Esteban and Hutchinson BMC Genomics 2011, 12:261 http://www.biomedcentral.com/1471-2164/12/261 poxviruses, the C-terminus (containing the BH domains) of the F1L family is highly conserved. There are 7 resi- dues under diversifying selection located in this domain (Figure 4B). Of those, 2 residues identified in this study are located in the BH domains. BH3 and BH1, primarily located on alpha helices a2 and a5, make up the BH3 binding pocket responsible for binding Bak [37]. One residue (A173 CPXV) identified in this study, corre- sponding to A144 in the F1L homolog of Modified Vac- cinia Ankara (MVA), is located in the binding pocket and was shown by mutagenesis to increase binding affi- nity if mutated to phenylalanine. M124 in CPXV (I95 in MVA) is located in a2 and therefore could be involved in ligand binding or in determining the shape of the pocket. Another 3 sites under diversifying selection are located immediately C-terminal to BH2. Overall, the capacity of the Bayes empirical Bayes analysis to identify residues known to be important in protein function, such as in the IL-18BP and mitochondrial associated apoptosis inhibitor, suggests that it may be valuable to test other predicted sites across the genome for their role in protein function. The identification of host-interacting genes in pox- viruses as ones experiencing adaptive molecular evolu- tion is consistent with the findings of several other studies identifying genes involved in the “host-pathogen arms race” or other co-evolutionary processes and is seen throughout nature. Chitinase and other plant defense proteins show evidence of diversifying selection, and mutagenesis studies have confirmed the functional importance of the identified sites [32,38]. The wsp gene of the bacterium Wolbachia, which encodes an outer membrane protein, shows evidence of diversifying selec- tion when in a parasitic relationship with arthropods, but not in a mutualistic relationship with nematodes [39]. In other viruses, all the major genes of HIV [40,41], and the capsid protein of Foot-and-Mouth Dis- ease virus (FMDV) [33] experience diversifying selection. Importantly, the amino acids identified computationally in the FMDV capsid are known to be antigenic sites identified by monoclonal antibody escape mutants [33]. In host species, diversifying selection has been shown in the antigen recognition sites of the major histocompat- ibility (MHC) gene [42,43]. As has been demonstrated with the FMDV capsid pro- tein [33], interaction between an antigen and the immune response can drive diversifying selection. Sev- eral studies have found evidence of diversifying selection in surface proteins on viruses, including HIV env, influ- enza hemagglutinin, and others [44-46]. Recent proteo- mics studies have identified the major and minor virion- associated proteins [16,17]. In this study, 39% of the genes identified are associated with the virion (Figure 3). Virion structural components and virion-associated Page 9 of 12 enzymes may have greater exposure to antibody responses, leading to diversifying selection. A few small whole genomes have been analyzed for diversifying selection, showing that diversifying selection can play strikingly different roles in the molecular evolu- tion of organisms. A study of Picornaviridae shows evi- dence of diversifying selection in structural proteins but not in non-structural proteins [47]. Only a few codons in the astrovirus genome are under diversifying selection [48], while 9-38% of sites in human rhinoviruses experi- ence diversifying selection [49]. The poxvirus genome is significantly larger than any other viral genome analyzed by this method, and the results indicate a more impor- tant role for diversifying selection in poxvirus genome evolution than in other viruses. This may be related to the large size of the poxvirus genome and the large number of accessory “non-essential” genes such as the host-response modifiers or the large number of encapsi- dated proteins. Over 1700 genes of the Streptococcus genome were analyzed and approximately 8% of the genes were found to be under diversifying selection [50]. Of those, 29% are related to virulence, and many others show tissue specific expression during invasive disease. A large fraction of poxvirus genes under diversifying selection are also known to be virulence factors, echoing the findings in Streptococcus. In Streptococcus, several essential core function genes were identified, indicating that virulence is more com- plex than simply the presence of pathogen associated genes [50]. Similarly, analysis of E. coli genomes found 29 genes common to pathogenic and non-pathogenic strains which showed evidence of diversifying selection, and many of which are involved in functions like DNA metabolism and nutrient acquisition [51]. Several core poxvirus genes, not typically thought of as virulence fac- tors, were also found to be under diversifying selection in the poxvirus genome. Some (but not all) of these are packaged in the virion and may be exposed to the host antibody response. However, this suggests that other poxvirus systems, in addition to manipulation of the host response, may be important in virulence. In attempting to explain virulence differences between strains of poxviruses, it may therefore be important to consider not only major genomic differences such as gene complement, but also diversifying selection in well conserved genes. Conclusions The identification of 79 genes in the orthopoxviruses that experience diversifying selection implicates this as a major mechanism of poxvirus evolution. Because many of these genes either interact with host defense mechan- isms or may be targets of the immune response, interac- tion with the host may be the basis for adaptive