BACKGROUND
avipoxviruses have been shown to naturally infect  <dig> of the approximately  <dig>  described species of both wild and domestic birds  <cit> , yet relatively little is known about the host range and genetic diversity of these viruses.
there are currently only ten defined- and three tentative avipoxvirus species  <cit>  with species names assigned according to the bird species which they infect or from which they were isolated  <cit> .
this current method of taxonomy and classification has been criticized because it has been found that multiple avian species can be infected by a single viral species and/or multiple viral species can infect a single avian species  <cit> .
phylogenetic relationships of avipoxviruses have been analyzed based on the gene corresponding to vaccinia virus  p4b   <cit> , indicating that all avipoxvirus strains cluster into  <dig> major clades, namely, a -like), b -like) and c .
clade a can be further divided into seven subclades  and clade b is comprised of three subclades   <cit> .
thus far, the genomes of only three avipoxviruses have been published; a pathogenic american strain of fowlpox virus   <cit> , an attenuated european strain of fowlpox virus derived from european fwpv hp <dig> passaged over  <dig> times in chick embryo fibroblasts   <cit> , and a virulent canarypox virus isolated from a canary   <cit> .
comparison of the cnpv and fwpv genomes reveals overall synteny in genome arrangement with similar genetic complements.
they do however, exhibit significant differences in the terminal, variable regions as well as in three internal, variable regions which is in contrast to the conservation of central genomic regions in other chordopoxviruses .
these variable regions within the conserved central region of the genomes occur near the junctions of areas that were identified in fwpv as rearranged relative to other chpvs and contain genes involved in virus-host interactions  <cit> .
avipoxviruses are considerably divergent from other chpvs  <cit>  and may constitute a separate subfamily within the poxviridae family  <cit> .
fp <dig>  fpvus and cnpv have large genomes of  <dig>   <dig> and  <dig> kbp encoding  <dig>   <dig> and  <dig> open reading frames  respectively  <cit> .
<dig> genes have been found to be conserved amongst all chpvs and to comprise the minimum essential genome  <cit> .
the remainder of the genetic component of avian poxviruses is largely made up of immunomodulatory and host specific genes located in the terminal regions of the genome, that have allowed the viruses to take advantage of their unique hosts.
laidlaw and skinner  <cit>  compared virulent  and hp <dig> ) and attenuated, tissue culture adapted ) fwpv strains.
instead of the predicted changes in immunomodulators as the mechanism of attenuation, members of multigene families, especially those encoding ankyrin repeat proteins were seen to be the drivers of attenuation  <cit> .
ankyrin repeat proteins are thought to be involved in poxvirus host-range  <cit> , and have previously been implicated in the attenuation of sheeppox virus  <cit> .
over 49%  of the cnpv genome and 38%  of the fwpv genome are comprised of genes belonging to gene families,  <dig> and  <dig> of which are orfs containing ankyrin repeats respectively  <cit> .
these and other differences in immunomodulatory gene families encoded by avipoxviruses may account for the extensive variability in virulence, host range and host interaction  <cit> .
fpvus and cnpv are significantly divergent with amino acid identity between orf homologues  being similar to that observed between different chpv genera  <cit> .
the cnpv genome contains  <dig> orfs not present in fwpv,  <dig> of which encode unique, hypothetical proteins.
cnpv contains orfs coding for two additional proteins involved in nucleotide metabolism , tnfr , an il- <dig> like protein , cellular ubiquitin , a protein tyrosine phosphatase , a thioredoxin binding protein , and two rep like proteins .
fwpv contains  <dig> orfs not present in cnpv,  <dig> of which encode hypothetical proteins.
homologues of fpv <dig> and fpv <dig> are notably absent from cnpv, and are similar to orfs from insect baculoviruses, and from avian herpes- and adenoviruses respectively.
members of the columbidae family, consisting of pigeons and doves are susceptible to avipoxvirus infection and cases have been reported in several columbiform species including the mourning dove  <cit> , laughing dove  <cit> , white-tailed laurel-pigeon  <cit> , rock pigeon  <cit> , feral pigeon  <cit>  and others  <cit> .
due to the host species based approach to avipoxvirus taxonomy, poxviruses infecting the columbiformes are designated as pigeonpox viruses .
this study focuses on two avipoxviruses, one isolated from a feral pigeon  from port elizabeth, south africa  in  <dig> and the other from an african penguin  housed at sanccob, a seabird rehabilitation centre, following an oil spill off the coast of cape town, south africa  in  <dig>  <cit> .
these locations are 790 km apart.
isolation and preliminary characterisation of pepv has been previously described  <cit> .
poxvirus infections have not been observed in wild african penguins living in coastal waters around cape town .
mosquito netting was introduced at sanccob in  <dig> to protect housed penguins from contracting avian malaria and there has been no evidence of pox infections since then, suggesting that the initial infections were transmitted by biting insects.
it is worth noting that many pigeons nest in the sanccob enclosure and could well have been the primary host for this virus, which could have been transmitted to penguins via biting insects.
it is therefore possible that the african penguin is not the primary host of pepv.
when grown on the chorioallantoic membranes of embryonated 10– <dig> day old chicken eggs, the pigeonpox virus fep <dig> caused greater cell proliferation and immune cell infiltration as compared to pepv, cnpv, fwpv and some of the other avipoxvirus isolates investigated in our laboratory  <cit> .
furthermore, based on phylogenetic analysis of four conserved genetic regions, fep <dig> groups in clade a, subclade a <dig> and pepv belongs to clade a, sub-clade a <dig>  fpvus and fp <dig> group in clade a <dig> and cnpv groups in clade b <dig>  <cit> .
poxviruses isolated from the african, magellanic and humboldt penguins group in clades a <dig>  a <dig>  and b <dig>  respectively  <cit> .
columbiformes have been shown to be susceptible to viruses from clade a   <cit>  and clade b   <cit> .
in this study we have sequenced the genomes of two novel south african avipoxviruses using the  <dig>  and ion torrent  platforms.
we present a purpose-designed bioinformatics pipeline for analysis of next generation sequence data of avian poxviruses and compare the different avipoxviruses sequenced to date with particular attention to the viruses isolated from a feral pigeon  and african penguin .
