BACKGROUND
since their discovery in the last years of the xix century up to our days, viruses have never ceased to puzzle biologists, especially those studying their evolution.
because of their nature as entities at the border between the living and the non-living, their origin has been the matter of passionate debate.
opinions range between the two extreme "virus-early" and "virus-late" hypotheses.
the first postulates that viruses originated before modern cells  <cit> .
on the contrary, the second hypothesis proposes that viruses originated either by the escape of genetic material and proteins from cells, or by a dramatic reductive evolution from cellular forms that lost all the "cellular paraphernalia" unnecessary for the parasitic lifestyle  <cit> .
as a consequence, the position of viruses within the tree of life is also a subject of disagreement.
whereas some scientists fervently advocate that viruses should have a place in the tree of life  <cit> , many other consider that, being acellular and lacking any kind of carbon and energy metabolism, viruses cannot be properly compared with true living beings  and, therefore, do not belong to the tree of life  <cit> .
moreover, there is compelling evidence for the polyphyletic origin of viruses, which further complicates the issue  <cit> .
these debates have recently gained an unprecedented impetus when raoult and co-workers published the description of the genome of mimivirus, a giant virus first detected as a parasite of amoeba of the genus acanthamoeba, which is endowed with the biggest viral genome known to date:  <dig>  mbp containing  <dig> genes  <cit> .
a number of genomic signatures strongly support that mimivirus belongs to the ncldv  virus family.
the mimiviral genome contains genes frequently found in viruses, as those involved in genome replication, but also, and for the first time in a virus, a number of genes coding for proteins involved in transcription and translation.
therefore, at least from the point of view of gene content, mimivirus appears somehow closer to a typical cell than any other described virus.
moreover, raoult et al.
identified seven mimiviral proteins that have closely related eukaryotic homologues.
their phylogenetic analysis in a multi-protein concatenation supported the emergence of mimivirus as a sister-group of eukaryotes in a tree including representatives from the three domains of life  <cit> .
therefore, it was hypothesised that this virus would define a new branch distinct from the three domains of life: bacteria, archaea and eukaryotes  <cit> .
such an unexpected result had the potential to revolutionise our conceptions on the diversity and evolution of life, up to now based on the tripartite scheme of the three domains  <cit> .
raoult et al.
further suggested that the ncldv family may have played a role in the origin of eukaryotes  <cit> .
this would agree with previous hypotheses stating that viruses could be at the origin of many eukaryotic genes  <cit>  or even the eukaryotic nucleus  <cit> .
the study of viral evolution using phylogenetic analysis is most often a difficult task due to several peculiarities of viruses related to their parasitic lifestyle, such as rapid evolutionary rates and the propensity to be involved in horizontal gene transfer  events  <cit> .
as recently shown  <cit> , the emergence of mimivirus at the base of the eukaryotic branch in the seven-protein study by raoult et al.
was the result of an accumulation of artefacts due to the simultaneous analysis of several markers that had been profusely exchanged between distant species by hgt  <cit> .
that was the case for two aminoacyl-trna synthetases: methyonyl-trna  and tyrosyl-trna  synthetases, which are known to have been intensely affected by hgt  <cit> .
for example, the proteobacterium escherichia coli has a met-rs of archaeal origin and a tyr-rs transferred from gram-positive firmicutes  <cit> .
therefore, a single species can have aminoacyl-trna synthetases with at least two very different evolutionary histories and, consequently, their simultaneous analysis in a multi-protein concatenation will inevitably lead to a wrong phylogenetic tree, especially when a restricted taxonomic sampling is used .
when coping with datasets affected by hgt, the most reliable way to avoid artefacts is to carry out independent analyses for each marker.
in fact, when independent phylogenetic trees of the proteins used by raoult et al.
were carried out with a rich taxonomic sampling, the results were completely different  <cit> .
not only hgt events were detected, but mimivirus did no longer form an independent branch at the base of the eukaryotes, but emerged well nested within them.
moreover, for certain markers such as the tyr-rs, it branched as a close relative of several species of amoeba  <cit> .
this result showed that, instead of being the ancestral source of eukaryotic genes, mimivirus has incorporated these genes from its eukaryotic host, the amoeba, into its own genome.
while these studies were based on only a handful of genes, we sought here to understand how extensive was the role of hgt in shaping the whole mimivirus genome, and which were the sources of the transferred genes.
these questions were recently addressed using surrogate methods, those that do not require inference of phylogenetic trees, such as the analysis of blast scores.
using this approach, ogata et al.
