BACKGROUND
human rhinovirus  is the most frequent cause of infection across all age groups of the population  <cit> .
replication is often restricted to the upper respiratory tract leading to self-limited illnesses such as the common cold.
however, hrv infections can also exacerbate pre-existing airway disorders, invade the lower respiratory tract and lead to serious complications  <cit> .
hrvs are single positive-stranded rna viruses of approximately  <dig> base pairs.
they belong to the picornaviridae family and are closely related to hevs, another genus of the same family.
the genome organization of picornaviridae is conserved among the family with a long 5'-untranslated region , a single open reading frame  encoding a polyprotein, a short 3'utr, and a poly tail  <cit> .
the 5'-terminal ump of the viral rna is covalently linked to the small viral protein vpg  <cit> .
the 5'utr contains two structural elements  <cit> .
one is the 5'-cloverleaf structure involved in the plus-strand rna synthesis and in the process of switching from translation to replication  <cit> .
the other is the internal ribosomal entry site  which promotes translation of the polyprotein.
the 3'-utr is necessary for efficient rna replication, but the exact mechanism is still not well understood  <cit> .
in addition, species-specific internal cis-acting replication elements  have been identified in hev  <cit> , hrv-a  <cit>  and hrv-b  <cit> .
hrv strains have been classified into  <dig> serotypes  <cit>  based on the ability of a given serum to neutralize virus growth of a given strain in cell culture, although several serotypes share significant antigenic cross-reactivity  <cit> .
according to nucleotide sequence relatedness of some serotypes  <cit>  and to sequence comparison of all serotypes in the vp <dig>  <cit>  and vp4-vp <dig> capsid protein-coding regions  <cit> , the  <dig> serotypes segregate in two different groups:  <dig> belong to the hrv-a species and  <dig> to the hrv-b species.
in addition to the division of hrvs into two species, they have also been classified into major and minor groups according to receptor usage.
the major group ofhrvs  binds icam <dig>  whereas the minor group viruses  bind preferentially to ldl receptors  <cit> .
the existence of multiple serotypes within each of these two lineages and different receptor usage support the hypothesis of significant differences at the protein level.
surprisingly, despite the fact that hrvs are the major cause of human respiratory infections, little is known about their genome variability at the full-length scale.
to the best of our knowledge, part of the vp4-vp <dig>  <cit>  and vp1-2a  <cit>  regions have been sequenced for all serotypes and half of them for the 3d regions  <cit> , but full-length sequences of only  <dig> serotypes are publicly available in the picornaviridae database  <cit>    <cit> .
while the present manuscript was in the process to be accepted, kistler and coworkers published additional hrv-a and hrv-b full-length sequences increasing significantly the number of sequences available  <cit> .
among the picornaviridae, hevsare the closest relatives of hrvs and, as for hrvs, humans are the only known reservoir.
phylogenetic analyses of vp1-2a hrv and hev sequences suggest thathrvs and hevs could be considered members of the same genus  <cit> .
in addition, hrv- <dig> presents a high sequence similarity to hevs and was recently reclassified as ev- <dig>  <cit> .
yet, the exact relation between hev and hrv remains ambiguous without full-length genome comparison.
at the phenotype level, however, hrv and hev are clearly distinct: in vitro, cell tropism, ph tolerance and optimal growth temperature are significantly different; in vivo, the site of infection, organ tropism and the ability to disseminate are well-established characteristics that differentiate hevs from hrvs.
hrvs infections are restricted to the respiratory tract , whereas most hevs have the ability to replicate predominantly in the gastrointestinal tract .
a large proportion can also disseminate, causing viremia and potentially invading the central nervous system  <cit> .
full-length genome comparison of these two genera helped us to identify genomic features and divergences at the amino acid level that might explain some biological differences.
we have sequenced  <dig> full-length genomes of different hrv serotypes and we present here the comparative analysis of  <dig> prototype hrv strains  and  <dig> publicly available hev strains that identifies the key elements differentiating these medically important viruses.
