BACKGROUND
macaque monkeys are an important animal model in biomedical research, particularly in infectious diseases.
they are used in vaccine and infectivity studies of viruses that either do not infect or do not produce realistic pathogenic features in small animal models – viruses such as human immunodeficiency virus  where simian immunodeficiency virus  or the human envelope substituted version  can be studied as a surrogate in macaques.
recently, the limited availability of indian rhesus macaques in north america and elsewhere has led to the development of resources and standards for the use of cynomolgus macaques and chinese rhesus macaques  <cit> , the former in particular being an excellent model of hiv-1/siv infection  <cit> .
though relatively closely related, divergence between populations or species of macaques and their corresponding cytomegaloviruses  strains is substantial enough as to preclude direct replacement in any study.
in fact, with increased usage in research, it has been questioned whether cynomolgus macaques may be in need of taxonomic re-classification  <cit> .
captive populations of cynomolgus macaques are frequently interbred with little concern as to their origins.
however, the genetic divergence between geographically distinct populations of cynomolgus macaques rivals that found between indian and chinese rhesus macaques, a factor that has the potential to differently influence experimental results  <cit> .
for example, disease susceptibility is highly variable between isolated-tightly knit groups of macaques found across the sunda shelf of southeast asia.
macaque genome sequencing has enabled a better understanding of their disease susceptibility from an evolutionary and conservation standpoint, and provided insight into population structure and patterns of migration .
interest in nonhuman primate herpesviruses, and cmvs in particular, has increased in recent years in recognition of the unique immune response that they evoke.
specifically, cmv evokes a type of effector memory t cell response, which intermittently is boosted by reactivated virus throughout the life of its immunocompetent host.
this type of immune memory qualitatively is different from conventional t cell memory in that it does not require priming and can respond immediately to antigen.
thus cmvs are also of interest to vaccinologists who are examining ways to expand the duration of rapidly inducible t cell responses to block primary viral infection.
the evaluation of cmvs in rhesus and more recently, in cynomolgus macaques, has become a priority in order to facilitate the use of these animal models for vaccine research and development.
cmv infects many primate and non-primate hosts including humans, baboons, green monkeys, chimpanzees, squirrel monkeys, macaques, oysters and rodents  <cit> .
since previous studies have demonstrated only limited mixing and horizontal transmission between mammalian populations  <cit> , it is thought that cmv, like other herpesviruses, diversified via co-evolution with their host species  <cit> .
during speciation, pathogens may co-speciation with their host  <cit> .
herpesviruses, for instance, co-evolve with their mammalian hosts  <cit> .
herpesviruses were present in primates  <dig> mya  <cit> , and have been used as surrogates to track mammalian – including human – evolution and migration  <cit> .
herpesviruses, including cmvs, were also present in the most recent common ancestor of rhesus and cynomolgus macaques, and are presumed to have undergone geographic differentiation similar to their host species  <cit> .
however, macaques can become infected with multiple strains of cmv and these cmv strains may experience recombination.
hosts co-infected with two cmv strains occasionally release infectious viral particles  from multiple strains , but generally have a dominant strain that makes up the majority of shed virus  <cit> .
interaction among populations of cynomolgus macaques can facilitate the exchange of cmv strains  <cit>  but cross infection between species is generally rare  <cit> .
in the present study, we sequenced a new strain of cycmv from a cynomolgus macaque from mauritius and compared it to three other strains – one from a cynomolgus macaque that originated in the philippines   <cit> , and two from indian rhesus macaques , rhcmv 68– <dig>  <cit>  and rhcmv  <dig>   <cit> .
we also characterized variation in gene content among these macaque cmvs.
our results indicate that evolutionary relationships over the complete genomes of cynomolgus cmv strains matches those of their hosts, thereby supporting their co-evolution and also the further use of cmvs in the study of mammalian biogeography and phylogeny.
we also found phylogenetic correspondence between cmvs and their hosts in a broader phylogenetic analysis that included several other cmv strains.
however, phylogenetic relationships among some genomic regions of the macaque cmvs deviated from the expected relationship, and this can be explained either by intra-strain recombination or alternatively could reflect phylogenetic error.
RESULTS
cycmv mauritius is a unique cmv from mauritian cynomolgus macaques
in the present study, we characterized a novel cmv strain, isolated from a mauritian cynomolgus macaque, and compared its sequence to that of three other cmv strains, including that of cycmv ottawa  <cit> , and two strains from rhesus macaques: rhcmv 68– <dig>  <cit>  and rhcmv  <dig>   <cit> .
our analyses indicate that the sequence of cycmv mauritius is highly similar to the sequence of cycmv ottawa, and also that both are derived from a more recent common ancestor than either is with one of the rhesus macaque strains.
our assembly of illumina reads from cycmv mauritius produced three contigs that were then connected by sanger sequencing.
the assembly had an average 280x fold coverage that was  <dig> 200 bp in length, and had  <dig>  % gc content.
in comparison, cycmv ottawa is 841 bp longer and has a similar gc content .
rhcmv 68– <dig> and rhcmv <dig>  are both similar in length and gc content, with rhcmv  <dig>  being the shortest and rhcmv 68– <dig> the longest of the four viruses.
compared to other mammalian cmvs, all macaque cmvs examined are shorter than chimpanzee cmv  heberling strain and shorter on average than human cmv  strains ad <dig> and han <dig>  of the cmvs that have been sequenced thus far, these four macaque cmvs are most similar in length to aotine  cmv and african green monkey cmv.table  <dig> select sequenced primate cmv strains
cycmv mauritius and cycmv ottawa share  <dig>  % identity by linear full genome alignment while rhcmv 68– <dig> and rhcmv  <dig>  share  <dig>  % identity.
cycmv mauritius and rhcmv  <dig>  have  <dig>  % identity and cycmv mauritius and rhcmv 68– <dig> have  <dig>  % identity.
cycmv ottawa has  <dig>  % identity with rhcmv 68– <dig> and  <dig>  % identity with rhcmv  <dig> .
due to the possibility of rearrangements in these viral genomes we evaluated sequence synteny using the progressive mauve multiple genome alignment algorithm  <cit> .
this analysis reveals a local co-linear region around 160– <dig> kbp where the rhcmv 68– <dig> sequence is reversed in comparison to cycmv mauritius, cycmv ottawa and rhcmv  <dig>  .
this region is immediately adjacent to the ul <dig> to ul <dig> deletion found in rhcmv 68– <dig>  there are two locally collinear blocks whose order are reversed between rhesus  and cynomolgus  macaque cmv genomes.
both collinear blocks are short and located immediately upstream of the reversed region in rhcmv 68– <dig> fig.
<dig> multiple genome alignment of cycmv mauritius, cycmv ottawa, rhcmv 68– <dig>  and rhcmv  <dig> .
genome homology and rearrangement are presented using the mauve multiple genome alignment algorithm.
locally collinear blocks are indicated differentiated by colour and connected by linking line.
within collinear blocks column height indicates average conservation of base pairs locally between homologous collinear blocks in other strains, full columns indicate regions fully conserved between species while empty columns indicate unique regions to the particular genome or absences.
collinear blocks shown above the x-axis are in the same orientation, with those shown below reversed in comparison to the same co-linear blocks in other species
phylogenetic relationships among cmv strains match those of their hosts
evolutionary relationships were estimated for cycmv mauritius, cycmv ottawa, rhcmv  <dig>  and rhcmv 68– <dig> using hcmv ad <dig> as an outgroup .
evolutionary relationships among these complete macaque cmv genome sequences match those of their hosts .
within the cynomolgus macaque, the viral strains cycmv mauritius and cycmv ottawa are diverged by  <dig>  substitutions per site , whereas within the rhesus macaque, the viral strains rhcmv  <dig>  and rhcmv 68– <dig> are diverged by only  <dig>  sps.
thus, divergence between cmv strains of cynomologous macaques is greater than between the cmv strains of rhesus macaques, and this may also be true of the respective host populations.fig.