<cit>  studied the distribution of blast scores for  <dig> mimivirus orfs, searched against a database containing homologous sequences from the amoeba entamoeba histolytica, metazoa, fungi and plants.
their analysis showed that only five mimivirus orfs  are more similar to their e. histolytica homologues than to the sequences from other eukaryotic groups.
a similar analysis for the entire set of mimivirus orfs showed that about  <dig> of them have eukaryotic or bacterial sequences as best matches  <cit> .
however, blast searches, as also other surrogate methods, offer only a very rough estimate of the phylogenetic affinity of a gene.
indeed, very often the best blast hit does not correspond to the closest evolutionary relative.
for example, a blast search of the orf mimi_r <dig>  retrieves several fungi as best hits, whereas phylogenetic analysis supports its relationship with bacterial homologues .
this can be explained by several factors that can reduce the accuracy of blast searches, in particular the heterogeneity of evolutionary rates among species  <cit> .
in that sense, several studies have shown that surrogate methods are clearly inferior to phylogenetic analysis in inferring the evolutionary origin of genes  <cit> .
in addition, phylogenetic analyses can provide a very precise identification of the donors of genes acquired by hgt, especially in the case of recent transfer events, and they can also provide statistical measures of the support of the inferred phylogenetic relationships .
therefore, we carried out a detailed phylogenetic analysis of all the mimivirus genome orfs with well-identified cellular homologues in order to determine whether these genes have a viral or a cellular origin.
in the latter case, phylogenetic analyses can be helpful to identify cell-to-mimivirus hgt events and the corresponding gene donors.
RESULTS
accurate phylogenetic reconstruction requires a correct degree of conservation among the sequences analysed.
therefore, we focused only on the set of  <dig> mimiviral proteins ascribed to cog families  <cit> .
mimivirus orf homologues have an extremely patchy taxonomic distribution
for each protein, we retrieved by blastp all homologues available in the protein non-redundant  database and studied their distribution in the three domains of life.
we considered that homologues of a mimiviral orf are 'present' in a domain only if they are widely distributed across different phyla of the domain or, at least, in most species of one major phylum .
for  <dig> orfs out of the  <dig> starting proteins, we did not retrieve any clear homologue, some of these orfs most likely corresponding to erroneous annotations .
among the remaining  <dig> orfs, the most abundant class  was that of orfs present only in bacteria and eukaryotes , followed by those present in the three domains  or only in eukaryotes .
smaller proportions of orfs were found only in bacteria and archaea , bacteria  or archaea and eukaryotes .
a more detailed inspection revealed that the distribution within each domain was in certain cases very unequal.
for example, some orfs were only found in animals , in bacteria and fungi , or in a number of very diverse combinations of taxa .
interestingly, we found that only a very small fraction of the  <dig> mimiviral orfs has homologues in other members of the ncldv family of viruses, even in those with relatively big genomes.
for example, the phycodnaviruses emiliania huxleyi virus  <dig>  and paramecium bursaria chlorella virus  <dig>  contain only  <dig> and  <dig> homologues of the  <dig> mimiviral orfs , respectively.
this represents less than 15% of these  <dig> orfs, and could be partially explained by the smaller genome size of those phycodnaviruses.
however, the values are significantly smaller than those expected just by the difference in genome size: ~100– <dig> homologues should be retrieved in these phycodnaviruses.
in addition, these data show a very disparate taxonomic distribution of genes among the different viruses of the ncldv family, a situation that is frequently a clear symptom of hgt  <cit> .
moreover, our phylogenetic analyses support a viral origin for only  <dig> orfs among the  <dig> mimiviral orfs studied .
this observation, together with the fact that the mimiviral orfs that have homologues in the three domains of life are not the dominant class of orfs in mimivirus , is extremely difficult to reconcile with the hypothesis that ncldv viruses may define a fourth major lineage of life .
in fact, genomes among the smallest ones found in archaea  and bacteria , which are significantly smaller than the mimivirus genome, share many more genes with their archaeal and bacterial relatives than the mimivirus does with the other ncldv.
moreover, these archaeal and bacterial species with highly reduced genomes have a much larger repertoire of typical cellular genes than the mimivirus  <cit> .
if the "fourth domain" hypothesis  <cit>  was correct, the actual taxonomic distribution shown by the mimiviral orfs could only be explained by an extremely massive loss of ancestral genes in the different ncldv viruses but, even in that case, the majority of gene phylogenies should support a clear separation of the mimiviral sequences from those of the other domains.
on the contrary, if the "gene acquisition by hgt" hypothesis  <cit>  was correct, the majority of gene phylogenies should support an emergence of the mimivirus orfs that have cellular homologues within one of the three domains .
we have explored these two possibilities by detailed phylogenetic analyses, applying maximum likelihood  and bayesian inference  methods, of all the  <dig> mimiviral orfs with cellular homologues and with an adequate degree of sequence conservation.
phylogenetic analysis: mimiviral orfs of prokaryotic origin
we detected a single orf related to archaeal homologues, the dna-directed rna polymerase mimi_r <dig> , and a much larger number of orfs related to bacterial sequences.
