BACKGROUND
members of the herpesviridae have a linear double-stranded dna genome, 120– <dig> kbp in length, and cause significant morbidity and mortality in diverse groups of animals.
members are further classified into three subfamilies; the alpha-, beta- and gammaherpesvirinae.
reports of herpesvirus infections in the marsupialia date back to the 1970s.
the first isolation of a marsupial herpesvirus was from a fatal outbreak of severe respiratory disease and systemic organ failure in a zoological collection of parma wallabies   <cit> .
the isolation of this alphaherpesvirus, designated macropodid herpesvirus  <dig> , was closely followed by the isolation of a second, related, herpesvirus  from fatal cases of disease in several vulnerable macropod species  <cit> .
the macropodid viruses were detected in animals displaying some clinical signs of disease similar to those caused by human herpesviruses  <dig> and  <dig>  infection, such as conjunctivitis and vesicular anogenital lesions, but also included hepatic disease  <cit> .
in the following  <dig> years, before the identification of additional marsupial herpesviruses, evidence of herpesvirus infection in metatherians was largely observed through electron microscopy or sero-epidemiological surveys.
these sero-prevalence studies measured neutralising antibodies to mahv- <dig>  which were detected in both wild  and captive  populations of marsupials  <cit> .
since  <dig>  eleven additional marsupial herpesviruses have been identified, including a closely-related alphaherpesvirus, macropodid herpesvirus  <dig> , in free-ranging eastern grey kangaroos  with clinical signs of respiratory and possible neurological disease  <cit>  and two gammaherpesviruses from macropods .
a further eight herpesviruses have been identified in other  marsupial species, though little sequence data are available for these viruses .
despite its classification as a simplexvirus, early genome hybridization studies of mahv- <dig> identified a type d genome structure  of approximately  <dig> kbp in length, containing a short unique  region, flanked by large inverted repeat sequences  joined to a long unique  region  <cit> .
mahv- <dig> occurs as only two equimolar genomic isomers  <cit> .
these genomic features are characteristic of varicelloviruses such as varicella zoster virus  and pseudorabies virus  and contrast with those of mahv- <dig>  mahv- <dig> has a type e genome arrangement, more typical of the simplexviruses, and occurs as four equimolar genomic isomers  <cit> .
to date mahv- <dig> is the only alphaherpesvirus that encodes both icp <dig>   and the host-derived oncogene thymidylate synthase  <cit> .
sequence analysis of two conserved orfs in mahv- <dig>  − <dig> and − <dig>  as well as analyses of their antigenic relationships, has clustered these macropodid viruses closely with the primate simplexviruses  <cit> .
as metatherian and eutherian mammals are believed to have diverged over  <dig> million years ago  <cit> , this viral phylogenetic grouping differed from the typical virus-host co-evolutionary pattern observed within the herpesviridae  <cit>  and was instead suggestive of a recent and complex speciation event.
this study aimed to sequence and analyse the full genome of the metatherian alphaherpesvirus, mahv- <dig>  with particular attention to novel genomic features.
RESULTS
whole genome sequence analysis
the genome of mahv- <dig> is the first metatherian herpesvirus to be sequenced.
excluding the genomic termini, which remained unresolved, the final genome length of mahv- <dig> was approximately  <dig>  kbp  , larger than previously predicted.
this difference appears to be due to a larger than predicted inverted repeat region  <cit> .
this included a  <dig>  kbp ul region and a  <dig>  kbp us region flanked by  <dig> kbp inverted repeat sequences .
the mahv- <dig> genome had a g + c content of  <dig>  %, but had a higher g + c content  within the irs/trs regions.
the final genome assembly had a mean depth of  <dig>  reads per bp  and approximately 95 % of reads had a quality score of at least phred <dig>  three origins of replication were identified.
the origin of lytic replication  was located between ul <dig> and ul <dig> in the ul region and the oris was located within the irs/trs regions.
thus two copies of oris were present, as in the genomes of hhv- <dig> and − <dig> fig.