<dig> phylogenetic tree comparison of macaque cmv genome and select genes.
trees were generated comparing cycmv mauritius, cycmv ottawa, rhcmv 68– <dig> and rhcmv  <dig>  using hcmv ad <dig> as an out-group.
trees were generated using mr. bayes, following mafft alignment, using a model of evolution selected by jmodel test a) from a full genome global alignment; b) from an alignment of putative gb  orf, a typical marker of phylogeny; c) from an alignment of us <dig>  an mhc-i down-regulatory gene necessary for superinfection of cmv, chimpanzee cmv is included; d) from an alignment of us <dig> genes, for which  <dig> copies are encoded in each macaque cmv .
cycmv genes cyus28a-cyus28e and rhcmv genes rh214-rh <dig> are relabeled as us28a – us28e, inclusive of the rhcmv 68– <dig> pseudogene homologous to other macaque cmv us28c, based on order in genome.
numbers at loci indicate posterior probability with color scaled according to probability; genetic distances measured in substitutions per site  are given by scale below
all four-macaque cmvs share similar genome architecture.
macaque cmv and the cmv of closely related primates, like green monkey cmvs  colburn and  <dig>  have terminal repeats, as well as unique long and unique short regions in genome structure, but lack the internal repeat regions of other primate cmvs  <cit> .
cycmv genomes show diversity in gene conservation
viral genes that are important for host specificity may tend to be more diverged among strains in different hosts.
to explore these possibilities, we compared divergence and gene content of the four completely sequenced macaque cmv strains in order to better understand how these factors varied among strains.
annotation of the sequenced cycmv mauritius genome identified  <dig> putative open reading frames .
by contrast, cycmv ottawa has  <dig> putative orfs  <cit> , rhcmv 68– <dig> has  <dig> orfs  <cit> , and rhcmv  <dig>  has  <dig> orfs  <cit> .
to better understand the conservation of individual orfs in these cmv genomes we used bit-score, a log-scaled measure indicating the size of a random search string required to find an equivalently or more similar sequence than the observed match.
bit-scores were generated by comparing orfs in each of the six pairwise comparisons between the four macaque cmv genomes.
bit-scores comparing homologous orfs of cycmv mauritius and cycmv ottawa, rhcmv 68– <dig> or rhcmv  <dig>  were examined grouped by gene family .
the average bit-score for genes in each family, but not the bit-score of each individual gene, is higher for the comparison of genes between the two cycmvs than between cycmv mauritius and rhcmv  <dig>  or rhcmv 68– <dig>  the only exception to this is the cox- <dig> family, which is absent from cycmv ottawa.fig.
<dig> conservation of orfs of known gene families.
bit-scores are plotted for orfs of cycmv mauritius to homologous orfs of rhcmv 68– <dig> and rhcmv  <dig>   and to homologous orfs of cycmv ottawa  grouped by gene gamily.
each point represents a single bit-score comparison for a single orf.
horizontal line indicates the mean ± standard deviation
two-dimensional bit-score plots were used to further visualize variation in individual gene conservation in the pairwise comparisons between the four macaque cmv strains , as in  <cit> .
when plotted this way, orfs found along the x = y diagonal have equivalent sequence conservation in both pairwise comparisons.
genes that are equally well conserved between the two rhcmvs and between the two cycmvs  cluster along the diagonal.
however, this analysis of pairwise comparisons highlights a non-uniform level of sequence conservation in some genes.
the cyclooxygenase- <dig> gene , for example, is absent in cycmv ottawa.
similarly cytrl <dig>  an epithelial cell tropism factor  <cit> , is more highly conserved between the rhcmvs than between the two cycmvs.
there was a larger discordance between the bit-scores generated comparing cycmv genes and those comparing rhcmv for genes with a lower maximum bit-score.
of those genes with known functions, those involved in immune modulation or as temperance factors, and membrane proteins are more dispersed than other groups with many individual genes being more highly conserved in viruses from one species of macaque than the other.
this could suggest that these viral genes face differing evolutionary pressures in the two macaque species.fig.
<dig> two-dimensional bit-score plots between cmv strains.
graph represents comparisons of gene homologue bit-scores across cycmv mauritius versus cycmv ottawa and rhcmv 68– <dig> versus rhcmv  <dig> .
bit-score among rhcmv homologues of the indicated gene are tracked on the y-axis while cycmv homologues are tracked on the x-axis.
a high bit-score indicated a high level of similarity between genes, while a bit-score of zero indicates one or both species in the comparison lacked a copy of the gene.
genes that fall on the diagonal are equally conserved in all comparisons, for example cyul <dig>  and cyul <dig> ; genes that fall to one side of the diagonal are better conserved between viruses of one species than between viruses of the other species, for example cox- <dig>  which is highly conserved between rhcmv  <dig>  and rhcmv 68– <dig>  but absent from cycmv ottawa, and cyul <dig> .
genes are coloured according to known gene family.
select genes are annotated according to their cycmv mauritius names.
where genes belong to multiple families only one is indicated, for breakdown of chart by gene family see additional file 8: figure s <dig> or additional file 9: figure s <dig>  additional file 10: figure s <dig> and additional file 11: figure s <dig> for alternative pairwise comparisons
when grouped by gene function, capsid, dna binding/nuclear, and tegument groupings contain highly conserved genes and less deviation in bit-score from the diagonal is observed .
in each of these functional categories, genes deviate towards a higher bit-score in cycmv mauritius versus cycmv ottawa and cycmv mauritius versus rhcmv 68– <dig>  and towards a lower bit-score in cycmv mauritius versus rhcmv  <dig> .
a similar trend for higher bit-scores in cycmv mauritius versus cycmv ottawa and cycmv mauritius versus rhcmv 68– <dig>  and a lower bit-score in cycmv mauritius versus rhcmv  <dig>  was observed with orfs involved in viral temperance, though the average bit-score was lower, and was observed to a lesser absolute extent with immune regulatory, entry, and membrane associating orf groupings.fig.