the six mimiviral orfs mimi_l <dig>  mimi_l <dig>  mimi_r <dig>  mimi_r <dig>  mimi_r <dig>  and mimi_r <dig> are shared exclusively with bacteria .
they likely correspond to genes that have been acquired by the virus by hgt from bacteria.
interestingly, the three uncharacterised proteins mimi_r <dig>  mimi_r <dig> and mimi_r <dig> show very similar taxonomic distributions across bacteria and similar phylogenies.
these three mimiviral orfs emerge within cyanobacteria , suggesting a single hgt from a cyanobacterial donor to the mimivirus.
in addition to them, our phylogenetic analyses detected  <dig> additional genes of bacterial origin among the mimiviral orfs shared with bacteria and other domains .
some of these are related to homologues from gram positive firmicutes: mimi_l <dig>  and mimi_r <dig> .
the others are closer to proteobacterial sequences: e.g., orfs mimi_l <dig> , mimi_l <dig> , and mimi_r <dig> .
the source of these bacterial-related orfs is intriguing.
the most appealing possibility is that mimivirus has acquired them from bacteria that co-infect its same eukaryotic hosts.
indeed, amoebas harbour a variety of intracellular bacteria, including proteobacteria, gram positives , chlamydiae and bacteroidetes  <cit> .
our phylogenetic trees show that several mimiviral orfs are clearly related to the corresponding homologues from bacterial species that are typical inhabitants of amoeba.
that is the case of two of the orfs cited above: mimi_l <dig>  related to legionella pneumophila  and mimi_r <dig>  related to campylobacter spp.
, which are proteobacteria frequently found in different amoebas, including acanthamoeba  <cit> .
a recent article stresses the role that amoebas may have played to facilitate the exchange of genes between different intracellular bacterial species, a phenomenon that might have been important in their adaptation to life within eukaryotic cells  <cit> .
our results suggest that the situation may be even more complex, since the intracellular bacteria appear to have transferred genes also to the mimiviral genome.
some of these genes are probably involved in the parasitic adaptations of the mimivirus, such as the campylobacter-like outer membrane lipoprotein cited above .
mimiviral orfs of eukaryotic origin: amoebas and evidence for unknown hosts
most of the  <dig> mimiviral orfs that have homologues in cellular species useful for phylogenetic analysis can be found in eukaryotes .
our phylogenetic analyses inferred a eukaryotic origin for  <dig> of these orfs.
interestingly, several orfs of eukaryotic origin are closely related to homologues found in different amoebas, as in the case of mimi_l <dig>  already reported  <cit> .
we inferred a clear amoebal origin also for mimi_r <dig> , mimi_l <dig> , mimi_l <dig> , mimi_r <dig> , mimi_l <dig> , mimi_l <dig> , mimi_r <dig> , mimi_l <dig> , mimi_r <dig> , mimi_r <dig> , mimi_r <dig>  mimi_r <dig> and mimi_r <dig> .
to sum up, our trees support that ~10% of the mimiviral orfs with eukaryotic homologues were acquired from amoeba.
these orfs are involved in a variety of processes and, in some cases, their acquisition by hgt was followed by duplication events, such as the kinases mimi_r <dig>  mimi_r <dig> and mimi_r <dig> .
certain mimiviral orfs are exclusively shared by this virus and its amoebal hosts and they represent probable additional host-to-virus hgt events.
this is the case of the  <dig> mimiviral orfs proteins containing the fnip motif detected by song et al.
<cit> .
it is important to note that all these orfs of amoebal origin represent a minimal number since others might also have an amoebal origin but did not produce well resolved trees, a problem that can be due to different causes: lack of phylogenetic signal, small number of positions useful for phylogenetic analyses of several orfs, tree reconstruction artefacts due to unequal evolutionary rates among taxa, and/or missing data concerning amoebas .
in addition to all the orfs likely acquired by hgt from the amoebal hosts, we detected a few ones that support a close phylogenetic relationship between the mimivirus and eukaryotes unrelated to the amoebozoa.
the phylogenetic analysis of the two hsp <dig> homologues found in mimivirus is a remarkable example.
whereas mimi_l <dig>  an endoplasmic reticulum-type hsp <dig>  is strongly related to amoebozoan homologues, the cytosolic-type hsp <dig> mimi_l <dig> is clearly related to species of the genera naegleria and sawyeria .
these species are flagellated amoebas, many of them parasitic, which belong to the heterolobosea  <cit> .
this group is possibly related to the euglenozoa, forming a large assemblage called the discicristata, very distant from amoebozoa  <cit> .
the presence of giant virus-like particles in the cytoplasm of naegleria fowleri was already noticed in the 1970s  <cit> , and it was recently hypothesised that these particles might be related to mimivirus  <cit> .