<dig> schematic representation of the genome arrangement of macropodid herpesvirus  <dig>  herpesviral conserved orfs are annotated in grey using the hhv- <dig> and hhv- <dig> orf nomenclature.
novel orfs  are given a pw prefix.
unique long genome region , unique short genome region , trs/irs = inverted repeat flanking unique short region , oril/s = origins of replication
conserved alphaherpesvirus orfs
the ul region of the mahv- <dig> genome encoded  <dig> orfs common to other herpesviruses .
the predicted protein sequences of these orfs shared between 41 % and 73 % aa pairwise identity  with hhv- <dig> and − <dig> homologues.
in the us region the mahv- <dig> genome encoded seven orfs common to other simplexviruses , with the predicted protein sequences sharing between  <dig> and 59 % aa pairwise identity  with hhv- <dig> and − <dig> homologues.
the irs/trs regions encoded five orfs, including those for thymidylate synthase, icp <dig> and icp <dig> .
there were no homologues of ul <dig>  ul <dig>  ul <dig> or us <dig>  identified in the mahv- <dig> genome.
also, the us <dig>  homologue was predicted to be non-functional, as the orf was prematurely truncated .
this is consistent with previous published sequence data reporting a truncation of the mahv- <dig> gg orf  <cit> .
phylogenetic analyses using translated protein sequences of three core herpesvirus genes  are shown in fig.
<dig>  these analyses show that mahv- <dig> clusters most closely with other macropodid herpesviruses , as well as with the simplexviruses that infect primates.
it also groups with the herpesvirus of an indonesian pteropodid bat.
comparison of other viral core genes yielded similar clustering patterns.
comparison of the mahv- <dig> ul <dig> and ul <dig> orfs with those of the recently sequenced fruit bat herpesvirus  <dig>   showed that these orfs shared  <dig> and 67 % pairwise aa identity, respectively .
this similarity is comparable to that seen between mahv- <dig> and hhv-1/hhv- <dig> , which may offer some insight into their evolutionary relationship, for example, may suggest transmission of herpesviruses from primates to bats, and then to marsupials.
sequencing of herpesviruses from other metatherians, as well as other australasian mammals, will be needed to determine the significance of this clustering.table  <dig> predicted open reading frames  identified in different structural regions of the macropodid herpesvirus  <dig> genome and percentage pairwise amino acid identity to orfs in related alphaherpesviruses
§putative function of encoded polypeptides in mahv-1
#amino acid identities with homologues in human herpesvirus  <dig> and  <dig>  and macropodid herpesviruses  <dig> and  <dig>
fig.
<dig> the relationship between macropodid herpesvirus  <dig>  and other viruses from the alphaherpesvirinae subfamily.
neighbour-joining distance trees were generated using the translated protein sequences of conserved herpesvirus orfs: a glycoprotein b, gb ; b dna polymerase, dpol ; and c glycoprotein d, gd .
bootstrap values  are shown for each branch, with gallid herpesvirus  <dig>  as an outgroup.
the abbreviations and genbank accession details are: macropodid herpesvirus  <dig>  , macropodid herpesvirus  <dig>  , macacine herpesvirus  <dig>  , fruit bat herpesvirus  <dig>  , gallid herpesvirus  <dig>  , gallid herpesvirus  <dig>  , human herpesvirus  <dig>  , human herpesvirus  <dig>  , human herpesvirus  <dig>  , saimiriine herpesvirus  <dig>  , bovine herpesvirus  <dig>  , bovine herpesvirus  <dig>  , bovine herpesvirus  <dig>  , canine herpesvirus  <dig>  , felid herpesvirus  <dig>  , equine herpesvirus  <dig>  , equine herpesvirus  <dig>  , suid herpesvirus  <dig>
although ul <dig> and ul <dig> are conserved in all other alphaherpesviruses examined to date, gene deletion studies in the human simplexviruses have found that deletion of ul <dig> and ul <dig> does not affect viral replication or cell-to-cell spread in vitro  <cit> .
in vivo functions of the ul <dig> and ul <dig> encoded accessory proteins are not well understood, but they colocalise and directly interact with the transcriptional repressor icp <dig>  encoded by us <dig>  in small dense nuclear bodies and may also be involved in the late phase of viral replication .
the absence of gj is also of note.
this is the third simplexvirus found to lack an orf encoding a gj homologue, which is otherwise conserved in the simplexvirus genus.
the other two simplexviruses lacking gj are leoporid herpesvirus  <dig> and fbhv- <dig>  <cit> .
in other herpesviruses gj inhibits host cell apoptosis by inducing an increase in concentrations of reactive oxygen species in the host cell  <cit> .
it is unclear whether the absence of ul <dig>  ul <dig> and gj might be related to adaptation to a new host  or whether it may be the result of virus passage in vitro.