RESULTS
the fep <dig> and pepv genomes were assembled into contiguous sequences of  <dig>  bp and  <dig>  bp respectively.
because the terminal hairpin loops were not sequenced, for each genome the left most nucleotide was arbitrarily nominated base  <dig>
phylogenetic analysis of fep <dig> and pepv
phylogenetic analysis was performed using the dna polymerase gene, a concatenated amino acid sequence of  <dig> translated orfs and the entire genomic sequence .
all three analyses produced comparable tree topologies and the tree based on dna polymerase is given in figure  <dig>  fep <dig> and pepv are most closely related to one another, grouping on the fwpv branch , and separate from cnpv .
comparisons of fep <dig>  pepv, fwpv and cnpv dna
the four genomic sequences of fep <dig>  pepv, fwpv and cnpv were compared for nucleotide percentage identity.
pepv and fep <dig> are more closely related to one another  than they are to either fwpv  or cnpv .
dot plot analysis reveals a large deletion of  <dig> kbp in fep <dig> compared to both of pepv and fwpv .
like the other avipoxviruses, fep <dig> and pepv have a/t rich genomes, both with  <dig> % a + t content.
inverted terminal repeats
the fep <dig> and pepv genomes, like most other poxviruses, contain two identical inverted repeated sequences at their termini .
these are  <dig>  bp in fep <dig> and  <dig>  bp in pepv, both of which are shorter than the itrs of the other sequenced avipoxviruses .
like fpvus and cnpv, their a + t content within the itrs is lower than average -  <dig> % for fep <dig> and  <dig> % for pepv.
at the terminal ends of each itr in fep <dig> exists a 257 bp repeat region with  <dig>  and  <dig>  copies respectively of a 34 bp and 55 bp tandem repeat.
in contrast, pepv has a 165 bp repeat region containing  <dig>  copies of a 47 bp tandem repeat.
fpvus contains a  <dig>  kbp region with  <dig> copies of a 31-32 bp tandem repeat and cnpv contains at least  <dig>   <dig>  and  <dig> copies of 17-, 41-, and 48-bp tandem repeats respectively within the terminal regions of each itr  <cit> .
the repeated sequences are unique for the different avipoxviruses.
overall arrangement of orfs in genome
the genomes of fep <dig> and pepv have been compared to fpvus, fp <dig> and cnpv in figure  <dig>  figure  <dig> provides a multiple sequence alignment and comparative orf map of fep <dig>  pepv and fpvus genomes.
clearly, there is conservation amongst the  <dig> sequenced avipoxviruses, but, at the same time, there are major differences between them.
unlike the orthopoxviruses, the avipoxviruses exhibit variation within the central core region where blocks of avipoxvirus genes have been inverted and/or transposed, suggesting that this genus is the most divergent of the chpvs  <cit> .
comparison of gene orthologues indicate overall genomic synteny between fep <dig>  pepv and fpvus and genomic organisation is similar to that of other sequenced chpvs  <cit> .
additional file 1: table s <dig> lists all open reading frames  potentially coding for proteins of > <dig> amino acids for fep <dig> and pepv, and figures  <dig> and  <dig> represent these graphically.
fep <dig> contains  <dig> orfs which have been annotated as putative genes and as orthologues of fpvus and other genes .
these orfs represent an approximate coding density of  <dig> % and encode proteins between  <dig> and  <dig>  amino acids .
as published by hendrickson et al.,  <cit> , an orf was annotated as a whole gene if intact at its 5’ end with at least 80% the length of its orthologue.
an orf intact at its 5’ end but less than 80% of its orthologue length has been annotated as a truncated gene.
any orf that has been disrupted at its 5’ end has been annotated as a fragmented gene, which is not expected to be transcribed and/or translated into a functional gene product  <cit> .
of these  <dig> orfs,  <dig> have been annotated as truncated genes .
a further  <dig> of these orfs are fragmented forms of  <dig> larger orthologous genes .
therefore a total of  <dig> orfs have been annotated as full length putative genes.
pepv contains  <dig> orfs which have been annotated here as putative genes  representing an approximate coding density of  <dig> % and encoding proteins ranging between  <dig> and  <dig>  amino acids long .
a total of  <dig> of these orfs have been annotated as whole genes,  <dig> as truncated genes  and  <dig> as fragments of larger genes .
where a gene is deleted or disrupted in only one virus , then the status of the other is given as either intact , truncated , fragmented  or translocated .
putative functions for the deleted genes have been given where possible.
relative to the fpvus genome, fep <dig> and pepv have  <dig> and  <dig> deleted orfs respectively .
fep <dig> contains  <dig> inserted orfs relative to fpvus, comprising  <dig> fragments of larger genes,  <dig> truncated genes and  <dig> intact genes .
similarly, pepv contains  <dig> inserted orfs, which include  <dig> gene fragments,  <dig> truncated genes and  <dig> intact genes .
a total of  <dig> intact orfs are shared between pepv and fep <dig> throughout the genome and  <dig> of these do not have any functional orthologue in fpvus .
amongst pepv, fep <dig> and fwpv, the most conserved orf is the orthologue of fpv <dig>  which encodes the dna binding virion core phosphoprotein and shares 100% amino acid identity amongst the three viruses.
inserted genes have been listed if they are present in fep <dig> or pepv in an unexpected site as predicted by genomic synteny with the fpvus.
where no homology to fpvus genes is present, the name of the best blastp hit has been used.
both pepv and fep <dig> lack sequences similar to the reticuloendotheliosis virus  observed in fpvus and other avipoxvirus strains  <cit> .although the fep <dig> and pepv genomes are closely related and syntenic to the fwpv genome overall , when compared to each other, and to fwpv, these viruses display differences in gene content throughout their genomes.
of particular note is the large  <dig> kbp deletion in fep <dig> relative to both pepv  and fpvus .
conserved genes
there are  <dig> genes that are conserved amongst all five sequenced avipoxviruses, which have been highlighted in blue in figure  <dig>  these include genes that lie within the more variable terminal regions of the genome.
these avipoxvirus core genes were defined as such if intact orthologues of the genes were present at least once in all five avipoxvirus sequences.
the  <dig> apv core genes include the  <dig> core genes conserved in all chpvs which are involved in essential functions such as replication, transcription and virion assembly  <cit>  .
there are an additional  <dig> genes conserved in all avipoxviruses with a variety of different functions .
gene families
avipoxviruses contain extensive gene families, which vary greatly between different species.
in cnpv, these comprise over 49% of the genome, whereas they encompass 38% of the fwpv genome  <cit> .
in cnpv and fwpv, these gene families account for much of the variation between the two genomes  <cit> .
intact members of gene families comprise 33% of the fep <dig> and pepv genomes.
table  <dig> summarises the differences in the number of intact gene family proteins found in the five sequenced avipoxvirus genomes.
a summary of the numbers of intact gene family proteins in pigeonpox virus , penguinpox virus , fowlpox virus us strain , fowlpox virus fp <dig> strain , canarypox virus  and vaccinia virus .