RESULTS
the  <dig> newly sequencedhrv genomes have been deposited in genbank .
they vary in sequence length from  <dig> nucleotides  to  <dig> nucleotides  which is similar to the length range of previously sequenced genomes .
the average size ofhrvs type a  is smaller than the average size of hrvs type b , whereas the average size of  <dig> hevs analysed in this study is  <dig>
hrv-b is more closely related to hev than to hrv-a
phylogenetic analysis
the phylogeny of hrv-a, hrv-b and hev was reconstructed by using maximum-likelihood phylogenetic method for the full polyproteins , as well as for each individual protein, using simian picornavirus  as the outgroup.
the whole-polyprotein phlyogenetic analysis suggests the hypothesis that hrv-b and hev lineages radiated from a common ancestor after its separation from hrv-a , where the percentage of bootstrap support of each grouping reflects the statistical confidence.
yet, when the analysis is conducted at the level of each individual protein, the subsequent reconstructed tree topology does not always support the same conclusion .
in the region of vp <dig>  vp <dig>  3b and 3c proteins, the analysis supports the alternative hypothesis that hrv-b and hrv-a radiated from a common ancestor after separation from hev.
in the region of vp <dig>  vp <dig> and 3a proteins, the analysis cannot discriminate the phylogenetic relationships between these three virus groups.
the bootscanning experiment  presented in figure 2d is consistent with all the above-mentioned findings and also supports the hypothesis that in some parts of the capsid hrv-a and hrv-b share the last ancestor after their separation from hev.
protein product similarity
for each individual protein cleavage product, we also quantified pair-wise sequence identities among the hrv-a, hrv-b, and hev genomes under consideration.
figure  <dig> shows the arrangement of these proteins along the picornavirus genomes and the corresponding protein identity matrices.
over all these three picornavirus , vp <dig> is the least conserved protein  and vp <dig> the most conserved .
globally, these comparisons are consistent with the phylogenetic analysis and confirm that the 2a, 2b, 2c, and 3d percentage of sequence similarities are higher between hrv-b and hev than between hrv-a and b .
in contrast, at the level of vp <dig>  vp <dig>  vp <dig>  3b and 3c sequences, hrv-a and hrv-b have a higher percentage of homologies compared to hev .
between the hrv-a and hrv-b groups, vp <dig> shows the highest overall conservation  and 2a protein exhibits the lowest .
within each of the species , the vp <dig> protein appears as the least conserved  and vp <dig> as the most conserved  .
protein 3b also shows poor conservation among hrv-b serotypes, but this may be an artefact due to its small size.
rna structural elements
5'- and 3'- utrs
analysis of the 5'utr of hrv-a, hrv-b and hev shows that the well known 5' cloverleaf cre element as well as the ires structure are highly conserved throughout the three groups .
the cloverleaf structure was originally discovered in polioviruses  <cit> .
this secondary structure is deposited in the rfam database  under the accession number .
the corresponding consensus structure of hrv-a, hrv-b and hev, which was recovered without prior knowledge of it by comparative sequence analysis  matches well this rfam consensus structure.
in the same line, the ires structure for hrv-a, hrv-b and hevs is also very similar to the rfam picornaviridae consensus structure   <cit> , except for the presence of two additional small helices .
the picornavirus 3'-utr encodes astem-loop structure that may play a role in replication efficiency  as well as in polyadenylation of genomic rna  <cit> .
in contrast to the 5'utr structures, the 3'utrs structures are not universally conserved in sequence and position among the three groups studied.
the length of the 3' utr between the groups varies between  <dig> nt ,  <dig> nt  and  <dig> nt .
furthermore, both hrv-a and hrv-b contain a stable stem-loop structure of  <dig> nt at the 3' end of the 3'utr.
hev also contains a  <dig> nt stem-loop which is located closer to the middle of the 3'utr.
nevertheless, there is a large amount of sequence variability within this whole group which leads to a less stable consensus stem-loop for all the analyzed hev sequences.
in addition, there is a conserved stem-loop structure in hrv-a located close to the 3'utr, yet the corresponding region in hrv-b and hev suggest different structures, and the overall high sequence conservation in the region could give a misleading signal of structural conservation .
internal cis-acting elements
besides the 5' and 3'utr, disparate internal cre elements have been previously described among various rhinoviral serotypes of both hrv-a and hrv-b  <cit> , and have been identified by our comparative analysis.
hrv-a cre
the hrv- <dig> 2a internal cre motif   <cit>  is conserved among all hrv-a genomes analysed in this study, but has not been identified in any hrv-b or hev viruses .
the same region of hrv-b also folds into a conserved secondary structure that seems specific to this group .