<dig> two-dimensional bit-score plots between cmv strains.
graphs represent comparisons of gene homologue bit-scores across three strains simultaneously.
comparison of cycmv mauritius versus cycmv ottawa and cycmv mauritius versus rhcmv 68– <dig> , cycmv mauritius versus rhcmv  <dig>  and cycmv mauritius versus rhcmv 68– <dig> , and cycmv mauritius versus rhcmv  <dig>  and cycmv mauritius versus cycmv ottawa .
genes are coloured according to known function.
immune orfs are involved in host immune regulation or evasion, entry orfs are known cell entry factors, temperance orfs are involved in temperance of viral growth, membrane orfs are presented in cell membrane on infected cells or in virions, dna/nuclear are known to interact with dna or shuttle to the cell nucleus, capsid orfs form the virus capsid, tegument proteins are found in the viral tegument, the function of orfs with other known function vary, and the function is unknown at the time of writing for orfs plotted as unknown.
individual orf may have several functions; see additional file 3: figure s <dig>  additional file 4: figure s <dig>  additional file 5: figure s <dig> and additional file 6: figure s <dig> for visualization without overlap.
select genes are annotated according to their cycmv mauritius names
though closely related to cycmv ottawa, cycmv mauritius is less genetically diverged from both rhcmvs than cycmv ottawa is at several individual genes, including homologues of cmv genes rl11b, ul <dig>  ul83b, ul <dig> and a homologue of mammalian cox- <dig> .
this suggests a non-uniform rate of evolution of some genes  and could reflect differences in selective pressure on the cmv strains in different hosts.
the high average bit-score of capsid, dna binding/nuclear, and tegument orfs indicate a slow rate of evolution of these genes.
immune regulatory, viral entry, and membrane associated orfs, in contrast, exhibit signs of more rapid evolution.
this may be because the proteins these genes encode interact directly with the host immune system and may consequentially face greater directional or diversifying selection.
macaque cmv genomes lack an internal repeat region between the unique long  and unique short  regions.
we explored the effect of this feature of genome architecture on the genomic location of genes using gene bit-scores.
our analyses indicate that there is more divergence between genes from different macaque cmv strains that are closer to the terminal regions of both the ul and us genome regions than between genes in different strains that are far from these genomic features .
this decrease in gene conservation near the ul/us border was observed even in the absence of an internal terminal repeat region between the ul and us regions.
additionally, these terminal areas contain a higher proportion of genes that are strain specific.
the center of the ul region contains a region with a high density of highly conserved genes.
this is expected since in general, the rate of evolution of genes in a virus is slower in key genes, faster in less important genes, and fastest in non-coding and non-regulatory regions  <cit> , and because the ul region includes a cluster of core genes that are conserved across all herpesviruses.
during cmv replication, the genome circularizes and large genome segments may be reordered, reversed or lost  <cit> .
this mechanism could explain why the ul128-ul <dig> region is absent in rhcmv 68– <dig>  but present in rhcmv  <dig>   <cit> , and why rh12-rh <dig> is absent from cycmv mauritius and cycmv ottawa but present in both rhcmvs.
loss of the ul128-ul <dig> region is typical of attenuation  in fibroblast cell lines, and demonstrates an inability to infect endothelial and epithelial cell lines.
thus our findings do not necessarily prove an absence of this region in the original wild-type rhcmv 68– <dig>  <cit> .
how reversal and reordering of cmv genome segments  <cit>  affect the evolution of cmv in wild macaque populations is yet to be fully understood.
gene content of cycmv mauritius is distinguished from other strains
several cmv genes found in cycmv ottawa, rhcmv 68– <dig> and rhcmv  <dig>  are absent in cycmv mauritius.
in particular, nine cycmv ottawa orfs lack homologues in cycmv mauritius whereas  <dig> cycmv mauritius orfs lack homologues in cycmv ottawa, and of the latter, nine have homologues that are present in both rhcmv strains .
in all,  <dig> genes of rhcmv 68– <dig> and rhcmv  <dig>  are not identified in cycmv mauritius .
similarly, comparison to human cmv data, identified  <dig> orfs present in strains of hcmv but absent in cycmv mauritius .
the majority of genes absent in all macaque cmv strains but present in hcmv strains have unknown functions though some, such as ul <dig> and ul <dig>  are known to have effects on cmv growth kinetics .table  <dig> rhcmv genes absent from cycmv mauritius
footnotes:
abased on study of gene mutation in hcmv towne  <cit>
bsimilarity annotation based on comparisons of rhcmv  <dig>  and hcmv in  <cit>
cfunction annotated based on studies of hcmv  <cit>
footnotes:
afunction annotated based on studies of hcmv  <cit>  unless otherwise indicated
bbased on study of gene mutation in hcmv towne  <cit>
cbased on study of us <dig> in hcmv ad <dig>  <cit>
anomalous gene trees reveal patterns of gene family evolution
analysis of individual genes reveals several interesting features of these viral genomes.
the bayesian phylogeny for the important viral surface glycoprotein b gene , for example, indicates an atypically high level of divergence among these strains as compared to the whole genomes .
this is of interest since it is a target of immune responses.
in the analysis of the ul <dig> gene, cycmv mauritius and cycmv ottawa do not cluster together, and cycmv mauritius instead is inferred to be more closely related to the rhcmvs than to cycmv ottawa.
cycmv mauritius ul <dig> is diverged from cycmv ottawa by  <dig>  sps but diverged from rhcmv 68– <dig> and rhcmv  <dig>  by only  <dig>  and  <dig>  sps respectively.
figure 2c shows a phylogenic estimate for the us <dig> gene, which encodes an mhc down regulatory protein essential for superinfection  <cit> .
included in the analysis are multiple gene homologs of us <dig> generated by gene duplication in various cmvs.
inferred evolutionary relationships within us <dig> paralogs again indicate a closer evolutionary relationship between cycmv mauritius and the rhcmvs than to cycmv ottawa, although with weak statistical support .
interestingly, rh <dig> is not present in rhcmv  <dig> .
figure 2d shows results of a phylogenetic analysis of the multiple copies of us <dig>  a macaque cmv capsid protein that induces cox- <dig> in target cells upon entry.
cycmv mauritius genes were named following the synteny-based system established for cycmv ottawa  <cit> .
the five copies of cycmv ottawa, cycmv mauritius and rhcmv  <dig> , as well as the four copies and single pseudogene of rhcmv 68– <dig>  cluster first by synteny and then by species.
this likely indicates that divergence of all five copies occurred before speciation and that loss of one copy in rhcmv 68– <dig> occurred after speciation.
unlike the other us <dig> genes, however, phylogenetic sequence analysis of us28e  in rhcmv <dig> , rh <dig> in rhcmv 68– <dig>  cyus28e  suggests that cycmv mauritius and cycmv ottawa are each more closely related to rhcmv us28e than to cycmv us28e.
cycmv mauritius is diverged from cycmv ottawa by  <dig>  sps and diverged from rhcmv  <dig>  and rhcmv 68– <dig> by  <dig>  sps and  <dig>  sps, respectively.
cycmv ottawa is diverged from rhcmv  <dig>  and rhcmv 68– <dig> by  <dig>  sps and  <dig>  sps, respectively.
phylogenetic relationships were further estimated for several cmv genes known to have homology to mammalian host genes .
strain specific absence of several human and rhesus homologues supports independent evolution of each strain.
this is evidenced, for example, by the absence of the cox- <dig> gene in cycmv ottawa.
similarly, a cox- <dig> homologue , which promotes the formation of arachidonic acid in infected cells  <cit> , is present in cycmv mauritius, rhcmv  <dig>  and rhcmv 68– <dig>  but absent in cycmv ottawa  <cit> .
viral cox- <dig>  is necessary for the infection of endothelial cells  <cit>  and it has been shown that viral cox- <dig>  but not cellular cox- <dig>  protein is expressed when rhcmv 68– <dig> infects cells  <cit> .
since cmv has been implicated in vascular inflammation , the study of rh <dig>  which confers cellular tropism and its selection in macaques, is of particular interest.
us <dig> is a viral capsid protein capable of inducing host cox- <dig> expression in target cells  <cit> .
cycmv mauritius, cycmv ottawa and rhcmv  <dig>  have five copies each while rhcmv 68– <dig> has only four copies of us <dig> genes and a us <dig> pseudogene.
the pseudogene in rhcmv 68– <dig> shows that it, similar to other macaque cmvs, had five copies of us <dig>  and that the fifth copy was likely pseudogenized after speciation.
the number of copies of us <dig> is variable across mammalian cmvs.
baboon cmv, similar to macaque cmv, has four distinct copies, hcmv has a single copy and both rcmv and mcmv lack us <dig> homologues  <cit> .
these multiple copies are more divergent from each other within a genome than they are from corresponding homologues between macaque species  and can be assumed to have emerged prior to the speciation of these macaques.fig.
