Esteban and Hutchinson BMC Genomics 2011, 12:261 http://www.biomedcentral.com/1471-2164/12/261 Page 7 of 12 Figure 3 Functional categories of the genes under diversifying selection . A) Functions of the 25 genes identified by both models. B) Functions of 43 non-virion-associated proteins identified by model M8, B) Functions of 36 virion-associated proteins identified by model M8. content and gene sequences [2]. Many terminally located genes are host response modifiers that directly interact with components of the host immune response or cellular response to infection. Grouping the genes identified in this study into broad functional categories (Figure 3), we identified several host response modifiers and host range genes that have sites under diversifying selection. These may be sites that are involved in host- specific interactions, demonstrating adaptation to the virus’s particular host. Previous studies have also demonstrated diversifying selection in poxvirus host response modifying genes [19-21]. The current study uses the largest dataset providing the most comprehen- sive analysis of diversifying selection in poxvirus gen- omes. Among the host response modifiers identified were several secreted immunomodulators (IL-18 binding protein, IL-1 beta receptor and IFN gamma receptor) that are known to contribute to virulence [22-24]. Genes that modulate the cellular response to infection were also identified, including the mitochondrial asso- ciated apoptosis inhibitor, an inhibitor of the protein kinase R (PKR) response to double stranded RNA, a ubi- quitin ligase, and an inhibitor of Toll-like receptor sig- naling [25-28]. In addition to host response modifying genes, we also showed, using model M8, that genes involved in viral replication and virion structure experience diver- sifying selection, possibly due to their protein products being packaged in the virion. A proteomics study [17] identified 75 viral proteins in the VACV virion, 18 of which are present at abundances greater than 1% of the weight of the virion. Of those, 6 were identified in the current study, including the major core protein (A4), the most abundant protein in the virion by weight. Further, the immune response to poxvirus infection induces the formation of neutralizing antibo- dies to several virion membrane proteins [29,30]. We identified 3 known major targets of neutralizing anti- bodies: an immature virion membrane protein (A7, CPXV-BR-154), carbonic anhydrase (D8, CPXV-BR- 129) and an enveloped virion protein (B5, CPXV-BR- 205). Thus detection of diversifying selection is not limited to host response modifying genes but more broadly to genes whose products interact with the host, such as major antigens. Designation of families in the VOCs database was based on a BLAST expect value of 10-17, thus families are likely to be composed of orthologs [2]. Horizontal gene transfer (HGT) is recognized as a factor in evolu- tion of poxvirus genomes. It is possible that some of the genes within a gene family may not be orthologs if they arose through multiple independent horizontal gene transfer events. Several lines of evidence are needed to demonstrate HGT, including phylogenetic clustering of the gene in taxa unrelated to the genome under study. The origins of most poxvirus genes are unknown, although most chordopoxvirus genes show greater simi- larity to eukaryotic genes than to other viral genes [31]. Full phylogenies of each poxvirus gene family are needed to determine if multiple horizontal gene transfer events have occurred within the family. Such research is underway and will be valuable in further interpretation of the current data.