“0” is given where no member of the gene family is present in the respective virus.
gene translocations and duplications
analysis of the fep <dig> and pepv genomes revealed several occurances of gene duplication and translocation relative to fwpv.
in some instances the postion of the gene was closer to that observed in cnpv than fwpv.
a second copy of the ankyrin repeat family gene, fpv <dig> orthologue  is found in the equivalent place in the left hand region of each african avipoxvirus genome.
disrupted and deleted genes
relative to fpvus, fep <dig> contains  <dig> truncated fpvus genes and the fragmented forms of  <dig> fpvus orthologues.
pepv contains  <dig> truncated genes and the orf fragments of  <dig> fpvus orthologues .
both fep <dig> and pepv contain truncated or fragmented remains of orfs with similarity to cnpv .
relative to the fpvus genome, fep <dig> and pepv have  <dig> and  <dig> deleted orfs respectively  that are absent from any potential coding regions of their genomes.
inserted genes
relative to fwpv, the pepv and fep <dig> genomes both contain several inserted genes .
pepv, but not fep <dig> or fwpv  <cit> , has orthologues of cnpv <dig>  which encodes a protein similar to tumour necrosis factor receptor   <cit>  and a  <dig> amino acid long orthologue of ubiquitin  which is most similar to human ubiquitin, s5a ubiquitin-interacting motif- <dig>    <cit> .
both pepv  and fep <dig>  contain orthologues of thymidylate kinase   .
fep <dig> and pepv both encode a putative interleukin- <dig>  gene with  <dig> % amino acid identity between them  and limited similarity to the cnpv orthologue  .
amino acid identity between all available avian il- <dig> genes and pepv and fep <dig> fall between  <dig> % and  <dig> %, however for cnpv the identities are significantly lower .
the pepv, fep <dig> and cnpv il- <dig> genes are most similar to that of a collared flycatcher  sharing  <dig> %,  <dig> % and  <dig> % amino acid identity respectively .
fwpv does not encode an il- <dig> gene  <cit> .
the respective positions of fep <dig>  pepv and cnpv il- <dig> orthologues were analysed to determine the possibility of these genes existing in the three genomes as a result of independent horizontal gene transfer events .
based on this analysis, it is possible that there were two independent transfer events into avian poxviruses, one into cnpv and another into the progenitor of pepv and fep <dig>
pepv and fep <dig> both encode homologues of tanapox virus  and yaba-like disease virus  67r which are orthologues of vacv c7l.
these c7l-like genes  are in the equivalent genome positions, between orthologues of fpv <dig>  and fpv <dig>  .
comparison of fep <dig> and pepv to attenuation and lineage specific mutations
in order to determine lineage specific mutations, laidlaw and skinner  <cit>  determined the sequence of hp <dig> , the virulent parent of fp <dig>  at all loci where fp <dig> differed from fpvus   <cit> .
here we compared nucleotide changes in pepv and fep <dig> to  <dig> genomic positions influencing amino acid composition of  <dig> open reading frames between european and american strains as previously identified  <cit> .
in  <dig> and  <dig> of the places, fep <dig> and pepv were the same as hp <dig>  respectively.
in  <dig> and  <dig> of the places, fep <dig> and pepv were the same as fpvus  respectively.
pepv and fep <dig> differ from both hp <dig> and fpvus in  <dig> and  <dig> places respectively.
discussion
genomic sequences have been determined for two novel south african avipoxviruses.
the dna sequences of pepv and fep <dig> are significantly different from those of cnpv  and fwpv .
in orthopoxviruses , the internal region of different species share at least 96% identity when compared at the nucleotide level, while different strains from the same species share at least 99% nucleotide sequence identity  <cit> .
these identities have been listed as criteria for establishing poxvirus taxonomy  <cit> .
therefore, according to the identities between pepv, fep <dig> and fpvus genomes , as well as the differences in phylogenetics and growth characteristics between these two viruses  <cit> , one can deduce that pepv and fep <dig> are separate species of apv.
laidlaw and skinner  <cit>  identified geographical lineage-specific mutations which distinguished european fowlpox viruses, fp <dig> and hp <dig> from the american strain fpvus.
a comparison of pepv and fep <dig> with the published fwpv sequences at these sites shows that pepv and fep <dig> share lineage-specific mutations with both hp <dig> and fpvus.
the south african viruses are no more closely related to either the american or the european virus.
these results suggest that fep <dig> and pepv originate from a common ancestor that diverged relatively recently from a fwpv-like progenitor which was more distantly related to the cnpv-like progenitor.
it is important to note that the divergence between avipoxvirus species is comparable to that between some poxvirus genera and the different species within the opv genus are highly similar.
fep <dig> and pepv are most closely related to fwpv.
despite this, relative to fwpv, the pepv and fep <dig> genomes both contain several genes that are more closely related to cnpv throughout their genomes.
fep <dig> and pepv encode  <dig> and  <dig> intact cnpv orthologues respectively , five of which are not in fwpv, including a tnfr-like protein  and a tmpk protein  .
the remainder are more similar to cnpv orthologues but are present in fwpv.
rather than being acquired through horizontal gene transfer or recombination, these genes are most probably from a common ancestor.
their existence suggests that fep <dig> and pepv viruses diverged prior to the present day fwpv, somewhere between the cnpv and fwpv branches.
several of these cnpv-like genes exist as gene fragments , which is indicative of their gradual loss through progressive mutation, rearrangements and/or deletions over many rounds of virus replication.
there are several instances of gene duplication and translocation in fep <dig> and pepv relative to fwpv .
most of these involve genes containing ankyrin repeats.
virulent fwpv contains  <dig> ankyrin repeat containing genes and attenuated fp <dig> contains  <dig> of these genes.
pepv contains  <dig> ankyrin repeat-containing genes whereas fep <dig> contains only  <dig> .
it is postulated that loss of these genes may be responsible for the attenuation of the virus  <cit> .
thus the reduced numbers of intact ankyrin repeat genes observed in fep <dig> compared to pepv would suggest that this virus is less virulent.
on the other hand, the expansion of the number of ankyrin repeat genes in certain avipoxvirus strains could represent the forming of adaptive genomic accordions, similar to that of the k3l gene in opv, the formation of which has been described to play an adaptive role in these viruses  <cit> .
the most striking difference between fep <dig> and the fwpv-like avipoxviruses, is a large deletion of ~ <dig> kbp from the central, usually conserved, region of the genome.
orfs corresponding to fpv <dig> , fpv <dig> and fpv <dig> , fpv <dig>  and fpv <dig>  are deleted in fep <dig> at this site.
the existence of gene fragments and insertions on the borders of this deletion, as well as in the equivalent regions in fwpv and pepv, suggests that this is a “hotspot” of genetic change, the mechanism of which is uncertain.
relative to the fpvus genome, fep <dig> and pepv have  <dig> and  <dig> deleted orfs respectively .
most of the genes deleted in pepv and fep <dig> are members of multi-gene families, the disruption of which has been implicated in the attenuation of poxviruses  <cit> , however there are several other deletions of significant orfs.
of interest, the orthologue of fpv <dig> encoding a dnase ii has been deleted in fep <dig> but is in intact in pepv.
in fwpv, fpv <dig> represents the large subunit of cellular dnase ii  <cit>  and cellular dnase ii is thought to function in dna catabolism during apoptosis  <cit> .
additionally, the orthologue of fpv <dig> encoding one of the two α-snap genes present in fwpv, is absent from fep <dig> but intact in pepv .
eukaryotic α-type soluble nsf attachment proteins  are involved in vesicular transport through the golgi apparatus and for exocytosis  <cit> .
the fpv <dig> gene has been shown to be conserved in fwpv strains but non-essential to viral replication and it is thought to be involved in virus-host interactions  <cit> .
although the fpv <dig> gene orthologue has been deleted in fep <dig>  a second α-snap-like gene  exists in the genome which exhibits  <dig> % and  <dig> % amino acid identity with fpv <dig> and pepv <dig> respectively.
fwpv contains two genes with homology to cellular β-nerve growth factor  .
in fep <dig>  the orthologue of fpv <dig> is completely deleted and the orthologue of fpv <dig>  is fragmented .
conversely, in pepv, the orthologue of fpv <dig> is completely deleted and the orthologue of fpv <dig>  is fragmented .
an additional β-ngf-like gene is observed in a truncated form in fep <dig>  and as a fragmented gene in pepv  .
these are most similar to the cnpv gene cnpv <dig>  which exists as a fragment in fwpv  <cit> .
the fep <dig> orf, fep <dig> is truncated to  <dig> amino acids by the introduction of a premature stop codon, where the intact cnpv <dig> orthologue is  <dig> amino acids.
it is unknown whether this protein would be functional in fep <dig> but it is unlikely that pepv encodes a functional β-ngf protein.