<dig> phylogenetic tree comparison of cmv genes with mammalian homologues.
trees were prepared comparing cycmv mauritius, cycmv ottawa, rhcmv 68– <dig>  rhcmv  <dig> , ccmv heberling and hcmv ad <dig> with mammalian homologue genes using the relevant homo sapiens homologue as the outgroup.
trees were generated by mr. bayes following mafft alignment using a model of evolution selected by jmodel test comparing a) cycox-2/rh <dig> putative protein product to its mammalian homologue prostaglandin-endoperoxide synthase  <dig> ; b) ul <dig> and ul146-like cmv genes with mammalian homologue cxc chemokine ligand  <dig> ; c) ul <dig> with mammalian homologue tumor necrosis factor receptor superfamily  <dig> .
numbers at loci indicate posterior probability with color scaled according to probability; genetic distances measured in substitutions per site  are given by scale below
cox- <dig> homologues are absent from cycmv ottawa and hcmv but present in both rhcmv 68– <dig> and rhcmv  <dig>   <cit>  interestingly, however, the cycmv mauritius genome has three orfs with homology to cox-2: cycox-2a, cycox-2b, and cycox-2c.
it is perhaps not coincidental that rhcmv 68– <dig>  which has a cox- <dig> homologue the least diverged from mammalian cox- <dig>  is also the strain with fewest functioning us <dig> genes.
meanwhile, a high degree of divergence between cox- <dig> homologues in the three strains that retain it suggests a more recent integration or only a low fitness advantage.
the absence of the cox- <dig> homologue in cycmv ottawa and the fact that rhcmv 68– <dig> and cycmv mauritius are genetically less distant than rhcmv 68– <dig> and cycmv ottawa may again suggest a faster rate of divergence of cycmv ottawa.
comparison of cox- <dig> homologues  showed a clustering of cmv homologues that are substantially diverged from their macaque homologs, prostaglandin endoperoxide synthase  <dig>  in macaca fascicularis and macaca mulatta .
there is no hcmv equivalent of ptgs <dig>  it can be inferred that the cox- <dig> gene was copied from a mammalian host once to an ancestral cmv prior to cycmv and rhcmv speciation.
a mammalian origin of the vcox- <dig> genes is further evidenced by the presence of introns in these genes.
perhaps the unique mhc haplotypes of mauritian macaques favor the retention of an immune-modulatory gene, such as cox- <dig> homologue in cycmv mauritius.
similarly, mammalian cxc chemokine ligand  <dig>  genes substantially diverged from cmv encoded ul146-like genes .
interestingly, viral chemokine-encoding ul146-like genes such as ccmv ul <dig> clustered with rhcmv rh <dig>  whereas hcmv ul <dig> clustered with cycmv cy <dig> with weak support.
two interesting observations were noted.
first, both ccmv ul <dig> and hcmv ul <dig> are more diverged from mammalian cxcl <dig> genes  than they are from multiple macaque cmv genes .
second, the cycmv mauritius gene cy <dig> is less diverged from all the mammalian cxcl <dig> genes than it is from any cmv genes .
this probably reflects variation in the rate of evolution among these genes and among cmv strains, with the cycmv mauritius being slower than the others .
the clustering of ccmv and hcmv with other cmvs may indicate the acquisition of a viral cxcl <dig> homologue in a common ancestral cmv strain followed by gene duplication and mutation to create the ul146-like genes.
this phylogeny supports at least two independent gene duplication events before diversification of old world primates.
possibly after this, other gene duplication events generated additional gene copies in macaques.
alternatively, more than two gene duplication events occurred in the ancestor of old world primates followed by more extensive gene loss in apes than macaques.
it is interesting that no ul146-like genes were identified in rhcmv <dig> .
figure 6c shows a well supported close relationship between cmv ul <dig> genes of cycmv mauritius, cycmv ottawa, rhcmv 68– <dig> and ccmv heberling to the exclusion of the homologous tumor necrosis factor receptor superfamily  <dig>  genes in their macaque and human hosts, which is again consistent with a single copying event in the ancestor of these cmv strains.
cmvs and their hosts share similar evolutionary patterns
in order to test for co-evolution between host species and the cmvs that they harbor over a broader phylogeny, we performed a phylogenetic analysis of  <dig> available primate cmv viral genomes .
the resulting phylogenetic tree of these cmvs was then compared to a previously estimated phylogenetic tree of mammalian genomes  <cit> .
phylogenetic relationships among multiple primate cmvs exhibit an identical pattern of cmv and host diversification ;  <cit> .
there is evidence of attenuation in some sequenced cmvs caused by growth of the isolated strains in tissue culture prior to sequencing.
this can result in the mutation or loss of viral genes necessary for entry or growth in certain cell types, as is the case with the deletion of the ul128-ul <dig> region in rhcmv 68– <dig>  <cit> , and other laboratory adapted cmv strains  <cit> .
despite this, there is no indication of cross species contact or attenuation of some strains during cmv diversification prior to laboratory isolation.
dna polymerase sequence comparisons have suggested a speciation for cycmv ottawa and rhcmv 68– <dig> of around  <dig>  mya  <cit> .
this places speciation and divergence from a common cmv strain at ~ <dig>  mya, in line with y-dna segregation and the suspected end to macaque migration between islands of the sunda shelf  <cit> .fig.
<dig> bayesian phylogenetic tree of selected cmv strains.
generalized time reversible  tree with proportion invariant sites and gamma distribution of multiple sequenced primate cmv strains.
whole genome sequences were aligned using mafft with scoring matrix of 200pam/k =  <dig> a gap open penalty of  <dig>  and offset value of  <dig> .
new world primates, owl monkey  s34e and squirrel monkey  sqshv were used as the out-group to root the tree.
rhcmv 68– <dig>  rhcmv  <dig> , cycmv ottawa, cycmv mauritius, chimpanzee cmv  heberling, human cmv  ad <dig>  human cmv  han <dig>  african green monkey cmv  colburn and  <dig>  and baboon cmv  ocom4- <dig> were compared.
numbers at loci indicate posterior probability with color scaled according to probability; genetic distances measured in substitutions per site  are given by scale below
it is possible for multiple cmv strains to co-exist within a single macaque, or within a single macaque population, because cmv can superinfect seropositive individuals  <cit> .
however, co-infection of a host may only be possible for closely related cmv strains – cynomolgus macaques, for example, are not susceptible to infection or co-infection by a rhcmv  <cit> .
the correspondence between viral and host evolutionary relationships suggests further study of cmv would serve as a molecular tool to understand primate evolution.