our detection of a mimiviral orf phylogenetically related to homologues from naegleria and sawyeria strongly supports the hypothesis that mimivirus can also infect these amoeboid species, even if they are very distant from the hosts, such as acanthamoeba spp., known up to date.
in addition to this orf, we detected three other orfs  that are related to homologues from different protist species belonging to the euglenozoa, more specifically to the kinetoplastida, which are flagellates with both free-living and parasitic members, such as trypanosoma and leishmania.
since the heterolobosean flagellate amoebas, as naegleria, are likely related to the euglenozoa , the possibility exists that these orfs have also been acquired from naegleria relatives but that we could only detect a phylogenetic affinity with the kinetoplastida because the corresponding sequences from naegleria are not available in current databases.
that could also be the case for the orfs that branch as sisters of the kinetoplastid sequences: mimi_l <dig>  and mimi_l <dig> .
however, for the orf mimi_r <dig> , our phylogenetic analyses strongly support the emergence of mimivirus within the kinetoplastida, indicating that it acquired this orf from a kinetoplastid species .
this suggests that not only protists with amoeboid cell structures, but also typical flagellates such as the kinetoplastids may be hosts of mimiviruses.
nevertheless, we cannot discard the possibility that hgt from these flagellates to mimivirus occurred within amoebas since kinetoplastid parasites  have been detected in several amoebal species  <cit> .
as in the case of several mimiviral orfs closely related to homologues from parasitic bacteria , these flagellates may have transferred genes to the mimivirus infecting the same amoebal hosts.
a less parsimonious alternative hypothesis would be that these orfs have been transferred to mimivirus by an unidentified third partner, for example viruses infecting kinetoplastids and heterolobosea that could have recombined with mimivirus.
however, the fact that particles very similar to mimivirus have been observed in naegleria supports the hypothesis of direct gene acquisition at least from heterolobosean hosts.
the amoebas as complex ecosystems promoting hgt
the number of bacterial species that have been characterised as stable inhabitants of diverse amoebas seems to be far from being completely known  <cit> .
as commented above, the promiscuity of these bacterial species within amoebas may have facilitated hgt and the adaptation to parasitic lifestyles  <cit> .
several eukaryotic parasites, such as the kinetoplastid perkinsiella, can also be found in amoebas  <cit> .
moreover, there is increasing evidence that the use of amoebas as a reservoir can be a key factor in the selection of virulent strains of eukaryotic parasites of mammals, in particular several pathogenic dimorphic fungi  <cit> .
mimiviruses infecting amoebas can thus coexist in a confined environment with a variety of other parasites.
in addition to the dna from the amoebal host, the sporadic cell lysis of these parasites provides dna that can be a source of new genes for the virus.
the acquisition of genes from the host and from its bacterial and eukaryotic parasites may be significant in the development of virulence traits of the virus, but also in its opportunistic pre-adaptation to alternative hosts, including humans.
this has been shown to be an important process in other pathogens.
for example, the parasitic bacterium l. pneumophila has acquired by hgt several eukaryotic genes involved in a variety of cell functions, in particular two serine/threonine protein kinases  <cit> .
it has been shown in several pathogens that these protein kinases are responsible of inhibiting phagosome-lysosome fusion, allowing intracellular survival, but also of disrupting the host defence by interfering with the eukaryotic signal transduction pathways  <cit> .
interestingly, mimivirus possesses three serine/threonine protein kinases  that have been acquired from its amoebal hosts .
this is also the case for the ras gtpase mimi_r <dig>  the presence of these proteins suggests that mimivirus can regulate the host cell cycle to its benefit.
this is an example of the crucial role that hgt may have had in the evolution of the virulence strategy of mimivirus.
CONCLUSIONS
most of the genes in mimivirus with homologues in cellular organisms appear to have been acquired by hgt to the virus.
this strongly supports that mimivirus does not define a new domain of life.
mimivirus certainly acquired most of these genes either from its eukaryotic hosts or from other parasites coexisting in the same host.
in that sense, this virus appears to have followed a similar strategy as other parasites to interfere with host cellular processes through the modification and expression of genes acquired from the host by hgt.
our data also suggest that the primary hosts for mimivirus are the amoebozoa, as most of the mimiviral orfs of eukaryotic origin with a well resolved phylogeny are closely related to homologues from this group.
nevertheless, we detect hgt from putative alternative hosts, such as heterolobosea  and kinetoplastids.
despite the fact that mimivirus appears to have the capacity to infect also humans  <cit> , we identified only a single case of a probable gene acquisition from animals, the glycosyltransferases mimi_l <dig> and mimi_r <dig> .
this small number suggests that the recently reported human infection by this virus may be a relatively rare or very recent event.