β-ngf has proinflammatory properties and is produced by cells in response to infection, injury and stress  <cit> .
fpv encoded β-ngf is thought to be involved in promoting survival in infected cells and could have a role in inhibiting antiviral immune responses in the host  <cit> .
previously, it was speculated that the absence of a β-ngf gene in an avipoxvirus genome could be partly responsible for the decreased inflammatory response observed in pepv-infected cams as compared to fep <dig>  <cit> .
an orthologue of fpv <dig> encoding an il- <dig> binding protein has been deleted in pepv.
in fep <dig> this orf  has been truncated due to a 1 bp deletion resulting in a premature stop codon.
il- <dig> is a multifunctional pro-inflammatory cytokine that induces interferon gamma  production, th- <dig> responses and nk cell activity  <cit> .
it is modulated in vivo by il- <dig> binding protein  via a negative feedback mechanism  <cit> .
il18bp homologues are encoded by many poxviruses  <cit>  and have been found to inhibit il-18-dependent ifn-γ production  <cit> .
in fwpv, fpv <dig> is thought to have an anti-inflammatory function  <cit> .
pepv contains an orthologue of cnpv <dig>  which encodes a protein similar to tnf receptor  <cit> .
this orf is absent from fep <dig>  and fwpv  <cit> .
tumour necrosis factor  is a pro-inflammatory cytokine which is involved in induction of cytokines, cell proliferation, differentiation, necrosis and apoptosis  <cit> .
tnf -induced cellular responses are mediated by one of two types of tnf receptors  <cit> .
several poxviruses encode tnfr homologues which function to modulate tnf-induced anti-viral responses  <cit> .
pepv  and fep <dig>  both contain an orthologue of thymidylate kinase  , which is absent in fwpv  <cit> .
this is the only difference found with respect to the complement of nucleotide metabolism genes.
interestingly, despite being within the conserved central region of the genome, the tmpk gene orthologues occur within a region of pepv and fep <dig> that is highly variable compared to fwpv.
in addition, the cnpv orthologue insertions that occur within this particular variable region in fep <dig> and pepv occur in different sites across the cnpv genome.
the higher than average amino acid identity shared between fep <dig> and pepv  and the cnpv genomes , suggests that this tmpk gene is necessary and conserved in these viruses.
cnpv, fep <dig> and pepv are the only poxvirus species outside of the opv genus to contain a tmpk homologue.
vacv encodes tmpk  that is 42% identical to human tmpk  <cit> .
tmpk catalyzes an important step in the biosynthesis of  thymidine triphosphate and is essential for cell metabolism  <cit> .
in cnpv, fep <dig> and pepv, the presence of a tmpk gene suggests a different optimization of cellular nucleotide pools as compared to fwpv and probably affects cell and tissue tropism in the different viruses  <cit> .
pepv contains a  <dig> amino acid long orthologue of ubiquitin  which is most similar to human ubiquitin , s5a ubiquitin-interacting motif- <dig> .
cnpv also contains an intact ubiquitin orthologue    <cit> .
fep <dig>  and fpvus  encode the fragmented remains of an ubiquitin gene but no open reading frame is observed in either genome  <cit> .
in all four genomes these ubiquitin orfs or sequence remains are in the same location .
ubiquitin is a highly conserved  <dig> amino acid protein with diverse cellular functions effected by means of marking proteins for intracellular signalling  <cit> .
ubiquitin proteins are thought to affect the diverse ubiquitination-mediated cellular functions, which may impact on cnpv and pepv virulence and host range  <cit> .
other than cnpv and pepv, ubiquitin genes have only been found in two insect poxviruses, melanoplus sanguinipes   <cit>  and amsacta moorei   <cit> .
virus-encoded ubiquitin genes have also been identified in the baculoviridae, including in the autographa californica nuclear polyhedrosis virus   <cit> .
in acnpv, disruption of the ubiquitin gene was shown to have no effect on virus viability but to cause a decrease in virion budding and total infectious particle production  <cit> .
the ubiquitin encoding genes in pepv and cnpv may have an effect on virus production and budding but this remains to be determined.
fep <dig> and pepv both encode a putative interleukin- <dig>  gene .
putative orthologues of il- <dig> are also found in orf virus, bovine papular stomatitis virus   <cit> , lumpy skin disease virus   <cit>  and yaba-like disease virus   <cit> .
il- <dig> is a cytokine that has both immunostimulatory and immunosuppressive functions  <cit>  and the orf virus il- <dig> orthologue has been shown to be immunomodulatory in function  <cit> .
the il- <dig> genes encoded by pepv and fep <dig> may also be involved immune evasion, however, because of the divergence from host il- <dig> proteins , like cnpv <dig>  these genes may have a novel biological function  <cit> .
the il- <dig> gene is likely to have been incorporated into poxvirus genomes via horizontal gene transfer   <cit> .
bratke and mclysacht  <cit>  made use of comparative genomic information and synteny conservation to investigate hgt in pox genomes  <cit> .
here they found that the il- <dig> orthologue encoded by cnpv was in an unexpected location as compared to other poxviruses  but because of the lack of conservation at this region, no conclusion could be drawn about the transfer event into cnpv.
an analysis of the genome synteny of avipoxviruses in the region of the il- <dig> gene shows that the cnpv il- <dig> is in a different location despite being in a similar region to fep <dig> and pepv .
although this region is not well conserved between genera, it appears to be relatively well conserved between avian poxviruses making it possible that there were two independent transfer events into avian poxviruses, one into cnpv and one into the common ancestor of fep <dig> and pepv.
pepv and fep <dig> both encode a gene with homology to tanapox virus  and yaba-like disease virus  67r which are orthologues of vacv c7l and not found in fwpv or cnpv.
the poxvirus c7l family of host range genes functions by mediating poxvirus host range and antagonising the host defence system  <cit> .
vacv c7l orthologues are found in all orthopoxviruses and most mammalian poxviruses, with the exception of molluscum contagiosum virus and parapoxviruses  <cit> .
this is the first report of a c7l-like gene in avipoxviruses.
in mammalian cells, c7l has been shown to inhibit apoptosis  <cit> , antagonise the anti-viral effects of type  <dig> interferons  and interferon regulatory factor  <dig>   <cit>  and can antagonize the dsrna-activated protein kinase  pathway by inhibiting the phosphorylation of eif2a  <cit> .
it has previously been suggested that c7l orthologues are an important adaptation of mammalian poxviruses for replication in mammalian hosts  <cit> .
the presence of c7l orthologues in pepv and fep <dig> confounds these previous observations but the function of this gene in these two avipoxviruses remains to be determined.
the limited amino acid identity may suggest a novel function of these genes as compared to other c7l orthologues.
however, yldv 67 l shows only 28-30% identity to the vacv c7l protein but has been shown to function equivalently in supporting vacv replication in mammalian hosts  <cit> .