in respect to gj, the former scenario could be more likely, as the absence of this orf in other non-primate herpesviruses shows that it is not strictly conserved within the genus.
sequencing of other marsupial alphaherpesviruses, particularly field isolates, would help to resolve this finding.
unique or hypothetical orfs
seven unique hypothetical orfs were identified; one in the ul region, two located in the irs/trs regions, and four in the us region.
viral transcript analyses by qrt-pcr confirmed that six of the seven predicted orfs were transcribed at both  <dig> and 12 h post infection  under in vitro conditions .
no transcripts for these six orfs were detected in the uninfected cell controls at any time point.
the seventh predicted orf, which was located in the large inverted repeat region flanked by icp <dig> and icp <dig> , was excluded from further analyses as qrt-pcr targeting this orf could not confirm transcription.
the six orfs for which transcription was confirmed were annotated pw <dig> to pw <dig>  pw <dig> was encoded in the trs/irs repeat region , and no significant structural or sequence domains or motifs were identified within it.
four novel orfs, pw <dig> to pw <dig>  were encoded in the us region as a cluster downstream of us <dig> .
no putative conserved domains were detected in the polypeptides encoded by pw <dig> and  <dig>  although pw <dig> and  <dig> had increased internal hydrophobicities, suggestive of transmembrane domains.
structural prediction analyses using i-tasser suggested potential structural homologues for pw <dig>  and pw <dig> .
no significant structural homologues were predicted for any of the other novel orfs.
analysis of the predicted amino acid sequence of pw <dig> detected a microneme/rhoptry antigen domain .
there was only one novel predicted orf within the ul region, pw <dig>  this orf was identified downstream of ul <dig>  analysis of the sequence of pw <dig> did not detect any conserved functional domain or motifs, but a hydrophobic domain near the carboxyl terminus was identified, suggestive of a transmembrane domain.
the mahv- <dig> genome lacked an identifiable ul <dig> homologue.
studies in hhv- <dig> have shown that ul <dig> encodes a tegument protein involved in relocalising ubiquitin ligase nedd <dig> in hhv- <dig> infected cells, and has a role in intracellular virion transport and/or virion release from the cell surface  <cit> .
ul <dig> polypeptide interacts and complexes with ul <dig> polypeptide as they co-localise in the golgi apparatus and in aggresome-like structures  <cit> .
in hhv- <dig>  ul <dig> is dispensable for virus growth in vitro, but deletion of it results in reduced production of cell-free infectious virus  <cit> .
in vivo, the presence of ul <dig> is important for pathogenicity of hhv- <dig>  with deletion mutants having reduced neuroinvasiveness  <cit> .
the hydrophobic c-terminal region of ul <dig> is particularly important for pathogenicity  <cit> .
a similar hydrophobic region was identified in the c-terminus of pw <dig>  which was encoded directly downstream of ul <dig>  this may indicate that pw <dig> is a distant ul <dig> homologue, although it only shared 17 % aa pairwise identity  with hhv- <dig> ul <dig>  however, at this stage any structural or functional similarities between pw <dig> and ul <dig> remain unclear, particularly as preliminary analyses of predicted tertiary structures did not identify significant structural similarities.
in the absence of conserved motifs or domains, the sequences of pw <dig> to pw <dig> provide no indication of the potential functions of these novel polypeptides.
the identification of a rhoptery antigen domain in pw <dig> may suggest an association with organelles, but little else can be inferred.
high relative levels of transcript of pw <dig> to pw <dig> at  <dig> hpi may indicate that they are transcribed at an early stage of the replication cycle, but further studies are necessary to better differentiate the kinetics of expression of these orfs.
it is not clear whether these genes are important for in vitro replication or in vivo pathogenicity.
gene deletion studies or functional studies of the products of these orfs would be necessary to elucidate their function.
the clustering of novel orfs identified in the us/ir region, pw <dig> to pw <dig>  suggest that they may have been acquired in a single event, possibly from an unknown host or another virus during virus speciation.
sequence comparisons with other marsupial herpesviruses would help determine whether the novel orfs are unique to mahv- <dig>  or are instead orfs common to herpesviruses infecting metatherians.
CONCLUSIONS
this is the first genome sequence of a herpesvirus that infects metatherians, a taxonomically unique mammalian clade.
members of the simplexvirus genus are remarkably conserved, so the absence of orfs otherwise conserved in eutherian and avian alphaherpesviruses contributes to our understanding of the alphaherpesvirinae more generally.
together with the sequence similarities observed to the human herpesviruses, these conclusions indicate that further study of metatherian herpesvirus genetics and pathogenesis will provide a unique approach to understanding herpesvirus-mammalian interactions.