 since their discovery in the last years of the xix century up to our days, viruses have never ceased to puzzle biologists, especially those studying their evolution. because of their nature as entities at the border between the living and the non-living, their origin has been the matter of passionate debate. opinions range between the two extreme "virus-early" and "virus-late" hypotheses. the first postulates that viruses originated before modern cells. on the contrary, the second hypothesis proposes that viruses originated either by the escape of genetic material and proteins from cells, or by a dramatic reductive evolution from cellular forms that lost all the "cellular paraphernalia" unnecessary for the parasitic lifestyle. as a consequence, the position of viruses within the tree of life is also a subject of disagreement. whereas some scientists fervently advocate that viruses should have a place in the tree of life, many other consider that, being acellular and lacking any kind of carbon and energy metabolism, viruses cannot be properly compared with true living beings and, therefore, do not belong to the tree of life. moreover, there is compelling evidence for the polyphyletic origin of viruses, which further complicates the issue. these debates have recently gained an unprecedented impetus when raoult and co-workers published the description of the genome of mimivirus, a giant virus first detected as a parasite of amoeba of the genus acanthamoeba, which is endowed with the biggest viral genome known to date: mbp containing genes. a number of genomic signatures strongly support that mimivirus belongs to the ncldv virus family. the mimiviral genome contains genes frequently found in viruses, as those involved in genome replication, but also, and for the first time in a virus, a number of genes coding for proteins involved in transcription and translation. therefore, at least from the point of view of gene content, mimivirus appears somehow closer to a typical cell than any other described virus. moreover, raoult et al. identified seven mimiviral proteins that have closely related eukaryotic homologues. their phylogenetic analysis in a multi-protein concatenation supported the emergence of mimivirus as a sister-group of eukaryotes in a tree including representatives from the three domains of life. therefore, it was hypothesised that this virus would define a new branch distinct from the three domains of life: bacteria, archaea and eukaryotes. such an unexpected result had the potential to revolutionise our conceptions on the diversity and evolution of life, up to now based on the tripartite scheme of the three domains. raoult et al. further suggested that the ncldv family may have played a role in the origin of eukaryotes. this would agree with previous hypotheses stating that viruses could be at the origin of many eukaryotic genes or even the eukaryotic nucleus. the study of viral evolution using phylogenetic analysis is most often a difficult task due to several peculiarities of viruses related to their parasitic lifestyle, such as rapid evolutionary rates and the propensity to be involved in horizontal gene transfer events. as recently shown, the emergence of mimivirus at the base of the eukaryotic branch in the seven-protein study by raoult et al. was the result of an accumulation of artefacts due to the simultaneous analysis of several markers that had been profusely exchanged between distant species by hgt. that was the case for two aminoacyl-trna synthetases: methyonyl-trna and tyrosyl-trna synthetases, which are known to have been intensely affected by hgt. for example, the proteobacterium escherichia coli has a met-rs of archaeal origin and a tyr-rs transferred from gram-positive firmicutes. therefore, a single species can have aminoacyl-trna synthetases with at least two very different evolutionary histories and, consequently, their simultaneous analysis in a multi-protein concatenation will inevitably lead to a wrong phylogenetic tree, especially when a restricted taxonomic sampling is used. when coping with datasets affected by hgt, the most reliable way to avoid artefacts is to carry out independent analyses for each marker. in fact, when independent phylogenetic trees of the proteins used by raoult et al. were carried out with a rich taxonomic sampling, the results were completely different. not only hgt events were detected, but mimivirus did no longer form an independent branch at the base of the eukaryotes, but emerged well nested within them. moreover, for certain markers such as the tyr-rs, it branched as a close relative of several species of amoeba. this result showed that, instead of being the ancestral source of eukaryotic genes, mimivirus has incorporated these genes from its eukaryotic host, the amoeba, into its own genome. while these studies were based on only a handful of genes, we sought here to understand how extensive was the role of hgt in shaping the whole mimivirus genome, and which were the sources of the transferred genes. these questions were recently addressed using surrogate methods, those that do not require inference of phylogenetic trees, such as the analysis of blast scores. using this approach, ogata et al. studied the distribution of blast scores for mimivirus orfs, searched against a database containing homologous sequences from the amoeba entamoeba histolytica, metazoa, fungi and plants. their analysis showed that only five mimivirus orfs are more similar to their e. histolytica homologues than to the sequences from other eukaryotic groups. a similar analysis for the entire set of mimivirus orfs showed that about of them have eukaryotic or bacterial sequences as best matches. however, blast searches, as also other surrogate methods, offer only a very rough estimate of the phylogenetic affinity of a gene. indeed, very often the best