hrv-b cre
similarly, the internal cre motif reported for the hrv- <dig> vp <dig>  a member of hrv-b, is present in all  <dig> hrv-b serotypes and is notably absent in all hrv-a and hev analyzed .
furthermore, the availability of new hrv-b sequences allowed us to identify another conserved cre motif within the hrv-b 2c coding sequence  that has the typical r1nnnaar2nnnnnnr <dig> cre motif  <cit>  in all hrv-b serotypes analysed , with the exception of hrv- <dig> that has a u instead of an r at position r <dig>  more importantly, the newly identified hrv-b 2c cre corresponds to the hev 2c cre, previously identified in several hevs  <cit> .
gc content
the gc composition is an important genomic factor that can be evolutionary optimized for adaptation to multiple environmental constraints .
the gc content varies substantially between the groups of hev, hrv-a and hrv-b , where hrv-b exhibits lowest values, hev exhibits the highest values, and hrv-b is intermediate.
this holds not only globally, but also locally, for each of the sliding windows along the whole genomes.
these trends are statistically significant as the two-sided kolmogorov-smirnov test rejects the hypothesis that gc contents of hrv-a, hrv-b and hev can be drawn from the same underlying distribution: hrv-a vs. hrv-b p-value < 10-15; hrv-a vs. hev p-value < 10-15; hrv-b vs. hev p-value < 10- <dig>
discussion
hrvs were first classified into two groups based on a differential sensitivity to a variety of antiviral compounds targeting vp <dig>  <cit> .
the members of the hrv-a group were susceptible to most of these antiviral compounds, whereas the hrv-b were not.
this classification was then confirmed by nucleotide sequence relatedness in the vp <dig>  <cit>  and vp4-vp <dig> capsid protein-coding regions of all serotypes  <cit> .
analysis of other regions like the 3c protease has been restricted to a limited number of serotypes  <cit> .
whole genome comparisons have not been conducted since only one full-length hrv-b genome  as well as a limited number of hrv-a genomes were available.
complete sequencing and analysis of additional hrv-b and hrv-a genomes allowed us to describe their phylogeny and the similarity of individual proteins between the two hrv groups and hev.
for example, proteins such as 2a show a particularly pronounced difference in inter- versus intra-group conservation.
conversely, surface proteins such as vp <dig>  are better conserved across all groups.
it appears that hrv-b share a common ancestor with hev as shown by the whole-genome phylogenetic analysis, which suggests that rhinovirus is not monophyletic.
this observation is reinforced by the identification of a new hrv-b 2c cre that is lacking in all hrv-a genomes studied.
this cre consists of a hairpin structure with a conserved r1nnnaar2nnnnnnr <dig> motif in the loop  <cit>  and was previously only known in hev 2c gene.
the first two as in this motif serve as the template for the vpg uridylylation reaction by the viral polymerase.
uridylylated vpg then serves as a primer for rna synthesis  <cit> .
although the question is still open to debate, it has been suggested that the polya tail may serve as template for vpg uridylylation and synthesis of the minus strand rna, whereas internal cre are necessary for plus strand synthesis  <cit> .
hrv- <dig> vp <dig> cre, as well as hrv- <dig> 2a cre, were shown to be functionally equivalent to poliovirus rna 2c cre in in vitro uridylylation assays with poliovirus vpg and polymerase  <cit> .
further studies are underway to define whether the hrv-b 2c cre identified in this study plays a role in vpg uridylylation or can be considered as an evolutionary leftover of hev 2c cre.
concerning the replacement of an r position by a u in hrv- <dig> cre, it should be noted that the effect of a similar substitution on replication efficiency could not be studied for hrv- <dig> 2c cre since it results in the introduction of a stop codon  <cit> .
however, for coxsackiesvirus b <dig> 2c cre, a substitution at the r <dig> position by a u was shown permissive for replication  <cit> .
besides this putative new cre element in the 2c region of hrv-b, we could also identify the already known elements  in the 5' and 3' utrs from all studied genomes.
the cloverleaf structure and the ires are highly conserved.
interestingly, we identified many compensatory mutations in the sequences of these structures, which points out that the selective pressure is "working" on the structural level.
the functionality of these elements is therefore more determined by their structure than by their primary sequence.
the observation that hrv-b and hevs are more closely related to each other than either is to hrv-a seems paradoxical, given that hrvs differentiate themselves from most hevs at the phenotypic level.