CONCLUSIONS
the genome sequences of fep <dig> and pepv have greatly added to the limited repository of genomic information available for the avipoxvirus genus.
in the comparison of fep <dig> and pepv to existing sequences, fwpv and cnpv, we have established insights into african avipoxvirus evolution.
although fep <dig> and pepv are more closely related to fwpv, the presence of whole or disrupted genes with similarity to cnpv genes that are absent in fwpv, suggests that fep <dig> and pepv originate from the common ancestor of cnpv and fwpv.
the presence of an intact gene in cnpv, fep <dig> and pepv where the fwpv counterpart is fragmented into two orfs further supports this theory as fragmented genes represent the gradual loss of genetic information during the process of virus evolution.
additional genome sequences of avipoxviruses would help to define avipoxvirus evolution as a whole.
the ongoing search for an ideal vaccine vector makes this work relevant.
future work could focus on how these two avipoxviruses differ from the well characterized fwpv and cnpv with respect to immune activation and foreign gene expression.

 avipoxviruses have been shown to naturally infect of the approximately described species of both wild and domestic birds, yet relatively little is known about the host range and genetic diversity of these viruses. there are currently only ten defined- and three tentative avipoxvirus species with species names assigned according to the bird species which they infect or from which they were isolated. this current method of taxonomy and classification has been criticized because it has been found that multiple avian species can be infected by a single viral species and/or multiple viral species can infect a single avian species. phylogenetic relationships of avipoxviruses have been analyzed based on the gene corresponding to vaccinia virus p4b, indicating that all avipoxvirus strains cluster into major clades, namely, a -like), b -like) and c. clade a can be further divided into seven subclades and clade b is comprised of three subclades. thus far, the genomes of only three avipoxviruses have been published; a pathogenic american strain of fowlpox virus, an attenuated european strain of fowlpox virus derived from european fwpv hp passaged over times in chick embryo fibroblasts, and a virulent canarypox virus isolated from a canary. comparison of the cnpv and fwpv genomes reveals overall synteny in genome arrangement with similar genetic complements. they do however, exhibit significant differences in the terminal, variable regions as well as in three internal, variable regions which is in contrast to the conservation of central genomic regions in other chordopoxviruses. these variable regions within the conserved central region of the genomes occur near the junctions of areas that were identified in fwpv as rearranged relative to other chpvs and contain genes involved in virus-host interactions. avipoxviruses are considerably divergent from other chpvs and may constitute a separate subfamily within the poxviridae family. fp fpvus and cnpv have large genomes of and kbp encoding and open reading frames respectively. genes have been found to be conserved amongst all chpvs and to comprise the minimum essential genome. the remainder of the genetic component of avian poxviruses is largely made up of immunomodulatory and host specific genes located in the terminal regions of the genome, that have allowed the viruses to take advantage of their unique hosts. laidlaw and skinner compared virulent and hp ) and attenuated, tissue culture adapted ) fwpv strains. instead of the predicted changes in immunomodulators as the mechanism of attenuation, members of multigene families, especially those encoding ankyrin repeat proteins were seen to be the drivers of attenuation. ankyrin repeat proteins are thought to be involved in poxvirus host-range, and have previously been implicated in the attenuation of sheeppox virus. over 49% of the cnpv genome and 38% of the fwpv genome are comprised of genes belonging to gene families, and of which are orfs containing ankyrin repeats respectively. these and other differences in immunomodulatory gene families encoded by avipoxviruses may account for the extensive variability in virulence, host range and host interaction. fpvus and cnpv are significantly divergent with amino acid identity between orf homologues being similar to that observed between different chpv genera. the cnpv genome contains orfs not present in fwpv, of which encode unique, hypothetical proteins. cnpv contains orfs coding for two additional proteins involved in nucleotide metabolism, tnfr, an il- like protein, cellular ubiquitin, a protein tyrosine phosphatase, a thioredoxin binding protein, and two rep like proteins. fwpv contains orfs not present in cnpv, of which encode hypothetical proteins. homologues of fpv and fpv are notably absent from cnpv, and are similar to orfs from insect baculoviruses, and from avian herpes- and adenoviruses respectively. members of the columbidae family, consisting of pigeons and doves are susceptible to avipoxvirus infection and cases have been reported in several columbiform species including the mourning dove, laughing dove, white-tailed laurel-pigeon, rock pigeon, feral pigeon and others. due to the host species based approach to avipoxvirus taxonomy, poxviruses infecting the columbiformes are designated as pigeonpox viruses. this study focuses on two avipoxviruses, one isolated from a feral pigeon from port elizabeth, south africa in and the other from an african penguin housed at sanccob, a seabird rehabilitation centre, following an oil spill off the coast of cape town, south africa in. these locations are 790 km apart. isolation and preliminary characterisation of pepv has been previously described. poxvirus infections have not been observed in wild african penguins living in coastal waters around cape town. mosquito netting was introduced at sanccob in to protect housed penguins from contracting avian malaria and there has been no evidence of pox infections since then, suggesting that the initial infections were transmitted by biting insects. it is worth noting that many pigeons nest in the sanccob enclosure and could well have been the primary host for this virus, which could have been transmitted to penguins via biting insects. it is therefore possible that the african penguin is not the primary host of pepv. when grown on the chorioallantoic membranes of embryonated 10– day old chicken eggs, the pigeonpox virus fep caused greater cell proliferation and immune cell infiltration as compared to pepv, cnpv, fwpv and some of the other avipoxvirus isolates investigated in our laboratory. furthermore, based on phylogenetic analysis of four conserved genetic regions, fep groups in clade a, subclade a and pepv belongs to clade a, sub-clade a fpvus and fp group in clade a and cnpv groups in clade b. poxviruses isolated from the african, magellanic and humboldt penguins group in clades a a and b respectively. columbiformes have been shown to be susceptible to viruses from clade a and clade b. in this study we have sequenced the genomes of two novel south african avipoxviruses using the and ion torrent platforms. we present a purpose-designed bioinformatics pipeline for analysis of next generation sequence data of avian poxviruses and compare the different avipoxviruses sequenced to date with particular attention to the viruses isolated from a feral pigeon and african penguin.  