blast hit does not correspond to the closest evolutionary relative. for example, a blast search of the orf mimi_r retrieves several fungi as best hits, whereas phylogenetic analysis supports its relationship with bacterial homologues. this can be explained by several factors that can reduce the accuracy of blast searches, in particular the heterogeneity of evolutionary rates among species. in that sense, several studies have shown that surrogate methods are clearly inferior to phylogenetic analysis in inferring the evolutionary origin of genes. in addition, phylogenetic analyses can provide a very precise identification of the donors of genes acquired by hgt, especially in the case of recent transfer events, and they can also provide statistical measures of the support of the inferred phylogenetic relationships. therefore, we carried out a detailed phylogenetic analysis of all the mimivirus genome orfs with well-identified cellular homologues in order to determine whether these genes have a viral or a cellular origin. in the latter case, phylogenetic analyses can be helpful to identify cell-to-mimivirus hgt events and the corresponding gene donors.  accurate phylogenetic reconstruction requires a correct degree of conservation among the sequences analysed. therefore, we focused only on the set of mimiviral proteins ascribed to cog families. mimivirus orf homologues have an extremely patchy taxonomic distribution for each protein, we retrieved by blastp all homologues available in the protein non-redundant database and studied their distribution in the three domains of life. we considered that homologues of a mimiviral orf are'present' in a domain only if they are widely distributed across different phyla of the domain or, at least, in most species of one major phylum. for orfs out of the starting proteins, we did not retrieve any clear homologue, some of these orfs most likely corresponding to erroneous annotations. among the remaining orfs, the most abundant class was that of orfs present only in bacteria and eukaryotes, followed by those present in the three domains or only in eukaryotes. smaller proportions of orfs were found only in bacteria and archaea, bacteria or archaea and eukaryotes. a more detailed inspection revealed that the distribution within each domain was in certain cases very unequal. for example, some orfs were only found in animals, in bacteria and fungi, or in a number of very diverse combinations of taxa. interestingly, we found that only a very small fraction of the mimiviral orfs has homologues in other members of the ncldv family of viruses, even in those with relatively big genomes. for example, the phycodnaviruses emiliania huxleyi virus and paramecium bursaria chlorella virus contain only and homologues of the mimiviral orfs, respectively. this represents less than 15% of these orfs, and could be partially explained by the smaller genome size of those phycodnaviruses. however, the values are significantly smaller than those expected just by the difference in genome size: ~100– homologues should be retrieved in these phycodnaviruses. in addition, these data show a very disparate taxonomic distribution of genes among the different viruses of the ncldv family, a situation that is frequently a clear symptom of hgt. moreover, our phylogenetic analyses support a viral origin for only orfs among the mimiviral orfs studied. this observation, together with the fact that the mimiviral orfs that have homologues in the three domains of life are not the dominant class of orfs in mimivirus, is extremely difficult to reconcile with the hypothesis that ncldv viruses may define a fourth major lineage of life. in fact, genomes among the smallest ones found in archaea and bacteria, which are significantly smaller than the mimivirus genome, share many more genes with their archaeal and bacterial relatives than the mimivirus does with the other ncldv. moreover, these archaeal and bacterial species with highly reduced genomes have a much larger repertoire of typical cellular genes than the mimivirus. if the "fourth domain" hypothesis was correct, the actual taxonomic distribution shown by the mimiviral orfs could only be explained by an extremely massive loss of ancestral genes in the different ncldv viruses but, even in that case, the majority of gene phylogenies should support a clear separation of the mimiviral sequences from those of the other domains. on the contrary, if the "gene acquisition by hgt" hypothesis was correct, the majority of gene phylogenies should support an emergence of the mimivirus orfs that have cellular homologues within one of the three domains. we have explored these two possibilities by detailed phylogenetic analyses, applying maximum likelihood and bayesian inference methods, of all the mimiviral orfs with cellular homologues and with an adequate degree of sequence conservation. phylogenetic analysis: mimiviral orfs of prokaryotic origin we detected a single orf related to archaeal homologues, the dna-directed rna polymerase mimi_r, and a much larger number of orfs related to bacterial sequences. the six mimiviral orfs mimi_l mimi_l mimi_r mimi_r mimi_r and mimi_r are shared exclusively with bacteria. they likely correspond to genes that have been acquired by the virus by hgt from bacteria. interestingly, the three uncharacterised proteins mimi_r mimi_r and mimi_r show very similar taxonomic distributions across bacteria and similar phylogenies. these three mimiviral orfs emerge within cyanobacteria, suggesting a single hgt from a cyanobacterial donor to the mimivirus. in addition to them, our phylogenetic analyses detected additional genes of bacterial origin among the mimiviral orfs shared with bacteria and other domains. some of these are related to homologues from gram positive firmicutes: mimi_l and mimi_r. the others are closer to proteobacterial