CONCLUSIONS
in this study, we generated a novel genome sequence from a cmv strain isolated from a cynomolgus macaque from mauritius.
when analyzed with other cmv genomes from macaques and from other host species, inferred phylogenetic relationships among the viruses generally matched those among their hosts.
comparisons of this new genome with other macaque cmvs identify several functional categories of genes with atypically high levels of divergence, variation in gene content, and several genes with inferred phylogenetic relationships that differed from the genome-wide estimate.
these results have implications for use of cmv as a vaccine vector and molecular tool, in cmv pathogenesis studies, as well as providing a tool to assist in tracing both viral and macaque migration and distribution.

 macaque monkeys are an important animal model in biomedical research, particularly in infectious diseases. they are used in vaccine and infectivity studies of viruses that either do not infect or do not produce realistic pathogenic features in small animal models – viruses such as human immunodeficiency virus where simian immunodeficiency virus or the human envelope substituted version can be studied as a surrogate in macaques. recently, the limited availability of indian rhesus macaques in north america and elsewhere has led to the development of resources and standards for the use of cynomolgus macaques and chinese rhesus macaques, the former in particular being an excellent model of hiv-1/siv infection. though relatively closely related, divergence between populations or species of macaques and their corresponding cytomegaloviruses strains is substantial enough as to preclude direct replacement in any study. in fact, with increased usage in research, it has been questioned whether cynomolgus macaques may be in need of taxonomic re-classification. captive populations of cynomolgus macaques are frequently interbred with little concern as to their origins. however, the genetic divergence between geographically distinct populations of cynomolgus macaques rivals that found between indian and chinese rhesus macaques, a factor that has the potential to differently influence experimental results. for example, disease susceptibility is highly variable between isolated-tightly knit groups of macaques found across the sunda shelf of southeast asia. macaque genome sequencing has enabled a better understanding of their disease susceptibility from an evolutionary and conservation standpoint, and provided insight into population structure and patterns of migration. interest in nonhuman primate herpesviruses, and cmvs in particular, has increased in recent years in recognition of the unique immune response that they evoke. specifically, cmv evokes a type of effector memory t cell response, which intermittently is boosted by reactivated virus throughout the life of its immunocompetent host. this type of immune memory qualitatively is different from conventional t cell memory in that it does not require priming and can respond immediately to antigen. thus cmvs are also of interest to vaccinologists who are examining ways to expand the duration of rapidly inducible t cell responses to block primary viral infection. the evaluation of cmvs in rhesus and more recently, in cynomolgus macaques, has become a priority in order to facilitate the use of these animal models for vaccine research and development. cmv infects many primate and non-primate hosts including humans, baboons, green monkeys, chimpanzees, squirrel monkeys, macaques, oysters and rodents. since previous studies have demonstrated only limited mixing and horizontal transmission between mammalian populations, it is thought that cmv, like other herpesviruses, diversified via co-evolution with their host species. during speciation, pathogens may co-speciation with their host. herpesviruses, for instance, co-evolve with their mammalian hosts. herpesviruses were present in primates mya, and have been used as surrogates to track mammalian – including human – evolution and migration. herpesviruses, including cmvs, were also present in the most recent common ancestor of rhesus and cynomolgus macaques, and are presumed to have undergone geographic differentiation similar to their host species. however, macaques can become infected with multiple strains of cmv and these cmv strains may experience recombination. hosts co-infected with two cmv strains occasionally release infectious viral particles from multiple strains, but generally have a dominant strain that makes up the majority of shed virus. interaction among populations of cynomolgus macaques can facilitate the exchange of cmv strains but cross infection between species is generally rare. in the present study, we sequenced a new strain of cycmv from a cynomolgus macaque from mauritius and compared it to three other strains – one from a cynomolgus macaque that originated in the philippines, and two from indian rhesus macaques, rhcmv 68– and rhcmv. we also characterized variation in gene content among these macaque cmvs. our results indicate that evolutionary relationships over the complete genomes of cynomolgus cmv strains matches those of their hosts, thereby supporting their co-evolution and also the further use of cmvs in the study of mammalian biogeography and phylogeny. we also found phylogenetic correspondence between cmvs and their hosts in a broader phylogenetic analysis that included several other cmv strains. however, phylogenetic relationships among some genomic regions of the macaque cmvs deviated from the expected relationship, and this can be explained either by intra-strain recombination or alternatively could reflect phylogenetic error.  cycmv mauritius is a unique cmv from mauritian cynomolgus macaques in the present study, we characterized a novel cmv strain, isolated from a mauritian cynomolgus macaque, and compared its sequence to that of three other cmv strains, including that of cycmv ottawa, and two strains from rhesus macaques: rhcmv 68– and rhcmv. our analyses indicate that the sequence of cycmv mauritius is highly similar to the sequence of cycmv ottawa, and also that both are derived from a more recent common ancestor than either is with one of the rhesus macaque strains. our assembly of illumina reads from cycmv mauritius produced three contigs that were then connected by sanger sequencing. the assembly had an average 280x fold coverage that was 200 bp in length, and had  % gc content. in comparison, cycmv ottawa is 841 bp longer and has a similar gc content. rhcmv 68– and rhcmv are both similar in length and gc content, with rhcmv being the shortest and rhcmv 68– the longest of the four viruses. compared to other mammalian cmvs, all macaque cmvs examined are shorter than chimpanzee cmv heberling strain and shorter on average than human cmv strains ad and han of the cmvs that have been sequenced thus far, these four macaque cmvs are most similar in length to aotine cmv and african green monkey cmv.table select sequenced primate cmv strains cycmv mauritius and cycmv ottawa share  % identity by linear full genome alignment while rhcmv 68– and rhcmv share  % identity. cycmv mauritius and rhcmv have  % identity and cycmv mauritius and rhcmv 68– have  % identity. cycmv ottawa has  % identity with rhcmv 68– and  % identity with rhcmv. due to the possibility of rearrangements in these viral genomes we evaluated sequence synteny using the progressive mauve multiple genome alignment algorithm. this analysis reveals a local co-linear region around 160– kbp where the rhcmv 68– sequence is reversed in comparison to cycmv mauritius, cycmv ottawa and rhcmv. this region is immediately adjacent to the ul to ul deletion found in rhcmv 68– there are two locally collinear blocks whose order are reversed between rhesus and cynomolgus macaque cmv genomes. both collinear blocks are short and located immediately upstream of the reversed region in rhcmv 68– fig. multiple genome alignment of cycmv mauritius, cycmv ottawa, rhcmv 68– and rhcmv. genome homology and rearrangement are presented using the mauve multiple genome alignment algorithm. locally collinear blocks are indicated differentiated by colour and connected by linking line. within collinear blocks column height indicates average conservation of base pairs locally between homologous collinear blocks in other strains, full columns indicate regions fully conserved between species while empty columns indicate unique regions to the particular genome or absences. collinear blocks shown above the x-axis are in the same orientation, with