 members of the herpesviridae have a linear double-stranded dna genome, 120– kbp in length, and cause significant morbidity and mortality in diverse groups of animals. members are further classified into three subfamilies; the alpha-, beta- and gammaherpesvirinae. reports of herpesvirus infections in the marsupialia date back to the 1970s. the first isolation of a marsupial herpesvirus was from a fatal outbreak of severe respiratory disease and systemic organ failure in a zoological collection of parma wallabies. the isolation of this alphaherpesvirus, designated macropodid herpesvirus, was closely followed by the isolation of a second, related, herpesvirus from fatal cases of disease in several vulnerable macropod species. the macropodid viruses were detected in animals displaying some clinical signs of disease similar to those caused by human herpesviruses and infection, such as conjunctivitis and vesicular anogenital lesions, but also included hepatic disease. in the following years, before the identification of additional marsupial herpesviruses, evidence of herpesvirus infection in metatherians was largely observed through electron microscopy or sero-epidemiological surveys. these sero-prevalence studies measured neutralising antibodies to mahv- which were detected in both wild and captive populations of marsupials. since eleven additional marsupial herpesviruses have been identified, including a closely-related alphaherpesvirus, macropodid herpesvirus, in free-ranging eastern grey kangaroos with clinical signs of respiratory and possible neurological disease and two gammaherpesviruses from macropods. a further eight herpesviruses have been identified in other marsupial species, though little sequence data are available for these viruses. despite its classification as a simplexvirus, early genome hybridization studies of mahv- identified a type d genome structure of approximately kbp in length, containing a short unique region, flanked by large inverted repeat sequences joined to a long unique region. mahv- occurs as only two equimolar genomic isomers. these genomic features are characteristic of varicelloviruses such as varicella zoster virus and pseudorabies virus and contrast with those of mahv- mahv- has a type e genome arrangement, more typical of the simplexviruses, and occurs as four equimolar genomic isomers. to date mahv- is the only alphaherpesvirus that encodes both icp and the host-derived oncogene thymidylate synthase. sequence analysis of two conserved orfs in mahv- − and − as well as analyses of their antigenic relationships, has clustered these macropodid viruses closely with the primate simplexviruses. as metatherian and eutherian mammals are believed to have diverged over million years ago, this viral phylogenetic grouping differed from the typical virus-host co-evolutionary pattern observed within the herpesviridae and was instead suggestive of a recent and complex speciation event. this study aimed to sequence and analyse the full genome of the metatherian alphaherpesvirus, mahv- with particular attention to novel genomic features.  whole genome sequence analysis the genome of mahv- is the first metatherian herpesvirus to be sequenced. excluding the genomic termini, which remained unresolved, the final genome length of mahv- was approximately kbp, larger than previously predicted. this difference appears to be due to a larger than predicted inverted repeat region. this included a kbp ul region and a kbp us region flanked by kbp inverted repeat sequences. the mahv- genome had a g + c content of  %, but had a higher g + c content within the irs/trs regions. the final genome assembly had a mean depth of reads per bp and approximately 95 % of reads had a quality score of at least phred three origins of replication were identified. the origin of lytic replication was located between ul and ul in the ul region and the oris was located within the irs/trs regions. thus two copies of oris were present, as in the genomes of hhv- and − fig. schematic representation of the genome arrangement of macropodid herpesvirus herpesviral conserved orfs are annotated in grey using the hhv- and hhv- orf nomenclature. novel orfs are given a pw prefix. unique long genome region, unique short genome region, trs/irs = inverted repeat flanking unique short region, oril/s = origins of replication conserved alphaherpesvirus orfs the ul region of the mahv- genome encoded orfs common to other herpesviruses. the predicted protein sequences of these orfs shared between 41 % and 73 % aa pairwise identity with hhv- and − homologues. in the us region the mahv- genome encoded seven orfs common to other simplexviruses, with the predicted protein sequences sharing between and 59 % aa pairwise identity with hhv- and − homologues. the irs/trs regions encoded five orfs, including those for thymidylate synthase, icp and icp. there were no homologues of ul ul ul or us identified in the mahv- genome. also, the us homologue was predicted to be non-functional, as the orf was prematurely truncated. this is consistent with previous published sequence data reporting a truncation of the mahv- gg orf. phylogenetic