sequences: e.g., orfs mimi_l, mimi_l, and mimi_r. the source of these bacterial-related orfs is intriguing. the most appealing possibility is that mimivirus has acquired them from bacteria that co-infect its same eukaryotic hosts. indeed, amoebas harbour a variety of intracellular bacteria, including proteobacteria, gram positives, chlamydiae and bacteroidetes. our phylogenetic trees show that several mimiviral orfs are clearly related to the corresponding homologues from bacterial species that are typical inhabitants of amoeba. that is the case of two of the orfs cited above: mimi_l related to legionella pneumophila and mimi_r related to campylobacter spp., which are proteobacteria frequently found in different amoebas, including acanthamoeba. a recent article stresses the role that amoebas may have played to facilitate the exchange of genes between different intracellular bacterial species, a phenomenon that might have been important in their adaptation to life within eukaryotic cells. our results suggest that the situation may be even more complex, since the intracellular bacteria appear to have transferred genes also to the mimiviral genome. some of these genes are probably involved in the parasitic adaptations of the mimivirus, such as the campylobacter-like outer membrane lipoprotein cited above. mimiviral orfs of eukaryotic origin: amoebas and evidence for unknown hosts most of the mimiviral orfs that have homologues in cellular species useful for phylogenetic analysis can be found in eukaryotes. our phylogenetic analyses inferred a eukaryotic origin for of these orfs. interestingly, several orfs of eukaryotic origin are closely related to homologues found in different amoebas, as in the case of mimi_l already reported. we inferred a clear amoebal origin also for mimi_r, mimi_l, mimi_l, mimi_r, mimi_l, mimi_l, mimi_r, mimi_l, mimi_r, mimi_r, mimi_r mimi_r and mimi_r. to sum up, our trees support that ~10% of the mimiviral orfs with eukaryotic homologues were acquired from amoeba. these orfs are involved in a variety of processes and, in some cases, their acquisition by hgt was followed by duplication events, such as the kinases mimi_r mimi_r and mimi_r. certain mimiviral orfs are exclusively shared by this virus and its amoebal hosts and they represent probable additional host-to-virus hgt events. this is the case of the mimiviral orfs proteins containing the fnip motif detected by song et al.. it is important to note that all these orfs of amoebal origin represent a minimal number since others might also have an amoebal origin but did not produce well resolved trees, a problem that can be due to different causes: lack of phylogenetic signal, small number of positions useful for phylogenetic analyses of several orfs, tree reconstruction artefacts due to unequal evolutionary rates among taxa, and/or missing data concerning amoebas. in addition to all the orfs likely acquired by hgt from the amoebal hosts, we detected a few ones that support a close phylogenetic relationship between the mimivirus and eukaryotes unrelated to the amoebozoa. the phylogenetic analysis of the two hsp homologues found in mimivirus is a remarkable example. whereas mimi_l an endoplasmic reticulum-type hsp is strongly related to amoebozoan homologues, the cytosolic-type hsp mimi_l is clearly related to species of the genera naegleria and sawyeria. these species are flagellated amoebas, many of them parasitic, which belong to the heterolobosea. this group is possibly related to the euglenozoa, forming a large assemblage called the discicristata, very distant from amoebozoa. the presence of giant virus-like particles in the cytoplasm of naegleria fowleri was already noticed in the 1970s, and it was recently hypothesised that these particles might be related to mimivirus. our detection of a mimiviral orf phylogenetically related to homologues from naegleria and sawyeria strongly supports the hypothesis that mimivirus can also infect these amoeboid species, even if they are very distant from the hosts, such as acanthamoeba spp., known up to date. in addition to this orf, we detected three other orfs that are related to homologues from different protist species belonging to the euglenozoa, more specifically to the kinetoplastida, which are flagellates with both free-living and parasitic members, such as trypanosoma and leishmania. since the heterolobosean flagellate amoebas, as naegleria, are likely related to the euglenozoa, the possibility exists that these orfs have also been acquired from naegleria relatives but that we could only detect a phylogenetic affinity with the kinetoplastida because the corresponding sequences from naegleria are not available in current databases. that could also be the case for the orfs that branch as sisters of the kinetoplastid sequences: mimi_l and mimi_l. however, for the orf mimi_r, our phylogenetic analyses strongly support the emergence of mimivirus within the kinetoplastida, indicating that it acquired this orf from a kinetoplastid species. this suggests that not only protists with amoeboid cell structures, but also typical flagellates such as the kinetoplastids may be hosts of mimiviruses. nevertheless, we cannot discard the possibility that hgt from these flagellates to mimivirus occurred within amoebas since kinetoplastid parasites have been detected in several amoebal species. as in the case of several mimiviral orfs closely related to homologues from parasitic bacteria, these flagellates may have transferred genes to the mimivirus infecting the same amoebal hosts. a less parsimonious alternative hypothesis would be that these orfs have been transferred to mimivirus by an unidentified third partner, for example viruses infecting kinetoplastids and heterolobosea that could have recombined with mimivirus. however, the fact that particles very similar to mimivirus have