Genomic sequences have been determined for two novel South African avipoxviruses. The DNA sequences of PEPV and FeP2 are significantly different from those of CNPV (approx. 63% identity) and FWPV (approx. 85% identity). In Orthopoxviruses (OPVs), 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 [27–33]. These identities have been listed as criteria for establishing poxvirus taxonomy . Therefore, according to the identities between PEPV, FeP2 and FPVUS genomes (94.4% between PEPV and FeP2, 85.3% between PEPV and FPVUS and 84.0% between FeP2 and FPVUS), as well as the differences in phylogenetics and growth characteristics between these two viruses , one can deduce that PEPV and FeP2 are separate species of APV.
Laidlaw and Skinner  identified geographical lineage-specific mutations which distinguished European fowlpox viruses, FP9 and HP1 from the American strain FPVUS. A comparison of PEPV and FeP2 with the published FWPV sequences at these sites shows that PEPV and FeP2 share lineage-specific mutations with both HP1 and FPVUS. The South African viruses are no more closely related to either the American or the European virus. These results suggest that FeP2 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.
FeP2 and PEPV are most closely related to FWPV. Despite this, relative to FWPV, the PEPV and FeP2 genomes both contain several genes that are more closely related to CNPV throughout their genomes. FeP2 and PEPV encode 16 and 15 intact CNPV orthologues respectively (Table 3), five of which are not in FWPV, including a TNFR-like protein (cnpv086, pepv132) and a TMPK protein (cnpv170, fep126, pepv133) (Additional file 1: Table S1 and Table 3). 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 FeP2 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 (Table 2), 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 FeP2 and PEPV relative to FWPV (Figures 3 and 4). Most of these involve genes containing ankyrin repeats. Virulent FWPV contains 31 ankyrin repeat containing genes and attenuated FP9 contains 22 of these genes. PEPV contains 33 ankyrin repeat-containing genes whereas FeP2 contains only 26 (Additional file 1: Table S1; Table 3). It is postulated that loss of these genes may be responsible for the attenuation of the virus [9, 10]. Thus the reduced numbers of intact ankyrin repeat genes observed in FeP2 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 FeP2 and the FWPV-like avipoxviruses, is a large deletion of ~16 kbp from the central, usually conserved, region of the genome. ORFs corresponding to fpv121 (cc-chemokine family), fpv122 and fpv123 (VAR B22R family), fpv124 (N1R/p28 family) and fpv125 (V-type Ig domain gene family) are deleted in FeP2 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, FeP2 and PEPV have 36 and 25 deleted ORFs respectively (Table 1). Most of the genes deleted in PEPV and FeP2 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 fpv032 encoding a DNase II has been deleted in FeP2 but is in intact in PEPV. In FWPV, fpv032 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 fpv033 encoding one of the two α-SNAP genes present in FWPV, is absent from FeP2 but intact in PEPV (pepv038). Eukaryotic α-type soluble NSF attachment proteins (α-SNAP) are involved in vesicular transport through the Golgi apparatus and for exocytosis . The fpv033 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 fpv033 gene orthologue has been deleted in FeP2, a second α-SNAP-like gene (fpv011, pepv012) exists in the genome which exhibits 34.0% and 33.8% amino acid identity with fpv033 and pepv038 respectively.
FWPV contains two genes with homology to cellular β-nerve growth factor (β-NGF) (fpv072 and fpv076). In FeP2, the orthologue of fpv076 is completely deleted and the orthologue of fpv072 (fep074) is fragmented (Table 1). Conversely, in PEPV, the orthologue of fpv072 is completely deleted and the orthologue of fpv076 (pep079) is fragmented (Table 1). An additional β-NGF-like gene is observed in a truncated form in FeP2 (fep213) and as a fragmented gene in PEPV (pepv221) (Table 2). These are most similar to the CNPV gene cnpv279, which exists as a fragment in FWPV . The FeP2 ORF, fep213 is truncated to 67 amino acids by the introduction of a premature stop codon, where the intact cnpv279 orthologue is 169 amino acids. It is unknown whether this protein would be functional in FeP2 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 FeP2 .