the fep and pepv genomes were assembled into contiguous sequences of  bp and  bp respectively. because the terminal hairpin loops were not sequenced, for each genome the left most nucleotide was arbitrarily nominated base phylogenetic analysis of fep and pepv phylogenetic analysis was performed using the dna polymerase gene, a concatenated amino acid sequence of translated orfs and the entire genomic sequence. all three analyses produced comparable tree topologies and the tree based on dna polymerase is given in figure  fep and pepv are most closely related to one another, grouping on the fwpv branch, and separate from cnpv. comparisons of fep pepv, fwpv and cnpv dna the four genomic sequences of fep pepv, fwpv and cnpv were compared for nucleotide percentage identity. pepv and fep are more closely related to one another than they are to either fwpv or cnpv. dot plot analysis reveals a large deletion of kbp in fep compared to both of pepv and fwpv. like the other avipoxviruses, fep and pepv have a/t rich genomes, both with % a + t content. inverted terminal repeats the fep and pepv genomes, like most other poxviruses, contain two identical inverted repeated sequences at their termini. these are  bp in fep and  bp in pepv, both of which are shorter than the itrs of the other sequenced avipoxviruses. like fpvus and cnpv, their a + t content within the itrs is lower than average - % for fep and % for pepv. at the terminal ends of each itr in fep exists a 257 bp repeat region with and copies respectively of a 34 bp and 55 bp tandem repeat. in contrast, pepv has a 165 bp repeat region containing copies of a 47 bp tandem repeat. fpvus contains a kbp region with copies of a 31-32 bp tandem repeat and cnpv contains at least and copies of 17-, 41-, and 48-bp tandem repeats respectively within the terminal regions of each itr. the repeated sequences are unique for the different avipoxviruses. overall arrangement of orfs in genome the genomes of fep and pepv have been compared to fpvus, fp and cnpv in figure  figure  provides a multiple sequence alignment and comparative orf map of fep pepv and fpvus genomes. clearly, there is conservation amongst the sequenced avipoxviruses, but, at the same time, there are major differences between them. unlike the orthopoxviruses, the avipoxviruses exhibit variation within the central core region where blocks of avipoxvirus genes have been inverted and/or transposed, suggesting that this genus is the most divergent of the chpvs. comparison of gene orthologues indicate overall genomic synteny between fep pepv and fpvus and genomic organisation is similar to that of other sequenced chpvs. additional file 1: table s lists all open reading frames potentially coding for proteins of > amino acids for fep and pepv, and figures  and represent these graphically. fep contains orfs which have been annotated as putative genes and as orthologues of fpvus and other genes. these orfs represent an approximate coding density of % and encode proteins between and amino acids. as published by hendrickson et al.,, an orf was annotated as a whole gene if intact at its 5’ end with at least 80% the length of its orthologue. an orf intact at its 5’ end but less than 80% of its orthologue length has been annotated as a truncated gene. any orf that has been disrupted at its 5’ end has been annotated as a fragmented gene, which is not expected to be transcribed and/or translated into a functional gene product. of these orfs, have been annotated as truncated genes. a further of these orfs are fragmented forms of larger orthologous genes. therefore a total of orfs have been annotated as full length putative genes. pepv contains orfs which have been annotated here as putative genes representing an approximate coding density of % and encoding proteins ranging between and amino acids long. a total of of these orfs have been annotated as whole genes, as truncated genes and as fragments of larger genes. where a gene is deleted or disrupted in only one virus, then the status of the other is given as either intact, truncated, fragmented or translocated. putative functions for the deleted genes have been given where possible. relative to the fpvus genome, fep and pepv have and deleted orfs respectively. fep contains inserted orfs relative to fpvus, comprising fragments of larger genes, truncated genes and intact genes. similarly, pepv contains inserted orfs, which include gene fragments, truncated genes and intact genes. a total of intact orfs are shared between pepv and fep throughout the genome and of these do not have any functional orthologue in fpvus. amongst pepv, fep and fwpv, the most conserved orf is the orthologue of fpv which encodes the dna binding virion core phosphoprotein and shares 100% amino acid identity amongst the three viruses. inserted genes have been listed if they are present in fep or pepv in an unexpected site as predicted by genomic synteny with the fpvus. where no homology to fpvus genes is present, the name of the best blastp hit has been used. both pepv and fep lack sequences similar to the reticuloendotheliosis virus observed in fpvus and other avipoxvirus strains.although the fep and pepv genomes are closely related and syntenic to the fwpv genome overall, when compared to each other, and to fwpv, these viruses display differences in gene content throughout their genomes. of particular note is the large kbp deletion in fep relative to both pepv and fpvus. conserved genes there are genes that are conserved amongst all five sequenced avipoxviruses, which have been highlighted in blue in figure  these include genes that lie within the more variable terminal regions of the genome. these avipoxvirus core genes were defined as such if intact orthologues of the genes were present at least once in all five avipoxvirus sequences. the apv core genes include the core genes conserved in all chpvs which are involved in essential functions such as replication, transcription and virion assembly. there are an additional genes conserved in all avipoxviruses with a variety of different functions. gene families avipoxviruses contain extensive gene families, which vary greatly between different species. in cnpv, these comprise over 49% of the genome, whereas they encompass 38% of the fwpv genome. in cnpv and fwpv, these gene families account for much of the variation between the two genomes. intact members of gene families comprise 33% of the fep and pepv genomes. table  summarises the differences in the number of intact gene family proteins found in the five sequenced avipoxvirus genomes. a summary of the numbers of intact gene family proteins in pigeonpox virus, penguinpox virus, fowlpox virus us strain, fowlpox virus fp strain, canarypox virus and vaccinia virus. “0” is given where no member of the gene family is present in the respective virus. gene translocations and duplications analysis of the fep and pepv genomes revealed several occurances of gene duplication and translocation relative to fwpv. in some instances the postion of the gene was closer to that observed in cnpv than fwpv. a second copy of the ankyrin repeat family gene, fpv orthologue is found in the equivalent place in the left hand region of each african avipoxvirus genome. disrupted and deleted genes relative to fpvus, fep contains truncated fpvus genes and the fragmented forms of fpvus orthologues. pepv contains truncated genes and the orf fragments of fpvus orthologues. both fep and pepv contain truncated or fragmented remains of orfs with similarity to cnpv. relative to the fpvus genome, fep and pepv have and deleted orfs respectively that are absent from any potential coding regions of their genomes. inserted genes relative to fwpv, the pepv and fep genomes both contain several inserted genes. pepv, but not fep or fwpv, has orthologues of cnpv which encodes a protein similar to tumour necrosis factor receptor and a amino acid long orthologue of ubiquitin which is most similar to human ubiquitin, s5a ubiquitin-interacting motif-. both pepv and fep contain orthologues of thymidylate kinase. fep and pepv both encode a putative interleukin- gene with % amino acid identity between them and limited similarity to the cnpv orthologue. amino acid identity between all available avian il- genes and pepv and fep fall between % and %, however for cnpv the identities are significantly lower. the pepv, fep and cnpv il- genes are most similar to that of a collared flycatcher sharing %, % and % amino acid identity respectively. fwpv does not encode an il- gene. the respective positions of fep pepv and cnpv il- orthologues were analysed to determine the possibility of these genes existing in the three genomes as a result of independent horizontal gene transfer events. based on this analysis, it is possible that there were two independent transfer events into avian poxviruses, one into cnpv and another into the progenitor of pepv and fep pepv and fep both encode homologues of tanapox virus and yaba-like disease virus 67r which are orthologues of vacv c7l. these c7l-like genes are in the equivalent genome positions, between orthologues of fpv and fpv. comparison of fep and pepv to attenuation and lineage specific mutations in order to determine lineage specific mutations, laidlaw and skinner determined the sequence of hp, the virulent parent of fp at all loci where fp differed from fpvus. here