been observed in naegleria supports the hypothesis of direct gene acquisition at least from heterolobosean hosts. the amoebas as complex ecosystems promoting hgt the number of bacterial species that have been characterised as stable inhabitants of diverse amoebas seems to be far from being completely known. as commented above, the promiscuity of these bacterial species within amoebas may have facilitated hgt and the adaptation to parasitic lifestyles. several eukaryotic parasites, such as the kinetoplastid perkinsiella, can also be found in amoebas. moreover, there is increasing evidence that the use of amoebas as a reservoir can be a key factor in the selection of virulent strains of eukaryotic parasites of mammals, in particular several pathogenic dimorphic fungi. mimiviruses infecting amoebas can thus coexist in a confined environment with a variety of other parasites. in addition to the dna from the amoebal host, the sporadic cell lysis of these parasites provides dna that can be a source of new genes for the virus. the acquisition of genes from the host and from its bacterial and eukaryotic parasites may be significant in the development of virulence traits of the virus, but also in its opportunistic pre-adaptation to alternative hosts, including humans. this has been shown to be an important process in other pathogens. for example, the parasitic bacterium l. pneumophila has acquired by hgt several eukaryotic genes involved in a variety of cell functions, in particular two serine/threonine protein kinases. it has been shown in several pathogens that these protein kinases are responsible of inhibiting phagosome-lysosome fusion, allowing intracellular survival, but also of disrupting the host defence by interfering with the eukaryotic signal transduction pathways. interestingly, mimivirus possesses three serine/threonine protein kinases that have been acquired from its amoebal hosts. this is also the case for the ras gtpase mimi_r the presence of these proteins suggests that mimivirus can regulate the host cell cycle to its benefit. this is an example of the crucial role that hgt may have had in the evolution of the virulence strategy of mimivirus.  most of the genes in mimivirus with homologues in cellular organisms appear to have been acquired by hgt to the virus. this strongly supports that mimivirus does not define a new domain of life. mimivirus certainly acquired most of these genes either from its eukaryotic hosts or from other parasites coexisting in the same host. in that sense, this virus appears to have followed a similar strategy as other parasites to interfere with host cellular processes through the modification and expression of genes acquired from the host by hgt. our data also suggest that the primary hosts for mimivirus are the amoebozoa, as most of the mimiviral orfs of eukaryotic origin with a well resolved phylogeny are closely related to homologues from this group. nevertheless, we detect hgt from putative alternative hosts, such as heterolobosea and kinetoplastids. despite the fact that mimivirus appears to have the capacity to infect also humans, we identified only a single case of a probable gene acquisition from animals, the glycosyltransferases mimi_l and mimi_r. this small number suggests that the recently reported human infection by this virus may be a relatively rare or very recent event. 
on the contrary, our phylogenetic trees strongly suggest that mimivirus acquired most of these genes by horizontal gene transfer either from its amoebal hosts or from bacteria that parasitise the same hosts. the detection of hgt events involving different eukaryotic donors suggests that the spectrum of hosts of mimivirus may be larger than currently known. in fact, the first phylogenetic analyses of a few mimivirus genes that are also present in cellular lineages suggested that it could define an independent branch in the tree of life in addition to the three domains, bacteria, archaea and eucarya. we tested this hypothesis by carrying out detailed phylogenetic analyses for all the conserved mimivirus genes that have homologues in cellular organisms. however, the recent publication of the genome sequence of the mimivirus, a giant virus that parasitises amoebas, strengthened the idea that viruses should be included in the tree of life. the large number of genes acquired by mimivirus from eukaryotic and bacterial sources suggests that hgt has been an important process in the evolution of its genome and the adaptation to parasitism.
 although capable to evolve, viruses are generally considered non-living entities because they are acellular and devoid of metabolism. however, the recent publication of the genome sequence of the mimivirus, a giant virus that parasitises amoebas, strengthened the idea that viruses should be included in the tree of life. in fact, the first phylogenetic analyses of a few mimivirus genes that are also present in cellular lineages suggested that it could define an independent branch in the tree of life in addition to the three domains, bacteria, archaea and eucarya.  we tested this hypothesis by carrying out detailed phylogenetic analyses for all the conserved mimivirus genes that have homologues in cellular organisms. we found no evidence supporting mimivirus as a new branch in the tree of life. on the contrary, our phylogenetic trees strongly suggest that mimivirus acquired most of these genes by horizontal gene transfer either from its amoebal hosts or from bacteria that parasitise the same hosts. the detection of hgt events involving different eukaryotic donors suggests that the spectrum of hosts of mimivirus may be larger than currently known.  the large number of genes acquired by mimivirus from eukaryotic and bacterial sources suggests that hgt has been an important process in the evolution of its genome and the adaptation to parasitism. 