An orthologue of fpv073 encoding an IL-18 binding protein has been deleted in PEPV. In FeP2 this ORF (fep075) has been truncated due to a 1 bp deletion resulting in a premature stop codon. IL-18 is a multifunctional pro-inflammatory cytokine that induces interferon gamma (IFN-γ) production, Th-1 responses and NK cell activity . It is modulated in vivo by IL-18 binding protein (IL18BP) via a negative feedback mechanism . IL18BP homologues are encoded by many poxviruses [9, 11, 41, 42] and have been found to inhibit IL-18-dependent IFN-γ production . In FWPV, fpv073 is thought to have an anti-inflammatory function .
PEPV contains an orthologue of cnpv086 (pepv132) which encodes a protein similar to TNF receptor . This ORF is absent from FeP2 (Additional file 1: Table S1) and FWPV . Tumour Necrosis Factor (TNF) 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 [11, 45–47].
PEPV (pepv133) and FeP2 (fep126) both contain an orthologue of thymidylate kinase (TMPK) (cnpv170; VACV A48R), 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 FeP2 that is highly variable compared to FWPV. In addition, the CNPV orthologue insertions that occur within this particular variable region in FeP2 and PEPV occur in different sites across the CNPV genome. The higher than average amino acid identity shared between FeP2 and PEPV (90.8%) and the CNPV genomes (81.6% and 80.2% respectively), suggests that this TMPK gene is necessary and conserved in these viruses. CNPV, FeP2 and PEPV are the only poxvirus species outside of the OPV genus to contain a TMPK homologue. VACV encodes TMPK (A48R) that is 42% identical to human TMPK . TMPK catalyzes an important step in the biosynthesis of (deoxy) thymidine triphosphate and is essential for cell metabolism . In CNPV, FeP2 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 77 amino acid long orthologue of Ubiquitin (pepv074) which is most similar to human ubiquitin (97% BLASTp identity), S5a ubiquitin-interacting motif-1 (UIM-1)(Accession: 1YX5_B). CNPV also contains an intact ubiquitin orthologue (cnpv096) (86.1% amino acid identity to pepv074) . FeP2 (nt81,771-81,622) and FPVUS (nt74,550-74,220) 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 (between orthologues of fpv070.5 and fpv071). Ubiquitin is a highly conserved 76 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 (MSEV)  and Amsacta moorei (AMEV) . Virus-encoded ubiquitin genes have also been identified in the Baculoviridae, including in the Autographa californica nuclear polyhedrosis virus (AcNPV) . 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.
FeP2 and PEPV both encode a putative Interleukin-10 (IL-10) gene (Figures 3 and 4). Putative orthologues of IL-10 are also found in ORF virus, Bovine Papular Stomatitis Virus (BPSV) , Lumpy skin disease virus (LSDV)  and Yaba-Like disease virus (YLDV) . IL-10 is a cytokine that has both immunostimulatory and immunosuppressive functions  and the ORF virus IL-10 orthologue has been shown to be immunomodulatory in function . The IL-10 genes encoded by PEPV and FeP2 may also be involved immune evasion, however, because of the divergence from host IL-10 proteins (20-31% amino acid identities to various avian IL-10 genes), like cnpv018, these genes may have a novel biological function .
The IL-10 gene is likely to have been incorporated into poxvirus genomes via horizontal gene transfer (HGT) . Bratke and McLysacht  made use of comparative genomic information and synteny conservation to investigate HGT in pox genomes . Here they found that the IL-10 orthologue encoded by CNPV was in an unexpected location as compared to other poxviruses (yatapox and capripox) 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-10 gene shows that the CNPV IL-10 is in a different location despite being in a similar region to FeP2 and PEPV (Figure 5). 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 FeP2 and PEPV.
PEPV and FeP2 both encode a gene with homology to Tanapox virus (TANV) and Yaba-Like Disease virus (YLDV) 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 1 interferons (IFNs) and Interferon Regulatory Factor 1 (IRF-1)  and can antagonize the dsRNA-activated protein kinase (PKR) 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 FeP2 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 .