analyses using translated protein sequences of three core herpesvirus genes are shown in fig.  these analyses show that mahv- clusters most closely with other macropodid herpesviruses, as well as with the simplexviruses that infect primates. it also groups with the herpesvirus of an indonesian pteropodid bat. comparison of other viral core genes yielded similar clustering patterns. comparison of the mahv- ul and ul orfs with those of the recently sequenced fruit bat herpesvirus showed that these orfs shared and 67 % pairwise aa identity, respectively. this similarity is comparable to that seen between mahv- and hhv-1/hhv-, which may offer some insight into their evolutionary relationship, for example, may suggest transmission of herpesviruses from primates to bats, and then to marsupials. sequencing of herpesviruses from other metatherians, as well as other australasian mammals, will be needed to determine the significance of this clustering.table predicted open reading frames identified in different structural regions of the macropodid herpesvirus genome and percentage pairwise amino acid identity to orfs in related alphaherpesviruses §putative function of encoded polypeptides in mahv-1 #amino acid identities with homologues in human herpesvirus and and macropodid herpesviruses and fig. the relationship between macropodid herpesvirus and other viruses from the alphaherpesvirinae subfamily. neighbour-joining distance trees were generated using the translated protein sequences of conserved herpesvirus orfs: a glycoprotein b, gb; b dna polymerase, dpol; and c glycoprotein d, gd. bootstrap values are shown for each branch, with gallid herpesvirus as an outgroup. the abbreviations and genbank accession details are: macropodid herpesvirus, macropodid herpesvirus, macacine herpesvirus, fruit bat herpesvirus, gallid herpesvirus, gallid herpesvirus, human herpesvirus, human herpesvirus, human herpesvirus, saimiriine herpesvirus, bovine herpesvirus, bovine herpesvirus, bovine herpesvirus, canine herpesvirus, felid herpesvirus, equine herpesvirus, equine herpesvirus, suid herpesvirus although ul and ul are conserved in all other alphaherpesviruses examined to date, gene deletion studies in the human simplexviruses have found that deletion of ul and ul does not affect viral replication or cell-to-cell spread in vitro. in vivo functions of the ul and ul encoded accessory proteins are not well understood, but they colocalise and directly interact with the transcriptional repressor icp encoded by us in small dense nuclear bodies and may also be involved in the late phase of viral replication. the absence of gj is also of note. this is the third simplexvirus found to lack an orf encoding a gj homologue, which is otherwise conserved in the simplexvirus genus. the other two simplexviruses lacking gj are leoporid herpesvirus and fbhv-. in other herpesviruses gj inhibits host cell apoptosis by inducing an increase in concentrations of reactive oxygen species in the host cell. it is unclear whether the absence of ul ul and gj might be related to adaptation to a new host or whether it may be the result of virus passage in vitro. in respect to gj, the former scenario could be more likely, as the absence of this orf in other non-primate herpesviruses shows that it is not strictly conserved within the genus. sequencing of other marsupial alphaherpesviruses, particularly field isolates, would help to resolve this finding. unique or hypothetical orfs seven unique hypothetical orfs were identified; one in the ul region, two located in the irs/trs regions, and four in the us region. viral transcript analyses by qrt-pcr confirmed that six of the seven predicted orfs were transcribed at both and 12 h post infection under in vitro conditions. no transcripts for these six orfs were detected in the uninfected cell controls at any time point. the seventh predicted orf, which was located in the large inverted repeat region flanked by icp and icp, was excluded from further analyses as qrt-pcr targeting this orf could not confirm transcription. the six orfs for which transcription was confirmed were annotated pw to pw pw was encoded in the trs/irs repeat region, and no significant structural or sequence domains or motifs were identified within it. four novel orfs, pw to pw were encoded in the us region as a cluster downstream of us. no putative conserved domains were detected in the polypeptides encoded by pw and although pw and had increased internal hydrophobicities, suggestive of transmembrane domains. structural prediction analyses using i-tasser suggested potential structural homologues for pw and pw. no significant structural homologues were predicted for any of the other novel orfs. analysis of the predicted amino acid sequence of pw detected a microneme/rhoptry antigen domain. there was only one novel predicted orf within the ul region, pw this orf was identified downstream of ul analysis of the sequence of pw did not detect any conserved functional domain or motifs, but a hydrophobic domain near the carboxyl terminus was identified, suggestive of a transmembrane domain. the mahv- genome lacked an identifiable