those shown below reversed in comparison to the same co-linear blocks in other species phylogenetic relationships among cmv strains match those of their hosts evolutionary relationships were estimated for cycmv mauritius, cycmv ottawa, rhcmv and rhcmv 68– using hcmv ad as an outgroup. evolutionary relationships among these complete macaque cmv genome sequences match those of their hosts. within the cynomolgus macaque, the viral strains cycmv mauritius and cycmv ottawa are diverged by substitutions per site, whereas within the rhesus macaque, the viral strains rhcmv and rhcmv 68– are diverged by only sps. thus, divergence between cmv strains of cynomologous macaques is greater than between the cmv strains of rhesus macaques, and this may also be true of the respective host populations.fig. phylogenetic tree comparison of macaque cmv genome and select genes. trees were generated comparing cycmv mauritius, cycmv ottawa, rhcmv 68– and rhcmv using hcmv ad as an out-group. trees were generated using mr. bayes, following mafft alignment, using a model of evolution selected by jmodel test a) from a full genome global alignment; b) from an alignment of putative gb orf, a typical marker of phylogeny; c) from an alignment of us an mhc-i down-regulatory gene necessary for superinfection of cmv, chimpanzee cmv is included; d) from an alignment of us genes, for which copies are encoded in each macaque cmv. cycmv genes cyus28a-cyus28e and rhcmv genes rh214-rh are relabeled as us28a – us28e, inclusive of the rhcmv 68– pseudogene homologous to other macaque cmv us28c, based on order in genome. numbers at loci indicate posterior probability with color scaled according to probability; genetic distances measured in substitutions per site are given by scale below all four-macaque cmvs share similar genome architecture. macaque cmv and the cmv of closely related primates, like green monkey cmvs colburn and have terminal repeats, as well as unique long and unique short regions in genome structure, but lack the internal repeat regions of other primate cmvs. cycmv genomes show diversity in gene conservation viral genes that are important for host specificity may tend to be more diverged among strains in different hosts. to explore these possibilities, we compared divergence and gene content of the four completely sequenced macaque cmv strains in order to better understand how these factors varied among strains. annotation of the sequenced cycmv mauritius genome identified putative open reading frames. by contrast, cycmv ottawa has putative orfs, rhcmv 68– has orfs, and rhcmv has orfs. to better understand the conservation of individual orfs in these cmv genomes we used bit-score, a log-scaled measure indicating the size of a random search string required to find an equivalently or more similar sequence than the observed match. bit-scores were generated by comparing orfs in each of the six pairwise comparisons between the four macaque cmv genomes. bit-scores comparing homologous orfs of cycmv mauritius and cycmv ottawa, rhcmv 68– or rhcmv were examined grouped by gene family. the average bit-score for genes in each family, but not the bit-score of each individual gene, is higher for the comparison of genes between the two cycmvs than between cycmv mauritius and rhcmv or rhcmv 68– the only exception to this is the cox- family, which is absent from cycmv ottawa.fig. conservation of orfs of known gene families. bit-scores are plotted for orfs of cycmv mauritius to homologous orfs of rhcmv 68– and rhcmv and to homologous orfs of cycmv ottawa grouped by gene gamily. each point represents a single bit-score comparison for a single orf. horizontal line indicates the mean ± standard deviation two-dimensional bit-score plots were used to further visualize variation in individual gene conservation in the pairwise comparisons between the four macaque cmv strains, as in. when plotted this way, orfs found along the x = y diagonal have equivalent sequence conservation in both pairwise comparisons. genes that are equally well conserved between the two rhcmvs and between the two cycmvs cluster along the diagonal. however, this analysis of pairwise comparisons highlights a non-uniform level of sequence conservation in some genes. the cyclooxygenase- gene, for example, is absent in cycmv ottawa. similarly cytrl an epithelial cell tropism factor, is more highly conserved between the rhcmvs than between the two cycmvs. there was a larger discordance between the bit-scores generated comparing cycmv genes and those comparing rhcmv for genes with a lower maximum bit-score. of those genes with known functions, those involved in immune modulation or as temperance factors, and membrane proteins are more dispersed than other groups with many individual genes being more highly conserved in viruses from one species of macaque than the other. this could suggest that these viral genes face differing evolutionary pressures in the two macaque species.fig. two-dimensional bit-score plots between cmv strains. graph represents comparisons of gene homologue bit-scores across cycmv mauritius versus cycmv ottawa and rhcmv 68– versus rhcmv. bit-score among rhcmv homologues of the indicated gene are tracked on the y-axis while cycmv homologues are tracked on the x-axis. a high bit-score indicated a high level of similarity between genes, while a bit-score of zero indicates one or both species in the comparison lacked a copy of the gene. genes that fall on the diagonal are equally conserved in all comparisons, for example cyul and cyul; genes that fall to one side of the diagonal are better conserved between viruses of one species than between viruses of the other species, for example cox- which is highly conserved between rhcmv and rhcmv 68– but absent from cycmv ottawa, and cyul. genes are coloured according to known gene family. select genes are annotated according to their cycmv mauritius names. where genes belong to multiple families only one is indicated, for breakdown of chart by gene family see additional file 8: figure s or additional file 9: figure s additional file 10: figure s and additional file 11: figure s for alternative pairwise comparisons when grouped by gene function, capsid, dna binding/nuclear, and tegument groupings contain highly conserved genes and less deviation in bit-score from the diagonal is observed. in each of these functional categories, genes deviate towards a higher bit-score in cycmv mauritius versus cycmv ottawa and cycmv mauritius versus rhcmv 68– and towards a lower bit-score in cycmv mauritius versus rhcmv. a similar trend for higher bit-scores in cycmv mauritius versus cycmv ottawa and cycmv mauritius versus rhcmv 68– and a lower bit-score in cycmv mauritius versus rhcmv was observed with orfs involved in viral temperance, though the average bit-score was lower, and was observed to a lesser absolute extent with immune regulatory, entry, and membrane associating orf groupings.fig. two-dimensional bit-score plots between cmv strains. graphs represent comparisons of gene homologue bit-scores across three strains simultaneously. comparison of cycmv mauritius versus cycmv ottawa and cycmv mauritius versus rhcmv 68–, cycmv mauritius versus rhcmv and cycmv mauritius versus rhcmv 68–, and cycmv mauritius versus rhcmv and cycmv mauritius versus cycmv ottawa. genes are coloured according to known function. immune orfs are involved in host immune regulation or evasion, entry orfs are known cell entry factors, temperance orfs are involved in temperance of viral growth, membrane orfs are presented in cell membrane on infected cells or in virions, dna/nuclear are known to interact with dna or shuttle to the cell nucleus, capsid orfs form the virus capsid, tegument proteins are found in the viral tegument, the function of orfs with other known function vary, and the function is unknown at the time of writing for orfs plotted as unknown. individual orf may have several functions; see additional file 3: figure s  additional file 4: figure s  additional file 5: figure s and additional file 6: figure s for visualization without overlap. select genes are annotated according to their cycmv mauritius names though closely related to cycmv ottawa, cycmv mauritius is less genetically diverged from both rhcmvs than cycmv ottawa is at several individual genes, including homologues of cmv genes rl11b, ul ul83b, ul and a homologue of mammalian cox-. this suggests a non-uniform rate of evolution of some genes and could reflect differences in selective pressure on the cmv strains in different hosts. the high