this could be explained by recombination events that would have taken place soon after the divergence of hev and hrv-a and during which regions were exchanged between hrv-a and the hev ancestor of hrv-b.
the protein identity plots and the bootscanning suggest that the recombining region may have included the capsid region.
this is consistent with the fact that recombination breakpoints have been found to be largely restricted to nonstructural regions of the genome, mostly in the 2a-2c parts, and between the 5' utr and the capsid-encoding region  <cit> .
recombination has been extensively documented as a driving force for the evolution of some picornaviridae  <cit> , although only hypothesized forhrvs  <cit> .
interspecies in vivo recombination was also suspected for hevs  <cit> .
our hypothesis is that early recombination events may have occurred between hrv and hev.
although these two virus species have often different tropism in vivo, both can easily infect the respiratory tract, an event that could provide opportunities for recombination.
since vp <dig> is responsible for recognizing the receptor on the host cell surface, the hypothesis of capsid sequence transfer from hrv-a to hev to yield hrv-b could explain a tissue tropism and a disease pattern similar to that of hrv-a rather than hev.
in addition, the similar gc content observed between hrv-b and hrv-a may account for some of these phenotypic differences.
the relatively lower gc content of hrv-b may reflect an adaptation to the environment of the upper respiratory tract, whereas the higher gc content of hevs might reflect convergent adaptation to the gastrointestinal tract of the central nervous system environment .
CONCLUSIONS
the analysis of new hrv full-length genomes statistically supports  the current classification of hrv into two distinct species.
hrv-b seems to be phylogenetycally more closely related to hev, another important member of the picornaviridae family, than it is to hrv-a.
however, our observations suggest that this species classification accurately reflects the capsid type, but not all parts of the genome.
we have shown that hrv-a and hrv-b differ significantly at the protein level and in the composition and structure of their cis-acting sequences.
one of the evolutionary scenarios that would explain the differential grouping of hrv-b with hevs or with hrv-a along the genome is that of an ancient recombination between hrv-a and hev lineages, given that the hrv-b closer relation with hevs is overall more statistically sound.
however, without additional data, this remains only a hypothesis.
the genomic features highlighted in our study help to contribute to our understanding of why these viruses maintain different phenotypic variations in humans, thereby enabling a more accurate analysis of their relationship.

 human rhinovirus is the most frequent cause of infection across all age groups of the population. replication is often restricted to the upper respiratory tract leading to self-limited illnesses such as the common cold. however, hrv infections can also exacerbate pre-existing airway disorders, invade the lower respiratory tract and lead to serious complications. hrvs are single positive-stranded rna viruses of approximately base pairs. they belong to the picornaviridae family and are closely related to hevs, another genus of the same family. the genome organization of picornaviridae is conserved among the family with a long 5'-untranslated region, a single open reading frame encoding a polyprotein, a short 3'utr, and a poly tail. the 5'-terminal ump of the viral rna is covalently linked to the small viral protein vpg. the 5'utr contains two structural elements. one is the 5'-cloverleaf structure involved in the plus-strand rna synthesis and in the process of switching from translation to replication. the other is the internal ribosomal entry site which promotes translation of the polyprotein. the 3'-utr is necessary for efficient rna replication, but the exact mechanism is still not well understood. in addition, species-specific internal cis-acting replication elements have been identified in hev, hrv-a and hrv-b. hrv strains have been classified into serotypes based on the ability of a given serum to neutralize virus growth of a given strain in cell culture, although several serotypes share significant antigenic cross-reactivity. according to nucleotide sequence relatedness of some serotypes and to sequence comparison of all serotypes in the vp and vp4-vp capsid protein-coding regions, the serotypes segregate in two different groups: belong to the hrv-a species and to the hrv-b species. in addition to the division of hrvs into two species, they have also been classified into major and minor groups according to receptor usage. the major group ofhrvs binds icam whereas the minor group viruses bind preferentially to ldl receptors. the existence of multiple serotypes within each of these two lineages and different receptor usage support the hypothesis of significant differences at the protein level. surprisingly, despite the fact that hrvs are the major cause of human respiratory infections, little is known about their genome variability at the full-length scale. to the best of our knowledge, part of the vp4-vp and vp1-2a regions have been sequenced