main factors that account for codon usage bias in different organisms . 
   enc - gc plot was considered as a part of the general strategy to investigate patterns of synonymous codon usage [ 9 , 18 , 26 ] . 
   herein , all the plots are laid below the expected curve , suggesting that codon usage bias in all these 17 hmpv genomes was principally influenced by mutation bias , which consistent with that mutation pressure rather than natural selection is the most important determinant of the codon usage in human rna virus [ 8 , 2731 ] . 
   this observation can be explained as the mutation rates in rna viruses much higher than those in dna viruses . 
   mutation pressure is the main force in shaping synonymous codon usage bias of rna virus . 
   however , based on correspondence analysis , we observed an interesting phenomenon that codon usage bias in hmpv showed distinct differences among different phylogenetic types . 
   it might suggest that codon usage bias plays an important role in hmpv evolution history . 
   this similar phenomenon also observed in several other viruses , this might reflect that phylogenetic difference is a common influencing factor in shaping codon usage bias [ 25 , 27 , 3034 ] . 
   therefore , we hypothesize that the difference of codon usage bias might influence its virulence in different genotypes .   in this study , we also observed that hmpv showed a complementary trend with human cells by comparing the codon usage . 
   this complementary will be benefit for the survive of virus , which can keep replication by using the nonpreferred codons in the host cell without competition , and this could be one of the mechanisms of virus persistent infection in the human environment . moreover , this pattern might also be caused by the longitude selection and evolution between human hosts with virus . 
   therefore , this characteristic is important for hmpv keeping balanced with their host on the codon usage side , and also for understanding the cell - host interaction and viral evolution . 
   in summary , we firstly reported the synonymous codon usage pattern in hmpv genomes and revealed that mutation pressure is the main force in shaping its codon usage bias . 
   phylogenetic difference and host factors are also discussed , and this information can provide better understanding on the molecular evolution and its pathogenicity of hmpv .
summary: human metapneumovirus ( hmpv ) is an important agent of acute respiratory tract infection in children , while its pathogenicity and molecular evolution are lacking . herein , we firstly report the synonymous codon usage patterns of hmpv genome . 
   the relative synonymous codon usage ( rscu ) values , effective number of codon ( enc ) values , nucleotide contents , and correlation analysis were performed among 17 available whole genome of hmpv , including different genotypes . 
   all preferred codons in hmpv are ended with a / u nucleotide and exhibited a great association with its high proportion of these two nucleotides in their genomes . 
   mutation pressure rather than natural selection is the main influence factor that determines the bias of synonymous codon usage in hmpv 
   . the complementary pattern of codon usage bias between hmpv and human cell was observed , and this phenomenon suggests that host cells might be also act as an important factor to affect the codon usage bias . 
   moreover , the codon usage biases in each hmpv genotypes are separated into different clades , which suggest that phylogenetic distance might involve in codon usage bias formation as well . 
   these analyses of synonymous codon usage bias in hmpv provide more information for better understanding its evolution and pathogenicity .