we compared nucleotide changes in pepv and fep to genomic positions influencing amino acid composition of open reading frames between european and american strains as previously identified. in and of the places, fep and pepv were the same as hp respectively. in and of the places, fep and pepv were the same as fpvus respectively. pepv and fep differ from both hp and fpvus in and places respectively. discussion genomic sequences have been determined for two novel south african avipoxviruses. the dna sequences of pepv and fep are significantly different from those of cnpv and fwpv. in orthopoxviruses, the internal region of different species share at least 96% identity when compared at the nucleotide level, while different strains from the same species share at least 99% nucleotide sequence identity. these identities have been listed as criteria for establishing poxvirus taxonomy. therefore, according to the identities between pepv, fep and fpvus genomes, as well as the differences in phylogenetics and growth characteristics between these two viruses, one can deduce that pepv and fep are separate species of apv. laidlaw and skinner identified geographical lineage-specific mutations which distinguished european fowlpox viruses, fp and hp from the american strain fpvus. a comparison of pepv and fep with the published fwpv sequences at these sites shows that pepv and fep share lineage-specific mutations with both hp and fpvus. the south african viruses are no more closely related to either the american or the european virus. these results suggest that fep and pepv originate from a common ancestor that diverged relatively recently from a fwpv-like progenitor which was more distantly related to the cnpv-like progenitor. it is important to note that the divergence between avipoxvirus species is comparable to that between some poxvirus genera and the different species within the opv genus are highly similar. fep and pepv are most closely related to fwpv. despite this, relative to fwpv, the pepv and fep genomes both contain several genes that are more closely related to cnpv throughout their genomes. fep and pepv encode and intact cnpv orthologues respectively, five of which are not in fwpv, including a tnfr-like protein and a tmpk protein. the remainder are more similar to cnpv orthologues but are present in fwpv. rather than being acquired through horizontal gene transfer or recombination, these genes are most probably from a common ancestor. their existence suggests that fep and pepv viruses diverged prior to the present day fwpv, somewhere between the cnpv and fwpv branches. several of these cnpv-like genes exist as gene fragments, which is indicative of their gradual loss through progressive mutation, rearrangements and/or deletions over many rounds of virus replication. there are several instances of gene duplication and translocation in fep and pepv relative to fwpv. most of these involve genes containing ankyrin repeats. virulent fwpv contains ankyrin repeat containing genes and attenuated fp contains of these genes. pepv contains ankyrin repeat-containing genes whereas fep contains only. it is postulated that loss of these genes may be responsible for the attenuation of the virus. thus the reduced numbers of intact ankyrin repeat genes observed in fep compared to pepv would suggest that this virus is less virulent. on the other hand, the expansion of the number of ankyrin repeat genes in certain avipoxvirus strains could represent the forming of adaptive genomic accordions, similar to that of the k3l gene in opv, the formation of which has been described to play an adaptive role in these viruses. the most striking difference between fep and the fwpv-like avipoxviruses, is a large deletion of ~ kbp from the central, usually conserved, region of the genome. orfs corresponding to fpv, fpv and fpv, fpv and fpv are deleted in fep at this site. the existence of gene fragments and insertions on the borders of this deletion, as well as in the equivalent regions in fwpv and pepv, suggests that this is a “hotspot” of genetic change, the mechanism of which is uncertain. relative to the fpvus genome, fep and pepv have and deleted orfs respectively. most of the genes deleted in pepv and fep are members of multi-gene families, the disruption of which has been implicated in the attenuation of poxviruses, however there are several other deletions of significant orfs. of interest, the orthologue of fpv encoding a dnase ii has been deleted in fep but is in intact in pepv. in fwpv, fpv represents the large subunit of cellular dnase ii and cellular dnase ii is thought to function in dna catabolism during apoptosis. additionally, the orthologue of fpv encoding one of the two α-snap genes present in fwpv, is absent from fep but intact in pepv. eukaryotic α-type soluble nsf attachment proteins are involved in vesicular transport through the golgi apparatus and for exocytosis. the fpv gene has been shown to be conserved in fwpv strains but non-essential to viral replication and it is thought to be involved in virus-host interactions. although the fpv gene orthologue has been deleted in fep a second α-snap-like gene exists in the genome which exhibits % and % amino acid identity with fpv and pepv respectively. fwpv contains two genes with homology to cellular β-nerve growth factor. in fep the orthologue of fpv is completely deleted and the orthologue of fpv is fragmented. conversely, in pepv, the orthologue of fpv is completely deleted and the orthologue of fpv is fragmented. an additional β-ngf-like gene is observed in a truncated form in fep and as a fragmented gene in pepv. these are most similar to the cnpv gene cnpv which exists as a fragment in fwpv. the fep orf, fep is truncated to amino acids by the introduction of a premature stop codon, where the intact cnpv orthologue is amino acids. it is unknown whether this protein would be functional in fep but it is unlikely that pepv encodes a functional β-ngf protein. β-ngf has proinflammatory properties and is produced by cells in response to infection, injury and stress. fpv encoded β-ngf is thought to be involved in promoting survival in infected cells and could have a role in inhibiting antiviral immune responses in the host. previously, it was speculated that the absence of a β-ngf gene in an avipoxvirus genome could be partly responsible for the decreased inflammatory response observed in pepv-infected cams as compared to fep. an orthologue of fpv encoding an il- binding protein has been deleted in pepv. in fep this orf has been truncated due to a 1 bp deletion resulting in a premature stop codon. il- is a multifunctional pro-inflammatory cytokine that induces interferon gamma production, th- responses and nk cell activity. it is modulated in vivo by il- binding protein via a negative feedback mechanism. il18bp homologues are encoded by many poxviruses and have been found to inhibit il-18-dependent ifn-γ production. in fwpv, fpv is thought to have an anti-inflammatory function. pepv contains an orthologue of cnpv which encodes a protein similar to tnf receptor. this orf is absent from fep and fwpv. tumour necrosis factor is a pro-inflammatory cytokine which is involved in induction of cytokines, cell proliferation, differentiation, necrosis and apoptosis. tnf -induced cellular responses are mediated by one of two types of tnf receptors. several poxviruses encode tnfr homologues which function to modulate tnf-induced anti-viral responses. pepv and fep both contain an orthologue of thymidylate kinase, which is absent in fwpv. this is the only difference found with respect to the complement of nucleotide metabolism genes. interestingly, despite being within the conserved central region of the genome, the tmpk gene orthologues occur within a region of pepv and fep that is highly variable compared to fwpv. in addition, the cnpv orthologue insertions that occur within this particular variable region in fep and pepv occur in different sites across the cnpv genome. the higher than average amino acid identity shared between fep and pepv and the cnpv genomes, suggests that this tmpk gene is necessary and conserved in these viruses. cnpv, fep and pepv are the only poxvirus species outside of the opv genus to contain a tmpk homologue. vacv encodes tmpk that is 42% identical to human tmpk. tmpk catalyzes an important step in the biosynthesis of thymidine triphosphate and is essential for cell metabolism. in