for all serotypes and half of them for the 3d regions, but full-length sequences of only serotypes are publicly available in the picornaviridae database. while the present manuscript was in the process to be accepted, kistler and coworkers published additional hrv-a and hrv-b full-length sequences increasing significantly the number of sequences available. among the picornaviridae, hevsare the closest relatives of hrvs and, as for hrvs, humans are the only known reservoir. phylogenetic analyses of vp1-2a hrv and hev sequences suggest thathrvs and hevs could be considered members of the same genus. in addition, hrv- presents a high sequence similarity to hevs and was recently reclassified as ev-. yet, the exact relation between hev and hrv remains ambiguous without full-length genome comparison. at the phenotype level, however, hrv and hev are clearly distinct: in vitro, cell tropism, ph tolerance and optimal growth temperature are significantly different; in vivo, the site of infection, organ tropism and the ability to disseminate are well-established characteristics that differentiate hevs from hrvs. hrvs infections are restricted to the respiratory tract, whereas most hevs have the ability to replicate predominantly in the gastrointestinal tract. a large proportion can also disseminate, causing viremia and potentially invading the central nervous system. full-length genome comparison of these two genera helped us to identify genomic features and divergences at the amino acid level that might explain some biological differences. we have sequenced full-length genomes of different hrv serotypes and we present here the comparative analysis of prototype hrv strains and publicly available hev strains that identifies the key elements differentiating these medically important viruses.  the newly sequencedhrv genomes have been deposited in genbank. they vary in sequence length from nucleotides to nucleotides which is similar to the length range of previously sequenced genomes. the average size ofhrvs type a is smaller than the average size of hrvs type b, whereas the average size of hevs analysed in this study is hrv-b is more closely related to hev than to hrv-a phylogenetic analysis the phylogeny of hrv-a, hrv-b and hev was reconstructed by using maximum-likelihood phylogenetic method for the full polyproteins, as well as for each individual protein, using simian picornavirus as the outgroup. the whole-polyprotein phlyogenetic analysis suggests the hypothesis that hrv-b and hev lineages radiated from a common ancestor after its separation from hrv-a, where the percentage of bootstrap support of each grouping reflects the statistical confidence. yet, when the analysis is conducted at the level of each individual protein, the subsequent reconstructed tree topology does not always support the same conclusion. in the region of vp vp 3b and 3c proteins, the analysis supports the alternative hypothesis that hrv-b and hrv-a radiated from a common ancestor after separation from hev. in the region of vp vp and 3a proteins, the analysis cannot discriminate the phylogenetic relationships between these three virus groups. the bootscanning experiment presented in figure 2d is consistent with all the above-mentioned findings and also supports the hypothesis that in some parts of the capsid hrv-a and hrv-b share the last ancestor after their separation from hev. protein product similarity for each individual protein cleavage product, we also quantified pair-wise sequence identities among the hrv-a, hrv-b, and hev genomes under consideration. figure shows the arrangement of these proteins along the picornavirus genomes and the corresponding protein identity matrices. over all these three picornavirus, vp is the least conserved protein and vp the most conserved. globally, these comparisons are consistent with the phylogenetic analysis and confirm that the 2a, 2b, 2c, and 3d percentage of sequence similarities are higher between hrv-b and hev than between hrv-a and b. in contrast, at the level of vp vp vp 3b and 3c sequences, hrv-a and hrv-b have a higher percentage of homologies compared to hev. between the hrv-a and hrv-b groups, vp shows the highest overall conservation and 2a protein exhibits the lowest. within each of the species, the vp protein appears as the least conserved and vp as the most conserved. protein 3b also shows poor conservation among hrv-b serotypes, but this may be an artefact due to its small size. rna structural elements 5'- and 3'- utrs analysis of the 5'utr of hrv-a, hrv-b and hev shows that the well known 5' cloverleaf cre element as well as the ires structure are highly conserved throughout the three groups. the cloverleaf structure was originally discovered in polioviruses. this secondary structure is deposited in the rfam database under the accession number. the corresponding consensus structure of hrv-a, hrv-b and hev, which was recovered without prior knowledge of it by comparative sequence analysis matches well this rfam consensus structure. in the same line, the ires structure for hrv-a, hrv-b and hevs is also very similar to the rfam picornaviridae consensus structure, except for the presence of two additional small helices. the picornavirus 3'-utr encodes astem-loop structure that may play a role in replication