average bit-score of capsid, dna binding/nuclear, and tegument orfs indicate a slow rate of evolution of these genes. immune regulatory, viral entry, and membrane associated orfs, in contrast, exhibit signs of more rapid evolution. this may be because the proteins these genes encode interact directly with the host immune system and may consequentially face greater directional or diversifying selection. macaque cmv genomes lack an internal repeat region between the unique long and unique short regions. we explored the effect of this feature of genome architecture on the genomic location of genes using gene bit-scores. our analyses indicate that there is more divergence between genes from different macaque cmv strains that are closer to the terminal regions of both the ul and us genome regions than between genes in different strains that are far from these genomic features. this decrease in gene conservation near the ul/us border was observed even in the absence of an internal terminal repeat region between the ul and us regions. additionally, these terminal areas contain a higher proportion of genes that are strain specific. the center of the ul region contains a region with a high density of highly conserved genes. this is expected since in general, the rate of evolution of genes in a virus is slower in key genes, faster in less important genes, and fastest in non-coding and non-regulatory regions, and because the ul region includes a cluster of core genes that are conserved across all herpesviruses. during cmv replication, the genome circularizes and large genome segments may be reordered, reversed or lost. this mechanism could explain why the ul128-ul region is absent in rhcmv 68– but present in rhcmv, and why rh12-rh is absent from cycmv mauritius and cycmv ottawa but present in both rhcmvs. loss of the ul128-ul region is typical of attenuation in fibroblast cell lines, and demonstrates an inability to infect endothelial and epithelial cell lines. thus our findings do not necessarily prove an absence of this region in the original wild-type rhcmv 68–. how reversal and reordering of cmv genome segments affect the evolution of cmv in wild macaque populations is yet to be fully understood. gene content of cycmv mauritius is distinguished from other strains several cmv genes found in cycmv ottawa, rhcmv 68– and rhcmv are absent in cycmv mauritius. in particular, nine cycmv ottawa orfs lack homologues in cycmv mauritius whereas cycmv mauritius orfs lack homologues in cycmv ottawa, and of the latter, nine have homologues that are present in both rhcmv strains. in all, genes of rhcmv 68– and rhcmv are not identified in cycmv mauritius. similarly, comparison to human cmv data, identified orfs present in strains of hcmv but absent in cycmv mauritius. the majority of genes absent in all macaque cmv strains but present in hcmv strains have unknown functions though some, such as ul and ul are known to have effects on cmv growth kinetics.table rhcmv genes absent from cycmv mauritius footnotes: abased on study of gene mutation in hcmv towne bsimilarity annotation based on comparisons of rhcmv and hcmv in cfunction annotated based on studies of hcmv footnotes: afunction annotated based on studies of hcmv unless otherwise indicated bbased on study of gene mutation in hcmv towne cbased on study of us in hcmv ad anomalous gene trees reveal patterns of gene family evolution analysis of individual genes reveals several interesting features of these viral genomes. the bayesian phylogeny for the important viral surface glycoprotein b gene, for example, indicates an atypically high level of divergence among these strains as compared to the whole genomes. this is of interest since it is a target of immune responses. in the analysis of the ul gene, cycmv mauritius and cycmv ottawa do not cluster together, and cycmv mauritius instead is inferred to be more closely related to the rhcmvs than to cycmv ottawa. cycmv mauritius ul is diverged from cycmv ottawa by sps but diverged from rhcmv 68– and rhcmv by only and sps respectively. figure 2c shows a phylogenic estimate for the us gene, which encodes an mhc down regulatory protein essential for superinfection. included in the analysis are multiple gene homologs of us generated by gene duplication in various cmvs. inferred evolutionary relationships within us paralogs again indicate a closer evolutionary relationship between cycmv mauritius and the rhcmvs than to cycmv ottawa, although with weak statistical support. interestingly, rh is not present in rhcmv. figure 2d shows results of a phylogenetic analysis of the multiple copies of us a macaque cmv capsid protein that induces cox- in target cells upon entry. cycmv mauritius genes were named following the synteny-based system established for cycmv ottawa. the five copies of cycmv ottawa, cycmv mauritius and rhcmv, as well as the four copies and single pseudogene of rhcmv 68– cluster first by synteny and then by species. this likely indicates that divergence of all five copies occurred before speciation and that loss of one copy in rhcmv 68– occurred after speciation. unlike the other us genes, however, phylogenetic sequence analysis of us28e in rhcmv, rh in rhcmv 68– cyus28e suggests that cycmv mauritius and cycmv ottawa are each more closely related to rhcmv us28e than to cycmv us28e. cycmv mauritius is diverged from cycmv ottawa by sps and diverged from rhcmv and rhcmv 68– by sps and sps, respectively. cycmv ottawa is diverged from rhcmv and rhcmv 68– by sps and sps, respectively. phylogenetic relationships were further estimated for several cmv genes known to have homology to mammalian host genes. strain specific absence of several human and rhesus homologues supports independent evolution of each strain. this is evidenced, for example, by the absence of the cox- gene in cycmv ottawa. similarly, a cox- homologue, which promotes the formation of arachidonic acid in infected cells, is present in cycmv mauritius, rhcmv and rhcmv 68– but absent in cycmv ottawa. viral cox- is necessary for the infection of endothelial cells and it has been shown that viral cox- but not cellular cox- protein is expressed when rhcmv 68– infects cells. since cmv has been implicated in vascular inflammation, the study of rh which confers cellular tropism and its selection in macaques, is of particular interest. us is a viral capsid protein capable of inducing host cox- expression in target cells. cycmv mauritius, cycmv ottawa and rhcmv have five copies each while rhcmv 68– has only four copies of us genes and a us pseudogene. the pseudogene in rhcmv 68– shows that it, similar to other macaque cmvs, had five copies of us and that the fifth copy was likely pseudogenized after speciation. the number of copies of us is variable across mammalian cmvs. baboon cmv, similar to macaque cmv, has four distinct copies, hcmv has a single copy and both rcmv and mcmv lack us homologues. these multiple copies are more divergent from each other within a genome than they are from corresponding homologues between macaque species and can be assumed to have emerged prior to the speciation of these macaques.fig. phylogenetic tree comparison of cmv genes with mammalian homologues. trees were prepared comparing cycmv mauritius, cycmv ottawa, rhcmv 68– rhcmv, ccmv heberling and hcmv ad with mammalian homologue genes using the relevant homo sapiens homologue as the outgroup. trees were generated by mr. bayes following mafft alignment using a model of evolution selected by jmodel test comparing a) cycox-2/rh putative protein product to its mammalian homologue prostaglandin-endoperoxide synthase; b) ul and ul146-like cmv genes with mammalian homologue cxc chemokine ligand; c) ul with mammalian homologue tumor necrosis factor receptor superfamily. numbers at loci indicate posterior probability with color scaled according to probability; genetic distances measured in substitutions per site are given by scale below cox- homologues are absent from cycmv ottawa and hcmv but present in both rhcmv 68– and rhcmv interestingly, however, the cycmv mauritius genome has three orfs with homology to cox-2: cycox-2a, cycox-2b, and cycox-2c. it is perhaps not coincidental that rhcmv 68– which has a cox- homologue the least diverged from mammalian cox- is also the strain with fewest functioning us genes. meanwhile, a high degree of divergence between cox- homologues in the three strains that retain it suggests a more recent integration or only a low fitness advantage. the absence of the cox- homologue in cycmv ottawa and the fact that rhcmv 68– and cycmv