cnpv, fep and pepv, the presence of a tmpk gene suggests a different optimization of cellular nucleotide pools as compared to fwpv and probably affects cell and tissue tropism in the different viruses. pepv contains a amino acid long orthologue of ubiquitin which is most similar to human ubiquitin, s5a ubiquitin-interacting motif-. cnpv also contains an intact ubiquitin orthologue. fep and fpvus encode the fragmented remains of an ubiquitin gene but no open reading frame is observed in either genome. in all four genomes these ubiquitin orfs or sequence remains are in the same location. ubiquitin is a highly conserved amino acid protein with diverse cellular functions effected by means of marking proteins for intracellular signalling. ubiquitin proteins are thought to affect the diverse ubiquitination-mediated cellular functions, which may impact on cnpv and pepv virulence and host range. other than cnpv and pepv, ubiquitin genes have only been found in two insect poxviruses, melanoplus sanguinipes and amsacta moorei. virus-encoded ubiquitin genes have also been identified in the baculoviridae, including in the autographa californica nuclear polyhedrosis virus. in acnpv, disruption of the ubiquitin gene was shown to have no effect on virus viability but to cause a decrease in virion budding and total infectious particle production. the ubiquitin encoding genes in pepv and cnpv may have an effect on virus production and budding but this remains to be determined. fep and pepv both encode a putative interleukin- gene. putative orthologues of il- are also found in orf virus, bovine papular stomatitis virus, lumpy skin disease virus and yaba-like disease virus. il- is a cytokine that has both immunostimulatory and immunosuppressive functions and the orf virus il- orthologue has been shown to be immunomodulatory in function. the il- genes encoded by pepv and fep may also be involved immune evasion, however, because of the divergence from host il- proteins, like cnpv these genes may have a novel biological function. the il- gene is likely to have been incorporated into poxvirus genomes via horizontal gene transfer. bratke and mclysacht made use of comparative genomic information and synteny conservation to investigate hgt in pox genomes. here they found that the il- orthologue encoded by cnpv was in an unexpected location as compared to other poxviruses but because of the lack of conservation at this region, no conclusion could be drawn about the transfer event into cnpv. an analysis of the genome synteny of avipoxviruses in the region of the il- gene shows that the cnpv il- is in a different location despite being in a similar region to fep and pepv. although this region is not well conserved between genera, it appears to be relatively well conserved between avian poxviruses making it possible that there were two independent transfer events into avian poxviruses, one into cnpv and one into the common ancestor of fep and pepv. pepv and fep both encode a gene with homology to tanapox virus and yaba-like disease virus 67r which are orthologues of vacv c7l and not found in fwpv or cnpv. the poxvirus c7l family of host range genes functions by mediating poxvirus host range and antagonising the host defence system. vacv c7l orthologues are found in all orthopoxviruses and most mammalian poxviruses, with the exception of molluscum contagiosum virus and parapoxviruses. this is the first report of a c7l-like gene in avipoxviruses. in mammalian cells, c7l has been shown to inhibit apoptosis, antagonise the anti-viral effects of type interferons and interferon regulatory factor and can antagonize the dsrna-activated protein kinase pathway by inhibiting the phosphorylation of eif2a. it has previously been suggested that c7l orthologues are an important adaptation of mammalian poxviruses for replication in mammalian hosts. the presence of c7l orthologues in pepv and fep confounds these previous observations but the function of this gene in these two avipoxviruses remains to be determined. the limited amino acid identity may suggest a novel function of these genes as compared to other c7l orthologues. however, yldv 67 l shows only 28-30% identity to the vacv c7l protein but has been shown to function equivalently in supporting vacv replication in mammalian hosts.  the genome sequences of fep and pepv have greatly added to the limited repository of genomic information available for the avipoxvirus genus. in the comparison of fep and pepv to existing sequences, fwpv and cnpv, we have established insights into african avipoxvirus evolution. although fep and pepv are more closely related to fwpv, the presence of whole or disrupted genes with similarity to cnpv genes that are absent in fwpv, suggests that fep and pepv originate from the common ancestor of cnpv and fwpv. the presence of an intact gene in cnpv, fep and pepv where the fwpv counterpart is fragmented into two orfs further supports this theory as fragmented genes represent the gradual loss of genetic information during the process of virus evolution. additional genome sequences of avipoxviruses would help to define avipoxvirus evolution as a whole. the ongoing search for an ideal vaccine vector makes this work relevant. future work could focus on how these two avipoxviruses differ from the well characterized fwpv and cnpv with respect to immune activation and foreign gene expression. 
we present a purpose-designed bioinformatics pipeline for analysis of next generation sequence data of avian poxviruses and compare the different avipoxviruses sequenced to date with specific emphasis on their evolution and gene content. the fep and pepv genomes encode and open reading frames respectively and are more closely related to one another than to either fowlpox virus or canarypox virus. fep and pepv both encode orthologues of vaccinia virus c7l and interleukin pepv contains a amino acid long orthologue of ubiquitin sharing 97% amino acid identity to human ubiquitin. the most striking difference between fep and the fwpv-like avipoxviruses is a large deletion of ~ kbp from the central region of the genome of fep deleting a cc-chemokine-like gene, two variola virus b22r orthologues, an n1r/p28-like gene and a v-type ig domain family gene. two novel avipoxviruses from south africa have been sequenced, one from a feral pigeon and the other from an african penguin. the genome sequences of fep and pepv have greatly added to the limited repository of genomic information available for the avipoxvirus genus. our data supports the independent evolution of these south african avipoxviruses from a common ancestral virus to fwpv and cnpv. overall, fep pepv and fwpv have syntenic gene arrangements; however, major differences exist throughout their genomes. in the comparison of fep and pepv to existing sequences, fwpv and cnpv, we have established insights into african avipoxvirus evolution.
 two novel avipoxviruses from south africa have been sequenced, one from a feral pigeon and the other from an african penguin. we present a purpose-designed bioinformatics pipeline for analysis of next generation sequence data of avian poxviruses and compare the different avipoxviruses sequenced to date with specific emphasis on their evolution and gene content.  the fep and pepv genomes encode and open reading frames respectively and are more closely related to one another than to either fowlpox virus or canarypox virus. overall, fep pepv and fwpv have syntenic gene arrangements; however, major differences exist throughout their genomes. the most striking difference between fep and the fwpv-like avipoxviruses is a large deletion of ~ kbp from the central region of the genome of fep deleting a cc-chemokine-like gene, two variola virus b22r orthologues, an n1r/p28-like gene and a v-type ig domain family gene. fep and pepv both encode orthologues of vaccinia virus c7l and interleukin pepv contains a amino acid long orthologue of ubiquitin sharing 97% amino acid identity to human ubiquitin.  the genome sequences of fep and pepv have greatly added to the limited repository of genomic information available for the avipoxvirus genus. in the comparison of fep and pepv to existing sequences, fwpv and cnpv, we have established insights into african avipoxvirus evolution. our data supports the independent evolution of these south african avipoxviruses from a common ancestral virus to fwpv and cnpv. avipoxviruspoxviruspigeonpoxpenguinpoxgenomevectors 