efficiency as well as in polyadenylation of genomic rna. in contrast to the 5'utr structures, the 3'utrs structures are not universally conserved in sequence and position among the three groups studied. the length of the 3' utr between the groups varies between nt, nt and nt. furthermore, both hrv-a and hrv-b contain a stable stem-loop structure of nt at the 3' end of the 3'utr. hev also contains a nt stem-loop which is located closer to the middle of the 3'utr. nevertheless, there is a large amount of sequence variability within this whole group which leads to a less stable consensus stem-loop for all the analyzed hev sequences. in addition, there is a conserved stem-loop structure in hrv-a located close to the 3'utr, yet the corresponding region in hrv-b and hev suggest different structures, and the overall high sequence conservation in the region could give a misleading signal of structural conservation. internal cis-acting elements besides the 5' and 3'utr, disparate internal cre elements have been previously described among various rhinoviral serotypes of both hrv-a and hrv-b, and have been identified by our comparative analysis. hrv-a cre the hrv- 2a internal cre motif is conserved among all hrv-a genomes analysed in this study, but has not been identified in any hrv-b or hev viruses. the same region of hrv-b also folds into a conserved secondary structure that seems specific to this group. hrv-b cre similarly, the internal cre motif reported for the hrv- vp a member of hrv-b, is present in all hrv-b serotypes and is notably absent in all hrv-a and hev analyzed. furthermore, the availability of new hrv-b sequences allowed us to identify another conserved cre motif within the hrv-b 2c coding sequence that has the typical r1nnnaar2nnnnnnr cre motif in all hrv-b serotypes analysed, with the exception of hrv- that has a u instead of an r at position r more importantly, the newly identified hrv-b 2c cre corresponds to the hev 2c cre, previously identified in several hevs. gc content the gc composition is an important genomic factor that can be evolutionary optimized for adaptation to multiple environmental constraints. the gc content varies substantially between the groups of hev, hrv-a and hrv-b, where hrv-b exhibits lowest values, hev exhibits the highest values, and hrv-b is intermediate. this holds not only globally, but also locally, for each of the sliding windows along the whole genomes. these trends are statistically significant as the two-sided kolmogorov-smirnov test rejects the hypothesis that gc contents of hrv-a, hrv-b and hev can be drawn from the same underlying distribution: hrv-a vs. hrv-b p-value < 10-15; hrv-a vs. hev p-value < 10-15; hrv-b vs. hev p-value < 10- discussion hrvs were first classified into two groups based on a differential sensitivity to a variety of antiviral compounds targeting vp. the members of the hrv-a group were susceptible to most of these antiviral compounds, whereas the hrv-b were not. this classification was then confirmed by nucleotide sequence relatedness in the vp and vp4-vp capsid protein-coding regions of all serotypes. analysis of other regions like the 3c protease has been restricted to a limited number of serotypes. whole genome comparisons have not been conducted since only one full-length hrv-b genome as well as a limited number of hrv-a genomes were available. complete sequencing and analysis of additional hrv-b and hrv-a genomes allowed us to describe their phylogeny and the similarity of individual proteins between the two hrv groups and hev. for example, proteins such as 2a show a particularly pronounced difference in inter- versus intra-group conservation. conversely, surface proteins such as vp are better conserved across all groups. it appears that hrv-b share a common ancestor with hev as shown by the whole-genome phylogenetic analysis, which suggests that rhinovirus is not monophyletic. this observation is reinforced by the identification of a new hrv-b 2c cre that is lacking in all hrv-a genomes studied. this cre consists of a hairpin structure with a conserved r1nnnaar2nnnnnnr motif in the loop and was previously only known in hev 2c gene. the first two as in this motif serve as the template for the vpg uridylylation reaction by the viral polymerase. uridylylated vpg then serves as a primer for rna synthesis. although the question is still open to debate, it has been suggested that the polya tail may serve as template for vpg uridylylation and synthesis of the minus strand rna, whereas internal cre are necessary for plus strand synthesis. hrv- vp cre, as well as hrv- 2a cre, were shown to be functionally equivalent to poliovirus rna 2c cre in in vitro uridylylation assays with poliovirus vpg and polymerase. further studies are underway to define whether the hrv-b 2c cre identified in this study plays a role in vpg uridylylation or can be considered as an evolutionary leftover of hev 2c cre. concerning the replacement of an r position by a u in hrv- cre, it should be noted that the effect of a similar substitution on replication efficiency could not be studied for hrv- 2c cre since it results in the introduction of a stop codon. however, for coxsackiesvirus b 2c cre, a substitution at the r position by a u was shown permissive for replication. besides this