mauritius are genetically less distant than rhcmv 68– and cycmv ottawa may again suggest a faster rate of divergence of cycmv ottawa. comparison of cox- homologues showed a clustering of cmv homologues that are substantially diverged from their macaque homologs, prostaglandin endoperoxide synthase in macaca fascicularis and macaca mulatta. there is no hcmv equivalent of ptgs it can be inferred that the cox- gene was copied from a mammalian host once to an ancestral cmv prior to cycmv and rhcmv speciation. a mammalian origin of the vcox- genes is further evidenced by the presence of introns in these genes. perhaps the unique mhc haplotypes of mauritian macaques favor the retention of an immune-modulatory gene, such as cox- homologue in cycmv mauritius. similarly, mammalian cxc chemokine ligand genes substantially diverged from cmv encoded ul146-like genes. interestingly, viral chemokine-encoding ul146-like genes such as ccmv ul clustered with rhcmv rh whereas hcmv ul clustered with cycmv cy with weak support. two interesting observations were noted. first, both ccmv ul and hcmv ul are more diverged from mammalian cxcl genes than they are from multiple macaque cmv genes. second, the cycmv mauritius gene cy is less diverged from all the mammalian cxcl genes than it is from any cmv genes. this probably reflects variation in the rate of evolution among these genes and among cmv strains, with the cycmv mauritius being slower than the others. the clustering of ccmv and hcmv with other cmvs may indicate the acquisition of a viral cxcl homologue in a common ancestral cmv strain followed by gene duplication and mutation to create the ul146-like genes. this phylogeny supports at least two independent gene duplication events before diversification of old world primates. possibly after this, other gene duplication events generated additional gene copies in macaques. alternatively, more than two gene duplication events occurred in the ancestor of old world primates followed by more extensive gene loss in apes than macaques. it is interesting that no ul146-like genes were identified in rhcmv. figure 6c shows a well supported close relationship between cmv ul genes of cycmv mauritius, cycmv ottawa, rhcmv 68– and ccmv heberling to the exclusion of the homologous tumor necrosis factor receptor superfamily genes in their macaque and human hosts, which is again consistent with a single copying event in the ancestor of these cmv strains. cmvs and their hosts share similar evolutionary patterns in order to test for co-evolution between host species and the cmvs that they harbor over a broader phylogeny, we performed a phylogenetic analysis of available primate cmv viral genomes. the resulting phylogenetic tree of these cmvs was then compared to a previously estimated phylogenetic tree of mammalian genomes. phylogenetic relationships among multiple primate cmvs exhibit an identical pattern of cmv and host diversification;. there is evidence of attenuation in some sequenced cmvs caused by growth of the isolated strains in tissue culture prior to sequencing. this can result in the mutation or loss of viral genes necessary for entry or growth in certain cell types, as is the case with the deletion of the ul128-ul region in rhcmv 68–, and other laboratory adapted cmv strains. despite this, there is no indication of cross species contact or attenuation of some strains during cmv diversification prior to laboratory isolation. dna polymerase sequence comparisons have suggested a speciation for cycmv ottawa and rhcmv 68– of around mya. this places speciation and divergence from a common cmv strain at ~ mya, in line with y-dna segregation and the suspected end to macaque migration between islands of the sunda shelf.fig. bayesian phylogenetic tree of selected cmv strains. generalized time reversible tree with proportion invariant sites and gamma distribution of multiple sequenced primate cmv strains. whole genome sequences were aligned using mafft with scoring matrix of 200pam/k =  a gap open penalty of and offset value of. new world primates, owl monkey s34e and squirrel monkey sqshv were used as the out-group to root the tree. rhcmv 68– rhcmv, cycmv ottawa, cycmv mauritius, chimpanzee cmv heberling, human cmv ad human cmv han african green monkey cmv colburn and and baboon cmv ocom4- were compared. numbers at loci indicate posterior probability with color scaled according to probability; genetic distances measured in substitutions per site are given by scale below it is possible for multiple cmv strains to co-exist within a single macaque, or within a single macaque population, because cmv can superinfect seropositive individuals. however, co-infection of a host may only be possible for closely related cmv strains – cynomolgus macaques, for example, are not susceptible to infection or co-infection by a rhcmv. the correspondence between viral and host evolutionary relationships suggests further study of cmv would serve as a molecular tool to understand primate evolution.  in this study, we generated a novel genome sequence from a cmv strain isolated from a cynomolgus macaque from mauritius. when analyzed with other cmv genomes from macaques and from other host species, inferred phylogenetic relationships among the viruses generally matched those among their hosts. comparisons of this new genome with other macaque cmvs identify several functional categories of genes with atypically high levels of divergence, variation in gene content, and several genes with inferred phylogenetic relationships that differed from the genome-wide estimate. these results have implications for use of cmv as a vaccine vector and molecular tool, in cmv pathogenesis studies, as well as providing a tool to assist in tracing both viral and macaque migration and distribution. 
in this study we sequenced the ~ kb genome of a cytomegalovirus isolated from a mauritius cynomolgus macaque, cycmv mauritius, and compared it to previously sequenced cytomegaloviruses from a cynomolgus macaque of filipino origin and two from indian rhesus macaques. though more closely related to cycmv ottawa, cycmv mauritius is less genetically distant from both rhcmv strains than is cycmv ottawa. several individual genes, including homologues of cmv genes rl11b, ul ul83b, ul and a homologue of mammalian cox- show a closer relationship between homologues of cycmv mauritius and the rhcmvs than between homologues of cycmv mauritius and cycmv ottawa. a broader phylogenetic analysis of cmv strains from eight species recovers evolutionary relationships among viral strains that mirror those amongst the host species, further demonstrating co-evolution of host and virus. phylogenetic analyses of rhesus and cynomolgus macaque cmv genome sequences demonstrate co-speciation of the virus and host. cynomolgus macaquecytomegalovirusphylogeneticsco-speciationco-evolutionhttp://dx.doi.org/http://dx.doi.org/ /501100000024canadian institutes of health research107743http://dx.doi.org/http://dx.doi.org/ /501100000085ontario hiv treatment networkissue-copyright-statement© the author 2016
 cytomegaloviruses belong to a large, ancient, genus of dna viruses comprised of a wide array of species-specific strains that occur in diverse array of hosts. methods in this study we sequenced the ~ kb genome of a cytomegalovirus isolated from a mauritius cynomolgus macaque, cycmv mauritius, and compared it to previously sequenced cytomegaloviruses from a cynomolgus macaque of filipino origin and two from indian rhesus macaques.  though more closely related to cycmv ottawa, cycmv mauritius is less genetically distant from both rhcmv strains than is cycmv ottawa. several individual genes, including homologues of cmv genes rl11b, ul ul83b, ul and a homologue of mammalian cox- show a closer relationship between homologues of cycmv mauritius and the rhcmvs than between homologues of cycmv mauritius and cycmv ottawa. a broader phylogenetic analysis of cmv strains from eight species recovers evolutionary relationships among viral strains that mirror those amongst the host species, further demonstrating co-evolution of host and virus.  phylogenetic analyses of rhesus and cynomolgus macaque cmv genome sequences demonstrate co-speciation of the virus and host. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords cynomolgus macaquecytomegalovirusphylogeneticsco-speciationco-evolutionhttp://dx.doi.org/http://dx.doi.org/ /501100000024canadian institutes of health research107743http://dx.doi.org/http://dx.doi.org/ /501100000085ontario hiv treatment networkissue-copyright-statement© the author 2016 