ul homologue. studies in hhv- have shown that ul encodes a tegument protein involved in relocalising ubiquitin ligase nedd in hhv- infected cells, and has a role in intracellular virion transport and/or virion release from the cell surface. ul polypeptide interacts and complexes with ul polypeptide as they co-localise in the golgi apparatus and in aggresome-like structures. in hhv- ul is dispensable for virus growth in vitro, but deletion of it results in reduced production of cell-free infectious virus. in vivo, the presence of ul is important for pathogenicity of hhv- with deletion mutants having reduced neuroinvasiveness. the hydrophobic c-terminal region of ul is particularly important for pathogenicity. a similar hydrophobic region was identified in the c-terminus of pw which was encoded directly downstream of ul this may indicate that pw is a distant ul homologue, although it only shared 17 % aa pairwise identity with hhv- ul however, at this stage any structural or functional similarities between pw and ul remain unclear, particularly as preliminary analyses of predicted tertiary structures did not identify significant structural similarities. in the absence of conserved motifs or domains, the sequences of pw to pw provide no indication of the potential functions of these novel polypeptides. the identification of a rhoptery antigen domain in pw may suggest an association with organelles, but little else can be inferred. high relative levels of transcript of pw to pw at hpi may indicate that they are transcribed at an early stage of the replication cycle, but further studies are necessary to better differentiate the kinetics of expression of these orfs. it is not clear whether these genes are important for in vitro replication or in vivo pathogenicity. gene deletion studies or functional studies of the products of these orfs would be necessary to elucidate their function. the clustering of novel orfs identified in the us/ir region, pw to pw suggest that they may have been acquired in a single event, possibly from an unknown host or another virus during virus speciation. sequence comparisons with other marsupial herpesviruses would help determine whether the novel orfs are unique to mahv- or are instead orfs common to herpesviruses infecting metatherians.  this is the first genome sequence of a herpesvirus that infects metatherians, a taxonomically unique mammalian clade. members of the simplexvirus genus are remarkably conserved, so the absence of orfs otherwise conserved in eutherian and avian alphaherpesviruses contributes to our understanding of the alphaherpesvirinae more generally. together with the sequence similarities observed to the human herpesviruses, these conclusions indicate that further study of metatherian herpesvirus genetics and pathogenesis will provide a unique approach to understanding herpesvirus-mammalian interactions. 
further study of metatherian herpesvirus genetics and pathogenesis provides a unique approach to understanding herpesvirus-mammalian interactions. this is the first genome sequence of a herpesvirus that infects metatherians, a taxonomically unique mammalian clade. while many placental herpesvirus genomes have been fully sequenced, the complete genome of a marsupial herpesvirus has not been described. here we present the first genome sequence of a metatherian herpesvirus, macropodid herpesvirus. members of the simplexvirus genus are remarkably conserved, so the absence of orfs otherwise retained in eutherian and avian alphaherpesviruses contributes to our understanding of the alphaherpesvirinae. we identified six novel orfs and confirmed their transcription by rt-pcr.
 while many placental herpesvirus genomes have been fully sequenced, the complete genome of a marsupial herpesvirus has not been described. here we present the first genome sequence of a metatherian herpesvirus, macropodid herpesvirus.  the mahv- viral genome was sequenced using an illumina miseq sequencer, de novo assembly was performed and the genome was annotated. the mahv- genome was kbp in length and clustered phylogenetically with the primate simplexviruses, sharing 67 % nucleotide sequence identity with human herpesviruses and the mahv- genome contained predicted open reading frames homologous to those in other herpesvirus genomes, but lacked homologues of ul ul ul and glycoprotein j. this is the first alphaherpesvirus genome that has been found to lack the ul and ul homologues. we identified six novel orfs and confirmed their transcription by rt-pcr.  this is the first genome sequence of a herpesvirus that infects metatherians, a taxonomically unique mammalian clade. members of the simplexvirus genus are remarkably conserved, so the absence of orfs otherwise retained in eutherian and avian alphaherpesviruses contributes to our understanding of the alphaherpesvirinae. further study of metatherian herpesvirus genetics and pathogenesis provides a unique approach to understanding herpesvirus-mammalian interactions. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords marsupialherpesvirusgenomewildlifemacropodkangarooherpes simplexissue-copyright-statement© the author 2016 