putative new cre element in the 2c region of hrv-b, we could also identify the already known elements in the 5' and 3' utrs from all studied genomes. the cloverleaf structure and the ires are highly conserved. interestingly, we identified many compensatory mutations in the sequences of these structures, which points out that the selective pressure is "working" on the structural level. the functionality of these elements is therefore more determined by their structure than by their primary sequence. the observation that hrv-b and hevs are more closely related to each other than either is to hrv-a seems paradoxical, given that hrvs differentiate themselves from most hevs at the phenotypic level. this could be explained by recombination events that would have taken place soon after the divergence of hev and hrv-a and during which regions were exchanged between hrv-a and the hev ancestor of hrv-b. the protein identity plots and the bootscanning suggest that the recombining region may have included the capsid region. this is consistent with the fact that recombination breakpoints have been found to be largely restricted to nonstructural regions of the genome, mostly in the 2a-2c parts, and between the 5' utr and the capsid-encoding region. recombination has been extensively documented as a driving force for the evolution of some picornaviridae, although only hypothesized forhrvs. interspecies in vivo recombination was also suspected for hevs. our hypothesis is that early recombination events may have occurred between hrv and hev. although these two virus species have often different tropism in vivo, both can easily infect the respiratory tract, an event that could provide opportunities for recombination. since vp is responsible for recognizing the receptor on the host cell surface, the hypothesis of capsid sequence transfer from hrv-a to hev to yield hrv-b could explain a tissue tropism and a disease pattern similar to that of hrv-a rather than hev. in addition, the similar gc content observed between hrv-b and hrv-a may account for some of these phenotypic differences. the relatively lower gc content of hrv-b may reflect an adaptation to the environment of the upper respiratory tract, whereas the higher gc content of hevs might reflect convergent adaptation to the gastrointestinal tract of the central nervous system environment.  the analysis of new hrv full-length genomes statistically supports the current classification of hrv into two distinct species. hrv-b seems to be phylogenetycally more closely related to hev, another important member of the picornaviridae family, than it is to hrv-a. however, our observations suggest that this species classification accurately reflects the capsid type, but not all parts of the genome. we have shown that hrv-a and hrv-b differ significantly at the protein level and in the composition and structure of their cis-acting sequences. one of the evolutionary scenarios that would explain the differential grouping of hrv-b with hevs or with hrv-a along the genome is that of an ancient recombination between hrv-a and hev lineages, given that the hrv-b closer relation with hevs is overall more statistically sound. however, without additional data, this remains only a hypothesis. the genomic features highlighted in our study help to contribute to our understanding of why these viruses maintain different phenotypic variations in humans, thereby enabling a more accurate analysis of their relationship. 
in contrast to hev viruses, hrv-a and hrv-b share also markedly lower gc content along the whole genome length. here we report complete genome sequences of hrv-a and hrv-b serotypes, more than doubling the current number of available hrv sequences. the whole-genome maximum-likelihood phylogenetic analysis suggests that hrv-b and human enteroviruses diverged from the last common ancestor after their separation from hrv-a. nevertheless they differ significantly at the phenotypic level.
 human rhinoviruses, the most frequent cause of respiratory infections, include different serotypes segregating into two species, a and b. rhinoviruses share extensive genomic sequence similarity with enteroviruses and both are part of the picornavirus family. nevertheless they differ significantly at the phenotypic level. the lack of hrv full-length genome sequences and the absence of analysis comparing picornaviruses at the whole genome level limit our knowledge of the genomic features supporting these differences.  here we report complete genome sequences of hrv-a and hrv-b serotypes, more than doubling the current number of available hrv sequences. the whole-genome maximum-likelihood phylogenetic analysis suggests that hrv-b and human enteroviruses diverged from the last common ancestor after their separation from hrv-a. on the other hand, compared to hev, hrv-b are more related to hrv-a in the capsid and 3b-c regions. we also identified the presence of a 2c cis-acting replication element in hrv-b that is not present in hrv-a, and that had been previously characterized only in hev. in contrast to hev viruses, hrv-a and hrv-b share also markedly lower gc content along the whole genome length.  our findings provide basis to speculate about both the biological similarities and the differences of these three groups of viruses. 